METHOD FOR PRODUCING HIGH-CONCENTRATION COLLAGEN FOR USING AS MEDICAL MATERIAL

Abstract

Disclosed is a method of producing high-concentration collagen for use as a medical material, including: washing tissue of a mammal; subjecting the washed tissue to crushing and immersion in ethyl alcohol; subjecting the tissue to enzymatic treatment with stirring in purified water containing phosphoric acid and pepsin; adding sodium chloride to the collagen subjected to enzymatic treatment, performing stirring, and aggregating collagen; dissolving the aggregated collagen in purified water to give a collagen solution, which is then filtered using a filter and concentrated by removing the pepsin, low-molecular-weight material, and sodium chloride from the collagen solution using a tangential flow filtration device; subjecting the concentrated collagen to sterile filtration, aggregating the collagen using a pH solution in a neutralization tank, and concentrating the collagen by removing a non-aggregated solution; and concentrating the concentrated collagen using a centrifuge and stirring the concentrated collagen using a mixer.

Claims

1. A method of producing high-concentration collagen for use as a medical material, comprising: washing tissue of a mammal; subjecting the washed tissue to crushing and immersion in ethyl alcohol; subjecting the tissue to enzymatic treatment with stirring in purified water containing phosphoric acid and pepsin; adding sodium chloride to the collagen subjected to enzymatic treatment, performing stirring, and aggregating collagen; dissolving the aggregated collagen in purified water to give a collagen solution, which is then filtered using a filter and concentrated by removing the pepsin, low-molecular-weight material and sodium chloride from the collagen solution using a tangential flow filtration device; subjecting the collagen concentrated using the tangential flow filtration device to sterile filtration, aggregating the collagen using a pH solution in a neutralization tank, and concentrating the collagen by removing a non-aggregated solution; and concentrating the concentrated collagen using a centrifuge and stirring the concentrated collagen using a mixer.

2. The method of claim 1, wherein the crushed tissue of the mammal is immersed in 70% ethyl alcohol for at least 72 hr and is then reacted with pepsin for at least 72 hr in a solution titrated to a pH of 1.5˜2.5 so that a virus is inactivated and collagen is extracted.

3. The method of claim 1, wherein the collagen subjected to enzymatic treatment is added with sodium chloride at a concentration of 0.5˜0.9 M and stirred to thus aggregate collagen, the non-aggregated solution is removed, and the aggregated collagen is dissolved in purified water and then filtered using a filter having a pore size of 2.0˜0.5 μm.

4. The method of claim 1, wherein the tangential flow filtration device comprises a 50˜150 kDa molecular-weight-cutoff filtration membrane, purified water is added in an amount corresponding to an amount of the solution that is removed so as to maintain flowability, materials smaller than a pore size of the filtration membrane are removed, and collagen is concentrated to 5 mg/mL or less so as to pass through a filter having a pore size of 0.22 μm.

5. The method of claim 1, wherein the collagen concentrated using the tangential flow filtration device is allowed to stand for 4 hr˜24 hr under a condition that the collagen is adjusted to a neutral pH (6.0˜8.0) and is maintained at a temperature of 25˜35° C., whereby the aggregated collagen is recovered and centrifuged using a centrifuge, thereby being concentrated.

6. The method of claim 1, wherein the collagen concentrated through neutralization is centrifuged at a gravitational acceleration of 4,000˜6,000 g, thereby being concentrated, and is stirred using a mixer, thus obtaining high-concentration collagen of 120 mg/mL.

Description

DESCRIPTION OF DRAWINGS

[0029] FIG. 1 shows a process of filtering collagen using a tangential flow filtration (TFF) device according to the present invention;

[0030] FIG. 2 shows a tangential flow filtration membrane according to the present invention;

[0031] FIG. 3 shows the structure of a neutralization tank according to the present invention; and

[0032] FIG. 4 shows a mixer.

DESCRIPTION OF THE REFERENCE NUMERALS IN THE DRAWINGS

[0033] 10: tangential flow filtration membrane device 11:

[0034] storage tank

[0035] 12: pump 13: tangential flow filtration membrane

[0036] 14: valve 15: pressure gauge

[0037] 16: recovery of non-passed material 17: discarding of passed material

[0038] 18: purified water

[0039] 21: solution flow direction 22: pressure direction

[0040] 23: tangential flow filtration membrane 24: non-passed material

[0041] 25: passed material 31, a: gasket

[0042] 32, b: fastener 33: pH titration solution inlet

[0043] 34: pH electrode c: rotary shaft sealing device

[0044] d: pH electrode

BEST MODE

[0045] The present invention is characterized in that high-concentration liquid collagen, the biological safety of which is ensured and which has high purity, is extracted from the tissue of a mammal so as to be used as a medical material.

