The effect of freezing conditions on freeze-drying process and characteristics of product containing a model protein substance

Liofilizacija je proces, v katerem vodo najprej zamrznemo, v procesu primarnega sušenja jo večinsko odstranimo s procesom sublimacije, med sekundarnim sušenjem pa z desorpcijo zmanjšamo njeno vsebnost na minimalno sprejemljivo vrednost. Omenjeni proces izvajamo na vzorcih z namenom odstranitve vode brez termične obremenitve vzorca, z namenom stabilizacije materiala in upočasnitve razgradnih reakcij, z namenom olajšanja transporta in podaljšanja roka uporabnosti proizvoda. Vsi proteini, ki jih liofiliziramo, se oblikujejo v formulaciji s pomožnimi snovmi, ki stabilizirajo in ščitijo protein pri obremenitvah med procesom ali pa omogočajo ustrezen izgled končnega izdelka. V našem primeru smo se osredotočili na optimizacijo faze zamrzovanja, ki je kritičnega pomena v liofilizacijskem procesu. Po zamrznitvi vzorca se ohranita struktura in oblika vzorca, z nastankom velikih kristalov ledu in posledično velikih por za sublimacijo vodne pare med sušenjem pa lahko vplivamo na skrajšanje časa primarnega sušenja, ki je stroškovno in časovno najpotratnejša faza liofilizacije. V generična liofilizacijska cikla smo vpeljali dodatna koraka temperiranja vzorca in vzdrževanja temperature podhladitve raztopine ter poizkušali optimizirati hitrost zamrzovanja. Primerjali smo procesne grafe vseh izvedenih liofilizacijskih ciklov, kjer nas je najbolj zanimal temperaturni profil produkta. Z nobenim izmed izvedenih ciklov nismo uspeli skrajšati časa primarnega sušenja v primerjavi z osnovnim konzervativnim ciklom, iz katerega smo izhajali, a smo vseeno dobili številne uporabne informacije s stališča optimizacije faze zamrzovanja in sestave formulacij. V ciklih z dodanima korakoma temperiranja vzorca in vzdrževanja podhladitve raztopine smo določili optimalni temperaturi vzdrževanja omenjenih faz pri dani sestavi formulacije. Vpliv temperiranja vzorca v našem primeru težje vrednotimo v agresivnem kot v konzervativnem liofilizacijskem ciklu. Podaljšanje časa temperiranja vzorca in povišanje deleža kristaliničnega polnila nista skrajšala časa primarnega sušenja. Tudi sprememba hitrosti ohlajanja v ciklih z vzdrževanjem podhladitve raztopine ni vplivala na skrajšanje časa sušenja. Cikel, v katerem smo združili koraka temperiranja vzorca in vzdrževanja podhladitve raztopine je dajal najobetavnejše rezultate s stališča temperaturnega profila produkta in lastnosti končnega izdelka. V vseh izvedenih ciklih smo dobili liofilizate z ustreznim izgledom pogače, sprejemljivimi časi rekonstitucije in optimalno vsebnostjo rezidualne vode. Liofilizati izkazujejo zadostno fizikalno in kemijsko stabilnost med shranjevanjem pri sobni temperaturi in celo višjih temperaturah.Freeze-drying, also known as lyophilization, is a process in which the water is first frozen and then, during primary drying, removed from the sample with the process of sublimation. During secondary drying, the water content is then reduced to the minimum acceptable value with desorption. This process is widely applied to the samples in order to remove the water from heat-sensitive compounds without excessive damage, to enhance product stability by reducing chemical or physical degradation reactions, to simplify transport and to improve long-term stability of the product. Nearly all freeze-dried pharmaceutical proteins are formulated in an excipient system, which allows protein stabilization against the stress of freeze-drying and satisfactory appearance of the final product. In this study, we focused on the freezing step, which is presumably the most critical step in the freeze-drying process. Moreover, the microstructure established by the freezing process usually represents the microstructure of the final dried product. With the formation of large ice crystals and large pores the primary drying can be accelerated, which with all aspects represents the costliest portion of the aforementioned process. Into the generic freeze-drying cycles, additional annealing and supercooling holding steps were introduced. Furthermore, investigation of the cooling rate was carried out. The comparison of the cycles was made especially in terms of product temperature profile. However, we couldn’t manage to significantly shorten the time of primary drying with none of the studied cycles, as compared to the conventional freeze-drying cycle from which we originated, but we got some useful information about optimizing the freezing step and formulation composition. We determined optimum maintaining temperature at the given formulation composition in the cycles with additional annealing and supercooling holding step. It was more difficult to evaluate the impact of annealing in aggressive than in conventional freeze-drying cycle in our study. Prolonging the annealing time and increasing the weight fraction of glycine didn’t shorten the time of primary drying. In addition, a change in the cooling rate in cycles with supercooling holding also didn’t shorten the drying time. The cycle in which we combined annealing and supercooling holding step showed the most promising results from the standpoint of the product temperature profile and the characteristics of the final product. The cycle resulted in the formation of dried samples with uniform cake appearance, acceptable reconstitution times and optimal residual moisture content. The lyophilized material consisted of adequate chemical or physical long-term stability during storage at ambient or even higher temperatures