Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria.

Efficient protein secretion is very important in biotechnology as it provides active and stable enzymes, which are an essential prerequisite for successful biocatalysis. Therefore, optimizing enzyme-producing bacterial strains is a major challenge in the field of biotechnology and protein production. In this study, the Gram-positive model bacterium Bacillus subtilis was optimized for heterologous protein secretion using a novel approach. Two lipolytic enzymes, cutinase from Fusarium solani pisi and a cytoplasmatic esterase of metagenomic origin, were chosen as reporters for heterologous protein secretion. In a systematic screening approach, all naturally occurring (non-lipoprotein) Sec-type signal peptides (SPs) from B. subtilis were characterized for their potential in heterologous protein secretion. Surprisingly, optimal SPs in cutinase secretion were inefficient in esterase secretion and vice versa, indicating the importance of an optimal fit between the SP and the respective mature part of the desired secretion target proteins. These results highlight the need for individually optimal signal peptides for every heterologous secretion target. Therefore, the SP library generated in this study represents a powerful tool for secretion optimization in Gram-positive expression hosts.

Efficiency improvement of an antibody production process by increasing the inoculum density.

Increasing economic pressure is the main driving force to enhance the efficiency of existing processes. We developed a perfusion strategy for a seed train reactor to generate a higher inoculum density for a subsequent fed batch production culture. A higher inoculum density can reduce culture duration without compromising product titers. Hence, a better capacity utilization can be achieved. The perfusion strategy was planned to be implemented in an existing large scale antibody production process. Therefore, facility and process constraints had to be considered. This article describes the initial development steps. Using a proprietary medium and a Chinese hamster ovary cell line expressing an IgG antibody, four different cell retention devices were compared in regard to retention efficiency and reliability. Two devices were selected for further process refinement, a centrifuge and an inclined gravitational settler. A concentrated feed medium was developed to meet facility constraints regarding maximum accumulated perfundate volume. A 2-day batch phase followed by 5 days of perfusion resulted in cell densities of 1.6 × 10(10) cells L(-1) , a 3.5 fold increase compared to batch cultivations. Two reactor volumes of concentrated feed medium were needed to achieve this goal. Eleven cultivations were carried out in bench and 50 L reactors showing acceptable reproducibility and ease of scale up. In addition, it was shown that at least three perfusion phases can be combined within a repeated perfusion strategy.

Optimizing capacity utilization by large scale 3000 L perfusion in seed train bioreactors.

Increasing capacity utilization and lowering manufacturing costs are critical for pharmaceutical companies to improve their competitiveness in a challenging environment. Development of next generation cell lines, improved media formulations, application of mature technologies and innovative operational strategies have been deployed to improve yields and capacity utilization. This article describes a large-scale perfusion strategy for the N-1 seed train bioreactor that was successfully applied to achieve higher inoculation cell densities in the production culture. The N-1 perfusion at 3,000-L scale, utilizing a inclined settler, achieved cell densities of up to 158 × 10(5) cell mL(-1) at perfusion rates of 2950 L day(-1) and a retention efficiency of >85%. This approach increased inoculation cell densities and decreased cultivation times by ~20% in a CHO-based, fed-batch antibody manufacturing process while providing comparable culture performance, productivity, and product quality. The strategy therefore yielded significant increase in capacity utilization and concomitant cost improvement in a large scale cGMP facility. Details of the strategy, the cell retention device, and the cell culture performance are described in this article.