[0046] Specifically, the tissue of a mammal is washed with clean water and alcohol and then kept frozen. As such, the water is preferably purified water from which microorganisms and ions have been removed, and the alcohol is preferably 70% ethyl alcohol for disinfection.

[0047] Before extraction, animal tissue is pretreated in a manner of finely crushing the tissue using a crusher and then immersing the tissue in 70% ethyl alcohol for 24 hr.

[0048] The pretreated animal tissue is rinsed with purified water and then extracted.

[0049] For primary extraction, enzymatic treatment is performed by placing the tissue and a protease in purified water at a pH of 1.5 to 2.5 and then performing stirring for 72 hr or more.

[0050] Here, pH titration may be carried out using any acidic solution such as phosphoric acid or hydrochloric acid, and the protease is preferably pepsin, which is able to remove the terminus of collagen for causing an immune response in the human body without damaging the triple-helix structure of collagen.

[0051] If the pH for enzymatic treatment is less than 1.5, it is unsuitable for chemical resistance of a filter for use in the subsequent filtration process. On the other hand, if the pH exceeds 2.5 or the reaction time is less than 72 hr, the virus inactivation effect may decrease.

[0052] The pretreatment process including immersion in ethyl alcohol for 72 hr or more and the enzymatic treatment process at a pH of 2.5 or less for 72 hr or more enable the inactivation of viruses that may be present in the animal tissue, thus ensuring biological safety.

[0053] For secondary extraction and purification, the solution subjected to enzymatic treatment undergoes a salt treatment process in a manner such that it is reacted with sodium chloride at a concentration of 0.5˜0.9 M. This is because each protein is able to aggregate at a specific salt concentration and also because collagen may aggregate and float at the above salt concentration, whereby the other non-aggregated impurities are discarded to thus increase the purity of collagen.

[0054] The aggregated collagen is dissolved again in purified water so as to perform a filtration process.

[0055] For tertiary purification, the solution is first filtered using a filter having a pore size of 2.0˜0.5 μm, and is then treated using a TFF device so that low-molecular-weight materials such as pepsin, sodium chloride, etc. may be removed from the solution.

[0056] The TFF device preferably includes a 50˜150 kDa molecular-weight-cutoff (MWCO) filtration membrane. The 150 kDa or less MWCO filtration membrane functions to prevent the loss of collagen of about 300 kDa, and the 50 kDa or more MWCO filtration membrane functions to remove pepsin of about 35 kDa.

[0057] The filtration process using the TFF device is performed using a storage tank 11, a pump 12, a filtration membrane 13, a pressure gauge 15 and a valve 14. The solution in the storage tank is transferred to the filtration membrane via the pump so that collagen larger than the pore size of the filtration membrane is not passed but is recovered into the storage tank and also so that impurities smaller than the pore size are passed through the pores and thus removed. The purified water is added in an amount corresponding to the amount of the impurities removed through the pores in the filtration membrane. This procedure is repeated, whereby the purity of collagen may be increased while maintaining the flowability of the collagen solution in the storage tank (FIGS. 1 and 2). The results of purification using the TFF device may be confirmed through various methods, among which the removal of sodium chloride can be found through osmotic measurement (Example 3).

[0058] The TFF device is used for a concentration process, in addition to the purification process. When the filtration is repeated without the addition of purified water at a time point at which the removal of impurities is completed, water is removed via the pores, and thus the amount of collagen in the storage tank is increased. Accordingly, the amount of collagen may be concentrated to 10 mg/mL, and collagen thus obtained may be utilized in fields that do not require high concentration, such as those of cosmetic materials.

[0059] However, since the viscosity of collagen is increased with an increase in the concentration thereof, when concentration process to 10 mg/mL or more is carried out, collagen may accumulate on the filtration membrane, and the flowability of the collagen solution may significantly decrease, thus lowering the yield and requiring a long processing time. In order to produce collagen for medical use, it is concentrated to 5 mg/mL or less, thereby ensuring flowability and viscosity suitable for sterile filtration.

