Methods for sterilizing preparations of glycosidases

Abstract

Methods are disclosed for sterilizing preparations of glycosidases to reduce the level therein of one or more active biological contaminants or pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, single or multicellular parasites, prions or similar agents responsible, alone or in combination, for TSEs. These methods involve sterilizing preparations of glycosidases, such as alpha-glucosidase or alpha-galactosidase, with irradiation.

Description

BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The present invention relates to methods for sterilizing preparations of glycosidases to reduce the level therein of one or more active biological contaminants or pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, prions or similar agents responsible, alone or in combination, for TSEs and / or single or multicellular parasites. The present invention particularly relates to methods of sterilizing preparations of glycosidases, such as alpha-glucosidase or alpha-galactosidase, with irradiation. [0003] 2. Background of the Related Art [0004] The principal foods upon which an organism, such as a human, survives can be broadly categorized as carbohydrates, fats and proteins. These substances, however, are useless as nutrients without the process of digestion to break down foods into chemical compone...

AI Technical Summary

Benefits of technology

[0036] In accordance with these and other objects, a first embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising irradiating the preparation of one or more glycosidases with radiation for a time effective to sterilize the material at a rate effective to sterilize the material and to protect the material from radiation.

[0038] Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising: (i) reducing the residual solvent content of a preparation of one or more glycosidases to a level effective to protect the preparation of one or more glycosidases from radiation; and (ii) irradiating the preparation of one or more glycosidases with radiation at an effective rate for a time effective to sterilize the preparation of one or more glycosidases.

[0039] Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising: (i) reducing the temperature of a preparation of one or more glycosidases to a level effective to protect the preparation of one or more glycosidases from radiation; and (ii) irradiating the preparation of one or more glycosidases with radiation at an effective rate for a time effective to sterilize the preparation of one or more glycosidases.

[0040] Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising: (i) applying to the preparation of one or more glycosidases a stabilizing process selected from the group consisting of: (a) reducing the residual solvent content of a preparation of one or more glycosidases, (b) adding to the preparation of one or more glycosidases at least one stabilizer, and (c) reducing the temperature of the preparation of one or more glycosidases; and (ii) irradiating the preparation of one or more glycosidases with radiation at an effective rate for a time effective to sterilize the preparation of one or more glycosidases, wherein the stabilizing process and the rate of irradiation are together effective to protect the preparation of one or more glycosidases from radiation.

[0041] Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising: (i) applying to the preparation of one or more glycosidases at least two stabilizing processes selected from the group consisting of: (a) reducing the residual solvent content of a preparation of one or more glycosidases, (b) adding to the preparation of one or more glycosidases at least one stabilizer, and (c) reducing the temperature of the preparation of one or more glycosidases; and (ii) irradiating the preparation of one or more glycosidases with radiation at an effective rate for a time effective to sterilize the preparation of one or more glycosidases, wherein the stabilizing processes may be performed in any order and are together effective to protect the preparation of one or more glycosidases from radiation.

Problems solved by technology

These substances, however, are useless as nutrients without the process of digestion to break down foods into chemical components that are sufficiently small to be absorbable in the digestive tract.

People who lack peptic activity in the stomach will experience poor absorption of ingested meats because there is poor penetration by these other glycosidases.

For example, a given glycosidase may be ineffective when sialic acid is present on N-linked oligosaccharides.

Roughly 1% of one's red blood cells are replaced each day, which means that a significant amount of Gb3 requires degradation each day.

As a result, Gb3 accumulates in lysosomes throughout the patient's body, which impairs (clogs blood vessels with built-up Gb3) organs and body parts that depend on proper functioning of small blood vessels, such as kidneys, heart, nervous system, and skin.

In addition, many patients with Fabry disease are unable to perspire, which causes further discomfort with exercise or exposure to high temperatures.

Thus, common problems include heart and circulatory malfunctions, such as high blood pressure, heart attack, heart failure, mitral valve prolapse, cardiac arrhythmia, stroke, and kidney malfunctions, such as renal failure requiring dialysis of the patient.

Normally, acid alpha-glucosidase breaks down excess glycogen in a cell; however, this function is blocked in cells of the GSD II patient to produce abnormal, excess accumulation of glycogen, which causes the muscle failure in respiratory muscles and in heart muscle.

Preparations of glycosidases that are prepared for human, veterinary, diagnostic and / or experimental use may, however, contain unwanted and potentially dangerous biologically active contaminants or pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, single or multicellular parasites, prions or similar agents responsible, alone or in combination, for TSEs.

Such procedures, however, are not always reliable and are not able to detect the presence of certain viruses, particularly in very low numbers, and in the case of as yet unknown viruses or other contaminants or pathogens that may be in blood.

This reduces the value or certainty of the test in view of the consequences associated with a false negative result.

False negative results can be life threatening in certain cases, for example in the case of Acquired Immune Deficiency Syndrome (AIDS).

Furthermore, in some instances it can take weeks, if not months, to determine whether or not the preparation is contaminated.

Moreover, to date, there is no reliable test or assay for identifying prions within a biological material that is suitable for screening out potential donors or infected material.

Thus the products of unicellular natural or recombinant organisms or tissues carry a risk of pathogen contamination.

In addition to the risk that the producing cells or cell cultures may be infected, the processing of these and other biological materials creates opportunities for environmental contamination.

Interestingly, even products from species as different from humans as transgenic plants carry risks, both due to processing contamination as described above, and from environmental contamination in the growing facilities, which may be contaminated by pathogens from the environment or infected organisms that co-inhabit the facility along with the desired plants.

Indeed, such rodents are notoriously difficult to control, and may gain access to a crop during sowing, growth, harvest or storage.

Likewise, contamination from overflying or perching birds has to potential to transmit such serious pathogens as the causative agent for psittacosis.

Thus any biological material, regardless of its source, may harbour serious pathogens that must be removed or inactivated prior to the administration of the material to a reicipient.

This is a result of safety concerns for the workers conducting the tests, and the difficulty and expense associated with the containment facilities and waste disposal.

Heat treatment requires that the product be heated to approximately 60° C. for about 70 hours which can be damaging to sensitive products.

In some instances, heat inactivation can actually destroy 50% or more of the biological activity of the product.

Unfortunately, this method may also remove products that have a high molecular weight.

Further, in certain cases, small viruses and similarly sized contaminants and pathogens, such as prions, may not be removed by the filter.

This procedure requires that unbound sensitizer is washed from products since the sensitizers are toxic, if not mutagenic or carcinogenic, and cannot be administered to a patient.

The published literature in this area, however, teaches that gamma radiation can be damaging to radiation sensitive products, such as blood, blood products, enzymes, protein and protein-containing products.

In particular, it has been shown that high radiation doses are injurious to red cells, platelets and granulocytes (Leitman).

Unfortunately, many sensitive biological materials, such as monoclonal antibodies or enzymes, may lose viability and activity if subjected to freezing for irradiation purposes and then thawing prior to administration to a patient.

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