Probing the Tryptophan Environment in Therapeutic Proteins: Implications for Higher Order Structure on Tryptophan Oxidation.

Abstract:

:Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs. Size-exclusion chromatography with UV and intrinsic Trp fluorescence detection was demonstrated to be a useful method to monitor Trp oxidation levels in mAbs. Furthermore, the Trp oxidation levels measured by size-exclusion chromatography with UV and intrinsic Trp fluorescence detection were found to be in agreement with the values obtained from tryptic peptide mapping by liquid chromatography with mass spectrometric detection and correlate with the total solvent accessible surface area of the exposed Trp residues from in silico modeling. Finally, near-UV circular dichroism and Raman spectroscopy were used to evaluate the impact of Trp oxidation on HOS and identify specific oxidation products, respectively. This work demonstrates that protein HOS is altered on Trp oxidation in mAbs and multiple spectroscopic markers can be used to monitor the molecule-dependent Trp oxidation behavior.

journal_name

J Pharm Sci

authors

Barnett GV,Balakrishnan G,Chennamsetty N,Hoffman L,Bongers J,Tao L,Huang Y,Slaney T,Das TK,Leone A,Kar SR

doi

10.1016/j.xphs.2018.12.027

subject

Has Abstract

pub_date

2019-06-01 00:00:00

pages

1944-1952

issue

6

eissn

0022-3549

issn

1520-6017

pii

S0022-3549(19)30006-1

journal_volume

108

pub_type

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