Implementacija reometrije v mikrokanalu in iskanje parametrov za napoved viskoznosti proteinskih formulacij

• Main Author: Komatar, Silvo
• Other Authors: Plazl, Igor
• Format: Master Thesis
• Language: Slovenian
• Published: 2021
• Subjects: agregacija; mikrokanal; proteini; razvoj bioloških zdravil; viskoznost; aggregation; biopharmaceuticals; microchannel; proteins; viscosity; sami
• Online Access: https://repozitorij.uni-lj.si/IzpisGradiva.php?id=127773; https://repozitorij.uni-lj.si/Dokument.php?id=144302&dn=; https://plus.si.cobiss.net/opac7/bib/70478083?lang=sl

Uporaba proteinov predstavlja nov način zdravljenja bolezni ter s tem postaja pomembna veja sodobne farmacije. V primerjavi s klasičnimi zdravili, so gradniki biofarmacevtskih učinkovin precej drugačni. Biološka zdravila imajo kompleksno 3D strukturo z visoko molekulsko maso. Proteinske molekule se v primeru neustreznih pogojev utegnejo sprijeti v večje skupke podobnih velikosti (agregate). Z namenom zagotavljanja kakovosti terapevtskih proteinov se čedalje večjo pozornost namenja spremljanju lastnosti in stabilnosti proteinov ter sami interakciji med njimi. Ena izmed temeljnih lastnosti raztopin proteinov je njihova viskoznost. Merjenje viskoznosti v zgodnjih fazah razvoja zdravilne učinkovine je vedno bolj zanimivo tudi z vidika vse natančnejšega napovedovanja makroskopskega vpliva reverzibilnega povezovanja proteinskih molekul končnega produkta. Poznavanje natančnih vrednosti viskoznosti različnih formulacij prispeva k razumevanju in izboljševanju matematičnih modelov agregacijskih poti. Delo opisuje uspešno implementacijo analitske metode za določanje viskoznosti raztopine proteinov na napravi VROC Initium, ki glede na predhodne metode prinaša številne prednosti. Gre za avtomatski viskozimeter, ki temelji na uporabi mikrokanala in je prilagojen na mikrotitrske plošče ali viale. Meritve so v primerjavi s predhodno uporabljeno reometrijo hitrejše, natančnejše ter porabijo manjše količine materiala. V delu je prihranek proteinskega materiala prikazan na primeru iskanja ustreznih ekscipientov za zmanjševanje viskoznosti formulacije. Poleg tega so bili uporabljeni novi, manjši vsebniki, zato je bila znanstveno potrjena tudi ustreznost membran za filtracije in dializo. Nova analitska metoda je bila uporabljena še za potrditev korelacij med viskoznostjo raztopin proteina visokih koncentracij in interakcijami med proteini pomerjenimi pri nizkih koncentracijah. Rezultati meritev viskoznosti treh različnih razvojnih proteinov ustrezno korelirajo z meritvami dinamičnega interakcijskega parametra (kD). Umetno tvorjeni večji agregati mikrokanala niso mašili. Nova analitska metoda je tako uspešno nadomestila prej uporabljeno reometrijo. Recombinant therapeutic proteins are an advanced way of treating various life-threatening conditions and thus becoming an important branch of modern pharmacy. Compared to conventional drugs, the building blocks of biopharmaceuticals are quite different. Biological drugs have a complex 3D structure with high molecular weight. When they are exposed to different stress conditions, protein molecules may adhere to larger clusters of similar size (aggregates). In order to ensure quality, increasing emphasis is placed on monitoring the properties and stability of proteins together with interactions between them. One of the fundamental properties of protein solutions is their viscosity. Measurement of viscosity in the early stages of drug development is also becoming increasingly interesting for progressively accurate prediction of the macroscopic impact of final product reversible self-association. Knowing the exact viscosity values of different formulations contributes to understanding and improving mathematical models of the aggregation process. The thesis describes a successful implementation of an analytical method for determining the viscosity on a VROC Initium device. It is an automatic viscometer based on the use of a microchannel and adapted to microtiter plates or vials. Measurements are fast, very accurate and consume small amounts of liquid material. Viscometer VROC enabled many experiments that were not possible before or simply not to such an extent. The saving of protein material is shown in the case of examining suitable viscosity reducing additives (VRA). Reduced sample volume needed for measurement enabled the use of smaller plastic containers. The suitability of their membranes for filtration and dialysis was scientifically confirmed. Furthermore, a new analytical method was used to confirm the correlations between the viscosity of high-concentration protein solutions and the interactions between proteins measured at low concentrations. The results of viscosity measurements of three different proteins correlate accordingly with the measurements of the dynamic interaction parameter (kD). Artificially generated larger aggregates haven't clogged the microchannel. The method successfully replaced the previously used rheometry.