Abstract
Monoclonal antibody drug conjugate (ADCs) have received much attention as pharmaceutical agents for treating serious diseases such as cancer. However, it is difficult to separate them on the basis of the drug to antibody ratio, DAR. Hydrophobic chromatography (HIC) is commonly used for the analysis of the drug to antibody ratio, DAR. The retention of ADCs on HIC can be controlled by the hydrophobic nature of ADCs, depending on the mobile phase conditions. They are sometimes performed at the restricted conditions where the solubility is too low. Ion exchange chromatography (IEC) using electrostatic interaction is an orthogonal method to HIC. IEC is widely used because of its higher capacity than HIC. We investigated the retention behavior of the protein conjugated with surrogate drugs on IEC. The surrogate drugs employed are 7-diethylamino-3-(4’-maleimidylhenyl) 4-methylcoumarin (CPM), N-(1-pyrenyl) maleimide (NPM). Bovine serum albumin (BSA) was used as a model protein. The molar ratio (CPM and NPM to protein) was set to 3. The maleimide group of CPM and NPM reacts with the thiol group of the proteins. On the linear gradient elution experiments, the elution salt concentrations of the conjugated and non-conjugated proteins were measured to obtain chromatographic parameter of the number of binding sites, B.
Highlights
PEGylated proteins and monoclonal antibody drug conjugates (ADC) are considered as generation biopharmaceuticals
The additional peaks were observed around 400 nm with respect to Bovine serum albumin (BSA)-N-(1-pyrenyl) maleimide (NPM) reaction mixture, and around 340 nm with BSA-CPM, respectively
These wavelengths were empoyed to investigae the BSA conjugates elution profiles on the chromatography column in the following
Summary
PEGylated proteins and monoclonal antibody drug conjugates (ADC) are considered as generation biopharmaceuticals. The same chemical reactions can be employed in the preparation of PEGylated proteins and ADCs (Gordon et al, 2015; Fee and van Alstine, 2006). Since multiple lysine residues of proteins are involved in the reactions, the product is heterogeneous in the number of modified PEGs or drugs per protein. For the site-specific conjugation, the reactions between cysteine residues and the derivatives of malaimide and iodoacetamide, (Bonora and Drioli, 2009) can be employed since the number of cysteine residues on a protein surfaces is much smaller than that of lysine ones. The retention time of PEGylated proteins on IEC becomes shorter than that of unmodified ones because of the charge shielding and steric hindrance effects caused by the conjugated PEG.
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