Validacija metode za merjenje biološke aktivnosti pri celično posredovani citotoksičnosti, odvisni od protiteles : magistrski študij laboratorijske biomedicine

Ukmar, Matic (author), Černe, Darko (mentor), Čepeljnik, Tadej (co-mentor)

Uporaba analitskih metod v farmacevtski industriji omogoča sledenje različnih parametrov, ki so kazalniki uspešnosti in ustreznosti proizvodnega procesa. Pri izvajanju metod v laboratoriju sledimo predpisanim analitskim postopkom. Zelo pomembno je, da so vse metode validirane, pred izvajanjem rednega analitičnega dela za sproščanje. Validacijo je treba opraviti zato, da se dokaže ustreznost metode za njen namen in zmožnost pridobivanja zanesljivih in ponovljivih rezultatov, s katerimi potrdimo varnost, učinkovitost in kakovost proizvedenih zdravil. S tem namenom so regulatorni organi izdali smernice, v katerih so opisani parametri, ki jih je pri validaciji priporočeno ovrednotiti. Smernice Mednarodne konference o harmonizaciji podaja usklajen pristop regulatornih organov Evrope, Japonske in Združenih držav Amerike. V okviru magistrske naloge smo obravnavali validacijo metode merjenja biološke aktivnosti modelnega biološkega zdravila, ki spada med protitelesa. Predhodno je bilo ugotovljeno, da je za določanje biološke aktivnosti tega protitelesa najbolj reprezentativna metoda celično posredovane celične citotoksičnosti. Pri tej metodi merimo količino sproščenega barvila, ki se sprosti iz tarčnih celic, ko se nanje posredno, s pomočjo monoklonskih protiteles vežejo efektorske celice in povzročijo apoptozo. Predpisali smo si naslednje validacijske parametre, ki smo jih glede na namen metode oblikovali skladno s priporočil smernic. To so specifičnost, linearnost, delovno območje, točnost, natančnost (ponovljivost in vmesna natančnost), robustnost in zmožnost metode za sledenje stabilnosti vzorca. Na podlagi rezultatov zaključujemo, da je metoda specifična, saj vplivov matriksa nismo zaznali; linearna (točna in ponovljiva) v območju 50–200 % relativne biološke aktivnosti in robustna. V vseh primerih smo dobili rezultate, kjer je bila točnost znotraj 8-odstotnega odstopanja od pričakovane vrednosti, variabilnost pa tudi največ 8 %. Izbrana metoda izpolnjuje vse zahteve pri vrednotenih parametrih ter je tako ustrezna za pridobivanje zanesljivih rezultatov. V prihodnosti se bo pri vsakdanjem delu uporabljala za merjenje biološke aktivnosti modelnega biološkega zdravila.

• Language: Slovenian
• Keywords: monoklonsko protitelo celično posredovana citotoksičnost biološki test validacija biološka aktivnost
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [M. Ukmar]
• Year: 2017
• Number of pages: IX, 46 f.
• PID: 20.500.12556/RUL-120963
• UDC: 616-074:615.32(043.3)
• COBISS.SI-ID: 4325489
• Publication date in RUL: 28.09.2020

Secondary language

• Language: English
• Title: Method validation for measuring biological activity in antibody dependent cell mediated cytotoxicity

Abstract

Use of analytical methods in pharmaceutical industry enables monitoring of different parameters that indicate successful and suitable production process. When using a method in a laboratory, prescribed analytical procedures need to be followed. It is very important that the methods are validated before using them for routine use, e.g. release testing. Validation has to be performed in order to prove the suitability of the method for its intended purpose and the capability of producing reliable and reproducible results which confirm safety, effectiveness and quality of the produced medicines. For this purpose, the regulatory authorities issued several guidelines describing the parameters, to be evaluated during validation. The International conference on harmonization guidelines provide a coordinated approach by regulatory authorities from Europe, Japan and the United States of America. In this master's thesis method validation for biological activity measurement of a model antibody biologics is discussed. It had been determined that the most important mode of action, and consequently the release method for measuring biological activity of this specific antibody was antibody dependent cell mediated cytotoxicity. Using this method, we measure the quantity of released dye from target cells after effector cells kill them with the help of monoclonal antibodies. According to the guidelines, the following validation parameters were defined based on the intended purpose of the method: specificity, linearity, range, accuracy, precision (repeatability, intermediate precision), robustness and stability indicating properties. Based on the results it can be concluded, that the selected method is specific, as no matrix interferences were detected; linear (accurate and precise) within the 50–200 % range of the relative biological activity, and robust. In all tests the accuracy of the results was within 8 % of the expected value with variability of 8 % or less. The method met all the requirements during the validation and is therefore suitable for its intended use. In the future the method will be used routinely for determining biological activity of a model antibody biologics.

• Keywords: biopharmaceuticals monoclonal antibody antibody dependent cell citotoxicity bioassay biological activity validation