A method of protein egg phosphatidyl modification

Abstract

The application belongs to the field of protein medicine bioengineering and technology, and discloses a protein lecithinization modification method. The method comprises the following steps: mixing a buffer system containing inorganic salt, a modified protein and an organic solution of phosphatidylcholine active ester at 4 DEG C to 30 DEG C, and standing for modification for 0.1 h to 96 h. The protein lecithinization modification method can improve the lecithinization modification efficiency of the protein, and improve the yield and purity of the lecithinized protein.

Description

Technical Field

[0001] This invention belongs to the field of protein pharmaceutical bioengineering and technology, and specifically relates to a method for protein lecithinization modification. Background Technology

[0002] The development of modern biotechnology has made the large-scale production of protein drugs a reality, and the number of these drugs used in clinical practice is increasing. Protein drugs include peptides and genetically engineered drugs, monoclonal antibodies and genetically engineered antibodies, and recombinant vaccines. Compared with traditional small-molecule drugs, protein drugs have the characteristics of high activity, strong specificity, low toxicity, well-defined biological functions, and are beneficial for clinical application. With the development of protein chemistry and molecular biology, the research and application of protein drugs for the treatment of various diseases has become a hot topic in the development of the biopharmaceutical industry. Peptides and protein drugs have few side effects, strong activity, and the ability to treat both the symptoms and the root cause. Moreover, due to their low cost, high success rate, and safety, they have become an important part of pharmaceutical products.

[0003] Precursor proteins expressed by different host cells or bacteria through gene recombination technology possess only complete amino acid sequences and are inactive. They often require a series of post-translational modifications to become functional, mature proteins. These modifications and processes are diverse and numerous, including but not limited to glycosylation, phosphorylation, phospholipidation, ubiquitination, acetylation, and methylation. Phospholipidation generally refers to the process by which phosphatidylcholine (lecithin, abbreviated as PC, with higher grades being PPC) is combined with drugs through processing to form passively targeted drugs; this is also known as protein lecithinization. Protein lecithinization can also improve the stability of protein drugs.

[0004] Currently, there are few reported methods for protein lecithinization in the literature. For example, Japanese Patent Publication No. CN101035891 discloses a lecithinized superoxide dismutase composition and its preparation method. However, the yield and purity of this method are both low. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to address the deficiencies of the prior art by providing a method for protein lecithinization modification, so as to improve the yield and purity of lecithinized proteins.

[0006] To achieve the objectives of this invention, the following technical solution is adopted:

[0007] A method for protein lecithinization modification involves mixing an inorganic salt-containing buffer system, the protein to be modified, and an organic solution of phosphatidylcholine active ester at 4℃-30℃, and allowing the mixture to stand for 0.1h-96h.

[0008] The organic solution of the phosphatidylcholine active ester is a solution obtained by dissolving the phosphatidylcholine active ester in an organic solvent.

[0009] Preferably, the buffer system containing inorganic salts is selected from phosphate buffer (PB), borate buffer, sodium citrate buffer, sodium acetate buffer, and Tris hydrochloric acid buffer, with a pH value of 6-9. In some embodiments, the buffer system is a Tris hydrochloric acid buffer system.

[0010] The inorganic salt in the buffer system containing inorganic salt is selected from potassium chloride, sodium chloride, ammonium sulfate, and sodium sulfate, with a concentration of 5–100 mM. In some embodiments, the inorganic salt is 20 mM sodium chloride.

[0011] In the method of this invention, the organic solvent for dissolving the phosphatidylcholine active ester is selected from isopropanol, acetone, methanol, ethanol, n-butanol, N,N-dimethylacetamide, sulfolane, dimethyl sulfoxide, and polyethylene glycol. The concentration of the organic solvent is preferably 10%-99.99%, more preferably 30%-99%. In some embodiments, the organic solvent for dissolving the phosphatidylcholine active ester is 98% ethanol; in some embodiments, the organic solvent for dissolving the phosphatidylcholine active ester is 99% ethanol.

[0012] In the method of the present invention, the mass ratio of the phosphatidylcholine active ester to the modified protein is preferably (0.12-2.0):1, more preferably 0.16±0.02:1. In some embodiments, the mass ratio of the phosphatidylcholine active ester to the modified protein is 0.16:1.

