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
Results and Discussion
A universal medium that can support transfection, selection, and amplification, along with cell culture optimization for high
productivity, has time and cost advantages. Such a medium not only reduces overall timelines for cell culture development
but also provides a uniform process development platform by minimizing the changes caused by intermittent adaptation processes.
A concerted effort to develop a Medarex proprietary medium, MTCM A, as part of an integrated cell line and cell culture process
development, was carried out for a HuMAb CHO production platform. This medium was further optimized by adjusting the levels
of nutrient requirements for our cell lines, and an improved second-generation medium, MTCM B, was derived. These two versions
of MTCM media, with minor changes in composition, can support transfection, amplification, subcloning, and production, and
were successfully used for the development of different but related CHO cell lines for HuMAb production. The genealogy of
the CHO host cell lines that are generally used for recombinant expression is depicted in Figure 1.
Figure 1. Genealogy of commonly used CHO host cell lines