Hydrolysates (peptones) are widely used in biopharmaceutical manufacturing to enhance cellular growth and production. Ultrafiltration is often utilized to remove large molecular weight entities including endotoxin from hydrolysates. While minimization of endotoxin is a high priority in biomanufacturing, the necessity of ultrafiltration of raw materials has not been firmly established.
Some data suggests that ultrafiltration negatively impacts hydrolysate performance in cell culture media by removing small molecular weight entities that act as growth factors in the media. Moreover, the necessity of hydrolysate ultrafiltration in cases where the material is already low in endotoxin is unclear, despite unsubstantiated claims in the industry that ultrafiltered material supports superior cell culture performance.
LucraTone™ hydrolysates are manufactured by Solabia for exclusive distribution by Millipore. Evaluation of multiple hydrolysate lots indicates that manufacture of LucraTone Soy hydrolysates yields consistently low endotoxin levels, as well as low levels of large molecular weight entities, despite the lack of ultrafiltration in the manufacturing process.
The lack of ultrafiltration of this product provided an opportunity to evaluate the impact of ultrafiltration of hydrolysates on mammalian cell culture performance. In this case study, multiple lots of both ultrafiltered and nonultrafiltered LucraTone Soy P hydrolysates were evaluated to determine their impact on mammalian cell culture performance. Results of our study suggest that there is no impact of hydrolysate ultrafiltration on cell culture performance.
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.