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- Title
Insulin nucleation kinetics in an oscillatory flow-based platform: Protein crystallization as a highly reproducible separation process.
- Authors
Ferreira, Joana; Araújo, Sofia; Ferreira, António; Teixeira, José; de Campos, João Moreira; Rocha, Fernando; Castro, Filipa
- Abstract
Insulin is a human therapeutic protein that has been used to treat diabetes for almost a century. Its crystallization has been of significant interest since the protein is commonly administered by subcutaneous injections of crystalline formulations, where crystal size distribution (CSD) has a critical role in product release and injectability. Herein, insulin crystallization was investigated in a unique platform based on oscillatory flow mixing technology. Assays were carried out at different supersaturation ratios (i.e. , insulin concentrations) in the presence and absence of acetone, and turbidity of the crystallization solution was monitored over time by UV-Vis spectrophotometry. The results show the impact of both supersaturation ratio and acetone on nucleation kinetics, as well as on CSD and growth rate. As the initial supersaturation increases, the nucleation rate increases, and the growth rate and mean crystal size decrease. The presence of acetone allows a faster nucleation event, a narrower CSD and a larger mean crystal size. The kinetic parameter A derived from the Classical Nucleation Theory (CNT) also indicates the kinetics of molecular attachment acceleration in the presence of acetone. These findings contribute to a better understanding of insulin crystallization processes under oscillatory flow. Thus, the described strategy and oscillatory flow-based systems are very promising for optimizing protein crystallization processes to be used during the downstream separation of bioproducts. [Display omitted] • Standardized apparatus for in-line monitoring and investigation of crystallization processes. • Unique platform for protein crystallization experimentation at the meso scale. • Experimental determination of nucleation kinetics and growth rate. • Acetone plays a major role in insulin nucleation kinetics. • Short operation time, narrow CSD, and high crystallization process yield.
- Subjects
SUPERSATURATION; INSULIN; NUCLEATION; CRYSTALLIZATION; INSULIN receptors; CRYSTALLIZATION kinetics; RATE of nucleation; DISCONTINUOUS precipitation; SUBCUTANEOUS injections
- Publication
Chemical Engineering Research & Design: Transactions of the Institution of Chemical Engineers Part A, 2024, Vol 203, p453
- ISSN
0263-8762
- Publication type
Article
- DOI
10.1016/j.cherd.2024.01.057