Fast separations of intact and reduced monoclonal antibodies (mAbs) were achieved with the use of an Agilent ZORBAX Rapid Resolution High Definition (RRHD) 300 diphenyl reversed-phase column and optimized chromatographic conditions. The unique diphenyl phase and robust rapid resolution high definition column technology, in combination with optimized gradient conditions, provided ultra fast separations and delivered excellent peak shapes demonstrating utility for high throughput mAb charac terizations.
This document presents an application note detailing the use of the Agilent ZORBAX Rapid Resolution High Definition (RRHD) 300 diphenyl reversed-phase column for the rapid separation of intact and reduced monoclonal antibodies (mAbs). The study evaluates the column's unique diphenyl phase and robust technology to achieve ultra-fast separations with excellent peak shapes for high-throughput mAb characterization.
The note systematically optimizes gradient conditions at elevated temperatures to separate intact mAbs and reduced light and heavy chain variants. It compares results from antibodies expressed by Chinese Hamster Ovary (CHO) and CDH media cell lines. Additionally, the document assesses the column's operational tolerance, longevity, and reproducibility under elevated pressures (> 900 bar) and temperatures (75 °C).
| Column Dimensions (Long) | 2.1 x 100 mm |
|---|---|
| Column Dimensions (Short) | 2.1 x 50 mm |
| Operating Temperature | 75 °C |
| Detection Wavelength | 280 nm |
| Flow Rate (Reduced) | 0.5 mL/min |
| Flow Rate (Intact) | 1.0 mL/min |
The Agilent ZORBAX Rapid Resolution High Definition (RRHD) 300 diphenyl reversed-phase column.
The column was tested at elevated operating pressures (> 900 bar) and temperature (75 °C) for 1,000 injections.
A 2-minute post-run wash with 90% iso-propanol is added to re-equilibrate the column.