Cell Xpress™ Technology Facilitates High - Producing Recombinant Cell Line

Generation Using Glutamine Synthetase Gene Expression System

Genova Richardson1, Nan Lin1, Kimberly Lacy1, Lynn Davis2, Misa Gray2, Jennifer Cresswell1, Mark Gerber1, Matthew Caple1 and Kevin Kayser1
Cell Sciences & Development, SAFC Biosciences 12909 Laclede Avenue, Saint Louis, MO 63103, 213804 W. 107th Street, Lenexa, Kansas 66215
ESACT 2007

Introduction Correlations of Productivity and IgG HC, LC and GS Relative mRNA Levels

Anti-Rabies GS Expression Vector Cell Line Generation Work Flow 30 0.7

Relative Heavy Chain mRNA Level

Number of 37B12
37B12
SV40 Poly A 25 0.6 33G1
transfectants / 84B1

Relative GS mRNA Level

Transfection clones
(-) Gln , (+) MSX 20 0.5 32B1
Heavy Chain CDS AmpR “Masterwell” (MW) Transfectants >200
>200 36B12
15 0.4
ELISA Productivity Screen 31
10 0.3
Promoter 3 Characterization of MW Transfectants 13
Secretion Population Heterogeneity Analysis 5 0.2
Growth and Productivity
4
GS cDNA
0 0.1
Promoter 1 Single - Cell Cloning
-5
0.0
0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700
SV40 Poly A Characterization of Single - Cell Clones 57 Peak Volumetric Productivity (mg/L)
SV40 Poly A Secretion Population Heterogeneity Analysis Peak Volumetric Productivity (mg/L)
Growth and Productivity
Relative mRNA Level Analysis (HC, LC and GS) Work in
Light Chain CDS
Gene Copy Number Analysis (HC, LC and GS)
progress 120 160
1a Promoter 2 1b Clone Expansion Secretion Dynamic Analysis
32B1
32B1 37B12
37B12
140

Relative Light Chain mRNA Level

Relative Light Chain mRNA Level
100
Figure 1 120
80 32B1
32B1
Secretion Population Heterogeneity Analysis Using Cell Xpress™ 84B1
84B1 100
60
80 84B1
84B1
Secreted IgG 40
60
20
40
0
Detection Reagent 20

IgG
IgG--Secreting
- Secreting
Cell
Cell -20 0
0 100 200 300 400 500 600 700 0 5 10 15 20 25 30
((CellTracker
(CellTracker
Green
Green
Stained)
Stained)
Peak Volumetric Productivity (mg/L) Relative Heavy Chain mRNA Level

Figure 4: Linear correlations of peak volumetric productivity of the single-cell clones and relative mRNA levels of heavy chain (Upper Left), light
Plate surface Capture Reagent chain (Lower Left) and GS (Upper Right). Lower right panel is linear correlation of heavy and light chain mRNA levels. Correlation line is solid. 95%
Assay time 20 hours confidence interval of the correlation is marked by the dashed lines. The clones 37B12 and 32B1 (marked on graphs) are outliers. Their mRNA
might be more efficiently utilized comparing to other clones, which may contribute to higher productivity. They are undergoing further molecular
“Halo” characterization.

Figure 2a: The LEAP system (Laser-Enabled Analysis and Processing, Cyntellect, San Diego, CA) is a unique laser-based cell processing system
that combines cell imaging and laser-mediated cell manipulation in an automated and high-throughput manner via large field-of-view optics and
galvanometer steering. It allows the user to efficiently identify, select and monitor expansion of high recombinant protein secreting clones. It also Secretion Dynamics During Single-Cell Clone Expansion
enables analysis of IgG-secretion heterogeneity and purification of transfected populations by eliminating non-producers.
Green fluorescence indicates CellTracker Green (Molecular Probes) stained live cells. Extracellular red fluorescence, or “halos”, indicates secreted IgG.
Glutamine Synthetase (GS) expression vector and cell line generation work flow. The GS System (expression vectors and CHO K1SV parental cell line) is
licensed from Lonza Biologics.

