KR101212499B1 - Method for purifying bone morphogenetic protein - Google Patents

Abstract

The present invention relates to a process for purifying BMP, in particular BMP7. The present invention further relates to BMP, preferably BMP7, which has been purified with high purity and high recovery rate according to the above purification method.

Description

Purification method of bone formation protein {METHOD FOR PURIFYING BONE MORPHOGENETIC PROTEIN}

The present invention relates to a process for purifying BMP, in particular BMP7. The present invention also relates to BMP purified with high purity and high recovery according to the above method.

BMP (Bone Morphogenetic Protein) is a protein belonging to the TGF-β superfamily of secretory growth and differentiation factors, including BMP2, BMP3, BMP3b, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP10, BMP11, BMP12, BMP13, BMP-14, BMP-15 and the like. BMPs can form homodimers consisting of monomers with a single BMP phase or heterodimers consisting of monomers with two different BMP phases, which disulfide in a head-to-tail arrangement Connected by a bond.

BMPs function to regulate the growth and differentiation of cells of the same type as osteogenic cells, in particular BMP2, BMP4, BMP5, BMP6, BMP7 and BMP8 can induce bone formation alone in ectopic sites in vitro and in vivo Known growth and differentiation factors. It has also been reported that heterodimers consisting of BMP4 and BMP7 are potential inducers of mesoderm, and BMP dimers such as BMP2 homodimers and BMP2 / BMP7 heterodimers are reported to be activated in vivo. In particular, BMP7 plays an important role in the transformation of mesenchymal cells into bone and cartilage and may be involved in bone homeostasis, which has been proposed as an active ingredient for treating diseases such as chronic renal failure and infertility.

As a result, various studies have been conducted on BMPs, which play an important role in regeneration of bone tissue. Accordingly, the industry needs to purify BMP with high purity and high recovery rate by removing impurities from cell culture medium containing BMP. It is becoming.

Conventionally, in order to obtain a high recovery rate of BMP from a culture medium such as cells, and to obtain a high recovery rate, several steps of chromatography based on charge, ligand, hydrophobicity or size, etc. have been combined and used in the kind and sequence of binding. As a result, there are many differences in purity and recovery rate.

However, the longer the time required to purify the BMP and the more the purification step, the lower the final recovery rate of the BMP because the final BMP is to be degraded or lost by the protease in the intermediate step . At this time, even if the number of steps for purifying BMP and the time required for purification are shortened to solve the problem, the purity of the finally recovered BMP should not be lowered.

MEANS TO SOLVE THE PROBLEM AND SOLUTION To solve this problem, the present inventors have identified the chemical and physical characteristics of BMP, the type of chromatography, the resin used in each chromatography, the type of buffer, wash or eluent, concentration and pH, etc. As a result of performing various experiments, the present invention has been completed to shorten the steps and time required for purification and to obtain high purity BMP with high recovery rate.

The present invention provides a method for purifying BMP comprising the following steps:

a) applying affinity chromatography to a solution containing BMP; And

b) applying hydrophobic interaction chromatography to the solution obtained in step a).

In a preferred embodiment, the BMP is BMP7, and the solution containing the BMP is CHO cell culture.

In a preferred embodiment, the affinity chromatography of step a) uses a cellulose pin resin, in particular i) equilibrating the column with a buffer containing Tris, ii) a solution containing BMP in the column. Loading, and iii) eluting the solution containing BMP with a buffer containing Tris and arginine. Optionally, between the steps ii) and iii), the step of washing the column with a buffer containing Tris and arginine may be additionally included.

The buffer used in the affinity chromatography of step a) has a pH in the range of 5-9, preferably 6-8, more preferably 7-8.

The concentration of Tris used in the affinity chromatography of step a) is 5 to 500 mM, preferably 50 to 100 mM, more preferably 50 mM.

The concentration of arginine used in the affinity chromatography of step a) is 0.1 to 5M, preferably 0.3 to 1M, more preferably 0.3 to 0.8M.

