Current Strategies for Cell Line Development

Baisong Mei May 25, 2011

Introduction
Cell line development is one of the most critical steps in recombinant protein manufacture
- Process productivity Cost of Goods (COG) - Product quality, safety and efficacy

Cell line can determine product comparability to the marketed counterpart Cell line development is time consuming and labor intensive Despite significant progress over the last 25 years, the science of cell line development is still not well understood

B. Mei, May 25, 2011

Significant Progress Achieved Over Last 25 Years

Cell specific productivity increased from <10 pg/cell/day to 50-100 pg/cell/day Cell line improvement and medium / upstream process development helped titer increase from 0.05 g/L to 5 g/L

(Wurm, 2004, Nature Biotechnology) B. Mei, May 25, 2011

Cell Line Development Processes and Timeline

Molecular Cloning

Transfection/ Drug Electroporation selection

Single Cell Cloning

CharacteriProcess zation optimization

cGMP Cell Banking

Timeline (month)
0.5 0.5 0.5-1

2-4 1-2 1-2 0.5-1

Processes typically take 6-11 months depending host cell lines and nature of the recombinant proteins Some steps can be overlapped to save time and increase efficiency

B. Mei, May 25, 2011

Cell Line Development: Key Considerations

Expression vector systems Host cell lines Cell clone selection

Cell line characterization

Process optimization Platform technologies and cGMP requirements

B. Mei, May 25, 2011

Expression Vector Systems

Selection markers
Dihydrofolate Reductase (DHFR) Glutamine Synthetase (GS) Antibiotics CMV EF-1 alpha Inducible promoters Scalfold/Matrix Attachment Regions (S/MAR) Chromotin opening elements Locus control regions Insulators Viral terminal repeats Transcription Processing Export
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pUC origin Intron S/MAR CMV promoter

Promoters:
-

bGH polyA
GOI

GOI 2

Vector of GOI
7583 bp

Other genetic elements:

-

SV40 poly A

EF-1a promoter

S/MAR

Drug resistance gene

Introns enhancing mRNA

-

B. Mei, May 25, 2011

Intron Facilitates mRNA Export

FISH Analysis of b-globin mRNA

Ratio of Cytoplasmic and Nuclear mRNA

With Intron

Without Intron

Intron:

(Valencia et al, 2008, PNAS)

B. Mei, May 25, 2011

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) PER.C6 (human embryonic retinal cells) CAP (human amniocytes) HKB11 (hybrid of HEK293 and human B cell)

Other cell lines

-

Host cell pre-adaptation Cell line engineering

Anti-apoptosis Chaperones DHFR knock out/knock down GS knock out

B. Mei, May 25, 2011

Targeted Gene Integration

Currently introduction of transgene to host cell line is a random integration process Random integration can lead transgene to genomic area where transcription is not active or inhibited Targeted integration can guide transgene to the desired active genomic 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 to recombinases)

Zinc finger nucleases Meganucleases

Transcription activatorlike effector nucleases (TALEN)

B. Mei, May 25, 2011

Targeted Integration Technologies

Involves site specific cleavage of genomic DNA by nuclease DNA binding domain provides specificity of the cleavage (for insertion or inactivation) Can be used for gene knock in (targeted integration) as well as gene knock out (cell line engineering) Selection of integration sites (hot spots) remains challenging

DNA Cleavage Domain DNA Binding Domain

ZFN (Sigma and Sangamo)

Gene Insertion Knock In

B. Mei, May 25, 2011

Gene Inactivation Knock Out

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Single Cell Cloning and Clone Selection

A very small fraction of single cell clones provide high and stable expression Extensive cell line cloning and screening required

Productivity

Single Cell Clones

(Mei et al, 2006, Mol Biotechnology)

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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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Single Cell Cloning and Clone Selection

Clone selection criteria
- Productivity
Cell specific productivity Volumetric productivity

- Cell performance - Product quality

Single Cell Cloning

Limited dilutions FACS ClonePix LEAP

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Single Cell Cloning Technologies

ClonePix
- Fluorescence label high producing cells - Desired single cell colonies picked up by robotic arm

http://www.genetix.com

LEAP
- Laser Enabled Analysis and Processing - Fluorescence label high producing cells - Laser eliminates unwanted cells
http://www.cyntellect.com

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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 Copy Number High producing clones

mRNA Level

(Chusainow et al, 2009, Biotechol Bioeng)

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Intraclonal Heterogeneity
Each single cell clone has its own characteristics and interclonal heterogeneity is expected Single cell cloning is necessary Cells from a clonal cell line can also be heterorgeous Intraclonal heterogeneity is caused by factors in addition to cell size and cell cycle Intraclonal variation nay not be heritable Excessive rounds of single cell cloning may not be beneficial
(Pilbrough, Munro, Gray, 2009, PLoS One) B. Mei, May 25, 2011

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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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Cell Line Quality: Productivity vs COG

Cost of goods (COG) perspective:

COG distribution by unit operation for MAb manufacture

(Costioli et al, 2010, BioPharm Intl)

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Cell Line Quality: Productivity vs COG

Improvement of cell line productivity not always proportionally translated to COG reduction Product quality is one of the most important criteria for cell clone selection
COG Distributions vs. MAb Bioreactor Titer
6 120% USP USP 4 3 2 1 0 0.1 0.3 1.0 Bioreactor Titer (g/L) 5.0 10.0 Other Other 80% 60% 40% 20% 0% 100%

Unit Production Cost

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

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% Distribution

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 in bioreactor before preparing GMP cell bank
Medium Feeds:

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

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Successful High Quality Cell Lines

Product Quality Expression Vector Robustness

Upstream Process

Host Cell

Scalability

High Quality Cell lines

Media Clone Selection

Stability

Characterization

Specific Productivity

Cell Growth

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Platform Technologies and GMP Requirements

Platform technologies
Important for cell line quality Improves process efficiency Arrays of tools necessary No one size fits all solution Host Cell Lines Cell Cloning Characterization Media and USP Invest upfront Same cell line for clinical and commercial Expression Vectors

Platform Tool Boxes

Front-loaded model
-

Guidelines and regulations from ICH and regulatory agencies cGMP compliance (documentation, Quality control, Quality and Regulatory inspection and audit)

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Summary
Cell line development is one of critical steps in manufacture of protein therapeutics
Multiple factors contribute to development of high quality cell lines

Product quality is one of the most important criteria for a successful cell line
Employ new technologies available for cell line development

Platform technologies can improve cell line quality and process efficiency

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