A reversed-phase HPLC assay for plasminogen activators

ACTIVASE(R) is the recombinant form of human tissue-type plasminogen activa tor (r-tPA), used in the management of acute myocardial infarction and pulm onary embolism. ACTIVASE(R) is also now approved for treating ischemic stro ke. It is produced by expressing the complementary DNA (cDNA) for natural h uman tPA in Chinese hamster ovary (CHO) cells. TNK-tPA is a genetically eng ineered variant of r-tPA with enhanced efficacy and lower incidence of blee ding compared with activase. It was created by three site-directed mutation s (T103N, N117Q and KHRR296-299AAAA) and, is also cloned and expressed in C HO cells. CHO cells biosynthesize endogenous hamster tPA called CHO-PA. The amino acid sequence of CHO-PA is highly homologous (80% identical) to that of r-tPA. All three thrombolytic proteins exist as heterogeneous isoforms, mainly due to proteolysis/hydrolysis and differential glycosylation. In th is report, a reversed-phase HPLC method was developed to support manufactur ing process development. This method not only has the ability to resolve th e three plasminogen activators from each other, but also is capable of iden tifying and quantifying different isoforms of each molecule.