Chapter 1

Protein Purification: An Overview

Nikolaos E. Labrou

Abstract
Biological macromolecules such as proteins constitute an important class of products in the food,
biotechnology, pharmaceutical, and cosmetics industries. The growing need to develop efficient and rapid
protein purification methods is driving research and growth in this area. Advances and progress in the
methods and techniques of protein purification have been such that one can reasonably expect that any
protein of a given order of stability may be purified to currently acceptable standards of homogeneity.
However, protein production cost remains extremely high, with downstream processing constituting a
substantial proportion of the overall cost. Understanding of the methods and optimization of experimental
conditions have become critical to the manufacturing industry in order to minimize production costs while
satisfying all regulatory requirements. New purification protocols exploiting specific, effective, and robust
methods and materials are expected to guide the future of the protein purification area.

Key words Chromatography, High-resolution methods, Low-resolution methods, Market analysis,

Protein precipitation, Protein purification

1 Introduction

As scientists complete sequencing the genomes of hundreds of

species, they embarked on the next great challenge toward under-
standing at molecular level the biology of the cell [1]. As they
attempt to understand the vast amount of genetic information,
proteomics has become a major focus in the molecular biology.
If sequencing the human genome has been a major technological
challenge, characterizing the proteome promises to be an even
great one [1–3]. With thousands of potential genes identified from
sequencing projects, protein biochemistry seeks to define the role
and biological function of the gene products. Biotechnology seeks
to develop new protein-based applications and their commercial
exploitation. The first requirement for achieving these goals is the
development of efficient and effective purification methods and
materials [4].
The various steps in the downstream processing protocol
separate the protein and nonprotein parts of the mixture and finally

Nikolaos E. Labrou (ed.), Protein Downstream Processing: Design, Development and Application of High and Low-Resolution Methods,
Methods in Molecular Biology, vol. 1129, DOI 10.1007/978-1-62703-977-2_1, © Springer Science+Business Media, LLC 2014

3
4 Nikolaos E. Labrou

Table 1
Physicochemical basis of common bioseparation methods

Separation Basis of separation Resolution

Precipitation
Ammonium sulfate Solubility Low
Organic solvents Solubility Low
Polyethyleneimine Charge, size Low
Polyethylene glycol Solubility Low
Isoelectric Solubility, pI Low
Affinity precipitation Molecular recognition, solubility Low
Phase partitioning
Aqueous two-phase partition Solubility/hydrophobicity Low/medium
Three phase partitioning Solubility/hydrophobicity Low/medium
Chromatography
Ion exchange Charge, charge distribution High
Hydrophobic interaction Hydrophobicity High
Reverse-phase HPLC Hydrophobicity, size High
Affinity chromatography Molecular recognition High
Gel filtration/size exclusion Size, shape High

separate the desired protein from all other proteins while retaining
the biological activity and chemical integrity of the polypeptide.
This last step is typically the most laborious and difficult aspect of
protein purification. The purified protein should be free not only
of contaminants (e.g., nucleic acids, viruses, pyrogens, residual
host cell proteins, cell culture media, leachates from the separation
media) but also of the presence of various isoforms, originating
from posttranslational modifications [5].
Separation steps may exploit differences in chemical/structural/
functional properties between the target protein and other pro-
teins in the crude mixture (Table 1). These properties include
size, shape, charge, isoelectric point, charge distribution, hydro-
phobicity, solubility, density, ligand-binding affinity, metal
binding, reversible association, posttranslational modifications,
and specific sequences or structures. By exploiting these tremen-
dous variations in physical and chemical properties among pro-
teins, several different fractionation and chromatographic steps
can usually be exploited to design a workable purification scheme
[6]. However, some proteins may be very challenging to purify in
an active and stable form, for example, integral membrane pro-
teins, unstable protein complexes, proteins produced as insoluble
aggregates, and proteins with a specific set of posttranslational
modifications. The challenges and difficulties in protein purifica-
tion make worthwhile to gain solid knowledge about protein puri-
fication so that the available methods can be selected and applied
in an optimal way.
 Protein Purification 5