Optionally, the buffer used in the affinity chromatography of step a) may further comprise 0.1 to 2 M NaCl.

In a preferred embodiment, the hydrophobic interaction chromatography of step b) is butyl sepharose chromatography, more preferably butyl sepharose S fast flow resin is used.

In particular, the hydrophobic interaction chromatography of step b) comprises i) equilibrating the column with a buffer containing Tris and NaCl, ii) loading the solution obtained in step a), and iii) Tris , Eluting with a buffer containing arginine and propylene glycol.

The buffer used in the hydrophobic interaction chromatography of step b) has a pH in the range of 5-9, preferably 6-8, more preferably 7-8.

The concentration of Tris used in the hydrophobic interaction chromatography of step b) is 5 to 500 mM, preferably 50 to 100 mM, more preferably 50 mM.

The concentration of arginine and NaCl used in the hydrophobic interaction chromatography of step b) is 0.1 to 5M, preferably 0.3 to 1M, respectively.

In a preferred embodiment, prior to applying hydrophobic interaction chromatography in step b), 0.2-5 M, preferably 0.5-2 M, more preferably 1 M NaCl is added to the solution obtained in step a). And then performing filtration is additionally performed.

Purification method of the BMP according to the present invention has been simplified and reduced compared to the conventional purification method, there is an advantage that can be purified faster BMP and reduce the purification cost.

In addition, the longer the time to purify BMP and the more the step of purifying BMP, the problem that BMP is degraded by protease in the intermediate stage and lost during the purification process is solved, thereby BMP by the method according to the present invention. The final recovery rate of is increased.

Moreover, in the process according to the invention, the purity of the recovered purified BMP can be achieved at a high level, although the purification step is reduced.

1A and 1B show SDS-PAGE results and Western blotting results with different compositions of equilibration buffer and elution buffer in affinity chromatography using cellulose pin resin and with or without adding a wash step. Are respectively shown (see Example 2 below).
2A and 2B show the results of SDS-PAGE and Western blotting, respectively, when varying the volume of a sample injected into a column in hydrophobic interaction chromatography using butyl sepharose S resin. See example 4).
FIG. 3 shows the results of SDS-PAGE when affinity chromatography and hydrophobic interaction chromatography are performed together and when hydrophobic interaction chromatography alone is performed (see Example 7 below).
4A, 4B, and 4C show SDS-PAGE results, Western blotting results, and RP-HPLC (reverse phase high performance liquid chromatography) results when affinity chromatography and hydrophobic interaction chromatography were performed together. (See Example 8 below).

In the following, specific examples of purifying BMP7 among the methods for purifying BMP according to the present invention are shown. However, the scope of the present invention is not limited by the following examples, and those skilled in the art will be able to perform various modifications within the meaning of the present invention.

Example 1

The present inventors compared and analyzed the recovery and purity of purified BMP at different concentrations of NaCl contained in the equilibration buffer and the elution buffer in affinity chromatography. Affinity chromatography was performed using cellulose pin sulfate resin under the following conditions.

Flow rate: 1 ml / min

Column volume: about 0.8 ml

Samples injected into the column: 20 CV of CHO cell culture medium containing BMP7

Linear gradient: 0-100%.

In particular, the composition of the equilibration buffer and the elution buffer used in this example and the recovery rate of BMP7 are shown in Tables 1 to 3 below. The eluate fractions 1, 2 and 3 shown in Table 1 and Table 2 do not collect the eluates at once and measure the recovery rate, but mean that the eluates are continuously divided into fractions to measure the recovery rates for each. In addition, the present inventors used an ELISA assay to measure the recovery of BMP.

