Sustained productivity in recombinant Chinese hamster ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype.

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.

Development and characterization of a Chinese hamster ovary cell-specific oligonucleotide microarray.

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.

Microarray and proteomics expression profiling identifies several candidates, including the valosin-containing protein (VCP), involved in regulating high cellular growth rate in production CHO cell lines.

A high rate of cell growth (micro) 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.

Proteomic profiling of CHO cells with enhanced rhBMP-2 productivity following co-expression of PACEsol.

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.

Transcriptional profiling of gene expression changes in a PACE-transfected CHO DUKX cell line secreting high levels of rhBMP-2.

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.

The Hsp70 and TRiC/CCT chaperone systems cooperate in vivo to assemble the von Hippel-Lindau tumor suppressor complex.

The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell. Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex. VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells. Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells. Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70. VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4, a subunit of the TRiC/CCT complex. Analysis of the VHL-chaperone interactions in these strains revealed that the cct4ts mutation decreased binding to TRiC but did not affect the interaction with Hsp70. In contrast, loss of Hsp70 function disrupted the interaction of VHL with both Hsp70 and TRiC. We conclude that, in vivo, folding of some polypeptides requires the cooperation of Hsp70 and TRiC and that Hsp70 acts to promote substrate binding to TRiC.

Mammalian cell line developments in speed and efficiency.

Mammalian cell expression systems are the dominant tool today for producing complex biotherapeutic proteins. In this chapter, we discuss the basis for this dominance, and further explore why the Chinese hamster ovary (CHO) cell line has become the prevalent choice of hosts to produce most recombinant biologics. Furthermore, we explore some of the innovations that are currently in development to improve the CHO cell platform, from cell line specific technologies to overarching technologies that are designed to improve the overall workflow of bioprocess development.

Microarray expression profiling identifies genes regulating sustained cell specific productivity (S-Qp) in CHO K1 production cell lines.

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.

Predicting cell-specific productivity from CHO gene expression.

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 (RMSE(CMV)). 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.

Large scale microarray profiling and coexpression network analysis of CHO cells identifies transcriptional modules associated with growth and productivity.

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.

The direct binding of the catalytic subunit of protein phosphatase 1 to the PKR protein kinase is necessary but not sufficient for inactivation and disruption of enzyme dimer formation.

The PKR protein kinase is among the best-studied effectors of the host interferon (IFN)-induced antiviral and antiproliferative response system. In response to stress signals, including virus infection, the normally latent PKR becomes activated through autophosphorylation and dimerization and phosphorylates the eIF2alpha translation initiation factor subunit, leading to an inhibition of mRNA translation initiation. While numerous virally encoded or modulated proteins that bind and inhibit PKR during virus infection have been studied, little is known about the cellular proteins that counteract PKR activity in uninfected cells. Overexpression of PKR in yeast also leads to an inhibition of eIF2alpha-dependent protein synthesis, resulting in severe growth suppression. Screening of a human cDNA library for clones capable of counteracting the PKR-mediated growth defect in yeast led to the identification of the catalytic subunit (PP1(C)) of protein phosphatase 1alpha. PP1(C) reduced double-stranded RNA-mediated auto-activation of PKR and inhibited PKR transphosphorylation activities. A specific and direct interaction between PP1(C) and PKR was detected, with PP1(C) binding to the N-terminal regulatory region regardless of the double-stranded RNA-binding activity of PKR. Importantly, a consensus motif shared by many PP1(C)-interacting proteins was necessary for PKR binding to PP1(C). The PKR-interactive site was mapped to a C-terminal non-catalytic region that is conserved in the PP1(C)2 isoform. Indeed, co-expression of PP1(C) or PP1(C)2 inhibited PKR dimer formation in Escherichia coli. Interestingly, co-expression of a PP1(C) mutant lacking the catalytic domain, despite retaining its ability to bind PKR, did not prevent PKR dimerization. Our findings suggest that PP1(C) modulates PKR activity via protein dephosphorylation and subsequent disruption of PKR dimers.