Agilent Buffering Advisor Software  

General Principles: 

Ion exchange uses an insoluble matrix that carries ionic charges that retard the movement of molecules of opposite charge. There are 2 types, one where the solid phase is negatively charged (cation exchange resin) or positively charged (an anion exchange resin). 

IEX has been a platform for antibody purificaiton for many years. Because antibodies have a more basic isoelectric point that the majority of other serum or contaiminating proteins, IEX is useful in purifying anitobdies regardless of isotype. The general strategy is to keep the pH below the isoelectric point for antibodies so that they will not bind to the AEX such as DEAE modified resin or, alternaitvely to raise the pH above the pI where the antibodies will bind to the DEAE groups. The opposit strategy works for cation exchanges. The bound antibodies are commonly eluted with a salt or pH gradient. (Josic “Analytical and Preparative methods for purification of antibodies” in “methods foor purificaiton of antibodies, Food technol. biotechnol. 39 (3), 215-226 (2001). 

IEX is based on differential adsorption of charged substances at oppositely charged surfaces of porous chromatogrpahic media. The strenght of such interactions and the adsorption capacity of the IEX media are assumed to vary inversely with conductivity.  Protein dynamic binding capacities on IEX are typically expected to decrease with increasing conductivity and decreasing prtoein charge. However, several models describe the complex adsorption mechanisms in IEX and departures from expected IEX behavior have been reported Harinaryayan (Biotechnol Bioeng. 2006, 95(5) 775-87).

Binding conditions: 

a. Generally/optimization: 

In ion exchange chromatography, proteins are separated based on their charge. Proteins consist of various amino acids whose side chains may usually carry, in addition to uncharged radicals, also acidic and basic radicals and which thus contribute to the total charge of the protein. At low pH, below the isoelectric point of the protein, the total charge is positive, due to protonation of the charged side chains. At higher pH it is negative due to deprotonation. Since proteins carry a multiplicity of charged groups whose actual charge depends on the pH and also the environment of the individual amino acid, separation according to charge is a powerful method of separating proteins. In a ion exchange chromatography, the pH is chosen so as to enable the protein of choice to bind to the matirx. In the case of anion exchangers, this pH is usually at least one pH unit above the isoelectric point of the protein (pH at which the protein has a net charge of 0). In the case of cation exchangers it is below the isoelectric point. Binding to the matrix takes place via electrostatic interavctions. Proteins which do not bind to the matrix are washed out with buffer (WO2005/100394). Protein purification using ion-exchange chromatography usually employs positively charged anion exchangers because the majority of proteins are negative charged at neutral pH (i.e., have a low isoelectric point).

IEX chromatography steps are widely applied in protein purificaiton processes because of their high capacity, selectivity, robust operations, and well-understood principles. Optimziation of IEX steps typically involves resin screening and selection of the pH and coutnerion concentraitons of the load, wash, and elution (Kelley, Biotechnology and Bioengineering, 100(5), 2008). 

In summary, the key determinant for adsorption to an ion-exchange matrix is the charge of a peptide or protein. Thus, a protein has an affinity for an anion-exchange matrix if the protein has an overall negative charge, and conversely, a cation exchange matrix binds a positively charge protein. Because of the ionization state of surface amino acids, the net charge of a protein or peptide varies with the pH of the buffer. The pH is referred to as the protein’s isoelectirc point (pI) when the total number of + charges on a protein equals the number of – charges, in other words, when the protein’s net charge is zero. A protein is negatively charged at a pH above its pI and positively charged at a pH below its pI. For most separations, a pH that is 1 U from the pI of the protein is best for achieving the reversible binding required in ion exchange chromatography (see, Bollage, “Ion-Exchange Chromatography” ch. 2 in “Methods in Molecular Biology, Vol 36, Peptide Analysis Protocols”.)

