Razvoj in optimizacija procesa liofilizacije formulacij z biološkimi učinkovinami - Bjelošević, Maja (2021)
V zadnjih letih so biološka in biološko podobna zdravila na farmacevtskem področju doživela velik razcvet, zato ne preseneča dejstvo, da na letni ravni predstavljajo največji obseg prodaje zdravil. Gre za skupino zdravil, ki kot aktivno zdravilno učinkovino vsebujejo različne proteinske molekule, pri čemer velja omeniti monoklonska protitelesa (mAb) kot eno najpomembnejših in najbolj zastopanih skupin. Biološke molekule kot nosilke zdravilnega učinka so spremenile dosedanje načine zdravljenja številnih bolezni in s tem prispevale k njegovemu napredku. Biološka in biološko podobna zdravila imajo številne prednosti pred klasičnimi sinteznimi zdravili, vendar pa je njihova proizvodnja v primerjavi s proizvodnjo klasičnih zdravil veliko bolj kompleksna in dolgotrajna. Zagotavljanje stabilnosti proteinskih molekul je bistvenega pomena, saj neposredno vpliva na varnost, kakovost in učinkovitost zdravila in tako predstavlja enega izmed najbolj kritičnih korakov procesa izdelave bioloških zdravil. Nestabilnost proteinskih molekul, skupaj z njihovo molekulsko maso, podvrženostjo encimski razgradnji in kratkim razpolovnim časom so razlogi za običajno zelo majhno biološko uporabnost bioloških makromolekul po peroralni aplikaciji, zato med biološkimi zdravili prevladujejo parenteralne farmacevtske oblike v obliki raztopin, suspenzij ali liofilizatov. Liofilizacija oz. sušenje z zamrzovanjem je tehnološki postopek, ki temelji na fizikalnem pojavu sublimacije, pri čemer z uporabo ustrezno nizkih temperatur in tlaka iz vzorca odstranimo vodo. V osnovi je liofilizacija proces, ki je sestavljen iz treh zaporednih, medsebojno prepletajočih se faz, in sicer sledita začetnemu zamrzovanju še primarno in sekundarno sušenje. Znano je dejstvo, da je liofilizacija dolgotrajen in energetsko visoko potraten proces, zato je za zagotavljanje upravičenosti stroškov nujno potrebna njena optimizacija. Slednja temelji predvsem na uporabi agresivnih pogojev primarnega sušenja kot najbolj dolgotrajne faze procesa, za optimizacijo pa je predpogoj ustrezna sestava formulacije. Pomožne snovi, kot so stabilizatorji, polnila, površinsko aktivne snovi in pufrne soli, predstavljajo osnovo formulacij s proteini, pri čemer z vidika optimizacije procesa igrajo bistveno vlogo polnila. Vključitev kristaliničnih polnil, kot sta manitol in glicin, v proteinske formulacije omogoča izvedbo primarnega sušenja pri temperaturah, višjih od kritičnih temperatur formulacij. Kristalinična polnila zagotavljajo ustrezno strukturo liofilizata in ohranitev kritičnih atributov kakovosti produkta, tudi če je med primarnim sušenjem presežena temperatura steklastega prehoda kritično koncentrirane zamrznjene raztopine (Tg') in v nekaterih najnovejših sistemih tudi temperatura kolapsa (Tc). Pozitivna vloga kristaliničnih polnil na področju liofilizacije je bila do sedaj raziskana predvsem v povezavi s proteinskimi molekulami z manjšo molekulsko maso, medtem ko je razumevanje mehanizmov delovanja polnil v povezavi z mAb še pomanjkljivo. Kljub temu da je intravenska aplikacija najpogostejši način dajanja zdravil z mAb, pa se v zadnjem času srečujemo s porastom formulacij za subkutano aplikacijo. Slednja izkazuje številne prednosti, med katerimi so najpomembnejše zmanjšanje stroškov oskrbe na račun manj pogoste aplikacije, ki jo v določenih primerih izvedejo kar bolniki sami, posledično pa se z uporabo tovrstnih zdravil povečuje komplianca s strani bolnikov. S tehnološkega vidika gre za pripravo formulacij, pri kateri se srečamo z mnogimi izzivi, predvsem zaradi majhnih volumnov, ki so posledica vrste aplikacije, zato na področju zdravil za subkutano uporabo, govorimo o visokokoncentriranih oblikah formulacij. Na tem mestu velja izpostaviti tudi povečano viskoznost raztopin za subkutano injiciranje, ki nemalokrat omejuje injektabilnost tako pripravljenih formulacij. Pomembno je, da se je uporaba liofilizacije razširila tudi na področje visokokoncentriranih formulacij, kjer se prav tako soočamo z vrsto procesnih izzivov. V prvem delu doktorske naloge smo se osredotočili na načrtovanje in optimizacijo liofilizacijskih ciklov. Pripravili smo dve različni formulaciji, in sicer formulacijo, ki