Publicação
Overcome challenges in influenza virus-like particles downstream process
| Resumo: | The development of new vaccines for influenza virus introduced a new generation of vaccines using virus-like particles (VLPs). The lack of genetic material, possibility of production on cell lines and presence of antigens with immunogenicity are the main advantages over the traditional vaccines. The development of a cost-effective downstream process while maintaining the high purity, potency and quality of VLPs is a challenge. In this thesis, several purification steps – clarification, concentration, chromatography, polishing and sterile filtration – were studied to develop a new downstream proves for influenza VLPs. In clarification step, a strategy using D0HC followed by Opticap XL SHC filters presented the best result. For concentration step, the cassette with cut-off of 300 kDa presented a higher yield on hemagglutinin recovery and the lowest process time. For chromatography step, the membrane Sartobind Q and the resin HiTrap Q HP were evaluated, concluding that resin HiTrap presented higher dynamic binding capacity and better resolution on elution. For polishing step, size-exclusion chromatography and multimodal chromatography operate in flow-through mode were compared. The last presented higher recovery yield on hemagglutinin and it was select due to the non-limitation for scale-up. Different materials were analysed for the final sterile filtration. A proof of concept run was performed were the optimized conditions and best devices were evaluated. In the end of process, it was obtained influenza VLPs with concentration and quality enough to advance for animal in vivo studies and for clinical phase I. Additionally, a new tool – magnetic sulphated cellulose particles – was evaluated with the goal to obtain purified and concentrated samples to use in characterization techniques. Overall, this thesis contributes to introduce a new tool and a novel cost-effective downstream purification process with high purity, potency and quality for the next generation of influenza vaccines - VLPs. |
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| Autores principais: | Ferreira, Hugo Miguel Lambuça |
| Assunto: | purification process influenza vacines vírus-like particles magnetic sulphated cellulose particles processo de purificação vacinas de influenza partículas semelhantes a vírus partículas magnéticas de celulose sulfatada |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | The development of new vaccines for influenza virus introduced a new generation of vaccines using virus-like particles (VLPs). The lack of genetic material, possibility of production on cell lines and presence of antigens with immunogenicity are the main advantages over the traditional vaccines. The development of a cost-effective downstream process while maintaining the high purity, potency and quality of VLPs is a challenge. In this thesis, several purification steps – clarification, concentration, chromatography, polishing and sterile filtration – were studied to develop a new downstream proves for influenza VLPs. In clarification step, a strategy using D0HC followed by Opticap XL SHC filters presented the best result. For concentration step, the cassette with cut-off of 300 kDa presented a higher yield on hemagglutinin recovery and the lowest process time. For chromatography step, the membrane Sartobind Q and the resin HiTrap Q HP were evaluated, concluding that resin HiTrap presented higher dynamic binding capacity and better resolution on elution. For polishing step, size-exclusion chromatography and multimodal chromatography operate in flow-through mode were compared. The last presented higher recovery yield on hemagglutinin and it was select due to the non-limitation for scale-up. Different materials were analysed for the final sterile filtration. A proof of concept run was performed were the optimized conditions and best devices were evaluated. In the end of process, it was obtained influenza VLPs with concentration and quality enough to advance for animal in vivo studies and for clinical phase I. Additionally, a new tool – magnetic sulphated cellulose particles – was evaluated with the goal to obtain purified and concentrated samples to use in characterization techniques. Overall, this thesis contributes to introduce a new tool and a novel cost-effective downstream purification process with high purity, potency and quality for the next generation of influenza vaccines - VLPs. |
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