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Current Strategies for Cell Line Development

Baisong Mei

May 25, 2011


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B. Mei, May 25, 2011 2

Introduction

Cell line development is one of the most critical steps inrecombinant protein manufacture

- Process productivity Cost of Goods (COG)

- Product quality, safety and efficacy

Cell line can determine product comparability to themarketed counterpart

Cell line development is time consuming and labor intensive

Despite significant progress over the last 25 years, thescience of cell line development is still not well understood


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B. Mei, May 25, 2011 3

(Wurm, 2004, Nature Biotechnology)

Significant Progress Achieved Over Last 25 Years

Cell specific productivity increased from


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B. Mei, May 25, 2011 4

Processoptimization

MolecularCloning

Transfection/Electroporation

Single CellCloning

Drugselection

Characteri-zation

cGMP CellBanking

Processes typically take 6-11 months depending host cell lines andnature of the recombinant proteins

Some steps can be overlapped to save time and increase efficiency

Cell Line Development Processes and Timeline

Timeline (month)

1-2

0.5

2-4

0.5-11-2

0.5-10.5


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Cell Line Development: Key Considerations

Expression vector systems

Host cell lines

Cell clone selection

Cell line characterization

Process optimization

Platform technologies and cGMP requirements


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Expression Vector Systems

Selection markers

- Dihydrofolate Reductase (DHFR)- Glutamine Synthetase (GS)

- Antibiotics

Promoters:- CMV

- EF-1 alpha

- Inducible promoters

Other genetic elements:- Scalfold/Matrix Attachment

Regions (S/MAR)

- Chromotin opening elements

- Locus control regions

- Insulators

- Viral terminal repeats

Introns enhancing mRNA- Transcription

- Processing

- Export

Vector of GOI

758 3 bp

Drug resistance gene

GOI

GOI 2

Intron

S/MAR

S/MAR

SV40 poly A

bGH polyA

CMV promoter

EF-1a promoter

pUC origin


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(Valencia et al, 2008, PNAS)

Ratio of Cytoplasmicand Nuclear mRNA

With Intron Without Intron

FISH Analysis of -globin mRNA

Intron: +

Intron Facilitates mRNA Export
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2265164/figure/F1/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2265164/figure/F1/


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Host Cell Lines

Commonly used mammalian cell lines

- CHO (Chinese hamster ovary)

DHFR(-): DG44; DUKX B11

DHFR(+): CHO-K1 and derivatives

- NS0 (mouse myeloma)

- BHK21 (baby hamster kidney)

- HEK293 (human embryonic kidney) Other cell lines

- PER.C6 (human embryonic retinal cells)

- CAP (human amniocytes)

- HKB11 (hybrid of HEK293 and human B cell)

Host cell pre-adaptation

Cell line engineering

- Anti-apoptosis

- Chaperones

- DHFR knock out/knock down

- GS knock out


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Targeted Gene Integration

Currently introduction of transgene to host cell line is a randomintegration process

Random integration can lead transgene to genomic area wheretranscription is not active or inhibited

Targeted integration can guide transgene to the desired activegenomic area for maximum gene expression

Ideal for stable, high level protein expression

Human genomic sequence available human cell lines

CHO genomic sequencing is becoming available

Several targeted integration technologies available (in addition torecombinases)

- Zinc finger nucleases

- Meganucleases

- Transcription activatorlike effector nucleases (TALEN)


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Gene Insertion

Knock In

Gene Inactivation

Knock Out

Involves site specific cleavage of genomic DNA by nuclease

DNA binding domain provides specificity of the cleavage (forinsertion or inactivation)

Can be used for gene knock in (targeted integration) as well asgene knock out (cell line engineering)

Selection of integration sites (hot spots) remains challenging

DNA CleavageDomain

DNA BindingDomain ZFN (Sigma and

Sangamo)

Targeted Integration Technologies


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(Mei et al, 2006, Mol Biotechnology)

Single Cell Cloning and Clone Selection

A very small fraction of single cell clones provide high andstable expression

Extensive cell line cloning and screening required

Single Cell Clones

Productivity


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Best Strategies To Select Single Cell Clones?

Find a needle in a haystack what are the best strategies?


