Dr. Christina Morris, Senior Scientist and HCP-MS expert at BioPharmaSpec says, “Bringing mass spectrometry to HCP detection can generate meaningful data and enhance the understanding of HCP profiles by providing an orthogonal analysis to extend ELISA-derived information. I often present this table to illustrate the advantages and disadvantages of these two complementary techniques.”
Quantitation of HCPs can be achieved using both labelling and label-free methods like multiple-reaction-monitoring (MRM) or sequential window acquisition of all theoretical mass spectra (SWATH-MS). The SWATH strategy has seen an increase in popularity, benefitting from simple, fast and cost-effective workflows. Through a combination of IDA (Information Dependent Acquisition) and SWATH MS acquisitions of proteolytic digests of product material, HCP identification and quantitation is performed by matching the MS/MS spectra to theoretical masses of equivalent peptide fragments in an HCP-specific sequence database.
To make sense of the large and highly complex HCP-MS datasets generated from these workflows, bioinformatics tools are used for peak-picking and filtering of potentially thousands of HCP identifications. The algorithms that sort through these spectra are critical to the HCP-MS strategy in terms of time-efficiency, but a degree of manual assessment (as is recognized in this new USP chapter) is an important cross-check to look for any HCP mis-assignments or false positives and is especially important in identifying low-abundance peptides.
With the new HCP-MS General Chapter 1132.1 proposal, the USP clearly recognizes the unique role that MS-based HCP analysis can play in the understanding of product impurity profiles. The ability to identify and quantitate HCPs provides a means to understand the product to a greater extent by generating complementary and orthogonal data to the ELISA-derived information and, through protein identification, accumulate useful information for manufacturing process development and ultimately improved product quality.
BioPharmaSpec’s approach to HCP-MS is flexible and proceeds through clearly defined stages, meaning that you receive data that is useful and applicable at your stage of product development and allows you to meet your specific objectives.