Yih Yean Lee

Transcriptome and Proteome Profiling to Understanding the Biology of High Productivity CHO Cells

A combined transcriptome and proteome analysis was carried out to identify key genes and proteins differentially expressed in Chinese hamster ovary (CHO) cells producing high and low levels of dhfr-GFP fusion protein. Comparison of transcript levels was performed using a proprietary 15 K CHO cDNA microarray chip, whereas proteomic analysis was perfomed using iTRAQ quantitative protein profiling technique. Microarray analysis revealed 77 differentially expressed genes, with 53 genes upregulated and 24 genes downregulated. Proteomic analysis gave 75 and 80 proteins for the midexponential and stationary phase, respectively. Although there was a general lack of correlation between mRNA levels and quantitated protein abundance, results from both datasets concurred on groups of proteins/genes based on functional categorization. A number of genes (20%) and proteins (45 and 23%) were involved in processes related to protein biosynthesis. We also identified three genes/proteins involved in chromatin modification. Enzymes responsible for opening up chromatin, Hmgn3 and Hmgb1, were upregulated whereas enzymes that condense chromatin, histone H1.2, were downregulated. Genes and proteins that promote cell growth (Igfbp4, Ptma, S100a6, and Lgals3) were downregulated, whereas those that deter cell growth (Ccng2, Gsg2, and S100a11) were upregulated. Other main groups of genes and proteins include carbohydrate metabolism, signal transduction, and transport. Our findings show that an integrated genomic and proteomics approach can be effectively utilized to monitor transcriptional and posttranscriptional events of mammalian cells in culture.

Engineering mammalian cells in bioprocessing – current achievements and future perspectives

Over the past 20 years, we have seen significant improvements in product titres from 50 mg/l to 5–10 g/l, a more than 100‐fold increase. The main methods that have been employed to achieve this increase in product titre have been through the manipulation of culture media and process control strategies, such as the optimization of fed‐batch processes. An alternative means to increase productivity has been through the engineering of host cells by altering cellular processes. Recombinant DNA technology has been used to over‐express or suppress specific genes to endow particular phenotypes. Cellular processes that have been altered in host cells include metabolism, cell cycle, protein secretion and apoptosis. Cell engineering has also been employed to improve post‐translational modifications such as glycosylation. In this article, an overview of the main cell engineering strategies previously employed and the impact of these strategies are presented. Many of these strategies focus on engineering cell lines with more efficient carbon metabolism towards reducing waste metabolites, achieving a biphasic production system by engineering cell cycle control, increasing protein secretion by targeting specific endoplasmic reticulum stress chaperones, delaying cell death by targeting anti‐apoptosis genes, and engineering glycosylation by enhancing recombinant protein sialylation and antibody glycosylation. Future perspectives for host cell engineering, and possible areas of research, are also discussed in this review.

Low-Glutamine Fed-Batch Cultures of 293-HEK Serum-Free Suspension Cells for Adenovirus Production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low‐glutamine fed‐batch cultures of serum‐free, suspension cells in a 5‐L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell‐exclusion system that allows for aseptic, cell‐free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose‐free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed‐batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed‐batch cultures. In all cases, fed‐batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed‐batch culture using adenovirus‐green fluorescent protein (Ad‐GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed‐batch culture showed that there is an approximately 10‐fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed‐batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.

Overexpression of heat shock proteins (HSPs) in CHO cells for extended culture viability and improved recombinant protein production

It has been widely reported that CHO cells undergo apoptosis in culture, despite supplementation of nutrients through fed-batch strategies. Improvement of cell viability in culture can effectively improve recombinant protein yield through extension of the cultures production lifespan, especially at high cell densities. Heat shock proteins (HSPs) have been reported to demonstrate anti-apoptotic effects against a wide range of physical and chemical stimuli through their ability to bind and act as antagonists to critical apoptotic molecules. CHO-IFN-gamma cells, expressing recombinant human interferon-gamma (IFN-gamma), were engineered to overexpress two HSPs (HSP27 and HSP70) either individually or in combination. In fed-batch bioreactor cultures, the engineered cell lines exhibited a more gradual viability loss and extension of culture times of 36-72h, with corresponding delays in escalation of caspases 2, 3, 8 and 9 activities, compared to the control cultures utilizing cells transfected with the vector backbone. The extension in culture times translated to a 2.5-fold improvement in IFN-gamma production over controls in fed-batch cultures. These results suggest that overexpression of HSPs represents a promising generic strategy for the development of robust CHO cell lines resistant to apoptotic insults and possessing improved culture characteristics to enhance recombinant glycoprotein yields.

