title: Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization.
abstract: OBJECTIVE
  Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregation.
  
  
  METHODS
  We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography and orthogonal analytical methods, including small-angle X-ray scattering and dynamic light scattering and supplemented the experimental data with crystal structure-based spatial aggregation propensity (SAP) calculations.
  
  
  RESULTS
  We revealed distinct solution behaviors between the three mAb models: At acidic pH IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were identified, which may lead to two distinct pathways of reversible and irreversible aggregation, respectively.
  
  
  CONCLUSIONS
  We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Skamris
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Xinsheng
authors.initials: X
authors.lastname: Tian
authors.affiliation: Global Research Unit, Novo Nordisk A/S, Novo Nordisk Park 1, 2760, Måløv, Denmark.
authors.forename: Matthias
authors.initials: M
authors.lastname: Thorolfsson
authors.affiliation: Global Research Unit, Novo Nordisk A/S, Novo Nordisk Park 1, 2760, Måløv, Denmark.
authors.forename: Hanne Sophie
authors.initials: HS
authors.lastname: Karkov
authors.affiliation: Global Research Unit, Novo Nordisk A/S, Novo Nordisk Park 1, 2760, Måløv, Denmark.
authors.forename: Hanne B
authors.initials: HB
authors.lastname: Rasmussen
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Annette E
authors.initials: AE
authors.lastname: Langkilde
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark. bente.vestergaard@sund.ku.dk.
authors.forename: Bente
authors.initials: B
authors.lastname: Vestergaard
mesh_terms: D000143:Acids / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D055503:Protein Multimerization; D012996:Solutions / Q000737:chemistry
chemical_list: D000143:Acids; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D012996:Solutions
keywords: biopharmaceutics; formulation; monoclonal antibody; protein stability; small-angle X-ray scattering (SAXS)