CA1104127A - Method for preparing pyrogen free collagen - Google Patents
Method for preparing pyrogen free collagenInfo
- Publication number
- CA1104127A CA1104127A CA307,883A CA307883A CA1104127A CA 1104127 A CA1104127 A CA 1104127A CA 307883 A CA307883 A CA 307883A CA 1104127 A CA1104127 A CA 1104127A
- Authority
- CA
- Canada
- Prior art keywords
- corium
- collagen
- aqueous
- hypochlorite
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 239000002510 pyrogen Substances 0.000 title claims abstract description 27
- 102000008186 Collagen Human genes 0.000 title claims abstract description 23
- 108010035532 Collagen Proteins 0.000 title claims abstract description 23
- 229920001436 collagen Polymers 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000005406 washing Methods 0.000 claims abstract description 14
- 239000000126 substance Substances 0.000 claims abstract description 11
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- 229910052784 alkaline earth metal Inorganic materials 0.000 claims abstract description 5
- -1 alkaline earth metal hypochlorites Chemical class 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 23
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Inorganic materials Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 claims description 22
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 19
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 229960005076 sodium hypochlorite Drugs 0.000 claims 8
- 238000012545 processing Methods 0.000 abstract description 4
- 239000003513 alkali Substances 0.000 abstract description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 22
- 229960005188 collagen Drugs 0.000 description 14
- 239000012153 distilled water Substances 0.000 description 11
- 239000000463 material Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011046 pyrogen test Methods 0.000 description 3
- 230000001698 pyrogenic effect Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000003899 bactericide agent Substances 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 229940127554 medical product Drugs 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000490025 Schefflera digitata Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 210000003372 endocrine gland Anatomy 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 235000015250 liver sausages Nutrition 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09H—PREPARATION OF GLUE OR GELATINE
- C09H1/00—Pretreatment of collagen-containing raw materials for the manufacture of glue
- C09H1/04—Pretreatment of collagen-containing raw materials for the manufacture of glue of hides, hoofs, or leather scrap
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Materials For Medical Uses (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Treatment And Processing Of Natural Fur Or Leather (AREA)
- Compositions Of Macromolecular Compounds (AREA)
- Cosmetics (AREA)
- Peptides Or Proteins (AREA)
- Detergent Compositions (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Abstract of the Disclosure Collagen, such as corium, is freed of pyrogenetic substances by processing green corium with aqueous solutions of alkali or alkaline earth metal hypochlorites, washing with aqueous ethanol and subsequently washing with pyrogen free water.
Description
~4~2~
This ln~ention relates to the manufacture of pyrogen-free collagen, such as, for example, ~orium, adapted for use in the manufacture of collagen derived products intended for medical uses, particularly in surgical procedures.
In the preparation of a wide ~ariety of collagen derived products for medical uses, it is necessary that the products be free of microorganisms, such as bacteria, yeasts and molds. As is well known, these microorganisms may be destroyed or rendered innocuous readily by sterilization, as by subjecting the collagen source material and/or collagen derived product to radiation, bactericides, moldicides, various gases and heat treatment.
Pyrogens, on the other hand, are not living organisms and are not rendered innocuous by bactericides, moldicides and gases, and are thermostable. Pyrogens are generally considered to be thermostable products o-E the growth of strains of bacteria, yeasts and molds, some being soluble and others being insoluble and filterable. In addition to their fever producing effects, pyrogens have physiologic effects on the circulatory system, the endocrine glands and metabolic processes. The rise in body temperature is only one of the manifestations to the introduction of minute quantities of pyrogenetic substances into the animal ~ody and the specific effects will be dependent upon the individual subject. Hence, it is essential that pyro-geneti~ substances be remo~ed. The microorganiqms may be rendered innocuous by a sterilization treatment of the final product.
B
Animal hides constitute the principal source of corium, commonly termed green corium, the collagen raw material for the preparation of various medical grade products. For example, a hemostatic mass of fibers consisting of ioniæable, partial salts of collagen con-taining about 50 to about 90% of the stoichiometric amounts of acid, calculated as ~ICl, may be prepared from such corium, as described in the U. S. patent No. 3,7~2,955, dated July 3, 1973.
