Analysis of intact biomacromolecules is essential if molecular diversity and related action is to be fully characterized and understood. Compositional characterization of intact biomacromolecules is achieved using selective separation in combination with high-resolution MS. CE has been coupled to MS to reveal the micro-heterogeneity of monoclonal antibodies (mAbs). Using a middle-up approach detailed information on glycosylation and C-terminal lysine formation was obtained (Figure). This analytical platform was also used for the elucidation of antibody-drug conjugate structures revealing antibodies with different drug loads and allowing quantitative determination of the specific and overall drug-antibody ratio. A SEC-MS method for the separation and analysis of large mAb fragments was also developed. An MS-compatible mobile phase for achieving resolution of mAb heavy and light chains, and F(ab)2 and Fc fragments was achieved. MS detection allowed mass determination of mAb fragments and detailed assignment of different glycoforms of the heavy chain and Fc fragments.
Capillary gel electrophoresis (CGE) has emerged as a quality-control method for the determination of the size heterogeneity of therapeutic mAbs. As CGE is relatively slow, possibilities to speed up analysis were investigated leading to of a four-fold decrease of analysis times by optimizing parameters such as voltage and capillary length. The coupling of CGE to MS is explored in order to permit identification of unknown mAb fragments or contaminants.
Affinity capillary electrophoresis (ACE) was explored for the assessment of protein-protein interactions. ACE offers the unique possibility to simultaneously study the interaction of multiple proteins (e.g. proteoforms) with a target receptor under near-physiological conditions. A new ACE-MS method was developed simultaneously providing some unique features: (a) assignment of the molecular weight of all protein sample components, including modified forms, other variants and impurities, (b) protein-variant-selective determination of multiple dissociation constants (Kds), and (c) establishment of protein-complex stoichiometries. The ACE-MS method also showed useful for the fast screening of high/low-affinity components in heterogeneous protein samples.
Combination of the protein separation power of CE with label-free affinity detection by SPR could be very useful. This would allow affinity evaluation of different proteoforms in one sample. A new CE-SPR flow cell with integrated electrodes was developed allowing the closure of the electrical circuit required for CE. Immobilization of human serum albumin (HSA) on a CMD5 sensor in the flow cell, allowed detection of binding of anti-HSA. Different flow cell designs are currently tested.
A novel and relatively unexplored property of cyclodextrin complexes is their ability to aggregate to form amorphous nanoparticles. These constructs hold the promise of formulating difficult-to-formulate drugs as well as specifically target their delivery topically, orally as well as parenterally. In collaboration with Janssen we started a project on understanding how these aggregates are formed. CE and MS methods are developed to quantitavely investigate binding of drugs to heterogeneous cyclodextrines and to assess aggregate formation and stability.