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Selecting Columns and Initial Conditions for IEX and SEC Biomolecule Analysis

Dr. Dawn Watson, CHROMacademy Technical Expert

Biopharmaceutical compounds are genetically engineered from living cells and have molecular weights in the range 2,000-2,000,000 Da with 10-2,000 reactive groups and are often comprised of a mixture of closely related variants; therefore, there is a wider degree of variability (a degree of heterogeneity) in comparison with small molecule pharmaceuticals. 

The combination of all of these factors make biopharmaceutical compounds incredibly complex and present unique analytical challenges in their separation and characterization.  Although the wider degree of variability and heterogeneity exhibited by biopharmaceuticals renders them complex in terms of synthesis, purification, and characterization, this trait also leads to the ability to produce compounds which mimic (biosimilars) or improve on (biobetters) the properties of the parent biopharmaceutical (i.e. improvements in efficacy, safety, drug disposition etc.).

In order to fully characterize biopharmaceutical products an array of analytical techniques will be employed, commonly reversed phase (RP), hydrophilic interaction (HILIC), ion exchange (IEX), and size exclusion (SEC) chromatography.

The previous ‘Essentials’ has already focused on the use of RP and HILIC for biomolecule analysis.   

Ion Exchange Chromatography (IEX)

Ion exchange chromatography (IEX) has been widely used in the analysis of biomolecules and remains one of the gold standard strategies for charge variant analysis. The most common cause of charge variants is deamidation, isomerization, succinimide formation, oxidation, silylation, N-terminal pyroglutamic acid, or C-terminal lysine clipping. This technique separates compounds based on the electrostatic interaction of the ionic groups on the protein surface or the monoclonal antibody (mAb) 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.

IEX is of interest for the analysis of biomolecules as the separation is performed under non-denaturing conditions, unlike RP HPLC. Elution of compounds in IEX is achieved by running a salt or pH gradient to interrupt the compound/stationary phase interactions.

The selection of stationary phase and mobile phase pH strongly depends on the species being analyzed. The conditions have to be selected such that the protein and the stationary phase are in their charged forms (Figure 1).

IEX stationary phase

Figure 1: IEX stationary phase selection.

A generic salt gradient for intact mAb analysis is detailed in Table 1.1  There is an increasing interest in pH gradient cation exchange chromatography for the characterization of mAbs.  A generic pH gradient for intact mAb analysis is detailed in Table 2.2

Column 4.6 x 100 mm, 5.0 μm non-porous SCX
Temperature 30 °C
Detector Fluorescence 280-360 nm
Mobile phase A: 10 mM MES pH 5.7
B: 10 mM MES pH 5.7 + 1 M NaCl
0-20%B, 20 min
Flow rate 0.6 mL/min

Table 1: Generic salt gradient method for the characterization of intact mAbs. 

Column 4.6 x 100 mm, 5.0 μm non-porous SCX
Temperature 30 °C
Detector Fluorescence 280-360 nm
Mobile phase A: CX-1 buffer A pH 5.6
B: CX-1 buffer B pH 10.2
0-100%B, 20 min
Flow rate 0.6 mL/min

Table 2: Generic pH gradient method for the characterization of intact mAbs. 

Comparison of the elution order for salt and pH gradient experiments show that the mAb compounds elute in a similar order under both sets of conditions with variations seen for only a few mAbs.  The peak capacity (resolving power) for each method was also calculated and was found to be higher under salt gradient conditions.  It should also be noted that the buffer system used in the pH gradient IEX is more expensive; therefore, it may be of more interest to use salt gradient IEX chromatography when characterizing mAbs.

IEX salt and pH gradients can also be applied to the analysis of antibody fragments (~50 kDa, Fab (fragment antibody) and Fc (fragment crystallizable)) which result from papain digestion of intact mAbs.  This type of analysis is used to assess the charge variance.

Cation exchange chromatography is particularly suited to the analysis of mAbs due to their basic nature.  It has been shown that cation IEX can be used to identify post-translation modifications such as aspartate isomerization, asparagine deamidation, oxidation, and fragmentation etc.3

Size Exclusion Chromatography (SEC)

Size exclusion chromatography is used for separating and quantifying protein mixtures, this includes measuring aggregates (dimers, trimers, tetramers etc.), separating low molecular weight excipients and impurities from the larger molecular weight proteins, and determining changes to the molecule such as clipping and other post-translational modifications. It is important to understand and be able to control aggregation in biomolecules as it will affect their efficacy, lifetime, and can produce an immunological response. In size exclusion chromatography (SEC) there is no interaction between the analyte and the surface and separation is primarily by means of the degree of inclusion or exclusion from the pores within the stationary phase.

