The L&K Process Guide

L&K Biosciences
Solutions for biotechnology
The L&K Process Guide

March 2009
The L&K ProcessGuide at www.lk-processguide.com is a web based tool for biopharmaceutical process design taking all aspects of the chemistry, manufacture and control (CMC) package into consideration. The aim of introducing this database is to shorten time to market by introducing better process design with strong focus on safety, process robustness, and Good Manufacturing Practices. The database is probably the most comprehensive tool for process design. The Guide is password protected and a license from L&K Biosciences must be obtained before access is granted. A short version is available at www.lk-processguide.com for free. The Guide consists of documents written specifically for industrial applications. It is organised in a way that reflects the biopharmaceutical drug development programme including categories describing cell line development, expression systems, process design, quality control, quality assurance, and documentation helping the reader to plan for later critical stages, such as scale up and process validation. Good planning will reduce the risk of process redesign, failed batches and costly delays. Documents are based on more than 30 years of experience in the biotech industry and carefully selected peer reviewed papers (cited in each document). The Guide is undergoing continuous assessment and documents are updated on a regular basis making the Guide an up-to-date tool for anyone involved in process design strategies and execution of activities. The short version available at www.lk-processguide.com provides an excellent overview of the guide structure and items handled. This is a pri9nted version of the web site.
L&K Biosciences ApS Sophienberg Vaenge 30 DK-2960 Rungsted Kyst Denmark ls@lk-biosciences.com +45 2023 2346
L&K Biosciences ApS 2008-2009
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Contents
1.0 Biopharmaceutical drug development...............................................................7 Introduction..................................................................................................................7 Expression systems .....................................................................................................7 Process design ............................................................................................................7 Protein stability ............................................................................................................8 Scale up and manufacture ...........................................................................................8 Quality control..............................................................................................................9 Quality assurance ...................................................................................................... 10 Documentation .......................................................................................................... 11 Pre-clinical studies ..................................................................................................... 11 The Investigational New Drug Application (INDA)....................................................... 12 Clinical trials .............................................................................................................. 12 Phase 1 clinical trials .............................................................................................. 12 Phase 2 clinical trials .............................................................................................. 12 Phase 3 clinical trial................................................................................................ 12 Phase 4 trials ......................................................................................................... 13 The license application .............................................................................................. 13 2.0 Expression systems ......................................................................................... 14 Introduction................................................................................................................ 14 Expression systems ................................................................................................... 14 E coli ...................................................................................................................... 14 Yeast ..................................................................................................................... 16 Insect cells ............................................................................................................. 17 Mammalian cells .................................................................................................... 17 Transgenic animals ................................................................................................ 18 Cell lines.................................................................................................................... 19 Cell banks ................................................................................................................. 19 Research cell banks ............................................................................................... 19 Master cell banks ................................................................................................... 20 Working cell banks ................................................................................................. 20 3.0 Process design ................................................................................................. 21 Introduction................................................................................................................ 21 Upstream ................................................................................................................... 22 Introduction ............................................................................................................ 22 Batch and fed batch mode ...................................................................................... 22 Perfusion mode ...................................................................................................... 22 Harvest ...................................................................................................................... 22 Downstream .............................................................................................................. 23 Introduction ............................................................................................................ 23 Buffers and co-solvent............................................................................................ 24 Techniques ............................................................................................................ 24
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Formulation ............................................................................................................... 32 Introduction ............................................................................................................ 32 Freeze-thaw ........................................................................................................... 33 Lyophilization ......................................................................................................... 33 Development program ............................................................................................ 34 Excipients .............................................................................................................. 34 Fill & finish ................................................................................................................. 34 4.0 Protein stability ................................................................................................. 36 Introduction................................................................................................................ 36 Temperature .............................................................................................................. 36 Factors affecting protein stability ................................................................................ 37 pH .......................................................................................................................... 37 Temperature .......................................................................................................... 38 Conductivity ........................................................................................................... 38 Redox potential ...................................................................................................... 38 Protein concentration ............................................................................................. 38 Holding and storage time........................................................................................ 39 Co-solvents ............................................................................................................ 39 Other proteins ........................................................................................................ 39 Shear forces........................................................................................................... 40 Protein stability during processing .............................................................................. 40 Upstream ............................................................................................................... 40 Downstream ........................................................................................................... 40 Formulation ............................................................................................................ 40 Control of protein stability ....................................................................................... 40 Protein stability during storage ................................................................................... 41 Intermediary compound stability studies ................................................................. 41 Drug substance stability studies ............................................................................. 41 Drug product stability studies.................................................................................. 41 Stability studies of products used for phase I-III clinical trials .................................. 42 Analytical testing .................................................................................................... 42 Accelerated and stress conditions .......................................................................... 42 Physical instability...................................................................................................... 42 Denaturation .......................................................................................................... 42 Aggregation............................................................................................................ 43 Precipitation ........................................................................................................... 43 Chemical instability .................................................................................................... 43 Proteolysis ............................................................................................................. 43 Deamidation ........................................................................................................... 44 Oxidation................................................................................................................ 44 Carbamylation ........................................................................................................ 44 -elimination............................................................................................................. 45 Racemization ......................................................................................................... 45 Cysteine residues ................................................................................................... 45 Hydrolysis .............................................................................................................. 45 5.0 Scale up and manufacture................................................................................ 46 Introduction................................................................................................................ 46
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When, how and why?................................................................................................. 47 Technical batches...................................................................................................... 48 Toxicology batches .................................................................................................... 49 GMP batches............................................................................................................. 49 6.0 Quality control .................................................................................................. 51 Introduction................................................................................................................ 51 Cell banks ................................................................................................................. 51 Raw materials............................................................................................................ 51 In process control ...................................................................................................... 51 Control of drug substance and drug product ............................................................... 52 Reference material .................................................................................................... 53 Analytical methods..................................................................................................... 53 Amino acid composition.......................................................................................... 53 Amino acid sequence ............................................................................................. 53 Bicinchoninic assay ................................................................................................ 54 Bioburden .............................................................................................................. 54 Biuret assay ........................................................................................................... 54 Bradford assay ....................................................................................................... 54 Capillary electrophoresis ........................................................................................ 54 Circular dicroism .................................................................................................... 54 Differential scanning caloriemetry ........................................................................... 54 ELISA .................................................................................................................... 54 Fluorescence spectrometry .................................................................................... 54 High performance chromatography......................................................................... 54 Infrared spectrometry ............................................................................................. 55 Isoelectric focusing ................................................................................................. 55 Kjeldahl analysis .................................................................................................... 55 Limulus amebocyte lysate ...................................................................................... 55 Lowry Assay ........................................................................................................... 55 Mass spectroscopy................................................................................................. 55 Microbial testing ..................................................................................................... 55 Native electrophoresis ............................................................................................ 55 Nuclear magnetic resonance .................................................................................. 56 O-pthalaldialdehyde assay ..................................................................................... 56 Peptide mapping .................................................................................................... 56 Polymerase chain reaction ..................................................................................... 56 SDS-PAGE ............................................................................................................ 56 2D-electrophoresis ................................................................................................. 56 UV-absorbance ...................................................................................................... 56 X-ray diffraction ...................................................................................................... 56 Impurities................................................................................................................... 57 Host cell related impurities...................................................................................... 57 Process related impurities ...................................................................................... 58 Product related impurities ....................................................................................... 58 7.0 Quality assurance ............................................................................................. 60 Introduction................................................................................................................ 60 Regulatory affairs....................................................................................................... 60
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The role of Quality Assurance .................................................................................... 61 Specification .............................................................................................................. 61 Process validation...................................................................................................... 61 Process validation prerequisites ............................................................................. 61 Process validation .................................................................................................. 63 Stability studies.......................................................................................................... 63 8.0 Documentation.................................................................................................. 64 Introduction................................................................................................................ 64 Authentication............................................................................................................ 64 Planning .................................................................................................................... 64 Project plan ............................................................................................................ 64 Process validation plan........................................................................................... 64 Process design and development .............................................................................. 64 Laboratory notebooks ............................................................................................. 64 Process design rationale ........................................................................................ 65 Development reports .............................................................................................. 65 Analytical ................................................................................................................... 66 Analytical method protocols .................................................................................... 66 Analytical development reports ............................................................................... 66 Analytical validation reports .................................................................................... 66 Reference material ................................................................................................. 66 Cell lines.................................................................................................................... 66 Master production protocols ................................................................................... 66 Batch production record ......................................................................................... 66 Certificate of analysis ............................................................................................. 66 Deviation reports .................................................................................................... 66 Disposition document ............................................................................................. 66 Manufacture .............................................................................................................. 66 Master production protocols ................................................................................... 66 Batch production record ......................................................................................... 67 Certificate of analysis ............................................................................................. 67 Deviation reports .................................................................................................... 67 Disposition document ............................................................................................. 67 Stability...................................................................................................................... 67 Forced degradation stability protocols and reports .................................................. 67 Accelerated protocols and reports .......................................................................... 67 Short-term stability protocols and reports ................................................................ 67 Long-term stability protocols and reports ................................................................ 67 Process validation...................................................................................................... 67 Process validation plan........................................................................................... 67 Development reports .............................................................................................. 67 Process characterization reports............................................................................. 68 Process validation protocol ..................................................................................... 68 Process validation report ........................................................................................ 68 Virus validation protocols and reports ..................................................................... 68 Applications ............................................................................................................... 68 Investigational new drug application ....................................................................... 68
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Biological license application .................................................................................. 68
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1.0 Biopharmaceutical drug development

Introduction The L&K Process Guide focuses on biopharmaceutical drug development of recombinant derived globular proteins. Typical drugs belonging to this category are hormones, monoclonal antibodies, growth factors, certain vaccines, cytokines and blood clotting agents such as factor VIII. The biopharmaceutical drug program comprises specified activities relating to cell banking, process design (upstream, downstream, formulation), fill & finish, scale up, manufacturing of technical, tox, and GMP batches, stability studies, in process control, quality control of drug substance and drug product. Further, a test program comprising toxicology studies (animal studies), phase 1 (healthy volunteers), phase 2 (small group of selected patients) and phase 3 (large patient population) is carried out to address safety and efficacy of the product before entering the market. The size and extent of the program is scaring and one must expect a time to market ranging from 8-12 years at cost in between 400 to 800 million USD. Careful planning from day one is considered a must (project plan and master validation plan) and the entire program must be managed by experts in the field. When entering the drug development phase, research is no longer an issue. Several biotech companies operate with strict transfer criteria between research and development realizing the cultural difference between the areas and the cost implications to move into development. It should be mentioned that most projects never make it to the market; approximately 150 products have been approved since the biotech industry started about 30 years ago. The biotech industry is by and large a private industry aiming for shortest possible time to market at lowest possible costs This objective is no way in contrast to the demands of the public health care system and both parties share a common interest in providing safe and reliable products to the patients. In order to assure a common safe platform health care authorities have established regulatory bodies of which the Food and Drug Administration (FDA), the European Medicines Evaluation Agency (EMEA) and the Japan Ministry of Health and Welfare (MHV) are major drivers in the field. Recently US, Japan and EU have agreed to apply to the Conference on Harmonisation (ICH) guidelines as part of the harmonization initiative. A short description of the entire development program is given below, however the guides focus is process design thus excluding the pre-clinical and clinical part of the program. Expression systems Several systems have been used to express recombinant proteins: bacteria, fungi, yeast, insect cells, mammalian cells, transgenic animals and transgenic plants. The most popular systems are E. coli, S. cerevisiae and mammalian cells cultured in large bioreactors. They perform differently with respect to time, expression levels, economy, impurity profiles, product complexity and safety aspects, which makes selection of the expression system a major strategic event. Process design A typical drug development program commence with defining the overall project strategy taking the market size, the administration form (e.g. parental), the device, the final formulation of the drug product, the composition of the drug substance (purified bulk), shelf life, storage conditions, safety aspects, dose, expression system, cell line and economy into consideration. This is no easy task, as many questions cannot be answered in early development. However, each item has great impact on process design
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and continuous assessment of unit operation rationales is strongly recommended. The next step is to establish a research cell bank, which is used for making the master cell bank and for providing vials for experimental process design comprising upstream, downstream and formulation development. Each unit operation is carefully investigated and optimized before the entire process is tested in small scale. Meanwhile preliminary specifications (appearance, identity, potency, purity, quantity, pH and bioburden) are defined making it possible to test the purified bulk against specifications. The first stability studies are initiated in this phase. The needed analytical methods (for in process and quality control) are developed in parallel with process design and testing. The methods need not be validated at this point, but they should be qualified for their intended purpose. Various techniques are used to culture cells (batch, fed batch perfusion), to purify the product (precipitation, folding, filtration, chromatography, crystallization), and to formulate the product (addition of excipients, lyophilization, crystallization, immobilization). Although several purification patterns are common, variations due to expressions system, target protein nature and dose will occur. It should be mentioned that globular proteins are only marginally stable in aqueous solutions resulting in both chemical and physical degradation. Chemical degradation is typically de-amidation, oxidation, scrambling of disulfide bridges, proteolytical cleavage and -elimination. Chemical degradation will take place during processing and during storage as well. Physical instability results in partially unfolding of the protein leading to reversible or irreversible aggregation and loss of product. Surface-surface contact (including at the air-liquid interface), shear forces, high protein concentration, storage, denaturing agents, and removal of stabilizing components during purification trigger physical instability. Protein stability Proteins are very complex molecules having their tertiary structure (the native biologically active state) maintained by a complicated interplay of van der Waals forces, hydrophobic, eletrostatic interactions and hydrogen bonds. The state is thermodynamically favorable by a few kcal/mol making globular proteins prone to physical degradation. Even small changes in structure may result in aggregation and loss of product. Chemical instability leads to formation of des-amido, oxidized, cleaved, and carbamylated forms; byproducts which may be biologically inactive and may raise immunogenic responses. Protein stability should be addressed at two levels: during manufacture and during storage. In principle the specific biological activity should remain constant throughout manufacture and storage. However, in practice, analytical methods may not be available for determination of the specific biological activity in early process stages. Scale up and manufacture It is common practice to define stop/go criteria for process scale up. At this point the process must comply with a certain maturity level, as large scale process redesign is time consuming and costly. Some issues to address are process robustness, specifications, process economy, and scalability. If the process is found applicable for pilot scale, several technical batches are performed in a non-GMP environment mainly to address technical issues and to optimize unit operations. The resulting drug substance is used for formulation and stability studies. When the technical batch program is successfully completed, toxicology batches are manufactured with the purpose of producing material for animal studies. Toxicology batches are normally manufactured in non-GMP facilities, but some companies prefer to run the toxicology studies with the same GMP material used for phase 1-2 clinical studies. The manufacturing process for the toxicology material should be close to identical to the following GMP manufacturing process in order to avoid costly comparability studies. It is, therefore, important that all major process related issues are addressed and solved before toxicology manufacture is initiated.
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Material used for clinical studies must be manufactured under current Good Manufacturing Practices (cGMP). GMP defines the criteria for the manufacturing unit with respect to design, flows, cleaning, training of personnel, equipment qualification, ventilation systems, room classification etc. The manufacturing process is a priori defined in a master protocol approved by the quality assurance department. The document is an extensive description of the manufacturing process including templates for filling in data (e.g. buffer schemes) and no room is left for improvisations the process is locked. Any deviation from the protocol must be recorded and it is the responsibility of the quality assurance department that a report on the incident is provided. Major changes from the protocol may result in batch rejection. The resulting drug substance is tested against specifications and a certificate of analysis is issued. If specifications are met, the batch can be approved provided no major process changes took place during manufacture. The entire process is documented in the batch record. The drug substance (purified bulk) is not intended for human use. Two issues must be addressed: Is it stable and is any change in formulation needed before administration? Most drugs are not stable in liquid formulation making freeze drying or crystallization procedures necessary to implement. Several vaccines require adjuvant to increase the immune response and most parental products must be isotonic and at physiological pH. Further, the drug product must be sterile and provided in safe containers (vials). The drug product is like the drug substance undergoing intensive testing although different issues relating to formulation and sterility may be addressed. Quality control The quality of the final drug product is assessed and controlled on several levels: Raw materials, cell banks, during process, intermediary compounds, drug substance, drug product and by means of stability studies. Raw materials should preferable be of EP, USP or JP grade and of non-animal origin. They shall apply to manufacture under GMP and only trusted, qualified vendors (operating under ISO 9000) should be used. It is common practice to include suited raw materials in early development in order to avoid changes of raw materials during manufacture. Cell banks should be tested and released before entering GMP manufacture. The test programs are extensive and may take several months to carry out. The task is often outsourced to highly specialized laboratories. One of the most significant changes in the quality concept has been the introduction of Process Analytical Technology (PAT) emphasizing the need for better process understanding and control. PAT is considered to be a system for designing, analyzing, and controlling manufacturing through timely measurements (i.e. during processing) of critical quality and performance attributes of raw and in-process materials and processes with the goal of ensuring final product quality. Each process parameter should be addressed and its criticality for product quality should be assessed. For manufacturing reasons, parameters are defined in acceptable ranges allowing for predetermined variations around a fixed set point. This set-up allows for use of statistical approaches (factorial designs, design of experiments) during process qualification. Further, a series of in process analyses are carried out (in process control) to assure that bioreactors have not been contaminated, to address quantity and to specifically address issues of importance (e.g. endotoxin and DNA levels) throughout processing. The drug substance and the drug product will undergo intensive testing for identity, potency, purity, quantity, pH, bioburden (drug substance), sterility (drug product) and appearance prior to release for its intended purpose. The analytical data obtained (issued as a certificate of analysis) must comply with specifications, which are defined a priori based on earlier experience. Specification may be changed during the development program, but a rationale for the change must be provided.