[0013] The mass ratio of the phosphatidylcholine active ester to the modified protein, calculated in g / L, is (0.12–2.0):1; the volume ratio of the buffer system containing inorganic salts to the organic solvent dissolving the phosphatidylcholine active ester, calculated in g / L, is (0.75–0.1):(0.1–0.75).

[0014] The method described in this invention can modify proteins containing free primary amine groups with lecithin, such as superoxide dismutase, bovine serum albumin, hemocyanin, immunoglobulins (e.g., IgG, IgM, IgA), idursulfase, imiglucerase, α-galactosidase, α-glucosidase alfa, insulin and its variants, and coagulation factors (F VII, F VIII, etc.) with lecithinization.

[0015] The modification temperature is preferably 4℃-30℃, more preferably 15℃-25℃. The modification time is preferably 0.1h-96h, more preferably 2h-36h.

[0016] Preferably, the mixing step in the method of the present invention is performed in one of the following orders:

[0017] (1) Add the buffer system containing inorganic salts, the organic solution of phosphatidylcholine active ester, and the modified protein in that order and mix well.

[0018] (2) Add the modified protein, the buffer system containing inorganic salts, and the organic solution of phosphatidylcholine active ester in that order and mix well.

[0019] (3) Add the organic solution of phosphatidylcholine active ester, the buffer system containing inorganic salts, and the modified protein in that order and mix well.

[0020] (4) Add the buffer system containing inorganic salts, the modified protein, and the organic solution of phosphatidylcholine active ester in sequence and mix well.

[0021] More preferably, the buffer system containing inorganic salts, the modified protein, and the organic solution of phosphatidylcholine active ester are added in sequence and then mixed.

[0022] In some embodiments, the mixing specifically involves mixing the buffer system containing inorganic salts with the modified protein at a stirring speed of 30 rpm to 600 rpm until homogeneous, then adding an organic solution of phosphatidylcholine active ester at a rate of 10 ml / min to 120 ml / min, and finally mixing at a stirring speed of 30 rpm to 600 rpm until homogeneous after complete addition.

[0023] Preferably, the stirring speed is 50 rpm to 200 rpm. In some embodiments, the stirring speed is 70 rpm ± 10 rpm.

[0024] Preferably, the flow rate of the organic solution of the phosphatidylcholine active ester is 20 ml to 100 ml / min. More preferably, it is 40 ml to 80 ml / min. In some embodiments, the flow rate is 60 ml / min.

[0025] Furthermore, in some embodiments, the protein lecithinization modification method of the present invention further includes the steps of ion exchange column chromatography purification and ultrafiltration to replace the organic solvent with an inorganic salt buffer system.

[0026] The chromatographic column packing material used for ion exchange column purification is either anion exchange packing material or cation exchange packing material.

[0027] Preferably, the chromatographic column packing material used for ion exchange column purification is anion exchange packing material. In some embodiments, the packing material is Cellulofinesf A-500 packing material.

[0028] In some embodiments, the purification specifically involves loading the modified protein solution into a chromatography column, first separating the unmodified SOD using 20% ​​mobile phase B, and then eluting and collecting the PC-SOD using 100% mobile phase B.

[0029] The mobile phase A consists of a buffer solution containing 5-100 mM inorganic salts and ethanol, with the ethanol content being 30%-70% and the buffer solution pH being 6-9. The mobile phase B consists of a buffer solution containing 100-400 mM inorganic salts and ethanol, with the ethanol content being 30%-70% and the buffer solution pH being 6-9.

[0030] In some embodiments, mobile phase A is a 40mM Tris buffer containing 20mM NaCl, with a pH of 8.0 and an ethanol content of 60%, i.e., the volume ratio of the NaCl-containing Tris buffer to ethanol is 40%:60%; mobile phase B is a 40mM Tris buffer containing 200mM NaCl, with a pH of 8.0 and an ethanol content of 60%, i.e., the volume ratio of the NaCl-containing Tris buffer to ethanol is 40%:60%.

[0031] In some embodiments, the purification process of the present invention first uses mobile phase A to pre-equilibrate the chromatography column packed with anion exchange packing material (Cellulofinesf A-500), then loads the prepared lecithin-modified protein onto the column. After all the protein has been loaded, it is re-equilibrated using mobile phase A. Then, 20% mobile phase B is used to separate the unmodified protein, and then 100% mobile phase B is used to elute the lecithin-modified protein.

