Dual Data-Independent Acquisition Approach Combining Global HCP Profiling and Absolute Quantification of Key Impurities during Bioprocess Development

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.