| Abstract | Upalna bolest crijeva (engl. Inflammatory Bowel Disease, IBD) je kronična upalna bolest gastrointestinalnog
trakta, a dijeli se na Crohnovu bolest, ulcerozni kolitis i nedeterminirani kolitis. IBD je najviše zastupljena u
visoko razvijenom dijelu svijeta, a incidencija joj raste. Patofiziologija IBD-a je složena te uključuje različite
citokine od kojih je za terapijsku primjenu najviše iskorištena signalizacija faktora tumorske nekroze α (engl.
Tumor Necrosis Factor, TNF). Razvijeni su biološki lijekovi koji su po svojoj strukturi monoklonska
protutijela, a djeluju kao anti-TNF-α inhibitori. Ti lijekovi se koriste za liječenje različitih bolesti uključujući
i IBD. Biološki lijekovi su velike, složene molekule osjetljive na različite uvjete. Važno je osigurati im
kvalitetu, sigurnost i učinkovitost, a za to je potrebno koristiti složene analitičke tehnike.
Za određivanje sadržaja anti-TNF-α inhibitora adalimumaba i njegovih razgradnih produkata korištena je
kromatografija isključenjem po veličini (engl. Size Exclusion Chromatography, SEC) koja se temelji na
razdvajanju molekula s obzirom na veličinu čestica. Uzorci korišteni za razvoj metode dobiveni su prisilnom
razgradnjom uz korištenje stresnih uvjeta koji su uključivali kisele, bazične uvjete, povišenu temperaturu i
dnevno svjetlo. Razgradnja lijeka dogodila se pri svim uvjetima, najintenzivnije pri kiselim i bazičnim
uvjetima, a produkti razgradnje na 280 nm utvrđeni su nakon provedene kisele, bazične i razgradnje pri
povišenoj temperaturi. Svi produkti razgradnje pokazali su duža vremena zadržavanju u odnosu na
adalimumab. Ovom metodom uspješno su odvojeni ishodni analit i njegova onečišćenja te je eksperimentalno
utvrđeni sadržaj bio gotovo identičan onom deklariranom. Stoga je SE-HPLC prikladna metoda za
određivanje sadržaja bioloških lijekova u prisutnosti njihovih produkata razgradnje. |
| Abstract (english) | Inflammatory bowel disease (IBD) is a chronical inflammatory disease of gastrointestinal tract, and it is
divided into Crohn's disease, ulcerative colitis, and indeterminate colitis. IBD is mostly prevalent in the highly
developed part of the world, and its incidence is rising. The pathophysiology of IBD is complex, and it
includes various cytokines, of which tumor necrosis factor α (TNF-α) signalling is the most used for
therapeutic application. Biological drugs have been developed that are monoclonal antibodies by their
structures, and act as TNF-α inhibitors. These drugs are used for treatment of various diseases, including IBD.
Biological drugs are big, complex molecules that are sensitive to different conditions. It is important to ensure
their quality, safety, and efficiency, and for this it is necessary to use complex analytical techniques.
For determining the content of the anti-TNF-α inhibitor adalimumab and its degradation products, size
exclusion chromatography (SEC) was used, which is based on the separation of molecules regarding particle
size. The samples used for the development of the method were obtained by forced degradation using stressconditions that included acidic, basic conditions, elevated temperature, and daylight. Degradation of the drug
occurred under all conditions, the most intensely under acidic and basic conditions, and degradation products
at 280 nm were determined after acid, basic degradation, and degradation under elevated temperature. All
degradation products had longer retention times compared to adalimumab. Using this method, the initial
analyte and its impurities were successfully separated, and the experimentally determined content was almost
identical to the declared one. Therefore, SE-HPLC is a suitable method for determining the content of
biological drugs in the presence of their degradation products. |
