The selection of clones displaying a high rate of cell growth is an essential component of Chinese hamster ovary (CHO) cell line development. In recent years various "omics" technologies have been utilised to understand the mechanisms underlying bioprocess phenotypes. In this study, gene expression analysis using a CHO-specific microarray was conducted for a panel of CHO-K1 MAb-secreting cell lines spanning a range of growth rates that were derived from a single cell line development project. In-silico functional analysis of the resulting transcriptomic data revealed the overrepresentation of biological processes such as cell cycle and translation within those genes upregulated during fast growth, while genes associated with cellular homeostasis were downregulated. Using differential expression and correlation analysis we identified a high priority group of 416 transcripts (190 upregulated; 226 downregulated) associated with growth rate. Expression changes of eight of these genes were independently confirmed by qPCR. Finally, we demonstrate the enrichment of predicted mRNA targets of miR17-92, a microRNA (miRNA) cluster known to be upregulated during rapid proliferation, within downregulated transcripts.

We have evaluated a more cell-intensive approach to both cell line adaptation and production culture as a means of achieving high integrated viable cell density (IVCD) in order to improve cell culture process yields. Shake flask based Fed-batch (FB) production cultures were seeded at multiple cell densities. Increases in seed density in the fed batch up to 8x control seed density resulted in significantly higher IVCD and titer with a 2–3 fold benefit relative to control seeded cultures. Whereas the initial increments in production seed density resulted in a linear increase in IVCD and titer, further increments showed diminishing returns. It appeared that the highest seed density production cultures were limited by nutrient supply and so richer medium and a higher feeding rate were then tested, resulting in further improvements in IVCD and titer. We also tested the effect of adapting the cells to high densities for over 50 generations prior to production cultures. For the production assessment, both the high- and standard-density adapted cells were seeded at multiple cell densities. Effect of the adaptation was modest and variable. By combining high-seed adaptation, high production culture seed densities, enriched media and high feeding rate titer yields of up to 9 g/L were achieved.

Fed batch culture processes are often characterized by decreasing cell culture performance as the process continues, presumably through the depletion of vital nutrients and the accumulation of toxic byproducts. We have similarly observed that cellular productivity (Qp) often declines during the course of a fed batch process; however, it is not clear why some cell lines elicit this behavior, while others do not. We here present a transcriptomic profiling analysis of a phenotype of sustained Qp (S-Qp) in production Chinese hamster ovary (CHO) culture, in which a marked drop in Qp levels ("non-sustained" (NS) phenotype) in two cell lines irrespective of viability levels was compared to two cell lines that consistently displayed high Qp throughout the culture ("sustained" (S) phenotype). Statistical analysis of the microarray data resulted in the identification of 22 gene transcripts whose expression patterns were either significantly negatively or positively correlated with long-term maintenance of Qp over the culture lifespan. qPCR analysis of four of these genes on one of each (NS2, S2) of the cell lines examined by microarray analysis confirmed that two genes (CRYAB and MGST1) both replicated the microarray results and were differentially regulated between the NS and S phenotypes.

BACKGROUND:To study the role of microRNA (miRNA) in the regulation of Chinese hamster ovary (CHO) cell growth, qPCR, microarray and quantitative LC-MS/MS analysis were utilised for simultaneous expression profiling of miRNA, mRNA and protein. The sample set under investigation consisted of clones with variable cellular growth rates derived from the same population. In addition to providing a systems level perspective on cell growth, the integration of multiple profiling datasets can facilitate the identification of non-seed miRNA targets, complement computational prediction tools and reduce false positive and false negative rates.RESULTS:51 miRNAs were associated with increased growth rate (35 miRNAs upregulated and 16 miRNAs downregulated). Gene ontology (GO) analysis of genes (n=432) and proteins (n=285) found to be differentially expressed (DE) identified biological processes driving proliferation including mRNA processing and translation. To investigate the influence of miRNA on these processes we combined the proteomic and transcriptomic data into two groups. The first set contained candidates where evidence of translational repression was observed (n=158). The second group was a mixture of proteins and mRNAs where evidence of translational repression was less clear (n=515). The TargetScan algorithm was utilised to predict potential targets within these two groups for anti-correlated DE miRNAs.CONCLUSIONS:The evidence presented in this study indicates that biological processes such as mRNA processing and protein synthesis are correlated with growth rate in CHO cells. Through the integration of expression data from multiple levels of the biological system a number of proteins central to these processes including several hnRNPs and components of the ribosome were found to be post-transcriptionally regulated. We utilised the expression data in conjunction with in-silico tools to identify potential miRNA-mediated regulation of mRNA/proteins involved in CHO cell growth rate. These data have allowed us to prioritise candidates for cell engineering and/or biomarkers relevant to industrial cell culture. We also expect the knowledge gained from this study to be applicable to other fields investigating the role of miRNAs in mammalian cell growth.

