METHOD FOR SEPARATING AND PURIFYING ALPHA2-MACROGLOBULIN FROM COHN FRACTION IV PRECIPITATION

Abstract

The present invention discloses a method for separating and purifying 2-macroglobulin from Cohn Fraction IV precipitation, in which Cohn Fraction IV precipitation is treated by ammonium sulfate precipitation, zinc ion affinity chromatography, gel filtration, and ultrafiltration and concentration sequentially and thereby 2-macroglobulin is obtained finally. With the method provided in the present invention, purified 2-macroglobulin plasma protein that has a clinical application value is obtained, the Cohn Fraction IV precipitation is changed from a discarded material into a valuable material, and plasma is utilized comprehensively. In addition, the method is easy and simple to use, easy to scale up, and suitable for separation and purification of 2-macroglobulin at a large scale.

Claims

1. A method for separating and purifying 2-macroglobulin from a Cohn Fraction IV precipitation, subjecting the Cohn Fraction IV precipitation to ammonium sulfate precipitation, zinc ion affinity chromatography, gel filtration, and ultrafiltration and concentration sequentially, thereby obtaining 2-macroglobulin, wherein the ultrafiltration and concentration step utilizes an ultrafiltration membrane having a molecular weight cutoff within a range of 30-500 kD.

2. The method according to claim 1, wherein, in the ammonium sulfate precipitation, the saturation of the ammonium sulfate is 40-60%, preferably is 45%-55%.

3. The method according to claim 1, wherein, the ammonium sulfate precipitation is a single precipitation, wherein after ammonium sulfate is added, the mixture is stirred for 1 h and then centrifuged for 15 min.

4. The method according to claim 1, wherein, in the zinc ion affinity chromatography, the purification medium is zinc ion chelated high flow-rate agarose medium (Zn-IDA QZT 6FF).

5. The method according to claim 1, wherein, in the zinc ion affinity chromatography, the eluent may be one of 0.1M Na.sub.2EDTA with pH=6.5-7.0, 20 mM Tris+0.1M NaCl+0.1-0.5M imidazole with pH=7.4, 0.02M Tris+0.5M NaCl at pH=4.5-5.0, and 20 mM Tris+0.1M NaCl+0.01-0.05M histidine with pH=7.4.

6. The method according to claim 1, wherein, in the gel filtration, the filtering medium may be Polyacrylamide-dextran gel Sephacryl-200 HR, Polyacrylamide-dextran gel Sephacryl-300 HR, agarose gel (Superose prep grade or Superose 12), or cross-link dextran (G-200 Sephadex G-200).

7. The method according to claim 1, wherein, in the ultrafiltration and concentration, the ultrafiltration membrane has molecular weight cutoff within a range of 50-100 kD.

8. The method according to claim 1, comprising the following steps: a) ammonium sulfate precipitation which comprises: dissolving Cohn Fraction IV precipitation in distilled water in volume equal to 8-12 times of the volume of the Cohn Fraction IV precipitation, obtaining filtrate through compression filtration, adding ammonium sulfate to the filtrate till the saturation is 40%-60%, centrifuging the resultant mixture to obtain precipitate and supernatant, and then discarding the supernatant; b) zinc ion affinity chromatography which comprises: dissolving the precipitate obtained in the step a) in equilibrium buffer to obtain a solution of dissolved precipitate, treating the solution by zinc ion affinity chromatography, stopping elution when a protein peak drops to the baseline, and then collecting eluent produced through affinity chromatography; wherein, zinc ion chelated high flow-rate agarose medium (Zn-IDA QZT 6FF) is selected as the purification medium, and preferably the volume of the equilibrium buffer used to dissolve the precipitate is of the volume of the supernatant in the step a); c) gel filtration which comprises: filtering the eluent obtained through affinity chromatography in the step b) by gel filtration, and collecting eluent with the first protein peak after gel filtration; d) ultrafiltration and concentration which comprises: concentrating the protein concentration in the eluent obtained through gel filtration in the step c) to 4.5-5.5%, and then using a 0.22 m filter to carry out sterile filtration, so as to obtain the target protein; preferably, the equilibrium buffer used to dissolve the precipitate in the step b) is buffer solution 0.02M Tris+0.5M NaCl with pH=6.0-7.0; the eluent in the step b) is 0.1M Na.sub.2EDTA with pH=6.5-7.0.

