Assay Improvement for Protein Therapeutics at Clinical Development Stage

Assay Improvement for Protein
Therapeutics at Clinical
Development Stage
BPI China 2015 Conference - August 12, 2015
Weijun Li
Global Biological Development

Disclaimer
• The contents in this presentation are based on presenter’s
personal opinions and experiences. The statements and
approaches do not represent the official position of Bayer
Pharmaceuticals. The actual results may differ materially
from the contents of the presentation due to various
factors. The presenter and Bayer have no obligation to
update the contents contained in this presentation.

Overview
Dynamic assay development and continuing assay
improvement from early to late stage development
Focused on GMP routine release/stability assay
development
Bayer Berkeley site – where are we?
Phase-dependent assay development
Causes for assay improvements
Risk based assay improvements (optimization and
changes) and relevant regulatory consideration
Case studies with HPLC assay development

• Development Principles in GBD • November 2014

Biological Products Supported by
Bayer R&D in Berkeley
Recombinant
Factor VIII for the
treatment of
hemophilia A
First company to
offer a treatment
option for Multiple
Sclerosis- an
autoimmune
disease
Treatment for wet
age-related macular
degeneration
(AMD). Owned by
Bayer and
Regeneron

Biological CMC Analytical Development
Preclinical Phase I
Clinical
Phase II
Clinical
Phase III
Clinical
BLA
/MAA
Product
Launch
Release assays
Method
Development
Supporting early
stage clinical
trial
Method
Validation
Release and Stability testing
Extended characterization / Comparability
GLP Tox Testing
Supporting late
stage clinical
trial
Method
Validation
Support Process Development
Dev Testing
Release and Stability testing /
Extended Characterization / Comparability
GMP Testing
Assay
Improvement if
needed
Method
Development
Full validation per
ICH Q2
Method Transfer to
commercial
production
Method
Validation
Process early development, process change, process comparability study, and process transfer
Process Development in Parallel with Analytical Development
Post-approval Change
Method Transfer to different
sites
Method Life Cycle
Management

Evolvement from Platform Assays to
Product Specific Assays
• Benefit of applying platform assays in early stage
development: fast to the clinical trial and cost-effective
• Similarities between the products (e.g., monoclonal
antibodies) and existing experiences justify the use of
platform assays at early development stage:
- Size distribution: SEC-HPLC
- Impurity testing: Host Cell DNA, HCP, residual Protein A
- Purity: SDS-CGE, RP-HPLC
- Glycosylation: Oligosaccharide mapping
- Charge Variants: CIEF, CEX-HPLC, CZE
• Assay improvement towards late stage development
needed

Factors Triggering the Assay
Improvement
Cause for Assay
Improvement
Subclasses Example Cases
Supplier issue • Supplier quality change
• Product or service
discontinuation
• Assay reagent/kit
• Agilent HPLC models 1100 vs.
1260
Better
technology
• Using modern technology in
accordance with current GMP
• SDS-PAGE vs. SDS-CGE
• HPLC vs. UHPLC
Process change • Different API profile or strength
• Different impurity level
• Different sample matrix
• Change of cell line
• Change of purification process
• Change of product formulation
Assay
performance
issue
• Robustness issue / high invalid
rates
• User friendly feature
• Failing the system suitability
• Portable NIR vs. FTIR
Regulatory
consideration
• Product specific assays at late
stage
• HCP platform Ab vs. cell line
specific Ab

Assay Improvement:
Optimization vs. Change
• Change to report a different CQA
• Change to a different methodology
• Change to nonequivalent critical reagents
/columns
• Revise critical assay conditions
• Revise critical data analysis procedure
• Revise system suitability or control
parameters
• Improve instrument maintenance
• Revise noncritical assay conditions
• Equivalent instrument models
• Noncritical reagents
• Administrative changes
Assay Change
(High Risk)
Assay
Optimization
(Low Risk)
RiskAssessment

Assay Change: FDA Requirement
21 CFR 610.9(a) requires that the applicant should
present evidence in the license application, or a
supplement to the application “…demonstrating
that the modification will provide assurances of the
safety, purity, potency, and effectiveness of the
biological product equal to or greater than the
assurances provided by the method or process
specified in the general standards or additional
standards for the biological product.”

