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Stabilnost zdravilne učinkovine vpliva na njeno varnost in učinkovitost, zato je ključna pri razvoju zdravil. Pri razvoju zdravil je potrebno testiranje stabilnosti in identifikacija znatnih razgradnih produktov zdravilne učinkovine. Za poglobljeno razumevanje stabilnosti zdravilne učinkovine se izvaja dolgoročno in pospešeno testiranje stabilnosti. Pospešeno testiranje traja 6 mesecev dolgoročno pa 12 mesecev, kar znatno podaljšuje razvoj zdravila. Zgodnje napovedovanje in identifikacija možnih nečistot v zdravilu je zato pomembna pri farmacevtskem razvoju. Stresni testi zdravilne učinkovine lahko pomagajo pri zgodnji identifikaciji možnih razgradnih produktov, kar pripomore pri določevanju razgradnih poti in intrinzične stabilnosti molekule. S stresnimi testi si pomagamo tudi pri razvoju analiznih metod za testiranje stabilnosti zdravilne učinkovine. Pred izvedbo stresnih testov navadno skušamo napovedati razgradne produkte, pri čemer si pomagamo z literaturnimi podatki in kemijskim znanjem. Vedno bolj se uporabljajo tudi napovedna orodja oz. in silico orodja, ki napovedujejo razgradne produkte in nam dajo začetno sliko glede stabilnosti molekule pod različnimi pogoji. Taka orodja so lahko v pomoč tudi pri identifikaciji razgradnih produktov, pridobljenih s stresnim testiranjem. Venetoklaks je selektiven zaviralec anti-apoptotične beljakovine limfoma celic B (Bcl)-2. Je prva registrirana zdravilna učinkovina s to proteinsko tarčo. Leta 2016 je bil odobren za zdravljenje bolnikov s kronično limfocitno levkemijo v Združenih državah Amerike. Ker je venetoklaks relativno nova zdravilna učinkovina, v literaturi še ni bilo veliko znanega glede njegove stabilnosti. Prav tako ni bilo moč najti analiznih metod za testiranje stabilnosti venetoklaksa. Kot edina zdravilna učinkovina v skupini zaviralcev proteina Bcl-2 ima strukturo, ki se razlikuje od drugih znanih zdravilnih učinkovin. Tako je tudi stabilnost molekule na prvi pogled težje napovedljiva. Namen našega dela je bila določitev razgradnih produktov venetoklaksa z uporabo kombiniranega pristopa z in silico orodji in stresnimi testi ter razvoj analizne metode za spremljanje venetoklaksa in njegovih razgradnih produktov. Na podlagi struktur razgradnih produktov smo nato želeli določiti kemizme razgradnje venetoklaksa. Cilj je bil tudi vključitev principa razvoja analizne metode z vgrajeno kakovostjo (AQbD) in s tem izboljšanje robustnosti analizne metode in boljše razumevanje vplivov posameznih parametrov analizne metode na ključne odzive analizne metode. V uvodnem poglavju smo predstavili venetoklaks in njegovo potencialno široko terapevtsko uporabnost v obliki preglednega znanstvenega članka, ki zavzema pregled znanstvene in patentne literature. Poglavje smo dopolnili s teoretičnim ozadjem testiranja stabilnosti ter AQbD. V prvem poglavju raziskovalnega dela je predstavljen razvoj analizne metode reverzno-fazne tekočinske kromatografije za določevanje stabilnosti venetoklaksa z uporabo principov AQbD. Razvoj metode je potekal na stresnih vzorcih venetoklaksa. Celoten proces razvoja analizne metode je bil sestavljen in začetnih poskusov po principu spreminjanja enega faktorja naenkrat (OFAT), definicijo tarčnega profila analizne metode (ATP), izbiro kritičnih atributov metode (CMA), oceno tveganja, identifikacijo kritičnih parametrov metode (CMP), začetnih presejalnih poskusov, optimizacije, testiranja robustnosti, določitve območja delovanja metode (MODR) in postavitve kontrolne strategije. Metodo smo kvalificirali v delovni točki, kjer smo potrdili tarčni profil analizne metode: zmožnost določanja venetoklaksa ob prisotnosti razgradnih produktov v koncentracijskem območju od 80 – 120% tarčne koncentracije s točnostjo 100 ± 2% in ponovljivostjo z ? 2% relativnega standardnega odklona (RSD). Metoda, ki smo jo razvili je ustrezna za določanje venetoklaksa ob prisotnosti glavnih razgradnih produktov pridobljenih s stresnimi testi in jo lahko uporabljamo za določanje stabilnosti venetoklaksa. V drugem poglavju raziskovalnega dela smo opisali izvedbo stresnih testov, izolacijo glavnih razgradnih produktov in določevanje strukture razgradnih produktov. Predpostavili smo glavne kemizme razgradnje venetoklaksa. Identificirali smo štiri glavne razgradne produkte venetoklaksa ob dodatku močne kisline, tri glavne razgradne produkte venetoklaksa ob dodatku močne baze in en razgradni produkt venetoklaksa, ki nastane z oksidacijo. Pri tem smo poleg predvidene hidrolize N-acilsulfonamiden vezi, določili še dekarboksilacijo kislinskega fragmenta, ki nastane iz omenjene hidrolize, dimerizacijo venetoklaksa preko metilenskega mostička ob prisotnosti dimetil sulfoksida, ciklizacijo o-nitroanilinskega dela, substitucijo amina na 2-nitro-substituiranem benzenu s hidroksilno skupino in oksidacijo dušika piperazinskega obroča. Tretje poglavje obsega in silico napovedovanje razgradnih produktov venetoklaksa. Razgradne produkte smo s programskim orodjem napovedovali pri različnih eksperimentalnih pogojih. Primerjali smo rezultate eksperimentalno pridobljenih razgradnih produktov z napovedanimi. Uporabljeno in silico orodje je uspešno napovedalo tri od sedmih eksperimentalno določenih razgradnih produktov venetoklaksa. Drug stability is crucial in drug development since it affects the