Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies
In this work, a high‐resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single‐component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using reco...
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| Veröffentlicht in: | Electrophoresis 2013-09, Vol.34 (18), p.2754-2759 |
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| creator | Espinosa-de la Garza, Carlos E. Perdomo-Abúndez, Francisco C. Campos-García, Víctor R. Pérez, Néstor O. Flores-Ortiz, Luis F. Medina-Rivero, Emilio |
| description | In this work, a high‐resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single‐component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon‐β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r2) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon‐β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines. |
| doi_str_mv | 10.1002/elps.201300232 |
| format | Article |
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Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon‐β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r2) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon‐β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines.</description><identifier>ISSN: 0173-0835</identifier><identifier>EISSN: 1522-2683</identifier><identifier>DOI: 10.1002/elps.201300232</identifier><identifier>PMID: 23857606</identifier><language>eng</language><publisher>Germany: Blackwell Publishing Ltd</publisher><subject>CGE ; Electrophoresis ; Electrophoresis, Capillary - methods ; Humans ; Inclusion bodies ; Inclusion Bodies - chemistry ; Inclusions ; Interferon beta-1b ; Interferon-beta - chemistry ; Interferon-β 1b ; Linear Models ; Mathematical models ; Models, Chemical ; Proteins ; Purity ; Recombinant ; Recombinant Proteins - analysis ; Recombinant Proteins - chemistry ; Recombinant Proteins - isolation & purification ; Repeatability ; Reproducibility ; Reproducibility of Results ; Sensitivity and Specificity ; Solubility ; Validation</subject><ispartof>Electrophoresis, 2013-09, Vol.34 (18), p.2754-2759</ispartof><rights>2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim</rights><rights>2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4111-cc1a18738320a49762f06b160fab43a9437f3d8d075d688957ee5d3b8cd46d3b3</citedby><cites>FETCH-LOGICAL-c4111-cc1a18738320a49762f06b160fab43a9437f3d8d075d688957ee5d3b8cd46d3b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Felps.201300232$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Felps.201300232$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1416,27922,27923,45572,45573</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23857606$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Espinosa-de la Garza, Carlos E.</creatorcontrib><creatorcontrib>Perdomo-Abúndez, Francisco C.</creatorcontrib><creatorcontrib>Campos-García, Víctor R.</creatorcontrib><creatorcontrib>Pérez, Néstor O.</creatorcontrib><creatorcontrib>Flores-Ortiz, Luis F.</creatorcontrib><creatorcontrib>Medina-Rivero, Emilio</creatorcontrib><title>Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies</title><title>Electrophoresis</title><addtitle>ELECTROPHORESIS</addtitle><description>In this work, a high‐resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single‐component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon‐β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r2) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon‐β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines.</description><subject>CGE</subject><subject>Electrophoresis</subject><subject>Electrophoresis, Capillary - methods</subject><subject>Humans</subject><subject>Inclusion bodies</subject><subject>Inclusion Bodies - chemistry</subject><subject>Inclusions</subject><subject>Interferon beta-1b</subject><subject>Interferon-beta - chemistry</subject><subject>Interferon-β 1b</subject><subject>Linear Models</subject><subject>Mathematical models</subject><subject>Models, Chemical</subject><subject>Proteins</subject><subject>Purity</subject><subject>Recombinant</subject><subject>Recombinant Proteins - analysis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Repeatability</subject><subject>Reproducibility</subject><subject>Reproducibility of Results</subject><subject>Sensitivity and