In accordance with the present invention, the hides and the collagen raw material, such as corium, derived from the hides and contaminated with microorganisms are processed to produce pyrogen free collagen by mechanically removing foreign substances from the surfaces of the col-lagen while in contact with an aqueous solution of alkali or alkaline earth metal hypochlorite, for example, sodium hypochlorite, washing the collagen with an aqueous ethanol solution, and thereafter washing the collagen with pyrogen free water.
In a practical operation, the hides following skinning are trimmed to remove tails and pates and washed, as in a drum washer, with copious amounts of water containing sodium hypochlorite so as to remove dirt and other foreign matter.
The water may contain from 50 to 250 ppm, preferably 50 to 100 ppm, of sodium hypochlorite. The hypochlorite may be obtained from commercial solutions generally containing 5.25% sodium hypochlorite. A~ter the hides are clean, they may be transferred from the drum washer to a holding tank and maintained in water containing from about 50 to 100 ppm sodium hypochlorite until a batch of a desired quantity is obtained. The green corium is then obtained by removal of the flesh and grain sides of the hide as by splitting operations. The foregoing processing is performed at an abattoir. Generally, the preparation of the medical product is conducted at a location remote from the abattoir.
In such instance, the corium is blast frozen and transported in the frozen state to the desired location f~r conversion into a desired product.
The hypochlorite treatment at the concentrations utilized in producing the corium contro~s the prolifera-tion of bacterial growth before freezing. It is necessary that the subsequent processlng of the corium into the source material for the preparation of the final product be effected under treatment conditions to produce a pyrogen free source material. Where the corium is received in a frozen state, it is thawed by placing in preferably pyrogen free, distilled water which may contain between about 50 and 250 ppm, preferably 50 to 100 ppm, sodium hypochlorite, the solution having a pH between 7 and 9, preferably pH 7-8. Obviously, where the corium has not been frozen it is maintained in such solution. The sur-faces of the sheets are then scraped mechanically while in contact with the hypochlorite solution so as to loosen and/or remove substances such as dirt, loose protein and other foreign material on or embedded in the surfaces. A
convenient method of scraping involved brush washing by drawing the corium sheet between -two rotating, stiff bristle brushes (stiff nylon bristles) under a copious stream of pyrogen free, distilled water, preferably con-taining the hypochlorite.
Following scraping or brush washing the sheets maybe cut into s~rips for convenience in subse~uent process-ing. ~he sheets or strips are rinsed thoroughly with pyrogen free distilled water containing the h~pochlorite and placed in such solution until required for use.
In the preparation of the collagen derived products, the sheets are removed from the hypochlorite solution and the coriu~ separated from excess solution, as by pressing or centrifugation. The corium is washed, as by immersing in a mixture of ethanol and water containing preferably from 30 - 90% ethanol by volume, more preferably containing about 70 volumes of ethanol and 30 volumes of pyrogen free, distilled water, and the mass agitated for about 15 to 30 minutes. The excess liquid is separated from the cor,ium and treatment with the aqueous alcohol repeated at least once. Preferably, -three such aqueous alcohol treatments or washing s-teps are utilized. ~he corium is finally washed with copious amounts of pyrogen free water and is used in the preparation of the desired products~ All processing is preferably conducted at temperatures not exceeding about ~0C., more preferably 'be-tween -the freezing point of the liquid and about 25C.
The removal of pyrogenetic substances from ~reen corium received in frozen state from an abattoir is illustrated by the following e~amples:
Example 1 A 100 gm~ sample of thawed, green corium strips (35%
solids) was placed in 2000 gm pyrogen free, distilled water to which had been added 1.96 gm of a 5.25% aqueous solution of sodium hypochlorite~ The solution contained approximately 50 ppm sodium hypochlorite and was at a temperature of about 35C. I~he mass was stirred gently for about 15 minutes. The skrips were scraped with a knife edge, washed with a like solution and placed in a fresh solution of sodium hypochlorite. The strips were removed subsequently, squeezed to remove excess liquid and used in performing a pyrogen test.
