Multi-dimensional fractionation and characterization of crude protein mixtures: Toward establishment of a database of protein purification process development parameters

T. Ahamed, M.W.H. Pinkse, L.A.M. van der Wielen, P. Verhaert, G. Dedem, M.H.M. Eppink, E. van de Sandt, M. Ottens

Research output: Contribution to journalArticleAcademicpeer-review

31 Citations (Scopus)

Abstract

A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC). In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while. the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody (mAb) purification from crude hybridoma cell culture supernatant (HCCS) was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior HTS of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation.
Original languageEnglish
Pages (from-to)3070-3083
JournalBiotechnology and Bioengineering
Volume109
Issue number12
DOIs
Publication statusPublished - 2012

Keywords

  • ion-exchange chromatography
  • expert-systems
  • separations
  • adsorption
  • optimization
  • displacers
  • selection
  • design

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