The MS/MS data analysis of the collision-induced dissociation (CID) of the new peak is shown in Figure ?Figure33B

The MS/MS data analysis of the collision-induced dissociation (CID) of the new peak is shown in Figure ?Figure33B. similar primary structures along with similar purity, heterogeneity profiles, antigen binding activity, Fc receptor binding affinity, and Fc-effector functions. Biosimilar KX1-004 candidate 2 (BC2), which had an amino acid replacement at a constant region, a different values of MabThera and two biosimilar candidates. cIEF analysis followed the product monograph (USP Medicines Compendium). In icIEF analysis, rituximab was mixed with Pharmalyte 3C10, Pharmalyte 8C10.5, 1% methyl cellulose, pmarker (7.0/10.0), and distilled water. The mixture was loaded onto an iCE3 icIEF instrument (Protein Simple) and resolved by prefocusing for 1 min at 1500 V and focusing for 4.5 min at 3000 V. The pof the sample peaks were determined using a linear regression between two or three pmarker peaks. Rituximab samples were treated with carboxypeptidase B (CpB) (100:1 717.3581 6+, eluted at 38.28 min, was detected in the chymotryptic peptide mapping chromatographic profiles of BC2. This peak did not match the desired rituximab sequence according to the data analyzed by UNIFI 1.9. The new peak molecular weight was 28 Da heavier than the semi-digested chymotryptic C17 peptide of Fd (ICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELL, 712.6864, 6+) (Figure ?Figure33A). The increased mass of the C17 peptide complied with the mass shift in the subunit analysis. The MS/MS data analysis of the collision-induced dissociation (CID) of the new peak is shown in Figure ?Figure33B. The MS/MS spectrum of the C17 chymotryptic peptide of BC2 showed a mass shift of +28 Da in the b18 ion relative to the theoretical b18 ion mass. The C17 chymotryptic peptide was collided with higher energy to improve the fragmentation. The identifaction of the b15, b16, and b17 ions illustrated that alanine (A) at 219 of HC was replaced by valine (V). The A219V mutation, which is located in the constant region 1 of HC, was an allotype of IgG1. Open KX1-004 in a separate window Figure 2 Comparison of the peptide map profiles of the RP, BC1, and BC2 by UPLC. Due to coelution with other peptides, HC CDR3 was not labeled in peptide mapping. Open in a separate window Rabbit Polyclonal to MRPL44 Figure 3 Extracted ion chromatograms (XIC) spectrum and MS/MS spectrum of the C17 peptide precursor ion (717.3581, 6+). (A) Comparison of the C17 peptide XIC spectra of RP and BC2. (B) MS/MS spectrum of the C17 peptide with A219 V mutation. Table 1 Retention Times and Masses of CDRs for the RP and Its Biosimilar Candidates Detected by UPLC-QDa values of the main peak obtained from the RP, BC1, and BC2 were 9.29, 9.36, and 9.35, respectively, which satisfied the KX1-004 criteria of 9.1C9.5 in accordance with the USP MC (Figure ?Figure44A). The main peak pvalues from the two biosimilars differed by no more than 0.1 punits from the corresponding peak of the RP. Compared with that in the RP, a higher level of basic variants was found in BC1, whereas slightly higher levels of acidic and basic variants were observed in BC2. Open in a separate window Figure 4 cIEF (A) and icIEF (B) profiles of MabThera and its two biosimilar candidates for the determination of their p9.33 and 9.43, was higher in BC1 than in the RP, whereas the percentage of acidic variants was higher in BC2 than in the RP in compliance with the results from cIEF. CEX is a technique orthogonal to cIEF, given that only the solvent-exposed part of the protein can interact with the stationary phase, whereas the pvalue in cIEF described the pH at which the net charge is equal to 0.16 In CEX analysis, charge variants were successively eluted in acidic peaks, main peak, and basic peaks (Figure ?Figure55). After the removal of lysine with CpB, BC1 exhibited similar levels of basic and acidic variants compared with the RP,.