The Chemistry of Monoclonal Antibodies

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