Intact protein quantification in biological samples by liquid chromatography – high-resolution mass spectrometry: somatropin in rat plasma

Bults, Peter; Sonesson, Anders; Knutsson, Magnus; Bischoff, Rainer; Merbel, Nico C. van de

doi:10.1016/j.jchromb.2020.122079

Cited by 11 publications

(3 citation statements)

References 18 publications

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“…Optimal results with regard to the amount of beads and antibody, temperature and duration of the capture, and elution steps are presented in Table 2 . The final procedure resulted in a reproducible immunocapture recovery of 70%, which is comparable to the findings of Bults et al with the same capture antibody for recombinant human GH [ 10 ]. The antibody also captured the GH2 isoform, albeit to a lower extent (30–60%).…”

Section: Resultssupporting

confidence: 79%

“…So far, the experience with LC-MS for GH quantification has been limited. Two published methods target the recombinant form of GH1 (22 kDa), called somatropin, and these methods were developed for supporting preclinical pharmacokinetic studies in rats [9,10]. Two other methods were described for the GH quantitative analysis in human plasma or serum.…”

Section: Introductionmentioning

confidence: 99%

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Selective quantification of the 22-kDa isoform of human growth hormone 1 in serum and plasma by immunocapture and LC–MS/MS

et al. 2022

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The human growth hormone GH1 (22 kDa) is a commonly measured biomarker for diagnosis and during treatment of growth disorders, but its quantification by ligand binding assays may be compromised by the occurrence of a number of isoforms. These can interfere in the assays and lead to differences in results between laboratories and potentially even in the treatment of patients. We present an LC–MS/MS method that is able to distinguish the major growth hormone isoform (GH1, 22 kDa) from other isoforms and quantify it without any interference across the clinically relevant concentration range of 0.5 to 50 ng/mL. Analysis involves purification of a 100-µL serum sample by immunocapture using an anti-GH-directed antibody, tryptic digestion, and LC–MS/MS quantification of an isoform-specific signature peptide for GH1 (22 kDa). A tryptic peptide occurring in all GH isoforms is monitored in the same 16-min analytical run as a read-out for total GH. Stable-isotope-labeled forms of these two peptides are included as internal standards. Full validation of the method according to recent guidelines, against a recombinant form of the analyte in rat plasma calibrators, demonstrated intra-assay and inter-assay imprecision below 6% across the calibration range for both signature peptides and recoveries between 94 and 102%. An excellent correlation was found between nominal and measured concentrations of the WHO reference standard for GH1 (22 kDa). Addition of up to 1000 ng/mL biotin or the presence of a 100-fold excess of GH binding protein did not affect the measurement. Equivalent method performance was found for analysis of GH in serum, EDTA, and heparin plasma. Analyte stability was demonstrated during all normal sample storage conditions. Comparison with the IDS-iSYS GH immunoassay showed a good correlation with the LC–MS/MS method for the isoform-specific signature peptide, but a significant positive bias was observed for the LC–MS/MS results of the peptide representing total GH. This seems to confirm the actual occurrence of other GH isoforms in serum. Finally, in serum from pregnant individuals, no quantifiable GH1 (22 kDa) was found, but relatively high concentrations of total GH. Graphical abstract

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Section: Resultssupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

Selective quantification of the 22-kDa isoform of human growth hormone 1 in serum and plasma by immunocapture and LC–MS/MS

et al. 2022

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“…This technique is traditionally used for qualitative purposes, and its detection sensitivity is often perceived as less favorable than that of the standard triple-quadrupole mass spectrometer. However, its usefulness for intact protein quantification at trace levels in biological samples has been demonstrated, such as for the 22 kDa biopharmaceutical somatropin, which could be quantified down to 10 ng/ml in rat plasma, when combined with immunoaffinity extraction [22]. Here, it should be noted that when protein isoforms have small or no differences in mass, HRMS may not be able to distinguish the different forms, and some sort of chromatographic resolution is essential to separate the different forms prior to their detection.…”

Section: Separation and Detectionmentioning

confidence: 99%

Large Molecule Bioanalysis by LC–MS: Beyond Simply Quantifying

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Intact quantitative bioanalytical method development and fit-for-purpose validation of a monoclonal antibody and its related fab fragment in human vitreous and aqueous humor using LC-HRMS

et al. 2022

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Intact protein quantification in biological samples by liquid chromatography – high-resolution mass spectrometry: somatropin in rat plasma

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Cited by 11 publications

References 18 publications

Selective quantification of the 22-kDa isoform of human growth hormone 1 in serum and plasma by immunocapture and LC–MS/MS

Selective quantification of the 22-kDa isoform of human growth hormone 1 in serum and plasma by immunocapture and LC–MS/MS

Large Molecule Bioanalysis by LC–MS: Beyond Simply Quantifying

Intact quantitative bioanalytical method development and fit-for-purpose validation of a monoclonal antibody and its related fab fragment in human vitreous and aqueous humor using LC-HRMS

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