Detection and immunogenicity evaluation of recombinant monoclonal antibodies’ structural variants

Ignjatović, Janko (avtor), Štrukelj, Borut (mentor), Švajger, Urban (komentor)

Background: Development of new recombinant biotechnology products has greatly expanded in the field of modern pharmacy and medicine. Since biological recombinant molecules are sensitive, simple, or composed proteins, their function is heavily dependent on their structure. In addition to their efficacy, biological medicinal products could show side effects such as immunogenicity. Therefore, detection and characterization of protein structural variants is essential during development and quality control of therapeutic proteins that might trigger immune response in organism. Methods: Doctoral dissertation includes proposed detection and characterization of aggregated as well as other modified forms of monoclonal antibodies (mAb) by using selected chromatographic and spectrometric methods. Additionally, the selected mAb’s aggregates and modified structural variants of monoclonal antibodies were subjected to the immature monocyte-derived dendritic cells’ (DC) examination experiment for monitoring of activated DC cells in order to determine potential immunogenicity of mAb structural variants. Furthermore, potential innate immune response of peripheral blood mononuclear cell (PBMC) cultures to mAb aggregates was also evaluated by measuring pro-inflammatory cytokine response during early exposure of PBMC to different mAb samples and by determining the effect of mAb aggregates on PBMC proliferation during long-term cultures. Results and conclusion: All developed and proposed analytical methods and immunological in vitro DC and PBMC assays could be used as a platform for complementary analytical characterization and determination of potential for immunogenicity for all biopharmaceutical products, which contain monoclonal antibodies as active pharmaceutical ingredients.

• Jezik: Angleški jezik
• Ključne besede: Monoclonal antibodies, stress, aggregation, characterization, immunogenicity, in vitro tests, DC assay, PBMC assay
• Vrsta gradiva: Doktorsko delo/naloga
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: BF - Biotehniška fakulteta
• Založnik: [J. Ignjatović]
• Leto izida: 2019
• PID: 20.500.12556/RUL-106393
• UDK: 616-097:616-097.3(043.3)
• COBISS.SI-ID: 9175161
• Datum objave v RUL: 21.02.2019

IGNJATOVIĆ, Janko, 2019, Detection and immunogenicity evaluation of recombinant monoclonal antibodies’ structural variants [na spletu]. Doktorska disertacija. J. Ignjatović. [Dostopano 3 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=106393

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Detekcija in ocena imunogenosti strukturnih variant rekombinantnih monoklonskih protiteles

Izvleček

Ozadje: Razvoj novih rekombinantnih biotehnoloških izdelkov se je močno razširil v sodobno farmacijo in medicino. Ker so biološke rekombinantne molekule občutljive in kompleksne beljakovine, je njihova funkcija močno odvisna od njihove strukture. Poleg njihove učinkovitosti lahko biološka zdravila povzročijo neželene učinke, kot je imunogenost. Zato je odkrivanje in karakterizacija strukturnih variant beljakovin bistvenega pomena pri razvoju in nadzoru kakovosti terapevtskih proteinov, ki bi lahko sprožili imunogeni odziv v organizmu. Metode: Doktorska disertacija vključuje predlagano detekcijo in karakterizacijo agregiranih in drugih modificiranih oblik monoklonskih protiteles (mAb) z uporabo izbranih kromatografskih in spektrometričnih metod. Poleg tega smo vzorce agregatov in modificiranih strukturnih variant monoklonskih protiteles izpostavili testu z nezrelimi dendritičnimi celicami (DC), ki izhajajo iz monocitov, za spremljanje aktiviranih celic DC in ugotavljanje potencialne imunogenosti strukturnih variant monoklonskega protitelesa. Ovrednotili smo tudi potencialni prirojeni imunogenski odziv kulture mononuklearnih celic periferne krvi (PBMC) v stiku z agregati mAb, in sicer z merjenjem vnetnega citokinskega odziva pri zgodnji izpostavitvi PBMC-jev različnim strukturnim oblikam monoklonskega protitelesa in z določanjem učinka agregatov mAb na proliferacijo dolgoživeče kulture PBMC-jev. Rezultati in sklep: Vse razvite in predlagane analitske metode in imunološka in vitro testiranja s celicami DC in PBMC bi se lahko uporabljala kot platforma za komplementarno analitsko karakterizacijo in določitev potenciala za imunogenost za vse biofarmacevtske izdelke, ki vsebujejo monoklonska protitelesa kot aktivne farmacevtske sestavine.

• Ključne besede: Monoklonska protitelesa, stres, agregacija, karakterizacija, imunogenost, in vitro testi, DC assay, PBMC assay