Recombinant CHO Cell Line Research Articles

Strain-specific requirements of susceptibility to rhesus enteric calicivirus infection.

Recently, we identified the coxsackie and adenovirus receptor (CAR) as the entry receptor for rhesus enteric calicivirus (ReCV) isolate FT285 and demonstrated that co-expression of the CAR and the type B histo-blood group antigen (HBGA) is required to convert the resistant CHO cell line susceptible to infection. To address whether the CAR is also the functional entry receptor for other ReCV isolates and the requirement for specific HBGAs or other glycans, here we used a panel of recombinant CHO cell lines expressing the CAR and the type A, B, or H HBGAs alone or in combination. Infection studies with three diverse ReCV strains, the prototype GI.1 Tulane virus (TV), GI.2 ReCV-FT285, and GI.3 ReCV-FT7, identified that cell surface expression of the CAR is an absolute requirement for all three strains to promote susceptibility to infection, while the requirement for HBGAs varies among the strains. In addition to the CAR, ReCV-FT285 and TV require type A or B HBGAs for infection. In the absence of HBGAs, TV, but not Re-CV FT285, can also utilize sialic acids, while ReCV-FT7 infection is HBGA-independent and relies on CAR and sialic acid expression. In summary, we demonstrated strain-specific diversity of susceptibility requirements for ReCV infections and that CAR, type A and B HBGA, and sialic acid expression control susceptibility to infection with the three ReCV isolates studied. Our study also indicates that the correlation between in vitro HBGA binding and HBGAs required for infection is relatively high, but not absolute. This has direct implications for human noroviruses.IMPORTANCEHuman noroviruses (HuNoVs) are important enteric pathogens. The lack of a robust HuNoV cell culture system is a bottleneck for HuNoV cell culture-based studies. Often, cell culture-adapted caliciviruses that rapidly replicate in conventional cell lines and recapitulate biological features of HuNoVs are utilized as surrogates. Particularly, rhesus enteric caliciviruses (ReCVs) display remarkable similarities, including the primate host, clinical manifestation of gastroenteritis, genetic/antigenic diversity, and reliance on histo-blood group antigens (HBGAs) for attachment. While the HuNoV entry receptor(s) is unknown, the coxsackie and adenovirus receptor (CAR) has recently been identified as the ReCV entry receptor. Here, we identified the CAR, the type A and B HBGAs, and sialic acids as critical cell surface molecules controlling susceptibility to ReCV infections. The CAR is required for all ReCV isolates studied. However, the requirement for the different carbohydrate molecules varies among different ReCV strains. Our findings have direct implications for HuNoVs.

Efficient site-specific integration in CHO-K1 cells using CRISPR/Cas9-modified donors.

Conventional methods applied to develop recombinant CHO (rCHO) cell line as a predominant host for mammalian protein expression are limited to random integration approaches, which can prolong the process of getting the desired clones for months. CRISPR/Cas9 could be an alternative by mediating site-specific integration into transcriptionally active hotspots, promoting homogenous clones, and shortening the clonal selection process. However, applying this approach for the rCHO cell linedevelopment depends on an acceptable integration rate and robust sites for the sustained expression. In this study, we aimed at improving the rate of GFP reporter integration to the Chromosome 3 (Chr3) pseudo-attP site of the CHO-K1 genome via two strategies; these include the PCR-based donor linearization and increasing local concentration of donor in the vicinity of DSB site by applying the monomeric streptavidin (mSA)-biotin tethering approach. According to the results, compared to the conventional CRISPR-mediated targeting, donor linearization and tethering methods exhibited 1.6- and 2.4-fold improvement in knock-in efficiency; among on-target clones, 84% and 73% were determined to be single copy by the quantitative PCR, respectively. Finally, to evaluate the expression level of the targeted integration, the expression cassette of hrsACE2 as a secretory protein was targeted to the Chr3 pseudo-attP site by applying the established tethering method. The generated cell pool reached 2-fold productivity, as compared to the random integration cell line. Our study suggested reliable strategies for enhancing the CRISPR-mediated integration, introducing Chr3 pseudo-attP site as a potential candidate for the sustained transgene expression, which might be applied to promote the rCHO cell linedevelopment.

