Identification of autocrine growth factors secreted by CHO cells for applications in single-cell cloning media.

Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However, regulatory authorities have discouraged the use of FBS to reduce the risk of contamination by adventitious agents from animal-derived components. In this study, we demonstrated how a complementary mass spectrometry-based shotgun proteomics strategy enabled the identification of autocrine growth factors in CHO cell-conditioned media, which has led to the development of a fully defined single-cell cloning media that is serum and animal component-free. Out of 290 secreted proteins that were identified, eight secreted growth factors were reported for the first time from CHO cell cultures. By supplementing a combination of these growth factors to protein-free basal media, single cell growth of CHO cells was improved with cloning efficiencies of up to 30%, a 2-fold improvement compared to unsupplemented basal media. Complementary effects of these autocrine growth factors with other paracrine growth factors were also demonstrated when the mixture improved cloning efficiency to 42%, similar to that for the conditioned medium.

Profiling of N-glycosylation gene expression in CHO cell fed-batch cultures.

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N-glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N-glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed-batch culture of a CHO cell line producing recombinant human interferon-gamma (IFN-gamma). Of the 24 N-glycosylation genes examined, 21 showed significant up- or down-regulation of gene expression as the fed-batch culture progressed from exponential, stationary and death phase. As the fed-batch culture progressed, there was also an increase in less sialylated IFN-gamma glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN-gamma. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed-batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N-glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible "bottlenecks" or "compromised" pathways in N-glycosylation and subsequently allow for the development of strategies to improve glycosylation quality.

Enhancement of plasmid DNA yields during fed-batch culture of a fruR-knockout Escherichia coli strain.

Well-characterized derivatives of Escherichia coli K12 such as DH5alpha are the host strains commonly used for plasmid DNA production. Owing to the prospective clinical demand for large quantities of plasmid DNA for gene therapy and DNA vaccination, existing plasmid production processes need to be optimized to attain higher plasmid yields. To date, nearly all production optimization efforts are focused on media or fermentation process design. Although there has been a simple empirical evaluation of the available host strains, there is a lack of systematic effort at engineering these host strains for improved plasmid DNA production. In view of this, we engineered DH5alpha WT (wild-type) cells carrying a DNA vaccine plasmid by knocking out the fruR (fructose repressor) [also known as the Cra (catabolite repressor activator)] global regulator gene and evaluated the growth and plasmid yields of these P+ (plasmid-bearing) fruR cells (fruR-knockout cells) during fed-batch cultures with exponential feeding. The P+ fruR cells showed a more rapid accumulation of plasmid DNA towards the end of the fed-batch cultures compared with the P+ WT cells. As a result, the specific plasmid yield of the P+ fruR cells was 21% higher than that of the P+ WT cells [19.2 versus 15.9 mg/g DCW (dry cell weight)]. These results demonstrate that, from an initial high-yielding fermentation process, the knockout of the fruR global regulator gene in E. coli DH5alpha further improves plasmid yields during fed-batch culture.

Inactivating FruR global regulator in plasmid-bearing Escherichia coli alters metabolic gene expression and improves growth rate.

The introduction of plasmids into Escherichia coli is known to impose a metabolic burden, which diminishes the growth rate. This effect could arise from perturbation of the central metabolic pathways, which supply precursors and energy for macromolecule synthesis. We knocked out a global regulator of central metabolism, FruR (also called Cra), to assess its phenotypic effect in E. coli carrying plasmids. During bioreactor runs, a higher specific growth rate of 0.91h(-1) was observed for the plasmid-bearing fruR knockout (P+ fruR) cells compared to its parental plasmid-bearing wildtype (P+ WT) cells (0.75h(-1)), while both the plasmid-free cells displayed similar growth rates (1.0h(-1), respectively). To investigate gene expression changes possibly related to the growth rate recovery, quantitative reverse transcriptase PCR and 2DE proteomic studies were performed. In P+ fruR cells, expression of enzymes involved in sugar catabolism, glycolysis and transcription/translation processes were upregulated, while those related to gluconeogenesis, tricarboxylic acid cycle and stress response were downregulated. Our findings demonstrate that the inactivation of FruR global regulator in recombinant E. coli alters metabolic gene expression and significantly reduces growth retardation from the burden of maintaining a plasmid. This study represents the first attempt to explore the role of a global regulatory gene on plasmid metabolic burden.

Mammalian Systems Biotechnology Reveals Global Cellular Adaptations in a Recombinant CHO Cell Line.

Effective development of host cells for therapeutic protein production is hampered by the poor characterization of cellular transfection. Here, we employed a multi-omics-based systems biotechnology approach to elucidate the genotypic and phenotypic differences between a wild-type and recombinant antibody-producing Chinese hamster ovary (CHO) cell line. At the genomic level, we observed extensive rearrangements in specific targeted loci linked to transgene integration sites. Transcriptional re-wiring of DNA damage repair and cellular metabolism in the antibody producer, via changes in gene copy numbers, was also detected. Subsequent integration of transcriptomic data with a genome-scale metabolic model showed a substantial increase in energy metabolism in the antibody producer. Metabolomics, lipidomics, and glycomics analyses revealed an elevation in long-chain lipid species, potentially associated with protein transport and secretion requirements, and a surprising stability of N-glycosylation profiles between both cell lines. Overall, the proposed knowledge-based systems biotechnology framework can further accelerate mammalian cell-line engineering in a targeted manner.

Identification of cellular objective for elucidating the physiological state of plasmid-bearing Escherichia coli using genome-scale in silico analysis.

