Abstract
Although Staphylococcus Protein A (SpA) affinity chromatography is the state of the art capture step for antibody purification, non-affinity methods are more economical. We used two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) to evaluate the purification of a recombinant IgG(1) antibody from cultured cells, with two different processes: (1) SpA capture followed by cation-exchange chromatography (CEX); and (2) CEX capture, followed by anion exchanger, then hydrophobic interaction chromatography. Efficiencies were similar in sodium dodecylsulphate polyacrylamide gel electrophoresis and size-exclusion chromatography; however, 2-D DIGE revealed higher efficiency with SpA than with CEX capture. Thus, 2-D DIGE is a valuable tool for downstream process development.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antibodies, Monoclonal / chemistry
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Antibodies, Monoclonal / genetics
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Antibodies, Monoclonal / isolation & purification*
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Antibodies, Monoclonal / metabolism
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CHO Cells
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Chromatography / methods*
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Chromatography, Affinity
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Cricetinae
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Cricetulus
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Electrophoresis, Gel, Two-Dimensional / methods*
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Fluorescence
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Immunoglobulin G / chemistry
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Immunoglobulin G / genetics
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Immunoglobulin G / isolation & purification*
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Immunoglobulin G / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Staphylococcal Protein A / chemistry
Substances
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Antibodies, Monoclonal
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Immunoglobulin G
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Recombinant Proteins
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Staphylococcal Protein A