je vsebovala samo saharozo in formulacijo, ki je poleg saharoze vsebovala tudi glicin (polnilo) ter ju liofilizirali pod agresivnimi in konzervativnimi pogoji. Z uvedbo agresivnih pogojev sušenja smo dokazali, da lahko primarno sušenje izvajamo pri višjih temperaturah in tlakih ter s tem dosežemo skrajšanje sušenja za do 54 %, brez vpliva na kritične lastnosti produkta, kot so videz liofilizata, rekonstitucijski čas ter delež rezidualne vlage. V naslednji stopnji smo ocenili vpliv agresivnih pogojev sušenja na stabilnost modelnega mAb in z različnimi analiznimi tehnikami ugotovili, da le-ti ne vplivajo na njegovo stabilnost. Z rezultati smo potrdili, da so agresivni pogoji ustrezna alternativa konzervativnim pogojem sušenja, pri čemer le-ti omogočajo skrajšanje primarnega sušenja in zagotavljajo ustrezne kritične atribute kakovosti produktov. V drugem delu smo proučili vpliv razmerja med mAb in stabilizatorjem (saharoza) in vpliv koncentracij mAb na stabilnost modelnega mAb. Osredotočili smo se na določanje fizikalne in kemijske stabilnosti testiranega mAb, in sicer pred in po liofilizaciji ter v okviru stresne stabilnostne študije. Najpogostejši pojav nestabilnosti proteinov se kaže v okviru fizikalne nestabilnosti, in sicer je najpogostejša oblika agregacija, kot posledica popolnega ali delnega razvitja proteinskih molekul. Na začetku smo pripravili formulacije s štirimi različnimi molarnimi razmerji mAb/ saharoza, in sicer 1:140, 1:280, 1:420 in 1:850 ter tremi različnimi koncentracijami mAb (10, 20 in 30 mg/mL). Pridobljeni rezultati kažejo, da večji deleži saharoze vodijo k boljši fizikalni in kemijski stabilnosti, in sicer takoj po procesu liofilizacije, kot tudi po 1 in 3 mesecih stresne stabilnostne študije. Nasprotno, z višanjem koncentracije mAb, narašča delež agregatov, pri čemer višja koncentracija mAb pomeni manj molekul saharoze na molekulo proteina. Enak trend smo opazili tudi pri vrednotenju kemijske stabilnosti, pri čemer se le-ta z zmanjševanjem molarnega razmerja med saharozo in mAb in zviševanjem koncentracije mAb, slabša. Nadalje smo v študijo vključili tudi višje koncentracije mAb (60, 90, 120, 150 mg/mL) in ugotovili, da ob primernem razmerju s saharozo, mAb med in po procesu liofilizacije ohranijo ustrezno stabilnost. Kot nadgradnjo proučevanja vpliva pomožnih snovi smo podrobneje ovrednotili vpliv manitola in njegovih polimorfov na stabilnost mAb in kritične lastnosti kakovosti liofilizatov ter s spreminjanjem sestave formulacije in procesnih spremenljivk ugotavljali, kakšni pogoji vodijo v nastanek polimorfne oblike manitol hemihidrata (MHH). Dokazali smo, da slednji nastaja v primeru, ko je razmerje med saharozo in manitolom nizko ter da višja koncentracija mAb inhibira kristalizacijo v MHH. Nadalje smo dokazali, da z višanjem temperature sekundarnega sušenja in temperiranja omogočimo popolno dehidratacijo MHH ter da na nastanek MHH primarno sušenje nima vpliva. Zaključili smo z ugotovitvijo, da MHH ne vpliva na stabilnost mAb, medtem ko na stabilnost vpliva prisotnost manitola. Zadnji del doktorske naloge smo namenili področju formulacij za subkutano uporabo, pri čemer smo želeli ovrednotiti vpliv visokih koncentracij mAb na viskoznost raztopin ter rekonstitucijski čas liofilizatov. Podrobneje smo proučili vpliv agresivnih pogojev sušenja na formulacije z različnimi koncentracijami mAb, in sicer 30, 60, 90 in 120 mg/mL. Slednje smo najprej ovrednotili z vidika termičnih lastnosti in viskoznosti, in sicer je zvišanje koncentracije mAb nakazovalo trend zvišanja Tg' ter eksponentno naraščanje viskoznosti. Na podlagi analiz smo dokazali, da višanje koncentracije mAb ne vpliva na stabilnost mAb zaradi dovolj velikega deleža saharoze. Primerjava med agresivnimi in konzervativnim ciklom je pokazala, da z uporabo agresivnih pogojev dosežemo skrajšanje cikla za do 80 % in s tem ne vplivamo na kritične lastnosti produkta. V raziskovalno študijo smo nadalje vključili različne aminokisline (polnila) in ugotovili, da je potencialna alternativa uporabi manitola za nizkokoncentrirane formulacije izolevcin, medtem ko njegova uporaba v formulacijah z visokimi koncentracijami mAb še vedno ostaja vprašljiva. In recent years, biopharmaceuticals in the pharmaceutical field have experienced a great expansion, so it is not