A GFP-based method facilitates clonal selection of transfected CHO cells.

TL;DR: A method based on the co‐expression of enhanced green fluorescent protein (EGFP) that allows clonal selection in standard 96‐well cell culture plates and is an alternative to the identification of high‐producer clones using various cell sorting methods is presented.

Abstract: The identification of highly expressing clones is a crucial step in the development of cell lines for production of recombinant proteins. Here we present a method based on the co-expression of enhanced green fluorescent protein (EGFP) that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the protein of interest are linked by an internal ribosome entry site and thus are transcribed into the same mRNA but are translated independently. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein for each clone. By expressing recombinant growth factors in CHO cells, we demonstrate the robustness and performance of this technique. The method is an alternative to the identification of high-producer clones using various cell sorting methods, as it can be performed with standard laboratory equipment.

Summary

Introduction

• A key step in the generation of cell lines for the efficient production of recombinant proteins is the selection of high producer clones following transfection and selection [1].
• The process of randomly picking a large number of clones produced, e.g. by limited dilution, and assaying them individually by ELISA is tedious, time consuming, and reportedly not always very effective [2,3].
• Moreover, this method does not account for differences in either cell density or media volume between wells.
• The enhanced green fluorescent protein (EGFP) has been suggested in the past as co-expressed indicator protein for applications in FACS-based cell separation, but also for the screening of cultures in multiwell plates [8-10].
• The titres are assumed to be linked.

Materials and Methods

• Materials 96-well plates used for screening were from Nunc, Wiesbaden, Germany (96 F TC Nunclon) and BD-Falcon, Heidelberg, Germany (Microtest 96).
• Chemicals were from established suppliers such as Sigma Aldrich and used as received.
• High quality water was produced by a Millipore unit.
• Cell culture Chinese Hamster Ovary cells (cell line CHO-K1 (CCL-61, ATCC)) were maintained in growth medium (R10: RMPI 1640 medium supplemented with L-glutamine (2 mM), penicillin/streptomycin (0.1 mg/mL) and 10 % foetal calf serum) in an atmosphere of 5.0 % CO2 and at 37 °C (incubator: Forma Steri-Cult or Forma Direct-Heat, ThermoFisher Scientific, Dreieich, Germany).
• Prior to the spinner (IIBS, Chur, Switzerland) experiments, the adherent cells were adapted to cultivation in suspension.

Plasmid construction

• The coding regions of the human growth factors VEGFA and IGF1A were amplified by RTPCR as follows, using total human RNA from Saos-2 and HEK293 as template.
• The PCR products were purified and subcloned into pGEMT-Easy (Promega, Mannheim, Germany).
• After 48 h, the cells were collected by trypsinisation and the transfection efficiency was roughly estimated via the EGFP fluorescence by flow cytometry in a Cytomics FC500 (Beckman Coulter, Krefeld, Germany).
• The basis for this method is a dilution of the cell suspension to a point were statistically less than one cell per well (here 0.6) are plated.
• The concentration of hVEGFA and hIGF1A in cell culture supernatants were determined using the specific ELISA development kits from Peprotech (London, UK) and from R&D systems (Wiesbaden, Germany) according to the manufacturer’s instructions.

Results and discussions

• In the pEGFP-N1-based gene constructs used here, the genes encoding for the recombinant target protein and for EGFP are linked by an internal ribosome entry site (IRES).
• Moreover, the use of pEGFP-N1 for transgene expression besides good product titres also makes possible the screening strategy developed in this contribution Determination of optimal screening parameters.
• Both examined types of transparent microplates exhibited a rather high background fluorescence, if compared to values given by the suppliers for the special fluorescence plates.
• The detected fluorescence values were in the range of BFplate, indicating that the contribution of the non-transfected cells to the measured fluorescence is low and does not depend on the number of cells per well.
• Nunc plates were thus used in the subsequent measurements.

Screening for high producers

• In order to test the method for clone screening, CHO cells were transfected with pEGFPhVEGFA or pEGFP-hIGF1A.
• Subsequently, the transfected cells were cultivated for two weeks under selection pressure before being subjected to limited dilution into 96-well plates.
• The intensity of the EGFP fluorescence was in the same range for all the investigated clones, the quantity of secreted hVEGFA was ten-fold higher than that of hIFG1A.

Conclusions

• The use of a recombinant bicistronic plasmid to express the protein of interest along with EGFP allows for a fast screening for “high producers” via the EGFP fluorescence.
• The screening can be performed in standard transparent microtitre plates and less than 3000 EGFP expressing cells can be detected.
• The new screening method allows an efficient detection of good producer clones at an early stage of the process development.
• As it does not rely on fluorescence-activated cell sorting, nor does it require the availability of an antibody specific for the therapeutic protein to be expressed, it can be easily implemented in any laboratory and stage of the cell line development process.
• Eva Weiss and Nicole Andersen provided technical support.

