Screening approaches for product quality to enable ... · Case study 3 –MEDI-FP2 summary....
Transcript of Screening approaches for product quality to enable ... · Case study 3 –MEDI-FP2 summary....
Screening approaches for
product quality to enable
attribute-driven cell line
development with an eye
towards commercialization
23rd January 2017CMC Strategy Forum
Chris Sellick
Senior Scientist
Cell Culture and Fermentation Sciences
TiterProduct
quality
• Over the past six years the biologics pipeline has tripled in size and now
comprises about 50% of AstraZeneca’s pipeline
The changing pipeline
Non-mAb formats have
increased from 20% to 50%
of the Research Portfolio
utilising up to 13 different
technologies/formats
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No
n-m
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2010 2011 2012 2013 2014 2015
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Combined Functional and Developability Focus
• Molecular design with functional focus & developability as a
fundamental input
Developability
Target
Profile
Functional
Target
Profile
Target
Molecule
Physiochemical properties
Manufacturability
Stability and integrity
PK/PD
Bioactivity
Specificity
Species cross-reactivity
Pre-Clinical/
Clinical
Launch &
PLC/LCMDiscovery
Pre-Clinical
DevelopmentEarly
Development
• Product knowledge sharing between Research and BPD- Structure/Function relationships- Mechanism of Action- Developability
• “What are the quality attributes we need to get right?”
• “The product defines the process and required analytics”
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Project R&D Interface: A Critical Change
Research CMCResearch CMC
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Attribute Focused Analytical Development
Define initial pCQAs
Evaluate methods for each
pCQA
Implement method to support
CLD and process development
Define method purpose &
requirements for each pCQA
Gain Early Product Knowledge
from R&D Interface &
Developability
Identify likely cell line /process-
dependent attributes
DevelopmentResearch
Overview of Cell Line Development (CLD)
Launch &
PLC/LCMDiscovery
Pre-Clinical
DevelopmentEarly
DevelopmentPre-Clinical/
Clinical
CLD Project Start
Vector
Construction
Creation of Pools and
Parental Cell Lines
Clonal Cell Line
Isolation
Cell Line
Characterisation
CLD generates clonal cell lines to produce product suitable for GMP manufacture
~20 weeks
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Current CLD platform
Shaking 24 well-plate
overgrows
BULK
Expression
vector
CHO Host
Cell Line
AMBR
Research Cell Banks (RCB) for
top cell lines
Titre screening to select the best producing cell lines
ClonePix
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Requirements for process analytics
Process Stage
Early multi well screen
AMBR
Number of
vessels
200 to
400
1 to 8
24 to 48
Method
Requirements
• Analysis in cell
culture media
• Sample volume
<0.5ml
• Concentration
potentially
<1mg/ml
• Rapid
turnaround
3L to 500L Bioreactor
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Case study 1 – Changing a cell line to deliver improved yield
• Case study
– In-licensed human IgG2 monoclonal antibody
– Original cell line produced 0.9 ± 0.1 g/L average titer at 2000 L scale for
Phase I and II studies
– For commercial considerations a new cell line was required
Attribute Assay Criteria
Titre Protein A HPLC ≥ 4 g/L in a 14-day fed-batch process
Phenotypic Stability Protein A HPLC ≤ 30 % drop in titer between 40-60 PDL from RCB
Charge variants IECWithin target range (64% - 84%)
