A carregar...
Publicação
Continuous alkaline lysis of plasmid-containing E. colicells using an oscillatory flow reactor
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 1.62 MB | Adobe PDF |
Autores
Orientador(es)
Resumo(s)
Avanços na terapia genética e vacinação com DNA, aumentam a necessidade de produção em larga escala de DNA plasmídico (pDNA). Uma etapa crítica neste processo é a lise celular, geralmente por lise alcalina, é desafiante na produção industrial de pDNA e na adaptação à produção em contínuo. Durante a lise, a libertação de DNA aumenta a viscosidade do lisado, dificultando a mistura e exigindo controlo de pH e tempo de residência para desnaturar o DNA cromossómico sem degradar o pDNA, evitando extremos de pH localizados e forças de cisalhamento. É ainda necessário separar uma quantidade significativa de precipitado do lisado.
Este estudo avalia a viabilidade da lise alcalina em contínuo num reator de fluxo oscilatório (OFR), testando diferentes condições de amplitude e frequência de oscilação, para mistura adequada e maximização do rendimento pDNA. Demonstrou-se que a frequência de oscilação afeta o rendimento de pDNA e a amplitude de oscilação afeta a percentagem de pDNA superenrolado (sc). O rendimento de pDNA foi semelhante ou superior ao processo em batch, com o maior rendimento obtido a uma amplitude de 1 mm e frequência de 5 Hz, recuperando mais de 60% de pDNA sc. Alta amplitude e frequência de oscilação geram uma mistura mais intensa, diminuindo o rendimento e percentagem de pDNA sc.
Avaliou-se ainda o impacto do tempo de residência e concentração de células. Um tempo de residência mais curto de 2,5 minutos no OFR não afetou o rendimento de pDNA, no entanto, tempo de residência de 10 minutos resultou em agregados e menor rendimento de pDNA. Aumentando a concentração de células até DO600nm de 59, o pDNA recuperado foi 4 vezes superior processo em batch, que se manteve consistente independentemente do aumento da concentração celular.
Embora sejam necessários mais estudos, lise alcalina contínua pode mitigar limitações na produção industrial de pDNA.
Advances in gene therapy and DNA vaccination, have increased the demand for large-scale plasmid DNA (pDNA) production. A critical step in this process is cell lysis, commonly by alkaline lysis, which remains a bottleneck for industrial-scale pDNA production and is challenging to adapt to continuous manufacturing. During lysis, the DNA release increases in viscosity of the lysate impacting mixing characteristics and requiring pH and residence to denature chromosomal DNA but not degrade pDNA, avoiding local pH extremes and shear forces. Moreover, a significant amount of precipitate requires separation from the lysate. This study evaluates the viability of continuous alkaline lysis in an oscillatory flow reactor (OFR), testing different conditions of amplitude and frequency of oscillation for appropriate mixing and maximising pDNA yield. It was demonstrated that oscillation frequency impacts pDNA yield and oscillation amplitude affects supercoiled (sc) pDNA percentage. pDNA yield was similar or higher to that in batch mode, the highest yield obtained at an oscillation amplitude of 1mm and frequency of 5Hz, recovering more than 60% of sc pDNA in all cases. High amplitude and frequency of oscillation generate more intense mixing, decreasing yield and sc pDNA percentage. Furthermore, the impact of residence time and cell concentration was assessed. A shorter residence time of 2.5 minutes in the OFR did not affect pDNA yield, however, a residence time of 10 minutes resulted in aggregate formation and lower pDNA yield. Increasing cell concentration up to an OD600nm of 59, there was a 4-fold increase in recovered pDNA compared to the batch process, which remains consistent regardless of the increase in cell concentration. Although further studies are required, continuous alkaline lysis can mitigate constraints of in large-scale industrial production of pDNA.
Advances in gene therapy and DNA vaccination, have increased the demand for large-scale plasmid DNA (pDNA) production. A critical step in this process is cell lysis, commonly by alkaline lysis, which remains a bottleneck for industrial-scale pDNA production and is challenging to adapt to continuous manufacturing. During lysis, the DNA release increases in viscosity of the lysate impacting mixing characteristics and requiring pH and residence to denature chromosomal DNA but not degrade pDNA, avoiding local pH extremes and shear forces. Moreover, a significant amount of precipitate requires separation from the lysate. This study evaluates the viability of continuous alkaline lysis in an oscillatory flow reactor (OFR), testing different conditions of amplitude and frequency of oscillation for appropriate mixing and maximising pDNA yield. It was demonstrated that oscillation frequency impacts pDNA yield and oscillation amplitude affects supercoiled (sc) pDNA percentage. pDNA yield was similar or higher to that in batch mode, the highest yield obtained at an oscillation amplitude of 1mm and frequency of 5Hz, recovering more than 60% of sc pDNA in all cases. High amplitude and frequency of oscillation generate more intense mixing, decreasing yield and sc pDNA percentage. Furthermore, the impact of residence time and cell concentration was assessed. A shorter residence time of 2.5 minutes in the OFR did not affect pDNA yield, however, a residence time of 10 minutes resulted in aggregate formation and lower pDNA yield. Increasing cell concentration up to an OD600nm of 59, there was a 4-fold increase in recovered pDNA compared to the batch process, which remains consistent regardless of the increase in cell concentration. Although further studies are required, continuous alkaline lysis can mitigate constraints of in large-scale industrial production of pDNA.
Descrição
Tese de mestrado, Engenharia Farmacêutica, 2024, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Plasmid DNA Continuous manufacturing Oscillatory flow reactor Downstream processing Alkaline lysis Teses de mestrado - 2024