No thanks! I would like to know more about CHROMacademy

 Over 3000 E-Learning topics / 5000 Articles & Applications
HILIC, IEX, and SEC for the Analysis of Biomolecules

Biopharmaceuticals offer great hope in treating medical conditions which are currently poorly served at best by traditional pharmaceuticals. 

It is estimated that there are over 400 biopharmaceuticals in clinical trials for in excess of 200 disease areas. 

The enhanced complexity and variability that comes from the size of biopharmaceuticals, allied with the intricacy of the production process, mean chromatography is employed to a much greater extent during production and release testing.  


HILIC Analysis - Glycans

As well as the amino acid composition it is important to characterize the glycosylation of proteins; including the sequence, composition, branching, linkage etc.  It is important to keep in mind that glycosylation is one of the major sources of mAb variability.  This is somewhat surprising as the amount of glycan by weight only accounts for about 3% of the total mAb weight. 

Glycans are generally composed of sugar units such as mannose, sucrose, galactose etc. 
There are two types of glycans
1) o-glycans which are most commonly attached to the peptide chain through serine (Ser) and threonine (Thr) residues via a hydroxyl group and
2) n-glycans which are covalently attached to a protein at asparagine (Asn) residues via the amide group to form an N-glycosidic bond. 

Glycosylation can be revealed at both the protein and peptide level but in reality this is quite complicated and full characterization of the carbohydrates can only be achieved after removal from the protein or peptide backbone.  Like amino acids, native glycans are very hydrophilic, therefore, they cannot be sufficiently retained under RPLC conditions, hence, HILIC is commonly employed to provide retention of these highly hydrophilic species. 

A stationary phase bonded with an amide group is generally the best choice of HILIC column for glycan analysis.  Glycans cannot be detected using spectrophotometric detectors; therefore, a labelling protocol using 2-aminobenzamide (2-AB) is used to allow their detection using spectrophotometric detection.  Figure 1 shows the successful identification of glycans labelled with 2-AB and analyzed under HILIC conditions.

Figure 1: Glycan 2-AB labelling and HILIC analysis.


Figure 2 details the workflow used for glycan analysis.  The first step involves the glycans being cleaved from the antibody using an enzyme.  Following this the glycans are labelled using 2-aminobenzamide via reductive amination to allow analysis using HILIC coupled with UV, fluorescence, or MS detection as appropriate. 


Figure 2: N-glycan analysis workflow.


Figure 3 shows the N-glycan analysis of four different batches of mAb.  It can be seen that each lot exhibits a different glycosylation profile.  The method used has allowed structural isomers to be resolved i.e. G1Fa and G1Fb which only differ in the position of the galactose residues on the 1,3 or 1,6 branch of the complex glycan. 

Lot number 2, 3, and 4 are very similar, however, batch 1 is quite different.  It can be seen that lot 1 contains a higher amount of G2F as well as some low quantities of some other glyco forms. 


Figure 3: N-glycan analysis of four production lots of mAb.


Under HILIC conditions the analytes are eluted by increasing the water concentration in the mobile phase.  In HILIC water is the ‘strong’ solvent, whereas, in RPLC this is the weak solvent.


Ion Exchange Chromatography (IEX) - Proteins

Ion exchange chromatography (IEX) has been widely used in the analysis of biomolecules and remains as one of the gold standard strategies.  This technique allows separation of compounds based on the electrostatic interaction of the charged side chains of the proteins or the mAb and the opposite charge on the surface of the stationary phase. 

Weak (WCX) or strong cation exchange (SCX) materials, which contain carboxylic or sulfonic acid groups respectively, are generally employed for the analysis of biomolecules.  In the case of cation exchange the most acidic compounds are eluted first as can be seen in Figure x, due to the electrostatic repulsion between the compound and the cation exchange surface, whereas, the most basic compounds are retained and elute later due to attractive interactions with the cation exchange material.  The chromatogram in Figure 4 exhibits numerous peaks, whereas, under RPLC analysis of the same mAb only 4 peaks were observed; clearly demonstration the superiority of IEX over RPLC for charge variance characterization.

Elution of compounds in IEX is achieved by increasing the salt content of the mobile phase, or more commonly, by adjusting the pH to interrupt the compound/stationary phase interactions.  


Figure 4: Charge variant analysis of mAb by SCX.


