Immunoconjugates are antibodies to which potent cytotoxic agents are attached, for example, antibody-drug conjugates (ADCs). One way to attach cytotoxic agents to antibodies is through the use of heterobifunctional linkers. In this study, we used N-succinimidyl 4-(2-pyridylthio)butyrate as the linker. During the first step of the manufacturing process, the N-succinimidyl end of the linker reacts with the antibody, while in the second step, the cytotoxic agent is attached to the linker. To ensure that the attached linkers are conjugated with cytotoxic agents, there is a need for a suitable analytical assay. For this purpose, two different methods were developed and evaluated. One method is based on mass spectrometric analysis of the immunoconjugate products, while the other method is based on chromatographic analysis of 2-mercaptopyridine that was generated through the reduction of the unreacted linkers that were attached to the antibody. The results indicated that the chromatographic method is more sensitive than the mass spectrometric method and is suitable for the analysis of samples obtained during process optimization, but it is more labor intensive. The mass spectrometric assay is more convenient for the analysis of samples in the early stages of process development.
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