Transgene copy number distribution profiles in recombinant CHO cell lines revealed by single cell analyses

Clonally derived recombinant cell lines are highly desired to achieve consistent production of recombinant biotherapeutics. Despite repeated rounds of cloning by limiting dilution or single cell cloning, the resulting cell lines have often been observed to diverge, becoming a heterogeneous population and losing productivity over long-term sub-culturing. To understand the underlying molecular mechanisms, we developed quantitative polymerase chain reaction (qPCR) assays for the analysis of transgene copy number distribution in single recombinant cells isolated from Chinese hamster ovary (CHO) cell lines. Single cells were obtained by fluorescence activated cell sorting (FACS) technology and lysed directly in 96-well plates. qPCR assays were then applied to analyze the quantity and distribution of transgenes in those single cells. Results revealed multiple types of transgene copy number distribution profiles from those clonally derived CHO cell lines. The cell lines that maintained productivity over time displayed relatively constant and homogeneous transgene copy number distributions; while most of those cell lines exhibiting a loss of productivity over time showed varying degrees of transgene copy number heterogeneity and distribution drift with passaging. Some cell lines showed the existence of a significant portion of cells lacking the transgenes (referred to as negative cells in this study) and the percentage of those negative cells increased with subsequent generations. Criteria based on transgene copy number distribution profiles were developed to assess cell line suitability for clinical applications, which include (i) percentage of negative cells; (ii) standard deviation of qPCR threshold cycle (C(t) ) value, a measure of population heterogeneity; (iii) mean C(t) changes during aging, a measure of population drift. By implementing these criteria, undesirable cell lines were eliminated for further clinical and commercial applications.     

Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.     

Recombinase‐mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line

To meet product quality and cost parameters for therapeutic monoclonal antibody (mAb) production, cell lines are required to have excellent growth, stability, and productivity characteristics. In particular, cell line generation stability is critical to the success of a program, especially where high cell line generation numbers are required for large in‐market supply. However, a typical process for developing such cell lines is laborious, lengthy, and costly. In this study, we applied a FLP/FRT recombinase‐mediated cassette exchange (RMCE) system to build a site‐specific integration (SSI) system for mAb expression in the commercially relevant CHOK1SV cell line. Using a vector with a FRT‐flanked mAb expression cassette, we generated a clonal cell line with good productivity, long‐term production stability, and low mAb gene‐copy number indicating the vector was located in a ‘hot‐spot.’ A SSI host cell line was made by removing the mAb genes from the ‘hot‐spot’ by RMCE, creating a ‘landing pad’ containing two recombination cassettes that allow targeting of one or two copies of recombinant genes. Cell lines made from this host exhibited excellent growth and productivity profiles, and stability for at least 100 generations in the absence of selection agents. Importantly, while clones containing two copies had higher productivity than single copy clones, both were stable over many generations. Taken together, this study suggests the use of FLP‐based RMCE to develop SSI host cells for mAb production in CHOK1SV offers significant savings in both resources and overall cell line development time, leading to a shortened ‘time‐to‐clinic’ for therapeutic mAbs. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1645–1656, 2015     

Selection of CHO host cell subclones with increased specific antibody production rates by repeated cycles of transient transfection and cell sorting

Optimization of host cell lines both for transient and stable protein production is typically hampered by the inherent heterogeneity of cells within a population. This heterogeneity is caused not only by “hard fact” gene mutations, but also by subtle differences in the cellular network of regulation, which may include epigenetic variations. Taking advantage of this heterogeneity, we sorted for naturally occurring variants of CHO‐K1 and CHO‐S host cells that possess an improved cellular machinery for transient antibody production. The long‐term goal of this study was both to identify host cells that yield recombinant cell lines with on average higher productivity, but also to study the molecular differences that characterize such cells, independent of the site of gene integration or gene amplification. To identify such cells we optimized the procedure for transient transfection by electroporation to a degree that gave uniform transfer of plasmid DNA into nearly 100% of the cells and resulted in reproducible average productivities, with a standard deviation of 16% between independent experiments. Using this optimized protocol, the 1% of cells with the highest specific productivity was sorted and subcloned with a cold capture secretion assay. Upon re‐transfection, the resulting subclones showed the same specific productivity as their respective parental cell line. To enrich for cells with potentially stable improved properties, the 1% highest producers were sorted three times, 2 days after transient transfection each, and the enriched population was again sorted into microtiter plates for subcloning. For each of the two parental cell lines tested, three subclones were obtained that had a threefold higher specific productivity after transient transfection. This property was stable for approximately 3 months, indicating that the changes in productivity were regulatory and not mutational. Biotechnol. Bioeng. 2011;108: 386–394. © 2010 Wiley Periodicals, Inc.     

