Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I − Optimization of separation conditions

Abstract

Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be characterized during development and for further quality control. The coupling of on-line HIC x RPLC to high resolution mass spectrometry can be considered as a very efficient analytical method, providing extensive information on ADC sample, within a reduced time scale. Our intention in this first paper is to present the approach used to rationally optimize the numerous conditions that can affect the quality of the 2D-separation. HIC and RPLC conditions were therefore optimized to prevent salt precipitation due to solvent mixing and to enhance sensitivity, while limiting the total analysis time. We demonstrated that adding salt in the sample solvent before HIC injection allows a significant peak shape improvement. The gradient profile was also carefully optimized in both dimensions, leading to a two-step gradient in HIC and bracketed gradient in RPLC. This study shows that on-line HIC x RPLC hyphenated to high resolution mass spectrometry is a useful method to obtain rapid and extensive structural information on the peaks observed in the first HIC dimension, thereby leading, in a single step requiring 75min, to the precise determination of the average drug-to-antibody ratio (DAR) by HIC as well as the knowledge of the drug load distribution for a particular DAR. The structural characterization of ADC fragments by RPLC-QTOF will be discussed in the second part of this two-part series.

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

“…An on-line HIC x RPLC-MS method has recently been reported for the thorough characterization of brentuximab vedotin [71,72]. The purpose was to get relevant information on both drug loaded profile and structural information on positional isomers of each DAR of the cysteine-linked commercial ADC.…”

Optimizing separations in online comprehensive two‐dimensional liquid chromatography

“…HIC has only recently come back into fashion and there are few examples of its use in LC × LC. Heinisch et al used HIC × RPLC-MS for separating antibody-drug conjugates[145,146].…”

Recent Advances in Chromatography for Pharmaceutical Analysis

“…In this context, several contributions have arisen in recent years, in response to this analytical demand, using heart-cutting, 137 multiple heart-cutting, 138 and comprehensive 2D-LC. 139 HIC × RPLC-HRMS was performed by Sarrut et al 139 Recently, the thorough and simultaneous characterization of both small and large molecules (drug payloads decorating mAbs in ADC formats) has also stimulated interest in the applications of 2D-LC. 136,137,139 As practical examples, Venkatramani et al 141 coworkers, 144 by analyzing several commercial mAbs and ADCs.…”

“…139 HIC × RPLC-HRMS was performed by Sarrut et al 139 Recently, the thorough and simultaneous characterization of both small and large molecules (drug payloads decorating mAbs in ADC formats) has also stimulated interest in the applications of 2D-LC. 136,137,139 As practical examples, Venkatramani et al 141 coworkers, 144 by analyzing several commercial mAbs and ADCs. It was found that the UHP-SEC columns were equivalent in terms of mAb characterization performance, since the average resolution for each column was between 1.9 -2.1 for the monomer and dimer forms of 10 different commercial mAbs.…”