Secretion Heterogeneity Analysis Workflow

Image Acquisition Raw values by well

(12 - 20 replicated wells that contain 2400 - 4000 cells) Grubb’s test (Z value < 1.67)
Exclude outlier wells

Image Analysis to Collect Secretion

Area Intensity and Secretion Area Use 90 percentile value from the parental population
Average Intensity of “Halos” (background) to normalize the raw values

Review Well Images for Image Quality Exclude normalized values that are below
background (<1)
Statistical Data Processing
Figure 5a: Cells from MW31H3 culture were stained by CellTracker Green and plated in a 384-well C-lect plate at 2.5 cell/well. Well mages were
Normalized background subtracted values acquired right after plating to confirm clonality. Single-cell clones were expanded for 7 days, triturated and sampled to another C-lect plate for
secretion analysis. Representative well images in single-cell stage (Day 0) and after expansion (Day 7) are shown above.
Figure 2b
70000

60000
Secretion Area Average Intensity

Results
Background Subtracted

50000

40000
Secretion Population Heterogeneity Analysis of the Anti-Rabies CHO-GS Single-Cell Clones
30000
10
Normalized Secretion Area Average Intensity

9 20000

8
10000
7
0
6
C5
C7
C21
D20
E3
E12
F7
F15
F17
G4
G9
H10
J22
K18
L19
L21
H9

5
Figure 5b: Scatter plots and mean of Day 7 secretion area average intensity (denoted by the red lines) of the clones. The non-expressers were
4 excluded based on background fluorescence from a well containing no PE fluorescing cells.
3
120.00%
120.00% y == -3E -05x ++1.158
-3E -05x 1.158
2 R2 2 = 0.7963*
0.7963*
100.00%
100.00% E12
E12
1
CV%
Secretion CV%

80.00%
80.00%
0
Well Secretion
32B1
33G11
36B1
37B12
MW4F3
77A7
77E2
78A7
80F5
81G12
82C3
82D5
83H4
83H8
84B1
84G10
85A12
31-H3

60.00%
60.00%
F17
F17
40.00%
40.00% C5
C5
Figure 3a: Cell Xpress™ secretion heterogeneity analysis results from 16 single-cell clones derived from two “Masterwell” transfectant populations
(Red scatter plots – MW4-F3 derived clones; Black scatter plots – MW31-H3 derived clones). The MW transfectants (blue and gray scatter plats) 20.00%
20.00%
are included as control. Mean values are indicated by black or red lines. Secretion Area Aveage Intensity is normalized to parental CHOK1SV as
described in Figure 2B. 0.00%
0.00%
0.0E+00

5.0E+03

1.0E+04

1.5E+04

2.0E+04

2.5E+04

3.0E+04

3.5E+04

4.0E+04
0.0E+00

5.0E+03

1.0E+04

1.5E+04

2.0E+04

2.5E+04

3.0E+04

3.5E+04

4.0E+04

Correlation of Normalized Secretion Area Average Intensity and Peak Volumetric Productivity and Mean Secretion
Mean Secretion Area
Area Intensity,
Intensity, Background
Background SubtractedSubtracted
(RFU) (RFU)
Representative Well Images
Figure 5c: Linear correlation of secretion area average intensity vs. coefficient of variation (%) of each clone. Heterogeneity may contribute to the
6
y = 0.006x + 1.8143 overall secretion level of a clone. *R2 is calculated excluding clone C5. The clones shown in Figure 5a are marked.

R 2 = 0.8414
5
Normalized Secretion Area Average

Conclusions
4
82D5 ™
• Cell Xpress enables selection of high-IgG secreting clones as well as visualization and analysis of secretion population
Intensity

3 heterogeneity on a cell-by-cell basis.

• Secretion area average intensity acquired by Cell Xpress™ correlates to productivity in non-fed batch cultures. Such
2
correlation may allow prediction of clone performance in a high-throughput fashion.
32B1
1 • In two of the high-producing clones, IgG mRNA levels do not correlate with secreted protein levels. These clones require
further studies on post-transcription levels.
0
0 200 400 600 800 • Single-cell clones may develop secretion heterogeneity during expansion, which may contribute to post-expansion
productivity.
Peak Volumetric Productivity (mg/L)

Figure 3b: Correlation of normalized secretion area average intensity and HPLC IgG peak volumetric productivity from 16 single-cell clones.
Growth and productivity experiments were performed in Erlenmeyer flasks non-fed batch culture in proprietary media. Representative well images
of clone 32B1 and 82D5 were shown.

02943-021104