20.500.12556/RUL-141345 Mehanizem, termodinamika in kinetika agregacije terapevtskih monoklonskih protiteles Mechanism, thermodynamics and kinetics of aggregation of therapeutic monoclonal antibodies Monoklonska protitelesa so eno izmed najhitreje rastočih razredov zdravilnih učinkovin. Čeprav je njihovo aminokislinsko zaporedje skoraj popolnoma človeško, je zaradi invazivne aplikacije še vedno veliko pozornosti potrebno nameniti omejevanju števila neželenih imunskih odzivov. Ti so največkrat posledica nastanka različnih agregatov v zdravilu. Že zgodaj v razvoju biološkega zdravila je zato potrebno poiskati pogoje, ki učinkovito zavirajo nastanek agregatov, da se zagotovi njegova učinkovitost, varnost in kakovost do konca roka uporabe. Vrednotenje in optimizacija dolgoročne stabilnosti je torej ključen korak v razvoju bioloških zdravil, ki pa je še vedno dolgotrajen in vnaša precej tveganja v razvoj. Do danes namreč še ni bilo nedvoumno pokazano, da povečanje termodinamske in koloidne stabilnosti, kar je v zadnjih letih glavna strategija, vodi v zmanjšanje hitrosti agregacije. Po drugi strani se želi razvoj pospešiti tako, da se primerja prirast agregatov v različnih formulacijah po inkubaciji pri povišanih temperaturah. Tak pristop pa praviloma ne odraža sprememb do konca roka uporabe pri temperaturi shranjevanja, ponavadi tri leta pri 5 °C. V tem delu predstavljam nekaj različnih pristopov za skrajšanje časa potrebnega za vrednotenje dolgoročne agregacije in s tem pospešitev optimizacije formulacije, s ciljem zmanjšanja hitrosti agregacije prav pri pogojih shranjevanja. Za množico protiteles, ki se uporabljajo v onkologiji, imunološki terapiji, revmatologiji, pri zdravljenju osteoporoze in krvnih bolezni, sem z uporabo kinetičnega mehanizma agregacije, ki vsebuje dve poti, nizko- in visokotemperaturno pot, uspešno opisal podatke agregacije v širokem temperaturnem in koncentracijskem območju. Model natančno napove delež agregatov po treh letih z uporabo podatkov dobljenih v mnogo krajšem časovnem obdobju. Na obstoj dveh različnih od temperature odvisnih poti razvejan kinetični mehanizem agregacije nakazuje le posredno, zato sem izoliral različne frakcije agregatov izbranega protitelesa mAb1. Analiza kemijskih modifikacij protitelesa v posameznih frakcijah z metodama cIEF in PepMap-MS ter vrednosti aktivacijskih energij je pokazala, da se agregati nastali pri temperaturi značilni za nizkotemperaturno pot pomembno razlikujejo od agregatov nastalih pri temperaturi značilni za visokotemperaturno pot. Poleg razlik v nivoju oksidacije in deamidacije, ti rezultati s pomočjo vpogleda v kristalno strukturo človeškega protitelesa IgG kažejo na možnost, da visokotemperaturno agregacijo spremlja tudi delno razvitje protitelesa. Analiza agregacije po dveh poteh zahteva obširno študijo za vsako evalvirano formulacijo, kar je za farmacevtski razvoj zelo nepraktično. Poenostavljen kinetični model, ki obravnava agregacijo kot reakcijo (psevdo) prvega reda, omogoča veliko pospešitev in poenostavitev pri razvoju formulacij z vidika agregacije, kadar je koncentracija protitelesa v vrednotenih formulacijah enaka. Na podlagi stabilnostnih podatkov dobljenih pri vsaj treh temperaturah, pri katerih visokotemperaturna pot ne prispeva k agregaciji, lahko izračunamo dolgoročno hitrost agregacije pri poljubni temperaturi z zadovoljivo natančnostjo. Pri dovolj nizki temperaturi lahko formulacije primerjamo med sabo tudi neposredno in tako kar najhitreje zožimo nabor pomožnih snovi in pogojev, ki uspešno upočasnjujejo agregacijo pri temperaturi shranjevanja. Navidezna standardna Gibbsova prosta energija denaturacije, dobljena z analizo kemijske denaturacije protitelesa, predstavlja termodinamsko stabilnost, ki je dober pokazatelj nagnjenosti protitelesa k agregaciji. Dobljeni fazni prostor agregacije je razkril, da pri protitelesih z nizko termodinamsko stabilnostjo povečanje le-te bistveno vpliva na upočasnitev agregacije preko zmanjšanja začetne koncentracije intermediata, ki agregira. Pri protitelesih z visoko termodinamsko stabilnostjo pa s spremembo termodinamske stabilnosti vplivamo predvsem na kinetično konstanto nastanka tega intermediata. Posledično je vpliv termodinamske stabilnosti na hitrost agregacije veliko manjši. Tudi analiza toplotne denaturacije ponudi uporabno informacijo za farmacevtski razvoj, in sicer nudi oceno temperature, do katere agregacija po visokotemperaturni poti ne poteka, ki je približno 15 oz. 25 °C nižja od temperatur prehoda pri toplotni denaturaciji domen CH2 in Fab. Strategija, ki združuje kinetično in termodinamično analizo povečuje učinkovitost razvoja in proizvodnje bioloških zdravil ter prispeva tudi k boljšemu razumevanju molekularnih mehanizmov agregacije protiteles. Monoclonal antibodies are one of the fastest growing classes of pharmaceutics. Although they possess an almost completely human aminoacidic sequence, special attention needs to be paid to limit adverse immune responses, which are, due to invasive application, most