title: Nonnative aggregation of an IgG1 antibody in acidic conditions, part 2: nucleation and growth kinetics with competing growth mechanisms.
abstract: Aggregation mechanisms as a function of pH were assessed for the IgG1 antibody described in Part 1 (Brummitt RK, Nesta DP, Chang L, Chase SF, Laue TM, Roberts CJ. Non-native aggregation of an IGG1 antibody in acidic conditions: 1. Unfolding, colloidal interactions, and high molecular weight aggregate formation. J Pharm Sci. In press). Aggregation kinetics along with static light scattering and size-exclusion chromatography indicated that the aggregate nucleus was a dimer for all conditions tested, and this was semiquantitatively consistent with scaling of the characteristic time scale for nucleation (τ(n)) versus protein concentration at pH 4.5 and pH 5.5. Changing pH significantly altered the mechanism of aggregate growth, as well as the size and solubility of aggregates that were formed. Aggregates at pH 3.5 grew primarily by monomer addition and remained small and soluble. Aggregates at pH 4.5 grew first by chain polymerization, followed by condensation polymerization, leading ultimately to large insoluble particles. At pH 5.5, monomer loss resulted primarily in insoluble aggregate formation, with only low levels of soluble aggregate intermediates detected at early times. The influence of pH on aggregate solubility and the reversibility of aggregate phase separation were confirmed via cloud point titrations. Qualitatively, the global aggregation behavior was consistent with reduction of charge-charge repulsions as a primary factor in promoting larger aggregates and aggregate phase separation.
authors.affiliation: Department of Chemical Engineering and Center for Molecular and Engineering Thermodynamics, University of Delaware, Newark, Delaware 19716, USA.
authors.forename: Rebecca K
authors.initials: RK
authors.lastname: Brummitt
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Nesta
authors.forename: Liuquan
authors.initials: L
authors.lastname: Chang
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Kroetsch
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Roberts
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D052160:Benzothiazoles; D001702:Biopharmaceutics / Q000379:methods*; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D012542:Scattering, Radiation; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D013844:Thiazoles; C009462:thioflavin T