Analysis of purity

In approaches to protein purification, the concepts of yield and purity are routinely used, but are often difficult to define in absolute terms. To some extent the purity of a protein sample will be defined by the final designated use of the product. In most cases, analyses will involve measurement of the mass of the protein in the sample and quantitation of specific property of the target molecule (e.g. activity) to provide values for the yield. Thus, the calculation of specific activity of given fractions through the purification process provides a valuable indication of the level of the purity attained. Although, conventionally, specific activity is used in the purification of enzymes, due to the availability of sensitive and specific assays, recent developments in fast chromatographic separations and protein mass spectrometry have led to application of these techniques to address the purity of a wider variety of biomolecules. The level of purity for any protein product requires several factors to be taken into consideration. Besides the intended use, the source of the protein will dictate the extent of analyses required, since the level of impurities present in the final product will depend not only on the purification process used but also on the starting source material. For bulk enzyme preparations (for use e.g. in biotransformations or related applications) it may only be necessary to ensure the product is free of any contaminating activities which could effect the outcome of these types of applications. For proteins required for physical studies (protein crystallography, primary sequence analysis), the purity criteria are more stringent, particularly, since the lack of purity (including sample microheterogeneity) can drastically influence the outcome of such studies. Alternatively, proteins intended for therapeutic use will have purity considerations significantly different, constrained not only by regulatory requirements but also by clinical responses that may arise from the presence of any contaminants. The nature of these contaminants, as mentioned earlier, will depend on the starting source of the target molecule (i.e. animal tissue, human serum, recombinant micro-organisms [prokaryotes, eukaryotes], and hybridomas).

Purification by exploitation of activity

Proteins carry out their biological functions through one or more binding activities and, consequently, contain binding sites for interaction with other biomolecules, called ligands. Ligands may be small molecules such as substrates for enzymes or larger molecules such as peptide hormones. The interaction of a binding site with a ligand is determined by the overall size and shape of the ligand as well as the number and distribution of complementary surfaces. These complementary surfaces may involve a combination of charged and hydrophobic moieties and exhibit other short-range molecular interactions such as hydrogen bonds. This binding activity of a protein, which is stereoselective and often of a high affinity, can be exploited for the purification of the protein in a technique commonly known as affinity chromatography. The operation of affinity chromatography involves the following steps: (a) Choice of an appropriate ligand. (b) Immobilization of the ligand onto a support matrix. (c) Contacting the protein mixture of interest with the matrix. (d) Removal of non-specifically bound proteins. (e) Elution of the protein of interest in a purified form. At best, affinity chromatography is the most powerful technique for protein purification since its high selectivity can, in principle, allow purification of a single protein of low abundance from a crude mixture of proteins at higher concentrations. Secondly, if the affinity of the ligand for the protein is sufficiently high, the technique offers simultaneous concentration from a large volume. In practice, such single-step purifications are not common and successful affinity chromatography requires careful consideration of a number of parameters involved. The remainder of this chapter attempts to guide the experimenter in the selection and use of affinity adsorbents for protein purification. For more extensive information on this technique the reader is advised to consult the many excellent texts on this subject as well as proceedings of symposia. The construction of an affinity adsorbent for the purification of a particular protein involves three major factors: (a) Choice of a suitable ligand. (b) Selection of a support matrix and spacer. (c) Attachment of the ligand to a support matrix. The criteria for making these decisions are discussed in the following sections.

