title: Antibody solubility behavior in monovalent salt solutions reveals specific anion effects at low ionic strength.
abstract: Protein solubility was measured using the crystalline precipitate of a recombinant therapeutic antibody, in monovalent salt solutions containing KF, KCl, and KSCN (up to ∼ 0.7 M) at different pH conditions. For all three anions, the antibody solubility demonstrated complex behavior, both monotonic and nonmonotonic, with dependence on pH and salt concentration. At pH 7.1, close to the isoelectric point (pI) of 7.2, a typical salting-in behavior was observed with the salting-in constants of 12.7, 8.0, and 2.8 M for KSCN, KCl, and KF, respectively, suggesting that the anions follow the order of SCN(-) > Cl(-) > F(-) for increasing antibody solubility. Nonmonotonic behavior, as described by an initial solubility decrease followed by a solubility increase with ionic strength, was observed at pH 5.3, far below its pI. The effectiveness of the anion for reducing the solubility followed the order of SCN(-) > Cl(-) > F(-) . After the solubility reached the minimum, the anion's effectiveness for raising the antibody solubility was in agreement with that at pH 7.1. The mechanisms for the above phenomena are discussed based upon specific binding of the anions to the antibody surface. The mechanistic view of anion binding and its charge neutralization effect at pH 5.3 was supported by the results from the effective charge and zeta-potential measurements.
authors: Department of Analytical and Formulation Sciences, Amgen Inc., Thousand Oaks, California 91320-1799, USA.
authors: Le
authors: L
authors: Zhang
authors: Helming
authors: H
authors: Tan
authors: R Matthew
authors: RM
authors: Fesinmeyer
authors: Cynthia
authors: C
authors: Li
authors: David
authors: D
authors: Catrone
authors: David
authors: D
authors: Le
authors: Richard L
authors: RL
authors: Remmele
authors: Jifeng
authors: J
authors: Zhang
mesh_terms: D000838:Anions / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D005459:Fluorides / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012492:Salts / Q000737:chemistry*; D012995:Solubility; D013861:Thiocyanates / Q000737:chemistry
chemical_list: D000838:Anions; D007074:Immunoglobulin G; D017680:Potassium Compounds; D011994:Recombinant Proteins; D012492:Salts; D013861:Thiocyanates; D011189:Potassium Chloride; C033320:potassium fluoride; D005459:Fluorides; C009941:potassium thiocyanate