Full text

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A GFP-based method facilitates clonal selection of
transfected CHO cells
Denise Freimark, Valérie Jérôme, Ruth Freitag
To cite this version:
Denise Freimark, Valérie Jérôme, Ruth Freitag. A GFP-based method facilitates clonal selec-
tion of transfected CHO cells. Biotechnology Journal, Wiley-VCH Verlag, 2009, 4 (10), pp.24.
�10.1002/biot.200800264�. �hal-00514309�

For Peer Review
A GFP-based method facilitates clonal selection of
transfected CHO cells
Journal:
Biotechnology Journal
Manuscript ID:
BIOT-2008-0264.R2
Wiley - Manuscript type:
Technical Report
Date Submitted by the
Author:
22-Jun-2009
Complete List of Authors:
Freimark, Denise; University of Bayreuth, Chair for Process
Biotechnology
Jérôme, Valérie; University of Bayreuth, Chair for Process
Biotechnology
Freitag, Ruth; Lehrstuhl Bioprozesstechnik, Universität Bayreuth
Keywords:
growth factor, high producer, mammalian cell culture, recombinant
protein production, transfection
Wiley-VCH
Biotechnology Journal

For Peer Review
Page 1 of 19
Technical Report ((4783 words))
A GFP-based method facilitates clonal selection of transfected
CHO cells
Denise Freimark, Valérie Jérôme and Ruth Freitag*
Chair for Process Biotechnology University of Bayreuth, Germany
Key words: growth factor, high producer, mammalian cell culture, recombinant protein
production, transfection
*corresponding author: Chair for Process Biotechnology, University of Bayreuth, 95440
Bayreuth, Germany, phone: 0049 921 55-7371, Fax: 0049 921 55-7375, e-mail:
bioprozesstechnik@uni-bayreuth.de
Abbreviations:
BSA: Bovine serum albumin
CHO: Chinese hamster ovary
EGFP: Enhanced green fluorescent protein
FACS: Fluorescence assisted cell sorting
G418: Geneticin (antibiotic used for screening)
hIGFIA: Human insulin-like growth factor 1A
IRES: Internal ribosome entry site
hVEGFA: Human vascular endothelial growth factor A
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Abstract
The identification of highly expressing clones is a crucial step in the development of cell lines
for production of recombinant proteins. Here we present a method based on the co-expression
of enhanced GFP that allows clonal selection in standard 96-well cell culture plates. The
genes encoding the EGFP protein and the protein of interest are linked by an internal
ribosome entry site and thus are transcribed into the same mRNA but are translated
independently. Since both proteins arise from a common mRNA, the EGFP expression level
correlates with the expression level of the therapeutic protein for each clone. By expressing
recombinant growth factors in CHO cells, we demonstrate the robustness and performance of
this technique. The method is an alternative to the identification of high-producer clones using
various cell sorting methods, as it can be performed with standard laboratory equipment.
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Introduction
A key step in the generation of cell lines for the efficient production of recombinant proteins
is the selection of high producer clones following transfection and selection [1]. Most often,
successfully transfected cells pass through a subcloning stage using 96-well plates. A robust
method for screening at this level is valuable, as it helps to focus on good producers early on.
Analysis of the product titre in cell culture supernatant by ELISA is commonly used at this
stage to identify clones secreting high levels of recombinant protein. However, the process of
randomly picking a large number of clones produced, e.g. by limited dilution, and assaying
them individually by ELISA is tedious, time consuming, and reportedly not always very
effective [2,3]. Moreover, this method does not account for differences in either cell density
or media volume between wells. Apart from the already mentioned tediousness, this approach
may therefore not accurately predict the clones with high specific productivity.
High throughput screening methods based, e.g., on fluorescence-activated cell sorting (FACS)
or magnetic bead-based cell separation are faster [4,5], but require, especially in case of
FACS, not only expensive equipment but also highly skilled operators. Hence such methods
tend to be restricted to labs in frequent need of cell sorting. Moreover, unless the target
molecule itself shows fluorescence, labelling or co-expression of a fluorescent or otherwise
detectable molecule is required [6,7]. The enhanced green fluorescent protein (EGFP) has
been suggested in the past as co-expressed indicator protein for applications in FACS-based
cell separation, but also for the screening of cultures in multiwell plates [8-10]. In this
approach the recombinant cells express both the fluorescent and the target protein, the titres
are assumed to be linked. However, mostly the genes for EGFP and the target protein were
co-transfected into the cells via individual plasmids, e.g. [9,11]. Hence no physical connection
between the expression of the two proteins existed.
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