Whitepaper: Characterising antibody-drug conjugates using micro pillar array columns combined with mass spectrometry (μPAC™-MS)

The use of micro pillar array columns (μPAC™) for characterising antibody-drug conjugates is presented. Kadcyla® (ado-trastuzumab emtansine) and, for comparison, Herceptin® (trastuzumab) tryptic digests were analysed on a 200 cm μPAC™ C18 column and peaks eluting were detected by ultraviolet (UV) spectroscopy and high-resolution mass spectrometry (MS).

The successes of monoclonal antibodies (mAbs) as therapeutics have triggered the development of various next generation formats. In oncology, antibody-drug conjugates (ADCs) are particularly promising, since they synergistically combine a specific mAb linked to a biologically active cytotoxic drug via a stable linker. The promise of ADCs is that highly toxic drugs can selectively be delivered to tumor cells thereby substantially lowering side effects as typically experienced with classical chemotherapy. Currently two ADCs are marketed, namely brentuximab vedotin (Adcetris®) and ado-trastuzumab emtansine (Kadcyla^®) and over 30 are in clinical trials. From a structural point of view, ADCs possess an unsurpassed complexity since the heterogeneity of the initial antibody is superimposed with the variability associated with the conjugation strategy. Conjugation typically takes place on the amino groups of lysine residues or on the sulphydryl groups of interchain cysteine residues as is the case in, respectively, ado-trastuzumab emtansine and brentuximab vedotin. With 80-100 lysine and only 8 interchain cysteine residues available, lysine conjugation yields a more heterogeneous mixture of species compared to cysteine conjugation this despite the similar overall level of drugs incorporated per antibody (between 0 and 8 with an average of 3-4). This application note demonstrates how micro pillar array columns (μPAC™) in combination with ultraviolet (UV) spectroscopy and high-resolution mass spectrometry (MS) comes in as a very powerful tool to assess drug conjugation sites.