The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

Generating stable, high-producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum-free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high-producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB-mediated gene delivery is expected to reduce the extent of recombinant cell line screening.     

Novel insect cell line capable of complex N-glycosylation and sialylation of recombinant proteins

Paucimannose or oligomannose structures are usually attached to glycoproteins produced by insect cells, while mammalian glycoproteins usually have complex glycans. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system (BEVS), despite its high productivity and versatility. The availability of cell lines capable of complex glycosylation can overcome such a problem and potentially increase the utility of BEVS. In this work the capability of two novel cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1), to produce and glycosylate a recombinant protein (secreted human placental alkaline phosphatase, SeAP) was assessed. SeAP produced by Tn5B1-4 cells at a low passage number (<200) was utilized for comparison. The optimal conditions for the production of SeAP by DpN1 cells were defined, and the glycosylation profiles of SeAP produced by the cell lines were quantitatively determined. Both the A7S and the DpN1 cells produced lower concentrations of SeAP than the Tn5B1-4 cells. Less than 5% of the glycans attached to SeAP produced by the Tn5B1-4 cells had complex forms. Glycans attached to SeAP from A7S cells contained 4% hybrid and 8% complex forms. Galactosylated biantennary structures were identified. Glycans attached to SeAP produced by the DpN1 cell line had 6% hybrid and 26% complex forms. Of the complex forms in SeAP from DpN1, 13% were identified as sialylated glycans. The galactosyltransferase activity of the three cell lines was measured and correlated to their ability to produce complex forms. Even though neither novel cell line produced as much recombinant protein as the Tn5B1-4 cells, the glycosylation of SeAP expressed by both cell lines was more complete. These novel cell lines represent interesting alternatives for the production of complex glycosylated proteins utilizing the BEVS.     

Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production

Recombinant Chinese hamster ovary (rCHO) cells producing erythropoietin (EPO) and rCHO cells producing follicle-stimulating hormone (FSH) showed a significant increase in specific productivity (q) when grown at 32 degrees C compared to 37 degrees C. However, low culture temperature suppressed cell growth, and therefore, did not increase volumetric productivity as much as q. In an attempt to increase the volumetric productivity through improvement of hypothermic growth, EPO producing rCHO (CHO-EPO) cells and FSH producing rCHO (CHO-FSH) cells were adapted at 32 degrees C in a repeated batch mode using spinner flasks. Cell growth of both CHO-EPO and CHO-FSH gradually improved during adaptation at 32 degrees C. Specific growth rates of CHO-EPO and CHO-FSH cells at 32 degrees C, through adaptation, were increased by 73% and 20%, respectively. During adaptation at 32 degrees C, mRNA levels of cold-inducible RNA-binding protein (CIRP) of both rCHO cell lines did not change significantly, suggesting that CIRP expression may not be the only cause for growth suppression at low culture temperature. Unlike cell growth, the recombinant protein production of both rCHO cell lines was not increased during adaptation due to decreased specific productivities. The specific EPO productivity and specific FSH productivity were decreased by 49% and 22%, respectively. Southern blot analyses showed that the decreased specific productivities were not due to the loss of foreign gene copies. Taken together, improvement of hypothermic cell growth by adaptation does not appear to be applicable for enhanced recombinant protein production, since specific productivity decreases during adaptation to the low culture temperature.     

