KR101759506B1 - Production method of bmp7 complex - Google Patents

Abstract

The present invention relates to a process for producing a complex of BMP7 prodomain and mature BMP7 dimer in high yield and high purity.

Description

PRODUCTION METHOD OF BMP7 COMPLEX < RTI ID = 0.0 >

The present invention relates to a method of producing a complex of BMP7 prodomain and mature BMP7 dimer.

BMP (Bone Morphogenetic Protein) is one of the growth factors belonging to TGF-β super family. BMP generally functions to promote the growth and differentiation of osteocytes and chondrocytes, and up to now, BMP2, BMP3, BMP3b, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP10, BMP11, BMP12, BMP13, BMP15 and the like are known.

Among BMPs, BMP7 (Bone Morphogenetic Protein 7: bone formation protein 7) is known to act as an important factor for cell growth and differentiation in relation to bone formation. BMP7 is also known to play an important role in kidney development and normal development of the eye (Nature. 2008 Aug 21; 454 (7207): 1000-4., Genes Dev. 1995 Nov 15; 9 (22): 2795 -807., Int J Dev Biol. 2009; 53 (4): 597-603). In this connection, studies have recently been reported that BMP7 inhibits or reduces scarring and wound formation in the eyeball or cornea (US Patent Publication No. 2006-0122109, Laid-open Patent Publication No. 10-2014-0098999).

The structure of BMP7 is specifically disclosed in Fig. 1 of the applicant's patent registration No. 10-1279145. BMP7 expressed in BMP7-expressing cells is generally present in the form of proBMP7 initially composed of a prodomain and a mature BMP7 domain. The two proBMP7 are bound to each other through a dimerization reaction. The proBMP dimer thus formed is cleaved by the Paired basic amino acid cleaving enzyme (PACE), resulting in the formation of a dimer in which two mature BMP7 domains are bound. The dimer in which two mature BMP7 domains are bound can exhibit activity intracellularly or in vivo.

The present applicant has developed a method for purifying a mature BMP7 dimeric form of BMP7 (Patent Registration No. 10-1212499). The purified BMP7 is in the form of a mature dimer having activity in vivo or in a cell. However, BMP7 obtained according to such a purification method was unsatisfactory as a therapeutic agent in terms of yield and maintenance of activity after purification. It has also been observed that some of the buffer solution used in the purification process may cause some irritation on the ocular surface when administered directly to the cornea or eye. Accordingly, for the purpose of effectively treating or suppressing the formation of eye wounds or scars that may occur in patients undergoing eye surgery such as LASIK surgery, LASEK surgery, or eye surgery such as cataract, glaucoma, and macular degeneration, An alternative is needed for the structural stability and activity of the BMP7 protein used and the composition of the buffer used in the purification process.

Under these circumstances, recent studies have reported that BMP7 binds to the prodomain and becomes structurally more stable when it forms a complex (J Biol Chem. 2016 Jun 10; 291 (24): 12732-46. Doi: 10.1074 / jbc. M115.704734. Epub 2016 Apr 8). Other studies have also reported that the BMP7 pro domain affects the solubility, stability and activity of BMP7 as it affects the structure of the BMP7 complex (J Biol Chem. 2005 Jul 29; 280 (30): 27970-80, Epub 2005 Jun 1., Development 2000 Jan; 127 (2): 343-54).

Thus, the applicant has been required to produce a complex of BMP7 prodomain and mature BMP7 dimer as a substance to inhibit or reduce scarring and wound formation in the eyeball or cornea, but to further increase the production yield and purity of the manufacturing process. There is also a need to develop stabilizers that allow the complex to be safely applied to the cornea or eye.

Patent Registration No. 10-1212499 (December 14, 2012)

In order to produce a complex of the BMP7 prodomain and the mature BMP7 dimer with high yield and high purity, the present inventors have found that the kind of the purification process, the type of resin, buffer, washing solution or eluent used for chromatography, As a result of carrying out various experiments, the present invention has been completed.

The present invention provides a method for producing a complex of a BMP7 prodomain and a mature BMP7 dimer.

The process according to the invention comprises the following steps:

a) applying affinity chromatography to a solution containing BMP7; And

b) eluting the complex of BMP7 prodomain and mature BMP7 dimer by applying hydrophobic interaction chromatography to the eluate obtained in step a).

