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This document provides an outline of a presentation and is incomplete without the accompanying oral commentary and discussion. Conclusions and/ or potential strategies contained herein are NOT necessarily endorsed by Pfizer
management. Any implied strategy herein would be subject to management, regulatory and legal review and approval before implementation.
Leveraging Process and Product Understanding to
Develop and Implement a Holistic Control Strategy
Ranga Godavarti
WCBP, Washington DC
27 January, 2015
Presentation Outline
• Control Strategy Concepts
• Pfizer’s Lifecycle Approach to Control Strategy
• Case Study 1: Late Phase implementation of Control
Strategy for high molecular mass species CQA (HMMS)
– Focus on enhanced process understanding
• Case Study 2: ‘Fit-for-purpose’ or lifecycle implementation
of control strategy for HMMS
– Focus on enhanced product and process understanding
• Future Directions
• Conclusions
Pfizer Confidential │ 2
3
Comprehensive Approach to Control Strategy
Delineates Various Control Elements
‘Fit-for-purpose’/Lifecycle Approach to Control Strategy Continuum:
Control attributes consistent with level of knowledge and clinical stage
Pfizer Confidential │ 4
Discovery
• Molecular Assessments
• Select best candidate by engineering out high risk attributes
Early Clinical
Phase 1/2
• Identify potential CQAs based on prior knowledge, literature
• In-vitro/non-clinical studies for certain product variants
• Understand mechanism of action (MOA)
Post-POC/Ph 3
• Revise CQAs based on non-clinical, in-vivo studies as well as MOA understanding
• Develop control strategy for CQAs and non-CQAs
Launch/BLA Post-market
• Finalize DS
and DP control
strategy
• Testing and
monitoring plan
• Specs based
on clinical
exposure,
structure-
function and
non-clinical data
• Life cycle
management
Process Development
Drug Safety/Tox
Research Units
Process Development
Tech Services Manufacturing
Pfizer Confidential │ 5
Ele
men
t
Description
Drug Substance Drug Product
Via
l T
haw
Sh
ake
Fla
sks
& C
ult
ure
Bag
s
See
d B
iore
acto
rs
Pro
du
ctio
n F
ed-B
atch
Bio
reac
tor
Har
ves
t
Pro
tein
A
Lo
w p
H
TM
AE
HiC
ap
VR
F
UF
/DF
Fin
al F
iltr
atio
n
Fre
eze
Sto
rage
Tra
nsp
ort
Th
aw
Po
ol
Man
nit
ol A
dd
itio
n
Dil
uti
on
Ste
rile
Fil
ter
Ste
rile
Ho
ld
Fil
l &
Sto
pp
er
Cap
Insp
ect
Sto
re
1 Direct In-process Monitoring or
Control of Product QA None None
2 Monitoring or Control of PP or MA
Related to Product QA a a a b b
3 Direct In-process Monitoring or
Control of PPA N/A
4 Monitoring or Control of PP or MA
Related to PPA N/A
5 Drug Substance & Drug Product
Testing (Routine & Non-routine)c QA Controlled with Acceptance Criteria QA Controlled with Acceptance Criteria
6 Stability Testingc QA Controlled with Acceptance Criteria QA Controlled with Acceptance Criteria
7 Control of Raw Material Related to
QA or PPA None
8 Facility and Equipment Controls None
a Culture viability during inoculum train controlled to prevent sub-selection of cells which could lead to altered product quality. b Quality attribute controlled by process design of this unit operation with no specific PP or MA related to quality attribute. c Drug substance and drug product testing and stability monitoring only performed on the final material and not in-process.
QA: Quality Attribute; PP: Process Parameter; PPA: Process Performance Attribute; MA: Material Attribute; N/A: Not applicable
Areas that are in yellow indicate the controls in place.
Case Study 1: Initial Control Strategy for HMMS Relies on
Process Design, DS and DP Release and Stability Testing
Enhanced Process Understanding for Control of
HMMS: mAb Purification Process
Protein A
Clarified CHO Culture
Anion exchange
Virus filter
UF/DF
Capture column
Single polishing step (“weak partitioning mode”)
Assurance of viral safety
Buffer exchange and concentration
Weak Partitioning Chromatography: A high
capacity, high performance polishing option
Log Cl -
Lo
g K
p
Bind Flowthrough
Weak
Partitioning
• Pool collection - isocratic
• Binds 1-20 g/L of product
• Maximize purity with acceptable yield p C
Q K = =
[free]
[bound]
Total Cl-
pH
Kp >10
Kp 10
Kp 3.0
Kp 1.0
Kp 0.3
Kp
Enhanced Process Characterization Reveals HMMS
and Impurity Breakthrough on AEX step
Fraction Turbidity % HMMS HCP (LRV)
Load 28.1 3.5 -
1 4.9 0.9 >3.0
2 18.6 2.1 1.1
3 24.8 2.8 0.9
4 25.3 3.2 0.9
5 21.4 3.2 0.9
1200
1300
1400
1500
1600
1700
1800
mAU
55.0
60.0
65.0
70.0
75.0
80.0
mS/cm
500 1000 1500 2000 2500 ml
F2 F3 F4 F5 F6 F7
Impurity Breakthrough
1 2 3 4 5 Control Limit
(250 mg/mL)
HMMS breakthrough correlates to other impurities: HCP, DNA, virus
Impurity breakthrough also correlates to turbidity increase
Dynamic Light Scattering of AEX Load suggests
presence of large multimeric species
• ~25 nm multimeric
species present-
confirmed as protein
• Levels higher in early
Protein A (TMAE load)
cycles
• Higher levels result in
higher turbidity of
TMAE load
Larger species
~ 25 nm.