Developing selection strategy for CHO-K1 cell line that secretes scfv-Fc fusion antibodies using ClonePix2

In the pharmaceutical industry, biopharmaceuticals (biologics) are gaining market share. There has been a dramatic increase in the sale and market penetration of monoclonal antibodies in particular. Typically, therapeutic antibodies are produced using high-expression, clonal, or recombinant CHO cell lines. CHO cells dominate the market as a commercial production host due to their ease of use, built-in regulatory records, and security profiles. While traditional limiting-dilution and cloning-ring regulations are frequently used to select mammalian cell lines that produce high levels of proteins, they have a number of drawbacks. ClonePix2 is a fully automated, single cell-based clone selector that significantly increases the likelihood of rapidly selecting high-production clones with high monoclonality. Scfv-Fc recombinant antibody structures with a variety of therapeutic advantages have gained prominence in recent years. Single cell cloning of CHO cells expressing the scfv-Fc fusion protein, which differs from the classical immunoglobulin structure, was performed in situ using the ClonePix2 device using FITC-tagged anti-Fc and anti-H+L antibodies. The fluorescent intensity parameters of the resulting cell clones were analyzed. Additionally, ELISA was used to determine the production capacities of the best clones. As a result, it was established that anti-Fc antibody recognizes the scfv-Fc fusion protein in a semi-solid environment, enabling the identification of higher production clones.

Functional Trimeric SARS-CoV-2 Envelope Protein Expressed in Stable CHO Cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. One of the three membrane-bound envelope proteins is the spike protein (S), the one responsible for docking to the cellular surface protein ACE2 enabling infection with SARS-CoV-2. Although the structure of the S-protein has distinct similarities to other viral envelope proteins, robust and straightforward protocols for recombinant expression and purification are not described in the literature. Therefore, most studies are done with truncated versions of the protein, like the receptor-binding domain. To learn more about the interaction of the virus with the ACE2 and other cell surface proteins, it is mandatory to provide recombinant spike protein in high structural quality and adequate quantity. Additional mutant variants will give new insights on virus assembly, infection mechanism, and therapeutic drug development. Here, we describe the development of a recombinant CHO cell line stably expressing the extracellular domain of a trimeric variant of the SARS CoV-2 spike protein and discuss significant parameters to be considered during the expression and purification process.

Global phosphoproteomic study of high/low specific productivity industrially relevant mAb producing recombinant CHO cell lines

Abstract Understanding the biology of CHO cells at a cellular level is extremely valuable to the pharmaceutical industry, as it presents the opportunity to rationally engineer clonally derived cell lines (CDCLs) with elevated recombinant specific productivity (Qp). To date, relatively little is understood about the intracellular pathways which influence desirable phenotypes in CHO cell lines. In this study we identified phosphoproteins and total proteins which are differentially expressed between high and low Qp IgG4 producing industry CHO cell lines, some of which upon further investigation, could potentially be used as targets to engineer high Qp CDCLs. We highlighted the importance of phosphoproteins and total proteins associated with amino acid sensing in high Qp cell lines. We also identified increased expression of proteins which promote progression through the cell cycle in low Qp cell lines, suggesting that lower Qp CDCLs are focusing their translational machinery on translating cell cycle associated mRNAs.

Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines.

Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS−/− CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10–19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500‐L and 1000‐L bioreactors replicated laboratory results using 5‐L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

All-trans retinoic acid in combination with sodium butyrate enhances specific monoclonal antibody productivity in recombinant CHO cell line.

The effects of all-trans retinoic acid (RA) and sodium butyrate (NaBu) on growth, viability and antibody production of two types of transfected Chinese hamster ovary cell lines (CHO-K1 and CHO-S) were investigated using a batch mode cell culture. By adding 0.5mM NaBu in the CHO-K1 cell culture, the cell specific productivity (Qp) and antibody concentration increased by five- and threefold, respectively. The optimal concentration of RA was 100nM which resulted in twofold increase in antibody production. In a combination model, RA applied at early growth phase of CHO-K1 cells followed by addition of NaBu with lowering culture temperature at the end of stationary phase resulted in two- and threefold increase in Qp and final antibody concentration, respectively. The latter strategy was also applied on suspended CHO-S cells with enhanced Qp and antibody concentration, but to a lesser extent than the CHO-K1 cells. In conclusion, our results demonstrate that the addition of RA and NaBu along with lowering the culture temperature can increase cell culture period as well as Qp and the final concentration of recombinant monoclonal antibody in both CHO-K1 and CHO-S cells without any significant change in binding affinity of the mAb.

