Application of a caprylate/chromatography purification process for production of a high potency rabies immune globulin from pooled human plasma.

BACKGROUND: Human rabies immunoglobulin (RIG) is an integral part of post-exposure prophylactic treatment of rabies (along with rabies vaccination). Infiltration of most, if not all, of the RIG dose at the wound site is recommended. RIG produced by a caprylate/chromatography manufacturing process (RIG-C; HyperRAB) increased the potency and purity of this product over the existing licensed RIG from a solvent/detergent process (RIG-S/D; HyperRAB-S/D). METHODS: A series of studies were conducted to characterize the content and purity of RIG-C. A single-dose pharmacokinetic study in rabbits was performed to compare intramuscular (IM) immunoglobulin products manufactured by two different purification processes, solvent/detergent (IGIM-S/D) and caprylate/chromatography (IGIM-C). RESULTS: RIG-C was found to be a highly purified IgG formulation with high monomer content and formulated with twice the anti-rabies potency of RIG-S/D while maintaining the same overall protein concentration. RIG-C facilitates IM administration at the wound site by halving the injection volume. The new caprylate/chromatography process eliminated detectible levels of pro-coagulant impurities and IgA that were carried through in the prior S/D process. These impurities have been associated with thrombotic complications and allergic reactions in susceptible patients. After single dose administration, IGIM-C was pharmacokinetically equivalent to IGIM-S/D in rabbits. CONCLUSION: RIG-C is a more potent RIG formulation with less impurities yielding a safer and more convenient product with similar pharmacokinetic profile.

Thrombolysis with plasmin: implications for stroke treatment.

Plasmin is a direct-acting thrombolytic agent with a striking hemostatic safety advantage over plasminogen activators in animal models of thrombolysis and bleeding. In contradistinction to plasminogen activators, which risk bleeding at any effective thrombolytic dose, plasmin is tolerated without bleeding at several-fold higher amounts than those needed for thrombolysis. Plasmin has been safe in a current trial in patients with peripheral arterial or graft occlusion, and efforts are now directed toward therapy of stroke caused by cerebral artery occlusion. A rabbit (4 kg body weight) model of 2-hour, thrombin-induced middle cerebral artery occlusion using angiographic documentation of vascular patency and recanalization was used to perform a dose-ranging study of plasmin, delivered by catheter over a median duration of 10 minutes. Plasmin induced early recanalization in all animals (3 per group) within 10 minutes after discontinuation of 3, 2, or 1 mg of agent infusion. Control saline infusion failed to induce recanalization in 3 of 3 subjects. Plasmin rapidly induces middle cerebral artery recanalization, as determined in an angiogram-based animal model of arterial occlusion. Based on these data and other information, a phase I/IIa clinical trial of plasmin in human middle cerebral artery ischemic stroke has been initiated.

Production and characterization of a novel human recombinant alpha-1-antitrypsin in PER.C6 cells.

Alpha-1-antitrypsin (A1PI) is a proteinase inhibitor of the serpin superfamily and circulates in plasma at about 1-2 g/L. A1PI deficiency in humans often results in organ damage, particularly to the lungs and liver. Current augmentation therapies rely entirely on A1PI isolated from human plasma, thus prompting an evaluation of alternate sources. We have co-expressed recombinant A1PI and α-2,3-sialyltransferase in the human cell line, PER.C6. The requirement for sialyltransferase overexpression in PER.C6 and the essential contribution of sialic acid glycan capping on pdA1PI and recA1PI to prevent rapid A1PI plasma elimination is shown. Using assays to predict high levels of A1PI production and sialylation, stably transfected PER.C6 cells were screened through two rounds of cell cloning to ensure monoclonality. Fed-batch culturing was used to evaluate recA1PI production and cell line characteristics, identifying subclones expressing over 2.5 g/L recA1PI. Cell stability was assessed over 50 generations, verifying subclone stability during continuous culture. Finally, data are presented showing that recA1PI and pdA1PI are equivalent in their ability to block elastase activity in functional cell-based assays and their pharmacokinetic properties. These data show that recombinant human A1PI recovered from PER.C6 cells offers a reliable source of functionally active A1PI for augmentation therapies and, potentially, other diseases.