Applications

Several different applications of affinity membranes have been described. Typical examples of their use for the separation and purification of bio-molecules are shown in Table 2.

The most common application is the separation and purification of biomolecules and especially proteins for large scale production. A common example is the separation of immunoglobulins from blood-serum or plasma or from cell culture supernatants. Hollow-fibre cartridges with immobilized protein A or pseudobiospecific ligands have been used for this purpose. Figure 2 shows a chromatogram from a case study of immunoglobulin G separation from human plasma using a small, developmental-scale (28 cm2 surface area) poly(ethylene-co-vinyl alcohol) hollow-fibre membrane cartridge. The pseudobiospecific affinity ligand histidine was immobilized on to the membrane after activation with butanediol dig-lycidyl ether, thus introducing a spacer arm. Serum was injected 10-fold diluted in cross-flow filtration mode. Weakly retained and entrapped proteins were then removed by washing the lumen and the outer shell of the fibres, as well as the pores in back-flushing mode. Adsorbed immunoglobulins were subsequently eluted with a buffered solution of 0.4molL~1 NaCl in back-flushing mode. The eluted fraction contained 93% immunoglobulins (82% IgG, 10.8% IgM). The dynamic binding capacity of the membrane for immunoglobulin G was determined to be 1.9 gm~2. The process could then be scaled up by using a cartridge with 1 m2 membrane surface area.

A related application is the final polishing of an already pure product. For example, the removal of bacterial endotoxins from contaminated solutions of monoclonal antibodies has been demonstrated using membrane-bound pseudobiospecific ligands.

Table 2 Examples for the use of affinity membranes for isolation and purification of biomolecules

Isolated substance

Affinity ligand

Membrane material

Configuration

Application

Human serum amyloid protein

Heparin

Human IgG

Recombinant protein G

Trypsin (porcine)

Glucose-6-phosphate dehydrogenase

Human IgG

Autoantibodies

Lysozyme, cytochrome c, ribonuclease A

Anti-hSAP Ab (polyclonal)

Poly-l-lysine

Recombinant protein A

Human IgG

Soybean trypsin inhibitor Cibacron blue

Histidine

IDA-Cu2

Cellulose

Cellulose diacetate poly(ethylene-co-vinyl alcohol), coated polyethylene

Poly(caprolactam) Modified poly(caprolactam) Polysulfone-coated hydroxyethyl cellulose Glycidyl methacrylate-co-ethylene dimethacrylate Modified cellulose

Nylon

Poly(ethylene-co-vinyl alcohol)

Glass

Flat sheets

Hollow fibres

Hollow fibres Hollow fibres, flat sheet Hollow fibres

Discs

Spiral wound sheet

(radial flow) Stack of flat sheets

Hollow fibres

Hollow fibres

Extracorporeal circuit, removal of amyloid from blood Extracorporeal circuit, removal of heparin from blood

Purification

Purification

Purification

Purification from clarified yeast homogenate Purification from blood plasma and serum Removal from blood plasma in extracorporeal circuit Purification

Figure 2 Separation of immunoglobins from human serum using a poly(ethylene-co-vinyl alcohol) hollow-fibre cartridge with immobilized l-histidine. (a) Immunoglobulin adsorption in cross-flow filtration mode; (b) lumen wash; (c) shell wash; (d) back-flush wash; (e) back-flush elution. (Adapted from JournalofMembrane Science 117, Bueno SMA, Legallais C, Haupt K and Vijayalakshmi MA, Experimental kinetic aspects of hollow-fiber membrane-based pseudobioaffinity filtration: Process for IgG separation from human plasma, pp. 45-56, Copyright 1996, with permission from Elsevier Science.)

Figure 2 Separation of immunoglobins from human serum using a poly(ethylene-co-vinyl alcohol) hollow-fibre cartridge with immobilized l-histidine. (a) Immunoglobulin adsorption in cross-flow filtration mode; (b) lumen wash; (c) shell wash; (d) back-flush wash; (e) back-flush elution. (Adapted from JournalofMembrane Science 117, Bueno SMA, Legallais C, Haupt K and Vijayalakshmi MA, Experimental kinetic aspects of hollow-fiber membrane-based pseudobioaffinity filtration: Process for IgG separation from human plasma, pp. 45-56, Copyright 1996, with permission from Elsevier Science.)

Affinity membranes have also been suggested for use in extracorporeal circuits, for the removal of toxic substances such as certain metabolites or antibodies from blood. For example, exogenous human serum amyloid P component, a substance associated with Alzheimer's disease, has been removed from whole rat blood in an extracorporeal circulation system. This model system used a polyclonal antibody coupled to cellulose flat-sheet membranes. The bio-compatibility of the membrane was also demonstrated. A similar application is the removal of autoan-tibodies from human plasma, using membrane-bound affinity ligands in extracorporeal circuits.

Apart from preparative applications, small cartridges with membrane discs or continuous membrane rods should be useful for analytical-scale separations and affinity solid-phase extraction, for example for immunoextraction.

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