Optimization of medium with perfusion microbioreactors for high density CHO cell cultures at very low renewal rate aided by design of experiments

Abstract

A novel approach of design of experiment (DoE) is developed for the optimization of key substrates of the culture medium, amino acids, and sugars, by utilizing perfusion microbioreactors with 2 mL working volume, operated in high cell density continuous mode, to explore the design space. A mixture DoE based on a simplex‐centroid is proposed to test multiple medium blends in parallel perfusion runs, where the amino acids concentrations are selected based on the culture behavior in presence of different amino acid mixtures, and using targeted specific consumption rates. An optimized medium is identified with models predicting the culture parameters and product quality attributes (G0 and G1 level N‐glycans) as a function of the medium composition. It is then validated in runs performed in perfusion microbioreactor in comparison with stirred‐tank bioreactors equipped with alternating tangential flow filtration (ATF) or with tangential flow filtration (TFF) for cell separation, showing overall a similar process performance and N‐glycosylation profile of the produced antibody. These results demonstrate that the present development strategy generates a perfusion medium with optimized performance for stable Chinese hamster ovary (CHO) cell cultures operated with very high cell densities of 60 × 106 and 120 × 106 cells/mL and a low cell‐specific perfusion rate of 17 pL/cell/day, which is among the lowest reported and is in line with the framework recently published by the industry.

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells

“…Perfusion (PER) culture is a continuous culture mode, in which fresh medium is continuous to be added into the bioreactor and the product is continuously harvested. Therefore, PER culture is a relatively new technique to maintain cell viability and harvest high-quality proteins (Liang et al 2023 ; Schwarz et al 2023 ). PER culture also provides a narrower residence time distribution for products, especially unstable RTPs like FVIII, limiting exposure to extracellular enzymes (e.g., proteases, glycosidases, sialidases) as well as the extracellular environment (pH, temperature) (Coronel et al 2020 ).…”

Advanced cell technologies: Making protein, cell, and gene therapies a reality

“…Interestingly they report a positive impact of cellular "organized stress" in the seed bioreactor on the production performance. Schwarz et al (2023) implemented a design of experiment approach to design an optimal CHO culture medium capable of supporting the operation of microbioreactors in perfusion at HCD and low specific perfusion rates while maintaining constant specific product quality attributes such as N-glycosylation profile of the produced antibody.…”

Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells

“…Detailed descriptions of the Mobius ® Breez system (Sigma Aldrich, St. Louis, MO, USA) with relevant methodologies have been previously described [23]. Prior to performing the automated pH calibration procedure, EX-CELL ® Advanced HD Perfusion Medium (Sigma Aldrich, St. Louis, MO, USA) was characterized to determine the relationship between pH and dissolved CO 2 .…”