Monoclonal antibodies (mAbs) have complex structures that are susceptible to degradation through various mechanisms. Their structures consist of primary, secondary, tertiary, and quaternary levels that give them their shape and biological functions. Common degradation pathways for mAbs include aggregation, oxidation, hydrolysis, and denaturation. Many steps involved in preparing and handling mAbs, such as temperature changes, shaking, dilution, and interaction with containers, can accelerate degradation by introducing factors like metal ions, oxygen, shear forces, and adsorption to surfaces. Care must be taken to minimize degradation risks through practices like avoiding excessive temperature changes and forces during preparation, handling, and storage.

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