Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) has been widely used to separate and quantify monoclonal antibody (mAb) fragments and impurities. Two fragments with different masses smaller than monomer were observed in the CE-SDS analysis of a recombinant IgG1 mAb treated at 40 °C for 28 days. Reversed phase liquid chromatography mass spectrometry (RP–LC–MS) and trypsin digestion peptide mapping followed by LC–MS/MS analysis were employed to conclusively demonstrate that these antibody fragments were the degradation products derived from a Ser–Ser peptide bond cleavage at the heavy chain complementarity determining region (CDR). The total percentage of the cleaved antibody heavy chain was increased from 1.1% in the control sample to 4.6% in the forced degradation sample stored at 40 °C for 28 days by using CE-SDS analysis. Native size exclusion liquid chromatography (SEC-HPLC) was used to analyze the fragment profile of this treated mAb but did not reveal fragments smaller than the monomer. However, a small fragment was detected using denaturing size exclusion liquid chromatography (dSEC-HPLC) and was confirmed to be the N-terminal fragment of the heavy chain cleaved at the CDR region using RP–LC–MS analysis. These results suggested that this N-terminal fragment may be closely associated with the rest of the cleaved antibody molecule through non-covalent bond interactions. Moreover, when dSEC-HPLC and RP–LC–MS were used to analyze the aggregate and monomer samples individually collected from SEC-HPLC, both samples contained the same amount of the cleaved N-terminal fragment. These results suggested that the CDR region cleaved fragments likely not be involved in aggregates formation of this antibody. This case study reiterated that a combination of orthogonal analytical methods plays a crucial role in comprehensively analyzing and characterizing antibody fragmentation.
In assisted reproductive technology (ART), controlled ovarian hyperstimulation (COS) plays an important role in guiding treatment progress and determining success. Many ovulation-inducing agents are used in this process, including human menopausal gonadotropin (HMG), recombinant follicle-stimulating hormone (r-FSH), recombinant luteinizing hormone (r-hLH), urinary human chorionic gonadotropin (u-hCG), and recombinant human chorionic gonadotropin (r-hCG). HMG, which comes from the urine of postmenopausal women, mainly contains FSH, LH, and hCG. While bioactivity-based potency units and immunoassay-based ELISA methods remain the gold standard for quantifying protein therapeutics, LC–MS/MS is emerging as a game-changing technology. Its unique strengths are exceptional specificity and absolute quantification capabilities, independence from specialized reagents, powerful multiplexing potential, and ability to provide structural insights, establishing it as an essential tool for both orthogonal verification and cutting-edge research in the field. This study used reverse-phase liquid chromatography with mass spectrometry (RP-LC–MS/MS) and a label-free quantitative proteomics method to analyze imported r-hFSH, r-hLH, r-hCG (Gonal-F, Luveris, and Ovidrel), three domestic HMG products (Le Baode, Man Fuxin, and Xin Yunle), and u-hCG (Li Zhu). The results showed that the purity of the main component in imported gonadotropin drugs was higher than 98.55%. On the other hand, the main element of domestic u-HMG is FSH, with small amounts of hCG and very little LH. These results reveal significant differences in the composition of imported and domestic gonadotropin products, underscoring the need to further evaluate their biological activity and clinical effectiveness. This study provides important insights into drug selection for ART, with practical value for improving the quality of HMG drugs and making better use of them in clinical settings.
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