Pub Date : 2011-01-01DOI: 10.1002/9780470939932.ch14
Conan J Fee, James M Van Alstine
Separation of PEGylated proteins is challenging because PEG itself is a relatively inert, neutral, hydrophilic polymer and the starting point for PEGylation is a pure protein. Thus, other than molecular weight and size, differences in the physicochemical properties typically used to fractionate proteins may be slight between different PEGylated forms of a protein. The usual properties of electrostatic charge and molecular weight (size) form the basis of the most commonly used separation techniques, particularly IEC, SEC, and ultrafiltration. The main effect of PEGylation on ion-exchange separations is to shield the electrostatic charges on the protein surface and to reduce the strength of interactions with higher PEG chain molecular weight or higher PEGylation extent. Thus, ion exchange can be used very effectively to separate on the basis of PEGylation extent for low extents, but as N increases, the effectiveness of separation rapidly diminishes. Separation of positional isomers is possible by RPC or ion exchange at analytical scale, but it is problematic at the preparative scale due to the small size of the differences in electrostatic interactions between isomers. PEGylation imparts significant changes in molecular weight with each chain added to a protein and there are corresponding increases in molecular size, so SEC and ultrafiltration (and dialysis) are effective methods for separating native and PEGylated proteins. However, the relative size difference between variants differing in PEGylation extent by one adduct reduces with N, so that efficient SEC separation between PEGylated species differing by one PEG chain is not likely to be economic at the preparative scale for N > 3. This holds true even for PEG proteins produced with large PEG polymers (Mr > or =20 kDa). For small PEG polymers (Mr = 2 kDa), only native and PEGylated species can be separated effectively. At the analytical scale, with proper calibration, SEC can provide valuable information on PEGylation extent. Membranes can be used to reduce the concentration of smaller molecular weight species by dialysis but cannot fully remove them, and an operational trade-off between purity and yield is required. Gel electrophoresis can confirm PEGylation reactions have proceeded and indicate the relative purity of products, but its use to confirm PEGylation extent requires further research. The main drawback of separations based solely upon molecular size is that they cannot differentiate between positional isomers. Capillary electrophoresis is an exception, quantitatively combining any or all of size, shape, conformational freedom, and small differences in protein surface properties to separate by both PEGylation extent and positional isomerism. Relative hydrophobicity is a useful property for analytical separations using RPC, but HIC, which is used routinely for production-scale purification of proteins, does not appear to be particularly useful for separation of PEGylated species
{"title":"Purification of pegylated proteins.","authors":"Conan J Fee, James M Van Alstine","doi":"10.1002/9780470939932.ch14","DOIUrl":"https://doi.org/10.1002/9780470939932.ch14","url":null,"abstract":"<p><p>Separation of PEGylated proteins is challenging because PEG itself is a relatively inert, neutral, hydrophilic polymer and the starting point for PEGylation is a pure protein. Thus, other than molecular weight and size, differences in the physicochemical properties typically used to fractionate proteins may be slight between different PEGylated forms of a protein. The usual properties of electrostatic charge and molecular weight (size) form the basis of the most commonly used separation techniques, particularly IEC, SEC, and ultrafiltration. The main effect of PEGylation on ion-exchange separations is to shield the electrostatic charges on the protein surface and to reduce the strength of interactions with higher PEG chain molecular weight or higher PEGylation extent. Thus, ion exchange can be used very effectively to separate on the basis of PEGylation extent for low extents, but as N increases, the effectiveness of separation rapidly diminishes. Separation of positional isomers is possible by RPC or ion exchange at analytical scale, but it is problematic at the preparative scale due to the small size of the differences in electrostatic interactions between isomers. PEGylation imparts significant changes in molecular weight with each chain added to a protein and there are corresponding increases in molecular size, so SEC and ultrafiltration (and dialysis) are effective methods for separating native and PEGylated proteins. However, the relative size difference between variants differing in PEGylation extent by one adduct reduces with N, so that efficient SEC separation between PEGylated species differing by one PEG chain is not likely to be economic at the preparative scale for N > 3. This holds true even for PEG proteins produced with large PEG polymers (Mr > or =20 kDa). For small PEG polymers (Mr = 2 kDa), only native and PEGylated species can be separated effectively. At the analytical scale, with proper calibration, SEC can provide valuable information on PEGylation extent. Membranes can be used to reduce the concentration of smaller molecular weight species by dialysis but cannot fully remove them, and an operational trade-off between purity and yield is required. Gel electrophoresis can confirm PEGylation reactions have proceeded and indicate the relative purity of products, but its use to confirm PEGylation extent requires further research. The main drawback of separations based solely upon molecular size is that they cannot differentiate between positional isomers. Capillary electrophoresis is an exception, quantitatively combining any or all of size, shape, conformational freedom, and small differences in protein surface properties to separate by both PEGylation extent and positional isomerism. Relative hydrophobicity is a useful property for analytical separations using RPC, but HIC, which is used routinely for production-scale purification of proteins, does not appear to be particularly useful for separation of PEGylated species","PeriodicalId":18579,"journal":{"name":"Methods of biochemical analysis","volume":"54 ","pages":"339-62"},"PeriodicalIF":0.0,"publicationDate":"2011-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470939932.ch14","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30170894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2011-01-01DOI: 10.1002/9780470939932.ch2
J. Janson, J. Jönsson
A simple introduction to chromatography, considering thin-layer, column and gas-liquid methods, explaining the factors governing choice of one or the other. Basic references and suppliers of equipment are included.