9. The method according to claim 8, wherein, the zinc ion affinity chromatography in the step b) mainly comprises the following steps: i) column loading: loading the purification medium into a chromatography column; ii) purification medium buffering: buffering the purification medium with the equilibrium buffer; iii) sample loading: pumping the solution of dissolved precipitate into the chromatography column; iv) rinsing: leaching off the protein that is not coupled to the purification medium with the equilibrium buffer; v) elution: eluting off the proteins coupled to the purification medium with the eluent; the gel filtration in the step c) mainly comprises the following steps: i) column loading: loading the filtering medium into a chromatography column; ii) filtering medium buffering: buffering the filtering medium with the equilibrium buffer; iii) sample loading: pumping the eluent obtained through affinity chromatography in the step b) into the chromatography column; iv) leaching: leaching off the target protein 2-macroglobulin with the equilibrium buffer; preferably, the equilibrium buffer used in the purification medium buffering and leaching in the step b) and the equilibrium buffer used in the filtering medium buffering and leaching in the step c) are buffer solution 0.02M Tris+0.5M NaCl with pH=6.0-7.0.

10. The method according to claim 8, wherein, before the step b) is executed, the precipitate obtained in the step a) is dissolved in a dialysis solution for desalination by ultrafiltration, wherein, the dialysis solution is buffer solution 0.02M Tris+0.5M NaCl with pH=6.0-7.0; preferably, the ultrafiltration membrane used in the desalination by ultrafiltration has molecular weight cutoff within a range of 30-500 kD, preferably is an ultrafiltration membrane that has 50-100 kD pore size.

11. 2-macroglobulin obtained by separation and purification from Cohn Fraction IV precipitation with the method according to claim 1, at purity not lower than 94%, wherein, the recovery rate of the method is not lower than 45%.

12. 2-macroglobulin obtained by separation and purification from Cohn Fraction IV precipitation, wherein, the separation and purification are executed with the method according to claim 1.

Description

DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a chromatogram of the 2-M obtained by separation and purification through zinc ion affinity chromatography;

[0029] FIG. 2 is a chromatogram of the 2-M obtained by separation and purification through gel filtration;

[0030] FIG. 3 is a non-reduced electrophoregram of the 2-M obtained in SDS-PAGE identification.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The method for separating and purifying 2-macroglobulin from Cohn Fraction IV precipitation provided in the present invention employs a process in which Cohn Fraction IV precipitation is treated by ammonium sulfate precipitation, zinc ion affinity chromatography, gel filtration, and ultrafiltration and concentration sequentially and thereby 2-macroglobulin is obtained finally.