Concentration of the extract

A concentration step is frequently required after a clarified solution of the protein has been obtained, in order to aid subsequent purification steps. This is particularly important when the protein is obtained in culture medium from cells (e.g. bacteria or tissue culture cells). Concentration of the protein solution results in a decreased volume, as well as a higher protein concentration. Clearly a smaller volume of solution is easier to handle in subsequent steps, such as precipitation or loading onto a chromatography column. Higher protein concentration minimize protein losses by non-specific adsorption to container walls or column matrices. In addition many subsequent purification steps require a minimum protein concentration to be effective, for example, precipitation is more efficient at concentrations above 100 μg/ml, whilst for adsorption chromatography (e.g. ion exchange or affinity) the concentration of protein must be greater than the dissociation constant. Concentration is achieved by removal of water and other small molecules: (a) By addition of a dry matrix polymer with pores that are too small to allow entry of the large protein molecules (Section 2). (b) By removal of the small molecules through a semi-permeable membrane which will not allow the large molecules through (i.e. ultrafiltration, Section 3). (c) By removal of water in vacua (i.e. lyophilization, Section 4). Precipitation can also be used to concentrate proteins if the pellet is redissolved in a smaller volume, and in addition often results in some degree of purification of the protein of interest. However, as mentioned above precipitation is more effective if the total protein concentration is above 100 μg/ml (see Section 6). Two-phase aqueous extraction can also be used to concentrate the protein, with an associated degree of purification (see Section 7). This is one of the simplest and quickest methods of concentrating solutions of proteins, requiring minimal apparatus. A dry matrix polymer, such as Sephadex, is added to the protein solution and allowed to absorb the water and other small molecules; the pores within the matrix are too small to allow the protein to be absorbed.

Clarification techniques

Proteins can be produced by a number of different routes such as fermentation, tissue culture, and by extraction from plasma or plants. Whatever route is chosen, the raw protein-bearing stream is likely to be a complex mixture containing both dissolved species and particulate material. The target protein will be present at very low concentration and with a host of contaminants such as cells or cell debris, DNA, proteins and polysaccharides, and a large quantity of water. Such a mixture is very difficult to treat using the highly selective processes that are required to obtain the target product at high purity since the presence of particulate material impairs their function. The first challenge of protein purification is therefore to convert the complex fermentation broth which is a mixture of dissolved and suspended solids into a form that is amenable to further purification. Although there is much interest in direct recovery of protein from such materials, the most frequent first step currently is to clarify the raw protein source to remove suspended matter. It is then possible to use a range of highly selective techniques to purify the target protein. There are a number of clarification techniques that can be adopted and the choice of which to use depends on both the source of raw feed and the scale of operation. There are two main classes of process; sedimentation and filtration. Sedimentation can be carried out under normal gravity conditions or, as is almost always the case for biological streams, using a centrifuge. Filtration can be performed using either conventional filter media or using membrane filters for removal of finer particles. The aim of this chapter is to describe these methods, and their underlying principles, the advantages of each are discussed, and examples of equipment are presented. Practical advice is presented on how and when to use each technique. Sedimentation processes operate primarily on the basis of density differences between the various components of a mixture. They are most commonly applied to suspensions of solid in liquid, but also to disengage immiscible liquids. If there is no density difference between particulates and the suspending medium, sedimentation cannot occur.

Cell disintegration and extraction techniques

Disruption of cells is necessary when the desired product is intracellular. Many commercial intracellular products are proteins, such as soluble enzymes or soluble genetically engineered peptides. However, a number of genetically engineered proteins in E. coli are present in the cell as insoluble inclusion bodies; although this may be due to the chosen lysing conditions. This chapter concentrates on the extraction of soluble enzymes. To achieve a good yield of an intracellular product, it is generally a good idea to minimize the number of steps involved in the purification, as there is loss of material associated with each step. Since cell disruption is an extra procedure which itself may demand a further clarification step, it may be worth investigating if extraction of the protein can be made directly from the cell lysate (the disrupted material) without a cell debris clarification step. It may also be possible to manipulate the cell so that it excretes the protein into the surrounding growth medium, and thus, not require an extraction procedure (if the subsequent dilution of the product by the broth can be tolerated). The choice of disruption method usually follows one of the two directions: (a) Can a given disrupter be used for a particular cell type? (b) Which is the best method of extracting a product? The former often occurs in a laboratory context, whilst the latter is a question to be asked during scale-up of a process when costs are paramount. This chapter tries to accommodate both questions. Whatever type of disruption process is adopted, there are some key questions to be addressed. These are briefly discussed in the remaining sections of this overview. A recent comprehensive review of many disruption techniques is given by Middelberg. It is well known that some enzymes are more stable than others. The disruption method can impose great physical and chemical stress on the enzyme. Enzymes which are stable in the cell, perhaps by virtue of being attached to membranes, will be released into the medium during disruption.

