Serum-Free Transient Protein Production System Based on Adenoviral Vector and PER.C6 Technology : High Yield and Preserved Bioactivity
Identifieur interne : 003474 ( Main/Exploration ); précédent : 003473; suivant : 003475Serum-Free Transient Protein Production System Based on Adenoviral Vector and PER.C6 Technology : High Yield and Preserved Bioactivity
Auteurs : M. J. E. Havenga [Pays-Bas] ; L. Holterman [Pays-Bas] ; I. Melis [Pays-Bas] ; S. Smits [Pays-Bas] ; J. Kaspers [Pays-Bas] ; E. Heemskerk [Pays-Bas] ; R. Van Der Vlugt [Pays-Bas] ; M. Koldijk [Pays-Bas] ; G. J. Schouten [Pays-Bas] ; G. Hateboer [Pays-Bas] ; K. Brouwer [Pays-Bas] ; R. Vogels [Pays-Bas] ; J. Goudsmit [Pays-Bas]Source :
- Biotechnology and bioengineering [ 0006-3592 ] ; 2008.
Descripteurs français
- Pascal (Inist)
- Wicri :
- topic : Production, Technologie, Homme.
English descriptors
- KwdEn :
Abstract
Stable El transformed cells, like PER.C6, are able to grow at scale and to high cell densities. El-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an ΔE1/ΔE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed AE1/AE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The ΔE1/ΔE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/ cell) of ΔE1/ΔE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that ΔE1/ΔE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.
Affiliations:
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J. E." last="Havenga">M. J. E. Havenga</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Holterman, L" sort="Holterman, L" uniqKey="Holterman L" first="L." last="Holterman">L. Holterman</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Melis, I" sort="Melis, I" uniqKey="Melis I" first="I." last="Melis">I. Melis</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Smits, S" sort="Smits, S" uniqKey="Smits S" first="S." last="Smits">S. Smits</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Kaspers, J" sort="Kaspers, J" uniqKey="Kaspers J" first="J." last="Kaspers">J. Kaspers</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Heemskerk, E" sort="Heemskerk, E" uniqKey="Heemskerk E" first="E." last="Heemskerk">E. 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Van Der Vlugt</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Koldijk, M" sort="Koldijk, M" uniqKey="Koldijk M" first="M." last="Koldijk">M. 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Schouten</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Hateboer, G" sort="Hateboer, G" uniqKey="Hateboer G" first="G." last="Hateboer">G. Hateboer</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Brouwer, K" sort="Brouwer, K" uniqKey="Brouwer K" first="K." last="Brouwer">K. Brouwer</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Vogels, R" sort="Vogels, R" uniqKey="Vogels R" first="R." last="Vogels">R. Vogels</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Goudsmit, J" sort="Goudsmit, J" uniqKey="Goudsmit J" first="J." last="Goudsmit">J. Goudsmit</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Crucell Holland BV, PO Box 2048</s1><s2>2301CA Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>11 aut.</sZ><sZ>12 aut.</sZ><sZ>13 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author></analytic><series><title level="j" type="main">Biotechnology and bioengineering</title><title level="j" type="abbreviated">Biotechnol. bioeng.</title><idno type="ISSN">0006-3592</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Biotechnology and bioengineering</title><title level="j" type="abbreviated">Biotechnol. bioeng.</title><idno type="ISSN">0006-3592</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Adenoviridae</term><term>Biological activity</term><term>Human</term><term>Production</term><term>Serum free medium</term><term>Serum protein</term><term>Technology</term><term>Vector</term><term>Yield</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Protéine sérique</term><term>Milieu sans sérum</term><term>Production</term><term>Vecteur</term><term>Technologie</term><term>Adenoviridae</term><term>Rendement</term><term>Activité biologique</term><term>Homme</term></keywords><keywords scheme="Wicri" type="topic" xml:lang="fr"><term>Production</term><term>Technologie</term><term>Homme</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Stable El transformed cells, like PER.C6, are able to grow at scale and to high cell densities. El-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an ΔE1/ΔE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed AE1/AE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The ΔE1/ΔE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/ cell) of ΔE1/ΔE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that ΔE1/ΔE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.</div></front></TEI><affiliations><list><country><li>Pays-Bas</li></country><region><li>Hollande-Méridionale</li></region><settlement><li>Leyde</li></settlement></list><tree><country name="Pays-Bas"><region name="Hollande-Méridionale"><name sortKey="Havenga, M J E" sort="Havenga, M J E" uniqKey="Havenga M" first="M. J. E." last="Havenga">M. J. E. Havenga</name></region><name sortKey="Brouwer, K" sort="Brouwer, K" uniqKey="Brouwer K" first="K." last="Brouwer">K. Brouwer</name><name sortKey="Goudsmit, J" sort="Goudsmit, J" uniqKey="Goudsmit J" first="J." last="Goudsmit">J. Goudsmit</name><name sortKey="Hateboer, G" sort="Hateboer, G" uniqKey="Hateboer G" first="G." last="Hateboer">G. Hateboer</name><name sortKey="Heemskerk, E" sort="Heemskerk, E" uniqKey="Heemskerk E" first="E." last="Heemskerk">E. Heemskerk</name><name sortKey="Holterman, L" sort="Holterman, L" uniqKey="Holterman L" first="L." last="Holterman">L. Holterman</name><name sortKey="Kaspers, J" sort="Kaspers, J" uniqKey="Kaspers J" first="J." last="Kaspers">J. Kaspers</name><name sortKey="Koldijk, M" sort="Koldijk, M" uniqKey="Koldijk M" first="M." last="Koldijk">M. Koldijk</name><name sortKey="Melis, I" sort="Melis, I" uniqKey="Melis I" first="I." last="Melis">I. Melis</name><name sortKey="Schouten, G J" sort="Schouten, G J" uniqKey="Schouten G" first="G. J." last="Schouten">G. J. Schouten</name><name sortKey="Smits, S" sort="Smits, S" uniqKey="Smits S" first="S." last="Smits">S. Smits</name><name sortKey="Van Der Vlugt, R" sort="Van Der Vlugt, R" uniqKey="Van Der Vlugt R" first="R." last="Van Der Vlugt">R. Van Der Vlugt</name><name sortKey="Vogels, R" sort="Vogels, R" uniqKey="Vogels R" first="R." last="Vogels">R. Vogels</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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