A new fused-core particle designed specifically for the separation of proteins and monoclonal antibodies with 3.4 µm particle
size and a thin 0.2 µm porous shell demonstrates high resolution of the heavy and light chain fragments of IgG1.
The focus of many pharmaceutical companies has shifted to larger biotherapeutic molecules as potential treatments for disease.
These biomolecules present a new set of challenges compared to their small molecule predecessors when complete characterization
is considered. Variants can be found through different glycans, deamidations, enzymatic clipped polypeptides, and so on, not
to mention the potential for aggregate formation. Rapid reversed-phase LC analysis is convenient for its ability to be interfaced
to online mass spectrometry. The HALO Protein C4 bonded phase is ideal for this application with its optimized design for
high recovery and stability at the high temperatures and low pH required by the analysis.
Column: 100 mm × 2.1 mm Halo Protein C4; gradient: 29–32% B in 20 min; mobile phase A: 0.5% (v/v) formic acid with 20 mM ammonium
formate; mobile phase B: 45% acetonitrile, 45% isopropanol/0.5% (v/v) formic acid with 20 mM ammonium formate; temperature:
80 °C; flow rate: 0.4 mL/min; instrument: Shimadzu LCMS 2020; detection: 280 nm; MS-2020 single quadrupole MS instrument using
ESI at +4.5 kV capillary voltage, 2 pps scan rate from 500 to 2000 amu m/z. Component masses for these measurements were determined by deconvolution using MagTran software, v. 1.02, based on the ZScore
algorithm developed by Zhang and Marshall (1).
The addition of a small amount of ammonium formate to formic acid in the mobile phase improves the peak shapes. An optimized,
shallow gradient allows resolution of the variants of both the heavy and light chain fragments of IgG1 as shown in Figure
1. The MS spectra can then be deconvoluted to determine the masses associated with each peak.
Figure 1: LC–MS separation of reduced IgG1. Column: 2.1 mm i.d × 100 mm HALO Protein C4; Flow rate: 0.4 mL/min. Mobile phase
A: 0.5 % formic acid with 20 mM ammonium formate; mobile phase B: 45% AcN/45% IPA/ 0.5 % formic acid with 20 mM ammonium formate;
gradient: 29–32% B in 20 min.; temperature: 80 °C; detection: 280 nm; MS conditions: Shimadzu LCMS-2020, ESI +4.5 kV, 2 pps,
HALO Protein C4 demonstrates the low pH and high temperature stability that is required to analyze reduced and alkylated IgG1
using mass-spec friendly mobile phase. The use of 80 °C enables improved peak shape while the high resolution allows complete
analysis of the IgG1 fragments that are present.
(1) Z. Zhang and A.G. Marshall, J. Am. Soc. Mass Spectrom.
9, 225 (1998).
MAC-MOD Analytical, Inc.
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