0
10
20
30
40
50
60
70
80
1 10 100
TMAE Load pools
DLS at 10 mg/mL
R(nm)
Cycle # 2
Cycle # 6
Cycle # 10
Scatt
ering I
nte
nsity (
%)
R (nm)
Pro A
Culture
AEX
Depth
filtration
Depth Filtration of AEX Load Provides
Significant Reduction of HMMS and Turbidity
• Depth filtration of AEX load
– Significant reduction in HMMS and turbidity
– Capacity was regained to >1000 mg/mL
AEX Pool Purity
Assay No-Depth Filter Post Depth Filter
Turbidity (NTU) 26 5
HMW (%) 4.3 1.0
HCP (LRV) <1.0 >3
Pfizer Confidential │ 11
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 10 20 30 40 50 60
HM
MS
NTU
HMMS Multimer (%)
HMMS Dimer (%)
Practically eliminated
After
Depth
Filter
Before Depth Filter of
Pro A Peak Pools
~ 2x reduction
Turbidity Measurement is a Surrogate for
multimeric HMMS: PAT to Control HMMS
• Reduction of turbidity (NTU)
post-depth filtration correlates to
reduction of HMMS
• PAT control:
• NTU measured on
manufacturing floor
• Results within 1 minute
• ~10 NTU correlates to < 0.2%
multimeric HMMS
• Two-fold reduction in dimeric
HMMS
• AEX step downstream
designed to provide additional
HMMS reduction
• If post-filtration NTUs >10, re-
filter
Pfizer Confidential │ 12
Ele
men
t
Description
Drug Substance Drug Product
Via
l T
haw
Sh
ake
Fla
sks
& C
ult
ure
Bag
s
See
d B
iore
acto
rs
Pro
du
ctio
n F
ed-B
atch
Bio
reac
tor
Har
ves
t
Pro
tein
A
Lo
w p
H
TM
AE
HiC
ap
VR
F
UF
/DF
Fin
al F
iltr
atio
n
Fre
eze
Sto
rage
Tra
nsp
ort
Th
aw
Po
ol
Man
nit
ol A
dd
itio
n
Dil
uti
on
Ste
rile
Fil
ter
Ste
rile
Ho
ld
Fil
l &
Sto
pp
er
Cap
Insp
ect
Sto
re
1 Direct In-process Monitoring or
Control of Product QA None None
2 Monitoring or Control of PP or MA
Related to Product QA a a a b b c
3 Direct In-process Monitoring or
Control of PPA N/A
4 Monitoring or Control of PP or MA
Related to PPA N/A
5 Drug Substance & Drug Product
Testing (Routine & Non-routine)d None QA Controlled with Acceptance Criteria
6 Stability Testingd None QA Controlled with Acceptance Criteria
7 Control of Raw Material Related to
QA or PPA None
8 Facility and Equipment Controls None
a Culture viability during inoculum train controlled to prevent sub-selection of cells which could lead to altered product quality. b Quality attribute controlled by process design of this unit operation with no specific PP or MA related to quality attribute. c In-process PAT control via turbidity testing of TMAE load. d Drug substance and drug product testing and stability monitoring only performed on the final material and not in-process.
QA: Quality Attribute; PP: Process Parameter; PPA: Process Performance Attribute; MA: Material Attribute; N/A: Not applicable
Areas that are in yellow indicate the controls in place.
