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
5. How do mAbs differ from traditional chemical molecules?
Size
Aspirin Growth hormone IgG Antibody
21 atoms 3,000 atoms 25,000 atoms
Complexity
150 parts 14,000 parts 6,000,000 parts
9. Structure of Monoclonal Antibody!
Antigen specific • Therapeutic mAbs
binding 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
Cell receptor region
specific binding
10. Structure of Monoclonal Antibody!
Antigen specific • Therapeutic mAbs
binding 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
Cell receptor region
specific binding
11. The importance of structure on the mechanism of action!
An#body
dependant
cellular
cytotoxicity
Mechanism of action
include:
• Cell death via ADCC
• Cell death CDCC
• Inducing apoptosis
• Neutralization of
soluble molecules
• Mediating cellular
activity
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
23. Processes contributing to degradation mAbs
Chemical degradation - Deamidation
- Change in conformation
- Change in bonding
- Change in bonding - Change in conformation
- 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
aggregated protein
• Affect fluid dynamics in organ systems
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
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
39. Processes contributing to degradation of mAbs
Native protein
Chemical Stability Physical stability/Aggregation
Oxidation Conformational Stability Colloidal Stability
(2ry, 3ry, 4ry structure) (multimers, sub-visible/visible particles)
Deamidation
Free energy change Intermolecular
Hydrolysis interactions
Proteolysis
Unfolded states
Aggregates
Biologics specific techniques Traditional assay 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. Follow us at - mabstalk.com
26th September 2012
Maria Connolly!
Director of Quality & Compliance!
