Exploring Liquid Chromatography Method Development Strategies: Enhancing Analytical Performance in Pharmaceutical Applications
Typ
Examensarbete för masterexamen
Master's Thesis
Master's Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2024
Författare
Habibollahi, Porya
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
From discovery to the final phase of drug development, Liquid Chromatography (LC) is considered one of the core separation techniques. Its broad capabilities in identifying and quantifying complex compound mixtures find application in quality control, formulation studies, pharmacokinetics, environmental monitoring, and purity determination [1]. This 60-credit thesis aims to develop and optimize LC methods through exploration of three individual studies, each detailed in separate chapters.Chapter one is focused on evaluating separation performance under various conditions using a selected compound mixture as reference. This study examined factors such as buffer selection, mobile phase composition, and instrument compatibility based on the separation of these reference compounds. Despite high separation capability of trifluoroacetic acid (TFA) due to its strong ion pairing strength, its significant drawbacks include signal suppression in mass spectrometry (MS) and environmental impacts. The obtained results suggest that difluoroacetic acid (DFA) and ammonium acetate (AA) by showing acceptable performance, can be potential replacements for TFA. In chapter two, the LC method development process for separating impurities of metoprolol was examined. Fusion QbD software was employed in this section to assist in the design of experiments (DoE), analysis of screening experiments, and optimization of the separation methods. Ultimately, the investigation revealed a method using a 10 mM potassium phosphate buffer with a pH of 8.1 and a gradient from 15 to 42% acetonitrile over 7 minutes at 50°C
yielded the most optimal separation of the target compound from its impurities. Furthermore, the study compared the performance of an MS-friendly buffer, ammonium acetate, with potassium buffers based on the parameters of the optimized method.In the final chapter, an optimized experimental procedure for assessing the stability of Inclisiran, a commercial siRNA used for reducing low-density lipoprotein cholesterol (LDL C), was studied. This exploration focused on measuring the melting point (Tm) and involved testing various parameters such as the number of thermal cycles, the effectiveness of silicone oil, and the choice of temperature ramp. In the end, it was demonstrated that setting only one cycle, adding 10 µL of silicone oil, and using a temperature ramp of 0.5°C/min were the optimal parameters for obtaining reliable data. On the other hand, experiments assessing Inclisiran stability in the TEA/HFIP system revealed that TEA concentration did not significantly impact stability at 5 and 15 mM, but adverse effects were observed at 100 mM TEA, resulting in a lowering of the melting point by 11.6°C. LC separation experiments provided additional insights into Inclisiran's behaviour, showing complete denaturation at 65°C but non denaturation conditions at lower temperatures.
Beskrivning
Ämne/nyckelord
Liquid Chromatography, Analytical Method Development, Alternative LC Buffers, Fusion QbD, Metoprolol Impurity Profiling, Inclisiran Stability, siRNA Melting Temperature