In one embodiment, the solution containing BMP7 is a cell culture medium such as a CHO cell culture. Preferably, the cell culture liquid is collected after cell culture for 3 to 7 days. More preferably, the cell culture medium is collected after cell culture for 7 days.

In one embodiment, affinity chromatography in step a) employs a cellulphin sulfate resin.

In one embodiment, the affinity chromatography of step a) comprises i) equilibrating the column with a buffer containing histidine and NaCl, ii) loading a solution containing BMP7 in the column, iii) Washing the column with a buffer containing NaCl and arginine hydrochloride, and iv) eluting the column with a buffer containing histidine, NaCl and arginine hydrochloride.

The pH of the buffer used in affinity chromatography in step a) is in the range of 4 to 8, preferably 5 to 7, more preferably 6.

The concentration of histidine used in affinity chromatography in step a) is 2 to 200 mM, preferably 10 to 100 mM, more preferably 20 mM.

The concentrations of NaCl and arginine hydrochloride used in affinity chromatography in step a) are 0.1 to 5 M, preferably 0.1 to 1 M, respectively.

In one embodiment, the hydrophobic interaction chromatography in step b) above uses butyl HP resin.

In one embodiment, the hydrophobic interaction chromatography of step b) comprises i) equilibrating the column with a buffer containing histidine and NaCl, ii) loading the eluate obtained in step a) into the column, iii) ) Washing the column with a buffer containing histidine and NaCl, and iv) eluting the column with a buffer containing histidine, NaCl and propylene glycol.

The pH of the buffer used in the hydrophobic interaction chromatography of step b) is in the range of 4 to 8, preferably 5 to 7, more preferably 6.

The concentration of histidine used in the hydrophobic interaction chromatography of step b) is 2 to 200 mM, preferably 10 to 100 mM, more preferably 20 mM.

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

In one embodiment, prior to step b), further comprises filtering the effluent obtained in step a). Preferably, 0.5 M NaCl is added to the eluate obtained in the step a), and filtered. At this time, filtration can be performed using a filter having a size of 0.2 mu m.

The complex eluted in the step b) may be contained in the stabilizing liquid and used for an application to the eye or the cornea.

Preferably, the stabilizing liquid does not contain arginine but contains glycine.

Complexes of BMP7 prodomains and mature BMP7 dimers are produced according to the methods of the present invention. The complex appears to be more active than the mature BMP7 dimer because the BMP7 prodomain is bound to the mature BMP7 dimer and is structurally complex to affect the availability and stability of the mature BMP7 dimer.

When such a highly active complex is produced according to the method of the present invention, it can be obtained with high yield and purity, and thus it is possible to significantly increase the efficiency of the production process.

Such complexes produced with high yield and purity can be used to effectively treat eye wounds or scars that may occur in patients undergoing eye surgery such as LASIK surgery, LASEK surgery, or eye surgery such as cataract, glaucoma, and macular degeneration Or to inhibit the formation.

In addition, since the contact lens which is in direct contact with the eyeball can be worn and detached, scarring may occur in the eyeball, and further, inflammation, redness, visual loss and visual loss may occur. It can prevent the same phenomenon and increase the rate of eye scar repair.

In addition, the stabilizing liquid according to the present invention can ensure safety to the eye when used in combination with the complex.

Figure 1 shows the structure for a complex of BMP7 prodomain and mature BMP7 dimer. It is believed that the BMP7 dimer has a structure in which two BMP7 monomers are linked by a cysteine covalent bond, and the BMP7 prodomain has a structure connected by a non-covalent bond with a BMP7 dimer. The covalent bond between the two BMP7 monomers is indicated by a solid line, and the non-covalent bond between the BMP7 prodomain and the BMP7 dimmer is indicated by a dotted line. The BMP7 prodomain and BMP7 dimer appear to be present in a ratio of about 2: 1 in the complex.
Figure 2 is an HPLC graph showing the composition and purity of the complex obtained according to one embodiment of the production process of the present invention (see Example 1).
Figures 3 to 5 show the results of SDS-PAGE performed on the final products produced according to the present invention.
6 shows the results of performing gel filtration chromatography (GF) on a mixture of the product of Example 1 and BMP2 dimer.
FIG. 7 shows the result of SDS-PAGE performed on the eluate shown in FIG.
8 is a graph showing the results of measurement of ALP activity of the product of Example 1. Fig.
9 is a graph showing the survival rate of ATDC5 epithelial cells according to each test group.