UNLABELLEDCoexpression analysis is a powerful, widely used methodology for the investigation of underlying patterns in gene expression data. This "guilt-by-association" approach aims to find groups of genes with closely correlated expression profiles. Observation of consistent correlations across phenotypically diverse samples indicates that these genes have a shared function. We have recently described the application of weighted gene coexpression network analysis (WGCNA) to a 295 sample production CHO cell line microarray dataset and elucidated groups of genes related to growth rate and cell-specific productivity (Qp). In this study, we present the CHO gene coexpression database (CGCDB), a web-based system, designed specifically for researchers in the CHO community to provide user-friendly access to these gene-gene coexpression patterns. In addition to correlation between genes, the direct correlations between probesets and either growth rate or Qp are provided. Results are presented to the user via an interactive network diagram and in a downloadable tabular format. It is hoped that this resource will allow researchers to prioritize cell line engineering and/or biomarker candidates to enhance CHO-based cell culture for the production of biotherapeutics.AVAILABILITYwww.cgcdb.org.

Weighted gene coexpression network analysis (WGCNA) was utilised to explore Chinese hamster ovary (CHO) cell transcriptome patterns associated with bioprocess relevant phenotypes. The dataset set used in this study consisted of 295 microarrays from 121 individual CHO cultures producing a range of biologics including monoclonal antibodies, fusion proteins and therapeutic factors; non-producing cell lines were also included. Samples were taken from a wide range of process scales and formats that varied in terms of seeding density, temperature, medium, feed medium, culture duration and product type. Cells were sampled for gene expression analysis at various stages of the culture and bioprocess-relevant characteristics including cell density, growth rate, viability, lactate, ammonium and cell specific productivity (Qp) were determined. WGCNA identified six distinct clusters of co-expressed genes, five of which were found to have associations with bioprocess variables. Two coexpression clusters were found to be associated with culture growth rate (1 positive and 1 negative). In addition, associations between a further three coexpression modules and Qp were observed (1 positive and 2 negative). Gene set enrichment analysis (GSEA) identified a number of significant biological processes within coexpressed gene clusters including cell cycle, protein secretion and vesicle transport. In summary, the approach presented in this study provides a novel perspective on the CHO cell transcriptome.

Improving the rate of recombinant protein production in Chinese hamster ovary (CHO) cells is an important consideration in controlling the cost of biopharmaceuticals. We present the first predictive model of productivity in CHO bioprocess culture based on gene expression profiles. The dataset used to construct the model consisted of transcriptomic data from 70 stationary phase, temperature-shifted CHO production cell line samples, for which the cell-specific productivity had been determined. These samples were utilised to investigate gene expression over a range of high to low monoclonal antibody and fc-fusion-producing CHO cell lines. We utilised a supervised regression algorithm, partial least squares (PLS) incorporating jackknife gene selection, to produce a model of cell-specific productivity (Qp) capable of predicting Qp to within 4.44 pg/cell/day root mean squared error in cross model validation (RMSECMV). The final model, consisting of 287 genes, was capable of accurately predicting Qp in a further panel of 10 additional samples which were incorporated as an independent validation. Several of the genes constituting the model are linked with biological processes relevant to protein metabolism.

The Chinese hamster ovary (CHO) cell line is one of the most widely used mammalian cell lines for biopharmaceutical production. We have developed and characterized a gene expression microarray (WyeHamster2a) specific for CHO cells that has enabled the study of ~3,500 sequences. Analysis of multiple sets of replicate scans showed that data derived from the WyeHamster2a array is highly reproducible confirming it as a robust tool for profiling. Twelve gene sequences were selected for follow-up RT-qPCR to confirm the accuracy and precision of the microarray results. In all but the most subtle gene expression differences, the microarray proved to be a reliable measure of differential gene expression. Finally, we were able to quantify the difference between using a bona fide CHO-specific microarray for profiling CHO cells versus an alternate, commercially available, rodent microarray such as a mouse or rat-specific format.

