Razvoj metode peptidnega kartiranja za uporabo v biofarmacevtiki

Rukavina, Andrea (author), Podgornik, Aleš (mentor), Progar, Vasja (comentor)

Razvoj bioloških in biološko podobnih zdravil, ki so proizvedena s tehnikami molekulske in celične biologije, v zadnjih desetletjih hitro narašča. Le-ta so proteinske narave, med njimi se vse bolj uveljavljajo rekombinantna monoklonska protitelesa (mAbs). Zaradi dolgoletne uporabe in zgodovine regulatornih odobritev se za proizvodnjo rekombinantnih mAbs najpogosteje uporabljajo celične linije ovarijskih celic kitajskega hrčka, pri katerih je tehnologija transfekcije, amplifikacije in selekcije klona dobro vpeljana in okarakterizirana. Pri razvoju celične linije in bioprocesa za proizvodnjo rekombinantnih proteinov se uporabljajo različne analitske metode za spremljanje kritičnih atributov kakovosti (CQA). Vrednosti posameznih atributov morajo ustrezati določenim vnaprej postavljenim mejam, saj lahko le na tak način zagotavljamo ustrezno varnost in učinkovitost terapevtskega proteina. Tradicionalno se uporabljajo specifične analitske metode, za katere velja princip: en atribut – ena metoda. V zadnjem času pa tradicionalne analitske metode nadomešča multiatributna metoda (MAM), ki največkrat temelji na peptidnem kartiranju (Pepmap). Njena glavna prednost pred tradicionalnimi analitskimi metodami je v tem, da lahko v eni analizi spremljamo več atributov hkrati, s čimer prihranimo veliko časa in sredstev. Namen raziskovalne naloge je bil vzpostaviti in optimizirati MAM, pri čemer smo uporabljali tehniko tekočinske kromatografije visoke ločljivosti, sklopljene z masno spektrometrijo, ki temelji na Orbitrap tehnologiji. Razvili smo uporabno orodje za spremljanje številnih CQA, ki se je izkazalo kot odlična alternativa številnim konvencionalnim metodam.

• Language: Slovenian
• Keywords: celična linija CHO, rekombinantna terapevtska mAbs, CQA, MAM, Pepmap, LC-MS
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [A. Rukavina]
• Year: 2020
• PID: 20.500.12556/RUL-123513
• UDC: 606:62:602:543.51(043.2)
• COBISS.SI-ID: 44461571
• Publication date in RUL: 20.12.2020

RUKAVINA, Andrea, 2020, Razvoj metode peptidnega kartiranja za uporabo v biofarmacevtiki [online]. Master’s thesis. A. Rukavina. [Accessed 19 May 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=123513

Secondary language

• Language: English
• Title: Development of the peptide mapping method for use in biopharma industry

Abstract

The development of biologics and biosimilars, which are produced by molecular and cell biology techniques, has been growing rapidly in recent decades. These are proteins among which recombinant monoclonal antibodies (mAbs) are gaining ground. Due to the long-term use and history of regulatory approvals, Chinese hamster ovary cell lines are most commonly used for the production of recombinant mAbs, in which transfection, amplification and clone selection is well established and characterized. Various analytical methods for monitoring critical quality attributes (CQA) are used during development of the cell line and bioprocess for the production of recombinant mAbs. The value of individual attribute must meet certain predefined criteria, as this is the only way to ensure adequate safety and efficacy of the therapeutic protein. Traditionally, specific analytical single-attribute methods are used for monitoring of CQA. Recently, however, traditional analytical methods have been replaced by the multi-attribute method (MAM), which is most often based on peptide mapping (Pepmap). Its main advantage over traditional analytical methods is monitoring of multiple attributes simultaneously in one analysis, thus saving a lot of time and resources. The main scope of our work was the establishment and optimization of MAM by using high-resolution liquid chromatography coupled with Orbitrap-based mass spectrometry technique. We have developed a useful tool for monitoring of multiple attributes simultaneously in one analysis, which could be an excellent alternative to conventional chromatographic and electrophoretic techniques.

• Keywords: CHO cell lines, recombinant therapeutic mAbs, CQA, MAM, Pepmap, LC-MS