Multimodal Interaction Chromatography for the Investigation and Separation of Proteins

Kristian Kallberg

Research output: ThesisDoctoral Thesis (compilation)

Abstract

Minor structural changes on the surface of proteins can have tremendous impact on their biological properties. When producing therapeutic proteins it is therefore important to verify that the generated protein is the desired one. Small variations such as glycosylations or amino acid substitutions can mean the difference between a functional, defective or even toxic product.

This thesis describes the characterization and use of a multimodal pH-tunable hydrophobic interaction chromatography media for separating proteins with small structural differences. The media is constructed by attaching copolymer ligands on the surface of spherical agarose support beads. From the multimodal character of the polymeric ligand it is possible to allow several interaction types to be in effect at the same time, a property which makes it sensitive to small protein surface modifications. The chromatography media presented here is mainly hydrophobic but will also participate in electrostatic interactions and form hydrogen bonds. In addition, the media will react to the pH of the surrounding solvent and change its hydrophobicity. At low pH it will be more hydrophobic than at neutral pH. Both pH-dependent behavior and how polymer length effects separation have been explored.

The pH-responsiveness of the media provides opportunities to fine-tune the separation process to virtually any protein desirable. This property was used in the work presented here for purifying delicate recombinant hemoglobin. The high selectivity that was obtained from the multimodality was used to separate and investigate the stability of the haptoglobin-hemoglobin complex. Additionally, the media was able to separate glycosylated IgG variants which traditional HIC media could not. This property enabled developing a rapid monitoring method for antibody producing cell systems.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Pure and Applied Biochemistry
Supervisors/Advisors
  • Bülow, Leif, Supervisor
  • Svensson Bonde, Johan, Supervisor
Award date2013 Apr 29
ISBN (Print)978-91-7473-478-2
Publication statusPublished - 2013

Bibliographical note

Defence details

Date: 2013-04-29
Time: 10:30
Place: Lecture hall C at the Centre for Chemistry and Chemical Engineering, Getingevägen 60, Lund

External reviewer(s)

Name: Jungbauer, Alois
Title: Professor
Affiliation: University of Natural Resources and Life Sciences, Vienna, Austria

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Subject classification (UKÄ)

  • Biochemistry and Molecular Biology

Free keywords

  • Hydrophobic interaction chromatography
  • Multimodal chromatography
  • Glycosylation
  • Antibody
  • Hemoglobin
  • Protein purification.

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