PMID- 36586502 OWN - NLM STAT- MEDLINE DCOM- 20230113 LR - 20230214 IS - 1096-0309 (Electronic) IS - 0003-2697 (Print) IS - 0003-2697 (Linking) VI - 663 DP - 2023 Feb 15 TI - Fingerprinting trimeric SARS-CoV-2 RBD by capillary isoelectric focusing with whole-column imaging detection. PG - 115034 LID - S0003-2697(22)00494-8 [pii] LID - 10.1016/j.ab.2022.115034 [doi] AB - Because the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is the immunodominant antigen, the S protein and its receptor-binding domain (RBD) are both targets currently to be genetically engineered for designing the broad-spectrum vaccine. In theory, the expressed protein exists as a set of variants that are roughly the same but slightly different, which depends on the protein expression system. The variants can be phenotypically manifested as charge heterogeneity. Here, we attempted to depict the charge heterogeneity of the trimeric SARS-CoV-2 RBD by using capillary isoelectric focusing with whole-column imaging detection (cIEF-WCID). In its nature form, the electropherogram fingerprints of the trimeric RBD were presented under optimized experimental conditions. The peaks of matrix buffers can be fully distinguishable from peaks of trimeric RBD. The isoelectric point (pI) was determined to be within a range of 6.67-9.54 covering the theoretical pI of 9.02. The fingerprints of three batches of trimeric RBDs are completely the same, with the intra-batch and batch-to-batch relative standard deviations (RSDs) of both pI values and area percentage of each peak no more than 1.0%, indicating that the production process is stable and this method can be used to surveillance the batch-to-batch consistency. The fingerprint remained unchanged after incubating at 37 degrees C for 7 d and oxidizing by 0.015% H(2)O(2). In addition, the fingerprint was destroyed when adjusting the pH value to higher than 10.0 but still stable when the pH was lower than 4.0. In summary, the cIEF-WCID fingerprint can be used for the identification, batch-to-batch consistency evaluation, and stability study of the trimeric SARS-CoV-2 RBD, as part of a quality control strategy during the potential vaccine production. CI - Copyright (c) 2022 Elsevier Inc. All rights reserved. FAU - Du, Jialiang AU - Du J AD - NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, Division of Monoclonal Antibody Products, National Institutes for Food and Drug Control, Beijing, 102629, China. FAU - Wu, Gang AU - Wu G AD - NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, Division of Monoclonal Antibody Products, National Institutes for Food and Drug Control, Beijing, 102629, China. FAU - Chen, Quanyao AU - Chen Q AD - NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, Division of Monoclonal Antibody Products, National Institutes for Food and Drug Control, Beijing, 102629, China; School of Pharmacy, Yantai University, Yantai, 264005, China. FAU - Yu, Chuanfei AU - Yu C AD - NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, Division of Monoclonal Antibody Products, National Institutes for Food and Drug Control, Beijing, 102629, China. FAU - Xu, Gangling AU - Xu G AD - NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, Division of Monoclonal Antibody Products, National Institutes for Food and Drug Control, Beijing, 102629, China. FAU - Liu, Anhui AU - Liu A AD - School of Pharmacy, Yantai University, Yantai, 264005, China. FAU - Wang, Lan AU - Wang L AD - NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, Division of Monoclonal Antibody Products, National Institutes for Food and Drug Control, Beijing, 102629, China. Electronic address: wanglan@nifdc.org.cn. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20221228 PL - United States TA - Anal Biochem JT - Analytical biochemistry JID - 0370535 RN - BBX060AN9V (Hydrogen Peroxide) SB - IM MH - Humans MH - *SARS-CoV-2 MH - Isoelectric Focusing/methods MH - Capillary Isoelectric Focusing MH - Hydrogen Peroxide MH - *COVID-19 MH - Protein Binding PMC - PMC9794521 OTO - NOTNLM OT - Isoelectric point OT - Receptor-binding domain OT - SARS-CoV-2 OT - Spike OT - cIEF-WCID COIS- Declaration of competing interest The authors declare that they have no conflict of interest. EDAT- 2023/01/01 06:00 MHDA- 2023/01/14 06:00 PMCR- 2022/12/28 CRDT- 2022/12/31 19:14 PHST- 2022/11/08 00:00 [received] PHST- 2022/12/20 00:00 [revised] PHST- 2022/12/27 00:00 [accepted] PHST- 2023/01/01 06:00 [pubmed] PHST- 2023/01/14 06:00 [medline] PHST- 2022/12/31 19:14 [entrez] PHST- 2022/12/28 00:00 [pmc-release] AID - S0003-2697(22)00494-8 [pii] AID - 115034 [pii] AID - 10.1016/j.ab.2022.115034 [doi] PST - ppublish SO - Anal Biochem. 2023 Feb 15;663:115034. doi: 10.1016/j.ab.2022.115034. Epub 2022 Dec 28.