Agilent Community
Agilent Community
  • User
  • Site
  • Search the Community
  • User
Consumables
  • Technical Areas
Consumables
Wiki Hydrophobic Interaction Chromatography of Proteins and mAbs
  • Announcements
  • Forum
  • Files
  • Wiki
  • More
  • Cancel
  • Consumables
  • A Beginner’s Guide to Hydrophobic Interaction Chromatography
  • A simple tip to protect your columns
  • A Tip for Preparing Robust and Consistent Mobile Phases
  • Achieve accurate and repeatable gas flow meter measurements
  • AdvanceBio Columns Blog Series
  • Agilent 123 Meter Sodium probe maintenance
  • Agilent Collection of Columns, Supplies, and Standards Resources
  • Analysis of microplastics in the environment
  • Analyzing ADCs by HIC
  • Avoiding downtime in the lab: top tips for GC/MS success
  • Best Practices for Aqueous Mobile Phases
  • Best Practices for Making Good Connections
  • Bio LC Column User Guides
  • Bursting Tubing and Columns (GC and HPLC)
  • Calculate the Maximum Allowable Working Pressure for Tubing
  • Calculating Column Volume
  • Cannabis Potency Testing: a Reliable, Cost-Effective Method
  • Carbohydrate Analyses in LC
  • Checking Your Intuition - Sub 2 µm vs Superficially Porous
  • Choosing the right GC Injection Technique
  • Choosing the right pore size for size exclusion chromatography
  • Columns, Supplies, and Standards Knowledgebase
  • Consumables Applications and Workflows
  • Consumables Recommended Supplies Lists for Agilent.com
  • Custom Product Request
  • Extreme Makeover – Derivatizations in Chromatography – Part 1 GC
  • Extreme Makeover – Derivatizations in Chromatography – Part 2 LC
  • Fake It Until You Make It: When BioInert Isn’t an Option
  • Flipping Amino Acid Analysis on Its Head
  • Glycans at a glance:  Analyzing therapeutic glycoproteins
  • Handle and Care of Syringes
  • Help! My Peaks Look Strange - Fronting and Tailing in GC
  • Help! My Peaks Look Strange - Saddle Points - LC/GC Troubleshooting
  • How do I select a Split/Splitless liner?
  • Hydrophobic Interaction Chromatography of Proteins and mAbs
  • Importance of Silica Particle Strength for Sub-2 µm SEC Columns
  • KB: Ferrules recommended for GC self-tightening column nut
  • LC and LC/MS Columns - USP Designations
  • LC Column User Guides
  • LC Method Translation - the Dwell Volume
  • Minimize spectroscopy workflow disruptions
  • Minimizing Metals for Best HILIC Results
  • More than just a drink: Analyzing the elemental composition of beer
  • Multi-Attribute Methods – Peptide Mapping Part IV
  • Must See Webinars
  • Nomenclature of CFC's/Freons/Halons/Coolants
  • Oligonucleotide Analysis - Unexpected Details Matter
  • Optimizing Bonding Chemistry for Sub-2 µm SEC Particles
  • Pass the Salt, Please – Mobile Phase Preparation for HIC
  • Pesticides and their stability during GC analyses
  • Pre-Columns - the forgotten art of using retention gaps
  • Problematic polar analytes? Hello HILIC…
  • Protecting your laboratory productivity
  • Recommended Reading
  • Sample Prep Pointers - Peptide Mapping Part I
  • Save your results with sample filtration
  • Simplified cone inspection with the new Agilent LED measuring magnifier
  • Software - Supported Method Development - The Scanview Application
  • Software tool for the ADM Flow Meter (G6691A)
  • Stay Safe: A Win-Win for Solvent Storage
  • Streamline your sample processing
  • The importance of chemical composition for vial performance
  • Tips & Tricks for Amino Acid Analysis – Part I
  • Tips & Tricks for Amino Acid Analysis – Part II
  • Tips & Tricks for Amino Acid Analysis – Part III
  • Tips & Tricks for Amino Acid Analysis – Part IV
  • Tips for Smooth Sailing with HIC
  • Troubleshooting HPLC autosamplers
  • Troubleshooting HPLC degassers
  • Troubleshooting Sequence Coverage – Peptide Mapping Part III
  • UltiMetal Plus Flexible Metal Ferrule
  • UV, MS, TFA, and Formic Acid – What to use? Peptide Mapping Part II
  • What are the typical % Gain or EHT values for hollow cathode lamps?
  • You Need Lamps or Chemical Standards for Atomic Absorption Single-Element Analyses?
Still Need Help?

