Coagulation factor IX conjugates
A technology of conjugates and factors, applied in the direction of drug combinations, blood diseases, biochemical equipment and methods, etc., can solve the problem of attachment of half-life extension parts that are not suitable for highly functionalized
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[0207] In one embodiment, the FIX polypeptide conjugated to HEP is wild-type FIX.
[0208] In one embodiment, the FIX polypeptide conjugated to HEP is wild-type FIX(a).
[0209] In another embodiment, the FIX polypeptide conjugated to HEP is an analog or variant having >95% sequence identity to wild-type FIX or FIX(a).
[0210] In one embodiment, the FIX of the HEP-FIX Polypeptide Conjugate is mutated such that it has enhanced proteolytic activity.
[0211] In one embodiment, the HEP polymer conjugated to the FIX polypeptide has a molecular weight of 5 to 15 kDa.
[0212] In one embodiment, the HEP polymer conjugated to the FIX polypeptide has a molecular weight of 15 to 25 kDa.
[0213] In one embodiment, the HEP polymer conjugated to the FIX polypeptide has a molecular weight of 25 to 35 kDa.
[0214] In one embodiment, the HEP polymer conjugated to the FIX polypeptide has a molecular weight of 35 to 45 kDa.
[0215] In one embodiment, the HEP polymer conjugated to the F...
Embodiment 1
[0354] Example 1: Quantitative method
[0355] The purity of the conjugates of the invention was analyzed by HPLC. HPLC was also used for conjugate quantification. Quantitation is based on the integration of the area under the curve using the absorption spectrum at a wavelength of 280 nm. Use manufactured by Wyeth Pharmaceuticals Inc. Recombinant coagulation factor IX was used as reference. A Zorbax 300SB-C3 column (4.6x50 mm; 3.5 μm Agilent, Cat. No.: 865973-909) was used. The column was operated on an Agilent 1100 Series HPLC equipped with a fluorescence detector (Ex 280nm, Em 348nm). The column temperature was 30 °C, the sample injection volume was 5 μg and the flow rate was 1.5 ml / min. The column was eluted with a water (A)-acetonitrile (B) solvent system containing 0.1% trifluoroacetic acid. The gradient program was as follows: 0 min (25% B); 4 min (25% B); 14 min (46% B); 35 min (52% B); 40 min (90% B); 40.1 min (25% B).
Embodiment 2
[0356] Embodiment 2: SDS-PAGE analysis
[0357] SDS PAGE analysis was performed using precast Nupage 7% tris-acetate gels, NuPage tris-acetate SDS running buffer and NuPage LDS sample buffer all from Invitrogen. Samples were denatured (70°C, 10 min) prior to analysis. HiMark HMW (Invitrogen) was used as standard. Electrophoresis was run at 150V, 120mA for 80min in an XCell Surelock Complete (Invitrogen) with power station. Gels were stained using SimplyBlue SafeStain from Invitrogen.
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