The presence of multiple copies of plasmids in Escherichia coli could induce a complex cascade of physiological changes known as the metabolic burden response. In this work, the physiological effect of such plasmid metabolic burden on E. coli metabolism was investigated by constraint-based genome-scale flux modeling. We systematically applied three cellular objectives: (a) maximizing growth rate, (b) maximizing plasmid production, and (c) maximizing maintenance energy expenditure to quantify in silico flux distributions. These simulated results were compared with experimental flux information to identify which of these cellular objectives best describes the physiological and metabolic states of plasmid-bearing (P+) E. coli. Unlike the wild-type E. coli cells that have directed the metabolism toward an optimum growth rate under the nutrient-limited condition, the maximum growth rate objective could not correctly predict the metabolic state of recombinant P+ cells. Instead, flux simulations by maximizing maintenance energy expenditure showed good consistency with experimental observation, indicating that the P+ cells are energetically less efficient and could require higher maintenance energy. This study demonstrates that the cellular objective of maximizing maintenance energy expenditure provides a better description of the underlying physiological state in recombinant microorganisms relevant to biotechnological applications.

Identification of HUGT1 as a potential BiP activator and a cellular target for improvement of recombinant protein production using a cDNA screening system.

The development of a high-throughput functional genomic screening provides a novel and expeditious approach in identifying critical genes involved in specific biological processes. Here we describe a cell-based cDNA screening system to identify the transcription activators of BiP, an endoplasmic reticulum (ER) chaperone protein. BiP promoter contains the ER stress element which is commonly present in the genes involved in unfolded protein response (UPR) that regulates protein secretion in cells. Therefore, the positive regulators of BiP may also be utilized to improve the recombinant protein production through modulation of UPR. Four BiP activators, including human UDP-glucose:glycoprotein glucosyltransferase 1 (HUGT1), are identified by the cDNA screening. Overexpression of HUGT1 leads to a significant increase in the production of recombinant erythropoietin, interferon gamma, and monoclonal antibody in HEK293 cells. Our results demonstrate that the cDNA screening for BiP activators may be effective to identify the novel BiP regulators and HUGT1 may serve as an ideal target gene for improving the recombinant protein production in mammalian cells.

Targeting early apoptotic genes in batch and fed-batch CHO cell cultures.

Based on the transcriptional profiling of CHO cell culture using microarray, four key early apoptosis signaling genes, Fadd, Faim, Alg-2, and Requiem, were identified and CHO GT (Gene Targeted) cell lines were developed by targeting these four genes. Two were CHO GT(O) cell lines overexpressing anti-apoptotic genes, Faim and Fadd DN and two were CHO GT(KD) cell lines involving knockdown of Alg-2 and Requiem which are pro-apoptotic genes using small interfering RNA (siRNA) technology. Comparisons of these CHO GT cell lines with the parental cell line in batch culture (BC) and fed-batch culture (FBC) were performed. Compared to parental cells, the CHO GT cell lines showed apoptosis resistance as they significantly delayed and/or suppressed initiator caspase-8 and -9 and executioner caspase-3 activities during culture. FBC of CHO GT cell lines reached significantly higher maximum viable cell densities (up to 9 x 10(6) cells/mL) compared with the parental cell line (5 x 10(6) cells/mL). The recombinant interferon gamma (IFN-gamma) yields were increased by up to 2.5-fold. Furthermore, it was observed that the IFN-gamma was more highly sialylated.

Transcriptional profiling of apoptotic pathways in batch and fed-batch CHO cell cultures.

Chinese Hamster ovary (CHO) cells are regarded as one of the "work-horses" for complex biotherapeutics production. In these processes, loss in culture viability occurs primarily via apoptosis, a genetically controlled form of cellular suicide. Using our "in-house" developed CHO cDNA array and a mouse oligonucleotide array for time profile expression analysis of batch and fed-batch CHO cell cultures, the genetic circuitry that regulates and executes apoptosis induction were examined. During periods of high viability, most pro-apoptotic genes were down-regulated but upon loss in viability, several early pro-apoptotic signaling genes were up-regulated. At later stages of viability loss, we detected late pro-apoptotic effector genes such as caspases and DNases being up-regulated. This sequential regulation of apoptotic genes showed that DNA microarrays could be used as a tool to study apoptosis. We found that in batch and fed-batch cultures, apoptosis signaling occurred primarily via death receptor- and mitochondria-mediated signaling pathways rather than endoplasmic reticulum-mediated signaling. These insights provide a greater understanding of the regulatory circuitry of apoptosis during cell culture and allow for subsequent targeting of relevant apoptosis signaling genes to prolong cell culture.

Determination of interferon-gamma in Chinese hamster ovary cell culture supernatant by coupled-column liquid chromatography.

A robust tandem HPLC method coupling size-exclusion (Shodex Asahipak GS-320HQ) and reversed phase (Vydac 218TP54) columns with ultraviolet detection was developed for quantitative determination of interferon-gamma (IFN-gamma) in Chinese hamster ovary cell culture supernatant. The 2D-HPLC system was linked up by a 6-port 2-position low hold-up volume switch valve. Compared to a commercial ELISA kit for IFN-gamma, the coupled column LC approach was able to detect and quantify soluble IFN-gamma, regardless of the glycoprotein's molecular/conformational variability and sample background. Each LC-LC analysis took 90 minutes inclusive of column regeneration. The relative standard deviation of measurements (n = 5) was less than 3%. The limit of detection (LOD) was determined to be 0.35 microg IFN-gamma.