title: Effect of ions on agitation- and temperature-induced aggregation reactions of antibodies.
abstract: OBJECTIVE
  The impact of ions on protein aggregation remains poorly understood. We explored the role of ionic strength and ion identity on the temperature- and agitation-induced aggregation of antibodies.
  
  
  METHODS
  Stability studies were used to determine the influence of monovalent Hofmeister anions and cations on aggregation propensity of three IgG(2) mAbs. The C(H)2 domain melting temperature (T (m1)) and reduced valence (z*) of the mAbs were measured.
  
  
  RESULTS
  Agitation led to increased solution turbidity, consistent with the formation of insoluble aggregates, while soluble aggregates were formed during high temperature storage. The degree of aggregation increased with anion size (F(-) < Cl(-) < Br(-) < I(-) < SCN(-) ~ ClO(4) (-)) and correlated with a decrease in T (m1) and z*. The aggregation propensity induced by the anions increased with the chaotropic nature of anion. The cation identity (Li(+), Na(+), K(+), Rb(+), or Cs(+)) had no effect on T (m1), z* or aggregation upon agitation.
  
  
  CONCLUSIONS
  The results indicate that anion binding mediates aggregation by lowering mAb conformational stability and reduced valence. Our observations support an agitation-induced particulation model in which anions enhance the partitioning and unfolding of mAbs at the air/water interface. Aggregation predominantly occurs at this interface; refreshing of the surface during agitation releases the insoluble aggregates into bulk solution.
authors.affiliation: Department of Process and Product Development, AW2/D-3152, Amgen Court West, Seattle, Washington, 98119, USA.
authors.forename: R Matthew
authors.initials: RM
authors.lastname: Fesinmeyer
authors.forename: Sabine
authors.initials: S
authors.lastname: Hogan
authors.forename: Atul
authors.initials: A
authors.lastname: Saluja
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Brych
authors.forename: Eva
authors.initials: E
authors.lastname: Kras
authors.forename: Linda O
authors.initials: LO
authors.lastname: Narhi
authors.forename: David N
authors.initials: DN
authors.lastname: Brems
authors.forename: Yatin R
authors.initials: YR
authors.lastname: Gokarn
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004356:Drug Storage; D005079:Excipients / Q000737:chemistry*; D006358:Hot Temperature; D007074:Immunoglobulin G / Q000737:chemistry*; D008956:Models, Chemical; D009994:Osmolar Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D012995:Solubility; D013678:Technology, Pharmaceutical / Q000379:methods; D013997:Time Factors; D044366:Transition Temperature; D014867:Water / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D007074:Immunoglobulin G; D014867:Water