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Minimizing Carry-Over in an Online Pepsin Digestion System used for the H/D Exchange Mass Spectrometric Analysis of an IgG1 Monoclonal Antibody
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Minimizing Carry-Over in an Online Pepsin Digestion System used for the H/D Exchange Mass Spectrometric Analysis of an IgG1 Monoclonal Antibody
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Minimizing Carry-Over in an Online Pepsin Digestion System used for the H/D Exchange Mass Spectrometric Analysis of an IgG1 Monoclonal Antibody
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Journal Article
Minimizing Carry-Over in an Online Pepsin Digestion System used for the H/D Exchange Mass Spectrometric Analysis of an IgG1 Monoclonal Antibody
2012
Overview
Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~150 kDa protein, approximately 30 % of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5 %)/ isopropanol (5 %)/ acetic acid (20 %) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides.
Publisher
Springer-Verlag,Springer Nature B.V
Subject
/ Antibodies, Monoclonal - analysis
/ Antibodies, Monoclonal - chemistry
/ Antibodies, Monoclonal - metabolism
/ Chemistry and Materials Science
/ Chromatography, Liquid - instrumentation
/ Chromatography, Liquid - methods
/ Deuterium Exchange Measurement - instrumentation
/ Deuterium Exchange Measurement - methods
/ Immunoglobulin G - chemistry
/ Immunoglobulin G - metabolism
/ Pepsin
/ Peptide Fragments - analysis
/ Peptide Fragments - chemistry
/ Peptide Fragments - metabolism
/ Peptides
/ Proteins
/ Spectrometry, Mass, Electrospray Ionization - instrumentation
/ Spectrometry, Mass, Electrospray Ionization - methods
/ Washing