Scale-up considerations

Once a potential indication has been found for a given protein product, it is necessary to produce increasing quantities to satisfy the demands of market trial activities. In the majority of cases, this will lead to scale-up of the purification process to meet demand whilst maintaining the safety, efficacy, and quality of the product. The following chapter provides an overview of the key issues that will arise during scale-up and provides the reader with practical advice on process and equipment selection. The text examines the key issues in defining production scale, identifies critical scale-up and development issues on an overall process basis, presents practical tips on scale-up, and concludes with two industrial scale-up case studies. The final choice of purification scale must reflect the most cost-effective solution for the whole of an organization and as such there are a great many influences to be considered. As a result the development process is an iterative exercise in which the production demand and schedule are balanced against available resources. A step-by-step approach to choice of purification scale is developed below: (a) Step 1: define volume of product required and when it is needed. (b) Step 2: develop a preliminary scale-up schedule. (c) Step 3: match scale and production schedule to production resources. Each of these steps will now be developed further. From preliminary product trials it will be possible to develop a schedule of product demand against time which can be used as the building block for step 2. Typically for pharmaceutical products the schedule will included materials for pre-clinical trials, phases I-III clinical trials, and commercial manufacture. During trials a defined quantity of product will be required over a clearly defined period whilst, once a product has been approved, demand will be less well defined and generally increase gradually over time to market saturation. From the schedule developed in step 1 and a knowledge of the approximate process yields, a preliminary assessment of overall raw material throughput can be developed. This can then be broken down and combined with information on product shelf-life to assess the most appropriate production strategy for each stage of a product’s life.

Chromatography on the basis of size

Chromatography has been employed for the separation of proteins and other biological macromolecules on the basis of molecular size since the mid 1950s when Lathe and Rutheven employed modified starch as a media for separation. Porath and Flodin developed the technique further using cross-linked dextran and coined the term gel filtration. Some confusion over nomenclature has been created by the term gel permeation, used to describe separation by the same principle in organic mobile phases using synthetic matrices. It is now generally agreed that the terms gel filtration and gel permeation do not accurately reflect the nature of the separation. Size exclusion Chromatography (SEC) has been widely accepted as a universal description of the technique and in line with the IUPAC nomenclature this term will be adopted. The historical development of SEC for protein separation has been reviewed. SEC is a commonly used technique due to the diversity of the molecular sizes of proteins in biological tissues and extracts. In addition to isolating proteins from crude mixtures, SEC has been employed for many roles including buffer exchange (desalting), removal of non-protein contaminants (DNA, viruses), protein aggregate removal, the study of biological interactions, and protein folding. The principle of size exclusion is based on a solid phase matrix consisting of beads of defined porosity which are packed into a column through which a mobile liquid phase flows. The mobile phase has access to both the volume inside the pores and the volume external to the beads. Unlike many other chromatographic procedures size exclusion is not an adsorption technique. Separation can be visualized as reversible partitioning into the two liquid volumes. The elution time is dependent upon an individual protein’s ability to access the pores of the matrix. Large molecules remain in the volume external to the beads as they are unable to enter the pores. The resulting shorter flow path means that they pass through the column relatively rapidly, emerging early. Proteins that are excluded from the pores completely, elute in the void volume, V0. This is often determined experimentally by the use of a high molecular weight component such as blue dextran or calf thymus DNA.

Separation on the basis of chemistry

Most chromatographic separations are based on chemical interaction between the solute of interest or impurities to be removed and the separation medium. The exception is separations based upon physical properties such as size (e.g. size exclusion chromatography) or transport in a force field (e.g. electrochromatography). The chemical interaction may be weak (e.g. employing van der Waals forces) or very strong (e.g. involving formation of chemical bonds as in covalent chromatography). Whenever separation is based upon attractive forces between the solute and the separation medium, we talk about adsorption chromatography (also when the solute is merely retarded). The chemical interaction between the solute and the adsorbent (the chromatography medium) is governed by the surface properties of the solute and the adsorbent and is in most cases mediated by the mobile phase or additives to the mobile phase. Macromolecules such as proteins display a variety of properties and, ideally, a selected set of properties is utilized for obtaining the required selectivity (i.e. relative separation from other solutes) using a separation medium of complementary properties. This chapter briefly reviews the different types of forces of interaction between solutes and surfaces commonly employed for chromatographic purifications, important properties of solvents, and some basic surface chemical properties of proteins. This, together with a description of some common types of chromatography modes provides a basis for a rational selection of separation mechanism for the purification of proteins and the choice of mobile phase composition to regulate the relative influence of different interaction mechanisms. The separation mechanisms are focused to adsorptive modes with the exception of affinity chromatography which is discussed in Chapter 9. The different attractive forces acting between molecular and particle surfaces include (1): • dispersion forces • electrostatic dipole interactions • electron donor-acceptor forces • formation of covalent bonds All these forces are due to interactions between electric charges (permanent or induced). Dispersion, or London forces, are caused by induced dipole-induced dipole interactions and are thus classified as a non-specific interaction. This type of non-polar interaction is the dominant force promoting dissolution of non-polar solutes in organic solvents.

