1. Biological Drugs – practical considerations
for handling and storage
Dr Andrew 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. Stability indicating assays
5. Interpreting stability data
Topics Learning Objectives
• Describe general classes of biologicals
• Appreciate the chemistry and structure
• Receptor binding and mechanism of
action
• Describe risk to molecules from incorrect
handling
• Consequences of incorrect handling to
the product
• Describe correct handling
• Overview of elements of stability testing
processes.
• Understanding of how stability data can
be applied

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. Types of monoclonal antibodies
- mAb’s can be raised from a number of species
- Being protein based, they will be recognised by our immune system

10. Mechanisms of action
- mAb’s often act by triggering a series of biochemical pathways in a cell
- Firstly, this requires binding to their target receptor.
o Example: Herceptin
• Herceptin binds to Her2 receptor.
• Causes a series of intracellular
signalling events.
• Results in reduced cell
proliferation and increased cell
death.
Shape of the drug is essential for receptor binding → therapeutic effect!

11. 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!

12. Recap
 Monoclonal antibodies are a class of biological therapeutic
 mAb’s have a complex chemical structure
 1 structure (amino acid sequence) defines the mAb and is held
together by strong forces (amide bonds)
 2º, 3º and 4º structures determine the shape of the mAb and are held
together by weaker forces (hydrogen bonding)
 The shape of the mAb determines its biological properties
 Potency
 Serum half-life
 Immunogenicity
Degradation can result in changes to the shape and structure of the drug

13. 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

14. Native protein
Chemical Stability Physical stability/Aggregation
Oxidation
Deamidation
Hydrolysis
Proteolysis
Conformational Stability
(2º, 3º, 4º structure)
Colloidal Stability
(multimers, sub-visible/visible particles)
Unfolded states
Aggregates
Free energy change Intermolecular
interactions
Processes contributing to degradation of mAb’s
1º structure

15. Degradation of mAb’s
How many types of degradation do we contribute to when
preparing a product?
Lyophilized
powder
Infusion bag
Reconstituted
vial

16. Handling and Storage of Biologicals
Manufacturing process
• Temperature change
• Shaking
• Oxygen exposure
• Metals
• Filters
• Shearing
• Dilution

17. 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
Handling and Storage of Biologicals

18. Aggregation - Can form dimers, tetramers or larger
aggregates/particles
• Decreased bioactivity
• Increased immunogenicity (small aggregates)
• Affect fluid dynamics in organ systems (particles)
aggregated protein
Handling and Storage of Biologicals

19. Impact of degradation on safety
Bevacizumab
Aggregation
Silicon
Agitation
Storage
conditions
Light
exposure
Storage
device
Surfactant
Bevacizumab eye syringes as an example:
• Clinical problems resulting from
degradation can be specific for
indication and use
• Elevated intra-ocular pressure
thought to result from aggregate
particles

20. 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

21. 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

22. 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.

23. 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

24. Summary of Degradation Pathways
Physical
• Denaturation
• Aggregation
• Surface adsorption
Chemical
• Disulfide formation/exchange
• Non-reducible cross linking
• Deamidation
• Isomerization
• Oxidation
• Glycosylation
Cellular response
Primary structure
Secondary structure
Tertiary structure
Quaternary structure
Impact upon:

25. Aggregation and particles
0µm 0.5µm 10µm 25µm 50µm 500µm
partial unfolding aggregation
Wide dynamic range of aggregate types and sizes means that
multiple techniques are need to assess this form of degradation.
SEC DLS Microflow Visual inspection

26. SEC HPLC (Size-Exclusion)
Aggregates
Fragments
Lower MWHigher MW
Monomer
Validation (ICH Q2)
Selectivity
• Forced degradation studies
Linearity
• Calibration Curves
• Limit of detection
• Limit of quantification
Accuracy
• Over 3 concentrations levels (min 9
injections)
Precision
• Repeatability of 6 injections (%RSD <2%)

27. Dynamic Light Scattering (DLS)
• Sample is illuminated with a laser
• Measures time dependant fluctuations in the intensity of scattered light
• Fluctuations occur because particles are constantly undergoing Brownian motion
• Effectively measures velocity of molecules which can be converted on to an equivalent
hydrodynamic radius.
The size distribution obtained is a plot of the relative intensity of light scattered by particles
in various size classes.
monomer aggregate