[0032] Specifically, the method comprises the following steps: [0033] a) ammonium sulfate precipitation: dissolving Cohn Fraction IV precipitation in distilled water (the mass-to-volume ratio (g:mL) of Cohn Fraction IV precipitation to distilled water is 1:(8-12)), obtaining filtrate through compression filtration, adding ammonium sulfate to the filtrate slowly (for about 1 h) till the saturation is 40%-60%, preferably 45%-55%, centrifuging the resultant mixture at 8,000 g/min. centrifugal rate for 15 min. to obtain precipitate and supernatant, and then discarding the supernatant. [0034] b) zinc ion affinity chromatography: dissolving the precipitate obtained in the step a) with equilibrium buffer (0.02M Tris+0.5M NaCl with pH=6.0-7.0) to obtain a solution of dissolved precipitate (obtained by adding the precipitate into the equilibrium buffer and stirring), wherein, the volume of the equilibrium buffer is of the volume of the compression leachate obtained in the step a); then, performing zinc ion affinity chromatography, till the protein peak drops to the baseline; at that point, stopping the elution and collecting the eluent produced through affinity chromatography. [0035] The zinc ion affinity chromatography is performed with a protein purifier AKTA Purifier from GE, and mainly consists of the following steps: i) column loading: loading a purification medium into a chromatography column (the volume of the purification medium is 1 column volume (1 CV)), and connecting the chromatography column to the protein purifier; ii) purification medium buffering: buffering the purification medium with about 5CV equilibrium buffer; iii) sample loading: pumping the solution of dissolved precipitate into the chromatography column, so that the target protein 2-M can be coupled to the purification medium extensively; iv) rinsing/leaching leaching off the protein that is not coupled to the purification medium with the equilibrium buffer, till only protein coupled to the purification medium is left in the purification medium when the protein is leached to the baseline, wherein, usually about 8-10CV equilibrium buffer is required for the leaching; v) elution: eluting off the proteins coupled to the purification medium with the eluent, wherein, usually 1CV eluent is required. [0036] A zinc ion chelated high flow-rate agarose medium (Zn-IDA QZT 6FF) is selected as the purification medium, the volume ratio of the purification medium to the solution of dissolved precipitate is determined according to the concentration of proteins in the solution of dissolved precipitate; in the present invention, the volume ratio of the purification medium to the solution of dissolved precipitate is about 1:5. An equilibrium buffer (0.02M Tris+0.5M NaCl with pH=6.0-7.0) is used in the purification medium buffering and leaching procedures, and 0.1M Na.sub.2EDTA with pH=6.5-7.0 is used as the eluent. [0037] c) gel filtration: performing gel filtration of the eluent obtained through affinity chromatography in the step b). The gel filtration realizes separation and purification of proteins under a principle that the retention times of proteins in different molecular weights in the filtering medium are different from each other, i.e., the higher the molecular weight of a protein is, the earlier the protein is leached off. Owing to the fact that 2-M has the highest molecular weight among the proteins, only the first protein peak has to be collected. Similar to the zinc ion affinity chromatography process, this process also consists of column loading, filtering medium buffering, sample loading, and leaching steps, and is executed with the protein purifier AKTA Purifier from GE, which can detect the concentrations of proteins in the eluent and plot a protein peak diagram automatically according to the concentrations. In the operation, since the target protein 2-M is leached off first, the eluent with the first protein peak is collected according to the indication of protein peak in the instrument, and the collection is stopped once the second peak occurs. [0038] Wherein, the filtering medium is one of Polyacrylamide-dextran gel Sephacryl-200 HR, Polyacrylamide-dextran gel Sephacryl-300 HR, Superose 12 (agarose gel), Superose prep grade (agarose gel), and Sephadex G-200 (cross-link dextran G-200), the volume of the eluent produced through affinity chromatography usually is 1-3% of the volume of the filtering medium, and an equilibrium buffer (0.02M Tris+0.5M NaCl with pH=6.0-7.0) is used in the filtering medium buffering and the leaching. [0039] d) ultrafiltration and concentration: performing ultrafiltration and concentration of the eluent obtained through gel filtration in the step c), to concentrate to 4.5-5.5% protein concentration; then, performing sterile filtration with a 0.22 m filter; thus; the target protein 2-M product is obtained, and is in liquid state. [0040] The ultrafiltration membrane has a molecular weight cutoff within a range of 30-500 kD, and preferably is an ultrafiltration membrane that has 50-100 kD pore size. [0041] In the method described above, before the step b) is executed, preferably the precipitate obtained in the step a) is dissolved in a dialysis solution for desalination by ultrafiltration, wherein, the ultrafiltration membrane has molecular weight cutoff within a range of 30-500 kD, and the dialysis solution is buffer solution 0.02M Tris+0.5M NaCl with pH=6.0-7.0.

EXAMPLES

[0042] 2-M in Cohn Fraction IV precipitation is separated and purified with the method described above, and some parameters in the method are adjusted (see Table 1). Then, the obtained products are identified by mass spectrometry, as shown in Table 2. Next, the purity and activity recovery rate of the obtained 2-M are detected, and the results are shown in Table 3.

[0043] Taking Example 1 as an example, FIGS. 1-3 show chromatograms of 2-M obtained through separation and purification with the method in the example 1, wherein, FIG. 3 is a non-reduced chromatogram of 2-M identified by SDS-PAGE, M is molecular weight marker, the bands 1-3 respectively represent the compression leachate in the step a), the solution of dissolved precipitate in the step b), and the eluent obtained through affinity chromatography in the step b), and the band 4 represents the eluent obtained in the step c), i.e., the first peak of Sephacryl-200 HR.

