Effect of Gas Sparging in Mammalian Cell Bioreactors

One of the major problems in the operations of mammalian cell bioreactors is the detrimental effect of gas sparging. Since the most convenient way to oxygenate any bioreactor is by gas sparging, this adverse effect has often been one of the limiting oxygen transport problems in both laboratory and industrial mammalian cell bioreactors. When one examines the literature on the effect of gas sparging on the death of mammalian cells, a great deal of confusions has been reported. It is not clear from the published literature as to the leading cause for gas-sparged related cell death. These confusions prevent the rational design and operations of mammalian cell bioreactors. In our laboratory, we have attempted to address this problem both fundamentally as well as attempt to obtain a general understanding on the adverse effect of gas sparging. Our analyses first examined the fluid shear associated with the various sections that the gas bubbles encounter during entrance, passage through the bioreactor and the final exit of the gas bubbles. Our analyses showed that the major damage of the mammalian cells by gas bubbles is due to the burst of the bubbles when exiting the bioreactor. It was also our hypothesis that the entrained cells in the liquid boundary layer of the gas bubble upon bursting is the major cause for cell death. We have corroborated this hypothesis by correlating the liquid entrainment with the cell death rate using results from our laboratory as well as other studies. Pluonic F-68, a weak surfactant, has routinely been used in laboratory and industrial bioreactors. In the past, the protective effect of Pluronic F-68 has never been shown as to why it is effective. In our research, we have data using microphotography which clearly demonstrated and corroborated our entrainment hypothesis is the major reason for the effectiveness of Pluronic F-68 in protecting the cells from gas-sparged related cell death.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

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

Proteins in Mixed Solvents: A Molecular-level Perspective

We present a statistical mechanical approach for quantifying thermodynamic properties of proteins in mixed solvents. This approach, based on molecular dynamics simulations which incorporate all atom models and the theory of preferential binding, allows us to compute transfer free energies with experimental accuracy and does not incorporate any adjustable parameters. Specifically, we applied our approach to the model proteins RNase A and T1, and the solvent components water, glycerol, and urea. We found that the observed differences in the binding of glycerol and urea to RNase T1 and A are predominantly a consequence of density differences in the first coordination shell of the protein with the cosolvents, but the second solvation shell also contributes to the overall binding coefficients. The success of this approach in modeling preferential binding indicates that it incorporates the important underlying physics of proteins in mixed solvent systems and that the difficulty in quantitative prediction to date can be surmounted by explicitly incorporating the complex protein-solvent and solvent-solvent interactions.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

Simple and Versatile Route to the Synthesis of Anisotropic Bimetallic Core-Shell and Monometallic Hollow Nanostructures: Ag (AgCl)-Pt Core-Shell Nanocubes and Pt Nanoboxes

We report herewith a simple and versatile route for the preparation of anisotropic Ag(AgCl)-Pt core-shell nanocubes and Pt nanoboxes. The core-shell nanocubes were first synthesized through the simultaneous reduction method and then treated with bis-(p-sulfonatophenyl)-phenylphosphine (BSPP) to remove the core materials. The changes in morphology, structure and composition during these syntheses were carefully followed. We found that, BSPP, in addition to being an effective silver oxidant, is also a good solubilizer for AgCl nanoparticles at room temperature. This allowed us to prepare pure Pt nanoboxes easily from the as-synthesized Ag (AgCl)-Pt nanocubes using a greatly simplified post-treatment for AgCl, which is the perennial impurity byproduct in the preparation of hollow nanostructures by the replacement reactions.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

Understanding Oxidative Instability of Protein Pharmaceuticals

Mechanism of oxidation of methionine residues in protein pharmaceuticals by hydrogen peroxide was investigated via ab initio calculations. Specifically, two reactions, hydrogen transfer of hydrogen peroxide to form water oxide and the oxidation of dimethyl sulfide (DMS) by hydrogen peroxide to form dimethyl sulfoxide, were studied as models of these processes in general. Solvent effects are included both via including explicitly water molecules and via the polarizable continuum model. Specific interactions including hydrogen bonding with 2-3 water molecules can provide enough stabilization for the charge separation at the activation complex. The major reaction coordinates of the reaction are the breaking of the O-O bond of H₂O₂ and the formation of the S-O bond, the transfer of hydrogen to the distal oxygen of hydrogen peroxide occurring after the system has passed the transition state. Reaction barriers of the hydrogen transfer of H₂O₂ are in average of 10 kcal/mol or higher than the oxidation of DMS. Therefore, a two step oxidation mechanism in which the transfer of hydrogen atom occurs first to form water oxide and the transfer of oxygen to substrate occurs as the second step, is unlikely to be correct. Our proposed oxidation mechanism does not suggest pH dependence of oxidation rate within a moderate range around neutral pH (i.e. under conditions in which hydronium and hydroxide ions do not participate directly in the reaction), and it agrees with experimental observations over moderate pH values.Singapore-MIT Alliance (SMA

Biological Routes to Gold Nanoplates

Gold nanoplates are promising for optical and electronic applications; but their synthesis is complex, often requiring a seeded growth process or spherical to triangle morphology transformation. We have discovered a biological protocol to promote the anisotropic growth of different crystal planes under ambient conditions. Thin, flat, single-crystalline gold nanoplates were produced when aqueous chloroaurate ions reacted with the mycelia-free spent medium. While the exact mechanism for this shape-controlled synthesis is not clear at this time, the possibility of achieving nanoparticle shape control in a fungal based system is exciting.Singapore-MIT Alliance (SMA