Protein function examples: Antibodies bind to specific foreign particles, such as viruses and bacteria, to help protect the

body. Enzymes carry out almost all of the thousands of

chemical reactions that take place in cells. They also assist with the formation of new molecules by reading
the genetic information stored in DNA. Messenger proteins, such as some types of hormones,transmit signals to coordinate biological processes between different cells, tissues, and
organs. Structural These proteins provide structure and support for cells. On a larger scale, they also allow the body to move. Transport/storage These proteins bind and carry atoms
and small molecules within cells and throughout the body.
Antibody: type of Y-shaped protein that can bind to substances; can be used alone or to carry drugs, toxins, or radioactive substances directly to cells
Typical antibodies contain two heavy chains and two light chains ▪ The Fab domains have two variable regions (Fv) – gives antibody specificity in recognition ▪ Each variable domain has
three hypervariable regions called complementary determining regions (CDRs) Chimeric: part human, part mouse ▪ Humanized: antibodies further manipulated to increase human
composition ▪ Fully human: come from genetically engineered transgenic mice or phage display technologies
Polyclonal (pAb): recognize multiple epitopes on any one antigen; inexpensive and quicker to produce than mAb ▪ Monoclonal (mAb): detect only one epitope on the antigen; consist of
only one antibody subtype (IgG1, IgG2, IgG3) 5 immunoglobulin classes: IgG, IgD, IgA, IgE, IgM (Human 70-85% IgG)
Immune Response: Antigen: molecule capable of interacting with components of the immune system ▪ Immunogen: molecule capable of inducing an immune response; includes
proteins glycoproteins, lipoproteins, many polysaccharides, some nucleic acids.
Fc-Fusion protein: IgG Fc domain linked to another peptide; allows it to access specific areas of the host, carry an enzyme to specific site, or carry toxin to specific target for therapy
Antibody sources: native sources: human serum, grow cells in mouse, cells multiply and produce fluid in abdomen, harvest mAb from fluid, egg yolk. Recombinant sources (cell culture-
scales up easily): extracellular protein expression into supernatant or intracellular protein expression.
Proteins/antibody separation from: complex mixture usually cells, tissues, whole organisms. Depends on protein of interest: protein expression systems: e. coli: easiest, quickest,
cheapest. Yeast: grow to high density. Insect: can carry out post translational modifications than e coli and yeast, higher cost and longer duration. Mammalian cells: HEK or CHO,
complex proteins with proper post translational modifcations.
Upstream: Cell culture development ▪ Bioreactor production ▪ Clarification
Downstream: Affinity chromatography ▪ Viral inactivation ▪ Polishing chromatography ▪ Viral clearance ▪ Concentration and diafiltration ▪ Formulation ▪ Final filtration and filling
Factors in cell growth: ▪ Temperature: low temperature tends to reduce growth rate ▪ Acidity: optimal for bacteria usually 6.5-7.0; some bacteria excrete acid which affects the pH ▪
Oxygen: bacteria can be aerobes (survives and thrives in oxygenated environment) or anaerobes (does not require oxygen for growth) ▪ Micronutrients ▪ Toxins
Fermentation ▪ Two ways for cells to produce molecule of interest: ▪ Extracellular production: Protein is secreted from cells into surrounding fluid. The cells (biomass) are discarded at
beginning of separation. The medium (which contains the product) is kept for further processing Intracellular production: Protein is not secreted. Have to disrupt the cells (cell lysis)
containing the protein. Can do this by repeated freeze/thaw, sonication, homogenization by high pressure (French press), homogenization by grinding, permeabilization by detergents
and/or enzymes. Then, remove cell debris by centrifugation – proteins and other soluble compounds remain in the supernatant
Bioreactor design to provide proper environmental conditions required for optimal growth and/or product production under a contained system. ▪ Usually cylindrical▪ Tank geometry
influences homogeneity of the bioreactor ▪ Typically aspect ratio of 1:1 to 3:1 liquid height to diameter ▪ Temperature monitoring and control Rocking bioreactor: ▪ Non-invasive
rocking motion offers excellent mixing and gas transfer for cell growth ▪ Alternative to stainless steel ▪ Flexible ▪ Cost-effective DISPOSABLE ▪ Shift in industry toward disposable / single
use bioreactors ▪ Reduces cleaning time ▪ Eliminates autoclaving step ▪ No re-installation necessary ▪ Reduces risk of contamination
Mammalian Protein Production ▪ Fed-Batch: gradual addition of a fresh volume of selected nutrients during the growth–culture cycle to improve productivity and growth. The culture is
subsequently harvested and the product recovered. ▪ Advantage: simple ▪ Drawbacks: high start-up costs, large bioreactor capacity needed ▪ Typical cell densities: 5–25 x 106 cells/mL;
7-21 day production ▪ Perfusion: a continuous supply of fresh media is fed into the bioreactor while growth-inhibitory byproducts are constantly removed ▪ Advantage: high product
output from a smaller reactor size, advantages in scalability ▪ Drawbacks: more complicated – need cell retention device ▪ Typical cell densities: 30–100 x 106 cells/mL ▪ Can run
continuously for several months ▪ Fed-batch and perfusion are most common. Other strategies exist (Concentrated Fed-Batch, Concentrated Perfusion…)
Perfusion Production Alternating Tangential Flow (ATF) Repligen Xcell ATF system: diaphragm pump, stainless steel housing, single-use hollow fiber filter cartridge
How are proteins purified? ▪ Clarification, filtration ▪ Affinity chromatography ▪ Viral inactivation ▪ Polishing chromatography ▪ Viral clearance ▪ Concentration and diafiltration
Salting in / Salting out ▪ Salting in: Effect where increasing the ionic strength of a solution increases the solubility of some solute (such as a protein). Tends to be observed at lower
ionic strengths ▪ Salting out: Solubility decreases ▪ Kosmotropic (Order-making; Stabilizes water proteins); used to salt proteins out of solution ▪ Chaotropic salt (disorder-making;
disrupts water structure) ▪ Hofmeister series: classification of ions in order of their ability to salt out or salt in proteins