Transcriptome dynamics of transgene amplification in Chinese hamster ovary cells.

Dihydrofolate reductase (DHFR) system is used to amplify the product gene to multiple copies in Chinese Hamster Ovary (CHO) cells for generating cell lines which produce the recombinant protein at high levels. The physiological changes accompanying the transformation of the non-protein secreting host cells to a high producing cell line is not well characterized. We performed transcriptome analysis on CHO cells undergoing the selection and amplification processes. A host CHO cell line was transfected with a vector containing genes encoding the mouse DHFR (mDHFR) and a recombinant human IgG (hIgG). Clones were isolated following selection and subcloned following amplification. Control cells were transfected with a control plasmid which did not have the hIgG genes. Although methotrexate (MTX) amplification increased the transcript level of the mDHFR gene significantly, its effect on both hIgG heavy and light chain genes was more modest. The subclones appeared to retain the transcriptome signatures of their parental clones, however, their productivity varied among those derived from the same clone. The transcript levels of hIgG transgenes of all subclones fall in a narrower range than the product titer, alluding to the role of many functional attributes, other than transgene transcript, on productivity. We cross examined functional class enrichment during selection and amplification as well as between high and low producers and discerned common features among them. We hypothesize that the role of amplification is not merely increasing transcript levels, but also enriching survivors which have developed the cellular machinery for secreting proteins, leading to an increased frequency of isolating high-producing clones. We put forward the possibility of assembling a hyper-productivity gene set through comparative transcriptome analysis of a wide range of samples.

Dynamic gene expression for metabolic engineering of mammalian cells in culture.

Recombinant mammalian cells are the major hosts for the production of protein therapeutics. In addition to high expression of the product gene, a hyper-producer must also harbor superior phenotypic traits related to metabolism, protein secretion, and growth control. Introduction of genes endowing the relevant hyper-productivity traits is a strategy frequently used to enhance the productivity. Most of such cell engineering efforts have been performed using constitutive expression systems. However, cells respond to various environmental cues and cellular events dynamically according to cellular needs. The use of inducible systems allows for time dependent expression, but requires external manipulation. Ideally, a transgene's expression should be synchronous to the host cell's own rhythm, and at levels appropriate for the objective. To that end, we identified genes with different expression dynamics and intensity ranges using pooled transcriptome data. Their promoters may be used to drive the expression of the transgenes following the desired dynamics. We isolated the promoter of the Thioredoxin-interacting protein (Txnip) gene and demonstrated its capability to drive transgene expression in concert with cell growth. We further employed this Chinese hamster promoter to engineer dynamic expression of the mouse GLUT5 fructose transporter in Chinese hamster ovary (CHO) cells, enabling them to utilize sugar according to cellular needs rather than in excess as typically seen in culture. Thus, less lactate was produced, resulting in a better growth rate, prolonged culture duration, and higher product titer. This approach illustrates a novel concept in metabolic engineering which can potentially be used to achieve dynamic control of cellular behaviors for enhanced process characteristics.

Process engineering of natural killer cell-based immunotherapy.

Cell therapy offers the potential for curative treatment of cancers. Although T cells have been the predominantly used cell type, natural killer (NK) cells have attracted great attention owing to their ability to kill cancer cells and because they are naturally suitable for allogeneic applications. Upon stimulation by cytokines or activation by a target cell, NK cells proliferate and expand their population. These cytotoxic NK cells can be cryopreserved and used as an off-the-shelf medicine. The production process for NK cells thus differs from that of autologous cell therapies. We briefly outline key biological features of NK cells, review the manufacturing technologies for protein biologics, and discuss their adaptation for developing robust NK cell biomanufacturing processes.

Reaching the depth of the Chinese hamster ovary cell transcriptome.

The high-throughput DNA sequencing Illumina Solexa GAII platform was employed to characterize the transcriptome of an antibody-producing Chinese hamster ovary (CHO) cell line. More than 55 million sequencing reads were generated and mapped to an existing set of CHO unigenes derived from expressed sequence tags (ESTs), as well as several public sequence databases. A very significant fraction of sequencing reads has not been previously seen. The frequency with which fragments of a unigene were sequenced was taken as an estimate of the abundance level of the corresponding transcripts. A wide dynamic range of transcript abundance levels was observed, spanning six orders of magnitude. However, the distribution of coverage across transcript lengths was found to vary, from relatively uniform to highly variable. This observation suggests that more challenges are yet to be resolved before direct sequencing can be used as a true quantitative measure of transcript level and for differential gene expression analysis. With the depth that high-throughput sequencing methods can reach, one can expect that the entire transcriptome of this industrially important organism will be decoded in the near future.