Figure 5 shows the analysis of ten different mAb compounds using a generic salt gradient SCX method.  Acidic and basic mAb variants are eluted before and after the main peak respectively.  Using this method it was possible to rank the heterogeneity of the mAb compounds which is important when using IEX chromatography.  Charge variance was very limited for natalizumab (demonstrated by only a single peak in the chromatogram), whereas, cetuximab contains a greater degree of charge variance which is due to the fact that cetuximab contains two glycosylation sites.  To improve the resolution for a particular mAb in IEX chromatography the salt gradient as well as the mobile phase pH can be optimized. 


Figure 5: Generic salt gradient method for the characterization of 10 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: 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.1

There is an increasing interest in pH gradient cation exchange chromatography for the characterization of mAbs.  Figure 6 shows a generic pH gradient applied to the analysis of 10 intact mAbs.  The mAbs were well distributed over the pH gradient and the same conclusions on heterogeneity could be drawn from the pH and salt gradient experiments.


Figure 6: Generic pH gradient method for the characterization of 10 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.2


The elution order for the salt and pH gradient experiments were compared and show that the mAb compounds elute in a similar order under both sets of conditions with variation for only a few mAbs (adalimumab, denosumab, and bevacizumab Figure 7). 

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.


Figure 7: Comparison of elution order under salt and pH gradient cation exchange chromatography.


Finally, to prove the resolving power of cation exchange chromatography cetuximab was digested with papain to produce Fab and Fc fragments (~50 kDa) which were subsequently analyzed using salt and pH gradient IEX to determine the charge variance (Figure 8). 

It can be seen that up to 14 variants can be separated for digested cetuximab.  IEX is very interesting as a method for analyzing mAbs as the separation is performed under non-denaturing conditions, unlike RPLC.


Figure 8: IEX analysis of cetuximab digested with papain.

Size Exclusion Chromatography (SEC) - Proteins

In size exclusion chromatography (SEC) there is no interaction between the analyte and the surface.  Separation is primarily by means of the pores within the stationary phase having different accessibility for molecules of different sizes i.e. large molecules are poorly retained and elute first as they cannot access the pores within the stationary phase (Figure 9).  SEC is used to determine the level of aggregation within biomolecules.


Figure 9: SEC chromatogram.


Column length and particle size can be altered in SEC to improve resolution and throughput; however, this can cause some problems with biomolecules.  When the mobile phase temperature is increased the chromatographic peaks become more efficient, however, an increased amount of aggregates are observed. 

Therefore, increases in pressure and temperature can lead to an increase in aggregation on the column which would result in errors in quantitation when analyzing mAbs.  Therefore, care should be taken not to use high temperature and columns packed with small particles should be avoided.


Figure 10: Effect of temperature and pressure on SEC of mAbs. 


  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.

This article was based on our webcast
HPLC Techniques in Biopharmaceutical Analysis

You may also be interested in the following webcasts
Techniques Employed in Biopharmaceutical Analysis - Part I Reversed Phases and HILIC

Techniques Employed in Biopharmaceutical Analysis – Part II Ion Exchange and SEC

Reversed Phase HPLC for the Analysis of Biomolecules

loading data
loading data
loading data
loading data
loading data

group  subsCHROMacademy can deliver to corporate clients on a multi-user subscription basis.
Served up from secure servers to the corporate intranet or individual desktops.

  • Microsite - your own learning site powered by CHROMacademy
  • Your Landing Pages -with your logo and branding
  • Customized Assessments - Based on content agreed upon Certificate of Completion
  • Certification Programs - Offer your learners a goal to strive towards
  • LMS : Connect - Our Learning Management System is S.C.O.R.M. compliant and will connect to your system
  • Engagement Package - E-newsletter stimulation program derived from your content and ours
  • Full archive of Essential Guide webcasts & tutorials
  • 1000’s of eLearning topics - HPLC / GC / Sample Prep / Mass Spec
  • Ask the Expert - our experts will answer your chromatography questions within 24 hrs.
  • Assessments - test your knowledge
  • Application notes & LCGC articles
  • Troubleshooting and virtual lab tools

Request a quote


 Home | About UsContact Us | SubscribeTerms and Conditions | Advertise | Privacy Policy 

loading data

loading data

loading data


loading data

loading data