title: Production and application of high quality stable isotope-labeled human immunoglobulin G1 for mass spectrometry analysis.
abstract: Here, we describe the production of stable isotope-labeled human immunoglobulin G1 ([13C]-hIgG1) using [13C]-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 [13C]-hIgG1 was purified, and label incorporation was determined to be >99% at all lysine and arginine moieties. Sequence coverage was confirmed by peptide mapping. [13C]-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 (r2) 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. [13C]-hIgG1 was shown as a suitable IS for quantitative hIgG1 analysis in preclinical species by LC-MS/MS.
authors.affiliation: Novartis Pharma AG, BPRD-IBP, Basel, Switzerland.
authors.forename: Amsler
authors.initials: A
authors.lastname: Phillip
authors.affiliation: Novartis Pharma AG, DMPK, DMBA-BA, Basel, Switzerland.
authors.forename: Wolf
authors.initials: W
authors.lastname: Thierry
authors.affiliation: Novartis Pharma AG, DMPK, DMBA-BA, Basel, Switzerland.
authors.forename: Lanshoeft
authors.initials: L
authors.lastname: Christian
authors.affiliation: Novartis Pharma AG, BPRD-IBP, Basel, Switzerland.
authors.forename: Bettighofer
authors.initials: B
authors.lastname: Anja
authors.affiliation: Novartis Pharma AG, BPRD-IBP, Basel, Switzerland.
authors.forename: Eisfeld
authors.initials: E
authors.lastname: Jochen
authors.affiliation: Novartis Pharma AG, DMPK, DMBA-IL, Basel, Switzerland.
authors.forename: Moenius
authors.initials: M
authors.lastname: Thomas
authors.affiliation: Novartis Pharma AG, BPRD-IBP, Basel, Switzerland.
authors.forename: Probst
authors.initials: P
authors.lastname: Claudia
authors.affiliation: Novartis Pharma AG, BPRD-IBP, Basel, Switzerland.
authors.forename: Etter
authors.initials: E
authors.lastname: Coralie
authors.affiliation: Novartis Pharma AG, DMPK, DMBA-BA, Basel, Switzerland.
authors.forename: Heudi
authors.initials: H
authors.lastname: Olivier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis* / Q000097:blood / Q000737:chemistry*; D007553:Isotope Labeling; D013058:Mass Spectrometry; D051381:Rats
chemical_list: D007074:Immunoglobulin G
keywords: LC-MS/MS; SILAC; antibody; internal standard; quantitation