surprising that they represent the most commonly sold drugs on an annual basis. It is a group of drugs that contain various protein molecules as an active pharmaceutical ingredient, with monoclonal antibodies (mAbs) as one of the most important and most represented groups. Biological molecules have changed the current methods of treatment and thus contributed to its progress. Biopharmaceutical drugs have many advantages over conventional synthetic drugs, but their production is much more complex and time-consuming compared to the production of conventional drugs. Ensuring the stability of protein molecules is essential as it directly affects the safety, quality and efficacy of the drug and thus represents one of the most critical steps in the process of preparing biopharmaceuticals. The instability of protein molecules, together with their molecular weight, susceptibility to enzymatic degradation and low half-life, are reasons for the usually very low bioavailability of biological macromolecules after oral administration, so parenteral pharmaceutical forms in the form of solutions, suspensions or lyophilisates predominate among biological drugs. Lyophilisation or freeze-drying is a technological process based on the physical phenomenon of sublimation, where water is removed from the sample using appropriate temperatures and pressures. Basically, lyophilisation is a process consisting of three consecutive, intertwined phases, namely the initial freezing is followed by primary and secondary drying. It is a known fact that lyophilisation is a long-term and energy-intensive process, so in order to ensure the eligibility of costs, its optimisation is essential. The latter is mainly based on the use of aggressive primary drying conditions, as primary drying is the most intensive stage of the process, for which the appropriate composition of the formulation is a prerequisite. Excipients such as stabilisers, bulking agents, surfactants and buffers form the basis of protein formulations, and from the point of view of process optimisation, bulking agents play an essential role. The incorporation of crystalline bulking agents, such as mannitol and glycine into protein formulations allows primary drying to be performed at temperatures above the critical temperature of the formulations. Crystalline bulking agents ensure the proper structure of the lyophilisates and the preservation of critical quality attributes of the product, even if the glass transition temperature of maximally freeze-concentrated solutions (Tg') is exceeded during primary drying and, in some cases, also the collapse temperature (Tc). The positive role of crystalline bulking agents in the field of lyophilisation has been investigated mainly in connection with lower molecular weight proteins, while the understanding of the mechanisms of action of bulking agents in connection with mAb is still lacking. Despite the fact that the intravenous route of administration is the most common way of administering drugs with mAb, we have recently encountered an increase in formulations for subcutaneous application. The latter shows a number of advantages, the most important of which are the reduction of costs, namely due to less frequent application, which in some cases is performed by patients themselves, and consequently the use of such drugs increases patient compliance. From a technological point of view, it is a preparation of formulations that faces with many challenges, especially in terms of low volumes due to the type of application, so in the field of drugs for subcutaneous use, we are talking about highly concentrated formulations. At this point, it should be mentioned the increased viscosity of solutions for subcutaneous injection, which often limits the injectability of such formulations. Importantly, the use of lyophilisation has also spread to the field of highly concentrated formulations, where we also face a number of process challenges. In the first part of the doctoral thesis, we focused on the planning and optimisation of lyophilisation cycles. We prepared two different formulations, namely a formulation containing only sucrose and a formulation containing glycine (bulking agent) in addition to sucrose, and lyophilised them under aggressive and conventional