This article describes the optimization of selectivity for antibody separations using HPLC and LC/MS by increasing the percentage of n-propanol and acetonitrile in 0.1% trifluoroacetic acid using ZORBAX 300 Å SB-CN LC Columns.
This document describes a reversed-phase liquid chromatography/mass spectrometry (RPLC/MS) method developed for the analysis of reduced antibodies. The study focuses on separating light chains and heavy chain variants containing N-terminal glutamine or pyroglutamic acid.
The authors evaluated four different stationary phases available in the ZORBAX StableBond 300 A series: C3, C8, C18, and CN. Results indicate that the CN column provided the highest selectivity and resolution for separating heavy and light chains, as well as distinguishing between Q and pE variants of the heavy chain.
| Particle size | 3.5 um |
|---|---|
| Pore size | 300 A |
| Column dimensions (LC/MS) | 1 mm x 50 mm |
| Column dimensions (UV only) | 4.6 mm x 150 mm |
| Operating temperature (LC/MS) | 75 C |
| Flow rate (LC/MS) | 50 uL/min |
The ZORBAX StableBond 300 A CN column showed the highest selectivity and resolution.
The method used increasing percentages of n-propanol and acetonitrile in 0.1% trifluoroacetic acid.