Example_2 A 200 gm. sample of thawed, green corium strips (35%
solids) was placed in 1870 gm pyrogen free, distilled water containing approximately 50 ppm sodium hypochlorite at a temperature of about 35C. and the mass stirred gently for about 15 minutes. The strips were scraped with a knife edge, washed with a like solution, and placed in a fresh solution of sodium hypochlorite. The strips were removed, ~xcess liquid expressed by squeezing and the strips placed in 2000 ml of a mixture containing 70 volumes of ethanol and 30 volumes of pyrogen free, dis-tilled water. The mass was stirred for about 15 minut~s, the strips removed, squeezed to express excess liquid an placed in a fresh ethanol-water mixture. The mass was stirred for about 30 minutes, the strips removed subse-quently, squeezed and used in performing a pyrogen test~
The conventional test for pyrogens utilized the rabbit as the test animal and three rabbits are used in the test.
After establishin~ the normal temperature of each rabbit, 10 ml of a fluid to be tested per kg of body weight of the rabbit are injected into one marginal ear vein of the rabbit. The change in the rabbit temperature is deter-mined at l-hour intervals -for three hours~ A positive test results if any rabbit develops an individual temperature rise of 0~6CO or more at the end of any hour or if the total of the three rabbits' temperature elevation exceeds In the pyrogen test of the material prepared as des cribed in Example 1, a 10 gm sample of the treated corium was rins~d in 100 ml portions of pyrogen free, distilled water four times. The fourth rinse water was heated to 37C. prior to injecting 10 ml. of the rinse water per kg of body weight into each of thxee rabbits. The change in temperature as compared to the normal temperature of each rabbit was noted at l-hour intervals for each rabbit and is reported in Table 1 which follows:
Table 1 Temperature Chanqe, C.
Total Max.
Rabbit 1st hr. 2nd hr. 3rd hr. Change ~ +0.~ +0.6 +Oq5 +1.6 +0.6 B +0.2 +0.1 -0.1 +0.2 +OL20 C ~0.6 +0.6 +0.4~ +1~ ~0.6 Total for 3 rabbits +3.4 The test indicates the material to be pyrogenic.
Washing of the hypochlorite treated corium with pyrogen free water does not remove the pyrogenetic substances.
In a similar manner a 10 gm. sample of the material as described in Example 2 that had been subjected to the aqueous ethanol washing after contact with the hypochlorite solution was rinsed in 100 ml portions of pyrogen ~ree distilled water four time.s~ The fourth rinse water was heated to 37C prior to injecting 10 ml of the rinse water per kg of body weight into each of three rabbits and the s~
;27 change in the temperature of each rabbit noted as described above. The results are set forth in Table 2 which follows:
Table 2 Temperature Chan~, C.
Total Max.
Rabbit 1st hr. 2nd hr. 3rd hr. Chanqe Chanqe D +0.1+0.2 0.0 +0.3 +0.2 E 0.0~0.1 +0.1 ~0.2 ~0.1 0.00.0 ().0 0.0 0.0 Total for 3 rabbits +0.5 The test shows the material to be non-pyrogenic.
Washing after hypochlorite treated corium with the aqueous alcohol and pyrogen free water was effective in removing pyrogenetic substances~
The material prepared as described in Example 2 was subjected to a further pyrogen t~st by rinsing a 2 gm sample four times with 20 ml portions of pyrogen free, distilled water. After the fourth rinse, the sample was placed in 20 ml of pyrogen free, distilled water and held for 2 hours at 48C. The sample was removed and portions of the holding solution injected into three rabbits and changes in the body temperatures noted as described above. The temperature changes were as shown in Table 3.
Ta le 3 Temperature Change, C.
Total Max.
