The Impact of Cell Culture Medium on Cell Line and Process Development Timelines and Strategies
Cell culture medium plays a significant role in determining the cell density, viability, and productivity of a robust manufacturing process. Current regulatory requirements prefer bioprocesses that do not use any animal-derived components throughout cell line and cell culture development. However, using media containing serum for early stages of cell line development is still a common practice for deriving stable production cell lines for manufacturing. The ideal medium should be able to support all activities in the cell line and process development stages for maintaining consistent cell populations and product quality. Here, we describe such performance by an in-house-developed, universal animal component free (ACF) medium that can support transfection, amplification, subcloning, banking, and production of the different Chinese hamster ovary (CHO) cell lines generally used for recombinant protein production. By using this ACF medium, designated as MTCM, there was an overall reduction in cell line development time for two distinct expression systems by eliminating the adaptation period between serum-containing medium to ACF medium. Furthermore, by using the same basal medium throughout cell line and process development, transitional changes in growth characteristics, productivity, and heterogeneity in the cell populations were simultaneously kept to a minimum. Finally, a robust and high yielding upstream production platform technology for human monoclonal antibodies (HuMAbs) was developed by using MTCM both as basal and feed media in fed batch cultures. This accelerated CHO cell culture optimization that led to 6 g/L HuMAb production and facilitated an efficient process integration of upstream and downstream.
The development of cell culture medium has been one of the major efforts contributing to the significant improvements in product titer in the past by supporting high cell densities and extending culture longevity in fed-batch mode. Often, production medium for high titer processes is different from that used during the earlier stages of cell line development, including transfection and subcloning, where single-cell derived colonies need to grow. Medium with serum is often used at these early cell line development stages for both adherent as well as suspension cell cultures. These recombinant cell lines are then slowly adapted to ACF media or chemically defined (CD) media during the later stages of development, for inoculum expansion and recombinant protein production. These transitional phases can take a considerable amount of time for cell lines to adapt to such medium shifts and for process development scientists to select stable, high producers from a diversified population. Here, we describe upstream process development using a proprietary medium throughout the cell line and process development phases.
Materials and Methods
Transfection and Cell Line Development
Transfections of different CHO host cell lines were carried out using a single expression plasmid containing the heavy and light chain genes of a human antibody. A BioRad Gene pulser was used to electroporate the DNA into either a dhfr-deficient (dhfr–, Expression System I) or dhfr-containing (dhfr +, Expression System II) CHO cell line. Transfected cells were plated in 96-well plates and antibody levels in media with selection pressure were measured using a human IgG kappa/gamma sandwich ELISA. The highest producing cell lines were expanded and single-cell cloned by limiting dilution.
Subcloning and Cell Bank Preparation
An aliquot of actively growing cells was diluted in complete growth media to yield 0.25, 0.5, or 1.0 cell/well, and 10–20 x 96-well flat-bottom tissue culture plates were plated. Distinct clones that were confirmed to have originated from single cell/well were evaluated to identify the highest IgG expressing clones. A clone possessing the highest productivity was expanded to prepare a research cell bank. Master cell banks and manufacturer's working cell banks were further established from research cell banks.
Cell Culture Process and Analysis
Recombinant CHO cells were grown in shake flasks or 5- to 85-L scale stirred tank bioreactors (from Applikon, Bellco, or Bioengineering) in batch and fed-batch modes. The temperature and CO2 level were controlled for cell culture incubators. The pH, dissolved oxygen (DO), agitation, and gas flows were also controlled if cells were cultured in bioreactors. In fed-batch mode, feed media were added according to the feeding schedules that were tailored to the specific recombinant cell line. Cell density and cell viability were measured using Cedex (Innovatis), Vicell (Beckman Coulter) cell counters or by a manual hand count using the Trypan blue exclusion method. Antibody concentrations were determined by Protein A HPLC (Waters).
Medarex proprietary media for research, development, and large-scale manufacturing were formulated in both powder and liquid forms from a media vendor.
