qNMR characterization of potential peptide calibrators for isotope dilution LC–MS quantification of a SARS-CoV-2 IgG monoclonal antibody

Abstract

Abstract The year 2020 will be remembered for the most pernicious epidemic of modern history. The SARS-CoV-2 coronavirus has affected millions of people worldwide and has triggered an unprecedented race for the development of diagnostic tests and therapeutical vaccines. These technologies require befitting measurements that underpin reliability and patient safety. The level of blood-borne antibodies against SARS-CoV-2 is correlated initially with the extent of infection and subsequently with the degree of immunity attained. This renders their accurate measurement an important target for immunometric and liquid chromatography–mass spectrometry methods. Suitable reference materials and methods are being developed to ensure the reliability and consistency of such measurements. Solutions of purified monoclonal IgG may prove useful as primary calibrator materials for the development of calibration hierarchies for the measurement of blood-borne antibodies against SARS-CoV-2. Bottom-up approaches can be applied to the quantification of such structurally complex large molecules. These consist of trypsin and Lys C digestion and quantification of the resulting proteotypic peptides as surrogate analytes for an IgG monoclonal antibody. Establishing the metrological traceability of this procedure requires peptides whose purity has been determined accurately, for example with quantitative nuclear magnetic resonance (qNMR). The 1 H qNMR method allows for the mass fraction assignment of peptides by accurate quantification of 1 H resonance signals specific for both the peptide and for a certified reference material used as internal standard in the peptide solution. The current paper describes the qNMR characterization of five peptides that could be used in a double isotope dilution method for the quantification of SARS-CoV-2 IgG monoclonal antibodies in solution.

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