Maximum Viable Cell Concentration Research Articles

From single-cell cloning to high-yield influenza virus production - implementing advanced technologies in vaccine process development.

Innovations in viral vaccine manufacturing are crucial for pandemic preparedness and to meet ever-rising global demands. For influenza, however, production still mainly relies on technologies established decades ago. Although modern production shifts from egg-based towards cell culture technologies, the full potential has not yet been fully exploited. Here, we evaluate whether implementation of state-of-the-art technologies for cell culture-based recombinant protein production are capable to challenge outdated approaches in viral vaccine process development. For this, a fully automated single-cell cloning strategy was established to generate monoclonal suspension Madin-Darby canine kidney (MDCK) cells. Among selected cell clones, we could observe distinct metabolic and growth characteristics, with C59 reaching a maximum viable cell concentration of 17.3 × 106 cells/mL and low doubling times in batch mode. Screening for virus production using a panel of human vaccine-relevant influenza A and B viruses in an ambr15 system revealed high titers with yields competing or even outperforming available MDCK cell lines. With C113, we achieved cell-specific virus yields of up to 25,000 virions/cell, making this cell clone highly attractive for vaccine production. Finally, we confirmed process performance at a 50-fold higher working volume. In summary, we present a scalable and powerful approach for accelerated development of high-yield influenza virus production in chemically defined medium starting from a single cell.

Production of retroviral vectors in continuous high cell density culture

Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.Key points• A continuous high cell density process for MLV vector production was established• The tubular cell retention membrane allowed for continuous vector harvesting• The established process had a 18-fold higher space time yield compared to a batch

Accelerating HIV-1 VLP production using stable High Five insect cell pools.

Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production.

High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells.

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.

An efficient method to control high mannose and core fucose levels in glycosylated antibody production using deoxymannojirimycin

Glycosylation on the Fc region of recombinant Immunoglobulin G (IgG) therapeutic antibodies is a critical protein quality attribute which may affect the efficacy and safety of the molecule. During the development of biosimilar therapeutics, adjustment of the glycosylation profile is required in order to match the reference innovator profile. Deoxymannojirimycin (DMJ), a known inhibitor of mannosidase, was used in this study to modulate the glycosylation pattern of antibodies. The effect of DMJ, at concentrations of 5 μM - 500 μM, on non-fucosylated glycoform levels was tested in the biosynthesis processes of two different IgG1 (IgG1 #A and IgG1 #B) using two Chinese hamster ovary (CHO) cell lines (CHO-DXB-11 and CHOK1SV, respectively) in Erlenmeyer flasks and in lab scale bioreactors. DMJ affected glycan forms in a dose response manner. At the highest concentration tested, DMJ reduced N-linked complex glycoform and core fucose levels by 15 and 14 fold, respectively, and increased high mannose level by 21 fold. 10 μM DMJ decreased IgG1 #A core fucose level in CHO-DXB-11 from 92% to 73% and increased high mannose level from 4% to 22% in Erlenmeyer flasks. Furthermore, in lab scale bioreactors, 15 μM DMJ decreased IgG1 #A core fucose level from 95% to 84% and increased high mannose level from 3% to 13%. Core fucose level of IgG1 #B in CHOK1SV was decreased from 81% to 73% using 10 μM DMJ in lab scale bioreactors while high mannose was increased from 6% to 15%. While affecting core fucose and high mannose levels, DMJ decreased maximum viable cell concentration by 16% and did not significantly affect cell productivity (less than 10%). This study demonstrated that DMJ can enable the control of core fucosylated and high mannose levels of IgG1 antibodies in a defined range.

Development of hyper osmotic resistant CHO host cells for enhanced antibody production

Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328mOsm/kg) or hyper-osmolality (over 450mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process.

Strategies for improving production performance of probiotic Pediococcus acidilactici viable cell by overcoming lactic acid inhibition

Lactic acid bacteria are industrially important microorganisms recognized for fermentative ability mostly in their probiotic benefits as well as lactic acid production for various applications. Fermentation conditions such as concentration of initial glucose in the culture, concentration of lactic acid accumulated in the culture, types of pH control strategy, types of aeration mode and different agitation speed had influenced the cultivation performance of batch fermentation of Pediococcus acidilactici. The maximum viable cell concentration obtained in constant fed-batch fermentation at a feeding rate of 0.015 L/h was 6.1 times higher with 1.6 times reduction in lactic acid accumulation compared to batch fermentation. Anion exchange resin, IRA 67 was found to have the highest selectivity towards lactic acid compared to other components studied. Fed-batch fermentation of P. acidilactici coupled with lactic acid removal system using IRA 67 resin showed 55.5 and 9.1 times of improvement in maximum viable cell concentration compared to fermentation without resin for batch and fed-batch mode respectively. The improvement of the P. acidilactici growth in the constant fed-batch fermentation indicated the use of minimal and simple process control equipment is an effective approach for reducing by-product inhibition. Further improvement in the cultivation performance of P. acidilactici in fed-bath fermentation with in situ addition of anion-exchange resin significantly helped to enhance the growth of P. acidilactici by reducing the inhibitory effect of lactic acid and thus increasing probiotic production.

