doktorska disertacija
Ema Valentina Brovč (Avtor), Stane Pajk (Mentor), Janez Mravljak (Komentor)

Povzetek

Tekom razvoja končnih biofarmacevtskih izdelkov je glavni izziv razviti varno, učinkovito in kakovostno zdravilo s čim daljšim rokom uporabe. Večina bioloških zdravil na tržišču je terapevtskih proteinov, ki so v primerjavi s klasičnimi zdravili manj stabilni. Proteini so podvrženi kompleksnim razgradnim potem predvsem zaradi njihove velikosti in raznolike strukture. Z namenom, da bi jih zaščitili pred stresom na medfaznih površinah, in z namenom preprečitve agregacije in denaturacije proteinov v vodnih raztopinah, v formulacije dodajamo površinsko aktivne snovi. Polisorbati so najpogosteje uporabljene površinsko aktivne snovi v farmacevtski industriji in so prisotni v več kot 80 % komercialno dostopnih formulacijah z monoklonskimi protitelesi. V okviru doktorskega dela smo celovito ovrednotili polisorbate kot stabilizatorje bioloških zdravil. Razvili in optimizirali smo analizno metodo tekočinske kromatografije ultra visoke ločljivosti sklopljene z masno spektrometrijo visoke ločljivosti, ki omogoča nadzor kakovosti vhodnih polisorbatov, identifikacijo posameznih zvrsti polisorbatov, primerjavo med različnimi kakovostmi in proizvajalci polisorbatov, identifikacijo nečistot v vzorcih polisorbatov in spremljanje nastanka njihovih razgradnih produktov. Proste maščobne kisline so pomemben pokazatelj kakovosti intaktnih polisorbatov, indikator njihove razgradnje in eden od glavnih razlogov za tvorbo delcev v formulacijah s proteini. Ker je trenutno edina farmakopejska metoda, ki zazna prisotnost prostih maščobnih kislin v polisorbatih, nespecifična metoda za določanje kislinskega števila, smo razvili hitro in enostavno metodo jedrske magnetne resonance za kvantitativno določitev prostih maščobnih kislin v polisorbatih. Preučili smo vpliv sestave formulacije na stabilnost polisorbatov, zato smo spremljali njihovo stabilnost v najpogosteje uporabljenih biofarmacevtskih pufrih in vrednotili vpliv proteinov ter ostalih pomožnih snovi na njihovo razgradnjo. Ker so polisorbati podvrženi hidrolizi in oksidaciji, smo pojasnili vzroke in mehanizme za razpad polisorbatov v histidinijevem kloridnem pufru. S pomočjo razgradnih produktov smo odkrili do sedaj še neznano razgradno pot polisorbatov: aminolizo. Preučili smo histidinske purfne sisteme z različnimi protiioni in identificirali protiione, ob katerih ne poteče s histidinom katalizirana hidroliza polisorbatov. Mehanizem razgradnje polisorbatov v histidinskem pufru smo potrdili tudi s pomočjo kvantno mehanskih izračunov. Preverili smo smiselnost dodajanja kelatorjev v formulacije s polisorbati in terapevtskimi proteini. Z askorbatnim testom in peptidnim kartiranjem smo dokazali, da nekateri kelatorji v kombinaciji s kovinskim ioni, kisikom in askorbatom oksidacijo proteinov celo dodatno pospešijo. Zato mora biti odločitev za dodatek kelatorjev podprta z ustreznimi eksperimentalnimi rezultati in ne del običajne prakse. Na osnovi podatkov o stabilnosti polisorbatov, njihovih razgradnih poteh in vplivu ostalih pomožnih snovi v formulaciji, bo načrtovanje formulacij s polisorbati veliko bolj racionalno.

Ključne besede

polisorbati;razgradnja;razvoj formulacij;interakcije polisorbatov;liofilizacija formulacij;stabilnost polisorbatov;disertacije;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [E. V. Brovč]
UDK: 543.544:615.32(043.3)
COBISS: 73154307 Povezava se bo odprla v novem oknu
Št. ogledov: 140
Št. prenosov: 0
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Development and implementation of the analytical methods to study composition and degradation of polysorbates in media and formulations of therapeutic proteins
Sekundarni povzetek: During biopharmaceuticals development programme the main challeng is to develop safe, effective and high-quality drug product that have as long shelf-life as possible. The vast majority of biopharmaceuticals on the market are therapeutic proteins, and in contrast to conventional pharmaceuticals, these formulations are less stable. They have complex degradation pathways, mainly due to the large sizes and diverse structures of these proteins. To protect them from interfacial stress, and to counter their aggregation and denaturation in aqueous solutions, surfactants are added to these formulations. Polysorbates are the most commonly used surfactants in the pharmaceutical industry, and they are included in more than 80% of commercial monoclonal antibody formulations. As part of my doctoral project, polysorbates were comprehensively evaluated as stabilisers of biopharmaceuticals. Ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry was developed and optimised for quality control of input polysorbates, identification of individual polysorbate species, comparisons between different polysorbate qualities across different sources, identification of polysorbate impurities, and monitoring the formation of polysorbate degradation products. Free fatty acids are important indicators of the quality of intact polysorbates and their degradation, which is one of the main reasons for particle formation in protein formulations. As the only current pharmacopoeia method for evaluation of free fatty acids in polysorbates is a non-specific method that determines the acid value, a rapid and simple nuclear magnetic resonance method for quantitative evaluation of free fatty acids in polysorbates was designed and set up. The effects of formulation composition on the stability of polysorbates were also evaluated. The stabilities of polysorbates in the buffers most commonly used in biopharmaceuticals were determined and the influence of proteins and other excipients on the degradation of polysorbates was investigated. While polysorbates can undergo hydrolysis and oxidation, the mechanisms for polysorbates degradation in a histidine chloride buffer were further investigated. According to the polysorbates degradation products, a new degradation pathway of polysorbates was defined: aminolysis. Histidine buffer systems with different counterions were investigated and counterions which prevented histidine catalyzed hydrolysis of polysorbates were identified. The degradation mechanism of polysorbates in histidine buffer was also confirmed by quantum mechanics calculations. The addition of chelators to formulations with polysorbates and therapeutic proteins was examined. The use of an ascorbate redox system assay and peptide mapping demonstrated that in combination with metal ions, oxygen and ascorbate, some chelators can even accelerate the protein oxidation, instead of preventing it. Therefore, inclusion of chelators in such pharmaceutical formulations should be based on experimental data, and not on »common practice«. Based on the polysorbates stability data and their degradation pathways, and based on the influence of other excipients on polysorbates degradation in such formulations, their design can now follow a more rational approach.
Sekundarne ključne besede: Biološka zdravila;Analizna kemija;Kromatografska analiza;Jedrska magnetna resonanca;Tekočinska kromatografija;
Vrsta dela (COBISS): Doktorska disertacija
Komentar na gradivo: Univ. v Ljubljani, Fak. za farmacijo
Strani: 230 str.
ID: 15504089