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A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: Process development and product quality assessment
ABSTRACT Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in...
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Published in: | Biotechnology and bioengineering 2015-05, Vol.112 (5), p.977-986 |
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Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS‐KO host cell line. By creating a bi‐allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS‐KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)‐mediated transfections were performed at high cell density (4 × 106 cells/mL) followed by immediate growth arrest at 32°C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre‐complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7‐day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc‐fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7‐day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS‐KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293. Biotechnol. Bioeng. 2015;112: 977–986. © 2014 Wiley Periodicals, Inc.
Transient gene expression in CHO cells has typically required cell line and vector engineering al |
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Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS‐KO host cell line. By creating a bi‐allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS‐KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)‐mediated transfections were performed at high cell density (4 × 106 cells/mL) followed by immediate growth arrest at 32°C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre‐complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7‐day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc‐fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7‐day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS‐KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293. Biotechnol. Bioeng. 2015;112: 977–986. © 2014 Wiley Periodicals, Inc.
Transient gene expression in CHO cells has typically required cell line and vector engineering along with long fed‐batch processes to achieve high protein titers. Here, the authors demonstrated that high protein can be achieved from CHO without cell line or vector engineering by transfecting at high cell density, optimizing DNA and PEI addition, culturing cells at 32°C and using DMA along with proprietary feeds. Moreover, product quality analysis showed good correlation between transient CHO and stable CHO pool material.</description><identifier>ISSN: 0006-3592</identifier><identifier>EISSN: 1097-0290</identifier><identifier>DOI: 10.1002/bit.25514</identifier><identifier>PMID: 25502369</identifier><identifier>CODEN: BIBIAU</identifier><language>eng</language><publisher>United States: Blackwell Publishing Ltd</publisher><subject>Animals ; Antibodies, Monoclonal - genetics ; Biotechnology ; Cell Count ; Cells ; CHO Cells - cytology ; CHO Cells - metabolism ; Cricetulus ; Density ; Deoxyribonucleic acid ; DNA ; DNA - administration & dosage ; DNA - genetics ; Gene Expression ; Gene Knockout Techniques ; Glutamate-Ammonia Ligase - genetics ; GS CHO ; Humans ; mammalian cells ; Mathematical analysis ; Optimization ; Plasmids ; Polyetherimides ; polyethyleneimine ; Polyethyleneimine - metabolism ; Proteins ; Recombinant Proteins - genetics ; transfection ; Transfection - instrumentation ; transient gene expression ; Vectors (mathematics)</subject><ispartof>Biotechnology and bioengineering, 2015-05, Vol.112 (5), p.977-986</ispartof><rights>2014 Wiley Periodicals, Inc.</rights><rights>Copyright Wiley Subscription Services, Inc. May 2015</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4944-18c7de84acfcde48c52647cee7488e2424e365a9838fad35bb48731694a36e63</citedby><cites>FETCH-LOGICAL-c4944-18c7de84acfcde48c52647cee7488e2424e365a9838fad35bb48731694a36e63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25502369$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Rajendra, Yashas</creatorcontrib><creatorcontrib>Hougland, Maria D.</creatorcontrib><creatorcontrib>Alam, Riazul</creatorcontrib><creatorcontrib>Morehead, Teresa A.</creatorcontrib><creatorcontrib>Barnard, Gavin C.</creatorcontrib><title>A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: Process development and product quality assessment</title><title>Biotechnology and bioengineering</title><addtitle>Biotechnol. Bioeng</addtitle><description>ABSTRACT
Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS‐KO host cell line. By creating a bi‐allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS‐KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)‐mediated transfections were performed at high cell density (4 × 106 cells/mL) followed by immediate growth arrest at 32°C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre‐complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7‐day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc‐fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7‐day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS‐KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293. Biotechnol. Bioeng. 2015;112: 977–986. © 2014 Wiley Periodicals, Inc.
Transient gene expression in CHO cells has typically required cell line and vector engineering along with long fed‐batch processes to achieve high protein titers. Here, the authors demonstrated that high protein can be achieved from CHO without cell line or vector engineering by transfecting at high cell density, optimizing DNA and PEI addition, culturing cells at 32°C and using DMA along with proprietary feeds. Moreover, product quality analysis showed good correlation between transient CHO and stable CHO pool material.