EMA/CHMP/BWP/534898/2008 (Biological)

CT-1 refers to this guidance for IMPD amendment:
CHMP/QWP/185401/2004 final (Drug)
• Presentation Title • Date
Substantial assay change

Assay Improvement-Planning Stage
• Review the target assay profile (TAP) based on the
target product profile (TPP), critical quality attributes
(CQA) and critical process parameters (CPP)
• Stability indicating vs. non-stability indicating
assays
• Regulatory requirements for commercial assay
change may apply to the clinical products in principle
• Impact on the process development data package

• Experiences or lessons learned from the old assay
• Assay robustness
• Use diverse materials representing the process
variation
• For stability-indicating assays, plan early
evaluation on the actual stability samples (including
accelerated and stressed conditions) in addition to
forced degradation materials
Assay Improvement- Development Stage

Assay Improvement Case Study:
Protein SEC and RP-HPLC

Points to Consider in Development of
Size Exclusion Chromatography (SEC)
 SEC: High molecular weight
or low molecular weight
impurities
 Stability-indicating
 Tailing: protein secondary
interaction with column
 Excipient interference
 Sample stability
 Column robustness
 Peak identification

Points to Consider in Development
of Reversed Phase HPLC (RP-HPLC)
 RP-HPLC: Product-related
impurities
 Stability-indicating
 Gradient design
 Sample stability-less a
concern
 Column robustness
 HPLC models
 Carry-over issues
 Peak identification

Early Version of Monoclonal Antibody (mAb)
SEC Assay
Robustness issues:
The separation with Column S does not show sufficient robustness in long
term use. The aggregate level is below control limit due to the variability in
column performance (tailing issue) and secondary interaction in failing run.
Passing run Failing run

Optimize the mAb SEC Mobile Phase
with Different Composition
 mAb Product
#1 separation
with different
Mobile
Phases.
Retention time
were adjusted
for comparison
min7 8 9 10 11 12 13 14 15 16
mAU
0
5
10
15
20
25
DAD1B,Sig=280,8Ref=330,8(CV031814BCV031814B2014-03-1814-46-44062-0120.D)
*DAD1B,Sig=280,8Ref=330,8(CV031914ACV031914A2014-03-1916-13-41072-0108.D)
min2.5 5 7.5 10 12.5 15 17.5
mAU
0
100
200
300
400
DAD1B,Sig=280,8Ref=330,8(CV031914ACV031914A2014-03-1916-13-41072-0108.D)
Buffer A
Buffer B
Buffer C
Buffer D
Tailing
Column T1
min7 8 9 10 11 12 13 14
mAU
0
5
10
15
20
min2.5 5 7.5 10 12.5 15 17.5
mAU
0
100
200
300
400
500
600
Buffer A
Buffer B
Buffer C
Buffer D
Column T2
Tailing

Optimize the mAb SEC Mobile Phase
with Different Salt Concentration
 mAb Product #2 separation with different NaCl concentration in the
Mobile Phase. Retention time were adjusted for comparison purposes
min8 9 10 11 12 13 14
mAU
0
2
4
6
8
min8.5 9 9.5 10 10.5 11 11.5 12 12.5
mAU
0
100
200
300
400
500
600
Mobile Phase w/o NaCl
w/ NaCl (Level A)
w/ NaCl (Level B)
w/ NaCl (Level C)
w/ NaCl (Level D)
w/ NaCl (Level E)
w/ NaCl (Level F)
Tailing

Reference: Human IgG2 antibodies display disulfide-mediated structural isoforms.
Wypych et al., J. Biol. Chem. 2008, 283(23):16194-16205
Typical mAb IgG2 Isoforms
• IgG2-A independent Fab domains and hinge region
• IgG2-B symmetrical arrangement of covalently linked complex between
both Fab regions and the hinge.
• IgG2-A/B intermediate form with asymmetrical covalent linkage of one Fab
arm to the hinge

Early Version of IgG2 Isoform RP-HPLC assay
0 5 10 15 20 2 5 30
LU
0
5
10
15
20
25
F LD 1 A, Ex= 276, E m=3 40 ( RV0 5071 4A 201 4-0 5-0 7 13 -32 -33 043- 06 01.D )
A
rea
:96.78
67
A
rea:
1089.6
A
rea:
1012
.45
A
rea
:133
.703
A
rea:15
3.279
4.964
5.482
6.682
7.700
9.320
Robustness issues:
The separation window is within 2% of mobile phase B change over 20 min
(0.1%B change/min). Gradient is too shallow!
0 5 10 15
LU
0
5
10
15
20
25
F LD 1 A, Ex= 276, E m=3 40 ( RV0 5281 4A 201 4-0 5-2 8 14 -34 -57 033- 08 01.D )
Passing run Failing run

