Priprava in karakterizacija mestno specifičnih pegiliranih konjugatov interferona alfa-2b : doktorska disertacija

Kusterle, Mateja (author), Štrukelj, Borut (mentor), Gaberc-Porekar, Vladka (comentor)

Proteini se vse bolj uveljavljajo kot zdravilne učinkovine v terapiji mnogih patofizioloških stanj. Navadno imajo biološka zdravila poleg svoje velike učinkovitosti tudi veliko neugodnih lastnosti, zaradi katerih jih ne moramo obravnavati kot optimalne zdravilne učinkovine. Biofarmacevtiki prve generacije so zdravilne učinkovine, ki nadomeščajo naravne proteinske molekule in izkazujejo kemijsko in fizikalno nestabilnost, omejeno topnost, kratek razpolovni čas, proteolitsko nestabilnost, imunogenost in toksičnost. Vse navedene pomanjkljivosti navadno izboljša konjugacija proteina s PEG verigo - pegilacija. Slaba lastnost pegiliranih konjugatov je skoraj vedno zmanjšana biološka učinkovitost ˝in vitro˝, ki pa jo kompenzirajo v pogojih ''in vivo'' navadno zelo izboljšane farmakokinetične lastnosti. Podaljšanje razpolovnega časa v telesu temelji predvsem na povečanju hidrodinamskega radija proteina zaradi pripete PEG verige. Pripravili smo serijo različnih pegiliranih oblik rekombinantenga humanega inerferona alfa 2b (IFN), pridobljenega v bakteriji E. coli, in pegilirane konjugate preučili. Z uporabo N -terminalne mestno specifične pegilacije smo se izognili nastanku številnih pozicijskih izomer in vplivu prekrivanja različnih delov proteinske molekule s PEG verigo, kar omogoča lažje in natančnejše preučevanje zgolj vpliva velikosti in oblike PEGa. Specifičnost pegilacije za N-konec smo dokazali s peptidnim kartiranjem. V prvem delu naloge je predstavljena analiza velikosti oziroma hidrodinamskega radija ali navidezne molekulske mase pegiliranih konjugatov z različnimi metodami: gelska kromatografija (SE-HPLC), gelska elektroforeza (SDS-PAGE) ter dinamično sipanje svetlobe (DLS). Uporabili pa smo tudi za ta namen na videz manj primerni metodi reverzno fazno kromatografijo visoke ločljivosti (RP-HPLC) in analitsko kationsko kromatografija (CE-HPLC). Ugotovili smo, da najpogosteje uporabljena standardna analitska metoda za ugotavljanje hidrodinamskega radija – SE-HPLC, ne detektira razlik v velikosti med konjugati z linearno in razvejan PEG verigo, medtem ko smo z metodo DLS določili za konjugate z razvejanimi PEGi manjši hidrodinamski radij, kot pri konjugatih z linearnimi PEGi z isto molekulsko maso. Metoda CE-HPLC, ki temelji na ionskih interakcijah, je pokazala linearno odvisnost retenzijskega časa od dolžine linearnega PEG-IFN konjugata, medtem ko so konjugati z razvejano PEG verigo od te linearnosti izrazito odstopali. Konjugati, pegilirani na istem mestu, imajo isti neto naboj, razlike v retenzijskem času konjugatov pa so pokazale na izrazit vpliv velikosti in razvejanosti PEG verige, kar je verjetno posledica senčenja površine proteina in s tem manjše interakcije z matriksom. RP-HPLC metoda, ki temelji na hidrofobnih interakcijah z molekulami, je dala nepričakovan rezultat, saj so se pegilirani konjugati, ki naj bi bili zaradi vezave vode na PEG verigo izrazito hidrofilni, pokazali povečano retenzijo glede na povečano molekulsko maso PEG verige. Raziskave smo nadaljevali s študijem razmerij med strukturo in funkcijo, pri čemer smo velikost in obliko pegiliranih konjugatov interferona povezovali z ugotovitvami testiranj biološke aktivnosti ˝in vitro˝ in farmakokinetičnim obnašanjem, določenim na živalskem modelu (podgana). Poskusi ''in vivo'' so pokazali, da sta farmakokinetična profila Pegasya® (interferon α-2a nespecifično monopegiliran z 40 kDa razvejanim PEGom) in našega konjugata, konjugiranega s 45 kDa razvejanim PEG reagentom, primerljiva. Izrazito linearno povezavo med prej naštetimi analitskimi metodami in meritvami biološke aktivnosti ˝in vitro˝ smo v seriji N-terminalno pegiliranih konjugatov IFN ugotovili zgolj za metodo CE-HPLC. Ta ugotovitev kaže, da je senčenje površine proteina ključen faktor za biološko aktivnost ˝in vitro˝ in da je za serijo specifično monopegiliranih konjugatov z metodo CE-HPLC možno biološko aktivnost ˝in vitro˝ napovedati.