2 Historical Aspects

In the eighteenth century, proteins were known as a distinct class

of biological molecules by Antoine Fourcroy and others. They
distinguished these molecules by their ability to coagulate under
treatment with heat or acid (e.g., albumin from egg whites, blood
serum albumin). However, techniques for protein isolation and
purification were developed by Edwin Joseph Cohn during World
War II [7]. He carried out pioneering work on fractionation of
plasma proteins. The solubility properties, precipitation, and
crystallization dominated the design of early purification studies.
The next major milestone in protein purification was chromatog-
raphy [8]. Ion exchangers began to be used and have become
indispensible in protein purification. Whatman introduced cellu-
lose-based ion exchangers followed by introduction of dextran-
based ion exchangers by Pharmacia. In 1910, Emil Starkenstein
described the concept of affinity chromatography [9]. This was an
important milestone. He studied the separation of a macromole-
cule (liver α-amylase) via its interactions with an immobilized sub-
strate (starch). The term affinity chromatography introduced in
1968 by Pedro Cuatrecasas, Chris Anfinsen, and Meir Wilchek in
an article that briefly described the technique of enzyme purifica-
tion via immobilized substrates and inhibitors [10]. This progress
was some kind of a mini-revolution in protein purification. For a
couple of decades, a typical protein purification protocol invariably
consisted of precipitation by ammonium sulfate, one or two ion
exchange steps, gel filtration, and finally an affinity chromatogra-
phy step.

3 Low- and High-Resolution Protein Purification Methods

Downstream processing has been challenged with demands of

high yields, resolving power, and cost efficiency. This has triggered
remarkable developments in improvising process tools and innova-
tive strategies for protein separation. A wide variety of protein
purification methods that can be combined to generate a suitable
purification scheme are available. Usually, one executes a series of
purification steps, and only rarely proteins can be purified in a
single step, even when this step is based on an exquisitely specific
biological characteristic [11, 12]. Early steps combine low-
resolution and high-capacity methods (when large amounts of pro-
tein is present) with higher-resolution and lower-capacity ones
(when less protein is present) at later stages of purification scheme.
For low-resolution protein purification, methods such as fractional
precipitation and two-phase partition systems usually employed [13].
For applications requiring the highest purity and relatively small
6 Nikolaos E. Labrou

amounts of protein, chromatography can be chosen to selectively

purify the target protein [14].
Chromatography is certainly the principal and commonly used
operation in downstream processing [15]. This can be explained
by certain advantages of chromatography over other unit operations.
For example, chromatography displays high-resolution efficiencies
which allow the resolution of complex crude mixtures with very
similar molecular properties. In addition, chromatography is ideal
for capturing molecules from the dilute solutions encountered in
bioprocessing.
Among all chromatographic techniques, affinity chromatography
plays a major role [16]. In fact, affinity chromatography is the most
specific and effective protein purification technique, providing a
rational basis for the purification of target proteins. It exploits the
principle of biomolecular recognition, that is, the ability of biologi-
cally active macromolecules to form specific and reversible com-
plexes with affinity ligands. As conventional purification protocols
for high-value proteins are replaced by more sophisticated proce-
dures based on affinity chromatography, the focus is shifted toward
designing and selecting ligands of high affinity and specificity [17, 18].
Affinity ligands are distinguished in biological and synthetic
ligands. The former, although they display high affinity and bind-
ing capacity, they suffer seriously from low chemical and biological
stability and unfavorable economics since they show low-adsorbent
lifetime and high manufacturing costs. Synthetic ligands appear to
tackle effectively most of the problems mentioned above.
The accumulated knowledge of structures obtained from X-ray
crystallography, NMR and homology studies, the impressive
growth of bioinformatics and molecular docking techniques, the
defined and combinatorial chemical synthesis, the display tech-
niques based on biological/genetic packages, and the technologi-
cal advances in high-throughput screening has made the design
and selection of high-affinity synthetic ligands faster and more
effective [16–18]. There are three main methods for generating a
ligand, the rational, the combinatorial, and the structure-guided
combinatorial. The decision depends primarily on the information
accessible at the outset. Nevertheless, there are concerns regarding
toxicity and biocompatibility of synthetic ligands. To this end,
experimental studies on the toxicity of synthetic ligands may be
necessary for creating proper Drug Master Files (DMF) or
Regulatory Support Files (RSF).

