For a Phase 1 antibody project, the primary objective of our cell line development group is to deliver a stable, high performing, clonal cell line in an aggressive timeframe, leaving little time to react to the challenges associated with cell line instability, such as dramatically reduced product expression. Our current cell line development timeline allows for 6.5 months from transfection to the establishment of a cell bank for a lead clone, which is expected to deliver 1–3 g/L in a fed-batch process. Our cell line selection process consists of numerous rounds of successive screening, initially assessing hundreds of clones. During that time, clones are eliminated because of poor performance in scaled-down models of the fed-batch process or because of a loss of product expression over time. When such instability in product expression occurs for a lead clone at a late stage in the timeline, both upstream and downstream process groups must quickly identify alternatives so that the material needs of the project will be satisfied and deadlines are still met.
This manuscript presents examples of cell line instability that we have encountered during Phase 1 antibody projects, and the operational consequences that have ensued. It describes the different types of instability we have observed, our attempts to analyze the presumptive underlying genotypic causes of these different types of instability, and the general methods and tools that we use to investigate cell line instability. Finally, we describe the recent development of an RT-PCR–based assay that allows us to eliminate potentially unstable cell lines from consideration early in the cell line development timeline.
There are various approaches to developing a therapeutic antibody product, and many development organizations now use platform, or standard procedures, (see Figure 1 for an example) because this approach works well for achieving the objectives of delivering a stable, high performing, clonal cell line in a short timeframe. The goal of using a platform approach is to enable a greater number of molecular entities through the pipeline, and to decrease both the length of time and the level of investment in developing these molecular entities before they have proven efficacy in human clinical trials. Cell line development platforms rely primarily on multiple rounds of successive screening of large numbers of clones with the recognition that clones will be eliminated over the course of development because they have not performed sufficiently well in a scaled-down model of the fed-batch production process or they have lost expression of the protein product, even in the presence of selective pressure. During the cell line adaptation period shown in Figure 1, cell line expression characteristics can be evaluated and cell banks for each clone can be generated at some frequency. Hence, an enabling aspect of the design of the platform shown in Figure 1 is that the phenotypic stability of a clone can be established before its selection as the production cell line. By considering stability as part of the cell line selection and cell banking paradigm, we can ensure that the problems encountered and described here during the cell line development window are not observed during the clinical, or future commercial, manufacturing window.
Although our cell line development group has been successful in meeting our project goal of generating Chinese hamster ovary (CHO) Phase 1 cell lines that can deliver >1 g/L in a platform production process, cell line instability occurring at a late stage in the adaptation period timeline has resulted in both upstream and downstream process groups scrambling to deal effectively with last-minute changes to meet deadlines. Hence, we have developed specific methods and approaches to enable an increased understanding of the root causes of our observed cell line instability. This knowledge potentially could be used for the early, proactive identification of unstable clones or the elimination of cell line instability altogether. This would be highly beneficial to the cell line and cell culture process development organization, thus ensuring sustainable success in meeting deliverables and timelines.
This current work describes the types of instability we observe: "acute," a precipitous loss of product expression occurring over a short time span of approximately 2–4 weeks, and "gradual," a slow decline of product expression occurring over the months of the cell line adaptation period shown in Figure 1. In addition, we describe our attempts to analyze the presumptive underlying genotypic causes of these different manifestations of instability, which include, but are not limited to, DNA rearrangements and DNA methylation. We discuss the general methods and tools that we use to investigate cell line instability, including Southern and northern blot analysis, PCR-based methods, and an assay to detect methylated DNA, and how, when, and why we use these methods. Based on results from an intensive study examining the most frequent causes of cell line instability at the molecular level in our Phase 1 antibody projects, we have developed and implemented an RT-PCR–based cell line screening assay that enables the elimination of unstable cell lines from consideration early in the platform process.
Figure 1. A prototype platform cell line development timeline for a Phase 1 antibody project. Following transfection and selection procedures, clonal transfectants are isolated and subjected to successive high-throughput screens. The top performers from this screening process then undergo a cell line adaptation period, consisting of several months. During this time, cell line stability information is collected, and the clones are screened in a series of production fed batches, first at small scale, and then in benchtop bioreactors. A production clone is selected, the final production process is defined, and a master cell bank is generated.