Recombinant Protein Production Using CHO cells

Approaches to Improve Recombinant Protein Production Using
CHO Cells
Dr Ng Say Kong Recombinant Protein Production Using CHO cells Expression Vector Design
•  Improving selection stringency
Expression
•  IRES-mediated expression vectors
Vector
•  Anti-silencing promoters
Transfection and
Selection
Cell Engineering
•  Glycosylation mutants
CHO Cells cell lines
•  Apoptosis-resistant
Working
Cell Bank
Amplification and Limiting
Dilution Cloning
Cell Pool
Master
Cell Bank
Large-Scale Production
•  Small-Scale Studies
•  Stability Studies
•  Medium Optimization
•  Process Development
Candidate Clones
Media and Process Development
•  Proprietary chemically defined protein-free
media
•  Custom media for improved culture
performance
•  High density fed-batch process
-Omics characterization
•  Genomics
•  Metabolomics
•  Transcriptomics
•  Bioinformatics
•  Proteomics
Expression Vector Design
Improving Selection Stringency
Linearized Expression Vector
Gene of Interest
Normal Selector
Transfection
Linearized Expression Vector
Gene of Interest
Weakened Selector
Transfection
Typical Transfected Cell
Typical Transfected Cell
High Producing Cell High Producing Cell è  Weaken selector gene è Increased selection stringency è  Higher expression of GOI in selected / amplified cell pool
Expression Vector Design
Application of Destabilizing Sequences
• 
Target selector for degradation è Lower selector level
IFNγ
DHFR
PEST
• 
ARE
PEST
–  Destabilize protein
–  Clusters rich in proline (P), glutamate (E), serine (S), and threonine (T)
–  Commonly found in rapidly degraded proteins1 –  Sequence from C-terminus of mouse ornithine decarboxylase2
• 
AU-rich elements (ARE) –  Destabilize mRNA
–  In 5-8% of transcribed human mRNA3
–  Regulate diverse functionality3 e.g. cell growth, signal transduction
–  Nine bp basic sequence4
1. Nucleic Acids Res. 31(1): 421–423. 2003
2. Mol. Cell. Biol. 15(4): 2219-2230.1995
3. Science. 234: 364-368. 1986
4. J. Biol. Chem. 266(15): 11213-11220.1991
Expression Vector Design
Application of Destabilizing Sequences
• 
Target selector for degradation è Lower selector level
IFNγ
DHFR
PEST
ARE
IFNγ qp (10-3 pg/cell.day)
0 nM MTX
50 nM MTX
Control
2.4
0.03
ARE
4.0
104.3
26X
PEST
15.8
167.2
10X
PEST+ARE
31.9
1051.7
33X
13X
è Compatible with MTX amplification
è Improved specific productivity
Say Kong Ng, Daniel I.C. Wang, Miranda G.S. Yap. Application of destabilizing sequences on selection marker for improved
recombinant protein productivity in CHO-DG44. Metabolic Eng. 2007; 9(3): 304-316.
Expression Vector Design
Vector Fragmentation
3.2 kbp
BamHI
(2 cutter)
NheI
(Non-cutter)
Southern blotting of transfected
EGFP and dhfr genes in stably
transfected CHO-DG44 cell pool
è  Decoupling of dhfr cistron observed in Bands A2, D2 and D3
è  28% and 14% occurrence rate in transient and stable single cell clones
Say Kong Ng, Wenyu Lin, Rohit Sachdeva, Daniel I.C. Wang, Miranda G.S. Yap. Vector Fragmentation: Characterizing Vector
Integrity in Transfected Clones by Southern Blotting. Biotechnol Prog. 2010; 26(1):11-20.
