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.

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

“…65 Three replicate sets of H/D-exchange data were processed using HDExaminer (Sierra Analytics, California). Each set of data was manually curated independently to minimize bias by the analyst.…”

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

“…Three independent replicates for both protein concentrations were prepared and analyzed. To minimize peptide carryover in the immobilized pepsin column, the column was washed between samples following a cleaning procedure described previously 80 except that 2 cycles of pepsin column wash were used after each injection. Deuteration was measured using a time-of-flight mass spectrometer (Agilent 6220, Santa Clara, CA) equipped with a standard electrospray ionization source operated in positive mode.…”

Assessment of differences in the conformational flexibility of hepatitis B virus core‐antigen and e‐antigen by hydrogen deuterium exchange‐mass spectrometry

“…[16][17][18][19][20][21][22][23], in the a2b helix, and the junction with the a3 helix (peptide [38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55], and in the junction between a4b and a5 helices (peptide 104-116). Peptides were not identified for the central region of the a3 helix (residues [56][57][58][59][60][61][62][63][64][65][66][67][68] and the N-terminal half of the a5 helix (residues 121-125), hence the deuterium incorporation of these regions is unknown. Viewed in the context of the folded structure, the overall pattern of incorporation is clear [ Fig.…”

“…52,[58][59][60] Unfortunately, observation of EX1-type kinetics is often an artifact attributable either to chemical perturbation of the proteins, or to experimental error, such as carryover between injections. 61,62 Carryover was specifically avoided in our experiments by extensive washing of the pepsin and LC columns between sample injections (see Materials and Methods). In our HDX study, we identified in both Cp149 d and reduced Cp(210)149 d identical specific structural regions exhibiting a mixture of EX1-type and EX2-type kinetics.…”

“…To minimize sample carry over from the pepsin column, three blank injections were performed between each sample injection. 62 These consisted of:…”