EA045327B1 - ENZYME COMPOSITIONS WITH REDUCED VIRAL AND MICROBIAL CONTAMINATION - Google Patents

Description

Cross-reference to related applications

This invention claims priority under Provisional Application US 62/314,048 filed March 28, 2016, Provisional Application US 62/452,746 filed January 2017, and Provisional Application US 62/454,184 filed February 3, 2017. , which are incorporated herein by reference in their entirety.

Field of technology

The present invention relates to enzyme preparations derived from animal tissues, pharmaceutical compositions containing such enzyme preparations, and methods for reducing the risk of viral or microbial contamination of such preparations and compositions. Examples of enzyme compositions include pancreatic extracts suitable for therapeutic use, for example for the treatment and/or prevention of digestive disorders, in particular digestive disorders due to exocrine pancreatic insufficiency in animals and humans.

Prerequisites

Products derived from animal tissue may contain viral and/or microbial contamination. Some biological contaminants, such as bacterial or protozoal contaminants, may be inactivated during the manufacturing process. However, other biological contaminants, such as non-enveloped viruses, are resistant to conventional contaminant reduction or decontamination techniques. In particular, the problem is the deactivation or removal of viruses from enzyme compositions derived from animal tissues, without destroying or altering the activity of the enzymes in the process.

Common methods for inactivating viruses include, for example, pasteurization, dry heat, steam, solvent/detergent treatment, and lowering the pH. The choice of virus inactivation methods used depends on the nature and contamination of the product, the purification method used, if any, and the nature of the viral contaminant. For example, solvent or detergent treatment can disrupt the lipid membrane of enveloped viruses and thus can be used to inactivate enveloped viruses. However, many non-enveloped viruses are not usually inactivated by solvent or detergent treatment.

Heat, particularly dry heat, is another common method for inactivating viruses. Heat treatment, which can inactivate even highly resistant viruses such as non-enveloped viruses, requires long periods of time (up to several hours) and controlled moisture content. Moreover, heat treatment may impair the desired biological activity of the product, particularly if the product is an enzyme composition.

Pancreatic enzyme products have long been used to treat exocrine pancreatic insufficiency associated with gallbladder fibrosis (CF), chronic pancreatitis, pancreatic or bile duct obstruction (eg, due to neoplastic disease), surgical procedures such as pancreatectomy or gastrointestinal surgery. bypass, as well as with other diseases and disorders. The pancreas secretes enzymes, including lipases, proteases, and amylases, into the proximal lumen of the duodenum, where they assist in the hydrolysis of essential nutrients. Amylases and proteases are secreted by organs other than the pancreas, and they contribute to the processing of carbohydrates and proteins. However, only a small proportion of lipase from sources other than the pancreas is involved in lipid processing. In summary, patients with untreated exocrine pancreatic insufficiency typically have difficulty processing fats and may suffer symptoms of indigestion or malnutrition or both, with deficiencies of essential fatty acids and fat-soluble vitamins, weight loss, constipation, flatulence, bloating, and fatty, foul-smelling liquid stools (steatorrhea). For patients with CF, improper treatment can have serious consequences, since normal nutritional status is directly related to proper lung function.

Pancreatic enzyme therapy cures and/or prevents malabsorption and promotes recovery and development in patients with exocrine pancreatic insufficiency. In patients with CF, mucus clogs the pancreatic ducts, just as it does in the lungs. Digestive enzymes from the pancreas are not released into the intestines, and therefore the processing of starch, fat and protein is impaired. Insufficient processing leads to steatorrhea, abdominal pain and weight loss, among other things.

The basis of digestive disorders in animals and humans is usually a deficiency of digestive enzymes, in particular a deficiency of endogenous lipase, as well as protease and/or amylase. If pancreatic insufficiency is pathological, it can be congenital or acquired. Acquired pancreatic insufficiency, for example, can be a consequence of alcoholism. Congenital pancreatic insufficiency can be, for example, a consequence of fibrosis of the gallbladder. The consequences of digestive enzyme deficiency can be severe symptoms of malnutrition or poor nutrition, which may be accompanied by an increasing susceptibility to secondary diseases.