Rabbit 1st hr. 2nd hr. 3rd hr. Chanqe Chanqe G 0.0 0.0 +Oa3 +0~3 +0~3 H +0~1 0.0 +0.2 ~0.3 +0.2 I ~0.2 +0.3 ~0.3 ~0~8 -~0.3 Total for 3 rabbit,s +1.4 The test confirmed that the sample was non-pyrogenic.
.. . . . . .
Although the total change in temperature of the rabbits was ~1.4C., it will be noted that the maximum temperature change for any rabbit in a one hour period was only ~0.3C.
Although the invention has been illustrated specifi-cally as applied to corium, the method is equally applicable to the treatment of other collagen containing materials, such as, for example, sinews, tendons, cartilage and bones, and to medical products other than the fluffy fibrous pro-duct of the above-mentioned patent.
~B 8 -
This ln~ention relates to the manufacture of pyrogen-free collagen, such as, for example, ~orium, adapted for use in the manufacture of collagen derived products intended for medical uses, particularly in surgical procedures.
In the preparation of a wide ~ariety of collagen derived products for medical uses, it is necessary that the products be free of microorganisms, such as bacteria, yeasts and molds. As is well known, these microorganisms may be destroyed or rendered innocuous readily by sterilization, as by subjecting the collagen source material and/or collagen derived product to radiation, bactericides, moldicides, various gases and heat treatment.
Pyrogens, on the other hand, are not living organisms and are not rendered innocuous by bactericides, moldicides and gases, and are thermostable. Pyrogens are generally considered to be thermostable products o-E the growth of strains of bacteria, yeasts and molds, some being soluble and others being insoluble and filterable. In addition to their fever producing effects, pyrogens have physiologic effects on the circulatory system, the endocrine glands and metabolic processes. The rise in body temperature is only one of the manifestations to the introduction of minute quantities of pyrogenetic substances into the animal ~ody and the specific effects will be dependent upon the individual subject. Hence, it is essential that pyro-geneti~ substances be remo~ed. The microorganiqms may be rendered innocuous by a sterilization treatment of the final product.
B
Animal hides constitute the principal source of corium, commonly termed green corium, the collagen raw material for the preparation of various medical grade products. For example, a hemostatic mass of fibers consisting of ioniæable, partial salts of collagen con-taining about 50 to about 90% of the stoichiometric amounts of acid, calculated as ~ICl, may be prepared from such corium, as described in the U. S. patent No. 3,7~2,955, dated July 3, 1973.
In accordance with the present invention, the hides and the collagen raw material, such as corium, derived from the hides and contaminated with microorganisms are processed to produce pyrogen free collagen by mechanically removing foreign substances from the surfaces of the col-lagen while in contact with an aqueous solution of alkali or alkaline earth metal hypochlorite, for example, sodium hypochlorite, washing the collagen with an aqueous ethanol solution, and thereafter washing the collagen with pyrogen free water.
In a practical operation, the hides following skinning are trimmed to remove tails and pates and washed, as in a drum washer, with copious amounts of water containing sodium hypochlorite so as to remove dirt and other foreign matter.
The water may contain from 50 to 250 ppm, preferably 50 to 100 ppm, of sodium hypochlorite. The hypochlorite may be obtained from commercial solutions generally containing 5.25% sodium hypochlorite. A~ter the hides are clean, they may be transferred from the drum washer to a holding tank and maintained in water containing from about 50 to 100 ppm sodium hypochlorite until a batch of a desired quantity is obtained. The green corium is then obtained by removal of the flesh and grain sides of the hide as by splitting operations. The foregoing processing is performed at an abattoir. Generally, the preparation of the medical product is conducted at a location remote from the abattoir.
In such instance, the corium is blast frozen and transported in the frozen state to the desired location f~r conversion into a desired product.
