Utilization of non-AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines

Here we describe a method that couples flow cytometric detection with the attenuated translation of a reporter protein to enable efficient selection of CHO clones producing high levels of recombinant proteins. In this system, a small cell surface reporter protein is expressed from an upstream open reading frame utilizing a non-AUG initiation (alternate start) codon. Due to the low translation initiation efficiency of this alternate start codon, the majority of translation initiation events occur at the first AUG of the downstream open reading frame encoding the recombinant protein of interest. While translation of the reporter is significantly reduced, the levels are sufficient for detection using flow cytometric methods and, in turn, predictive of protein expression from the gene of interest since both ORFs are translated from the same mRNA. Using this system, CHO cells have been sorted to obtain enriched pools producing significantly higher levels of recombinant proteins than the starting cell population and clones with significantly better productivity than those generated from limiting dilution cloning. This method also serves as an effective screening tool during clone expansion to enable resources to be focused solely on clones with both high and stable expression.     

Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.     

Cloning and expression in Escherichia coli of cDNA for serine: pyruvate aminotransferase of rat liver

Cloned cDNAs for rat liver serine: pyruvate aminotransferase were obtained by screening of a cDNA expression bank of rat liver with an antibody against the enzyme. Nineteen clones were isolated from 33 000 transformants and most of them had common fragments of cDNA on analysis by digestion with some restriction enzymes. These clones were identified as those containing cDNA for serine:pyruvate aminotransferase by the following criteria. (a) At the nucleic acid level, a 500-base-pair fragment of cDNA prepared by digestion of cDNAs with EcoRI and PstI hybridized with the mRNA coding for serine:pyruvate aminotransferase as judged by hybrid-selected and hybrid-arrested translations. (b) Specific proteins were detected in nine bacterial clones, a 40-kDa protein in one clone and a 39-kDa protein in eight clones. Among them only the 40-kDa protein was found to be solubilized from the cell by sonication, and this protein was immunoprecipitated with an antibody against serine:pyruvate aminotransferase of rat liver. (c) High activity of serine:pyruvate aminotransferase was expressed both in whole cell suspension and sonicated extract prepared from the transformant producing the 40-kDa protein, and 99% of the activity was immunoreactive with the antibody. Two types of mRNA for serine:pyruvate aminotransferase were detected on the RNA blot analysis by using cloned cDNA fragment as a probe. The larger mRNA (approximately 1600 nucleotides) was glucagon-inducible while the smaller one (approximately 1500 nucleotides) was not affected by the hormone.     

Development of a novel ER stress based selection system for the isolation of highly productive clones

Most biotherapeutic drugs are recombinant monoclonal antibodies which are mostly produced in monoclonal cell lines derived from Chinese hamster ovary (CHO) cells. Various clones expressing a monoclonal recombinant antibody were analyzed and a correlation of the antibody concentration and the relative mRNA level of calreticulin (CALR), glucose‐regulated protein 78 and 94 kDa (GRP78, GRP94) and spliced X‐box binding protein 1 (XPB1) was observed. By means of these results we were motivated to establish a novel selection system based on endoplasmic reticulum (ER) stress, which allows the rapid identification and isolation of high‐expressing clones out of a pool mainly consisting of low‐ and medium‐producing cells. Several ER stress responsive elements were tested with the aid of a recombinase mediated cassette exchange (RMCE) procedure. Very surprisingly, only GRP78 reporter constructs were strongly stimulated upon antibody expression. Furthermore we found that GRP78 reporter constructs are very suitable to reflect the level of antibody expression (IgG) in recombinant CHO cells. Based on these results, it is concluded, that the novel ER stress based selection system developed during this study is suitable to identify and isolate clones with a high level of antibody expression. Biotechnol. Bioeng. 2012; 109: 2599–2611. © 2012 Wiley Periodicals, Inc.     

Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30–35, and 50–55 of the stability program. At generations 0 and 30–35, LC gene expression level was higher than HC gene, whereas at generation 50–55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).     

Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells

Mammalian cells are often used for the expression of recombinant proteins. The process of screening transfected cells randomly for high producing clones is tedious and time consuming. We evaluated using green fluorescent protein (GFP) for selection of high producing clones by fluorescence-activated cell sorter (FACS) to reduce screening effort. We expressed neurotrophin-3 (NT3), deoxyribonuclease (DNase), or vascular endothelial growth factor (VEGF) with GFP in Chinese hamster ovary cells. The vector expressed the desired secreted protein and the selectable marker, dihydrofolate reductase, in one expression unit and the intracellular GFP in a second expression unit. Transfected cells were grown in selection medium and sorted by FACS. High fluorescence clones were obtained and found to produce high amounts of the desired protein; VEGF productivity correlated well with GFP fluorescence in 48 clones. Further studies demonstrated that productivity correlated very well with RNA of the desired protein. For comparison, we randomly picked and screened 144 VEGF clones, and the highest producing VEGF clone obtained produced 0.7 pg/cell/day. In contrast, the highest producing VEGF clone obtained by FACS sorting produced 4.4 pg/cell/day. FACS sorting therefore selected high producing clones efficiently. Since an assay for the desired protein is not required, high producing clones for a protein of unknown function can be obtained by FACS sorting followed by measuring the RNA level of the desired protein in the highly fluorescent clones.     

