Protein Purification I: Liquid Chromatography

From individual academic laboratories to Big Pharma manufacturing plants, small- and large-scale protein purification usually requires some type of liquid chromatography. Most purification techniques have been in use for decades, but the development of new resins has improved the time-tested methods that exploit proteins' physical and chemical properties to effect separations. This profile examines four techniques—gel filtration (GF), ion exchange (IEX), hydroxyapatite (HAP), and hydrophobic interaction (HI) chromatography. Affinity chromatography and HPLC (high-performance liquid chromatography) will be discussed in future articles. For the novice protein purifier, finding a place to start might seem overwhelming, but fortunately the process can be approached systematically. Andrew Mitchell, technical consultant for Amersham Biosciences in Piscataway, NJ, explains that protein purification by liquid chromatography generally takes place in three phases: a capture step, in which the desired protein is separated from other cellular components such as DNA and RNA; an intermediate step, in which proteins are isolated from contaminants similar in size or other physical/chemical properties; and a polishing step, in which the sample is readied for use. Each purification stage has certain chromatography techniques and bead sizes that are best suited to it. The initial capture step typically involves protein isolation from a crude cell lysate and requires a resin with a high capacity and high flow rate. "Fast flow" resins with a large bead size and large bead size range (the range can vary widely from the average bead size) are ideal for this purpose. Fast flow resins also help prevent proteolytic cleavage of the target protein, because they allow faster separation of proteins from contaminants. The intermediate step requires higher resolution for better separation of components. Generally, bead size correlates inversely with resolution; smaller bead sizes are thus more appropriate at this stage. Adsorptive techniques, such as IEX and HI, are generally used in these first two stages of purification. Gel filtration is usually reserved for the polishing step, in which a small, highly concentrated sample is applied to the column. Mitchell notes that the sample volume should be between 1% and 4% of the column bed volume for gel filtration. Two factors to consider when selecting a resin are its selectivity for the desired protein and its efficiency, as demonstrated by the peak width of eluting components, says Mitchell. Selectivity refers to the ability of the resin to interact with and bind the protein of interest. IEX and HI chromatography involve interactions between the target molecule and separation matrix; the selectivity of GF is dependent on the fractionation range of the resin (discussed below). Resin efficiency refers to the ability of the chromatography matrix to elute components in distinct, rather than broad, peaks. "Even if you get good selectivity, the resolution will diminish if you have broad peaks," explains Mitchell. The larger the bead size, the broader the elution peaks, and the lower the column's efficiency. High efficiency is essential when purifying closely eluting proteins; the combination of high selectivity and high efficiency yields high resolution. Specific information about resins offered by a variety of companies is available in the product tables accompanying this article online. Gel Filtration Chromatography While arguably the most simple column chromatography technique, gel filtration (GF) chromatography is one of the most flexible, because it can be performed under a variety of physical and chemical conditions and generally does not involve an elaborate protocol.1,2 GF (also called size-exclusion chromatography) separates globular proteins according to their molecular weights. An aqueous solution of proteins is, with help of an eluent, passed through a solid phase or matrix composed of beads containing a range of pore sizes, chosen on the basis of the target protein's size. The liquid volume "seen" by the column consists of a mobile phase (the liquid surrounding the beads, or "void volume") and a stationary phase (the liquid contained within the beads' pores). Molecules that are too large to enter the bead pores will only come in contact with the void volume and will therefore elute first from the column. However, the smallest molecules will diffuse into the pores and come into contact with the total column volume and will elute last, with molecules of intermediate size eluting between the void volume and the total column volume. Amersham Biosciences' Mitchell explains that an ideal resin is one in which the target molecule elutes somewhere near the midpoint between the void volume and the total column volume, in the matrix's "separation range." One factor to consider when choosing a GF medium is the exclusion limit, or the molecular weight limit, of the pores; proteins above this limit will be completely excluded from the pores and will not be separated. The type of resin used is also important. A variety of resins exists, ranging from silica-based to polymeric. Silica-based gels have a pH limit of 2.5-7.5, but most purifications tend to be run in this range. Some companies offer cross-linked resins, which are advantageous for high-pressure purifications because they do not compress and lose porosity under high-pressure conditions. However, non-cross-linked resins are suitable for routine purifications. GF has a number of advantages over other types of liquid chromatography, according to Jack Kelley, technical representative for Montgomeryville, Pa.