CN1767851A - Hemoglobin conjugate and its preparing method and use - Google Patents

Abstract

Present invention provides a hemoglobin and human serum albumin conjugate and its preparation. The conjugates has Mr of between 100-300KD, comprising 1-3 hemoglobin molecules crosslinked intermolecularly or intramolecularly and 1-3 human serum albumin molecules, which gained by following steps: prepared stroma-free hemoglobin, then coupled hemoglobin to human serum albumin and purified products using anion exchange chromatography. The protein conjugate have good characteristic using as a blood substitute.

Description

Conjugated hemoglobin and its preparation method and use

technical field

The invention relates to a conjugate of hemoglobin, in particular to a conjugate of hemoglobin and human serum albumin used as a blood substitute, and a preparation method of the conjugate.

Background technique

Blood transfusion is an indispensable medical method for clinical operations, disaster relief and battlefield rescue. Relying on people to donate blood not only faces the problem of shortage of blood sources, but also due to the complexity of blood types, blood transfusion must be strictly matched. Security is threatened. In recent years, the demand for blood has been increasing, but safe and effective blood sources have become increasingly scarce. Therefore, in recent decades, human blood substitutes have always been a research and development focus in the international scientific and business circles.

Human blood substitutes, that is, human red cell substitutes, are artificial preparations that have the function of oxygen transfer, maintain blood osmotic pressure and acid-base balance, and expand capacity. An ideal blood substitute requires the oxygen transfer function, biocompatibility, safety and stability of natural red blood cells. It has a wide range of application prospects, not only for the treatment of clinical surgery, acute anemia, and massive bleeding in peacetime and wartime, but also for the treatment of various diseases, such as routine use in the perioperative period, trauma treatment , hemodilution, perfusion of ischemic tissue, septic shock, multiple antibody patients, tumor treatment, etc.

Existing human blood substitutes mainly include polymer perfluorocarbons synthesized by organic chemistry and hemoglobin and red blood cell blood substitutes prepared by biotechnology. Among them, because perfluorocarbons do not have the biological oxygen-carrying properties of natural hemoglobin, their research and development are no longer the focus; the red blood cells converted by blood type have shortcomings such as short storage period and difficult transportation, and their research and application are also extremely popular. However, hemoglobin (Hb) derived from red blood cells itself has the function of efficiently carrying oxygen and maintaining colloid osmotic pressure. At present, all countries are focusing on the development of oxygen-carrying agents based on hemoglobin, mainly including chemically modified hemoglobin. And liposome-encapsulated hemoglobin and other hemoglobin-containing microcapsules (Winslow RM. Hemoglobin-based red cell substitute. The John Hopkins University Press, Baltimore and London. 1992: 39).

Stroma-free hemoglobin is easily oxidized or dissociated into monomers and dimers in the body, causing nephrotoxicity, and has defects such as high oxygen affinity, short circulation half-life, and high colloid osmotic pressure. Cross-linking and increasing the molecular weight of hemoglobin can solve these problems to some extent. The hemoglobin molecule is composed of 4 subunits. Intramolecular cross-linking enhances the stability of the tetramer. Increasing the molecular weight of hemoglobin can make it difficult to pass through the kidney directly and be metabolized by the circulation, thus prolonging its circulation half-life in the body . But not all chemical conjugations reduce the immunogenicity of hemoglobin. Therefore, the modification requires on the one hand to increase the molecular weight of hemoglobin and eliminate the nephrotoxic side effects of the product in the process of metabolism in the body; on the other hand, the modification must also maintain the biological activity of hemoglobin and eliminate the immunogenicity of the product. Several hemoglobin modification methods commonly used at present include hemoglobin self-polymerization (Rausch C W, Gawryl M S, Light WR et al. PEG (Davis F, NHO K, ZALIPSKY S. Chemically modified hemoglobin as an effective, stable non-immunogenic red blood cell substitute. US 5234903.1993-08-10), and hemoglobin and polysaccharide or protein coupling (Adamson G J. Hemoglobin-polysaccharide conjugate .US 6500930.2002-11.31)