{"title":"Introduction to chromatography.","authors":"J. Janson, J. Jönsson","doi":"10.1002/9780470939932.ch2","DOIUrl":"https://doi.org/10.1002/9780470939932.ch2","url":null,"abstract":"A simple introduction to chromatography, considering thin-layer, column and gas-liquid methods, explaining the factors governing choice of one or the other. Basic references and suppliers of equipment are included.","PeriodicalId":18579,"journal":{"name":"Methods of biochemical analysis","volume":"262 1","pages":"25-50"},"PeriodicalIF":0.0,"publicationDate":"2011-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85933120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Introduction to protein purification.","authors":"Bo Ersson, Lars Rydén, Jan-Christer Janson","doi":"","DOIUrl":"","url":null,"abstract":"","PeriodicalId":18579,"journal":{"name":"Methods of biochemical analysis","volume":"54 ","pages":"3-22"},"PeriodicalIF":0.0,"publicationDate":"2011-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30170999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2011-01-01DOI: 10.1002/9780470939932.ch7
Lennart Kågedal
{"title":"Immobilized metal ion affinity chromatography.","authors":"Lennart Kågedal","doi":"10.1002/9780470939932.ch7","DOIUrl":"https://doi.org/10.1002/9780470939932.ch7","url":null,"abstract":"","PeriodicalId":18579,"journal":{"name":"Methods of biochemical analysis","volume":"54 ","pages":"183-201"},"PeriodicalIF":0.0,"publicationDate":"2011-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470939932.ch7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30171005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2011-01-01DOI: 10.1002/9780470939932.ch16
Pier Giorgio Righetti, Elisa Fasoli, Sabina Carla Righetti
{"title":"Conventional isoelectric focusing. In gel slabs and capillaries and immobilized pH gradients.","authors":"Pier Giorgio Righetti, Elisa Fasoli, Sabina Carla Righetti","doi":"10.1002/9780470939932.ch16","DOIUrl":"https://doi.org/10.1002/9780470939932.ch16","url":null,"abstract":"","PeriodicalId":18579,"journal":{"name":"Methods of biochemical analysis","volume":"54 ","pages":"379-409"},"PeriodicalIF":0.0,"publicationDate":"2011-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470939932.ch16","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30170896","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"High throughput screening techniques in protein purification.","authors":"Karol M Lacki, Eggert Brekkan","doi":"","DOIUrl":"","url":null,"abstract":"","PeriodicalId":18579,"journal":{"name":"Methods of biochemical analysis","volume":"54 ","pages":"489-506"},"PeriodicalIF":0.0,"publicationDate":"2011-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30170900","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2011-01-01DOI: 10.1007/978-1-60327-261-2
F. Batista‐Viera, J. Janson, J. Carlsson
{"title":"Affinity chromatography.","authors":"F. Batista‐Viera, J. Janson, J. Carlsson","doi":"10.1007/978-1-60327-261-2","DOIUrl":"https://doi.org/10.1007/978-1-60327-261-2","url":null,"abstract":"","PeriodicalId":18579,"journal":{"name":"Methods of biochemical analysis","volume":"1 1","pages":"221-58"},"PeriodicalIF":0.0,"publicationDate":"2011-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81196253","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号