The recovery at the concentration of 0.1M NaCl and the recovery at the 0.25M (fraction 1 + 2 + 3) were found to be about 50% or more (see Table 1 and Table 2). On the other hand, when the concentration of NaCl was 0.5M, the recovery was lower, about 24% (see Table 3). Accordingly, it can be seen that the lower the concentration of NaCl contained in the equilibration buffer and the elution buffer, the higher the recovery rate of BMP7 in affinity chromatography using cellulose pin sulfate resin.

Example 2

We compared the recovery and purity of purified BMP with different compositions of equilibration buffer and elution buffer in affinity chromatography, and with or without washing steps. Affinity chromatography was performed using cellulose pin sulfate resin under the following conditions.

Flow rate: 2.5 ml / min

Column volume: about 15 ml

Samples injected into the column: 50 ml of BMP7-containing CHO cell culture medium

In particular, the composition of the equilibration buffer, the wash buffer, the elution buffer used in this example and the recovery rate of BMP7 accordingly are shown in Tables 4 to 7 below.

In affinity chromatography using cellulose pin sulphate, the recovery was about 74% when BMP7 was eluted with a buffer containing 0.8 M arginine without undergoing a washing step (see Table 4). On the other hand, in the case of additional washing with a buffer containing 0.3M arginine, the recovery of BMP7 in the wash was about 42%, and the recovery of BMP7 in the eluate was about 34% (see Table 5).

In this regard, FIG. 1A shows the SDS-PAGE results and FIG. 1B shows the Western blotting results. Line 4 of FIG. 1A (eluate with a buffer containing 0.8M arginine without undergoing a washing step) and line 5 (washing solution with a buffer containing 0.3M arginine) of FIG. 1A show similar bands. This result is that impurities including the premature form of BMP7 to be removed in the present invention (which is not a dimer and forms a band around about 60 kDa) can be mostly removed by washing with a buffer containing 0.3 M arginine. Means that.

In addition, compared to line 5 of FIG. 1B (washing solution with a buffer containing 0.3M arginine), line 6 of FIG. 1B (elution with a buffer containing 0.8M arginine after the washing step) was placed at around 30 kDa. You can find a much darker band. This result means that the dimer BMP7 corresponding to the final form to be obtained in the present invention, that is, the mature form, can be obtained in high purity after the washing step.

Therefore, since most of the non-mature form of BMP7 was removed in the washing step with 0.3 M arginine buffer, the final purified BMP7 was recovered even if the recovery rate of BMP7 in the eluate was lower than the recovery rate without the washing step. The purity of is much higher.

Example 3

We compared the recovery of BMP to be purified when different resins were used in hydrophobic interaction chromatography. The types of resins used in this example, the composition of the buffer solution, and the like are shown in Table 8 below.

Butyl Sepharose S Phenyl Sepharose 6FF Butyl Sepharose 4FF Column volume (CV) 5ml 5ml 4ml Flow rate 3ml / min Same as left Same as left Equilibration buffer 50 mM Tris + 1.2 M NaCl, pH 7.3 Same as left Same as left Elution buffer 50 mM Tris + 0.8 M arginine + 10% propylene glycol, pH 7.3 Same as left Same as left

In Table 8 above, the column volume represents the volume of the fixed phase resin used in the chromatography. Equilibration buffer is a buffer used for equilibration to bind the BMP-containing cell culture solution to the stationary phase resin. Elution buffer is a buffer used to elute BMP bound to a fixed phase resin.

In this example, purification was performed according to the following protocol:

1) Equilibrate the chromatography column with equilibration buffer.

2) Initialize Auto Zero UV detector to check protein peak with UV.

3) CHO cell culture solution 5 CV is injected into the column to bind to the fixed phase resin.

4) Wash the column using 10 CVs of buffer of the same composition as the equilibration buffer. This process is necessary to equilibrate the UV peaks again.

5) Elution buffer 10 CV is used to elute and recover BMP7 bound to a fixed phase resin.

Table 9, Table 10 and Table 11 below shows the results of ELISA analysis when butyl sepharose S resin, phenyl sepharose 6FF resin and butyl sepharose 4FF resin were used, respectively. In particular, Table 9 and Table 10 show the results of the experiments performed twice, respectively, at the upper and lower ends.