Although few of the blood substitutes developed so far have the disadvantages of natural blood, there is no blood substitute that has all the advantages of blood. As far as the current research level is concerned, there are still many unresolved problems in blood substitutes, among which the safety and effectiveness of blood substitutes are the most important. Some side effects of blood substitutes (such as vasoconstriction and high blood pressure) limit their use, especially in the elderly with various diseases. Secondly, the product has a short retention time in the internal circulation (as short as a few hours, the longest life span is 70 hours, while the life span of natural red blood cells is 120 days), it can only provide short-term oxygen supply function, and it is different from normal blood function. The gap is large.

Human serum albumin (HSA) has a molecular weight of 67,000 and is the main protein in plasma. In the human body, it has the functions of maintaining blood osmotic pressure and carrying various ligands (including fatty acids, amino acids, steroids, metal ions and drugs) in the blood to exchange with tissues, etc. Physiological function, and can carry accumulated toxic substances such as bilirubin, so it has detoxification function. In medicine, human serum albumin is used as an important clinical drug for surgical blood transfusion and fluid rehydration for critically ill patients.

Contents of the invention

The object of the present invention is to provide a conjugate of hemoglobin and human serum albumin. The conjugate of the present invention is used as a blood substitute and has the functions of carrying oxygen, maintaining osmotic pressure and transporting nutritional components of relatively complete blood.

Another object of the present invention is to provide a method for preparing the conjugate of hemoglobin and human serum albumin.

The conjugate of hemoglobin and human serum albumin of the present invention preferably has a molecular weight distribution between 100KD and 300KD. The number of hemoglobin molecules in the conjugate is 1-3, preferably 1-2, and the number of human serum albumin molecules is 1-3, preferably 1. The hemoglobin in the conjugate is intramolecularly crosslinked or not intramolecularly crosslinked, preferably intramolecularly crosslinked hemoglobin. The hemoglobin of the conjugate of the present invention comes from human or other mammals, preferably red blood cells of pigs, cattle, sheep, horses and dogs.

The preparation method of the conjugate of hemoglobin and human serum albumin of the present invention comprises the preparation of stroma-free hemoglobin, the coupling of hemoglobin and human serum albumin and the purification of the coupling product.

The stroma-free hemoglobin can be prepared by methods known in the art for preparing electrophoretic or chromatographically pure stroma-free hemoglobin.

Preferably, the integrated purification of hemoglobin using membrane filtration and ion exchange chromatography results in electrophoretic or chromatographically pure stroma-free hemoglobin. The washed red blood cells were swollen and ruptured under low osmotic pressure (Doczi J. Injectable stroma free hemoglobin and its method of manufacture.1976, US3991181), and the obtained red blood cell lysate was microfiltered through a 0.22-0.65 μm membrane, and then passed through a 10-30KD Membrane ultrafiltration, preferably 0.45μm membrane microfiltration, and then 30KD membrane ultrafiltration for pretreatment. The obtained hemoglobin solution is further purified by permeation anion exchange chromatography, the chromatographic medium is preferably DEAE Sepharose Fast Flow, QMA Spherosil LS, Q Sepharose Big Beads (Amersham phamacia, Sweden), and the pH value of the chromatography is 6.6 to 8.5, preferably It is a phosphate buffer solution with a pH value of 7.0-7.8, and the concentration of the buffer solution is 10mM-100mM, preferably 20-50mM. PEG concomitant chromatography is adopted, PEG400-PEG4000 is selected, the concentration is 0.25%-10%, preferably 0.5-5%, and the recovery rate of the chromatography is greater than 95%. The operating temperature of each purification unit is 4-10°C. Obtained for cross-linking with human serum albumin.

The coupling agent used in the present invention is a bifunctional crosslinking agent that reacts with protein amino groups, sulfhydryl groups or hydroxyl groups, preferably with aldehyde groups, succinimide (NHS) groups, epoxy groups, maleimide Homo- or hetero-bifunctional cross-linking agent for group, imidate group.

