A Novel Strategy to Rapidly Profile and Quantify High-Risk Host Cell Proteins Using Integrated DDA-PRM-dMRM Mode

Host cell proteins (HCPs) are critical process-related
impurities
in biotherapeutics that threaten drug stability and safety. The substantial
dynamic range (>5 orders of magnitude) between therapeutic proteins
and residual HCPs often leads to difficulty in detecting high-risk
species. Herein, we developed a novel strategy integrating data-dependent
acquisition (DDA), parallel reaction monitoring (PRM), and multiple
reaction monitoring (MRM) techniques to comprehensively profile and
accurately quantify high-risk HCPs. We constructed a Chinese hamster
ovary (CHO) cell spectral library by DDA that covers all potential
HCPs present in downstream purification processes. Based on the constructed
library, 38 reported high-risk HCPs were focused and their unique
peptides and transitions were predicted. PRM and MRM were performed
to cross-validate the existing high-risk HCPs in CHO cell samples,
and 28 high-risk HCPs with 47 peptides and 141 transitions were validated.
A new dynamic MRM (dMRM) method was established and validated to simultaneously
quantify 28 high-risk HCPs. We applied this strategy to analyze five
purified monoclonal antibody process samples, using the DDA method
for comprehensive profiling of unknown HCPs and the dMRM method for
rapid quantification of 28 known high-risk HCPs. Overall, this strategy
enables thorough analysis of known and unknown HCPs, optimizing biopharmaceutical
process development.