title: Significance of unfolding thermodynamics for predicting aggregation kinetics: a case study on high concentration solutions of a multi-domain protein.
abstract: OBJECTIVE
  To enable aggregation rate prediction over a broad temperature range for complex multi-domain proteins at high concentrations.
  
  
  METHODS
  Thermal unfolding, non-isothermal kinetics and storage stability studies were conducted on a model multi-domain protein (MDP) at moderate to high concentrations (25-125 mg/mL) over a broad temperature range (4-40°C).
  
  
  RESULTS
  Storage stability studies indicated the aggregation of MDP in solution to be a second order process. Application of Arrhenius kinetics to accelerated stability data resulted in underestimation of the aggregation rate under refrigerated conditions. Additional studies undertaken to understand the mechanism of the aggregation process highlighted the association of the monomer (or the aggregation competent species) to be the rate-limiting step for aggregation over the temperature range studied. Thermal unfolding studies in the presence of urea were used to calculate the heat capacity change upon unfolding (Δcp,un). The resulting value of Δcp,un when used in the extended Lumry-Eyring model resulted in a more accurate and a conservative estimate of the aggregation rate under refrigerated condition. Some complicating factors for the aggregation rate prediction for multi-domain proteins at high concentration are discussed.
  
  
  CONCLUSIONS
  The work highlights (i) the significance of incorporating unfolding thermodynamics in protein aggregation rate prediction, (ii) the advantages and challenges associated with the use of the extended Lumry-Eyring (ELE) model for rate prediction and (iii) the utility of using the Arrhenius and the ELE models in tandem during product development.
authors.affiliation: Drug Product Science and Technology, Bristol-Myers Squibb Co., One Squibb Drive, New Brunswick, New Jersey, 08903, USA.
authors.forename: Atul
authors.initials: A
authors.lastname: Saluja
authors.forename: Vikram
authors.initials: V
authors.lastname: Sadineni
authors.forename: Amol
authors.initials: A
authors.lastname: Mungikar
authors.forename: Vishal
authors.initials: V
authors.lastname: Nashine
authors.forename: Andrew
authors.initials: A
authors.lastname: Kroetsch
authors.forename: Charles
authors.initials: C
authors.lastname: Dahlheim
authors.forename: Venkatramana M
authors.initials: VM
authors.lastname: Rao
mesh_terms: D000465:Algorithms; D004356:Drug Storage; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D058767:Protein Unfolding / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012996:Solutions; D013816:Thermodynamics
chemical_list: D011993:Recombinant Fusion Proteins; D012996:Solutions