The hypochlorite treatment at the concentrations utilized in producing the corium contro~s the prolifera-tion of bacterial growth before freezing. It is necessary that the subsequent processlng of the corium into the source material for the preparation of the final product be effected under treatment conditions to produce a pyrogen free source material. Where the corium is received in a frozen state, it is thawed by placing in preferably pyrogen free, distilled water which may contain between about 50 and 250 ppm, preferably 50 to 100 ppm, sodium hypochlorite, the solution having a pH between 7 and 9, preferably pH 7-8. Obviously, where the corium has not been frozen it is maintained in such solution. The sur-faces of the sheets are then scraped mechanically while in contact with the hypochlorite solution so as to loosen and/or remove substances such as dirt, loose protein and other foreign material on or embedded in the surfaces. A
convenient method of scraping involved brush washing by drawing the corium sheet between -two rotating, stiff bristle brushes (stiff nylon bristles) under a copious stream of pyrogen free, distilled water, preferably con-taining the hypochlorite.
Following scraping or brush washing the sheets maybe cut into s~rips for convenience in subse~uent process-ing. ~he sheets or strips are rinsed thoroughly with pyrogen free distilled water containing the h~pochlorite and placed in such solution until required for use.
In the preparation of the collagen derived products, the sheets are removed from the hypochlorite solution and the coriu~ separated from excess solution, as by pressing or centrifugation. The corium is washed, as by immersing in a mixture of ethanol and water containing preferably from 30 - 90% ethanol by volume, more preferably containing about 70 volumes of ethanol and 30 volumes of pyrogen free, distilled water, and the mass agitated for about 15 to 30 minutes. The excess liquid is separated from the cor,ium and treatment with the aqueous alcohol repeated at least once. Preferably, -three such aqueous alcohol treatments or washing s-teps are utilized. ~he corium is finally washed with copious amounts of pyrogen free water and is used in the preparation of the desired products~ All processing is preferably conducted at temperatures not exceeding about ~0C., more preferably 'be-tween -the freezing point of the liquid and about 25C.
The removal of pyrogenetic substances from ~reen corium received in frozen state from an abattoir is illustrated by the following e~amples:
Example 1 A 100 gm~ sample of thawed, green corium strips (35%
solids) was placed in 2000 gm pyrogen free, distilled water to which had been added 1.96 gm of a 5.25% aqueous solution of sodium hypochlorite~ The solution contained approximately 50 ppm sodium hypochlorite and was at a temperature of about 35C. I~he mass was stirred gently for about 15 minutes. The skrips were scraped with a knife edge, washed with a like solution and placed in a fresh solution of sodium hypochlorite. The strips were removed subsequently, squeezed to remove excess liquid and used in performing a pyrogen test.
Example_2 A 200 gm. sample of thawed, green corium strips (35%
solids) was placed in 1870 gm pyrogen free, distilled water containing approximately 50 ppm sodium hypochlorite at a temperature of about 35C. and the mass stirred gently for about 15 minutes. The strips were scraped with a knife edge, washed with a like solution, and placed in a fresh solution of sodium hypochlorite. The strips were removed, ~xcess liquid expressed by squeezing and the strips placed in 2000 ml of a mixture containing 70 volumes of ethanol and 30 volumes of pyrogen free, dis-tilled water. The mass was stirred for about 15 minut~s, the strips removed, squeezed to express excess liquid an placed in a fresh ethanol-water mixture. The mass was stirred for about 30 minutes, the strips removed subse-quently, squeezed and used in performing a pyrogen test~
The conventional test for pyrogens utilized the rabbit as the test animal and three rabbits are used in the test.
After establishin~ the normal temperature of each rabbit, 10 ml of a fluid to be tested per kg of body weight of the rabbit are injected into one marginal ear vein of the rabbit. The change in the rabbit temperature is deter-mined at l-hour intervals -for three hours~ A positive test results if any rabbit develops an individual temperature rise of 0~6CO or more at the end of any hour or if the total of the three rabbits' temperature elevation exceeds In the pyrogen test of the material prepared as des cribed in Example 1, a 10 gm sample of the treated corium was rins~d in 100 ml portions of pyrogen free, distilled water four times. The fourth rinse water was heated to 37C. prior to injecting 10 ml. of the rinse water per kg of body weight into each of thxee rabbits. The change in temperature as compared to the normal temperature of each rabbit was noted at l-hour intervals for each rabbit and is reported in Table 1 which follows:
Table 1 Temperature Chanqe, C.
