Primerjava različnih gojitvenih plaftorm celic CHO pri razvoju celičnih linij

Kopač, Urša (author), Zupan, Janja (mentor), Gaber, Rok (comentor)

V zadnjih nekaj desetletjih so se za gojenje ovarijskih celic kitajskega hrčka (celice CHO) na laboratorijskem nivoju za potrebe razvoja uporabljale rastne plastenke (SF). Zaradi vse večjih potreb po (podobnih) bioloških zdravilih, biofarmacevtska industrija teži k čim večjim izkoristkom proizvodnje in čim krajšim časovnicam razvoja omenjenih zdravil. To je razlog, da SF niso več ustrezni za gojenje celic CHO, saj ne omogočajo visoke zmogljivosti dela. V praksi neučinkovite SF sedaj nadomeščajo plošče 24DW, ki so zelo primerne za avtomatizacijo in robotizacijo postopkov v celičnem laboratoriju. S ploščami 24DW dosegamo boljše izkoristke v smislu časa, števila proizvedenih celičnih klonov, obsega dela, porabljenih surovin in financ. Še boljše izkoristke in višje zmogljivosti laboratorija nam omogočajo plošče 96DW. Z avtomatiziranimi postopki in uporabo robotskih pipetirnih sistemov v 96DW gojimo več tisoč klonov hkrati brez povečanja obsega dela. Z njimi izvedemo učinkovitejše presejanje klonov ter tako hitreje pridobimo visoko producirajoče celične linije. Pri razvoju bioprocesa se večinoma uporablja miniaturizirane bioreaktorske sisteme (npr. Ambr 15), ki omogočajo gojenje do 48 celičnih linij hkrati pri skrbno kontroliranih in uravnavanih pogojih. Sistem Ambr 15 zahteva dokaj visok finančni vložek na izveden bioproces, a je visoko zmogljiv in avtomatiziran ter odlično posnema obnašanje celične linije v velikih bioreaktorjih. V sklopu magistrske naloge smo želeli ovrednotiti gojenje celic CHO v novih, učinkovitejših platformah (24DW, 96DW) glede na uveljavljene SF. Zaradi izrazito višjih časovnih, materialnih in finančnih izkoristkov novejših platform smo želeli preveriti, ali bi slednje lahko nadomestile neučinkovite SF pri gojenju celic CHO za potrebe razvoja produkcijskih celičnih linij. Prav tako smo želeli preveriti, kako platforme v zgodnjem razvoju (SF, 24DW, 96DW) napovejo dogajanje v Ambr 15. Parametri, ki smo jih tekom magistrske naloge vrednotili, so bili: število živih celic, viabilnost ter celična produktivnost (titer). V analize smo vključili tako meritve, pridobljene med 14-dnevnimi šaržami z dohranjevanjem kot tudi meritve med 6-dnevnimi šaržami brez dohranjevanja. Primerjani parametri v 24DW in 96DW dobro korelirajo z obstoječimi SF (ρ > 0,87 za vrednosti titra). Slabše, vendar vseeno zadovoljive korelacije smo dobili med meritvami parametrov v sistemu Ambr 15 in ostalimi platformami (ρ > 0,63 za vrednosti titra). Hkrati smo z eksperimenti pokazali, da lahko s 6-dnevno šaržo brez dohranjevanja dobro napovemo dogajanje po koncu 14-dnevne šarže z dohranjevanjem. Vse bolj se zavedamo tudi pomembnosti poznavanja, modeliranja in kontrole N-glikozilacije monoklonskih protiteles oz. Fc-fuzijskih proteinov. Glikanski profili namreč vplivajo na aktivnost biofarmacevtika, na njegove farmakokinetične in farmakodinamične lastnosti ter na varnost njegove uporabe. V sklopu magistrske naloge smo zato s pomočjo interferometrije z biološkimi plastmi (analiza BLI) ovrednotili tudi glikanske profile proteina v različnih platformah. Ugotovili smo, da se glikanski profil proteina ohrani ne glede na uporabljeno platformo. Dobro ohranjenost glikanskega profila smo pokazali tudi med 14-dnevno šaržo z dohranjevanjem in 6-dnevno šaržo brez dohranjevanja.

• Language: Slovenian
• Keywords: celice CHO, rastna plastenka, 24DW, 96DW, Ambr 15
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-127866
• Publication date in RUL: 25.06.2021

Secondary language

• Language: English
• Title: Comparison of different CHO cells cultivation platforms used in cell line development

Abstract

In the last few decades shake flasks (SF) were traditionally used for small scale Chinese hamster ovary cell (CHO cell) cultivation in development of biopharmaceuticals. The need for novel biopharmaceuticals is increasing and biopharmaceutical industry is striving for high efficiency of production processes and shorter timelines for drug development. Due to above mentioned reasons conventional SFs are not sufficient anymore as they do not provide high-throughput workflow. In recent years SFs were replaced by high-throughput 24DW plates that are suitable for automation and robotization. By using 24DW we are able to reach higher efficiency regarding costs, time, number of produced clones and workload. Even better efficiencies are reached when using 96DW plates. With highly automated and robotized laboratory procedures we can handle thousands of clones in 96DW plates in parallel. Clone screening in this platform is very effective and the final production clone is selected quicker. In up-stream process development the use of miniaturized bio-reactor systems (e.g. Ambr 15) is increasing. This platform provides cultivation of 48 different clones simultaneously in highly controlled and regulated conditions. It requires decently high financial input; however, it is mainly automated, high-throughput system that perfectly mimics cell line behavior in large-scale bioreactors. In the present master thesis we evaluated cultivation of CHO cells in novel, high-throughput platforms (24DW, 96DW) and traditional SFs. Due to increasing development costs, need for higher resource and time efficiency we investigated whether novel platforms could replace the insufficient SFs while culturing CHO cells on laboratory scale. We also examined whether platforms in early development could mimic the bioprocess in Ambr 15. We analyzed different parameters such as viable cell density, viability and cell productivity (titer). In the evaluation of platforms we included data gathered during 14-day fed-batch as well as 6-day simple batch experiments. We observed very good correlation between 24DW, 96DW and conventional SFs cultivations (ρ > 0,87 for titer values). Correlations were slightly worse between Ambr 15 and other platforms (ρ > 0,63 for titer values). We showed that results from 6-day simple batch cultures predict the parameters at the end of 14-day fed-bach cultures in a fairly good manner. N-glycosylation of monoclonal antibodies and fusion proteins is particularly important nowadays, as it affects protein’s effector function, pharmacokinetics and pharmacodynamics as well as its safety in patient use. In this master thesis we evaluated whether the glycoprofile is preserved during CHO cell cultivation in different novel and already established platforms. Glycoprofiles were determined using bio-layer interferometry (BLI) measurements. We showed the glycan structure on expressed protein is mainly preserved regardless of the platform or cultivation regime used in experiments.

• Keywords: CHO cells, shake flask, 24DW, 96DW, Ambr 15