Main peak within historical range
GlycosylationOligosaccharide
profile
Man5/6 levels within historical range
Similar profile to Ref Std, no new peaks
Disulfides Peptide map Similar profile to Ref Std
Purity HPSEC ≥ 95.0%
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Case study 1 – Cell line development
Static
Top 62 cell lines
Shaking
Top 9 cell lines
Top 9 Clones 3-7 fold titre improvement
Historiccell line
AMBR
Analysis of product from
the cell lines to assess:
• Purity
• Charge variants
• Glycosylation
• Disulphide profiles
• Phenotypic stability
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Case study 1 – Cell line and product attributes
HPSEC
Gal:Agal ratio within 10% of Ref Std
Mannose ≤1SD from Ref Std
Glycans
Clone ID % Pre-peak% Main
peak
% Post-
peak
In
Spec?Main peak
SD range
Ref Std 20.4 72.0 8.3 1
F 16.3 71.7 12.0 1-2
G 18.2 71.4 10.5 1-2
E 20.4 69.1 10.5 2-3
H 19.7 67.5 12.8 2-3
I 20.5 66.7 12.8 2-3
C 22.4 64.7 13.0 >3
D 17.9 65.7 16.5 >3
A 21.3 63.4 15.3 X >3
B 18.4 63.2 18.3 X >3
IEC
64.0% to 84.0% main peak
≥ 95% monomer
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Case study 1 – Summary of cell line performance
• Cell line G chosen based on:
– Product quality attributes most comparable to reference standard
– Titre and phenotypic stability meet acceptance criteria
CloneIEC
(% main peak)
Oligo(% high
mannose)
Disulfide profiles
Purity (%
monomer)
40 PDL Titer (g/L)
Phenotypic Stability Loss
(%)
Target range 64 - 84 2.3 - 4.0 Similar ≥ 95 ≥ 4 ≤ 30
Reference 72.0 2.3 N/A 98.9 ≤ 0.8 N/A
G 71.4 2.8 Similar 98.8 6.0 11
E 69.1 7.6 Similar 98.5 5.9 15
H 67.5 2.8 Similar 98.8 6.5 8
I 66.7 3.9 Similar 98.7 6.6 46
C 64.7 4.3 Similar 98.8 5.4 6
A 63.4 3.4 Similar 97.8 4.5 29
D 65.7 7.0 Similar 98.7 NT NT
F 71.7 5.3 Similar 99.1 NT NT
B 63.2 5.4 Similar 98.2 NT NT
Case study 1 – Summary
• New clonal cell line has comparable product attributes
– Used in-house expression vectors and CHOK1SV host
– New platform process (feed and media formulation changes)
– Achieved 6 g/L in AMBR and subsequently at 5 L & 50 L scale
• Comparable product quality
– This was achieved through the use of micro-scale bioreactor and cell line
product evaluation at critical point in the platform process
– Integrated analytics and cross-functional working was key to the successful
switch to a new cell line
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Case study 2 – MEDI-FP1
• Highly glycosylated Fc-fusion protein
• Molecule characteristics
– Potential fragmentation (LMWS) and aggregation (HMWS)
– Contains sialylated glycans
• N- and O-glycans
• Sialylation crucial for half life of molecule
• Aim
– Minimise LMWS and HMWS
– Achieve consistent glycosylation with high sialylation
– Understand the effects of cell line and process on molecule
characteristics
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Case study 2 – Understanding the impact of cell line
and process on the product
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0
200
400
600
800
1000
1200
1400
Cell Line 1 Cell Line 2 Cell Line 3 Cell Line 4 Cell Line 5
Tit
re (
mg
/L)
Media A Media B Media B +Temp Shift
• Productivity dependent on
cell line
• Cell lines vary significantly
in their response to media
and feed
• Productivity only part of the
story, product quality needs
to be controlled
Assay
requirements Titre Sialylation Fragmentation Aggregation
Assay Octet ??? SDS-CE (GXII) UPLC-SEC
HT
Process
samples
Low volume
Case Study 2 – Analytical requirements and method
development
• Can methods be developed for high throughput measurement of molecule
sialylation?
