No abstract

Host cell proteins (HCP) are a major class of impurities derived from recombinant protein production processes. While HCP are usually monitored by ELISA, mass spectrometry (MS)-based approaches are emerging as promising orthogonal methods. Here, we developed an original method relying on data-independent acquisition (DIA) coupling global HCP amount estimation (Top 3) and absolute quantification with isotope dilution (ID). The method named Top 3-ID-DIA was benchmarked against ELISA and a gold-standard selected reaction monitoring assay (ID-SRM). Various samples generated at different steps and conditions of the purification process, including different culture durations, harvest procedures, and purification protocols were used to compare the methods. Overall, HCP were quantified over 5 orders of magnitude and down to the sub-ppm level. The Top 3-ID-DIA strategy proved to be equivalent to the gold-standard ID-SRM in terms of sensitivity (1-10 ppm), accuracy, and precision. Moreover, 81% of the Top 3 estimations were accurate within a factor of 2 when compared to ID-SRM. Thus, our approach aggregates global HCP profiling for comprehensive process understanding with absolute quantification of key HCP within a single analysis and provides an improved support for bioprocess development and product purity assessment.

No abstract

Mass spectrometry has proven to be a valuable tool for the accurate quantification of proteins. In this study, the performances of three targeted approaches, namely selected reaction monitoring (SRM), parallel reaction monitoring (PRM) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH‐MS), to accurately quantify ten potential biomarkers of beef meat tenderness or marbling in a cohort of 64 muscle samples were evaluated. So as to get the most benefit out of the complete MS2 maps that are acquired in SWATH‐MS, an original label‐free quantification method to estimate protein amounts using an I‐spline regression model was developed. Overall, SWATH‐MS outperformed SRM in terms of sensitivity and dynamic range, while PRM still performed the best, and all three strategies showed similar quantification accuracies and precisions for the absolute quantification of targets of interest. This targeted picture was extended by 585 additional proteins for which amounts were estimated using the label‐free approach on SWATH‐MS; thus, offering a more global profiling of muscle proteomes and further insights into muscle type effect on candidate biomarkers of beef meat qualities as well as muscle metabolism.