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Production and application of high quality stable isotope‐labeled human immunoglobulin G1 for mass spectrometry analysis
Author(s) -
Phillip Amsler,
Thierry Wolf,
Christian Lanshoeft,
Anja Bettighofer,
Jochen Eisfeld,
Thomas Moenius,
Claudia Probst,
Coralie Etter,
Olivier Heudi
Publication year - 2017
Publication title -
journal of labelled compounds and radiopharmaceuticals
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.432
H-Index - 47
eISSN - 1099-1344
pISSN - 0362-4803
DOI - 10.1002/jlcr.3486
Subject(s) - chemistry , chromatography , peptide , mass spectrometry , lysine , calibration curve , arginine , quantitative analysis (chemistry) , stable isotope ratio , tandem mass spectrometry , detection limit , amino acid , biochemistry , physics , quantum mechanics
Here, we describe the production of stable isotope‐labeled human immunoglobulin G1 ([ 13 C]‐hIgG1) using [ 13 C]‐L‐lysine/arginine–labeled hIgG1. The fermentation process was run in shake flasks containing labeled arginine and lysinethat were incorporated into the produced recombinant hIgG1. The [ 13 C]‐hIgG1 was purified, and label incorporation was determined to be >99% at all lysine and arginine moieties. Sequence coverage was confirmed by peptide mapping. [ 13 C]‐hIgG1 was then used as an internal standard (IS) for the development of a liquid chromatography–tandem mass spectrometry method applicable to the quantitative analysis of all human types of hIgG1 in rat serum. Four conserved peptides, namely, GPSVFPLAPSSK, TTPPVLDSDGSFFLYSK, VVSVLTVLHQDWLNGK, and FNWYVDGVEVHNAK, originating from different parts of the fraction crystallizable region of hIgG1, were used for quantitation of hIgG1 in rat serum. The calibration curves with a coefficient of determination (r 2 ) between 0.9950 and 0.9962 resulting from the peak area ratio of each peptide to its respective labeled IS were reproducible. A mean bias within ±20.0% of the nominal values and a precision of ≤20.0 % were obtained for the calibration standards and quality control samples for each peptide. [ 13 C]‐hIgG1 was shown as a suitable IS for quantitative hIgG1 analysis in preclinical species by LC‐MS/MS.