BACKGROUND:The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture.RESULTS:Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction.CONCLUSION:These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

Cell Stability, Animal Martin S. Sinacore, Martin S. Sinacore Genetics Institute, Andover, MassachusettsSearch for more papers by this authorTimothy S. Charlebois, Timothy S. Charlebois Genetics Institute, Andover, MassachusettsSearch for more papers by this authorDenis Drapeau, Denis Drapeau Genetics Institute, Andover, MassachusettsSearch for more papers by this authorMark Leonard, Mark Leonard Genetics Institute, Andover, MassachusettsSearch for more papers by this authorScott Harrison, Scott Harrison Genetics Institute, Andover, MassachusettsSearch for more papers by this authorS. Robert Adamson, S. Robert Adamson Genetics Institute, Andover, MassachusettsSearch for more papers by this author Martin S. Sinacore, Martin S. Sinacore Genetics Institute, Andover, MassachusettsSearch for more papers by this authorTimothy S. Charlebois, Timothy S. Charlebois Genetics Institute, Andover, MassachusettsSearch for more papers by this authorDenis Drapeau, Denis Drapeau Genetics Institute, Andover, MassachusettsSearch for more papers by this authorMark Leonard, Mark Leonard Genetics Institute, Andover, MassachusettsSearch for more papers by this authorScott Harrison, Scott Harrison Genetics Institute, Andover, MassachusettsSearch for more papers by this authorS. Robert Adamson, S. Robert Adamson Genetics Institute, Andover, MassachusettsSearch for more papers by this author First published: 15 April 2010 https://doi.org/10.1002/9780470054581.eib597 Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Factors that Affect Genetic Stability of Endogenous Genes Oncogenic Transformation, Aneuploidy, and Genetic Stability Molecular Regulation of Genetic Stablity Genotypic and Phenotypic Stability of Recombinant Cell Lines Stable Integration of Heterologous Genes in Animal Cell Genomes Phenotypic Heterogeneity and Population Dynamics Genetic and Epigenetic Manipulation of Cell Phenotypes Consequences of Genetic Instability on Recombinant Protein Quality Genotypic Characterization and Validation of Genetic Stability Gross Structural Evaluation of Genes and Transcripts Sequence Analysis of Genes and Transcripts Encoding the Product Genetic Stability References Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell TechnologyBrowse other articles of this reference work:BROWSE A-Z RelatedInformation

A high rate of cell growth (µ) leading to rapid accumulation of viable biomass is a desirable phenotype during scale up operations and the early stages of production cultures. In order to identify genes and proteins that contribute to higher growth rates in Chinese hamster ovary (CHO) cells, a combined approach using microarray and proteomic expression profiling analysis was carried out on two matched pairs of CHO production cell lines that displayed either fast or slow growth rates. Statistical analysis of the microarray and proteomic data separately resulted in the identification of 118 gene transcripts and 58 proteins that were differentially expressed between the fast‐ and slow‐growing cells. Overlap comparison of both datasets identified a priority list of 21 candidates associated with a high growth rate phenotype in CHO. Functional analysis (by siRNA) of five of these candidates identified the valosin‐containing protein (VCP) as having a substantial impact on CHO cell growth and viability. Knockdown of HSPB1 and ENO1 also had an effect on cell growth (negative and positive, respectively). Further functional validation in CHO using both gene knockdown (siRNA) and overexpression (cDNA) confirmed that altered VCP expression impacted CHO cell proliferation, indicating that VCP and other genes and proteins identified here may play an important role in the regulation of CHO cell growth during log phase culture and are potential candidates for CHO cell line engineering strategies. Biotechnol. Bioeng. 2010; 106: 42–56. © 2010 Wiley Periodicals, Inc.

We have previously reported on the development and use of a proprietary CHO-specific microarray and proteomics technology platforms to interrogate large and diverse sets of CHO cell samples, which embodied several industrially relevant phenotypes. In a collaboration between Wyeth BioPharma and NICB/Dublin City University, we have completed the analysis of 375 individual samples representing 29 different recombinant CHO cell lines expressing monoclonal antibody, receptor-Fc fusion molecules, coagulation factors or growth factors. In this paper, we will present key learnings and insights that we have made upon analysis of the large dataset generated by the collaboration. Of note is that, despite sometimes large differences in observed phenotypes, the changes that we measured in the genomics and proteomics experiments were relatively subtle. Lastly, functional validation is important to confirming the relevance of any particular target. The development of a robust, comprehensive, and high-throughput validation workflow will be discussed.

