Determination of immunogenic proteins in biopharmaceuticals by UHPLC\u2013MS amino acid analysis

Abstract

Nowadays, there is a growing interest in innovative and more efficient therapeutics—biopharmaceuticals, based on peptides or proteins. There are increased demands on quality control of such therapeutics. One of the methods usually used for characterization and quantification of biopharmaceuticals is amino acid analysis. In this work, a modern advanced analytical method based on precolumn derivatization and reversed-phase ultra high-performance liquid chromatography in combination with single quadrupole mass spectrometer was developed for amino acid analysis in different protein samples—model sample of bovine serum albumin, sample of strong immunogenic protein keyhole limpet hemocyanin, and sample of drug etanercept present in commercially available biopharmaceutical Enbrel. The method used isotopically labeled internal standards and was validated according to the International Council for Harmonisation guideline. The developed method was characterized by favorable performance and validation parameters, such as time of analysis (6 min), specificity, linearity (r2 ≥ 0.99), limit of detection (0.009–0.822 µM), limit of quantification (1–2.5 µM), accuracy (recovery in the range 90–102.8%), intra-day (RSD in the range 0.25–11.97%) and inter-day precision (RSD in the range 1.67–11.57%), or stability (RE ≤ 12%). According to these findings, the developed amino acid analysis approach is suitable for routine use in areas of peptide/protein quantification, such as quality control laboratories of biopharmaceutical companies.