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Chromatography is the science of separating molecules of interest to identify, quantify or even purify them. Properties of the target molecules such as their solubility, size or hydrophobicity give rise to different modes of interaction with the stationary phase, or different types of chromatographic separations, such as reverse phase (RP), size exclusion chromatography (SEC) or hydrophobic interaction chromatography (HIC).
What is ion exchange chromatography (IEX chromatography)?
Ion exchange chromatography, or IEX, is a class of liquid chromatography (LC) used to separate organic and inorganic molecules. One area in which it is particularly useful, and the focus of this article, is the separation of charged biomolecules including amino acids, proteins, carbohydrates and nucleic acids. Molecules such as proteins have a unique 3D structure and hence specific ionizable groups on their surfaces. As a result, the net surface charge at any given pH is unique to a molecule. This characteristic is exploited to separate molecules based on their interaction with the oppositely charged particles of the stationary phase and subsequent release from the column by modifying the pH or the ionic strength of the mobile phase.
What's the difference between ion exchange chromatography vs ion chromatography?
In literature, the terms &#;ion exchange chromatography&#; and &#;ion chromatography&#; (IC) are often used interchangeably, thanks to the wide use of IEX, and refer to the separation of polar or ionic species. However, IC is really an umbrella term that includes IEX along with ion exclusion chromatography (IEC) and ion pair (IP) chromatography. IEX, which involves preferential binding of charged analytes to the oppositely charged functional groups of the stationary phase, is the predominant mechanism utilized in the separation of biomolecules. 1 IEX uses buffers containing salt for its mobile phase; separation is brought about by changing the ionic strength or pH of the eluent and detection is performed using ultraviolet (UV) or fluorescence detectors. On the other hand, IC more broadly is also used for the separation of inorganic ions, small organic acids, amines, alcohols, aldehydes and carbohydrates by any of the three modes, IEX, IEC or IP. For these types of analytes, electrolytic solutions are used for elution and suppressed conductivity measurement is the most commonly used detection technique for IC.
Ion pair chromatography can also be considered as a type of partition chromatography where analytes of interest are eluted from a neutral stationary phase with the help of oppositely charged reagent ions, or an ion pairing reagent. By changing the concentration of the ion pairing reagent in the mobile phase, the retention times of the analytes are varied, leading to their separation.
How does ion exchange chromatography work?
Biomolecules have functional groups that ionize in solution and impart a specific net charge to the molecule. For instance, proteins are made up of amino acids that have both amino (-NH2) and carboxylic acid (-COOH) functional groups. The 3D structure of a protein determines which of the amino acid residues are exposed on its surface. Depending on the pH of the medium, these residues ionize, giving the molecule positive and negative surface charges. At low pH values, more amino groups are protonated and protein molecules carry a net positive charge. On the other hand, at higher pH values, more carboxylic acid groups are deprotonated and the resulting anions make the surface of the protein molecules negatively charged. The total number of ionized functional groups present on the surface determines the net surface charge and each molecule has a unique net surface charge at any given pH value. A protein is electrically neutral at its isoelectric point (pI), which is a specific pH value at which it has no net charge on the molecule.
Figure 1 shows a mechanism of separation by IEX. When the polar or charged analytes are loaded into an ion-exchange column, they are electrostatically bound to the oppositely charged ions present on the surface of the stationary phase particles. The greater the positive or negative charges on the surface of the analyte molecules, the stronger the electrostatic attraction to the oppositely charged particles of the stationary phase will be. Retention time of the target analyte also depends on the number of interactions with the stationary phase. Aqueous mobile phases containing buffers and salts are used to elute the bonded analytes by varying the pH or ionic strength.
Figure 1: Schematic diagram showing analyte separation in IEX by increasing the ionic strength of the elution buffer. Credit: Technology Networks.
There are five main steps to the process of IEX:2
1. Equilibration &#; As a first step, the stationary phase is washed with the start buffer (initial buffer composition) until the baseline is stabilized and eluent pH remains constant. This step ensures that the ionizable groups on the column are available to interact with the charged analyte molecules.
2. Sample loading &#; Samples dissolved in start buffer or buffers of the same pH as the start buffers are injected into the column. The pH and ionic strength of the buffer are adjusted such that the analytes bind to the column while impurities do not.
3. Wash &#; The column is washed once again with the starting buffer to remove uncharged molecules as well as molecules with the same charge as the stationary phase. The baseline stabilizes once the impurities are washed away.