[0032] The method of the present invention obtains purified lecithin-modified protein by ultrafiltration of an inorganic salt-containing buffer system after purification to replace the organic solvent.

[0033] Preferably, hollow fiber ultrafiltration or tangential flow membrane ultrafiltration is used for replacement.

[0034] In the above ultrafiltration step, the inorganic salt concentration in the buffer system containing inorganic salts is preferably 5–100 mM, and the pH of the buffer system is preferably 6–9. Those skilled in the art will understand that the inorganic salts and the buffer system in the buffer system containing inorganic salts are the same as those used in the lecithinization modification. The buffer system can be selected from phosphate buffer (PB), borate buffer, sodium citrate buffer, sodium acetate buffer, and Tris hydrochloric acid buffer, with a pH of 6–9; the inorganic salts can be selected from potassium chloride, sodium chloride, ammonium sulfate, and sodium sulfate, with a concentration of 5–100 mM.

[0035] In some embodiments, the buffer system in the ultrafiltration step is a Tris buffer system, and the inorganic salt is 20 mM sodium chloride.

[0036] As can be seen from the above technical solution, the present invention provides a method for protein lecithinization modification, wherein a buffer system containing inorganic salts, the protein to be modified, and an organic solution of phosphatidylcholine active ester are mixed at 4℃-30℃ and allowed to stand for modification for 0.1h-96h. The protein lecithinization modification method of the present invention can improve the efficiency of protein lecithinization modification, while providing the yield and purity of the modified lecithinized protein. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0038] Figure 1 The RP-HPLC results of Example 1 are shown after modification according to the modification method of the patent with publication number CN101035891;

[0039] Figure 2 Example 1 shows the RP-HPLC results after modification according to the modification method of the present invention;

[0040] Figure 3 Example 2 shows the RP-HPLC purity results of the patent with publication number CN101035891;

[0041] Figure 4 Example 2 shows the RP-HPLC purity results of the present invention;

[0042] Figure 5 Example 6 shows the RP-HPLC chromatogram of BSA reference standard;

[0043] Figure 6 Example 6 shows the PC-BSA RP-HPLC chromatogram. Detailed Implementation

[0044] This invention discloses a method for protein lecithinization modification. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and product of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0045] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0047] Example 1: PC-modified SOD

[0048] (1) Input ratio: SOD protein content: phosphatidylcholine active ester (APC): buffer system containing inorganic salts: organic phase solution = 1g: 0.16g: 0.1L: 0.1L

[0049] (2) Take 800ml of SOD stock solution with a concentration of 75mg / ml, and the total protein content is 60g.

[0050] (3) Preparation of buffer system containing inorganic salts: Prepare 6L of 40mM-Tris and 20mM-NaCl, and adjust the pH to 8.0±2.0 with hydrochloric acid.

[0051] (4) Preparation of organic phase solution: Weigh 9.6g APC and dissolve it in 6L 98% ethanol.

[0052] (5) At room temperature, first add 5.2 L of buffer system containing inorganic salts to the reactor, then pour 800 ml of SOD stock solution into the reactor, and start stirring at 70 rpm ± 10 rpm to mix. Finally, add the organic phase solution to the reactor at a rate of 60 ml / min. After the organic phase solution is completely added, stir at 70 rpm ± 10 rpm for 30 min, and then let it stand for more than 2 h to obtain the modified PC-SOD.

[0053] (6) The PC-SOD obtained above was determined by high performance liquid chromatography (RP-HPLC) purity determination method and compared with the PC-SOD obtained by patent publication number CN101035891. The specific RP-HPLC determination method is as follows:

[0054] Preparation of test solution: The PC-SOD sample was diluted with ultrapure water to 5 mg / ml, and the SOD physicochemical reference standard was diluted to 1 mg / ml. Both were then analyzed by HPLC.

[0055] Chromatographic conditions: Measurement wavelength: 220 nm

[0056] Column: TOSOH TSKgel Phenyl-5PW 4.6mm ID × 75mm

[0057] Column temperature: room temperature

[0058] Flow rate: 0.8 ml / min

[0059] Injection volume: 10 μl

[0060] Mobile phase A: 0.1% TFA - 20% acetonitrile

[0061] Mobile phase B: 0.075% TFA - 90% acetonitrile

[0062] Elution gradient:

[0063]

[0064]

[0065] The PC-SOD content is calculated using the following formula based on the total area (St) excluding the solvent peak and the PC-SOD peak area (Si).