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The stability of the drug substance/product is addressed in controlled studies over time (up to several years) using carefully selected stability indicating analytical methods to address product degradation at different temperatures. The drug product stability studies are used to define the products shelf life. A variety of analytical methods are used to address product quality in terms of identity, potency, purity, and quantity. Commonly used identity methods are peptide mapping, SDS-PAGE, iso-electric focusing, and high performance chromatography (retention time). Potency assays are very specific; they are handled on a case-by-case basis. Typical purity assays are high performance chromatography (product related impurities), LAL (endotoxins), Q-polymerase chain reaction (DNA), insulin ELISA, protein A ELISA, and host cell protein ELISA. Quantity is usually addressed by measurement of A280, but other methods like refractive index, Bradford, and Lowry have been used. Analytical methods are developed, qualified and validated by means of well-characterized reference materials. Besides undergoing the above mentioned tests, reference materials are further characterized with respect to primary, secondary and tertiary structure as well as post-translational modifications. Quality assurance Quality assurance comprises a variety of subjects related to the over all quality of the product, such as release of raw materials assurance that the product has been manufactured according to GMP validation of analytical methods process validation virus validation assurance that the master protocol has been followed and that all signatures are correct release of product handling of deviations cleaning validation training of personnel Material to be used for clinical trials must be manufactured under GMP and can only be released by a trained quality assurance officer from the quality assurance department. Companies in lack of a quality assurance department cannot release and handle such material on their own. This fact is relevant for small biotech companies, who rely on external partners for handling of drug product for clinical use. Analytical methods used for quality control of drug substance/product must be qualified and later validated for their intended used. As the active pharmaceutical ingredient and/or the buffer components may affect the outcome of the analytical method, even standard methods (e.g. SDS-PAGE) must be proven qualified on a case-by-case basis. Analytical method qualification begins with the early phase development program and each method will gradually develop with time until a fully validated method is available at the time of phase 3 manufacture. The demands to method validation vary with the purpose and different programs are needed for identity, purity, and quantity analyses. An important aspect of quality assurance is process validation, where the process is documented robust and to consistently produce high quality drug product in comparable yields. Like validation of analytical methods, process validation is an ongoing activity initiating during process development following a process validation master plan. Major prerequisites for the validation program are: process description, analytical methods, risk assessment, process characterization including statistical analyses and definition of critical parameters, cell lines, equipment, facilities and training programs. The validation program addresses unit operations, viral clearance (not for microbial expression systems), clearance of impurities, process consistency, resin and membrane lifetime. The
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process validation program is usually linked to phase 3 manufacture, where the process is finally locked. Viruses are infectious at doses far below the lower detection limit making final testing of the drug substance/product obsolete in many cases. However, the risk of a viral infection cannot be ruled out as viruses are used for transfecting animal cells, may be introduced during cell culturing or be introduced during downstream by cross contamination. An extra safety measure has, therefore, been introduced for insect cell, mammalian cell and transgenic animal expression systems. The downstream operation must assure a certain total virus removal factor, normally expressed in logarithmic terms. No fixed reduction factor can be provided, but ranges from 10-15 have commonly been reported. The virus validation program addresses each selected unit operation by addition of live virus to the sample to be purified and measurement of the resulting virus titer after purification. The experiments are carried out in small scale (downscale studies) in special designed facilities. The virus reduction factor of each unit operation is additive (except if 1 or close to 1) and a rule of thumb says that a reduction factor of more than 10 should be obtained. Virus validation studies are carried out prior to phase 1 clinical trials (one enveloped and one non-enveloped virus) and again at phase 3 manufacture (usually 4 different viruses). Documentation Special attention should be given to a costly and time-consuming activity, documentation. In essence everything in the entire drug development program should be documented in a traceable manner. Regulatory authorities regard experiments and information not documented as non-existent and lack of important information may halt the program. Documents should be properly authenticated and the contents should clearly reflect the purpose of the document. Documentation relates to the different drug development categories. Two major documents define the development program: the project plan and the master validation plan. The manufacture and release of cell banks will be reported separately (the master and working cell banks are produced and stored under Good Manufacturing Practices (GMP)). The process development work (upstream, downstream and formulation) will typically be reported in separate development reports eventually supported by a process design rationale and specific unit operation descriptions. Day to day experimental work is described in laboratory books. Pilot, technical, toxicology and GMP batches will typically operate from a master protocol building on the development reports and gradually evolving to the final manufacturing protocol. After manufacture the work carried out will be documented in batch records and a certificate of analysis will be issued. Analytical methods are described in analytical protocols and method applicability is reported in analytical development reports ending with the final validation report. Process and virus validation are documented in separate reports. Pre-clinical studies Pre-clinical activities serve the purpose of testing the drugs safety and efficacy, using cell based assays and animal models. It is essential that any potential risk to the safety of patient is identified during this phase of product development to assure that the product is safe for use in clinical trials. There are three major types of pre-clinical studies: pharmacodynamics, pharmacokinetics and toxicity testing. Pharmacodynamics addresses the therapeutic effect of the active pharmaceutical ingredient (API). The effective dose is determined in this stage. Pharmacokinetic studies describe how the body will affect the target molecule with respect to absorption, distribution, metabolism and excretion (ADME). Toxicity testing
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reveals information about the drugs harmfulness to the patient (maximal tolerable dose and side-effects). The test program comprises toxicity, carcinogenicity, genotoxicity and reproductive toxicology studies. Several animal species are used in the above mentioned test programs (e.g. mice, rats and monkeys), a procedure of great concern to the public. Although animal models can be unreliable, it is considered that animal studies cannot entirely be replaced by cell based in vitro assays. However, functional genomics may help in design of better animal models thus reducing the number of animals used in a given study and the use of computer simulations may in the future provide ADME information without use of animals. Animal studies and associated bioanalyses should be conducted under Good Laboratory Practices (GLP). The Investigational New Drug Application An Investigational New Drug Application (IND) is a request for authorization from the Food and Drug Administration (FDA) to administer an investigational drug or biological product to humans. An IND (or like application) must be submitted to FDA (or like regulatory body) before a clinical study is initiated. The application is a comprehensive document comprising information about the drug, an investigational plan, protocols for each planned study, preclinical information and information on chemistry, manufacturing and controls (CMC). FDA may withhold the application and request supportive data before the clinical trials can start. Otherwise the phase 1 study may begin 30 days after the submission of the application. The IND is a live document undergoing continuous updating in close collaboration with FDA. Clinical trials Phase 1 clinical trials The purpose of a phase 1 study is to address drug safety in healthy human volunteers or patients who are in relative stable condition with their disease. A phase 1 study typically involves from 20 to 80 volunteers and the study may last from 6 to 12 months. A feature of the phase 1 study is to determine the maximum tolerated dose (MTD) for the drug. The MTD must be higher than the expected therapeutic dose in order to continue the study. The next step is to conduct a multiple rising dose (MRD) or multiple ascending dose (MAD) study and ADME studies often using radioactive labeled compounds. A primary objective of a phase 1 study is to determine the maximum safe dose; hence, side effects are common in a phase 1 study. Drugs used for phase 1 clinical trials must be manufactured under GMP. Phase 2 clinical trials The purpose of a phase 2 clinical trial is to address drug safety and efficacy in its targeted disease group. It is not, as some people assume, to prove that a drug candidate is an effective treatment. Efficacy studies can be divided into 2a and 2b trials, with 2b as an extension to the safety and efficacy studies performed under 2a. A phase 2 study is larger than phase 1 clinical trials: 100-300 patients are typically enrolled in a phase 2 study. The study is conducted as a randomized, double blind study, where the drug is compared to another already existing drug or against a placebo given to a second control group of patients. A double blind trial is a trial where both investigator and patients are unaware of which patients are in control and active groups. The time frame of a phase 2 study is typically from 1 to 2 years. Phase 3 clinical trial The combined phase 1 and 2 clinical trial data should provide enough safety and efficacy data to justify initiation of phase 3 clinical trials (FDA should be consulted before entering a phase 3 study). A phase 3 study comprises from 500 to 1000 patients and it may last
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for several years. The study is very expensive to perform. However, the average successrate is high. The purpose of a phase 3 clinical trial is to confirm drug efficacy tested during phase 2 in a large patient group usually in multiple locations around the globe. Phase 4 trials Phase 4 trials monitor the efficacy and side effects of a drug in real patients in uncontrolled situations. Phase 4 studies serve several purposes: evaluation of long term effects, test of new indications, new routes of delivery or implications due to change of manufacturing process. The license application The pre-clinical, clinical and biological data are collected in the biological license application (BLA) or in the New Drug Application (NDA), unique documents carefully reviewed by the Food and Drug Administration (FDA) before a license can be issued.
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2.0 Expression systems

Introduction Six different expression systems are used for manufacture of biopharmaceuticals: bacteria, yeast, insect cells, mammalian cells, transgenic animals and transgenic plants. Two systems dominate the biotech industry, E. coli and the mammalian Chinese hamster ovary (CHO) cells. However, Bacillus subtilis, Lactococcus lactis, Saccharomyces cerevisiae and Pichia pastoris have been used with success. Transgenic animals have had limited success due to concern about presence of prions in the final product. Transgenic plants are making some progress, but few biotech products are commercialized via this route. Any expression system used in the biotech industry must be well characterized and it should be provided with a solid tracking record. As an example strict regulatory demands to cell line history, safety, genetic stability, expression levels, cell densities and cell viability must be met in order to assure that mammalian expression systems are accepted by regulatory authorities. Selecting the right expression system for a given protein is one of the most important decisions in the entire drug development program as time to market, process economy and safety is closely linked to the system used. The decision-making is complicated. Expression systems vary in their ability to express recombinant proteins, to handle posttranslational modifications, and very different safety concerns are linked to the various expression systems. Time is also a factor as expression system development and culture times vary considerably. Expression systems E coli The E. coli expression system has a proven track record over more than 30 years. It was the first expression system introduced in the biotech industry (recombinant insulin) and gram negative bacteria is still used for expression of a wide range of biopharmaceutical products. E. coli offers fast proliferation and the fermentation period is a few days only, offering high utilization degree of the upstream facility. The expression level is high, 1-5 g/L cell culture (intra-cellular expression) often resulting in formation of insoluble inclusion bodies. The intra-cellular protein is typically expressed with an extra N-terminal methionyl residue, which may have immunogenic consequences. E. coli cannot express post translational modified proteins (e.g. glycosylated forms) restricting the use of the expression system. E. coli is typically cultured in batch or fed batch mode using more or less defined cell culture media. The trend is to make use of well-defined media, only, with additional supply of ammonia and glucose nutrients in fed batch cultures. In its simplest form a microbial culture comprises three phases: A lag phase, an exponential growth phase and a stationary phase. A major difference between typical bacterial cultivations and E. coli is the technique of separating growth and production phases. This method takes advantage of regulated promoters to achieve high cell densities in the first phase (promotor off) and high rate of target protein expression in the second phase (promotor on). One way to shift from expansion to production mode is to change the temperature. In order to obtain high protein expression levels, it is important to establish a state of balanced exponential growth by supplying all nutrients in optimum amounts (above the limiting concentration but below the toxic concentration). The cells respond by growing exponentially. E. coli cell cultures may be infected with other bacteria, fungi and/or phages. The cells are typically harvested at decreased viability by centrifugation followed by homogenization at high pressure (several hundred bars) and further separation of the cell debris and the extracted target protein. If inclusion bodies are formed, this fraction can be
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separated and considerable purity can be obtained after washing of inclusion bodies. The extraction buffer must be designed according to the nature of the target protein if the protein comprises cysteine residues both denaturing and reducing agent must be present to assure a monomeric unfolded protein fraction. It may be advantageous to further purify this fraction prior to refolding using for example anion exchange chromatography or immobilized metal affinity chromatography, which also operate under denaturing conditions. The in vitro refolding must be carried out at alkaline pH and in presence of a mercapto reagent (e.g. cysteine) if formation of disulfide bonds is required. Low protein concentrations (e.g. 0.2 mg/ml) will reduce the risk of aggregate formation during folding and this less cost efficient method is often required in order to obtain reasonable yields. Formation of incorrectly formed disulfide bonds lead to scrambled products difficult to separate from the target protein. The refolded product is further purified using ion exchange, hydrophobic interaction, hydroxyapatite or reversed phase chromatography or combinations hereof. Size exclusion chromatography may be used as the final chromatographic step, as this procedure is the only chromatographic operation offering total flexibility in selection of drug substance buffer and further excellent separation of polymeric and monomeric target protein forms. The draw back of this method is the restricted sample application volume (< 5% v/v). An N-terminal methionine is associated with intra-cellular E. coli expression. The extra amino acid is not easily removed (in order to obtain the correct primary sequence) and specific extension and cleavage technologies must be utilized. One system makes use of a tag specifically designed for subsequent cleavage with diaminopeptidyl aminopeptidase (Catepsin C). This principle has been used to produce human growth hormone for therapeutic use. The E. coli process design must take into consideration the high amount of endotoxins (from the cell wall), host cell proteins and nucleic acids present. Viruses are of no concern with this expression system.
Advantages
Simple, well-understood genetics Established regulatory track record Rapid cell growth Inexpensive culture media Fermentation easy to scale up Inclusion bodies may be easy to purify No unintended glycosylation No viral or prion contamination risk
Disadvantages
Intra-cellular expression in Gram-negative bacteria (although some systems have been designed for periplasmatic expression of the target protein) Expression of Met-protein at the N-terminus in Gram-negative bacteria High endotoxin and host cell protein levels in initial extracts when Gram-negative bacteria have been used No post-translational modifications possible (cannot express glycosylated, acetylated and amidated proteins) In vitro folding often necessary
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Yeast The yeast species Saccharomyces cerevisiae and Pichia pastoris have gained considerable interest as production organisms for commercial biopharmaceutical products. The expression systems offer high efficiency in terms of short doubling times, high cell densities, high yields and low fermentation costs. The production levels ranges from milligrams product per liter of culture to several grams, depending on choice of yeast strain, type of product and culture conditions. Yeast is easy to grow in large scale with simple nutritional demands that lower the media cost. In contrast to E. coli, yeast can express correctly folded proteins directly to the medium, which greatly facilitates purification (low level of host cell proteins, non-viscous solution, low level of DNA). The rigid cell wall renders the use of all sorts of bioreactors possible regardless of stirring and shaking mechanisms. Yeast cell cultures may be infected with bacteria and fungi. Filtration, centrifugation and expanded bed technologies are used to harvest the cell culture. Expanded bed technology combines separation of cells and product and capture as the target protein binds to the suspended resin, while cells and culture medium are passing through the column. If filtration or centrifugation is used, the supernatant is applied directly to the capture column or after ultra-filtration with the purpose of sample concentration. Hydrophobic interaction chromatography should be avoided in the capture operation, as anti-foam agents added during fermentation will bind to the column thus reducing the binding capacity. The following purification and polishing steps are straightforward and yeast is regarded as one of the most safe and simple expressions in use. As with E. coli, this expression system is not able to produce post-translational modified proteins. Viruses are of no concern with this expression system and relative low levels of host cell proteins, nucleic acids and endotoxins (e.g. microbial contamination during downstream operations) should be expected.
Easy and cheap to grow in large scale Good track record Relative inexpensive fermentation media Well understood genetics Able to provide some posttranslational modifications Short doubling time High cell densities achievable Generally regarded as safe (GRAS) status Gives high yields No endotoxin release from host cell organism Extra-cellular expression to low viscosity cell culture medium Minor secretion of host cell proteins No specialized bioreactor required Safer than working with mammalian tissue or cell lines Problems with correct glycosylation Over glycosylation can ruin protein bioactivity Yeast glycosylation is not identical to mammalian glycosylation Extensive proteolysis of target protein Extensive intra-cellular proteolysis of some target proteins
Advantages
Disadvantages
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Insect cells Insect cells constitute a promising alternative to bacterial and yeast expression systems for a wide range of target proteins requiring proper post-translational modifications. The insect cell technology has for several reasons been slow to penetrate the biotech industry despite the safety of the expression system, ease of scale up, use of relatively cheap serum free media, reasonable expression levels, rapid cell line development and the ability to produce post-translational modified proteins. However, the rapid improvement in stable cell lines, better expression cassettes, and better understanding of cell culture conditions will probably result in a widespread use of the technology in the future. The classical baculovirus/insect cell system suffers from a number of drawbacks, and therefore novel expression vectors have been used to transform Lepidopteran insect cells into high-level expression systems.
Advantages
The protein is secreted to the medium in its native form Expresses post-translational modified proteins Expression vectors are commercially available The system is suited for expression of cell toxic products since the cells can be grown in a healthy state before infection The baculovirus vectors are harmless to humans.
Disadvantages
Minimal regulatory track record Semi-expensive culture media Cell line stability Cells are killed during infection releasing intra-cellular proteins (Lepidoteran species) Inactivation of the secretory pathway results in low expression yields Presence of immunogenic host cell proteins Inability to produce eukaryoic glycoproteins with complex Nlinked glycans Inability to proper processing of proteins that are initially synthesized as larger inactive precursor proteins (e.g. peptide hormones, neuropeptides, growth factors, matrix metalloproteases) Risk of infection with mammalian viruses Sensitive to shear forces.
Mammalian cells Since the introduction of continuous mammalian cell lines for the production of recombinant biopharmaceuticals (e.g. interferon), this expression system has been the second most used despite relatively high fermentation costs, slow growth, relatively low yields and potential risk for viral infections. This major achievement was made possible by a combination of technical progress (bioreactors) and strict demands to cell line safety and characterization, use of tested raw materials, good manufacturing practices, in process control and drug substance/product quality control programs. Mammalian cell cultures have found widespread use for expression of monoclonal antibodies, but also complex recombinant proteins such as tissue plasminogen activator,
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Factor VII and Factor VIII have successfully been purified from mammalian cell cultures. The relative low expression levels combined with high prices on culture media and expensive quality control programs makes it generally more expensive to produce recombinant proteins in animal cells than in microbial systems. However, complex proteins cannot be expressed in microbial systems leaving transgenic animals or plants as the only alternative. Mammalian cells have with success been cultured in perfusion cultures with a flow of 1-2 bioreactor volumes per day. At best these cultures can continue for up to 50 days in production mode and produce 50 times more than a traditional fed batch culture of the same volume. As the recombinant protein is expressed to the culture medium, cells can be removed from the target protein by filtration, centrifugation and expanded bed technology. Relative mild conditions must be used, as the mammalian cell is sensitive to sheer stress. The cell free filtrate/supernatant is then subject to the first chromatographic purification step, which for monoclonal antibodies typically is protein A resins. Mammalian cells are prone to virus infections and for safety reasons a virus inactivation step (e.g. low pH) is introduced into the first part of the downstream process and a virus filtration step into the final part of the process. Removal of endotoxins, nucleic acids and host cell proteins is not considered a major problem as mammalian cultures are free of microbial agents and most cell cultures are terminated at a cell viability of more than 80%. However, it is recommended to maintain the principle of minimum three different chromatographic unit operations to secure a high virus reduction factor and a low content of product related impurities.
