Purification of Biologics Flashcards
(40 cards)
How can post-translational modifications affect proteins?
-Direct proteins to different regions in the cell (Ubiquitylation to the lysosome, proteins with disulfide bridges to the cell membrane)
-Glycosylation is specific for an organism (yeast, Ecoli, mammalian) and will determine stability and half-life in the human body
Which sugars are created in different expression systems?
-> Yeast will produce Mannose and will be cleared quickly in the human body
-> Plant cells create Fucose, causing an immunogenic reaction
-> Sialic acid is created in mammalian cells -> have a longer half-life bc closer to human
Which expression system would be the best to produce complex PTMs?
Mammalian systems
-Chinese Hamster Ovary (CHO) cells
-HEK cells
What happens after protein synthesis and PTM?
Protein folding by chaperons into tertiary or quarternary structures
What happens to misfolded proteins in humans, or misfolded proteins produced in E. coli (biopharmaceutical industry)?
-humans: degraded or may form extracellular aggregates or Amyloids (e.g. beta-amyloid in Alzheimer’s)
-isolation from E.coli and refolding with guanidinium chloride
Compare generics and biosimilars
Biosimilars: structural variability from batch to batch due to PTMs
Generic: identical structure
What causes differences between biologics and Biosimilars?
-the difference in PTMs, but the primary structure is the same!
-no meaningful clinical difference between biologics and biosimilar
-no clinical difference from batch to batch within the same biologic drug
What is the most important requirement that the FDA asks for in biosimilars?
-Biosimilars must have the same bioefficacy as the reference biological drug
-small differences for clinically inactive ingredients are accepted (f.e. bioequivalence -> PK pattern)
What are the differences from batch to batch for small molecule drugs?
-Purity
-one batch may have a purity of the drug of 90%, and the other one may have a purity of 95%
What are the different steps of protein drug production?
- Cloning the DNA into vectors and transfer into an expression system
- upstream: optimizing cell conditions to produce the maximum amount of cells and protein product
- downstream: isolation and purification of the product + packaging of the drug
How is DNA created to produce the protein drugs?
-synthetically from a gene bank
OR
-with reverse transcriptase (from retroviruses) -> creating hybrid RNA-DNA from mRNA -> RNAse cleaves off the RNA -> DNA polymerase completes the ds-DNA
What is the difference between cDNA and native DNA?
cDNA is shorter bc the introns are removed and only the gene of interest is present
How is the created DNA amplified before cloning it into vectors?
Through PCR
What are the steps of PCR?
-Denaturation: Separation of strands
-Annealing: Strands are coming together
-Elongation: Bases are being attached to the complementary strand
What is the most common expression system for heterologous protein production? (proteins different from the host cell)
Ecoli
What are the advantages of E.coli as an expression system?
-well-understood genetics/biology: optimal conditions are known
-very productive (30% of total proteins are the protein of interest)
-easy to grow; simple, and cheap growth media
-doubling time is 30 minutes
-GRAS (generally regarded as safe)
What are the disadvantages of E.coli?
-the proteins are aggregated into inclusion bodies because of hydrophobic interactions -> (a tertiary structure is lost)
-no PTMs in E.coli -> not great for monoclonal antibodies
-lipopolysaccharides (LPS) -> PYROGENS can cause an immune reaction (antigenic)
Why is E.coli not the best expression system for monoclonal antibodies?
Because monoclonal Ab contains glycosylation, and E.coli is not able to create PTM
-sometimes E.coli is used for antibody fragments, bc they are smaller and the structure is less complex
How are proteins from inclusion bodies in E.coli renatured?
With chemicals like guanidinium chloride
Advantages of yeast as an expression system
-f.e. Saccharomyces cerevisiae or Pichia pastoris (eucaryotic but with a cell wall)
-Fast growth (90 min doubling time); easy to work with; inexpensive media
-bc of the cell wall they are resistant to handling (compared to mammalian cells)
-PTM, glycosylation is possible though (Mannose -> immunogenic)
-No inclusion bodies (like in E.coli)
-GRAS listed
Disadvantages of yeast as an expression system
-high mannosylation
-cleared from the body a lot faster than sialic acid-attached (mammalian) proteins
-protein expression is low: ~ 5% (compared to E. coli)
What are the advantages of mammalian cells as expression systems?
-f.e. Chinese Hamster Ovary (CHO) cells; baby hamster kidney (BHK)
-Glycosylation patterns are mammalian (sialic acid- tagged; advantage) -> Long half-life (bc more similar to the human system)
What are the disadvantages of mammalian cells as expression systems?
- Complex nutritional requirements (sometimes from natural sources -> increasing the chance of contamination (BSE)
-handling is harder (more sensitive to damage, bc no cell wall)
-Slow growth; long doubling times (12-24 h)
-High production costs (decontamination of nutrients (filtration, sterilization, irradiation)
Why are mammalian cells usually used for monoclonal antibodies?
Because monoclonal antibodies have complex structures (Y-shaped antibody) and require glycosylation
