Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatography, part 1: optimization of the mobile phase

J Pharm Biomed Anal. 2016 Jan 25;118:393-403. doi: 10.1016/j.jpba.2015.11.011. Epub 2015 Nov 14.


The goal of this work is to provide some recommendations for method development in HIC using monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) as model drug candidates. The effects of gradient steepness, mobile phase pH, salt concentration and type, as well as organic modifier were evaluated for tuning selectivity and retention in HIC. Except the nature of the stationary phase, which was not discussed in this study, the most important parameter for modifying selectivity was the gradient steepness. The addition of organic solvent (up to 15% isopropanol) in the mobile phase was also found to be useful for mAbs analysis, since it could provide some changes in elution order, in some cases. On the contrary, isopropanol was not beneficial with ADCs, since the most hydrophobic DAR species (DAR6 and DAR8) cannot be eluted from the stationary phase under these conditions. This study also illustrates the possibility to perform HIC method development using optimization software, such as Drylab. The optimum conditions suggested by the software were tested using therapeutic mAbs and commercial cysteine linked ADC (brentuximab-vedotin) and the average retention time errors between predicted and experimental retention times were ∼ 1%.

Keywords: Antibody-drug-conjugate; Brentuximab-vedotin; Hydrophobic interaction chromatography; Method development; Monoclonal antibody.

MeSH terms

  • Antibodies, Monoclonal / analysis*
  • Brentuximab Vedotin
  • Chemistry, Pharmaceutical / methods*
  • Chemistry, Pharmaceutical / trends
  • Chromatography, Ion Exchange / methods
  • Chromatography, Ion Exchange / trends
  • Hydrophobic and Hydrophilic Interactions*
  • Immunoconjugates / analysis*


  • Antibodies, Monoclonal
  • Immunoconjugates
  • Brentuximab Vedotin