Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins
ABSTRACT Purpose Ascorbic acid has been considered as a potential radical scavenging excipient for pharmaceutical formulations. However, under certain circumstances, ascorbic acid can generate reactive oxygen species via redox cycling. The objective of this study was to investigate ascorbic acid-ind...
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| Veröffentlicht in: | Pharmaceutical research 2016-02, Vol.33 (2), p.526-539 |
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| creator | Kryndushkin, Dmitry Rao, V. Ashutosh |
| description | ABSTRACT
Purpose
Ascorbic acid has been considered as a potential radical scavenging excipient for pharmaceutical formulations. However, under certain circumstances, ascorbic acid can generate reactive oxygen species
via
redox cycling. The objective of this study was to investigate ascorbic acid-induced oxidative carbonylation of therapeutic proteins and correlate the increase in carbonylation with protein aggregation.
Methods
An optimized ELISA for quantifying carbonyl levels was used to compare the oxidizing potentials of ascorbic acid and hydrogen peroxide by testing four pharmaceutically-relevant proteins (human serum albumin, immunoglobulin G, granulocyte-colony stimulating factor and calcitonin). Several transition metals at micromolar concentrations were evaluated for their ability to enhance ascorbic acid-induced protein carbonylation. Protein aggregation under oxidative conditions, with or without free radical scavengers, was measured by aggregate binding fluorescent dye and confirmed by microfluidic imaging.
Results
Addition of ascorbic acid alone resulted in higher increases in carbonylation than addition of hydrogen peroxide. The presence of trace amounts (>75 ppb) of copper enhanced oxidative effects of ascorbic acid, whereas other tested metals did not comparably promote oxidation. During oxidation, protein destabilization indicated by loss of the full-length protein, positively correlated with the increase in protein aggregation. However, levels of aggregation did not always correlate with the levels of protein carbonylation. At comparable carbonylation levels, addition of copper produced greater protein destabilization and aggregation than addition of iron.
Conclusions
The results strongly suggest that ascorbic acid with traces of metals, especially copper, can promote therapeutic protein carbonylation and potentially aggregation. At similar carbonylation levels, some oxidative conditions may lead to greater protein destabilization than others. |
| doi_str_mv | 10.1007/s11095-015-1807-y |
| format | Article |
| fullrecord | <record><control><sourceid>gale_proqu</sourceid><recordid>TN_cdi_proquest_miscellaneous_1837298054</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A747439404</galeid><sourcerecordid>A747439404</sourcerecordid><originalsourceid>FETCH-LOGICAL-c472t-227f63504c01eb507aec29c7305d60c6c1efa9dbdc62f2a54de334033c30f6613</originalsourceid><addsrcrecordid>eNqFkl1rFDEUhgdR7Fr9Ad7IgDfeTD35msxclqXWQqWCFbwL2czJmjKTrElWnP76Ztj6iSKBhJw875tz4K2q5wROCIB8nQiBXjRAREM6kM38oFoRIVnTA__0sFqBpLzpJCdH1ZOUbgCgIz1_XB3Rlvc942xVhXWYdjrq7L5ifWYtmpzqYOt3mPXYrHXZ51sc6qtvbnC3zm_rD3PKOBXI12sdN8HPY1GXm_ZDfeEzbqPL8-Jx_Rmj3uE-O1O_jyGj8-lp9cjqMeGz-_O4-vjm7Hr9trm8Or9Yn142hkuaG0qlbZkAboDgRoDUaGhvJAMxtGBaQ9DqftgMpqWWasEHZIwDY4aBbVvCjqtXB99dDF_2mLKaXDI4jtpj2CdFOiZp34Hg_0dlCz2lgrQFffkHehP20ZdBCiWEFBJE95Pa6hGV8zbkqM1iqk4ll5z1HJZvT_5ClTXg5EzwaF2p_yYgB4GJIaWIVu2im3ScFQG15EEd8qBKHtSSBzUXzYv7hvebCYcfiu8BKAA9AKk8-S3GXyb6p-sdlRq_Zw</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1755757058</pqid></control><display><type>article</type><title>Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins</title><source>MEDLINE</source><source>SpringerLink Journals</source><creator>Kryndushkin, Dmitry ; Rao, V. Ashutosh</creator><creatorcontrib>Kryndushkin, Dmitry ; Rao, V. Ashutosh</creatorcontrib><description>ABSTRACT
Purpose
Ascorbic acid has been considered as a potential radical scavenging excipient for pharmaceutical formulations. However, under certain circumstances, ascorbic acid can generate reactive oxygen species
via
redox cycling. The objective of this study was to investigate ascorbic acid-induced oxidative carbonylation of therapeutic proteins and correlate the increase in carbonylation with protein aggregation.
