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Complex Separations Made Simple

Principles of Ion-Exchange Chromatography for Buffer Optimization

Elena Komkova, Ph.D. Senior Scientist Natrix Separations Inc.
Technical Note TN1001 rev.0610

Introduction

Ion-exchange chromatography (IEX) is currently the most popular method for protein purification. This method of separation relies on the attraction of the charged sites of the target proteins with the surface charge of an ion-exchange matrix. In general, polymeric based matrices for use in ion-exchange applications are very robust. They are stable in strong acidic or basic solutions and are resistant to chemical agents such as urea and guanidineHCl. However, as IEX methods rely on the electrostatic attraction of oppositely charged ionic sites, the method is susceptible to changes in the surrounding environment namely changes in the nature of the mobile phase. Alteration of the ionic strength of the mobile phase by the addition of electrolytes or salts can impede any mutual attraction of the stationary phase with the molecule of interest. In addition, as the molecules of interest are proteins which are composed of a variety of differing chemical groups, changes to the pH can result in the protein being of one net charge or another. The following background information is provided to enable the user to select the correct type of salt or buffer, in the correct concentrations, to control these interactions for the Natrix Separations adseptTM membranes.1

Net surface charge and pH

Most biological compounds are either net positively or negatively charged when dissolved on a solution at a specific pH. In many instances, proteins can simultaneously contain both individually positive and negative charged sites depending on the chemical nature of each individual group that the protein is composed of. Thus, proteins and other large bio-molecules are known as amphoteric materials. Specific sites of the proteins can be composed of hydrophilic amino acid residues including for example, arginine, lysine, aspartic acid, and glutamic acid. At pH 7, the side chains of these amino acids carry charges positive for arginine and lysine, negative for aspartic acid and glutamic acid. As pH becomes more basic (pH > 7), lysine and arginine begin to lose their positive charge and at pH values > 12 they are mainly neutral. In contrast, as pH decreases, aspartic acid and glutamic acid begin to lose their negative charges and at a pH values of < 4 they are mainly neutral. Overall at a specific pH value, the surface of a protein has a net charge that depends on the number and identities of these amino acids groups. When the positive and negative charges are equal in number, and the net charge on the protein is zero and the protein is considered to be at its isoelectric point (pI). For most proteins this point occurs in the pH range of 5.5 to 8.0. At a pH values higher than the pI, the protein of interest is negatively charged and will bind (or be attracted to) to a positively charged stationary phase or anion exchange solid phase. At a pH value lower than pI, the protein is positively charged and will bind to a negatively charged stationary phase, referred to as a cation exchange solid phase. The correct balancing of this amphoteric phenomenon is essential to successful IEX chromatographic methods being developed.

1. The term buffer is often used to describe any solution that contains dissolved ionic material. For the purposes of this document the term refers only to solutions in which the dissolved salts directly affect or control the pH of the solution, whereas salts only affect the ionic strength of the solution and do not alter or interfere with the pH.
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Complex Separations Made Simple

Natrix Separations offers a unique ion-exchange media a new type of membrane produced by anchoring a functional porous structured hydrogel to a support material. The desired functional groups are covalently incorporated into the porous hydrogel matrix to create an ion exchanger. This unique approach enables the adseptTM membrane IEX materials to have superior binding capabilities, high process flow rates and linear scalability. Additionally, the hydrophilic nature of the hydrogel dramatically reduces fouling of the membranes by avoiding non-specific adsorption of aliphatic materials. Two broad categories of IEX materials have been produced by Natrix Separations; adseptTM cation and anion exchange membrane materials. For either class, both strong and weak sub-class materials are also available. Strong IEX materials typically work over very broad pH ranges and weak materials which are relatively easily converted to their chemically neutral forms. For example, a strong cation exchanger (S-type) has a sulphonate functional group whereas a weak cation exchangers functional group is a carboxylic acid (C-type). Likewise, the anion exchangers are derivatives of either secondary or tertiary amines (weak) or quaternary amines (strong or Q-type). During chromatographic separations, proteins of similar net charge attractively interact with the opposite charges of the stationary phase, leaving other proteins to be washed downstream with the flow of the mobile phase. The bound proteins can then be eluted from the stationary phase by changing the pH of the eluted buffer, thereby weakening the electrostatic forces. More specifically, in the case of cation exchange chromatography, raising the pH of the mobile phase will cause the protein molecule to become less protonated and hence less positively charged. This change results in the protein being less capable of forming a strong ionic interaction with negatively charged cation exchanger see Figure 1 below. A similar chain of events occurs with anion exchange media. At a lower pH of the mobile phase the target molecule becomes more protonated and hence positively charged. The result is that the target molecule no longer has the capability to form a strong ionic interaction with the positively charged anion exchanger which causes the molecule to elute from stationary phase. In some instances where multiple materials are bound under a specific pH condition, a pH gradient can be used to chromatographically separate the bound materials but while this approach is less often used, it is not necessarily inferior to salt gradients which are discussed below.

