|From one to four comprehensive separation dimensions to characterize antibody drug conjugates
|(R)evolutions in Biopharmaceutical Analysis (KVCV)
|Dr Davy GUILLARME
|Mr Alexandre Goyon
Dr Balazs Bobaly
Prof Jean-Luc Veuthey
Dr Szabolcs Fekete
|University of Geneva
Abstract Information :
The characterization of antibody-drug conjugates (ADCs), combining the specificity of a mAb with a potent cytotoxic agent covalently bound via a linker to the antibody, is a tremendous challenge to state-of-the-art analytical technologies. Indeed, subtle changes in these large (> 150 kDa) molecules can have profound effects on efficacy and pharmacokinetic properties.
The aim of this work was to highlight the possibility offered by hydrophobic interaction chromatography (HIC) for the characterization of cysteine linked ADC. In this context, brentuximab vedotin (Adcetris®) has been taken as a case study and various separation dimensions were employed to obtain the most relevant information from the ADC sample:
- One dimension separation: HIC was successfully employed to determine the average drug-to-antibody (DAR) ratio of brentuximab vedotin. However, care should be taken when selecting the mobile phase conditions (addition of organic solvent or not), and the gradient profile (linear or logarithmic) for elution of the DAR species.
- Three dimensions separation: A comprehensive 2D-LC approach involving HIC in the first dimension and RPLC in the second dimension was employed in combination with high resolution mass spectrometry (HRMS) for the structural identification of the species observed on the HIC trace. A one-month and two-months stressed samples of brentuximab vedotin were also evaluated using this approach.
- Four dimensions separation: To avoid the denaturation of cysteine linked ADC under RPLC conditions, a comprehensive 2D setup was employed, including HIC and SEC in the first and second dimensions, respectively. Then, ion mobility spectrometry (IMS) was activated prior to HRMS detection to gain additional information. This non-denaturing strategy allows obtaining easy-to-interpret data on ADC.