We have evaluated CZE-ESI-MS/MS for detection of trace amounts of host cell protein impurities in recombinant therapeutics. Compared to previously published procedures, we have optimized the buffer pH used in the formation of a pH junction to increase injection volume. We also prepared a 5-point calibration curve by spiking 12 standard proteins into a solution of a human mAb. A custom CZE-MS/MS system was used to analyze the tryptic digest of this mixture without depletion of the antibody. CZE generated a ∼70-min separation window (∼90-min total analysis duration) and ∼300-peak capacity. We also analyzed the sample using ultra-performance LC-MS/MS. CZE-MS/MS generated approximately five times higher base peak intensity and more peptide identifications for low-level spiked proteins. Both methods detected all proteins spiked at ∼100 ppm level with respect to the antibody.
Keywords: Bottom-up proteomics; CZE; Host cell proteins.
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