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.

In pursuit of the optimal fed-batch process for monoclonal antibody production.

Fed-batch culture currently represents the most attractive choice for large scale production for monoclonal antibodies (MAbs), due to its operational simplicity, reliability, and flexibility for implementation in multipurpose facilities. Development of highly productive cell lines, maximization of cell culture longevity, and maintenance of high specific antibody secretion rates through genetic engineering techniques, nutrition supplementation, waste product minimization, and control of environmental conditions are important for the design of high-yield fed-batch processes. Initially simple supplementation protocols have evolved into sophisticated serum-free multi-nutrient feeds that result in MAb titers on the order of 1-2 g/L. Limited research has been published to date on the effect of various culture parameters on potentially important quality issues, such as MAb glycosylation and stability. Although most fed-batch protocols to date have relied on relatively simple control schemes, increasingly sophisticated algorithms must be applied in order to take full advantage of the potentially additive effects of manipulating nutrient and environmental parameters to maximize fed-batch process productivity.

Monoclonal antibody process development using medium concentrates.

A fed-batch process using concentrated medium was evaluated for its ability to improve cell culture longevity and final monoclonal antibody (MAb) titers for two monoclonal antibody producing cell lines. It was found to result in up to 7-fold increases in final antibody titers compared to batch culture controls. Although the development cell line specific fed-batch protocols is critical to the development of cost-efficient large-scale production processes, the use of complete medium concentrates provided us with a quick and simple method for producing large quantities of antibodies in the early stages of process development, thus accelerating early work on purification process development, analytical development, biochemical characterization, and safety studies. Insights gained from the concentrated medium fed-batch approach were valuable for the development of refined, cell line specific feeding strategies yielding final MAb titers on the order of 1-2 g/L. Process development data on the effects of inhibitory growth byproducts, medium osmolarity, and the mode of nutrient feed addition on culture longevity and MAb production and information on culture metabolic behavior were successfully incorporated in the development of the optimized fed-batch protocols.

Use of a structured kinetic model of antibody synthesis and secretion for optimization of antibody production systems: I. Steady-state analysis.

Steady-state simulations using our previously developed structured kinetic model of antibody synthesis and secretion by hybridoma cells are used here in conjunction with factorial design analysis to identify intracellular parameters important in determining the specific antibody secretion rate and predict the dependence of this rate on cell specific growth rate. Simulation results suggest that the specific growth rate, the assembly rate of the heavy and light chains and the heavy- and -chain gene dosage can significantly affect the rate of antibody secretion. Based on these results, environmental and/or genetic manipulation approaches are proposed for maximizing the specific antibody secretion rate and the antibody volumetric productivity in large-scale antibody production systems.

Use of a structured kinetic model of antibody synthesis and secretion for optimization of antibody production systems: II. Transient analysis.

The dynamic behavior of the monoclonal antibody (MAb) secretory pathway is studied by transient simulations using our previously developed structured kinetic model for antibody synthesis and secretion by hybridoma cells. The response of the secretory pathway to blocks in specific pathway steps and step changes in characteristic pathway parameters is presented in order to gain a better understanding of pathway dynamics and identify possible rate-limiting steps in the pathway. Model simulations suggest that the step of antibody assembly in the endoplasmic reticulum (ER) is a very good candidate for a rate-limiting step in the antibody secretory pathway in fast-growing hybridoma cells, whereas translation of the heavy and light chains is most likely rate-limiting in slowly growing or stationary phase cells. Transient simulation results are compared with experimentally observed transient changes in specific antibody secretion rates and used to suggest strategies for optimizing antibody secretion in large-scale production systems.

Evidence for posttranscriptional stimulation of monoclonal antibody secretion by L-glutamine during slow hybridoma growth.

The addition of 5-40 mM L-glutamine to batch cultures of a murine hybridoma following the cessation of rapid growth significantly stimulated monoclonal antibody (mAb) synthesis and secretion per cell. Stimulation of mAb secretion following the cessation of rapid growth was also observed in response to addition of mitochondrial intermediates of glutamate oxidation and was not found to be the result of release of transiently stored mAb. Less than 1% of the secreted mAb was detected by ELISA in isolated hybridoma lysosomes. This stimulation was posttranscriptional and not the result of enhancement of levels of mAb mRNAs or stabilization of heavy (H) or light (L) chain encoding message. Sub-inhibitory levels of lysosomotrophic weak bases stimulated release of lysosomal contents but did not result in release of intact or partially degraded mAb. Inhibition of aspartic proteinase activity secreted by the hybridoma did not enhance mAb secretion even though a high level of mAb degrading proteinase activity was continuously secreted during both rapid and slow growth. These responses indicate that during slow growth, the addition of L-glutamine increases the availability of cellular ATP generated by mitochondrial respiration which stimulates some posttranscriptional step in the pathway of mAb secretion such as the rate of H or L chain translation, chain assembly, interorganelle transport or vesicular transport from the Golgi to the cell membrane.

A model of interorganelle monoclonal antibody transport and secretion in mouse hybridoma cells.

A three compartment model (ER --> Golgi --> extracellular medium) is used here to describe the interorganelle transport and final secretion of an IgG(2a) monoclonal antibody (MAb) in 9.2.27 murine hybridoma cells. Model simulations of pulse-chase and continuous labeling experiments are used to gain a better understanding of the kinetics of MAb interorganelle traffic. Simulation results for the continuous labeling case compare well with experimental data obtained during continuous labeling of 9.2.27 hybridoma cells. Incorporation of this compartmental transport model into our previously developed model of MAb synthesis and assembly can provide a useful tool for analyzing the dynamics and regulation of the complete antibody secretory pathway under different growth and/or nutritional conditions.

A structured model for monoclonal antibody synthesis in exponentially growing and stationary phase hybridoma cells.

An initial structured unsegregated kinetic model describing monoclonal antibody synthesis by a murine hybridoma cell line (9.2.27) grown in 1 liter batch cultures is described. The model is based on the intracellular balances of the heavy and light chain coding mRNAs, the intracellular balances of heavy and light chains and the description of the kinetics of heavy and light chain assembly. Model parameters were varied with specific growth rate in order to account for changes in the rates of antibody synthesis and secretion with entrance of the cells from the exponential into the stationary phase of growth. The parameters were varied based. on experimental data obtained in our laboratory on the variation of total cellular RNA content and the half-lives of heavy (H) and light (L) chain mRNAs with specific growth rate, and data from other investigators on immunoglobulin synthesis and secretion. The model successfully predicts the experimentally observed decrease in the intracellular heavy and light chain mRNA levels with entrance of 9.2.27 cells from the exponential into the stationary phase of growth, as well as the extracellular accumulation of antibody (IgG(2a)) during batch culture.