Post your question in our User Forum or Contact Support.

Hydrophobic Interaction Chromatography of Proteins and mAbs

Created by anne_blackwell anne_blackwell over 2 years ago | Last modified by Agilent Agilent over 2 years ago

Method development tips for HIC separations of proteins

 

Now that Sandeep has detailed how to make these salty mobile phases for HIC, let’s turn our attention to other method development details. Here’s where my colleague Andy is going to take the lead.

 

---

 

Hydrophobic Interaction Chromatography of Proteins and mAbs

 

Hydrophobic interaction chromatography (HIC) is rather unique in that it allows separation of molecules based on differences in their hydrophobicity, while they remain intact in their native state. This unique capability means it is possible to separate variants that are difficult to resolve by any other technique. For example, it can separate variants in oxidation state, deamidation, and other species that can arise during post-translational modification.

 

The most commonly used mobile phase for HIC of proteins uses high concentrations of ammonium sulfate in dilute phosphate buffer. The buffer maintains a steady pH, and the ammonium sulfate ensures that proteins are more likely to adsorb onto the HIC stationary phase but are unlikely to precipitate. Starting at a high concentration of ammonium sulfate and performing gradient elution to a low concentration ensures that molecules elute in order of increasing hydrophobicity (in much the same way reversed-phase chromatography, RPLC).

 

Water is quite viscous but adding modifiers such as organic solvents (for RPLC) or high concentrations of ammonium sulfate (for HIC) can increase the viscosity still further. This increased viscosity will lead to higher operating pressures and potentially to poor peak shape due to reduced mass transfer. The most commonly used tool to alleviate this problem is to run the HPLC experiment at a higher temperature.

 

For RPLC, higher temperatures generally have the desired effect: Peaks become sharper as mass transfer improves, and the operating pressure is reduced. Frequently the retention time of the molecules reduces too, however this shift is not usually problematic.

 

However, in HIC, increasing the temperature can have a deleterious effect. Instead of sharper peaks and shorter retention times, we often see an increase in retention time and broader peaks, which results in loss of resolution:

 

In practice, better resolution is obtained by reducing the operating temperature. It is important to note the severe deterioration in the peak shape of cytochrome C at even the moderately elevated temperature (40 to 45 °C) used in this experiment.

 

For more practical applications such as oxidized variants of monoclonal antibodies, it is possible to use oxidants to force degrade samples for use in method development. This approach should be used with care since the level of oxidation will vary considerably with choice of oxidant, concentration, and length of exposure time. A typical monoclonal antibody may contain 16 individual methionine residues distributed throughout the molecule (both heavy and light chains); some of these residues will be located in more exposed regions and will therefore react more quickly.

 

Heavy Chain and Light Chain Amino Acid Sequence of NIST mAb (RM 8671)

 

The methionine residues found in the RM 8671 NIST mAb are shown in Figure 2, highlighted in bold. Those in red (the Fc region of the heavy chain, and those in the light chain) have been observed as oxidized variants.

 

By exposing the NIST mAb sample to tert-butylhydroperoxide (t-BHP), an increase in the number of earlier eluting oxidized variants are seen:

 

For additional information on oxidation analysis, please see Agilent Application Note 5994-0199EN.

 

--

 

Thanks, Andy! Next time Andy will expand his method development tips to measuring the drug to antibody ratio of ADCs.

 

Keywords: Bio columns, liquid chromatography, hydrophobic interaction chromatography, HIC, method development, AdvanceBio blog

  • hic
  • method development
  • bio columns
  • advancebio blog
  • hydrophobic interaction chromatography
  • Biopharma LC Columns & Consumables
  • liquid chromatography
  • Share
  • History
  • More
  • Cancel
Anonymous
Related

Agilent Community Feedback

Agilent Community Feedback

×
We are glad this was helpful! We are sorry this was not helpful. If you still need assistance please create a community post or contact support. To help us improve, please provide any additional feedback. For full details of how we will treat your information, please view our privacy policy.
Submit Cancel
Submit Cancel
Recommended
Privacy Statement
Terms of Use
Contact Us
Site Help