Purification of Biologics

Question 1

How can post-translational modifications affect proteins?

Answer 1

-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

Question 2

Which sugars are created in different expression systems?

Answer 2

-> 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

Question 3

Which expression system would be the best to produce complex PTMs?

Answer 3

Mammalian systems
-Chinese Hamster Ovary (CHO) cells
-HEK cells

Question 4

What happens after protein synthesis and PTM?

Answer 4

Protein folding by chaperons into tertiary or quarternary structures

Question 5

What happens to misfolded proteins in humans, or misfolded proteins produced in E. coli (biopharmaceutical industry)?

Answer 5

-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

Question 6

Compare generics and biosimilars

Answer 6

Biosimilars: structural variability from batch to batch due to PTMs

Generic: identical structure

Question 7

What causes differences between biologics and Biosimilars?

Answer 7

-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

Question 8

What is the most important requirement that the FDA asks for in biosimilars?

Answer 8

-Biosimilars must have the same bioefficacy as the reference biological drug

-small differences for clinically inactive ingredients are accepted (f.e. bioequivalence -> PK pattern)

Question 9

What are the differences from batch to batch for small molecule drugs?

Answer 9

-Purity
-one batch may have a purity of the drug of 90%, and the other one may have a purity of 95%

Question 10

What are the different steps of protein drug production?

Answer 10

1. Cloning the DNA into vectors and transfer into an expression system
2. upstream: optimizing cell conditions to produce the maximum amount of cells and protein product
3. downstream: isolation and purification of the product + packaging of the drug

Question 11

How is DNA created to produce the protein drugs?

Answer 11

-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

Question 12

What is the difference between cDNA and native DNA?

Answer 12

cDNA is shorter bc the introns are removed and only the gene of interest is present

Question 13

How is the created DNA amplified before cloning it into vectors?

Answer 13

Through PCR

Question 14

What are the steps of PCR?

Answer 14

-Denaturation: Separation of strands
-Annealing: Strands are coming together
-Elongation: Bases are being attached to the complementary strand

Question 15

What is the most common expression system for heterologous protein production? (proteins different from the host cell)

Answer 15

Ecoli

Question 16

What are the advantages of E.coli as an expression system?

Answer 16

-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)

Question 17

What are the disadvantages of E.coli?

Answer 17

-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)

Question 18

Why is E.coli not the best expression system for monoclonal antibodies?

Answer 18

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

Question 19

How are proteins from inclusion bodies in E.coli renatured?

Answer 19

With chemicals like guanidinium chloride

Question 20

Advantages of yeast as an expression system

Answer 20

-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

Question 21

Disadvantages of yeast as an expression system

Answer 21

-high mannosylation
-cleared from the body a lot faster than sialic acid-attached (mammalian) proteins
-protein expression is low: ~ 5% (compared to E. coli)

Question 22

What are the advantages of mammalian cells as expression systems?

Answer 22

-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)

Question 23

What are the disadvantages of mammalian cells as expression systems?

Answer 23

• 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)

Question 24

Why are mammalian cells usually used for monoclonal antibodies?

Answer 24

Because monoclonal antibodies have complex structures (Y-shaped antibody) and require glycosylation

Question 25

Protein production in animals - Pharm animals

Answer 25

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

Question 26

Plants as an expression system

Answer 26

-f.e. tobacco plants creating 3 mAbs against the ebola virus
-not enough data yet to prove it efficacy

Question 27

Upstream processing

Answer 27

-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

Question 28

Upstream: Cell banking

Question 29

Why is the culture optimized for cell production preserved and not used for actual cell production?

Answer 29

-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

Question 30

What is the difference between a bacterial and a mammalian bioreactor?

Answer 30

-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

Question 31

What is special about bioreactors for mammalian cells?

Answer 31

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

Question 32

How are fixed-bed and hollow fibers different from those with propellers?

Answer 32

-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

Question 33

What is the disadvantage of using nutrients from a natural source?

Answer 33

-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

Question 34

What are the different phases of growth in cells?

Answer 34

-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

Question 35

In which phase of cell culture growth are cells harvested and the downstream process initiated?

Answer 35

Stationary Phase

Question 36

Downstream processes

Answer 36

-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

Question 37

What are the specific steps in the downstream process?

Answer 37

-refolding steps with guanidinium chloride for inclusion proteins
-removal of pyrogens (E.coli)
-using different filters (pore sizes) for contaminants of bacteria and viruses

Question 38

What are ways to separate the proteins from other cell material?

Answer 38

-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

Question 39

How are proteins eluted in ion-exchange chromatography?

Answer 39

+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

Question 40

In which condition can medical products be stored well?

Answer 40

-lyophilized -> more stable bc the water is removed
-Deep freezing: apply low pressure (vacuum) -> water sublimes (from solid to gaseous form)