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture.

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15mm) and agitated (working volume: 35, 50, 65mL, inoculum volume percentage: 10, 30% and agitation speed: 25, 60rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50mL working volume and the inoculum volume percentage was not significant in the range under study (10-30%) at 25rpm agitation. Agitation speed at 60rpm did not change the main kinetic parameters with respect to those observed for 25rpm. These results allowed for a schedule to produce more than 4×10(9) BHK-21 cells from 4×10(6) cells in 13day with 1,051mL culture medium.

Does earlier use of productivity enhancers during cell line selection lead to the identification of more productive cell lines?

Background After selection of a recombinant cell line for the production of a therapeutic protein, attempts are often made to increase volumetric productivity. Various techniques have been employed during the production process when trying to increase product concentration. Some aim to increase the time integral of viable cell concentration (IVC; the number of hours viable cells are available to produce the product) by increasing the maximum viable cell concentration and maintaining high viabilities. Techniques include (i) optimization of media and feeds, (ii) optimization of feeding strategies and (iii) genetic manipulation. Others aim to increase specific production rate (QP) by the deliberate inhibition of cell growth (controlled proliferation). Three methods commonly used to control proliferation are use of (i) chemical agents, (ii) hypothermic conditions and (iii) genetic manipulation. In this study, we focused on increasing volumetric productivity by manipulating QP; in particular, by the use of chemical agents. As a cell line has typically already been selected as a manufacturing cell line prior to assessing such methods to increase QP, the likelihood of success is not predictable: resulting in the frequently heard comment that results are ‘cell line specific’. But what if we were to look at these methods with many cell lines, at a much earlier stage of development (before the final cell line has been selected)? It could be that the cell line with the highest product concentration is typically a non-responder. The work described looks to answer the questions: (1) To what extent does the response to such methods vary in a large panel of cell lines producing the same antibody? and (2) Would their use in an earlier stage of cell line development enable the selection of a ‘better’ manufacturing cell line?

Effect of constitutively active ras overexpression on cell growth in recombinant chinese hamster ovary cells

Constitutively active Ras (CA-Ras) is known to enhance cell growth through the induction of various signaling cascades including the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ERK signaling pathways, although the cellular response is highly dependent on the cell type. To evaluate the effect of CA-Ras overexpression on cell growth in recombinant Chinese hamster ovary (rCHO) cells, an erythropoietin (EPO)-producing rCHO cell line with regulated CA-Ras overexpression (EPO-off-CA-Ras) was established using the Tet-off system. The CA-Ras expression level in EPO-off-CA-Ras cells was tightly regulated by doxycycline addition. Although CA-Ras overexpression slightly increased the viable cell concentration during the late exponential phase, it did not increase the maximum viable cell concentration or specific growth rate to a significant degree. Unexpectedly, CA-Ras overexpression in rCHO cells led only to the enhancement in the activation of the MAPK/ERK signaling pathway and not the PI3K/Akt signaling pathway. Taken together, CA-Ras overexpression in rCHO cells did not significantly affect cell growth; it also had no critical impact on viable cell concentration or EPO production, possibly due to a failure to activate the PI3K/Akt signaling pathway.

Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design

BackgroundSerum-containing medium (SCM), which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs), a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis.ResultsFrom a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM.ConclusionsThe use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

Performance of a vortex flow bioreactor for cultivation of CHO-K1 cells on microcarriers

In biochemical processes involving cultivation of animal cells, the design of a bioreactor is important because it should enable the provision of appropriate oxygen and nutrients to the culture medium at low shear stress. The aim of this study is to evaluate a new bioreactor designed and built based on the concept of Taylor vortex flow (Taylor vortex flow bioreactor – TVFB) employed in the cultivation of animal cells. The overall mass transfer volumetric coefficient ( K L a) was determined in order to evaluate the oxygen transfer. Mass transfer correlation under Taylor vortex flow regimes were estimated considering the overall volumetric oxygen transfer coefficient operating conditions and the geometry of the bioreactor. Experimental cultures were performed with CHO-K1 ( Chinese Hamster Ovary) cells attached to microcarriers in TVFB. Cell culture carried out in the bioreactor exhibited a maximum viable cell concentration ( X max ) of 1.0 × 10 6 cells/mL with μ max = 2 × 10 −2 h −1. Nevertheless, the results of this study suggest that Taylor vortex flow regimes provide an effective oxygen transfer required for animal cell culture with low hydrodynamics shear.

