PMID- 33556683 OWN - NLM STAT- MEDLINE DCOM- 20210316 LR - 20210316 IS - 1873-3778 (Electronic) IS - 0021-9673 (Linking) VI - 1640 DP - 2021 Mar 15 TI - Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry. PG - 461945 LID - S0021-9673(21)00069-8 [pii] LID - 10.1016/j.chroma.2021.461945 [doi] AB - The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The alpha-subunit structure, hCGalpha, has 2 N-glycosylation sites, while the beta subunit, hCGbeta, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle(R), as standard. It allowed the detection of about 30 hCGalpha glycoforms, but didn't allow the detection of hCGbeta glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 microl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGalpha and hCGbeta glycoforms, which allowed for the first time the detection of 33 hCGbeta glycoforms at intact level. In addition, a higher number of hCGalpha glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations. CI - Copyright (c) 2021. Published by Elsevier B.V. FAU - Al Matari, Amira AU - Al Matari A AD - Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, Chemistry, Biology and Innovation (CBI) UMR 8231, ESPCI Paris PSL, CNRS, PSL Research University, Paris, France. FAU - Goumenou, Anastasia AU - Goumenou A AD - Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, Chemistry, Biology and Innovation (CBI) UMR 8231, ESPCI Paris PSL, CNRS, PSL Research University, Paris, France. FAU - Combes, Audrey AU - Combes A AD - Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, Chemistry, Biology and Innovation (CBI) UMR 8231, ESPCI Paris PSL, CNRS, PSL Research University, Paris, France. FAU - Fournier, Thierry AU - Fournier T AD - Universite de Paris, INSERM, UMR-S1139, <>, 3PHM, F-75006 Paris, France. FAU - Pichon, Valerie AU - Pichon V AD - Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, Chemistry, Biology and Innovation (CBI) UMR 8231, ESPCI Paris PSL, CNRS, PSL Research University, Paris, France; Sorbonne Universite, France. FAU - Delaunay, Nathalie AU - Delaunay N AD - Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, Chemistry, Biology and Innovation (CBI) UMR 8231, ESPCI Paris PSL, CNRS, PSL Research University, Paris, France. Electronic address: nathalie.delaunay@espci.fr. LA - eng PT - Journal Article DEP - 20210128 PL - Netherlands TA - J Chromatogr A JT - Journal of chromatography. A JID - 9318488 RN - 0 (Chorionic Gonadotropin, beta Subunit, Human) RN - 0 (Glycoprotein Hormones, alpha Subunit) SB - IM MH - Animals MH - CHO Cells MH - Chorionic Gonadotropin, beta Subunit, Human/*analysis/chemistry MH - Chromatography, Liquid/*methods MH - Cricetulus MH - Glycoprotein Hormones, alpha Subunit/*analysis MH - Glycosylation MH - Humans MH - Mass Spectrometry/*methods MH - Nanotechnology/*methods OTO - NOTNLM OT - Glycoform OT - Human chorionic gonadotropin OT - Intact protein OT - Mass spectrometry OT - Nano-liquid chromatography COIS- Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. EDAT- 2021/02/09 06:00 MHDA- 2021/03/17 06:00 CRDT- 2021/02/08 20:14 PHST- 2020/11/12 00:00 [received] PHST- 2021/01/11 00:00 [revised] PHST- 2021/01/25 00:00 [accepted] PHST- 2021/02/09 06:00 [pubmed] PHST- 2021/03/17 06:00 [medline] PHST- 2021/02/08 20:14 [entrez] AID - S0021-9673(21)00069-8 [pii] AID - 10.1016/j.chroma.2021.461945 [doi] PST - ppublish SO - J Chromatogr A. 2021 Mar 15;1640:461945. doi: 10.1016/j.chroma.2021.461945. Epub 2021 Jan 28.