Development of a stability indicating analytical approach for evaluation of lactoferrin

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

Stability-Indicating Analytical Approach for Stability Evaluation of Lactoferrin

Lactoferrin is a multifunctional iron-binding glycoprotein in milk. Due to its potential for the treatment of various diseases, interest in products containing lactoferrin is increasing. However, as a protein, it is prone to degradation, which critically affects the quality of products. Therefore, the main purpose of our work was to develop a stability-indicating analytical approach for stability evaluation of lactoferrin. We were focused on two complementary methods: reversed-phase and size-exclusion chromatography. The stability-indicating nature of the selected methods was confirmed. They were successfully validated by following the ICH guidelines and applied to preliminary lactoferrin stability studies. Up to three degradation products, as well as aggregates and fragments of lactoferrin, were detected in various samples using complementary reversed-phase and size-exclusion chromatographic methods. The analytical approach was additionally extended with three spectroscopic techniques (absorbance, intrinsic fluorescence, and bicinchoninic acid method), which may provide valuable complementary information in some cases. The presented analytical approach allows the stability evaluation of lactoferrin in various samples, including the ability to detect differences in its degradation mechanisms. Furthermore, it has the potential to be used for the quality control of products containing lactoferrin

Stability-indicating analytical approach for stability evaluation of lactoferrin

Lactoferrin is a multifunctional iron-binding glycoprotein in milk. Due to its potential for the treatment of various diseases, interest in products containing lactoferrin is increasing. However, as a protein, it is prone to degradation, which critically affects the quality of products. Therefore, the main purpose of our work was to develop a stability-indicating analytical approach for stability evaluation of lactoferrin. We were focused on two complementary methods: reversed-phase and size-exclusion chromatography. The stability-indicating nature of the selected methods was confirmed. They were successfully validated by following the ICH guidelines and applied to preliminary lactoferrin stability studies. Up to three degradation products, as well as aggregates and fragments of lactoferrin, were detected in various samples using complementary reversed-phase and size-exclusion chromatographic methods. The analytical approach was additionally extended with three spectroscopic techniques (absorbance, intrinsic fluorescence, and bicinchoninic acid method), which may provide valuable complementary information in some cases. The presented analytical approach allows the stability evaluation of lactoferrin in various samples, including the ability to detect differences in its degradation mechanisms. Furthermore, it has the potential to be used for the quality control of products containing lactoferrin

The search for novel proline analogs for viscosity reduction and stabilization of highly concentrated monoclonal antibody solutions

Administration of monoclonal antibodies (mAbs) is currently focused on subcutaneous injection associated with increased patient adherence and reduced treatment cost, leading to sustainable healthcare. The main bottleneck is low volume that can be injected, requiring highly concentrated mAb solutions. The latter results in increased solution viscosity with pronounced mAb aggregation propensity because of intensive protein-protein interactions. Small molecule excipients have been proposed to restrict the protein-protein interactions, contributing to reduced viscosity. The aim of the study was to discover novel compounds that reduce the viscosity of highly concentrated mAb solution. First, the chemical space of proline analogs was explored and 35 compounds were determined. Viscosity measurements revealed that 18 proline analogs reduced the mAb solution viscosity similar to or more than proline. The compounds forming both electrostatic and hydrophobic interactions with mAb reduced the viscosity of the formulation more efficiently without detrimentally effecting mAb physical stability. A correlation between the level of interaction and viscosity-reducing effect was confirmed with molecular dynamic simulations. Structure rigidity of the compounds and aromaticity contributed to their viscosity-reducing effect, dependent on molecule size. The study results highlight the novel proline analogs as an effective approach in viscosity reduction in development of biopharmaceuticals for subcutaneous administration