ABSTRACT
Essentials Glycosylation heterogeneity of recombinant proteins affects pharmacokinetics and immunogenicity. N-glycomics/glycoproteomics of plasma-derived Factor VIII and 6 recombinant FVIIIs were compared. Depending on cell line, significant differences to plasma-derived FVIII were observed. Recombinant FVIIIs expressed distinct and immunologically relevant epitopes. SUMMARY: Background/Objective Human factor VIII (FVIII) is a plasma glycoprotein, defects of which result in hemophilia A. Current substitution therapy uses FVIII products purified from human plasma or from various cell lines (recombinant FVIII) with different levels of B-domain deletion. Glycosylation is a post-translational protein modification in FVIII that has a substantial influence on its physical, functional and antigenic properties. Variation in glycosylation is likely to be the reason that FVIII products differ in their pharmacokinetics, pharmacodynamics and immunogenicity. However, the literature on FVIII glycosylation is inconsistent, preventing assembly into a coherent model. Seeking to better understand the glycosylation mechanisms underlying FVIII biology, we studied the N-glycosylation of human plasma-derived (pd)FVIII and six rFVIII products expressed in CHO, BHK or HEK cell lines. Methods FVIII samples were subjected to head-to-head detailed glycomic and glycoproteomic characterization using a combination of MALDI-MS and MS/MS, GC-MS and UPLC-UV-MSE technologies. Results/Conclusion The results of our study detail the N-glycan repertoire of pdFVIII to an unprecedented level, and for the first time, provide evidence of N-glycolylneuraminic acid (NeuGc) found on pdFVIII. Although site-specific glycosylation of rFVIII proved consistent with pdFVIII regardless of the expression system, the entire N-glycan content of each sample appeared significantly different. Although the proportion of biologically important epitopes common to all samples (i.e. sialylation and high-mannose) varied between samples, some recombinant products expressed distinct and immunologically relevant epitopes, such as LacdiNAc (LDN), fucosylated LacdiNAc (FucLDN), NeuGc, LewisX/Y and Galα1,3 Gal epitopes. rFVIII expressed in HEK cells showed the greatest glycomic differences to human pdFVIII.
ABSTRACT
UNLABELLED: Essentials ADAMTS-13-deficiency is a cause of thrombotic thrombocytopenic purpura (TTP). Preclinical safety of recombinant human ADAMTS-13 (BAX930) was shown in animal models. Preclinical efficacy of BAX930 was shown in a mouse model of TTP. BAX930 showed advantageous efficacy over fresh frozen plasma, the current standard of care. Click to hear Dr Cataland and Prof. Lämmle present a seminar on Thrombotic Thrombocytopenic Purpura (TTP): new Insights in Pathogenesis and Treatment Modalities. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) is a rare blood disorder characterized by microthrombosis in small blood vessels of the body, resulting in a low platelet count. Baxalta has developed a new recombinant ADAMTS-13 (rADAMTS-13) product (BAX930) for on-demand and prophylactic treatment of patients with hereditary TTP (hTTP). Objectives To evaluate the pharmacokinetics, efficacy and safety of BAX930 in different species, by use of an extensive preclinical program. Methods The prophylactic and therapeutic efficacies of BAX930 were tested in a previously established TTP mouse model. Pharmacokinetics were evaluated after single intravenous bolus injection in mice and rats, and after repeated dosing in cynomolgus monkeys. Toxicity was assessed in rats and monkeys, safety pharmacology in monkeys, and local tolerance in rabbits. Results BAX930 was shown to be efficacious, as demonstrated by a stabilized platelet count in ADAMTS-13 knockout mice that were thrombocytopenic when treated. Prophylactic efficacy was dose-dependent and comparable with that achieved by treatment with fresh frozen plasma, the mainstay of hTTP treatment. Therapeutic efficacy was treatment interval-dependent. Safety pharmacology evaluation did not show any deleterious effects of BAX930 on cardiovascular and respiratory functions in monkeys. The compound's pharmacokinetics were similar and dose-proportional in mice, rats, and monkeys. BAX930 was well tolerated in rats, monkeys, and rabbits, even at the highest doses tested. Conclusions These results demonstrate that BAX930 has a favorable preclinical profile, and support the clinical development of rADAMTS-13 for the treatment of hTTP.