Advantages
Extra-cellular expression The protein is expressed in its native form Usually expresses and secretes even complex posttranslational modified proteins Expression vectors are commercially available Good regulatory track record
Disadvantages
Relative long cell expansion phase 8 Density rarely exceeding 10 cells/ml Expensive culture media and complex nutrient requirements Relative low productivity The culture media may contain bovine proteins and allergens Require extensive characterization Possible virus infections Sensitive to shear forces and extremes in osmolarity Safety concerns about transformed cell lines
Transgenic animals Transgenic animals are one of the most promising recombinant protein expression systems in the biopharmaceutical industry. Even complex post-translational modified
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proteins are successfully expressed in their native biologically active form, thus making it possible to produce plasma proteins, human antibodies and other proteins not easily derived from other sources at industrial scale. It takes 18-33 months from introduction of gene to production at usable levels. The animal husbandry and milking procedures are known technologies upgraded to Good Agricultural Practices (GAP). The target protein is usually expressed in the mammary gland, often at high protein concentrations (5-50 g/L milk) far exceeding other expression systems. The harvest procedure used for cell-based cultures is replaced by fat and casein removal operations known from the dairy industry. Two adventitious agents are of concern: viruses and prions; the latter have never been observed in milk and for that reason the safety risk is expected low. Never the less, the safety of transgenic animal derived products has been continuously challenged and is perhaps the main reason why this expression system has not found widespread usage. The downstream process very much resembles that of mammalian cell expression systems including virus inactivation and filtration steps. Very high expression levels Capable of complex protein processing Dairy technology is known and tested Easy scale-up Low cost production Relative long period from introduction of gene to production at usable levels Little regulatory experience Unknown viral contamination risk Unknown prion contamination risk Co-expression of animal protein
Advantages
Disadvantages
Cell lines Microbial, insect and mammalian expression systems are based on well-characterized cell lines constructed with the specific purpose of expressing the desired protein in sufficient quantities. Cultures usually grow in stainless steel reactors in batch, fed-batch or perfusion mode (upstream processing). Typically, the gene of interest (usually the gene coding for the target protein) is characterized and inserted into a suitable vector (e.g. plasmid), which is inserted into the host cell (e.g. E. coli, Pichia pastoris, Chinese hamster ovary fibroblasts). The transformed host cell is now cloned and cells that produce the target protein in sufficient quantities are selected and tested in small scale eventually with subsequent purification of the protein in order to address drug substance quality. It is important to document the origin of the cell line and vector for each step in the procedure of cell transformation and cloning (cell line history) as this information will be requested by regulatory authorities. Cell banks Research cell banks Once a sub-clone of the transformed cell line has been selected it should be tested for genetic stability and then cultured in quantities sufficient to lay down 30-50 vials (the research cell bank, RCB), which are stored at -80C or in liquid nitrogen. Vials from the RCB are used for upstream development and sometimes even for manufacture of tox batches.
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Master cell banks Vials from the RCB are used to produce the master cell bank (MCB), approximately 200 vials of cells with proven safety profiles, genetic stability, expression levels, cell density and viability. A MCB is a homogenate pool of the production cell line dispensed in multiple containers, by which each aliquot is representative for each other vial, stored under defined conditions (liquid nitrogen). The MCB offers the ability of having a characterized common starting source for each production lot even over a time span of several years. Before use, the MCB is thoroughly tested addressing identity, purity, stability, and viruses for insect and mammalian cell lines. The test program, which is very extensive, takes months to complete. A released MCB is a precious collection of vials laying the foundation of the coming years manufacture of batches for licensed product. Working cell banks In order to save MCB vials, it is common practice to establish a working cell bank (WCB) derived from one or a few MCB vials. Each WCB typically comprises 200 vials and usually one or two vials are used for production of one GMP batch (phase 3 or licensed product). The MCB and WCB may differ from each other in certain respects (e.g. culture components and culture conditions). There is generally little requirement to perform extensive testing of the WCB if the MCB has been thoroughly characterized. However, this should be defined on a case-by-case basis according to the nature of the cell line, handling procedures, and the MCB tests performed.
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3.0 Process design

Introduction Designing a biopharmaceutical process intended for manufacture of drug product is a complicated discipline. In contrast to the often simple and shortsighted set up used during the research phase, the design of a manufacturing process must take safety, economy, scale, environment, product shelf life, administration, good manufacturing practices and several other factors into consideration. It is common practice to address the mentioned issues in early development in order to minimize the number of late process changes, especially after scale up. A rationale for the resulting design should be provided and it is important to include the administration form (e.g. parental), the device and the expected shelf life, as these factors will influence the drug substance/product compositions and thereby the design of the upstream and downstream processes. The entire process typically comprises upstream, harvest, downstream, formulation and fill/finish. Upstream procedures are linked to expression of the target protein in for example microbial cells, mammalian cells, or in transgenic animals (milk). Harvest procedures links upstream to downstream in preparing the sample for the capture operation (e.g. protein A chromatography). The purpose of the downstream operations is to purify the target protein to a level defined in drug substance specifications. The resulting purified bulk (drug substance) is formulated to the drug product, which finally is filled into vials or specific devices under sterile conditions. After labeling and packaging the released drug can be administered to humans. Three major decisions must be taken in early development in order to design a proper process: the expression system, the drug product formulation and the nature of the drug product. The choice of expression system dictates to great extend the downstream process (e.g. virus removal related to mammalian cell expression or protein folding related to E.coli expression). The drug formulation depends on its purpose, is it a vaccine, an intra-venous drug or meant for subcutaneous injection. Finally, should the drug be stored as a liquid formulation, a lyophilized product or immobilized to for example alhydrogel? The process design should address robustness to assure that the process continuously will provide the expected yield and the drug substance will meet specifications. A rule of thumb dictates that at least three different chromatographic unit operations are needed regardless of expression system. As soon as information about expression system, drug substance composition, drug product formulation, administration device and route is available, the process designer can initiate work on process design and its rationale. It is an important, but often overlooked activity, to integrate upstream, downstream and formulation activities in the original design to avoid conflicts between the three categories. An improved expression level may, for example, be of considerable interest for the upstream group. However, if the optimized cell culture results in less product following downstream operations due to protein instability, little is gained. If cellular expression is used, a decision between batch, fed-batch or perfusion mode must be taken based on careful considerations between process economy and scalability. Downstream wise, it must be considered whether in vitro protein folding, virus inactivation and virus filtration should be a part of the process, which typically comprises 3-4 different chromatographic unit operation to assure efficient removal of impurities. The resulting drug substance should allow for an easy transfer to formulation. The drug product may be formulated as a liquid, a lyophilized powder, or crystal suspensions using a variety of excipients to improve protein stability during formulation and storage. Throughout, process robustness, process economy, scalability, and maintenance of protein stability should be addressed.
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Upstream Introduction The term upstream is associated with cell cultures be it bacteria, yeast, insect cells or mammalian cells. In small scale, cell cultures are grown in shakers, spinners, roller bottles, cubes, wave bags or stainless steel bioreactors. Large-scale manufacture typically takes place in stainless steel reactors (100-20000L) or in wave bags (up to 500L). Large-scale facilities are very expensive to build, maintain and operate and small biotech companies rely on contract manufacturers for such services. The purpose of the upstream operations is to express the target protein in sufficient quantities to allow for subsequent purification and formulation in order to provide enough product to maintain the clinical programs. All upstream development and manufacture programs are run under well-defined conditions setting strict demands to use of tested cells and suited raw materials. Cells are usually provided as research cell banks (RCB) or master cell banks (MCB). Preferred raw materials are those intended used for the Good Manufacturing Practices (GMP) manufacture. Batch and fed batch mode A batch culture is in essence a closed system with a fixed culture volume in which the cells grow until maximum cell density depending on medium nutrients, product toxicity, waste product toxicity and other essential factors are reached. The cells follow classic kinetics with a log phase of rapid proliferation where some products are produced and a stationary phase where the amount of cells does not change and where other products are produced. If the desired product is produced in the log phase, it can be prolonged by manipulating the growth conditions but only for a very limited time, and if the desired amount is not produced that quickly, it will be reasonable to choose another culture method. When the batch culture is terminated, the entire batch is harvested in one operation. Another solution is to conduct the fermentation in a fed-batch mode. Medium is added in fixed volumes throughout the process thus increasing the volume of the cell culture with time. Neither cells nor medium are leaving the bioreactor. In this way the sugar levels can be kept more or less constant for a long time and it is possible to switch from one substrate to another thus rendering the use of inducible promotors possible. The process is usually performed at low growth rates, the key control parameter being the feed rate whose upper limit is dictated by the oxygen transfer limit and cooling strategies available. The feed rate can be subjected to feedback control strategies using for instance measurement of the glucose concentration, dissolved oxygen (DO), biomass production or heat generation. It is common practice to run microbial cells as either batch or fed batch cultures, while mammalian cells may also be run as perfusion cultures. Perfusion mode An alternative approach to batch cultivation is to continuously add fresh medium to the bioreactor and to remove equivalent amounts of medium (with or without cells). A controlled perfusion bioreactor offers tight control of the growth conditions and cells can be kept in their productive phase for several months, if required. However, there is an increased risk of contamination due to poor equipment design and the complexity of operation. Although the productivity might be lower per liter of culture medium, the constant flow of high quality product, which can be harvested at short intervals often compensate for the extra media costs. Harvest Harvest links upstream and downstream processing serving the purpose of conditioning cell cultures for the first chromatographic operation (capture). Cells are usually separated
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from the culture medium by centrifugation, filtration (dead-end, micro-filtration or ultrafiltration) or combinations hereof. In case intra-cellular expression is used, cells are isolated for further processing. If extra-cellular expression is performed, the supernatant/filtrate is collected and carefully adjusted to match the conditions for the capture operation. Ultra-filtration is often used to concentrate the sample before application to the capture column. Specific considerations apply to harvest of perfusion cultures due to the prolonged culture times. It is common practice to collect and store harvest from 4-7 days of operation and partially purify this part, which is later poled under strict control with similar fractions. Downstream Introduction The purpose of the downstream operations is to purify the expressed protein from the cell culture, milk or plant extracts. The term purity relates to the content of host cell, process and product related impurities in the drug substance (final bulk). The acceptable content of each contaminant must be specified for toxicology and GMP batches, and it must be proven that the purified product meet specifications. Typical host cell related impurities are host cell proteins, nucleic acids, endotoxins, and in some cases viruses. Examples of process related impurities are reagents used in cell culture (e.g. insulin, anti-foam agents), specific raw materials used during downstream processing (e,g, protein A) or endotoxins/viruses stemming from contamination during processing. Notice that water is a major contaminant and great effort is paid to remove water during capture. Product related impurities are product derivatives such as cleaved forms, des-amido forms, oxidized forms or polymeric forms. The downstream design must address reduction of any impurity compromising safety and a rationale should be provided in order to assure that te process serves its purpose. The downstream operations are divided into three categories: capture, purification and polishing. Capture follows immediately after harvest serving the purpose of removing water and host cell derived impurities, while at the same time condition the target protein solution for further purification. Affinity chromatography is commonly used as the first chromatographic principle as high purity can be obtained in one operation (more than 90% purity is often observed after affinity chromatography operations). Use of hydrophobic interaction and reversed phase chromatography should be avoided as antifoam agents added during cell culture will bind strongly to such matrices thus lowering the binding capacity of the target protein. Resins used for capture operations are typically from 65-300 m to allow for acceptable flows and column back-pressure. The purpose of the purification unit operations is to further remove host cell related impurities, to reduce potential infectious agents such as viruses and to condition the target protein solution for polishing steps. The resin particle size is typically from 65-120 m. The chromatographic polishing step may operate with small particle size resins (15-35 m) allowing for separation of the target protein from its closely related derivatives (e.g. des-amido and oxidized forms). The latter resins are expensive and they are used for well-conditioned samples, only. The capture, purification and polishing concept is a stepwise approach to increased purity making use of decreased resin particle size, more uniform spheres providing better packing, and more resistant resins to cleaning agents (e.g. NaOH). The resulting three chromatographic unit operations, which should be different, comprise the basics of most biopharmaceutical purification programs, although more chromatographic steps may be needed. Various filtration techniques are used in downstream processing. 0.45 m dead end filtration is commonly used for cell debris removal, removal of microbial agents and to separate precipitates from supernatants. Also, buffers and application samples are typically filtered to lower microbial burden during downstream processing. Micro-filtration may be used for removal of cell debris and is often used to replace centrifugation. Ultra-filtration is used for buffer exchange and to concentrate the sample.
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Buffers and co-solvent The biotech industry tends to be fairly conservative with respect to choice of buffers. Typical examples are Na-acetate, Na-citrate, Na-phosphate, Tris, Na-bicarbonate, ammonium bicarbonate and glycine. Other co-solvents serve specific purposes such as precipitation (e.g. ammonium sulfate), organic modifiers (e.g. ethanol), excipients (e.g. mannitol) or denaturants (e.g. urea). It is strongly recommended that all raw materials used are suitable for Good Manufacturing Practices (GMP) manufacture. Techniques Sample preparation The unit operation starting material is usually an aqueous solution of the target protein. The sample should be clear in appearance and free from particulate matter. The sample composition and characteristics are important for the outcome of the unit operation, making strict control of co-solvents, protein concentration, pH, ionic strength, temperature, redox potential, and holding time, a must in order to ensure process compliance. Various techniques are used to condition the sample for further purification (centrifugation, filtration, micro-filtration, ultra-filtration, desalting, precipitation). Precipitation Several precipitation techniques are available for large-scale operations. Typical techniques are salting out (e.g. ammonium sulfate), isoelectric precipitation, addition of organic solvents (e.g. PEG). Large-scale precipitation operations may be difficult to handle and it should be considered to replace such unit operations with chromatographic principles in early development.
Advantages
Volume reduction Removal of specific impurities The protein stability is increased when using certain cosolvents (e.g. ammonium sulfate)
Disadvantages
Precipitated protein may be difficult to re-dissolve Environmental issues disposal of waste Expensive technology when precipitation of protein from large volumes is necessary Use of explosion proof facilities are needed if organic solvents are used Some co-solvents may decrease the protein stability Space requirement for equipment may be significant Labor intensive technology The technique is inefficient at low protein concentrations
Centrifugation Centrifugation is commonly used to separate cells from the cell culture following batch or perfusion cultures and to separate precipitate and supernatant following precipitation. Large-scale separation is very much different from small-scale operations and considerable technical expertise is required.
Advantages
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Well known technology My be the only option
Disadvantages
Labor intensive Technical complicated operations in large scale Cleaning in place
Affinity chromatography The separation principle in affinity chromatography (AC) is the bio-specific interaction between a ligand and the target protein using structural recognition as the basic principle. The dissociation constant must be in a range of 10-5 10-7 M, allowing for later elution of the protein either by changing pH, ionic strength, polarity or by addition of specific cosolvents. AC offers high selectivity but poor resolution between the protein and closely related derivatives, making the principle an excellent choice for capture operations where large volumes of highly complex samples are dealt with. Due to the high selectivity, desorption is, in most cases, carried out by step elution after washing away impurities. It has been common practice to express the target protein with a fusion partner and to purify the protein complex using a specific ligand towards the fusion protein. Few of the fusion systems have been used in large scale, mainly because of restrictions related the cleavage of the fusion protein.
Very specific for the target protein Mild elution conditions
Advantages
Disadvantages
Cannot separate the target protein and it derivatives The unit operation may be expensive Cleaning in place with NaOH is not always possible Protein A is toxic and some leakage from the resin should be expected
Immobilized metal affinity chromatography A specific feature of affinity chromatography is immobilized metal affinity chromatography (IMAC). The principle is based on differences in the affinity of protein for metal ions in complex with a chelating group (e.g. iminodiacetic acid). The strength of the complexes formed varies from protein to protein which, in many cases, gives rise to the high specificity. Exposure of certain amino acid residues (e.g. histidine, cysteine, tryptophan) is required for binding, exemplified in the extensive use of histidine tagged proteins expressed in E. coli with subsequent purification on metal chelating columns. The principle applies even under denaturing conditions (e.g. 7-8 M urea) and simple ionic adsorption can be suppressed by buffers of high ionic strength. The binding is influenced by pH, where low pH often leads to desorption. IMAC is preferably used for capture or first intermediary purification operations, where good separation between the protein, cell culture components and impurities is needed. Protein adsorption is remarkably unaffected by the presence of salts, denaturing agents, detergents, glycol and organic solvents, such as ethanol, making most samples
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applicable to the technique. Similar to other affinity-based techniques, IMAC should mainly be used during capture and intermediary purification. Presence of metal ions in product pool may cause regulatory concerns. The metal ions must be removed in subsequent purification steps.
Can provide high selectivity for the protein of interest Mild elution conditions Application sample may contain denaturants, detergents, glycol and ethanol Binding at high ionic strength is possible
Advantages
Disadvantages
Cannot separate derivatives from the protein of interest Potential leakage of metal ions
Ion exchange chromatography Ion exchange chromatography is the most utilized technique in protein purification. The reasons for the techniques popularity in industrial scale include its robustness, straight forward purification principle, general high resolving power, general high capacity, effective cleaning in place procedures, general large number of cycles possible and ease of performance. Although little is known about the fundamental mechanism behind protein binding to charged surfaces, good predictions can be made from knowledge of the protein properties (pI, Mr), the charge, the ion exchange matrix, the pH and the conductivity of the solvent. Anion exchangers are basic ion exchangers containing positive ligands binding negatively charged proteins (or counter ions, anions). They are traditionally divided into two groups, weak and strong anion exchangers based on the pKa of the charged group. The trend goes towards use of the strong anion exchangers (quaternary amines) capable of binding proteins in the pH range 2-12 depending on the pI of the protein (a protein of pI 3.1 is expected to bind at pH 4.1, while a protein of pI 6.1 is expected to bind at pH 7.1). A pH change in the interval of 2-12 will consequently not affect the net charge of a strong anion exchanger. The corresponding pH interval for a weak anion exchanger is 2-9. As a rule, a negatively charged protein will bind to the anion exchanger making adsorption strongly pH dependent. At pH values far from the isoelectric point, proteins bind strongly. Near the isoelectric point, weaker binding may be expected and, at the isoelectric point, local charge distributions may occur even if the net charge is neutral. The pH of the solution is, therefore, a very essential parameter in ion exchange chromatography, and a change of pH may be used to elute the protein. Cation exchangers are acidic ion exchangers containing negative ligands binding positively charged proteins (or counter ions, cations). They are traditionally divided into two groups, weak and strong cation exchangers based on the pKa of the charged group. The trend goes towards use of the strong cation exchangers (sulfopropyl, methyl sulfonate) capable of binding proteins in the pH range 4-13, depending on the pI of the protein (a protein of pI 6.1 is expected to bind at pH 5.1, while a protein of pI 4.1 is expected to bind at pH 3.1). A pH change in the interval of 3-14 will consequently not affect the net charge of a strong cation exchanger. The corresponding pH range for weak cation exchangers is 6-13. As a rule, a positively charged protein will bind to the cation exchanger making adsorption strongly pH dependent. At pH values far from the isoelectric point, proteins bind strongly. Near the isoelectric point, weaker binding may be expected and, at the isoelectric point, local charge distributions may account for binding even if the net charge is neutral. The pH of the solution is, therefore, a very essential
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parameter in ion exchange chromatography, and a decrease in pH may be used to elute the protein.