The present invention will be described in more detail based on the following examples, but it is not intended to limit the scope of the present invention. In addition, those skilled in the art will appreciate that various modifications and changes may be made thereto without departing from the spirit of the invention.

Example  One

This embodiment shows a representative example of the production method according to the present invention.

In order to produce a complex consisting of a BMP7 pro domain and a mature BMP7 dimer from a CHO cell culture containing BMP7, the present inventors performed affinity chromatography using a cellulphin sulfate resin as a first step.

The conditions for the affinity chromatography were as follows:

- Flow rate: 4 ml / min

- Column volume: about 20 ml

- Sample injected into the column: CHO cell culture containing BMP7 5 CV

Step gradient: 0.2 M arginine, 0.7 M arginine.

The compositions of equilibration buffer, wash buffer and elution buffer used in each step of the affinity chromatography are shown in Table 1 below.

Buffer composition  Volume (CV) 1) Equilibration 20 mM histidine + 0.1 M NaCl pH 6.0 5 2) Washing 20 mM histidine + 0.1 M NaCl pH 6.0 3 20 mM histidine + 0.5 M NaCl + 0.2 M arginine HCl pH 6.0 3 3) Elution 20 mM Histidine + 0.5 M NaCl + 0.7 M Arginine HCl pH 6.0 3

More specifically, the affinity chromatography was performed by equilibrating the column with 5 times (5 CV) of the volume of the column equilibrated buffer to the column packed with the cellulphin sulfate resin, and then the CHO cell culture solution containing BMP7 The column was then poured with 3CV of base buffer. Subsequently, 3CV of washing buffer was flowed and 3CV of elution buffer was flown to obtain an eluate.

Thereafter, 0.5 M NaCl was added to the eluate, and the mixture was filtered through a filter having a size of 0.2 mu m.

As the next step, hydrophobic interaction chromatography was subsequently carried out. Butyl HP resin was used. The compositions of equilibration buffer, washing buffer and elution buffer in each step of hydrophobic interaction chromatography are shown in Table 2 below.

Buffer composition Volume (CV) 1) Equilibration 20 mM histidine + 1 M NaCl pH 6.0 5 2) Washing 20 mM histidine + 1 M NaCl pH 6.0 3 3) Elution 20 mM histidine + 0.3 M NaCl + 10% propylene glycol pH 6.0 3

More specifically, the column packed with butyl HP resin was equilibrated with an equilibration buffer of 5 times the column volume (5 CV), and the result obtained in the previous step was injected into the column. Then 3CV of wash buffer was flushed, followed by 3CV of elution buffer.

The reason for not using Tris in place of histidine in each buffer in the above series is that Tris has recently been reported to be limited to the human body. If the end product (i. E., A complex of BMP7 prodomain and mature BMP7 dimer) is used in the human eye for inhibiting corneal scar formation, it should be separately demonstrated that Tris is not contained in the final product to ensure human safety So complex procedures or experiments must be added. However, when the buffer of a specific composition is combined with a chromatography using a specific resin as in the present invention, the procedure for verifying the content of Tris in the final product is not necessary since Tris is not used in the entire process, , And the yield and purity of the final product are rather superior, as will be described later, without using Tris.

An ELISA analysis method was used to measure the recovery of the complex of BMP7 prodomain and mature BMP7 dimer obtained through the above series of procedures. The recovery rates at each step are shown in Table 3 below.

sample Dilution factor
(Dilution Factor) density
(μg / ml) volume
(ml) BMP7 complex
(mg) Recovery rate
(%) BMP7 medium 40000 51.15 90 4.60 100.0 Solution discharged without being bound to the cellulphin sulfate resin column 4000 0.00 90 0.00 0.0 Cellulose sulfate resin wash 4000 0.51 15 0.01 0.2 Cellulphin sulfate resin column effluent 320000 290.57 15 4.36 94.7 Butyl HP resin column 4000 0.02 20 0.00 0.0 Butyl HP resin column effluent 160000 118.30 20 2.37 51.4

According to the above Table 3, it was confirmed that the composite recovery rate after the final purification step was 51.4%.

