Biology subjects

Thorlaksen, C.

Publications and source records attributed to Thorlaksen, C..

3 recordsLinked to original sources

bioRxiv2022

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 {micro}m) aggregates were found to be more immunogenic than sub-micron (0.1-1 {micro}m) 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 provides a universal workflow for reliable particle analysis in biotherapeutics.

immunology↗

bioRxiv2022

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 related to e.g., 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 a solvent 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 solvent 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 solvent exchange, 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.

biophysics↗

bioRxiv2020

Label free fluorescence quantification of hydrolytic enzyme activity on native substrates reveal 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 industry. Due to 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 measurement of pH variations or visible changes on material properties, like hydration, and often require high amount 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 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 enzymatic reaction is charged.

biochemistry↗