EF - Glycosylation of Biologics II

Question 1

Q1: Why is correct glycosylation critical in biologics? (7)

Answer 1

• Ensures conformational stability of proteins.
• Promotes proper protein folding.
• Increases thermal stability.
• Enhances solubility due to hydrophilic sugar chains.
• Improves pharmacokinetics (e.g., half-life, circulation time).
• Provides protection against proteolytic degradation.
• Is essential for biological activity and therapeutic function.

Question 2

Q3: How was glycoengineering used to improve erythropoietin (EPO)? (6)

Answer 2

• EPO is a glycoprotein that stimulates erythropoiesis (red blood cell production).
• Human EPO contains two N-glycosylation sequons: ‘NIT’ and ‘NSS’.
• rEPO is only functional if glycosylated, produced in mammalian cells.
• NESP (novel erythropoiesis stimulating protein) was engineered with two added sequons (‘NET’), increasing total N-glycans from 3 to 5.
• Higher sialic acid content leads to a longer half-life and improved efficacy.
• Enhanced pharmacokinetic profile makes NESP more effective in vivo.

Question 3

Q2: Why is glycoengineering required in biologic drug production? (3)

Answer 3

• Glycosylation patterns vary with health and age, affecting drug consistency.
• Regulatory bodies demand consistency of glycoforms to minimize immunogenicity.
• Glycoengineering ensures control over sugar residue structures to maintain therapeutic quality.

Question 4

Q4: Why are mammalian cell lines preferred for biopharmaceutical production? (2)

Answer 4

• They produce human-compatible N-glycans, reducing immunogenicity.
• They allow for complex PTMs, including proper sialylation and branching.

Question 5

Q5: How do glycosylation profiles differ across cell systems? (5)

Answer 5

• Bacteria – No glycosylation capability; unsuitable for most therapeutic proteins.
• Yeast – Produce high-mannose N-glycans, mildly immunogenic but generally tolerated.
• Mammalian – Generate human-like N-glycans, with minor variations.
• Insect – Add α(1-3) core fucose, which is immunogenic in humans.
• Plant – Add β(1-3) xylose and galactose + α(1-3) fucose, all of which are immunogenic.

Question 6

Q6: What are examples of top-selling monoclonal antibody therapies and their targets? (3)

Answer 6

• Keytruda (pembrolizumab) – targets PD-1; used in cancer immunotherapy.
• Humira (adalimumab) – targets TNF-α; treats autoimmune diseases like RA.
• Dupixent (dupilumab) – targets IL-4Rα; used in allergic diseases.

Question 7

Q7: What are the therapeutic mechanisms of action of monoclonal antibodies? (5)

Answer 7

• Signal blockade leading to cell cycle arrest.
• Direct induction of apoptosis.
• Sensitization to chemotherapy.
• Complement-mediated cytotoxicity (CMC).
• Antibody-dependent cellular cytotoxicity (ADCC) – key mechanism in cancer therapy.

Question 8

Q8: How does ADCC function in antibody therapy? (4)

Answer 8

1. Antibody binds antigen on cancer cell.
2. FcγRIIIa (CD16a) on NK cells binds to Fc region of the antibody.
3. NK cells release granzymes and perforin.
4. Target cell undergoes apoptosis.

Note: Tighter Fc-FcγRIIIa binding = higher efficacy, lower dose needed.

Question 9

Q9: How do Fc glycans influence mAb function? (3)

Answer 9

• Fc region has two N-glycans at Asn297 (CH2 domain).
• Increased sialylation enhances half-life and stability.
• Core α(1-6) fucosylation reduces binding to FcγRIIIa, thus lowering ADCC potency.

Question 10

Q10: How does removal of core fucose improve mAb efficacy? (3)

Answer 10

• Non-fucosylated antibodies bind more strongly to FcγRIIIa on NK cells.
• Result: Enhanced ADCC at 10–100x lower concentrations.
• Example: Non-fucosylated Rituximab shows much higher tumor cell killing.

Question 11

Q11: What are the two main strategies to remove core fucosylation? (7)

Answer 11

Engineered non-mammalian cells (e.g. yeast) with mammalian glycosylation enzymes:

• Introduce glycosyltransferases, glycosidases, and sugar transporters.
• Yeast can produce non-fucosylated complex N-glycans with sialylation.

Inactivation of fucosylation in mammalian cells:

• Knockdown or knockout of FUT8 gene (α-1,6 fucosyltransferase).
• Target GDP-fucose transport pathway into Golgi.
• Approaches include:
  • siRNA silencing of FUT8
  • Disruption of FUT8 gene (FUT8–/– cell lines)
  • Co-expression of GnTIII (MGAT3) and Golgi α-mannosidase II to block FUT8 access

Question 12

Q12: What are alternative methods for generating non-fucosylated antibodies? (3)

Answer 12

In vitro chemical/enzymatic synthesis – high precision, but extremely costly and unsuitable for large-scale use.

Use of glycomimetics (e.g. 8β, a fucose analog):

• Metabolized into GDP-carbafucose inside cells.
• Competes with GDP-fucose but isn’t recognized by FUT8.
• Simple, effective, and usable across many cell lines.

Question 13

Q13: What are the key conclusions regarding glycoengineering in biologics? (4)

Answer 13

• Glycosylation affects efficacy, half-life, and safety of biologics.
• Glycoengineering allows fine control over therapeutic performance.
• Core fucosylation impairs ADCC, so removing it enhances anti-cancer efficacy.
• Strategies must balance efficacy, scalability, and cost in biomanufacturing.