PhD defence B. (Baubek) Spanov

Bioanalytical methodology to study the in vivo biotransformation of therapeutic proteins

Therapeutic proteins (TPs) have emerged as an important class of pharmaceuticals for the treatment of various severe diseases. TPs are highly specific and present fewer unwanted side effects than traditional small molecules. A variety of chemical and enzymatic modifications that occur during expression, purification, and long-term storage significantly contribute to the heterogeneity of TPs. The increased heterogeneity poses challenges for analytical methods to identify, characterize, and quantify changes in TPs sequence and structure.

The work described in the thesis of Baubek Spanov aimed to develop analytical methodologies to study modifications that occur in the therapeutic monoclonal antibodies trastuzumab and pertuzumab upon in vitro stressing and in vivo biotransformation. To study in vitro modifications and in vivo biotransformation products by LC-MS, first of all, a powerful separation method is needed. pH gradient cation-exchange chromatography can fulfill this requirement and its application to the analysis of charge variants is shown in several chapters of this thesis. Bottom-up LC-MS is a widely used approach for the identification and characterization of modification sites in proteins. The bottom-up approach was extensively used in this thesis to link modifications to the corresponding proteoforms. Along with separation and characterization, it is also important to understand how various types of modifications that are due to in vitro stressing can affect therapeutic mAbs. For this purpose, the binding characteristics of stressed trastuzumab and pertuzumab to the target receptor and various types of Fcγ receptors were studied.

Promotors: prof. dr. R.P.H. Bischoff, prof. dr. N.C. van de Merbel