ELISA Analysis Results Using Butyl Sepharose S Resin Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7 0.79 25 19.78 100.0 Solution drained without being bound to the column 0.28 36 9.91 50.1 Eluate 0.31 24 7.49 37.9 Cell culture medium containing BMP7 0.79 25 19.78 100.0 Solution drained without being bound to the column 0.26 36 9.29 47.0 Eluate 0.31 24 7.55 38.2

ELISA Assay Using Phenyl Sepharose 6FF Resin Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7 0.79 25 19.78 100.0 Solution drained without being bound to the column 0.00 42 0 0.0 Eluate 0.11 24 2.70 13.6 Cell culture medium containing BMP7 0.79 25 19.78 100.0 Solution drained without being bound to the column 0.00 42 0 0.0 Eluate 0.19 24 4.66 23.5

ELISA Analysis Results Using Butyl Sepharose 4FF Resin Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7 0.79 20 15.82 100.0 Solution drained without being bound to the column 0.00 42 0.14 0.7 Eluate 0.14 24 3.36 17.0

The recovery of purified BMP7 using phenyl sepharose 6FF resin was only up to 23.5% for the sample of cell culture initially injected (see Table 10), and the recovery of purified BMP7 using butyl sepharose 4FF resin was It can be seen that only 17.0% (see Table 11).

In contrast, when butyl Sepharose S resin was used, the recovery of purified BMP7 was about 38% (see Table 9). Therefore, it can be seen that BMP7 can be recovered to the highest level when using butyl sepharose S resin among hydrophobic interaction chromatography.

Example 4

We performed hydrophobic interaction chromatography using butyl sepharose S resin under the following conditions, varying the volume of the sample injected into the column.

Equilibration buffer: 50mM tris + 1.2M NaCl

Elution buffer: 50 mM tris + 0.8 M arginine + 10% propylene glycol

Flow rate: 3 ml / min

Column volume (CV): about 8 ml

Sample: Cell culture medium containing BMP7 (fixed with 1.2 M NaCl, V / V)

Volume of sample injected into the column: 20 CV (160ml), 40 CV (320ml)

SDS-PAGE results for 20 CV and 40 CV samples are shown in FIG. 2A. BMP7 bands with the dimeric form of dimers near 30 kDa of line 1 (positive control), line 4 (eluate of 20 CV samples) and line 11 (eluate of 40 CV samples) in FIG. Was confirmed. On the other hand, almost similar bands were identified in line 2 (cell culture medium containing BMP7) and line 3 (solution discharged without binding to the column) of FIG. 2A.

The results of western blotting for 20 CV and 40 CV samples are shown in FIG. 2B. In Figure 2b, the positive control (line 1) using carrier free BMP7 purchased from R & D systems showed a number of bands. In addition, in line 4 (eluate of 20 CV samples) and line 11 (eluate of 40 CV samples) of FIG. 2B, bands that could not be confirmed from the results of the SDS-PAGE of FIG. 2A were found around 50 to 60 kDa. . Here, it is believed that the reason why such band was not confirmed in western blotting but only in SDS-PAGE is that SDS-PAGE is more sensitive than western blotting. This band, identified around 50-60 kDa, can be seen to represent an immature form before maturation to BMP7 of the dimer, which is the active form.

Example 5

We compared the recovery and purity of purified BMP with different compositions of elution buffer in hydrophobic interaction chromatography. Hydrophobic interaction chromatography was performed using butyl sepharose S resin under the following conditions.

Equilibration buffer: 50 mM tris + 1.2 M NaCl, pH 7.3

Flow rate: 3 ml / min

Column volume: about 8 ml

Samples injected into the column: BMP7-containing CHO cell culture medium (1.2 M NaCl added) 5 CV (40 ml)

In particular, the composition of the elution buffer used in this example and the recovery rate of BMP7 are shown in Tables 12 to 15 below.

