Liquid composition of modified factor vii polypeptides
A technology of composition and factor, applied in the direction of drug combination, coagulation/fibrinolytic factor, specific peptide, etc.
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[0107] Preparation and purification of factor VII polypeptide:
[0108] Purified human factors suitable for use in the present invention are preferably prepared by recombinant DNA techniques, e.g., as described by Hagen et al., Proc. VIIa. It is also possible to prepare Factor VII using the methods described by Broze and Majerus, J. Biol. Chem. 255(4):1242-1247, 1980 and Hedner and Kisiel, J. Clin. Invest. These methods produce Factor VII without detectable amounts of other coagulation factors. Including an additional gel filtration as a final purification step, it is possible to obtain even purer Factor VII preparations. Factor VII is then converted to activated Factor Vila by known methods, for example using several different plasma proteins, such as Factor XIIa, IXa or Xa. Alternatively, pass it through an ion exchange column such as Mono as described by Bjoern et al. (Pharmacia Fine Chemicals), etc., or self-activation in solution, may activate Factor VII.
[0109]...
Embodiment 1
[0120] Analytical method
[0121] Aggregate content was determined by non-denaturing size exclusion HPLC. The content of the oxidized form was determined by RP-HPLC. The content of the enzymatically degraded form was determined by RP-HPLC.
[0122] Native size exclusion chromatography was performed on a Waters Protein Pak 300SW column 7,5 x 300mm, using 0.2M ammonium sulfate, 5% 2-propanol pH 7,0 as the mobile phase. Flow rate: 0.5ml / min. Detection: 215nm. Loading: 25 μg FVIIa.
[0123] In particle size 5μm, pore size Reverse-phase HPLC was performed on a patented 4,5x250mm butyl-bonded silica column. Column temperature: 70°C. A-buffer: 0.1% v / v trifluoroacetic acid. B-buffer: 0.09% v / v trifluoroacetic acid, 80% v / v acetonitrile. The column was eluted with a linear gradient from X to (X+13)% B in 30 minutes. X is adjusted so that FVIIa elutes at a retention time of approximately 26 minutes. Flow rate: 1.0ml / min. Detection: 214nm. Loading: 25 μg FVlla.
Embodiment 2
[0125] composition preparation
[0126] In general, aqueous FVIIa composition samples for analysis in these experimental examples were prepared from purified stock solutions by exchanging buffer through gel filtration columns. Composition additives are included in the elution buffer, or added to the eluate, in final proportions. The resulting solution was aseptically filtered using a sterile membrane filter (0.2 μm pore size or equivalent), filled into a sterile glass bottle, and tightly capped and sealed with a butyl rubber stopper and a flip-off aluminum cap.
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