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Zaščitna vloga pegilacije pri stabilnosti zelo hidrofobnega proteina v tekoči farmacevtski obliki : magistrska naloga
Čerkić, Karmen (Author), Urleb, Uroš (Mentor) More about this mentor... This link opens in a new window

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Abstract
Razvoj tekoĉe farmacevtske oblike za zelo hidrofobne proteine je zahteven proces, saj so zaradi slabe topnosti nagnjeni k agregaciji. Agregati so pogosto imunogeni in njihova dovoljena vsebnost v biofarmacevtskih izdelkih je strogo omejena. Dodajanje ekscipientov (na primer sladkorjev) je najpogostejši naĉin stabilizacije proteinov v tekoĉi farmacevtski obliki. Drug uspešen pristop bi lahko bila PEGilacija. V prvi vrsti je PEGilacija proteinov namenjena podaljšanju razpolovnega ĉasa v telesu, kar privede do izboljšanih farmakokinetiĉnih in farmakodinamiĉnih lastnosti molekule. Vendar konjugacija proteina z verigo polietilenglikola (PEG) prinaša tudi druge prednosti, kot je izboljšana topnost v vodi oziroma izbrani formulaciji. To je še posebej pomembno pri zelo hidrofobnih proteinih. V magistrski nalogi smo izvedli študijo stresne stabilnosti in na modelnem zelo hidrofobnem proteinu preuĉili zašĉitno vlogo PEGilacije na nastanek agregatov. Dodatno smo preuĉili tudi uĉinek ekscipienta, sladkorja trehaloze. Naša izhodišĉna hipoteza je bila, da bo PEGilacija izboljšala stabilnost modelnega proteina. Modelni protein (Protein_t), v optimirani tekoĉi farmacevtski obliki z dodatkom trehaloze smo primerjali s PEGiliranim proteinom (PEGprotein) v dveh razliĉnih tekoĉih farmacevtskih oblikah in sicer z dodatkom trehaloze (PEGprotein_t) in brez (PEGprotein_a). Stabilitetna študija je vkljuĉevala temperaturno stabilnost (-60 °C, 4 °C, 25 °C in 40 °C), cikle zamrzovanja / odmrzovanja in striţne sile (stresanje). Stabilnost vzorcev je bila ocenjena s standardnimi tehnikami HPLC RPC in CEC. Agregacija je bila ovrednotena s tehniko dinamiĉnega sipanja svetlobe (DLS) in tehniko HPLC SEC, sklopljeno z merjenjem statiĉnega sipanja svetlobe (SEC-MALS). Ocenili smo tudi vpliv stresnih pogojev na biološko aktivnost in vitro. Razlike v stabilnosti smo zaznali pri pogojih pospešene stabilnosti, pri 25 °C in 40 °C. Glede na analize RPC in CEC, je bil nastanek razgradnih produktov v obeh PEGiliranih razliĉicah poĉasnejši in analiza SEC je nakazala poĉasnejšo agregacijo PEGiliranih molekul. Kombinacija PEGilacije in dodatka trehaloze je prinesla dodaten stabilizirajoĉi uĉinek. Z DLS smo v vzorcih Protein_t v povpreĉju zaznali veĉje število populacij razliĉnih velikosti kot v vzorcih PEGprotein, kar ravno tako kaţe na veĉjo stabilnost vzorcev PEGprotein. Raziskava je potrdila našo izhodišĉno hipotezo, PEGilacija ima zašĉitno vlogo v stabilnosti zelo hidrofobnega proteina v tekoĉi farmacevtski obliki. Vsi vzorci, tako Protein_t kot tudi PEGprotein, so po izpostavitvi temperaturnemu stresu ohranili biološko aktivnost in vitro.

Language:Slovenian
Keywords:biološka zdravila hidrofobni proteini pegilacija stabilnost proteinov analizna kromatografija
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:FFA - Faculty of Pharmacy
Year:2012
Publisher:[K. Čerkić]
Number of pages:VII, 61 f.
UDC:542+661.12
COBISS.SI-ID:3246961 This link opens in a new window
Views:204
Downloads:46
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Secondary language

Language:English
Title:Protective role of pegylation on stability of very hydrophobic protein in liquid pharmaceutical formulation
Abstract:
Very hydrophobic proteins are especially demanding regarding the development of liquid pharmaceutical formulations, mainly due to their low solubility which makes them prone to aggregation. Aggregates may elicit immune response in patients and their acceptable content in the final drug product is strictly limited. The most common method for protein stabilization in liquid formulations is addition of excipients (e.g. sugars). Another successful approach could be PEGylation. Protein PEGylation is primarily being introduced in cases where improved pharmacokinetic and pharmacodynamic properties arising from the prolonged elimination half-life are envisaged. But protein conjugation to the polyethylene glycol (PEG) chain also brings other benefits, such as increased solubility, which is particularly important when dealing with very hydrophobic proteins. In the MSc thesis we have performed a stress stability study of a highly hydrophobic model protein and tested a protective role of PEGylation on aggregation. Additionally, the effect of sugar excipient (trehalose) was tested. Our initial hypothesis was that PEGylation will improve stability of the model protein. The model protein (Protein_t) in an optimised liquid pharmaceutical formulation containing trehalose was compared to the PEGylated protein (PEGprotein) in two different liquid pharmaceutical formulations, with (PEGprotein_t) and without (PEGprotein_a) trehalose. The stability studies included temperature stability (-60 °C, 4 °C, 25 °C and 40 °C), freeze/thaw cycles and sheer forces (shaking). Stability of the tested samples was evaluated with standard HPLC techniques RPC and CEC. Aggregation was evaluated using dynamic light scattering (DLS) and HPLC technique SEC coupled to static light scattering (SEC-MALS). The influence of stress conditions to the in vitro biological activity was also evaluated. Differences in stability were observed at accelerated stability conditions, 25 °C and 40 °C. According to RPC and CEC the formation of degradation products was slower for both PEGprotein variants and SEC indicates slower formation of aggregates in PEGylated molecules. A combination of PEGylation and the addition of trehalose proved to ensure an even greater stabilizing effect. DLS in average detected a larger number of different size populations in the Protein_t samples than in the PEGprotein samples, which also indicates a higher stability of PEGprotein samples. The research confirmed our initial hypothesis, PEGylation plays a protective role in the stability of the very hydrophobic protein in the liquid pharmaceutical formulation. Both Protein_t and PEGprotein samples retained their in vitro biological activities after exposure to temperature stress.


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