(597a) Experimental and Computational Analysis of Chinese Hamster Ovary Stable Transfectants Grown in Fed-Batch Culture

With positive effects in medical treatments, therapeutic drugs that are derived from cells (biologics) have become one of the fast growing drug groups in the pharmaceutical market [1]. To date, Chinese Hamster Ovary (CHO) cells are the most common industrial hosts for therapeutic recombinant protein (rProtein) production. Despite the high specificity and medical efficacy of biologics, there is limited information on the metabolic activities of the cultured organisms during rProtein synthesis, while their production yields are often low. An increasing demand for the production of rProteins such as monoclonal antibodies (mAbs) places a necessity to understand the intracellular events, as well as to search for ways to control cell viability and improve productivity.

To increase the cell-specific production rate of mAbs (q_mAb), variations in culture environments are being integrated into fed-batch cultures. Mild hypothermic conditions (30 – 34 ^oC) in particular have been shown to enhance protein titres in CHO cell cultures [2]. Despite the increase in protein productivity in mild hypothermic cultures, this effect is dependent on host cell type, temperature variation and time of temperature shift. Therefore thorough understanding of the way host cells regulate their dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimisation. In this study, CHO cells expressing a mAb were cultured at physiological temperature (36.5^oC) and with a temperature shift to 32 ^oC at 3 days or 6 days post inoculation. We then compared culture performance with respect to cell growth, extracellular metabolite concentrations, stability of mAb mRNA and assembly intermediates, as well as secretion rates.

We found that at hypothermic conditions, cells grow at a reduced rate compared to those at physiological temperature. Cell metabolic activities also vary at hypothermic culture conditions. Regardless of the induction time of temperature shift, there is an overall reduction in glucose consumption. Decrease in lactate production was only observed in CHO cells that were cultured at 32^oC from day 6 onwards. Our results correlate with those observed in Nam et al. [3] where consumption and production of metabolites are reduced.

In the case of mAb synthesis, our experimental results show that the specific mAb productivity of cells that are cultured under hypothermic conditions is highly dependent on the induction time of the temperature shift. Reducing culture temperature early in exponential phase (day 3) has no beneficial effect on the overall mAb titre. However a small increase in q_mAb is observed when the temperature is reduced to 32 ^oC at late exponential phase (Day 6) when compared to those at 36.5 ^oC. Similar results are observed at mRNA level. CHO cells that experience temperature shift on day 6 achieve an overall higher mRNA gene copy number of heavy chain and an increase in intracellular H₂L₂ intermediates when compared to the other two cases. This is in agreement with Marchant et al. [4], who have shown that when cells are cultured at a cold-stress condition, the residence time of the protein in the ER increases and proteins are processed more efficiently. However, concentrations of intracellular H₂ and H₂L assembly intermediates remain lower at 32 ^oC when compared to those at physiological temperature.

In addition to experimental analysis of fed-batch CHO cultures, model-based techniques were introduced with an aim to identify bottlenecks and guide future experiments. In this respect, a hybrid unstructured/structured cell culture model was being developed for CHO cell culture grown in commercially-available media and supplemented with commercial feeds. Model parameters were estimated using datasets for culture under physiological temperature and with the switch to mild hypothermia. Simulation results provide good fits for cell growth, metabolite concentrations, concentrations of intracellular mRNA and mAb assembly intermediates, as well as secreted monoclonal antibody titres, when compared to experimental data for the three scenarios examined. We envisage that this model-based tool will provide a means of design of experiments, which aids in bioprocess optimisation.

Reference

[1]. Harris M. Market-Leading Biotechnology Drugs 2009: Blockbuster Dynamics in an Ailing Economy. BioWorld, Atlanta, GA. 2009.

[2]. Wulhfard S et al. Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells. Biotechnol. Prog. 2008; 24: 458-465.

[3]. Nam JH et al. The effects microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions. Cytotech. 2009; 59: 81-81.

[4]. Marchant, RJ et al. Metabolic rates, growth phase, and mRNA levels influence cell-specific antibody production levels from in vitro-cultured mammalian cells at sub-physiological temperatures. Mol. Biotech. 2008; 39 (1): 69-77.