The method for coupling human serum albumin and hemoglobin of the present invention can be a liquid-phase one-step coupling method or a two-step coupling method, or a method in which protein is adsorbed on a solid phase medium for coupling.

One-step coupling method: dissolve the protein in phosphoric acid, HEPES, boric acid, borax-sodium hydroxide or sodium carbonate buffer solution with pH 6-12, preferably pH 7.5-9.5, the protein concentration is 1-150 mg/ml, preferably protein The concentration is 10-100mg/ml, adding a cross-linking agent, the molar ratio of cross-linking agent to protein is 3:1-600:1, preferably 10:1-200:1, and the reaction temperature is controlled at 4-55°C, preferably 25-37°C, the reaction time is 0.1-48 hours, preferably 0.5-10 hours.

Two-step cross-linking method: first activate hemoglobin or first activate human serum albumin, dissolve it in pH 6-12, preferably pH 7.0-9.5 buffer solution of phosphoric acid, HEPES, boric acid, borax-sodium hydroxide or sodium carbonate , the protein concentration is 1-150mg/ml, preferably the protein concentration is 5-60mg/ml, the reaction temperature is controlled between 4-55°C, preferably 25-37°C, adding a cross-linking agent, the molar ratio of cross-linking agent to protein 3:1-600:1, preferably 10:1-500:1, and the reaction time is 0.1-48 hours, preferably 0.5-10 hours. After the cross-linking agent reacts with the active group of hemoglobin or human serum albumin (the active group can be sulfhydryl or amino), pass through Sephadex G～25 desalting column or low-temperature dialysis to remove the modification agent, and adjust the pH value of the buffer at the same time, so that The pH is the same or different from the first step reaction pH, but the pH range is still 6-12, preferably 7.5-9.5, and then another protein is added in an equal amount to make the cross-linking agent and its active group (the active group can be mercapto or amino) to continue the reaction for 1 to 48 hours.

Coupling on solid phase medium: the medium is an anion exchange medium or a cation exchange medium, preferably DEAE Sepharose Fast Flow, Q Sepharose Big Beads, Q Sepharose Fast Flow, SP Sepharose Fast Flow and CM Sepharose Fast Flow (Amershamphamacia, Sweden). Use 50mM pH4.0-8.5, preferably pH4.5-7.5 phosphate or HEPES buffer to equilibrate the chromatography medium, add 0.5-5mg/ml hemoglobin or human serum albumin solution, so that the protein is first adsorbed on the medium. Then add a cross-linking agent so that the molar ratio of cross-linking agent to protein is 30:1-600:1, preferably 10:1-500:1, and the reaction time is 0.1-12 hours, preferably 0.5-10 hours. After washing the column with the above-mentioned equilibration buffer to remove excess cross-linking agent, the activated protein with cross-linking agent was eluted with the buffer containing 0.5M NaCl, and the protein peak was collected. The NaCl in the solution was removed by Sephadex G-25 gel filtration column. Collect the protein-containing eluate, concentrate it by ultrafiltration until the protein concentration is 1-5 mg/ml, add an equimolar amount of another protein, and adjust the pH value of the buffer at the same time to make the pH the same or different from the reaction pH of the first step. The pH range is still 6 to 12, preferably 7.5 to 9.5, and the activated protein crosslinking agent is reacted with another protein active group (the active group can be a sulfhydryl group or an amino group) for 1 to 24 hours to obtain A conjugate with a molar ratio of hemoglobin to human serum albumin of 1:1.

When the coupling product prepared in the present invention is purified, the preferred method is selected from ion exchange chromatography, ultrafiltration and gel filtration chromatography purification, which can be any one of the methods, and two or three methods are used simultaneously. Remove components with no coupling reaction and molecular weight greater than 300KD and less than 100KD.

Adjust the pH value of the purified coupling product to 7.4. For hemoglobin affected by 2,3-diphosphoglycerate, it is necessary to add 2,3-DPG or pyridoxal phosphate to the solution to adjust the covalent oxygen affinity of hemoglobin. Conditioner. The final product has good properties as a blood substitute.

