Middle-down analysis of monoclonal antibodies with electron transfer dissociation orbitrap fourier transform mass spectrometry.
Anal Chem
; 86(6): 3005-12, 2014 Mar 18.
Article
em En
| MEDLINE
| ID: mdl-24588056
ABSTRACT
The rapid growth of approved biotherapeutics, e.g., monoclonal antibodies or immunoglobulins G (IgGs), demands improved techniques for their quality control. Traditionally, proteolysis-based bottom-up mass spectrometry (MS) has been employed. However, the long, multistep sample preparation protocols required for bottom-up MS are known to potentially introduce artifacts in the original sample. For this reason, a top-down MS approach would be preferable. The current performance of top-down MS of intact monoclonal IgGs, though, enables reaching only up to â¼30% sequence coverage, with incomplete sequencing of the complementarity determining regions which are fundamental for IgG's antigen binding. Here, we describe a middle-down MS protocol based on the use of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS), which is capable of digesting IgGs in only 30 min. After chemical reduction, the obtained â¼25 kDa proteolytic fragments were analyzed by reversed phase liquid chromatography (LC) coupled online with an electron transfer dissociation (ETD)-enabled hybrid Orbitrap Fourier transform mass spectrometer (Orbitrap Elite FTMS). Upon optimization of ETD and product ion transfer parameters, results show that up to â¼50% sequence coverage for selected IgG fragments is reached in a single LC run and up to â¼70% when data obtained by distinct LC-MS runs are averaged. Importantly, we demonstrate the potential of this middle-down approach in the identification of oxidized methionine residues. The described approach shows a particular potential for the analysis of IgG mixtures.
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Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
/
Anticorpos Monoclonais
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
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