Safc Biosciences Scientific Posters - An Evaluation Of The Intrinsic Igg Production Capabilities Of Different Chinese Hamster Ovary Parental Cell Lines
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An Evaluation of the Intrinsic IgG Production Capabilities of Different Chinese Hamster Ovary Parental Cell Lines Genova A. Richardson*, Daniel W. Allison, Nan Lin, Matthew V. Caple and Kevin J. Kayser Cell Sciences & Development, SAFC Biosciences, Saint Louis, Missouri 63103 *Corresponding author
Transient GFP Expression Evaluation
Introduction
b)
a)
Multiple strains of Chinese Hamster Ovary (CHO) parental cell lines are currently used for biotherapeutic protein production. While it has been established that each of these parental lines possess unique characteristics (e.g. DHFR–) that can influence recombinant protein productivity, the mechanisms that control the differences are poorly understood. Potential mechanisms may include predisposition for integration into highly transcriptionally active loci within the genome, variations in transgene copy number, transcription levels and variations in chaperones or other protein modification and secretion machinery. Analysis of potential protein production or secretion bottlenecks in each of these parental cells could allow us to gain a better understanding of the limitations of each line and would permit tailored parental cell line engineering. To better characterize such differences in expression and secretion capacity, we quantitatively analyzed production of Green Fluorescent Protein (GFP) and secretion of recombinant human IgG in transiently transfected CHOK1SV, ECACC K1 and CHO DG44 parental cell lines. By analyzing these production trends in a transient transfection system, we were able to compare recombinant protein production and secretion between the different CHO parental cell lines independent of integration site effects.
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CHOK1SV cells were provided by Lonza Biologics. CHO DG44 cells were obtained from Invitrogen. Both CHOK1SV and DG44 cells were cultured according to the manufacturer’s directions. CHOK1 cells were obtained from the European Collection of Cell Cultures (cells designated as ECACC K1). ECACC K1 cells were gradually weaned from serum and adapted to EX-CELL™ 325 Serum-Free Medium (SAFC Biosciences) supplemented with 4 mM L-glutamine. GFP was expressed using a proprietary expression vector. Human IgG anti-rabies SO57 heavy chain (HC) and light chain (LC) coding sequences were generated using total gene synthesis. These coding regions were then cloned into a single vector obtained from Lonza Biologics. Both HC and LC were driven with the same proprietary promoter and also had the same 5’UTR and polyA tail.
GFP Protein Production Analysis • At 48 hours post electroporation, stain cells with CellTracker Orange (CTO, Molecular Probes) and seed into a 384-well C-lect plate • Laser-Enabled Analysis and Processing (LEAP™) evaluation of single cell GFP production
• Electroporate GFP or IgG plasmid into CHO parental cells • At 24 hours post electroporation, isolate total RNA from cells • Perform Sybr Green qRT-PCR to determine GFP, HC, LC and housekeeping gene mRNA levels
CH0K1SV n=5019
ECACC K1 n=4680
DG44 n=2585
Relative GFP Protein Production Frequencies
0.3
CH0K1SV ECACC K1 DG44
0.25 Relative Frequency
Plasmid Delivery and Expression Analysis
c)
CH0 DG44
70000
ECACC K1
Materials and Methods
Per Cell Level of GFP Production
3.5
CH0K1SV
Relative GFP mRNA
GFP relative mRNA levels
0.2 0.15 0.1 0.5
• At 24 hours post electroporation, seed cells into a 384 well plate with IgG capture matrix, allow to incubate for 20 hours • Stain cultures with IgG detection reagent and Cell Tracker Green (CTG, Molecular Probes) • Perform Cell Xpress™ analysis of single cell IgG secretion
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IgG Protein Secretion Analysis
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0
Figure 3: CHOK1SV and ECACC K1 cells transiently express and produce GFP at higher levels than DG44 cells. Figure 3a: Relative normalized levels of GFP mRNA. Values were normalized using β2-microglobulin mRNA and based upon transfection efficiency. Figure 3b: LEAP™ analysis of GFP protein production levels. Each point on the plot represents the GFP fluorescence of a single cell 48 hours post electroporation. The black line in each column denotes the mean fluorescence of the population. Figure 3c: A relative frequency histogram of GFP protein production. Note the biphasic distribution of GFP production. A higher proportion of DG44 GFP producing cells are in the low fluorescence peak, and a higher proportion of CHOK1SV and ECACC K1 cells are in the high fluorescence peak.
