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Ion-Exchange Chromatography01:09

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Ion-exchange chromatography, or IEC, is a technique for separating ions based on their affinity for the stationary phase. The stationary phase is a cross-linked polymer resin with covalently attached ionic functional groups. The functional groups can be either positively charged (cation exchangers) or negatively charged (anion exchangers). A cation exchanger consists of a polymeric anion and active cations, while an anion exchanger is a polymeric cation with active anions. The choice of...
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The role of the detectors in High-Performance Liquid Chromatography (HPLC) is to analyze the solutes as they exit from the chromatographic column. The detector recognizes the solute's property and generates corresponding electrical signals, which are converted into a readable graph of the detector's response versus elution time called a chromatogram at the computer. There are several types of HPLC detectors, each with its own advantages and limitations, depending on the analyte...
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Capillary electrophoretic separations offer various modes, each with unique applications. These modes include capillary zone electrophoresis, capillary gel electrophoresis, capillary array electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
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High-Performance Liquid Chromatography: Instrumentation00:57

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High-performance liquid chromatography, or HPLC, is an analytical technique that separates liquid samples under high pressures. An HPLC instrument consists of glass bottles for storing solvents called mobile phase reservoirs. HPLC-grade solvents are used to maintain high purity, and the dissolved gases are removed using a degasser, such as a vacuum pumping system or sparging with helium. The solvents are then pumped into the analytical column using a screw-driven syringe or reciprocating pumps.
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Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
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Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Published on: September 21, 2011

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ADC Analysis by Hydrophobic Interaction Chromatography.

Ryan Fleming1

  • 1Antibody Discovery and Protein Engineering, Biologic Therapeutics, AstraZeneca, Gaithersburg, MD, USA. ryan.fleming@astrazeneca.com.

Methods in Molecular Biology (Clifton, N.J.)
|October 24, 2019
PubMed
Summary
This summary is machine-generated.

Hydrophobic interaction chromatography (HIC) offers mild, nondenaturing protein analysis. This study presents a generic HIC protocol for antibody-drug conjugate (ADC) characterization, including optimization and troubleshooting.

Keywords:
Antibody–drug conjugates (ADCs)Drug-to-antibody ratio (DAR)Hydrophobic interaction chromatography (HIC)HydrophobicityPayloadRetention time

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Chromatography

Background:

  • Hydrophobic interaction chromatography (HIC) is a standard technique for protein separation and purification.
  • Antibody-drug conjugates (ADCs) are increasingly prevalent in clinical trials, necessitating robust analytical methods.
  • HIC preserves the native structure and activity of proteins, making it ideal for analyzing complex molecules like ADCs.

Purpose of the Study:

  • To describe a generic HIC protocol for the screening, analysis, and characterization of ADCs.
  • To address parameters influencing data quality and interpretation in ADC HIC analysis.
  • To provide recommendations for method optimization and troubleshooting in ADC HIC.

Main Methods:

  • Utilized hydrophobic interaction chromatography (HIC) for protein analysis.
  • Employed an ammonium sulfate buffer system.
  • Used a high-pressure liquid chromatography (HPLC) system for ADC characterization.

Main Results:

  • A generic HIC protocol was successfully developed for ADC analysis.
  • Key parameters affecting data quality and interpretation were identified.
  • Recommendations for method optimization and troubleshooting were provided.

Conclusions:

  • HIC is a valuable technique for characterizing ADCs under native conditions.
  • The presented generic HIC protocol facilitates ADC screening and analysis.
  • Optimization and troubleshooting guidance enhance the reliability of HIC for ADC studies.