Replacement with exogenous digestive enzymes or mixtures of digestive enzymes (ie, pancreatic enzyme therapy) has been shown to be an effective therapy for endogenous digestive enzyme deficiency. The most commonly used pharmaceuticals for pancreatic enzyme therapy are those containing porcine pancreatin (also known as enzyme replacement therapy). For such pharmaceuticals, the active ingredient evaluated in clinical trials is lipase, and the dosage in commercially available products is expressed in units of lipase. However, such mixtures of digestive enzymes obtained from porcine pancreas contain lipases, amylases and proteases and can be effectively used in pancreatic enzyme therapy in humans due to the great similarity of the enzymes and related substances they contain with the composition of human pancreatic juice. Pancreatic enzymes are usually administered orally as solid preparations.

Pancreases can be obtained from animals, such as pigs, raised and slaughtered for meat. Regulatory regulations often require that pancreases be sourced from a single species of slaughterhouse (i.e., no other species can be slaughtered in the facility), limiting the availability of raw materials. Widespread contamination of equipment with infectious agents can lead to production curtailments and supply shortages. Performing ongoing testing helps identify contaminated lots, and rejecting such lots places further restrictions on an already limited supply of feedstocks.

Methods have been developed for obtaining enzyme(s) from animal pancreas. For example, US 4,623,624 describes methods by which pancreatin is produced by autolysis of an aqueous isopropanol-containing tissue suspension.

The presence of infectious agents and, in particular, viruses is noted in the pig pancreas used in the production of pancreatin. In fact, the majority of pig populations are infected with porcine parvovirus (PVV), which is highly resistant to inactivation. In pancreatin, PVA is identified as an infectious agent. Although PVA is not believed to be pathogenic in humans, it is desirable to receive pancreatin with a reduced PVA load. Moreover, PVA is a common model virus; it is difficult to deactivate using standard methods, such as chemical or thermal treatment.

US 2010/0119654 concerns the irradiation of an alcoholic or aqueous biological extract that contains a solid phase in suspension. The radiation used in US 2010/0119654 is ultraviolet (UV) radiation, X-ray, β-ray or γ-ray. UV irradiation of the pancreatin intermediate dissolved in 40% isopropanol leads to a decrease in M2 phage content by up to 4 log ₁₀ . Pancreatin gamma irradiation (PAI) produces approximately a 40% reduction in lipase activity at 27 kGy and a 13% reduction in lipase activity at 5 kGy. Bacteria are reduced by more than 2.5 log ₁₀ , but no virus inactivation is reported. When pancreatin (API) is subjected to β-irradiation, more than 85% of the enzyme activity is reported to be retained, but microbial counts are reduced by only approximately 1.5 log ₁₀ .

Publication WO 2003/020324 relates to irradiation sterilization of digestive enzymes such as trypsin, α-galactosidase and iduronate 2-sulfatase. Lyophilized or liquid enzymes (trypsin, glycosidase or sulfatase) are irradiated as such or in the presence of a stabilizer. Irradiation with γ-radiation is carried out using a ⁶⁰ Co source. Virus inactivation has not been reported.

Publication WO 2007/014896 describes reducing the concentration of one or more biological, in particular viral, contaminants of pancreatin by heating the pancreatin.

In US 2009/0233344, heat treatment of pancreatin at 80°C for 32 hours provides a reduction in the viral titer of PVA by 2.5 log ₁₀ , also accompanied by a 20% loss of lipase activity. Heat treatment of pancreatin at 100°C for 8 hours provides a reduction in the viral titer of PVA by more than 3 log ₁₀ , accompanied by approximately 50% loss of lipase activity.

Thus, process steps that may be effective against difficult-to-deactivate viruses such as PVA significantly alter the nature of the pancreatin product by degrading or reducing pancreatic enzymes, particularly lipase, to unacceptable levels. Such changes in activity may reduce or alter the efficacy profile of the final product. It is therefore desirable to maintain enzymatic activity, especially lipase activity, during the production process.

