Safc Biosciences Scientific Posters - An Evaluation Of The Intrinsic Igg Production Capabilities Of Different Chinese Hamster Ovary Parental Cell Lines
This document was uploaded by user and they confirmed that they have the permission to share it. If you are author or own the copyright of this book, please report to us by using this DMCA report form. Report DMCA
Overview
Download & View Safc Biosciences Scientific Posters - An Evaluation Of The Intrinsic Igg Production Capabilities Of Different Chinese Hamster Ovary Parental Cell Lines as PDF for free.
More details
- Words: 1,153
- Pages: 1
Cell Xpress™-Assisted Analysis of Clone Stability in Recombinant Chinese Hamster Ovary Cells Mark Gerber, Kimberly Lacy, Jennifer Cresswell, Nan Lin, Kevin Kayser, and Matthew Caple Cell Sciences & Development, SAFC Biosciences, Saint Louis, Missouri 63103 ESACT 2007 Reduced levels of IgG HC and LC expression correlate with lower productivity in less stable clonal populations after extended culture.
Introduction Clone Generation for Cell Line Stability Studies
Transcript Level, Ratio to Normalizer
160
Clone A2b
140
HC
LC
120 100
Clone A2
Clone A2
Clone A1
Clone A1
80 60 40 20
Clone A1b
10 wk culture
Initial Clone
1 wk culture Clone B1b Clone B1
Clone B1
Clone B2
Clone B2
0 A1
A1b
A2
A2b
B1
B1b
B2
B2b
Figure 3: Quantitative RT-PCR was used to measure IgG heavy chain (HC) and light chain (LC) mRNA levels among clonal cultures used in this study, compared to control mRNA levels (β2-microglobulin). As observed with secreted IgG production, Clone A2 maintains a higher level of HC and LC transcript expression for the duration of the culture period, while Clone B2 fails to maintain the higher level of expression after extended culture.
Clone A and B have no differences in relative copy number
Relative Copy Number, Normalized to Control
Clone B2b
Results
20.00 15.00
Fluorescence, (dRn)
Figure 1: Clones A and B were isolated from an initial IgG producing clonal CHO-DG44 line using Cell Xpress™ technology. Following clone expansion, Clones A and B were re-processed with Cell Xpress™ to isolate the highest 25% of secreting clones within each respective population. After expansion and banking of the individual isolates, a vial of each clone was thawed and cultured for 10 weeks (~60 population doublings). A second vial from each frozen bank was then thawed and cultured for 1 week. Resulting cultures were used for multiple analyses, including secretion productivity, Cell Xpress™ analysis of individual cell secretion, IgG heavy chain (HC) and light chain (LC) transcript quantitation, copy number determination, and chromatin immunoprecipitation (ChIP) studies. All clonal cultures were maintained under selection in the same proprietary SAFC Biosciences media formulation for the duration of these studies.
25.00 17.95
17.90
Clone A
Clone B
10.00 5.00 0.00
igG LC, Clone A igG LC, Clone B β2M, Clone A β2M, Clone B
350 D3
D5
2
D7
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
Figure 4: Quantitative PCR results of IgG light chain and β-2 microglobulin (β2M) from Clone A and Clone B genomic DNA. 0.2 ng of genomic DNA was amplified using SYBR-Green Jumpstart qPCR kit (Sigma, S4438). Amplification plots and subsequent quantitation demonstrate that relative copy number of stably integrated light chain cDNA do not differ from Clone A to Clone B. Quantitation of technical triplicates, normalized to (β2M), is shown in the inset panel.
250 200 150
Reduced Histone-H4 Acetylation of IgG HC and LC coding regions correlates with lower productivity in less stable clonal populations after extended culture
100
Acetyl-Histone H4, Normalized
50
1.20
0 A1
A1b
A2
A2b
B1
B1b
B2
B2b
Figure 2a: HPLC quantitation of day 3, day 5 and day 7 productivity for cultures of the clonal isolates obtained via the strategy depicted in Figure 1. Clone A2 exhibits a more stable production phenotype than Clone B2, when compared to their original clonal counterparts, Clone A1 and Clone B1 (compare differences between A2 and A2b and B2 and B2b).
A2
A2b
B2
B2b
LC
Figure 2b: Cell Xpress™ images of Clones A2 and A2b, and Cones B2 and B2b. Cells are cultured in the presence of capturing matrix, and secreted antibodies are captured in the proximity of the secreting cell (red fluorescence). Viable cells are indicated by uptake of Cell Tracker Green dye (Molecular Probes). Consistent with the productivity data shown in the top panel, Clone A2 retains increased single-cell productivity following extended culture when compared to Clone B2. Representative well images are shown for each sample set analyzed.
