Development of methods for ion determination in biopharmaceuticals with ion chromatography

Trg bioloških zdravil v zadnjih nekaj desetletjih izjemno hitro raste in pričakovati gre, da se bo trend nadaljeval tudi v prihodnosti. Eden od glavnih izzivov za razvoj novega biofarmacevtika je zagotoviti dolgotrajno stabilnost terapevtskega proteina v končni farmacevtski obliki. Zaradi njihove kompleksne strukture in nepredvidljive narave lahko na proteine v formulacijah vpliva vsaka sestavina. Ker ti učinki variirajo s koncentracijami, moramo biti sposobni kvantitativno določiti vsako pomožno snov. Ionske lastnosti nekaterih pomožnih snovi, npr. soli in pufrov, lahko izkoristimo z ionsko kromatografijo. Naš namen v magistrski nalogi je bil razviti analitske metode za določanje najpogostejših ionskih pomožnih snovi v bioloških zdravilih z ionsko kromatografijo ob uporabi dušilca in detekcije električne prevodnosti. Razvoj dveh metod, ene za anione in druge za katione, smo izvajali ločeno, a smo v obeh primerih postopali enako. V prvem koraku smo optimizirali kromatografske pogoje tako, da smo v najkrajšem možnem času uspešno ločili vse ione. To smo v glavnem dosegli s spreminjanjem ionske moči mobilne faze. Potem ko smo postavili končne kromatografske pogoje, smo pričeli z validacijo metode v skladu z internim validacijskim protokolom. Za vsak posamezen ion smo preverili linearnost, točnost in znotraj-analizno natančnost v izbranem koncentracijskem območju. Kasneje smo raziskali še med-analizno natančnost metode. Potrdili smo tudi enotedensko stabilnost vseh analiznih raztopin pri 2–8 °C. Analitsko metodo za anione smo v celoti validirali za določanje acetata, klorida, sukcinata in fosfata. Za uspešno ločbo vseh smo morali uporabiti gradient kalijevega hidroksida. Metodo za katione smo v celoti validirali za določanje natrija. Zadostovali so izokratski pogoji z metansulfonsko kislino. Tris(hidroksimetil)aminometana oz. trometamina nam ni uspelo ločiti od natrija. Končna metoda zato ni bila primerna za vzorce, ki so vsebovali tako natrij kot tudi trometamin. Po zaključeni validaciji smo v okviru različnih projektnih študij na našem analitskem oddelku izvedli tudi nekaj rednih analiz. V vseh vzorcih smo izmerili vsebnosti ionov, ki so bile v okviru pričakovanj. Izpolnjeni so bili vsi kriteriji za sprejemljivost analize, s katerimi smo vrednotili ustreznost delovanja inštrumenta in priprave standardnih raztopin.The biopharmaceuticals market has experienced a rapid growth over the last few decades and this trend is expected to continue for the foreseeable future. One of the main challenges in developing a new biopharmaceutical is to assure long-term stability of a therapeutic protein in the final dosage form. Due to their complex structure and unpredictable nature, proteins in formulations can be affected by every constituent. Since these effects vary with concentrations, we must be able to quantitatively determine each excipient. Ionic properties of some excipients, e.g. salts and buffers, can be exploited with ion chromatography. Our purpose in this master\u27s thesis was to develop analytical methods for determination of the most common ionic excipients in biopharmaceuticals with ion chromatography, using suppressed conductometric detection. The development of two methods, for anions and cations respectively, was performed separately, though the process was identical in both cases. First, we optimized the chromatographic conditions in a way that would allow a successful separation of all ions within the shortest possible time. We achieved this mostly by varying the ionic strength of the mobile phase. After the final chromatographic conditions were established, we began validating the method in accordance with the intern validation procedure. We assessed linearity, accuracy and intra-assay precision in the selected concentration range for each individual ion. Later, we investigated the method\u27s intermediate precision as well. One-week stability of all analytical solutions at 2–8 °C was also confirmed. The analytical method for anions was fully validated for determination of acetate, chloride, succinate and phosphate. For their successful separation, a gradient of potassium hydroxide had to be utilised. The method for cations was fully validated for determination of sodium. Isocratic conditions with methanesulfonic acid were sufficient. We failed to separate tris(hydroxymethyl)aminomethane, also known as tromethamine, from sodium. The final method was thus deemed unsuitable for samples containing both sodium and tromethamine. After validation we also performed a few regular analyses within the scope of several project studies at our analytical department. The determined ion contents in all of the samples were within expectations. All acceptance criteria, which were used to examine the instrument\u27s performance and standard solution preparation, were met