[0060] After the completion of purification and concentration using the TFF device, the resulting solution is filtered through a filter having a pore size of 0.22 μm and then transferred to a neutralization tank. Here, the sterile filter and the neutralization tank should be in a sterile state. In particular, the neutralization tank should be able to be sealed using a gasket 31 and a fastener 32 and should include a pH titration solution inlet, a pH measurement electrode, and a jacket for adjusting the temperature of the solution (FIG. 3).

[0061] The collagen solution from which microorganisms have been removed through sterile filtration is purified and concentrated once more using a neutralization process. The neutralization process is performed in a manner in which the pH of the collagen solution is adjusted to 6.0˜8.0 using a pH titration solution such as sodium hydroxide or hydrochloric acid and the temperature of the solution is adjusted to 25˜35° C. to thereby aggregate collagen.

[0062] During the neutralization process, if the pH of the solution falls out of the above range or the temperature of the solution is low, collagen may not sufficiently aggregate. On the other hand, if the temperature of the solution is high, the triple-helix structure may unravel due to thermal degradation.

[0063] Collagen is aggregated through the neutralization process, and the solution, which is not aggregated, is removed, whereby the amount of a concentrating target may be decreased before a concentration process using centrifugation, making it easy to separate water and collagen from each other, and the concentration process may be performed within a short time using a low centrifugal force, ultimately constructing an apparatus that enables mass production at relatively low cost.

[0064] The collagen, which is aggregated through neutralization, is placed in a sterilized centrifugation vessel, and is then concentrated for 5 min at a gravitational acceleration of 4,000˜6,000 g using a centrifuge.

[0065] If the above process is conducted using a lower centrifugal force or for a shorter time, the yield of collagen may decrease or the final concentration value may decrease. On the other hand, if higher gravitational acceleration is applied for a long time, a high-performance expensive centrifuge suitable therefor is required, and the production time may increase.

[0066] The collagen thus concentrated is placed in a mixer (FIG. 4) having a closed structure and is then homogeneously mixed, thus obtaining high-concentration collagen for use in a medical material. Here, the concentration of collagen may be adjusted by adding water from which microorganisms and exothermic materials have been removed, depending on the desired purposes, and collagen having a high concentration of 120 mg/mL may be obtained under the above conditions.

[0067] During the above mixing process, an acidic solution such as hydrochloric acid is added as the pH titration solution so that the pH of the solution is adjusted to 1.5˜5.5. The solution having an adjusted pH may be stored stably in a homogenous state for a long period of time. If the pH thereof is too low, it is difficult to titrate the pH to a neutral value in order to prepare a medical product. On the other hand, if the pH thereof is higher than 5.5, collagen may aggregate again, making it difficult to maintain a uniform concentration.

[0068] Depending on the needs, the collagen, obtained through extraction, purification, concentration and mixing processes, may be mixed and diluted with a mixing solvent such as water, an isotonic solution, platelet-rich plasma (PRP), or hyaluronic acid to thus give a liquid product, and the concentrated collagen may be provided in the form of a solid product through lyophilization.

[0069] In particular, a liquid product may be provided in a state in which biological safety is ensured in the absence of viruses and microorganisms within a minimal clean-room facility that enables mixing, filling, and packing.

EXAMPLE 1

[0070] Preparation and Use of Medical Collagen Using Pig Skin Tissue

[0071] 1) Pig skin tissue is washed with purified water and alcohol and then kept frozen at −20° C. or lower.

[0072] 2) The pig skin tissue is finely crushed.

[0073] 3) The crushed skin tissue is immersed in 70% ethyl alcohol for 24 hr.

[0074] (Primary Virus Inactivation)

[0075] 4) The tissue is rinsed by being placed in purified water, titrated to an acidic pH (pH 1.5˜2.5) using phosphoric acid, and is then reacted with stirring for 72 hr or more by the addition of pepsin. (Secondary virus inactivation)

[0076] Here, the amount of pepsin is ¼˜ 1/10 of the weight of the skin tissue.

[0077] 5) The collagen is added with sodium chloride at a concentration of 0.5˜0.9 M, stirred and aggregated, after which the non-aggregated solution is removed.