Comparative production of a monoclonal antibody specific for enrofloxacin in a stirred-tank bioreactor

A hybridoma producing monoclonal antibody (MAb) specific for enrofloxacin was cultivated in a 2-L stirred-tank bioreactor in various modes and the performances of each mode were compared. In batch mode, a maximum viable cell and MAb concentration of 9.21 × 10 5 cells mL −1 and 67.3 mg L −1, respectively, were obtained. When the hybridoma was cultivated in a fed-batch culture with the addition of specific nutrients, no improvement in either the viable cell number or MAb concentration was observed. On the other hand, an increase in the production of toxic metabolites, i.e. ammonia and lactate, was observed with growth inhibition of the hybridoma cells occurring at ammonia and lactate concentrations of 2.0 mM and 2.0 g L −1, respectively. However, the best performance of hybridoma cultivation was achieved in a perfusion culture mode using a spin filter, which was installed in the stirred-tank reactor as a cell retention device with a perfusion rate of 0.80 vvd. Under these conditions a steady viable cell concentration of 1.57 × 10 6 cells mL −1 was obtained within five days with an overall productivity and yield of 73.7 mg L −1 d −1 and 61.4 mg d −1, respectively, which was a significant increase over that attained with the batch process.

Enhanced growth in NS0 cells expressing aminoglycoside phosphotransferase is associated with changes in metabolism, productivity, and apoptosis

Prior work has reported that cotransfecting a gene of interest with the selectable marker neo can seriously perturb a number of cellular processes. In this study the influence of the neo gene on the growth, death, and metabolism of a murine myeloma NS0 cell line, expressing a chimeric antibody, was investigated. A pool of neo transfectants, 6A1-NEO, was selected with 500 microg/mL G418. Quantitative PCR analysis revealed that 6A1-NEO contained, on average, three copies of the neo gene per cell. Batch cultivation of 6A1-NEO showed that there was a 36% increase in maximum viable cell concentration, a 20% increase in the maximum apparent growth rate, and a 134% increase in cumulative cell hours as compared with the parent, 6A1-(100)3. Batch cultivation of five randomly selected clones illustrated that 6A1-NEO's advantage over the parent was not due to clonal variation. Neither the use of G418 during the selection process nor the cultivation of cells in the presence of G418 were responsible. This implied that the neo gene product, APH(3')-II, was causing the changes in proliferative capacity. Analysis of the cell cycle revealed that there were no differences in the distribution of cells in the G(1), S, and G(2) phases. When cell growth was synchronized, there were no observed differences in cell-cycle duration. 6A1-NEO resisted the onset of apoptosis during the growth phase. Consequently, there was a larger viable population of 6A1-NEO cells available for proliferation as compared with the parent. However, 6A1-NEO died at the same rate as the parent when resuspended in spent media or after treatment with staurosporin. Expression of the anti-apoptotic protein Bcl-2 was upregulated in 6A1-NEO, indicating that APH(3')-II could be acting by modulating endogenous gene expression. Analysis of key metabolites showed that 6A1-NEO's specific glucose consumption rate was 133% higher, whilst its specific glutamate consumption rate was 45% lower than the parent. 6A1-NEO's efficient utilization of glutamate and shift towards glucose metabolism may have contributed to the rise in proliferative capacity. However, this was accompanied by a 70% drop in the specific antibody production rate. These results show that the increase in growth rate and proliferative capacity caused by the expression of recombinant APH(3')-II was associated with changes in metabolism, apoptosis, and endogenous gene expression.

Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0°C

To investigate the effect of culture pH in the range of 6.85-7.80 on cell growth and erythropoietin (EPO) production at 32.5 and 37.0 degrees C, serum-free suspension cultures of recombinant CHO cells (rCHO) were performed in a bioreactor with pH control. Lowering culture temperature from 37.0 to 32.5 degrees C suppressed cell growth, but cell viability remained high for a longer culture period. Regardless of culture temperature, the highest specific growth rate (mu) and maximum viable cell concentration were obtained at pH values of 7.00 and 7.20, respectively. Like mu, the specific consumption rates of glucose and glutamine decreased at 32.5 degrees C compared to 37.0 degrees C. In addition, they increased with increasing culture pH. Culture pH at 32.5 degrees C affected specific EPO productivity (q(EPO)) in a different fashion from that at 37 degrees C. At 37 degrees C, the q(EPO) was fairly constant in the pH range of 6.85-7.80, while at 32.5 degrees C, the q(EPO) was significantly influenced by culture pH. The highest q(EPO) was obtained at pH 7.00 and 32.5 degrees C, and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0 degrees C. The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture temperature and pH. Although cell viability rapidly decreased in death phase at both 32.5 and 37.0 degrees C, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was observed only at 37.0 degrees C. Taken together, through the optimization of culture temperature and pH, a 3-fold increase in maximum EPO concentration and a 1.4-fold increase in volumetric productivity were obtained at pH 7.00 and 32.5 degrees C when compared with those at 37.0 degrees C. These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells.