TABLE-US-00001 TABLE 1 Results of 2-M Separated and Purified from Cohn Fraction IV Precipitation with Different Parameters Example Parameter 1 2 3 4 5 6 7 8 9 Step a) Cohn Fraction IV 100 1000 300 500 800 1000 1000 500 500 precipitation (g) Distilled water (mL) 800 11000 2700 4500 7200 10000 10000 4500 4500 Saturation of 40 60 45 50 55 50, 50, 50, 40, 30 70 ammonium sulfate (%) 50, 40, 55 55 Conditions of 8000 g/min 8000 g/min, 7000 g/min, 6000 g/min, 8000 g/min 7000 g/min centrifuge 15 min 10 min 15 min 20 min 20 min 15 min Desalination by Pore size (kD) 30 500 100 300 300 500 500 300 300 ultrafiltration Step b) pH of equilibrium buffer 7.0 6.0 6.4 6.5 6.8 6.0 6.0 6.5 6.5 pH of eluent 7.0 6.5 6.8 6.8 6.8 6.8 6.8 6.8 6.8 Eluent 0.1M Na.sub.2EDTA Purification medium Zn-IDA QZT 6FF Zn-Sepharose CL6B Zn-IDA QZT 6FF Step c) pH of equilibrium buffer 6.0 7.0 6.2 6.5 6.8 None None 6.5 Filtering medium Sephacryl-200 HR Sephacryl-300 HR Sephacryl-300 HR Step d) Concentration 4.5 5.5 5.5 4.8 5.2 5.2 5.0 4.9 5.3 of protein (%) Ultrafiltration 30 100 100 300 300 100 100 300 300 pore size (kD)

[0044] The purpose of mass spectrometry is to identify whether the prepared protein is the target protein, by analyzing the protein in the band 4 shown in FIG. 3, comparing the peptide fragments of the protein with the peptide fragments in the database, and sorting by the degree of matching. Higher degree of matching indicates higher probability that the protein obtained through purification is the matching protein. The number on the left is the gi no. of the corresponding peptide fragment on the right, and the gi no. may be searched in pubmed to obtain specific information of the corresponding peptide fragment. The results are obtained as shown in Table 2. According to Table 2: the three peptide fragments at the highest degree of matching belong to human 2-macroglobulin (the second gi no. and the third gi no, are searched in pubmed to obtain relevant information), the keratin identified by the seventh gi no. and the eighth gi no. is protein in the finger skin attached to the sample, and is an experimental error rather than a protein prepared in the experiment. All other identified proteins are essentially 2-macroglobulin with other properties. Hence, it can be ascertained through the experiment that the protein obtained through purification with the method described above is right 2-macroglobulin.

TABLE-US-00002 TABLE 2 Translation Result of Gi Nos. in the Mass Spectrometry Result of the Band 4 in FIG. 3 1 gi|177870 2-M precursor (human) 2 gi|377656551 Chain A, 2-M 3 gi|224053 2-M 4 gi|426371570 2-M analogue (gorilla) 5 gi|332232615 2-M (black gibbon) 6 gi|28317 Unnamed protein (human) 7 gi|11935049 Keratin (human) 8 gi|375314779 Keratin (human) 9 gi|403269387 2-M analogue (saimiri) 10 gi|390467482 2-M (marmoset)

[0045] The mass spectrometry identification result in Table 2 indicates that the band 4 is the 2-M obtained through purification with the method provided in the present invention. The chromatograms and mass spectrometry identification results of 2-M obtained through separation and purification with the methods in examples 2-5 are the same as those in the FIGS. 1-3 and Table 2, and will not be detailed further here.

