doktorska disertacija
Metka Stantič (Author), Aleš Podgornik (Mentor), Matevž Pompe (Thesis defence commission member), Igor Plazl (Thesis defence commission member), Aleš Ručigaj (Thesis defence commission member), Hrvoje Petković (Thesis defence commission member), Mojca Benčina (Thesis defence commission member), Gregor Gunčar (Co-mentor)

Abstract

Protitelesa in rekombinantne učinkovine druge generacije, kamor sodijo tudi Fc-fuzijski proteini, predstavljajo visok delež na svetovnem trgu bioloških učinkovin, saj obstaja široko področje njihove uporabe za številne indikacije. Ker biološka zdravila proizvajajo živi organizmi, ki so po svoji naravi spremenljivi, lahko zdravilna učinkovina v končnem biološkem zdravilu izraža določeno manjšo stopnjo variabilnosti (mikroheterogenosti), ki pa mora biti znotraj sprejemljivega razpona, da bi dosledno zagotavljali varnost in učinkovitost. Ena izmed pomembnejših posttranslacijskih modifikacij, ki ključno vpliva na njihovo strukturo, topnost, stabilnost, funkcijo, lokalizacijo, zvijanje in na interakcije z drugimi proteini, je glikozilacija. Proizvodnja terapevtskih glikoproteinov tako poteka pod nadzorovanimi pogoji, ki jih je treba konstantno spremljati, da bi zagotovili dosleden, ponovljiv in predvidljiv vzorec glikozilacije. S tem namenom smo želeli razviti kromatografske tehnike za določanje različnih izooblik protiteles, ki bi jih lahko vključili v sledenje bioprocesa. V prvem delu doktorske disertacije smo okarakterizirali lektinske ligande za afinitetno kromatografijo. V ta namen smo lektine modificirali za uspešnejšo vezavo na nosilce in boljšo izpostavljenost aktivnega mesta. Biološko aktivnost smo preverjali z interferometrijo z biološkimi plastmi (ang. Biolayer interferometry oz. kratica BLI). Modificiran lektin rPA-ILNME6, ki smo ga poimenovali rPE6, je, imobiliziran na nosilec, izkazoval boljšo kinetiko vezave v primerjavi s prvim. V drugem delu doktorske disertacije smo v nadaljevanju določali pogoje za delno razvijanje protiteles, ki je potrebno, da se ogljikovi hidrati izpostavijo in s tem postanejo dostopni za vezavo lektinov. Protitelesa smo inkubirali pri povišanih temperaturah ob prisotnosti reducenta in detektirali spremembe s pomočjo merjenja intrinzične fluorescence triptofana, DLS-meritev, gelsko izključitvene kromatografije in ionsko izmenjevalne kromatografije. Glede na rezultate meritev intrinzične fluorescence in BLI-meritev je do razvijanja protiteles sicer prišlo, vendar sta bila delež razvitih molekul, pa tudi njihova stabilnost nizka, saj jih nismo uspeli zaznati s kromatografskimi metodami. Vzporedno smo zato kot predstavnike glikoproteinov pri razvoju kromatografskih nosilcev z lektini uporabljali Fc-fuzijske proteine, z znanimi glikanskimi strukturami, ki so izpostavljene na zunanji strani proteina. Na ta način smo lahko v tretjem delu doktorske disertacije okarakterizirali poliHIPE-nosilec z vezanim lektinom rPE6. Kapaciteto nosilca smo ocenili na 0,57 mg/ml, mejo detekcije pa med 0,19 mg/ml in 8 mg/ml. S pomočjo BLI-metode smo pokazali, da je vezavna kinetika imobiliziranega lektina rPE6 dovolj hitra, da nima vpliva na vezavo Fc-fuzijskega proteina. To omogoča uporabo razvitega nosilca za hitro in učinkovito ločbo izooblik glikoproteinov. Čas ločbe smo optimizirali na 10 min, pri pretoku mobilne faze 8 ml/min. PoliHIPE-nosilec z vezanim lektinom rPE6 je ohranjal stabilnost vsaj še pet mesecev po izvedeni imobilizaciji. V četrtem delu doktorske disertacije smo prikazali še konkretni primer uporabe tega nosilca za spremljanje perfuzijskega bioprocesa proizvodnje Fc- fuzijskega proteina. Vzorec dobljenih koncentracij s poliHIPE-nosilcem se je ujemal z rezultati, dobljenimi s pomočjo izolacije s kolono, z imobiliziranim protienom A. Komponente gojišča, kot so HCP-ji in DNK-molekule, niso motila detekcije, zaradi česar je razviti nosilec primeren za ločevanje izooblik glikoproteinov, ki imajo v glikanski strukturi mono-/disaharide, ki jih veže lektinski ligand, v našem primeru terminalno vezani galaktozo in N-acetillaktozamin. Nosilec smo uporabili za metodo določanja debeline sloja z merjenjem padca tlaka, ki omogoča sledenje nastajanja agregatov Fc-fuzijskega proteina, vendar je bila njihova koncentracija pod mejo detekcije, smo pa s to metodo pokazali agregacijo lektina med imobilizacijo.

