The production of recombinant glycoproteins from mammalian cell cultures requires robust processes that can achieve high protein yield while ensuring the efficacy of these proteins as human therapeutics. We describe two approaches currently being developed in our group to genetically engineer cell lines with desirable characteristics for recombinant protein production. To enhance the degree of sialylation in the glycoprotein product, we propose to increase intracellular sialic acid availability by overexpressing the CMP-sialic acid transporters. We are also interested in engineering mammalian cells that can proliferate at reduced cultivation temperatures. Low temperature cultivation of mammalian cells has been shown to enhance glycoprotein production but reduces cell growth. It is hypothesized that a mutant cell line that can proliferate at low temperatures may be coupled with low temperature cultivation to improve recombinant protein production. / Singapore-MIT Alliance (SMA)

Thesis (MScEng)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Recombinant glycoproteins are important biopharmaceuticals, providing solutions for numerous previously untreatable illnesses, in everything from cancer to infertility. Most recombinant biopharmaceuticals are produced in mammalian cells due to their ability to provide the correct post-translational processing for use in humans. The post-translation processing influences many of the protein’s properties including pharmacokinetics, bioactivity, secretion, half-life, solubility, recognition and antigenicity. The aim of this thesis is to further study the upstream production of a glycosylated recombinant protein produced by Chinese hamster ovary (CHO) cells on production scale within the confines of an existing process. The process in question uses adherent CHO cells to produce a glycosylated recombinant hormone. As with most recombinant protein production processes, this process has two sections to the upstream production: a seed train to grow enough cells to inoculate production, and a production section, which focuses on the production of a recombinant protein. The seed train is predominantly conducted in roller bottles, while the production section takes place in perfusion bioreactors, where the cells are attached to microcarriers, with spin-filters for cell retention. The whole process uses medium with serum. There are two process challenges regarding an existing recombinant-protein production process: 1. The gradual increase, over the past several campaigns, of the final population doubling level of the cells (which must remain within certain specified limits) at the end of the seed train. 2. The low glycosylation levels of the product seen in certain campaigns, which meant that a certain number of final product batches were below the specified acceptable glycosylation limits. Following a literature survey several controlled process variables were chosen for investigation and hypotheses made on their effect on the seed train or glycosylation. To investigate their effect on the PDL and cell growth in the seed train: - Medium volume: decreasing the medium volume will yield a lower PDL due to slower cell growth caused by lower glucose availability. - Seeding density: if cells obtain confluence by the time they are harvested, decreasing the seeding density will yield a higher PDL. - Cultivation temperature: decreasing the temperature ought to decrease the growth rate. - Medium feed temperature: there will be no significant difference to the cell culture when pre-heated or cold medium is used. Aeration: using vent caps will increase the oxygen content of the medium in the roller bottles and the cell growth, yielding a higher PDL. To investigate their effect on glycosylation during production: - pH: better glycosylation will be seen at pH 6.9, than at pH 6.7. - Perfusion rate: a higher perfusion rate will lead to better glycosylation due to increased glucose and glutamine concentrations. In the seed train, the only factor that significantly influenced the final PDL was the seeding density. Cell growth was inhibited once cells reached confluence, so lowering the seeding density lead to a higher PDL. It is recommended to use a high seeding density to ensure a lower PDL. Historic data indicated that the seeding density was not the cause of the apparent increase of the final PDL, as all previous campaigns had been seeded with a high seeding density. What then became apparent was that the final PDL remained relatively constant during a campaign and that the increase in final PDL occurred between campaigns. It appears that the apparent increase in the final PDL is due to differences in cell counting between operators as each new campaign was managed by different operators. It is recommended that a mechanical cell counter be used to verify cells counts and to maintain a standard between campaigns. In the bioreactors, varying the pH proved to have no significant effect on the glycosylation levels. However, both the initial perfusion rate and the specific perfusion rate proved to be important from both historical data and the data generated during these experiments. Lower levels of the initial perfusion rate lead to better glycosylation and it is recommended that an initial perfusion rate of 1.0 volumes/day be used. The relationship between the specific perfusion rate and the glycosylation appears to be non-linear and requires further study, for now it is recommended that the