Active Hypothermic Growth: A Novel Means For Increasing Total Interferon-γ Production by Chinese Hamster Ovary Cells

When grown under hypothermic conditions, Chinese Hamster Ovary (CHO) cells become growth arrested in the G₀/G₁ phase of the cell cycle and also often exhibit increased recombinant protein production. In this study, we have validated this hypothesis by stimulating hypothermic growth using basic fibroblast growth factor and fetal bovine serum supplementation. This method led to 7.7- and 4.9-fold increase in total production compared to the 37°C and 32°C control cultures, respectively. This proof-of-concept study will motivate the creation of cell lines capable of growing at low temperatures for use in industrial processes.Singapore-MIT Alliance (SMA

Prediction of Glycerol-Effect on Antigen-Antibody Binding Affinity from Molecular Dynamics Simulations

Many biological and biotechnological processes are controlled by protein-protein interactions in solution. In order to understand, predict and optimize such processes, it is valuable to understand how additives such as salts, sugars, polyols and denaturants affect protein-protein interactions. Currently, no methodology to foretell the effect of additives on protein-protein interactions has been established and frequently and extensive empirical screening to identify additives beneficial to the protein process is resorted to. In this work, we developed a methodology enabling the prediction of the additive-effect on the protein reaction equilibrium. The only prerequisite is that the atomic structure of the protein reactants and products are known. The methodology is based on the thermodynamic model for preferential interactions and makes use of molecular dynamics simulations to gauge additive-protein interactions. In order to validate our methodology, the change in binding affinity of the antibody fragment Y32S Fv D1.3 for lysozyme in the presence of varying glycerol concentrations is being calculated and the results will be compared with experimental data from literature. Finally, our methodology will be used to predict the glycerol effect on the binding affinity of wild type Fv D1.3 and various mutants.Singapore-MIT Alliance (SMA

Reduced Temperature Production of Recombinant Proteins to Increase Productivity in Mammalian Cell Culture

The production of recombinant proteins from an industrial perspective has one of its main goals is to increase the product concentration whether in batch, fed-batch or continuous perfusion bioreactor systems. However, a major problem trying to achieve high product concentration over prolonged cultivation is the loss of cell viability leading to reduced production rate and lower product quality. One possible means to achieve high product concentration and main high cell viability is to perform the bioreactor operations at a reduced temperature than that traditional used for mammalian cell cultivation. A collaborative research project between MIT and the Bioprocessing Technology Institute (BTI) was established where the MIT Ph.D. candidate (S.R. Fox) performed his research in Singapore with the assistances of BTI personnel. The goal of this project was the production of recombinant gamma interferon (γ -IFN) in Chinese Hamster Ovary (CHO) cells by operating the bioreactor at 32°C in contrast to cultivating the CHO cells at the traditional temperature of 37°C. By reducing the cultivation temperature to 32°C, we have found that the specific γ -IFN productivity can be increased to 400% as compared to the higher temperature (37°). This increase was the result of two factors. First the cell death was reduced at the lower temperature and second, the mRNA for the γ -IFN gene was greater (presumably through decreased mRNA degradation). However, at the reduced temperature, the cell’s specific growth was also impaired. Mutation and selection for higher growth rate strain at the reduced temperature was successful but we are concerned with the genetic stability of such mutants. Therefore a new collaborative project has been initiated using molecular genetics to engineer new CHO strains with higher growth rate at the reduced temperatures. The preliminary findings from this new project will be presented as a poster in this Symposium by Mr. Hong Kiat Tan.Singapore-MIT Alliance (SMA

