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

Introduction Transient GFP Expression Evaluation

Multiple strains of Chinese Hamster Ovary (CHO) parental cell lines are currently used for biotherapeutic protein production. a) b)
While it has been established that each of these parental lines possess unique characteristics (e.g. DHFR–) that can influence GFP relative mRNA levels Per Cell Level of GFP Production
recombinant protein productivity, the mechanisms that control the differences are poorly understood. Potential mechanisms 3.5 70000
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. 3 60000
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

Relative GFP mRNA

2.5 50000
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
2 40000
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.
1.5 30000

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Materials and Methods
0.5 10000
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 0 0
Cell Cultures (cells designated as ECACC K1). ECACC K1 cells were gradually weaned from serum and adapted to EX-CELL™ CH0K1SV ECACC K1 DG44

CH0K1SV

ECACC K1

CH0 DG44
325 Serum-Free Medium (SAFC Biosciences) supplemented with 4 mM L-glutamine. n=5019 n=4680 n=2585
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 c) Relative GFP Protein Production Frequencies
and polyA tail. 0.3
CH0K1SV
ECACC K1
0.25 DG44
Plasmid Delivery and GFP Protein Production Analysis

Relative Frequency
Expression Analysis • At 48 hours post electroporation, stain cells 0.2
• Electroporate GFP or IgG plasmid with CellTracker Orange (CTO, Molecular
into CHO parental cells Probes) and seed into a 384-well C-lect plate
0.15
• At 24 hours post electroporation, • Laser-Enabled Analysis and
isolate total RNA from cells Processing (LEAP™) evaluation of single cell
GFP production 0.1
• Perform Sybr Green qRT-PCR to
determine GFP, HC, LC and 0.5
housekeeping gene mRNA levels
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0
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IgG Protein Secretion Analysis
• At 24 hours post electroporation, seed
cells into a 384 well plate with IgG capture Figure 3: CHOK1SV and ECACC K1 cells transiently express and produce GFP at higher levels than DG44 cells.
matrix, allow to incubate for 20 hours Figure 3a: Relative normalized levels of GFP mRNA. Values were normalized using β2-microglobulin mRNA and based upon transfection efficiency.
• Stain cultures with IgG detection reagent 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
and Cell Tracker Green (CTG, Molecular electroporation. The black line in each column denotes the mean fluorescence of the population.
Probes) 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
• Perform Cell Xpress™ analysis of single
peak.
cell IgG secretion

Cell Xpress™ Capture and Detection Transient IgG Secretion Evaluation Using Cell Xpress™

CH0K1SV ECACC K1 DG44

Secretion
Halo

Red Secretion “Halo”

Detection Reagent 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.

IgG -Secreting Transient IgG Expression Evaluation

Cell Secreted IgG
IgG HC mRNA IgG LC mRNA 3.00
Normalized IgG secretion (halo intensity)

a) b)
levels levels
16 35 2.75
14 2.50
30
Plate surface Capture Matrix
LC mRNA, normalized
HC mRNA normalized

12 2.25
25
10 2.00
20
Figure 1a: Image of secreted IgG detection.
8 1.75
Figure 1b: Schematic of extracellular IgG capture and detection 15
6 1.50
10
4 1.25

Results 2 5 1.00
CH0K1SV ECACC K1 DG44
Transient GFP Production Evaluation Using LEAP ™ 0 0 n=2641 n=1750 n=1056
ECACC K1

CH0 DG44

ECACC K1
CH0 DG44
CH0K1SV

CH0K1SV

CHOK1SV ECACC K1 CHO DG44

Figure 5: Transiently transfected CHOK1SV and ECACC K1 cells secrete more IgG than CHO DG44 cells.
GFP
Figure 5a: Relative normalized levels of HC and LC mRNA. Values were normalized using β2-microglobulin mRNA levels and based upon
producing % transfection efficiency. Note that the level of LC is very similar between DG44 and ECACC K1 cells.
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
• Differences in transient GFP and IgG expression was observed in the three parental CHO cell lines examined, using the same
CTO
expression constructs and electroporation conditions.
positive
cells • 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