Methods
An optimized ELISA for quantifying carbonyl levels was used to compare the oxidizing potentials of ascorbic acid and hydrogen peroxide by testing four pharmaceutically-relevant proteins (human serum albumin, immunoglobulin G, granulocyte-colony stimulating factor and calcitonin). Several transition metals at micromolar concentrations were evaluated for their ability to enhance ascorbic acid-induced protein carbonylation. Protein aggregation under oxidative conditions, with or without free radical scavengers, was measured by aggregate binding fluorescent dye and confirmed by microfluidic imaging.
Results
Addition of ascorbic acid alone resulted in higher increases in carbonylation than addition of hydrogen peroxide. The presence of trace amounts (>75 ppb) of copper enhanced oxidative effects of ascorbic acid, whereas other tested metals did not comparably promote oxidation. During oxidation, protein destabilization indicated by loss of the full-length protein, positively correlated with the increase in protein aggregation. However, levels of aggregation did not always correlate with the levels of protein carbonylation. At comparable carbonylation levels, addition of copper produced greater protein destabilization and aggregation than addition of iron.
Conclusions
The results strongly suggest that ascorbic acid with traces of metals, especially copper, can promote therapeutic protein carbonylation and potentially aggregation. At similar carbonylation levels, some oxidative conditions may lead to greater protein destabilization than others.</description><identifier>ISSN: 0724-8741</identifier><identifier>EISSN: 1573-904X</identifier><identifier>DOI: 10.1007/s11095-015-1807-y</identifier><identifier>PMID: 26499343</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Albumin ; Amino acids ; Animals ; Ascorbic Acid - pharmacology ; Biochemistry ; Biomedical and Life Sciences ; Biomedical Engineering and Bioengineering ; Biomedicine ; Biopharmaceutics ; Carbon ; Catalysis ; Comparative analysis ; Comparative studies ; Copper - chemistry ; Drug therapy ; Enzyme-linked immunosorbent assay ; Ethylenediaminetetraacetic acid ; Excipients - pharmacology ; Free Radical Scavengers - pharmacology ; Health aspects ; Humans ; Hydrogen peroxide ; Immunoglobulin G ; Medical Law ; Metals ; Microfluidics ; Oxidants - pharmacology ; Oxidation ; Oxidation-Reduction - drug effects ; Pharmaceuticals ; Pharmacology/Toxicology ; Pharmacy ; Protein Aggregates - drug effects ; Protein Carbonylation - drug effects ; Protein Stability - drug effects ; Proteins ; Proteins - chemistry ; Rabbits ; Research Paper ; Salmon ; Tositumomab</subject><ispartof>Pharmaceutical research, 2016-02, Vol.33 (2), p.526-539</ispartof><rights>Springer Science+Business Media New York (outside the USA) 2015</rights><rights>COPYRIGHT 2016 Springer</rights><rights>Springer Science+Business Media New York 2016</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c472t-227f63504c01eb507aec29c7305d60c6c1efa9dbdc62f2a54de334033c30f6613</citedby><cites>FETCH-LOGICAL-c472t-227f63504c01eb507aec29c7305d60c6c1efa9dbdc62f2a54de334033c30f6613</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/s11095-015-1807-y$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s11095-015-1807-y$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27903,27904,41467,42536,51298</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26499343$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kryndushkin, Dmitry</creatorcontrib><creatorcontrib>Rao, V. Ashutosh</creatorcontrib><title>Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins</title><title>Pharmaceutical research</title><addtitle>Pharm Res</addtitle><addtitle>Pharm Res</addtitle><description>ABSTRACT
Purpose
Ascorbic acid has been considered as a potential radical scavenging excipient for pharmaceutical formulations. However, under certain circumstances, ascorbic acid can generate reactive oxygen species
via
redox cycling. The objective of this study was to investigate ascorbic acid-induced oxidative carbonylation of therapeutic proteins and correlate the increase in carbonylation with protein aggregation.
Methods
An optimized ELISA for quantifying carbonyl levels was used to compare the oxidizing potentials of ascorbic acid and hydrogen peroxide by testing four pharmaceutically-relevant proteins (human serum albumin, immunoglobulin G, granulocyte-colony stimulating factor and calcitonin). Several transition metals at micromolar concentrations were evaluated for their ability to enhance ascorbic acid-induced protein carbonylation. Protein aggregation under oxidative conditions, with or without free radical scavengers, was measured by aggregate binding fluorescent dye and confirmed by microfluidic imaging.