In addition, the purity of the final product was quantitatively analyzed using an RP-HPLC (reverse phase high performance liquid chromatography) method. The results are shown in Fig. When calculated from the area of the peaks, the mature BMP7 dimer showed a content of 36.92% and the BMP7 prodomain had a content of 63.08% in the final product, and the complex in which the mature BMP7 dimer and the BMP7 prodomain were combined showed 100% As shown in Fig.

Example  2

This example is intended to show that in the final product produced according to the present invention BMP7 is a dimer of the mature form in which the prodomain is truncated.

The present inventors performed SDS-PAGE for each step of the product obtained in Example 1 above. The results are shown in Figures 3, 4 and 5.

In Figure 3, lane 6 represents the final product obtained by sequential sequencing of the cellulphon sulfate chromatography and the butyl HP chromatography procedure, wherein the bands at 30-40 kDa show that the prodomain cleaved BMP7 dimer .

In FIG. 4, the lane 2 is a 10-fold concentration of the final product in order to visually identify the band that appears dim due to low protein concentration. At this time, the band at the upper end of the second lane represents the BMP7 pro domain, and the band at the lower end represents the prodomain cleaved BMP7 dimer.

Particularly, in the lane 4 in which the reducing treatment is performed in FIG. 4, the band disappears at 30 to 40 kDa and appears at about 15 kDa in comparison with the lane 2 which is not treated with reducing. This proves that the reducing BMP7 dimer is separated into BMP7 monomers so that in the end product according to the invention BMP7 is present in homodimeric form.

Fig. 5 shows the result of purification of the cell culture obtained by varying the number of days of culture in each of the two groups according to the method of Example 1. Fig. SDS-PAGE was performed on each of the thus-obtained final products. As a result, when the cell culture liquid for 7 days was cultured, the BMP7 pro domain and the BMP7 dimer were clearly separated from the cell culture fluid on the 3rd day of culture. This is because the longer the cell culture period, the longer the time for BMP7 to be cleaved into the prodomain and mature BMP7 domains by PACE, which is present in a small amount in the cells, and the two bands become clearer.

Example  3

This example is intended to show that BMP7 pro domain and BMP7 dimer are present in complex form in the final product produced according to the present invention.

The mature dimer forms of BMP2 and BMP7 are almost identical in size, all at 30 kDa. Therefore, when gel filtration chromatography (GF: Gel filtration), which is a separation technique according to the size of proteins, is applied, BMP2 dimer and BMP7 dimer are detected together at the same time.

However, as a result of actually performing GF on the mixture of the final product and the BMP2 dimer produced according to the present invention (Example 1 above), the final product produced according to the present invention was eluted first, as shown in FIG. 6, BMP2 dimer was eluted. The result is that the BMP7 dimer in the final product produced according to the present invention is not present in isolated form, but in the form of a complex bound to the BMP7 pro domain, The size is larger.

When the elution time of FIG. 6 is compared with the standard protein elution time in Standard GF (not shown), it is estimated that the size of the complex eluted first in FIG. 6 is about 95-100 kDa.

FIG. 7 shows the result of SDS-PAGE performed on the eluate shown in FIG.

The lane 1 in Figure 7 is a void peak that generally appears initially for protein aggregates. 7 shows two bands in the lane 2 and the lane 3. The BMP7 pro domain and the BMP7 dimer are bonded to each other so that the BMP7 prodomain and the BMP7 dimmer are not represented as one band. This is because the noncovalent binding force between the BMP7 prodomain and the BMP7 dimer was not strong and the BMP7 pro domain and BMP7 dimer were separated during SDS-PAGE. This can be confirmed similarly in FIGS. 4 and 5. On the other hand, the BMP2 dimmer appears only as a single band in lane 4 of FIG.

7, it can be seen that the bands located at about 30 kDa in the lanes 2 and 3 move to about 15 kDa, indicating that the end products produced according to the present invention exhibit BMP7 Is dimmer.

Example  4

This example is to confirm the activity of the complex of BMP7 prodomain and mature BMP7 dimer.

An ALP assay was used for this. One of the widely used assays for determining the biological activity of BMP proteins in vitro is the Alkaline phosphatase assay (alkaline phosphatase assay). The principle of the ALP assay is that the activity of ALP (Alkaline phosphatase), which is an indicator of osteoblast differentiation, is increased when the BMP protein is treated to embryo cells or stem cells. ALP can be quantitatively expressed by reacting with substrate and quantitatively using unit unit / ml. This ALP value indirectly confirms the biological activity and degree of BMP recombinant protein.

