8/14/2019 SAFC Biosciences Scientific Posters - The Effects of Media Formulations on the Biochemical Profile of IgG Expresse…

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

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

Secretion Dynamics During Single-Cell Clone Expansion

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 wereacquired right after plating to confirm clonality. Single-cell clones were expanded for 7 days, triturated and sampled to another C-lect plate forsecretion analysis. Representative well images in single-cell stage (Day 0) and after expansion (Day 7) are shown above.

Figure 5b: Scatter plots and mean of Day 7 secretion area average intensity (denoted by the red lines) of the clones. The non-expressers wereexcluded based on background fluorescence from a well containing no PE fluorescing cells.

Figure 5c: Linear correlation of secretion area average intensity vs. coefficient of variation (%) of each clone. Heterogeneity may contribute to theoverall secretion level of a clone. *R2 is calculated excluding clone C5. The clones shown in Figure 5a are marked.

Conclusions

• Cell Xpress™ enables selection of high-IgG secreting clones as well as visualization and analysis of secretion populationheterogeneity on a cell-by-cell basis.

• Secretion area average intensity acquired by Cell Xpress™ correlates to productivity in non-fed batch cultures. Suchcorrelation may allow prediction of clone performance in a high-throughput fashion.

• In two of the high-producing clones, IgG mRNA levels do not correlate with secreted protein levels. These clones requirefurther studies on post-transcription levels.

• Single-cell clones may develop secretion heterogeneity during expansion, which may contribute to post-expansionproductivity.

Introduction

Anti-Rabies GS Expression Vector Cell Line Generation Work Flow

Figure 1

Secretion Population Heterogeneity Analysis Using Cell Xpress™

Figure 2a: The LEAP system (Laser-Enabled Analysis and Processing, Cyntellect, San Diego, CA) is a unique laser-based cell processing systemthat combines cell imaging and laser-mediated cell manipulation in an automated and high-throughput manner via large field-of-view optics andgalvanometer steering. It allows the user to efficiently identify, select and monitor expansion of high recombinant protein secreting clones. It alsoenables 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) islicensed from Lonza Biologics.

Secretion Heterogeneity Analysis Workflow

Figure 2b

Results

Secretion Population Heterogeneity Analysis of the Anti-Rabies CHO-GS Single-Cell Clones

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)are included as control. Mean values are indicated by black or red lines. Secretion Area Aveage Intensity is normalized to parental CHOK1SV asdescribed in Figure 2B.

Correlation of Normalized Secretion Area Average Intensity and Peak Volumetric Productivity andRepresentative Well Images

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 imagesof clone 32B1 and 82D5 were shown.

Cell Xpress™ Technology Facilitates High - Producing Recombinant Cell LineGeneration 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

02943-021104

SV40 Poly A

Heavy Chain CDS

Promoter 1

SV40 Poly A

Light Chain CDS

Promoter 2

SV40 Poly A

GS cDNA

Promoter 3

AmpR >200>200

5757

3131

Transfection

“Masterwell” (MW) Transfectants

Single - Cell Cloning

Characterization of MW TransfectantsSecretion Population HeterogeneityAnalysis

Growth and Productivity

Characterization of Single - Cell ClonesSecretion Population HeterogeneityAnalysis

Growth and Productivity

Relative mRNALevelAnalysis (HC, LCand GS)

Gene CopyNumberAnalysis (HC, LCand GS)

Clone Expansion Secretion Dynamic Analysis

(-) Gln, (+)MSX

44

Work inprogress

ELISA Productivity Screen

1313

Number oftransfectants / 

clones

1a 1b

Image Acquisition

(12 - 20 replicated wells that contain 2400 - 4000 cells)

Image Analysis to Collect Secretion

Area Intensity and Secretion Area

Average Intensity of “Halos”

Raw values by well

Grubb’s test (Z value < 1.67)

Exclude outlier wells

Review Well Images for Image Quality

Use 90 percentile value from the parental population

(background) to normalize the raw values

Exclude normalized values that are below

background (<1)

Normalized background subtracted values

Statistical Data Processing

   N  o  r  m  a   l   i  z  e   d   S  e  c  r  e   t   i  o  n   A  r  e  a   A  v  e  r  a  g  e   I  n   t  e  n  s   i   t  y