Phosphate feeding improves high-cell-concentration NS0 myeloma culture performance for monoclonal antibody production.

Phosphorus depletion was identified in high-cell-concentration fed-batch NS0 myeloma cell cultures producing a humanized monoclonal antibody (MAb). In these cultures, the maximum viable and total cell concentration was generally ca. 5 x 10(9) and 7 x 10(9) cells/L, respectively, without phosphate feeding. Depletion of essential amino acids, such as lysine, was initially thought to cause the onset of cell death. However, further improvement of cell growth was not achieved by feeding a stoichiometrically balanced amino acid solution, which eliminated depletion of amino acids. Even though a higher cell viability was maintained for a longer period, no increase in total cell concentration was observed. Afterwards, phosphorus was found to be depleted in these cultures. By also feeding a phosphate solution to eliminate phosphorus depletion, the cell growth phase was prolonged significantly, resulting in a total cell concentration of ca. 17 x 10(9) cells/L, which is much greater than ca. 7 x 10(9) cells/L without phosphate feeding. The maximum viable cell concentration reached about 10 x 10(9) cells/L, twice as high as that without phosphate feeding. Apoptosis was also delayed and suppressed with phosphate feeding. A nonapoptotic viable cell population of 6.5 x 10(9) cells/L, as compared with 3 x 10(9) cells/L without phosphate feeding, was obtained and successfully maintained for about 70 h. These results are consistent with the knowledge that phosphorus is an essential part of many cell components, including phospholipids, DNA, and RNA. As a result of phosphate feeding, a much higher integral of viable cell concentration over time was achieved, resulting in a correspondingly higher MAb titer of ca. 1.3 g/L. It was also noted that phosphate feeding delayed the cell metabolism shift from lactate production to lactate consumption typically observed in recombinant NS0 cultures. The results highlight the importance of phosphate feeding in high-cell-concentration NS0 cultures.

Development of a serum-free medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44) using a statistical design: beneficial effect of weaning of cells.

To develop serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44), a statistical optimization approach based on a Plackett-Burman design was adopted. DG44 cells which were normally maintained in 10 serum medium were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium. A basal medium was prepared by supplementing Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Twenty-eight different supplements were selected as variables on the basis of their growth-promoting abilities. From statistical analysis, leucine, tryptophan, lysine, proline, histidine, hydrocortisone, ethanolamine, and phosphatidylcholine were identified as important components showing positive effects on cell growth. A new SF medium (SF-DG44) was formulated by supplementing the basal medium with these components. When the weaned cells were inoculated at 1.0 x 10(5) cells/ml, a maximum viable cell concentration of 6.4 x 10(3)) cells/ ml was achieved in SF-DG44 medium. In contrast, when the unweaned cells were used, a concentration of only 4.1 x 10(5) cells/ml was reached under the same culture conditions, indicating that weaning of cells improves cell growth in SF medium. In summary, we found that development of a novel SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.

Influence of Agitation and Sparging on the Growth Rate and Infection of Insect Cells in Bioreactors and a Comparison with Hybridoma Culture

Sf9 insect cells have been cultivated in paired agitated 1.4 L bioreactors with and without Pluronic F-68 and with and without sparging. The results are reported and, as appropriate, compared to those of hybridoma culture. Without sparging, the cells grew well at agitator speeds from 100 to 400 rpm (equivalent to a realistic range of specific energy dissipation rates). At the higher speeds, a small reduction in growth rate and maximum viable cell concentration was observed. This reduction was shown not to be dependent on the energy dissipation rate but to be due to bubble entrainment. These results are rather similar to those found with hybridoma cells. However, unlike hybridoma cells which grow like unsparged controls when sparged at 0.007 vvm, these insect cells at such a sparge rate show a significant reduction in growth rate and in maximum cell number. This reduction due to the presence of bubbles, whether from head space entrainment or from sparging, could be eliminated by the presence of Pluronic F-68. These insect cells have then also been infected with recombinant or wild type AcNPV. Agitation had no effect on the rate of infection, but higher levels of MOI enhanced it. Both findings support a diffusion mechanism as previously. The presence of Pluronic F-68 did not have any impact on the rate of infection and was able to prevent cell damage while agitating and sparging infected cells.

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10(9) cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.