Comparing nitric acid treatment and microwave digestion for efficiency of metal extraction from bioprocess samples.

ABSTRACT

Metal ions may act as enzyme cofactors and influence the kinetics of biochemical reactions that may also influence the biological production of therapeutic proteins and quality attributes such as glycosylation. Because sample preparation is a significant step in the reliable analysis of metals, we compared two sample preparation procedures for metal analysis of bioreactor culture media samples by ICP-MS (i) samples were diluted in 2 % nitric acid (treatment with nitric acid, TNA); and (ii) samples were mixed with equal volume of 5 % nitric acid and closed vessel digestion was performed in a microwave (closed vessel digestion, CVD). In the comparison of extraction efficiencies between TNA and CVD procedures, CVD showed better extraction for Ca and Cu among bulk metals (∼30 %) and for Ni among the trace metals (∼65 %) for the bioreactor broth supernatant samples. For the cell pellet samples, the CVD procedure was found to be better for extraction of Fe (∼65 % more) among bulk metals, Zn (∼20 % more) among minor metals and Co (∼60 % more) and Ni (∼45 % more) among trace metals. Differences between the two procedures were less than 10 % and TNA was better for all other metals quantified from both supernatant samples and cell pellet samples. The current study helps bring more clarity to the methodology on comprehensive metal analysis to monitor and maintain trace metal content for biologics production.