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

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

Creatinine imprinted poly(hydroxyethyl methacrylate) based cryogel cartridges

Creatinine imprinted cryogel (MIP) cartridge was prepared with functional monomer N-methacryloyl-(L)-histidinemethylester (MAH) under frozen conditions. Creatinine adsorption studies and selectivity of MIP cryogel were evaluated in aqueous solution and artificial urine sample. Maximum adsorbed amount of creatinine was calculated as 6.83 mg/g polymer for MIP cryogel. Langmuir and Freundlich adsorption isotherm models were used to investigate the adsorption behaviour of creatinine. In the artificial urine sample; recovery amounts of creatinine were found 34.7–46.2%. Creatinine imprinted cryogel (MIP) cartridge recognized creatinine, 4.58 and 4.37 times greater competitive molecules. MIP cryogel catridge was repeatedly used many times for adsorption desorption cycles. © 2017 Taylor & Francis Group, LLC

Synthesis of poly[(hydroxyethyl methacrylate)-co-(methacrylamidoalanine)] membranes and their utilization as an affinity sorbent for lysozyme adsorption

Garipcan, Bora/0000-0002-1773-5607;WOS: 000172273200002Various adsorbent materials have been reported in the literature for protein separation. We have developed a novel and new approach to obtain high protein-adsorption capacity utilizing a 2-methacrylamidoalamne-containing membrane. An amino acid ligand 2-methacrylamidoalanine (MAAL) was synthesized from methacrylochloride and alanine. Then, poly[(2-hydro-xyethel methacrylate)-co-(2-methacrylamidoalanine)] [p(HEMA-co-MAAL)] membranes were prepared by UV-initiated photopolymerization of HEMA and MAAL. The synthesized MAAL monomer was characterized by NMR spectrometry. p(HEMA-co-MAAL) membranes were characterized by swelling studies, porosimeter, scanning electron microscopy,FT-IR spectroscopy and elemental analysis. These membranes have large pores; the micropore dimensions are around 5-10 mum. p(HEMA-co-MAAL) affinity membranes with a swelling ration of 198.9%, and containing 23.9 (mmol MAAL).m(-2) were used in the adsorption of lysozyme (0.1-3.0 mg.ml(-1)) and at different pH values (4.0-8.0). The effect of Cu(II) incorporation on lysozyme adsorption was also studied. The non-specific adsorption of lysozyme on the pHEMA membranes was 0.9 mug-cm(-2). Incorporation of MAAL molecules into the polymeric structure significantly increased the lysozyme adsorption up to 2.96 mg.cm(-2). The lysozyme-adsorption capacity of the membranes incorporated with Cu(III) (9.98 mg.cm(-2)) was greater than that of the p(HEMA-co-MAAL) membranes. More than 85% of the adsorbed lysozyme was desorbed in 1 h in the desorption medium containing 1.0 M NaCl. The p(HEMA-co-MAAL) membranes are suitable for repeated use for more than 5 cycles without noticable loss of capacity. These features make p(HEMA-co-MAAL) membrane a very good candidate for bioaffinity adsorption. Adsorption rates of lysozyme: MAAL loading: 23.9 mmol.m(-2); pH: 7.0; temperature: 20 degreesC; total external membrane surface area in each batch: 100 cm(2).l(-1); Each point is the average of five parallel studies