Embed Size (px)
Citation preview
Efficient stable cell pool/cell line development for large-scale antibody
and protein production
Hangxing YuOct 22, 2015
GenScript
Make Research Easy CONFIDENTIAL 2
Overview
Stable Cell Line Introduction
GenScript CHO-Glutamine Synthetase
Capability & Case Study
Summary
Stable Cell Line Introduction
Make Research Easy CONFIDENTIAL 4
Stable Cell Line Generation
Expression
Vector
Plasmid Construction
SelectionTransfection
Clone Expansion
MTX/MSX
Fed-batch cultureCell line generation & Process development
Make Research Easy CONFIDENTIAL
Chinese hamster ovary (CHO) cells Easily adapt to production medium
Grow to high cell density
Yield stable glycosylation of antibodies
DUXB11, DG44: lack of dihydrofolate reductase (DHFR) activity
CHOK1: with endogenous DHFR activity
NS0 murine myeloma cells: Non-Ig secreting and cholesterol auxotroph
Lack of endogenous glutamine synthetase enzyme activity
5
Cell Line
Make Research Easy CONFIDENTIAL
Origin of Replication:SV 40 origin, f1 origin, pBR322 origin
Promoter/Enhancer:1) Cytomegalovirus (CMV) promoter2) Elongation factor alpha (EF1) promoter
Intron Sequence in 5’ untranslated region: Increase export of transcribed mRNA to the cytoplasm from the nucleus.
3’ polyadenylation signal sequence: Maximize mRNA levels
Kozak Sequence GCC GCC (A/G)CC:Enhance the translation initiation
6
Expression Vector
Make Research Easy CONFIDENTIAL
Selectable Marker Selective Reagents
Metabolic Selectable Marker
Dihydrofolate reductase (DHFR) Methotrexate (MTX)
Glutamine Synthetase Methionine Sulphoximine (MSX)
Antibiotic Selectable Marker
Puromycin acetyltransferase Puromycin
Aminoglycoside phosphotransferase Neomycin (G418)
Hygromycin phospotransferase Hygromycin
7
Selection Marker
Make Research Easy CONFIDENTIAL
Random Integration of foreign genes
Disruption of the genome by gene amplification systems
Cell clones are highly heterogeneous!
Active region supporting high gene expression in the chromosome is rare.
High producing clones are rare!
Screening Method:
Serial limiting dilution
Multiple rounds of serial subcloning
Single cell cloning
ELISA for antibody titering measurement
8
Clone Screening
Make Research Easy CONFIDENTIAL
Medium Development:
Component titration determine the “dose response” to various media components
Media blending a mixture of different media
Spent medium analysis medium changes during the culture process.
Automated screening focus on throughput
9
Feeding Strategy
GenScript CHO-Glutamine Synthetase
Make Research Easy CONFIDENTIAL 11
Principle of Glutamine SynthetaseExpression
Many mammalian cells require exogenous glutamine for survival.
Expression vector encoding GOI + Glutamine Synthetase gene allows glutamine synthesis.
CHO cells express sufficient Glutamine Synthetase to survive without exogenous glutamine.
CHO cell endogenous Glutamine Synthetase inhibited by MSX necessitating engineered Glutamine Synthetase activity.
ATP
ADP + Pi
Glutamine Synthetase Enzyme
NH4+ + Glutamate
Glutamine
Methionine Sulfoximine
(MSX) is an inhibitor
which binds Glutamine
Synthetase irreversibly
Make Research Easy CONFIDENTIAL 13
Objective
CHO K1 Suspension Cells
CHO- Glutamine Synthetase Vector
Cell Line Development
Media & Feed Optimization
GenScript CHO-Glutamine Synthetase
High producing
cell line
To develop a mammalian expression platform that would lead to rapid, efficient and high quality biologics production
Make Research Easy CONFIDENTIAL 14
GenScript CHO-Glutamine Synthetase
De novo synthesized expression vector
Developed in-house CHO-K1 clone as expression host
Validated system and developed case studies
Launched CHO-Glutamine Synthetase in April 2015 for
guaranteed gram level expression of rAbs in 4
months
Make Research Easy CONFIDENTIAL 15
Vector
Genes encoding Ab codon optimized.