Results
Addition of ascorbic acid alone resulted in higher increases in carbonylation than addition of hydrogen peroxide. The presence of trace amounts (>75 ppb) of copper enhanced oxidative effects of ascorbic acid, whereas other tested metals did not comparably promote oxidation. During oxidation, protein destabilization indicated by loss of the full-length protein, positively correlated with the increase in protein aggregation. However, levels of aggregation did not always correlate with the levels of protein carbonylation. At comparable carbonylation levels, addition of copper produced greater protein destabilization and aggregation than addition of iron.
Conclusions
The results strongly suggest that ascorbic acid with traces of metals, especially copper, can promote therapeutic protein carbonylation and potentially aggregation. At similar carbonylation levels, some oxidative conditions may lead to greater protein destabilization than others.</description><subject>Albumin</subject><subject>Amino acids</subject><subject>Animals</subject><subject>Ascorbic Acid - pharmacology</subject><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedical Engineering and Bioengineering</subject><subject>Biomedicine</subject><subject>Biopharmaceutics</subject><subject>Carbon</subject><subject>Catalysis</subject><subject>Comparative analysis</subject><subject>Comparative studies</subject><subject>Copper - chemistry</subject><subject>Drug therapy</subject><subject>Enzyme-linked immunosorbent assay</subject><subject>Ethylenediaminetetraacetic acid</subject><subject>Excipients - pharmacology</subject><subject>Free Radical Scavengers - pharmacology</subject><subject>Health aspects</subject><subject>Humans</subject><subject>Hydrogen peroxide</subject><subject>Immunoglobulin G</subject><subject>Medical Law</subject><subject>Metals</subject><subject>Microfluidics</subject><subject>Oxidants - pharmacology</subject><subject>Oxidation</subject><subject>Oxidation-Reduction - drug effects</subject><subject>Pharmaceuticals</subject><subject>Pharmacology/Toxicology</subject><subject>Pharmacy</subject><subject>Protein Aggregates - drug effects</subject><subject>Protein Carbonylation - drug effects</subject><subject>Protein Stability - drug effects</subject><subject>Proteins</subject><subject>Proteins - chemistry</subject><subject>Rabbits</subject><subject>Research Paper</subject><subject>Salmon</subject><subject>Tositumomab</subject><issn>0724-8741</issn><issn>1573-904X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><recordid>eNqFkl1rFDEUhgdR7Fr9Ad7IgDfeTD35msxclqXWQqWCFbwL2czJmjKTrElWnP76Ztj6iSKBhJw875tz4K2q5wROCIB8nQiBXjRAREM6kM38oFoRIVnTA__0sFqBpLzpJCdH1ZOUbgCgIz1_XB3Rlvc942xVhXWYdjrq7L5ifWYtmpzqYOt3mPXYrHXZ51sc6qtvbnC3zm_rD3PKOBXI12sdN8HPY1GXm_ZDfeEzbqPL8-Jx_Rmj3uE-O1O_jyGj8-lp9cjqMeGz-_O4-vjm7Hr9trm8Or9Yn142hkuaG0qlbZkAboDgRoDUaGhvJAMxtGBaQ9DqftgMpqWWasEHZIwDY4aBbVvCjqtXB99dDF_2mLKaXDI4jtpj2CdFOiZp34Hg_0dlCz2lgrQFffkHehP20ZdBCiWEFBJE95Pa6hGV8zbkqM1iqk4ll5z1HJZvT_5ClTXg5EzwaF2p_yYgB4GJIaWIVu2im3ScFQG15EEd8qBKHtSSBzUXzYv7hvebCYcfiu8BKAA9AKk8-S3GXyb6p-sdlRq_Zw</recordid><startdate>20160201</startdate><enddate>20160201</enddate><creator>Kryndushkin, Dmitry</creator><creator>Rao, V. Ashutosh</creator><general>Springer US</general><general>Springer</general><general>Springer Nature B.V</general><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>3V.</scope><scope>7RV</scope><scope>7TK</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>K9.</scope><scope>KB0</scope><scope>M0S</scope><scope>M1P</scope><scope>NAPCQ</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>7X8</scope><scope>7U7</scope><scope>C1K</scope></search><sort><creationdate>20160201</creationdate><title>Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins</title><author>Kryndushkin, Dmitry ; Rao, V. Ashutosh</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c472t-227f63504c01eb507aec29c7305d60c6c1efa9dbdc62f2a54de334033c30f6613</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Albumin</topic><topic>Amino acids</topic><topic>Animals</topic><topic>Ascorbic Acid - pharmacology</topic><topic>Biochemistry</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedical Engineering and Bioengineering</topic><topic>Biomedicine</topic><topic>Biopharmaceutics</topic><topic>Carbon</topic><topic>Catalysis</topic><topic>Comparative analysis</topic><topic>Comparative studies</topic><topic>Copper - chemistry</topic><topic>Drug therapy</topic><topic>Enzyme-linked immunosorbent assay</topic><topic>Ethylenediaminetetraacetic acid</topic><topic>Excipients - pharmacology</topic><topic>Free Radical Scavengers - pharmacology</topic><topic>Health aspects</topic><topic>Humans</topic><topic>Hydrogen peroxide</topic><topic>Immunoglobulin