Three analytical techniques are essential for effectively evaluating the stability of monoclonal antibodies (MABs): 1. Size-exclusion chromatography provides quantification of MAB monomers and aggregates as well as purity assessment. Validation is needed to establish linearity, accuracy, precision, and robustness. 2. Dynamic light scattering characterizes polydispersity and size distributions to detect aggregation. It can resolve oligomers and high molecular weight aggregates. 3. Microflow imaging counts and characterizes insoluble particles above 1um, 2um, and 10um, allowing separation of aggregates from air bubbles and oil droplets. Together these techniques provide a stability-indicating profile to monitor changes in primary,

1. MABs Stability – Analytical Techniques
Andrew G Watts PhD.
Medicinal Chemistry Group
Department of Pharmacy and Pharmacology
September 2012

2. Disclosure
Glythera Ltd.
Founder and Scientific Advisor
Bath ASU
Scientific Consultant

3. Key questions which need to be answered
Which analytical techniques do we need to use to effectively
evaluate the stability of MABs ?
What do we need to consider when interpreting the results from
these techniques?
What amount/type of degradation is significant/important?
- Acceptance criteria

4. 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 Proceedures and Acceptance criteria for
bioltechnological/biological products
Guidelines generally aimed at the licensing of new drug products.

5. Scope of Q5C
Q5c
-­‐
characterised
proteins
and
polypeptides
their
derivatives
and
products
of
which
they
are
components
and
which
are
isolated
from
tissues,
body
fluids,
cell
cultures,
or
produced
using
rDNA
technology
Ø cytokines
(interferons,
interleukins,
colony-­‐stimulating
factors,
tumour
necrosis
factors)
Ø erythropoietins
Ø plasminogen
activators
Ø blood
plasma
factors
Ø growth
hormones
and
growth
factors
Ø insulins
Ø monoclonal
antibodies
Ø vaccines
consisting
of
well-­‐characterised
proteins
or
polypeptides

6. ICH Q5C :
DOES NOT : Provide us with a ‘recipe’ for how to analyse or interpret
MAB stability
DOES : Outline criteria for a sound approach to designing protocols
for determining stability.
• Primary data to support a requested storage period, long-term, real-time
and real-condition stability studies
• Preferable NOT to use accelerated/stressed stability testing

7. 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 :
Primary structure
Identity
Chemical Secondary structure
Purity analysis Tertiary structure
Quaternary structure
Biological
Potency Cellular response
activity
Other characteristics
§ the determination of which tests should be included will be product-
specific

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

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

10. Aggregation
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.

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

12. SEC HPLC (Size-Exclusion)
Day
HMW-4-% HMW-3-% HMW-2-% HMW-1-% Monomer-%
A
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
1.76 ± 0.24
98.25 ± 0.24
B
0.00 ± 0.00
0.00 ± 0.00
0.21 ± 0.02
2.18 ± 0.10
97.61 ± 0.12
C
0.00 ± 0.00
0.00 ± 0.00
0.25 ± 0.02
2.40 ± 0.06
97.36 ± 0.09
D
0.00 ± 0.00
0.00 ± 0.00
0.18 ± 0.11
2.14 ± 0.41
97.68 ± 0.52
E
0.00 ± 0.00
0.00 ± 0.00
0.21 ± 0.01
2.25 ± 0.03
97.51 ± 0.05
F
0.00 ± 0.00
0.00 ± 0.00
0.19 ± 0.01
2.27 ± 0.02
97.54 ± 0.02
G
0.00 ± 0.00
0.00 ± 0.00
0.23 ± 0.01
2.34 ± 0.06
97.44 ± 0.07
H
0.00 ± 0.00
0.00 ± 0.00
0.22 ± 0.01
2.30 ± 0.03
97.48 ± 0.04
I
0.18 ± 0.36
0.17 ± 0.01
0.82 ± 0.04
1.39 ± 0.08
97.46 ± 0.34
J
0.00 ± 0.00
0.21 ± 0.01
1.00 ± 0.04
1.54 ± 0.07
97.25 ± 0.08
Linearity of response and LOD, LOQ
are crucial.
Low molecular weight aggregates
can be reversible
- constant temperature