28. Microflow Imaging (Particle counting)
Particles (1000/ml) ± SD
X mg/ml > 1µm >2µm >10µm >25µm
Day A 275 ± 102 45 ± 29 5 ± 3 0.0 ± 0.1
Day B 441 ± 14 194 ± 10 10 ± 2 0.0 ± 0.2
Day C 166 ± 21 56 ± 14 0.3 ± 3 0.0 ± 0.4
micro-air bubble
silicone oil
aggregated protein
MFI uses high resolution camera to detect insoluble particles in a flow
cell
• Captures numerous frames per second
• Software crops particles from each raw image and produces collage
• Results must be corrected using statistical filters to distinguish silicone
oil droplets and micro air bubbles

29. Circular Dichroism
• Measures difference in absorbance
left hand and right hand circularly
polarised light
• Far-UV range used for analysis of
secondary structure (200-260nm)
• Near-UV range used for analysis of
tertiary structure (260-350nm)
• Use computer program to estimate
ratios of secondary structures β-
sheets, α-helices etc.
• Compare spectra between time
points and also examine thermal
stability

30. SDS – Page (Elecrophoresis)
Native (intact mAb)
Tris Acetate 3-8% polyacrilamide gel
Better separation of high molecular weight
proteins (300-100kDa)
Coomassie Staining:
- Maximum loading of 0.5ug protein
Reducing (individual H & L chains)
Bis-Tris 10% polyacrilamide gel
Superior separation of mid-low molecular
weight proteins
Silver staining:
- Maximum loading of 1ng protein
Qualitative at best. Method of visualisation (stain) is very important.
150 kDa
50 kDa
25 kDa

31. Liquid Chromatography – Mass spectroscopy
Core sugar is primarily complex bi-antennary
type structure
Licensed recombinant mAb’s are generally
core fucosylated with low levels of
galactosylation
Outer arms have variable addition of fucose,
galactose, bisecting N-acetylglucosamine and
sialic acid on the antenna
Can directly measure the atomic mass of the protein (mixture)

32. Analytical
method
Principle Information/use Advantages/ Disadvantages
SEC Size Quantify mAb and relative purity + separation of main isoforms
- Limited mass resolution
SDS-PAGE Size Estimate purity and molecular mass + Cheap, fast
- Limited information
- Limited mass resolution
DLS Size Polydispersity, size distribution,
detection of high molecular weight
aggregates
+ Highly sensitive technique
- High polydispersity will affect accuracy
- Cannot resolve short oligomers
CD Shape / 3D
structure
Estimate ratios of secondary
structures, detect changes in
tertiary structure/ conformation
+ speed and ease of use
+ spectra can be obtained with small volume
and concentration of antibody
- Expensive instrument
- Diluents and excipients may show significant
absorption in far-UV
Microflow Size +
morphology
Count and characterisation of
aggregates
+ characterisation allows removal of air and oil
droplets
- Reliable shape information above 4µm
diameter
pH Hydrogen
potential
Hydrogen potential of solution + indicative of degradation processes
- Limited information
LC-MS Polarity + mass Charge to mass ratio, separation,
characterisation, quantification of
mAb isoforms
+Online desalting and fragment separation
+ highly detailed information
- Significant method optimization
- Expensive and requires trained analyst
Summary of Techniques

33. Potency
Receptor
Studies
Cell Line
Studies
Clinical
Effect

34. ELISA
- Receptor binding studies
- Allow one to measure changes
in binding ability of Fc and
variable region.
- DOES NOT tell us whether
this binding leads to a cellular
effect.
- Can be carried out quickly at a
range of temperatures and
concentrations.
- Real system???

35. Cell-based methods
- As we want the assay to be representative of clinical effect,
measurement of ultimate effect of the mAb is preferred.
- For Herceptin, we can directly measure cell death
- Other pathways higher up the cascade could also be monitored.
- Measure changes in pAkt
- Changes in cell proliferation

36. Application of stability data
Stability data can be used as a measure for QC, safety, or for
assignment of extended shelf-life.
We need to be certain the stability testing has:
• encompassed all aspects of degradation
o Chemical pathways, physical pathways
• demonstrated efficacy
• real time/ real temperature
• product specific
• specific for the manufacturing process used