Expression Vector Design
Application of Internal Ribosome Entry Site (IRES)
Dual promoter vector
DNA
GOI
IRES vector
DHFR
GOI
IRES
DHFR
DHFR
GOI
IRES
DHFR
Transcription
mRNA
GOI
No transcription, unless
in-frame integration
downstream of
endogenous promoter
(rare event)
Translation
Protein
GOI
DHFR
Separate integration and
expression of genes
GOI
DHFR
Expression Vector Design
IRES-linked Destabilized DHFR Expression System
MTX amplification
sDectin-1
Attenuated
IRES
DHFRPEST
MTX Concentration (nM)
MTX Concentration (nM)
0
0
10
50
250
500
10
50
250 500
Transcription
30kDa
5’ cap
AAAAA
sDectin-1 Attenuated
IRES
3’
cHis pool
DHFRPEST
Viable cell density (x106 cells/ml)
5
55 120
120
300
4
44
100
100
250
3
33
80
80
60
60
2
22
40
40
1
11 20
20
0
0
è High producing cell pools in 12 – 17 weeks
1
2
3
4
5
Days
6
7
00 0
8 00
200
150
100
50
0
11
2 2 3
54
34
Days
65
cHis pool
nHis pool
Titer = 0.25 g/L
Expo qp = 4.3 pcd
Titer = 0.16 g/L
Expo qp = 4.9 pcd
7 6 8
0
7
8
Say Kong Ng, Tessa Rui Min Tan, Yang Wang, Daniel Ng, Lin-Tang Goh, Muriel Bardor, Victor Vai Tak Wong, Kong Peng Lam.
Production of functional soluble Dectin-1 glycoprotein using an IRES-linked destabilized-dihydrofolate reductase expression vector.
PLoS One. 2012; 7(12):e52785
sDectin-1 concentration (mg/l)
PEST driven
degradation
nHis-‐sDectin-‐1
cHis-‐mDectin-‐1
Culture viability (%)
dhfr-PEST
nHis pool
Shake flask
batch
cHis-‐sDectin-‐1
Translation
sDectin-1
30kDa
Culture Medium Characterization
Autocrine Growth Factors in Conditioned Media (CM)
Secretion into CM
1
Growth Factor and
Receptor Synthesis
2
Autocrine
Stimulation
3
Post Receptor
Signal
Transduction
2
Receptor
Growth Factor
1
3
Nucleus
-CM improved the growth of low density CHO cell cultures (Dhulipala et al. 2011)
-NSO cells (Haggstrom et al. 2005); Hybridoma cells (Ozturk et al. 1990); Insect cells
(Eriksson et al. 2005)
•  Proteomics to identify what autocrine growth factors are secreted in CM
Culture Medium Characterization
Label-Free 2D LC-MS Approach
8 secreted growth
factors
2512 proteins Culture Medium Characterization
Verification of Autocrine Growth Factors in CHO-K1 for Medium Supplementation
Ligand Gene Name Transcript Western Blot Brain-‐derived neurotrophic factor Bdnf + + Fibroblast growth factor 8 Fgf8 Growth regulated α protein + + + Hepatocyte growth factor Hgf + + Hepatoma-‐derived growth factor + + Leukemia inhibitory factor Lif + + Macrophage colony-‐
sImulaIng factor 1 + + Vascular endothelial growth factor C Cxcl1 + Hdgf Csf1 Vegfc + + Membrane Receptors Trkb tyrosine kinase Fibroblast growth factor receptor 3 Fibroblast growth factor receptor 4 C-‐x-‐c chemokine receptor 1 C-‐x-‐c chemokine receptor 2 Hepatocyte growth factor receptor -‐ Leukemia inhibitory factor receptor Macrophage colony-‐sImulaIng factor receptor Vascular endothelial growth factor receptor 2 Vascular endothelial growth factor receptor 3 Gene Name Transcript Western Blot Trkb -‐ -‐ Fgfr3 + + Fgfr4 -‐ -‐ Cxcr1 Cxcr2 -‐ -‐ -‐ -‐ Hgfr + + -‐ -‐ Lifr + + Mcsfr -‐ -‐ Vegfr2 + + Vegfr3 -‐ -‐ •  Shortlisted autocrine growth factors: FGF-8, HGF, LIF & VEGF-C
Culture Medium Characterization
Functional Testing of Autocrine Growth Factors by MTT assay
-Recombinant human FGF8, HGF, VEGFc and LIF -Vary growth factor concentrations (0.1, 0.5, 1, 10, 50, 100 and 250 ng/ml)
- CHOK1 seeded at 1E4 cells/ml
- MTT assay, 72 hr incubation CHOK1
**
*
**
**
*
*
*
*
**
*
*
*
**
Students T-Test
* P<0.05 **P<0.001 •  Optimal concentration: FGF-8 (100ng/ml); HGF (10ng/ml); VEGF-C (100ng/ml)
Culture Medium Characterization
Synergistic Effects of Autocrine Growth Factors
A Combinatorial screen for FGF8, HGF, VEGF-C. B & C. Output from MTT Assay after 72 hours incubation at a seeding density of 1E4
cells/ml
A
B
C
1
2
3
CHOK1
expt FGF8
HGF
VEGF-C
Viable cell density
no.