1725
1566
1445
1878
1647
1341
1482
1586
Sample Size
60000
HC
1.00 0.80 0.60 0.40 0.20 0.00
Secretion Area Average Intensity
4
300
Ratio of Acetylated Histone H4
IgG Volumetric Productivity, mg/L
Production stability for two clonal isolates from the same original clone
A2/A2b
B2/B2b
A2b/B2b
Figure 5: Ratio of acetylated histone H4 enrichment in the coding regions of IgG heavy and light chain, from clones late in culture to those early in culture. Following chromatin crosslinking, sheared chromatin was immunoprecipitated with anti-Acetylated histone H4 (Millipore) and levels of precipitated chromatin and input chromatin were measured via quantitative PCR. Ratio is expressed as enrichment of specifically immunoprecipitated chromatin over input chromatin, normalized to β-2 microglobulin controls. Consistent with the observed reduction in expression of both IgG HC and LC mRNA in the unstable Clone B2, the ratio of acetyl-Histone H4 (an indicator of actively transcribed genes) after extended culture to short-term culture is significantly lower as compared to the negligible decrease observed in the relatively stable Clone A2. As shown in the third column, the levels of H4 acetylation in the less stable B2 clone are slightly lower than those observed for A2 at one week in culture, possibly an indication that the process of epigenetic silencing has already begun in the less stable of the two clones.
Conclusions
55000
• Cell Xpress™ can be used to analyze and select clonal populations to achieve clones with higher levels of IgG production compared to original single-cell isolates.
50000
• Sub-clones from an original single-cell clones can exhibit distinct differences in long-term IgG production stability.
45000
• Loss of production stability in some clones does not appear to be dependent upon gene copy number, as individual clones with varying long-term stability have a nearly identical relative copy number. • Concomitant loss of histone H4 acetylation is observed in the IgG coding regions in the clonal isolate that exhibits reduced production stability, suggesting a potential role for chromatin modifications in the regulation of expression of these transcripts during extended culture.
40000 35000
• Epigenetic modifications and pathways leading to such modifications could be important markers for production cell line stability.
30000 25000 20000 15000 10000 A1
A1b
A2
A2b
B1
B1b
B2
B2b
Figure 2c: Aligned dot plot of Secretion Area Average Intensity values for each clonal population. Data for each analyzed cell and its corresponding “halo” is shown as an individual dot in an aligned dot plot. Population average values for each population are shown with a horizontal black line. As seen with the volumetric productivity, Clone A2 maintains a stable average productivity in comparison to Clone B2 (compare with “b” cultures for each respective set). The number of cells measured in each sample set is shown at the top of each column. For statistical analysis, 10 wells of a 384-well plate were used for data collection for each sample set, and Grubb’s z-test was applied to remove outliers with a cutoff of +/-1.67.
02971-021104
Introduction Clone Generation for Cell Line Stability Studies
Transcript Level, Ratio to Normalizer
160
Clone A2b
140
HC
LC
120 100
Clone A2
Clone A2
Clone A1
Clone A1
80 60 40 20
Clone A1b
10 wk culture
Initial Clone
1 wk culture Clone B1b Clone B1
Clone B1
Clone B2
Clone B2
0 A1
A1b
A2
A2b
B1
B1b
B2
B2b
Figure 3: Quantitative RT-PCR was used to measure IgG heavy chain (HC) and light chain (LC) mRNA levels among clonal cultures used in this study, compared to control mRNA levels (β2-microglobulin). As observed with secreted IgG production, Clone A2 maintains a higher level of HC and LC transcript expression for the duration of the culture period, while Clone B2 fails to maintain the higher level of expression after extended culture.
Clone A and B have no differences in relative copy number
Relative Copy Number, Normalized to Control
Clone B2b
Results
20.00 15.00
Fluorescence, (dRn)
Figure 1: Clones A and B were isolated from an initial IgG producing clonal CHO-DG44 line using Cell Xpress™ technology. Following clone expansion, Clones A and B were re-processed with Cell Xpress™ to isolate the highest 25% of secreting clones within each respective population. After expansion and banking of the individual isolates, a vial of each clone was thawed and cultured for 10 weeks (~60 population doublings). A second vial from each frozen bank was then thawed and cultured for 1 week. Resulting cultures were used for multiple analyses, including secretion productivity, Cell Xpress™ analysis of individual cell secretion, IgG heavy chain (HC) and light chain (LC) transcript quantitation, copy number determination, and chromatin immunoprecipitation (ChIP) studies. All clonal cultures were maintained under selection in the same proprietary SAFC Biosciences media formulation for the duration of these studies.
