The Chemistry of Monoclonal Antibodies

Maria Connolly
Director of Quality & Compliance
Bath ASU

Transcript

1. Maria Connolly! Director of Quality & Compliance! The Chemistry of Monoclonal Antibodies 26th September 2012
2. Introduction Some of the questions I started with two years ago: •  How do mAbs differ from traditional molecules? •  How do they degrade? •  What factors affect mAb stability? •  How safe are the degradation products? •  What is an acceptable degradation limit? •  How can we detect degradation products?
3. Overview •  What are mAbs? •  How are they constructed? •  How do they breakdown? •  What is it that drives degradation? •  Examples of degradation? •  Assessing mAb stability
4. What are mAbs?
5. How do mAbs differ from traditional chemical molecules? Size Complexity 150 parts Aspirin 21 atoms Growth hormone 3,000 atoms IgG Antibody 25,000 atoms 14,000 parts 6,000,000 parts
6. Types of Monoclonal Antibody
7. TGN1412 Immunogenicity
8. How are they constructed?
9. Structure of Monoclonal Antibody! Antigen specific binding Cell receptor specific binding •  Therapeutic mAbs predominantly of IgG1 class and subtype •  IgG consist of 2 heavy and light chains •  Around 150kDa in size •  Chains held together by disulfide bond between conserved cysteine residues at the hinge region •  Fc region binding cell surface Ig receptors •  Antigen binding variable region
10. Structure of Monoclonal Antibody! Antigen specific binding Cell receptor specific binding •  Therapeutic mAbs predominantly of IgG1 class and subtype •  IgG consist of 2 heavy and light chains •  Around 150kDa in size •  Chains held together by disulfide bond between conserved cysteine residues at the hinge region •  Fc region binding cell surface Ig receptors •  Antigen binding variable region
11. The importance of structure on the mechanism of action! Mechanism of action include: •  Cell death via ADCC •  Cell death CDCC •  Inducing apoptosis •  Neutralization of soluble molecules •  Mediating cellular activity An#body  dependant  cellular  cytotoxicity  
12. The importance of structure on the mechanism of action! ELISA studies are not sufficient as they: •  only tell you they are binding •  do not demonstrate biological activity Cell based studies •  demonstrate biological activity •  may require multiple functional studies to assess various modes of action
13. Specificity is derived from protein structure (shape)! Primary Structure – the amino acid sequence linked via covalent peptide bonds Secondary Structure – linking of sequences of amino acids by non covalent interactions (Alpha helices, Beta sheets)
14. Specificity is derived from protein structure (shape)! Primary Structure – the amino acid sequence Secondary Structure – linking of sequences of amino acids by hydrogen bonding (beta sheets, alpha helices) Tertiary Structure – attractions between beta sheets and alpha helices to give 3-D structures Quaternary Structures – protein consisting of more than one amino acid chain (complex of protein molecules)
15. Monoclonal antibody quaternary structure! •  Y shaped Quaternary structure. •  Functionality relies on quaternary structure •  Interchain disulfide bonds at the hinge region and non covalent interactions between CH3 domains stabilise the structure •  CH2 domain is overlaid by an oligosaccharide covalently attached at Asn297
16. Glycans influence Monoclonal antibody function! Glycosylation is a critical quality attribute •  CH2 domain is overlaid by an oligosaccharide covalently attached at Asn297 •  Small contribution to mAb size •  Influence t ½ •  Stability to degradation •  Influence protein folding •  Solubility •  Changes can alter functional activity •  Immunogenicity
17. How many species!!
18. How do they break down?
19. Processes contributing to degradation of mAbs! Native protein Chemical Stability Physical stability/Aggregation Oxidation Deamidation Hydrolysis Proteolysis Conformational Stability (2ry, 3ry, 4ry structure) Colloidal Stability (multimers, sub-visible/visible particles) Unfolded states Aggregates Free energy change Intermolecular interactions
20. Processes contributing to degradation mAbs! Chemical degradation - Oxidation Reduction in pH Extra bonds
21. Processes contributing to degradation mAbs! Chemical degradation - Oxidation Changes to hydrogen bonding and conformation
22. Degradation products! Chemical degradation - Hydrolysis
