Laktoferin je glavni glikoprotein, ki veže železo v človeškem mleku. Deluje protimikrobno, protivnetno, imunomodulatorno, protirakavo in ima še številne druge fiziološke vloge. Laktoferin in drugi proteini so kompleksne molekule, zato za njihovo vrednotenje potrebujemo kombinacije primernih analiznih metod. Primarni namen magistrske naloge je bil razviti stabilnostno indikativno analizno metodologijo, ki omogoča vrednotenje stabilnosti laktoferina v predformulaciji in v končnih izdelkih.
Osredotočili smo se na reverznofazno in izključitveno kromatografijo, ki sta komplementarni metodi. Preizkusili smo štiri različne reverznofazne in dve izključitveni kromatografski koloni. Metode smo optimizirali s spreminjanjem posameznih kromatografskih pogojev. Z analizo stresnih vzorcev laktoferina smo med razvitimi metodami izbrali najprimernejšo z vidika ločbe in oblike kromatografskega vrha za laktoferin. Izbrane kromatografske metode smo uspešno validirali v skladu s smernicami ICH, potrdili smo stabilnostno indikativnost teh metod in ovrednotili njihovo vlogo pri testiranju stabilnosti laktoferina. Kromatografske metode smo nato primerjali z UV spektroskopijo in fluorescenco. Za kvalitativno in kvantitativno določanje laktoferina so primernejše kromatografske metode, ki jih lahko glede na namen analize dopolnjujemo s spektroskopskimi, saj jih odlikuje hitrost analize in cenovna dostopnost.
Pri analizi realnih vzorcev smo ugotovili, da topilo za pripravo vzorca vpliva na določanje laktoferina. Za kvantifikacijo je primernejša reverznofazna kromatografija, ki je boljša od izključitvene kromatografije z vidika validacijskih parametrov in manjšega vpliva topila vzorca na odziv laktoferina. Zato smo z njo preverili vsebnost laktoferina v treh komercialnih izdelkih in šestih polizdelkih ter tudi potrdili ustreznost metode za predviden namen uporabe.
V okviru stresnih testov smo ugotovili, da je laktoferin najbolj občutljiv na bazični medij ter povišano temperaturo, najmanj pa na izpostavitev svetlobi. Potrdili smo veljavnost Arrheniusove enačbe za laktoferin med 60 in 80 °C. Ugotovili smo, da obstaja razlika v stabilnosti laktoferina v končnem izdelku v primerjavi s čistim laktoferinom. Vpeljan stabilnostno indikativni analizni pristop omogoča vrednotenje stabilnosti laktoferina vključno s sposobnostjo zaznave razlik v mehanizmu pretvorbe laktoferina.Lactoferrin is the main iron-binding glycoprotein in human milk. It has anti microbial, anti inflammatory, immunomodulatory, anticancer and many other physiological activities. Lactoferrin and other proteins are complex moleculestherefore a combination of appropriate analytical methods for their evaluation is needed. The main purpose of our work was to develop a stability indicating analytical methodology that would serve for stability evaluation of lactoferrin in preformulation studies as well as in final products.
We were focused on two complementary methods: reversed-phase and size-exclusion chromatography. Methods were optimized on four different reversed-phase and two size exclusion columns by modifying individual chromatographic conditions. The final methods were selected in a forced degradation study based on the resolution ability and the chromatographic peak shape of lactoferrin. The selected methods were successfully validated according to ICH guidelines, also confirming their stability indicating nature. Chromatographic methods were then compared to UV-spectroscopy and fluorescence and they proved to be more appropriate for qualitative and quantitative evaluation of lactoferrin. However, the complementary information from simpler, faster and more accessible spectroscopic techniques may be valuable in some cases.
During the real sample analyses it was observed that the sample media affect lactoferrin quantification. The reversed-phase chromatography was shown to be a more suitable option for quantitative analysis of lactoferrin compared to the size-exclusion chromatography because of its better validation performance and lesser sample media influence on the chromatographic response. The established reversed-phase method was afterwards applied for determination of lactoferrin in three commercially available products and six intermediate products confirming the method’s suitability for the intended purpose.
In stress testing, it was shown that lactoferrin is most prone to degradation under thermal and alkaline conditions and is the least affected by exposure to light. It was proved that the Arrhenius equation describes lactoferrin’s degradation kinetic between 60 °C and 80 °C. A difference in stability of a final product containing lactoferrin compared to pure lactoferrin was also noticed. The presented stability indicating analytical approach allows the stability evaluation of lactoferrin, including the ability to detect differences in its degradation mechanisms
