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By Zachary W. Deeds, Ashley D. Smith, Benjamin J. Cutak, C. Steven Updike, Mindy S. Wilson, Daiva Dailide, Dennis W. Cooley, Barry R. Drew, and Matthew V. Caple

Protein hydrolysates are commonly utilized in cell culture processes either as a component of a complete medium formulation or as part of a fed-batch bioreactor process. It is well documented that hydrolysates can have a substantial positive impact on cell growth and/or protein production. Given the undefined nature and the lot-to-lot variability associated with hydrolysates, there exists a need to mitigate these risks with a chemically defined (CD) alternative that can maintain the desired performance. In order to create a CD alternative to protein hydrolysates, a two-pronged approach was taken to capture the associated effects. The first experimental path utilized standard analytical techniques to identify the nutritive components supplied by hydrolysates (i.e. amino acids, vitamins). A "basal" supplement was designed to supply these nutrients, as this is a significant portion of the functionality of hydrolysates. The second path was based upon Reverse Phase HPLC fractionation of multiple different hydrolysate types. Cell culture screening with Chinese Hamster Ovary (CHO) cells led to the identifi cation of "bioactive" fractions. Subsequent identification of the components contained within the fractions led a greater understanding of the effects of hydrolysates. The learnings from both approaches were utilized to generate a chemically defined supplement, EX-CELLTM CD Hydrolysate Fusion, which is capable of replacing the functions of hydrolysates in many CHO cell systems.