Early Scouting in New Assay Development
• Target assay profile (TAP):
A RP-HPLC assay to provide consistent IgG2 isoform profiles with a robust gradient
at no less than 0.5 % mobile phase B change / minute.
• Example assay parameters to be evaluated:
Parameters Range of evaluation Comment
Column type
robustness
• Column A, B and C • Three types of RP-HPLC columns evaluated
Injection
volume at 5
mg/mL
• 1 mL, 2 mL, 5 mL • High injection volume may increase the signal/noise ratio,
however, may compromise the resolution due to the peak
broadening
Flow rate • 0.5 – 1.0 mL/min • Slow flow may allow better partition between the analyte
and stationary phase, while fast flow may minimize the
mass transfer limit for large molecules
In-line filter/
guard column
• With or without • In-line filter /guard column ensures better column life,
however, may compromise the resolution
Sample dilution
buffer
• Product formulation
buffer or mobile
phase
• Mobile phase A may reduce the potential miscibility issue
on the columns, however, the analyte may be less stable
in it than formulation buffer

Final Optimization of the RP-HPLC Gradient
Side by side comparison study to optimize the gradients and mobile phases
• %B change/min in 6 gradient conditions:
A, 1.1%,  B, 1.0%,  C, 0.9%,  D, 0.8%,  E, 0.9%
• Test on 3 different column lots to confirm the robustness
Acquisition Methods
RP_Isoform_A RP_Isoform_B RP_Isoform_C RP_Isoform_D RP_Isoform_E
Time
(min)
% B
Time
(min)
% B
Time
(min)
% B
Time
(min)
% B
Time
(min)
% B
0.00 13.0 0.00 13.0 0.00 13.0 0.00 13.0 0.00 11.0
2.00 13.0 2.00 13.0 2.00 13.0 2.00 13.0 2.00 11.0
19.00 32.0 21.00 32.0 23.00 32.0 26.00 32.0 25.00 32.0
21.00 100.0 23.00 100.0 25.00 100.0 28.00 100.0 27.00 100.0
26.00 100.0 28.00 100.0 30.00 100.0 33.00 100.0 32.00 100.0
27.00 13.0 30.00 13.0 32.00 13.0 35.00 13.0 34.00 11.0
42.00 13.0 45.00 13.0 47.00 13.0 50.00 13.0 49.00 11.0

 Method “D” showed the best profiles between columns, with RT shift of ~0.4
min. Within the same run, there is no significant shift of retention time
Column Lot 1
Column Lot 2
Column Lot 3

 The protein load per injection, not the injection volume, causes the
retention time shift in the Isoform RP-HPLC
Effect of Protein Load on Retention Time

 Is the improvement considered as high risk improvement (assay
change) or low risk (assay optimization)?
 Does the new assay report new product/process-related impurities or
new active substances?
 Purpose of assay comparability study (if needed)
- Assay Comparability Study ≠ Assay Equivalency Study
- Is the new method equivalent, superior or non-inferior to the old
method?
- If stability-indicating, does the new method have the same trend of
stability as the old method?
Assay Improvement-Implementation Stage

 Assay Comparability Study Plan
- Assay technical comparison (intended use, assay design)
- Assay validation (better use the same sample lots by both assays for
comparison)
- Test sufficient range of product lots to cover possible process variation
- Stability sample testing needed (if stability-indicating)
 Example statistical tool for assay comparison
- Two one sided t-test (TOST) for homogenous samples
- Paired t-test for heterogeneous samples
 The effect of assay change on the product specification and existing
process development data package (small scale validation, design
space, column life cycle studies, etc.)
Assay Improvement-Implementation Stage

Summary
Assay improvement is common in biological product
development. Assay developers build up understanding of
assay performance and product quality attributes as
projects advance from early stage to late stage
development and product launch.
The approach for assay change should be product-
specific and phase-appropriate given the complexity of
biological product development.

References
• FDA Guidance (2015): Analytical Procedures and Methods Validation
for Drugs and Biologics
• ICH Q2 (R1): Validation of Analytical Procedures
• FDA Draft Guidance (2003): Comparability Protocols -Protein Drug
Products and Biological Products - Chemistry, Manufacturing, and
Controls Information
• ICH Q5E: Comparability of Biotechnological/Biological products Subject
to Changes in Their Manufacturing Process
• F. Diana et al., “Analytical Method Comparability in Registration and
Post-Approval Stages: A Risk-Based Approach”, Pharmaceutical
Technology, 2014, Volume 38, Issue 10

Acknowledgement
 Chemistry and Protein Chemistry Assay Development
Carolina Vega, Roselle Visaya, Quan Yuan, Susan Chen,
Xue-Min Zhou, Rich Ohnmeiss
 Biomolecular Interaction Assay Development
Richard Hatch, Eric Helgeson
 Analytical Development
Lisa Regan
 QC Protein Characterization
Patrick Hillas

Assay Improvement for Protein Therapeutics at Clinical Development Stage

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Assay Improvement for Protein Therapeutics at Clinical Development Stage

Similar to Assay Improvement for Protein Therapeutics at Clinical Development Stage (20)

Recently uploaded

Recently uploaded (20)

Assay Improvement for Protein Therapeutics at Clinical Development Stage