The advantages of preparing blood substitutes by coupling hemoglobin and human serum albumin are very obvious. On the one hand, hemoglobin is the main oxygen-carrying protein, and albumin can maintain blood osmotic pressure, transport nutrients, and carry accumulated toxic substances such as bilirubin to have a detoxification function. Therefore, this new blood substitute is different from existing blood substitutes. In comparison, will have a more complete function of the blood. On the other hand, coupling human serum albumin greatly increases the molecular weight of hemoglobin, and the intramolecular cross-linking at the same time can enhance the stability of the tetramer, and HSA is negatively charged in vivo, and it is not easy to pass through the membrane due to the effect of charge, so it is more stable. Further prolong the circulation half-life of the conjugate in vivo. And it is easier to form a cross-linked product with a relatively uniform molecular weight, avoid the aggregation of polymers, and eliminate the side effects of nephrotoxicity. More importantly, unlike serum albumin from other animals, human-derived serum albumin will not produce immunogenicity, and if coupled with animal hemoglobin, it will also greatly weaken or eliminate allogeneic hemoglobin input into humans Immunogenicity caused in vivo. Therefore, the present invention adopts the method of coupling hemoglobin from animals such as humans or pigs, cows, horses, sheep, dogs and human serum albumin to prepare a safe and effective new blood substitute.

Description of drawings

Figure 1A is the electrophoresis scanning pattern of hemoglobin swelling and rupture solution

Figure 1B is the electrophoresis scanning pattern of purified hemoglobin

Figure 2 is the high performance gel filtration liquid chromatogram of purified hemoglobin

The chromatographic column is TSK 3000SW, the detection wavelength is 280nm

Figure 3 is the separation and purification of conjugates by DEAE Sepharose Fast Flow anion exchange method

Figure 4 shows the identification of conjugates by Superdex 200 gel filtration

a is the gel filtration column on the protein peak collected by ion exchange method

b is the gel filtration column on the reaction mixture

Figure 5 is the spectrum of the conjugate identified by SDS-PAGE

1 is the standard molecular weight protein, 2 is the conjugate, 3 is the standard bovine serum albumin, 4 is the purified stroma-free hemoglobin

Figure 6 shows the oxygen-carrying activity of the coupling product

Figure 7 High performance gel filtration chromatogram of coupled products

Figure 8 Survival of rats in hemorrhagic shock

Detailed ways

Embodiment 1: Preparation of pure matrix-free bovine hemoglobin by electrophoresis

Take a certain volume of washed fresh bovine red blood cells and suspend them with twice the volume of cold swelling solution (20 mmol/L KH ₂ PO ₄ /Na ₂ HPO ₄ (PBS) buffer containing 0.6% NaCl, pH 7.4), 4 Shake at ℃ for 1 hour; pump 20 mmol/LPBS buffer solution (pH 7.4) twice the volume of red blood cells at a rate of 10% of the total volume of the solution per minute, adjust the NaCl salt concentration to 0.9% after shaking for 1 hour, and obtain red blood cell swelling and rupture solution . The rupture fluid was pretreated by a Millipore Pellicon cross-flow membrane filtration system. First, use 0.22μm membrane microfiltration to remove cell debris and macromolecular impurities, and then use membrane ultrafiltration with a molecular weight cut-off of 10KD to remove small molecular impurities. The obtained hemoglobin solution was further purified by DEAE Sepharose Fast Flow anion exchange chromatography. Adopt PEG concomitant chromatography, use 20mM phosphate buffer solution containing 5% PEG400, adjust the pH value to 7.4 and carry out chromatography, collect and pass through the hemoglobin peak and detect as a band by SDS-PAGE electrophoresis (Fig. 1A and B). Chromatographic recovery was 97%. The operating temperature was 10°C. The 50% oxygen saturation (P ₅₀ ) of the purified hemoglobin detected by a HEMOX blood gas analyzer was 25.6 mmHg, and the Hill coefficient was 2.41.

