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Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method
Published on: September 19, 2018
Analysis of immunoglobulin G using a capillary electrophoretic affinity assay with protein A and laser-induced
R Lausch1, O W Reif, P Riechel
1Institut für Biochemie, Abteilung Biotechnologie, Westfälische Wilhelms-Universität Münster, Germany.
Insights
This study introduces a fast and sensitive capillary electrophoresis method to measure immunoglobulin G (IgG) in cell culture. Recombinant protein A fragments offer superior resolution for accurate antibody monitoring.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Immunology
Background:
- Accurate quantification of immunoglobulin G (IgG) is crucial for bioprocess monitoring.
- Existing methods for IgG determination in complex matrices like cultivation media can be time-consuming or lack sensitivity.
- Development of rapid and specific assays is needed for efficient biopharmaceutical production.
Purpose of the Study:
- To develop and validate a rapid and sensitive affinity assay for immunoglobulin G (IgG) determination in cultivation media.
- To evaluate the efficacy of protein A and its recombinant fragments as affinity ligands in capillary electrophoresis.
- To demonstrate the application of the developed assay for monitoring monoclonal antibodies during cultivation processes.
Main Methods:
- Capillary electrophoresis coupled with an affinity assay using fluorescently labeled protein A or its recombinant fragments.
- Evaluation of different protein A conjugates (fluorescein diisocyanate, dichlorotriazinyl-aminofluorescein) as affinity ligands.
- Comparison of whole protein A versus recombinant fragments for zone resolution in capillary electrophoresis.
Main Results:
- Successful formation of a fluorescing complex between immunoglobulin G and the protein A affinity ligand.
- Demonstration that recombinant protein A fragments provide baseline resolution, superior to whole protein A.
- Accurate determination of immunoglobulin concentrations across a two-order-of-magnitude range.
- Specificity of the assay allows analysis in the presence of other cultivation media components.
Conclusions:
- Capillary electrophoretic affinity assay using recombinant protein A fragments is a rapid, sensitive, and specific method for IgG determination.
- The assay is suitable for monitoring monoclonal antibodies in complex cultivation media.
- This technique offers a valuable tool for bioprocess monitoring and optimization.
Abstract:
A method for the rapid and sensitive determination of immunoglobulin G (IgG) in cultivation media by an affinity assay using capillary electrophoresis is presented. For that purpose we evaluated protein A conjugated with a fluorescent dye such as fluorescein diisocyanate or dichlorotriazinyl-aminofluorescein as an affinity ligand. The ligand formed a fluorescing complex with immunoglobulin G in the sample and rapid separation from excess protein A was performed by capillary zone electrophoresis. However, only partial resolution of the zones was achieved when protein A as a whole molecule was utilized. In contrast, baseline resolution of the zones was obtained when recombinant fragments of protein A were used as affinity ligands. Immunoglobulin concentrations in the range of two orders of magnitude were determined. Due to the specificity of protein A for immunoglobulin G, analysis can be carried out even in the presence of high concentrations of other components and in cultivation media. Thus, the capillary electrophoretic affinity assay was successfully applied to monitor monoclonal antibodies in a cultivation process.
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