Chemical caspase inhibitors enhance cell culture viabilities and protein titer

Mammalian cell cultures are integral to the production of therapeutic and diagnostic proteins. A common problem encountered in culturing these cell lines, however, is a loss in viability at later stages of the cell culture process. In this study the effects of three newly synthesized chemical caspase inhibitors were investigated for their capacity to inhibit cell death. Findings show that these protease inhibitors were successful in prolonging cellular viabilities among anchorage-dependent CHO-K1 and HEK-293 cells lines. Cells treated with one inhibitor, 7312, performed as well or better when compared with the commercially known caspase inhibitor, zVAD.fmk. Suspension CHO cells producing an IgG were used to investigate the capacity of 7312 to improve protein production. Treatment of cells with 7312 increased integrated cell densities by 33% with treated cells having a higher maximum cell density and higher viability. As a result, monoclonal antibody titers increased by 20% and higher in spinner flask experiments. Increasing productivity in mammalian cell cultures has key implications for the pharmaceutical and biotechnology sectors, which are presently focused on developing methods to enhance cell performance in bioreactor environments.     

Thermal adaptation in<Emphasis Type="Italic">Drosophila serrata</Emphasis> under conditions linked to its southern border: Unexpected patterns from laboratory selection suggest limited evolutionary potential

To investigate the ability ofDrosophila serrata to adapt to thermal conditions over winter at the species southern border, replicate lines from three source locations were held as discrete generations over three years at either 19‡C (40 generations) or temperatures fluctuating between 7‡C and 18δC (20 generations). Populations in the fluctuating environment were maintained either with an adult 0‡C cold shock or without a shock. These conditions were expected to result in temperature-specific directional selection for increased viability and productivity under both temperature regimes, and reduced development time under the fluctuating-temperature regime. Selection responses of all lines were tested under both temperature regimes after controlling for carry-over effects by rearing lines in these environments for two generations. When tested in the 19‡C environment, lines evolving at 19‡C showed a faster development time and a lower productivity relative to the other lines, while cold shock reduced development time and productivity of all lines. When tested in the fluctuating environment, productivity of the 7–18‡C lines selected with a cold shock was relatively lower than that of lines selected without a shock, but this pattern was not observed in the other populations. Viability and body size as measured by wing length were not altered by selection or cold shock, although there were consistent effects of source population on wing length. These results provide little evidence for temperature-specific adaptation inD. serrata —although the lines had diverged for some traits, these changes were not consistent with a priori predictions. In particular, there was no evidence for life-history changes reflecting adaptation to winter conditions at the southern border. The potential forD. serrata to adapt to winter conditions may therefore be limited.     

Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines. To overcome these bottlenecks, we have explored the use of various growth factors to enhance specific productivity and titers in the context of transient gene expression. For that purpose, several growth factors were cloned and screened for their effect on transient gene expression in HEK293E and CHO-DG44 cells. In particular, acidic fibroblast growth factor (aFGF) was able to increase specific productivity by 60% and recombinant protein titers by 80% in HEK293E cells, while FGF9 increased titers by 250% in CHO-DG44 cells.     

Descriptive parameter evaluation in mammalian cell culture

Several methods exist for assessing population growth and protein productivity in mammalian cell culture. These methods were critically examined here, based on experiments with two hybridoma cell lines. It is shown that mammalian cell culture parameters must be evaluated on the same basis. In batch culture mode most data is obtained on a cumulative basis (protein product titre, substrate concentration, metabolic byproduct concentration). A simple numerical integration technique can be employed to convert cell concentration data to a cumulative basis (cell-hours). The hybridoma lines used in this study included a nutritionally non-fastidious line producing low levels of MAb and a nutritionally fastidious hybridoma with high productivity. In both cases the cell-hour approach was the most appropriate means of expressing the relationship between protein productivity and cell population dynamics. The cell-hour approach could be used as the basis for all metabolic population parameter evaluations. This method has the potential to be used successfully for both prediction and optimization purposes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Integration of cell line and process development to overcome the challenge of a difficult to express protein

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi‐prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench‐top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10‐fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1201–1211, 2015     

Characterization of the stability of recombinant protein production in the GS-NS0 expression system