4. Elution &#;A salt gradient is used to elute the bound analytes as the ions in the elution buffer compete for and replace the analytes on the charged sites on the column surface. At low ionic strength, weakly bound analytes (molecules having lower surface charge densities), start eluting from the column. As the salt concentration is increased, strongly bound molecules with increasingly higher surface charge densities elute sequentially from the column. Alternatively, a pH gradient can be used to elute the bound analytes which are released from the column at their respective pI value. To elute cations, the pH of the eluting buffer is increased, whereas, anions are eluted from the anion exchange column by decreasing the pH of the eluting buffer. A pH gradient cannot be used if a molecule precipitates at its pI value.
5. Column regeneration &#; Finally, the column capacity is restored for the next run by washing out any molecules bound on the column. To achieve this, a high ionic strength buffer is allowed to flow through the column until the baseline and pH of the eluent stabilize. The column is then conditioned with the starting buffer prior to the next run.
Although, UV or fluorescence detectors are most frequently used in IEX, other detectors such as mass spectrometers, refractive index (RI) or multi-angle light scattering (MALS) detectors have also been used.
What's the difference between anion exchange chromatography vs cation exchange chromatography?
Based on the charge on the ions to be separated, two types of IEX chromatography, namely anion exchange or cation exchange, are used. The anion exchange columns have positively charged functional groups covalently bonded to the stationary phase particles and cation exchangers have negatively charged functional groups bonded to stationary phase particles. As their names suggest, anion exchange chromatography is used for the separation of anions such as deprotonated or &#;acidic&#; protein molecules that have more carboxylic acid groups on their surface, while cation exchange columns are used for the separation of cations such as protonated &#;basic&#; proteins that have more amino groups on their surface.
The ion exchange columns are further classified as strong and weak anion and cation exchangers depending on their ion exchange capacity. Strong exchangers retain their ion exchange capacity over a wide pH range as they do not take up or lose protons with changes in the mobile phase pH. Whereas, weak exchangers are effective over a narrow pH range where they are ionized. Table 1 shows some examples of different kinds of IEX columns and their effective pH ranges.
Table 1: Examples of ion-exchange columns.
Column type
Column
Functional group
pH range
Strong anion exchanger (SAX)
Quaternary ammonium salts (Q)
(CH3)3 N+CH2O-
0-14
Weak anion exchanger (WAX)
Diethylamino ethyl (DEAE)
(C2H5)2N CH2CH2O-
2-9
Strong cation exchanger (SCX)
Sulfopropyl (SP)
-OCH2CH2CH2SO3-
0-14
Weak cation exchanger (WCX)
Carboxymethyl (CM)
&#;OCH2COO-
2-9
Mixed-mode chromatography, which involves two separation mechanisms such as RP and IEX or IEX and HIC, using a single column is implemented to increase the resolution of the analytes.Mixed-mode chromatography, which involves two separation mechanisms such as RP and IEX or IEX and HIC, using a single column is implemented to increase the resolution of the analytes.
Ion exchange resin selection
The IEX columns are packed with porous or non-porous, inert, polymeric resin or gel beads that have functional groups covalently bonded to them. These groups impart charge to the surface in their ionized state depending on the pH of the buffer flowing through the column. The beads are made of materials such as dextran, agarose or cellulose. They have high physical stability and uniform size which supports high flow rate and ensures that the column volume does not change at high pH or salt concentrations. The size and porosity of the support beads plays an important role in the resolution of the charged species.
In IEX, choosing the right stationary phase is crucial to achieving effective separations. The choice of the stationary phase is dependent on the pI and stability of the analytes of interest. If the target molecules are stable at pH values lower than their pI values, then a cation exchanger is preferred, but if the target molecules are stable above their pI values, then an anion exchanger is preferred. Either type can be used if the target molecules are stable over a wide pH range.
During method development, strong exchangers are used to enable the use of a broad pH range. This helps speed up the process. Although the use of weak exchangers helps to improve resolution when strong exchangers are not effective, these have limitations such as variations in sample load capacity with pH and longer equilibration times. When using IEX for purification, the chosen column should retain either the target analyte or the impurities due to the different net surface charges on the target molecules and impurities. The choice of media will depend on the extent of the purification desired, ranging from sample capture to final polishing.
Ion exchange chromatography considerations, strengths and limitations
Parameters affecting the separation include:
pka
While there are several advantages of IEX, the technique has some limitations as well (Table 2) that must be factored in when selecting this technique for separation, purification or quantification of biomolecules.