specific perfusion rate be kept below 0.3 volumes/day/109 cells. Probable reasons for the unsatisfactory glycosylation seen in certain runs could also be proposed from these two factors: • RP33-133 : Very high specific perfusion rate • RP32-135 : High initial perfusion rate and very high specific perfusion rate • RP32-138 : High initial perfusion rate • RP33-139 : High initial perfusion rate Further research is recommended into the effect of the specific perfusion rate as well as the specific glucose consumption rate and the specific glutamine concentration on the glycosylation. / AFRIKAANSE OPSOMMING: Rekombinante glikoproteïene is baie belangrike biofarmaseutiese produkte wat oplossings bied vir talle voorheen ongeneeslike siektes in alles van kanker tot onvrugbaarheid. Meeste rekombinante farmaseutiese produkte word gemaak deur diere-selle as gevolg van hulle bevoegtheid om die korrekte na-translasie stappe te volg sodat die produkte in mense gebruik kan word. Die na-translasie stappe beïnvloed baie van die proteïene se karaktertreke insluitende die farmakokinetika, bioaktiwiteit, uitskeiding, half-leeftyd, oplosbaarheid, herkenbaarheid and antigeniciteit. Die doel van hierdie tesis is om die stroomop produksie van ‘n rekombinante glikoproteïene vervaardig deur Chinese hamster ovariale (CHO) selle verder te bestudeer binne die grense van ‘n bestaande proses op grootskaalse vlak. Die huidige proses gebruik CHO selle om ‘n rekombinante glikohormoon te produseer. Soos meeste prosesse wat rekombinante proteïene produseer bestaan die stroomop gedeelte van die proses uit twee dele: ‘n saad trein wat genoeg selle maak vir produksie en ‘n produksie gedeelte wat fokus op die vervaardiging van die glikoproteïen. Die saad trein bestaan hoofsaaklik uit roller bottels terwyl produksie plaasvind in perfusie bioreaktors waar die selle op “microcarriers” groei, met spin-filters om die selle binne die bioreaktors te hou; die hele proses gebruik medium met serum. Daar is twee probleme in die stroomop gedeelte van die bestaande proses: 1. Die geleidelike toename oor die afgelope paar jaar van die finale verdubbelingsvlak van die selle aan die einde van die saad trein 2. Die lae glukosilering van die eindproduk wat veroorsaak dat sekere lotnommers buite spesifikasie is Na ‘n literatuur studie, was seker beheerde proses parameters gekies om verder te bestudeer en hipotesisse gemaak oor hulle effek op die saad trein of die vlak van glukosilering. Die volgende faktore is bestudeer vir hulle effek op die finale verdubbelingsvlak van die selle in die saad trein: - Medium volume: ‘n laer medium volume sal lei tot a laer verdubbelingsvlak van die selle as gevolg van stadige groei - Konsentrasie van selle vir inokulasie: as die selle konfluent is teen die tyd wat hulle versamel word sal ‘n laer konsentrasie selle lei tot ’n hoër verdubellingsvlak. - Temperatuur: laer temperatuur behoort te lei tot ‘n stadiger groei koers van die selle - Medium voer-temperatuur: die voer-temperatuur van die medium sal geen beduidende verskil maak - Belugting: die gebruik van “vent-caps” sal die suurstof inhoud van die roller bottels verhoog Die volgende faktore is bestudeer vir hulle effek op die glukosilering tydens produksie: - pH: beter glukosilering word verwag by by pH 6.9 dan by pH 6.7 - Perfusie koers: ‘n hoër perfusie koers sal lei tot beter glukosilering as gevolg van hoër glukose en glutamien konsentrasies Die konsentrasie van die selle wat gebruik word vir inokulasie blyk die enigste faktor te wees wat die finale verdubbelingsvlak van die selle en die groei van die selle in die saad trein beïnvloed het. Die groei van die selle was beprek wanneer die selle konfluent geraak het en dus het ‘n laër sel konsentrasie by inokulasie gelei tot ‘n hoër sel verdubbelingsvlak. Dit word aanbeveel dat ‘n hoë sel konsentrasie by inokulasie gebruik word. Die geleidelike toename van die finale verdubbelingsvlak van die selle in die saad trein is waarskynlik as gevolg van die variasie in sel tellings tussen verskillende operateurs eerder as as gevolg van die beheerde proses parameters. Dit word aanbeveel dat ‘n meganiese sel-teller gebruik word om die verskil in sel tellings tussen operateurs te kontroleer en om ‘n standaard te handhaaf tussen produksie lotte. In die bioreaktors, het die pH geen beduidende invloed gehad op die glukosilering maar uit historiese data en die huidige data van hierdie eksperimente blyk albei die begin perfusie koers en die spesifieke perfusie koers ‘n belangrike invloed te hê op die glukosilering. Laër vlakke van die begin perfusie koers lei tot beter glikosilsie en dit word aanbeveel dat elke produksielot ‘n begin perfusie koers het van 1.0 volume/dag. Die verhouding tussen die glukosilering en die spesifieke perfusie koers blyk om nie-liniêr te wees nie. Nog navorsing hieroor word aanbeveel, maar vir nou word dit aanbeveel dat die spesifieke perfusie koers onder 0.3 volumes/dag/109 selle gehou word. Hierde twee faktore blyk die oorsaak te wees vir die lae glukosilering wat in sekere produksielopies gevind was: • RP33-133 : baie hoë spesifieke perfusie koers • RP32-135 : hoë begin perfusie koers en baie hoe spesifieke perfusie koers • RP32-138 : hoë begin perfusie koers • RP33-139 : hoë begin perfusie koers Dit word aanbeveel dat verdere navorsing gedoen word op die effek van die spesifieke perfusie koers asook die spesifieke koers van glukose verbruik en die spesifieke glutamien konsentrasie op die glukosilering van die produk.