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CN-121975004-A - Method for extracting unimodal ulinastatin from human urine

CN121975004ACN 121975004 ACN121975004 ACN 121975004ACN-121975004-A

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a method for extracting unimodal ulinastatin from human urine. The method sequentially comprises the steps of carrying out primary capturing and elution on human urine through ZGA313,313 resin chromatography, carrying out ultrafiltration desalination on eluent by taking purified water as buffer solution, carrying out specific affinity purification on the desalted liquid by using a monoclonal antibody chromatographic column with a ligand of a part area antibody of a Kunitz structural domain II, carrying out pasteurization on the collected liquid, carrying out selective removal on multimers generated in virus inactivation by using the difference of the number of hydrophobic sites of the multimers and monomers, and finally carrying out ultrafiltration to replace the buffer solution to obtain the unimodal kunitding solution. The invention has the advantages of synergistic process step design, high specificity and pertinence, capability of obviously improving the purity and yield of the product, effective removal of key impurities including polymers, guarantee of the safety of the product, simple operation, short period and suitability for large-scale production.

Inventors

  • YU BIN
  • LIU JIA
  • ZHAO WENMENG
  • ZHANG QI
  • LI ZHIWEI
  • Zhong Youhua

Assignees

  • 河南美森药业有限公司

Dates

Publication Date
20260505
Application Date
20260204

Claims (10)

  1. 1. A method for extracting unimodal ulinastatin from human urine is characterized by comprising the following steps: s1, loading human urine to ZGA313,313 resin chromatographic columns, sequentially washing with washing liquid containing sodium chloride, eluting with eluent containing sodium chloride, and collecting the eluent; S2, performing ultrafiltration desalination on the eluent obtained in the step S1 by using purified water as a buffer solution to obtain a desalted liquid; S3, after the pH value of the desalted liquid is regulated, loading the desalted liquid to a monoclonal antibody chromatographic column, and sequentially washing and eluting the monoclonal antibody chromatographic column to collect eluent, wherein the ligand of the monoclonal antibody chromatographic column is an antibody of a part region II of a Kunitz structural domain of ulinastatin; S4, performing pasteurization on the eluent obtained in the step S3 to obtain a virus inactivated solution; S5, after the pH value of the solution after virus inactivation is regulated, loading the solution to an RPC30 chromatographic column, sequentially washing and eluting, and collecting eluent, wherein the RPC30 chromatography selectively removes multimers generated in the virus inactivation process based on the difference of the number of hydrophobic sites between ulinastatin monomers and multimers; and S6, performing ultrafiltration on the eluent in the step S5 and replacing the buffer solution to obtain the unimodal ulinastatin solution.
  2. 2. The method for extracting unimodal ulinastatin from human urine as claimed in claim 1, wherein in the step S1, the loading volume is not more than 200 times of the column volume of ZGA313 resin chromatographic columns; In the step S3, the loading capacity is not more than 0.25 hundred million IU/L monoclonal antibody chromatographic packing; In the step S5, the loading capacity is not more than 0.25 hundred million IU/L RPC30 chromatographic packing.
  3. 3. The method for extracting unimodal ulinastatin from human urine as claimed in claim 1, wherein in step S1, the washing solution is water containing 1-5 g/L sodium chloride, and the eluent is water containing 45-60g/L sodium chloride.
  4. 4. The method according to claim 1, wherein in the step S3, the pH is adjusted to 5.5-6.5, the washing solution used in the washing is a citric acid buffer solution of 0.005.+ -. 0.0005 mol/L and the pH is 5.5-6.5, and the eluting solution used in the eluting is a buffer solution containing 0.005.+ -. 0.0005 mol/L citric acid and 0.3.+ -. 0.03 mol/L NaCl and the pH is 2.5-3.5.
  5. 5. The method for extracting unimodal ulinastatin from human urine as claimed in claim 1, wherein in step S4, the heating treatment is maintained at 60-62 ℃ for 10-11 hours.
  6. 6. The method for extracting unimodal ulinastatin from human urine as claimed in claim 1, wherein in step S5, the pH is adjusted to 6.0-7.0, and the washing solution used in the washing is a buffer solution containing 0.05+/-0.005 mol/L sodium acetate and 5+/-0.5% (v/v) alcohol, and the pH is 6.0-7.0; the eluting solution is buffer solution containing 0.05+ -0.005 mol/L sodium acetate and 15+ -1.5% (v/v) alcohol, and has pH of 6.0-7.0.
  7. 7. The method for extracting unimodal ulinastatin from human urine as claimed in claim 1, wherein in step S2, ultrafiltration is performed by using an ultrafiltration membrane with a molecular weight cut-off of 10kDa or 30kDa, and the desalting end point is controlled to have a conductivity of 1-5 mS/cm.
  8. 8. The method of claim 1, wherein in step S6, ultrafiltration is performed by using an ultrafiltration membrane with a molecular weight cutoff of 10kDa or 30kDa, the ultrafiltration buffer is 0.2.+ -. 0.02 mol/L NaCl solution, and ultrafiltration is repeated for 20 times or more, and the titer of the finally obtained unimodal ulinastatin solution is controlled to be not lower than 10 ten thousand IU/ml.
  9. 9. The method according to claim 1, wherein the sample flow rate of ZGA resin chromatography in step S1 is 8-12 column volumes/hr, the wash and elute flow rate is 0.8-1.2 column volumes/hr, the sample flow rate of monoclonal antibody chromatography in step S3 is 4-6 column volumes/hr, the wash and elute flow rate is 0.8-1.2 column volumes/hr, and the sample flow rate of RPC30 chromatography in step S5 is 4-6 column volumes/hr, and the wash and elute flow rate is 0.8-1.2 column volumes/hr.
  10. 10. The method of claim 1, wherein in step S1, the urine after ZGA313 resin chromatography is returned to the sewage treatment system.

