Quality Control

Raw Material, In-process, Final

Product, Lot Release, and Stability
1
QUALITY CONTROL IN REGULATED
BIOTECHNOLOGY
UNIVERSITY OF HOUSTON

Outline
2

Definitions
Raw Material Tests
In-Process Tests
Final Product and Release Tests
Stability Tests
Specific Assays (commonly used assays)
Reference

Q6B: Specifications : Test Procedures and Acceptance Criteria

for Biotechnological/Biological Products @ www.ich.org

Definitions
3

Contaminants materials unintentionally introduced during the

process and not suppose to be in the final drug substance/drug

product

Ex. Microbes, viruses, leachables

Drug product the protein in its final formulation in the labeled
syringe or vial or other final packaging
Drug substance the protein after the last purification step before
filling
Impurities naturally occurring from the process

Ex. Cell culture media, host cell protein, host cell DNA, product
related proteins (aggregates, misassembled protein, proteolytic
fragments)
Purity quantitative measurement of the desired protein

Raw Materials Testing

4

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

RM (contd)
5
Quality requirements for raw materials are met somewhat differently in

production of clinical batches (process evolution) and manufacturing of

licensed product

The requirements become stricter as you move from Phase I through Phase III and into
commercial production

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
Raw materials must be quarantined, identified, and released by an
authorized person and their identity proven by specific assays (often
available from the vendor)

Certificates of analysis (COA) should be received for each lot of raw material
Each vendor must undergo a vendor qualification program
It is recommended, where possible, to use raw materials of USP or EP grade

In-Process Testing
6

The safety and thereby the quality of biopharmaceutical

drug products is linked to the process design, process

robustness, process compliance with cGMP and
extensive quality control programs including product
specifications, process specifications, in process
control, drug substance and drug product testing,
regulatory policies and scientific understanding
For this class, discussion of in-process testing is limited
to the analytical testing of intermediary compounds

That is, samples taken anywhere during any process involved in

making the final biological product

The purpose of the in process analytical control program

is to ensure the quality of intermediary compounds

During initial development this part of the program is the most

important part of process development as parameter intervals have
not been defined at this early stage

It is common practice at this stage to make use of an

extended analytical program, which is then later reduced

as process validation progresses.
Typical in process control analyses are test for microbial
agents, test for fungi, test for mycoplasmas, test for
viruses, test for endotoxins, 1D-SDS, HP-IEC, HP-RPC,
HP-SEC, and ELISA

Release Tests - Specifications

8

Identity
Quantity (related to potency)
Physicochemical (related to identity)
Purity, impurities and contaminants
Potency - biological activity (may include

immunochemical properties)

Identity
9

Assays which prove the protein is the correct protein

and not a related one or another product made at the

facility

The assay should be unique for that protein.

The test lot is run in the assay along with reference

standard
Acceptable assays - Peptide map, Ion exchange
chromatography, Immunoblots
Inappropriate assays SEC, SDS gels

Quantity
10

This is the measurement of the amount of drug

substance/product.
Normally A280 is used for proteins one of the most
accurate measurements with S.D. <3%
If there is a buffer component that interferes with A280,
then an alternative protein quantification method is
used Bradford or BCA
If the protein is filled and/or dosed based on activity,
then the activity assay is used. However activity is often
reported as U/mg so a protein quantification assay is
still required.

Physicochemical
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Characterize the protein

% Monomer vs. % fragment or % aggregate
Deamidation
Oxidation
Glycosylation
Phosphorylation

Purity, Impurities and Contaminants

12

Purity quantitate the product and product related

substance
Impurities assays for proteins known to be added
into cell culture/media, host cell proteins, proteins
used in chromatography (ex. Protein A if a protein A
column is used) and DNA
Contaminants microbes covered in QC
Microbiology

Potency
13

Enzymes enzyme activity

Inhibitors inhibition of enzyme activity
Antibodies cell based assay demonstrating

inhibition or activation and an assay demonstrating

the antibody binds the specific antigen (ELISA or
BIAcore)
Cytokines inhibition or activation in a cell based
assay
Some proteins require an animal model to measure
potency ex. FSH

Stability Tests
14

Stability testing is performed on drug product and drug

substance
Performed on a regular basis (every three months) on at least 3
lots for 2 4 years
Tests look at the quality of the drug over time

Potency
Quantity
Purity

Aggregation -SEC and SDS gels

Charge distribution (Deamidation or oxidation) IEF, IEC, RPHPLC
Peptide map

Usually do not test for glycosylation, impurities such as host cell

protein content and DNA, or identity

Specific Tests
15

Electrophoresis

SDS reducing and denaturing

SDS non-reducing and denaturing
Isoelectric Foccusing (IEF)
Capillary electrophoresis

HPLC assays

SEC aggregation
IEX charge
RP HPLC on intact protein
Peptide map (RP HPLC)

ELISAs
Immunoblots (westerns)

Electrophoresis
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SDS PAGE gel

Non-reducing gels
Reducing gel

Isoelectic focussing gel

Capillary Electrophoresis

SDS -PAGE
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Sodium Dodecyl Sulfate

Polyacrylamide Gel Electrophoresis

SDS PAGE
18

The physics behind this is friction.

