In vitro stability of lyophilized and reconstituted recombinant activated factor VII formulated for storage at room temperature.

Background: Recombinant activated factor VII (rFVIIa) is indicated to treat bleeding episodes or prevent bleeding related to surgery in patients with hemophilia A or B who have antibodies to coagulation factors VIII or IX. The first-generation rFVIIa formulation is stable when stored under refrigeration. A new formulation has been developed for storage at room temperature, which may improve patients' access to treatment during bleeding episodes.

Objective: These in vitro experiments were conducted to evaluate the stability of the new formulation of rFVIIa, both lyophilized and reconstituted, under the expected storage conditions, as well as at higher temperatures.

Methods: The stability of the new rFVIIa formulation when stored under various conditions before and after reconstitution was evaluated in terms of retained activity (clotting assay), rFVIIa content (high-performance liquid chromatography [HPLC]), and rFVIIa degradation products (HPLC), including aggregates (dimer/oligomer). Activity was analyzed within specific limits representing the allowable minimum/maximum for each test parameter at the end of the product's shelf-life, as adopted by the European Medicines Agency and the US Food and Drug Administration. Before reconstitution, vials from 9 lots of the new rFVIIa formulation, 3 of each size (1, 2, and 5 mg/vial), were stored at refrigerated temperature (5 degrees C) and at room temperature (25 degrees C) for 24 months, and at 30 degrees C for 12 months. To simulate short-term exposure to temperatures higher than recommended, samples were stored at 40 degrees C for 6 months, followed by storage at 25 degrees C for 12 months. To simulate the home setting, in which the product may be alternately stored in and out of the refrigerator, samples were stored at 30 degrees C for 8 hours and then at 5 degrees C for 16 hours, repeated daily for 5 days. To analyze the effect of storage at extremely elevated temperatures, samples were exposed to temperatures of 50 degrees C, 60 degrees C, and 70 degrees C for 12 hours. After reconstitution, samples were maintained at 25 degrees C for up to 6 hours or at 5 degrees C for up to 24 hours.

Results: The specific activity and rFVIIa content of the new lyophilized formulation remained stable after storage for 24 months at 5 degrees C and 25 degrees C, and for 12 months at 30 degrees C; after 5 days of daily alternation between storage at 5 degrees C and 30 degrees C; and after storage for 6 months at 40 degrees C followed by 12 months at 25 degrees C. When stored at 50 degrees C and 60 degrees C for 12 hours, activity remained constant, whereas rFVIIa aggregates increased within the specified limits; after storage at 70 degrees C for 12 hours, rFVIIa activity decreased in parallel with the formation of aggregates, which exceeded the specified limit for the 5-mg product. After reconstitution, samples of all vial sizes of the new rFVIIa formulation retained their activity when stored at 25 degrees C for 6 hours and at 5 degrees C for 24 hours.

Conclusions: In these in vitro experiments, the new lyophilized formulation of rFVIIa was stable when stored for 24 months at 25 degrees C, 12 months at 30 degrees C, 6 months at 40 degrees C, and 12 hours at 50 degrees C and 60 degrees C without compromise to its activity or rFVIIa content. The reconstituted product retained activity when stored at 25 degrees C for 6 hours and at 5 degrees C for 24 hours.