Protein isolation using affinity chromatography

T. Besselink

Research output: Thesisinternal PhD, WU

Abstract

Many product or even waste streams in the food industry contain components that may have potential for e.g. functional foods. These streams are typically large in volume and the components of interest are only present at low concentrations. A robust and highly selective separation process should be developed for efficient isolation of the components. Affinity chromatography is such a selective method. Ligands immobilized to a stationary phase (e.g., a resin or membrane) are used to bind the component of interest. Affinity chromatography is, however, a costly process, due to the batch-wise operation, the large amount of solvents required and the high costs of the ligands and stationary phases. Therefore, its current use is mainly limited to lab-scale purifications and pharmaceutical applications.

The aim of this research was to investigate the potential of affinity chromatography for the isolation of minor protein in the food industry. The discovery of the VHH ligand, based on the binding domain of a llama antibody, has led to a new class of highly selective ligands, which can be produced on a large scale. We studied the chromatography process to measure productivity, but also to develop a rational protocol for decisions on suitable stationary phases and process configurations. The research presented in this thesis provides insights in the opportunities and challenges for large-scale affinity chromatography.

The isolation of protein using affinity chromatography requires several stages: adsorption, washing, and desorption. In Chapter 2, we studied these stages for the isolation of bovine serum albumin (BSA) from pure BSA solutions with high and low concentration and from actual feedstock, in this case cheese whey. A small-scale packed bed column was used to investigate the yield and productivity. BSA was retrieved in highly pure and concentrated form in the desorption stage. Furthermore, we found that the productivity of the system strongly depended on the point at which the adsorption stage is terminated.

Acids or salts are commonly used to disrupt the bond between ligand and target protein during desorption. This results in the use of large quantities of chemicals, whilst the potential of other methods for desorption, such as an increase in temperature, is not fully explored. In Chapter 3 we measured the thermodynamics of the adsorption reaction between BSA and the VHH ligand with isothermal titration calorimetry (ITC). Temperature and pH were varied to find other conditions for desorption. A buffer with high pH could be used for desorption, and an increase of temperature seemed to weaken the bond between protein and ligand. However, the acidic buffer would in this case still be most effective.

Apart from the bond between ligand and target protein, the stationary phase to which the ligand is immobilised plays a key role in the chromatography process. Many supports are available, of which we investigated a selection of resins for packed bed chromatography in Chapter 4. We found that some resins were unsuitable for our process due to their low adsorption capacity. A ranking and weighing method was presented to determine the optimal resin depending on the requirements of the process.

An important issue we found for all the resins investigated, was the low adsorption capacity compared to other types of adsorptive chromatography processes, such as ion exchange chromatography. Therefore, we studied the immobilization of the ligand to three resins in more detail in Chapter 5. The efficiency of ligand immobilization depended on the ligand concentration used in the immobilization procedure. However, only approximately one out of five immobilized ligands was able to bind to the target. Improvement of ligand immobilization is therefore a potential route to increase the feasibility of affinity chromatography for large-scale processes. 

Eventually the lab-scale process has to be scaled-up to industrial scale. The commonly used axial flow column, essentially a cylinder filled with resin through which the feed flows in the axial direction, can have problems at scale-up, because of increased pressure drop as the column is lengthened. Therefore, scale-up usually takes place by widening the column. Another option is to use a radial flow column, in which the resin is confined between two concentric cylinders and liquid flows from the outside inwards or from the inside outwards. The radial flow column can be scaled up in height instead of width. In Chapter 6 we compared axial and radial flow affinity chromatography both experimentally and theoretically. We found that the differences in performance were minimal, because the process was limited by diffusion inside the resin particle. At a small process scale, radial flow columns are impractical in terms of size, but at a larger process scale they may compete with axial flow columns because of their smaller foot print and possibly lower construction costs.

The research in this thesis was focused on a defined ligand-protein system and commercially available resins in packed-bed configuration. The potential of other stationary phases, such as non-porous (magnetic) particles, membranes and monoliths was therefore discussed in Chapter 7. We found that currently the packed bed of porous resin beads still seems to be the most suitable configuration. A radial flow column with porous affinity resin is in theory capable of isolating a low-concentrated protein from a large feed of 10 m3/h. However, the relatively low capacity of the resin, the limited liquid velocity, as well as large buffer usage and the current costs remain important issues to resolve to further expand the opportunities of affinity chromatography for minor protein isolation.

 

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Boom, Remko, Promotor
  • Janssen, Anja, Co-promotor
Award date7 Dec 2012
Place of PublicationS.l.
Print ISBNs9789461734266
Publication statusPublished - 2012

Keywords

  • protein extraction
  • isolation
  • affinity chromatography
  • resins
  • ligands
  • bovine serum albumin
  • waste treatment
  • waste utilization
  • industrial applications

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