Advantages
Can be used during capture, intermediary purification and polishing High resolution possible only surpassed by reversed phased chromatography (RPC) The principle applies in buffers with organic solvents, in urea buffers, and in non-ionic detergent buffers. High flexibility with several types of different ion exchangers available Samples with low protein concentration can be applied directly to the column (provided pH, conductivity etc. is correct) without significant losses. A high concentration factor can be obtained
Disadvantages
Binding is often poor at medium to high ionic strength High ionic strength in eluted fractions when salt desorption is used Local pH extremes during elution can affect protein stability Uncontrolled pH during elution may cause precipitation in the column Some glycoproteins may exhibit a very complex purification pattern
Hydroxyapatite chromatography Hydroxyapatite is, unlike most matrices, both the support and the ligand. The matrix is a poly-calcium phosphate [Ca10(PO4)6(OH)2] comprising positively charged pairs of crystal calcium ions and clusters of 6 negatively charged oxygen atoms associated with triplets of crystal phosphates. The crystal structure is degraded at pH 5 and below and in presence of chelating agents. Besides that, hydroxyapatite media are resistant to sodium hydroxide, detergents, organic solvents and denaturing agents. Binding occurs both by nonspecific attraction between the positive charges of the protein and negatively charged hydroxyapatite and by specific complexing of protein carboxyls with calcium loci on the mineral. Elution can take place either as a result of the nonspecific ion screening of charges or by specific displacement of protein groups from the sites of the column with which they have complexed. Neither the principles nor the outcome of HAC can be compared to that of ion exchange chromatography (IEC), and the principle should be regarded as a chromatographic joker, sometimes offering surprisingly good results and excellent separation even between very closely related protein derivatives. The adsorption depends on pH and ionic strength. The elution behavior is a function of the isoelectric point of the protein leading to three different classes: 1. Basic proteins, which elute at similar, moderate concentrations of phosphate, fluoride, chloride or thiocyanate (0.1-0-3 M). Alternative low concentrations (< 0.003 M) of Ca2+ or Mg2+ are used to elute the protein. 2. Acidic proteins, which elute at about equal moderate concentrations of phosphate and fluoride but do not elute with Ca2+ and usually not with chloride.
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3. Neutral proteins, which elute with phosphate, fluoride and chloride and which do not elute with Ca2+ or thiocyanate. The hydroxyapatite chromatography techniques are used equally well in capture, intermediary purification or polishing mode at neutral to alkaline pH. Proteins may bind even at moderate to high ionic strength depending on their isoelectric pH offering high process flexibility. The availability of different size uniform stable particles in the range from 10 to 100 m favors intermediate purification and polishing operations, thus providing an excellent alternative to immobilized metal affinity, ion exchange, hydrophobic and reversed phase chromatography.
Advantages
Can be used during capture, intermediary purification and polishing Sometimes a very unique separation is obtained even between closely related compounds The principle applies in buffers with organic solvents Mixed mode purification principle
Disadvantages
The matrix is not stable at acidic pH and in presence of chelating agents
Hydrophobic interaction chromatography Hydrophobic interaction chromatography (HIC) is based on interaction between hydrophobic patches on the protein surface and hydrophobic ligands of the matrix. The interaction can be explained in the same way as the interaction between non-polar solutes in water, where the driving force is the increase in free entropy originating from water molecules leaving the more ordered structure around the non-associated solutes for the more unstructured bulk water. The binding strongly depends on the proteins tendency to avoid the solvent (solvophobic effect). This is why the high ionic strength of the solvent will favor binding, as will salts that give high surface tension. However, the use of high salt concentration in the application sample introduces the risk of salting out the protein. The conductivity of fermentation samples is often relatively high (10-30 mS/cm), making HIC an excellent capture step for hydrophobic proteins. However, the binding capacity may be decreased by presence of anti-foam agents added during fermentation and batch-to-batch variations may occur. Ligand optimization is a central issue as the large volumes handled and environmental concerns prohibit addition of extra salt. If HIC is used in intermediate purification, it is an excellent step following ion exchange chromatography, where high salt concentrations have been used to elute the target protein.
Advantages
Proteins bind at high salt concentrations and elute at low salt concentrations High recovery of most proteins Complementary to ion exchange chromatography Generally, a high binding capacity is obtained
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Disadvantages
Protein precipitation at high salt concentrations is a risk Environmental considerations must be included in large-scale operations Binds detergents
Reversed phase chromatography The hydrophobic effect can provide important selectivity options unavailable to other separation methods that are based on differences in size or charge. The separation mechanism in reversed phase chromatography (RPC) depends on the hydrophobic interaction between the solute molecules in the mobile phase and the ligand (the stationary phase). In contrast to hydrophobic interaction chromatography, where the ligands interact individually with the solutes, the RPC absorbent can be regarded as a continuous hydrophobic phase with the solute molecules partitioning between the mobile phase and the stationary phase. The separation depends on the reversible absorption/desorption of solute molecules with varying degree of hydrophobicity to a hydrophobic stationary phase. The technique of RPC offers excellent separation, even with closely related molecules. It is a close to perfect polishing technique, but rarely used in intermediary purification and not in the capture mode. Presence of organic solvent in the eluted fractions makes it often necessary to introduce a subsequent buffer exchange step.
Very high resolution possible between closely related compounds Samples of high ionic strength can be applied
Advantages
Disadvantages
The presence of organic solvents may destabilize/denature the protein of interest Protein solubility may be affected by the presence of organic solvents Generally the technique is not applicable for proteins with molecular weight above 25 kD High ligand density may result in protein denaturation
Size exclusion chromatography In SEC (gel filtration, gel permeation chromatography, molecular sieving chromatography), the solute molecules are separated on the basis of the different fractions of the intra-particle volume (pore volume) that, for steric reasons, are available for solutes of different size. Large molecules, which cannot enter the intra particle volume are not retained, while small molecules, able to enter the particles and diffuse into a larger volume, are retained. The elution volume of a non-retained solute is equal to the inter particle volume (the void volume, V0). The ratio between the intra particle volume (Vi) and V0 is called the permeability of the chromatographic medium. Sample molecules are added in solution as a zone to the top of the bed. The sample zone moves down the bed as buffer is added to the top. The small molecules, which diffuse into the bed, are delayed in contrast to large molecules, which cannot enter the particles. The result is a separation according to molecular size (or approximately
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molecular weight). The resolution is determined by the size difference between the solutes, the selectivity of the gel and parameters such as load, flow rate, particle size and column dimensions. Due to the reduced capacity (an application volume not exceeding 5% v/v of the column volume), SEC is rarely used in capture and intermediary operations. However, SEC is recommended as the final downstream process step for several reasons: 1. The method does not affect protein stability. Often a stabilizing effect can be observed. 2. Di- and polymer protein products can be effectively removed by this technique. No other chromatographic technique offers this general separation ability. 3. The drug substance formulation can be changed as the formulation work proceeds without altering the separation capability of the final step; a fact of enormous practical importance. 4. The composition of the resulting bulk material is defined, and batch-to-batch variations are minor
Effective removal of di- and polymers Method development is simple and fast Mild method unlikely to affect protein stability
Advantages
The buffer can be exchanged independent of the chromatographic technique
Disadvantages
Low capacity application sample volume should not exceed more than 5% v/v of the column volume The unit operation may be costly due to large column volumes Long cycle times due to long beds and low flow velocities The sample is diluted by the SEC procedure
Filtration The biopharmaceutical industry relies heavily on filtration technology for clarifying, purifying and sterilizing samples, buffers, cell culture media, bulk products and final aseptic fill. Filtration is a pressure driven separation process that uses membranes to separate components in a liquid solution or suspension based on their size and charge differences. In normal flow filtration, the fluid is convected directly towards the membrane under applied pressure. Particulates that are too large to pass the filter accumulate at the membrane surface, while smaller molecules pass through. Depending on the size of the filter, the different tasks can be divided into subclasses: Pre-filtration (5 m) Clarification (1 m) Filtration (0.45 m) Sterilization (< 0.1 m). In tangential flow filtration, the fluid is pumped tangentially along the surface of the membrane. An applied pressure serves to force a portion of the fluid through the membrane. Molecules or particulates too large to pass the membrane are retained and swept along by the tangential flow, thus avoiding the build up of particulate matter or molecules. The tangential flow filtration is traditionally divided into micro-filtration and ultra-filtration. Micro-filtration is usually used upstream in the recovery process to separate intact cells or cell debris from the harvest. Membrane pore size cut-off values are typically in the range from 0.05 1.0 m. Ultra-filtration is used to separate protein
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from buffer components (buffer exchange), sample concentration or virus filtration. Depending on the protein to be retained, the nominal molecular weight limits are from 1 1000 kD. Nano-filtration (0.05 m) is used to assure effective removal of virus particles. Filtration technology is an integrated part of the downstream processing with focus on sample conditioning prior to chromatographic procedures (e.g. concentration or buffer exchange). Micro-filtration of cell cultures (prior to the capture operations) is an alternative to centrifugation or expanded bed technology. The filtrate, including the target protein, comprises the sample for the first chromatographic unit operation. The need for buffer exchange using dia-filtration may arise anywhere in the process and tangential flow or hollow fibers are commonly used in parallel with chromatographic desalting procedures. However, it should be kept in mind that the shear forces applied might affect protein stability. Hence, if used as the final process step, the technique should be used with uttermost care. Ultra-filtration is also used for sample concentration (e.g. prior to size exclusion chromatography or precipitation).
Excellent technique for large scale operations Cost efficient technology May be simpler and cheaper to execute than centrifugation Efficient technology Good in process control tool
Advantages
Disadvantages
Fouling and thereby change in membrane properties Difficult to validate filters Membrane leaks may have severe consequences Blocking of filters
Protein folding In vitro protein folding (renaturation) is closely related to the E. coli expression system. The high intra-cellular expression levels often result in expression of denatured forms of the target protein and formation of inclusion bodies. It is common practice to dissolve the target protein into denaturing buffers at alkaline pH (e.g. urea or guanidinium chloride) in order to bring the protein on its monomeric form. However, the unfolded protein must be brought back to its native and biologically active state by in vitro renaturation. One of the most difficult tasks is to re-establish the correct disulfide bond pattern. Carefully designed folding buffers and fine-tuned parameter intervals (protein concentration, pH, conductivity, temperature and redox potential) are needed to assure even reasonable yields and to reduce the amount of incorrectly folded (scrambled) forms and aggregates. The redox potential of the folding solution is a critical operational parameter if the target protein comprises cysteine amino acid residues and mixtures of reduced and oxidized agents (e.g. -mercaptoethanol, cysteine) are added to facilitate folding. The folding mechanism is expressed in the following equilibrium: PS + R-S-S-R = P-S-S-P + RS where PS- is the protein on its reduced form
-
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R-S-S-R is an oxidizing agent P-S-S-P is the oxidized protein RS- is the corresponding reduced agent The reaction takes place at alkaline pH (usually the interval 7.5-9.5 is used), as the deprotonated form of cysteine is required. Several methods can be used for transfer of the target protein from the initial denaturation buffer to the folding buffer: dilution, dialysis, hollow fiber, size exclusion, immobilization and ultra-filtration. Crystallization Crystallization is a powerful purification method applicable only to proteins, which can crystallize. The relative long-term stability of crystallized proteins make this form suited for formulation of the final product (e.g. insulin) or as intermediary compound during purification. The crystallization window is often limited and it can take considerable time to define key crystallization parameters. Be aware that modern crystallization technology allows most proteins to crystallize.
Advantages
Effective purification Crystals are easy to isolate using either filtration or centrifugation Generally good long-term stability
Disadvantages
Not always applicable Narrow crystallization window
Formulation Introduction Formulation of the purified bulk (drug substance) serves several purposes: making it possible to administer the active pharmaceutical ingredient into humans, increase the shelf life, stimulation of the immune system (e.g. vaccines) and making the final drug product fit to the device used for administration. Drug products are typically formulated as liquid solutions (e.g. insulin pen systems), lyophilized products (human growth hormone) or suspensions (several vaccines). The final formulation very much depends on the stability of the drug product, which is generally enhanced by lyophilization or immobilization (e.g. alhydrogels). The most dominant destabilizing factors are aggregation, oxidation, de-amidation and loss of tertiary structure resulting in decreased potency. Excipients can be added in order to increase protein stability upon freezing, lyophilization and storage. Typical stress factors are agitation, temperatures near or above the melting temperature, TM, temperatures near or below the critical temperature, TC, freeze/thaw procedures, lyophilization, surface interactions and removal of protectants. Exposure to any of these factors may induce protein unfolding or denaturation resulting in uncontrolled aggregation during processing and upon storage. A successful formulation strategy builds on solid knowledge on drug administration, delivery devices, dose, intended shelf life, single versus multi-dose and storage conditions. The starting point for any formulation is the drug substance defined as the active pharmaceutical ingredient (API) bulk resulting from the purification process. The
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composition of the drug substance is of uttermost importance for the preceding formulation development study as co-solvents present in the drug substance may affect protein stability. Freeze-thaw Freezing of intermediary compounds, drug substances and drug products is a commonly accepted procedure in the biopharmaceutical industry for several reasons: protein degradation reaction rates are decreased, the shelf life of the final drug product is increased, shipping procedures are simplified, and finally freezing is part of the lyophilization procedure. However, the procedure is not without drawbacks and great care should be taken to assure that no damage is induced to the active pharmaceutical ingredient. A typical liquid formulation comprises the active pharmaceutical ingredient, water, inorganic anions/cations, stabilizer(s), oxygen, nitrogen, carbon dioxide and perhaps free radicals. The active pharmaceutical ingredient is often present in high concentrations (150 mg/ml) far exceeding the in vivo concentration. Upon freezing bulk water freezes out of the solution as pure ice crystals severely reducing the accessible volume in which the solutes now are present in much higher concentrations. At some point a solute becomes super saturated and releases a latent heat causing a slight increase in temperature. That point is known as the eutectic point (Te). A consequence of reducing the accessible volume is reduced mobility of the solutes, and increased viscosity changing the physical state from an elastic liquid to amorphous solid glass. That point is known as the glass transition temperature (Tg). In practice frozen solutions should be kept well below Tg, where mobility is severely reduced. Solutes may precipitate or crystallize as the accessible volume decreases. This is exemplified in the change in pH observed when freezing phosphate buffered solutions. Dibasic phosphate is removed faster from the solution than mono and tri basic forms resulting in a radical shift in pH, which may damage the target protein. The negative effects of freezing procedures can be compensated for by addition of cryoprotectants such as glycerol and ethylene glycol. It should be noted that cryoprotectants may be poor lyoprotectants. Lyophilization Water impacts protein stability and the importance of residual water in lyophilized biopharmaceuticals are well recognized in the biotech industry. If proteins were stable in aqueous solution few if any would enter into the complicated and expensive freeze drying procedure. A lyophilized product often maintains both physical and chemical stability during shipping and upon storage even at ambient temperatures. Despite the complexity of the freezing and lyophilization process, a rational designed development program should assure a relative rapid progress. A successful lyophilized formulation is characterized by its relative increased stability, a strong, elegant cake structure, a TG exceeding the storage temperature, and a low water content (<1% w/w). The lyophilization process comprises 4 steps: freezing, annealing, primary drying and secondary drying. The freezing procedure serves the purpose of forming ice crystals, a prerequisite for the following sublimation procedure. The purpose of the annealing step is to avoid uncontrolled moisture release by removing a fraction of the excipient from the amorphous phase and thereby increase the collapse temperature, Tg. In the primary phase ices is sublimated from the product. The drying is driven by pressure reduction and applying heat to the chamber. It is important that the drying temperature is below the collapse and eutectic melting temperatures (Tg and Te) of amorphous and crystalline solutes, respectively. During the secondary drying stage moisture is removed from the product. The temperature and extent of secondary drying influences the final moisture content of the product.
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It is common practice to add bulking agents (e.g. mannitol) in order to assure an elegant cake structure and to add lyoprotectants (e.g. sucrose) for stabilization of the protein during lyophilization. Acetic acid and ammonium bicarbonate are commonly used buffer ingredients as they undergo sublimation during the lyophilization procedure. Development program The aim of pharmaceutical formulation development is to design a quality drug product produced by a robust and controlled manufacturing process. This is no different from the purpose of upstream and downstream processing, however, much less attention has been paid to formulation development resulting in late and inadequate programs. Fortunately a paradigm shift among large biotech companies has resulted in addressing administration of biopharmaceutical drugs, devices, drug product formulation, single versus multi-dose, storage conditions and shelf life very early in the drug development program. This approach has been deemed necessary as the downstream process and drug substance composition is affected by dosage (acceptable limits for impurities) and presence of co-solvents (influence on protein stability). A liquid drug product formulation is totally different from a lyophilized product and entirely different formulation development programs should be carried out. Every protein has unique physicochemical properties, which must be addressed in order to ensure a stable product with a reasonable shelf life (usually 1-2 years). The major factors of concern are aggregation, de-amidation, oxidation and loss of conformational stability resulting in reduced potency. All factors mentioned contribute to product degradation always observed regardless of formulation principle (liquid formulation, lyophilized product, spray dried product, micro-spheres, or suspensions). The complexity of drug formulation and the complicated decision trees associated with drug formulation development, can to some extent be systemized by addressing administration form, dosage, protein solubility, protein hydrophobicity, air-water interfaces, reactive residues, response to stress, tonicity, shelf life, storage temperature, and costs in the development program. Excipients Excipients are compounds added to assure the optimal protein stability in solution, during freezing/thaw, during lyophilization and during re-hydration of the dried product. They further serve the purpose of improving the cake structure of lyophilized products, assure tonicity, reduce surface activated protein destabilization, and improve resistance to heat. Excipients are classified as follows:
Excipients
Bulking agents (e.g. maltose) Solution stabilizers (e.g. ammoniumsulphate) Cryoprotectants (glycerol) Lyoprotectants (e.g. sucrose) Surfactants (e.g. Pluronic 68) Heat protectants (e.g. sorbitol) Anti oxidants (e.g. cystein)
Fill & finish The drug product must be stored in a suited vial or device in order to assure stability during the entire storage period. Most administration forms require sterile drugs, hence a
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sterile filtration and/or sterilization procedure is carried out before vialing. Each vial must contain specified information about lot number, content, volume, expiry date etc.