Because each of the previously tested viral contaminant clearance methods that have some effectiveness against difficult-to-deactivate viruses (e.g., RPVs) results in a significant loss of enzymatic activity, including lipase activity, manufacturers have expressed skepticism that a reasonable level of ratio can be achieved inactivation/purification without degrading product quality. In particular, manufacturers have expressed skepticism that an acceptable independent viral purification step can be developed without negatively affecting the chemical, physical or pharmaceutical properties of pancreatin. For example, see letter from Scientific Protein Laboratories to the FDA dated June 22, 2004, in file number 2003B-0206.

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Brief description of the invention

The present invention relates to an enzyme preparation isolated from an animal tissue source. The isolated enzyme preparation includes one or more enzymes, has reduced viral and/or microbial contamination compared to the original animal tissue, and retains at least one biological activity of the animal tissue source. In particular embodiments, the enzyme preparation is prepared by exposing the original animal tissue to irradiation, preferably electron beam radiation, and then isolating one or more enzymes from the irradiated tissue. In some embodiments, the original animal tissue is uninfected tissue. In some embodiments, the irradiated tissue exhibits a reduction in viral and/or microbial contaminants of at least 3 log ₁₀ , preferably at least 4 log ₁₀ , compared to non-irradiated parent animal tissue. In some embodiments, additional independent virus reduction steps are used (eg, during the steps of isolating one or more enzymes from irradiated tissue). In some embodiments, an enzyme preparation isolated from irradiated tissue has a biological activity corresponding to at least 50%, preferably at least 90% of the biological activity of a comparator enzyme preparation, such as an enzyme preparation isolated from a non-irradiated animal tissue source. In some embodiments, the biological activity is lipase activity. In some embodiments, the irradiated tissue exhibits a reduction in viral and/or microbial contaminants of at least 3 log ₁₀ , preferably at least 4 log ₁₀ , compared to non-irradiated parent animal tissue, and the enzyme preparation isolated from the irradiated tissue has biological activity, corresponding to at least 50%, preferably at least 90%, of the biological activity of a reference enzyme preparation, such as an enzyme preparation isolated from a non-irradiated animal tissue source.

Another aspect of the present invention relates to a method for producing an enzyme preparation derived from animal tissue, wherein the enzyme preparation has reduced viral and/or microbial contamination compared to the original animal tissue. The method includes a treatment sufficient to achieve a viral and/or microbial contaminant level of at least 3 log ₁₀ , preferably at least 4 log ₁₀ , compared to the original animal tissue. In some embodiments, the treatment includes exposing an intact animal tissue source. Exposure to radiation, preferably electron beam, to produce irradiated animal tissue. In some embodiments, electron beam exposure is sufficient to reduce viral and/or microbial contamination of the original animal tissue while maintaining at least one biological activity of the original animal tissue. In some embodiments, the biological activity is lipase activity. In some embodiments, one or more enzymes and/or proenzymes are extracted from irradiated animal tissue. In some embodiments, the method reduces the risk of infectious contamination of a drug derived from an animal source, or a pharmaceutical composition comprising a drug derived from an animal source, compared to an untreated sample.

Another aspect of the present invention relates to a pharmaceutical composition comprising a drug as disclosed herein. The pharmaceutical composition may be an oral pharmaceutical dosage form. In some embodiments, the pharmaceutical composition is used to treat or prevent a disease responsive to pancreatic enzyme replacement therapy, such as exocrine pancreatic insufficiency. Thus, another aspect of the present invention relates to a method of treating or preventing exocrine pancreatic insufficiency, comprising administering to a patient for treatment a dose of an enzyme preparation or pharmaceutical composition disclosed herein.

Another aspect of the present invention relates to kits that contain an enzyme preparation or pharmaceutical composition disclosed in the description.

These and other objects of the invention are disclosed in the following subsections. These objects should not be considered as limiting the scope of the invention.

