20.500.12556/RUL-172034 Optimizacija formulacij monoklonskega protitelesa IgG4 z vidika viskoznosti in stabilnosti Optimization of IgG4 monoclonal antibody formulations from the perspective of viscosity and stability Zdravljenje z monoklonskimi protitelesi (mAb) v obliki subkutanih injekcij je v zadnjih letih pridobilo na pomenu zaradi številnih prednosti pred intravensko aplikacijo, hkrati pa prinaša tudi specifične izzive. Omejen volumen subkutane aplikacije zahteva uporabo visokokoncentriranih raztopin mAb, kar lahko povzroči visoko viskoznost formulacij in slabšo stabilnost teh bioloških makromolekul. Cilj magistrske naloge je bil ovrednotiti vpliv izbranih testnih pomožnih snovi iz štirih različnih razredov, t.j. (i) aminokislin, (ii) mimetikov prolina, (iii) di- in tripeptidov s prolinom ter (iv) mimetikov prolina in nikotinske kisline na viskoznost formulacij z modelnim mAb tipa IgG4 in nadalje njihov vpliv na stabilnost testiranega mAb. Najprej smo optimizirali osnovno formulacijo mAb na podlagi izračuna interakcijskega difuzijskega koeficienta (kD) in določili njen optimalni pH, ki je bil 5,5. V nadaljevanju smo dokazali, da viskoznost formulacij narašča eksponentno z naraščajočo koncentracijo mAb. Z namenom znižanja viskoznosti smo v viskokokoncentrirane formulacije mAb (210–310 mg/mL) dodali izbrane testne pomožne snovi v koncentraciji 25 mM. Viskoznost smo merili z viskozimetrom na čipu in ugotovili, da so vse spojine (razen S13, ki je dipeptid s prolinom) v določenem obsegu znižale viskoznost raztopin. Pri tem so bile najbolj učinkovite tiste, ki omogočajo večvrstne interakcije z mAb, kot so vodikove vezi, hidrofobne interakcije ter π-π interakcije. Spremljali smo tudi pojav in prirast agregatov v formulacijah z metodami dinamičnega sipanja laserske svetlobe (DLS, MADLS) in velikostno izključitvene kromatografije (SEC). Shranjevanje vzorcev pri 4 °C ni povzročilo zaznavnega prirasta agregatov, ne glede na prisotnost testnih spojin. Nasprotno pa je trimesečno shranjevanje pri 40 °C pokazalo pomembne razlike med vzorci, kjer so spojine S05 (mimetik prolina), S15 (dipeptid s prolinom) in NaCl prispevale k večji agregaciji v primerjavi s kontrolo, druge pa so fizikalno stabilnost izboljšale (S10, S11, ki sta dipeptida s prolinom). Z metodo MADLS smo zaznali prisotnost večjih delcev v nekaterih formulacijah, kar smo kasneje potrdili tudi z analizo SEC. Statistična analiza je pokazala signifikantno korelacijo med obema metodama (r = –0,6796; p = 0,00378), kar pomeni, da je MADLS uporabna metoda za spremljanje zgodnjih znakov agregacije in s tem primerna podpora pri razvoju ter optimizaciji formulacij z mAb. Subcutaneous administration of monoclonal antibodies (mAbs) has gained importance in recent years due to the numerous advantages over intravenous administration, but it also has specific disadvantages. The limited volume of subcutaneous application requires the use of highly concentrated mAb solutions, which can lead to increased viscosity of formulation and reduced stability of these biological macromolecules. The aim of this master’s thesis was to evaluate the effect of selected test excipients from four different classes, i.e., (i) amino acids, (ii) proline mimetics, (iii) di- and tripeptides containing proline and (iv) proline and nicotinic acid mimetics, on the viscosity of formulations containing a model IgG4 mAb and further their impact on the stability of the tested mAb. First, we optimized the basic formulation based on the calculation of the interaction diffusion coefficient (kD) and determined its optimal pH which was 5.5. Furthermore we demonstrated that the viscosity of formulations increases exponentially with increasing mAb concentration. In order to reduce viscosity, selected excipients were added at a concentration of 25 mM and NaCl in concentrations 50 and 150 mM to highly concentrated mAb formulations (210–310 mg/mL). Viscosity was measured using a chip-based viscometer, and it was found that all compounds (except S13, a dipeptide containing proline) reduced solution viscosity to some extent. The most effective compounds were those capable of forming multiple types of interactions with the mAb, such as hydrogen bonds, hydrophobic interactions, and π-π interactions. The appearance and growth of aggregates in formulations was also monitored using dynamic light scattering methods (DLS, MADLS) and size exclusion chromatography (SEC). Storage of samples at 4 °C did not result in a detectable increase in aggregates, regardless of the presence of test compounds. In contrast, three month storage at 40 °C revealed significant differences between samples with compounds S05 (a proline mimetic), S15 (a proline-containing dipeptide), and NaCl contributing to increased aggregation compared to the control, while others improved physical stability (S10, S11, both proline-containing dipeptide). Using the MADLS method, the presence of larger particles was detected in some formulations, which was later confirmed by SEC analysis. Statistical analysis showed a significant correlation between the two methods (r = –0.6796; p = 0.00378), indicating that MADLS is a suitable method for monitoring early signs of aggregation and thus a valuable tool in the development and optimization of mAb formulations. Monoklonska protitelesa subkutana aplikacija zniževanje viskoznosti stabilnost agregacija Monoclonal antibodies subcutaneous administration viscosity reduction stability aggregation true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-09-05 08:45:33 2025-09-05 08:45:43 2025-10-02 03:33:37 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2026-09-04 117149 28180.pdf 28180.pdf 1 A1755700A09222E6E16B42A15DADDC72 db99816e5e3b1f26e0421593fd380fbeda86e48c4601c0ddd65aabe37aed78ad cb2d9cf9-8a23-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=215753 Fakulteta za farmacijo 0 0 0