• Assays need to be in place before the start of the CLD campaign
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Case study 2 – Octet-based lectin assay to screen
sialylated glycans
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Biosensor
Biosensor Biosensor
Biosensor Biosensor
Biosensor
Biosensor Biosensor
Biosensor
Case study 2 – Octet-based lectin assay to screen
sialylated glycans
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Lectin Solution
Assay
requirements Titre Sialylation Fragmentation Aggregation
Assay Octet ??? SDS-CE (GXII) UPLC-SEC
HT
Process
samples
Low volume
Case Study 2 – Analytical requirements and method
development
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Assay
requirements Titre Sialylation Fragmentation Aggregation
Assay OctetOctet-based
lectin assaySDS-CE (GXII) UPLC-SEC
HT
Process
samples
Low volume
Case Study 2 – Analytical requirements and method
development
• Process analytics in place before the start of the CLD campaign
– Allows informed attribute-led decisions to be made
• How and when should they be integrated into the platform process?
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Tailored CLD campaign
Shaking 24 well-plate
overgrows
BULK
ambr
Research Cell Banks (RCB) for
top cell lines
ClonePix
Titre screening to select high producing cell lines
MAL I/II binding assay to select optimal N- and O-glycan sialylation profiles
Non-reduced GXII to minimise fragmentation
UPLC-SEC to select the highest monomer purity21
Expression
vector
CHO Host
Cell Line
Case study 2 – PQ screening during CLD
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Terminal sialic acid
Aggregation
Fragmentation
• Early interactions between Research and Development ensured potential
critical quality attributes (pCQAs) were identified
• Early development of analytics to monitor the pCQAs enabled them to be
used as an input for cell line development and process optimisation
• Process analytics were critical for controlling the product quality from
early material supply to toxicology batches Enabled timely decision making
• Defining the pCQAs early in the project lifecycle was essential for the
rapid progression of this project Process development was completed 3 months ahead of schedule
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Case study 2 – MEDI-FP1 summary
Case study 3 – MEDI-FP2
• Large multimeric Fc-fusion protein
• Molecule characteristics
– Requires correct assembly to form multimer
– Potential fragmentation (LMWS) and aggregation (HMWS)
– Fc glycans and an additional glycosylation site
• N-glycans
• Aim
– Confirm correct assembly
– Minimise LMWS and HMWS
– Achieve consistent glycosylation with low Man5
– Determine if additional glycosylation site is required
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Assay
requirements Titre
Oligomeric
state Aggregation Mannose 5
Assay Octet UPLC-SEC UPLC-SEC
Glycan
analysis
(GXII)
HT
Process
samples
Purified
samples
Low volume
Analytical requirements and method development
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Case study 3 – Oligomeric assembly and aggregation
• UPLC-SEC on crude supernatant from different clones in AMBR
– Different amounts of aggregation
– Varying amounts of HCP’s
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Correct
assembly
Minus 2
subunitsHMWS
48 cell lines screened
Case study 3 – High throughput purification
• Atoll columns on a Tecan robot
• Protein A purification of up to 96 samples at a time
• Samples used directly for glycan analysis
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Case study 3 – Fc glycan screening
• Cell line screening in AMBR system
• Three clones from one parent show high (>95%) Man5
– None of the clones from this parent were progressed
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• Tailored analytics that monitor glycosylation, oligomeric state and
aggregation were used to support attribute-led cell line selection
• High Man5 cell lines were removed early in the process
• Selected cell lines produced high % of hexameric product
• Early implementation of attribute focused screening methods allowed for
accelerated project delivery
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Case study 3 – MEDI-FP2 summary
Conclusion
• Understanding the potential critical quality attributes early allows optimal
cell lines and processes to be selected
– CLD and PD with an eye towards commercialization
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Late Stage
DevelopmentProcess
Development
Cell Line
DevelopmentResearch
Likelihood of optimal
Developability characteristics
ResourceTime
CostMonitor pCQAs
Developability
assessment
Control of
pCQAs
Streamlined late
stage development
Develop HT
analytics
Define pCQAs
Attribute-based
cell line selection
Commercialization
Acknowledgements
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• BioProcess Analytics
• Cross-functional Analytical Development (CAD) team
• Research
• Cell Line Development
• Cell Culture and Fermentation Sciences
• Purification Process Sciences
• Analytical Biotechnology
Questions?
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