Bioreactor process improvements in a legacy perfusion-based process

The legacy manufacturing processes for production of the enzyme products at Genzyme include long-term bioreactor perfusion-based cell culture platform. For the process with tight filed ranges and limited characterization, a phased approach is being used to improve bioreactor productivity. In the first phase, short-term process changes that are within filed and historical ranges were implemented. In the second phase, long-term process improvements that are outside the filed ranges will be implemented for a significant improvement in bioreactor productivity. Results from lab and at-scale study have confirmed that there was no adverse impact of phase 2 process improvements on cell culture, downstream processes and product quality. After finalizing the regulatory strategy, process validation campaigns to qualify phase 2 process improvements are currently being planned

Development of antibody detection methods for active product at the cell culture stage

Development of new analytical methods to characterize the manufacturing process became necessary when the site wanted to implement process improvements to increase output from the bioreactor. The bioreactor process is monitored and controlled for dissolved oxygen content, glucose concentration, temperature, etc. and samples of the harvest are analyzed daily using an endpoint assay and a total protein method. Neither of the two methods can detect the total amount of product at the harvest stage; the endpoint assay will only detect active product and the total protein method will not discriminate between the cellular proteins and the active product. We have developed antibody based methods to overcome these challenges

Direct inoculation of a perfusion bioreactor with a frozen intermediate seed train

Flexibility in cell culture manufacturing via a reduction in the process duration is a key strategy to execute cell culture manufacturing campaigns quickly in a multi-product facility. A major bottleneck is the seed train, which can add weeks to the timeline of the production culture. Seeding production bioreactors with a direct, cryopreserved CHO cell inoculum could possibly eliminate the need for a lengthy continuous seed train and provide other numerous benefits. Previous efforts have shown that high-density (HD) cell banking can be an effective means to reduce the number of seed-train steps required and also improve operational success in seed-train processes. This study demonstrates that it is possible to remove the entire seed train during routine operations by using an intermediate frozen seed train. This involves cultivating cells to high cell density in a perfusion bioreactor, and cryopreserving cells in multiple disposable bags. Each run for a manufacturing campaign would then come from a thaw of one or more of these cryopreserved bags directed inoculated in the N-1 bioreactor. The data gathered during the development and optimization of the different steps during the generation of a frozen intermediate seed train using various approaches and technologies, will be presented. As well as the extensive data set that has been generated to demonstrate that the new process scheme delivered the same performance as the conventional seed train process

Preferentially selecting cellular metabolism and improving productivity by controlling do and Pco2

Cells utilize glucose as their main resource for deriving energy through ATP production. The quantity of ATP generated depends on the metabolic pathways that are employed, aerobic glucose metabolism or anaerobic glucose metabolism. Using our bench top bioreactor model, we have shown these two metabolic pathways can be preferentially selected by controlling the desired cell culture DO and pCO2, and productivity was increased as a result. The DO and pCO2 controlling strategy was implemented in at-scale bioreactors and yielded the expected metabolic and productivity outcom