Method for potency determination of haina polysaccharide (hps)

A method for potency determination of a haina polysaccharide (HPS), comprising the following steps:

• obtaining an HPS activity curve by a p-nitroaniline chromogenic assay, wherein the HPS activity curve uses a reciprocal of an HPS weight as an abscissa and a logarithm of a residual Factor Vill (FVIII) activity unit as an ordinate;
• and
• defining a weight of the HPS that inhibits an Intrinsic Factor Xase (iFXase) activity required for formation of 1 IU of an FVIII as one potency unit, and obtaining a potency of the HPS from the HPS activity curve;
• wherein
• the HPS is a fucosylated chondroitin sulfate polysaccharide;
• the HPS has a weight-average molecular weight of 90,000 to 130,000, and has a monosaccharide composition comprising glucuronic acid, N-acetyl-D-galactosamine, and fucose, wherein a molar ratio of the glucuronic acid, the N-acetyl-D-galactosamine, and the fucose is in a range of 1:0.8-1.2:0.5-0.8;
• and a sulfate group in the HPS has a mass percentage of 25% to 40%;
• wherein
• the obtaining the HPS activity curve comprises:
• preparing HPS working solutions with gradient concentrations;
• mixing the HPS working solutions, an FVIII solution, and an activation reagent solution, and conducting a first incubation to obtain a first incubation mixture, wherein the activation reagent solution comprises a Factor IXa (FIXa), a Factor IIa (FIIa), a phospholipid, a buffer, and CaCl2;
• mixing the first incubation mixture with a Factor X (FX) solution, and conducting a second incubation to obtain a second incubation mixture;
• mixing the second incubation mixture with a chromogenic substrate solution, conducting a third incubation, adding a stop solution, and measuring an absorbance of a resulting mixture at 405 nm to obtain an absorbance value, wherein a chromogenic substrate in the chromogenic substrate solution is SXa-11;
• and
• substituting the absorbance value into an FVIII complex activity standard curve, calculating the residual FVIII activity unit, and plotting the HPS activity curve using the reciprocal of the HPS weight as the abscissa and the logarithm of the residual FVIII activity unit as the ordinate;
• wherein the FVIII complex activity standard curve uses a logarithm of an FVIII concentration as an abscissa and the absorbance value as an ordinate.