safety and efficiency of the drug product. Stability testing and identification of significant degradation products of the active pharmaceutical ingredient (API) are required during drug development. Long-term and accelerated stability testing is performed to provide an in-depth understanding of the stability of the active substance. Accelerated testing lasts 6 months and long-term testing lasts 12 months, which significantly prolongs the development of the drug product. Therefore, early prediction and identification of possible impurities in drug product is important in pharmaceutical development. Stress tests of the active substance can help in the early identification of possible degradation products, which helps to determine degradation pathways and intrinsic stability of the molecule. Stress tests also help to develop stability-indicating analytical methods. Before performing stress tests, we usually try to predict degradation products, using literature data and chemical knowledge. Increasingly, in silico tools that predict degradation products are used, which give us an initial picture of the stability of the molecule under different conditions. Such tools can also be helpful in identifying degradation products obtained in stress testing. Venetoclax is a selective inhibitor of the anti-apoptotic B-cell lymphoma (Bcl)-2 protein, a first of its kind. It was approved for the treatment of patients with chronic lymphocytic leukemia in the United States in 2016. Because venetoclax is a relatively new active substance, not much is known about its stability in the literature and no stability-indicating analytical method for venetoclax could be found. As the only Bcl-2 protein inhibitor on the market, it has a structure that differs from other known active substances. Thus, the stability of the molecule is also more difficult to predict at first glance. The purpose of our work was to determine the degradation products of venetoclax using a combined approach with in silico tools and stress tests, as well as to develop an analytical method for monitoring venetoclax and its degradation products. Additionally, we wanted to determine the degradation pathways and degradation chemistry of venetoclax based on the structures of degradation products. The aim was also to include the principles of Analytical Quality by Design (AQbD) in analytical method development, thus improve the robustness of the analytical method, and better understand the effects of critical method parameters on the critical method attributes. In the introductory chapter, we presented venetoclax and its potentially broad therapeutic applicability in the form of a scientific review article covering the review of the scientific and patent literature. The chapter was expanded with a theoretical background of stability testing and AQbD. The first chapter of the research work presents the development of a reverse-phase liquid chromatography stability-indicating analytical method for venetoclax using the AQbD principles. Stress samples of venetoclax were utilized for the development process. The development process of the analytical method was composed of initial one-factor-at-a-time (OFAT) experiments, definition of the analytical target profile (ATP), selection of critical method attributes (CMAs), risk assessment, identification of critical method parameters (CMPs), initial screening experiments, optimization, robustness testing, method optimal design region (MODR) determination, and a proposition of a control strategy. The method was qualified in the working point, where we confirmed the analytical target profile: the ability to determine venetoclax in the presence of its degradation products over a range of 80% to 120% of the target concentration with an accuracy of 100% ± 2% and repeatability ⡤ 2% RSD. The method we have developed is suitable for the determination of venetoclax in the presence of major degradation products obtained by stress tests and can be used to determine the stability of venetoclax. In the second chapter of the research work, we described the implementation of stress tests, the isolation of major degradation products and the determination of the structures of the major degradation products. We hypothesized the main chemistry pathways of venetoclax degradation. We identified four major degradation products of venetoclax with the addition of a strong acid, three major degradation products of venetoclax with the addition of a strong base, and one degradation product of venetoclax formed by oxidation. In addition to the predicted hydrolysis of the N-acylsulfonamide bond, we determined decarboxylation of the acid fragment resulting from said hydrolysis, dimerization of venetoclax via a methylene bridge in the presence of dimethyl sulfoxide, cyclization of the o-nitroaniline moiety, substitution of the amine with a hydroxy group, and oxidation of nitrogen present in the piperazine ring moiety. The third chapter covers in silico predictions of venetoclax degradation products. Degradation products were predicted under different experimental conditions using a software tool. We compared the results of experimentally obtained degradation products with the predicted ones. The in silico tool successfully predicted three out of the seven experimentally determined degradation products of venetoclax.