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Current Strategies for Cell Line Development
Baisong Mei
May 25, 2011
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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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(Wurm, 2004, Nature Biotechnology)
Significant Progress Achieved Over Last 25 Years
Cell specific productivity increased from
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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