In vitro and in vivo immunogenicity assessment of protein aggregate characteristics.

The immunogenicity risk of therapeutic protein aggregates has been extensively investigated over the past decades. While it is established that not all aggregates are equally immunogenic, the specific aggregate characteristics, which are most likely to induce an immune response, remain ambiguous. The aim of this study was to perform comprehensive in vitro and in vivo immunogenicity assessment of human insulin aggregates varying in size, structure and chemical modifications, while keeping other morphological characteristics constant. We found that flexible aggregates with highly altered secondary structure were most immunogenic in all setups, while compact aggregates with native-like structure were found to be immunogenic primarily in vivo. Moreover, sub-visible (1-100 µm) aggregates were found to be more immunogenic than sub-micron (0.1-1 µm) aggregates, while chemical modifications (deamidation, ethylation and covalent dimers) were not found to have any measurable impact on immunogenicity. The findings highlight the importance of utilizing aggregates varying in few characteristics for assessment of immunogenicity risk of specific morphological features and may provide a workflow for reliable particle analysis in biotherapeutics.

Reproducible Formation of Insulin Superstructures: Amyloid-Like Fibrils, Spherulites, and Particulates.

Inducing protein aggregation in vitro under various formulation and stress conditions may lead to an increased understanding of the different association routes a protein can undergo. However, a range of factors can affect the aggregation process, often leading to heterogenous samples and experimental irreproducibility between labs. Here, we present detailed methods to reproducibly form homogenous samples of superstructures: amyloid-like fibrils, spherulites, and particulates from human insulin. We discuss pitfalls and good practice in the lab, with the aim of creating awareness on the potential sources of artefacts for protein stability and aggregation studies.

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.

Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature.

Lipases are important hydrolytic enzymes used in a spectrum of technological applications, such as the pharmaceutical and detergent industries. Because of their versatile nature and ability to accept a broad range of substrates, they have been extensively used for biotechnological and industrial applications. Current assays to measure lipase activity primarily rely on low-sensitivity measurements of pH variations or visible changes of material properties, like hydration, and often require high amounts of proteins. Fluorescent readouts, on the other hand, offer high contrast and even single-molecule sensitivity, albeit they are reliant on fluorogenic substrates that structurally resemble the native ones. Here we present a method that combines the highly sensitive readout of fluorescent techniques while reporting enzymatic lipase function on native substrates. The method relies on embedding the environmentally sensitive fluorescent dye pHrodo and native substrates into the bilayer of liposomes. The charged products of the enzymatic hydrolysis alter the local membrane environment and thus the fluorescence intensity of pHrodo. The fluorescence can be accurately quantified and directly assigned to product formation and thus enzymatic activity. We illustrated the capacity of the assay to report the function of diverse lipases and phospholipases both in a microplate setup and at the single-particle level on individual nanoscale liposomes using total internal reflection fluorescence (TIRF). The parallelized sensitive readout of microscopy combined with the inherent polydispersity in sizes of liposomes allowed us to screen the effect of membrane curvature on lipase function and identify how mutations in the lid region control the membrane curvature-dependent activity. We anticipate this methodology to be applicable for sensitive activity readouts for a spectrum of enzymes where the product of the enzymatic reaction is charged.