Protein isolation using affinity chromatography

154 Pages·2012·3.48 MB·English

Propositions

1. When comparing chromatographic media or configurations, at least one will
perform suboptimal. (this thesis)
2. A chromatographic monolith has advantages over a packed bed of porous
particles only when very small or very large molecules are to be adsorbed.
(this thesis)
3. More precise measurements do not always produce more precise results.
4. The word ‘science’ suggests a certainty that often does not exist.
5. You only truly understand something if you can explain it to others.
6. On‐line social media often result in asocial behaviour in real‐life.
7. Flexible working hours, especially when working from home, make it harder
to balance work and private life.

Propositions belonging to the thesis:
‘Protein isolation using affinity chromatography’
Tamara Besselink
Wageningen, 7 December 2012
Protein isolation using
affinity chromatography

Tamara Besselink
Thesis committee

Promotor
Prof. dr. ir. R.M. Boom
Professor of Food Process Engineering
Wageningen University

Co‐promotor
Dr. ir. A.E.M. Janssen
Assistant professor, Food Process Engineering Group
Wageningen University

Other members
Prof. dr. W.J.H. van Berkel, Wageningen University
Dr. ir. P.J.Th. Bussmann, TNO, Zeist
Prof. dr. ir. A.B. de Haan, Eindhoven University of Technology
Prof. dr. J.D. Mikkelsen, Technical University of Denmark

This research was conducted under the auspices of the graduate school VLAG
(Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health
Sciences).
Protein isolation using
affinity chromatography

Tamara Besselink

Thesis
submitted in fulfilment of the requirements for the degree of doctor
at Wageningen University
by the authority of the Rector Magnificus
Prof. dr. M.J. Kropff,
in the presence of the
Thesis Committee appointed by the Academic Board
to be defended in public
on Friday 7 December 2012
at 1.30 p.m. in the Aula.
Tamara Besselink
Protein isolation using affinity chromatography
146 pages.

PhD thesis, Wageningen University, Wageningen, NL (2012)
With references, with summaries in Dutch and English

ISBN 978‐94‐6173‐426‐6
Contents

Chapter 1 Introduction 1
Chapter 2 Isolation of bovine serum albumin from whey using affinity 9
chromatography
Chapter 3 The influence of pH and temperature on bovine serum albumin – 25
VHH ligand binding
Chapter 4 Comparison of activated chromatography resins for protein 49
immobilization
Chapter 5 Efficiency of protein‐based ligand immobilization for affinity 67
chromatography
Chapter 6 Are axial and radial flow chromatography different? 83
Chapter 7 General Discussion 109
Summary 131
Samenvatting 135
Dankwoord 139
Curriculum Vitae 141
Publications 143
Overview of completed training activities 145

Chapter 1
Introduction

1.1 A short history of chromatography
According to IUPAC (International Union of Pure and Applied Chemistry) the official
definition of chromatography is “a physical method of separation in which the
components to be separated are distributed between two phases, one of which is
stationary (stationary phase) while the other (the mobile phase) moves in a definite
direction” [1]. Literally, the word chromatography means ‘colour writing’ and dates
back to 1906. The Russian botanist Mikhail Tswett (his last name meaning ‘colour’ or
‘flowering’ in Russian) separated colourful plant pigments from each other using a
column packed with an adsorbent [2]. Tswett investigated many different adsorbents
(stationary phases) and solvents (mobile phases) to find an optimal separation
method. It took over thirty years before the value of Tswetts work was recognized [3].
This was mainly due to the work of Nobel Prize winners A.J.P. Martin and R.L.M. Synge
who described the chromatography process theoretically. From there gas
chromatography and new types of liquid chromatography were developed. For a long
time, chromatography was mainly used as an analytical tool using small sample sizes.
Later on, the preparative use of chromatography was rediscovered (e.g., [4]), with its
main applications in the pharmaceutical industry. The challenges in scale‐up of
chromatography processes, the batch‐wise nature of the process, the amount of
solvents (buffers) required, and the high costs of target‐specific chromatography
adsorbents are the main reasons why chromatography is not more widely used. For
pharmaceuticals, purity is crucial and high separation costs can be acceptable in this
field. A picture of a contemporary lab‐scale preparative chromatography system is
shown in Figure 1.1.
1
Chapter 1

Figure 1.1. Example of a lab scale preparative chromatography setup with an Äkta Purifier (GE Healthcare,
Uppsala, Sweden).
1.2 Chromatography adsorbents and ligands
Chromatography can be used to separate a mixture into its components. Two phases
can be distinguished: the mobile phase, which can be a solvent but also the feed
mixture itself, and the stationary phase, the adsorbent. The adsorbent or resin usually
consists of spherical particles made of agarose, cellulose, polymethacrylate, silica and
many other materials which are packed in a cylindrical column. The particles may be
porous or non‐porous. Membranes and monoliths can also be used as a stationary
phase [5].
Several methods of separation exist in chromatography. In size‐exclusion
chromatography, also known as gel filtration chromatography, components are
separated based on size. This method is mainly suitable for analytical purposes and
final polishing, because the column cannot be loaded with the mixture to be separated
for more than ~5% of the column volume.
Another possibility is to adsorb components to the stationary phase. This means that
after adsorption, a change in buffer is required to desorb the components from the
stationary phase. As an example, charged components can be separated using ion
exchange, a method that is frequently applied in the pharmaceutical industry, and for
example in the desalting of whey. To remove the adsorbed components, a salt gradient
is used. On small scales, it is used in mechanical dish washers to remove multivalent
ions from water.
The most specific method in chromatography is affinity chromatography. This type of
chromatography relies on specific interaction between the target component and a
2