Novel amino acid feeding strategy in perfusion cultures to enhance monoclonal antibody production

Amino acids represent an essential source of nutrients in all the cell culture media. The concentration of each individual amino acid in the media has a huge impact on the performance of the cell culture. Traditionally, their concentrations in commercially available basal media have been determined in laborious studies using batch and fed-batch experiments. Hence, the resulting amino acid composition in the culture media is optimized based on the metabolic requirements of a specific cell line in a batch process. These requirements are likely to be different when the cell line is changed and the medium is used in perfusion processes. In order to optimize the performance of a perfusion culture by means of increasing cell specific productivity, decreasing byproduct formation and minimizing the cell specific perfusion rate, we developed a screening procedure for different media with distinct amino acid compositions in pseudo-perfused spin tubes. The results obtained from a monoclonal antibody expressing CHO-K1 (GS) cell line in these media revealed significant differences in amino acid uptake rates, and led to diverse metabolic behaviors. Cell specific productivities varied in a range of 35 % and ammonium production was even completely ceased under certain conditions. The lactate production rates differed widely and was mostly influenced by the seed cell density. From the results of an initial screening we designed a modified medium to target the amino acid consumption to a desired metabolic state with maximized antibody productivity and minimized byproduct formation. The performance of the medium was assessed with a perfusion culture in an ATF bioreactor system, where the amino acid consumption was precisely controlled to the targeted value by the appropriate feeding of amino acids. This strategy can be potentially widely applied across various cell lines to define the optimum concentration of amino acids or other components in perfusion media

Small-scale end-to-end mAb platform with a continuous and integrated design

Fully continuous manufacturing of therapeutic proteins is an emerging trend in the biopharmaceutical industry. Integration of the upstream and downstream processes and conversion to continuous manufacturing leads to increased productivities, decreased equipment size, improved product quality, and overall reduced production costs. An integrated continuous bioprocess (ICB) from the early stages of process development can accelerate the transfer of a new drug candidate to commercial manufacturing. The process presented in this work is a proof-of-concept of an end-to-end monoclonal antibody (mAb) production platform at small scale. It was implemented by a 200 mL ATF perfusion bioreactor, integrated with a single lab-scale chromatography system, performing all purification steps for the mAb from the cell culture harvest in a continuous way. The downstream process consisted of a periodic twin-column capture with protein A resin, followed by a virus inactivation step, a cation exchange step in bind-elute mode, and an anion exchange step in flow-through mode. MAbs were produced for 17 days in a high cell density perfusion culture of CHO cells and purified continuously with a recovery yield of up to 60 % by the following downstream train. A 5-log reduction of host cell protein levels revealed that impurities were sufficiently removed. A consistent glycosylation pattern of the purified product was ensured by the steady-state operation of the process. With this proof-of-concept, we demonstrated the technical feasibility of a fully continuous end-to-end process with a compact design, integrating several unit operations in a single chromatography station and using small-size equipment for the upstream and downstream operations. The work presented here can become a useful development tool for the future of continuous bioprocesses

Raman spectroscopy for on-line monitoring of amino acid concentrations and antibody N- glycosylation in high cell density perfusion process

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Amino acids and antibody N-glycosylation based on Raman spectroscopy in high cell density perfusion culture

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Transcriptomics guided mechanistic metabolic model for perfusion culture process

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Transcriptomics and modelling to understand the benefits of low perfusion rate

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Workflow for mining process relevant knowledge from transcriptomics

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Pilot-scale integrated continuous biomanufacturing for monoclonal antibodies including mild pH

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Development and application of screening scale bioreactor systems for very high cell density perfusion of mammalian cells

The development of cultivation processes can be significantly more efficient and cost effective when performed in parallel screening systems operated at small scale. Although perfusion systems at screening scale have been proposed, none of them enables a real mimic of very high cell density perfusion bioreactors. The latter is however important to maximize the performance of processes at 80, 100 x 106 cells/mL, or higher cell densities, in particular to optimize the nutrient supply and feeding regime. The purpose of the present work was to develop such a system and to apply it to perfusion optimization. Chinese Hamster Ovary cells (CHO) and Human Embryonic Kidney cells, HEK293, both producing biopharmaceuticals (monoclonal antibody and non-antibody), were used for this development. Although CHO cells are the established workhorses of the biopharmaceutical industry, human HEK293 cells can be advantageous compared to rodent cells for the secretion of some biopharmaceuticals. However, HEK293 cells are more sensitive than CHO cells to the culture conditions and their culture at high cell density is more challenging. We evaluated two different stirred tank bioreactors of ≤ 250 mL with cell separation carried out either by Alternating Flow Filtration (ATF) or classical Tangential Flow Filtration (TFF). With this equipment, we developed perfusion processes for HEK293 cells and for CHO cells, stably achieving ≈ 80 to 100 x 106 cells/mL with very high viability. These systems were used to minimize the cell specific perfusion rate and the feeding of glucose and glutamine by a specific approach, reducing the generation of the by-products. The influence of the feeding regimes on the glycosylation patterns of the recombinant proteins was investigated. The effect of shear stress generated by the ATF and by the TFF was studied from a theoretical point of view indicating that ATF generates a lower shear stress, independently from the effect of the pump used for the recirculation in the TFF loop. In the experimental study supporting this theoretical result, the effect of shear stress on the cells was investigated by transcriptomics analysis