Morphological integrity of insulin amyloid-like aggregates depends on preparation methods and post-production treatments.

Protein aggregates are often varying extensively in their morphological characteristics, which may lead to various biological outcomes, such as increased immunogenicity risk. However, isolation of aggregates with a specific morphology within an ensemble is often challenging. To gain vital knowledge on the effects of aggregate characteristics, samples containing a single morphology must be produced by direct control of the aggregation process. Moreover, the formed aggregates need to be in an aqueous solution suitable for biological assays, while keeping their morphology intact. Here we evaluated the dependence of morphology and integrity of amyloid-like fibrils and spherulites on preparation conditions and post-treatment methods. Samples containing either amyloid-like fibrils or spherulites produced from human insulin in acetic acid solutions are dependent on the presence of salt (NaCl). Moreover, mechanical shaking (600 rpm) inhibits spherulite formation, while only affecting the length of the formed fibrils compared to quiescent conditions. Besides shaking, the initial protein concentration in the formulation was found to control fibril length. Surprisingly, exchanging the solution used for aggregate formation to a physiologically relevant buffer, had a striking effect on the morphological integrity of the fibril and spherulite samples. Especially the secondary structure of one of our spherulite samples presented dramatic changes of the aggregated ß-sheet content after exchanging the solution, emphasizing the importance of the aggregate stability. These results and considerations have profound implications on the data interpretation and should be implemented in the workflow for both fundamental characterization of aggregates as well as assays for evaluation of their corresponding biological effects.

Subtle pH variation around pH 4.0 affects aggregation kinetics and aggregate characteristics of recombinant human insulin.

Insulin is a biotherapeutic protein, which, depending on environmental conditions such as pH, has been shown to form a large variety of aggregates with different structures and morphologies. This work focuses on the formation and characteristics of insulin particulates, dense spherical aggregates having diameters spanning from nanometre to low-micron size. An in-depth investigation of the system is obtained by applying a broad range of techniques for particle sizing and characterisation. An interesting observation was achieved regarding the formation kinetics and aggregate characteristics of the particulates; a subtle change in the pH from pH 4.1 to pH 4.3 markedly affected the kinetics of the particulate formation and led to different particulate sizes, either nanosized or micronsized particles. Also, a clear difference between the secondary structure of the protein particulates formed at the two pH values was observed, where the nanosized particulates had an increased content of aggregated ß-structure compared to the micronsized particles. The remaining characteristics of the particles were identical for the two particulate populations. These observations highlight the importance of carefully studying the formulation design space and of knowing the impact of parameters such as pH on the aggregation to secure a drug product in control. Furthermore, the identification of particles only varying in few parameters, such as size, are considered highly valuable for studying the effect of particle features on the immunogenicity potential.