Since approximately the early 70's a variety of support systems has been developed for the immobilization of biologically active proteins, especially enzymes. Some are based on ion exchange forces for immobilization of enzymes, others use physical entrapment of the enzyme, and still others are based on covalent bond formation between a functional group on the support matrix and one on the enzyme, the latter one usually being an amino group. Although such support systems are, in principle, suitable for all varieties of biologically active proteins, certain uses place stringent operational demands on a support, demands which the prior art materials are not designed to meet. This is particularly true for immobilized antibodies.
For example, immunoaffinity chromatography is an especially useful procedure for the purification of products from a fermentation broth. In particular, immunoaffinity chromatography is quite well suited for the isolation of extracellular material expressed by a microorganism as the result of recombinant DNA incorporated into it. In such cases the desired product is viewed as an antigen, and an immobilized antibody specific to the desired product selectively and efficiently removes it from the fermentation broth by forming an insoluble antibody-antigen complex firmly held on the column of solid support to which the antibody is immobilized. When all the product has been removed, or when the support approaches saturation with the antigen, the antigen is eluted from the column by breaking the antibody-antigen complex and releasing pure antigen into the eluant. With removal of the antigen the antibody is regenerated, and the column once more can be used for immunoaffinity chromatography.
Since antibody-antigen complexes typically are quite strong their cleavage to release the antigen requires elution with a reagent often found to be deleterious to the immobilized antibody system. That is, eluants used often are not benign and often cause chemical disruption of the immobilized antibody system which may be a disruption of either the underlying support or the bound antibody. Such eluants are exemplified by strong acids, strong bases, detergents, and chaiotropic reagents. The result is that the antibody or some component of the underlying support matrix leaches into the eluant and ultimately contaminates the antigen-containing products. The presence of such contaminants in the product can be especially detrimental in purifying injectable materials, or when immunoaffinity chromatography with a bound antibody or enzyme is used for extracorporeal blood treatments.
The requirements of a support for antibody immobilization can be simply stated. The system must be low leaching, both in the context of solubilization of the antibody and of all components of the underlying support matrix. Low leaching with respect to the antibody requires that the latter be immobilized via a strong covalent bond. Low leaching with respect to the underlying support matrix means that all components as a whole must be very resistant to harsh chemical conditions. Additionally, the support matrix for an immobilized antibody is desirably incompressible so that it can accommodate high flow rates when used in, for example, a fixed bed. Because the immobilized antibody often may be used to purify or selectively remove a protein, it is imperative that the immobilized system minimize non-specific adsorption of protein. That is, a protein not an antigen for an immobilized antibody should not be absorbed by the immobilized antibody system via general and non-specific adsorption by the remainder of the support matrix, else there is a likelihood of such proteins being subsequently eluted upon release of the antigen with contamination of the product. Finally, there is a pragmatic need to maximize antibody loading (concentration of antibody immobilized per unit weight of support matrix) for optimizing process efficiency.
Immobilized antibodies or enzymes can be used for extracorporeal blood treatment if the immobilization system is covalent such that an immune response is not generated and non-thrombogenic. The system must not be chemically or enzymatically degraded by blood nor must it cause hemolysis, thrombosis or platelet removal. It also should not be toxic nor carcinogenic. In addition, the support should have low compressibility and good flow properties. In extracorporeal treatment, the serum fraction of the patient's blood containing the anti-coagulant heparin is passed directly over the immobilized protein. Enzymes such as asparaginase, bilirubin oxidase, phenylalanine ammonia lyase and heparinase have been somewhat successfully used to lower the serum concentrations of asparagine, bilirubin, phenylalanine and heparin, respectively, in animal models. M. D. Klein and R. Langer, Trends in Biotech., July, 1986, p. 179-186.
We have found a new family of support matrices which exhibit such favorable characteristics. In a sense these may be viewed as second generation systems for immobilization of biologically active proteins because they represent a significant advance in the operational sense as described above. Such materials satisfy needs not envisaged when the prior art support matrices were designed simply because the needs arose from demands imposed by new uses.
USPTO Backgrounds
USPTO Backgrounds