A study of protein aggregation processes using Dynamic Light Scattering : Validation of the technique and experimental trial with an active pharmaceutical ingredient

Protein pharmaceuticals is one of the fastest growing class of therapeutics today. However, they pose a lot of challenges in production lines due to their poor stability. Protein aggregation is one of the most common results of protein instability and is a risk factor regarding the quality of therapeutics. This master thesis at RISE focused on validating the techniques Dynamic Light Scattering (DLS) and multi angle DLS (MADLS) with respect to detection of aggregation. The model protein B-lactoglobulin was used to assess the robustness and accuracy of DLS. A comparison between two instruments from Malvern, Zetasizer Nano (2006) and Zetasizer Ultra (2018) was done with respect to DLS. It was determined that they were in many ways equivalent, but the newer model Ultra was favourable due to reduced noise and its ability to detect a lower concentration of aggregates. MADLS produced more precise results which is reflected in narrower distributions and has a higher sensitivity than DLS with regards to separating particles near in size. Both techniques proved sensitive enough to differentiate between aggregates and native protein. Experimental trials were performed with an active pharmaceutical ingredient, API. The experimental trials with the API aimed to investigate what conditions and surface-interfaces that might pose a risk for aggregation. Despite efforts put in creating an environment where aggregation could be monitored, aggregation could not be established. Measurements with the API generated less reliable results due to noisy data and a lack of reproducibility between individual measurements

A study of protein aggregation processes using Dynamic Light Scattering : Validation of the technique and experimental trial with an active pharmaceutical ingredient

Protein pharmaceuticals is one of the fastest growing class of therapeutics today. However, they pose a lot of challenges in production lines due to their poor stability. Protein aggregation is one of the most common results of protein instability and is a risk factor regarding the quality of therapeutics. This master thesis at RISE focused on validating the techniques Dynamic Light Scattering (DLS) and multi angle DLS (MADLS) with respect to detection of aggregation. The model protein B-lactoglobulin was used to assess the robustness and accuracy of DLS. A comparison between two instruments from Malvern, Zetasizer Nano (2006) and Zetasizer Ultra (2018) was done with respect to DLS. It was determined that they were in many ways equivalent, but the newer model Ultra was favourable due to reduced noise and its ability to detect a lower concentration of aggregates. MADLS produced more precise results which is reflected in narrower distributions and has a higher sensitivity than DLS with regards to separating particles near in size. Both techniques proved sensitive enough to differentiate between aggregates and native protein. Experimental trials were performed with an active pharmaceutical ingredient, API. The experimental trials with the API aimed to investigate what conditions and surface-interfaces that might pose a risk for aggregation. Despite efforts put in creating an environment where aggregation could be monitored, aggregation could not be established. Measurements with the API generated less reliable results due to noisy data and a lack of reproducibility between individual measurements

Optimering av en aerob biorening med avseende på minimering av filament

Deficiencies in biotreatment due to uncontrolled growth of filamentous microorganisms is a problem for wastewater plants worldwide. The project was assigned with the task of reducing the growth of filamentous bacteria at the paper mill SCA Munksund. 12 bacteria and 1 fungi species were presented to the project as problematic due to their filamentous properties. Operating parameters affecting filamentous growth was examined and presented. The information gathered showed that a decrease in filamentous growth for 5 especially problematic bacteria could possibly be achieved by raising the DO, increasing the concentration of nutrients and decreasing the sludge age. Other parameters affecting the growth of filamentous bacteria, such as pH, temperature and F/M were found to be too diverse among the examined species. Alternative solutions not focused on finding optimal operating parameters was also investigated. Promising results were treatment using a feast-famine approach, installation of an anaerobic selector and ozonation of return sludge. A statistical analysis was also performed on operating data. Methods used were Principal component analysis, Clustering and Logistic regression. These were used as a proof of concept rather than providing meaningful data for an operating window

Delineating functional and molecular impact of ex vivo sample handling in precision medicine

Consistent handling of samples is crucial for achieving reproducible molecular and functional testing results in translational research. Here, we used 229 acute myeloid leukemia (AML) patient samples to assess the impact of sample handling on high-throughput functional drug testing, mass spectrometry-based proteomics, and flow cytometry. Our data revealed novel and previously described changes in cell phenotype and drug response dependent on sample biobanking. Specifically, myeloid cells with a CD117 (c-KIT) positive phenotype decreased after biobanking, potentially distorting cell population representations and affecting drugs targeting these cells. Additionally, highly granular AML cell numbers decreased after freezing. Secondly, protein expression levels, as well as sensitivity to drugs targeting cell proliferation, metabolism, tyrosine kinases (e.g., JAK, KIT, FLT3), and BH3 mimetics were notably affected by biobanking. Moreover, drug response profiles of paired fresh and frozen samples showed that freezing samples can lead to systematic errors in drug sensitivity scores. While a high correlation between fresh and frozen for the entire drug library was observed, freezing cells had a considerable impact at an individual level, which could influence outcomes in translational studies. Our study highlights conditions where standardization is needed to improve reproducibility, and where validation of data generated from biobanked cohorts may be particularly important.Peer reviewe

Targeting SAMHD1 with hydroxyurea in first-line cytarabine-based therapy of newly diagnosed acute myeloid leukaemia : Results from the HEAT-AML trial

Background: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. Objectives: In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. Methods: Nine patients were enrolled and received at least two courses of ara-C (1 g/m2/2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1–2 g d1-5) and daunorubicin (60 mg/m2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. Results: The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level 1.0 × 109/L and to platelet recovery >50 × 109/L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9–20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. Conclusion: Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study