[0066] PC-SOD

[0067] Figure 1 and Figure 2 The results, as shown by the RP-HPLC chromatogram, indicate that after modification using the above method, the content of unmodified SOD is 19% lower than that of the method in patent publication number CN101035891. This means that the SOD purity obtained by correctly modifying with PC is 19% higher, which also reduces the pressure on subsequent purification processes and facilitates the subsequent purification process. Specific data are shown in Table 1 below:

[0068] Table 1. Comparison of HPLC-RP purity data

[0069]

[0070] Example 2: Purification of modified PC-SOD

[0071] (1) Preparation of purification mobile phase A: Prepare 40mM-Tris and 20mM-NaCl, adjust the pH to 8.0±0.2 with hydrochloric acid, and add ethanol to make the ratio of inorganic salt buffer to ethanol 40%:60%.

[0072] (2) Preparation of purification mobile phase B: Prepare 40mM-Tris and 200mM-NaCl, adjust the pH to 8.0±0.2 with hydrochloric acid, and add ethanol to make the ratio of inorganic salt buffer to ethanol 40%:60%.

[0073] (3) The chromatography column packed with anion exchange packing material (Cellulofinesf A-500) was pre-equilibrated for 3CV using mobile phase A, and then the PC-SOD obtained in Example 1 was loaded onto the column. After all the columns were loaded, the column was re-equilibrated for 3CV using mobile phase A.

[0074] (4) Use 20% mobile phase B to separate the unmodified SOD first, and then use 100% mobile phase B to elute the PC-SOD. Finally, use a tangential flow membrane to perform ultrafiltration to replace the buffer in 10mM Tris buffer to obtain the purified PC-SOD.

[0075] (5) The PC-SOD of the purified PC-SOD was determined by the high performance liquid chromatography (RP-HPLC) method of Example 1 and compared with the PC-SOD obtained by the patent with publication number CN101035891.

[0076] Figure 3 and Figure 4 The results, as shown by the RP-HPLC chromatogram, indicate that the purity of PC-SOD purified according to the method of this invention is approximately 13% higher than that obtained by the method in patent publication number CN101035891. Specific data are shown in Table 2 below:

[0077] Table 2 Comparison of HPLC-RP purity data

[0078]

[0079] (6) Comparison of recovery rates

[0080] The PC-SOD recovery rate was statistically analyzed, and the results are shown in Table 3.

[0081] Table 3 Comparison of Recovery Rates

[0082]

[0083] As can be seen from Table 3, the PC-SOD recovery rate obtained by the modification and purification method of the present invention is about 22% higher than that obtained by the method in the patent with publication number CN101035891.

[0084] (7) Comparison of PC-SOD protease activities

[0085] The activity of PC-SOD obtained by the method in patent CN101035891 and the method in this invention was determined using the non-protease activity assay (NBT method). The specific assay method is as follows:

[0086] The activities of SOD and PC-SOD were detected using the NBT activity assay kit from Wako Pure Chemical Industries, Japan. The experimental principle is the classic nitroblue tetrazolium (NBT) colorimetric method, which detects enzyme activity based on the different superoxide anion scavenging abilities of SOD and PC-SOD at different concentrations.

[0087] Reagent: SOD activity assay kit (Wako Pure Chemical Industries, Japan)

[0088] Preparation of standard solution: Dissolve one vial of SOD active reference standard in 1 ml of ultrapure water to prepare a standard solution containing an SOD concentration of 0.5 mg / ml. Perform a 3-fold serial dilution in 1.5 ml Eppendorf tubes, for a total of 8 dilutions, with 2-3 replicates for each dilution.

[0089] Preparation of test solution: The PC-SOD stock solution was serially diluted 3-fold starting from 1.0 mg / ml with ultrapure water, resulting in a total of 8 dilutions. Each dilution was replicated 2-3 times.

[0090] Assay: Take one bottle of enzyme stock solution from the SOD activity assay kit and mix it with one bottle of enzyme diluent to prepare the enzyme solution. Use within two weeks of preparation. Place the 96-well plate on a cold plate and add the sample and reagent solution to each well of the 96-well plate in the order shown in 1-7 below.