[0078] 6) The aggregated collagen is dissolved in water titrated at a pH of 1.5˜4.0 and then filtered using a filter having a pore size of 2.0˜0.5 μm.

[0079] 7) In order to obtain collagen suitable for medical use, low-molecular-weight materials such as pepsin and sodium chloride are removed from the solution using a TFF device.

[0080] The TFF device preferably includes a 50˜150 kDa MWCO filtration membrane, and purified water is added in an amount corresponding to the amount removed through the pores of the filtration membrane so that the amount of the collagen solution in the storage tank is maintained to thus ensure flowability.

[0081] 8) After the completion of removal of low-molecular-weight materials such as pepsin and sodium chloride, the supply of purified water is stopped and TFF is maintained, and thus a concentration process is carried out. As such, the concentration of collagen is adjusted to 5 mg/mL or less so as to pass through a filter having a pore size of 0.22 μm.

[0082] 9) To remove microorganisms, filtration is performed using a filter having a pore size of 0.22 μm.

[0083] 10) The solution from which microorganisms have been removed is transferred to a sterilized neutralization tank. The neutralization tank has to be sterilized and has to have a sealed structure able to maintain a sterile state, and includes a pH titration solution inlet and a pH measurement electrode.

[0084] 11) To aggregate collagen, the collagen solution is allowed to stand for a time period ranging from 4 hr to one day under the condition that it is titrated to an approximately neutral pH (pH 6.0˜8.0) using hydrochloric acid (HCl) and a sodium hydroxide solution (NaOH) and the temperature thereof is adjusted to 25˜35° C.

[0085] 12) The non-aggregated solution is discarded and the aggregated collagen is placed in a centrifugation vessel and then in a centrifuge, and is centrifuged at a gravitational acceleration of 4,000˜6,000 g, thereby concentrating collagen.

[0086] 13) Water is decanted from the centrifugation vessel and the concentrated collagen, having been separated from the water, is placed in a mixer and then stirred, thus obtaining collagen having a concentration of about 120 mg/mL.

[0087] As such, the collagen thus obtained is mixed with an acidic solution such as hydrochloric acid to adjust the pH thereof to 1.5˜5.5, whereby the collagen may be stored at a homogeneous concentration.

[0088] 14) The collagen, from which microorganisms have been removed and which has high purity, is prepared in a state in which the properties of collagen are maintained, using the above method, and is thus suitable for medical use, and may have a high concentration of 120 mg/mL and may thus be applied to various products.

[0089] For example, collagen may be provided in the form of a product that may be injected into the body in a manner in which the collagen is mixed with an additive so as to possess the same composition as the saline in the human body while maintaining a liquid phase and is then charged in a pre-filled syringe.

EXAMPLE 2

[0090] Verification of Virus Inactivation

[0091] The virus inactivation process during the production process of Example 1 was performed through three simulations to thus verify the virus inactivation.

[0092] Adopted as indicator viruses were PEDV (Porcine epidemic diarrhea virus), PRV (Porcine rotavirus), PPV (Porcine parvovirus), and Pseudorabies virus, depending on the genotypes, presence or absence of enveloped lipids, and resistivity.

[0093] The adopted four kinds of viruses are inoculated into the crushed pig tissue and then incubated in 70% ethyl alcohol for 24 hr and in an acidic solution having a pH of 2.5, adjusted by the use of phosphoric acid, for 72 hr in order to reproduce the pretreatment and enzymatic treatment procedures of the actual preparation method.

[0094] The samples in which the virus inactivation process was reproduced are compared before and after treatment through quantitative viral analysis.

[0095] The results of three simulations under the same conditions were the same. All viruses were detected to a detection threshold or less, and the log reduction factor for each virus inactivation process was measured to be a 2-log reduction factor or more corresponding to a typical standard that is regarded as effective for virus inactivation. The maximum cumulative log reduction factors of PEDV, PRV, PPV, and Pseudorabies virus per process were 6.75, 9.75, 8.75, and 8.75, respectively, from which strong virus inactivation effects can be concluded to have been exhibited.