G</topic><topic>Medical Law</topic><topic>Metals</topic><topic>Microfluidics</topic><topic>Oxidants - pharmacology</topic><topic>Oxidation</topic><topic>Oxidation-Reduction - drug effects</topic><topic>Pharmaceuticals</topic><topic>Pharmacology/Toxicology</topic><topic>Pharmacy</topic><topic>Protein Aggregates - drug effects</topic><topic>Protein Carbonylation - drug effects</topic><topic>Protein Stability - drug effects</topic><topic>Proteins</topic><topic>Proteins - chemistry</topic><topic>Rabbits</topic><topic>Research Paper</topic><topic>Salmon</topic><topic>Tositumomab</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kryndushkin, Dmitry</creatorcontrib><creatorcontrib>Rao, V. Ashutosh</creatorcontrib><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>Nursing & Allied Health Database</collection><collection>Neurosciences Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Nursing & Allied Health Premium</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>MEDLINE - Academic</collection><collection>Toxicology Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><jtitle>Pharmaceutical research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kryndushkin, Dmitry</au><au>Rao, V. Ashutosh</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins</atitle><jtitle>Pharmaceutical research</jtitle><stitle>Pharm Res</stitle><addtitle>Pharm Res</addtitle><date>2016-02-01</date><risdate>2016</risdate><volume>33</volume><issue>2</issue><spage>526</spage><epage>539</epage><pages>526-539</pages><issn>0724-8741</issn><eissn>1573-904X</eissn><abstract>ABSTRACT
Purpose
Ascorbic acid has been considered as a potential radical scavenging excipient for pharmaceutical formulations. However, under certain circumstances, ascorbic acid can generate reactive oxygen species
via
redox cycling. The objective of this study was to investigate ascorbic acid-induced oxidative carbonylation of therapeutic proteins and correlate the increase in carbonylation with protein aggregation.
Methods
An optimized ELISA for quantifying carbonyl levels was used to compare the oxidizing potentials of ascorbic acid and hydrogen peroxide by testing four pharmaceutically-relevant proteins (human serum albumin, immunoglobulin G, granulocyte-colony stimulating factor and calcitonin). Several transition metals at micromolar concentrations were evaluated for their ability to enhance ascorbic acid-induced protein carbonylation. Protein aggregation under oxidative conditions, with or without free radical scavengers, was measured by aggregate binding fluorescent dye and confirmed by microfluidic imaging.
Results
Addition of ascorbic acid alone resulted in higher increases in carbonylation than addition of hydrogen peroxide. The presence of trace amounts (>75 ppb) of copper enhanced oxidative effects of ascorbic acid, whereas other tested metals did not comparably promote oxidation. During oxidation, protein destabilization indicated by loss of the full-length protein, positively correlated with the increase in protein aggregation. However, levels of aggregation did not always correlate with the levels of protein carbonylation. At comparable carbonylation levels, addition of copper produced greater protein destabilization and aggregation than addition of iron.
Conclusions
The results strongly suggest that ascorbic acid with traces of metals, especially copper, can promote therapeutic protein carbonylation and potentially aggregation. At similar carbonylation levels, some oxidative conditions may lead to greater protein destabilization than others.</abstract><cop>New York</cop><pub>Springer US</pub><pmid>26499343</pmid><doi>10.1007/s11095-015-1807-y</doi><tpages>14</tpages></addata></record> |
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| subjects | Albumin Amino acids Animals Ascorbic Acid - pharmacology Biochemistry Biomedical and Life Sciences Biomedical Engineering and Bioengineering Biomedicine Biopharmaceutics Carbon Catalysis Comparative analysis Comparative studies Copper - chemistry Drug therapy Enzyme-linked immunosorbent assay Ethylenediaminetetraacetic acid Excipients - pharmacology Free Radical Scavengers - pharmacology Health aspects Humans Hydrogen peroxide Immunoglobulin G Medical Law Metals Microfluidics Oxidants - pharmacology Oxidation Oxidation-Reduction - drug effects Pharmaceuticals Pharmacology/Toxicology Pharmacy Protein Aggregates - drug effects Protein Carbonylation - drug effects Protein Stability - drug effects Proteins Proteins - chemistry Rabbits Research Paper Salmon Tositumomab |
| title | Comparative Effects of Metal-Catalyzed Oxidizing Systems on Carbonylation and Integrity of Therapeutic Proteins |
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