Elution buffer: 50 mM tris + 0.8 M arginine + 10% propylene glycol, pH 7.3 Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7 1.44 160 230.60 100.00 Solution drained without being bound to the column 0.58 200 115.08 49.90 Eluate 3.06 25 76.38 33.12

Elution buffer: 50 mM tris + 10% propylene glycol, pH 7.3 Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7 1.29 40 51.69 100.00 Solution drained without being bound to the column 0.27 100 27.07 52.38 Eluate 0.19 40 7.42 14.36

Elution buffer: 50 mM tris + 0.8 M arginine, pH 7.3 Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7 1.29 40 51.69 100.00 Solution drained without being bound to the column 0.28 100 28.00 54.17 Eluate 0.24 40 9.60 18.56

Elution buffer: 50 mM tris, pH 7.3 Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7 1.29 40 51.69 100.00 Solution drained without being bound to the column 0.26 100 25.69 49.71 Eluate 0.06 40 2.48 4.79

The combined recovery of 14.36% when using an elution buffer containing 10% propylene glycol (see Table 13) and 18.56% when using an elution buffer containing 0.8M arginine (see Table 14), 10 Considering the similarity of 33.12% recovery (see Table 12) when using an elution buffer containing both% propylene glycol and 0.8 M arginine, propylene glycol and arginine were obtained from the hydrophobic interaction chromatography of the present invention. It is believed that it must be contained in the elution buffer.

Example 6

We compared the recovery and purity of purified BMP with different compositions of equilibration buffer in hydrophobic interaction chromatography. Hydrophobic interaction chromatography was performed using butyl sepharose S resin under the following conditions.

1) When the concentration of NaCl in the equilibration buffer is increased to 2M

Equilibration buffer: 50 mM tris + 2M NaCl, pH 7.3

Elution buffer: 50 mM tris + 1 M arginine + 10% propylene glycol, pH 7.3

Flow rate: 2.3 ml / min (300 cm / hr)

Column volume: about 5 ml

Samples injected into the column: cell culture medium containing BMP7 (20 ml, incubated for 5 days and fixed with 2M NaCl)

2) 2M (NH ₄ ) ₂ SO ₄ in place of NaCl in equilibration buffer

Equilibration buffer: 50 mM tris + 2M ((NH ₄ ) ₂ SO ₄ , pH 7.3

Elution buffer: 50 mM tris + 1 M arginine + 10% propylene glycol, pH 7.3

Flow rate: 2.3 ml / min (300 cm / hr)

Column volume: about 5 ml

Samples injected into the column: cell culture medium containing BMP7 (20 ml, incubated for 5 days and fixed with 2M (NH ₄ ) ₂ SO ₄ )

As a result, when the concentration of NaCl contained in the equilibration buffer in 1) was increased to 2M, the recovery rate of BMP7 was increased, but there was a problem in that the purity was also lowered due to the increase in the content of impurities. In addition, when 2M (NH ₄ ) ₂ SO ₄ was used in place of NaCl as the equilibration buffer in 2), it was found that the purity decreased as agglomeration occurred, where BMP7 was lost in the buffer exchange process. Was considered to be.

Example 7

We compared the recovery and purity of purified BMP when affinity chromatography and hydrophobic interaction chromatography were performed together and hydrophobic interaction chromatography alone.

First, the conditions when affinity chromatography and hydrophobic interaction chromatography are performed together are as follows.

