Washing buffer and method for purifying bovine hemoglobin using the washing buffer

Abstract

The invention discloses a washing buffer solution. The washing buffer solution is composed of HEPES and a sulfhydryl compound, wherein the molar concentration of the HEPES in the buffer solution is 19-21 mM, the molar concentration of the sulfhydryl compound is 3-6 mM, and the pH value of the washing buffer solution is 7.5-7.8. The invention also discloses a method for purifying bovine hemoglobinby adopting the washing buffer solution. The washing buffer solution is convenient to prepare, can be used for purifying bovine hemoglobin by anion exchange chromatography, and can obtain high-puritybovine hemoglobin, and the purity can reach 99% or above. The method for purifying the bovine hemoglobin can simplify the anion exchange chromatography process, the purification method is simple, thehigh-purity bovine hemoglobin can be obtained by washing chromatography columns with the washing buffer solution, the yield can reach more than 90 percent, and the bovine hemoglobin can be directly used as a raw material for post-chemical cross-linking polymerization and has good industrial application prospect.

Description

technical field [0001] The invention belongs to the field of biological products, and in particular relates to a washing buffer, in particular, the invention also relates to a method for purifying bovine hemoglobin by using the washing buffer. Background technique [0002] Cross-linking hemoglobin through chemical methods, due to its long shelf life, smaller particle size (nano-scale), universal blood type, etc., can be used as a blood substitute, rescue of traumatic blood loss in the field, organ protection in surgery, stroke rescue, etc. Fields are widely used. Due to the large amount of clinical use of blood substitutes, if residual traces of foreign proteins or impurities participate in the later chemical cross-linking reaction, it may trigger a strong immune response in the patient. Only ultra-high purity hemoglobin raw materials can ensure that the cross-linked hemoglobin is used clinical use. At present, hemoglobin is mainly obtained by separation and purification f...

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Problems solved by technology

Among them, ion-exchange chromatography has the characteristics of low packing cost, easy scale-up, and low cost of cleaning and regeneration, and has been widely used. Among them, US5264555 discloses an anion-exchange flow-through mode method for purifying bovine hemoglobin. Although it is simple and fast, its The resolution is not high, and some related impurities such as carbonic anhydrase and other related impurities in erythrocytes may flow through together with hemoglobin, and the purity is difficult to meet the requirements of clinical grade raw materials. Guoyong Sun, Andre F. The anion exchange method (Preparation of ultrapure bovine and human hemoglobinby anion exchange Journal of Chromatography B, 867 (2008) 1–7), due to gradient elution has strict requirements on the column efficiency and sample volume, At the same time, the gradient elution function is required for the chromatography system, which increases the production cost. In addition, the elution time is relatively long, which increases the risk of methemoglobin and denaturation due to oxidation on the protein column. At the same time, the consumption of eluting solution increases the waste In addition, the final harvested hemoglobin contains sodium chloride, which requires further dialysis and fluid replacement during chemical cross-linking in the later stage, which increases the process steps and is not suitable for large-scale commercial production in the later stage. Therefore, it is necessary to develop a simple and practical Buffer system for commercial anion exchange chromatography purification of bovine hemoglobin

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