Development and optimization of growth media, feed solutions, and feed strategies is a complex project. More than 200 components
(amino acids, trace metals, vitamins, growth factors, carbon sources, etc.) can be found in various commercial growth media
formulations. A typical cell culture medium formulation may contain 50 to 60 individual components. Some of these may be critical
for cell growth or productivity, others may be toxic at certain levels, and many may be involved in complex interactions in
the same or competing pathways within the cell. Media and feed components typically are screened individually or in small
combinations in shake flasks, in experiments of a few dozen shake flasks in parallel. This method significantly limits the
ability to discover complex interactions among medium components.
A high-throughput medium development technology that allows the rapid development of optimized base growth media, feed solutions,
and feed strategies has been developed and was used in the following study with a proprietary Chinese hamster ovary (CHO)
cell line. Approximately 100 components were screened in titration experiments to select the top 16 components for feed optimization.
A 16-component central composite design with axial and center points was then implemented in a shaker plate model system.
Based on the productivity levels, two of the top combinations were selected for confirmation in shake flask studies. Using
a high-throughput approach, we were able to increase the viable cell density and volumetric productivity from 3.5 x 106/mL and 0.3 g/L to 8.0 x 106/mL and 1.2 g/L in two experiments. This article discusses the high-throughput technology and the experimental approach.
A recombinant CHO cell line engineered to express a proprietary antibody was provided by an outside party. Cells were grown
in a commercial serum-free medium with L-glutamine and insulin. Cell cultures were grown at 37°C with 5% CO2. Initially, optimal conditions were determined for the growth of CHO cells in a shaker plate model system, and it was shown
that cell growth was comparable to shake flasks. A high-throughput liquid handler was used to deliver the samples to the shaker
plates according to the combinations derived from a design of experiments software program. For this study, 23 amino acids
and derivatives, 4 carbon sources, 10 hydrolysates, 8 lipid solutions, 21 salts, 7 trace metals, 18 vitamin solutions, and
9 other components were prepared in the serum-free medium (without L-glutamine or insulin); pH was adjusted to 7.2 ± 0.2.
Cell count and the viability of cultures in flasks were determined using trypan blue exclusion dye. Viable cell counts for
the cultures in the shaker plate system were determined using a high-throughput plate-based imaging method in which a fluorescent
viability stain was added, and plates were incubated and scanned using a high-throughput fluorescent microscope. Protein concentration
was determined using a bead-based enzyme-linked immunosorbent assay (ELISA)-style immuno-competition assay. Streptavadin beads
were prepared with a biotinylated anti-immunoglobulin G (IgG) antibody. Unknowns were competed against a Cy5-labeled IgG and
read against an antibody standard.
For feed formulation development, titration experiments were performed with 100 components, from which 16 components were
selected based on their impact on viable cell days, specific productivity, and prior experience. A 16-component central composite
design with triplicates was performed in the shaker plate system. Shake flask studies were performed using the top two combinations
based on protein productivity from the 16-component design.
Figure 1. High throughput media development strategy