title: Characterization of Fc-fusion protein aggregates derived from extracellular domain disulfide bond rearrangements.
abstract: Aggregation of protein biotherapeutics has consequences for decreasing production and has been implicated in immunogenicity. The mechanisms of protein aggregation vary depending on the protein and the expression system utilized, making it difficult to elucidate the conditions that promote their formation. Nonnative aggregation of recombinant immunoglobulin G protein therapeutics from mammalian expression systems has been extensively studied. To better understand the mechanisms behind aggregation of glycosylated fusion proteins produced in Chinese hamster ovarian cells, we have examined the high-molecular-weight (HMW) species of activin receptor-like kinase 1 Fc fusion protein. Size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that two populations of aggregate exist: (1) nondisulfide-linked, higher-order aggregates and (2) disulfide-linked oligomers. The largest aggregated species have increased nonnative structure, whereas the smallest aggregated species maintain structure similar to monomer. The HMW species display decreased levels of O-linked glycosylation, higher occupancy of high-mannose N-linked oligosaccharide structures, and overall less sialylation as their size increases. Disulfide-linked aggregate species were found to associate through the extracellular domain. N-linked glycosylation on the extracellular domain (ECD) appears to discourage disulfide-linked aggregation. Elucidation of the specific mechanisms behind disulfide-linked aggregate formation may assist in designing processes that limit aggregate formation in cell culture, with implications for increased production.
authors.affiliation: Acceleron Pharma, Cambridge, Massachusetts 02139, USA.
authors.forename: James
authors.initials: J
authors.lastname: Strand
authors.forename: Chi-Ting
authors.initials: CT
authors.lastname: Huang
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
mesh_terms: D030301:Activin Receptors, Type II / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004220:Disulfides; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; C425364:ACVRL1 protein, human; D030301:Activin Receptors, Type II