Method of sterilization

Details Method of sterilization Method of sterilization

1999-06-03

2003-01-14

McKane, Elizabeth (Department: 1744)

Chemical apparatus and process disinfecting, deodorizing, preser

Process disinfecting, preserving, deodorizing, or sterilizing

Using disinfecting or sterilizing substance

C435S001100

Reexamination Certificate

active

06506339

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to sterilization processes and more specifically to sterilization processes which are particularly suited for biological materials, such as organ replacements, and which methods exhibit efficacy against difficult-to-kill bacteria and bacterial spores.
BACKGROUND OF THE INVENTION
Sterilization techniques are widely used and important in industries such as food processing and health care. Saturated steam at temperatures above 110° C. has frequently been used to destroy microorganisms, such as microbial spores. Certain articles, particularly those used for health care, cannot withstand the temperatures and moisture of steam sterilization, and oftentimes such articles are also considered not to be suitable for sterilization by ionizing radiation. As a result, gaseous sterilants have been developed which function at relatively low temperatures and thus offer an attractive alternative. One of the most commonly used gaseous sterilants is ethylene oxide, which is used for medical product sterilization and for other sterilization processes. However, in certain instances, the presence of residual ethylene oxide, even in small quantities, is considered to be detrimental, and accordingly improved sterilization processes, particularly for sterilization of medical products, have continued to be sought.
SUMMARY OF THE INVENTION
It has now been found that sterilization of items, including biological tissue, replacement organs and synthetic prosthetic materials, including polymers and metals, can be effectively carried out by treatment with a coupling agent, e.g. a water-soluble carbodiimide, that is capable of creating amide linkages between amines and carboxylic acids; such treatment has been proven to be bactericidal. Sterilization treatment is carried out at a temperature above ambient, and although it may employ an optional coupling enhancer, such is not felt necessary. Treatment may be carried out using an organic solution of an appropriate coupling agent or using an aqueous buffered solution that may optionally contain isopropyl alcohol or the like, but the presence of such an alcohol is not necessary to achieve effective sterilization. The residuals from such treatment are nontoxic, biocompatible, and water-soluble; they can generally be easily be washed off the tissue before implantation in a human body. Surprisingly, biological tissue which has been effectively sterilized using a water-soluble carbodiimide may exhibit enhanced resistance to degeneration and/or calcification following its implantation within a living body.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The term “coupling agent” is herein used to refer to a chemical reagent that facilitates the formation of amide bonds. Such bonds may be formed between reactive amines and reactive carboxyls on enzymes and proteins as well as between the reactive carboxyl or amine moieties located on and within bioprosthetic tissue. Those having skill in peptide synthesis and related arts will be familiar with some such reagents, e.g. water-soluble carbodiimides and succinimides; there are other known coupling agents that are soluble in organic solvents. When penetration of such coupling agents into the cells of microorganisms occurs, it results in sterilization, destroying bacteria, spores and possibly viruses and other infectious agents by internal cross-linking. When biological tissue is to be treated, the coupling agent chosen is preferably one that is water-soluble so the treatment can be effected in aqueous solution at a physiological pH. When other materials that are resistant to organic solvents are to be sterilized, e.g. synthetic polymeric materials, organic solutions of appropriately soluble coupling agents may be used. Any suitable carbodiimide can be used as the coupling agent; however, the preferred water-soluble coupling agent is 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide hydro-chloride (EDC). Other water-soluble carbodiimides include 1-Cyclohexyl-3(2-morpholinoethyl)carbodiimide, N,N′-Carbonyldiimidazole, Woodward's Reagent K, and mixtures of such carbodiimides. A list of such cross-linking coupling agents can be found in the book: Bioconjugate Techniques by Greg T. Hermanson published by Academic Press 1996, the relevant disclosure of which is incorporated herein by reference. When biological tissue is being treated, the water-soluble coupling agent EDC is preferably used. As indicated above, an optional enhancer, e.g. N-hydroxysulfosuccinimide (sulfo-NHS), might be included at a concentration between 0.5 mM and about 30 mM when EDC is used as the coupling agent; however, such is not considered necessary for effective sterilization.
The sterilization treatment is considered to be temperature-dependent, with a relatively low temperature of about 35-40° C. being preferred because of its lack of potential adverse effect upon the material being treated. The concentration of the coupling agent can be varied within a reasonable range, and treatment with higher concentrations of the coupling agent has been found to achieve sterilization within a shorter time of treatment. Although lower concentrations, e.g. 5-15 mM, may be effectively used, particularly when higher temperatures are employed, the coupling agent is preferably used in a concentration between about 25 millimolar (mM) and about 150 mM, more preferably between about 35 mM and about 100 mM, and most preferably at between about 50 mM and about 75 mM, in order to be certain of destroying all commonly encountered bacteria and spores within a reasonable duration of treatment.
Higher concentrations of coupling agent, so long as compatible with the material being sterilized, will generally reduce the duration of treatment needed. It has been found that effective sterilization is achieved when such treatment is carried out at a suitable temperature above ambient, e.g. at a temperature of at least about 35° C., for a minimum number of concentration-duration units, i.e. a multiple of coupling agent concentration and duration of exposure. By arbitrarily basing such units upon millimoles of the coupling agent and hours of sterilization treatment, it has been found that at a temperature of about 35-40° C., a minimum number of units equal to at least about 450 millimole hours should be employed. For example, effective sterilization may be achieved at a coupling agent concentration of about 50 mM for about 9-10 hours or alternatively at a concentration of about 25 mM for about 20-24 hours. At a concentration of about 120 mM, treatment at about 40° C. for about 6 hours should achieve sterilization. Although even higher concentrations, e.g. 150 mM, might be used, they are considered to be generally unnecessary and likely impractical from an economic standpoint. Moreover, by raising the temperature, e.g. to about 50-55° C., treatment for at least about 100-150 millimole hours should suffice, e.g. 3 hours with a concentration of 50 mM or 10 hours at 10 mM. Obviously, longer durations can be employed, and for purposes of safety, it may be desirable to employ such sterilization treatment for about 25-50% longer than the above-stated minimum that should achieve sterilization under normal conditions.
For purposes of this application, a particular sterilization treatment is deemed to be effective when it will effect a reduction of about 10
6
(6 log) when about 10
6
spores and/or microorganisms are inoculated in the test sample. This should assure that there will be no survivors in actual practice because biological tissue or other material that is being subjected to a sterilization treatment will not reasonably contain a level of microorganism contamination even approaching this magnitude. This treatment not only achieves sterilization without risk of damage to biological tissue that is to be implanted, but it may also make some contribution to stability of certain fixed biological tissue, e.g. the resistance of such biological tissue to degenerate and/or calcify within a living body may be enhanced.
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