Embodiment 2: Preparation of chromatographically pure matrix-free porcine hemoglobin

Take a certain volume of washed fresh porcine red blood cells and suspend them with twice the volume of cold swelling solution (20mmol/L KH ₂ PO ₄ /Na ₂ HPO ₄ (PBS) buffer containing 0.6% NaCl, pH7.4), 4 Shake at ℃ for 1 hour; pump 20 mmol/LPBS buffer solution (pH 7.4) twice the volume of red blood cells at a rate of 10% of the total volume of the solution per minute, adjust the NaCl salt concentration to 0.9% after shaking for 1 hour, and obtain red blood cell swelling and rupture solution . The rupture fluid was pretreated by a Millipore Pellicon cross-flow membrane filtration system. First, use 0.45μm membrane microfiltration to remove cell debris and macromolecular impurities, and then use membrane ultrafiltration with a molecular weight cutoff of 30KD to remove small molecule impurities. The obtained hemoglobin solution was further purified by Q Sepharose BigBeads anion exchange chromatography. The operating temperature was 4°C. Adopt PEG concomitant chromatography, use 20mM phosphate buffer containing 0.5% PEG4000, adopt 10mM phosphate buffer, adjust the pH value to be 7.8 and carry out chromatography, collect the hemoglobin peak that sees through through high-performance gel filtration liquid chromatography (HPLC ) was detected as a single peak (Figure 2), and the recovery of the chromatography was 95%.

Example 3: One-step cross-linking of human hemoglobin and human serum albumin by m-maleimide benzoic acid-N-hydroxysuccinimide ester (MBS)

Since the sulfhydryl group of the HSA molecule will react with MBS, thereby affecting the coupling reaction. Therefore, blocking of remaining sulfhydryl groups is required prior to activation of HSA with MBS. Slowly add 0.2ml of 30mM iodoacetamide to 10ml of 5mg/ml HSA solution (HEPES buffer at pH 7.8), and react at room temperature for 20 minutes. Then add 0.5ml of 30mM MBS solution (dissolved in dimethylformamide), and react at room temperature for 30 minutes. The reaction mixture was passed through a Sephadex G-25 gel filtration column to remove excess MBS and iodoacetamide, and the protein fraction was collected. Because the hemoglobin molecule has a reactive thiol group (the sulfhydryl group of β-93 cysteine), the activated HSA can directly react with the hemoglobin molecule. The collected protein fraction and 10 ml of 20 mg/ml hemoglobin solution were filled with nitrogen gas for 2 hours, mixed and reacted at room temperature for 2 hours under nitrogen protection. Subsequently, 0.2 ml of 30 mM iodoacetamide was added to terminate the coupling reaction.

Example 4: Ethylene glycol diglycidyl ether cross-linked sheep hemoglobin and human serum albumin in two steps

The hemoglobin concentration is 60 mg/ml, the solution system is 50 mM HEPES buffer solution with pH 7.5, the total solution system is 10 ml, the molar ratio of ethylene glycol diglycidyl ether to hemoglobin is 500:1, and the reaction is carried out on a water bath shaker at 37° C. for 10 hours. Remove the modifier by Sephadex G-25 gel filtration, change the buffer solution to 50mM borax-sodium hydroxide buffer solution with pH 9.5, add albumin according to hemoglobin:albumin=3:1 (molar ratio), and bathe at 37°C Shaker reaction for 48 hours. The cross-linked product was detected by SDS-PAGE electrophoresis, and the results showed that 83kD and 97kD bands were generated. The molecular weight of a single hemoglobin subunit is about 16kD, the molecular weight of albumin is 67kD, 83kD is a cross-linked product of a single hemoglobin subunit and serum albumin, and 97kD is a conjugate of two cross-linked hemoglobin subunits and a serum albumin. It shows that the conjugate of hemoglobin and serum albumin is generated.