The GS-NS0 system is an important mammalian expression system used largely within industry for the high-level expression of recombinant proteins for therapeutic use. It is essential that the productivity of this system remains stable throughout culture expansion for the successful long-term production of recombinant proteins. Here we present a study of the stability of recombinant protein production from unamplified GS-NS0 cell lines over extended period of continuous culture. The cell lines used in this study were generated by the transfection of NS0 cells with DNA encoding for a secreted recombinant protein and by two subsequent rounds of limiting dilution cloning prior to analysis of stability. The stability of recombinant protein production was assessed at intervals over a period of 134 days using repeated batch culture in shake flasks. Heterogeneous stability was identified. The productivity of some clones remained consistent throughout 134 days of continuous culture. Others exhibit rapid and progressive loss of productivity. Analysis of the causal relationships underlying stability indicates that the initial transfectant determines the susceptibility to loss or retention of productivity. Selection of production clones on the basis of growth and productivity alone will not predict stability during long-term culture. Our research indicates that stable high-producing clones can readily be obtained from use of the GS-NS0 system in the absence of amplification but there may be molecular features of the original transfectants that could serve as very important predictive indicators of the stability of recombinant protein production.     

Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria

Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications.     

Vaccinia virus-based expression of gp120 and EGFP: survey of mammalian host cell lines

Production of recombinant proteins with the vaccinia virus expression system in five mammalian cell lines (HeLa, BS-C-1, Vero, MRC-5, and 293) was investigated for protein yield and proper posttranslational modifications. Regulatory acceptance of the host cell line was taken into consideration, where Vero, MRC-5, and 293 were considered more acceptable to the regulatory authorities. Relevant process knowledge for ease of scale-up with the particular cell type was also considered. Two proteins were expressed, enhanced green fluorescent protein (EGFP) in the cytoplasm and gp120, an HIV envelope coat protein that is secreted into the culture medium. HeLa cells produced the most EGFP at 17.2 microg/well with BS-C-1 and 293 following. BS-C-1 produced the most gp120 at 28.2 microg/mL with 293 and Vero following. Therefore, of the three most appropriate cell lines (Vero, MRC-5, and 293) for production processes, the best results were obtained with 293 cells. Although MRC-5 had a very high productivity on a per cell basis, the low cell density and slow growth rate made the overall production insufficient. Because gp120 contained a significant amount of posttranslational modification, this protein, produced by the different cell lines, was further analyzed by PNGase digestion suggesting N-linked glycosylation modifications in all cell lines tested. On the basis of these results and overall process considerations, 293 cells are recommended for further production process optimization in a serum-free suspension system.     

Productivity enhancement of recombinant protein in CHO cells via specific promoter activation by oncogenes

To construct a recombinant protein highly producing cell lines, we have previously developed the Oncogene Activated Production (OAP) system by using BHK-21 cells. Here we verified the availability of the OAP system in CHO cells. We firstly generated ‘primed’ ras amplified CHO cells, ras clone I, by introducing human c-Ha-ras oncogene into CHO cells. This ras clone I enables quick and easy establishment of recombinant protein hyper producing cell lines by introduction reporter gene of interest. Then we generated I13 by introducing human interleukin 6 (hIL-6) gene as a reporter gene, which showed enhanced productivity rate as compared to A7 established by conventional method. Furthermore, we found that hIL-6 production level of I13 was slightly improved by raising the CO₂ concentration from 5 to 8% possibly because of the enhanced growth rate. We further introduced the E1A oncogene, which has been shown to have a synergistic effect on the recombinant protein production of the ras-amplified BHK-21 cells, then evaluated the productivity. When culture in 5% CO₂ condition, only the slight effect can be seen. However when cultured in 8% CO₂ condition, not only cell number, but also productivity increased significantly, resulted in great augmentation of hIL-6 production, maximum production being 88.6 μg/ml/3 days. This study demonstrates that recombinant protein production level reached commercially desirable level by utilizing our OAP system in CHO cells and optimizing the culture condition. This revised version was published online in August 2006 with corrections to the Cover Date.