title: A thermal-cycling method for disaggregating monoclonal antibody oligomers.
abstract: Non-native oligomeric forms of biopharmaceutical proteins are therapeutically inactive, and potentially toxic and immunogenic, and therefore undesirable in pharmaceutical formulations. Immunoglobulin G class of antibodies are known to form stable nonnative oligomers through Fab-Fab interactions. In this paper, we investigate thermal-cycling as a technique for disaggregating antibody oligomers. Aggregate containing monoclonal antibody (mAb) samples were exposed to rapid heating and cooling cycles in a thermal-cycler. The heating phase of the thermal-cycle resulted in partial unfolding of the Fab domain, leading to the release of monomer from the oligomer complexes, whereas the rapid cooling that followed led to refolding and minimized the probability of protein reaggregation. The extent of mAb oligomer disaggregation was determined by size-exclusion chromatography and hydrophobic interaction membrane chromatography, whereas protein refolding was assessed by circular dichroism spectroscopy. The thermal-cycling technique in addition to being suitable for disaggregating protein oligomer samples could also potentially be useful for studying the mechanisms of protein aggregation and disaggregation.
authors.affiliation: Department of Chemical Engineering, McMaster University, Hamilton, Ontario, L8S 4L7, Canada.
authors.forename: Rahul H
authors.initials: RH
authors.lastname: Sadavarte
authors.forename: Raja
authors.initials: R
authors.lastname: Ghosh
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015704:CD4 Antigens / Q000737:chemistry; D002942:Circular Dichroism; D019281:Dimerization; D004355:Drug Stability; D004356:Drug Storage; D006358:Hot Temperature; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010316:Particle Size; D054730:Protein Interaction Domains and Motifs; D058849:Protein Refolding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins
keywords: chromatography; circular dichroism; disaggregation; monoclonal antibody; oligomer; protein aggregates; protein folding/refolding; refolding; stability; thermal-cycling