title: Minimizing carry-over in an online pepsin digestion system used for the H/D exchange mass spectrometric analysis of an IgG1 monoclonal antibody.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA.
authors.forename: Ranajoy
authors.initials: R
authors.lastname: Majumdar
authors.forename: Prakash
authors.initials: P
authors.lastname: Manikwar
authors.forename: John M
authors.initials: JM
authors.lastname: Hickey
authors.forename: Jayant
authors.initials: J
authors.lastname: Arora
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
authors.forename: David D
authors.initials: DD
authors.lastname: Weis
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D041961:Deuterium Exchange Measurement / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D016014:Linear Models; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010446:Peptide Fragments; D010434:Pepsin A