Detailed Description of the Invention

This detailed description is intended solely to familiarize those skilled in the art with the present invention, its principles, its practical application so that those skilled in the art will be able to adapt and apply the invention in all its many forms so that best adapt it to the requirements of practical application. Descriptions and specific examples are for illustrative purposes only. Thus, the present invention is not limited to the embodiments disclosed herein and may be modified in various ways.

A. Definitions.

Definitions used in the description and in the attached claims, unless otherwise indicated

- 3 045327 other, have the following meanings given below.

The term API used refers to the active pharmaceutical ingredient. As stated, the preferred API is pancreatin, in particular porcine pancreatin, which is commonly used for therapeutic purposes, i.e. pancreatin conforming to the standards of pharmacopoeias, for example the European Pharmacopoeia and/or the United States Pharmacopoeia, and suitable for oral administration for the treatment or prevention of digestive disorders in mammals and in particular digestive disorders due to chronic exocrine pancreatic insufficiency, such as in patients suffering from gallbladder fibrosis, chronic pancreatitis, or in patients who have undergone surgery on the upper gastrointestinal tract.

The term crude as used refers to the crude composition or mixture containing enzymes and/or proenzymes as well as additional source tissue components. The crude preparation or mixture includes, but is not limited to, animal tissue.

The term enzyme preparation refers to any composition of the invention containing one or more enzymes in both active and inactive forms (ie, proenzymes or zymogens). The term includes extracts from cells or tissues, as well as preparations obtained from tissue or cellular material of animals. One example of an enzyme preparation is pancreatin, pancrelipase, an extract obtained from the pancreas of a mammal, preferably a pig.

The term extract, as it relates to present enzyme preparations, defines one or more enzymes and/or proenzymes that are isolated from at least one component of the tissue from which they originate. The extracted components may be in the form of an active enzyme or a proenzyme (zymogen), requiring subsequent conversion to the active form.

The term isolated substance, as it relates to these enzyme preparations, refers to one or more active enzymes that have been isolated from at least one component of the tissue from which they originate. Thus, in some embodiments, the isolated enzyme or isolated enzyme preparation includes one or more active enzymes that are converted from the corresponding proenzyme form by hydrolysis and/or autolysis. Hydrolysis and/or autolysis to convert the proenzyme into an active enzyme can occur before, during or after extraction.

As used herein, the terms pancreatic enzymes, pancreatin and pancrelipase refer to enzyme mixtures isolated from the pancreas of animals, including as major components digestive enzymes such as lipase, protease and amylase. In particular, the terms pancreatic enzymes, pancreatin and pancrelipase are used interchangeably herein and refer to extracts of the pancreas suitable for therapeutic use in accordance with standard pharmacopoeias and containing certain digestive enzymes, the properties of which are defined in known monographs, as indicated above. Through standard manufacturing methods, pancreatic enzymes, pancreatin and pancrelipase are generally available in powder form, such as pancreatin powder, sometimes referred to as pancreatic powder. The pancreatic enzymes, pancreatin and pancrelipase can also be, and preferably are, active pancreatic isolates. Pancreatin for pharmaceutical use is usually of bovine or porcine origin. Pork pancreatin is preferred. Pancrelipase in some sources is described as an enzyme preparation with increased (lipase) activity compared to pancreatin.

As used herein, the term pharmaceutical composition means a composition comprising the enzyme preparation disclosed herein and optionally one or more pharmaceutical excipients.

The term pharmaceutically acceptable is used as an adjective and means that the corresponding noun refers to a product suitable for pharmaceutical use or a part of a pharmaceutical product.

An orthogonal microbial and/or viral load reduction step refers to a specific method of reducing the content of microbes and/or viruses that may be present in a sample. The microbial and/or viral load reduction step may be orthogonal due to the fact that the method provides one or more additional microbial and/or viral load reduction steps. In certain embodiments, the orthogonal microbial and/or viral load reduction step has a mechanism substantially different from other microbial and/or viral load reduction steps used in the method such that the log ₁₀ kill achieved by the orthogonal step becomes additive with the cumulative log ₁₀ kill achieved by carrying out all other stages of reducing the microbial and/or viral load carried out during the production of the enzyme preparation.