1) Affinity Chromatography with Cellulpin Sulfate Resin

Equilibration buffer: 50 mM tris, pH 7.3

Wash buffer: 50 mM tris + 0.3 M arginine, pH 7.3

Elution buffer: 50 mM tris + 0.8 M arginine, pH 7.3

Flow rate: 2.5 ml / min

Column volume: about 4 ml

-Sample injected into the column: 40 ml of BMP7-containing CHO cell culture medium (sample cultured for 5 days after PACEsol treatment)

2) Hydrophobic Interaction Chromatography with Butyl Sepharose S Fast Flow Resin

Equilibration buffer: 50 mM tris + 1 M NaCl, pH 7.3

Elution buffer: 50 mM tris + 1 M arginine + 10% propylene glycol, pH 7.3

Flow rate: 2.3 ml / min (300 cm / hr)

Column volume: about 5 ml

Samples injected into the column: 7 ml of eluate obtained from the affinity chromatography (filtered after addition of 1 M NaCl (V / V))

In the two step purification process, NaCl was added to the hydrophobic interaction chromatography column before injecting the eluate obtained from the affinity chromatography. At this time, as the coagulation partially occurred, some coagulum was formed in the eluate, and filtration was performed using a filter having a pore size of 0.2 μm to remove it. ELISA analysis confirmed that BMP7 was not lost even though filtration was performed to remove aggregates.

The ELISA analysis results of affinity chromatography using cellulose pin sulfate resin during the purification process are shown in Table 16 below, and the ELISA analysis results of hydrophobic interaction chromatography using butyl sepharose S fast flow resin are shown in Table 17 below. Shown in

Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7
(Line 2 of FIG. 3) 5.92 40 236.86 100.00 Solution drained without being bound to the column
(Line 3 of FIG. 3) 0.05 44 2.20 0.93 Washing liquid
(Line 4 of FIG. 3) 8.70 10 87.00 36.73 Eluate
(Line 5 of FIG. 3) 5.62 12 67.50 28.50

Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Eluate Obtained from Affinity Chromatography with Cellulpin Sulfate Resin
(Line 9 of FIG. 3) 5.34 7 37.37 100.0 Solution drained without being bound to the column
(Line 10 in FIG. 3) 0.25 20 4.92 13.2 Eluate
(Line 11 of FIG. 3) 1.46 10 14.64 39.2

Next, the conditions when only hydrophobic interaction chromatography was performed alone using butyl sepharose S fast flow resin are as follows.

Equilibration buffer: 50 mM tris + 1 M NaCl, pH 7.3

Elution buffer: 50 mM tris + 1 M arginine + 10% propylene glycol, pH 7.3

Flow rate: 2.3 ml / min (70 cm / hr)

Column volume: about 5 ml

Samples injected into the column: 40 ml of BMP7-containing CHO cell culture medium (1M NaCl (V / V) was added)

Experimental results of the hydrophobic interaction chromatography are shown in Table 18 below.

Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7
(Line 6 of FIG. 3) 6.02 40 240.88 100.00 Solution drained without being bound to the column
(Line 7 of FIG. 3) 4.23 45 190.48 79.07 Eluate
(Line 8 of FIG. 3) 1.49 12 17.93 7.44

Compared with affinity chromatography and hydrophobic interaction chromatography alone, the recovery of BMP7 was lower when hydrophobic interaction chromatography alone was performed. In addition, as shown in line 11 of FIG. 3, when affinity chromatography and hydrophobic interaction chromatography were performed together, almost all impurities were removed, and as a result, the dimer form of BMP7 was very high in purity (about 97% or more).

Example 8

In this example, affinity chromatography and hydrophobic interaction chromatography were performed together. In particular, the filtration was performed with a 0.2 μm filter before injecting the CHO cell culture solution into the affinity chromatography column, and the filtration was performed with a 0.2 μm filter before injecting the eluate into the hydrophobic interaction chromatography column. Specific purification conditions are as follows.

1) Affinity Chromatography with Cellulpin Sulfate Resin

Samples injected into the column: BMP7-containing CHO cell cultures were filtered with a 0.2 μm filter. Thereafter, about 20 CVs of the resulting filtrate was injected into the column.

Equilibration buffer: 50 mM tris, pH 7.3

Wash buffer: 50 mM tris + 0.3 M arginine, pH 7.3

Elution buffer: 50 mM tris + 0.8 M arginine, pH 7.3

Eluent: about 2 CV obtained.

