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
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.
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.
Robin A. Heller-Harrison is the associate director and the corresponding author in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Robin A. Heller-Harrison
Kerstin Crowe is a research scientist II in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Kerstin Crowe
Cecilia Cooley is a research scientist I in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Cecilia Cooley
Megan Hone is scientist II in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Megan Hone
Kevin Mccarthy is a principal scientist I in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Kevin Mccarthy
Mark Leonard is the director in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Mark Leonard
Rate this page
Would you recommend this page to a friend?
Your original vote has been tallied and is included in the ratings results.