haku: @instructor Eerikäinen, Tero / yhteensä: 27
viite: 6 / 27
| Tekijä: | Valkama, Anniina |
| Työn nimi: | Optimization of lentiviral vector production |
| Lentivirusvektorien tuotannon optimointi | |
| Julkaisutyyppi: | Diplomityö |
| Julkaisuvuosi: | 2015 |
| Sivut: | 116 + 14 Kieli: eng |
| Koulu/Laitos/Osasto: | Kemian tekniikan korkeakoulu |
| Oppiaine: | Biotekniikka ja elintarviketekniikka (KE3002) |
| Valvoja: | Ojamo, Heikki |
| Ohjaaja: | Eerikäinen, Tero ; Heikura, Tommi ; Ylä-Herttuala, Seppo |
| Elektroninen julkaisu: | http://urn.fi/URN:NBN:fi:aalto-201512165690 |
| Sijainti: | P1 Ark Aalto 3400 | Arkisto |
| Avainsanat: | lentivirus lentiviral vectors fixed-bed bioreactor calcium phosphate transfection tangential flow filtration process optimization lentivirusvektorit kiinteä-petibioreaktori kalsiumfosfaatti transfektio tangentiaalivirtaussuodatus prosessin optimointi |
| Tiivistelmä (eng): | Lentiviral vectors have emerged as a promising alternative in the field of gene therapy. They present a stable and efficient method for gene transfer in humans. However, production with the conventional small-scale production methods does not meet the requirements for producing large amounts of vectors required for several treatments. The aim of this study was to optimize the production of lentiviral vectors in a 4 m2 fixed-bed bioreactor with calcium phosphate transfection, clarification of the product, and concentration with tangential flow filtration. In the upstream phase, the aim was to optimize the conditions, and to find feasible working methods suitable for large-scale production. In the downstream phase, the aim was to find a feasible harvest window, and to find a suitable filter and conditions for clarification and concentration. Upstream phase performed with the conventional transfection protocol resulted in approximately 5 liters of lentiviral vector product per run with an average yield of 1.4 TU/ml. The best yield obtained with the new transfection protocol designed for large-scale operation was 1.1 TU/ml. Based on the downstream results, the optimal harvest window for an adherent fixed-bed bioreactor lentiviral vector production is three days starting from the day after transfection. For clarification, depth filters with large pore size are preferable. In this study, clarification was found to be optimal with a depth filter with pore size of 0.6 - 1.2 µm. Sterile filtration in the early downstream phase decreases the recoveries. Concentration was conducted with tangential flow filtration, and followed by diafiltration. It was observed that relatively high transmembrane pressure of 1.2 bar combined with small nominal molecular weight cutoff of 100 kDa was optimal. Suitably large filtration area is essential to reduce the operation time. Up to 44 % total recovery was achieved with the optimized methods. |
| ED: | 2016-01-17 |
INSSI tietueen numero: 52813
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