Stochastic Fluctuations and Distributed Control of Gene Expression Impact Cellular Memory

Despite the stochastic noise that characterizes all cellular processes the cells are able to maintain and transmit to their daughter cells the stable level of gene expression. In order to better understand this phenomenon, we investigated the temporal dynamics of gene expression variation using a double reporter gene model. We compared cell clones with transgenes coding for highly stable mRNA and fluorescent proteins with clones expressing destabilized mRNA-s and proteins. Both types of clones displayed strong heterogeneity of reporter gene expression levels. However, cells expressing stable gene products produced daughter cells with similar level of reporter proteins, while in cell clones with short mRNA and protein half-lives the epigenetic memory of the gene expression level was completely suppressed. Computer simulations also confirmed the role of mRNA and protein stability in the conservation of constant gene expression levels over several cell generations. These data indicate that the conservation of a stable phenotype in a cellular lineage may largely depend on the slow turnover of mRNA-s and proteins.     

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

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.     

Lymphocyte HEV adhesion variants differ in the expression of multiple gene sequences

Lymphocyte adhesion to high endothelial venule cells in lymphoid organs of mice is mediated by several cell-surface glycoproteins, one of which, gp90MEL-14, is detected by the MEL-14 monoclonal antibody (mAb). The MEL-14 mAb was used to select two variants of the EL4 cell line, EL4MEL-14-hi and EL4-MEL-14-lo, that have disparate cell surface expression of this adhesion receptor. A cDNA library constructed from EL4MEL-14-hi mRNA was enriched for sequences present at higher levels in EL4MEL-14-hi cells than EL4MEL-14-lo cells. Quantitative analysis of candidate differential clones by RNA probe protection methods identified five clones whose steady-state mRNA levels were increased in the EL4MEL-14-hi cells. One of these clones, DIFF6, is derived from an RNA whose expression level is higher in several cell lines producing high amounts of MEL-14-reactive gp90, and absent or present at lower levels in several cell lines expressing low levels of this glycoprotein. However, DIFF6 does not encode gp90MEL-14. The nucleotide sequence of this clone predicts a relatively hydrophilic protein characteristic of a cytoplasmic or nuclear protein. Present experiments indicate that expression of gp90MEL-14, a cell-surface-adhesion receptor molecule, may be coregulated with additional cytoplasmic or nuclear factors.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.     

Efficient Recombinant Production in Mammalian Cells Using a Novel IR/MAR Gene Amplification Method

We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone.     

Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers

Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.     

Molecular cloning of a differentiation-related mRNA in the adipogenic cell line 1246.

The 1246 cell line is a C3H mouse teratoma-derived adipogenic cell line that can proliferate and differentiate in defined medium. We have constructed a recombinant phage library containing complementary DNAs (cDNAs) prepared from mRNA of differentiated 1246 cells. This library was screened using a differential hybridization technique. We have isolated five different cDNA clones corresponding to mRNAs that are induced during adipogenesis of 1246 cells and one cDNA clone corresponding to mRNA that is decreased during adipogenesis. Among the mRNAs expressed during adipose differentiation, some are not expressed in undifferentiated cells, whereas some are expressed at very low levels under these conditions. Moreover, the level of induction during differentiation and the temporal expression of the mRNAs corresponding to these cDNAs varied. Our results indicate that one of the cDNA clones isolated, called 154, which selects a 2.2-kilobase mRNA, was induced 100-fold at a very early time during the onset of the differentiation program in 1246 cells and also in adipocyte precursors in primary culture. Direct sequencing of 154 cDNA insert revealed no homology with sequences in GenBank and PIR protein databases. The expression of 154 mRNA was stimulated by accelerators of differentiation such as dexamethasone and isobutylmethylxanthine and inhibited by tumor necrosis factor alpha, transforming growth factor beta, and epidermal growth factor, which are known inhibitors of 1246 cell differentiation. In addition, 154 mRNA level in adipocytes was down-regulated by tumor necrosis factor alpha, but not by transforming growth factor beta or epidermal growth factor. These results suggest that the increase in 154 mRNA expression is related to the onset of adipose differentiation. Further analysis of this clone should allow characterization of a novel protein induced early during the process of differentiation.     

mRNA stability and antibody production in CHO cells: Improvement through gene optimization

The productivity of stably transfected cell lines is of critical importance for the manufacturing of therapeutic proteins. Various methods have been successfully implemented to increase the production output of mammalian cell cultures. Increasing evidence suggests that optimization of the gene coding sequences of an expression vector can improve specific cell line yield of the recombinant protein. Here we demonstrate that gene optimization substantially enhances antibody production in Chinese hamster ovary cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection. Elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggested that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization. Gene optimization also led to increased antibody production in stable clones.