Total Max.
Rabbit 1st hr. 2nd hr. 3rd hr. Change ~ +0.~ +0.6 +Oq5 +1.6 +0.6 B +0.2 +0.1 -0.1 +0.2 +OL20 C ~0.6 +0.6 +0.4~ +1~ ~0.6 Total for 3 rabbits +3.4 The test indicates the material to be pyrogenic.
Washing of the hypochlorite treated corium with pyrogen free water does not remove the pyrogenetic substances.
In a similar manner a 10 gm. sample of the material as described in Example 2 that had been subjected to the aqueous ethanol washing after contact with the hypochlorite solution was rinsed in 100 ml portions of pyrogen ~ree distilled water four time.s~ The fourth rinse water was heated to 37C prior to injecting 10 ml of the rinse water per kg of body weight into each of three rabbits and the s~
;27 change in the temperature of each rabbit noted as described above. The results are set forth in Table 2 which follows:
Table 2 Temperature Chan~, C.
Total Max.
Rabbit 1st hr. 2nd hr. 3rd hr. Chanqe Chanqe D +0.1+0.2 0.0 +0.3 +0.2 E 0.0~0.1 +0.1 ~0.2 ~0.1 0.00.0 ().0 0.0 0.0 Total for 3 rabbits +0.5 The test shows the material to be non-pyrogenic.
Washing after hypochlorite treated corium with the aqueous alcohol and pyrogen free water was effective in removing pyrogenetic substances~
The material prepared as described in Example 2 was subjected to a further pyrogen t~st by rinsing a 2 gm sample four times with 20 ml portions of pyrogen free, distilled water. After the fourth rinse, the sample was placed in 20 ml of pyrogen free, distilled water and held for 2 hours at 48C. The sample was removed and portions of the holding solution injected into three rabbits and changes in the body temperatures noted as described above. The temperature changes were as shown in Table 3.
Ta le 3 Temperature Change, C.
Total Max.
Rabbit 1st hr. 2nd hr. 3rd hr. Chanqe Chanqe G 0.0 0.0 +Oa3 +0~3 +0~3 H +0~1 0.0 +0.2 ~0.3 +0.2 I ~0.2 +0.3 ~0.3 ~0~8 -~0.3 Total for 3 rabbit,s +1.4 The test confirmed that the sample was non-pyrogenic.
.. . . . . .
Although the total change in temperature of the rabbits was ~1.4C., it will be noted that the maximum temperature change for any rabbit in a one hour period was only ~0.3C.
Although the invention has been illustrated specifi-cally as applied to corium, the method is equally applicable to the treatment of other collagen containing materials, such as, for example, sinews, tendons, cartilage and bones, and to medical products other than the fluffy fibrous pro-duct of the above-mentioned patent.
~B 8 -
Claims (12)
1. The method for producing pyrogen free collagen which comprises removing mechanically foreign substances from the surfaces of the collagen while in contact with an aqueous solution of an alkalie or alkaline earth metal hypochlorite, washing the collagen with an aqueous ethanol solution and thereafter washing the collagen with pyrogen free water.
2. The method as defined in claim 1 wherein the aqueous hypochlorite solution is an aqueous solution of sodium hypo-chlorite.
3. The method as defined in claim 2 wherein the aqueous solution contains from about 50 to 250 ppm of sodium hypo-chlorite.
4. The method as defined in claim 1 wherein the aqueous ethanol solution contains about 30 to 90% ethanol by volume and 70-10% water by volume.
5. The method as defined in claim 1 wherein the aqueous hypochlorite solution is an aqueous solution of sodium hypo-chlorite containing from about 50 to 250 ppm sodium hypo-chlorite and the aqueous ethanol solution contains about 30 to 90% ethanol by volume and 70 to 30% water by volume.
