CELL DETACHMENT BY PROLYL-SPECIFIC ENDOPEPTIDASE FROM <i>WOLFIPORIA COCOS</i>

Abstract

As requirements for Advanced Therapy Medicinal Product (ATMP) production differ from other production processes (e.g., therapeutic protein production), cell detachment is often a crucial step for the process success. In most cases, cell detachment is done enzymatically. Although many peptidases are established in cell culture in R&D, e.g., Trypsin as gold standard, many of them seem to be unsuitable in ATMP production processes. Therefore, the present study investigated a novel endopeptidase used in food biotechnology for its applicability in ATMP processes where cell detachment is needed. The Prolyl-specific Peptidase (PsP) is of nonmammalian origin and considered as safe for humans. PsP was purified from the supernatant of the fungus Wolfiporia cocos. The isolation and purification resulted in an enzyme solution with 0.19 U mg −1 prolylspecific activity. By in silico analysis it was confirmed that attachment-promoting proteins can be cleaved by PsP in a similar amount than with Trypsin. Further the proteolytic activity was determined for PsP and Trypsin by using the same enzymatic assay. Detachment with both enzymes was compared for cells used in typical therapeutic production processes namely a mesenchymal stem cell line (hMSC-TERT) as a model for a cell therapeutic, Vero and MA104 cells used for viral therapeutic or vaccine production. The cell detachment experiments were performed with comparable enzyme activities (1.6 U mL −1). hMSC-TERT detachment was faster with PsP than with Trypsin. For Vero cells the detachment with PsP was not only faster but also more efficient. For MA104 cells the detachment rate with PsP was similar to Trypsin. For all cell types, detachment with PsP showed less influence on cell growth and metabolism compared to standard Trypsin.Thus, three cell types used in ATMP, viral therapeutics or vaccine production can be detached efficiently and gently with PsP. Therefore, PsP shows potential for cell detachment in ATMP and viral/vaccine production processes.

The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

“…A promising and safer alternative to trypsin is the prolyl-specific peptidase (PsP) expressed natively and isolated from the fungus Wolfiporia cocos [58]. Allogenic hMSC-TERT cells were detached more rapidly with 1.6 U/mL PsP than trypsin, and the new enzyme also showed less severe effects on the growth and metabolism of re-cultivated cells.…”

“…The molecular basis of cell attachment depends on the microcarrier, so the choice of enzyme for detachment must take this into account. Furthermore, animal-derived enzymes such as pancreatic trypsin should be avoided [58,59]. Further aspects that must be addressed include the commercial availability of the enzyme, the final cell density, the incubation time and the temperature.…”