Abstract
Being able to measure the size and distribution of oligomers in solution is a critical issue in the manufacture and stability of insulin and other protein formulations. Measuring oligomers reliably can however be complicated, due to their fragile self-assembled structures, which are held together by weak forces. This can cause issues in chromatographic based methods, where dissociation or re-equilibration of oligomer populations can occur e.g. upon dilution in a different eluting buffer, but also for light scattering based methods like dynamic light scattering (DLS) where the size difference involved (often less than a factor 3) does not allow mixtures of oligomers to be resolved. Intrinsic fluorescence offers an attractive alternative as it is non-invasive, sensitive but also because it contains scattered light when implemented via excitation emission matrix (EEM) measurements, that is sensitive to changes in particle size.Here, using insulin at formulation level concentrations, we show for the first time how EEM can both discriminate and quantify the proportion of oligomeric states in solution. This was achieved by using the Rayleigh scatter (RS) band and the fluorescence signal contained in EEM. After validating size changes with DLS, we show in particular how the volume under the RS band correlated linearly with protein/oligomer molecular weight, in agreement with the Debye-Zimm relationship. This was true for the RS data from both EEM and polarized EEM (pEEM) measurements, the latter providing a stronger scatter signal, more sensitive to particle size changes.The fluorescence signal was then used with multivariate curve resolution (MCR) to quantify more precisely the soluble oligomer composition of insulin solutions. In conditions that promoted the formation of mainly one type of oligomer (monomer, dimer, or hexamer), pEEM-MCR helped identify the presence of small amounts of other oligomeric forms, while in conditions that were previously said to favour the insulin tetramer, we show that in the presence of zinc, these insulin samples were instead a heterogenous mixture composed of mostly dimers and hexamers. These MCR results correlated in all cases with the observed discrimination by principal component analysis (PCA), and deviations observed in the RS data. In conclusion, using pEEM scatter and emission components with chemometric data analysis provides a unique analytical method for characterising and monitoring changes in the soluble oligomeric state of proteins.
Highlights
Protein aggregation remains one of the biggest challenges affecting the manufacture and safe use of biopharmaceuticals, altering bioavailability [1] and potentially increasing the risk of adverse immune responses [2]
In conditions that promoted the formation of mainly one type of oligomer, polarized EEM (pEEM)-multivariate curve resolution (MCR) helped identify the presence of small amounts of other oligomeric forms, while in conditions that were previously said to favour the insulin tetramer, we show that in the presence of zinc, these insulin samples were instead a heterogenous mixture composed of mostly dimers and hexamers
The use of pEEM measurements was better at discriminating both types and mixtures of oligomers, because both the fluorescence emission and the Rayleigh scatter were sensitive to changes in protein structure
Summary
Protein aggregation remains one of the biggest challenges affecting the manufacture and safe use of biopharmaceuticals, altering bioavailability [1] and potentially increasing the risk of adverse immune responses [2]. The most difficult and important to measure are the small (nm) soluble oligomers, which can be formed by self-assembly from either a partially unfolded state as suspected in amyloid related diseases, or from a protein in its native state as frequently observed biologically via various environmental factors [6]. These often lack covalent linkages and instead are held together by a complex network of weak forces such as hydrophobic interactions, van der Waals forces, electrostatic, ionic interactions, and hydrogen bonds. Oligomers are fragile structures that exist in delicate equilibrium with other species, making their accurate measurement in solution difficult, especially if small size changes are involved
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