10

9

8

7

6

5

4

3

2

1

0

   3   2   B   1

   3

   3   G   1   1

   3   6   B   1

   3

   7   B   1   2

   M   W   4   F   3

   7   7   A   7

   7   7   E   2

   7   8   A   7

   8   0   F   5

   8

   1   G   1   2

   8   2   C   3

   8   2   D   5

   8   3   H   4

   8   3   H   8

   8   4   B   1

   8

   4   G   1   0

   8

   5   A   1   2

   3   1  -   H   3

Peak Volumetric Productivity (mg/L)0 100 200 300 400 500 600 700

   R  e   l  a   t   i  v  e   H  e  a  v

  y   C   h  a   i  n  m   R   N   A   L  e  v  e   l

-5

0

5

10

15

20

25

30

37B1237B12

   R  e   l  a   t   i  v  e   L   i  g   h   t   C   h  a   i  n  m   R   N   A   L  e  v  e   l 37B12

84B1

32B1

Peak Volumetric Productivity (mg/L)

0 100 200 300 400 500 600 700-20

0

20

40

60

80

100

120

37B12

84B1

32B1

33G184B1

32B1

36B12

Peak Volumetric Productivity (mg/L)

0 10 0 2 00 30 0 4 00 5 00 60 0 7 000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

33G184B1

32B1

36B12

   R  e   l  a   t   i  v  e   G   S  m   R   N   A

   L  e  v  e   l

32B1

84B1

Relative Heavy Chain mRNA Level

0 5 10 15 20 25 300

20

40

60

80

100

120

140

160

32B1

84B1

   R  e   l  a   t   i  v  e   L   i  g   h   t   C   h  a   i  n  m   R   N   A   L  e  v  e   l

70000

60000

50000

40000

30000

20000

10000

0

   C   5

   C   7

   C   2   1

   D   2   0

   E   3

   E   1   2    F   7

   F   1   5

   F   1   7

   G   4

   G   9

   H   1   0

   J   2   2

   K   1   8

   L   1   9

   L   2   1

   H   9

   S  e  c  r  e   t   i  o  n   A  r  e  a   A  v  e  r  a  g  e   I  n   t  e  n  s   i   t  y

   B  a  c   k  g  r  o  u  n   d   S  u   b   t  r  a  c   t  e   d

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

   0 .   0   E  +   0   0

   5 .   0   E  +   0   3

   1 .   0   E  +   0   4

   1 .   5   E  +   0   4

   2 .   0   E  +   0   4

   2 .   5   E  +   0   4

   3 .   0   E  +   0   4

   3 .   5   E  +   0   4

   4 .   0   E  +   0   4

Mean Secretion Area Intensity, Background Subtracted (RFU)

   W  e   l   l   S  e  c  r  e   t   i  o  n   C   V   % 

C5

E12

F17

y = -3E -05x + 1.158R 2 = 0.7963*

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

   0 .   0   E  +   0   0

   5 .   0   E  +   0   3

   1 .   0   E  +   0   4

   1 .   5   E  +   0   4

   2 .   0   E  +   0   4

   2 .   5   E  +   0   4

   3 .   0   E  +   0   4

   3 .   5   E  +   0   4

   4 .   0   E  +   0   4

Mean Secretion Area Intensity, Background Subtracted (RFU)

   W  e   l   l   S  e  c  r  e   t   i  o  n   C   V   % 

C5

E12

F17

y = -3E -05x + 1.158R 2 = 0.7963*

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

   0 .   0

   E  +   0   0

   5 .   0

   E  +   0   3

   1 .   0

   E  +   0   4

   1 .   5

   E  +   0   4

   2 .   0

   E  +   0   4

   2 .   5

   E  +   0   4

   3 .   0

   E  +   0   4

   3 .   5

   E  +   0   4

   4 .   0

   E  +   0   4

   W  e   l   l   S  e  c

  r  e   t   i  o  n   C   V   % 

C5

E12

F17

y = -3E -05x + 1.158

R 2 = 0.7963*

Mean Secretion Area Intensity, Background Subtracted (RFU)

Capture Reagent

Detection Reagent

IgG-Sec reting Cell(CellTracker Green Stained)

Secreted

Plate surface Capture Reagent

Detection Reagent

IgG-( Green Stained)

Secreted

Plate surface Capture Reagent

Detection Reagent

IgG - Secreting Cell(CellTracker Green Stained)

Secreted IgG

Plate surfaceAssay time 20 hours

“Halo”

y = 0.006x + 1.8143

R2 = 0.8414

0

1

2

3

4

5

6

0 200 400 600 800

Peak Volumetric Productivity (mg/L)

   N  o  r  m  a   l   i  z  e   d   S  e  c  r  e   t   i  o  n   A  r  e  a   A  v  e  r  a  g  e

   I  n   t  e  n  s   i   t  y

32B1

82D5

ESACT 2007