4 Current Trends

The gap between upstream and downstream processing is a big

problem for the biotechnology industry [19]. This is because the
progress toward producing more protein per unit volume of culture
 Protein Purification 7

medium has improved more rapidly than increases in the rates at

which these materials can be purified. Therefore the biotechnology
industry is suffering from a severe shortage of purification capacity
for proteins. Thus, there has been a general consensus that reduc-
ing the number of separation steps and maximizing the yield at
each step are necessary for an economical process.
The essential goal of the development and optimization of the
downstream processing protocol is the production of quality products
with sufficient purity while maintaining the biological activity in a con-
sistent manner and satisfying all regulatory requirements. Quality and
safety guidelines for biotechnology products have grown increasingly
stringent. Guidance from the International Organization for
Standardization (ISO) [20] and from the International Society of
Pharmaceutical Engineers (ISPE) [21] provides basic information for
production of safe and effective products.
For certain applications, some proteins can be used as crude
extracts with little purification. However, pharmaceutical proteins
typically require exceptional purity, making downstream process-
ing a critical component of the overall process with high costs [22,
23]. It is estimated that 50–80 % of protein manufacturing costs
are due to downstream processing. This is owing to the stringent
quality criteria imposed to protein products, such as defined
purity, efficacy, potency, stability, toxicity, and immunogenicity.
It is worth noting that the steps of the downstream processing
procedure are further complicated and influenced by the commer-
cial conditions, business plans, and patent rights [24, 25]. The
commercial competition to develop new protein products or to
decrease production cost of the patented product becomes very
important than ever, and cost reduction is now often more critical
than speed to market.
Recombinant DNA technology impacts the development of
protein purification methods in two ways: Firstly, the development
and availability of several different protein expression systems
meant that sources of proteins are not limited to naturally occur-
ring animals, plants, and microbes [26, 27]. Protein expression
systems are used to produce wild-type proteins in biotechnology
and industry and more recently to produce novel engineered vari-
ants of proteins that display improved properties. Commonly used
protein expression systems include those derived from bacteria,
yeast, baculovirus/insect, mammalian cells, and transgenic plants
and animals. Second, the use of “affinity tags” and production of
proteins in the form of fusion proteins became possible [28]. The
efficiency gained by the generic purification approaches based on
affinity tagging of the target protein has simplified protein purifi-
cation. Biotechnology companies have commercialized several
different protein-fusion systems available for biochemical research.
It should be noted, however, that these methods do not always
8 Nikolaos E. Labrou

provide sufficient purity, and additional physicochemical-based

chromatography methods, for example, gel filtration and ion
exchange, may thus have to be included to the protocol.

5 Market Analysis

The growing need to develop new methods and rapid purification

protocols is driving research and growth in the protein purification
market. The global market for engineered protein products is esti-
mated to be worth $168 billion by 2017, growing at an annual
growth rate of 10.94 % from 2012 to 2017 [29]. The purified
protein market is divided into three segments based on its applica-
tions, namely, therapeutic proteins, diagnostic proteins, and pro-
teins used in research applications. Therapeutic proteins are the
fastest growing segment with monoclonal antibodies with more
than 50 % market share. Since the early 1980s proteins have
emerged as a major new class of pharmaceuticals with ~200 mar-
keted products that are mainly therapeutics with a small number of
diagnostics and vaccines [30]. The biosimilars or “follow-on pro-
tein products” industry is growing at an annual growth rate of
around 52 % during 2010–2015 [31]. Concerning protein purifi-
cation market, affinity tags remain a significant driver of the chro-
matography market due to the ability to achieve high-purity
proteins in a single step [32]. In 2010, this market earned $341.7
million in revenue at 4.8 % growth.

6 Conclusions

Due to the enormous number of newly discovered “open reading

frames,” progress in the analysis and commercial exploitation of
the corresponding proteins depends on the ability to perform
efficient purification. The need to obtain a purified protein, eco-
nomically and in sufficient quantity, applies to any purification,
from preparation of an enriched protein extract for biochemical
characterization to large-scale production of a therapeutic recom-
binant protein. Downstream processing has thus been challenged
with demands of high yields, resolving power, and cost efficiency.
This has triggered remarkable developments in improvising process
tools and innovative strategies for protein separation. Therefore,
a plethora of emerging technologies for the development and
optimization of protein purification challenge are anticipated to
help address these needs. Innovations, possibly combined with
cost savings, exploiting specific, effective, and robust methods
and materials, are expected to guide the future of the protein
purification area.

Acknowledgements
This work was supported by the grants: AlgaeCom and AquaPhage
(funded by the European Union, FP7), BioExplore (09ΣΥΝ-
23-94, co-funded by the European Union—European Social
Fund & National Resources), and THALES (MIS380236, co-
funded by the European Union—European Social Fund &
National Resources).
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