Specificity</subject><subject>Solubility</subject><subject>Validation</subject><issn>0173-0835</issn><issn>1522-2683</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtv1DAUhS0EotOBLUvkJZsMfsX2LNGoHSpGUPEQEhvLcW6oIYlT2xHMv8ejlNlWsnRl-zvn2vcg9IqSDSWEvYV-ShtGKC8bzp6gFa0Zq5jU_ClaEap4RTSvL9BlSr8IIWIrxHN0wbiulSRyhY47O_m-t_GIf0KPoQeXY5juQoTkE-5CxPkO8P1sx-w772z2YcR2bPE0R5-PuIUMcfDjchE6HMGFoSkHY8ZTDBn8mLAfy3L9nE5QE1oP6QV61tk-wcuHukbfrq--7t5Xh0_7m927Q-UEpbRyjlqqFdecESu2SrKOyIZK0tlGcLsVXHW81S1RdSu13tYKoG55o10rZKl8jd4svuUx9zOkbAafHJQ_jxDmZKgQWgkly8geRzmTjOrSdI02C-piSClCZ6bohzJGQ4k5JWNOyZhzMkXw-sF7bgZoz_j_KAogFuCP7-H4iJ25Otx-EZrSIqsWmU8Z_p5lNv42UnFVm-8f92b_mR5uf2w_mJr_A2FtqvY</recordid><startdate>201309</startdate><enddate>201309</enddate><creator>Espinosa-de la Garza, Carlos E.</creator><creator>Perdomo-Abúndez, Francisco C.</creator><creator>Campos-García, Víctor R.</creator><creator>Pérez, Néstor O.</creator><creator>Flores-Ortiz, Luis F.</creator><creator>Medina-Rivero, Emilio</creator><general>Blackwell Publishing Ltd</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>7X8</scope><scope>7U5</scope><scope>8FD</scope><scope>L7M</scope></search><sort><creationdate>201309</creationdate><title>Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies</title><author>Espinosa-de la Garza, Carlos E. ; Perdomo-Abúndez, Francisco C. ; Campos-García, Víctor R. ; Pérez, Néstor O. ; Flores-Ortiz, Luis F. ; Medina-Rivero, Emilio</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4111-cc1a18738320a49762f06b160fab43a9437f3d8d075d688957ee5d3b8cd46d3b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>CGE</topic><topic>Electrophoresis</topic><topic>Electrophoresis, Capillary - methods</topic><topic>Humans</topic><topic>Inclusion bodies</topic><topic>Inclusion Bodies - chemistry</topic><topic>Inclusions</topic><topic>Interferon beta-1b</topic><topic>Interferon-beta - chemistry</topic><topic>Interferon-β 1b</topic><topic>Linear Models</topic><topic>Mathematical models</topic><topic>Models, Chemical</topic><topic>Proteins</topic><topic>Purity</topic><topic>Recombinant</topic><topic>Recombinant Proteins - analysis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Repeatability</topic><topic>Reproducibility</topic><topic>Reproducibility of Results</topic><topic>Sensitivity and Specificity</topic><topic>Solubility</topic><topic>Validation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Espinosa-de la Garza, Carlos E.</creatorcontrib><creatorcontrib>Perdomo-Abúndez, Francisco C.</creatorcontrib><creatorcontrib>Campos-García, Víctor R.</creatorcontrib><creatorcontrib>Pérez, Néstor O.</creatorcontrib><creatorcontrib>Flores-Ortiz, Luis F.</creatorcontrib><creatorcontrib>Medina-Rivero, Emilio</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>MEDLINE - Academic</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Technology Research Database</collection><collection>Advanced Technologies Database with Aerospace</collection><jtitle>Electrophoresis</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Espinosa-de la Garza, Carlos E.</au><au>Perdomo-Abúndez, Francisco C.</au><au>Campos-García, Víctor R.</au><au>Pérez, Néstor O.</au><au>Flores-Ortiz, Luis F.</au><au>Medina-Rivero, Emilio</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies</atitle><jtitle>Electrophoresis</jtitle><addtitle>ELECTROPHORESIS</addtitle><date>2013-09</date><risdate>2013</risdate><volume>34</volume><issue>18</issue><spage>2754</spage><epage>2759</epage><pages>2754-2759</pages><issn>0173-0835</issn><eissn>1522-2683</eissn><abstract>In this work, a high‐resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single‐component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon‐β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r2) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon‐β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. 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| subjects | CGE Electrophoresis Electrophoresis, Capillary - methods Humans Inclusion bodies Inclusion Bodies - chemistry Inclusions Interferon beta-1b Interferon-beta - chemistry Interferon-β 1b Linear Models Mathematical models Models, Chemical Proteins Purity Recombinant Recombinant Proteins - analysis Recombinant Proteins - chemistry Recombinant Proteins - isolation & purification Repeatability Reproducibility Reproducibility of Results Sensitivity and Specificity Solubility Validation |
| title | Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies |
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