CIRP Expression on Growth and Productivity of CHO Cells

Mammalian cell culture is typically operated at the physiological temperature of 37°C. Low temperature cell culture at 30-33°C, in particular for CHO cells, increased the specific productivity of many recombinant proteins amongst many other benefits. However, the cell density is lower, thus reducing the total protein yield. Of the 17 mammalian cold-stress genes reported to be up- or down-regulated at low temperature, CIRP shows potential as a gene target for improving recombinant protein production, as its expression levels were reported to affect both growth and specific productivity. In this study, it was shown that over-expression of the cold-stress gene CIRP did not cause growth arrest in CHO cells, in contrast to previous reports. However, over-expression of CIRP successfully improved the specific productivity and total yield of a recombinant interferon-γ CHO cell-line at 37°C by 25%.Singapore-MIT Alliance (SMA

Variability in the Stability and Productivity of Transfected Genes in Chinese Hamster Ovary (CHO) cells

In the field of biologics production, productivity and stability of the transfected gene of interest are two very important attributes that dictate if a production process is viable. To further understand and improve these two traits, we would need to further our understanding of the factors affecting them. These would include integration site of the gene, gene copy number, cell phenotypic variation and cell environment. As these factors play different parts in the development process, they lead to variable productivity and stability of the transfected gene between clones, the well-known phenomenon of “clonal variation”. A study of this phenomenon and how the various factors contribute to it will thus shed light on strategies to improve productivity and stability in the production cell line. Of the four factors, the site of gene integration appears to be one of the most important. Hence, it is proposed that work is done on studying how different integration sites affect the productivity and stability of transfected genes in the development process. For the study to be more industrially relevant, it is proposed that the Chinese Hamster Ovary dhfr-deficient cell line, CHO-DG44, is used as the model system.Singapore-MIT Alliance (SMA

The Effect of Culture Temperature on Recombinant IFN-γ Production and Quality

The goal of this research project is to analyze the effect of culture temperature on the production and quality of IFN-γ produced and secreted by suspension culture CHO cells.The effect of low temperature on IFN-γ glycosylation, which is under the control of a battery of enzymes whose activities will be influenced by temperature, is unknown. Work is focused on implementing a system for accurately monitoring the glycosylation of IFN-γ and then using the system for quantifying the effect of culture temperature on glycosylation. The system consists of immunoaffinity purification of IFN-γ , followed by capillary electrophoresis for determining glycosylation macroheterogeneity and MALDI-TOF MS and HPLC for determining glycosylation microheterogeneity. Initial results suggest that glycosylation macroheterogeneity is slightly decreased (~5%) at low temperature, thereby identifying a potential quality “bottleneck” for the use of low temperature to increase IFN-γ production. Low temperature (32°C) shifts the cells towards the non-growth, G1 portion of the cell cycle. In batch culture, if cells are shifted to low temperature once a reasonably high cell density is reached, an approximately 4-fold improvement in total IFN-γ production compared to 37°C culture is achieved. Pseudo-continuous culture was used to show that IFN-γ production is statistically significantly higher at 32°C compared to 37°C even when nutrient depletion is not a concern (p < 0.5). In fed-batch bioreactor culture, cells grown at low temperature display a short period of growth followed by a prolonged stationary phase of high specific IFN-γ productivity (~4-fold higher than compared to 37°C) whereas cells at 37°C grow rapidly, reach a peak cell density and then begin to die immediately. The net result is a 2-fold increase in total IFN-γ production at low temperature. Real-time RT-PCR was used to show that the amount of IFN-γ mRNA present during the 32°C stationary production phase is approximately 4-fold higher than the amount present during the exponential growth phase of the 37°C culture. To further explore the effect of low temperature on cell RNA levels, total RNA per cell was quantified during the course of batch cultures at 32°C and 37°C. Total RNA levels were found to be approximately 50% higher at 32°C than 37°C. The kinetics of the low temperature RNA concentration profile was modeled to obtain transcription (Ks) and degradation (Kd) rate constants and these were found to be consistent with literature values. This finding suggests that temperature shift may offer a novel approach for measuring RNA kinetic parameters in any cell system that can tolerate mild temperature changes.Singapore-MIT Alliance (SMA