-based Tosoh Biosep. First, there is a high upper limit on the size of the proteins that can be purified using this technique. Tosoh Biosep, for example, sells polymeric-based resins with an exclusion limit of up to 200,000 kD. Additionally, the pore shape is ideal for separating globular molecules such as proteins, and the technique does not require the use of protein-denaturing organic solvents. Also, GF chromatography is one of the oldest, most well-understood liquid chromatographic techniques.3 GF chromatography, however, can be difficult to fine-tune. In addition, protein resolution depends on the sample volume applied to the column, as mentioned above. Thus, dilute samples are difficult to purify by this technique. Kelley also notes that some proteins might be difficult to purify via GF; the resins used in this technique are slightly hydrophilic and proteins with a high charge density will sometimes stick to them. Finally, GF media is expensive, and GF chromatography is not directly scalable from an analytical to a bulk purification level—two disadvantages for large-scale purification. Ion Exchange Chromatography Because it offers a greater degree of control and specificity, ion exchange chromatography (IEX) is frequently used after GF chromatography. IEX relies on charge interactions between the protein of interest and the ion exchange matrix, which is generally composed of resins, such as agarose, dextran, and cross-linked cellulose and agarose, that are covalently bound to a charged group.4 Other options include Fractogel® resins from Madison, Wis.-based Novagen. These resins incorporate a "tentacle" technology in which linear polyelectrolyte chains are grafted onto a methacrylate bead. According to company literature, this technology provides higher resolution chromatography by decreasing steric hindrance and thereby increasing the number of sterically available ligands to which proteins can bind. Charged groups are classified according to type (cationic and anionic) and strength (strong or weak); the charge characteristics of strong ion exchange media do not change with pH, whereas with weak ion exchange media, sample loading capacity can change owing to loss of charge at varying pH, preventing protein binding.4 Amersham Biosciences' Mitchell explains that it is generally best when experimenting with IEX resin types to try a strong exchanger first, as these resins provide more reproducible results and allow optimization over a broader pH range. The most commonly used charged groups include diethylaminoethyl, a weakly anionic exchanger; carboxymethyl, a weakly cationic exchanger; quaternary ammonium, a strongly anionic exchanger; and methyl sulfonate, a strongly cationic exchanger. Other charged groups are available. Ion exchange resins selectively bind proteins of opposite charge; that is, a negatively charged resin will bind proteins with a net positive charge, and vice-versa. The technique takes place in five steps: equilibration of the column to pH and ionic conditions ideal for target protein binding; reversible adsorption of the sample to the column through counterion displacement; introduction of elution conditions that change the buffer's pH or ionic strength in order to displace bound proteins; elution of substances from the column in order of binding strength (weakly-bound proteins are eluted first); and re-equilibration of the column for subsequent purifications. Researchers can design IEX protocols so that the target protein is selectively bound to the column (allowing contaminants to pass through) or so that contaminants adsorb and the target protein is selectively excluded.4 Like GF chromatography, IEX is performed under aqueous conditions and requires no organic solvents. It is also easy to perform and requires a minimal amount of equipment—only a pump, column, injector, and detector (UV, fluorescent, or conductivity) are needed. However, unlike GF, IEX allows a greater degree of specificity. "There is a lot of control inherent in ion exchange chromatography that isn't necessarily there in gel filtration," explains Tosoh Biosep's Kelley. Researchers can tailor both the stationary phase (resin) and the mobile phase (buffer) to meet purification needs. Media used in IEX can be adapted to the target protein's size by controlling the resin's porosity; some of the resins used in gel filtration can be bound to an ion exchange ligand, allowing purification of high molecular weight proteins. Also, the buffer's pH and chemical composition can be controlled according to the properties of the protein of interest. One of IEX's main advantages is its scalability: IEX is directly upgradable from a small-scale to a process-scale level. Furthermore, IEX resins are relatively inexpensive and widely available in bulk quantities. Hydroxyapatite Chromatography Of the techniques discussed in this article, hydroxyapatite (HAP) chromatography is the least understood, historically. Hydroxyapatite is a crystalline form of calcium phosphate most frequently used in antibody purification. HAP may demonstrate low binding capacity due to excess phosphate,5 poor physical and chemical stability, and poor flow properties due to its crystalline form. However, manufacturers such as Hercules, Calif.