[0091]

[0092] The test samples can be standard solutions or individual samples; staining solutions, enzyme solutions, and stop solutions are provided with the kit. The stop solution is prone to solidification and should be placed in a 37°C water bath until completely dissolved before use. To ensure the stability of the enzyme reaction, the 96-well plate should be placed on a frozen cold plate to cool the bottom of the plate during operation. Each sample should be tested in triplicate. To ensure parallelism, all solutions except the sample can be added using a multichannel pipette.

[0093] Experimental data were processed using a computer program or a four-parameter regression method, and the results were calculated using the following formula:

[0094] Protein activity of the test sample

[0095] Protein specific activity of the test sample

[0096] In the formula, Pr represents the reference activity, U / ml;

[0097] Ds is the pre-dilution factor of the test sample;

[0098] Dr represents the pre-dilution factor of the reference standard;

[0099] Es is the dilution factor of the test sample relative to the half-effective dose of the reference sample;

[0100] Er is the dilution factor of the half-effective dose of the reference standard.

[0101] The results showed that the PC-SOD prepared according to the modification and purification method of this invention had a specific activity approximately 1000 U / mg higher than that prepared by the method in patent publication number CN101035891. Specific data are shown in Table 4 below.

[0102] Table 4 Specific Activity Results

[0103] Curve Name Well ID Y Formula Y X Specific activity U / ml Specific activity U / mg SOD activity control sample SPL1 0.171 0.171 45.679 3000 6000 PC-SOD in this invention SPL2 0.171 0.171 46.974 154253 4059 PC-SOD of the patented method SPL3 0.171 0.171 33.104 108706 3106

[0104] Example 3

[0105] The same procedures as in Examples 1 and 2 were used to prepare PC-SOD twice more. The results of the three preparation processes (purity of PC-SOD after PCification, purity of PC-SOD after purification, recovery rate, and specific activity) are summarized in the table below:

[0106] Table 5 Summary of Data

[0107] name first The second The third PC-SOD purity after PCification 90.5％ 91.10％ 90.20％ Purity of PC-SOD after purification 99.1％ 99.30％ 98.90％ Specific activity 4059U / mg 4013U / mg 4027U / mg Recovery rate 77％ 78％ 80％

[0108] Example 4: Comparison of results after modification with different APC ratios

[0109] Five different APC input ratios were used to prepare PC-SOD according to the operating methods in Examples 1 and 2.

[0110] Input ratio: SOD protein content: phosphatidylcholine active ester (APC): buffer system containing inorganic salts: organic phase solution

[0111] Input ratio ① = 1g : 0.12g : 0.1L : 0.1L

[0112] Input ratio ② = 1g : 0.14g : 0.1L : 0.1L

[0113] Input ratio ③ = 1g : 0.16g : 0.1L : 0.1L (Examples 1 and 2)

[0114] Input ratio ④ = 1g : 0.18g : 0.1L : 0.1L

[0115] Input ratio ⑤ = 1g : 0.20g : 0.1L : 0.1L

[0116] The results of the five preparation processes (PC-SOD purity after PCification, PC-SOD purity after purification, recovery rate, and specific activity) are summarized in the table below:

[0117] Table 6 Summary of Data

[0118] name Investment ratio ① Investment ratio ② Investment ratio ③ Investment ratio ④ Investment ratio ⑤ PC-SOD purity after PCification 81.7％ 87.3％ 90.5％ 90.3％ 91.3％ Purity of PC-SOD after purification 98.1％ 98.5％ 99.1％ 99.0％ 98.9％ Specific activity 3984U / mg 4002U / mg 4059U / mg 4021U / mg 4039U / mg Recovery rate 65％ 71％ 77％ 75％ 72％

[0119] The results showed that in ratio ①, the amount of APC used was relatively small, and after PCification, a large amount of SOD remained unmodified, resulting in a relatively low final yield. In ratio ⑤, the amount of APC used was relatively large, and after PCification, there was a greater amount of highly modified PC-SOD, which increased the hydrophobicity of the protein and affected the yield of subsequent purification. Ratio ③, the ratio used in this invention, showed the best overall results.

[0120] Example 5: Comparison of results from modifications using different ratios of inorganic and organic solutions.

[0121] PC-SOD was prepared using three different ratios of inorganic and organic solutions, following the procedures described in Examples 1 and 2.

