Immunoaffinity resin

Affinity chromatography techniques have shown less utility in analytical testing than in preparative separations for a variety of reasons, including cost and the difficulty of validating consistent operation as the column changes over time. Protein A affinity has been commonly used to quantitate the total antibody content of either ascites or cell culture fluids. To provide guidance in the development of a purification process, specific immunoaffinity resins are either available or can be readily prepared to quantitate the levels of unrelated protein contaminants. To rapidly determine what the active species in a mixture is, a monoclonal antibody that... 

FIGURE 21 Separation of mouse monoclonal IgM by Immunoaffinity chromatography on rat anti-jit-chain antibodies chemically immobilized on agarose beads (4.2 mg per milliliter of resin). Column 10 mm i.d.X 45 mm. Load of 5 mL of whole mouse ascitic fluid previously filtered. Buffer phosphate buffered saline, pH 7.2. Elution by lowering the pH to 2.8 with 0.2 M glycine - HCI buffer. The first peak did not contain IgM while eluted peak (arrow) contained about 7.8 mg of IgM total. 

Useofavidin-biotin technology in immunoaffinity chromatography can lead to improved performance of the affinity column, which translates both into yields of the target material and stability of the column (1,2). The presence of free biotin-binding sites of the avidin-4 esin allows secondary interaction with biotinylated antibody or its complexes with avi-din that may have leaked from the affinity resin. 

An IND, BIA, or NDA submission should list the source and country of origin for every animal- and human-derived raw material [3, 46]. Lot numbers and supplier information should be available on site during an inspection (21 CFR 211.184). It is also recommended to contact vendors to determine if less-obvious raw materials such as amino acids used in the basal tissue culture medium, enzymes used to make protein hydrolysates, cholesterol, and some detergents (e.g., polysor-bates) are animal-derived. In some cases, protein A (isolated to make chromatography resins) has been purified over human IgG immunoaffinity columns, and the use of these resins should also be tracked and reported (21 CFR 211.184) [46]. 

Tharakan, J., F. Highsmith, D. Clark, and W. Drohan (1992). Physical and biochemical characterization of five commercial resins for immunoaffinity purification of factor IX. /. Chromatogr. 595 103-111. 

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