-based Bio-Rad Laboratories have made improvements to the technology in recent years. Bio-Rad has developed a spherical, stable, porous, and ceramic hydroxyapatite (CHT™ ceramic hydroxyapatite) with an improved calcium:phosphate ratio, which overcomes HAP's various shortcomings. Bio-Rad offers CHT ceramic hydroxyapatite in two forms (types I and II), which are sintered at different temperatures to improve chemical and physical stability, control pore size, and increase purity. The mechanism of HAP is complicated and involves nonspecific interactions between negatively charged protein carboxyl groups and positively charged calcium ions on the resin, and positively charged protein amino groups and negatively charged phosphate ions on the resin.1,5 Basic or acidic proteins can be adsorbed selectively onto the column by adjusting the buffer's pH; elution can be achieved by varying the buffer's salt concentration. According to Randy Lowe, Bio-Rad's senior product manager for chromatography, HAP chromatography may separate proteins that are not readily separable by other techniques. Bio-Rad has developed a HAP technique that separates two proteins of similar pI, MW, and hydrophobicity, for example. HAP also does not involve the use of harsh elution buffers. The main drawback of HAP chromatography, according to Lowe, is that it is often difficult to predict the technique's effectiveness based on the target protein's chemical and physical properties. For example, knowledge of the protein's pI helps researchers determine the potential success of an IEX protocol. "With HAP, the outcome is less straightforward," Lowe says. In general, however, hydroxyapatite has a higher affinity for proteins with high pIs and a lower affinity for those with low pIs. Hydrophobic Interaction Chromatography Another technique that can be used in protein purification is hydrophobic interaction (HI) chromatography, which relies on interactions between exposed hydrophobic patches on the protein's surface and hydrophobic ligands attached to the resin. The mechanism of hydrophobic adsorption is not well understood, and several theories exist to explain the process, all of which have at their core the increased structure of water molecules surrounding the protein-ligand complex.6 The HI process involves the use of a high salt buffer, which unravels the protein to expose hydrophobic sites. The protein is retained by the hydrophobic ligands on the column, and is exposed to a gradient of buffers containing decreasing salt concentrations. As the salt concentration decreases, the protein returns to its native conformation and eventually elutes from the column. The selectivity of HI resins depends on the hydrophobic ligand's structure. Straight-chain alkyl ligands and aryl ligands are used, and in general, protein binding increases with increasing chain length.6 The ideal resin choice depends on the target protein's chemistry. Tosoh Biosep's Kelley warns against choosing a resin that binds the protein too tightly, as it "becomes a real headache to elute it." But finding the right hydrophobic ligand is an empirical process. Kelley recommends starting with an intermediate ligand such as phenyl and increasing the resin's hydrophobicity to increase its binding strength. To ease optimization, Amersham Biosciences offers a hydrophobic interaction selection kit, which contains five different resins. Scaling Up An important factor to consider when developing a protein purification procedure is the scalability of each individual step; many protocols are developed initially on a small-scale analytical level and then expanded to process-scale for bulk purification. Karsten Fjarstedt, marketing director for Amersham Biosciences' purification systems group, explains that most types of resins can be used for process-scale separations provided that they allow the needed flow rate. Fjarstedt adds that the key to scaling up is to scale outwards, not upwards; in other words, to maintain column height and increase column width while making sure that the linear flow rate (cross-sectional flow rate) and pH or salt gradient (if used) remains identical to the smaller-scale procedure. Smaller bead sizes can also be problematic when scaling up, as the smaller spacing between the beads will produce a higher backpressure, compressing the column particles. For this reason larger, and preferably more rigid beads, having larger spacing, higher flow rates, and lower backpressure should be used in process-scale procedures. Fjarstedt explains that an analytical preparation using small-sized beads (5 or 15 mm, for example) can be scaled up using a larger version of the same type of resin. Because liquid chromatography is such a well-known, widely used technique, a tremendous amount of easily accessible literature exists from many of the companies that supply resins for the above techniques. The Amersham Biosciences and Tosoh Biosep Web sites are excellent resources for novices and experts alike and offer detailed descriptions of a variety of purification methods; the Amersham Biosciences Web site, for example, provides detailed chromatography handbooks (see references below). St. Louis-based Sigma-Aldrich, which distributes a large number of resins, also provides technical information on its Web site. Aileen Constans (aconstans@the-scientist.com) is a contributing editor. References 1. C. Smith, "Liquid chromatography: Products in the protein chemist's tool chest," The Scientist, 12[16]:14, March 16, 1998. 2. Amersham Biosciences, "Gel filtration. Principles and methods," 1998. 3. J. Porath, P. Flodin, "Gel filtration: Method for desalting and group separation," Nature, 183:1657-1659, 1959. 4. Amersham Biosciences, "Ion exchange chromatography. Principles and methods." 5. P. Gagnon et al., "Ceramic hydroxyapatite: A new dimension in chromatography of biological molecules," Bio-Rad Laboratories, Hercules, Calif., Technical Bulletin #2156, 1996. 6. Amersham Biosciences, "Hydrophobic interaction chromatography. Principles and methods," 1993. Supplemental Materials Chromatography Media Suppliers Table: Ion Exchange Resins Table: Hydroxyapatite Resins Table: Hydrophobic Interaction Resins Table: Gel Filtration

Protein Purification I: Liquid Chromatography

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