MODIFICATION OF THERAPEUTIC GLYCOPROTEINSTHROUGH SPECIFIC ENZYMATIC OXIDATION

Atrium

Objective. To develop a method for chemical modification of glycoproteins by using galactose oxidase. Background. Despite growing interest in therapeutic biologics, there still are unmet needs in optimal delivery. Conjugation of other functional entities to therapeutic proteins has potential to change their disposition and increase efficacy. Among biologics, glycoproteins represent significant portion and often contain significant amount of terminal galactoses, which can be specifically modified to offer better control of biological activities. Methods. Using tPA as a model glycoprotein, two chemical modification schemes are compared using well-known thiolating agents: 1) Random modification through primary amino groups on lysines. tPA was treated with 5~10-fold molar access of N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP); and 2) enzymatic oxidation of terminal galactose by using galactose oxidase/horseradish peroxidase, followed by adding 3-[2-pyridyldithio] propionyl hydrazide (PDPH). The resulting thiolated tPAs were further conjugated with thiolated oligoanions, followed by ion-exchange chromatography. Enzyme activities of the conjugates were determined using indirect chromogenic assay with/without protamines to evaluate degrees of reversible control of enzyme activities. Results. Method 1 resulted in 2-3 thiolations/tPA whereas Method 2 resulted in ~1 thiolation/tPA. In both cases, retention of enzyme activity was ~95% of native tPA. Protamine treatment resulted in ~55% inhibition of tPA activity in Method 1 whereas ~90% inhibition was observed in Method 2. Upon removal of protamine by heparin, enzyme activities were fully recovered in both cases. Conclusion. Site-specific modification though enzymatic oxidation offered a superior control of enzyme activity of tPA. Grants. This work was supported in part by HPD Research Grant