[0122] Input ratio: SOD protein content: phosphatidylcholine active ester (APC): buffer system containing inorganic salts: organic phase solution

[0123] Input ratio ① = 1g : 0.16g : 0.75L : 0.1L

[0124] Input ratio ② = 1g : 0.16g : 0.1L : 0.1L (Examples 1 and 2)

[0125] Input ratio ③ = 1g : 0.16g : 0.1L : 0.75L

[0126] The results of the three preparation processes (purity of PC-SOD after PCification, purity of PC-SOD after purification, recovery rate, and specific activity) are summarized in Table 7 below.

[0127] Table 7 Summary of Data

[0128] name Investment ratio ① Investment ratio ② Investment ratio ③ PC-SOD purity after PCification 89.1％ 90.5％ 91.1％ Purity of PC-SOD after purification 98.1％ 99.1％ 99.3％ Specific activity 4042U / mg 4059U / mg 4018U / mg Recovery rate 74％ 77％ 69％

[0129] The results showed that ratio ③ had a relatively high proportion of organic phase. In the later stages of PC modification, this relatively high proportion of organic phase caused a small amount of protein precipitation, ultimately affecting the yield. Ratio ②, the ratio used in this invention, showed better overall results.

[0130] Example 6: PC-modified bovine serum albumin

[0131] (1) Input ratio: Bovine serum albumin (BSA): Phosphatidylcholine active ester (APC): Buffer system containing inorganic salts: Organic phase solution = 1g: 0.16g: 0.1L: 0.1L

[0132] (2) Preparation of buffer system containing inorganic salts: Prepare 1L of 40mM-Tris and 20mM-Nacl, and adjust the pH to 8.0±2.0 with hydrochloric acid.

[0133] (3) Preparation of organic phase solution: Weigh 1.6g APC and dissolve it in 1L 99% ethanol.

[0134] (4) Weigh 10g of BSA and dissolve it in 200mL of a buffer system containing inorganic salts to a concentration of 50mg / mL.

[0135] (5) At room temperature, first add 800 mL of buffer system containing inorganic salts to the reactor, then pour 200 mL of BSA into the reactor, and start stirring at 70 rpm ± 10 rpm to mix. Finally, add the organic phase solution to the reactor at a rate of 60 mL / min. After the organic phase solution is completely added, stir the reaction at 70 rpm ± 10 rpm for 30 min, and then let it stand for more than 2 h to obtain the modified PC-BSA.

[0136] (6) The PC-SOD and BSA reference standards obtained above were determined according to the high performance liquid chromatography (RP-HPLC) purity determination method described in Example 1. The RP-HPLC purity results are shown in [Figure 1]. Figure 5 and 6 Tables 8 and 9.

[0137] Table 8. RP-HPLC data for BSA reference standard

[0138] Peak Retention time area high area% 1 9.149 16031177 585820 100 total 16031177 585820 100

[0139] Table 9 PC-BSA RP-HPLC Data

[0140] Peak Retention time area high area% 1 9.623 85280 1392 0.417 2 12.332 263150 2688 1.286 3 16.875 20067690 342385 98.057 4 23.264 49306 797 0.241 total 20465427 347262 100

[0141] (7) SDS-PAGE electrophoresis purity results

[0142] The purity calculation results based on the integration of SDS-PAGE electrophoresis bands are shown in Table 10.

[0143] Table 10 SDS-PAGE Results

[0144] name BSA PC-BSA Molecular weight (KD) 54.949 71.065 purity 100％ 100％

[0145] The results show that the modification method in this invention can modify BSA with PC, and the purity of the modified PC-BSA is close to 100%.

[0146] Example 7

[0147] Referring to the method in Example 6, hemocyanin, immunoglobulin IgG, iduxitase, imiglucerase, α-galactosidase, α-glucosidase, insulin, and coagulation factor FVII were subjected to PC modification, respectively, and the dosage ratios were as follows:

[0148] The ratio of input to output is: hemocyanin: phosphatidylcholine active ester (APC): buffer system containing inorganic salts: organic phase solution = 1g: 0.15g: 0.1L: 0.1L;

[0149] The ratio of input is: Immunoglobulin IgG: Phosphatidylcholine active ester (APC): Buffer system containing inorganic salts: Organic phase solution = 1g: 0.17g: 0.1L: 0.1L;

[0150] The ratio of input to output is: Idusulfatase: Phosphatidylcholine active ester (APC): Buffer system containing inorganic salts: Organic phase solution = 1g: 0.15g: 0.1L: 0.8L;

