Method for producing high-concentration collagen for using as medical material
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.
1. A method of producing high-concentration collagen for use as a medical material, the method comprising the sequential steps of:
(a) washing tissue of a mammal;
(b) crushing the washed tissue, immersing the crushed tissue in 70% ethyl alcohol for 24 hours, placing the tissue to enzymatic treatment with stirring in purified water titrated to a pH of 1.5-2.5 using phosphoric acid, and allowing the tissue to react with an amount of pepsin having ¼- 1/10 of a weight of the tissue by stirring for at least 72 hours, thereby inactivating viruses and extracting collagen;
(c) adding sodium chloride to the extracted collagen at a concentration of 0.5-0.9 M, performing stirring, aggregating collagen, and removing a non-aggregated solution;
(d) dissolving the aggregated collagen in purified water to give a collagen solution, filtering using a filtration membrane having a pore size of 0.5-2.0 μm and concentrated by removing low-molecular-weight materials having a smaller size than pores of the filtration membrane, including pepsin and the sodium chloride, from the collagen solution using a tangential flow filtration device;
(e) subjecting the collagen concentrated using the tangential flow filtration device to sterile filtration, aggregating the collagen by adjusting pH to 6.0-8.0 in a neutralization tank, and concentrating the collagen by removing a non-aggregated solution; and
(f) further concentrating the concentrated collagen using a centrifuge, and then stirring the concentrated collagen using a mixer,
wherein the collagen concentrated through neutralization is centrifuged at a gravitational acceleration 4,000-6,000 g producing the concentrated collagen, and is stirred using a mixer, thus obtaining high-concentration collagen of at least 120 mg/mL.
2. 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, the low-molecular-weight 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 allow the low-molecular-weight materials to pass through a filter having a pore size of 0.22 μm.
3. The method of claim 1 , wherein the concentrated collagen produced using the tangential flow filtration device is allowed to stand for 4-24 hours 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.