Example 5: Two-step glutaraldehyde cross-linking of dog hemoglobin and human serum albumin

The concentration of albumin is 1mg/ml, the solution system is 50mM HEPES buffer solution with pH 7.0, the total solution system is 10ml, the molar ratio of glutaraldehyde to albumin is 100:1, react on a water bath shaker at 37°C for 1 hour, pass through Sephadex G-25 gel filtration to remove the modifier, change the buffer to 50mM boric acid-borax buffer at pH 8.5, add hemoglobin according to hemoglobin:albumin = 1:3, and react in a water bath shaker at 37°C for 10 hours. The cross-linked product was detected by SDS-PAGE electrophoresis, and the result showed that an 83kD band was generated. The molecular weight of hemoglobin single subunit is about 16kD, the molecular weight of albumin is 67kD, and 83kD is the cross-linked product of single hemoglobin subunit and serum albumin, indicating that the conjugated product of hemoglobin and serum albumin is generated.

Example 6: Glycolaldehyde solid-phase cross-linked horse hemoglobin and human serum albumin

Equilibrate 5ml of Q Sepharose Fast Flow medium with 50mM HEPES buffer (pH 6.6), and mix with 10ml of 2mg/ml serum albumin solution to make the protein adsorb on the medium. Add glycolaldehyde, the molar ratio of which to protein is 500:1, mix well and let stand at 10°C, and seal the column. After 2 hours, the column was washed with 300ml 50mM HEPES buffer (pH 6.6), and then eluted with 50mM HEPES buffer (pH 6.6) containing 0.5 MNaCl to collect the protein peak. The collection was then applied to a Sephadex G-25 gel filtration column equilibrated with 50mM HEPES buffer (pH 6.6) to remove NaCl in the solution. Collect the protein-containing eluate, concentrate it by ultrafiltration to a protein concentration of 5 mg/ml, add equimolar hemoglobin, and adjust the pH value of the buffer to 9.5, so that the cross-linking agent carried by the activated albumin and hemoglobin continue to react for another 24 hours , to obtain a conjugate of the two proteins.

Example 7: Glutaraldehyde solid-phase cross-linking of bovine hemoglobin and human serum albumin

Balance 5ml of DEAE Sepharose Fast Flow medium with 50mM phosphate buffer (pH 8.0), mix with 10ml of 5mg/ml hemoglobin solution to make the protein adsorb on the medium. Add the cross-linking agent glutaraldehyde, the molar ratio of which to protein is 10:1, mix well and let it stand at 4°C, and seal the column, the intramolecular cross-linking of hemoglobin under the action of glutaraldehyde is carried on at the same time. Crosslinkers with reactive ends. After 0.5 hours, the column was washed with 300ml 50mM phosphate buffer (pH 8.0), then eluted with 50mM HEPES buffer (pH 8.0) containing 0.5M NaCl, and the protein peak was collected. The collection was then applied to a Sephadex G-25 gel filtration column equilibrated with 50mM HEPES buffer (pH 8.0) to remove NaCl in the solution. Collect the protein-containing eluate, concentrate it by ultrafiltration until the protein concentration is 1 mg/ml, add equimolar albumin, and keep the pH value of the buffer at 8.0, so that the glutaraldehyde aldehyde group of activated hemoglobin can continue to react with albumin After 8 hours, a conjugate of the two proteins was obtained.

Embodiment 8: the purification of coupling product

Use 300KD and 100KD ultrafiltration membranes to remove components greater than 300KD and less than 100KD in the polymerization product, and the resulting product is a coupling product with a molecular weight distribution between 100KD and 300KD, that is, 1 to 3 intramolecular cross-linked or non-intramolecular A conjugate of cross-linked hemoglobin and 1 to 3 molecules of human serum albumin.