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4.0 Protein stability

Introduction One of the often-overlooked issues in the manufacture of recombinant proteins is the chemical and physical destabilization of the protein during fermentation and purification except for the relatively few cases where the protein is expressed in a non-native form such as inclusion bodies. It must be kept in mind that the protein is over-expressed in a foreign host organism usually stressing the cell and that the protein during purification is deprived its natural environment be it stabilizing lipids, lipoproteins and/or proteins. Further, as the protein is purified, its concentration usually far exceeds the in vivo concentrations. Other factors such as sheer stress during cell culture, co-solvents, pH, etc. may also influence the stability of the protein. Globular proteins are not very stable in aqueous solutions and are easily prone to denaturation, which in several cases lead to protein aggregation and thereby loss of product. However, also the native form of certain proteins (e.g. insulin, elongation factor) may be subject to aggregation due to hydrophobic interaction. Cystein containing proteins may form inter-molecular di-sulfide bonds also leading to aggregation. Chemical modifications involve proteolytic cleavage, deamidation, oxidation, elimination, racemization, carbamylation and acylation of the target protein. In contrast to the physical instability, where huge losses can be observed due to aggregation, chemical instability typically results in less than 10% loss. However, the derivatives may be difficult to separate from the target molecule due to almost similar physical and chemical properties. The stability of other proteins present in the harvest is of interest too. These proteins may precipitate under prolonged holding-times resulting in clotting of filters and/or chromatographic columns. It is, therefore, necessary to investigate sample holding-times typical of large scale operations. It is of interest to discuss protein stability from a statistical point of view. Protein stability is maintained if GNATIVE GUNFOLDED < 0. As G = GNATIVE GUNFOLDED = -RxTxln([N]/[U]), the criteria for a stable solution is that [N] > [U]. At [N] = [U] G = 0, which is the transition between the folded and the unfolded state. The corresponding temperature is called the melting temperature TM. A plot of G versus the temperature (see below) shows the correlation between protein stability and the ratio between native and unfolded molecules. Table: G versus temperature. Protein stability is maintained in the gray area. - G
TS
TM
Temperature
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The protein solution has the maximal stability at TS where the ratio [N]/[U] is at maximum value. As the temperature increases towards TM, the ratio [N]/[U] approaches 1. Thus, a protein solution always comprises a population of native molecules and subpopulations of more or less unfolded molecules. The ratio [N]/[U], is a measure of the stability of the solution very much depending on the unfolded forms ability to form irreversible aggregates. It should be pointed out that [N]/[U] in some cases also decreases with decreasing temperature; the phenomenon is called cold denaturation. In practice, the unit operation should be carried out near TS. Protein unfolding (for example induced by urea or GuCl) does not lead to loss of protein if the reaction natve protein <=> unfolded protein is reversible and if the unfolded forms do not aggregate under the unfolding conditions. Protein aggregation is typically a irreversible reaction of second order kinetics. Once the aggregation reaction is initiated, great risk is that more and more target protein molecules are dragged out of the solution by aggregation resulting in gelation of the entire sample in very short time intervals or formation of visible aggregated molecules (e.g. fibrils). Will the stability of the active pharmaceutical ingredient (API) affect the stability of the drug product? The answer relates to the distribution of folded and unfolded molecules of the API and the nature of unfolded sub-populations. Without knowledge of the said distribution very little can be predicted about the long-term stability of the drug substance. Factors affecting protein stability pH pH has a high impact on protein stability. The preferred working range is from pH 3.0 to 9.5 outside which severe damage to the protein should be expected due to hydrolysis of the peptide bond, protein unfolding, cleavage of disulfide bonds or racemization. Below is listed a range of pH dependent reactions: Deamidation should be expected above pH 5. The optimal working range in which to avoid deamidation is probably between 3.0 and 5.0. There is no specific pH range in which all enzymes are considered inactive; At slightly alkaline pH the non-specific enzymes of the vacuoles and lysozomes will be minimally active. Use strong buffers for extraction to prevent unintended shift in pH as a result of cell disruption. Some yeast enzymes are least active in the pH range 4-5, but active in the pH range 7-9. The oxidation rate is assumed low at slightly acidic pH. High pH will favor abstraction of the -hydrogen under formation of a carbanion leading to racemization or -elimination. pH should be kept below 10 and use of NaOH in concentration above 0.1 M should be avoided when adjusting pH. Minimum reactivity of sulfhydryl groups should be expected in the pH range 3-7. The reactivity of the SH group is at maximum above the pKa (8.5), where the group is de-protonated. In strongly acidic media the reaction is expected to take place via a sulfenium cation by an electrofil displacement. The Asp-peptide bonds are prone to degradation at acidic pH High pH should be avoided in order to prevent protein unfolding. Some proteins do change conformation as a function of pH and certain pH intervals should be avoided. Arg is converted to ornithine by OH in a concentration dependent manner Proteins tend to be most stable near the isoelectric point. The protein solubility is minimal near the iso-electric point The redox potential is pH dependent. An increase of one pH unit is equivalent to a decrease in redox potential of 60 mV.
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Temperature The operational temperature should always be kept near the TS to assure the maximum number of native molecules under the given conditions. The deamidation rate increases with increasing temperature Low temperature decreases the proteolytic activity. It is recommended to store harvest at 4-8 C or frozen Working at low temperatures decreases the rate of oxidation High temperature even at pH 4-8 results in -elimination The temperature should be kept low to prevent racemization The unfolding process is a function of temperature. Many proteins have optimal stability in the temperature range 10-30 C. Loss of structure should be expected both at low temperatures (cold denaturation) and at elevated temperatures. The reason why many protein biopharmaceuticals are stored at low temperatures is to minimize chemical degradation (e.g. deamidation). The temperature should be kept low (4-20C) to prevent cleavage of disulfide bonds at alkaline pH The aggregation reaction can be very fast High temperature increases the conformational flexibility and for example organic solvents may more easily penetrate the internal structure of the protein Conductivity It is generally recommended to work at low ionic strength in order to prevent precipitation, deamidation, and -elimination. The ionic strength of the solution should be kept low in order to prevent deamidation and -elimination. At high ionic strength the deamidation reaction can be fast even at neutral pH. High ionic strength normally results in protein precipitation. Keep the salt concentration low to moderate, but at the same time, be aware of the salting in effect. Ions such as NH4+ do stabilize the protein upon precipitation. Redox potential The redox potential of the solution influences the behavior of the sulfhydryl group. At reducing conditions free sulfhydryl groups (either as SH or S- depending on pH) should be expected, while intra-molecular or inter-molecular disulfide bonds are dominant at oxidizing conditions, the latter often leading to aggregation and loss of product. Reducing and oxidizing conditions may alter the disulfide bridge arrangement and state of free cysteine residues thus influencing the protein secondary and tertiary structure A high redox potential indicates presence of oxidizing agents Cystinyl rich proteins may decompose under formation of HS-, which will lower the redox potential of the solution. Reduction of disulfide bonds may result The redox potential is a function of pH (60 mV/pH unit) Cleavage of disulfide bonds should be expected under reducing conditions. A redox potential below 100 mV is considered unstable for some proteins (e.g. insulin). Not all proteins will undergo conformational changes upon reduction of the disulfide bond(s). Protein concentration Globular proteins may aggregate from the native, intermediary folded, and unfolded state in a reaction of second order kinetics. The higher the concentration, the faster is the aggregation reaction. A rule of thumb is therefore to maintain low protein concentrations during purification and storage, a principle unfortunately in conflict with both process economy and demand for highly concentrated drug product liquid formulations.
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The intra-molecular disulfide bond formation is a first order reaction and thus independent of protein concentration. Inter-molecular reactions via the cysteine residue may be affected by the protein concentration (aggregation). Low protein concentrations should be favored to avoid aggregation and to prevent precipitation.
Holding and storage time In general holding times should be kept as short as possible in order to keep proteolytic degradation, deamidation, oxidation, carbamylation, -elimination, racemization, hydrolysis aggregation and precipitation to a minimum. Presence of polymeric, cleaved, des-amido, oxidized, carbamylated or degraded forms is a commonly used marker for protein stability during storage. Co-solvents Presence of co-solvents is well known to influence protein stability. In general the buffer species and the buffer strength will influence the rate of deamidation. High solvent dielectrics favors deamidation In model peptides the protein stability was higher in Tris buffer than in phosphate buffer Avoid oxidizing agents and protect against light. Addition of chelating agents (EDTA, citric acid, thioglycolic acid), antioxidants (propyl gallate, Vitamin E) and/or reducing agents (Cys, dithiothreitol, methionine, ascorbic acid, sodium sulfite, thioglycolic acid, thioglycerol) may reduce oxidation Cyanate is able to react with amino, sulfhydryl, carboxyl, phenolic hydroxyl, imidazole and phosphate groups in proteins. The pH optimum for the reaction is about 7 Removal of divalent metal ions with EDTA will reduce -elimination Cystein (non animal origin) is recommended as a reducing agent for large-scale operations. Divalent metal ions should be removed by EDTA. Sucrose, mannose, glucose, glycine, alanine, glutamine, ammonium sulfate are examples of compounds acting as protein stabilizers (weak or no binding to the protein surface Magnesium sulfate, guanidinium sulfate, sodium chloride and other weakly interacting salts will exhibit an effect depending on protein charge and concentration Polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) act as stabilizers due to steric exclusion and repulsion from charged groups. Both PEG and MPD may destabilize the protein under certain circumstances, where binding is favored over exclusion. Co-solvents such as urea or guanidinium chloride, which binds strongly to the protein surface, are strong denaturants Denaturing or destabilizing agents (e.g. urea, certain alcohols, organic solvents) should be used with care. Detergents may prevent aggregation, but they often bind strongly to the protein. Typical protein precipitation agents are salts (e.g. ammonium sulfate), polyethylene glycols (e.g. PEG 20000), polyelectrolytes (e.g. carboxymethyl cellulose) and organic solvents (e.g. acetone) Phosphate may exhibit a stabilizing effect on proteins Other proteins Presence of other proteins in excess (e.g. albumin) will reduce the proteolytic damage.
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Shear forces Shear forces arising as a result of fermentation, tangential membrane filtration or pumps often result in protein destabilization and aggregation. Reduce such interactions to a minimum. Protein stability during processing Upstream Target proteins are generally expressed in their native form to the medium together with a limited number of host cell proteins. Although presence of other proteins may to some extent protect the target protein, shear stress from bioreactors may result in loss or product due to aggregation and host cell enzymes released upon cell apoptosis may result in degradation. The target protein concentration is generally low at this stage. In this respect, transgenic animals comprise an interesting expression system as the protein is expressed under relatively mild conditions (short expression time and no shear forces) into a well-defined medium (milk). Downstream The major objective of the downstream operations is to isolate the target protein from host, process and product related impurities. Early in the process removal of proteolytic enzymes is a major objective often accomplished after the capture step. It is important to optimize unit operation parameters in order to minimize formation of des-amido, oxidized, carbamylated, and acylated forms although the choice of stability parameters always will be in competition with purification parameters. However, extreme pH conditions should be avoided (3.0 < pH <9.5 is preferred) and operations should preferably be carried out near TS. Proteins with di-sulfide bonds should not be exposed to reducing conditions and care should be taken to expose the protein to oxidizing conditions if it contains free cysteine(s). Urea should be used with care as presence of cyanate results in carbamylation of primary amino groups. A specific, but important issue, is the reduced protein stability, which may follow in vitro refolding. The following chromatographic unit operation and separation conditions should therefore be selected with care to avoid protein aggregation. Formulation The drug substance, which is the resulting bulk material from the downstream processing, is rarely used directly for human injection. Instead, the substance is converted to the drug product taking injection form, device, stabilizing co-solvents, adjuvants, etc. into consideration. The formulation procedure may severely affect the stability of the drug product; hence separate stability programs for drug substance and product are carried out. Control of protein stability One of the major tasks during process development is to assure process robustness in the sense that the process repeatedly is doing what it is supposed to do. If the term building quality into the process should be taken rigorously, process quality should be demonstrated throughout the development. As traditional one-factor-at-a-time experiments do not effectively address critical parameters and interactions between variables, it is recommended to introduce statistical design of experiments such as response surface methodologies, factorial designs and statistical evaluation of model predictions. The typical unit operation experiment involve the study of the effect of several factors (e.g. pH, temperature, protein concentration, temperature) by for example investigating the effect from all combinations of factors at low and high level and determine critical parameters and their interactions. The effect of a factor is defined to be the change in response produced by a change in the level of the factor.
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The multivariate data analysis here proposed has several advantages over the onefactor-at-a-time approach typical for laboratory work: Critical parameters are statistically defined Parameter interactions can be determined The experimental work can be planned and delays stemming from endless searches for optimal conditions avoided Proven acceptable ranges, regulatory ranges and process ranges can be statistically justified A statistical approach will support process validation It is suggested that protein stability is made a response in the factorial design experiments carried out for a given unit operation. Protein stability during storage The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a re-test period for the drug substance or a shelf life for the drug product and recommended storage conditions (ICH Harmonized Tripartite Guideline Q1A(R2) of February 2003, Step 4). Several factors (e.g. temperature, light, oxidation, humidity, shear forces) can affect the molecular conformation and hence the potency and immunogenicity of a recombinant derived protein upon storage. It is, therefore, on a case-by-case basis necessary to thoroughly investigate the stability of critical intermediary compounds, the drug substance and the drug product as a function of time under conditions for the storage of said compounds. It is important the products are stored under defined conditions with respect to containers, closure systems, exposure to light, shipment procedures, etc. Appropriate analytical methods are used to monitor potency, purity and quality in order to support stability data for the representative storage period. Intermediary compound stability studies During manufacture of biotechnological products storage of intermediates may be required and thud the quality of intermediates may be critical to the production of the final product. In such cases the stability of said intermediates should be investigated and assured within the required time limits. Drug substance stability studies The drug substance is the bulk material resulting from the purification process before formulation, filling and packaging. If the drug substance is to be stored prior to formulation and final manufacturing (which is usually the case), stability data should be provided on at least 3 batches for which manufacture and storage are representative of the manufacturing scale of production. In general stability data should be provided for the entire storage period, but a minimum of 6 months stability data (at the time of submission) should be submitted in cases where storage periods greater than 6 months are requested. Data from pilot scale batches (typically at a reduced scale) may be used prior to the manufacturing scale long-term stability study. Drug product stability studies The drug product is the final product. Stability data should be provided on at least 3 batches of final container product. In general stability data should be provided for the entire storage period, but a minimum of 6 months stability data (at the time of submission) should be submitted in cases where storage periods greater than 6 months are requested. Data from pilot scale batches (typically at a reduced scale) may be used prior to the manufacturing scale long-term stability study.
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Stability studies of products used for phase I-III clinical trials Although there are no specific guidelines for stability testing of products used for phase IIII clinical studies, the issue should not be neglected. Stability data of drug substance and drug product are equally important and should as a minimum provide information of the potency, purity and quality for as long as the study takes to conduct. Strategic planning is needed, as the process often undergoes changes from early development and onwards. It is common practice to produce reference material, which is thoroughly tested and characterized and use it as a reference for the stability studies. Major process changes may require renewed stability studies and a delicate balance between need for change and conserved processes prevail. Many biotech companies tend to manufacture products for phase I and II in the same campaign in order to use said material for stability studies as well. However, it may be desirable to use the tox material instead, as this will provide more stability data in due time. This strategy makes it necessary to use similar processes for manufacture of tox- and cGMP batches. Analytical testing Stability test methods should be able to monitor those attributes of the active pharmaceutical ingridient (API) that are susceptible to change during storage and are likely to influence quality, safety and/or efficacy. It is the responsibility of the manufacturer to propose a stability indicating profile that provides assurance that changes in the identity, purity, and potency of the product will be detected. Examples of methods contributing to the test program are biological activity (potency), SDS-PAGE, immuno electrophoresis, IEF, Western blot, peptide mapping (identity), HP-IEC, HP-RPC, HPSEC, capillary electrophoresis (purity). Typically more than one purity method is being used to assess the quality. Other product characteristics such as visual appearance, visible particulates in solution, pH, moisture level of powders and lyophilized The specification must be defined on a case-by-case basis. Accelerated and stress conditions It s strongly recommended to supply the stability program with stability studies conducted on the drug substance and drug product under accelerated and stress conditions (e.g. elevated temperature). Such studies may provide useful data on impurity profiles and loss of native structure (e.g. aggregation), which can be used to set the expiry date, to specify indicative test parameters for product stability and to evaluate stability indicating methods. Physical instability Denaturation The native protein molecule is loosing its tertiary structure upon denaturation resulting in a population of partially unfolded molecules. In practice the denaturation process will lead to a mixture of more or less unfolded molecules comprising residual secondary structure elements (helix, -sheet, -turn, cis-trans isomery around the prolinyl residue). A population of random coil molecules should not be expected even under strong denaturing and reducing conditions. Upon denaturation the inner hydrophobic core of the protein molecule is exposed to the hydrophilic environment (solvent water) often resulting in (irreversible) aggregation of the target protein. The cooperativity of the denaturation process results in an abrupt transition from the native to the unfolded state within a narrow range of pH, temperature, ionic strength, and denaturant concentration meaning that protein denaturation may come fast and unexpected. As globular proteins are only marginally stable in aqueous solutions, parameter interactions should be well understood and described using for example factorial design experiments. Proteins with disulfide bonds may undergo unfolding under reducing conditions, where the covalent bond is cleaved. A denatured protein may be brought back to its native form by in vitro folding. The folding process is often slow and yields can be poor. As each protein is unique, the in vitro
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folding conditions must be determined case by case often using specific co-solvents as additives. An example is the group of proteins where disulfide bonds must be reestablished as part of the renaturation process. Aggregation Protein aggregation is a major problem in the purification and formulation of protein biopharmaceuticals. Two types of inter-molecular reactions dominate: aggregation resulting from hydrophobic interactions, and aggregation stemming from inter molecular disulfide bond formation between cysteine residues. Proteins exposed to even mildly denaturing conditions may partially unfold resulting in exposure of hydrophobic residues to the aqueous solvent favoring aggregation. The aggregation process is assumed to be controlled by the initial dimerization step in a second-order reaction. Consequently, high protein concentrations will increase the aggregation rate. Inter-molecular disulfide bond formation between cysteine residues takes place at alkaline pH under oxidizing conditions. Proteins with reactive free thiol groups should be purified under reducing conditions (typically 1-10 mM reducing agent) in the presence of EDTA. Even proteins with disulfide bonds may participate in inter-molecular disulfide bond reactions due to disulfide bond shuffling at neutral and alkaline pH. Expression of proteins in E. coli often results in the formation of insoluble aggregates called inclusion bodies, probably comprising fully or partially unfolded protein. Inclusion bodies are brought to their monomeric form by extraction with a denaturant (e.g. 8 M urea) under reducing conditions (e.g. 0.1 M cysteine). Precipitation Protein precipitates are aggregates large enough to be visible. However, in practice aggregates not visible by the naked eye may result in severe problems as filters and chromatographic columns can be blocked. Protein precipitation is typically observed at high ionic strength, in the presence of organic solvents or close to the isoelectric point, where solubility is low due to the zero net charge of the protein. Presence of precipitates is not always easily observed. Typical markers are presence of large often white particles or flocculates, a turboid appearance, fibrils or increased viscosity. Unintended precipitation can be difficult to predict as the effect depends on a combination of the distribution of hydrophilic and hydrophobic residues on the proteins surface, the pH, ionic strength, protein concentration, temperature and composition of the aqueous phase. A perfectly clear solution may gradually become turboid during application to a chromatographic column resulting in column blocking. Chemical instability Proteolysis In contrast to the cellular environment, where enzymatic degradation of proteins is highly controlled, extra cellular proteases is the cause of uncontrolled protein degradation. The result of the proteolytic attack may vary from complete hydrolysis, single breaks within the peptide chain, or loss of a few N- or C-terminal amino acid residues. Besides loosing the product, presence of truncated forms may seriously challenge the purification design. Proteolytic enzymes are released to the medium because of cell death, mechanical stress or induced cell lysis. Their presence should be expected during fermentation and initial downstream unit operations. Most enzymes of the vacuoles and lysozomes will be minimally active at slightly alkaline pH (7-9), a pH interval strongly recommended for extraction of proteins expressed in bacteria. Proteins are probably more resistant towards proteolytic attacks in their native state and stabilizing factors (e.g. co-factor, correct parameter interval, co-solvent) should always be considered optimized. Use of enzyme inhibitors is not recommended for safety reasons.
