Materials and Methods
Ultrafiltration of LucraTone Soy P: Five lots of LucraTone Soy P hydrolysates were prepared in 50 g/L stock solutions using MilliQ sterile water and 0.22 µm filtered. Stock solutions were separated into triplicate aliquots. Two aliquots were ultrafiltered either by normal flow or tangential flow ultrafiltration using a 5 kDa MWCO filter (Millipore). The third unfiltered aliquot served as a negative control. All three aliquots were 0.2 µm filtered, then stored at 2–8ºC prior to cell culture performance analyses. Endotoxin levels were determined before and after ultrafiltration using the LAL (Limulus amebocyte lysate) assay.
CHO Cell Culture: For this case study, a CHO cell line expressing a human IgG was grown in serum-free chemically defined medium. Exponentially growing CHO cells were harvested, counted by a Vi-Cell® counter employing the trypan blue exclusion technique, and seeded at 0.5x105 cells/mL into triplicate 125 mL shake flasks (30 mL/flask). Cells were grown in either basal CD-CHO media, CD-CHO media supplemented with 1g/L non-UF LucraTone Soy P, or CD-CHO media supplemented with 1g/L UF LucraTone Soy P.
In all instances, 8mM glutamine, 10 µg/mL puromcyin, 1X HT solution, and 0.2% (v/v) Pluronic F-68 were added to media used in the study. Protein production levels were quantified using a commercially available ELISA kit (Bethyl Laboratories) to detect human IgG. Cell densities were determined daily starting at study day four using a Vi-Cell cell counter. All experiments were performed twice, with each time point sampled in triplicate to demonstrate reproducibility. Error bars are based on one standard deviation of the mean value for the given time point. Data show representative results obtained for all five hydrolysate lots evaluated.