Embodiment 9: the purification of coupling product

The pH value of the coupled product in Example 6 was adjusted to 7.0 and put on a DEAE Sepharose Fast Flow anion exchange chromatography column, which had been equilibrated with 50mM HEPES buffer (pH7.0) containing 0.1M NaCl. Elute with 25ml of equilibration solution at a flow rate of 0.5ml/min. Then use 55ml of 50mM HEPES buffer (pH 7.0) containing 0.1-0.5M NaCl to elute, and the flow rate is 0.5ml/min. The eluate was detected at 280nm, and the spectrum is shown in Figure 3. The protein peaks containing conjugates were collected, concentrated and then applied to a Superdex 200 gel filtration column. With 50mM HEPES buffer (pH 7.0) as the mobile phase, the flow rate is 0.35ml/min. After elution, two elution peaks were detected at 280 nm, and the conjugate was in the first elution peak (Figure 4), which was collected and further identified by SDS-PAGE gel electrophoresis (Figure 5). The purified conjugate mainly contained two protein bands (lane 2) with molecular weights of approximately 16 kDa and 83 kDa. This is because hemoglobin is a tetrameric protein, which depolymerizes into 16kDa subunits after adding the loading buffer and boiling. After hemoglobin is coupled with HSA, three subunits in the tetrameric protein are dissociated, and the remaining one subunit combines with BSA molecule to form a protein with a molecular weight of 83kDa. Therefore, the protein was identified as a 1:1 binding HSA-hemoglobin conjugate.

Embodiment 10: The preparation and characteristics of the finished blood substitute

The pH of the purified coupling product was adjusted to 7.4. For hemoglobin affected by 2,3-diphosphoglycerate, covalent modulators such as 2,3-DPG or pyridoxal phosphate, which regulate the oxygen affinity of hemoglobin, need to be added to the solution. The P ₅₀ of the purified hemoglobin measured by the HEMOX blood gas analyzer of the coupling product in Example 7 was 26.8 mmHg, and the Hill coefficient was 2.30 ( FIG. 6 ).

The product was detected by high-performance gel filtration liquid chromatography (HPLC) coupled with a multi-angle laser scattering detector (MALLS, DAWN EOS, Wyatt Technology Co., USA), and the molecular weight of the single peak separated by HPLC was identified (Figure 7) , the chromatographic column is TSK 3000SW, and the detection wavelength is 280nm. The results showed that 88.5% were 1:1 conjugates of hemoglobin and human serum albumin with a weight average molecular weight (Mw) of 138kD, 4.3% were 2 to 4 protein conjugates with a weight average molecular weight of 202kD, and unconjugated albumin And hemoglobin accounted for only 4.5% and 0.5% respectively. The product has a single composition, and its colloid osmotic pressure (COP) is measured to be 21.5mmHg, which is close to the normal colloid osmotic pressure of human blood of 25mmHg. Other modified hemoglobin products have complex composition, wide molecular weight distribution, and large colloid osmotic pressure deviation from the normal value. For example, the COP of polymerized hemoglobin is generally below 10mmHg, and the COP of polyethylene glycol (PEG) or polyoxyethylene (POE) modified hemoglobin can reach Above 70mmHg, it will affect the osmotic pressure balance in the body. Element characteristic Element characteristic Hb-HSA 7(g%) ^Cl- 110～125mM Molecular weight of Hb-HSA 100～300kD Na ⁺ 136mM MetHb <3% K ⁺ 3.2～4.2mM P ₅₀ 25～30mmHg Ca++ 2.0mM Hill Coefficient 1.90～2.30 Mg ⁺⁺ 1mM pH 7.4 HCO ₃ ^- 25mM crystal osmotic pressure 300～320mOsm HPO ₄ ^- 0.5mM Virus Negative HPO ₄ ^2- 0.5mM bacteria Negative viscosity 3.0～8.0mPa.s Pyrogen Negative Miscellaneous protein Negative Lipid Negative Glutathione or other sulfhydryl donors Appropriate amount

Example 11: Abnormal Toxicity Test of Products

According to the 2000 edition of "China Biological Products Regulations", the abnormal toxicity of biological products is tested. The experimental animals were 5 ICR mice (18-22 grams) and 2 normal-grade guinea pigs (271.1-277.7 grams). The administration method is intraperitoneal injection, 0.5ml/mouse, 5ml/guinea pig, and observe for 7 days. During the observation period, all the animals survived without any abnormal reaction, and each animal's body weight increased at the end of the period, and the abnormal toxicity test of the product passed.

