Growth Factor Production by Creutzfeldt-Jakob Disease Cell Lines
Genetic Characterization of Production Cell Lines · Production Cell Lines 2017 CMC Strategy Forum....
Transcript of Genetic Characterization of Production Cell Lines · Production Cell Lines 2017 CMC Strategy Forum....
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Luhong He and Christopher Frye
Genetic Characterization of
Production Cell Lines
2017 CMC Strategy Forum
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Outlines
Overview
Genetic characterization strategy and case studies
•Transgene characterization for registration
•Transgene analysis during CLG
•Engineered host-target characterization
Looking forward – emerging technologies
Summary
Acknowledgements
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CHO Production Cell Lines Are
Populations of Cells
Production cell lines are clonally-derived
populations of cells
Product quality and manufacturing consistency
Absolute genetic homogeneity is not achievable!
•Transgenes • Extensively engineered
• Highly expressed
•CHO host • Genomic plasticity
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Minimize Potential Risk through
Characterization
Transgene analysis during CLG
Transgene characterizationfor registration (ICH Q5B)
Risk-based phase-appropriate characterization
CS FHD FRD FL
Characterization by ICH Q5B
Risk assessment
Minimize risks by screening
CLG
Engineered host-target characterization (no regulatory guidance)
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Transgene Characterization for
Registration
Demonstrate genetic stability in commercial process
MCB and End of Production Cells (EOPC) at limit for in vitro cell age
• In vitro cell age: critical in-process control parameter
• Not necessary to test genetic stability on WCBs if within the “window”
Characterization• Coding gene sequence confirmation by RT-PCR sequencing
• Gross structural integrity by Southern blot
• Transgene copy number analysis by qPCR
EOPC
~G60
MCB
G0
WCB
expansion production
extra passages
Manufacturing “window”
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Transgene Analysis during CLG:
Genetic Suitability
Primary goal is to eliminate cell lines showing significant population drift
• to avoid cell line change
Transgene population
characterization
Clonally-derived candidate cell lines
Population drift over extended culturing
Traditional assessment
phenotypic productivity
Titer @ different generations
0-20 -10 0 10 20 30 40 50 60 70
Generation number
Tit
er
Process A
Process B
Process C
Process D
Process E
Process F
Process F
Process F
Titer drop 7% ~ 60% depending on processes
Krebs, Gao & Frye, 2015, Bioprocessing J. 13(4):6-19
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Population Drift Post-Single Cell Cloning
Detected by sc-qPCR
Unsuitable cell lines were observed independent of transgene types
He, Winterrowd, Kadura & Frye, 2012, Biotechnol. Bioeng. 109(7):1713-22
Single cell qPCR
•48 single cells per condition
•Directly lysed, qPCR
•Statistical analysis of
transgene populations
Orthogonal methods: Intra-cellular staining /sc-RT-PCR
Cell line
genetic suitability
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Relatively Homogeneous Populations
Identified by Screening
~80% of candidate cell lines are suitable.
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Transgene Integrity Evaluation
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Transcription
mRNA
Protein
DNA
Translation
5’ leader Coding 3’ UTR
Mutation
Cryptic aberrant splicing
Sequence variants
Replication
Transcription
Mis-incorporation
Product-related impurities
Orthogonal evaluations by nucleic acid and protein characterization are needed to ensure DS integrity.
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Examples: intron reading through; in-frame/out-frame
Impacted molecules: LC/HC, bispecific, protein
Level: very low ~ 50%; ratio impacted by conditions
Detection: RT-PCR, Northern, LC/MS
Cryptic Aberrant mRNA Splicing
A…AAG…ACTCTC
A…AAG…ACCCTC
G…AAG…ACTCTG
..GGTGAAG
..GGTGAGG
..GGTGGGT
Donor region Acceptor region
None
150 bp “intron”
103 bp “intron”
Aberrant splice
The available splice-site recognition programs were unable
to identify those aberrant splice sites utilized in CHO cells.
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Culture conditions can impact the ratio of aberrant spliced vs full length
mRNA. It may impact the robustness of purification strategy.
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Cryptic Aberrant mRNA Splicing
Aberrant splice sites need to be “fixed” to ensure
robustness of DS manufacturing process.
Phase I Cell line Phase II / Commercial Cell Line
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Host-target Characterization
Objective of host-target engineering
• Improving process (business)
• GS-KO, stringent selection
• Landing pad for target integration
• Improving PQ (business /regulatory)
• Impurity, immunogenic HCP
• product stability
Characterization strategy
•Cell line generation
•Genetic/phenotypic package for both
•Further characterization
•PQ-related targets only
•Sanger sequence confirmation
0
10
20
30
40
50
RCB G 29 G 42 G 59
Mu
tati
on
Pro
file
Cell Line Generations
Stability of Engineered Host-target
Mu
tati
on
A
B
No wild type sequencesNo new type of mutations
Genetic/phenotypic confirmation obviate the need for release assay
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New Development and Challenges
New expression construct
(scaffolds)• Hetero mAbs
• Enzymes co-expressed with transgenes
Host cell engineering• New host cells for targeted integration
• Host engineering for PQ improvement
Sequence variants• Observed in DHFR/GS systems
• Genetic mutations vs. nutrient depletion
• Detection methods
• LC/MS, DNA sequencing
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Selection marker
ColE1 Ori
transgenePromoter
Selection
markerEnzyme
Selection marker
ColE1 Ori
mAb1 (LC, HC)Promoter
Selection
marker
mAB1’ (LC’, HC’)
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Looking Forward
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Emerging technologies
• Next-generation sequencing (NGS)
• 3rd generation sequencing
• Advanced in past 10-year
• Screening/investigational tools
Key considerations
• Sequencing error rate
• SV, acceptance level
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NGS as Investigational Tool for
Cell Line Characterization
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Plasmid 0% C
NGS %C 98.3% 98.2%
G-12
C A C A
cDNA
Sanger
G20
C A C A
Bulk
C A A A
6.5% 1.2% C
LC-MS detected a sequence variant: Q (CAA) > H (CAC)
C A A A
Bulk DNA
NGS offers potential additional characterization capabilities.
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Summary
Production cell lines are populations of cells exhibiting
various levels of genetic and phenotypic heterogeneity.
Risk-based, phase-appropriate strategy enables identification
of production cell lines ensuring product quality and
manufacturing consistency.
Comprehensive characterization should include three aspects
•Transgene integrity
•Cell line population drift
•Engineered host-target, especially PQ -related
Continue to evaluate new technologies, such as NGS, to
complement current genetic characterization capability.
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Acknowledgements
Eli Lilly and Company
•Bioprocess R&D and Lilly Biotechnology
Lonza Biologics (Slough, U.K.)
•Licensing of and permission to modify the GS-CHO
expression technology
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Backup
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Regulatory Guidance
FDA, Supplement to the Points to Consider in the Production and Testing of New
Drugs and Biologic & Produced by Recombinant DNA Technology: Nucleic Acid
Characterization and Genetic Stability (1992)
FDA, Points to Consider in the Characterization of Cell Lines Used to Produce
Biologicals (1993)
FDA, Points to Consider in the Manufacture and Testing of Monoclonal Antibody
Products for Human Use (1997)
ICH Q5D. Derivation and Characterization of Cell Substrates Used for Production
of Biotechnological/Biological Products (1998)
ICH Q5B. Quality of Biotechnological Products: Analysis of the Expression
Construct in Cells used for Production of r-DNA Derived Protein Products (1996)
EMA/CHMP/BWP. Guideline on the requirements for quality documentation
concerning biological investigational medicinal products in clinical trials (2012)