Danny Chee Furng Wong

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.

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‐γ (IFN‐γ). 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‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. 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. Biotechnol. Bioeng. 2010;107: 516–528.

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.

Elucidating the role of requiem in the growth and death of Chinese hamster ovary cells

Requiem, a hypothesized transcription factor with apoptosis-related activity, was previously shown to be a potential cell engineering gene target for improving recombinant protein production. Requiem suppression has resulted in improved viable cell density and extended culture viability, leading to an overall improvement in recombinant protein productivity. However, not much is known about the function of requiem. We found that requiem is highly conserved at both nucleotide and amino acid levels in Chinese hamster ovary (CHO) cells when compared to human and mouse sequences, suggesting that requiem’s functional role is evolutionary well conserved. Upon inducing requiem over-expression, proliferation rates of CHO cells were significantly decreased with doubling times increased by 26%. Interestingly, the over-expression of requiem did not decrease cell viability and could not induce apoptosis. However, requiem sensitized the cells to increased caspase-9 activities under staurosporine-induced apoptosis, suggesting that it has a role to play in mitochondria-mediated apoptosis under staurosporine treatment. The nuclear localization of REQUIEM in CHO cells and its conserved plant homeodomain (PHD) zinc fingers seem to further support the hypothesis that requiem encodes for a potential transcription factor. Upon requiem over-expression, we found that the differentially expressed genes involved in transcriptional regulation and cell proliferation and growth were associated both upstream and downstream of p53.

Simultaneous Targeting of Requiem & Alg-2 in Chinese Hamster Ovary Cells for Improved Recombinant Protein Production

Apoptosis is known to be the main cause of cell death in the bioreactor environment, leading to the loss of recombinant protein productivity. In a previous study, transcriptional profiling was used to identify and target four early apoptosis-signaling genes: FADD, FAIM, Alg-2, and Requiem. The resulting cell lines had increased viable cell numbers and extended culture viability, which translated to increased protein productivity. Combinatorial targeting of two genes simultaneously has previously been shown to be more effective than targeting one gene alone. In this study, we sought to determine if targeting Requiem and Alg-2 was more effective than targeting Requiem alone. We found that targeting Requiem and Alg-2 did not result in extended culture viability, but resulted in an increase in maximum viable cell numbers and cumulative IVCD under fed-batch conditions. This in turn led to an approximately 1.5-fold increase in recombinant protein productivity.

Simulation and Optimization of Essential Amino Acids in Dynamic Mammalian Cell Culture

Optimization in mammalian cell culture systems is currently exclusively experimental, which is an expensive and time-consuming process. Mathematical models have become established practice in many process industries. Herein, a systematic framework has been developed that allows the simulation and off-line optimization of fed-batch monoclonal antibody-producing hybridoma cell cultures. The model describes major cellular functions as well as the uptake of amino acids and how it affects growth and productivity. The model-based optimization approach was able to provide an estimation of the optimal dynamic profile of essential amino acids in order to improve the IFNγ yield.

Dynamic Optimisation of CHO-IFNγ Cell Culture Fed-Batch Time-Profile

Production of high-value biopharmaceuticals using mammalian cell cultures is an expensive process. A model of an IFNγ-producing CHO cell-line was constructed to simulate the culture in batch and fed-batch conditions, which included major cellular functions such as growth, death, glucose/amino acids uptake, IFNγ synthesis, and by-products formation. The model structure was analysed with global sensitivity analysis to reduce model complexity by identifying insensitive parameters. Using the model, dynamic optimisation results were obtained which enabled the identification of higher yield with the corresponding feeding strategies.

Elucidating apoptotic cell death in cho cell batch & fed-batch cultures

Chinese Hamster Ovary (CHO) cells are regarded as one of the industrial ‘work-horses’ for complex biotherapeutics production. In these processes, loss in culture viability occurs primarily via apoptosis, a genetically controlled form of cellular suicide. By applying microarray technology using our ‘in-house’ developed CHO cDNA array and a mouse oligonucleotide array for time profile expression analysis, the genetic circuitry that regulates and executes apoptosis induction can be carried out rapidly. We found that in both batch and fed-batch cultures, receptorand mitochondrial-mediated apoptosis pathways play important roles in apoptosis induction. There are also several other minor pro-apoptotic genes that appear to be upregulated during apoptosis induction in CHO cells. However, although these other genes had been implicated in apoptosis induction, their exact role in apoptosis induction has yet to be elucidated. By having a greater understanding of the regulatory circuitry of apoptosis relevant to cell culture processes, future effective strategies could be developed towards cell death prevention.

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.

Impact of apoptosis gene targeting on recombinant protein glycosylation

Background Chinese Hamster Ovary (CHO) cell is one of the major cell lines used for complex recombinant protein production. During CHO cell culture, loss in viability attributed to apoptosis often results in lower recombinant protein yield and affects protein quality. Through the individual targeting of four apoptosis genes identified via expression profiling studies, four apoptosis resistant CHO GT (Gene Targeted) cell lines were constructed. These cell lines enabled prolonged culture viability, higher maximum viable cell densities and significant increase in recombinant human interferon gamma (IFN-γ) yields. Furthermore, it was observed that the IFN-γ from CHO GT cells has a higher level of sialic acid content.