[0151] The ratio of input to output is: imiglucerase: phosphatidylcholine active ester (APC): buffer system containing inorganic salts: organic phase solution = 1g: 0.16g: 0.75L: 0.1L;

[0152] The ratio of input to output is: α-galactosidase: phosphatidylcholine active ester (APC): buffer system containing inorganic salts: organic phase solution = 1g: 0.18g: 0.1L: 0.9L;

[0153] The ratio of input to output is: α-glucosidase: phosphatidylcholine active ester (APC): buffer system containing inorganic salts: organic phase solution = 1g: 0.16g: 0.1L: 0.1L;

[0154] The ratio of insulin to active phosphatidylcholine ester (APC) to a buffer system containing inorganic salts to an organic phase solution is 1 g : 0.18 g : 0.1 L : 0.9 L.

[0155] The ratio of coagulation factor F VII to active phosphatidylcholine ester (APC) is: 1 g : 0.16 g : 0.85 L : 0.1 L;

[0156] The purity of the PC-based product obtained above was determined by high performance liquid chromatography (RP-HPLC) as described in Example 1. The RP-HPLC purity results are shown in Table 11.

[0157] Table 11 RP-HPLC Structure of PC-based Products

[0158] PC-based products Main Peak Retention Time area% PC-hemocyanin 17.257 95.178 PC-immunoglobulin IgG 16.598 94.268 PC-idosasulfatase 15.349 96.024 PC-imiglucosidase 13.298 93.142 PC-α-galactosidase 15.689 92.578 PC-α-glucosidase 16.345 96.586 PC-insulin 15.239 90.256 PC - Coagulation Factor F VII 16.213 90.249

[0159] As shown in the table above, the PC modification method described in this invention can be used to modify the relevant proteins to obtain high-purity PC-modified proteins. Furthermore, the purity of the PC-modified proteins can be calculated by integrating the SDS-PAGE electrophoresis bands, and the results show that the purity of the PC-modified proteins is close to 100%.

Claims

1. A method for superoxide dismutase lecithinization modification, characterized in that, The inorganic salt-containing buffer system, superoxide dismutase, and organic solution containing phosphatidylcholine active ester were mixed at room temperature. Then, the inorganic salt-containing buffer system, superoxide dismutase, and organic solution containing phosphatidylcholine active ester were added sequentially and mixed thoroughly. The mixture was allowed to stand for 2-36 hours for modification. The mass ratio of the phosphatidylcholine active ester to the superoxide dismutase was 0.16:

1. The volume ratio of the inorganic salt-containing buffer system to the organic solvent dissolving the phosphatidylcholine active ester was 0.1:0.1 (g / L). The superoxide dismutase was a protein containing free primary amine groups. The buffer system containing inorganic salts is a Tris hydrochloric acid buffer system with a pH of 8.0 ± 0.2; the inorganic salt is selected from sodium chloride with a concentration of 20 mM; and the organic solvent for dissolving the phosphatidylcholine active ester is 98% or 99% ethanol. After the modification is completed, the process also includes the steps of ion exchange column chromatography purification and ultrafiltration to replace the organic solvent with a buffer system containing inorganic salts; The ion exchange column used for purification is anion exchange packing material. Specifically, the purification involves loading the modified protein solution into the column, performing initial washing with mobile phase A, followed by elution and collection with mobile phase B. Mobile phase A consists of 40mM Tris buffer containing 20mM NaCl and ethanol, with an ethanol content of 60% and a buffer pH of 8.0±0.

2. Mobile phase B consists of 40mM Tris buffer containing 200mM NaCl and ethanol, with an ethanol content of 60% and a buffer pH of 8.0±0.

2. In the step of replacing the organic solvent with an inorganic salt-containing buffer system, the replacement method is tangential flow membrane ultrafiltration, and the inorganic salt-containing buffer system is a 5-10 mM Tris buffer solution with a pH of 6-9.

2. The method according to claim 1, characterized in that, The mixing process specifically involves mixing an inorganic salt-containing buffer system with superoxide dismutase and stirring at a speed of 30 rpm to 600 rpm until homogeneous. Then, an organic solution containing phosphatidylcholine active ester is added at a speed of 10 ml / min to 120 ml / min. After complete addition, the mixture is stirred at a speed of 30 rpm to 600 rpm until homogeneous.

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