Identification of Autocrine Growth Factors Secreted by CHO Cells for Applications in Single-Cell Cloning Media

Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However, regulatory authorities have discouraged the use of FBS to reduce the risk of contamination by adventitious agents from animal-derived components. In this study, we demonstrated how a complementary mass spectrometry-based shotgun proteomics strategy enabled the identification of autocrine growth factors in CHO cell-conditioned media, which has led to the development of a fully defined single-cell cloning media that is serum and animal component-free. Out of 290 secreted proteins that were identified, eight secreted growth factors were reported for the first time from CHO cell cultures. By supplementing a combination of these growth factors to protein-free basal media, single cell growth of CHO cells was improved with cloning efficiencies of up to 30%, a 2-fold improvement compared to unsupplemented basal media. Complementary effects of these autocrine growth factors with other paracrine growth factors were also demonstrated when the mixture improved cloning efficiency to 42%, similar to that for the conditioned medium