Facile And Green Fabrication Of Novel Membrane Adsorbers And Its Application In Protein Purification

Efficient bio-processing technology is the key requirement in the manufacture of biopharmaceuticals.The increasing need to reduce biopharmaceutical cost is driven by the business challenges such as the emerging bio-generics.The great advances in upstream technologies make bio-separation the major cost in bioprocessing.Developing efficient bio-separation technologies is therefore strongly desired.Membrane chromatography is a promising bio-separation technology which combines the advantages of membrane technology and chromatography,thus making high-throughput and high resolution bioprocesses feasible.This thesis focuses on the production of novel membrane adsorbers via a facile and green fabrication mechanism and its application for the purification of proteins.A versatile platform was created by functionalizing nylon membrane via a non-immunogenic bio-adhesive polymer,Alginate dialdehyde(ADA),without any organic solvent usage.Cation exchange(CEX),metal-affinity(M.A),histidine-affinity(His-Affinity)and peptide-affinity(Pep-affinity)membrane adsorbers were fabricated based on ADA-coated nylon membrane platform.By comparing the physiochemical properties of the nylon membrane and obtained membrane adsorbers,it was confirmed that different ligands were grafted on the membranes.All the membrane adsorbers exhibited a high selectivity in fractionation of IgG(immunoglobulin)/HSA(human serum albumin)mixture.Along with high purity and recovery of 100%and 94-99%respectively,a high IgG binding capacity of 30,24,21 and 28 mg/mL(membrane volume)was achieved by CEX,M.A,His-affinity and Pep-affinity membrane adsorbers respectively,which is superior to the reported membrane adsorbers.Furthermore,via ADA coating,non-specific adsorption on the nylon membrane was completely suppressed.The currently used Protein A agarose for the purification of IgG is challenged due to ligand leakage and high cost.Based on the ADA-coated nylon platform,a synergetic membrane adsorber(ion exchange and peptide-affinity)was constructed via immobilizing a peptide DWHW for the purification of IgG from real human plasma solution.The ADA coating time during adsorber fabrication and the operating pH during IgG static adsorption were optimized.The ADA carboxylic groups along with the peptide captured IgG synergistically with high recovery and purity of 99%and 98.6%respectively.Furthermore,a dynamic binding capacity(DBC)of 38.7 mg/mL was achieved for the newly fabricated adsorber,which was quite higher than the Protein A agarose(having a DBC of 24.4 mg/g).Usually the binding capacity of membrane adsorbers is lower for proteins with lower molecular weights as compared to the columns.Keeping in view this point a highly potent dual cation exchange membrane adsorber was developed based on the pre-coated ADA-nylon by the addition of sulphonic groups without any organic solvent usage.The influence of ADA coating time,pH,ionic concentration and initial lysozyme concentration on maximum lysozyme adsorption were examined,and the optimal conditions were obtained for lysozyme purification.Taking advantage of the abundant dual CEX(carboxylic&sulphonic)groups on sulphonic-AD A(S-ADA)ligands,this novel S-ADA-nylon membrane adsorber showed an unprecedented static binding capicity of 286 mg/mL for lysozyme adsorption.Meanwhile,the prepared membrane adsorber could be easily regenerated(complete protein elution)under mild conditions and be reused at least for five times.Featured with a unique selectivity,the S-ADA-nylon membrane also captured lysozyme from chicken egg white solution with a high purity(100%)and a high recovery of 98%.The purified lysozyme showed similar specific activity as commercial product.