Keywords

glikozilirani proteini;monoklonska protitelesa;Fc-fuzijski proteini;lektini;kromatografija;lektinske kromatografske kolone;interferometrija z biološkimi plastmi;doktorske disertacije;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [M. Stantič]
UDC: 66.081.3(043.3)
COBISS: 90194435 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Development of chromatographic techniques of process analytical technology for determination of monoclonal antibody isoforms
Secondary abstract: Antibodies and recombinant active substances of the second generation, which also include the Fc-fusion protein, represent a high percentage of the global market of biologically active substances as there is a wide range of their use in numerous applications. Because biological medicines are produced by living organisms, the healing substance in the final biological medicine can express a certain lower stage of variability (microheterogeneity), which has to be within a certain acceptable range. One of the more important post-translation modifications, that has a vital influence on their structure, solubility, stability, function, locality, folding, and interactions with other proteins, is glycosylation. The production of therapeutic glycoproteins thus takes place under controlled conditions, which must be constantly monitored to ensure a consistent, reproducible and predictable pattern of glycosylation. With this aim, we wanted to develop chromatographic techniques for determining different isoforms o antibodies, which we could include in a bioprocess analysis. In the first part of the doctorate dissertation, we characterized lectin ligands for affinity chromatography. For this purpose, we modified the lectins for a more successful immobilization to carriers and better exposure of the active region for binding. Biological activity of lectins was checked by biolayer interferometry (BLI). The modified lectin rPA-ILNME6, which we named rPE6, bonded to a carrier showed better bonding kinetics in comparison to the first. In the second part of the doctorate dissertation, we further determined the conditions for partial antibody unfolding, which is needed, for the carbohydrates to expose themselves and with that become accessible to bond with lectins. We exposed the antibodies to temperature stress with the presence of a reductant and detected changes with the help of tryptophane intrinsic fluorescent measurements, DLS measurements, size-exclusion chromatography, and ionic exchange chromatography. According to the results of intrinsic fluorescence and BLI measurements, the unfolding of antibodies was achieved, but the proportion of unfolded molecules, as well as their stability, was low, as they could not be detected by chromatographic methods. Because of the unsuccessful unfolding of the anty bodies we used Fc-fusion proteins, as representatives of glycoproteins, with known glycan structures exposed for bonding, for the development of chromatographic carriers with lectins. This way we were able to characterize a poliHIPE carrier with the bonded lectin rPE6 in the third part of the doctorate dissertation. We estimated the carrier capacity to 0.57 mg/ml and the limit of detection between 0.19 mg/ml and 8 mg/ml. We showed that the binding kinetics of the immobilized lectin rPE6 is fast enough so that it doesn't affect the Fc-fusion protein binding. This allows us to use the developed carrier for fast and efficient separation of glycoprotein isoforms. We optimized the separation time to 10 min, at a mobile phase rate of 8 ml/min, and the carrier maintained stability for a further four months after immobilization. In the fourth part of the doctorate dissertation, we showed an example of the use of this carrier for monitoring a perfusion bioprocess production of an Fc-fusion protein. The concentration pattern obtained with poliHIPE carrier matched with the results obtained with the protein A column isolation. The components of the medium, HCPs and DNA molecules did not interfere with the detection with our carrier, which makes it suitable for separating isoforms of glycoproteins that have oligosaccharides bound by lectin ligand in the glycan structure. The carrier was also used to determine layer thickens by measuring pressure drop, which allows us to track the aggregate formation of the Fc-fusion protein, but their concentration was below the limit of detection. However, we used this method to show lectin aggregation during immobilization.
Secondary keywords: lectins;monoclonal antibody;Fc-fusion protein;chromatography;biolayer interferometry;Kromatografija;Disertacije;
Type (COBISS): Doctoral dissertation
Study programme: 1000381
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo
Pages: XVIII, 100 str.
ID: 13854611