Purification of ADCs by Hydrophobic Interaction Chromatography.

Antibody-drug conjugate (ADC) in vitro potency has been shown to be dependent on drug load, with higher drug load providing lower IC50 values. However, in vivo potency is affected by intrinsic biological effects as well, such as plasma clearance, dose-limiting toxicity, etc. Developing a preparative HIC process for ADC purification to isolate species with a specific drug loading involves several steps including conjugation optimization, resin selection, solubility studies gradient screening, and step gradient development (buffer selection). In this chapter, the rationale and general considerations for developing a preparative hydrophobic interaction chromatography (HIC) method are described for isolation of an example ADC with specific drug load, e.g., two monomethyl auristatin E (MMAE) payloads (E2).

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.

Impurity rejection in the crystallization of ABT-510 as a method to establish starting material specifications.

Understanding impurity rejection in a drug substance crystallization process is valuable for establishing purity specifications for the starting materials used in the process. Impurity rejection has been determined for all known ABT-510 impurities and for many of the reasonable & conceivable impurities. Based on this study, a very high purity specification (e.g., > 99.7%) can be set for ABT-510 with a high level of confidence.