Cell-line Engineering of Chinese Hamster Ovary Cells for Low-temperature Culture

Developments in mammalian cell culture and recombinant technology has allowed for the production of recombinant proteins for use as human therapeutics. Mammalian cell culture is typically operated at the physiological temperature of 37°. However, recent research has shown that the use of low-temperature conditions (30-33°) as a platform for cell-culture results in changes in cell characteristics, such as increased specific productivity and extended periods of cell viability, that can potentially improve the production of recombinant proteins. Furthermore, many recent reports have focused on investigating low-temperature mammalian cell culture of Chinese hamster ovary (CHO) cells, one of the principal cell-lines used in industrial production of recombinant proteins. Exposure to low ambient temperatures exerts an external stress on all living cells, and elicits a cellular response. This cold-stress response has been observed in bacteria, plants and mammals, and is regulated at the gene level. The exact genes and molecular mechanisms involved in the cold-stress response in prokaryotes and plants have been well studied. There are also various reports that detail the modification of cold-stress genes to improve the characteristics of bacteria or plant cells at low temperatures. However, there is very limited information on mammalian cold-stress genes or the related pathways governing the mammalian cold-stress response. This project seeks to investigate and characterise cold-stress genes that are differentially expressed during low-temperature culture of CHO cells, and to relate them to the various changes in cell characteristics observed in low-temperature culture of CHO cells. The gene information can then be used to modify CHO cell-lines for improved performance in the production of recombinant proteins.Singapore-MIT Alliance (SMA

Enhancing Production of Recombinant Proteins from Mammalian Cells

The bio-manufacturing of recombinant proteins from mammalian cell cultures requires robust processes that can maximize protein yield while ensuring the efficacy of these proteins as human therapeutics. Recognizing that the challenge of improving protein yield and quality can be met through various approaches, this paper presents three strategies currently being developed in our group. A method for rapidly selecting subpopulations of cells with high production characteristics is proposed. This method combines the efficiency of green fluorescent protein/fluorescence-activated cell sorting (GFP/FACS)–based screening with homologous recombination to generate and select high-producing subclones. Next, the development of chemically defined, protein-free media for enhancing monoclonal antibody production is described. Analysis of culture media effects on the genome-wide transcriptional program of the cell is presented as a means to optimize the culture media and identify potential targets for genetic manipulation. Finally, we propose a method for increasing the extent of intracellular sialylation by improving the transport of CMP-sialic acid into the trans-Golgi. This is hypothesized to increase the sialic acid availability, and may enhance the degree of sialylation in the glycoprotein product.Singapore-MIT Alliance (SMA

Cell-line Engineering for Low-temperature Growth

In the chemical industry, the rates of reactions are usually enhanced by the use of high-temperature and high-pressure conditions. This chemical engineering approach is rarely applied in the biotechnology field. Firstly, most biochemical reactions take place in an aqueous phase, which makes them relatively insensitive to changes in pressure. Secondly, they form a tightly regulated network, with distinct pathways that operate optimally at relatively low temperatures of about of 25-40°C. Beyond this range, higher temperatures would denature the proteins in the cell, leading to eventual cell death. Mammalian cells are integral the biotechnology field for production of human therapeutics. Bio-reactors for mammalian cells are typically operated at 370C. The effects of temperature down-shifting have been well-investigated and documented for several mammalian cell-lines and recombinant products. Although the rate of growth of cells is reduced, the productivity of recombinant protein is increased at lower temperatures. Apoptosis and nutrient requirements are reportedly reduced at lower temperatures also. As such, it is advantageous to investigate the effects of mammalian cell culture at down-shifted temperatures, with the ultimate aim of improving recombinant protein production and quality.Singapore-MIT Alliance (SMA

Differential regulation of effector- and central-memory responses to Toxoplasma gondii infection by IL-12 revealed by tracking of Tgd057-specific CD8+ T cells

10.1371/journal.ppat.1000815PLoS Pathogens6