METHODS AND MATERIALS
The following methods and measurements were used to perform the analyses.
Cell Lines and Media
Table 1 shows the CHO cell lines tested as part of the development of the CHO Media Library. The media used were all animal-component
free media and some were chemically-defined media without hydrolysates. Stock cultures for the different cell lines were pre-adapted
to the test formulations or directly seeded into the test formulations without adaptation. All formulations and the two control
media were supplemented with 6 mM L-glutamine unless otherwise specified. The CHO cell lines were cultured in the 125-mL shake
flasks or 50-mL TPP tissue culture tubes in a Multitron incubator shaker (ATR Biotech). The cells were counted with a Cedex
cell counter (Flownamics, Inc.).
Table 1. CHO cell line models for CHO Media Library study
Alkaline Phosphatase Activity
Alkaline phosphatase activity was measured with an Alkaline Phosphatase Fluorescence Detection Kit (Sigma-Aldrich, AP-F) according
to the manufacturer's protocol, but with some adjustments. Briefly, 20 uL of cell supernatant sample (diluted with deionized
water) was added to each well of a 96-well plate. Then, 180 uL of the bulk buffer solution consisting of dilution buffer and
fluorescent assay buffer at a 1:8 ratio was added to each well. Four uL of 2.5 mM substrate (4-methylumbelliferyl phosphate
disodium salt) was added to each well and mixed. The enzymatic activity was measured in a fluorometer at 360 nm excitation
and 440 emission using the kinetic reading mode.
Human hGH ELISA
hGH productivity was quantified with an hGH ELISA Detection Kit (Roche Product # 1585878) according to the manufacturer's
Human IgG HPLC Assay
IgG concentration was measured with a Protein G affinity chromatography as described.2
Metabolites were quantified with a BioProfile 400 Analyzer (Nova Biomedical) according to the manufacturer's instruction.
The effect of shifting culture temperature was examined by changing the culture temperature from 37 °C to 31 °C on cell culture
day six, a point at which cell growth had decreased such that the viable cell density increased <50% from the previous day.
As an efficient statistical methodology, DOE has been used in different areas, including drug discovery and cell culture media
development.3–6 Screening and mixing experimental designs (Figure 2) were developed and the data were analyzed using Design-Expert Version