The terms prevent, prevent or prevent refer to a method of preventing the onset of a condition, disorder or disease and/or the occurrence of symptoms associated therewith, or to prevent a subject from the occurrence of a condition, disorder or disease. As used herein, the terms prevent, prevent or prevent also include delaying the onset of a condition, disorder or disease and/or the onset of symptoms associated therewith and reducing a subject's risk of experiencing the condition, disorder or disease.

The term subject includes humans and other primates, as well as domestic and substantially domesticated animals, including, but not limited to, poultry, bees, cows, sheep, goats, pigs, horses, dogs, cats, rabbits, rats, mice and the like. The term poultry covers all types of birds, including, but not limited to, chickens, turkeys, ducks, geese, flightless birds and game birds. In certain embodiments, the subject is a human.

The term therapeutically effective amount means a sufficient amount of an enzyme preparation or pharmaceutical composition to treat a disorder or disease condition with a reasonable benefit/risk ratio applicable to any medical treatment. When used in medical treatment, a therapeutically effective amount of one of the enzyme preparations may be administered in extract or crude form. Alternatively, the enzyme composition may be administered as a pharmaceutical composition containing the target enzyme composition in combination with one or more pharmaceutically acceptable carriers.

The terms treat, therapeutic, and treatment refer to a method of partially relieving and suppressing the symptoms of a condition, disorder, or disease and/or its associated symptoms.

B. Enzyme preparations and methods of production.

In one embodiment, the present invention includes an enzyme preparation containing one or more enzymes and/or proenzymes of animal origin, preferably from mammalian tissue. In certain embodiments, the enzyme preparation includes a mixture of digestive enzymes and/or proenzymes. In certain embodiments, the enzyme preparation includes a lipase. In some embodiments, the enzyme preparation includes amylase. In some embodiments, the enzyme preparation includes a protease. In some embodiments, the enzyme preparation includes pancreatin. In some embodiments, the enzyme preparation includes a proenzyme such as prolipase or typsinogen. In some embodiments, the enzyme preparation is in a crude form. In some embodiments, the enzyme preparation includes one or more enzymes and/or proenzymes that have been extracted from animal tissue. In some embodiments, the enzyme preparation includes one or more enzymes that have been isolated from animal tissue.

In some embodiments, the isolated enzyme preparation has the same or substantially the same biological activity, but is less contaminated than the tissue from which it was isolated. In certain embodiments, the isolated enzyme preparation has the same or substantially the same biological activity as the reference enzyme preparation. In certain embodiments, the infectivity of the isolated enzyme preparation is reduced by at least three log ₁₀ , preferably by at least four log ₁₀ , compared to the infectivity of the tissue from which it was isolated. In certain embodiments, the infection that is reduced is a viral infection, in particular a non-enveloped virus infection and/or an enveloped virus infection. In certain embodiments, the biological activity of the isolated enzyme preparation is at least 50%, at least 60%, at least 70%, at least 80%, or preferably at least 90% of the biological activity of the reference enzyme preparation. In certain embodiments, the biological activity is an enzyme activity, such as protease activity, amylase activity, or preferably lipase activity.

In another embodiment, the enzyme preparation is isolated from a pretreated tissue source and has the same or substantially the same biological activity, but is less contaminated, than a comparison preparation isolated from an untreated tissue source. In certain embodiments, the contamination of the enzyme preparation is reduced by at least three log ₁₀ , preferably by at least four log ₁₀ relative to the comparator preparation. In certain embodiments, the infectivity of the pretreated tissue source is reduced by at least three log ₁₀ , preferably by at least four log ₁₀ relative to the untreated tissue source. In certain embodiments, the infection being reduced is a viral infection, especially a non-enveloped virus infection. In certain embodiments, the biological activity of the enzyme preparation is at least 50%, at least 60%, at least 70%, at least 80%, or preferably at least 90% of the biological activity of the reference drug isolated from an untreated tissue source. In certain embodiments, the biological activity is an enzyme activity, such as protease activity, amylase activity, or preferably lipase activity.