Table 2: Strengths and limitations of IEX chromatography.3
Strengths
Limitations
High loading capacity
Expensive buffers and instrumentation
High resolution group separation of molecules of interest from other molecules and impurities
Sample injection solvent must have low ionic strength and controlled pH which may require an additional buffer exchange step
Can separate proteins differing by only one charged amino acid or molecules with only small differences
Weak ion exchange columns are sensitive to pH changes and must be used within the prescribed range to maintain capacity and resolution
Fast separations as IEX can be carried out at high flow rates
Bio-inert LC systems may have to be used to prevent corrosion at high salt concentrations
High recoveries of target molecules are obtained during purification
Concentrating the solution post-separation to enhance protein recovery increases the salt concentration in the solution which may require buffer exchange prior to further analysis
Can be used as a pre-concentration or purification step ahead of second dimensional separation. The added advantage is that the buffers used do not denature the proteins
Coupling with mass spectrometry (MS) is limited due to high salt concentration in the eluent
Applications of ion exchange chromatography
Among the various LC techniques used for the analysis of biopharmaceuticals, IEX has been extensively applied for the separation and purification of monoclonal antibodies (mAbs) and proteins. Cation exchange chromatography with a salt gradient has been shown to be suitable for the separation of charge variants of the mAb cetuximab.4 A cation exchanger with a linear pH gradient has been used for the purification of a common light chain IgG-like bispecific antibody. This method has been shown to be successful in separating molecules differing in their sequence by a single charged amino acid or a pI difference of 0.1.5 Instead of conventional UV or fluorescence detectors, a MALS detector can be coupled with IEX to characterize protein populations such as short peptides, proteins with similar masses or oligomers that were not well resolved by SEC.6 Fractionation of crude protein mixtures and separation of seventeen proteins have been carried out using anion exchange chromatography with a linear pH gradient.7
To enhance the safety of therapeutic proteins and antibodies, IEX is used to remove contaminants such as viruses during downstream purification process. Cation exchange chromatography has been shown to be effective in the removal of xMulV, a model retrovirus, during the polishing step of mAb production.8 Not just the cells, but also cellular components have been separated for quantification or purification using IEX. Baseline separation and quantification of empty and full capsids in recombinant adeno-associated virus (rAAV) samples from multiple serotypes was achieved using QC-compatible anion exchange chromatography.9 Negatively charged extracellular vesicles (EV) in amniotic fluid were purified using weak cation exchange chromatography.10
In addition to proteins, sugars and nucleic acids have also been separated using IEX. Glycans have been fractionated according to their sialic acid content using a weak anion exchanger, prior to their desialylation, nano-LC separation and detection by both MS and nanofluorescence detectors.11 Short RNA oligomers (~20 mers) have been analyzed using mixed-mode chromatography involving two separation mechanisms, ion pair RP and anion exchange in a single column.12
Conclusion
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IEX, a type of ion chromatography, is a versatile technique for the purification, characterization and analysis of large biomolecules such as proteins, antibodies or nucleic acids. As the separation is based on net surface charge on these complex molecules, IEX is an important alternative to other techniques such as reverse phase chromatography, hydrophobic interaction chromatography or size exclusion chromatography.
References
1. Acikara ÖB. Ion-Exchange Chromatography and Its Applications. Chapter 2. Column Chromatography, Dean F. Martin and Barbara B. Martin. IntechOpen. . doi: 10./
2. Khan HU. The Role of Ion Exchange Chromatography in Purification and Characterization of Molecules, Chapter 14. Ion Exchange Technologies, Ayben Kilislioğlu. IntechOpen. . doi: 10./
3. Stanton P. Ion-Exchange Chromatography. In: Aguilar MI. (eds) HPLC of Peptides and Proteins. Methods in Molecular Biology&#;. Totowa, NJ:Springer;;251. doi: 10./1--742-4:23
4. Fekete S, Beck A, Fekete J, Guillarme D. Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: salt gradient approach. J Pharm Biomed Anal. ;102:33-44. doi: 10./j.jpba..08.035
5. Sharkey B, Pudi S, Wallace Moyer I, Zhong L, Prinz B, Baruah H, et al. Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients. MAbs. ;9(2):257-268. doi: 10./..
6. Amartely H, Avraham O, Friedler A, Livnah O, Lebendiker M. Coupling Multi Angle Light Scattering to Ion Exchange chromatography (IEX-MALS) for protein characterization. Sci Rep. May 2;8(1):. doi: 10./s-018--6. Erratum in: Sci Rep. Apr 11;9(1):.
