TY - JOUR

T1 - Morphological integrity of insulin amyloid-like aggregates depends on preparation methods and post-production treatments

AU - Thorlaksen, Camilla

AU - Stanciu, Adriana-Maria

AU - Neergaard, Martin Busch

AU - Hatzakis, Nikos S.

AU - Fodera, Vito

AU - Groenning, Minna

PY - 2022

Y1 - 2022

N2 - 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 beta-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.

AB - 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 beta-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.

KW - Spherulites

KW - Amyloid-like fibrils

KW - Human insulin

KW - Aggregates

KW - Solution exchange

KW - MONOCLONAL-ANTIBODY

KW - IMMUNOGENICITY

KW - FIBRILS

KW - FIBRILLATION

KW - HETEROGENEITY

KW - POLYMORPHISM

KW - SPHERULITES

KW - PARTICLES

KW - MECHANISM

KW - KINETICS

U2 - 10.1016/j.ejpb.2022.08.018

DO - 10.1016/j.ejpb.2022.08.018

M3 - Journal article

C2 - 36058445

VL - 179

SP - 147

EP - 155

JO - European Journal of Pharmaceutics and Biopharmaceutics

JF - European Journal of Pharmaceutics and Biopharmaceutics

SN - 0939-6411

ER -