Identification of Potential Sites for Tryptophan Oxidation in Recombinant Antibodies Using tert-Butylhydroperoxide and Quantitative LC-MS

Miriam Hensel,Juergen Fichtl,Michael Molhoj,Tilman Schlothauer,Dietmar Reusch,Frank Wedekind,Patrick Bulau,Rebecca Steurer,Andreas Petzold,Carsten Elger,Maxim Antopolsky

doi:10.1371/journal.pone.0017708

Abstract

Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies.

Highlights

The oxidative degradation of pharmaceutical proteins has been reviewed by several authors [1,2,3,4]
No significant differences in peak intensities were observed for the tryptic peptides containing light chain Met-4, heavy chain Met-34, heavy chain Met-83, and heavy chain Met-429 by LC-UV analysis
The existence of oxidized Met-253 and Trp32 was further suggested by liquid chromatography-mass spectrometry (LC-MS) analysis results, in which mass differences of +16 Da (LC fraction 1 versus 2) and +4 Da (LC fraction 3 versus 4) were observed for the detected peptides (Figure 1, inset)

Summary

Introduction

The oxidative degradation of pharmaceutical proteins has been reviewed by several authors [1,2,3,4]. Amino acid residues that are susceptible to oxidation include cysteine, methionine, tryptophan, histidine, and tyrosine, in that order. Since recombinant antibodies do not contain significant amounts of free cysteine, cysteine oxidation will not be addressed here. We focus on the oxidation of methionine (Met) and tryptophan (Trp). Oxidation of Met residues in the constant domains of recombinant antibodies has been demonstrated to impact the interaction with the neonatal Fc receptor and binding to the Fcc receptors [5]. Induction of Trp oxidation in the complementary-determining regions (CDRs) of a monoclonal antibody by photooxidation resulted in a progressive loss of target binding and biological activity [6]

Methods

Results

Conclusion