Agilent Community
Agilent Community
  • User
  • Site
  • Search the Community
  • User
Consumables
  • Technical Areas
Consumables
Wiki Analyzing ADCs by HIC
  • 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.

Analyzing ADCs by HIC

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

ADCs often post additional challenges beyond those posed by mAbs - learn what to watch out for and how to adjust your method.

 

Hello again, everybody. This week my colleague Andy is back to talk about HIC method development for ADCs. As if mAbs weren’t tough enough, ADCs present another level of complexity, and require special consideration to be sure that you get accurate results.

 

--

 

Analyzing ADCs by HIC

 

ADCs often post additional challenges beyond mAbs - learn what to watch out for and how to adjust your method

The incorporation of hydrophobic drug molecules into a monoclonal antibody (mAb) forming an antibody drug conjugate (ADC) results in numerous different molecular species with varying degrees of hydrophobicity. This means hydrophobic interaction chromatography (HIC) is an ideal technique for analyzing the all-important drug to antibody ratio (DAR).

 

However, the range of hydrophobicity presents certain important challenges:

  1. Sample preparation must result in complete dissolution of the sample.
  2. HIC gradient conditions must result in complete elution of all variants.

 

Too much ammonium sulfate in the sample solution could result in DAR 6 and DAR 8 variants not being fully dissolved which may result in an underestimated DAR value. Similarly, the gradient conditions need to ensure elution of all DAR species. This elution is achieved by including propan‑2‑ol as organic modifier.

 

Start by dissolving the sample in the dilute aqueous mobile phase buffer and then add the high ammonium sulfate mobile phase to dilute the sample to the required level. Do this in a clear container so that any signs of cloudiness (indicating sample precipitation) can be prevented by adding more of the high aqueous mobile phase buffer as required.

 

Following the recommendations in the Quick Start Guide (5991-9514EN), use a gradient of 1–0 M ammonium sulfate with a simultaneous gradient of 5–25 % propan-2-ol. This will ensure the complete elution of all species.  Because of the high eluent viscosity, a lower flow rate will be beneficial. A 10 cm column will give maximum resolution; a 3 cm column will enable faster separations.

 

By choosing the appropriate mobile phase and gradient conditions, it is possible to separate ADC DAR variants in a very short analysis time using new Agilent AdvanceBio HIC columns (Application Note 5994-0149EN has more details).

 

--

 

Thanks again to Andy and Sandeep! I’ll be back in two weeks to wrap up our HIC series with some tips and reminders to keep HIC running smoothly.

 

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

  • hic
  • adc
  • 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