Short- and long-term effects on mAb-producing CHO cell lines after cryopreservation.

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Qp ) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Qp at increased cell age: 17 of the 24 cell lines displayed ≥20% Qp decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Qp decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Qp decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463-477, 2018.

Development and validation of a cell based assay for the detection of neutralizing antibodies against recombinant insulins

Recombinant biopharmaceuticals can induce generation of anti-drug antibodies, which could potentially neutralize therapeutic drug activity. In this report, we describe development and validation of a cell-based assay for detection of neutralizing antibodies (Nab) against insulin and insulin analogues. In order to achieve clinically meaningful sensitivity the method used an early signalling event, insulin induced insulin receptor phosphorylation as the endpoint. Percentage insulin receptor phosphorylation in cell lysates was measured using ECL based ELISA. Presence of neutralizing antibodies (Nab) in samples will inhibit insulin induced receptor phosphorylation and consequently lead to a reduction in the percentage of phosphorylated insulin receptor. Additionally, usage of human insulin receptor overexpressing recombinant CHO cell line further improved the assay sensitivity by reducing the fixed drug (EC50) concentration used for induction of receptor phosphorylation. To ensure adequate free drug tolerance a pre-treatment step was introduced, where serum samples underwent acid dissociation and charcoal extraction before drug incubation. In order to distinguish ADA positive samples containing true Nab from samples containing non-antibody phosphorylation inhibitory serum factors, a confirmatory tier was integrated based on immunodepletion using protein AGL mix. Assay parameters including determination of screening and confirmatory cut-points, intra and inter assay precision, selectivity, specificity and stability were assessed during validation in accordance with recent regulatory guidelines and white papers. The advantage of selecting insulin receptor phosphorylation as assay endpoint made the assay capable of detecting Nab against insulin and insulin analogues.

The molecular weight and concentration of dextran sulfate affect cell growth and antibody production in CHO cell cultures.

To investigate the effect of dextran sulfate (DS), a widely used anti-aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM-0.025 and CS13-1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viability in the decline phase of growth. However, it increased mAb production only in the CS13-1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13-1.00 cells, and showed the highest maximum mAb concentration. For SM-0.025 cells, it significantly decreased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not negatively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its efficacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addition of DS did not affect N-glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13-1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3-1.0 g/L. Taken together, to maximize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1113-1122, 2016.

Components of yeast (Sacchromyces cervisiae) extract as defined media additives that support the growth and productivity of CHO cells

Yeast and plant hydrolysates are used as media supplements to support the growth and productivity of CHO cultures for biopharmaceutical production. Through fractionation of a yeast lysate and metabolic analysis of a fraction that had bioactivity equivalent to commercial yeast extract (YE), bioactive components were identified that promoted growth and productivity of two recombinant CHO cell lines (CHO-Luc and CHO-hFcEG2) equivalent to or greater than YE-supplemented media. Autolysis of the yeast lysate was not necessary for full activity, suggesting that the active components are present in untreated yeast cells. A bioactive fraction (3KF) of the yeast lysate was isolated from the permeate using a 3kDa molecular weight cut-off (MWCO) filter. Supplementation of this 3KF fraction into the base media supported growth of CHO-Luc cells over eight passages equivalent to YE-supplemented media. The 3KF fraction was fractionated further by a cation exchange spin column using a stepwise pH elution. Metabolomic analysis of a bioactive fraction isolated at high pH identified several arginine and lysine-containing peptides as well as two polyamines, spermine and spermidine, with 3.5× and 4.5× higher levels compared to a fraction showing no bioactivity. The addition of a mixture of polyamines and their precursors (putrescine, spermine, spermidine, ornithine and citrulline) as well as increasing the concentration of some of the components of the original base medium resulted in a chemically-defined (CD) formulation that produced an equivalent viable cell density (VCD) and productivity of the CHO-Luc cells as the YE-supplemented medium. The VCD of the CHO-hFcEG2 culture in the CD medium was 1.9× greater and with equivalent productivity to the YE-supplemented media.

Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification.

The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non-UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long-term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.

Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines

Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposons-piggyBac, Tol2, and Sleeping Beauty-for their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900 mg/L were obtained from polyclonal cell pools and up to 1.5 g/L from clonal cell lines using any of the three transposons. Biotechnol. Bioeng. 2016;113: 1234-1243. © 2015 Wiley Periodicals, Inc.

Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.

Effects of copper on CHO cells: cellular requirements and product quality considerations.

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes.

Development of an improved mammalian overexpression method for human CD62L

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5–30mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6months to produce. To shorten the construction time, we replaced the multi-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ∼5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines.

Establishment of stable, high producing, recombinant CHO cell lines using Rosa26 Bacterial Artificial Chromosomes for transgene delivery

Establishment of stable, high producing, recombinant CHO cell lines using Rosa26 Bacterial Artificial Chromosomes for transgene delivery

Utilization of recombinase mediated cassette exchange (RMCE) for the generation of recombinant CHO cell lines with defined expression properties

Utilization of recombinase mediated cassette exchange (RMCE) for the generation of recombinant CHO cell lines with defined expression properties

In search of expression bottlenecks in recombinant CHO cell lines—a case study

The efficient production of recombinant proteins such as antibodies typically involves the screening of an extravagant number of clones in order to finally select a stable and high-producing cell line. Thereby, the underlying principles of a powerful protein machinery, but also potential expression limitations, often remain poorly understood. To shed more light on this topic, we applied several different techniques to investigate a previously generated cell line (4B3-IgA), which expressed recombinant immunoglobulin A (IgA) with an unusually low specific productivity. Results were compared to the host cell line and to another recombinant CHO cell line (3D6-IgA) expressing another IgA that binds to an overlapping epitope. The low specific productivity of clone 4B3-IgA could not be explained by GCN or mRNA levels, but insufficiencies in protein maturation and/or secretion were determined. Despite the presence of free light chain polypeptides, they occasionally failed to associate with their heavy chain partners. Consequently, heavy chains were misassembled and accumulated to form intracellular aggregates, so-called Russell bodies. These protein deposits evoked the expression of increased amounts of ER-resident chaperones to combat the induced stress. Despite bottlenecks in protein processing, the cells' quality checkpoints remained intact, and predominantly correctly processed IgA was exported into the culture medium. The results of our study demonstrated that recombinant protein expression was impaired by heavy chain aggregation despite the presence of a disposable light chain and revealed elevated chaperone formation in combination with limited antibody assembly. Our studies suggest that the primary amino acid sequence and consequently the resulting structure of an expressed protein need to be considered as a factor influencing a cell's productivity.

The relationship between mTOR signalling pathway and recombinant antibody productivity in CHO cell lines

BackgroundHigh recombinant protein productivity in mammalian cell lines is often associated with phenotypic changes in protein content, energy metabolism, and cell growth, but the key determinants that regulate productivity are still not clearly understood. The mammalian target of rapamycin (mTOR) signalling pathway has emerged as a central regulator for many cellular processes including cell growth, apoptosis, metabolism, and protein synthesis. This role of this pathway changes in response to diverse environmental cues and allows the upstream proteins that respond directly to extracellular signals (such as nutrient availability, energy status, and physical stresses) to communicate with downstream effectors which, in turn, regulate various essential cellular processes.ResultsIn this study, we have performed a transcriptomic analysis using a pathway-focused polymerase chain reaction (PCR) array to compare the expression of 84 target genes related to the mTOR signalling in two recombinant CHO cell lines with a 17.4-fold difference in specific monoclonal antibody productivity (q p ). Eight differentially expressed genes that exhibited more than a 1.5-fold change were identified. Pik3cd (encoding the Class 1A catalytic subunit of phosphatidylinositol 3-kinase [PI3K]) was the most differentially expressed gene having a 71.3-fold higher level of expression in the high producer cell line than in the low producer. The difference in the gene’s transcription levels was confirmed at the protein level by examining expression of p110δ.ConclusionExpression of p110δ correlated with specific productivity (q p ) across six different CHO cell lines, with a range of expression levels from 3 to 51 pg/cell/day, suggesting that p110δ may be a key factor in regulating productivity in recombinant cell lines.