Regulation of XBP-1 signaling during transient and stable recombinant protein production in CHO cells.

X‐box binding protein 1 (XBP‐1) is a key regulator of cellular unfolded protein response (UPR). The spliced isoform of XBP‐1, XBP‐1S, is a transcription activator, which is expressed only when UPR is induced. However, the impact of recombinant protein production on the regulation of XBP‐1 signaling in CHO cells is not well understood. In this report, we cloned the Chinese hamster XBP‐1 homolog to aid the investigation of the interplay between protein productivity, culture conditions, and endogenous XBP‐1 signaling in CHO cells. Interestingly, expression of XBP‐1S is detected in the non‐producing and unstressed CHO‐K1 cells. Transient expression of recombinant erythropoietin reveals a positive correlation between XBP‐1 mRNA abundance and protein production level. However, such a correlation is not observed in batch cultivation of stable producing cell lines. The increased XBP‐1 splicing is detected in late‐phase cultures, suggesting that induction of XBP‐1S may be a result of nutrient limitations or other environmental stresses rather than that of increased intracellular accumulation of recombinant proteins. Our data suggest that XBP‐1 is a key determinant for the secretory capacity of CHO cells. Understanding its dynamic regulation hence provides a rational basis for cellular engineering strategies to improve recombinant protein secretion.

Modeling Amino Acid Metabolism in Mammalian Cells‐Toward the Development of a Model Library

Amino acids are necessary to mammalian cell cultures both for protein synthesis and as an energy source. In this study, we present an unstructured mathematical model describing (i) cell growth and death kinetics and (ii) metabolism of glucose and 19 amino acids for HEK‐293 and CHO IFN‐γ cell cultures. The proposed mathematical framework is in good agreement with experimental data for both cell lines. It accommodates the inclusion of expressions for other cellular activities, such as the production of recombinant viral vectors or proteins, and can be used as the basis for the development of a model library for mammalian cell cultures.

Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed‐batch bioreactors

CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures.

Identification of cellular genes critical to recombinant protein production using a Gaussia luciferase-based siRNA screening system

Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon gamma, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells.

TRANSCRIPTIONAL PROFILING OF BATCH AND FED-BATCH PROTEIN-FREE 293-HEK CULTURES USING DNA MICROARRAY

Dynamic nutrient feeding to control glutamine at low levels in protein-free fed-batch cultures of 293-human embryonic kidney (HEK) cells achieved cell concentrations of 6 x 10(6) cells/ml. This represented a 4-fold improvement in cell concentration compared to batch cultures. Reduction in glutamine and glucose consumption, as well as lactate and ammonia production, were also observed in these fed-batch cultures. High virus production titers of 3 x 10(11) pfu/ml were achieved in fed-batch cultures which were 10,000-fold higher than batch cultures. An investigation of the transcriptional regulation of the metabolic changes associated with the batch and the low-glutamine fed-batch cultures using DNA microarray was conducted. This analysis provides better understanding of the transcriptional regulatory mechanism resulting in the observed physiological changes. Transcriptional profiling of cells from the mid-exponential, late exponential and stationary phases of both the batch and fed-batch were undertaken using an 18,000 element human chip. Transcriptional profiles were ontologically classified to provide a global view of the genetic changes. Furthermore, a pathway-oriented analysis focusing on cellular metabolism was conducted to reveal the dynamic regulation of genes related to amino acid metabolism, tRNA synthetases, TCA cycle, electron transport chain and glycolysis.

Enhancing recombinant glycoprotein yield and quality using gene targeted CHO cells lines

Background It has been widely reported that CHO cells undergo apoptosis in culture, despite nutrient supplementation through fed-batch strategies. An understanding of apoptosis signaling can thus enable the identification of key genetic targets for the engineering of cell lines that could prolong culture viability and attain higher cell densities to effectively improve recombinant glycoprotein yield and quality.