Mammalian cell cultures for biologics manufacturing.

Biopharmaceuticals represent a growing sector of the pharmaceutical industry, and are used for a wide range of indications, including oncology and rheumatology. Cultured mammalian cells have become the predominant expression system for their production, partly due to their ability to complete the posttranslational modifications required for drug safety and efficacy. Over the past decade, the productivity of mammalian cell culture production processes has growth dramatically through improvements in both volumetric and specific productivities. This article presents an overview of the biologics market, including analysis of sales and approvals; as well as a review of industrial production cell lines and cell culture operations.

LDH-C can be differentially expressed during fermentation of CHO cells.

Expression of CHO mRNA was measured with special microarrays from the Consortium for Chinese Hamster Ovary (CHO) Cell Genomics led by Prof. Wei-Shou Hu of the University of Minnesota and Prof. Miranda Yap of the Bioprocess Technology Institute of A*STAR, Singapore (http://hugroup.cems.umn.edu/CHO/cho_index.html). Cultivation experiments were performed in small scale 2L stirred tank bioreactors. During fermentation a temperature shift of -3°C was performed. This was accompanied by a reduction of the cell specific lactate production rate. The analysis of transcriptome samples before and after the temperature shift with microarrays showed several changes in the expression of available gene markers. LDH-C expression raised about 2 fold after temperature shift. LDH-A did not change. As LDH-C is known to be a specialized isoenzyme in sperm cells for consuming lactate in a lactate containing milieu, LDH-C could be proposed as a target for genetic engineering, facilitating lactate consumption in the late phase of high cell density cultures and prolonging longevity of CHO production cultures by reducing lactate and base accumulation.

Transcriptome and proteome analysis of Chinese hamster ovary cells under low temperature and butyrate treatment.

Recombinant Chinese hamster ovary (CHO) cells selected for high productivity are capable of secreting immunoglobulin G (IgG) molecules at a level that rivals plasma cells in vivo. Following butyrate treatment at 33 degrees C, further increases in productivity are observed. To better understand the mechanisms by which this increased productivity is incurred, the transcriptional response of an antibody-producing cell line undergoing these treatments was investigated using oligo-DNA microarrays. Using distance calculations, more than 900 genes were identified as kinetically differentially expressed between the butyrate-treated 33 degrees C culture and the untreated culture. Furthermore, transcript levels of the heavy and light chain IgG genes increased following treatment. Using stable isotope labeling (SILAC), the secretion rate of IgG was investigated by tracking the decay of the isotope label upon switching to unlabeled medium. Both treated and untreated cultures exhibited very similar IgG secretion kinetics. In contrast, the intracellular IgG content was found to be elevated following treatment. This result suggests that increased productivity under treatment is attributable to elevated cellular secretory capacity, rather than shorter holding times in the secretory pathway. This hypothesis is further supported by the results of gene set enrichment analysis (GSEA), which revealed that elements of the secretory pathway, including Golgi apparatus, cytoskeleton protein binding and small GTPase-mediated signal transduction are enriched and thus may play a role in the increased recombinant protein production observed under butyrate treatment at 33 degrees C.

Developing genomic platforms for Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used in recombinant protein production, yet despite their importance in bioprocessing, few genomic resources have been developed for this cell line. Over the past several years, we have made considerable progress in the development of genomic tools for CHO. Using Sanger-based sequencing technology, we have accrued a sequence repertoire of more than 68,000 expressed sequence tags (ESTs), representing more than 28,000 unique CHO transcripts. Using closely related species, we have functionally annotated this sequence set and have currently achieved significant representation in a number of functional classes, including some closely tied to recombinant protein production. This sequence repository has been used to design custom CHO Affymetrix arrays for transcriptome analysis. Illumina Solexa deep sequencing technology was also applied to study the CHO cell transcriptome and survey the identity and expression of small RNAs. These applications demonstrate the utility of genomic tools, and illustrate the applicability of emerging next-generation sequencing technologies.