The ED50 (50% effective concentration) was calculated based on the ALP values for each experimental group. ED50 is referred to as a 50% effective concentration or a 50% effective dose, which is a dose estimated to produce a 50% effect when administered to a subject with a toxin or drug. The lower the ED50 is, the higher the activity of the drug is believed to be.

 The results are shown in Table 4 and FIG.

Experimental group ED50
(μg / ml) Mature BMP7 dimer 0.24 BMP7 commercially available from R & D 0.17 The final product produced according to this Example 1, a complex of BMP7 prodomain and BMP7 dimer 0.17

As can be seen in FIG. 8, the ALP value of the final product produced according to this Example 1 is higher than that of BMP7 commercially available from mature BMP7 dimer or R & D company.

Also, according to Table 4, the final product produced according to this Example 1 exhibits an ED50 of 0.17 μg / ml, whereas a mature BMP7 dimer, generally known to be active in vivo or intracellularly, is 0.24 μg / ml ED50.

Thus, the final product produced according to this Example 1 was found to be more active than the mature BMP7 dimer. That is, the complex consisting of the BMP7 pro domain and the mature BMP7 dimer is more active. It is believed that this is due to the fact that the BMP7 pro domain is structurally bound to the mature BMP7 dimer in the form of a complex, which affects the availability and stability of the mature BMP7 dimer.

Example  5

This example is intended to show the relative difference in yield when the resin of the hydrophobic interaction chromatography used in the second purification step in Example 1 was changed.

After affinity chromatography using a cellulose resin was carried out in the same manner, yields of the products were determined by different kinds of resins in hydrophobic interaction chromatography as shown in Tables 5 to 7 below.

sample Dilution factor
(Dilution Factor) density
(μg / ml) volume
(ml) BMP7 complex
(mg) Recovery rate
(%) Cellulphin sulfate elution 80000 128.69 25 3.22 100.0 Butyl-S 6 FF unbound 80000 108.24 25 2.71 84.1 Butyl-S 6 FF elution 4000 1.32 20 0.03 0.8

sample Dilution factor
(Dilution Factor) density
(μg / ml) volume
(ml) BMP7 complex
(mg) Recovery rate
(%) Cellulphin sulfate elution 80000 128.69 25 3.22 100.0 Butyl 4 FF unbound 80000 90.94 25 2.27 70.7 Butyl 4 FF Elution 32000 24.74 25 0.62 19.2

sample Dilution factor
(Dilution Factor) density
(μg / ml) volume
(ml) BMP7 complex
(mg) Recovery rate
(%) Cellulphin sulfate elution 80000 128.69 25 3.22 100.0 Butyl HP unbound 32000 14.52 30 0.44 13.5 Butyl HP elution 160000 134.59 20 2.69 83.7

As shown in Tables 5 and 6, the recovery rates of the BMP7 complexes were only 0.8% and 19.2%, respectively, despite the different kinds of resins in the hydrophobic interaction chromatography used in the second purification step.

In contrast, as shown in Table 7, in the hydrophobic interaction chromatography using butyl HP according to the present invention, the yield of the BMP7 complex was as high as 83.7%. The reason for the difference from the recovery rate calculated in Example 1 is considered to be that the expression of BMP7 protein in the starting CHO cell culture medium changes depending on conditions.

Example  6

This example is for confirming safety to the eye when the final product produced according to the present invention is administered to the eye.

The final product produced according to the present invention is a complex of BMP7 pro domain and mature BMP7 dimer. When the complex is administered to the eye to inhibit or reduce scarring and scarring of the eyeball or cornea, safety to the eye should be ensured. To confirm this, experiments were carried out to measure the cell survival rate when epithelial cells were treated with each test group.

