Intact Mass and Top-Down HPLC/MS Analysis of Monoclonal IgG Antibodies on Orbitrap

摘要

Molecular weight of intact monoclonal lgG antibody (150 kDa) was accurately determined on Orbitrap within +/- 2 Da by both direct infusion and on-line with reversed-phase HPLC. Both accurate mass and top-down fragmentation information was acquired within a short, 20-minutes reversed-phase HPLC/MS run, with Orbitrap alternating between a high mass range scan (for intact mass analysis) and a normal mass range scan with in-source fragmentation (for top-down analysis). The method can be used in pharmaceutical applications to establish identity and assess chemical modifications of therapeutic monoclonal IgG antibodies. This utility was illustrated by characterizing disulfide isoforms of an IgG2 antibody (Figure 5) and glutamine and pyroglutamate variants of a heavy chain (Figure 8). Glycosylation profile including several glycoforms with different numbers of terminal galactose residues (162 Da) can be determined from mass spectra of intact and reduced IgG antibodies. As expected, formic acid instead or in addition to TFA increased ion abundance and mass spectrometric resolution, but decreased HPLC resolution of the IgG2 disulfide isoforms and the heavy chain N-terminal forms. Acetonitrile instead of n-propanol increased abundance of heavy chains top-down fragments, but also decreased HPLC resolution. An initial evaluation indicated that HPLC/MS/MS of light and heavy chains of a reduced and alkylated IgG increased number of fragmentation sites and structural resolution of analysis compare to the intact analysis (Figure 7-8). This middle-down method was further developed and described in details on poster (4).