Comparability analysis of protein therapeutics by bottom-up LC-MS with stable isotope-tagged reference standards

MAbs
Anton V ManuilovDavid H Lee

Abstract

Comparability studies lie at the heart of assessments that evaluate differences amongst manufacturing processes and stability studies of protein therapeutics. Low resolution chromatographic and electrophoretic methods facilitate quantitation, but do not always yield detailed insight into the effect of the manufacturing change or environmental stress. Conversely, mass spectrometry (MS) can provide high resolution information on the molecule, but conventional methods are not very quantitative. This gap can be reconciled by use of a stable isotope-tagged reference standard (SITRS), a version of the analyte protein that is uniformly labeled (13)C6-arginine and (13)C6-lysine. The SITRS serves as an internal control that is trypsin-digested and analyzed by liquid chromatography (LC)-MS with the analyte sample. The ratio of the ion intensities of each unlabeled and labeled peptide pair is then compared to that of other sample(s). A comparison of these ratios provides a readily accessible way to spot even minute differences among samples. In a study of a monoclonal antibody (mAb) spiked with varying amounts of the same antibody bearing point mutations, peptides containing the mutations were readily identified and quantified at concentr...Continue Reading

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Citations

Nov 9, 2012·Analytical Chemistry·Alain BeckSarah Sanglier-Cianférani
Jan 15, 2016·Mass Spectrometry Reviews·Piliang HaoSiu Kwan Sze
Sep 29, 2012·Journal of Mass Spectrometry : JMS·Guillaume PicardVirginie Brun
Oct 23, 2012·Analytical Biochemistry·Xin ZhangJette Wypych

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