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Clone selection criteria

- Productivity

Cell specific productivity

Volumetric productivity

- Cell performance

- Product quality

Single Cell Cloning

- Limited dilutions

- FACS

- ClonePix

- LEAP

Single Cell Cloning and Clone Selection
http://www.bioexpress.com/mas_assets/image_cache/3/5/c/0/500x500_sku.t_3015_1_w.s_w.p.0.4_13760_file.jpeg


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http://www.genetix.com

Single Cell Cloning Technologies

ClonePix- Fluorescence label high

producing cells

- Desired single cell coloniespicked up by robotic arm

LEAP

- Laser Enabled Analysis and

Processing- Fluorescence label high

producing cells

- Laser eliminates unwanted cells

http://www.cyntellect.com


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(Chusainow et al, 2009, Biotechol Bioeng)

Stable High Producing Cell Lines

Cellular and molecular factors determine the quality of a cell line

High gene copy number may not result in a high producer

High producers usually produce high mRNA level

Gene silencing (e.g. methylation) is one of major reasons for cell line instability

Low producing clones High producing clones

mRNALevel

CopyNumber


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Intraclonal Heterogeneity

(Pilbrough, Munro, Gray, 2009, PLoS One)

Each single cell clone hasits own characteristics andinterclonal heterogeneity isexpected

Single cell cloning isnecessary

Cells from a clonal cell linecan also be heterorgeous

Intraclonal heterogeneity iscaused by factors inaddition to cell size andcell cycle

Intraclonal variation naynot be heritable

Excessive rounds of singlecell cloning may not bebeneficial
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2793030/figure/pone-0008432-g003/


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Cell Line and Product Quality

Many product quality attributes are inherent from production cell line The attributes often can not be changed via process optimization

Product comparability (quality) achieved early

Analytical investment for cell line development heavy and early

- Structure

- Charge variants

- Glycosylation

N- and O-glycosylation

Non-human glycans Neu5Gc, 1-3 alpha Gal

- Other PTM (e.g. Sulfation, phosphorylation)

- Aggregation

- Biological activity- Productivity

Justified PTM differences acceptable (safety, efficacy, consistency)

Weighted ranking for clone selection


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(Costioli et al, 2010, BioPharm Intl)

Cell Line Quality: Productivity vs COG

Cost of goods (COG) perspective: COG distribution by unit operation for MAb manufacture


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(Modified from Strube et al, 2007, Bioseparation and Bioprocessing)

COG Distributions vs. MAb Bioreactor Titer

0

1

2

3

4

5

6

0.1 0.3 1.0 5.0 10.0

Bioreactor Titer (g/L)

Unit

ProductionCost

0%

20%

40%

60%

80%

100%

120%

%

Distribution

USP Other

USP Other

Cell Line Quality: Productivity vs COG

Improvement of cell line productivity not always proportionallytranslated to COG reduction

Product quality is one of the most important criteria for cell cloneselection


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Media and Upstream Process

Media and fed strategies significantly improve productivity

Several types of media may be necessary to support

- Host cell maintanance

- Transfection/cloning

- Scale up

- Production

Evaluate and characterize final cell line candidates inbioreactor before preparing GMP cell bank

(from Yu et al, 2011, Biotechnol. Bioeng.)

Medium Feeds:


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Successful High Quality Cell Lines

High QualityCell lines

Product Quality

Stability

Cell Growth

Scalability

SpecificProductivity

RobustnessExpression

Vector

UpstreamProcess

Media Clone Selection

Characterization

Host Cell


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Platform Technologies and GMP Requirements

Expression Vectors

Host Cell Lines

Cell Cloning

Characterization

Media and USP

Platform technologies- Important for cell line quality

- Improves process efficiency

- Arrays of tools necessary

- No one size fits all solution

Front-loaded model

- Invest upfront

- Same cell line for clinical and commercial

Guidelines and regulations from ICH andregulatory agencies

cGMP compliance (documentation, Qualitycontrol, Quality and Regulatory inspectionand audit)

Platform Tool Boxes
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Summary

Cell line development is one of critical steps inmanufacture of protein therapeutics

Multiple factors contribute to development of high qualitycell lines

Product quality is one of the most important criteria for asuccessful cell line

Employ new technologies available for cell linedevelopment

Platform technologies can improve cell line quality and

process efficiency

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