Chromatographic use of Natrix AdseptTM IEX Membranes

Figure 1 Principles of an anion exchange chromatography: effect of pH. Lowering pH on the protein elution from positively charged anion-exchange membrane. Molecules with higher charge density bind much stronger to the membrane and consequently require greater change in pH to be released from the membrane surface.

TN1001 rev.0610

Complex Separations Made Simple

Alternatively, and more commonly, the materials can also be removed by increasing the ionic strength of the mobile phase. In many cases, the addition of a salt such as NaCl in concentrations of 500 1000 mM will enable an effective elution step without changing the pH of the mobile phase. This also allows for the selective separation of bound molecules with small differences in net charges. By a continuous or stepwise increase the ionic strength of the salt concentration, the bound proteins are displaced from the ion exchange membrane surface by a new counter ion. When initially loading a capture media, in most instances an initial binding or background salt concentration of 20 50 mM is suitable. As many proteins tend to aggregate in solutions close to the proteins pI these small amounts of salts impede this aggregation by screening the very weak ionic attraction between proteins. In addition, hydrophobic interactions between proteins and the stationary phase will also be avoided. Choice of background salt is often dictated by the requirements of the purification process. A second solution containing only the background salt and at the identical pH of the binding solution can be used to wash the loaded IEX materials and remove all unbound and unwanted materials. Once the capture media has been loaded and washed, an elution salt concentration must be chosen so that the target molecules are not co-eluted with unwanted contaminants that have also bound to the ion exchange stationary phase. While the most typical elution salt is often NaCl a number of other suitable salts can be used. The effectiveness of displacement for commonly used cations and anions can be presented in following order (which correlates with the Hofmeister series).2, 3 Cations: NH4+ > K+ > Na+ > Mg 2+ > Ca2+ Anions: SO42- > HPO42- > acetate > Cl Table 1 lists a series of common salts that can be readily used with Natrix Separations adseptTM membranes and devices thereof for binding and elution steps. Some of these materials also act as chemical buffers which can affect the pH of the solution. For most users, Natrix Separations recommends the use sodium chloride for elution. However, these other salts can be successfully used for many applications but the effectiveness must be assessed for each individual case.

Table 1 Salt types and concentrations commonly used for protein elution

Salt
Sodium chloride Potassium chloride Sodium sulphate Potassium sulphate Ammonium sulphate Sodium acetate Potassium phosphate Calcium chloride Magnesium chloride

Concentration (M)
0.5 - 1.0 0.5 - 1.0 0.2 - 0.3 0.25 0.5 0.5 1.0 0.5 1.0 0.5 1.0 0.5 -1.0 0.5 1.0

2. Protein purification: principles, high resolution, methods and applications. Edited by Jan-Christer Janson and Lars Ryden, 1998, John Wiley & Sons, New York, 695p. 3. Purifying proteins for proteomics: a laboratory manual. Edited by Richard J. Simpson, 2004, Cold Spring Harbor laboratory Press, New York, 801p.
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Complex Separations Made Simple

Figure 2 Principles of an anion-exchange separation: effect of ionic strength. High salt concentrations cause shielding of the charged groups on the protein surface and effective binding to ion exchanger can no longer take place. Molecules with higher charged density bind more strongly to the membrane. Sample molecules are released in the order of their strengths of binding, with the most weakly bound substances eluting first.

By considering the principles of ion exchange binding and elution, carefully selected conditions allows for the quick and simple development of separation tasks with Natrix Separations adseptTM membrane devices, whether at lab scale or in a process setting. The following statements should provide initial guidance:

Example 1: Binding and elution of all sample components:

Binding is achieved by choosing a low ionic strength salt solution (< 5 mS/cm) to allow all components to bind to ion-exchange stationary phase. Once bound the materials can be washed using continued flow. The increasing the ionic strength of the mobile phase through the addition of more salt will lead to the release of the bound material. The drawback of this approach is that the binding capacity of the ion exchanger for the target substance depends on the amount of contaminants in the sample. Strongly binding contaminants can also displace bound target protein if a large volume of sample is loaded.

Example 2: Enrichment of target protein

Binding is achieved by choosing chromatography conditions such as pH and ionic strength that promotes maximal binding of a target protein and suppresses binding of sample contaminants. Experiments are required to determine the most appropriate conditions that enable this selective binding. Collecting the target can be achieved in the same manner as indicated in example 1.

