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Ion Exchange Chromatography for the Analysis of Biomolecules

Ion exchange chromatography (IEX) has been widely used for the analyis of biomolecule such as antibodies, peptides, glycans, proteins, polynucleotides, and cell lysates, and remains one of the gold standard strategies for charge variant analysis. Some common causes of charge variants are deamidation, isomerization, succinimide formation, oxidation, silylation, N-terminal pyroglutamic acid, or C-terminal lysine clipping. 

Biomolecule IEX separates compounds based on electrostatic interactions between the ionic groups on the protein or monoclonal antibody (mAb) surface, and the oppositely charged ionic group on the surface of the stationary phase.  Cation exchange columns have a negatively charged surface - these columns are used at pH values below the isoelectric point (pI, the pH at which the protein charges are balanced and the net surface charge is 0) of the peptide or protein. 

Conversely, anion exchange columns have a positively charged stationary phase and are operated above the peptide or protein pI.  Weak (WCX) or strong cation exchange (SCX) materials, containing carboxylic or sulfonic acid groups respectively, are generally employed for the analysis of biomolecules.

Cation exchange chromatography is most commonly applied to the analysis of therapeutic proteins due to their basic nature.  Parameters such as column chemistry, mobile phase pH, and salt concentration gradient need to be optimized for each individual protein.  The number of charged sites on a protein increases with molecular weight and can be affected by post translational modifications - these modifications can cause addition or loss of charge, or changes in confirmation which can expose other charge residues.  Therefore, as molecular weight increases IEX chromatograms become more complex and resolution of charged variants can be lost.  Hence, intact but reduced, or digested proteins are commonly analyzed.

In cation exchange the most acidic compounds are eluted first (Figure 1), due to the electrostatic repulsion between the compound and the cation exchange surface.  The most basic compounds are retained and elute later due to attractive interactions with the cation exchange material.  The chromatogram in Figure 1 exhibits numerous peaks, whereas, reversed phase HPLC analysis of the same mAb reveals only four peaks; demonstrating the superiority of IEX over reversed phase HPLC for charge variance characterization.

Charge variant analysis of mAb by SCX

Figure 1: Charge variant analysis of mAb by SCX (representative chromatogram).

IEX is of interest for the analysis of biomolecules as the separation is performed under non-denaturing conditions, unlike reversed phase HPLC.  IEX is used for both characterization and release testing, and can be applied to preparative applications.

Some modifications have a direct effect on the charge of the protein such as C-terminal lysine truncation (loss of positive charge), deamidation (gain of acidic function), succinimide (loss of acidic function) which can help to explain elution behavior. 

Other modifications result in conformational changes which will expose different charged residues on the proteins surface.  For example, aspartate isomerization does not result in a change in net charge but causes structural alterations by introducing additional carbon into the peptide backbone.  Elution order of post translation modifications under cation exchange chromatography have been well characterized (Table 1).   Acidic and basic variants are eluted before and after the main peak respectively.

Modification Cation Exchange Chromatographya Effect on Protein Charge
Aspartate isomerization Variable Conformational changes
Asparagine deamidationb Post-peak Direct
Oxidation Variable Conformational changes
PyroGlu from Glu (–H2O) Post-peak Direct
PyroGlu from Gln (–NH3) Pre-peak Direct
Succinimide Post-peak Direct
Sugar Variable Direct in case of sialylation
C-terminal lysine Post-peak Direct
Aggregation - -
Fragmentation Variable Direct
Truncation and N-terminal glutamine cyclization Pre-peak Direct
Sialylation Pre-peak Direct
Glycation Pre-peak Direct
C-terminal amidation Post-peak Direct

Table 1: Elution order of common post-translational modifications relative to the main peak.1
a Situation is typically reversed when using anion exchange chromatography.  
Under certain conditions, asparagine deamidation gives rise to aspartate and isoaspartate which appear as a pre- and post-peak respectively.

Cation exchange chromatography is predominantly applied to the analysis of therapeutic proteins due to their basic nature.  However, anion exchange chromatography has been applied to the separation and characterization of more acidic proteins or the more basic oxidized variants.

Pros and cons of IEX

Pros Cons
Permits high flow rates Sample must be loaded at low ionic strength
(salt concentration)
Concentrates samples Clusters of positively charged residues can cause a
net-negatively charged protein to bind to a cation exchanger,
and vice versa
High yield Small changes in pH can greatly affect the binding profile
of an IEX phase
Non-denaturing conditions Particle size greatly influences resolution
  Not easily hyphenated with mass spectrometry
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