(307f) On the impact of Nutrient Supplementation Strategies on antibody producing CHO cell cultures

The production of recombinant proteins remains a fast growing market, expected to surpass $125 billion by 2020. Further improvements in manufacturing capacity and bioprocess yields are required to meet the increasing global demand for biologics. Fed batch cultures have been the dominant mode of production over the last 20 years due to increased productivity compared to batch cultures and relative simplicity compared to continuous cultures. Process intensification strategies for fed-batch cultures can be implemented primarily through manipulation of: (1) feed media composition, (2) mode of operation (e.g. concentrated fed batch with cell retention) and (3) feeding schedules (e.g. fixed vs variable volume additions). The development of sophisticated, highly customized cell culture media has arguably contributed the most to the high cell densities and titers attainable today. Nevertheless, due to the high associated costs, media development has been carried out primarily by the industrial sector, leading to a considerable amount of knowledge and know-how being either semi-empirical or kept in-house as intellectual property (IP). In order to unlock further improvement potential, an in depth understanding of cellular metabolism and its response to nutrient and co-factor perturbations through detailed omics analyses is required. The overall aim of this work is to investigate the metabolic response of antibody producing CHO cells to variations in feed media composition, mode of operation and feeding schedule in order to improve final titer.

Herein we present in detail the formulation of a basal chemically defined media (UCBE-CHO, University College London) and compare its performance against commercially available media (CD-CHO, Life Technologies, USA) in batch and fed batch cultures for three different CHO cell lines (IgG4 producing GS-CHO K1, parental GS-CHO K1 and CHO-S). Detailed statistical analysis of the experimental data using multivariate analysis techniques (MVA) revealed positive correlations between specific nutrient groups and cell growth, culture longevity and specific productivity. Based on this understanding, a series of fed-batch experiments that introduced perturbations in the concentrations of 15 nutrient groups in the feed media were performed and fully analyzed using both commercial (CD-CHO) and in-house (UCBE-CHO) media. Overall, the impact of nutrient perturbations during batch and fed batch cultures highlighted nutrient groups of particular interest for bioprocessing applications.

Subsequently, a series of fed-batch experiments to investigate the impact of different modes of operation (e.g. concentrated fed batch with cell retention) and different feeding schedules (e.g. fixed vs variable volume additions) on the performance characteristics of GS-CHO cell cultures were performed. Of particular interest is the performance observed during concentrated fed-batch (CFB) cultures which led up to a 5 fold volumetric increase in viable cell count (70x10⁶ cells mL^-1) and 11 fold increase in antibody titer (2 g L^-1 day^-1) compared to control fed-batch cultures of the same duration. The observed increases in volumetric productivity were due to synergies arising from the increased and sustained high viable cell densities (CFB) coupled with increases in specific productivity from improvements in feed media composition. The presented herein represents an effort to offer insights for the systematic design, development and optimization of metabolically balanced media and feeding strategies for improved cell growth and productivity.