Transient IgG Secretion Evaluation Using Cell Xpress™
Cell Xpress™ Capture and Detection
CH0K1SV
ECACC K1
DG44
Secretion Halo
Red Secretion “Halo” Detection Reagent IgG -Secreting Cell
Figure 4: Representative pictures of transient IgG production. Green fluorescence (CTG) indicates live cells. Extracellular red fluorescence indicates secreted IgG. The same exposure and gain settings were used for all images. Non-transfected controls (not shown) revealed no background IgG fluorescence.
Transient IgG Expression Evaluation IgG HC mRNA levels
35
™
Transient GFP Production Evaluation Using LEAP CHOK1SV
ECACC K1
CHO DG44
GFP producing cells
10 8 6 4
25 20 15 10
2
5
0
0
b)
3.00 2.75 2.50 2.25 2.00 1.75 1.50 1.25 1.00
CH0 DG44
Results
LC mRNA, normalized
Figure 1b: Schematic of extracellular IgG capture and detection
12
CH0 DG44
Figure 1a: Image of secreted IgG detection.
30
ECACC K1
Capture Matrix
CH0K1SV
Plate surface
HC mRNA normalized
14
IgG LC mRNA levels
ECACC K1
16
CH0K1SV
a)
Normalized IgG secretion (halo intensity)
Secreted IgG
CH0K1SV n=2641
ECACC K1 n=1750
DG44 n=1056
Figure 5: Transiently transfected CHOK1SV and ECACC K1 cells secrete more IgG than CHO DG44 cells. Figure 5a: Relative normalized levels of HC and LC mRNA. Values were normalized using β2-microglobulin mRNA levels and based upon % transfection efficiency. Note that the level of LC is very similar between DG44 and ECACC K1 cells. Figure 5b: Per cell level of IgG secretion. Every point on this plot represents the relative fluorescence intensity of the secreted IgG halo. The black line in each column denotes the average fluorescence of the population.
Conclusions CTO positive cells
• Differences in transient GFP and IgG expression was observed in the three parental CHO cell lines examined, using the same expression constructs and electroporation conditions. • Analysis of transient production of GFP in different parental cell lines reveals a direct correlation between GFP mRNA transcript and GFP protein production. • Although ECACC K1 and DG44 cells express similar levels of LC mRNA, ECACC K1 cells secrete almost two-fold more antibody.
Figure 2: Representative fluorescent images of transient GFP protein production. Top panel: GFP protein fluorescence. The same exposure and gain settings were used for all images. Non-transfected controls (not shown) revealed no background GFP fluorescence. Bottom panel: CTO staining for viable cells. Note the higher level of GFP protein fluorescence in CHOK1SV and ECACC K1 cells.
02968-021104
Transient GFP Expression Evaluation
Introduction
b)
a)
Multiple strains of Chinese Hamster Ovary (CHO) parental cell lines are currently used for biotherapeutic protein production. While it has been established that each of these parental lines possess unique characteristics (e.g. DHFR–) that can influence recombinant protein productivity, the mechanisms that control the differences are poorly understood. Potential mechanisms may include predisposition for integration into highly transcriptionally active loci within the genome, variations in transgene copy number, transcription levels and variations in chaperones or other protein modification and secretion machinery. Analysis of potential protein production or secretion bottlenecks in each of these parental cells could allow us to gain a better understanding of the limitations of each line and would permit tailored parental cell line engineering. To better characterize such differences in expression and secretion capacity, we quantitatively analyzed production of Green Fluorescent Protein (GFP) and secretion of recombinant human IgG in transiently transfected CHOK1SV, ECACC K1 and CHO DG44 parental cell lines. By analyzing these production trends in a transient transfection system, we were able to compare recombinant protein production and secretion between the different CHO parental cell lines independent of integration site effects.
3
60000
2.5
50000
2
40000
1.5
30000
1
20000
0.5
10000
0
0
CHOK1SV cells were provided by Lonza Biologics. CHO DG44 cells were obtained from Invitrogen. Both CHOK1SV and DG44 cells were cultured according to the manufacturer’s directions. CHOK1 cells were obtained from the European Collection of Cell Cultures (cells designated as ECACC K1). ECACC K1 cells were gradually weaned from serum and adapted to EX-CELL™ 325 Serum-Free Medium (SAFC Biosciences) supplemented with 4 mM L-glutamine. GFP was expressed using a proprietary expression vector. Human IgG anti-rabies SO57 heavy chain (HC) and light chain (LC) coding sequences were generated using total gene synthesis. These coding regions were then cloned into a single vector obtained from Lonza Biologics. Both HC and LC were driven with the same proprietary promoter and also had the same 5’UTR and polyA tail.