23. Processes contributing to degradation mAbs Chemical degradation - Deamidation - Change in conformation - Change in bonding - Change in conformation - Change in bonding - Change in pH - Change in bonding - Change in pH
24. Processes contributing to degradation mAbs Physical instabilities Fragmentation - Dissociation or cleavage of chains Loss of activity Denaturation - Change of shape/structure – alteration of bonds necessary for native conformation
25. Processes contributing to degradation mAbs Aggregation - Can form dimers, tetramers or larger aggregates/particles •  Decreased bioactivity •  Increased immunogenicity •  Affect fluid dynamics in organ systems aggregated protein
26. What is it that drives degradation?
27. Processes contributing to degradation mAbs Formulation – need to maintain mAb conformation •  Excipients - sucrose, trehalose, sodium chloride •  Surfactant - polysorbate 80, polysorbate 20 •  Buffers - Sodium phosphate, sodium citrate, HCl, L-histadine
28. Processes contributing to degradation mAbs Storage •  Degradation of excipients •  Heating •  Freezing/ thawing •  Light / photooxidation
29. Processes contributing to degradation mAbs Manufacturing process •  Shaking •  Oxygen exposure •  Metals •  Filters •  Shearing •  Dilution
30. Processes contributing to degradation mAbs Surface interactions • Adsorption - interact with all types of surfaces. Can potentially interact with devices during production and storage • Leaching – presence of solubilising agents in the formulation increases likelihood of leaching. •  Silicon – act as nucleation sites in certain circumstances silicone oil
31. Degradation examples
32. Bevacizumab aggregation Bevacizumab Aggregation Silicon Agitation Storage conditions Light exposure Storage device Surfactant
33. Side effects of licenced vs unlicenced products Eye disorders Very common Vitritis, vitreous detachment, retinal haemorrhage, visual disturbance, eye pain, vitreous floaters, conjunctival haemorrhage, eye irritation, foreign body sensation in eyes, lacrimation increased, blepharitis, dry eye, ocular hyperaemia, eye pruritus. Intraocular pressure increased Common Retinal degeneration, retinal disorder, retinal detachment, retinal tear, detachment of the retinal pigment epithelium, retinal pigment epithelium tear, visual acuity reduced, vitreous haemorrhage, vitreous disorder, uveitis, iritis, iridocyclitis, cataract, cataract subcapsular, posterior capsule opacification, punctuate keratitis, corneal abrasion, anterior chamber flare, vision blurred, injection site haemorrhage, eye haemorrhage, conjunctivitis, conjunctivitis allergic, eye discharge, photopsia, photophobia, ocular discomfort, eyelid oedema, eyelid pain, conjunctival hyperaemia. Uncommon Blindness, endophthalmitis, hypopyon, hyphaema, keratopathy, iris adhesion, corneal deposits, corneal oedema, corneal striae, injection site pain, injection site irritation, abnormal sensation in eye, eyelid irritation.
34. How safe are degradation products? Health and Drug Alert Epoetin alfa (Eprex): reports of pure red blood cell aplasia
35. Assessing mAb stability
36. Stability references International Conference for Harmonization (ICH) • ICH Q2 R1 Analytical validation • ICH Q5C Stability Testing of Biotechnological/Biological products • ICH Q6B Specifications Test Procedures and Acceptance Criteria for Biotechnological/ Biological Products
37. Stability references NHS guidelines (draft) require: •  Physical •  Chemical •  Functional •  Needs to be adopted, yellow cover
38. Different techniques will give you different information
39. Processes contributing to degradation of mAbs Native protein Chemical Stability Physical stability/Aggregation Oxidation Deamidation Hydrolysis Proteolysis Conformational Stability (2ry, 3ry, 4ry structure) Colloidal Stability (multimers, sub-visible/visible particles) Unfolded states Aggregates Free energy change Intermolecular interactions Traditional assay techniques Biologics specific techniques
40. Conclusion! •  Remember ICH guidance requires chemical, physical & functional activity for a reason! •  Many physical and chemical factors can affect product quality, efficacy & safety issues •  It is important to understand the chemistry of mAbs in order to: design stability studies which can effectively identify degradation products evaluate the impact on product quality and safety
41. Conclusion The less we know about a mAb the more stable we think it is!
42. Maria Connolly! Director of Quality & Compliance! Follow us at - mabstalk.com 26th September 2012