title: Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process.
abstract: Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used during the CE-SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE-SDS versus SDS-PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins.
authors.affiliation: Analytical and Formulation Sciences, Amgen, Thousand Oaks, CA 91320, USA.
authors.forename: Suminda
authors.initials: S
authors.lastname: Hapuarachchi
authors.forename: Szilan
authors.initials: S
authors.lastname: Fodor
authors.forename: Izydor
authors.initials: I
authors.lastname: Apostol
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D004220:Disulfides; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate