Fingerprinting trimeric SARS-CoV-2 RBD by capillary isoelectric focusing with whole-column imaging detection

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 °C for 7 d and oxidizing by 0.015% H₂O₂. 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.