Glutamine Synthetase and Ab gene on same plasmid.
Strong promoter (CMV) drives Ab gene expression.
Relatively weak promoter drives Glutamine Synthetase gene expression.
Favorable integration results in robust gene expression.
Make Research Easy CONFIDENTIAL
Cell Line
CHO K1 SP
ATCC clone
Several rounds of
optimization
Adapted to suspension &
chemically defined media
Selected for superior
growth and expression
Fully characterized
16
Host Cell Line
CHOK1SP: Derived from ATCC clone, optimized suspension in serum free media
CHO-Glutamine Synthetasetechnology facilitates rapid creation of high productivity cell lines coupled with robust growth characteristics
Several rounds of optimization conducted and 100s of clones screened
Ease of selection using glutamine-free medium
Make Research Easy CONFIDENTIAL 17
CHO-Glutamine Synthetase Cell Line Development
Codon optimization &Gene synthesis
Transfection
High-titer Pool selection
Cloning or limiting dilution
Repeat
Preliminary clone evaluation
& expansion
Clone evaluation & expansion
Cell banking
Further evaluationProcess
development
Stability study
cGMPMCB
cGMP manufacturing
Fed-batch culture
Capability & Case Study
Make Research Easy CONFIDENTIAL 19
Advantages
Productivity
Stability
Speed
Make Research Easy CONFIDENTIAL
0
1
2
3
4
5
6
Pool Single cellclone
Batchculture
Fed-batchround 1
Fed-batchround 2
Tite
r (g
/L)
mAb Expression level
mAb Expression level
20
Productivity
Use of CHO-Glutamine Synthetase results in high-producing cell lines.
GenScript has created cell lines producing >5g/L of recombinant antibody with production rates in the range 50-100 pg/cell/day.
High volumetric
productivity
Cost
Make Research Easy CONFIDENTIAL
Development stage crucial to success
GenScript CHO-Glutamine Synthetase Technology - for rapid selection of high-producing cell lines
Quick generation of cell lines suitable for cGMP
21
Speed
Gram level mAb productivity achieved in 14-17 weeks.
Milestone Weeks
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Pool
Single Cell Clone
Batch Culture
Fed-Batch Culture
Make Research Easy CONFIDENTIAL 22
Workflow and Timeline: DHFR vs Glutamine Synthetase
Gene synthesis and plasmids preparation
Transfection and High-titer pool
selection
Single clone selection
Fed-Batch culture
Stability evaluation
Research cell bank preparation
Week 2
Week 4
Week 8
Week 13
Week 17
Week 20
Week 21
Week 2
Week 4
Week 8
Week 20
Week 25
Week 29
Week 32
Week 33
Gene synthesis and plasmids preparation
Transfection and single cell clones
screening
MTX amplification
(gradient increase)
Subcloning for single subclones selection
Fed-Batch culture
Stability evaluation
Research cell bank preparation
DHFR Glutamine Synthetase
Make Research Easy CONFIDENTIAL 23
Stability
Glutamine Synthetase cell lines that are consistent in terms of product concentration, growth and product characteristics can be easily created.
0
5
10
15
20
25
30
35
40
45
50
55
60
65
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Tite
r(m
g/L)
& V
iab
le c
ell
den
sity
(x1
0^5
cells
/ml)
Generation
Titer
VCD
Make Research Easy CONFIDENTIAL
0
20
40
60
80
100
120
140
160
0-50 50-100 100-150 150-200 200-300
# o
f si
ngl
e ce
ll cl
on
es
Titer (mg/L)
Single Cell Clones
24
Case Study: Single Cell Clone Screening
Selected for batch culture
mAb X: Productivity of single cell clones in 96-well plates.
