Streamlined Characterization of an Antibody–Drug Conjugate by Two-Dimensional and Four-Dimensional Liquid Chromatography/Mass Spectrometry

Abstract

This study describes the use of a multidimensional HPLC (2D and 4D) system for a faster and more effective characterization of an antibody–drug conjugate (ADC) product, compared to the standard off-line approach of fraction collection and off-line variant characterization. The size variants of an interchain cysteine-linked ADC were characterized to understand the effect of the different drug-to-antibody ratio (DAR) species on aggregate formation. For this purpose, the ADC product and a full panel of stressed samples were analyzed. The dimeric ADC species were baseline resolved from the main peak (Rs = 2.7) by UHP-SEC (ultra-high-performance size exclusion chromatography) under nondenaturing conditions using a buffered mobile phase containing 5% 2-propanol. A 2D-LC (SEC-HIC) method was then developed to compare the average DAR values of the main peak species vs the aggregates. A 4D-LC/MS method (SEC-reduction-digestion-RPHPLC) was also developed to determine levels of potential critical quality attributes (pCQAs) including aggregation, average DAR, oxidation, and deamidation, in a 2 h run. An average DAR value of 3.5–3.6 was found for the main peak using both 2D-LC and 4D-LC methods, and these values were consistent with DAR determined by the in-house reference hydrophobic interaction chromatography (HIC) method. The multidimensional LC approaches also showed an increase in the content of high-DAR species in the SEC fractions containing the aggregates. Overall the entire workflow of data acquisition is completed within a day using the multidimensional on-line approach, in comparison to multiple days required with the traditional off-line approaches.

Recent Advances in Drug–Antibody Ratio Determination of Antibody–Drug Conjugates

“…To overcome these issues, on-line comprehensive two-dimensional liquid chromatography (LC × LC or 2D) emerged as an attractive technique. [72][73][74] 2D LC combines two different chromatography columns, and thereby enables a rapid and efficient analysis to provide greater resolution for the resulting chromatogram and obtain more information within a single injection. Furthermore, this combination system can be coupled with MS (2D LC/MS) to provide a wide variety of options for ADC characterization.…”

“…Furthermore, this combination system can be coupled with MS (2D LC/MS) to provide a wide variety of options for ADC characterization. 74) For example, the combination of HIC × SEC may provide ideal synergy for DAR determination with non-volatile salts in the first dimension (HIC) and the use of a second dimension (SEC) to desalt and remove the non-volatile salts prior to arrival at the MS chamber. 75,76) This complex but attractive LC/MS approach could address the analyses of complicated structures of new modalities such as complicated bioconjugates derived from novel drug delivery system, 77,78) which we expect will continue to diversify in the future.…”

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

“…It was not until much more recently that MS‐compatible comprehensive LC × LC strategies have been reported. Many groups have focused efforts on the use of multidimensional LC for characterization of protein therapeutics, especially antibodies [124–132]. Other multidimensional modes, including heart‐cutting and selective comprehensive modes have been developed and used very effectively to characterize different aspects of quality control, among other measures [133–135].…”

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

“…As an example, the characterization of five charge variants separated by IEC using off-line procedures can require up to 52 hours (17). In order to streamline this process, multi-dimensional LC-MS approaches have been developed and implemented to perform online fractionation followed by an in-line peptide mapping to characterize antibody variants separated by IEC or SEC (17)(18)(19)(20). The automated characterization of antibody variants by mD-LC-MS can be performed with a much faster turnaround (typically 9 vs 52 hours(17)) compared to conventional procedures (manual and/or off-line sample preparation and fraction collection), and has the potential to be used as a more integrated analytical platform for both upstream and downstream applications.…”