The pores have different accessibility for molecules of different sizes (hydrodynamic volume), i.e. large molecules are poorly retained and elute first as they cannot access the pores within the stationary phase, whereas smaller molecules will diffuse into the pores to a greater extent depending on their size and will be eluted later, with the smallest molecules eluting last. The ‘size’ of the biomolecule can depend upon the molecular weight, degree of folding and degree of hydration.

It should be noted that although elution order typically follows molecular weight, the true mechanism of SEC is based on size in solution. Proteins can come in several shapes, most are compact, however, some are cylindrical. Cylindrical proteins will have a larger hydrodynamic radius in solution and will elute earlier (even if they do not necessarily have the highest MW). Different mobile phases can also affect the elution order due to hydration of the proteins in solution resulting in changes in size (hydrodynamic radius or radius of gyration).

There are several factors which should be considered when selecting columns and conditions for SEC:

  • The stationary phase should be as insert as possible and silica based columns should be bonded with diol or a polymeric phase to prevent unwanted secondary interactions

  • The pore size of the SEC column should be selected to reflect the molecular weight (MW) range of the proteins being analyzed (Table 3)

  • Due to the fact that all compounds elute before the column dead time (t0), SEC columns often have large internal diameters (~7 mm) and are relatively long (25-30 cm)

  • Mobile phase flow rates are relatively low (0.5 mL/min) which generates a comparatively large t0

  • Mobile phase components should not interact with the solute proteins

  • For proteins a phosphate buffer with pH 7 is often selected as this is close to the pH of most proteins (it can also be considered as the buffer which most closely resembles physiological conditions)

  • Interactions between analytes and the column can be decreased by adjusting the salt concentration - at high salt concentration interaction between proteins and the stationary phase will be minimized
Pore Size (Å) MW Range (kDa)
100 0.1-100
150 0.5-150
300 5-1,250
500 15-5,000
1000 50-7,500
2000 >10,000
Table 3: SEC pore size in relation to analyte molecular weight (MW) range.

A generic method for initial protein analysis by SEC is given in Table 4.

Column 4.6 or 7.8 x 300 mm, 5.0 or 3.0 μm SEC with appropriate pore size
Temperature 10-30 °C (maximum 80 °C)
Mobile phase 150 mM phosphate buffer, pH 7
Isocratic in 30-60 min range
Flow rate 0.1-0.4 mL/min for 4.6 mm i.d. columns
0.1-1.25 mL/min for 7.8 mm i.d. columns
Sample size ≤ 5% of total column volume

Table 4: Initial SEC separation conditions.

After an initial chromatogram has been obtained the separation may require optimization to provide improved separation, maintain protein solubility, or to decrease sample interaction with the chromatographic media.  This can be done by optimizing the following parameters:

  • Addition of salt to alter the mobile phase ionic strength

    • 100-150 mM sodium chloride in 50 mM sodium phosphate, pH 7.0
    • 100-150 mM sodium sulfate in 50 mM sodium phosphate, pH 7.0
    • 50-100 mM urea in 50 mM sodium phosphate, pH 7.0
    • Other similar salts (e.g. KCl) and guanidine hydrochloride can also be used

  • pH can also be optimized by + 0.2 units. If further optimization is necessary, this range can be extended from pH 2.0-8.5

  • Addition of organic solvent

    • 5-10% ethanol (or similar solvents) in 50 mM sodium phosphate, pH 7.0
    • 5% DMSO in 50 mM sodium phosphate, pH 7.0

  • Temperature

    • Typically SEC separations are carried out at 20-30 °C.  Separation of proteins and peptides may require higher temperatures to improve resolution and recovery of proteins and hydrophobic peptides.  The maximum temperature of the SEC column should always be considered when increasing temperature.  Furthermore, increases in pressure and temperature can lead to an increase in aggregation on the column which would result in errors in quantitation


  1. Fekete, S.; Beck, A.; Fekete, J.; Guillarme, D. J. Pharm. Biomed. Anal. 2015, 102, 33-34.
  2. Fekete, S.; Beck, A.; Fekete, J.; Guillarme, D. J. Pharm. Biomed. Anal. 2015, 102, 282-289.
  3. Sandra, K.; Vandenheede, I.; Sandra, P. J. Chromatogr. A 2013, 1422, 27-33.
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