Comprehensive Mass Metals Balance in CHO Cell Processes

Cameron Stouffer 1, Sarah Wysor 1 and R. Kenneth Marcus 1

(2) Department of Chemistry, Clemson University

It is well established that the metal content in Chinese hamster ovary (CHO) cell culture media (CCM) significantly affects process productivity and critical quality attributes (CQAs). Metals exist in diverse chemical forms in CCM, which may change during the monoclonal antibody (mAb) production cycle. Naturally, it is postulated that the chemical speciation of these metals affects their uptake and metabolism. While the targeted forms of metals in media are company-specified, their commercial sources, as well as those of the organic media constituents, may result in concentrations and speciation that differ from the intended formulas. Therefore, there is a need for a method to determine the concentration of metals and their chemical forms. Presented here is a methodology to speciate inorganic-versus-ligated metals using high-performance liquid chromatography (HPLC) employing a polypropylene capillary-channeled (C-CP) fiber column with inductively coupled plasma mass spectrometry (ICP-MS) determinations of five target metals (Mn, Fe, Co, Cu, and Zn). A 50 µL injection of CCM supernatant is used for an effective quantification method to identify metal speciation and concentration deviations from reported formula levels. Two case studies as to the utility of the methodology are presented: shelf-life and chemical contamination.  The further development of this method will allow for improvement in quality control, identification of contaminants, assessment of media stability/degradation products, and measurement of whole cell metal uptake in these growth media.