Copy number analysis
Genomic DNA was purified from transfected and untransfected CHO cell lines. Gene-specific primers and a fluorescent probe for a TAQman method were developed by Applied Biosystems using the following two assays. A murine dhfr assay was used to determine the number of dhfr copies of the expression plasmid integrated into the CHO genome. A rodent gapdh assay was used to determine the number of genomes present in the same sample tested for dhfr for normalization. The reactions were thermal cycled and data was collected using the ABI Prism 7300 Sequence Detection System (Applied Biosystems).
Results and Discussion
Reduction in Cell Line Development Timelines
Conditions were then developed to accelerate the process in the commercial medium without serum right from the initiation of cell line development, a process that included optimizing transfection efficiency. This shortened the cell line development timeline by up to four months and eliminated the need for a lengthy serum-weaning process and one round of subcloning (Figure 2A). However, using only MTCM media during the entire process of cell line development would eliminate media transitions and potentially reduce the timeline to seven months for a 2 μM MTX amplified cell line (Figure 2A).
Expression System II with CHO dhfr+ host cell lines. A similar process could be carried out with Expression System II, which does not require amplification. Initial protocols used serum in a commercially available medium at the transfection stage, which was then removed during adaptation. In a manner similar to Expression System I above, the conditions were optimized using the same commercially available medium without serum throughout transfection and subcloning. As Figure 2B indicates, by using only MTCM media, we could accelerate the cell line development timeline to five months. In addition, further reduction in the timeline by another month could be achieved using bulk transfection and selection methods.
In both cases, pre-adapted host cell banks in MTCM media were generated in a serum-free freezing mixture. Transfection with these well-characterized host cell banks eliminated media transitions and reduced the timelines for cell line development, as illustrated in Figure 2.
Amplification Using MTCM Media
Subcloning Efficiency Using MTCM Media
Preparation of Cell Banks with MTCM Media
HuMAb Production with MTCM Media
Process Development Strategies and Advantages
The biotechnology industry depends on robust manufacturing cell lines to produce biologics. The timelines for deriving a stable manufacturing cell line suitable for consistent process scale-up requires thorough screening and subcloning of the initial transfectant population. In the case of suspension dhfr– CHO cell cultures, amplification procedures using MTX prolongs this process. However, the recombinant cell lines derived from Expression System II have a more straightforward development time, which can take approximately seven months to adapt from serum-containing transfection to serum removal and subcloning. Through careful development of MTCM medium that can support different activities, we were able to significantly reduce the timeline from transfection to cell line selection for both expression systems (Figure 2). Recombinant cell lines derived from both expression systems can use the same medium without serum right from the transfection stage. Using one medium throughout all upstream process development phases also helps retain the higher productivity carried from early clones by eliminating media shifts.
Overall, we significantly reduced resource requirements and substantially shortened cell line development timelines by optimizing a proprietary cell culture medium, MTCM, for all upstream process phases including transfection, amplification, subcloning, banking, and production. Medium changes are some of the most influential factors affecting the heterogeneity of cell populations with respect to post-translational or secondary modifications. Integrating medium screening into the process as early as the clone selection step focuses process development efforts, not only on deriving high productivity clones, but also on maintaining comparable biochemical profiles of the molecule for desired therapeutic potency.11
Using a single cell culture medium from early-stage cell line development all the way through high-titer production significantly affected the process development strategies to obtain stable, robust production cell lines and high yielding cell culture processes. It allows for more predictable feed streams for downstream processes and enables an efficient integration of process technologies.12 It also leads to effective regulatory strategies by supporting economical processes starting at early clinical phase production.
ALAHARI ARUNAKUMARI, PhD, is the senior director of process development and the corresponding author, XIAO-PING DAI, PhD, is the assistant director of bioprocess development, JOEL GOLDSTEIN, PhD, is the associate director of transfectoma development, CLAUDIA KLOTH, PhD, is the senior manager of upstream process validation, bioprocess development, HAILE GHEBREMARIAM is the assistant director, cell line development, GLENN MACISAAC is a scientist II, cell line development, and MELISSA WAGNER is a scientist I, cell line development, all at Medarex Inc., Bloomsbury, NJ, firstname.lastname@example.org
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