However, the behavior of proteins on ionic exchanges can be unpredictable as shown by Etzel (US 5,986,063). The isoelectric point is 4.2-4.5 for alpha-lactalbumin and 5.1 for beta-lactalbumin. As pH increases, one would the alpha-lactalbumin to elute first from a cation exchange because its net charge switches from positive to hegative at a lower pH than does beta-lactoglobulin which would elute second at a higher pH. However, this does not occur. Alpha-lactalbumin does not fully elute until about pH 6.5 which is 2-2.3 pH units above its PI, whereas the majority of the beta-lactoglobulin elutes at about pH 4.6 as expected, because this pH is close to its PI.

b. pH: 

Ansaldi (WO 99/62936) discloses a method for separating a polypeptide monomer form a mixture comprising dimers and/or multimers, where the method comprises applying the mixture to either a cation exchange or an anion exchange chromatography resin in a buffer, wherein if the resin is cation-exchange, the pH of the buffer is about 4-7, and wherein if the resin is anion-exchange, the pH of the buffer is about 6-9, and eluting the mixture at a gradient of about 0-1 M of an elution salt.

Hickman (WO 2010/048192) diclsoes the purification of antibodies using anion exchange in flow through mode where suitable equilibration conditions are 25 mM Tris, 50 mM sodium chloride at pH 8.0.

c. Conductivity:

In ion exchange chromatography, adsorbability is well known to decrease by an increase in electrical condutivity of a treatment liquid. Accordingly, in a sample haivng high electrical conductivity, the electrical conductivity needs to be decreased to 5 mS/cm by dilution or deminerlaization before applying adsorption treatment. In order to compesnate for such a disadvantage, achromatography media simultaneotusly haivng hydrophobic itneraction, hydrophilic interaction, a chelate interaction and so forth in addition to the electrostatic interaction has been developed (see mixed mode chromatography). (Matsumoto, US 2015/0344520; see also Matsumoto, US Patent Applicaiton No: 16/349,6127, published as US 2019/0345194) 

d. Addition of chemical compounds:

–EDTA: Charlton (WO2011/073235) disclsoes a method for purifying polypeptides by increasing the mount of polypeptide of interest that binds to an ion exchange matrix relative to the amount of the impurity. This is achieved by adding a chemical compound in the process which by also binding to the IEX due to a charge that is opposit to the charge of the IEX, reduces binding of the impurity more than the binding of the polypeptide of interest. 

Washing:

a. salt concentration/conductivity and/or pH: 

Emery (WO/2004/024866) discloses a method for purifying a polypeptide using an ion exchange resin with an equilibrium buffer having a first salt concentration/conductivity. The exchange is washed with a wash buffer until a predetermiend protein concentration is measured in the flowthrough and during the washing the salt concentration of the wash buffer increases from an initial, second salt concentration/conductivity that is greater than the salt concentration/conductivity of the equilibration buffer to a final third salt concentration.

Basey (WO99/57134) disclsoes a method for purifying a polypeptide by IEX which involves chaing the conductivity and/or pH of buffers during intermediate washings. Thus a polypeptide of interest is bound to the IEX at an initial conductivity or pH and then washed with an intermediate buffer at a different conductivity or pH or both. Then and contrary to standard practice, the IEX is washed with a wash buffer where the change in conductivity or pH or both from the itnermediate buffer is in an opposite direction. 

Elution: 

Elution is generally acheived by increasing the ionic strenght (i.e., conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to acheive elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (ste elution) (Rossi, WO/2005/049649). In IEC, NaCl has been the choice of the salt for elecution (Arakawa “Solvent Modulation of Column Chromatography” Protein & Peptide Letters, 2008, 15, 544-555).

–Ph and salt gradients: IEX with salt gradient has been used to separate N-terminus variants of recombinant Il-1beta, C-terminus variants of monoclonal antiboides, deamidated variants of mAbs and ribonuclease A. In generaly, a disadvantage of salt gradietn elution is the need for gradient mixing, which can be cumbersome to implement on a process sale. Moreover the salt may have to be removed before the next processing or formulation step. An alternative to salt gradient elution is elution with pH gradients. In this approach, ideally the charge variants are eluted in order of their pI, with more acidic proteins eluting earlier in cation excahngers and later in anion exchangers.  (Pabst, Biotechnol. Prog 2008, 24, 1096-1106).

Types: 

There are two main types of ion exchange chromatography:  a) cation exchange: and b) anion exchange resin: See more information on left panel.A strong cation exchanger contains a strong acid (such as a sulfopropyl group) that remains charged from pH 1-14 whereas a weak cation exchanger contains a weak acid (such as a carboxymethl grou) which gradually loses its charge as the pH decreases below 4 or 5.