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.

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.

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

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’)

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

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)