Rapid Development and Optimization of Cell Culture Media - - BioPharm International

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Rapid Development and Optimization of Cell Culture Media


BioPharm International
Volume 21, Issue 1

METHODS AND MATERIALS

The following methods and measurements were used to perform the analyses.

Cell Lines and Media


Table 1. CHO cell line models for CHO Media Library study
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.).

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 protocol.

Human IgG HPLC Assay

IgG concentration was measured with a Protein G affinity chromatography as described.2

Metabolite Profiling

Metabolites were quantified with a BioProfile 400 Analyzer (Nova Biomedical) according to the manufacturer's instruction.

Temperature Shift

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.

Statistical Analysis


Figure 2.
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 7.0.1 (Stat-Ease).


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