Application of EAD/CID mixed fragmentation mode supported by different data acquisition and processing strategies for Host Cell Proteins analysis.Authors: Rafał Szewczyk2, Maciej Stopa1, Katarzyna Krupczynska-Stopa2,3
IntroductionHost Cell Proteins (HCPs) are the common process-related impurities generated during biotherapeutic protein production. Due to the potential immunogenicity to humans and possible impact on drug stability the HCP level should be strictly monitored by the drug maker and is frequently on the target of regulatory authorities including FDA, EMA etc. The main challenge during HCP analysis in the scope of MS detection is the high dynamic range between the HCP impurities and therapeutic protein, typically from 100 ppm to far below 0.1 ppm. There is a strong demand for sensitive and robust methods for HCPs identification and quantitation by LC-MS applicable for protein therapeutic development. MethodsIn the presented work we have used micro-flow LC (SCIEX M5 MicroLC) in combination with the mixed fragmentation mode EAD/CID of SCIEX ZenoTOF 7600 mass spectrometer in both data dependent (DDA) as well as data independent (DIA) acquisition strategies to test the depth of HCPs identification in monoclonal antibody samples. The short, 20 minutes gradients were used on Waters CSH Peptide chromatographic column. In addition, the different data analysis modes were tested like library-free analysis vs DDA or gas-fractionated libraries. The accuracy of the HCP quantitation was tested using the spiked-in protein standard mix (UPS-1, Sigma-Aldrich). Finally, the high-resolution data were used to construct the targeted method on low-resolution QqQ instrument (SCIEX QTRAP 6500) for routine analysis. Preliminary resultsThe monoclonal antibody sample was prepared by spiking Waters Intact mAb Standard with UPS dynamic range proteomic standard. The application of mixed EAD/CID fragmentation mode results in ca 20% more peptide and protein identification, compared to single CID mode. As expected, the 6 gas-phase fractionated library contains the highest level of IDs, followed by library-free and DDA analysis. The raw data were processed by PEAKS software or DIA-NN (v.1.8) with processing in Skyline-daily. The quantitative performance of the developed method was superb – 86% and 94% of quantified peptides had CV<20% in Q-TOF and QqQ respectively. Using peptide spiked-in standard and iBAQ quantitation strategy the accuracy of USP-2 standard proteins was in the range of 40-170%, what is totally acceptable for general HCPs profiling method. Novel aspectWe present comprehensive optimization of the analytical method for HCPs analysis using novel EAD/CID mixed fragmentation using ZenoTOF platform. |
ASMS 2025 – Baltimore, USA, 1-5.06.2025 |
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