A protocol to transfer a fed-batch platform process into semi-perfusion mode: The benefit of automated small-scale bioreactors compared to shake flasks as scale-down model

Continuous processes such as perfusion processes can offer advantages compared to fed-batch or batch processes in bio-processing: improved product quality (e.g. for labile products), increased product yield, and cost savings. In this work, a semi-perfusion process was established in shake flasks and transferred to an automated small-scale bioreactor by daily media exchange via centrifugation based on an existing fed-batch process platform. At first the development of a suitable medium and feed composition, the glucose concentration required by the cells and the cell-specific perfusion rate were investigated in shake flasks as the conventional scale-down system. This lead to an optimized process with a threefold higher titer of 10 g/L monoclonal antibody compared to the standard fed-batch. To proof the suitability and benefit as a small-scale model, the established semi-perfusion process was transferred to an automated small-scale bioreactor with improved pH and dissolved oxygen control. The average specific productivity improved from 24.16 pg/(c*d) in the fed-batch process and 36.04 pg/c*d in the semi-perfusion shake flask to 38.88 pg/(c*d) in the semi-perfusion process performed in the controlled small-scale bioreactor, thus illustrating the benefits resulting from the applied semi-perfusion approach, especially in combination with controlled DO and pH settings. © 2019 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 35: e2757, 2019.     

Evaluation of cysteine ethyl ester as efficient inducer for glutathione overproduction in Saccharomyces spp.

Economical yeast based glutathione (GSH) production is a process that is influenced by several factors like raw material and production costs, biomass production and efficient biotransformation of adequate precursors into the final product GSH. Nowadays the usage of cysteine for the microbial conversion into GSH is industrial state of practice. In the following study, the potential of different inducers to increase the GSH content was evaluated by means of design of experiments methodology. Investigations were executed in three natural Saccharomyces strains, S. cerevisiae, S. bayanus and S. boulardii, in a well suited 50 ml shake tube system. Results of shake tube experiments were confirmed in traditional baffled shake flasks and finally via batch cultivation in lab-scale bioreactors under controlled conditions. Comprehensive studies showed that the usage of cysteine ethyl ester (CEE) for the batch-wise biotransformation into GSH led up to a more than 2.2 times higher yield compared to cysteine as inducer. Additionally, the intracellular GSH content could be significantly increased for all strains in terms of 2.29 ± 0.29% for cysteine to 3.65 ± 0.23% for CEE, respectively, in bioreactors. Thus, the usage of CEE provides a highly attractive inducing strategy for the GSH overproduction.     

Designing a fully automated multi-bioreactor plant for fast DoE optimization of pharmaceutical protein production

The identification of optimal expression conditions for state-of-the-art production of pharmaceutical proteins is a very time-consuming and expensive process. In this report a method for rapid and reproducible optimization of protein expression in an in-house designed small-scale BIOSTAT® multi-bioreactor plant is described. A newly developed BioPAT® MFCS/win Design of Experiments (DoE) module (Sartorius Stedim Systems, Germany) connects the process control system MFCS/win and the DoE software MODDE® (Umetrics AB, Sweden) and enables therefore the implementation of fully automated optimization procedures. As a proof of concept, a commercial Pichia pastoris strain KM71H has been transformed for the expression of potential malaria vaccines. This approach has allowed a doubling of intact protein secretion productivity due to the DoE optimization procedure compared to initial cultivation results. In a next step, robustness regarding the sensitivity to process parameter variability has been proven around the determined optimum. Thereby, a pharmaceutical production process that is significantly improved within seven 24-hour cultivation cycles was established. Specifically, regarding the regulatory demands pointed out in the process analytical technology (PAT) initiative of the United States Food and Drug Administration (FDA), the combination of a highly instrumented, fully automated multi-bioreactor platform with proper cultivation strategies and extended DoE software solutions opens up promising benefits and opportunities for pharmaceutical protein production.     

Employing QbD strategies to assess the impact of cell viability and density on the primary recovery of monoclonal antibodies

Quality by Design (QbD) is one of the most important tools for the implementation of Process Analytical Technology (PAT) in biopharmaceutical production. For optimal characterization of a monoclonal antibody (mAb) upstream process a stepwise approach was implemented. The upstream was divided into three process stages, namely inoculum expansion, production, and primary recovery, which were investigated individually. This approach enables analysis of process parameters and associated intermediate quality attributes as well as systematic knowledge transfer to subsequent process steps. Following previous research, this study focuses on the primary recovery of the mAb and thereby marks the final step toward a holistic characterization of the upstream process. Based on gained knowledge during the production process evaluation, the cell viability and density were determined as critical parameters for the primary recovery. Directed cell viability adjustment was achieved using cytotoxic camptothecin in a novel protocol. Additionally, the cell separation method was added to the Design of Experiments (DoE) as a qualitative factor and varied between filtration and centrifugation. To assess the quality attributes after cell separation, the bioactivity of the mAb was analyzed using a cell-based assay and the purity of the supernatant was evaluated by measurement of process related impurities (host cell protein proportion, residual DNA). Multivariate data analysis of the compiled data confirmed the hypothesis that the upstream process has no significant influence on the bioactivity of the mAb. Therefore, process control must be tuned towards high mAb titers and purity after the primary recovery, enabling optimal downstream processing of the product. To minimize amounts of host cell proteins and residual DNA the cell viability should be maintained above 85% and the cell density should be controlled around 15 × 10⁶ cells/ml during the cell removal. Thereby, this study shows the importance of QbD for the characterization of the primary recovery of mAbs and highlights the useful implementation of the stepwise approach over subsequent process stages.