Reference Example 1

[0046] The reference example 1 is the example 6. The specific operations are: 1,000 g Cohn Fraction IV precipitation is dissolved in 10,000 ml distilled water, the solution is stirred for 1 h and then filtered, and thereby 9,500 ml filtrate is obtained. Ammonium sulfate solution at 100% saturation in the same volume is added to the filtrate, and the mixture is stirred for 1 h and then aged overnight at 4 C. Precipitate A and supernatant A are obtained through centrifugal separation, the precipitate A is dissolved in 5,000 ml ammonium sulfate solution at 50% saturation, and the resultant solution is stirred for 1 h and then centrifuged, and thereby precipitate B and supernatant B are obtained. The precipitate B is dissolved in 4,000 ml ammonium sulfate solution at 50% saturation, and the resultant solution is centrifuged and thereby precipitate C and supernatant C are obtained; the precipitate C is dissolved in 3,000 ml distilled water, solid ammonium sulfate is added to the resultant solution at a ratio of 243 g/L, so that the saturation reaches 40%, and then the solution is treated by centrifugal separation, and thereby precipitate D and supernatant D are obtained. Solid ammonium sulfate is added to the supernatant D at a ratio of 97 g/L, so that the degree of saturation of ammonium sulfate reaches 55%, and then the solution is stirred for 1 h and centrifuged, the obtained precipitate E is dissolved in 1,000 ml buffer solution (0.02M Tris+0.15M NaCl, with pH=6.0), ultrafiltration is carried out with a 500 kDa ultrafiltration membrane for desalination, the obtained 600 ml trapped fluid is treated by zinc ion affinity chromatography, the material is leached further with the buffer solution after sample loading, till the protein peak is close to the baseline; then, the material is eluted with an eluent (0.1M Na.sub.2EDTA, with pH=6.8), till the protein peak drops to the baseline. About 150 ml eluent rich in 2-M is collected.

Reference Example 2

[0047] In the reference example 2, the method put forth by Qiming Peng et al. (separation and purification of 2-macroglobulin from human plasma, Progress in Biochemistry and Biophysics, 1986, 3:69-72) is used, and human plasma is used as the raw material; first, the plasma is processed by precipitation with 4%-12% polyethylene glycol, and then 2-macroglobulin is prepared through purification by zinc ion affinity chromatography, affinity chromatography with blue agarose, and secondary zinc ion affinity chromatography sequentially.

Reference Example 3

[0048] In the reference example 3, the method put forth by G. D. Virca et al. (Purification of Human 2-Macrolobulin by Chromatography on Cibacron Blue Sepharose, Analytical Biochemistry, 1978, 89:274-278), and haptoglobin 1-1 is used as the raw material, and treated by affinity chromatography with blue agarose and gel filtration sequentially.

Reference Example 4

[0049] In the reference example 4, 2-macroglobulin is prepared through ammonium sulfate fractional precipitation with the 2-macroglobulin preparation method specified in the China Requirements for Biological Products (Trial, Edition 1990).

[0050] The concentration, specific activity, recovery rate and purity of the proteins obtained in the examples 1-9 in the present invention are measured, and the detection results are recorded in Table 3. The activity of 2-M is measured on the basis of the quantity of trypsin inhibited by unit volume of sample (g/ml); the specific activity (g/mg) is equal to the activity divided by protein content, and represents the quantity of trypsin inhibited by per milligram of protein; the purity is measured with purity analysis software Band Scan 5.0.

TABLE-US-00003 TABLE 3 Summary of Purification Test Result Concentration Specific of Protein Activity Recovery Purity (%) (g/mg) Rate (%) (%) Example 1 4.5 14.4 55.3 95.1 Example 2 5.5 13.3 48.5 94.4 Example 3 5.0 14.6 53.2 96.4 Example 4 4.8 14.2 55.6 95.2 Example 5 5.5 13.7 58.8 94.8 Example 6/ 5.2 14.1 23.4 96.5 reference example 1 Example 7 5.3 10.3 39.4 70.5 Example 8 5.1 14.4 30.3 95.9 Example 9 5.0 11.7 65.5 80.5 Reference example 2 4.8 12.9 24.5 93.9 Reference example 3 4.8 9.8 70.0 67.8 Reference example 4 5.0 5.5 75.2 35.6

[0051] According to Table 3: the specific activity, recovery rate, and purity are stable in examples 1-5. For example, the protein concentration is about 5.0%; the specific activity, recovery rate, and purity are about 14.0 g/mg, 54%, and 95% respectively.

