Partial Replacement of Chemically Defined Media with Plant-Derived Protein Hydrolysates - - BioPharm International


Partial Replacement of Chemically Defined Media with Plant-Derived Protein Hydrolysates

BioPharm International Supplements

Setting Up a Test System

The effects of protein hydrolysates on the overall performance of a biopharmaceutical production system can be influenced by a number of factors including the specific cell line used, the raw material and process used to manufacture the hydrolysate, the hydrolysate dosage, and the composition of the basal medium. The effect of any supplement on the performance of a cell culture system is largely dependent on the formulation of the basal medium. Because CDM and hydrolysates share a number of common components, the additive effects of these components may reduce the performance of a given system as a result of unintentional "overdosing" of certain components. However, hydrolysate supplements also may provide a number of unique constituents that are beneficial for performance. Therefore, it is necessary to experimentally determine the proper hydrolysate dosage for a given hydrolysate medium combination that will provide the desired optimization effect.

Data for the figures in this article were collected using a sub-clone of CHO-K1 cells engineered to express secreted embryonic alkaline phosphate (SEAP) by means of a human cytomegalovirus (HCMV) promoter, and adapted to grow in suspension in a serum-free medium. Cultures were grown in 125-mL shakeflasks containing a final medium volume of 25 mL and various basal media were supplemented with 1 mg/mL G-418. Triplicate cultures were seeded at 3 x 105 cells/mL and incubated at 37 C, in 5% CO2 at 130 rpm for 12 days. Hydrolysate supplementation was achieved by using 100 g/L stock solutions prepared in each respective basal medium. The data shown are not reflective of all systems, but only depict certain examples from testing conducted using three different commercially available CDM.5,6

Cell Viability and Density

Figure 1
During preliminary investigations, a single chemically defined media (CDM-C) diluted to 80% strength with phosphate buffered saline was re-enriched using various soy, wheat, and cottonseed hydrolysates, and the cell viability was examined. It was observed that not all of the hydrolysates tested were able to fully overcome the medium dilution with respect to the overall performance in cell culture (data not shown). Particularly interesting results were obtained using the cottonseed-derived hydrolysate, HyPep 7504. This hydrolysate was tested at concentrations of 8 g/L with several commercially available CDM, both at full strength and diluted to 80% concentration with phosphate buffer.5 Adding HyPep to the media extended cell viability in both hydrolysate-supplemented treatments, as shown in Figure 1.

Figure 2
As Figure 2 shows, diluting the CDM resulted in a significant reduction in cell density, accompanied by a reduction in total SEAP produced (Figure 3). Adding HyPep to the full strength media resulted in a significantly higher maximum cell density as compared to the 100% CDM control. Adding the hydrolysate to the diluted medium more than doubled the amount of total SEAP produced. All hydrolysate-supplemented samples outperformed the 100% medium control in growth, viability, and production of the target protein, demonstrating a significant performance improvement of the cell culture by using this supplement.

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