Approaches for recombinant human factor IX production in serum-free suspension cultures
OBJECTIVE: To establish a serum-free suspension process for production of recombinant human factor IX (rhFIX) based on the human cell line HEK 293T by evaluating two approaches: (1) serum-free suspension adaptation of previously genetic modified cells (293T-FIX); and (2) genetic modification of cell...
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| Veröffentlicht in: | Biotechnology letters 2016-03, Vol.38 (3), p.385-394 |
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| creator | do Amaral, Robson Luis Ferraz de Sousa Bomfim, Aline de Abreu-Neto, Mário Soares Picanço-Castro, Virgínia de Sousa Russo, Elisa Maria Covas, Dimas Tadeu Swiech, Kamilla |
| description | OBJECTIVE: To establish a serum-free suspension process for production of recombinant human factor IX (rhFIX) based on the human cell line HEK 293T by evaluating two approaches: (1) serum-free suspension adaptation of previously genetic modified cells (293T-FIX); and (2) genetic modification of cells already adapted to such conditions (293T/SF-FIX). RESULTS: After 10 months, 293T-FIX cells had become adapted to FreeStyle 293 serum-free medium (SFM) in Erlenmeyer flasks. After 48 and 72 h of culture, 2.1 µg rhFIX/ml and 3.3 µg rhFIX/ml were produced, respectively. However, no biological activity was detected. In the second approach, wild-type 293T cells were adapted to the same SFM (adaptation process took only 2 months) and then genetically modified for rhFIX production. After 48 h of culture, rhFIX reached 1.5 µg/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 µg/ml with a biological activity of 0.3 IU/ml. CONCLUSION: The findings demonstrate that the best approach to establish an rhFIX production process in suspension SFM involves the genetic modification of cells already adapted to the final conditions. This approach is time saving and may better ensure the quality of the produced protein. |
| doi_str_mv | 10.1007/s10529-015-1991-1 |
| format | Article |
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RESULTS: After 10 months, 293T-FIX cells had become adapted to FreeStyle 293 serum-free medium (SFM) in Erlenmeyer flasks. After 48 and 72 h of culture, 2.1 µg rhFIX/ml and 3.3 µg rhFIX/ml were produced, respectively. However, no biological activity was detected. In the second approach, wild-type 293T cells were adapted to the same SFM (adaptation process took only 2 months) and then genetically modified for rhFIX production. After 48 h of culture, rhFIX reached 1.5 µg/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 µg/ml with a biological activity of 0.3 IU/ml. CONCLUSION: The findings demonstrate that the best approach to establish an rhFIX production process in suspension SFM involves the genetic modification of cells already adapted to the final conditions. This approach is time saving and may better ensure the quality of the produced protein.</description><identifier>ISSN: 0141-5492</identifier><identifier>EISSN: 1573-6776</identifier><identifier>DOI: 10.1007/s10529-015-1991-1</identifier><identifier>PMID: 26564408</identifier><language>eng</language><publisher>Dordrecht: Springer Netherlands</publisher><subject>Adaptation ; Applied Microbiology ; Biochemistry ; Biological ; Biomedical and Life Sciences ; Biotechnology ; blood proteins ; Cell culture ; Cell Culture Techniques - methods ; Cellular biology ; Culture ; Culture Media, Serum-Free ; Factor IX - genetics ; Factor IX - metabolism ; Gene expression ; genetic engineering ; Genetic modification ; Genetics ; Growth factors ; HEK293 Cells ; human cell lines ; Human factors ; Humans ; Life Sciences ; Microbiology ; Original Research Paper ; Recombinant ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism</subject><ispartof>Biotechnology letters, 2016-03, Vol.38 (3), p.385-394</ispartof><rights>Springer Science+Business Media Dordrecht 2015</rights><rights>Springer Science+Business Media Dordrecht 2016</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c532t-14c7c525c4399be8d4cadd9fc2c2db64f94662f782e781cdb788b84952d27cd03</citedby><cites>FETCH-LOGICAL-c532t-14c7c525c4399be8d4cadd9fc2c2db64f94662f782e781cdb788b84952d27cd03</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s10529-015-1991-1$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s10529-015-1991-1$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>315,782,786,27933,27934,41497,42566,51328</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26564408$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>do Amaral, Robson Luis Ferraz</creatorcontrib><creatorcontrib>de Sousa Bomfim, Aline</creatorcontrib><creatorcontrib>de Abreu-Neto, Mário Soares</creatorcontrib><creatorcontrib>Picanço-Castro, Virgínia</creatorcontrib><creatorcontrib>de Sousa Russo, Elisa Maria</creatorcontrib><creatorcontrib>Covas, Dimas Tadeu</creatorcontrib><creatorcontrib>Swiech, Kamilla</creatorcontrib><title>Approaches for recombinant human factor IX production in serum-free suspension cultures</title><title>Biotechnology letters</title><addtitle>Biotechnol Lett</addtitle><addtitle>Biotechnol Lett</addtitle><description>OBJECTIVE: To establish a serum-free suspension process for production of recombinant human factor IX (rhFIX) based on the human cell line HEK 293T by evaluating two approaches: (1) serum-free suspension adaptation of previously genetic modified cells (293T-FIX); and (2) genetic modification of cells already adapted to such conditions (293T/SF-FIX). RESULTS: After 10 months, 293T-FIX cells had become adapted to FreeStyle 293 serum-free medium (SFM) in Erlenmeyer flasks. After 48 and 72 h of culture, 2.1 µg rhFIX/ml and 3.3 µg rhFIX/ml were produced, respectively. However, no biological activity was detected. In the second approach, wild-type 293T cells were adapted to the same SFM (adaptation process took only 2 months) and then genetically modified for rhFIX production. After 48 h of culture, rhFIX reached 1.5 µg/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 µg/ml with a biological activity of 0.3 IU/ml. CONCLUSION: The findings demonstrate that the best approach to establish an rhFIX production process in suspension SFM involves the genetic modification of cells already adapted to the final conditions. This approach is time saving and may better ensure the quality of the produced protein.