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Deamidation Two amino acid residues are involved in the deamidation reaction: asparagyl and glutamyl. The conversion to the corresponding carboxylic acid residues results in a shift in net charge of the protein at pH above the pKa. As the deamidation may influence the biological activity and the stability of the molecule the maximal content of des-amido forms in bulk materials and in biopharmaceutical preparations is constantly being debated. The list of proteins undergoing deamidation is comprehensive and includes wellknown proteins such as insulin, human growth hormone and cytochrome C. Asparagine residues tend to be more susceptible to deamidation that glutamine residues. Further, the deamidation reaction is strongly sequence specific in model peptides with the half live of the -Asn-Pro- sequence being 100 fold greater than that of Asn-Gly. To some extent these observations can also be used on proteins taking the structural steric factors and nearby amino acid residues into consideration. Oxidation The amino acid residues histidyl, methionyl, cysteinyl, tryptophanyl and tyrosinyl are potential oxidation sites at neutral at slightly alkaline conditions. Oxidation of the said residues often results in loss of immunological and/or biological activity. The list of proteins that have been oxidized is comprehensive and includes biopharmaceutical products such as albumin, growth hormone, glucagons, interleukin-1 and interleukin 2. In many cases, the immunological and/or biological activity was only partially lost. In general, oxidation of methionyl to methionyl sulfoxide does not affect protein antigenicity, probably because the conformational structure of the oxidized protein is close to the native structure. On the other hand oxidation of a single amino acid residue often causes changes in the biological activity and all efforts should be taken to minimize oxidation reactions. Carbamylation Cyanate is able to react with amino, sulfhydryl, carboxyl, phenolic hydroxyl, imidazole and phosphate groups in proteins according to the general scheme RXH + HNCO = RXCONH2. Cyanate is easily soluble in water. Most reactions have a pH optimum around 7. Acidic pH should be avoided as acidic conditions are ideal for modifications of carboxyl groups. For the same reason reactions with cyanate should not be terminated with acid. At high concentrations cyanate may react with itself to form cyanuric acid and cyamelide and it is recommended to work at concentrations about 0.2M. Cyanate reacts rapidly with amino groups. At neutral pH and below the -amino group can be expected to react about 100 times faster than the -amino group. The resulting carbamoylamino groups are stable even in dilute NaOH. Typical reaction conditions are 3 mg/ml protein, 0.1 M cyanate at pH 8, 25C for 1 hour. Cyanate reacts even more rapidly with sulfhydryl groups than amino groups resulting in the formation of S-carbamylcysteine residues. Since cyanate reacts rapidly with sulfhydryl groups, labile disulfide bonds may be ruptured. The resulting carbamylmercaptans decompose readily to free mercaptan and cyanate at alkaline pH. Consequently, cyanate can be used as reversible blocking agent for SH groups. At acidic pH cyanate reacts with carboxylic groups under formation of a mixed anhydride, which can react with many nucleophiles (e.g. formation of amides). The reaction can be avoided entirely at pH 7 to 8. Aliphatic hydroxyl is resistant to carbamylation even at high cyanate concentrations at low pH. However, the reactive hydroxyl groups of chymotrypsin and other proteases react with cyanate to give urethans. Phenolic hydroxyl groups react more readily than aliphatic groups in a reversible reaction that is quite analogous to the one that occurs with SH groups.
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-elimination The -elimination reaction is caused by the abstraction of -hydrogen from cysteine, serine and threonine residues under alkaline conditions. The cystinyl residue decomposes as a result of -elimination under formation of HS and free sulfur thus affecting the redox potential of the solution. Several studies indicate that the rate of reaction is proportional to the hydroxide ion concentration and pH should consequently be kept low (use dilute NaOH solutions to adjust pH preferably below 0.1M NaOH). Racemization All amino acid residues except glycine are subject to racemization at alkaline pH resulting in formation of the D-enantiomers of the residue. Racemization is inevitably associated with conformational changes and thereby loss of function. The racemization of proteins has been described in several reports. Cysteine residues The reactive site of the cysteine residue is the thiol group, which is deprotonated at alkaline pH (pKa around 8.5). The residue is under oxidizing conditions (and neutral to alkaline pH) able to react with a similar residue under formation of a disulfide bond. Many proteins are stabilized by intra-molecular disulfide bonds (e.g. insulin, growth hormone, IGF-1), but inter-molecular bonds may also result from the reaction under formation of aggregates. In order to avoid unintended disulfide bond formation/cleavage, the redox potential of the solution must be monitored and controlled. In practice, aqueous buffers contain -molar amounts of dissolved oxygen assuring a redox potential of 200-600 mV, which is sufficient to maintain the intra-molecular disulfide bonds. Proteins with free cysteins may prefer slightly reducing conditions, which can be obtained by addition of molar amounts of reducing agent (e.g. cysteine or dithiothreitol). The number of proteins containing both SH groups and disulfide bonds are relatively small (e.g. albumin, lactoglobulin). In many cases the disulfide bond stabilization is essential for maintaining the biological activity. Ribonuclease, for example, loses almost all activity when the four disulfide bonds are reduced. Hydrolysis The peptide bond is not undergoing significant hydrolysis in the pH interval (3-9.5) usually used in industrial downstream processing. However, in dilute acid, where the carboxyl group of aspartyl residues is not dissociated, the peptide bond is cleaved 100 times faster than other peptide bonds and especially the Asp-Pro- sequence is prone for degradation. The guanidinium group of arginine is hydrolyzed by OH- to give ornithine and possibly some citrulline, depending on the nature of the protein.
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5.0 Scale up and manufacture

Introduction The transfer of a biopharmaceutical process from development to pilot is one of the major milestones in the drug program leading to manufacture of licensed product. The process is transferred from a pure development environment with many degrees of freedom to a strictly controlled environment where change procedures are costly and time consuming. The cultural differences between development and pilot departments are huge, the former building on scientific traditions, the latter on technical solutions and large scale operations. Over the time, most biotech companies have experienced multiple conflicts between the two cultures and complete process redesign upon transfer has been a far too common outcome. Lack of communication has been a major problem. There is no doubt that the development scientists have been far too optimistic in their design strategies including a number of small-scale solutions not fitting into a current good manufacturing practices (GMP) large-scale environment. On the other hand, the pilot community has not fully respected the protein chemistry background so important to produce a stable purified target protein, which can be used for human treatment. The result has been waste of time and money. The best biotech companies have responded to this challenge by the concept of design in, where issues related to process safety, robustness, GMP compliance, facility constraints, economy and time to market is build into the process at small-scale development. Further, the process is tested in small scale before transfer against predefined process maturity factors, such as parameter intervals, in process control points, critical parameters and their interactions, robustness, yield, documentation level, raw material qualification, and maturity of analytical procedures. An extensive tech transfer package is provided comprising the development documentation, process protocols, batch data, analytical method descriptions and data, stability data, and reference material supported with an extensive training program. The concept has constantly been challenged by stepwise scale up, where cell culture and initial purification steps (e.g. capture) are scaled up before the process is developed in small scale. The stepwise scale up procedure creates much confusion, is time consuming and invites to late process redesign. The reason why this concept is still popular is the constant pressure from upper management to gain time and to keep to unrealistic time schedules. The procedure cannot be recommended. It has been common practice within the biotech industry to outsource parts of the drug program to external contract service partners either due to lack of capacity or lack of expertise. Especially the contract manufacturing area has expanded over the last decade as more and more biotech companies entered the scene. However, the transfer of projects between sponsor and contract manufacturer has in many cases resulted in poor outcomes partly due to unrealistic sponsor expectations and partly because of bad communication. Again, the importance of proper process design, realistic time schedules and finance in early project phases cannot be emphasized enough. Sponsors should realize that the contract manufacturer delivers what they are asked to, and they do not take project responsibility. Process scale up serves two purposes: production of drug product for pre-clinical and clinical trials and as a preparation for manufacturing of licensed product. It is a major strategic decision to transfer the process into the manufacturing environment, as process changes from now on are costly and time consuming to introduce. However, the need for trial material is always imminent, and there is a limit to how far the small-scale experiments can go on.
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When, how and why? Large-scale operations are very different from the small-scale experiments carried out in the laboratory. Simple tasks such as buffer preparation, pH adjustment, sample application and transfer of material become complicated and time consuming activities having severe impact on the process itself and its economy. Process planning in the biotech industry is usually carried out backwards from initiation of clinical trials and even launch to market raising severe concerns amongst process development scientists and manufacturing staff. The lack of time for proper process development and scale up often results in too fast tech transfer to technical scale followed by costly and time consuming process redesign. Ironically, dead lines for initiation of clinical trials are not met, either. A solution to the dilemma is to bring in a rational approach to process scale up setting strict criteria for process maturity, tech transfer, and process validation prerequisites. In the end, a robust, safe and GMP compliant process is expected for manufacturing of licensed product. Hence, careful planning of needed activities can save time on an overall schedule, while some intraproject activities may be shortly delayed. In the past, many biotech companies tend to go for a fast approach and thus accept major process redesign at a later stage (usually between manufacture for phase 2 and 3 trials). However, a great part of these exercises failed due to poor process understanding, lack of technical batches and protein instability. In order to define the tech transfer package one needs to investigate the information, which can be provided in small and technical scales. The small-scale experiments serve the purpose of testing and optimizing the unit operations, suggest proven accept ranges for parameters, define critical, key and non-key parameters, define holding times, investigate filters and report clotting, testing and optimizing the entire process and to provide material for preliminary stability studies. Another important aspect of small scale process testing is to obtain data for setting specification accept criteria and to test whether already specified accept criteria can be met. This approach is very much in agreement with Process Analytical Technology (PAT) from the Food and Drug Administration (FDA). Some aspects cannot be tested in small scale as bioreactors, centrifuges, and precipitation technology differs. A small-scale process may work well in the laboratory, but may prove ineffective after scale up. It is, therefore, important that the process developers take technical matters into consideration during unit operation design and optimization. One aspect of process design and development, which is often neglected, is to provide the necessary data to support the process validation program. However, Process validation cannot take place before process characterization studies have been carried out thus defining proven acceptable ranges, critical, key and non-key parameters. These data are essential for manufacture, which cannot operate from set points, only, as in small-scale operations. For that reason, process characterization studies should be integrated into the basic process development activities. Small-scale activities are very cheap in comparison with those of technical, tox and GMP manufacture. So the incentive for early scale up is purely time driven and as no visible project milestones are associated with process development, the time and resources to be allocated are often chosen at random. One solution is to define scale up as a milestone in project development and to link this event with process quality identifiers. A proposal fro a tech transfer package is given below.
Process design Cell line Raw materials Rationale for unit operation and overall process design Research cell bank description and status List of raw materials and their rationales
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Flow sheet Unit operation descriptions Drug substance Drug product In process control Quality control Analytical methods Process characterization Process testing
Process flow sheet including an in process control sampling plan Documentation of the work carried out and a protocol Drug substance composition and its rationale storage conditions Drug product formulation and its rationale storage conditions List of in process control analyses Specification, accept criteria, and quality control package Method applicability must be shown Identification of proven acceptable ranges, critical parameters, key and non-key parameters Batch data from several batches run in small scale
Documentation
Process design rationale Flow sheet List of raw materials Unit operation descriptions Specification and accept criteria Batch data Process protocol Analytical method descriptions Process characterization report Development report
The tech transfer strategy laid out merely excludes technical scale experiments prior to tech transfer from the process development laboratory to pilot. This strategy need not be a time constraint if enough qualified resources are allocated for the process development work. it is important that tech transfer allows for process optimization in technical scale. Technical batches Technical batches are carried out in a non-GMP environment with the purpose of implementing the process in technical scale. Technical batches should serve no other purposes. It is advised to mimic the coming GMP manufacture operations as closely as possible with respect to use of technical equipment, raw materials, buffers, etc. in order to reduce the number of process changes. Technical batches are carried out in close collaboration with the small-scale developers. When testing a process in technical scale the forthcoming tox and GMP manufacture is taken into consideration. Not only is the process being tested and optimized, so is the use of utensils (bags, tubing, containers, filters, membranes), vials, stoppers, chromatographic media, and columns. Large-scale buffer preparation is introduced and tested and often 0.45 or 0.2 m filters are inserted in between unit operation in order to reduce bioburden (large scale operations are more time consuming and microbial growth
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must be kept to a minimum). Cleaning and sanitization are addressed as well in order to prepare for aseptic manufacture. It is advised to perform several technical batches before entering tox and GMP manufacture. Toxicology batches Manufacture of tox batches, usually performed under non-GMP conditions, serves several purposes: Testing of the master cell bank (MCB) Supply of tox material for pre-clinical trials The drug substance serves as a source for reference material Process fine adjustment and final optimization The master production record is drafted and tested Testing of manufacturing, analytical, release and deviation procedures Further testing of the up-scaled process Supply of drug substance for accelerated stability studies Supply of drug product for accelerated stability studies Data generation It is important that the process used to produce the drug substance resembles that of GMP material used for clinical trials. If major process differences are introduced, a comparability study is needed to assure that the tox drug substance and the GMP drug substance are having identical physical, chemical and biological properties. Some companies chose to use GMP material for pre-clinical trials, as well, in order to assure product homogeneity. Released MCB vials must be used for GMP manufacture and most companies prefer to test the MCB before entering the costly GMP facilities. At this stage raw materials should be identical to the raw materials intended for GMP manufacture. The in process control program should mimic the GMP manufacture program and it is strongly recommended to take retain samples wherever possible. Such samples are very valuable trouble shooting if later batches fail due to process malfunction. It should be mentioned that an alternative to manufacture of tox batches is to use the GMP material also for pre-clinical studies. GMP batches Manufacture of GMP batches, always performed under GMP, serves several purposes: Testing of the working cell bank (WCB) Supply of drug substance intended for formulation The GMP material may serve as a source for reference material The GMP material serves as a source for reference standard Process validation studies Virus validation studies (insect, and mammalian cell cultures) Supply of drug substance and drug product for accelerated and long term stability studies Data generation Notice the difference between supply of tox and GMP material. Usually the tox material drug substance is used for pre-clinical studies, while it is the formulated drug product used in clinical trials. The GMP manufacturing process is thus more complicated due to the added formulation procedure, but also because of demands for sterile final products, when used for human administration. A qualified person releases the GMP batch (drug substance and drug product) for its intended used based on raw materials, certificates of origin, master cell bank certificate,
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the certificate of analysis, the batch records, the deviation reports, and quality of analytical methods used for the quality control program.
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6.0 Quality control

Introduction Biopharmaceutical quality control is exerted on several levels in order to assure the highest possible safety and quality of the product. First, all the quality and identity of cell banks and raw materials must be documented. Secondly, key and critical operational parameters should be identified and interactions documented. Thirdly, in process analyses are carried to assure proper processing. Finally the drug substance and the drug product is undergoing an extensive test program comprising appearance, potency, identity, purity and quantity, where results are compared to specified accept criteria. A complete set of acceptance criteria cannot be disclosed a priori as some acceptance criteria are established and justified during development and production of pre-clinical and clinical test batches. Insect and mammalian expression systems are further undergoing virus validation demonstrating the downstream process ability to remove virus particles. Data collected during development, scale up and manufacture comprises a valuable repository of information, which can be used for trouble-shooting, comparability and equivalence studies. Control should be exerted on all levels and should constitute an integrated part of the total quality assurance. Cell banks Three types of cell banks are commonly used in the biotech industry: research cell banks, master cell banks and working cell banks. The research cell bank is the first cell bank established. It is used for research and development purposes, but rarely for manufacture of tox and GMP batches. The master cell bank is established from the research cell bank serving two purposes: manufacture of GMP batches intended for clinical trials and to generate the working cell bank. The working cell bank is used for manufacture of licensed product. Cell banks are tested for identity (phenotyping or genotyping may be used to confirm species and strain), stability (demonstrate that the cell line produces the desired product in a consistent quality and quantity), and purity (freedom from adventitious agents and adventitious cellular contaminants must be assessed). The purity test programs differ among the expression systems used. Raw materials Raw materials are biological or chemical substances used to manufacture the biopharmaceutical such as cell culture nutrients, serum components, inorganic salts, detergents, anti foam agents, enzymes, reagents, organic compounds, organic solvents, cleaning agents, growth factors, and chromatographic media. The basic quality concepts include assurance of the identity, purity, suitability and traceability of all the raw materials used in the manufacturing process. The quality of the raw materials used should meet standards appropriate for their intended use. Quality requirements for raw materials are met somewhat differently in production of clinical batches (process evolution) and manufacturing of licensed product. Special attention should be paid to raw materials of animal or human origin. They should, as a general rule, be avoided. Each raw material should be evaluated to determine its criticality to the process. In process control Recently the Food and Drug Administration (FDA) drafted a non-binding recommendation, which should help manufacturers develop and implement new efficient tools for use during pharmaceutical development, manufacturing, and quality assurance.
