Anion exchange chromatography is the most popular chromatographic method for plasmid separation.POROS R1 50 is a perfusio n chro mato graphic suppo rt w hich is a reversed phase matrix and is an alternative to co nven...Anion exchange chromatography is the most popular chromatographic method for plasmid separation.POROS R1 50 is a perfusio n chro mato graphic suppo rt w hich is a reversed phase matrix and is an alternative to co nventio nal o nes due to its mass transfer pro perties.The adso rptio n and elutio n o f the pIDKE2 plasmid o nto reversed phase POROS R1 50 w as studied.Langmuir iso therm mo del w as adjusted in o rder to get the max imum adso rptio n capacity and the disso ciatio n co nstant fo r POROS R1 50-plasmid DNA(pDNA) system.Breakthro ugh curves w ere o btained fo r vo lumetric flo w s betw een 0.69-3.33 mL/min,given dynamic capacity up to 2.3 times higher than tho se repo rted fo r io nic ex change matrix used during the purificatio n pro cess o f plasmids w ith similar size to that o f pIDKE2.The efficiency w as less than 45% fo r the flo w co nditio ns and initial co ncentratio n studied,w hich means that the suppo rt w ill no t be o perated under saturatio n circumstances.展开更多
The main component of the Center for Genetic Engineering and Biotechnology(CIGB)candidate vaccine against Hepatitis C virus(HCV)is the pIDKE2 plasmid.The current designed downstream process for the production of pIDKE...The main component of the Center for Genetic Engineering and Biotechnology(CIGB)candidate vaccine against Hepatitis C virus(HCV)is the pIDKE2 plasmid.The current designed downstream process for the production of pIDKE2 fulfils all regulatory requirements and renders the required quantities of pharmaceuticalgrade plasmid DNA(pDNA)with 95%purity.The advantages of this procedure include high plasmid purity and the elimination of undesirable additives,such as toxic organic extractants and animal-derived enzymes.However,yields and consequently the productivity of the process are low.Previous work demonstrated that the most critical step of the process is the reverse phase chromatography,where conventional porous particle resins are used.Therefore,to increase the process productivity,alternative technologies such as membranes and chromatographic monoliths were tested as alternative options for this critical step.Here,a comparison between the behaviors of CIM~ C4-HLD and Sartobind phenyl matrices was performed.To obtain higher productivities and purities,the dynamic binding capacities and selectivities were evaluated.The results showed that both matrices had a similar capacity for pIDKE2 plasmid,but the separation of pDNA isoforms using CIM~ technology was much better than that with Sartobind.Additionally,the optimal conditions for loading plasmid DNA on a CIMC4-HLD 800-mL monolithic column in a real production process were determined.These optimizations will allow production levels to satisfy the high plasmid consumption demanded by clinical trials.展开更多
文摘Anion exchange chromatography is the most popular chromatographic method for plasmid separation.POROS R1 50 is a perfusio n chro mato graphic suppo rt w hich is a reversed phase matrix and is an alternative to co nventio nal o nes due to its mass transfer pro perties.The adso rptio n and elutio n o f the pIDKE2 plasmid o nto reversed phase POROS R1 50 w as studied.Langmuir iso therm mo del w as adjusted in o rder to get the max imum adso rptio n capacity and the disso ciatio n co nstant fo r POROS R1 50-plasmid DNA(pDNA) system.Breakthro ugh curves w ere o btained fo r vo lumetric flo w s betw een 0.69-3.33 mL/min,given dynamic capacity up to 2.3 times higher than tho se repo rted fo r io nic ex change matrix used during the purificatio n pro cess o f plasmids w ith similar size to that o f pIDKE2.The efficiency w as less than 45% fo r the flo w co nditio ns and initial co ncentratio n studied,w hich means that the suppo rt w ill no t be o perated under saturatio n circumstances.
基金Center for Genetic Engineering and Biotechnology and BIA Separations Fund
文摘The main component of the Center for Genetic Engineering and Biotechnology(CIGB)candidate vaccine against Hepatitis C virus(HCV)is the pIDKE2 plasmid.The current designed downstream process for the production of pIDKE2 fulfils all regulatory requirements and renders the required quantities of pharmaceuticalgrade plasmid DNA(pDNA)with 95%purity.The advantages of this procedure include high plasmid purity and the elimination of undesirable additives,such as toxic organic extractants and animal-derived enzymes.However,yields and consequently the productivity of the process are low.Previous work demonstrated that the most critical step of the process is the reverse phase chromatography,where conventional porous particle resins are used.Therefore,to increase the process productivity,alternative technologies such as membranes and chromatographic monoliths were tested as alternative options for this critical step.Here,a comparison between the behaviors of CIM~ C4-HLD and Sartobind phenyl matrices was performed.To obtain higher productivities and purities,the dynamic binding capacities and selectivities were evaluated.The results showed that both matrices had a similar capacity for pIDKE2 plasmid,but the separation of pDNA isoforms using CIM~ technology was much better than that with Sartobind.Additionally,the optimal conditions for loading plasmid DNA on a CIMC4-HLD 800-mL monolithic column in a real production process were determined.These optimizations will allow production levels to satisfy the high plasmid consumption demanded by clinical trials.
