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Publication Date: 2010-Sep-28
Document File: 6 page(s) / 489K

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A ligand with affinity for alpha-1 antitrypsin was developed using Camelidae -derived single domain antibody fragments from the immune response of llamas towards the target molecule human alpha-1 antitrypsin. This protein ligand was coupled to cross-linked agarose beads and used for affinity capture of alpha-1 antitrypsin from human blood plasma.

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Separation of alpha-1 antitrypsin

A ligand with affinity for alpha-1 antitrypsin was developed using Camelidae - derived single domain antibody fragments from the immune response of lamas towards the target molecule human alpha-1 antitrypsin. The gene of the selected protein was cloned into an expression system, for example a yeast cell system, bacteria or mammalian or plant cells can be used.

The ligand is chemically or physically bound to a matrix. The ligand is preferably covalently bound. A hydrophilic spacer might be used to enhance accessibility of the ligand. The immobilization of the ligand can be made via aldehyde activation, NHS, CNBr, epoxy, allyl, haloalkyl or other known techniques for attachment of protein ligands.

The matrix is preferably porous providing a large surface area. The matrix can be in the format of a filter, membrane, expanded bed, or packed bed particulate chromatography resin. The matrix can be made from synthetic polymers like poly-styrene/divinylbenzene, polymethacrylates, and polyvinyl ethers or carbohydrate polymers like agarose, cellulose, cellulose derivates, hemicelluloses, dextrans or gums. Also inorganic materials like porous glass, silica, titania and zirconia can be used as matrix. The matrix might be magnetic and/or have an expanded surface area using surface extenders such as hydrophilic polymers for example dextran or grafted tentacles.

A typical example of such separation material is the product Alpha-1 Antitrypsin Select (GE Healthcare Life Sciences). Alpha-1 Antitrypsin Select is a chromatography medium based on porous spherical agarose particles (base matrix) with a covalently attached alpha-1 antitrypsin binding protein (ligand).

Table 1. Characteristics of Alpha-1 Antitrypsin Select

Base Matrix Highly cross-linked spherical agarose Average particle size1 75 ┬Ám (d50V)

Ligand Alpha-1 antitrypsin binding ligand
Ligand density Approx. 5.5 mg/ml of medium
Binding capacity2 Approx. 10 mg alpha-1 antitrypsin /ml medium
1. d50V is the median particle size of the cumulative volume distribution
2. Determined using a total capacity chromatography method

The general principle of affinity chromatography is that a biospecific ligand attached to a chromatography base matrix is exploited to the corresponding adsorbent under conditions favorable for specific binding. The target adsorbent thus binds specifically and reversibly to the ligand. Preferably, unbound substances are washed away and thereafter the target substance is recovered by applying conditions suitable for elution (e.g. a change in pH and/or ionic strength). Samples are concentrated during binding and the target protein is collected in a purified and concentrated form.

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The binding and elution conditions depend on the target molecule, feed composition and the chromatography medium, and these must be studied together with other chromatographic parameters such as sample load, flow velocity, bed height, regeneration and clean...