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The framework is named Process Analytical Technology (PAT). PAT is considered to be a system for designing, analyzing, and controlling manufacturing through timely measurements of critical quality and performance attributes of raw and in process materials and processes with the goal of ensuring final product quality. The goal of PAT is to understand and control the manufacturing process as quality cannot be tested into products, but should be build-in. This approach can be taken during the development phase extending the in process control program to not only include traditional analytical testing, but also real time monitoring of the process parameters and responses. A desired goal of the PAT framework is to design and develop processes that can consistently ensure a predefined quality at the end of the manufacturing process. Such procedures would be consistent with the basic tenet of quality by design and could reduce the risks to quality and regulatory concerns while improving efficiency. In process control is exerted on four levels: Identity, purity, quantity and activity analyses of samples and pools as required, end of production test of cell cultures, measurement of parameters and responses, evaluation of UV-diagrams, impurity profiles, and recoveries of target protein. Consequently, process specifications based on mechanistic understanding of how process factors affect product performance are defined in the same manner as drug substance and drug product specifications. Any significant deviation may indicate process failure and measures should be taken to correct the problem. Specific analytical assays are used to identify and quantify the target protein (e.g. ELISA, HP-RPC, HP-IEC, SDS-PAGE) and to create impurity profiles. The formation of productderived impurities should be consistent from batch to batch and derivations from the usual pattern may indicate changes in performance. One of the most suitable levels at which possible adventitious agent contamination can be detected is in the unprocessed bulk. The test program should include test for bacteria, fungi, mycoplasma and viruses (if insect cell, mammalian cells or transgenic animals have been used). Biopharmaceutical processes are complex multi-factorial systems and may be difficult or impossible to optimize without using multivariate mathematical approaches, such as statistical design of experiments, response surface methodologies, and pattern recognition tools. Traditional one-factor-at-a-time experiments are too time consuming and will not address interactions of parameters. In order to reduce the number of factors, it is common practice to investigate unit operations separately and to define process acceptance criteria for each step. A modern approach to process control is to exert extensive control of parameters at each unit operation including but not limited to measurement of oxygen, nitrogen, carbon dioxide supply, pH, temperature, conductivity, redox potential, holding times, flow, back pressure, cell densities, and protein concentration. Many of these measurements can be exerted in-line providing continuous assessment of process performance. Examples are continuous monitoring of pH and conductivity during chromatographic procedures. The information collected can become part of a knowledge database to be used for determination of patterns and to develop process simulation models. Control of drug substance and drug product The quality of the drug substance and the drug product is intensively controlled. Analytical data are compared to predefined specifications comprising appearance, potency, identity, purity, quantity, pH and bioburden. Appearance is typically a visual inspection of the solution or suspension addressing particles, turbidity, and color. The potency assay is a measurement of the drugs biological activity often reported as the specific biological activity (e.g. units/mg). Identity assays (e.g. SDS-PAGE, peptide maps) compares the drug with a reference material undergoing the same analytical treatment.
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One of the major safety aspects of a biopharmaceutical drug is to control and measure the level of impurities present in the final product. Some impurities, like adventitious agents, are well known and the content can be challenged against defined accept criteria. Other impurities are process specific and will be evaluated on a case-by-case basis. Virus infection risk is for example not of concern if a bacterial expression system has been used, but presence of scrambled forms of the target protein arising from the in vitro folding could be an issue. Therefore, protein purity should not be related to a few analytical methods, but rather be analyzed on basis of the expression system used, the process design and the derivatives of the target protein arising as a consequence of upstream and downstream processing. This view is further supported by the fact that many impurities may be harmful even in very small concentrations stressing the need for highly sensitive methods (e.g. ELISA). Removal of host, process and product related impurities must be demonstrated indicating that specific assays must be implemented for each impurity expected to be present be it from host cells/animals/plants, raw materials or generated during processing. Different analytical set ups should be expected depending on the target protein properties and the expression system used. Quantity methods (A280, reversed phase high performance chromatography, refractive index) are used to determine the protein concentration of the drug substance/product. The bioburden test provides information of microbial contamination of the drug substance. A similar sterility test is used for the drug product. Each tox and GMP batch is tested according to carefully designed programs. The test programs comprise chromatographic impurity profiles, content of polymers, endotoxins, nucleic acids, host cell proteins, microbial agents, and specific assays relating to raw materials used (e.g insulin, protein A). Batches are released for their intended purpose if specifications are met. A certificate of analysis will be issued. Reference material A well-characterized reference material is needed for qualification and validation of analytical methods. The reference material (often produced from the tox batch or from one of the GMP batches) is used for reference during drug substance/product quality control. The reference material is not only analyzed according to the quality control program, but also by means of amino acid analysis, sequencing, mass spectroscopy and structural analyses such as circular dicroism, X-ray diffraction, and nuclear magnetic resonance spectroscopy. Analytical methods Common biotech analytical methods and their intended use are listed below in alphabetic order. Amino acid composition Amino acid analysis is used for quantitative determination of protein content (primary data). The amino acid analytical data are used to calculate the extinction coefficient and for quantity determination. Amino acid sequence Amino acid sequencing is divided into N-terminal and C-terminal sequencing. N-terminal sequencing is carried out using Edman degradation. The analysis will provide information about the first 10-20 N-terminal residues. Carboxy-terminal (C-terminal) sequence analysis is used for confirmation of the Cterminal part of the peptide or protein. The assay is used to characterize the reference material.
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Bicinchoninic assay The assay is a modification of the biuret assay. The assay is used for semi-quantitative in process measurement of protein concentration. Bioburden Presence of microbial agents is tested in the bioburden assay by counting plaque forming units on specific substrates. The test is used in the drug substance quality control program. Biuret assay The assay is based on polypeptide chelation of cupric ion in strong alkaline solution. The assay is used for semi-quantitative in process measurement of protein concentration. Bradford assay The semi-quantitative assay is based on the dye, Coomassie brilliant blue G-250, which undergoes a shift from 465 nm to 595 nm when binding to peptide bonds under acidic conditions. The assay is used for semi-quantitative in process measurement of protein concentration. Capillary electrophoresis The basic principle to capillary electrophoresis is to apply high voltage to a fused silica capillary filled with an appropriate electrolyte and with both ends dipped in the same solution. The separation occurs due to the combination of electrophoretic migration and electro-osmotic flow. Capillary electrophoresis is used to address product related impurities. Circular dicroism Circular dicroism measures the secondary structure (helix and -sheet) of proteins. The method is used for structural analysis of the reference material. Differential scanning caloriemetry DSC can be used to determine the glass transition temperature, TG, the crystallization temperature, and the melting temperature, TM. The thermo analytical technique measures the difference in the amount of heat required to increase the temperature of the sample relative to that of a standard, which has well-defined heat capacity over the temperature range investigated. When the sample undergoes a phase transition more or less heat will flow to it relative to the reference standard at the same temperature making it possible to determine the said temperature from a plot of temperature versus the heat flow. ELISA Immunoassays use the binding specificity of an antibody for its specific antigen (e.g. host cell protein). In order to quantitate the reaction the antigen or antibody is labeled or attached to an enzyme with a high turn over number (e.g. horseradish peroxidase, alkaline phosphatase). ELISA is used for detection of residual host cell proteins, insulin, protein A and other like impurities as part of the quality control program. Fluorescence spectrometry Fluorometers measure the amount of fluorescent radiation produced by a sample exposed to monochromatic radiation. Fluorescent methods have three significant advantages over absorption spectrometry: First, two wavelengths are used in flourimetry, secondly the signal to noise ration is low compared to absorption spectrometry and third fluorescent methods have a greater range of linearity. High performance chromatography Analytical high performance liquid chromatography offers high level of resolution and precision making the technology available for identity, purity and quantity determinations. Ion exchange, reversed phase and size exclusion chromatography is commonly used to
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address product related impurities and polymeric forms as part of the dru substance/product quality control program. The methods may also be used for in process control. Infrared spectrometry Fourier transformed infrared spectroscopy (FTIR) is a form of vibrational spectroscopy, which can be used to monitor the amide hydrogen exchange rates in proteins. FTIR spectroscopy is suited for determination of structural features of proteins. The method is used to characterize the reference material. Isoelectric focusing In isoelectric focusing, the movement of the protein through the gel matrix is modulated by a pH gradient created by soluble ampholytes, which are small organic molecules with various isoelectric points and buffering capacities. The pH-gradient is produced when the soluble amphoyte migrate in the gel matrix until they reach their isoelectric points. Isoelectric focusing is the analytical method offering the highest resolution and separation of glycosylated protein forms is possible. The method is used for identity as part of the drug substance/product quality control program. Kjeldahl analysis The Kjeldahl analysis is used for quantitative determination of the nitrogen content in protein samples. The method is accurate and it can be used as primary source for quantity determination. Limulus amebocyte lysate The Limulus amebocyte lysate test is for used for the determination of endotoxin. There are currently three test methods in use: the gel clot test, the turbidimetric test and the chromogenic test method. All methods allow for reading after a fixed time interval (endpoint tests). The turbidimetric and the chromogenic test methods can also be read continuously (kinetic tests). Test for endotoxins is carried out during downstream processing and as part of the drug substance/product quality control program. Lowry Assay The Lowry assay is a colorimetric method based on cupric ions and the Folin-Ciocalteau reagent for phenolic groups. The semi-quantitative Lowry assay is typically used during development for in process control of total protein, preferentially in crude samples. Mass spectroscopy The molecular weight of peptides and proteins (< 100000 kDa) can be determined with mass spectroscopy. The molecular weight determination is useful for peptide and protein characterization (sequence, and secondary structure such as disulfide bonds). Besides used for reference material characterization, mass spectroscopy can be used to address proteolytcal degradation and the identity of product related impurities. Microbial testing Microbes are in this context defined as bacteria, yeast or molds. Two methods are recognized by pharmacopoeias: direct inoculation and membrane filtration. Cell banks and harvest samples are tested for presence of microbial agents. Native electrophoresis Native electrophoresis separates proteins according to charge, molecular weight, shape and other factors (samples are typically applied under conditions maintaining the tertiary structure). The method is typically used during downstream process development and optimization.
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Nuclear magnetic resonance Nuclear magnetic resonance provides information about the tertiary structure of protein into solution. The analysis is used to characterize the reference material. O-pthalaldialdehyde assay O-phtalaldehyde reacts with amines under formation of an adduct that fluoresces at 340 nm and which can be detected at 450 nm. The assay is complex including extensive sample preparation. The semi-quantitative method may be suited if interfering components prevent other assays from being used for in process testing. Peptide mapping Peptides generated from enzymatic degradation of proteins can be separated by reversed phased high performance chromatography. The resulting peptide map pattern can be compared with that of the reference material as part of the quality control identity analytical program. Polymerase chain reaction The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify (i.e., replicate) a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. The method is used for quantitative determination of DNA in the drug substance. SDS-PAGE One-dimensional SDS-PAGE offers separation of proteins according to their molecular weight. Samples run under denaturing, but non-reducing conditions, will provide information of presence of other molecular species and of disulfide intermolecular di- and polymers. Samples run under denaturing and reducing conditions will provide information on monomeric compounds. The analysis is used as an identity method in the drug substance/product quality control program and as in process control during development and scale up. 2D-electrophoresis 2D-electrophoresis separates proteins according to the proteins isoelectric point (first dimension) and its molecular weight (second dimension). The method is a combination of IEF and SDS-PAGE. The resulting coordinate (pI,Mw) provides a unique identification of the protein. Differences in post-translational modifications (e.g. phosphorylation) will often result in separate spots (slightly different pI and Mw). 2D-electrophoresis may be used as an identity method or fr characterization of the reference material. UV-absorbance The method is based on the absorbance of tyrosine, tryptophan and phenylalanine residues at 275-280 nm (ultraviolet region). Phenylalanine is only weakly absorbing and is usually neglected for most purposes. The protein structure is not affected by the method making on-line measurement a possibility. Pigments, organic cofactors and phenolic compounds interfere with the assay. The assay is used for quantity determination of drug substance/product and to monitor protein concentration during purification. X-ray diffraction X-ray diffraction determines the tertiary structure of crystallized proteins. The method is used in the protein characterization program.
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Impurities Host cell related impurities Endotoxins Endotoxins are highly negatively charged lipo-polysaccharides from the cell wall of gramnegative bacteria (e.g. E. coli). The endotoxin content is measured by the Limulus amoebocyte lysate (LAL) test using amoebocyte lysate from the horsoshoe crab (Limulus polyphemus or Tachypleus tridentatus or the human leukocytic pyrogen asay, which tests for endogenous pyrogens. Viruses There are three main sources of viral contamination, the host cell, the culture medium and infections during manufacture. The host cell may contain a genomic virus or virus vectors used to transform the cell line. The type of viral genome and/or vector depends on the cell line history. Continuous cell lines are extensively characterized but chronic or latent viruses may be present. The retroviruses associated with continuous cell lines are noninfectious, but oncogenic. Epstein-Barr virus or Sendai virus is often used for cell transformations. Contaminants such as reovirus, bovine leukaemia and polyoma virus should be expected from serum supplemented media. Viruses can be divided into enveloped and non-enveloped DNA or RNA viruses, single or double stranded. Their size ranges from several hundred nanometers (e.g. poxviridae) to a few deci nanometers (e.g. parvoviridae). Virus control is executed on several levels. The cell line history reveals all information on the origin and identity of the cell line and the host genome vectors used to establish the cell line. The master cell bank is extensively characterized using viral identity tests, in vitro tests and in vivo tests to assure freedom of adventitious viruses. The end of production test of the cell culture is carried out to assure that the cells are free of viruses. Low titers of virus cannot be detected in the drug substance/product. Instead virus inactivation and removal are tested in scale down studies resembling the procedures of the manufacturing process. Such validation studies are typically carried out during manufacture of phase 1 and phase 3 material. Prions Transmissible spongiform encephalopathies (TSE) include scrapie in sheep ad goats, chronic wasting disease in mule deer and elk, Bovine spongiform encephalopathy (BSE) in cattle, Kuru and Creutzfeldt-Jacob disease (CJD) in humans. The disease causing agents (prions) replicate in infected individuals generally without evidence of infection detectable by available diagnostic tests applicable in vivo. The major source of contamination of a recombinant product is the use of animal derived raw materials, which could harbor bovine prions (BSE agent). Semi-quantitative biochemical assays include Western blot, Capillary immunoelectrophoresis, Conformation-dependent immunoassay, and dissociationenhanced time-resolved fluoimmunoassay. Nucleic acids Nucleic acids is a common term for DNA and RNA - negatively charged polynucleotides (at neutral pH). Nucleic acids absorb light at 260 nm. The residual content in drug substance/product is usually measured by polymerase chain reaction or amplification techniques. Host cell proteins The host organism is the prime source of contaminating proteins, which must be removed due to their immunogenic properties. Hos cell proteins are detected by means of specific ELISA methods derived from extracts of the host cell organism or raised against bulk preparations lacking the active pharmaceutical ingredient.
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Lipids Lipids (lipoproteins, triglycerides, phospholipids, cholesterol) are brought to the medium by cell lysis. If transgenic animals are used, the protein is expressed into the milk containing up to 4% fat. Lipids are typically hydrophobic in nature and tend to stick to surfaces including filters, membranes and chromatographic media. Lipids are detected by gas chromatography and high performance reversed phase chromatography. Process related impurities Raw materials and leaked compounds Examples of impurities are components from the cell culture media (e.g. anti-foam agents, growth factors) and leaked ligands from chromatographic columns (e.g. protein A). Bacteria, fungi and mycoplasmas Bacteria are unicellular prokaryotic organisms. Fungi are plant protists that are nonphotosynthetic and that are devoid of chlorophyll. Fungi generally contain a mycelium and are frequently coenocytic. Mycoplasmas are the smallest and simplest prokaryotes. They depend on their host for many nutrients due to their limited biosynthetic capabilities. Viable cells can be identified by membrane filtration or by spread out of the cell suspension or sample solution on agar plates. Mycoplasmas are difficult to detect, the only reliable way of demonstrating infection is by agar plating, fluorescent dying of DNA, or by polymerase chain reaction. Product related impurities Product related impurities comprises cleaved, des-amido, oxidized, carbamylated, scrambled, aggregated and glycosylated forms. Product related impurities are often generated during processing and storage and carefully designed studies are required to minimize formation of said compounds. The impurities are detected by means of high performance chromatography. Cleaved forms The term cleaved forms is typically used for target protein derivatives with almost identical molecular weight (+/- a few hundred Daltons), where a peptide bond is cleaved resulting in loss of a N- or C-terminal site or where an internal peptide bond is cleaved while at the same time the resulting fragments are kept together by means of disulfide bonds. Des-amido forms Des-amido forms are target protein derivatives in which one or several of the glutamine or asparagine amino acid residues are converted to the corresponding acids. Oxidized forms Oxidized forms are target protein derivatives in which one or several Met, Cys, His, Trp, Tyr residues have been oxidized. The oxidation of cysteine residues results in formation of a disulfide bond (cystine residue). Carbamylated forms Carbamylated forms are target protein derivatives in which one or several primary amino, sulfhydryl, carboxyl, phenolic hydroxyl, imidazole and phosphate groups react with cyanate to form a derivative. The blocking may change the pI of the protein. Scrambled forms Scrambled forms are target protein molecules with a disulphide bond pattern different from that of the native molecule. Scrambled forms are typically formed during in vitro folding of proteins, but disulfide bond shuffling at neutral pH or above has been reported emphasizing the need for control of protein stability during downstream processing.
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Aggregated forms Aggregated forms are target protein derivatives in which two or more molecules is linked together either by covalent inter disulfide bonds or by hydrophobic interaction. Target protein aggregates are formed as a result of hydrophobic inter molecular reactions or because of inter molecular disulfide bond formation under oxidizing conditions. Aggregates are very often antigenic resulting in formation of target protein antibodies. Proteins exposed to even mildly denaturing conditions may partially unfold resulting in exposure of hydrophobic residues to the aqueous solvent favoring aggregation. The aggregation process is assumed to be controlled by the initial dimerization step in a second-order reaction. Consequently, high protein concentrations will increase the aggregation rate. Inter-molecular disulfide bond formation between cysteine residues takes place at alkaline pH under oxidizing conditions. Proteins with reactive free thiol groups should be purified under reducing conditions (typically 1-10 mM reducing agent) in the presence of EDTA. Even proteins with disulfide bonds may participate in inter-molecular disulfide bond reactions due to disulfide bond shuffling at neutral and alkaline pH. Glycosylated forms Yeast may express target protein derivatives with one or more glycosylation groups (e.g. mannose, galactose). Higher order expression systems may secrete a variety of glycosylated products resulting in a complex composition of the drug substance/product. The three major classes of covalently attached carbohydrate are either N-linked to asparagine residues in Asn-Xaa-Thr/Ser residues, O-linked to a subset of Thr/Ser residues or linked to the C-terminus of some membrane proteins as glycosylphosphatidyl inositol.
