Application case of Heparin affinity resin by Bestchrom

Sep 05,2024

Bestchrom

Existing in mast cells, Heparin is made by the liver, from which it gets its name. Commercially available preparations of heparin are mainly produced from bovine lung and porcine gastrointestinal mucosa. Heparin is a type of mucopolysaccharide composed of glucosamine, L-idosaccharide, n-acetylglucosamine and D-glucuronic acid.

Clinical data have shown its wide application in bioactivity and clinical application apart from its function as blood anticoagulant. Since heparin has affinity function with multiple bio-molecules, it is possible for Bestchrom to get family specific affinity chromatography resin——Heparin Bestarose FF by coupling heparin covalent on highly cross-linked agarose beads. The resin enjoys merits including good chemical/physical stability, less leached ligands, longer service life, wider application and easy scale-up.

Advantages of Heparin Bestarose FF resin:

· High DBC: ≥2.0mg bovine AT III/mL resin

· High selectivity: specific affinity ligands, efficient purification of target proteins.

·  Excellent stability: good tolerance towards washing with commonly used solutions, e.g. 8M urea, 6M GuHCl, 0.1M NaOH, 4M NaCl.

Application

Pharmaceuticals: Quite different from general affinity resins, Heparin possesses affinity interaction with nearly a hundred of proteins. Thus, Heparin Bestarose FF resin can be widely applied in purification of bio-molecules, especially enzymes including antithrombin III, thrombinoid, human clotting factor IX, XI, VIII, lipoprotein lipase, collagenase, DNA polymerase.

It can also bind with bio-macromolecules including human interleukin, human prostate growth factor, recombinant human vascular endothelial growth factor, cartilage growth factor, Basic fibroblast growth factor, recombinant human acid fibroblast growth factor, recombinant hepatocyte growth factor, recombinant mouse heparin cofactor II, recombinant human platelet factor IV, recombinant human endostatin, recombinant human keratinocyte growth factor.

Food: Heparin affinity resin can also be used in food processing sector. According to GB5009.299-2024 China National Food Safety Standard Detection of lactoferrin content in food (which is issued on 2024-02-08 and implemented on 2024-08-08). According to GB500.299-2024, when detecting lactoferrin content in food, extract sample with phosphate buffer, used affinity resin pre-packed column to accumulate and purify sample before carrying out final detection.

Application cases

Heparin Bestarose FF can specifically bind and purify antithrombin Ⅲ in plasma protein. Antithrombin Ⅲ has excellent anticoagulant effect and can effectively prevent blood clotting related diseases.

The purification of antithrombin Ⅲ in blood plasmid with Heparin Bestarose FF resin

Column and resin: EzFast 1mL，Heparin Bestarose FF

Sample: blood plasmid

Binding buffer: 0.1M Tris，0.15M NaCl，pH7.4

Washing buffer: 0.1M Tris，0.33M NaCl，pH7.4

Elution buffer: 0.1M Tris，2.0M NaCl，pH7.4

 Tips for using resin

Buffer:

Binding buffer: 20~50mM PB or Tris，pH7.4~8.0，alternatively, 0.15M NaCl restrain non-specific adsorption.

Elution buffer: 20~50mM PB or Tris，1~2M NaCl，pH7.4~8.0，concentration of NaCl shall be adjusted according to binding strength with target proteins.

Flow:

according to column bed height, set flow at 60~300cm/h. The higher column bed height is, the lower flow velocity will be.

Cleaning:

frequent cleaning can effectively prevent fouling and help resin have a stable performance. The following is the recommendation for CIP target different impurities and fouling:

· Removal of ion-bound proteins: First wash with 3~5CV of 2M NaCl, then wash with 3~5CV of pure water.

· Removal of precipitated or denatured proteins: First wash with 2CV of 0.1M NaOH, then wash with 5~10CV of purified water, alternatively, 6M guanidine hydrochloride or 8M urea can also be used.

· Removal of hydrophobically bound proteins: Wash with 0.1~0.5% non-ionic detergent, and then wash with 3~5CV of purified water.

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