Peptide purity assignment for antibody quantification by combining isotope dilution mass spectrometry and liquid chromatography

Abstract

Peptide purity assignment is necessary for quantification of antibody. Recently, isotope dilution mass spectrometry (IDMS) has been proposed as a primary method for an accurate antibody measurement with traceability. When using the IDMS method, antibodies are measured using their signature peptide as the calibration standard. Therefore, the purity assignments of their signature peptide are essential for the standardization and harmonization of the antibody measurement. In this study, SI‐traceable purity assignments of the signature peptides of an antibody were investigated by combining the IDMS and high performance liquid chromatography (HPLC) method. The final purity was calculated from the mass fraction of the total peptide determined by IDMS and the relative fraction of the target peptide determined by HPLC. The uncertainty was evaluated within the 95% confidence range. These peptides with the certified purity value can also be used for the quantification of antibodies for SARS‐CoV‐2.

Determination of [Glu1]-fibrinopeptide B purity by gas chromatography – isotope dilution mass spectrometry

“…Therefore, the accurate assessment of the purity of peptides and proteins is a necessity. 19 Common approaches to such analyses include mass balance, 20,21 isotope dilution mass spectrometry (IDMS) [19][20][21][22][23][24][25][26] and quantitative nuclear magnetic resonance. 19,[26][27][28] However, mass balance is only suitable for evaluating the purity of solid peptides and proteins while quantitative nuclear magnetic resonance can only be employed in the evaluation of peptides with molecular weights of less than 10 000 Da.…”

“…19,[26][27][28] However, mass balance is only suitable for evaluating the purity of solid peptides and proteins while quantitative nuclear magnetic resonance can only be employed in the evaluation of peptides with molecular weights of less than 10 000 Da. 26,27 Hence, IDMS [19][20][21][22][23][24][25][26] is oen employed to ascertain the purity of these compounds. In this technique, peptides or proteins undergo hydrolysis to yield their individual amino acids.…”

“…This technique also does not require large amounts of organic solvents, making it more environmentally friendly, although sample derivatization may be necessary. [30][31][32] To date, there have been numerous literature reports regarding the analysis of peptide and protein purity by LC-IDMS, [19][20][21][22][23][24][25][26] including assessments of bovine serum albumin, 24 human insulin, 22 bradykinin, 30 vitamin-D-binding protein and its isoforms 29 and so on. In contrast, there have been only a very limited number of papers regarding the assessment of peptide purity using GC-IDMS because of its complicated sample pretreatment.…”