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?
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
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
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
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)
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)
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
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
tetramers or larger aggregates/particles • Decreased bioactivity • Increased immunogenicity • Affect fluid dynamics in organ systems aggregated protein
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
Analytical validation • ICH Q5C Stability Testing of Biotechnological/Biological products • ICH Q6B Specifications Test Procedures and Acceptance Criteria for Biotechnological/ Biological Products
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