Abstract
Antibody–drug conjugates (ADCs) are heterogeneous biotherapeutics and differ vastly in their physicochemical properties depending on their design. The number of small drug molecules covalently attached to each antibody molecule is commonly referred to as the drug-to-antibody ratio (DAR). Established analytical protocols for mass spectrometry (MS)-investigation of antibodies and ADCs often require sample treatment such as desalting or interchain disulfide bond reduction prior to analysis. Herein, the impact of the desalting and reduction steps—as well as the sample concentration and elapsed time between synthesis and analysis of DAR-values (as acquired by reversed phase liquid chromatography MS (RPLC–MS))—was investigated. It was found that the apparent DAR-values could fluctuate by up to 0.6 DAR units due to changes in the sample preparation workflow. For methods involving disulfide reduction by means of dithiothreitol (DTT), an acidic quench is recommended in order to increase DAR reliability. Furthermore, the addition of a desalting step was shown to benefit the ionization efficiencies in RPLC–MS. Finally, in the case of delayed analyses, samples can be stored at four degrees Celsius for up to one week but are better stored at −20 °C for longer periods of time. In conclusion, the results demonstrate that commonly used sample preparation procedures and storage conditions themselves may impact MS-derived DAR-values, which should be taken into account when evaluating analytical procedures.
Highlights
Antibody–drug conjugates (ADCs) are a class of biotherapeutics, which have gained clinical importance in oncology [1]
As native-ESI may give rise to lower signals compared to denaturing ESI–mass spectrometry (MS) [19], this study focuses on denaturing ESI–MS for drug-to-antibody ratio (DAR) determination
Three dilution series were made from desalted trastuzumab and ADCs and reference samples
Summary
Antibody–drug conjugates (ADCs) are a class of biotherapeutics, which have gained clinical importance in oncology [1] They are constructed by three main parts: an antibody backbone, a small molecule drug (payload) and a linker covalently connecting the two. In denaturing ESI–MS it is often beneficial to include a reduction step prior to analysis in order to cleave any remaining disulfide bridges between the antibody’s heavy and light chains [15,32] The reason for this is two-fold; (i) free light and heavy chains typically display higher sensitivity than intact ADCs as smaller molecules are easier to ionize and (ii) in cysteine-linked ADCs, some light and heavy chains are associated only by non-covalent forces.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
