Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: Salt gradient approach

Szabolcs Fekete, Alain Beck, J. Fekete, Davy Guillarme

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

Ion exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge variants. When applying salt gradient IEX approach for monoclonal antibodies (mAbs) characterization, this approach is described as time-consuming to develop and product-specific. The goal of this study was to tackle these two bottle-necks. By modeling the retention of several commercial mAbs and their variants in IEX, we proved that the mobile phase temperature was not relevant for tuning selectivity, while optimal salt gradient program can be easily found based on only two initial gradients of different slopes. Last but not least, the dependence of retention vs. pH being polynomial, three initial runs at different pH were required to optimize mobile phase pH. Finally, only 9. h of initial experiments were necessary to simultaneously optimize salt gradient profile and pH in IEX. The data can then be treated with commercial modeling software to find out the optimal conditions to be used, and accuracy of retention times prediction was excellent (less than 1% variation between predicted and experimental values).Second, we also proved that generic IEX conditions can be applied for the characterization of mAbs possessing a wide range of p. I, from 6.7 to 9.1. For this purpose, a strong cation exchange column has to be employed at a pH below 6 and using a proportion of NaCl up to 0.2. M. Under these conditions, all the mAbs were properly eluted from the column. Therefore, salt gradient CEX can be considered as a generic multi-product approach.

Original languageEnglish
Pages (from-to)33-44
Number of pages12
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume102
DOIs
Publication statusPublished - Jan 5 2015

Fingerprint

Ion Exchange Chromatography
Chromatography
Cations
Ion exchange
Salts
Monoclonal Antibodies
Proton-Motive Force
Bottles
Neck
Software
Tuning
Polynomials
Temperature
Proteins
Experiments

Keywords

  • Cetuximab
  • Ion exchange
  • Method development
  • Monoclonal antibody
  • Salt gradient

ASJC Scopus subject areas

  • Analytical Chemistry
  • Drug Discovery
  • Pharmaceutical Science
  • Spectroscopy
  • Clinical Biochemistry
  • Medicine(all)

Cite this

Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I : Salt gradient approach. / Fekete, Szabolcs; Beck, Alain; Fekete, J.; Guillarme, Davy.

In: Journal of Pharmaceutical and Biomedical Analysis, Vol. 102, 05.01.2015, p. 33-44.

Research output: Contribution to journalArticle

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