6. The method as defined in claim 1 wherein the collagen is corium.
7. The method as defined in claim 6 wherein the foreign substances are removed from the corium surfaces by brush washing the corium surfaces while in contact with an aqueous solution of sodium hypochlorite.
8. The method as defined in claim 6 wherein the corium is derived from hides washed with an aqueous solution of sodium hypochlorite containing from 50 to 250 ppm sodium hypochlorite and the flesh and graid sides of the hide are removed to recover the corium.
9. The method as defined in claim 8 wherein foreign substances are removed from the corium surfaces by brush washing the surfaces while in contact with an aqueous solution containing from about 50 to 100 ppm sodium hypochlorite and the aqueous ethanol solution contains about 70% ethanol by volume and 30% water by volume, the solutions having a temperature not exceeding about 40°C.
10. The method as defined in claim 9 wherein the temperature of the solutions is between the freezing point and 25°C.
11. As an article of manufacture, pyrogen free collagen prepared by the method as defined in claim 1.
12. An article of manufacture as defined in claim 11 wherein the collagen is corium.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US82835077A | 1977-08-29 | 1977-08-29 | |
| US828,350 | 1977-08-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1104127A true CA1104127A (en) | 1981-06-30 |
Family
ID=25251559
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA307,883A Expired CA1104127A (en) | 1977-08-29 | 1978-07-21 | Method for preparing pyrogen free collagen |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPS5449301A (en) |
| CA (1) | CA1104127A (en) |
| DE (1) | DE2835220A1 (en) |
| FR (1) | FR2401973A1 (en) |
| GB (1) | GB2003158B (en) |
| IT (1) | IT1106868B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4221491C2 (en) * | 1992-06-29 | 1994-09-29 | Pohl Boskamp Gmbh Chem Pharma | Process for the preparation of soft gelatine capsules |
| US7083820B2 (en) * | 2000-09-29 | 2006-08-01 | Schilling Marvin L | Method for producing biologically active products |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR51337E (en) * | 1941-01-04 | 1942-04-15 | Process for the production of proteinaceous substances of commercial value, in particular glue and gelatin, from waste leather | |
| CH441621A (en) * | 1963-06-04 | 1967-08-15 | Bayer Ag | Process for the production of gelatin preparations which can be used as blood substitute fluid |
| US3634561A (en) * | 1968-03-18 | 1972-01-11 | Lever Brothers Ltd | Collagen products |
| US3742955A (en) * | 1970-09-29 | 1973-07-03 | Fmc Corp | Fibrous collagen derived product having hemostatic and wound binding properties |
| US4035483A (en) * | 1973-05-29 | 1977-07-12 | John Bunyan | Antiseptic and non-toxic substance and a method of making the same |
-
1978
- 1978-07-21 CA CA307,883A patent/CA1104127A/en not_active Expired
- 1978-08-07 GB GB7832436A patent/GB2003158B/en not_active Expired
- 1978-08-08 IT IT50660/78A patent/IT1106868B/en active
- 1978-08-11 DE DE19782835220 patent/DE2835220A1/en active Granted
- 1978-08-25 FR FR7824752A patent/FR2401973A1/en active Granted
- 1978-08-29 JP JP10544878A patent/JPS5449301A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| DE2835220A1 (en) | 1979-03-08 |
| IT7850660A0 (en) | 1978-08-08 |
| JPS5449301A (en) | 1979-04-18 |
| GB2003158A (en) | 1979-03-07 |
| GB2003158B (en) | 1982-03-24 |
| JPS6351184B2 (en) | 1988-10-13 |
| DE2835220C2 (en) | 1988-06-30 |
| FR2401973A1 (en) | 1979-03-30 |
| IT1106868B (en) | 1985-11-18 |
| FR2401973B1 (en) | 1982-07-23 |
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Legal Events
| Date | Code | Title | Description |
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| MKEX | Expiry |