Incorporating In-process PAT into Final Control Strategy
for HMMS can Enable Release Testing of DP only
Case Study 2: ‘Fit-for-purpose’ / Lifecycle
Approach to Control Strategy Implementation
• Background
– Typical monoclonal antibody
– Atypically high levels of high molecular mass species (HMMS) observed during early development
• HMMS Control Strategy – Early Clinical Phase
– ‘Force fit’ into mAb Platform process with significant yield loss and facility fit bottlenecks
• HMMS Control Strategy – Late Clinical Phase
– Increased titers; unexpected increase of HMMS at large scale
– Dramatic impact on process robustness, facility fit and overall yield/manufacturability
– Mechanistic investigation to understand the underlying cause of the HMMS issue
Early Stage Control Strategy for HMMS relies
on Low Yielding Platform Process
pH Inactivation
Harvest / Clarification
Centrifugation
Production Bioreactor
~ 1.6 g/L
11-24% HMMS
MabSelect Protein A
Chromatography
Planova 20 Viral Filtration
Viral clearance
≤ 200 L/m2
Ultrafiltration/Diafiltration
Final Concentration
AEX Weak Partitioning
≤ 60 mg/mL Loading
HMMS < 3%
Final Formulation
~100 mg/mL DS
HMMS < 3%
• Near platform process able to
remove high level of HMMS to
acceptable Phase I levels • 24% HMMS in harvest reduced to
< 3% in DS
• Acceptable process for Phase I
• Issues for Late Stage Process
• Low process recovery (~55%)
• Not suitable for commercial
manufacture due to low
productivity and lack of process
robustness
Late Stage Control Strategy: Reducing HMMS Level
at Harvest can Mitigate Downstream Bottlenecks
Theoretical process for high HMMS at harvest
Harvest/Clarification
MabSelect Protein A
AEX Chromatography
Polishing Column 2
~20-30% HMMS
10 cycles/batch
Theoretical HMMS 1 – 3%
Theoretical yield ~30 – 40%
Final Formulation
9 cycles/batch
Polishing steps:
Buffer volume: 3100 L/Kg
Processing time: 65 hr
Eliminate 2nd Polishing step
Theoretical process for low HMMS at harvest
Harvest/Clarification
MabSelect Protein A
AEX WP Chromatography
~2% HMMS
Theoretical HMMS <1%
Theoretical yield ~70 – 80%
Final Formulation
3 cycles/batch
Polishing step:
Buffer volume: 150 L/Kg
Processing time: 12 hr
Focus on Product Understanding: Reducing CGE
Showed Excess LC in the HMMS Enriched Sample
Sample Name % LC % HC %LC/%HC
Fully purified reference material 36.6% 63.4% 0.58
HMMS enriched sample 48.8% 51.2% 0.95
L Chain
L Chain H Chain
H Chain Fully purified reference material
HMMS enriched sample
Subunit analysis of HMMS Enriched Sample Revealed a
Significant Amount of LC with N-terminal Extension.
H Chain
AU
0.00
0.05
0.10
0.15
0.20
0.25
AU
0.00
0.05
0.10
0.15
Minutes
10.00 15.00 20.00 25.00 30.00 35.00 40.00
Fully purified reference material
HMMS enriched sample
L Chain
L Chain
H Chain
-19SVP…ARCcm-L Chain
-19Ac-SVP…ARCcm-L Chain
Full length signal peptide detected in ~50% of LC in HMMS enriched sample
Molecular design: Unexpected Involvement of a
Full Length Signal Peptide
• Based on historical experience, the finding of uncleaved full length
signal peptide 1 was unexpected.
• Could HMMS levels be lowered by using a different signal peptide?
Signal peptide 1 Framework 1
Framework 1
LC CDR1
LC CDR1 Signal peptide 1
-20
-20
1
1
24
24
34
34
mAb X
mAb Y
Expected
cleavage site
Identical aa sequence Identical aa sequence Different aa sequence
8 – 24% HMMS
~2% HMMS
Molecular Design: Clones with an Alternate Signal Peptide
Increased Expression by ~70% and Reduced HMMS to 2%
SEC-HPLC shows dramatically
reduced HMMS with alternate
signal peptide
Molecular and Process design results in an
efficient and elegant control strategy
• Robust process with consistent low levels of high molecular
weight aggregates
• Simpler process fit in manufacturing facilities; ‘greener process’
Pfizer Confidential │ 20
Process Initial Process
Final Commercial
Process
Buffer Usage (L/batch) 74000 6400
Buffer Usage (L/Kg product) 3100 150
Solvent usage (Ethanol) L/Kg
product 220 25
Conc. NaOH
(L/Kg product) 410 0
Disposable (filters/bags) waste
(Kg/Kg product) 14 1
Typical Current State mAb Release Spec
Future Directions: Continue to Develop and Implement
Technologies Enabling ‘Fit-for-Purpose’ Control Strategies
Can we get from here…….
Potential Future State mAb Release Spec
Key technology needs:
• Multi-attribute analytical release method
• Broad rapid bioburden technology
• Rapid adventitious viral test method
• Robust technologies BRX control
• In-line/at-line technologies
• Enhanced characterisation of inputs
To there…..?
mAb Enabling Trends & Opportunities – Online/At-Line - Multi-Attribute Testing, Statistical Models
34030627223820417013610268341
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Process Progress
La
cta
te (
g/
L)
NOVA
Raman
Time Series Plot of Lactate
Conclusions
• A comprehensive lifecycle approach to Control Strategy relies on parallel development of product, process and analytical knowledge
• Investment in fundamental product and process understanding and PAT tools can result in efficient control strategies that are ‘fit-for purpose’
• Understanding the mechanisms underlying mAb aggregation can lead to design solutions to control CQAs (i.e. HMMS)
• A robust control strategy is determined by an understanding of the manufacturing process and material attributes of the components in the product and confirmed by the appropriate specification criteria and analytical methods
Acknowledgments
Tim Iskra
Lucy Liu
Keith Johnson
Mary Switzer
Alice Ferguson
Dave Cirelli
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