• Language: Slovenian
• Keywords: biološka zdravila, interferon, pegintron, metode, konjugati, analiza, pegililacija interferona alfa
• Work type: Dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [M. Kusterle]
• Year: 2009
• Number of pages: XIV, 75 f.
• PID: 20.500.12556/RUL-127045
• UDC: 577
• COBISS.SI-ID: 2591857
• Publication date in RUL: 14.05.2021

KUSTERLE, Mateja, 2009, Priprava in karakterizacija mestno specifičnih pegiliranih konjugatov interferona alfa-2b : doktorska disertacija [online]. Doctoral dissertation. Ljubljana : M. Kusterle. [Accessed 2 June 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=127045

Secondary language

• Language: English
• Title: Preparation and characterization of site specific pegylated conjugates of interferon alfa-2b

Abstract

In the last decades numerous proteins and peptides have been proven as promising human therapeutics for treatment of various pathogenic states. Although very effective and safe, these macromolecules share a number of properties, which do not classify them as optimal drugs. These, so called first-generation biopharmaceuticals, which are protein drugs mimicking the natural protein molecules, frequently display low stability and solubility, short elimination half-life, susceptibility to proteolytic enzymes, immunogenicity, toxicity and other properties that can be improved by covalent attachment of polyethylene glycol chain(s) to a protein, usually called PEGylation. Although pegylation of protein is usually accompanied by decrease of biological activity (measured ''in vitro''), this drawback is successfully compensated ''in vivo'' by improved pharmacokinetic profile. Extension of elimination half life is due to increased hydrodynamic radius of pegylated conjugate. We have prepared a set of well defined pegylated conjugates of interferon alpha-2b (expressed in E. coli) with various PEGs attached to the protein N-terminus and we used prepared conjugates in various studies. Use of N terminal site specific conjugation enabled us to avoid numerous positional isomers with different physiochemical and biological properties and we were able to study conjugates regarding only PEG size and shape. Specificity of the attachment was confirmed by peptide mapping and mass spectroscopy. For characterization, various methods have been used, not only common methods for estimating molecular weights and sizes, such as size exclusion chromatography (SE-HPLC), electrophoretic mobility in polyacrylamide gel (SDS-PAGE) and dynamic light scattering (DLS), but also cation exchange (CE-HPLC) and reverse phase chromatographies (RP-HPLC). Our SE-HPLC experiments have not revealed any size differences among linear and branched PEGs of the same nominal weight. Our DLS experiments have shown smaller hydrodynamic radii for branched conjugates, which are indicative of more compact structure. CE-HPLC, which is based on ion interactions, has revealed linear relationship between the retention time and logarithm of molecular weight for linear IFN-PEG conjugates. Theoretically all our isolated conjugates have the same overall net charge, but obviously increased PEG size and branched structure have distinct influence on retention on cation exchange column. This is probably caused by PEG which is shielding the protein surface and lowers interaction of conjugate with matrix. Pegylated proteins are expected to be very hydrophilic due to relative high binding of water by PEG moiety; however RP-HPLC analysis resulted in increased retention of conjugates. Retention time of the conjugates depended only on PEGs´ size. In the second part of our research we studied conjugates´ relationship between their structure and function focused on the influence of molecular weight and shape of PEGylated conjugates on relative biological activity ''in vitro'' as well as on pharmacokinetic behaviour in animal model (rats). ''In vivo'' study on rats showed that pharmacokinetic profiles for Pegasys® (non selectively pegylated interferon α-2a with 40 kDa branched PEG) and our conjugate with 45 kDa branched PEG are comparable. Distinctive linear relationship between mentioned analytical methods and ''in vitro'' biological activity for N-terminally pegylated IFN was established only for CE-HPLC. This finding shows that shielding of protein surface is the main cause that affects ''in vitro'' biological activity. Another interesting conclusion is that CE-HPLC is a useful tool for prediction of ''in vitro'' biological activity for a set of site specific monopegylated conjugates.