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Protein A carrying monosize PMMA microbeads for the removal of HIgG from human plasma
E Pişkin1, H Ayhan, E V Bulmuş
1Hacettepe University, Chemical Engineering Department, Ankara, Turkey.
Abstract:
Protein A-incorporated polymethylmethacrylate (PMMA) microbeads were investigated for specific removal of HIgG from human plasma. The microbeads were prepared by a phase inversion polymerization, and activated by periodate oxidation. Protein A was then incorporated by covalent binding onto these microbeads through hydroxyl groups coming from the stabilizer. The amount of incorporated protein A was controlled by the initial concentrations of protein A in the immobilization medium and pH. The maximum protein A immobilization of 0.615 mg protein A/g PMMA, was observed at a pH of 9.5 corresponding to an initial protein A concentration of 0.1 mg/ml. There was no HIgG adsorption onto the plain PMMA microbeads, while high HIgG adsorptions of up to 32 mg HIgG/g PMMA were achieved with human plasma.
Insights
Researchers developed Protein A-functionalized polymethylmethacrylate (PMMA) microbeads for efficient removal of human immunoglobulin G (HIgG) from human plasma, achieving high adsorption capacities.
Area of Science:
- Biomaterials Science
- Affinity Chromatography
- Protein Immobilization
Background:
- Human immunoglobulin G (HIgG) removal is crucial for various therapeutic applications.
- Polymethylmethacrylate (PMMA) microbeads offer a potential scaffold for biomolecule immobilization.
- Protein A is a well-established ligand for specific binding of immunoglobulin G.
Purpose of the Study:
- To develop and characterize Protein A-incorporated PMMA microbeads for selective HIgG capture from human plasma.
- To optimize the immobilization conditions for maximizing Protein A loading on PMMA microbeads.
- To evaluate the adsorption capacity of the functionalized microbeads for HIgG.
Main Methods:
- PMMA microbeads synthesized via phase inversion polymerization.
- Periodate oxidation used for microbead activation.
- Covalent immobilization of Protein A onto hydroxyl groups of the PMMA stabilizer.
- Controlled Protein A loading by varying initial concentration and pH.
- HIgG adsorption studies using functionalized and plain PMMA microbeads in human plasma.
Main Results:
- Maximum Protein A immobilization achieved was 0.615 mg/g PMMA at pH 9.5 and 0.1 mg/ml initial Protein A concentration.
- Plain PMMA microbeads showed negligible HIgG adsorption.
- Protein A-functionalized PMMA microbeads demonstrated high HIgG adsorption capacity, up to 32 mg/g PMMA.
Conclusions:
- Protein A-functionalized PMMA microbeads are effective for specific HIgG removal from human plasma.
- The developed method allows for controlled Protein A immobilization and high adsorption capacity.
- These functionalized microbeads show promise for applications in plasma purification and diagnostics.