EF - Glycosylation of Biologics I Flashcards
(13 cards)
Q1: What two major scientific ideas laid the foundation for biopharmaceutical manufacturing? (2)
Cells can produce useful substances:
- Leeuwenhoek discovered single-celled organisms.
- Pasteur uncovered microbial fermentation.
- Fleming discovered penicillin.
- Sabin & Salk derived polio vaccines from monkey kidney cells.
Proteins can treat disease:
- Banting and Best discovered insulin from pancreatic extracts for diabetes.
Q2: What key event in 1973 marked the start of modern biopharmaceuticals? (1)
The founding of Genentech, which pioneered the use of recombinant DNA technology to produce therapeutic proteins like insulin in microbes instead of harvesting from animals.
Q3: Why was the development of recombinant insulin significant? (3)
- Eliminated reliance on animal pancreas, reducing cost and contamination risk.
- Enabled large-scale, consistent production.
- Made insulin therapy safer and more accessible to diabetic patients.
Q4: Why isn’t chicken insulin used for humans? (2)
- Only \~60% identical to human insulin.
- Chickens are not mammals, so their insulin lacks necessary functional similarity.
Q5: What is a cell line, and why is it critical in protein production? (3)
- A cell line is a population of cells that can be grown indefinitely in vitro.
- The choice of cell line affects:
- Functional protein folding
- Intramolecular bonding (e.g. disulfide bridges)
- Post-translational modifications (PTMs)
Q6: Why are bacteria often unsuitable for producing complex therapeutic proteins? (3)
- Bacteria lack machinery for complex protein folding.
- Cannot form intermolecular disulfide bonds required in antibodies.
- Cannot perform complex PTMs, like glycosylation.
Q7: What glycosylation-related PTMs occur in insulin? (4)
- O-glycosylation at serine/threonine residues in the B-chain and C-peptide.
- O-GalNAc (O-linked N-acetylgalactosamine) is the sugar attached.
- Many glycosylation sites are conserved, suggesting functional importance.
- Roles include protein processing, receptor interaction, distribution, and stability.
Q8: What is a drawback of producing recombinant insulin in bacteria or yeast? (1)
They lack human-specific glycosylation patterns, which may affect protein function or stability in patients.
Q9: What is the ‘glycome’ and why does it matter for biologics? (2)
- The glycome is the full set of glycan structures in a cell or organism.
- It’s essential for determining protein activity, half-life, stability, and immunogenicity.
Q10: What is N-linked glycosylation and where does it occur? (4)
- A type of PTM where a glycan is added to an Asn residue in the sequon -NXS/T-.
- OST (oligosaccharyltransferase) transfers the glycan to the asparagine.
- Takes place in the endoplasmic reticulum (ER) membrane.
- Requires the sequence:
Q11: How does ER quality control (ERQC) regulate N-glycosylation? (5)
- Glucosidases I & II trim three glucose units from the glycan.
- This signals interaction with calnexin and calreticulin chaperones.
- These chaperones prevent aggregation and ensure correct folding.
- If misfolded, Glc-T (glucosyltransferase) re-adds glucose to restart folding.
- If folding fails repeatedly, the protein is marked for degradation.
Q12: What happens to N-glycans after the ER as they pass through the Golgi? (4)
- cis-Golgi: N-glycans are primarily high-mannose/oligomannose types.
- medial-Golgi: Converted to complex/hybrid N-glycans.
- trans-Golgi and TGN: Glycans undergo final modifications.
- Final protein destinations include the plasma membrane, lysosomes, or secretion.
Q13: Why do biopharmaceutical manufacturers aim to minimize glycosylation? (2)
- Glycans can cause batch variability, immunogenicity, and instability.
- Some proteins (like insulin) do not require glycosylation for function, making production simpler.