For a Phase 1 antibody project, the primary objective of our cell line development group is to deliver a stable, high performing, clonal cell line in an aggressive timeframe, leaving little time to react to the challenges associated with cell line instability, such as dramatically reduced product expression. Our current cell line development timeline allows for 6.5 months from transfection to the establishment of a cell bank for a lead clone, which is expected to deliver 1-3 g/L in a fed-batch process. Our cell line selection process consists of numerous rounds of successive screening, initially assessing hundreds of clones. During that time, clones are eliminated because of poor performance in scaled-down models of the fed-batch process or because of a loss of product expression over time. When such instability in product expression occurs for a lead clone at a late stage in the timeline, both upstream and downstream process groups must quickly identify alternatives so that the material needs of the project will be satisfied and deadlines are still met. This manuscript presents examples of cell line instability that we have encountered during Phase 1 antibody projects, and the operational consequences that have ensued. It describes the different types of instability we have observed, our attempts to analyze the presumptive underlying genotypic causes of these different types of instability, and the general methods and tools that we use to investigate cell line instability. Finally, we describe the recent development of an RT-PCR-based assay that allows us to eliminate potentially unstable cell lines from consideration early in the cell line development timeline.

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant protein biopharmaceuticals. The purpose of this study was to investigate differences in the proteome of CHO DUKX cells expressing recombinant human bone morphogenetic protein‐2 (rhBMP‐2) (G5 cells) compared to cells also expressing soluble exogenous paired basic amino acid cleaving enzyme soluble paired basic amino acid cleaving enzyme (PACEsol) (3C9 cells), which has been previously found to improve the post‐translational processing of the mature rhBMP‐2 dimer. PACEsol co‐expression was also associated with a significant increase (almost four‐fold) in cellular productivity of rhBMP‐2 protein. Differential proteomic expression profiling using 2‐D DIGE and MALDI‐TOF MS was performed to compare 3C9 and G5 cells, and revealed a list of 60 proteins that showed differential expression (up/downregulated), with a variety of different cellular functions. A substantial number of these altered proteins were found to have chaperone activity, involved with protein folding, assembly and secretion, as well as a number of proteins involved in protein translation. These results support the use of proteomic profiling as a valuable tool towards understanding the biology of bioprocess cultures.

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry for the production of recombinant human proteins including complex polypeptides such as recombinant human bone morphogenic protein 2 (rhBMP-2). Large-scale manufacture of rhBMP-2 has associated production difficulties resulting from incomplete processing of the recombinant human protein due to insufficient endogenous levels of the paired basic amino acid cleaving enzyme (PACE) in CHO. In order to resolve this issue, CHO DUKX cells expressing rhBMP-2 were transfected with the soluble version of human PACE (PACEsol) resulting in improved amino-terminal homogeneity and a fourfold increase in rhBMP-2 productivity. In this article, we present a microarray expression profile analysis comparing the parental lineage to the higher producing subclone co-expressing PACEsol using a proprietary CHO-specific microarray. Using this technology we observed 1,076 significantly different genes in the high-productivity cells co-expressing PACEsol. Following further analysis of the differentially expressed genes, the Unfolded Protein Response (UPR) component of the endoplasmic reticulum stress response pathway was identified as a key candidate for effecting increased productivity in this cell system. Several additional ER- and Golgi-localised proteins were identified which may also contribute to this effect. The results presented here support the use of large-scale microarray expression profiling as a viable and valuable route towards understanding the behaviour of bioprocess cultures in vitro.

A novel soluble receptor:Fc fusion protein was designed as a recombinant therapeutic targeting human IL-13, a central mediator of asthma. This protein, sIL-13R, was expressed in stable CHO cell lines for the purpose of producing sufficient amounts of material to support early clinical trials. However, expression levels of sIL-13R were poor, and the protein was prone to misfolding and aggregation. Culturing the recombinant CHO cell lines at reduced temperatures significantly enhanced expression levels and lowered the levels of aggregated material. An even more dramatic improvement was observed when sIL-13R was coexpressed with its natural ligand, IL-13, in CHO cells. Protein expression was enhanced at both 37 degrees C and 31 degrees C, and product aggregation was significantly reduced. The sIL-13R protein secreted by the coexpressing cell line adopted a conformation that remained stable even after removal of the ligand during purification. Together, these results suggest a possible strategy for improving the expression of other difficult to express soluble proteins.