The development and delivery of high quality therapeutic products necessitates the need for highthroughput (HTP) process development tools. Traditionally, these works requires a combination of shake flask and small-scale stirred tank bioreactor (STR) which are labor and resource intensive and time-consuming. Here we demonstrate a strategy for rapid and robust cell culture process development by evaluating and implementing the use of a new HTP disposable micro bioreactor (MBR) called AMBR TM system (Advanced Microscale Bioreactor) that has the capabilities for automated sampling, feed addition, pH, dissolved oxygen (DO), gassing and agitation controls. In these studies the performance of two monoclonal antibody (MAb) producing cell lines (MAb1 and MAb2) was evaluated both in the AMBR system and 3-L STR. We demonstrated that cell culture performance (growth and viability, production titer and product quality) were similar in both vessel systems. Furthermore, process control and feed optimization were demonstrated in an additional cell line (MAb3) in the disposable MBR and its performance confirmed at STR scale. The results indicate that the AMBR system can be used to streamline the process development effort and facilitate a rapid and robust cell culture process development effort for MAb programs in a HTP manner.

Recent advances in high‐throughput (HTP) automated mini‐bioreactor systems have significantly improved development timelines for early‐stage biologic programs. Automated platforms such as the ambr® 250 have demonstrated the ability, using appropriate scale‐down approaches, to provide reliable estimates of process performance and product quality from bench to pilot scale, but data sets comparing to large‐scale commercial processes (>10,000 L) are limited. As development moves toward late stages, specifically process characterization (PC), a qualified scale‐down model (SDM) of the commercial process is a regulatory requirement as part of Biologics License Application (BLA)‐enabling activities. This work demonstrates the qualification of the ambr® 250 as a representative SDM for two monoclonal antibody (mAb) commercial processes at scales >10,000 L. Representative process performance and product quality associated with each mAb were achieved using appropriate scale‐down approaches, and special attention was paid to pCO2 to ensure consistent performance and product quality. Principal component analysis (PCA) and univariate equivalence testing were utilized in the qualification of the SDM, along with a statistical evaluation of process performance and product‐quality attributes for comparability. The ambr® 250 can predict these two commercial‐scale processes (at center‐point condition) for cell‐culture performance and product quality. The time savings and resource advantages to performing PC studies in a small‐scale HTP system improves the potential for the biopharmaceutical industry to get products to patients more quickly.

T he characterization of a batch cell culture process to produce a monoclonal antibody from a GS-NS0 mouse myeloma cell line is described. Productivity and cellular metabolism were monitored during scale-up to both characterize the process and aid in assessing cell culture stability. During fermentation scale-up studies, it was found that as culture generation number increased, productivity declined. In both flask and bioreactor cultures, declining production started abruptly at approximately generation 60. In this study, we assessed whether the decline in productivity was due to genetic instability of the cell line, which resulted in the generation of a non-producer sub-population, or a shift to a less productive state of cellular metabolism.Genetic stability of the cell line was assessed by determining the copy number of the genetic elements encoding the antibody heavy and light chains, at both early and late generations. Construct copy number instability could result in declining productivity if copy number fell as the culture aged. To address this possibility, Southern analysis techniques were developed to distinguish between single copy, tandem copy, and multiple copy integration possibilities. This analysis revealed that there was a single copy of the construct encoding the heavy and light chain antibody fragments, and copy number did not change in cultures that were passaged for 100 generations, relative to the master cell bank. Genetic instability was therefore not the cause for the drop in antibody production.FACS analysis was employed to determine whether productivity declines could be explained by the presence of a sub-population of non-producing cells, or by a shift in the metabolic state of the culture. Fluorescently labeled anti-IgG antibodies were used to assess intracellular levels of antibody, and a mitochondrial probe was used to assess the metabolic state of the cells. This analysis revealed no evidence of a sub-population of non-producing cells. We did find, however, evidence suggesting that the metabolic state of the culture is a key factor in determining productivity.Significant increases in both lactate production and glucose consumption occurred after cell cultures were passaged for 60 generations, suggesting that later generation cultures were utilizing oxygen less efficiently than earlier generation cultures. These late generation cultures displayed a higher specific lac-tate production rate and produced lower antibody titers. Those cultures with higher mitochondrial activity produced higher antibody titers, and individual cells with higher mitochondrial activity contained higher levels of intracellular antibody. As a whole, these studies demonstrate that shifts in cellular metabolism can occur as a culture ages, significantly impacting culture productivity.

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