Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Scleroproteins – e.g. – fibroin – elastin – silk – etc.
Reexamination Certificate
1998-09-29
2001-03-06
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Scleroproteins, e.g., fibroin, elastin, silk, etc.
C530S412000, C530S427000, C514S002600, C514S021800, C514S801000, C424S422000, C424S444000, C424S445000
Reexamination Certificate
active
06197935
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method of eliminating prion for collagen or collagen-derived products, particularly by dehydrothermal or chemical inactivation.
BACKGROUND OF THE INVENTION
Among biological materials, collagen, particularly type I collagen, is a major component of various connective tissues including bone. The replacement of human tissues with human- or animal-derived tissues such as skin or bone grafts results in the improvement of the wound healing process because of the presence of collagen. Therefore, the application of collagen-derived products as biomaterials has tremendous impact in biomedicine because of (i) the natural structure of these products as a biological support for cells and scaffold for tissue repair or regeneration, (ii) their biodegradability that obviates removal of implants, and (iii) their biocompatibility. Collagen has been used to design biomaterials such as wound dressings, artificial dermis, bone or tendon substitutes, tissue engineered devices, and injectable materials in plastic surgery
1-5
. One of the advantages of using animal collagen, particularly from bovine species, is the facility with which large quantities of pure type I or type I/III collagen can be produced.
The emergence of new viruses and the appearance of new infective diseases require increased vigilance concerning the safety of biologicals, especially since the discovery of transmission of protein such as the prion correlates with infectivity (i.e., bovine spongiform encephalopathy)
6
. The assessment of the safety of a biological product towards prion is complicated by the lack of a test capable of detecting scrapie-like agents in the starting material. Collagen purified from human sources could also be a vector for the human spongiform encephalopathies
7
and, perhaps, viral diseases (known and unknown).
The scrapie agent is extremely resistant to heat and physical inactivation
8
. Prolonged exposure to concentrated NaOH solutions and autoclaving at temperatures above 130° C. can be recommended for routine inactivation and disinfection of scrapie-like agents
9
. However, there is still a debate over whether this procedure simply extends the incubation period
10
. Furthermore, only NaOH can be applied to collagen because autoclave destroys collagen.
Treatment of collagen by NaOH at similar concentrations and incubation periods (as recommended) have been investigated towards the elimination of other infectious agents such as bacterium or viruses (RNA or DNA viruses). In a basic environment, we have shown indirectly by agarose gel that DNA and RNA are not fragmented enough to implement elimination of infectious disease and transmission.
In another study we have compared the effect of glutaraldehyde treatment as usually used to crosslink collagen products (i.e., Yannas' skin; cardiac biological valves; vessel grafts; and other biological implants). Two methods of treating collagen with glutaraldehyde solution have been tested. One is using water and the other diluted acetic acid as buffer to crosslink collagen. The latter condition is described in Yannas' patent U.S. Pat. No. 4,060,081 on his artificial skin. Many investigators claim that glutaraldehyde treatment of collagen and derived products (i.e., gelatin capsules) can sterilize the final crosslinked products. Using the two methods of glutaraldehyde treatment, our investigation (using agarose gel) shows clearly that DNA and RNA incorporated in our collagen are not completely broken down and subsequently the risk of transmission of viruses and bacteria is most probable.
Other treatments such as 8M urea have been also recommended. Nevertheless, partial breakdown of DNA and RNA was observed. This breakdown is partial enough to offer no warranty of virus-free products.
On the other hand, formic acid (FA), SDS, fluorinated alcohols, and trifluoroacetic acid (TFA) have dramatic effects on the prion (PrP27-30) secondary and tertiary structures which correlates with the inactivation of scrapie infectivity
11-12
. There is no suggestion in the art that collagen will resist to those treatments.
As mentioned above, a combination of NaOH and autoclave is currently used to eliminate prion. We have shown that NaOH by itself cannot warrant the breakdown of DNA or RNA. It is further known that autoclaving at about 130° C. destroys collagen. Other procedures making use of severe dehydrothermal treatment are used for crosslinking polymer components. Dehydrothermal treatment also has for effect to sterilize the products as well as crosslinking polymer components. Nobody has investigated as to whether heat sterilization may remove prions while preserving the integrity of collagen-comprising products.
In view of the foregoing, there is a need for a method of making a prion-free collagen-comprising product wherein prion is eliminated while collagen is not substantially denatured beyond a desirable or unavoidable extent.
STATEMENT OF THE INVENTION
It is an object of the present invention to provide a method to eliminate prion from collagen-comprising products while preserving substantially the integrity of those products.
In a particular embodiment of the invention, such a method makes use of a strong acid having a pH solution below about 2. In a preferred embodiment, the strong acid is pure trifluoroacetic acid or fluoroacetic acid (pH 1) applied directly on a lyophilized collagen-comprising product by impregnation. The time of reaction may vary from about 1 to 5 hours depending on the nature of the acid e.g. the more potent is the acid, lesser the time is necessary to eliminate prion without affecting the integrity of the product.
In another embodiment of the invention, dehydrothermal treatment substitutes for the chemical inactivations e.g. the strong acid. In that particular method, collagen a collagen-derivative such as gelatin is submitted to temperature and time conditions which are sufficient to eliminate prion without affecting substantially the integrity of collagen beyond a desirable extent. In a preferred embodiment, those conditions are a temperature of 110° C. and a period of 1 to 3 days, in a dry atmosphere (under high vacuum).
In another embodiment of the invention, the two above-sterilization methods are combined. First, collagen is treated with TFA for a period of time which is dependent on the desirability to convert collagen into gelatin. When a substantive conversion to gelatin is desirable, collagen may be treated with TFA or an acid having an equivalent action, for a period of time which is higher than about 5 hours, preferably between 6 to 12 hours. The collagen or collagen-derivative (e.g., for example, collagen, TFA-treated collagen or gelatin) are then submitted to a dehydrothermal treatment, which has for effect to eliminate prion, if any, and crosslink the product. The heat treatment has for dual effect to stabilize the product by crosslinking and eliminate prion.
It is another object of this invention to-provide products comprising collagen and collagen derivatives prepared by the acid and/or heat-inactivation above processes which have the advantage of achieving a safe prion-free collagen. Collagen as a starting material in the production of collagen products, collagen already/shaped as films, sponges, drug-delivery systems or wound dressings; collagen conjugated to other acid-stable molecules, and collagen derivatives or fragments, are all examples of materials which be subject to inactivation processes.
This invention will be described hereinbelow by way of specific examples and appended figures, which purpose is to illustrate and not to limit the scope of the invention.
REFERENCES:
patent: 4060081 (1977-11-01), Yannas et al.
patent: 4511653 (1985-04-01), Play et al.
patent: 4642117 (1987-02-01), Nguyen et al.
Safar et al.,Prot. Sci., vol. 2, pp. 2206-2216, Dec. 1993.
Doillon et al.,J. Biomed. Res., vol. 37, pp. 212-221, 1997.
Berg, R.A., “Determination of 3-and 4-Hydroxyproline,”Meth. Enzymol.82 Pt.A:372-398 (1982).
Brown, P., et al., “Resistance of Scrapie infectivity
Doillon Charles
Drouin Régen
LaRoche Gaétan
DiagnoCure, Inc.
Low Christopher S. F.
Mohamed Abdel A.
Sterne Kessler Goldstein & Fox P.L.L.C.
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