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7.0 Quality assurance

Introduction Product quality is addressed in several ways with the purpose of assuring product safety. It is the responsibility of the biopharmaceutical industry to produce safe products, a virtue the industry has taken very serious since the introduction of recombinant technology in the 1970s. Product safety is addressed by public regulatory bodies expressed in current Good manufacturing Practices (cGMP) dictating the manner in which biopharmaceuticals and medical devices are produced. Quality assurance elements addressed in the BioProcess Guide are specifications, raw materials, expression system characterization, process validation, virus validation, validation of analytical methods, reference materials, and stability studies. Examples of quality assurance elements not included are facility validation, equipment validation, cleaning validation, vendor certification, and vendor audits. The term validation means documented evidence that a process will consistently produce a substance that meets its predetermined specifications and quality attributes. Process validation links back to 1976, where the Food and Drug Administration (FDA) introduced its new current regulations. Before 1976, the concept of validation had been applied mainly to analytical test methods. The paradigm shift meant that by the mid 1980s validation principles advanced to cover all manufacturing processes and computer systems, which at the time was an entirely new discipline. GMP regulations state what must be done but do not stipulate how to do it. For that reason FDA compliance policies are expressed in guides and guidelines, which can be made effective at any time with or without public notice or hearings. Guides and guidelines are not legally binding and the industry can write its own guidelines, if it so elects. Other regulatory bodies do issue guides and guidelines as well raising the need for harmonization. The need was early recognized resulting in the International Conference on Harmonisation (ICH) guidelines now accepted by United States, Europe and Japan. Product quality is addressed on several levels: expression system, processing, drug substance, drug product using not only a wide range of analytical procedures but also strict in process control programs monitoring responses like, pH, temperature, conductivity, pO2, cell density, viability protein concentration, protein stability, UV-profiles, and back-pressure. The build in of quality reflected in the extensive in process control and testing has gained more and more attention over the years and is now considered as important as product testing (the Process Analytical Technology (PAT) initiative). The drug substance and drug product is tested against pre-defined specifications, which must be met before batches can be released. The test program comprises tests for appearance, identity, potency, purity, quantity, imunogenecity, and bioburden/sterility. Other quality attributes are well-designed processes, process robustness, consistent yields, stability of intermediary compounds, and drug product/substance stability. Regulatory affairs No biopharmaceutical product can be sold in US, Europe and Japan without a license. In order to obtain a license, drug manufactures must present substantial data on efficacy and safety to official regulatory agencies such as US Food and Drug Administration (FDA), European Medicines Evaluation Agency (EMEA) and Japan Ministry of Health and Welfare (MHV). The regulatory filing is made in accordance with guidelines undergoing continuous assessment. The guidelines of FDA, EMEA and MHV are supported by the World Health Organization (WHO) and the International Conference on Harmonization (ICH), the latter being a project bringing together the above authorities and experts from
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the pharmaceutical/biopharmaceutical industry to discuss technical and scientific aspects of drug registration. The role of Quality Assurance The release of biopharmaceutical drugs is handled by the Quality Assurance; an independent body responsible of assuring and maintaining the overall quality of the facility and associated activities. The primary activities includes Establish and maintain the overall quality system Establish policies, objectives and plans (e.g. master validation plans) Establish and maintain standard operation procedures Assessing risk Establish and maintain the documentation system Review and approve all procedures related to production and maintenance Review analytical procedures Review master production protocols Review batch production records Review certificate of analysis Release of raw materials Handle deviations Monitoring packaging and labeling processes Release of toxicology and clinical batches Perform internal and external audits Training Implementation of a comprehensive quality system will facilitate compliance with 21 CFR parts 210 and 211. Specification Specification is a list of test methods used for batch release. Each test of the specification is associated with an accept criteria, which must be met if the batch shall be released. The accept criteria are not given a priori, they result from data obtained during the development work, the dose, and experience. The specification must be carefully designed taking the expression system, raw materials, protein nature and processing conditions into consideration. A tox or clinical batch is tested according to specification. All accept criteria must be met before the batch can be released after issue of a certificate of analysis. Process validation The reason that several agencies, such as the United States Food and Drug Administration (FDA), European Medicines Evaluation Agency (EMEA), and Japanese Ministry of Health and Welfare (MHW) require validation is to ensure that safe and efficacious products are manufactured. The rationale has been not only to control the resulting drug product, but also to introduce control into the manufacturing process itself. Thus, process validation is beginning with the initial development work in order to build in safety, robustness and compliance into the process along with the data produced. Process validation is a continuous activity terminating only after the product has been removed from the market. A graded approach has been accepted by the regulatory agencies and full validation is not required until phase III manufacture. Process validation prerequisites Process validation cannot be executed before several aspects of current good manufacturing practices (cGMP) have been completed. In essence the rationale for choice of expression system, cell line characterization, raw materials release, process design and its rationale, process development, formulation development, analytical
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methods validation, process scale up, process characterization, and extractables must be completed before process validation is carried out. These process validation prerequisites are addressed during process development and they should be completed before initiating the process validation program usually carried out during phase 3 manufacture. Master or working cell banks must be released before process validation is initiated. Microbial expression systems are tested for identity, presence of bacteriophages, contaminating micro-organisms and fungi, insert characterization, resistance to antibiotics, and stability. Insect and mammalian expression systems are tested for identity (phenotyping or genotyping), freedom of microbial agents, freedom of mycoplasmas, freedom of contaminating cell lines, freedom of viral agents and stability. Raw materials must be carefully selected from trusted vendors. If raw materials are not of compendial grade, specifications should be defined. Raw materials of animal origin are usually not acceptable for manufacture of biopharmaceuticals. However, if unavoidable a Statement of Origin (e.g. TSE certificate of suitability) from the vendor must be provided. It is assumed that raw material intake is carried out according to cGMP with well established test and release procedures. A rationale for the process design should be provided taking the nature of target protein, the expression system, the cell line, raw materials, dose and formulation into consideration. Analytical control is exerted on two levels: In process control during manufacture and control of drug substance/drug product against a priori specified accept criteria for identity, potency, purity, quantity, pH, bioburden and appearance. Analytical method validation is a continuous process initiating in early development, where the methods are tested for their ability to analyze given samples. In a later stage the methods are verified and finally validated using for example the recommendations of the International Conference on Harmonization (ICH) guidelines. An analysis is validated for a specific sample type (e.g. the drug substance) and must be revalidated if other samples are applied (e.g. in process samples). In order to reduce the validation burden, only critical in process control analyses are validated. Assay validation will take place in cGMP laboratories according to approved standard operation procedures (SOPs). To execute process validation effectively, it is recommended to identify and examine those unit operations that may affect the safety, identity, potency and purity of the drug substance (risk assessment). Besides analytical in process control, the process is controlled by a variety of process parameters and responses (e.g. pH, conductivity, temperature, yield, UV-profiles). Parameters are usually defined in ranges specifying a lower and upper limit. It is an integrated part of the process development work to define and justify these intervals (proven acceptable ranges) and to recommend set-points. The limits are purposely varied in a systematic way in order to challenge process robustness. Design of Experiments (DoE) methodologies are often used to reduce the experimental work and to determine interactions between parameters. Responses are typically critical product quality attributes affecting safety, identity, potency, and purity. Examples of responses are yield, protein stability, endotoxin level, UV-profiles, and polymer content. Process characterization is resource and time consuming if not integrated in the experimental process design from early development. It must be assured that leachables from containers, bags, vials and stoppers are identified and tested for toxicity. Further, the influence of temperature on containment should be tested, not all stoppers fit to the vial at -80C. The stability of intermediary compounds, drug substance and drug product should be addressed using selected stability indicating analytical methods. It is common practice to make use of accelerated stability studies in early development. Process development includes development laboratories, pilot facilities, manufacturing facilities, external partners, analytical laboratories, formulation and fill & finish. Consistency is assured by introduction of a well-characterized reference material, which
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is used for analytical verification and validation, as reference in analytical assays and as a link between different process designs as the process develops. Reference materials are produced throughout development, while the reference standard is typically produced from the phase 3 GMP batches. Process validation The process validation studies comprise validation approaches, viral clearance, process consistency, process intermediary stability, process solution stability, fill, freeze, thaw, storage of drug substance/product, chromatographic resin and filter membrane lifetime. Mammalian cells, transgenic animals and to some degree insect cells may be contaminated with human or animal viruses (endogenous contamination). If human or animal serum has been used to propagate cells for cell banking or culture, exogenous contamination is a possibility, as is contamination during cell culture. Several measures are taken to reduce viral contamination of the final product: reduce use of human or animal serum to a minimum, include at least two orthogonal virus removal steps in the downstream process, work in closed systems, use of non-animal origin raw materials and introduce efficient cleaning in place procedures. Usually two very efficient virus inactivating/removal steps are by default introduced in purification schemes of mammalian, insect and transgenic expression systems: virus inactivation by means of low pH or solvent-detergent treatment in order to inactivate enveloped viruses and virus removal by means of nano-filtration. It is not possible to measure viruses in low concentrations and virus removal factors must be determined in downscaled studies with spiked viruses. Viruses used in clearance studies should be selected on the history of the cell line, the type of cell line and exposure to cell culture media and additives that may contain viruses. The first virus validation study comprising one enveloped and one nonenveloped spiking virus should be conducted prior to phase 1 clinical trials. An extended study comprising 4 different viruses are carried out during manufacture of phase 3 clinical material. It should be remembered that the virus test assays are based on live viruses, hence interference studies comprising possible interactions with either the target protein or the buffers used must be carried out prior to testing. Stability studies Product quality should be continuously addressed during storage. It is common practice to investigate the stability of intermediary compounds, the drug substance and the drug product. Stability studies are initiated in early development often using forced degradation in order to determine major by-products formed and thus give valuable input to analytical method development. Also accelerated studies at elevated temperature are carried out with the purpose of detecting major stability issues. Stability studies are closely linked to the process used and to the composition of the tested substance. Process changes may influence protein stability and thus initiate new stability studies. In addition to the initial development stability studies, extensive studies are carried out using the drug substance and the drug product. The drug substance is typically stored for a shorter period than that of the drug product, which is about two years in average. Typical stability test points are 0, 1, 3, 6, 9, 12, 24 and 36 months at which the API is tested in carefully selected stability indicating assays at different temperatures. A stability test program typically comprises appearance, pH, identity, presence of major impurities, and presence of polymers.
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8.0 Documentation
Introduction One of the basic rules in biopharmaceutical drug development is to document the work carried out and to make the documents produced easily accessible for regulatory bodies. The statement if it is not documented, it has not been carried out should be taken seriously. This implies that documents can be easily tracked, even years later, if need be. The documentation package comprises planning, development work, manufacture, analytical methods, stability studies, process validation and applications forwarded to regulatory authorities. Authentication Documents should be easily identified and easy to track. For that reason each document should contain information about author, date, status, title, version, type, company logo, name of site for work carried out, and project. Planning Project plan The project plan is an overall description of the work to be carried out prior to the experimental work is initiated. At its best the plan outlines rationales for choice of expression system, composition of the drug substance, and drug product formulation and the impact on process design. The cell line history may be included. The document comprises the upstream, downstream, and formulation rationale and strategy based on the information available at the time of writing. Suggestions for specification and analytical methods of the quality control program are included. The project plan includes time schedules and economical forecasts as well. Process validation plan The process validation plan (PVP) should be implemented with the initiation of the process development. The PVP assures a well thought through assessment of safety by addressing risk, control of quality, analytical method qualification strategy, in process control programs, virus validation, process validation, and stability of the active pharmaceutical ingredient (API) during processing and storage. The PVP should include a structured overview of the safety and quality documents produced. The PVP is regularly updated and embraces an entire life cycle, beginning in research and development ending only when the product is no longer commercial. A logical approach to validation will reduce the risk of submitting incomplete applications. Process design and development Laboratory notebooks The laboratory notebook is a written record of the laboratory work performed. The notebook must be kept according to strict rules relating to type, authentication, signatures, language, content as follows: 1. Keep all records in a bound notebook. Use the right pages for a description of what you have been doing including references to other records (samples, analytical data). Use the left pages for analytical data. The first page of the notebook may state the name of the principal investigator, the title of the study and the date of initiation. Other key persons may be listed as well. 2. Begin with a new set of pages every day and do not skip any pages or leave any set of pages blank.
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3. Use the final set of pages for an overview of the experiments described in the book. 4. Write in past tense and do it while doing the experiment. Do not use any other paper for writing down your notes. State the object, purpose, and results of each experiment clearly and concisely. 5. Use indelible ink (black ballpoint) that do not smear, run or fade, but is suitably photocopied. 6. Sign and date each notebook page on the date of entry. 7. If the entry involves more than one page, cross-reference the pages. 8. If data are not kept in the notebook, they must be checked, signed, dated, and identified in order to provide a reference back to the pertinent page of the laboratory notebook. 9. If a mistake has been made, draw a line through the entry, mark it with your initials and date it. 10. Draw a line through non-used left side pages and blank spaces. 11. Have each page of the notebook signed and dated by you and by a witness who has read and understood the entry. 12. If the experiment or reduction to practice has been witnessed, this fact should be recorded and signed by the witness, who must not be a co-inventor. 13. If the work was done under the supervision of somebody else, record this fact. 14. Do not make additions to pages that have been signed and dated. 15. Entries must describe with sufficient clarity and in details the work done in order to enable someone of ordinary skills in the subject to repeat the work. 16. Do not state or suggest that the work has been or will be abandoned. Improper kept notebooks may result in failure to claim priority dates towards the US patent authorities. Process design rationale The process outline is a product of the expression system used, demands for drug substance composition, drug product formulation, device and administration form. The rationale for the over-all design and each unit operation should be given. Development reports A unit operation report describes the development work related to a given unit operation. The aim is to collect all information of relevance in one document giving easy access to overview the rationale and experimental work associated with the unit operation. The report is a final document providing process information at the time of tech transfer from development to pilot. A summary report describes a study related to the process development, the development of an analytical method, or other specific development issues that need to be addressed during drug substance development. Usually, the summary report is a short and concise final document that summarizes the results from a development study shortly after the study has been performed. The development report is the over-all development document summarizing the drug development program. Besides including references to rationales, unit operation reports, summary reports, analytical reports, stability reports the final development report will summarize the entire development work carried out in small scale addressing over all yields, process robustness, critical parameters, specification and accept criteria, typical small scale batch data, and impurity profiles.
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Analytical Analytical method protocols Analytical methods for quality control of drug substance and drug product are carried out according to compendial procedures (e.g. EU or US pharmacopoeia) or internal protocols qualified for their intended use. Analytical protocols are part of the quality assurance system and changes must be approved by the Quality Assurance (QA) department. Analytical development reports Analytical methods are tested using a reference material. Test results and information about method accuracy, specificity, linearity, detection limits, range and sensitivity are recorded in analytical development reports. Several reports may result over time as the analytical method develops towards a fully validated state. Analytical validation reports The method qualification end point is the validation report assuring that the method has been validated in accordance with International Conference on Harmonization (ICH) guidelines. The report is expected due before quality control and release of material phase 3 clinical trials. Reference material The manufacture, characterization and stability of the reference material used for qualification of analytical methods must be documented in a specific report. If several reference materials are produced, each reference material must be associated with a report. Cell lines Master production records The master production protocol (MPR) describes the manufacturing procedure of the master cell bank (MCB) or working cell bank (WCB). The MPR is approved by Quality Assurance (QA) before manufacture. Batch production record The batch production record (BPR) describes in detail the manufacturing work carried out. The BPR template is the MPR, the difference being that the BPR comprises all data and signatures. The BPR is controlled by QA. Certificate of analysis The certificate of analysis (CoA) summarizes the quality control data and compares said data to the specification accept criteria. Analytical data may be documented in specific analytical reports comprising the underlying documentation. The CoA is controlled by QA. Deviation reports Deviations from the MPR should be reported. Deviations are controlled by QA. Disposition document QA may release or reject a given batch based on information from the CoA and deviations. The decision is stated in a disposition document, which also specifies what the manufactured material can be used for and to whom the batch is released. Manufacture Master production records The master production protocol (MPR) describes the manufacturing procedure. Typically separate MPRs are written for upstream, downstream, formulation and fill/finish. The MPRs are approved by QA before manufacture.
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Batch production record The batch production record (BPR) describes in detail the manufacturing work carried out. The BPR template is the MPR, the difference being that the BPR comprises all data and signatures. The BPR is controlled by Quality Assurance (QA). Certificate of analysis The certificate of analysis (CoA) summarizes the quality control data and compares said data to the specification accept criteria. Analytical data may be documented in specific analytical reports comprising the underlying documentation. The CoA is controlled by QA. Deviation reports Deviations from the master production record should be reported. Deviations are controlled by QA. Disposition document QA may release or reject a given batch based on information from the CoA and deviations. The decision is stated in a disposition document, which also specifies what the manufactured material can be used for and to whom the batch is released. Stability Forced degradation stability protocols and reports Forced degradation studies are meant to provide fast information about degradation products and the studies are usually not part of formalized stability studies. The results of the forced study are presented in a development report. Accelerated protocols and reports Accelerated stability studies are carried out at elevated temperature (e.g. 40C). Accelerated studies can be part of a formalized program in accordance with International Conference on Harmonization (ICH) guidelines. Is so, the study is controlled by the Quality Assurance department (approval of protocols and reports). Short-term stability protocols and reports Short-term stability studies of drug substance and drug product are carried out according to ICH guidelines. Such studies are controlled by the quality unit (approval of protocols and reports). Long-term stability protocols and reports Long-term stability studies of drug substance and drug product are carried out according to ICH guidelines. Such studies are controlled by quality unit (approval of protocols and reports). Process validation The process validation documentation is extensive comprising both process validation prerequisites and process validation studies. Several types of documents are used to document the process validation activities. Process validation plan The process validation strategy should be outlined in the master validation plan. The process validation plan outlines the rationale and scope for process validation. Development reports Early development work is described in unit operation reports and development reports. Such studies, which are prerequisites for process validation, not only provide rationales and data for decision-making, they may also serve as references during investigations of discrepancies.
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Process characterization reports The process characterization reports describes process performance by systematically identifying critical, key and non-key parameters for each unit operation in addition the proven acceptable ranges. Process validation protocol The protocols are written plans pre-approved by the quality unit. Information about how the studies will be conducted, sampling, analytical programs, accept criteria, production equipment, and operating ranges should be included. Process validation report The report summarizes the outcome of the process validation study (usually 3 consecutive GMP batches) with respect to specific tests performed, comparison of test results with protocol accept criteria, comparison of quality control test data with specification accept criteria and addresses deviations encountered during the study. The report is approved by the quality unit. Virus validation protocols and reports The virus validation studies should be documented in a specific report. Protocols and reports are usually provided by specific contract service partners having specialized in virus validation studies. Applications Investigational new drug application The Investigational new drug application (IND) is the formal application to conduct clinical trials in the US. The request, which is forwarded to the Food and Drug Administration (FDA), specifies whether the product is intended for phase 1 or later phase studies. The INDA is a continuously updated file following the project progress through the clinical phases. The chemistry, manufacturing and control (CMC) part is of special interest to drug producers. However, the main purpose of the application is to provide information about pre-clinical data, drug formulation descriptions, investigational plan, and clinical study protocols. Biological license application A biological license application (BLA) is a request to market a new biological product in the US. The document contains volumes of pre-clinical, clinical and biological data. References are made to CMC data.
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