GFP Protein Production Analysis • At 48 hours post electroporation, stain cells with CellTracker Orange (CTO, Molecular Probes) and seed into a 384-well C-lect plate • Laser-Enabled Analysis and Processing (LEAP™) evaluation of single cell GFP production
• Electroporate GFP or IgG plasmid into CHO parental cells • At 24 hours post electroporation, isolate total RNA from cells • Perform Sybr Green qRT-PCR to determine GFP, HC, LC and housekeeping gene mRNA levels
CH0K1SV n=5019
ECACC K1 n=4680
DG44 n=2585
Relative GFP Protein Production Frequencies
0.3
CH0K1SV ECACC K1 DG44
0.25 Relative Frequency
Plasmid Delivery and Expression Analysis
c)
CH0 DG44
70000
ECACC K1
Materials and Methods
Per Cell Level of GFP Production
3.5
CH0K1SV
Relative GFP mRNA
GFP relative mRNA levels
0.2 0.15 0.1 0.5
• At 24 hours post electroporation, seed cells into a 384 well plate with IgG capture matrix, allow to incubate for 20 hours • Stain cultures with IgG detection reagent and Cell Tracker Green (CTG, Molecular Probes) • Perform Cell Xpress™ analysis of single cell IgG secretion
65000
60000
55000
50000
45000
40000
35000
30000
25000
20000
15000
10000
IgG Protein Secretion Analysis
5000
0
0
Figure 3: CHOK1SV and ECACC K1 cells transiently express and produce GFP at higher levels than DG44 cells. Figure 3a: Relative normalized levels of GFP mRNA. Values were normalized using β2-microglobulin mRNA and based upon transfection efficiency. Figure 3b: LEAP™ analysis of GFP protein production levels. Each point on the plot represents the GFP fluorescence of a single cell 48 hours post electroporation. The black line in each column denotes the mean fluorescence of the population. Figure 3c: A relative frequency histogram of GFP protein production. Note the biphasic distribution of GFP production. A higher proportion of DG44 GFP producing cells are in the low fluorescence peak, and a higher proportion of CHOK1SV and ECACC K1 cells are in the high fluorescence peak.
Transient IgG Secretion Evaluation Using Cell Xpress™
Cell Xpress™ Capture and Detection
CH0K1SV
ECACC K1
DG44
Secretion Halo
Red Secretion “Halo” Detection Reagent IgG -Secreting Cell
Figure 4: Representative pictures of transient IgG production. Green fluorescence (CTG) indicates live cells. Extracellular red fluorescence indicates secreted IgG. The same exposure and gain settings were used for all images. Non-transfected controls (not shown) revealed no background IgG fluorescence.
Transient IgG Expression Evaluation IgG HC mRNA levels
35
™
Transient GFP Production Evaluation Using LEAP CHOK1SV
ECACC K1
CHO DG44
GFP producing cells
10 8 6 4
25 20 15 10
2
5
0
0
b)
3.00 2.75 2.50 2.25 2.00 1.75 1.50 1.25 1.00
CH0 DG44
Results
LC mRNA, normalized
Figure 1b: Schematic of extracellular IgG capture and detection
12
CH0 DG44
Figure 1a: Image of secreted IgG detection.
30
ECACC K1
Capture Matrix
CH0K1SV
Plate surface
HC mRNA normalized
14
IgG LC mRNA levels
ECACC K1
16
CH0K1SV
a)
Normalized IgG secretion (halo intensity)
Secreted IgG
CH0K1SV n=2641
ECACC K1 n=1750
DG44 n=1056
Figure 5: Transiently transfected CHOK1SV and ECACC K1 cells secrete more IgG than CHO DG44 cells. Figure 5a: Relative normalized levels of HC and LC mRNA. Values were normalized using β2-microglobulin mRNA levels and based upon % transfection efficiency. Note that the level of LC is very similar between DG44 and ECACC K1 cells. Figure 5b: Per cell level of IgG secretion. Every point on this plot represents the relative fluorescence intensity of the secreted IgG halo. The black line in each column denotes the average fluorescence of the population.
Conclusions CTO positive cells
• Differences in transient GFP and IgG expression was observed in the three parental CHO cell lines examined, using the same expression constructs and electroporation conditions. • Analysis of transient production of GFP in different parental cell lines reveals a direct correlation between GFP mRNA transcript and GFP protein production. • Although ECACC K1 and DG44 cells express similar levels of LC mRNA, ECACC K1 cells secrete almost two-fold more antibody.
Figure 2: Representative fluorescent images of transient GFP protein production. Top panel: GFP protein fluorescence. The same exposure and gain settings were used for all images. Non-transfected controls (not shown) revealed no background GFP fluorescence. Bottom panel: CTO staining for viable cells. Note the higher level of GFP protein fluorescence in CHOK1SV and ECACC K1 cells.
02968-021104