</description><subject>Adaptation</subject><subject>Applied Microbiology</subject><subject>Biochemistry</subject><subject>Biological</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnology</subject><subject>blood proteins</subject><subject>Cell culture</subject><subject>Cell Culture Techniques - methods</subject><subject>Cellular biology</subject><subject>Culture</subject><subject>Culture Media, Serum-Free</subject><subject>Factor IX - genetics</subject><subject>Factor IX - metabolism</subject><subject>Gene expression</subject><subject>genetic engineering</subject><subject>Genetic modification</subject><subject>Genetics</subject><subject>Growth factors</subject><subject>HEK293 Cells</subject><subject>human cell lines</subject><subject>Human factors</subject><subject>Humans</subject><subject>Life Sciences</subject><subject>Microbiology</subject><subject>Original Research Paper</subject><subject>Recombinant</subject><subject>Recombinant Proteins - 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methods</topic><topic>Cellular biology</topic><topic>Culture</topic><topic>Culture Media, Serum-Free</topic><topic>Factor IX - genetics</topic><topic>Factor IX - metabolism</topic><topic>Gene expression</topic><topic>genetic engineering</topic><topic>Genetic modification</topic><topic>Genetics</topic><topic>Growth factors</topic><topic>HEK293 Cells</topic><topic>human cell lines</topic><topic>Human factors</topic><topic>Humans</topic><topic>Life Sciences</topic><topic>Microbiology</topic><topic>Original Research Paper</topic><topic>Recombinant</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>do Amaral, Robson Luis Ferraz</creatorcontrib><creatorcontrib>de Sousa Bomfim, Aline</creatorcontrib><creatorcontrib>de Abreu-Neto, Mário Soares</creatorcontrib><creatorcontrib>Picanço-Castro, Virgínia</creatorcontrib><creatorcontrib>de Sousa Russo, Elisa Maria</creatorcontrib><creatorcontrib>Covas, Dimas Tadeu</creatorcontrib><creatorcontrib>Swiech, Kamilla</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Chemoreception Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Engineering Collection</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Engineering Collection</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>Biotechnology Research Abstracts</collection><jtitle>Biotechnology letters</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>do Amaral, Robson Luis Ferraz</au><au>de Sousa Bomfim, Aline</au><au>de Abreu-Neto, Mário Soares</au><au>Picanço-Castro, Virgínia</au><au>de Sousa Russo, Elisa Maria</au><au>Covas, Dimas Tadeu</au><au>Swiech, Kamilla</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Approaches for recombinant human factor IX production in serum-free suspension cultures</atitle><jtitle>Biotechnology letters</jtitle><stitle>Biotechnol Lett</stitle><addtitle>Biotechnol Lett</addtitle><date>2016-03-01</date><risdate>2016</risdate><volume>38</volume><issue>3</issue><spage>385</spage><epage>394</epage><pages>385-394</pages><issn>0141-5492</issn><eissn>1573-6776</eissn><abstract>OBJECTIVE: To establish a serum-free suspension process for production of recombinant human factor IX (rhFIX) based on the human cell line HEK 293T by evaluating two approaches: (1) serum-free suspension adaptation of previously genetic modified cells (293T-FIX); and (2) genetic modification of cells already adapted to such conditions (293T/SF-FIX). RESULTS: After 10 months, 293T-FIX cells had become adapted to FreeStyle 293 serum-free medium (SFM) in Erlenmeyer flasks. After 48 and 72 h of culture, 2.1 µg rhFIX/ml and 3.3 µg rhFIX/ml were produced, respectively. However, no biological activity was detected. In the second approach, wild-type 293T cells were adapted to the same SFM (adaptation process took only 2 months) and then genetically modified for rhFIX production. After 48 h of culture, rhFIX reached 1.5 µg/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 µg/ml with a biological activity of 0.3 IU/ml. CONCLUSION: The findings demonstrate that the best approach to establish an rhFIX production process in suspension SFM involves the genetic modification of cells already adapted to the final conditions. This approach is time saving and may better ensure the quality of the produced protein.</abstract><cop>Dordrecht</cop><pub>Springer Netherlands</pub><pmid>26564408</pmid><doi>10.1007/s10529-015-1991-1</doi><tpages>10</tpages></addata></record> |
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| subjects | Adaptation Applied Microbiology Biochemistry Biological Biomedical and Life Sciences Biotechnology blood proteins Cell culture Cell Culture Techniques - methods Cellular biology Culture Culture Media, Serum-Free Factor IX - genetics Factor IX - metabolism Gene expression genetic engineering Genetic modification Genetics Growth factors HEK293 Cells human cell lines Human factors Humans Life Sciences Microbiology Original Research Paper Recombinant Recombinant Proteins - genetics Recombinant Proteins - metabolism |
| title | Approaches for recombinant human factor IX production in serum-free suspension cultures |
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