[0052] When the eluent in the example 6 (i.e., reference example 1) is ultra-filtered to 20 ml, the concentration of the obtained 2-M protein is 5.2%, the specific activity is 14.1 g/mg, and the recovery rate is 23.4%. In the reference example 1, 2-M is prepared with the method disclosed in an English literature (Purification of Human Plasma 2 Macroglobulin and 1 Proteinase Inhibitor Using Zinc Chelate Chromatography, ANALYTICAL BIOCHEMISTRY, 1979, 99:415-420), except that the starting material used in the English document is plasma, while the starting material used in the reference example 1 is Fraction IV precipitation. The differences between the method in the reference example 1 and the method provided in the present invention mainly include: (1) in the present invention, a single pass of ammonium sulfate precipitation is used, and the reaction time in the ammonium sulfate precipitation step is shorter (1 h stirring followed by 15 min. centrifugation), a fractional ammonium sulfate precipitation method (i.e., several passes of ammonium sulfate precipitation) is used, and the reaction time is longer (overnight); the recovery rate in the reference example 1 is lower, because the activity 2-M may be deteriorated by ammonium sulfate; (2) though the method provided in the present invention has an additional step of gel filtration compared with the method in the reference example 1, the protein is not deactivated in the gel filtration step, because the reaction conditions of the gel filtration are mild. In summary, in the method in the reference example 1; at the beginning of purification, ammonium sulfate precipitation is used for several times to purify 2-M to achieve high purity before zinc ion affinity chromatography, but the loss of activity of 2-M is severe, and the reaction time is long. In contrast, in the present invention, only one pass of ammonium sulfate precipitation is used, and the reaction time is shorter. Though a gel filtration step is added after zinc ion affinity chromatography, the overall purification time is short, and the recovery rate is higher than that in the reference example 1.

[0053] Though the specific activity and purity achieved in the example 6 are comparable to those achieved in the examples 1-5 (14.1 g/mg and 96.5% respectively), the recovery rate in the example 6 is too low (only 23.4%). Therefore, the method in the example 6 is not suitable for mass production. Compared with the method in the example 6, the method in the example 7 simplifies the ammonium sulfate precipitation step, and, as a result, the activity and recovery rate are improved (to 39.4%), but the purity is relatively low (only 70.5%). In the examples 8 and 9, the experimental procedures in the present invention are used essentially, but the selected saturation of ammonium sulfate is too high or too low; consequently, a satisfactory result in terms of purity and recovery rate can't be obtained in the product. For example, the purity in the example 8 is comparable to that in the examples 1-5, but the recovery rate in the example 8 is too low; though the recovery rate in the example 9 is higher, the purity is too low, only 80.5%. In the reference examples 2-4, the same defect also exists, i.e., a satisfactory result in terms of purity and recovery rate can't be obtained; though the purity in the reference example 2 is comparable to those in the examples 1-5, the recovery rate is too low; though the recovery rate in the reference example 3 is higher, the purity is too low; the purity in the reference example 4 is even lower.

[0054] Hence, with the method provided in the present invention, 2-M plasma protein that has a clinical application value is obtained through purification from Cohn Fraction IV precipitation that is regarded as an industrial waste at present by salt precipitation, affinity chromatography and gel filtration in combination. Thus, the Cohn Fraction IV precipitation is changed from a discarded material into a valuable material, and plasma is utilized comprehensively. In addition, the method is easy and simple to use, easy to scale up, and suitable for separation and purification of 2-M at a large scale. With the method provided in the present invention, the recovery rate in the separation and purification is about 50%, and the purity is about 95%. In contrast, in the processes reported in other technical literatures, high recovery rate and high purity can't be achieved at the same time. Therefore, the present invention has bright application prospects.

INDUSTRIAL APPLICABILITY

[0055] With the method provided in the present invention, 2-macroglobulin plasma protein that has a clinical application value can be separated and purified from Cohn Fraction IV precipitation, and the Cohn Fraction IV precipitation is changed from a discarded material into a valuable material; in addition, the method is easy and simple to use, easy to scale up, and is suitable for separation and purification of 2-macroglobulin at a large scale and suitable for industrial application.