Impurity removal by ion-exchange membrane absorbers

Trnovec, Helena (author), Podgornik, Aleš (mentor)

A fully continuous, downstream process represents one of the most interesting novel purification approaches in the biosimilars industry. This would enhance the production output while reducing the costs of complex biopharmaceuticals. Since it generally involves several chromatographic steps, the selection of appropriate chromatographic columns is of utmost importance. In this study we compared several commercially available ion -exchange-membrane adsorbers (NatriFlo®, Sartobind® and Mustang®). In the first part of the thesis, basic characterisation of the selected membrane adsorbers was performed, whereas in the second part of the thesis the removal of host cell proteins (HCPs) and monoclonal antibody aggregates in the flow -through mode was evaluated. Dynamic binding capacity, ionic capacity and type of ligand were determined for individual membrane adsorbers, using simple, fast and non-invasive methods. Design of Experiments (DoE) was employed to determine the optimal pH and conductivity conditions. We demonstrated that all the anion-exchange-membrane adsorbers were capable of removing HCPs from monoclonal antibody mixtures below the required threshold across a wide range of sample pHs and conductivity values, and that the HCPs’ normalised outlet concentration increases almost linearly with loading, being independent of the HCPs’ concentration. No significant differences in the profile of the adsorbed HCPs with respect to the membrane adsorbers were observed based on 2D electrophoresis analysis data, although they exhibited different binding capacities. Cation-exchange-membrane adsorbers were also tested for the removal of aggregates. The Yamamoto model was used to determine the number of active binding sites and estimate the conductivity range for efficient removal of aggregates, while maintaining a high monoclonal antibody recovery. However, the obtained range had to be further fine-tuned experimentally, due to displacement phenomena. Differences in the trends of binding-site number with a change in the pH value for the tested cation-exchange adsorbers indicate slightly different adsorption mechanisms. To obtain optimal process performance, adjustments of the pH and conductivity were required between the anion- and cation-exchange steps.

• Language: English
• Keywords: membrane adsorbers, host cell proteins, aggregates, displacement, Yamamoto model
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2020
• PID: 20.500.12556/RUL-120070
• COBISS.SI-ID: 31891715
• Publication date in RUL: 15.09.2020

Secondary language

• Language: Slovenian
• Title: Odstranjevanje nečistoč z ionsko-izmenjevalnimi membranskimi adsorberji

Abstract

Kontinuirna kromatografija za čiščenje bioloških zdravil predstavlja eno izmed bolj zanimivih pristopov v farmacevtski industriji pridobivanja bioloških zdravil, s katero lahko povečamo produktivnost pridobivanja bioloških zdravil ob nižjih stroških proizvodnje. Postopki čiščenja so običajno sestavljeni iz večih kromatografskih korakov, pri katerih je potrebno izbrati ustrezno tehnologijo. V tej študiji smo primerjali več komercialno dostopnih ionsko izmenjevalnih membranskih adsorberjev (NatriFlo®, Sartobind® and Mustang®). V prvem delu doktorske dizertacije smo okarakterizirali same nosilce, medtem ko smo se v drugem delu osredotočili na odstranjevanje procesnih nečistoč in nečistoč povezanih s produktom, kot so proteinski ostanki CHO celic (HCP) in agregati monoklonskih protiteles (agregati) pri takih kromatografskih pogojih, kjer se nečistoče vežejo na nosilec, medtem ko željeni produkt preide preko kolone. Osnovna karakterizacija membranskih nosilcev je zajemala določitev dinamične vezavne kapacitete nosilca, ionske kapacitete nosilca in struktura liganda. Statistično orodje smo uporabili za določitev optimalnih pogojev pH in prevodnosti raztopine, pri katerih ustrezno odstranimo nečistoče. Dokazali smo, da vsi testirani anionsko-izmenjevalni adsorberji ustrezno odstranijo nečistoče (HCP) iz mešanice monoklonskih teles in prisotnih nečistoč v širokem območju pH in prevodnosti raztopin. Membranski adsorberji so ustrezno odstranili normalizirane vrednosti HCP tudi pri višjih nanosih na membranski nosilec. Glede na rezultate 2D elektroferograma smo ugotovili, da anionski membranski adsorberji ods tranjujejo HCP enake strukture, razlike so se pojavile v deležu odstranjenih HCP. Yamamotov model smo uporabili ne samo za določitev števila vezavnih mest kationskih membranskih adsorberjev, ampak smo z njegovo pomočjo tudi ocenili območje prevodnosti, kjer še ustrezno odstranjujemo agregate, pri tem pa ohranjamo visok izkoristek separacije. Kljub rezultatom modela je bila potrebna optimizacija procesa, zaradi opaženega fenomena izmenjave monoklonskega protitelesa z agregati monoklonskega telesa na membranski nosilec. Razlike v številu vezavnih mest kationskih izmenjevalcev pri različnih pH raztopin nakazujejo na nekoliko različen mehanizem adsorpcije za posamezni testirani membranski adsorber. Za doseganje optimalnega procesa čiščenja monoklonskih protiteles je bilo potrebno uravnati pH in prevodnost raztopine med obema kromatografskima korakoma (kationsko izmenjevalnim in anionsko izmenjevalnim korakom).

• Keywords: membranski adsorberji, proteinski ostanki CHO celic, agregati monoklonskih protiteles, izmenjava, Yamamotov model