Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations.
Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased β-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mw(agg)) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mw(agg) and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %).
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
Gregory V
GV
Barnett
Malvern Biosciences Inc., Columbia, MD 21046, USA.
Wei
W
Qi
Malvern Biosciences Inc., Columbia, MD 21046, USA.
Samiul
S
Amin
Malvern Biosciences Inc., Columbia, MD 21046, USA.
E
E
Neil Lewis
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA. Electronic address: cjr@udel.edu.
Christopher J
CJ
Roberts
D002919:Chymotrypsinogen / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D000067493:Dynamic Light Scattering; D006863:Hydrogen-Ion Concentration; D013059:Spectrum Analysis, Raman; D014783:Viscosity
D002919:Chymotrypsinogen
Aggregate mechanism; Protein aggregation; Protein structure; Viscosity