7. Ahamed T, Nfor BK, Verhaert PDEM, van Dedem GWK, van der Wielen LAM, Eppink MHM, et al. pH-gradient ion-exchange chromatography: An analytical tool for design and optimization of protein separations. J of Chromat A. ;(1&#;2):181-188. doi: 10./j.chroma..07.010
8. Connell-Crowley L, Nguyen T, Bach J, Chinniah S, Bashiri H, Gillespie R, et al. Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes. Biotechnol Bioengg. ;109:157-165. doi: 10./bit.
9. Khatwani SL, Pavlova A, Pirot Z. Anion-exchange HPLC assay for separation and quantification of empty and full capsids in multiple adeno-associated virus serotypes. Mol. Therapy - Methods & Clin. Dev.;21:548-558. doi: 10./j.omtm..04.003
10. Kosanović M, Milutinović B, Goč S, Mitić, N, Janković M. Ion-exchange chromatography purification of extracellular vesicles. BioTechniques.;63(2):65-71. doi: 10./
11. Kalay H, Ambrosini M, Chiodo F, van Kooyk Y, García-Vallejo JJ. Enhanced glycan nanoprofiling by weak anion exchange preparative chromatography, mild acid desialylation, and nanoliquid chromatography-mass spectrometry with nanofluorescence detection. Electrophoresis. ;34(16):-6. doi: 10./elps.
12. Biba M, Jiang E, Mao B, Zewge D, Foley JP, Welch CJ. Factors influencing the separation of oligonucleotides using reversed-phase/ion-exchange mixed-mode high performance liquid chromatography columns, J. of Chromat. A. ;: 69-77. doi: 10./j.chroma..06.050
In ion exchange chromatography, the support consists of tiny beads to which are attached chemicals possessing a charge. Each charged molecule has a counter-ion. The figure shows the beads (blue) with negatively charged groups (red) attached. In this example, the counter-ion is sodium, which is positively charged. The negatively charged groups are unable to leave the beads, due to their covalent attachment, but the counter- ions can be &#;exchanged" for molecules of the same charge. Thus, in cation exchange column, the chemical groups attached to the beads are negatively charged groups and will have positively charged counter-ions and positively charged compounds present in a mixture passed through the column will exchange with the counter-ions and &#;stick" to the negatively charged groups on the beads. Molecules in the sample that are neutral or negatively charged will pass quickly through the column. On the other hand, in anion exchange chromatography, the chemical groups attached to the beads are positively charged and the counter-ions are negatively charged. Molecules in the sample that are negatively charged will &#;stick" and other molecules will pass through quickly. To remove the molecules &#;stuck" to a column, one simply needs to add a high concentration of the appropriate counter-ions to displace and release them. This method allows the recovery of all components of the mixture that share the same charge.
Figure 3.4.3.1: Cation exchange chromatography
Usually, samples are loaded under low ionic strength conditions and bound material is eluted using either a step or gradient elution of buffer with higher ionic strength.
Generally speaking, a protein will bind to a cation exchange resin if the buffer pH is lower than the isoelectric point (pI) of the protein, and will bind to an anion exchange resin if the pH is higher than the pI.
Knowledge of the pI of the protein is therefore helpful in designing a purification protocol using ion exchange resins (however, you can always simply try different resins to see which works best).
Figure 3.4.3.2: Gradient maker
This will produce a linear gradient from low to high salt concentrations over the total volume of the gradient
Figure 3.4.3.3: Salt concentration and volume
If we know the concentration range of salt over which a protein of interest will elute we can simply elute with a buffer containing that concentration of salt. This is known as a step elution.
Step elutions are generally faster to run, and elute the protein in a smaller overall volume than with gradient elutions. They generally work best when contaminants elute at a significantly different salt concentration than the protein of interest.
Figure 3.4.3.4: Step elution
Note that after ion exchange chromatography the protein of interest will be in a buffer with a potentially high salt concentration. This must be taken into account before proceeding with the next step in the purification scheme.
Figure 3.4.3.5: Dialysis (https://en.wikipedia.org/wiki/Dialysis_(biochemistry)
Over time the concentration of low molecular mass solutes within the bag, and in the low salt buffer, will come achieve equilibrium. In practical terms (for the above case) salt molecules will diffuse out of the bag into the low salt buffer.
Figure 3.4.3.6: Salt diffusion
For both dialysis and concentration, it is essential that the membrane does not interact with the protein (i.e. has no affinity for, and will not bind, the protein)
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