Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates

Abstract

Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics. ADCs show unprecedented intrinsic heterogeneity and address new quality attributes which have to be assessed. Liquid chromatography is one of the most frequently used analytical method for the characterization of ADCs. This review summarizes recent results in the chromatographic characterization of ADCs and supposed to provide a general overview on the possibilities and limitations of current approaches for the evaluation of drug load distribution, determination of average drug to antibody ratio (DAR), and for the analysis of process/storage related impurities. Hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC) and multidimensional separations are discussed focusing on the analysis of marketed ADCs. Fundamentals and aspects of method development are illustrated with applications for each technique. Future perspectives in hydrophilic interaction chromatography (HILIC), HIC, SEC and ion exchange chromatography (IEX) are also discussed.

Advances in native high‐performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products

“…The characterization of ADCs and their payloads using comprehensive LC modes has been described in an excellent review by Bobaly et al. .…”

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

“…As such, DAR values are considered critical quality attributes of ADCs, necessitating the development of a range of analytical methods for their quantitative evaluation. For example, many separation techniques have been utilized to accurately derive DAR values, such as hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), reverse phase liquid chromatography (RPLC), capillary electrophoresis (CE), or capillary isoelectric focusing (cIEF) . As lysine‐conjugated ADCs typically exhibit greater heterogeneity and poorer chromatographic peak shapes than equivalent cysteine‐modified therapeutics, mass spectrometry (MS) is typically deployed for their DAR assessment .…”

High-throughput oxidation screen of antibody–drug conjugates by analytical protein A chromatography following IdeS digest

“…When applying the novel protein A method on the model ADC, strong tailing was observed, which increased with the DAR. This suggests that this phenomenon is caused by a non‐specific interaction by the payload with the stationary phase . MAb 2,on the other hand, showed a much stronger interaction with the protein A caused by the second binding site at the Fab fragment, which only eluted after the gradient reached 100% buffer B.…”

“…This calls for more sophisticated analytical techniques and results in extensive method development for every individual ADC. It has been reported that the payload of ADCs can have an impact on the elution profile and resolution and can cause poor recoveries . Commonly used organic modifiers such as IPA, DMSO, propylene glycol or ACN can be applied to reduce non‐specific interaction of the payload with the stationary phase.…”