کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1204811 965169 2010 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Efficient on-column conversion of IgG1 trisulfide linkages to native disulfides in tandem with Protein A affinity chromatography
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Efficient on-column conversion of IgG1 trisulfide linkages to native disulfides in tandem with Protein A affinity chromatography
چکیده انگلیسی

Protein trisulfide linkages are generated by the post-translational insertion of a sulfur atom into a disulfide bond. Molecular heterogeneity was detected in a recombinant IgG1 monoclonal antibody (mAb) and attributed to the presence of a protein trisulfide moiety. The predominant site of trisulfide modification was the bond between the heavy and light chains. The trisulfide was eliminated during purification of the IgG1 mAb via a cysteine wash step incorporated into Protein A affinity column chromatography. Analysis of the cysteine-treated mAb by electrophoresis and peptide mapping indicated that the trisulfide linkages were efficiently converted to intact disulfide bonds (13% trisulfide decreased consistently to 1% or less) without disulfide scrambling or an increase in free sulfhydryls. The on-column trisulfide conversion caused no change in protein folding detectable by hydrogen/deuterium exchange or differential scanning calorimetry. Consistent with this, binding of the mAb to its antigen in vitro was insensitive to the presence of the trisulfide modification and to its removal by the on-column cysteine treatment. Similar, high efficiency trisulfide conversion was achieved for a second IgG1 mAb using the column wash strategy (at least 7% trisulfide decreased to 1% or less). Therefore, trisulfide/disulfide heterogeneity can be eliminated from IgG1 molecules via a convenient and inexpensive procedure compatible with routine Protein A affinity capture.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1217, Issue 32, 6 August 2010, Pages 5225–5232
نویسندگان
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