The test groups are as follows, n = 5 respectively:

● Group 1 - ATDC5 epithelial cells only (control group)

● Group 2 - saline (0.9% NaCl)

Group 3 - New stable (1% mannitol, 2% glycine, 0.01% polysorbate 80 - pH 7.3)

Group 4 - Complexes of BMP7 prodomain and mature BMP7 dimer diluted to a concentration of 2 [mu] g / ml in fresh stable

Group 5 - Complexes of BMP7 prodomain and mature BMP7 dimer diluted to a concentration of 0.66 [mu] g / ml in fresh stable

Group 6 - complex of BMP7 prodomain and mature BMP7 dimer diluted to a concentration of 0.22 μg / ml in fresh stable

Group 7 - Normal stabilizer (1% mannitol, 1% sucrose, 3% arginine, 0.1% histidine, 0.01% CaCl 2, 0.01% polysorbate 80 - pH 7.3)

Group 8 - mature BMP7 dimer diluted to a concentration of 2 [mu] g / ml in normal stability

Group 9 - Matured BMP7 dimer diluted to a concentration of 0.66 μg / ml in normal stability medium

Group 10 - mature BMP7 dimer diluted to a concentration of 0.22 μg / ml in normal stability

ATDC5 epithelial cells were seeded at a concentration of 10,000 cells / well in a 96-well plate and cultured for 24 hours. Then, each test group was treated with 10 μl each. Then, the absorbance at 450 nm was measured at 1 hour, 3 hours, 5 hours, and 24 hours. From this, viable cell numbers of ATDC5 epithelial cells were measured and shown in Table 8 below, and the survival rate was calculated and shown in FIG.

Test group 1hr 3hr 5hr 24hr One 100,000 100,000 100,000 100,000 2 94,466 93,519 83,551 88,925 3 97,628 88,580 85,117 83,949 4 101,581 89,815 86,423 86,517 5 100,000 77,778 83,029 74,478 6 96,443 78,395 83,029 76,565 7 101,976 90,432 87,467 83,628 8 104,348 95,062 87,206 72,713 9 105,534 92,593 88,512 78,491 10 108,300 88,272 76,240 79,775

The stabilizers used in the groups 7 to 10 are conventionally used and include arginine. Arginine can be used as an injectable agent, but there are limitations to its use as eye drops against the eye. Therefore, when the final product according to the present invention is used as a therapeutic agent for corneal scarring or as a scar formation inhibitor, it is troublesome that the stabilizer buffer containing arginine is used in a limited manner or an experiment for securing safety to human body must be performed separately do. For this reason, arginine was excluded in the stabilizers used with the final product according to the present invention, i.e. in the third to sixth groups.

As can be seen from Table 8 and FIG. 9, when the materials of Groups 3 to 6 were treated with epithelial cells, the cell survival rate was similar to that of cells treated with the materials of Groups 7 to 10, , The safety of the cells is considered to be satisfactory.

In addition, when compared to Group 4 and Group 8, cell survival rates were similar at 1 hour, 3 hours, and 5 hours, but at 24 hours, cell survival rate in Group 4 Respectively. Considering these points, it is confirmed that the end product according to the present invention is excellent in safety against cells even when contained in the novel stable liquid, and that the new stable liquid does not contain arginine.

Claims (9)

A method of producing a complex of BMP7 prodomain and mature BMP7 dimer, comprising the steps of:
a) applying affinity chromatography using a cellulphin sulfate resin to a solution containing BMP7, wherein said affinity chromatography comprises i) equilibrating the column with a buffer containing histidine and NaCl, ii) , Iii) washing the column with a buffer containing histidine, NaCl and arginine hydrochloride, and iv) eluting the column with a buffer containing histidine, NaCl and arginine hydrochloride ; And
b) eluting the complex of BMP7 prodomain and mature BMP7 dimer by applying hydrophobic interaction chromatography using a butyl HP resin to the eluate obtained in step a), wherein the hydrophobic interaction chromatography comprises: i) And NaCl; ii) loading the eluate obtained in step a) into the column; iii) washing the column with a buffer containing histidine and NaCl; and iv) And eluting the column with a buffer containing histidine, NaCl and propylene glycol. The method according to claim 1,
Wherein the complex has a form in which the BMP7 prodomain is linked to the mature BMP7 dimer through a non-covalent bond. The method according to claim 1,
Wherein the solution containing BMP7 is a cell culture solution. The method of claim 3,
Wherein the cell culture liquid is collected after being cultured for 3 to 7 days. The method according to claim 1,
Further comprising, prior to step b), filtering the effluent obtained in step a). The method according to claim 1,
Wherein the complex eluted in step (b) is contained in a stable liquid and is used for application to the eye or cornea. The method according to claim 6,
Characterized in that the stabilizing liquid does not contain arginine but contains glycine. delete delete

Images