Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen

Abstract

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0.05) decreased relative to the amount of PA remained in the solution after passing through unmodified as well as protein A modified poly(AAm-AGE) cryogel columns, indicates efficient PA removal from spiked PBS over 60 min of circulation. The high adsorption capacity towards anthrax toxin PA of the cryogel adsorbents indicated potential application of these materials for treatment of Bacillus anthracis infection.

Enhancements in sugar immobilization in polymeric macroporous matrices for affinity capture

“…Figure shows the porous structure of the D ‐GlcNAc immobilized cryogel with interconnected pores varying from 20 to 100 μm, where the majority of the pores measuring from 20 to 40 μm, consistent with the observations of other authors . It was also observed that the activation process did not affect its macroporous nature, because obstruction of the pores was not observed.…”

“…An alternative is to transform such structures into adsorbents of specific affinity by grafting desirable radicals . Aiming to improve the process of using monolithic beds as support for immobilization and purification of biomolecules, different approaches have been reported for the immobilization of the affinity ligands …”

Challenges and Advances in the Fabrication of Monolithic Bioseparation Materials and their Applications in Proteomics Research

“…They attracted great interests for fast enrichment and purification of biomacromolecules including enzymes, coenzymes, cofactors, antibodies, antigens, and DNA, as well as isolation of microbial cells and virus particles . For example, both plant‐derived human monoclonal antibody and glycosylated human monoclonal antibody were chemically immobilized onto poly(acrylamide‐ co ‐allyl glycidyl ether) cryogels by Sandeman and co‐workers The latter exhibited higher binding capacities of 117 mg g −1 toward anthrax toxin protective antigen than the former, indicating potential application of cryogel adsorbents for treatment of Bacillusanthracis infection. Due to the specificity of lectins to various carbohydrates, lectins‐based affinity chromatography is one of the most commonly used approaches for purification of glycolipids and glycoproteins.…”

Poly (2-Hydroxyethyl Methacrylate) Macroporous Cryogel for Extracorporeal Medical Devices

“…Macroporous cryogels have previously been used for biomedical applications and protein purification [3,[13][14][15][16][17]. Cryogels have a unique combination of properties such as interconnected pores of 10 -200 µm size, porosity exceeding 90%, high mechanical and chemical stability, elasticity and sponginess, drying and rehydrating without impairing pore structures [1,18].…”