**4. Conclusions**

MEKC was used for determining the relative hydrophobicities of modified auristatins. Optimal separation was achieved using a mixed micellar system of 20 mM SDS and 40 mM SC as the pseudostationary phase. The hydrophobicity of the auristatins decreased in the order of MMAE > compound 2 > compound 1, and the same pattern was identified in the cytotoxic assays where the cytotoxicity decreased in the same order.

Hydrophilic modification (attachment of a glucuronic acid residue) at the norephedrine residue of MMAE (compound 1; MMAU) was accompanied by a significant decrease in the cytotoxicity. The addition of a hydrophilic glycolinker at the *N*-terminal, on the other hand, had only a slight effect on the cytotoxicity (compound 2), which is an interesting observation since an amide bond at this position is known to render the auristatins close to non-toxic.

The preliminary cytotoxic evaluation of the trastuzumab-MC-Val-Cit-PABC-auristatin conjugates 3 and 4 revealed IC50-values in the pM range. These values are comparable to other leading state-of-the-art ADCs. Furthermore, the MMAU-ADCs have certain advantages over the MMAE-ADCs, for example, prematurely cleaved linkers will be accompanied by less off-site harmful side effects due to the lower cytotoxicity of MMAU when compared to MMAE. Altogether, our results on the use of hydrophilic payload conjugates prove that novel opportunities exist for the future design of ADCs with previously neglected hydrophilic molecules.

**Author Contributions:** Conceptualization, F.S.E., S.-K.R., S.K.W.; Investigation, F.S.E., S.-K.R., M.R., V.P., A.V., J.S., J.H.; Resources, F.S.E., T.S., S.K.W.; Writing—Original Draft Preparation, F.S.E., S.-K.R., T.S., S.K.W.; Writing—Review and Editing, F.S.E., S.-K.R., S.K.W., T.S.; Visualization, F.S.E., S.-K.R., S.K.W.; Supervision, S.K.W.; Project Administration, F.S.E., S.K.W.; Funding Acquisition, F.S.E., S.K.W.

**Funding:** This research was funded by the Magnus Ehrnrooth Foundation (S.K.W.), the Ruth and Nils-Erik Stenbäck Foundation (F.S.E), The Finnish Society of Sciences and Letters (S.K.W.), and the Academy of Finland (S.K.W; project number 266342).

**Acknowledgments:** Jesper Långbacka is acknowledged for assistance with the CMC measurements.

**Conflicts of Interest:** The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; and in the decision to publish the results.