often caused by aggregates of different sizes. To ensure efficacy, safety, and quality of biological drug products until the end of their shelf life, it is therefore essential to find conditions that efficiently slow down aggregation already in the early development. Evaluation and optimization of long-term stability is thus a crucial step in development of biologics, which is still long-lasting and introduces substantial risk to the development. To date it hasn’t been unambiguously shown that increasing thermodynamic and colloidal stability, a primary strategy in the recent years, really leads to lower aggregation rate. On the other hand, with aim to accelerate development, aggregate increase in different formulations is being evaluated at higher temperatures, which doesn’t necessarily reflect changes until the end of shelf-life at storage temperature, usually three years at 5 °C. In this work, I present several approaches for shortening time needed for aggregation assessment and, thus, accelerating formulation optimization aimed specifically at slowing down aggregation at storage conditions. For the group of antibodies used in oncology, immunology, rheumatology, for therapy of osteoporosis, and blood diseases, I managed to successfully describe aggregation data from wide temperature and concentration range by applying a kinetic aggregation mechanism comprised of two pathways, low- and high-temperature pathway. Additionally, model accurately forecasts aggregate fractions up to three years based on data obtained in much shorter period. Kinetic aggregation mechanism only implies existence of two distinct, temperature-dependent pathways. Therefore, I isolated several aggregate fractions of the selected antibody mAb1. Analysis of antibody’s chemical modifications in separate fractions with cIEF and PepMap-MS methods and analysis of activation energy values revealed that aggregates formed at temperature typical for low-temperature aggregation importantly differ from aggregates formed at temperature typical for high-temperature aggregation. Besides differences in oxidation and deamidation level, these results, with aid of available crystal structure of human IgG antibody, indicate that high-temperature aggregation is accompanied by the partial antibody unfolding. Analysis of aggregation on two pathways requires extensive study for every assessed formulation, which is highly impractical in pharmaceutical development. Simplified model that considers aggregation as a (pseudo) first order kinetic reaction enables large acceleration and simplification in comparing aggregation susceptibility of formulations, when target concentration is known beforehand. Based on data from at least three temperatures, at which high-temperature pathways do not contribute to total aggregation, we can calculate long-term aggregation rate at any temperature with satisfactory accuracy. If we choose low enough temperature, we can directly compare formulations between themselves and thus in a most efficient way narrow down set of excipients and conditions successfully decreasing aggregation at storage temperature. Apparent standard Gibbs free energy of denaturation obtained by analysis of antibody chemical denaturation is a parameter of thermodynamic stability, which is a good indicator of antibody’s aggregation propensity. A constructed aggregation phase space revealed that we can effectively slow down aggregation of antibodies with low thermodynamic stability by its increase. This is explained by decreasing pool of initial aggregating intermediate. On the other hand, for antibodies with high thermodynamic stability, its value primarily affects kinetic constant for formation of this intermediate, with the effect being much weaker in comparison. Nevertheless, analysis of thermal denaturation offers a useful information for the development of biopharmaceuticals. Namely, temperature, up to which aggregation through high-temperature pathway doesn’t contribute substantially, is approximately 15 or 25 °C lower than the thermal denaturation transition temperatures of CH2 and Fab domains, respectively. Strategy combining kinetic and thermodynamic analysis not only improves efficiency of biologic drug product development and production but contributes to a better understanding of molecular mechanisms of aggregation as well. - - true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2022-09-28 15:14:59 2022-09-28 15:15:00 2023-02-09 12:57:32 0000-00-00 00:00:00 2022 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 5643 126075651 Bunc_Marko_-_Mehanizem,_termodinamika_in_kinetika_agregacije_terapevtskih_monoklonskih_protitele.pdf Bunc_Marko_-_Mehanizem,_termodinamika_in_kinetika_agregacije_terapevtskih_monoklonskih_protitele.pdf 1 8CD43E40F453C504458E9BC6B5AB9DFD 9c836ed2ee13be9e1a71edf3b9e61047f1c218023ae7993943968da4895fe02d 8d627220-3f2f-11ed-9c94-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=162381 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0