enoviti magistrski študij farmacija
Gašper Vrhunc (Author), Pegi Ahlin Grabnar (Mentor), Boris Brus (Co-mentor)

Abstract

Liofilizacija je proces sušenja, pri katerem raztopino najprej globoko zamrznemo, nato pa s procesoma sublimacije in desorpcije iz nje odstranimo topilo, da dobimo suh produkt v obliki pogače. Glede na to, da je liofilizacija časovno in energetsko potraten proces, je njegova optimizacija bistvenega pomena. Raziskave kažejo, da lahko z izbiro ustreznih pomožnih snovi, kot so stabilizatorji in polnila, proteinske formulacije izpostavimo višjim temperaturam primarnega sušenja, s tem pa še vedno zagotovimo ustrezen videz izdelka z ohranjeno aktivnostjo učinkovine in kratkim časom rekonstitucije. Izbor procesnih parametrov (temperatura in tlak) temelji na poznavanju kritičnih temperatur formulacije, med katere uvrščamo temperaturo steklastega prehoda maksimalno koncentrirane zamrznjene raztopine (Tg'), temperaturo evtektičnega taljenja (Teu) in temperaturo kolapsa (Tc), ki jo lahko razdelimo še na temperaturo, pri kateri na mikroskopu z zamrzovanjem in sušenjem (FDM) opazimo prve spremembe v strukturi (Tconset), in temperaturo, kjer opazimo popoln kolaps (Tcfull). Dolgo časa je veljajo dejstvo, da sušenje nad Tcfull, vodi h kolapsu produkta, a novejše raziskave kažejo, da dodatek ustreznih polnil in stabilizatorjev zagotavlja ustrezen končni izdelek tudi v primeru, ko temperatura produkta (Tp), dosežena tekom faze primarnega sušenja, preseže Tcfull. Namen naloge je z višanjem tlaka in temperature polic primarnega sušenja (t.i. agresivni pogoji) proučiti vpliv Tp na pojav kolapsa ter tako definirati najvišjo še sprejemljivo temperaturo in tlak primarnega sušenja. Izmerjene Tp smo po koncu vsakega cikla primerjali z vnaprej določenimi kritičnimi temperaturami formulacij, določenimi z diferenčno dinamično kalorimetrijo (DSC) in FDM. Z namenom vrednotenja vpliva pomožnih snovi na pojav kolapsa smo liofilizirali tri formulacije z modelnim proteinom (monoklonsko protitelo), in sicer eno s 6% m/V amorfne saharoze in dve delno kristalinični z 2% m/V saharoze in 4% m/V glicina oziroma manitola. Po koncu vsakega cikla smo liofilizate vizualno ovrednotili, jim določili rekonstitucijski čas, velikost delcev v disperziji z metodo dinamičnega sipanja svetlobe ter ocenili morfološke lastnosti liofilizacijske pogače z metodo vrstične elektronske mikroskopije (SEM). Uvedba agresivnih pogojev sušenja je vodila k zvišanju Tp, pri čemer je bila v vseh agresivnih ciklih le-ta višja od kritične temperature Tg'. Kljub temu da je Tp pri formulaciji s saharozo presegla Tcfull že v začetnem agresivnem ciklu, smo kolaps liofilizacijske pogače opazili šele z nadaljnjim višanjem temperature polic in tlaka v komori (agresivni cikel 6), pri čemer je bila Tp 5,7 °C nad Tcfull. Slednje pripisujemo temu, da določanje Tc s FDM predstavlja le dobro oceno temperature, pri kateri dejansko dosežemo kolaps med samim procesom sušenja. Zaradi prisotnosti kristalne komponente (glicin/manitol) v primeru ostalih dveh formulacij kolapsa kljub še agresivnejšim pogojem sušenja nismo opazili. Z uporabo agresivnih ciklov smo čas primarnega sušenja skrajšali za kar 80% glede na osnovni konzervativni cikel in s tem uspešno povečali časovno in stroškovno učinkovitost procesa liofilizacije.

Keywords

liofilizacija;proteinska učinkovina;kritične temperature;kolaps;optimizacija liofilizacijskega cikla;

Data

Language: Slovenian
Year of publishing:
Source: Ljubljana
Typology: 2.09 - Master's Thesis
Organization: UL FFA - Faculty of Pharmacy
Publisher: [G. Vrhunc]
UDC: 542.47(043.3)
COBISS: 4569457 Link will open in a new window
Views: 338
Downloads: 64
Average score: 0 (0 votes)
Metadata: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Other data

Secondary language: English
Secondary title: Determination of the critical temperature of the formulations with a model protein substance during the lyophilization process
Secondary abstract: Lyophilization is a process of drying where the solution is deeply frozen and then the solvent is removed from it by the processes of sublimation and desorption. Given the fact that lyophilization is time and energy consuming process, its optimizaton is essential. Research show, that with a choice of appropriate excipients, such as stabilizers and bulking agents, protein formulation can be exposed to higher temperatures of primary drying. Even though product appearance is acceptable and activity of the active substance and short time of reconstitution are preserved. The choice of process parameters (temperature and pressure) is based on knowledge of critical temperatures of formulation, among which we classify glass transition temperature of the maximally freeze-concentrated solution (Tg'), temperature of eutectic melting (Teu) and collapse temperature (Tc), which can be subdivided into temperature at which the first changes in the structure are observed with the freeze-drying microscopy (Tconset) and temperature where full collapse is observed (Tcfull). For a long time a fact, that drying above Tcfull will result in full collapse of product, was applied, but newer studies show, that the addition of appropriate bulking agents and stabilizers provides acceptable cake appearance even when product temperature (Tp) during primary drying exceeds Tcfull. The purpose of this thesis is to study influence of Tp on product collapse with increasing of pressure and shelf temperature of primary drying (agressive conditions) and to define the highest accaptable temperature and pressure of primary drying. Measured Tps at the end of each cycle were compared to critical temperature of the formulation, which were defined by differental scanning calorimetry (DSC) and freeze-drying microscopy (FDM). With the purpose to evaluate impact of excipients on product collapse, three formulations with protein substance (monoclonal antibody) were lyophilized. One with 6% m/V of amorphous succrose and two partially crystaline with 2% m/V succrose and 4% m/V glycine and mannitol respectively. At the end of every cycle products were visually evaluated, reconstitution time and particle size were determinded and scanning electron microscopy (SEM) was preformed to estimate structure morphology. Initiating of agressive conditions of drying led to increased Tp, which was higher than Tg' in every agressive cycle. Even though Tp of formulation with succrose exceeded Tcfull already in the first agressive cycle, product collapse was seen when shelf temperature and pressure were further increased (agressive cycle 6), Tp was then 5,7 °C above Tcfull. The possible reason for this is, that Tc measured with FDM is only a good estimation of temperature, at which collapse is achieved during process of drying. The collapse of other formulations was not seen, because of presence of the crystal component (glycine/manitol). Using more agressive cycles primary drying time was reduced for 80% according to the conservative cycle and thereby time- and cost-efficiency of the lyophilization process were successfully increased.
Secondary keywords: lyophilization protein substance critical temperatures collapse lyophilization cycle optimization;
Type (COBISS): Master's thesis/paper
Thesis comment: Univ. Ljubljana, Fak. za farmacijo
Pages: X, 58 f.
ID: 12042774