TABLE-US-00001 TABLE 1 Reduction factor (Log.sub.10TCID.sub.50) Preparation process PEDV PRV PPV Pseudorabies virus Pretreatment (70% ethyl ≧4.05 ≧5.55 ≧5.05 ≧5.05 alcohol, 24 hr) Enzymatic treatment ≧2.70 ≧4.20 ≧3.70 ≧3.70 (pH 2.5, 72 hr) Maximum cumulative ≧6.75 ≧9.75 ≧8.75 ≧8.75 log reduction factor

EXAMPLE 3

[0096] Removal of Sodium Chloride Using TFF

[0097] After the enzymatic treatment, treatment with sodium chloride, and filtration using a filter having a pore size of 2.0˜0.5 μm of Example 1, TFF was conducted using a 100 kDa MWCO filtration membrane.

[0098] Here, purified water was added in an amount corresponding to the amount removed through the pores in the filtration membrane, and this procedure was repeated, whereby the flowability of the collagen solution in the storage tank was ensured and low-molecular-weight materials such as pepsin and sodium chloride were removed, and the total amount of purified water that was added was five times the initial amount of the collagen solution.

[0099] The above TFF process was repeated three times and the results of removal of low-molecular-weight materials were verified by measuring osmotic pressure using sodium chloride, chosen because it is a typical material.

[0100] Consequently, individual removal rates were 96.2%, 96.1%, and 96.4%, and the average removal rate was determined to be 96.2%. Thereby, the removal of low-molecular-weight materials via TFF is effective, and sodium chloride shows an osmotic pressure of 31˜33 mOsm, which is lower than 285 mOsm, which is the plasma osmotic pressure, and may be used as a medical material in a mixture with an isotonic solution, as necessary.

TABLE-US-00002 TABLE 2 Osmotic pressure (mOsm) Case Before TFF After TFF Removal rate 1 840 32 96.2% 2 846 33 96.1% 3 861 31 96.4% Average 96.2%

EXAMPLE 4

[0101] Collagen concentration by stepwise concentration process

[0102] According to the preparation method of Example 1, 5 kg of pig skin and 1 kg of pepsin were used, and the concentration of collagen for each step was measured when a concentration process was performed to 1/2 of the initial amount upon TFF.

[0103] The concentration from the enzymatic treatment process to the filtration process using a filter having a pore size of 2.0˜0.5 μm fell in the range of 1.6˜2.4 mg/mL for each case, and the concentration was 3.2˜4.8 mg/mL after the removal of low-molecular-weight materials during TFF.

[0104] This is performed to realize passage through a sterile filtration filter having a pore size of 0.22 μm after TFF. When the concentration is adjusted to 2 mg/mL or less, the amount of the collagen solution is increased and thus the volume of the neutralization tank is enlarged. After the neutralization process, a long period of time is required to perform a concentration process using centrifugation, ultimately increasing production costs. On the other hand, when the concentration of the collagen solution was adjusted to 5 mg/mL or more, viscosity is increased, making it difficult to pass through the sterile filter having a pore size of 0.22 μm, whereby the sterile filter may be clogged early, undesirably causing the loss of collagen and using a large filter. For this reason, the concentration value before a sterile filtration process is preferably set to the range of 2˜5 mg/mL.

[0105] The water, which was not aggregated during the neutralization process, was primarily removed, whereby collagen could be concentrated to 5.6˜6.7 mg/mL from 3.2˜4.8 mg/mL. Accordingly, collagen is concentrated about 1.4- to 1.8-fold compared to the concentration value before the neutralization process. Thus, during the concentration process using centrifugation, initial capacity may be reduced, advantageously resulting in industrial applicability in which the centrifuge capacity and the processing time may be reduced.

[0106] Finally, the collagen solution aggregated after the neutralization process is concentrated using centrifugation, whereby high-concentration collagen may be obtained in the concentration range of 114.6˜122.3 mg/mL for each case, which is suitable for use as a material for a medical liquid collagen product having a concentration of 30˜60 mg/mL and which is also able to reduce the capacity of a lyophilizer and the operating time thereof when a solid formulation is produced using lyophilization.

TABLE-US-00003 TABLE 3 Production Collagen concentration per production process (mg/mL) process A B C D Case 1 1.6 3.2 5.6 114.6 Case 2 2.4 4.8 6.7 122.3 Case 3 2.1 4.1 5.9 121.1 * Note) A: enzymatic treatment~filtration (with a pore size of 2.0~0.5 μm) B: tangential flow filtration (concentration process after removal of low-molecular-weight material) C: neutralization (after removal of non-aggregated solution) D: concentration process using centrifugation

[0107] Although the specific embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, equivalents and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.