Example 12: Rescue test on rats with hemorrhagic shock

Anesthetized SD rats were fixed on an alcohol-sterilized operating table, and polyethylene tubes with a diameter of 0.5 mm filled with 0.3% heparin were respectively inserted into the left femoral artery and vein and the right femoral artery, and the right femoral artery was intubated with a multiconductor catheter. The physiological recorder is connected to monitor the blood pressure changes of the rats online. After drawing 30% of the original blood from the femoral artery at a speed of 0.5ml/min, it is stable for 10 minutes. The rat body compensates by itself, and then continues to draw blood to 60 % original blood, stable for 30min. At this time, the blood pressure of the rats dropped to about 25% of the original blood pressure. Then at a speed of 0.5ml/min, inject the same volume of conjugated product, purified hemoglobin solution, HSA solution, original blood, and three times the volume of lactate solution from the left femoral vein to rescue hemorrhagic shock rats. Rats were reared after the surgical site was sutured, and the growth status was observed. If they were still alive after 14 days, they were regarded as alive. There were 6 rats in each group. The results showed that only the rats in the control group that were reinfused with the conjugated product and the original blood survived for 14 days (Figure 8), indicating that this product has a rescue effect similar to that of the original blood on rats with hemorrhagic shock.

Embodiment 13: dog transfusion test

Six Beagle dogs were anesthetized, blood was drawn from the leg arteries with a peristaltic pump, and the conjugated product was infused from the leg veins, and the changes in arterial blood pressure were monitored. After 50% transfusion was monitored for 2 hours, the dog's blood pressure was basically maintained at the original blood pressure level. After suturing the surgical wound, feed and observe the growth status. The dog’s mental state returned to normal 2 days after the operation. After 20 days of feeding, there was no abnormal growth, no hemoglobinuria, and no abnormal blood routine indicators.

Claims (11)

1. A conjugate of hemoglobin, characterized in that the conjugate is a conjugate of hemoglobin and human serum albumin. 2. The hemoglobin conjugate according to claim 1, characterized in that the molecular weight distribution of the conjugate is between 100KD and 300KD. 3. The hemoglobin conjugate according to claim 1, characterized in that the number of hemoglobin molecules in the conjugate molecule is 1-3, and the number of human serum albumin molecules is 1-3. 4. The hemoglobin conjugate according to claim 3, characterized in that the number of hemoglobin molecules in the conjugate is 1-2, and the number of human serum albumin molecules is 1-2. 5. The hemoglobin conjugate according to claim 4, characterized in that the number of hemoglobin is 1, and the number of human serum albumin is 1. 6. The hemoglobin conjugate according to any one of claims 1-4, characterized in that the hemoglobin is intramolecularly cross-linked hemoglobin. 7. A method for preparing a conjugate of hemoglobin according to claim 1, comprising steps of preparation of stroma-free hemoglobin, coupling of hemoglobin and human serum albumin, and purification of the coupling product. 8. The preparation method of the conjugate of hemoglobin according to claim 7, characterized in that the preparation method of stroma-free hemoglobin is to integrate membrane filtration and ion exchange chromatography to purify hemoglobin, and the steps are as follows: 1) Microfiltration with a 0.22-0.65 μm membrane, and the obtained permeate is ultra-filtered with a 10-30KD membrane; 2) The ultra-filtered hemoglobin concentrate is subjected to permeation-type anion-exchange chromatography, the pH value of the chromatography is 6.6-8.5, the buffer concentration is 10mM-50mM, and the PEG concomitant chromatography is used, and the concentration is 0.25 to PEG400-PEG4000 %～10%, the operating temperature is 4～10℃. 9. The preparation method of the conjugate of hemoglobin according to claim 7, characterized in that the coupling of hemoglobin and human serum albumin is a one-step coupling method in which two proteins are directly reacted with a cross-linking agent in a liquid phase , or a two-step coupling method that first modifies one protein and then couples another protein, or adsorbs the protein on a solid phase medium for coupling. 10. The method for preparing the conjugate of hemoglobin according to claim 7, characterized in that the purification method of the coupling product is selected from one or both of ion exchange chromatography, ultrafiltration and gel filtration chromatography purification methods. one or three methods. 11. A use of the hemoglobin conjugate according to claim 1 in the preparation of blood substitutes.

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