Coat proteins with a negative detergent
Using an electric field move them through a crosslinked

acrylamide gel
Large proteins have more drag and can not move easily
through the gel.
Small proteins have less drag and move quickly down the gel.
The hydrodynamic size of the protein is being measured. This
is roughly molecular weight.
Sugars have a very large hydrodynamic size so glycosylated
proteins run larger than their molecular weight.

SDS -PAGE
19

SDS PAGE is a technique for separating proteins based on their

hydrodynamic size (~molecular weight).
Large proteins migrate slowly through the gel matrix
Small proteins migrate quickly through the matrix

The porous gel matrix is composed

of the polymer polyacrylamide

Chemistry used to form the Gels

20

polymerization

Link

Cross Link

The polymerization reaction is catalyzed by TEMED

(tetramethyl ethylenediamine and APS (ammonium persulfate)

Acrylamide Concentration
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The pore size is determined by the percentage of

acrylamide in the gel.

Large pores are in gels with low acrylamide and thus,
low crossslinking. These gels are very floppy. They are
used to separate large proteins.
Small pores are in gels with high acrylamide. They are
used to separate small proteins.
Gradient gels are used to separate mixtures of large
and small proteins.
Unreacted acrylamide is a neurotoxin WEAR Gloves!

Acrylamide Concentration
22

Concentrations range from 5% to 30%

Sodium Dodecyl Sulfate

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SDS is a detergent with a 12 carbon hydrophobic

end and negatively charged sulfate end

Purpose of SDS
24

The hydrophobic region of SDS binds to the

hydrophobic region of proteins and unfolds them.

This also coats the proteins with a negative charge.

All proteins now have a net negative charge and will

migrate down the gel towards the positive charge.
----------------------------SDS

-----------------------------

Non-reducing Gels
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S
S

SDS

-------------------

----------------------- - -

Non-reducing gels are run to see the intact molecular

weight of a protein especially for multimeric proteins

with disulfide bonded subunits.

Reducing Gels
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S
S

----------------------------SDS

----------------------------Plus Reductant

Reducing agents break the disulfide bonded multimeric protein

down into their monomers or subunits.

Disulfide bonds must be broken to completely unfold the protein.
Reducing agents/antioxidents are used to break the disulfide

bonds. Ex. DTT: Dithiothreitol and BME: - mercaptoethanol

Running a Gel
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Negatively charged protein samples are loaded

into wells at the top of the gel
An electric current is applied so that there is a positive charge at the bottom of the gel and a
negative charge at the top of the gel.
The negatively charged proteins will migrate to the bottom of the gel according to size.

Pre-Stained Protein Standards

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Protein standards

chosen are single

naked peptide chains
They are not disulfide
bonded multimers
They are not
glycosylated

Visualizing the Proteins

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Types of Stains:
1. Coomassie Blue Sensitivity of 0.1 g protein per band
Traditional method requires staining followed by destaining to remove
background gel staining.
GelCode Blue from Pierce does NOT stain the gel and requires no destaining.
Increased sensitivity of 25 ng protein per band
2. Silver Stain Sensitivity of 2 ng protein per band
Glassware and plasticware must be very clean to prevent stain from
precipitating out of solution.
Prone to high background due to impurities in the acrylamide and surface
artifacts such as fingerprints WEAR Gloves!

Comparison of the Sensitivity of

SDS PAGE Stains:

dentical SDS-polyacrylamide gels

were stained with

A) SYPRO Orange protein gel stain

B) SYPRO Red protein gel stain
C) silver stain
D) Coomassie brilliant blue stain

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Heating Samples to Run in Gels

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Proteins must be completely coated with SDS to run properly

often protein samples are heated. (377C, 607C or 10 07C) Samples

not coated completely will migrate slower than they should.
Some proteins must be heated to completely reduce. Again partial
reduction of a protein will result in a ladder on the gel from the
intact multimer to groups of subunits to individual monomers
The sample buffer with dye is a Tris based buffer. Tris is very
sensitive to temperature the higher the temperature the lower the
pH
Low pH will catalyze peptide bond breakage especially if there is a
DG in the sequence resulting in fragments of your protein instead
of intact protein.

Isoelectric focussing
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Separation of proteins based on their isoelectric

point
A pH gradient is set up in a gel with a solution of
ampholytes (molecules that have both negative and
positive charges)
These ampholytes are subjected to an electric
current and migrate to their equilibrium position
forming the pH gradient
Proteins are then applied and they migrate to a
position corresponding to their pI.

Isoelectric Focussing
33

Isoelectric Gels
34

Can be run vertically and loaded from the high top

high pH end (anode)

Can be run horizontally and the protein can be
loaded anywhere within the pH gradient
Sample must be in H2O or buffer at very low ionic
strength 10 mM with no salt

Example IEF gel

35

Legend
1

pI-Marker

native bovine fetuin

3
+
4

fetuin after GlycoCleave

neuraminidase treatment

asialofetuin (acid hydrolysis)

Bovine carbonic anhydrase,

pl=6.0

-Lactoglobulin B, pl=5.1

Phycocyanin (3 bands),
4.65,with
4.75
after pl=4.45,
treatment

Isoelectric focussing of bovine fetuin before and

neuraminidase. In lane 3 und 4 the shift of the pI due to the removal of terminal
sialic acids can be seen for the processed glycoprotein. Enzymatic reaction was
carried out at 37 C for 20 hours.