(100 ng/ml)
(10 ng/ml)
(100 ng/ml)
Control
2
3
4
5
6
7
8
-
-
-
-
+
+
+
+
-
-
+
+
-
-
+
+
-
+
-
+
-
+
-
+
Positive (Day2 Conditioned Media)
( x 10-5 cells/ml)
Stimulation
(%)
1.12 ± 0.21
1.27 ± 0.15
1.29 ± 0.25
1.32 ± 0.22
1.36 ± 0.15
1.42 ± 0.22
1.44 ± 0.12
1.47 ± 0.18
-
13.4
15.1
17.9
21.4
26.7
28.6
31.3
1.66 ± 0.21
48.2
•  Growth stimulation by growth factors:- FGF-8>HGF>VEGF-C
•  The best combination for CHO cell growth is FGF-8 + HGF + VEGF-C
Culture Medium Characterization
Applications in Single Cell Cloning
CHOK1
CHO m250-9
Screen Day 28
>40% confluence
Check for
single cells
+ FBS
+ CM
+ Growth factors
Medium
Medium
Medium
Medium
Control
+ FBS
+ CM
+ Growth factors
Control
Plating (1 cell/well in 96-well plates)
30 wells/media (n=2)
Students T-Test
* P<0.05 Culture Medium Characterization
Applications in Single Cell Cloning
*
+ FBS
+ Growth factors
42
30
+ FBS
40
0
0
*
*
20
*
*
60
+ CM
85
+ Growth factors
+ IGF-1 + insulin + EGF +HSA
Control
80
+ IGF-1 + insulin + EGF +HSA
+ Growth factors
+ FBS
*
+ CM
+ Growth factors
+ IGF-1 + insulin + EGF +HSA
80
+ IGF-1 + insulin + EGF +HSA
100
+ Growth factors
120
Control
% of wells positive for growth
140
+ FBS
+ Growth factors
Media
Media + (A; 100ng/ml FGF8 + 10ng/ml HGF + 100ng/ml VEGFC)
Media + (B; 100ng/ml IGF-1 + 10ug/ml Insulin + 10ng/ml EGF + 2g/l HSA)
Media + (A) + (B)
Media + CM
Media + FBS
Media + FBS + (A)
160
43
*
36
28
25
16
22
30
12
Students T-Test
* P<0.05 0
CHOK1
CHO DG44
Cell Line
U Ming Lim, Miranda Yap, Yoon-Pin Lim, Lin Tang Goh, Say Kong Ng. Identification of Autocrine Growth Factors Secreted by CHO
cells for Applications in Single Cell Cloning Media. Journal of Proteome Research. 2013; 12(7):3496–3510.
U Ming Lim, Lin Tang Goh, Say Kong Ng. Growth factor supplements for CHO cell culture. PCT/SG2014/000119.
Summary
•  Vector Design Approach
–  Improve selection stringency by applying destabilizing sequences
–  Apply IRES to link selection marker to recombinant protein gene
–  Further optimization by IRES attenuation and applying PEST
è High producing cell pools/ clones in research lab setting
•  Culture Medium Characterization Approach
–  Identify proteins in conditioned medium
–  Identify and validate secreted growth factors
è Single cell cloning supplement that can match the cloning efficiency of
conditioned medium
Acknowledgements
• 
Prof Lam Kong Peng
• 
Prof Miranda Yap
• 
Dr Victor Wong
• 
Dr Muriel Bardor
• 
Tessa Tan
• 
Yang Wang
• 
Daniel Ng
• 
Dr Lin-Tang Goh • 
Dr U-Ming Lim
ACT @ BTI

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