Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing
Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Reg...
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| Veröffentlicht in: | Journal of biotechnology 2000-01, Vol.76 (2), p.197-205 |
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| creator | Levy, M.S Collins, I.J Tsai, J.T Ayazi Shamlou, P Ward, J.M Dunnill, P |
| description | Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg
−1 plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane. |
| doi_str_mv | 10.1016/S0168-1656(99)00189-3 |
| format | Article |
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−1 plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.</description><identifier>ISSN: 0168-1656</identifier><identifier>EISSN: 1873-4863</identifier><identifier>DOI: 10.1016/S0168-1656(99)00189-3</identifier><identifier>PMID: 10656334</identifier><identifier>CODEN: JBITD4</identifier><language>eng</language><publisher>Lausanne: Elsevier B.V</publisher><subject>Biological and medical sciences ; Bioreactors ; Biotechnology ; Blotting, Southern ; Cell membranes ; Chemical Precipitation ; Chromatography, Ion Exchange - methods ; Chromosomal contamination ; Chromosomes, Bacterial - chemistry ; Collodion ; DNA vaccination ; Drug Industry - methods ; Escherichia coli - genetics ; Filtration ; Filtration - methods ; Fundamental and applied biological sciences. Psychology ; Gene therapy ; Genes ; Genetic engineering ; Impurities ; Membranes, Artificial ; Methods. Procedures. Technologies ; Nitrocellulose ; nucleic acids ; Nucleic Acids - isolation & purification ; Others ; Plasmid purification ; Plasmids - isolation & purification ; Polyethylene glycols ; Purification ; Removal ; RNA, Bacterial - isolation & purification ; Sodium Chloride - chemistry ; Vaccines ; Various methods and equipments</subject><ispartof>Journal of biotechnology, 2000-01, Vol.76 (2), p.197-205</ispartof><rights>2000 Elsevier Science B.V.</rights><rights>2000 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c452t-8a7cb0fbea0b446888ff2ea2b44d81b533b7ff7e549b7ea516cda2aad35d9c7f3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0168165699001893$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27903,27904,65309</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1407663$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10656334$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Levy, M.S</creatorcontrib><creatorcontrib>Collins, I.J</creatorcontrib><creatorcontrib>Tsai, J.T</creatorcontrib><creatorcontrib>Ayazi Shamlou, P</creatorcontrib><creatorcontrib>Ward, J.M</creatorcontrib><creatorcontrib>Dunnill, P</creatorcontrib><title>Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing</title><title>Journal of biotechnology</title><addtitle>J Biotechnol</addtitle><description>Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg
−1 plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.</description><subject>Biological and medical sciences</subject><subject>Bioreactors</subject><subject>Biotechnology</subject><subject>Blotting, Southern</subject><subject>Cell membranes</subject><subject>Chemical Precipitation</subject><subject>Chromatography, Ion Exchange - methods</subject><subject>Chromosomal contamination</subject><subject>Chromosomes, Bacterial - chemistry</subject><subject>Collodion</subject><subject>DNA vaccination</subject><subject>Drug Industry - methods</subject><subject>Escherichia coli - genetics</subject><subject>Filtration</subject><subject>Filtration - methods</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene therapy</subject><subject>Genes</subject><subject>Genetic engineering</subject><subject>Impurities</subject><subject>Membranes, Artificial</subject><subject>Methods. Procedures. Technologies</subject><subject>Nitrocellulose</subject><subject>nucleic acids</subject><subject>Nucleic Acids - isolation & purification</subject><subject>Others</subject><subject>Plasmid purification</subject><subject>Plasmids - isolation & purification</subject><subject>Polyethylene glycols</subject><subject>Purification</subject><subject>Removal</subject><subject>RNA, Bacterial - isolation & purification</subject><subject>Sodium Chloride - chemistry</subject><subject>Vaccines</subject><subject>Various methods and