Description

Method for extracting unimodal ulinastatin from human urine Technical Field The invention relates to the technical field of biological pharmacy, in particular to a method for extracting unimodal ulinastatin from human urine. Background Ulinastatin is a kind of glycoprotein inhibitor extracted from urine of healthy person and has important clinical value and may be used widely in treating acute pancreatitis, shock and post-operation auxiliary treatment. The conventional production process generally depends on a combination of various chromatographic techniques, for example, after the initial adsorption of chitin, the chitin is subjected to multi-step purification such as ion exchange, metal chelation or hydrophobic interaction chromatography. Although the methods can realize the extraction of the ulinastatin, the defects of long process flow, large loss of target protein in multiple material conversion and elution and low yield of the final product are generally existed. More importantly, such conventional processes have limited removal of specific impurities from the product. Complex components of human urinary origin, such as degraded fragments (especially small molecules containing only the Kunitz domain II) that are structurally similar to the target protein, and protein multimers generated during production, are difficult to isolate efficiently. This directly results in an end product with insufficient purity, with related substances easily exceeding standards, and increased risk of allergic and pyrogenic reactions in clinical applications. In addition, the existing purification route is not stable enough in removing impurities such as endotoxin, kininogenase and the like, so that the quality difference among different production batches is obvious. In terms of virus safety, the traditional pasteurization step effectively inactivates viruses and simultaneously often causes the ulinastatin molecules to aggregate to generate polymers. These polymers are not only impurities, but may also affect the safety and efficacy of the product. However, existing conventional chromatographic methods (e.g., hydrophobic chromatography based on ammonium sulfate salting out) have difficulty in removing such multimers efficiently and specifically, often requiring a tradeoff between yield and purity. Therefore, a new purification process for ulinastatin needs to be developed in the art. The process needs to be capable of simplifying the flow, improving the total yield, and having the capability of efficiently removing impurities such as endotoxin, kininogenase and the like, so that a high-purity and high-quality unimodal ulinastatin product is stably obtained. Disclosure of Invention In order to solve the defects in the background technology, the method for extracting unimodal ulinastatin from human urine provided by the invention comprises the following steps: s1, loading human urine to ZGA313,313 resin chromatographic columns, sequentially washing with washing liquid containing sodium chloride, eluting with eluent containing sodium chloride, and collecting the eluent; S2, performing ultrafiltration desalination on the eluent obtained in the step S1 by using purified water as a buffer solution to obtain a desalted liquid; S3, after the pH value of the desalted liquid is regulated, loading the desalted liquid to a monoclonal antibody chromatographic column, and sequentially washing and eluting the monoclonal antibody chromatographic column to collect eluent, wherein the ligand of the monoclonal antibody chromatographic column is an antibody of a part region II of a Kunitz structural domain of ulinastatin; S4, performing pasteurization on the eluent obtained in the step S3 to obtain a virus inactivated solution; S5, after the pH value of the solution after virus inactivation is regulated, loading the solution to an RPC30 chromatographic column, sequentially washing and eluting, and collecting eluent, wherein the RPC30 chromatography selectively removes multimers generated in the virus inactivation process based on the difference of the number of hydrophobic sites between ulinastatin monomers and multimers; and S6, performing ultrafiltration on the eluent in the step S5 and replacing the buffer solution to obtain the unimodal ulinastatin solution. Further, in the step S1, the loading volume is not more than 200 times of the volume of the ZGA313,313 resin chromatographic column; In the step S3, the loading capacity is not more than 0.25 hundred million IU/L monoclonal antibody chromatographic packing; In the step S5, the loading capacity is not more than 0.25 hundred million IU/L RPC30 chromatographic packing. In the step S1, the washing liquid is water containing 1-5 g/L sodium chloride, and the eluent is water containing 45-60g/L sodium chloride. Further, in the step S3, the pH is adjusted to 5.5-6.5, the washing liquid used for washing is a citric acid buffer solution with the pH of 0.005+/-0.0005 mol/L and is 5.5-6.5, and