conditions. By introducing aggressive drying conditions, we proved that primary drying can be performed at higher temperatures and pressures, thus shortening drying for up to 54%, without affecting critical properties of the product such as lyophilisate appearance, reconstitution time and residual moisture content. In the next step, we evaluated the influence of aggressive drying conditions on the stability of the model mAb and found that the latter did not affect the stability of the mAb. The results confirmed that aggressive drying conditions are a suitable alternative to conventional drying conditions, which enable shortening of primary drying and provide critical quality attributes of the product. In the second part, we examined the influence of the different mAb to stabiliser ratio (sucrose) and the influence of mAb concentrations on the stability of model mAb. We focused on determining the physical and chemical stability of the tested mAb, both before lyophilisation, after lyophilisation, and as part of a stress stability study. The most common phenomenon of protein instability is manifested in the context of physical instability, namely the most common form is aggregation, as a result of complete or partial unfolding of protein molecules. Initially, formulations were prepared with four different mAb / sucrose molar ratios, namely 1: 140, 1: 280, 1: 420 and 1: 850, and three different mAb concentrations (10, 20 and 30 mg / mL). The obtained results show that higher sucrose proportions lead to better physical and chemical stability, immediately after the lyophilisation process, as well as after 1 and 3 months of stability study. In contrast, as the mAb concentration increases, the proportion of aggregates increases, since a higher mAb concentration means less sucrose molecules per protein molecule. The same trend was observed in the evaluation of chemical stability, which is lower with a decrease in the molar ratio between sucrose and mAb and an increase in the concentration of mAb. Furthermore, higher concentrations of mAb (60, 90, 120, 150 mg / mL) were also included in the study and it was found that with the appropriate ratio of sucrose, mAb maintains adequate stability during and after the lyophilisation process. As an upgrade of the study, we evaluated in more detail the influence of mannitol and its polymorphism on mAb stability and critical quality properties of lyophilisates and determined the conditions leading to the formation of polymorphic form of mannitol hemihydrate (MHH) by changing the composition and process variables. We have shown that the MHH occurs when the ratio of sucrose to mannitol is low and that a higher concentration of mAb inhibits crystallisation in MHH. We further proved that by raising the temperature of the secondary drying and annealing, we enabled complete dehydration of MHH and that the formation of MHH is not affected by primary drying. We concluded that MHH does not affect the stability of mAb, whereas stability is affected by the presence of mannitol. The last part of the doctoral thesis was dedicated to the field of formulations for subcutaneous use, where we wanted to evaluate the effect of high mAb concentrations on the viscosity of solutions and the reconstitution time of lyophilisates. The effect of aggressive drying conditions on formulations with different mAb concentrations, namely 30, 60, 90 and 120 mg / mL, was studied in more detail. The latter were first evaluated in terms of thermal properties and viscosity, namely the increase in mAb concentration indicated an upward trend in Tg' and an exponential increase in viscosity. From the view point of stability, we demonstrated that the increase in mAb concentration does not affect the stability of mAb, due to the sufficiently large proportion of sucrose. A comparison between the aggressive and convenitonal cycles showed that by using aggressive conditions we achieve a shortening of the cycle by up to 80% with no affect on the critical properties of the product. We further included various amino acids (bulking agents) in the research study and found that isoleucine is a potential alternative to the use of mannitol for low-concentration formulations, while its use for highly concentrated mAb formulations still remains questionable.