Example 3: Binding of sample contaminants/impurities

Binding is achieved by choosing chromatography conditions such as pH and ionic strength that promotes binding of some or all contaminants, but allows the target substance to pass through the ion exchange stationary phase. The drawback of this approach is that the target substance is not concentrated and the amount of sample that can be applied to the ion exchanger depends on the amount of contaminants in the sample.
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Complex Separations Made Simple

Selection of buffer pH and ionic strength

Optimized binding solutions (typically < 5mS/cm) are used in ion-exchange chromatography in order to facilitate the ionic interaction between the charged moieties on the molecule and stationary phase. Often it is necessary to maximize the pH buffering power at the lowest possible ionic strength to ensure adsorption of a weakly binding protein. Selection of the buffer pH is based on the following rules: The pKa of the buffer should be not more than 0.7 units from the pH being used, and preferably not more than 0.3 unit away. One of the buffering species should be uncharged so as not to contribute to the ionic strength. The pH of the buffer must be between the pI of the charged molecule and the pKa of the charged groups on the stationary phase. In general, the buffering ion should carry the same charge as the ion exchange group. Buffering ions of opposite charge may take part in the ion exchange process and cause local disturbances in pH. Typical buffers and associated pKas are listed in Table 2 and 3 for use with cation and anion exchange materials respectively.

Table 2 Buffers for cation exchange chromatography

pH interval
1.4-2.4 2.6-3.6 2.6-3.6 3.3-4.3 3.3-4.3 3.7-4.7 5.1-6.1 4.3-5.3 5.2-6.2 5.6-6.6 6.7-7.7 7.0-8.0 7.8-8.8

Substance
Maleic acid Methyl malonic acid Citric acid Lactic acid Formic acid Succinic acid Succinic acid Acetic acid Methyl malonic acid MES Phosphate HEPES BICINE

Concentration
(mM) 20 20 20 50 50 50 50 50 50 50 50 50 50

Counter-ion
Na+ Na or Li+ Na+ Na+ Na+ or Li+ Na+ Na+ Na+ or Li+ Na+ or Li+ Na+ or Li+ Na+ Na+ or Li+ Na+
+

pKa (25C)*
1.92 3.07 3.13 3.86 3.75 4.21 5.64 4.75 5.76 6.27 7.20 7.56 8.33

* Ref: Handbook of chemistry and physics, 83rd edition, CRC, 2002-2003

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Complex Separations Made Simple

Table 3 Buffers for anion exchange chromatography

pH interval
4.3-5.3 4.8-5.8 5.5-6.5 6.0-7.0 6.2-7.2 8.6-9.6 7.3-8.3 7.6-8.6 8.0-9.0 8.0-9.0 8.4-9.4 8.4-9.4 9.0-10.0 9.2-10.2 10.0-11.0 10.6-11.6

Substance
N-Methylpiperazine Piperazine L-Histidine bis-Tris Bis-Tris propane

Concentration
(mM) 20 20 20 20 20 20 20 50 20 at pH 8.4 50 at pH 8.8 20 20 20 20 20

Counter-ion
ClCl- or HCOO ClClClCl- or CH3COO ClSO42Cl- or CH3COO ClClClClClCl-

pKa (25C)*
4.75 5.33 6.04 6.48 6.65 9.10 7.76 8.07 8.52 8.52 8.88 8.88 9.50 9.73 10.55 11.12

Triethanolamine Tris N-methyl-diethanolamine N-methyl-diethanolamine Diethanolamine Propane 1,3-Diamino Ethanolamine Piperazine Propane 1,3-Diamino Piperidine

All of the above buffers are compatible with Natrix Separations adseptTM membranes. The best choice of buffer for any application will be dictated by the needs of the user and of the process that is being implemented or developed. Natrix Separations can be contacted for specific recommendations if needed. As examples, the capture of lysozyme (pI of 9.2) with the strong S-type adseptTM membrane functional groups having a pKa of 1.2 is typically used with a 10mM Na-MES buffer at pH 5.5. The Na-MES buffer has a pKa of 6.2 falling in an acceptable range for the pH used, the counter-ion is Na+, and the buffering species are HMES (neutral) and MES- (non-interactive). In anion exchange chromatography a target molecule such as bovine serum albumin (BSA) with a pI of 4.7 would be used with a 25 mM TRIS/HCl mobile phase buffer at pH 8.3. When used with S-type adseptTM membranes bearing functional groups with a pKa of 10.3 these conditions will provide optimal binding of the target BSA. The TRIS-chloride buffer obeys all the rules: the counter-ion is Cl- , the pKa of TRIS is 8.1 and the buffering species are HTRIS+ (non-interactive) and TRIS, the latter being the neutral form.

Summary

Selection of salts for altering ionic strength and controlling the pH of the mobile phase through buffer selection provide excellent control of binding, washing and elution steps. Understanding these basic principles will enable very fast development of successful IEX process. In addition the principles developed at lab scale will apply directly to larger process scale with little adaptation for the increase in scale and aiding in the rapid development of a separations process. For further information please contact Natrix Separations or visit our website: www.natrixseparations.com
* Ref: Handbook of chemistry and physics, 83rd edition, CRC, 2002-2003
Natrix Separations Inc. 2010 Natrix Separations Inc. 5295 John Lucas Drive, Burlington, Ontario CANADA, L9P1K3 tel. +1(905)319-268 6 TN1001 rev.0610