equipments</subject><issn>0168-1656</issn><issn>1873-4863</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU9vFSEUxYmxsa_Vj6BhYYwuRmGYGZiVaWq1TRpN_LMmd-BSMTPMKzBN-u1l-l60u7eBQH7nHjiHkJecveeMdx9-lEVVvGu7t33_jjGu-ko8IRuupKga1YmnZPMPOSYnKf1hjDV9y5-RY87KpRDNhuTvOM13MNLZUTOHDJMPEDINixnRGwrG20SHexp8jrPBcVzGOSF1fswRsp8DtUv04YZuf0OcwOCSvYGxuolgkW5HSJO39NPXM7pd9SkV9jk5cjAmfLHfT8mvzxc_zy-r629frs7PrivTtHWuFEgzMDcgsKFpOqWUczVCXQ5W8aEVYpDOSWybfpAILe-MhRrAitb2RjpxSt7s5hbr2wVT1pNP6x8g4LwkLZlStaz5QZDLppUlzcOg6JjsalbAdgeaOKcU0elt9BPEe82ZXgvUDwXqtR3d9_qhQC2K7tXeYBkmtI9Uu8YK8HoPQCo5uwjB-PSfa4p_t875uMOw5HvnMepkPAaD1kc0WdvZH3jJX5L4ukw</recordid><startdate>20000121</startdate><enddate>20000121</enddate><creator>Levy, M.S</creator><creator>Collins, I.J</creator><creator>Tsai, J.T</creator><creator>Ayazi Shamlou, P</creator><creator>Ward, J.M</creator><creator>Dunnill, P</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20000121</creationdate><title>Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing</title><author>Levy, M.S ; Collins, I.J ; Tsai, J.T ; Ayazi Shamlou, P ; Ward, J.M ; Dunnill, P</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c452t-8a7cb0fbea0b446888ff2ea2b44d81b533b7ff7e549b7ea516cda2aad35d9c7f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Biological and medical sciences</topic><topic>Bioreactors</topic><topic>Biotechnology</topic><topic>Blotting, Southern</topic><topic>Cell membranes</topic><topic>Chemical Precipitation</topic><topic>Chromatography, Ion Exchange - methods</topic><topic>Chromosomal contamination</topic><topic>Chromosomes, Bacterial - chemistry</topic><topic>Collodion</topic><topic>DNA vaccination</topic><topic>Drug Industry - methods</topic><topic>Escherichia coli - genetics</topic><topic>Filtration</topic><topic>Filtration - methods</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene therapy</topic><topic>Genes</topic><topic>Genetic engineering</topic><topic>Impurities</topic><topic>Membranes, Artificial</topic><topic>Methods. Procedures. Technologies</topic><topic>Nitrocellulose</topic><topic>nucleic acids</topic><topic>Nucleic Acids - isolation & purification</topic><topic>Others</topic><topic>Plasmid purification</topic><topic>Plasmids - isolation & purification</topic><topic>Polyethylene glycols</topic><topic>Purification</topic><topic>Removal</topic><topic>RNA, Bacterial - isolation & purification</topic><topic>Sodium Chloride - chemistry</topic><topic>Vaccines</topic><topic>Various methods and equipments</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Levy, M.S</creatorcontrib><creatorcontrib>Collins, I.J</creatorcontrib><creatorcontrib>Tsai, J.T</creatorcontrib><creatorcontrib>Ayazi Shamlou, P</creatorcontrib><creatorcontrib>Ward, J.M</creatorcontrib><creatorcontrib>Dunnill, P</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Levy, M.S</au><au>Collins, I.J</au><au>Tsai, J.T</au><au>Ayazi Shamlou, P</au><au>Ward, J.M</au><au>Dunnill, P</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing</atitle><jtitle>Journal of biotechnology</jtitle><addtitle>J Biotechnol</addtitle><date>2000-01-21</date><risdate>2000</risdate><volume>76</volume><issue>2</issue><spage>197</spage><epage>205</epage><pages>197-205</pages><issn>0168-1656</issn><eissn>1873-4863</eissn><coden>JBITD4</coden><abstract>Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg
−1 plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.</abstract><cop>Lausanne</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>10656334</pmid><doi>10.1016/S0168-1656(99)00189-3</doi><tpages>9</tpages></addata></record> |
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| subjects | Biological and medical sciences Bioreactors Biotechnology Blotting, Southern Cell membranes Chemical Precipitation Chromatography, Ion Exchange - methods Chromosomal contamination Chromosomes, Bacterial - chemistry Collodion DNA vaccination Drug Industry - methods Escherichia coli - genetics Filtration Filtration - methods Fundamental and applied biological sciences. Psychology Gene therapy Genes Genetic engineering Impurities Membranes, Artificial Methods. Procedures. Technologies Nitrocellulose nucleic acids Nucleic Acids - isolation & purification Others Plasmid purification Plasmids - isolation & purification Polyethylene glycols Purification Removal RNA, Bacterial - isolation & purification Sodium Chloride - chemistry Vaccines Various methods and equipments |
| title | Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing |
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