</description><subject>Animals</subject><subject>Antibodies, Monoclonal - genetics</subject><subject>Biotechnology</subject><subject>Cell Count</subject><subject>Cells</subject><subject>CHO Cells - cytology</subject><subject>CHO Cells - metabolism</subject><subject>Cricetulus</subject><subject>Density</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA - administration & dosage</subject><subject>DNA - genetics</subject><subject>Gene Expression</subject><subject>Gene Knockout Techniques</subject><subject>Glutamate-Ammonia Ligase - genetics</subject><subject>GS CHO</subject><subject>Humans</subject><subject>mammalian cells</subject><subject>Mathematical analysis</subject><subject>Optimization</subject><subject>Plasmids</subject><subject>Polyetherimides</subject><subject>polyethyleneimine</subject><subject>Polyethyleneimine - metabolism</subject><subject>Proteins</subject><subject>Recombinant Proteins - genetics</subject><subject>transfection</subject><subject>Transfection - instrumentation</subject><subject>transient gene expression</subject><subject>Vectors (mathematics)</subject><issn>0006-3592</issn><issn>1097-0290</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><recordid>eNqNks1u1DAUhSMEokNhwQsgS2xgkdaxHcdm147oTKURRWIEEhvLcW4Yd_JX24HOQ_GOOKTtAgmpK1_L3z3nXvkkyesMn2QYk9PShhOS5xl7kiwyLIsUE4mfJguMMU9pLslR8sL763gtBOfPk6MIY0K5XCS_z9DO_tghA02DKui8DQcUnI4FdGGuajDB9h3yBx-gRXXvUNiB0wOMwRo0uD6A7RDcDg68n8hSe6hQLDRarq_Q6ku673qz78cwGzW2gw_os-tNbIi2P6Hph3Yy1F01CVajCehm1M00jvY-YtPzy-RZrRsPr-7O42R78XG7XKebq9Xl8myTGiYZSzNhigoE06Y2FTBhcsJZYQAKJgQQRhhQnmspqKh1RfOyZKKgGZdMUw6cHifvZtk4yc0IPqjW-mlw3UE_epXxopBcSCoeg1IqWYbzR6A8qmIuSUTf_oNe96Pr4sqTICbxH0kWqfczZVzvvYNaDc622h1UhtUUDBWDof4GI7Jv7hTHsoXqgbxPQgROZ-CXbeDwfyV1frm9l0znDhtjcfvQod1exaWLXH37tFIX3zFeb75yxekfqkDTIQ</recordid><startdate>201505</startdate><enddate>201505</enddate><creator>Rajendra, Yashas</creator><creator>Hougland, Maria D.</creator><creator>Alam, Riazul</creator><creator>Morehead, Teresa A.</creator><creator>Barnard, Gavin C.</creator><general>Blackwell Publishing Ltd</general><general>Wiley Subscription Services, Inc</general><scope>BSCLL</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>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7U5</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>201505</creationdate><title>A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: Process development and product quality assessment</title><author>Rajendra, Yashas ; Hougland, Maria D. ; Alam, Riazul ; Morehead, Teresa A. ; Barnard, Gavin C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4944-18c7de84acfcde48c52647cee7488e2424e365a9838fad35bb48731694a36e63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Animals</topic><topic>Antibodies, Monoclonal - genetics</topic><topic>Biotechnology</topic><topic>Cell Count</topic><topic>Cells</topic><topic>CHO Cells - cytology</topic><topic>CHO Cells - metabolism</topic><topic>Cricetulus</topic><topic>Density</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA - administration & dosage</topic><topic>DNA - genetics</topic><topic>Gene Expression</topic><topic>Gene Knockout Techniques</topic><topic>Glutamate-Ammonia Ligase - genetics</topic><topic>GS CHO</topic><topic>Humans</topic><topic>mammalian cells</topic><topic>Mathematical analysis</topic><topic>Optimization</topic><topic>Plasmids</topic><topic>Polyetherimides</topic><topic>polyethyleneimine</topic><topic>Polyethyleneimine - metabolism</topic><topic>Proteins</topic><topic>Recombinant Proteins - genetics</topic><topic>transfection</topic><topic>Transfection - instrumentation</topic><topic>transient gene expression</topic><topic>Vectors (mathematics)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rajendra, Yashas</creatorcontrib><creatorcontrib>Hougland, Maria D.</creatorcontrib><creatorcontrib>Alam, Riazul</creatorcontrib><creatorcontrib>Morehead, Teresa A.</creatorcontrib><creatorcontrib>Barnard, Gavin C.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biotechnology and bioengineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Rajendra, Yashas</au><au>Hougland, Maria D.</au><au>Alam, Riazul</au><au>Morehead, Teresa A.</au><au>Barnard, Gavin C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: Process development and product quality assessment</atitle><jtitle>Biotechnology and bioengineering</jtitle><addtitle>Biotechnol. Bioeng</addtitle><date>2015-05</date><risdate>2015</risdate><volume>112</volume><issue>5</issue><spage>977</spage><epage>986</epage><pages>977-986</pages><issn>0006-3592</issn><eissn>1097-0290</eissn><coden>BIBIAU</coden><abstract>ABSTRACT
Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS‐KO host cell line. By creating a bi‐allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS‐KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)‐mediated transfections were performed at high cell density (4 × 106 cells/mL) followed by immediate growth arrest at 32°C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre‐complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7‐day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc‐fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7‐day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS‐KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293. Biotechnol. Bioeng. 2015;112: 977–986. © 2014 Wiley Periodicals, Inc.
Transient gene expression in CHO cells has typically required cell line and vector engineering along with long fed‐batch processes to achieve high protein titers. Here, the authors demonstrated that high protein can be achieved from CHO without cell line or vector engineering by transfecting at high cell density, optimizing DNA and PEI addition, culturing cells at 32°C and using DMA along with proprietary feeds. Moreover, product quality analysis showed good correlation between transient CHO and stable CHO pool material.</abstract><cop>United States</cop><pub>Blackwell Publishing Ltd</pub><pmid>25502369</pmid><doi>10.1002/bit.25514</doi><tpages>10</tpages></addata></record> |
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subjects | Animals Antibodies, Monoclonal - genetics Biotechnology Cell Count Cells CHO Cells - cytology CHO Cells - metabolism Cricetulus Density Deoxyribonucleic acid DNA DNA - administration & dosage DNA - genetics Gene Expression Gene Knockout Techniques Glutamate-Ammonia Ligase - genetics GS CHO Humans mammalian cells Mathematical analysis Optimization Plasmids Polyetherimides polyethyleneimine Polyethyleneimine - metabolism Proteins Recombinant Proteins - genetics transfection Transfection - instrumentation transient gene expression Vectors (mathematics) |
title | A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: Process development and product quality assessment |
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