Capillary Electrophoresis (CE)

36

Quantitative way to separate analytes using an

electric field
Autosampler to inject samples and results are in
electropherograms, which can be integrated
Requires very low amounts of sample and solvents
Capillary gel electrophoresis (CGE)
Capillary isoelectric focussing (CIEF)

CE Instrument
37

Electropherogram of SDS Protein Complexes

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Protein Peaks:
apo A-I, apolipoprotein A-I (28100 Da,
constituent polypeptide of high density
lipoprotein HDL)
Alb, albumin (67000 Da)
Tf, apotransferrin (76000 Da)
IgG, immunoglobulin G (150000 Da); 2M,
alpha-2-macroglobulin (dimer, 320000 Da)

CIEF Electropherogram
39

HPLC Assays
40

Size Exclusion Chromatography - see K. Grant

8/3/05

% aggregate
% fragment

RP HPLC intact protein

% purity (detect oxidized species for human growth hormone)

Peptide map RP HPLC

Demonstrate identity
With mass spectrometry confirms sequence and any
modifications

RP HPLC
41

HP-RPC is based on highly specific analytical columns

comprising mono disperse particles with a diameter of

5-10 m separating proteins according to their
hydrophobicity
The high-resolution methodology is restricted to
analysis of hydrophilic or semi-hydrophobic proteins of
a molecular weight of 3000-100000
Retention of molecules of interest can be controlled by
manipulating the properties of the mobile phase, and
separation of molecules with only small differences in
hydrophobicity can be performed

RP HPLC
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The HP-RPC is used for detection of target protein

related compounds (e.g. des-amido forms, oxidized

forms, scrambled forms, cleaved forms), which may be
present in amounts from 1 part per thousand (ppt) and
upwards
The resulting UV-diagram is said to provide an impurity
profile within the relatively narrow window offered by
the technology, but it should be kept in mind that not all
impurities are detected by this or similar methods
Impurities present in 0.1% or higher should be fully
characterized no later than phase 3 manufacture.

Peptide map
43

May reduce (DTT or ME) and alkylate (iodoacetic acid).

Easier to digest disulfide bonded proteins and seals off

cysteines so they do not react.
If mapping disulfide bonds, do not reduce and alkylate.
Work at pH <7.5 so the bonds do not scramble
Choose a protease so that the N and C termini peptides
are at least 3 amino acids long. 5-10 is best.
Trypsin, LysC or AspN are commonly used proteases.
Reactions may be done in the presence of denaturants.
Trypsin and AspN work in 2 M urea and LysC works in 4
M urea

Example of a Peptide Map

44

HPLC peptide map of tryptic PEDF peptides

Tryptic peptides were separated by RP-HPLC on Aquapore RP-300. The fractions were collected manually and characterized by Edman
degradation and MALDI-MS (results not shown). The majority of the peptides displayed the expected mass (results not shown). Three
contained post-translational modifications, including (i) an N-terminal peptide containing Glp in fraction 21, (ii) a peptide containing
Cys242 in fraction 9 and (iii) the peptide containing Asn266 in fraction 26 (see also Table 1).
Biochemical Journal (2004) Volume 374

ELISAs
45

E nzyme
L inked
I mmuno
S orbent
A ssay

ELISA Method
46

Reading an ELISA
47

Colorless
substrate
TMB

Colored substrate
(Yellow initially, but
the reaction is
usually quenched
with acid turning it
blue.)

ELISAs 4 parameter curve fit

48

Samples should not fall on the plateaus, but in the

linear region
Upper plateau
Linear region

Lower Plateau

ELISAs are used to:

49

Demonstrate an antibody binds a particular antigen

Quanititate individual process impurities

ex. A specific protein added to the cell culture media such as

transferrin or HSA
ex. Protein A if a protein A column is used in the process

Measure the host cell proteins

Other facts about ELISAs

50

Run many samples at once on the 96 well plate

The assay is long hours to 2 days with incubations

after coating, addition of sample/standards, and the

addition of detection antibody
Results have >10% CV for single analyte assays and
>15% CV for multiple anlyte assays (host cell
proteins)

Immunoblots
51

Run the SDS gel colored molecular weight markers

Do not fix or stain the gel
Electrophoretically transfer the proteins from the gel

to a PVDF membrane
Wash and block the membrane
Add labeled antibody and wash
Chemical reagent to visualize on film
Line the membrane up with the film and mark
where the colored MW proteins are

Immunoblots - Westerns
52

Example of an Immunoblot
53

Q.C. Lab The Laboratory

54

Q.C. analysts trained on cGMP, general Q.C. SOPs,

the instruments and the specifice SOP of an assay

Instruments are validated IQ, OQ and PQ
Calibration and routine maintenance up to date
Assays may be new from development, qualified or
validated