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

Introduction Reduced levels of IgG HC and LC expression correlate with lower productivity
in less stable clonal populations after extended culture.

Clone Generation for Cell Line Stability Studies Transcript Level, Ratio to Normalizer
160
Clone A2b 140 HC LC
120
100
Clone A2 Clone A2
80
60
Clone A1 Clone A1 40
20
0
Clone A1b
Initial 10 wk culture A1 A1b A2 A2b B1 B1b B2 B2b
Clone
1 wk culture 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,
Clone B1b 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 B1 Clone B1
Clone A and B have no differences in relative copy number

Clone B2 Clone B2

Relative Copy Number, Normalized to Control

Clone B2b 25.00
17.95 17.90
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 10.00
expansion, Clones A and B were re-processed with Cell Xpress™ to isolate the highest 25% of secreting clones within each respective population. 5.00
After expansion and banking of the individual isolates, a vial of each clone was thawed and cultured for 10 weeks (~60 population doublings). 0.00
Clone A Clone B
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.

igG LC, Clone A

igG LC, Clone B
Results β2M, Clone A
β2M, Clone B

Production stability for two clonal isolates from the same original clone

350
IgG Volumetric Productivity, mg/L

D3 D5 D7 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
300
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
250 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
200 to (β2M), is shown in the inset panel.

150
Reduced Histone-H4 Acetylation of IgG HC and LC coding regions
100
correlates with lower productivity in less stable clonal populations after extended culture

50 Acetyl-Histone H4, Normalized

1.20
LC HC
0
Ratio of Acetylated

1.00
A1 A1b A2 A2b B1 B1b B2 B2b
Histone H4

0.80
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 0.60
Clone B1 (compare differences between A2 and A2b and B2 and B2b).
0.40

A2 A2b 0.20
B2 B2b
0.00
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
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
culture, possibly an indication that the process of epigenetic silencing has already begun in the less stable of the two clones.
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 Conclusions
60000
• Cell Xpress™ can be used to analyze and select clonal populations to achieve clones with higher levels of IgG production
55000 compared to original single-cell isolates.
Secretion Area Average Intensity

50000 • Sub-clones from an original single-cell clones can exhibit distinct differences in long-term IgG production stability.
• Loss of production stability in some clones does not appear to be dependent upon gene copy number, as individual clones
45000 with varying long-term stability have a nearly identical relative copy number.

40000 • 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
35000 transcripts during extended culture.

30000 • Epigenetic modifications and pathways leading to such modifications could be important markers for production cell line
stability.
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