This application note describes a method for achieving highly resolved ultrafast separations of reduced and alkylated monoclonal antibodies using an Agilent ZORBAX sub-2 µm Rapid Resolution High Definition (RRHD) 300SB-C8 reversed-phase column under optimized chromatographic conditions. The StableBond C8 phase and RRHD column technology, in combination with robust gradient elution conditions, enabled efficient, fast, and effective separation of light chain and two heavy chain monoclonal antibody variants in under 4 minutes. Additionally, alternate mobile phase compositions were employed, making separations flexible for ultra fast LC/MS analysis. At elevated operating pressures and temperature, the stable C8 phase and rugged column packing technology delivered highly reproducible separations and delivered excellent chromatographic run-to-run results.
This document describes the use of an Agilent ZORBAX Rapid Resolution High Definition (RRHD) 300SB-C8 reversed-phase column for the ultra-fast separation of reduced and alkylated monoclonal antibodies. The study highlights how the StableBond C8 phase and RRHD technology enable efficient separations in under four minutes at elevated temperatures.
The note details specific experimental procedures, including the reduction and alkylation of antibodies, and provides optimized UHPLC conditions using an Agilent 1290 LC Infinity system. It also explores mobile phase compositions suitable for LC/MS analysis, demonstrating flexibility with different ion pairing additives like formic acid and acetic acid.
| Column Dimensions | 2.1 x 100 mm |
|---|---|
| Particle Size | 1.8 ym |
| Operating Temperature | 75 °C |
| Flow Rate | 0.5 mL/min |
| Detection Wavelength | 225 nm |
The analysis was performed at an elevated temperature of 75 °C.
An Agilent 1290 LC Infinity system with auto injector, binary pump, thermostatted oven, and diode array detector was used.