Make Research Easy CONFIDENTIAL 25
Case Study: Batch Culture Evaluation
0
10
20
30
40
50
60
70
80
300-500 500-600 600-800 800-1,000
# o
f si
ngl
e ce
ll cl
on
es
Titer (mg/L)
Single Cell Clones
Selected for fed-batch culture, round 1
mAb X: Productivity of single cell clones in batch culture mode in shake flask.
Make Research Easy CONFIDENTIAL 26
Case Study: Fed-Batch Culture Evaluation, Round 1
0
10
20
30
40
50
60
70
80
90
0.5-1 1-2 2-3
# o
f si
ngl
e ce
ll cl
on
es
Titer (g/L)
Single Cell Clones
mAb X: Productivity of single cell clones in Fed-Batch culture mode in shake flask, round 1.
25 clones selected for fed-batch culture, round 2
• 25 clones at 2-3g/L were selected for round 2 fed batch culture• Productivity increased to 5-6 g/L after fed-batch culture optimization in round 2
Make Research Easy CONFIDENTIAL 27
Case Study: Fed-Batch Culture Evaluation, Round 2
0
5
10
15
20
25
30
5-6
# o
f si
ngl
e ce
ll cl
on
es
Titer (g/L)
Single Cell Clones
mAb X: Productivity of single cell clones in Fed-Batch culture mode, round 2.
Make Research Easy CONFIDENTIAL 28
Case Study: Fed-Batch culture evaluation growth profile
A typical single clone expressing mAb-X grown in Fed-Batch mode in shake flask after two rounds of process optimization.
Make Research Easy CONFIDENTIAL 29
Protein/Ab Characterization & QC
Attributes Technique
Molecular Weight SDS-PAGE, MS
Purity, Homogeneity HPLC, SEC
ConcentrationProtein assay, UV
spectrophotometer
Identity Western Blotting, MS
Aggregation, Homogeneity SEC
PTM MS
Activity & Function Biochemical Assay
Safety Endotoxin Testing
Summary
Make Research Easy CONFIDENTIAL 31
Summary
• Glutamine Synthetase based selection system
• High productivities without amplification
• Speed – no need for rounds of gene amplification
• Scalable
Glutamine Synthetase
• CHO derived host cell line for expression
• Adapted to suspension and AFCDM
• Selected for superior growth and expressionCHO-K1
• Fully optimized process
• Production concentrations of >5g/L achieved
• High probability of monoclonality
Cell Line Development
Make Research Easy CONFIDENTIAL
About GenScript
32
GenScript
Products & ServicesMake Research Easy
Gene
Peptide
Protein
Antibody
Discovery Biology
Cell Line
Make Research Easy CONFIDENTIAL
Thank you for your participation
We wish you all success in your research
33
For details on upcoming and archived webinars, visit http://www.genscript.com/webinars.html
Examining the components of your peptide sample with AccuPep QC – Yu Lu, Ph.D., Senior Scientist, GenScript. October 29, 2015
Optimizing soluble protein expression: codon optimization, RBS design, and expression vector design – Rachel Speer, Ph.D., Marketing Specialist, GenScript. November 11, 2015
Large scale genome editing for metabolic engineering of E. coli – Yifan Li, Ph.D., Senior Scientist, GenScript. November 5, 2015
Antibody drug development: challenges & solutions – Liusong Yin, Ph.D., Senior Scientist, GenScript. November 18, 2015
Thank you!
![A Stable Cranial Neural Crest Cell Line from Mouse · Neural crest cell culture Cranial neural crest cells labeled with Wnt1-Cre; R26R-GFP [7,11,12] were obtained from E8.5 mouse](https://img.pdfslide.us/doc/110x75/5f42417ff2821645233c9c4f/a-stable-cranial-neural-crest-cell-line-from-mouse-neural-crest-cell-culture-cranial.jpg)
![Clinical feasibility for cell therapy using human neuronal ... · Development of Human hNT2.17 Cell Line We subcloned a human neuronal cell line from the parental NT2 cell line [33]](https://img.pdfslide.us/doc/110x75/5e85441a5f8b2528e12f70b2/clinical-feasibility-for-cell-therapy-using-human-neuronal-development-of-human.jpg)