13. 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 monomer aggregate
various size classes.
Oligomeric High MW Oligomeric High MW
Z-Ave Species Aggregates Species Aggregates
(r.nm)
PdI
%PD
mean r.nm
mean r.nm
(%Volume)
(% Volume)
Day A
7.98
0.07
27.33
8.67
0.00
100.00
0.00
Day D
8.27
0.10
31.56
8.75
2584.67
100.00
0.00
Day C
8.03
0.07
25.84
8.69
0.00
100.00
0.00
Day D
8.05
0.08
28.27
8.79
0.00
100.00
0.00
Day E
7.99
0.05
23.15
8.53
0.00
100.00
0.00
Day F
8.02
0.07
26.12
8.67
0.00
100.00
0.00
Day G
8.87
0.17
41.58
9.63
2323.33
99.98
0.02
Day H
7.97
0.05
22.53
8.50
0.00
100.00
0.00
Day I
7.95
0.05
21.93
8.49
0.00
100.00
0.00
Day J
8.09
0.08
27.97
8.79
0.00
100.00
0.00

14. Microflow Imaging (Particle counting)
aggregated protein
silicone oil
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 micro-air bubble
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

15. SDS – Page (Elecrophoresis)
Native
Tris Acetate 3-8% polyacrilamide gel
Better separation of high molecular
weight proteins (300-100kDa)
Coomassie Staining
Maximum loading of 0.5ug protein
Reducing
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 is very important.

16. Liquid Chromatography – Mass spectroscopy
Core heptasaccharide is primarily complex
biantennary type structure
G0 = zero galactose residues
Licensenced recombinant MAbs generally core
G1 = one galactose residue
fucosylated with low levels of galactosylation
G2 = two galactose residues
Outer arms have variable addition of fucose
F denotes fucosylation
galactose, bisecting N-acetyleglucosamine sialic acid
on the antenna

17. Circular Dichroism
• Measures difference in absorbance left hand and
right hand circularly polarised light
• Molecules must be chiral
• 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

18. Variable Temperature Circular Dichroism
Can measure changes in secondary structure as
a function of changing temperature
Can identify the melting temperature of a MAB,
which give information as to the integrity of
secondary and tertiary structure. Ie. Can identify
the IMPACT of chemical changes
Temperature
25°C
37.5°C
50°C
62.5°C
75°C
α-helix
5.3
5.4
5.5
5.5
6.6
Antiparallel
40.1
39.9
39.7
39.4
36.4
Parallel
5.4
5.4
5.4
5.4
5.5
Beta-turn
17.7
17.7
17.7
17.7
18.1
Random Coil
34.8
34.8
34.8
34.8
34.9

19. Other
pH
Visual appearance
Colour
Turbidity

20. Potency
Receptor Cell Line Clinical
Studies Studies Effect

21. ICH Q6B
ELISA

22. 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???

23. Cell based activity studies
- Want to ensure that results of the assay are representative.
- Parameters that need to be identified/optimized.
- Cell lines: Need cells which express receptor specific to the MAB
- Cell numbers, Culture time, Passages
- Correct MAB concentration to see a change
- What cellular response are we looking for. Ie. Apoptosis
- Validation is essential. But no instructions on how to do this, but what are
some key considerations?

24. Which effect ? Trastuzumab
- As we want the assay to be representative of clinical effect,
measurement of ultimate effect of the MAB is preffered.
- Cell death
- Other pathways higher up the cascade could also be monitored.
- pAkt
- Cellular proliferation

25. Which effect? Alemtuzumab
- Multiple mechanisms of action contribute to clinical efficacy
- More than one mechanism should be tested.

26. Establishing an end point
Conc 1
Conc 2
Conc 3
Base line
Baseline
Days
- Determination of a baseline
- Comparison to baseline over time
- Meaningful comparison requires high level of confidence in the data
- Reducing standard deviation between points is key.

27. Thank you !
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