Primerjava klasičnih analitskih metod in modernega visoko zmogljivega multi-funkcionalnega analitskega instrumenta pri razvoju bioloških zdravil

Amidović, Tea (author), Kolar, Mitja (mentor)

V zadnjih letih so biološka zdravila postala primarni fokus raziskav in razvoja v biofarmacevtiki. Sodobna biološka zdravila se proizvajajo z uporabo rekombinantne tehnologije v bioloških sistemih, kot so bakterije, kvasovke ali celice sesalcev. Ta zdravila so večinoma proteini in se zaradi svoje občutljivosti na zunanje pogoje pogosto vnašajo v telo v tekoči obliki. Na stabilnost zdravilne učinkovine v tekoči obliki vplivajo različne pomožne snovi (ekscipienti), med katere spadajo tudi sladkorji in pufri. Sladkorji zagotavljajo dolgotrajno stabilnost ter ščitijo proteine v kriogenih pogojih in med liofilizacijo, po drugi strani pufri omogočajo in vzdržujejo ustrezno pH vrednost ter ščitijo proteinske delce pred degradacijo. Standardna praksa za analizo ekscipientov v biofarmacevtski proizvodnji vključuje uporabo visokozmogljive tekočinske kromatografije (HPLC). V okviru tega diplomskega dela je bil ocenjen potencial infrardeče spektroskopije s Fourierjevo transformacijo (FT-IR) za zagotavljanje primerljivih rezultatov pri analizi različnih ekscipientov. Predstavljeni so osnovni koncepti HPLC in FT-IR tehnik, zlasti s sodobnim analizatorjem MIRA, pri čemer so izpostavljene njihove glavne prednosti, slabosti, področja uporabe ter uporabljeni materiali in metode. Izvedena sta bila dva sklopa eksperimentov, oba z namenom vrednotenja vpliva različnih analitskih pristopov na proces razvoja bioloških zdravil. Prvi eksperiment je vključeval analizo sladkorjev (sukroza, trehaloza, sorbitol, manitol) pri različnih koncentracijah z obema metodama. Drugi eksperiment je bil osredotočen na primerjavo natančnosti in zanesljivosti med FT-IR in HPLC metodama pri analizi pufrov v različnih koncentracijskih območij (citrat, histidin, acetat). Rezultati so pokazali, da obe metodi ponujata primerljivo natančnost in zanesljivost, vendar je priprava vzorcev za HPLC zaradi dodatnih redčenj daljša, kar povečuje možnost napak pri pipetiranju. Na podlagi vseh pridobljenih eksperimentalnih rezultatov je ugotovljeno, da FT-IR omogoča hitrejšo kvantitativno in kvalitativno analizo v primerjavi z metodo HPLC.

• Language: Slovenian
• Keywords: biološka zdravila, ekscipienti, biofarmacevtika, tekočinska kromatografija visoke ločljivosti, infrardeča spektroskopija s Fourierjevo transformacijo (FT-IR)
• Work type: Bachelor thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2024
• PID: 20.500.12556/RUL-161071
• Publication date in RUL: 06.09.2024

Secondary language

• Language: English
• Title: Comparison of classical analytical methods and a modern high-throughput multi-functional analytical instrument in biosimilar drug development

Abstract

In recent years, biological drugs have become the primary focus of research and development in biopharmaceuticals. Modern biological drugs are produced using recombinant technology in biological systems such as bacteria, yeast, or mammalian cells. These drugs are predominantly proteins, and due to their sensitivity to external conditions, they are often dispensed in liquid form. The stability of the active pharmaceutical ingredient in liquid form is affected by various excipients, including sugars and buffers. Sugars ensure long-term stability and protect proteins during cryogenic freezing and lyophilization, while buffers maintain an appropriate pH value and protect protein particles from degradation. The standard practice for analyzing excipients in biopharmaceutical production involves the use of high-performance liquid chromatography (HPLC). This thesis evaluates the potential of Fourier-transform infrared spectroscopy (FT-IR) to provide comparable results in the analysis of various excipients. The basic concepts of HPLC and FT-IR techniques are presented, particularly with a modern MIRA analyzer, highlighting their main advantages, disadvantages, areas of application, and the materials and methods employed. Two experiments were conducted to evaluate the impact of different analytical approaches on the development process of biological drugs. The first experiment involved the analysis of sugars (sucrose, trehalose, sorbitol, mannitol) at various concentrations using both methods. The second experiment focused on comparing the accuracy and reliability of the FT-IR and HPLC methods in the analysis of buffers at different concentration ranges (citrate, histidine, acetate). The results indicated that both methods offer comparable accuracy and reliability; however, sample preparation for HPLC is more time-consuming due to additional dilutions, which increases the potential for pipetting errors. Based on the obtained results, it was concluded that FT-IR enables faster quantitative and qualitative analysis compared to the HPLC method.

• Keywords: biological drugs, excipients, biopharmaceuticals, high-performance liquid chromatography (HPLC), Fourier-transform infrared spectroscopy (FT-IR)