25.00 17.95
17.90
Clone A
Clone B
10.00 5.00 0.00
igG LC, Clone A igG LC, Clone B β2M, Clone A β2M, Clone B
350 D3
D5
2
D7
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
Figure 4: Quantitative PCR results of IgG light chain and β-2 microglobulin (β2M) from Clone A and Clone B genomic DNA. 0.2 ng of genomic DNA was amplified using SYBR-Green Jumpstart qPCR kit (Sigma, S4438). Amplification plots and subsequent quantitation demonstrate that relative copy number of stably integrated light chain cDNA do not differ from Clone A to Clone B. Quantitation of technical triplicates, normalized to (β2M), is shown in the inset panel.
250 200 150
Reduced Histone-H4 Acetylation of IgG HC and LC coding regions correlates with lower productivity in less stable clonal populations after extended culture
100
Acetyl-Histone H4, Normalized
50
1.20
0 A1
A1b
A2
A2b
B1
B1b
B2
B2b
Figure 2a: HPLC quantitation of day 3, day 5 and day 7 productivity for cultures of the clonal isolates obtained via the strategy depicted in Figure 1. Clone A2 exhibits a more stable production phenotype than Clone B2, when compared to their original clonal counterparts, Clone A1 and Clone B1 (compare differences between A2 and A2b and B2 and B2b).
A2
A2b
B2
B2b
LC
Figure 2b: Cell Xpress™ images of Clones A2 and A2b, and Cones B2 and B2b. Cells are cultured in the presence of capturing matrix, and secreted antibodies are captured in the proximity of the secreting cell (red fluorescence). Viable cells are indicated by uptake of Cell Tracker Green dye (Molecular Probes). Consistent with the productivity data shown in the top panel, Clone A2 retains increased single-cell productivity following extended culture when compared to Clone B2. Representative well images are shown for each sample set analyzed.
1725
1566
1445
1878
1647
1341
1482
1586
Sample Size
60000
HC
1.00 0.80 0.60 0.40 0.20 0.00
Secretion Area Average Intensity
4
300
Ratio of Acetylated Histone H4
IgG Volumetric Productivity, mg/L
Production stability for two clonal isolates from the same original clone
A2/A2b
B2/B2b
A2b/B2b
Figure 5: Ratio of acetylated histone H4 enrichment in the coding regions of IgG heavy and light chain, from clones late in culture to those early in culture. Following chromatin crosslinking, sheared chromatin was immunoprecipitated with anti-Acetylated histone H4 (Millipore) and levels of precipitated chromatin and input chromatin were measured via quantitative PCR. Ratio is expressed as enrichment of specifically immunoprecipitated chromatin over input chromatin, normalized to β-2 microglobulin controls. Consistent with the observed reduction in expression of both IgG HC and LC mRNA in the unstable Clone B2, the ratio of acetyl-Histone H4 (an indicator of actively transcribed genes) after extended culture to short-term culture is significantly lower as compared to the negligible decrease observed in the relatively stable Clone A2. As shown in the third column, the levels of H4 acetylation in the less stable B2 clone are slightly lower than those observed for A2 at one week in culture, possibly an indication that the process of epigenetic silencing has already begun in the less stable of the two clones.
Conclusions
55000
• Cell Xpress™ can be used to analyze and select clonal populations to achieve clones with higher levels of IgG production compared to original single-cell isolates.
50000
• Sub-clones from an original single-cell clones can exhibit distinct differences in long-term IgG production stability.
45000
• Loss of production stability in some clones does not appear to be dependent upon gene copy number, as individual clones with varying long-term stability have a nearly identical relative copy number. • Concomitant loss of histone H4 acetylation is observed in the IgG coding regions in the clonal isolate that exhibits reduced production stability, suggesting a potential role for chromatin modifications in the regulation of expression of these transcripts during extended culture.
40000 35000
• Epigenetic modifications and pathways leading to such modifications could be important markers for production cell line stability.
30000 25000 20000 15000 10000 A1
A1b
A2
A2b
B1
B1b
B2
B2b
Figure 2c: Aligned dot plot of Secretion Area Average Intensity values for each clonal population. Data for each analyzed cell and its corresponding “halo” is shown as an individual dot in an aligned dot plot. Population average values for each population are shown with a horizontal black line. As seen with the volumetric productivity, Clone A2 maintains a stable average productivity in comparison to Clone B2 (compare with “b” cultures for each respective set). The number of cells measured in each sample set is shown at the top of each column. For statistical analysis, 10 wells of a 384-well plate were used for data collection for each sample set, and Grubb’s z-test was applied to remove outliers with a cutoff of +/-1.67.
02971-021104
