Razvoj in validacija stabilnostno indikativne analizne metode za določanje agregatov v zdravilu s peptidno zdravilno učinkovino

Tršek, Anja (author), Trontelj, Jurij (mentor), Trdan Lušin, Tina (comentor)

Terapevtski peptidi so poseben razred zdravilnih učinkovin, ki združujejo nekatere prednosti majhnih molekul in terapevtskih proteinov. Glavna terapevtska področja njihove uporabe so metabolne bolezni, onkologija in kardiovaskularne bolezni. Za zagotavljanje varnosti, kakovosti in učinkovitosti peptidnih zdravil regulativne agencije zahtevajo vrednotenje oligomernih in agregacijskih stanj. V ta namen se najpogosteje uporablja velikostno izključitvena kromatografija. Cilj magistrske naloge je bil razviti analizno metodo na osnovi velikostno izključitvene kromatografije za določanje agregatov v zdravilu s peptidno zdravilno učinkovino. Ključni želeni lastnosti metode sta bili zadostna občutljivost ter ločljivost med agregati in zdravilno učinkovino. Preizkusili smo različne kromatografske kolone ter proučili vpliv osnovnih kromatografskih pogojev: temperature kolone, pretoka in sestave mobilne faze. Ugotovili smo, da je bila za kakovostno ločbo ključna izbira kolone s čim manjšim premerom delcev stacionarne faze in ustrezno velikostjo por, poleg tega pa sta nanjo v veliki meri vplivala delež trifluoroocetne kisline in acetonitrila v mobilni fazi. Odločili smo se za dokončni razvoj metode na koloni, namenjeni UHPLC analizam. Optimizacija je vključevala povečanje temperature kolone, pretoka in deleža organskega modifikatorja propan-1-ola v mobilni fazi. Končno analizno metodo smo validirali v skladu z internim načrtom in kriteriji podjetja Lek d. d. Določili smo ponovljivost, rigidnost, mejo zaznave, mejo določitve, linearnost, točnost, robustnost in selektivnost. Validirano analizno metodo smo uporabili za določanje agregatov v stabilnostnih vzorcih proučevanega zdravila. Ugotovili smo, da vsebnost agregatov s temperaturo in časom narašča, s čimer smo potrdili uporabnost in primernost metode za spremljanje stabilnosti peptidne učinkovine v končnem izdelku.

• Language: Slovenian
• Keywords: terapevtski peptidi, agregati, regulativne zahteve, velikostno izključitvena kromatografija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-160270
• Publication date in RUL: 24.08.2024

Secondary language

• Language: English
• Title: Development and validation of a stability-indicating analytical method for determination of aggregates in a medicinal product with a peptide drug substance

Abstract

Therapeutic peptides are a special class of medicinal agents that combine certain advantages of small molecules and therapeutic proteins. Their main therapeutic areas of application include metabolic diseases, oncology, and cardiovascular diseases. To ensure the safety, quality, and efficacy of peptide drugs, regulatory agencies require the evaluation of oligomeric and aggregation states. For this purpose, size-exclusion chromatography is most commonly used. The aim of this master's thesis was to develop a stability-indicating analytical method based on size-exclusion chromatography for determination of aggregates in a medicinal product with a peptide drug substance. The key desired properties of the method were sufficient sensitivity and resolution between the aggregates and the drug substance. We tested different chromatographic columns and studied the influence of basic chromatographic conditions: column temperature, flow rate, and mobile phase composition. We found that the key to quality separation was to choose a column with the smallest possible particle diameter of the stationary phase and appropriate pore size. Additionally, the content of trifluoroacetic acid and acetonitrile in the mobile phase had a significant impact. We decided to finalize the method development on a column intended for UHPLC analyses. Optimization involved increasing the column temperature, the flow rate and the content of the organic modifier propan-1-ol in the mobile phase. The final analytical method was validated according to the internal plan and criteria of Lek d. d. We determined repeatability, rigidity, limit of detection, limit of quantification, linearity, accuracy, robustness and selectivity. The validated analytical method was then used to determine the content of aggregates in stability samples of the studied drug product. The content of aggregates was found to increase with temperature and time, thus confirming the usefulness and suitability of the method for monitoring the stability of the peptide drug substance in the studied drug product.

• Keywords: therapeutic peptides, aggregates, regulatory requirements, size-exclusion chromatography