Purification of Biologics Flashcards
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
Protein production in animals - Pharm animals
f.e. in goats
-DNA is tagged to the casein gene (casein is found in milk) and implanted into goats’ egg cells
-the offspring goats produce the protein of interest in the milk and can be extracted from the milk
Plants as an expression system
-f.e. tobacco plants creating 3 mAbs against the ebola virus
-not enough data yet to prove it efficacy
Upstream processing
-Develop and select a cell line, culture media, optimize growth
parameters
-Optimize processes to achieve maximal growth and production
-Monitor and regulate nutrients, temperature, pH, and oxygen supply
-Develop sterilization protocols
-Maintain a contaminant-free environment
Upstream: Cell banking
Why is the culture optimized for cell production preserved and not used for actual cell production?
-Because mutation would occur from one generation to the other and might decrease the efficacy of protein production
-100s of Mastercultures are preserved (frozen) and single working cultures are seeded from master cultures to produce the proteins
What is the difference between a bacterial and a mammalian bioreactor?
-Bacterial bioreactor: has Impellers and Baffles (on the wall) for efficient mixing of bacterial cells (bacteria cells have cell walls and can be mixed more rigorously)
-mammalian bioreactor: marine type Impeller and no baffles for slow mixing and minimal turbulence to protect the cells
What is special about bioreactors for mammalian cells?
Fixed-bed and hollow fiber bioreactors
-the mammalian cells stick to beads and grow on them
-the cells use nutrients to produce the protein of interest
-the proteins are harvested from hollow fibers
How are fixed-bed and hollow fibers different from those with propellers?
-open system bioreactor (here the reactor must not be cleaned and replaced with new cells every 2 weeks)
-replacement of nutrients is possible
-harvesting the products in between is possible
What is the disadvantage of using nutrients from a natural source?
-nutrients: sugar, fat, water, amino acids, electrolytes, vitamins, serum (BSE contamination), trace minerals, hormones
-the purity is not known
-the risk of contamination
-> Instead synthetic nutrients can be used
What are the different phases of growth in cells?
-Lag phase
-Log phase: cells using up nutrients and are dividing
-stationary phase: stops doubling bc the limit of nutrient
the product is harvested
-Dead phase: cells are dying
In which phase of cell culture growth are cells harvested and the downstream process initiated?
Stationary Phase
Downstream processes
-initial recovery (extraction or isolation)
-purification (removal of most contaminants -> 80%-90% purity
-polishing (removal of specified low-density contaminants) -> very pure product
-if the product is secreted (mammalian): filtration and centrifugation of the broth
-lysis (f.e. with cell homogenizer) and clarification of cells for intracellular products
What are the specific steps in the downstream process?
-refolding steps with guanidinium chloride for inclusion proteins
-removal of pyrogens (E.coli)
-using different filters (pore sizes) for contaminants of bacteria and viruses
What are ways to separate the proteins from other cell material?
-Nucleases: removes nucleic acid from the proteins
-Column chromatography: washing away cell material with wash and elution buffer -> Size exclusion, larger molecules will elute first
-Ion exchange chromatography: separation by charge
-Antibody chromatography: separation by antibody-interaction
-Affinity-based chromatography: uses Ligands attached to stationary phase that will bind to the protein, everything else will be washed away
How are proteins eluted in ion-exchange chromatography?
+vely charged protein binds to -vely charged beads or vice versa
-> for elution decrease or increase the pH to change the charge of the protein so that it looses attraction to the beads on the stationary phase
In which condition can medical products be stored well?
-lyophilized -> more stable bc the water is removed
-Deep freezing: apply low pressure (vacuum) -> water sublimes (from solid to gaseous form)
