Preparation and purification of antibody-enzyme conjugates for therapeutic applications

Abstract

The successful use of antibody-enzyme conjugates for therapeutic applications requires conjugates of defined composition and low molecular weight, dictating the use of heterobifunctional coupling agents under carefully controlled and optimised conditions. The coupling chemistry chosen for the production of antibody-enzyme conjugates has been based almost exclusively on the use of thioether linkages, because of their greater stability in vivo compared with the disulphide linkages commonly used for the production of antibody-toxin conjugates. Using this type of conjugation chemistry, the modification of the proteins takes place in a semi-random fashion, any exposed modifiable amino acids being potential coupling sites, and there is no homogeneity of the product in the sense that location of modified residues is not controllable. Whilst the yield of the conjugation step is typically about 35–50%, purification is complicated by the changes in the charge properties of the proteins, which occur as a result of heterobifunctional and thiolation agents modifying positively charged lysine residues. This has meant that although affinity and ion exchange chromatography techniques have been used to some extent, the most common method of purifying conjugates is size exclusion chromatography. Incomplete separation means that the overall yields of purified product are typically only 10–15% at best. New technologies for the construction of antibody-enzyme conjugates are gradually emerging, which may eventually supplant the current chemical conjugation techniques, although it seems likely that chemical conjugation will continue to be of value for preliminary studies. Reverse proteolytic methods seek to extend the usefulness of chemical conjugation by achieving conjugation at defined sites on the proteins. The development of antibody-enzyme fusion proteins is becoming common, and bifunctional antibodies, based on an antigen binding arm together with an enzyme capture or catalytic antibody arm have also been described. These offer the prospect of homogeneity of product, simple purification using immunoglobulin binding proteins, and straightforward routes to the development of non-immunogenic proteins using conventional humanisation techniques.