Razvoj in potrditev metode kapilarne gelske elektroforeze na čipu za določanje čistosti in nečistot terapevtskih monoklonskih protiteles

Plaznik, Nejc (avtor), Jakopin, Žiga (mentor), Podergajs, Neža (komentor)

Biološko podobna zdravila, kot so monoklonska protitelesa in fuzijski proteini, kažejo velik potencial v svetu industrijske farmacije. Njihov prodor na trg omogoča lažje dostopna zdravila enake kakovosti kot referenčna biološka zdravila. Uspešen razvoj teh zdravil temelji na natančni karakterizaciji končno razvitega rekombinantnega proteina ter hitri in učinkoviti analitiki velikega števila vzorcev, ki se proizvede tekom razvoja bioprocesa. Za analizo velikega števila vzorcev tekom razvoja si pomagamo z analitskimi tehnikami višjih zmogljivosti, ki omogočajo ovrednotenje večjega števila vzorcev v krajšem času. V okviru magistrske naloge smo razvili metodo kapilarne gelske elektroforeze na čipu, s katero bo možno določiti čistost in prisotnost nečistot večini terapevtskih monoklonskih protiteles. Pri razvoju smo se osredotočili na pripravo vzorcev, saj instrumentalnih nastavitev na takšnemu sistemu ni mogoče spreminjati. Vzorce smo pripravili pod reducirajoči in nereducirajoči pogoji. Ugotovili smo, da je alkilirno sredstvo N-etilmaleimid primernejši za pripravo nereduciranih vzorcev kot jodacetamid. Poleg ditiotreitola smo kot reducent za pripravo reduciranih vzorcev testirali tudi 2-merkaptoetanol. Noben od njiju se ni izkazal za opazno učinkovitejšega, zato smo se na podlagi organoleptičnih lastnosti slednjega odločili za prvega. Z metodo kapilarne gelske elektroforeze na čipu smo uspeli identificirati določene vrhove fragmentom v vzorcu, vendar kompleksnost monoklonskih protiteles onemogoča vpeljavo generičnega poimenovanja, ki bi zadostilo vsem testiranim vzorcem. Novo razvito metodo smo potrdili na štirih monoklonskih protitelesih in enem fuzijskem proteinu. Izvedli smo teste stabilnosti, mejo kvantifikacije, linearnosti, točnosti, ponovljivosti in robustnosti. S tem smo zagotovili, da metoda ustreza svojemu namenu. Kompleksnost terapevtskih proteinov ne omogoča, da bo metoda ustrezno delovala na vseh proteinih. V prihodnosti se lahko zgodi, da bo za določen terapevtski protein potrebno metodo nekoliko prilagoditi. Potrjeno metodo visoke zmogljivosti smo primerjali s konvencionalno metodo kapilarne gelske elektroforeze. Priprava vzorcev se pri obeh metodah nekoliko razlikuje, poleg tega so drugačne tudi instrumentalne nastavitve. Kljub temu smo opazili podobnost med obema metodama, predvsem v profilih ločbe, imenovanih elektroferogrami. Čistost terapevtskih proteinov in prisotnost določenih fragmentov se pri obeh metodah nekoliko razlikujejo, vendar sta kljub temu obe primerni za analitiko vzorcev iz različnih faz razvoja bioprocesa.

• Jezik: Slovenski jezik
• Ključne besede: Biološko podobna zdravila, protitelesa, fuzijski proteini, analitika, kapilarna elektroforeza visoke zmogljivosti
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2019
• PID: 20.500.12556/RUL-107388
• Datum objave v RUL: 10.04.2019

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Development and qualification of chip-based capillary gel electrophoresis for determination of monoclonal antibody purity and fragmentation

Izvleček

Biosmilars, such as monoclonal antibodies and fusion proteins, indicate big potential in pharmaceutical industry. Their availability on the market enables easier access to medicines with the same quality as those of their reference biopharmaceuticals. Successful development of biosimilars depends on in-depth analytical characterization of finally developed recombinant protein and efficient characterization of many samples that are produced throughout development phase. High-throughput methods help us to evaluate large number of samples in shorter time period. Our purpose was to develop a capillary gel electrophoresis method on a chip, which would be able to determine purity and fragmentation of most therapeutic monoclonal antibodies. We focused on sample preparation since instrumental settings for sample injection and separation could not be changed. Samples were prepared under reduced and non-reduced conditions. We found out that N-ethylmaleimide is more suitable for preparing non-reduced samples than iodoaceamide. Reduced samples were prepared with dithiothreitol and 2-mercaptoethanol. Since there were no significant differences between them and due to the organoleptic characteristics of the latter we decided to use dithiothreitol. Capillary gel electrophoresis on a chip helped us to identify some of the peaks, representing fragments. Due to the complexity of monoclonal antibodies we could not implement a generic nomenclature for all IgG samples. Newly developed method was qualified on four different monoclonal antibodies and one fusion protein. We performed tests for sample stability, limit of quantification, linearity, accuracy, repeatability and robustness of the method. By fulfilling the criteria for all these tests, we assured that method is suitable for its purpose. Complex structure of therapeutic proteins does not assure that our method will be suitable for all proteins. In the future, we might have to adapt some steps of the method to specific protein. Qualified high-throughput method on a chip was compared to conventional capillary gel electrophoresis method. There are some differences in sample preparation and instrumental settings between both methods. Despite these differences, similar separation profiles, named electropherograms, were observed. Purity and fragmentation of therapeutic proteins between both methods is different to a certain degree. However, both methods are suitable for analytics of samples from different phases of bioprocess development.

• Ključne besede: Biosimilars, antibodies, fusion proteins, analytics, high-throughput capillary electrophoresis