2) Hydrophobic Interaction Chromatography with Butyl Sepharose S Fast Flow Resin

Sample injected into the column: 1 M NaCl (V / V) was added to about 2 CV of the eluate obtained from the affinity chromatography, followed by filtration with a 0.2 μm filter. Thereafter, about 5 CV of the resulting filtrate was injected into the column.

Equilibration buffer: 50 mM tris + 1 M NaCl, pH 7.3

Elution buffer: 50 mM tris + 1 M arginine + 10% propylene glycol, pH 7.3

Eluent: about 3 CV is obtained.

The ELISA analysis results of affinity chromatography using cellulose pin sulfate resin during the purification process are shown in Table 19 below. The ELISA analysis results of hydrophobic interaction chromatography using butyl sepharose S fast flow resin are shown in Table 20 below. Shown in

Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Cell culture medium containing BMP7 8.97 450 4035.29 100.00 Eluate 15.66 90 1409.01 34.92

Concentration of BMP7
(μg / ml) Volume of solution
(ml) Amount of BMP7
(μg) Recovery of BMP7
(%) Eluent obtained from affinity chromatography (1M NaCl) 14.32 30 429.56 100.0 Solution drained without being bound to the column 1.67 38 63.60 14.80 Eluate 16.61 18 298.90 69.58

As can be seen from the ELISA analysis, it was confirmed that the recovery rate of the final purified BMP7 was about 24% based on the initial cell culture sample.

The purity of the final purified BMP7 can be confirmed from the SDS-PAGE analysis of Figure 4a and Western blotting analysis of Figure 4b. In particular, the darkest color observed in lines 5 of FIGS. 4A and 4B represents the mature dimer BMP7, and the three bands next to the main band are isoform BMP7. It was confirmed to represent.

In addition, the purity of BMP7 was quantitatively analyzed using the RP-HPLC (reverse phase high performance liquid chromatography) method. BMP7 was shown as peak 1 in the graph of FIG. 4C, and the purity calculated from the area of the peak was 97.38%, which was confirmed to be a very high level.

Claims (15)

Purification method of BMP7 (Bone Morphogenetic Protein 7: Bone Formation Protein 7) comprising the following steps:
a) subjecting affinity chromatography with cellulose pin resin to a solution containing BMP7, wherein the affinity chromatography comprises i) equilibrating the column with a buffer containing Tris, ii) BMP7 on the column Loading the solution containing iii) rinsing the column with a buffer containing Tris and arginine, and iv) eluting the column with a buffer containing Tris and arginine; And
b) applying hydrophobic interaction chromatography using butyl sepharose S fast flow resin to the eluate obtained in step a). The method of claim 1,
The solution containing BMP7 is a cell culture solution. The method of claim 1,
The buffer used in the affinity chromatography of step a) has a pH range of 5 to 9, a concentration of Tris of 5 to 500 mM, and an arginine concentration of 0.1 to 5M. The method of claim 1,
Before applying the affinity chromatography in step a), further comprising the step of performing filtration using a 0.2 μm filter on a solution containing BMP7. The method of claim 1,
The hydrophobic interaction chromatography of step b) comprises i) equilibrating the column with a buffer containing Tris and NaCl, ii) loading the solution obtained in step a) into the column, and iii) Tris, arginine And eluting the column with a buffer containing propylene glycol. The method of claim 5,
The buffer used in the hydrophobic interaction chromatography of step b) has a pH range of 5 to 9, a concentration of Tris of 5 to 500 mM, and a concentration of NaCl and arginine of 0.1 to 5M. The method of claim 1,
Before applying the hydrophobic interaction chromatography in step b), and further comprising the step of adding 0.2 to 5M NaCl to the eluate obtained in step a) and performing filtration using a 0.2㎛ filter Purification method to do. delete delete delete delete delete delete delete delete

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