Development and validation of a HPLC method for the determination of free emtansine impurity in a therapeutic conjugate based on a recombinant targeted protein molecule

To reduce the adverse effects of cytotoxic agents used in cancer therapy on healthy tissues, antibody – drug conjugates are used, which provide targeted drug delivery to tumor tissues. An alternative to antibody-based drugs are conjugates of cytotoxic agents and targeted carriers with smaller molecule sizes, in particular, the DARPin G3 protein, which has high stability and specificity for HER2-positive cancer cells. The object of the study was a conjugate of a targeted protein specific to the HER2 receptor fused to the albumin-binding domain (DARPin G3-ABD) and the cytotoxic agent emtansine (DM1), which is currently in preclinical trials. Due to the risk of accumulation of unconjugated emtansine, which can cause untargeted toxicity, it is necessary to monitor its content. For this purpose, a simplified and affordable technique for the determination of free emtansine by HPLC has been developed, providing operational quality control of therapeutic conjugates. During sample preparation, the conjugate was coagulated in ethanol, then the supernatant was analyzed by micro column chromatography with UV detection on a ProntoSIL-120-5-C18 column under selected conditions. The proposed sample preparation allows for chromatographic analysis without using expensive columns to separate conjugate and free emtansine. The developed technique for determining the impurity of free emtansine in the composition of the therapeutic conjugate DARPin G3-ABD-DM1 has been validated by key indicators, and its metrological characteristics have been evaluated. The technique was tested in the analysis of laboratory conjugate samples: the content of free emtansine did not exceed 0.2%.

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