Separation of antibody drug conjugate species by RPLC: A generic method development approach

Abstract

This study reports the use of modelling software for the successful method development of IgG1 cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method development strategy was proposed including the optimization of mobile phase temperature, gradient profile and mobile phase ternary composition. For the first time, a 3D retention modelling was presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable, with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention models. For routine purpose, four to six initial experiments were required to build the 2D retention models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can therefore be considered as a good method for estimating the average DAR of an ADC, based on the observed peak area ratios of RPLC chromatogram of the reduced ADC sample.

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

“…Previous studies showed the power of quality by design (QbD) based method development approach [25][26][27][28][29][30][31]. This methodology is based on the prediction of retention times based on initial experimental runs as input data for retention models.…”

“…Higher temperature generally enhances mass transfer for proteins, leading to higher separation performance. In addition, temperature is a common parameter in chromatographic method development since selectivity and resolution can be tuned by changing this variable [25,27,29,31,39]. For the analysis of proteins under RPLC conditions, temperature affects solute adsorption on the stationary phase.…”

“…Previous studies showed that intact mAbs, as well as mAb subunits, show poor recovery in RPLC conditions when working at moderate temperature (e.g. ≤80 • C) [31,40,41], thus demonstrating the need to work at 80-90 • C to avoid adsorption issues and reach acceptable recovery (e.g. above 90% of the injected protein amount).…”

“…3A, second panel), but a lower recovery of the LC and the Fd glycovariants to 39% and 62% was observed at 40 • C. Brentuximab vedotin has several highly hydrophobic loaded fragments due to cytotoxic drugs attached to them (LC I, Fd I, Fd II and Fd III fragments, with I, II and III corresponding to the number of bound payloads). The proper recovery of these loaded subunits is important for the estimation of the average drug-to-antibody ratio (DAR), which is a critical quality attribute (CQA) of the antibody drug conjugates [31,[42][43][44]. Average DAR can be calculated based on the formula reported by Wagner-Rousset et al [45] and it is generally close to 4.0 [46,47].…”

“…Fig. 3B shows the chromatograms of NISTmAb, cetuximab and brentuximab vedotin subunits obtained at 40, 70 and 90 • C. Peaks were annotated based on previous studies [7,31]. Finally, recovery may not be sufficient at lower temperature and this behaviour also depends on the selected protein.…”