Impact of process conditions and composition of formulation with model protein substance on the crystallization of mannitol during lyophilization

Liofilizacija je tehnološki proces, ki se v farmaciji pogosto uporablja pri proizvodnji bioloških zdravil s proteinsko učinkovino. Omogoča nam sušenje termolabilnih snovi z namenom izboljšanja njihove stabilnosti in olajšanega rokovanja z njimi. Pri procesu liofilizacije material najprej zamrznemo, nato pa sledi primarno sušenje, med katerim pri znižanem tlaku odstranimo zamrznjeno vodo s sublimacijo. Za tem med sekundarnim sušenjem odstranimo še preostalo vodo z desorpcijo. Pri liofilizaciji uporabljamo različne pomožne snovi s katerimi stabiliziramo protein ter polnila za zagotovljanje ustreznega videza in lastnosti liofilizata. Eno najpogosteje uporabljenih polnil je manitol. Manitol med liofilizacijo kristalizira. Obstaja lahko v več polimorfnih oblikah, med katerimi je tudi psevdopolimorf manitol hemihidrat (MHH). Gre za metastabilno obliko in če je le-ta prisotna v končnem produktu, lahko pride do sprostitve vezane vode, kar lahko vpliva na kakovost izdelka. Namen magistrskega dela je bil proučiti vplive procesnih pogojev in sestave formulacije na kristalizacijo manitola, predvsem na tvorbo MHH. Izvedli smo osem liofilizacijskih ciklov, v katerih smo spreminjali temperaturo posamezne faze procesa. Formulacije, ki smo jih uporabili, so poleg proteina vsebovale manitol v različnih kombinacijah s saharozo. Z uporabo rentgenske praškovne difrakcije in termične analize smo določili prisotnost polimorfov manitola v dobljenih liofilizatih. S tem smo ocenili vplive na kristalizacijo manitola in posledično tudi na lastnosti končnega produkta. Te smo dodatno ovrednotili še z drugimi metodami, kot so merjenje rekonstitucijskega časa, določanje specifične površine z BET analizo, vsebnosti vode s KF metodo in določanje morfoloških značilnosti s SEM. Ugotovili smo, da manitol med liofilizacijo v prisotnosti proteina kristalizira predvsem kot &#948oblika, pa tudi kot manitol hemihidrat. Največ MHH nastane predvsem pri nižjih deležih manitola, kjer imamo v vzorcu večji delež amorfnih komponent. Vpliv proteina na kristalizacijo manitola je bolj kompleksen, saj do neke mere zavira kristalizacijo hemihidrata in favorizira kristalizacijo &#948oblike, pri višjih koncentracijah proteina pa lahko zaznamo porast MHH. Ugotovili smo, da manitol ob odsotnosti temperiranja kristalizira kot &#948oblika, pri višji temperaturi temperiranja (-8 °C) pa nastane manj MHH, kot pri -20 °C. Temperatura primarnega sušenja na kristalizacijo manitola ni imela bistvenega vpliva. Hemihidrat, ki nastane med procesom, lahko odstranimo z višjo temperaturo med sekundarnim sušenjem (45 °C).Lyophilization, also known as freeze-drying, is a technological procedure, commonly used in the production of biopharmaceuticals for drying of formulations with thermo-labile protein substances to improve stability of the final product. The procedure consists of three phases: freezing, primary drying and secondary drying. In primary drying, frozen water is removed by sublimation, while in secondary drying, residual non-frozen water is removed by desorption. Freeze-drying of proteins demands the use of different excipients that stabilize the protein and bulking agents that ensure the elegant appearance of the lyophilized cake. One of the most commonly used bulking agents is mannitol. Mannitol can crystallize into different polymorphs during the freeze-drying process and one of those is metastable mannitol hemihydrate (MHH). Emergence of MHH can reduce the stability of the product due to the spontaneous liberation of water bonded in its structure. Because of that, we want to avoid its presence in the final product. The aim of this thesis was to study the impact of process conditions and formulation composition on mannitol crystallization during lyophilization, especially on the emergence of MHH. We used five different formulations that all contained protein and mixture of mannitol and sucrose and performed eight cycles that differed from each other in temperature during annealing, primary drying and secondary drying phase. The mannitol polymorphs in lyophilizates were studied using X-ray powder diffraction and thermal analysis. Additionally, we performed different analyses to determine the quality of the cake, including measuring of reconstitution time, residual water content with Karl-Fischer method, BET analysis and scanning electron microscopy. The obtained results showed that during freeze-drying mannitol crystalized mainly as &#948form and also as MHH. Quantity of MHH was highest in samples which contained lower fraction of mannitol and highest fraction of amorphous components. Protein had a complex impact on mannitol crystallization: in low concentration, it inhibited crystallization of MHH and crystallization of &#948form was favored, while at high concentration of protein, an increase in MHH content was observed. Less hemihydrate emerged during annealing at -8 °C compared to standard cycle with annealing at -20 °C. Absence of the annealing step resulted in crystallization of &#948polymorph. Temperature of the primary drying phase did not have significant impact on mannitol crystallization. We were also able to reduce the hemihydrate content with higher temperature during secondary drying (45 °C)