Getting started

This chapter provides a more detailed overview of the equipment requirements for protein purification and the practical aspects of developing a purification strategy, including the ordering of unit operations, buffer preparation, and approaches to minimizing yield losses. A well-equipped laboratory is an essential prerequisite to successful protein purification and time spent in deciding needs and purchasing essential items will help to avoid panics mid-way through a protocol when a key piece of equipment is lacking. This said, one should avoid expensive purchases (unless budget is no object) as the majority of purifications can be achieved with fairly routine equipment. The few essential pieces of expensive equipment are a spectrophotometer, a centrifuge, and a chromatography set-up. In general, money is best spent in purchasing plenty of the cheaper items such as tubes, beakers, measuring cylinders, salts, and buffers. Chromatography equipment is an essential item for any purification laboratory. There are plenty of expensive chromatography set-ups available which can provide a remarkable saving in time for process development purposes. If you are not familiar with such equipment and are new to the world of protein purification, I suggest you make do with a simple chromatography set-up to start with, until you become familiar with the technique and learn more about your exact requirements and how alternative tailor-made process development kits differ. A protein purification laboratory should be equipped with supplies of tapwater, de-mineralized water, and distilled water. Electricity and sinks are taken for granted. Required equipment can be roughly grouped into three categories: • those for ancillary purposes • those for detection • those for separation Table 1 lists the essential items for the protein purification laboratory. Time and money spent wisely on buying adequate supplies of support materials will pay dividends. There is nothing more irritating than having to rush around in search of a clock or some salt half-way through a delicate purification with your enzyme degrading in front of your eyes! Key requirements are tubes, beakers, pipettes, stirrers, and timers. In addition essential chemicals include salts and buffers. Adjustable pipettes (e.g. Gilson) are essential and those suitable for sample volumes from 10 μl to 5000 μl are recommended.

Purification strategy

Practical approaches to protein purification contains detailed practical information on separations techniques and the chapters of Protein purification techniques cover the unit operations and analytical techniques involved in some detail while Protein purification applications provides details of how to approach purification from a selected number of typical sources. However a key element of every purification, whether in University research or as part of scaling-up an industrial process, is planning. Time spent at the outset in establishing the key goals of the process prior to going into the laboratory will invariably save much time and effort. Key points to consider from the outset are: (a) Why are you doing the work—what is the reason for the purification? (b) What are the key considerations in selecting how to purify? (c) What implications does this have on how you will approach the purification? At the start of a purification, the target protein may be a minor component among millions of other proteins and other contaminants. This presents boundless opportunities for miscalculations, blind-alleys, and wasted effort. Years of practical work by separation scientists have derived certain rules which will help to minimize such problems and ensure protein purifications are successful. Here are ten purification rules to consider: 1. Keep the purification simple—minimize the number of steps and avoid difficult manipulations which will not reproduce. 2. Keep it cheap—avoid expensive techniques where a cheaper one will do. 3. Adopt a step approach—and optimize each step as you go. 4. Speed is important—avoid delays and slow equipment. 5. Use reliable techniques and apparatus. 6. Spend money on simple bits and pieces—e.g. test-tubes, pipettes. 7. Write out your methods before you start and record what you have done accurately. 8. Ensure your assays are developed to monitor the purification. 9. Keep notes on yields and activity throughout. 10. Bear in mind your objectives—be it high yield, high purity, final scale of operation, reproducibility, economical use of reagents/apparatus, convenience, throughput. Protein purification uses time, money, effort, and valuable equipment. Therefore, it is always advisable to pause for some moments to consider the reasons for purification in the first place.