Hydrophobic interaction chromatography (HIC) method development and characterization of resolved drug-load variants in site-specifically conjugated pyrrolobenzodiazepine dimer-based antibody drug conjugates (PBD-ADCs).

Antibody drug conjugates (ADCs) are heterogeneous biopharmaceutical products that demand extensive characterization to ensure batch consistency, safety, and efficacy. Hydrophobic interaction chromatography (HIC) is the state-of-the-art analytical tool to monitor conjugation-related critical quality attributes (CQAs) e.g. drug-load distribution and Drug-to-Antibody Ratio (DAR). For the next generation site-specific PBD-ADCs (PBD: pyrrolobenzodiazepine dimer), denaturing RP-HPLC (reverse-phase high-performance chromatography) is the current method to determine average DAR. In this manuscript, we have utilized native HIC for the first time to understand conjugation related CQAs in PBD-ADCs. In terms of the method development, the type of stationary phase and salt, coupled with reduction of the reactive imine in the PBD drug-linker to an amine form in the sample preparation, have played a key role in achieving the best HIC resolution for the drug-load variants. The established HIC conditions resolved DAR 0, DAR 1, and two DAR 2 peaks for PBD-ADCs. Extended characterization of the DAR 2 peaks confirmed that they have retained characteristically distinct antibody Fc N-glycan distributions (Fc = Fragment crystallization region). Therefore, the results support that the HIC conditions established for PBD-ADCs is valuable in not only determining DAR values but also other important attributes including native drug-load distribution and unique DAR 2 conformations existed as a result of the N-glycan heterogeneity.

Water-soluble prodrugs of the human immunodeficiency virus protease inhibitors lopinavir and ritonavir.

We studied the synthesis, cleavage rates, and oral administration of prodrugs of the HIV protease inhibitors (PIs) lopinavir and ritonavir. Phosphate esters attached directly to the central hydroxyl groups of these PIs did not demonstrate enzyme-mediated cleavage in vitro and did not provide measurable plasma levels of the parent drugs in vivo. However, oxymethylphosphate (OMP) and oxyethylphosphate (OEP) prodrugs provided improved rates of cleavage, high levels of aqueous solubility, and high plasma levels of the parent drugs when dosed orally in rats and dogs. Dosing unformulated capsules containing the solid prodrugs led to plasma levels equivalent to those observed for dosing formulated solutions of the parent drugs. A direct synthetic process for the preparation of OMP and OEP prodrugs was developed, and the improved synthetic method may be applicable to the preparation of analogous soluble prodrugs of other drug classes with limited solubility.