MAb’s stability issues

Session: Risk management for sterile preparation in hospital pharmacies
Presented by Dr Andrew G Watts

Transcript

1. MAb’s stability issues Session: Risk management for sterile preparation in hospital pharmacies Dr Andrew G Watts Medicinal Chemistry Group Department of Pharmacy and Pharmacology University of Bath
2. Overview 1.  Chemistry and structure 2.  Mechanisms of action 3.  Mechanisms of degradation 4.  Minimising degradation 5.  Stability testing protocols
3. Biologicals : Differences to Small molecules Size Aspirin 21 atoms Growth hormone 3,000 atoms Monoclonal Antibody 25,000 atoms Structure 1º 1º, 2º, 3º, 4º Bonding Strong (covalent) Strong & Weak (covalent & non-covalent)
4. Classes of Biologicals -  ‘Biologics’ is a generic term used to refer to numerous types of peptide- and protein- based therapeutic molecules. -  Biologicals themselves can differ significantly in size and complexity. -  Examples: •  Small peptides: Insulin, Fuzeon •  Medium proteins: Epogen, Neupogen •  Large proteins: Herceptin, Avastin Increasing size and complexity
5. Structure of mAb’s 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) Strong forces Weak forces
6. Structure of mAb’s 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)
7. Quaternary structure of mAb’s •  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
8. The Structure of mAb’s 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
9. Mechanisms of action -  mAb’s can also act through multiple pathways, each involving binding of Fc or Fv domains to different targets, i.e. Alemtuzumab -  Fc binding to an effector cell → Antibody Dependant Cellular Cytotoxicity -  Fc binding to complement → Complement Dependant Cytotoxicity -  Fv binding to CD52 receptor → Direct apoptosis Shape of the drug is essential for receptor binding → therapeutic effect!
10. Recap Ø  Monoclonal antibodies are a class of biological therapeutic Ø  mAb’s have a complex chemical structure Ø  1 structure (amino acid sequence) defines the mAb and is held together by strong forces (amide bonds) Ø  2º, 3º and 4º structures determine the shape of the mAb and are held together by weaker forces (hydrogen bonding) Ø  The shape of the mAb determines its biological properties Ø  Potency Ø  Serum half-life Ø  Immunogenicity Degradation can result in changes to the structure and shape of the drug
11. Degradation of mAb’s The degradation of biological drugs is a spontaneous process and will always occur to some extent. However, many of the manipulations we perform will act to accelerate this process. Formulation – need to maintain mAb conformation. •  Excipients - sucrose, trehalose, sodium chloride •  Surfactant - polysorbate 80, polysorbate 20 •  Buffers - Sodium phosphate, sodium citrate, HCl, L-histadine
12. Degradation of mAb’s: Theory into practice How many types of degradation do we contribute to when preparing or manipulating a product? Lyophilized powder Infusion bag Reconstituted vial
13. Handling and Manipulation of Biologicals Manufacturing process •  Temperature change •  Shaking •  Oxygen exposure •  Metals •  Filters •  Shearing •  Dilution
14. Surface interactions (Containers) •  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 Storage of Biologicals
15. Aggregation - Can form dimers, tetramers or larger aggregates/particles •  Decreased bioactivity •  Increased immunogenicity (small aggregates) •  Affect fluid dynamics in organ systems (particles) aggregated protein Degradation of mAb’s
16. Degradation of mAb’s How many types of degradation do we contribute to when preparing a product? Temperature change Introducing metal ions, silicon, oxygen Shaking, interaction with container, excipient dilution, shearing forces Absorption, silicon, oxygen, excipient dilution, metal ions, leaching, filters, shearing forces, temperature changes Removing from the fridge Adding diluent Reconstituting Introducing to infusion bag Aggregation, precipitation Oxidation, catalysis, aggregation Denaturing, unfolding, aggregation, oxidation, hydrolysis, deamination Denaturing, unfolding, aggregation, oxidation, hydrolysis, deamination, precipitation
17. Minimising degradation Be aware of the contributing factors and take steps to minimize their impact! •  Avoid rapid temperature changes (gradually warm) •  Avoid multiple temperature cycles •  Don’t subject to excess force (shaking and dispensing from syringes) •  Be aware of your devices (needle gauge, contaminants, composition) •  When purchasing pre-prepared products: CHECK MANUFACTURERS STABILITY DATA
18. Stability Testing: Source Guidance •  International Conference for Harmonization (ICH) •  Harmonization of British, US, Japanese and European Pharmacopeia's •  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 Guidelines generally aimed at the licensing of new drug products.  
19. NHS Source Guidance (UK)
20. Stability-Indicating profile §  no single stability-indicating assay or parameter profiles the stability characteristics of a biotechnological/biological product §  the stability-indicating profile should provide assurance that changes in the : Identity Purity Potency Other characteristics §  the determination of which tests should be included will be product- specific Chemical analysis Biological activity Cellular response Primary structure Secondary structure Tertiary structure Quaternary structure