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.
Productivity enhancement by CHO cell culture to several grams/L has been revolutionizing the biomanufacture of recombinant
therapeutic antibodies.1 The development of antibodies and their derivatives with close to 200 clinical trials largely depends on the knowledge gathered
about CHO expression systems, which has enabled the biotech industry to accelerate the development of its pipeline candidates.
It is also forecasted that CHO production systems will play a crucial role in future biomanufacturing processes because of
their capability to produce high expression titers,1–3 in some cases equivalent to those of transgenic animals (approximately 10 g/L), along with desirable post-translational
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