In another embodiment, the enzyme preparation is obtained from tissue irradiated with an electron beam. In some embodiments, the tissue irradiated with the electron beam is mammalian tissue, preferably porcine tissue. In some embodiments, the enzyme preparation obtained from tissue irradiated with an electron beam includes pancreatin. In some embodiments, the enzyme preparation obtained from tissue irradiated with the electron beam includes a proenzyme such as prolipase or typsinogen. In some embodiments, the enzyme preparation obtained from tissue irradiated with an electron beam is in a crude form. In some embodiments, the enzyme preparation obtained from tissue irradiated with an electron beam includes one or more enzymes and/or proenzymes that have been extracted from the irradiated tissue. In some embodiments, the enzyme preparation obtained from tissue irradiated with an electron beam includes one or more enzymes that have been isolated from the irradiated tissue.

In another embodiment, the enzyme preparation is isolated from tissue irradiated with an electron beam and has the same or substantially the same biological activity, but is less contaminated, than a comparison preparation isolated from a non-irradiated tissue source. In some embodiments, the irradiated tissue is pancreatic tissue. In some embodiments, the irradiated tissue is detached pancreatic tissue, a whole pancreas, or a portion of a whole pancreas. In some embodiments, the non-irradiated tissue source is pancreatic tissue. In certain embodiments, the contamination of the enzyme preparation is reduced by at least three logios, preferably by at least four _logios , relative to the comparator preparation. In certain embodiments, the infection that is reduced is a viral infection, in particular, a non-enveloped virus infection and/or an enveloped virus infection. In some embodiments, the infection being reduced is infection with porcine parvovirus. In certain embodiments, the biological activity of the enzyme preparation is at least 50%, at least 60%, at least 70%, at least 80%, or preferably at least 90% of the biological activity of the comparator drug. In some embodiments, the biological activity is an enzyme activity, such as protease activity, amylase activity, or preferably lipase activity.

In another embodiment, the enzyme preparation includes proenzymes irradiated with an electron beam. In certain embodiments, the enzyme preparation is subjected to further processing, such as converting the irradiated proenzymes to their active form (eg, autolysis and/or hydrolysis).

The present enzyme preparations will be better understood in connection with the following methods, which illustrate examples of techniques by which these enzyme preparations can be prepared.

In one embodiment, the present invention includes a method of producing an enzyme composition consisting of exposing a source of proenzyme to irradiation, preferably irradiation with an electron beam. In certain embodiments, the source of the proenzyme is a group of cells. In some embodiments, the group of cells is intact tissue obtained from a mammalian gland or portion thereof. In some embodiments, the group of cells is a whole gland obtained from a mammal. In some embodiments, the group of cells is a portion of a gland obtained from a mammal. In some embodiments, the group of cells is a frozen block of tissue. In some embodiments, the cell group is intact animal tissue or exfoliated or crushed animal tissue.

In another aspect, the present invention includes a method for producing an enzyme preparation. The method involves exposing animal tissue to radiation, preferably an electron beam. In some embodiments, the animal tissue is intact tissue. In some embodiments, the intact animal tissue is a frozen block of tissue. In some embodiments, the intact animal tissue is exfoliated or shredded animal tissue.

In some embodiments, the method begins with animal tissue, preferably intact animal tissue. In some embodiments, the animal tissue is mammalian tissue, preferably porcine pancreatic tissue. In some embodiments, the porcine pancreatic tissue is obtained from a verified slaughterhouse, preferably a monospecies slaughterhouse.

In certain embodiments, the intact animal tissue includes intact pancreatic tissue. In certain embodiments, the intact pancreatic tissue includes the entire pancreas or a portion thereof, such as one or more lobes. In certain embodiments, the intact pancreatic tissue includes detached frozen tissue. In some embodiments, the intact pancreatic tissue includes a frozen tissue block that can be mechanically processed. In some embodiments, intact pancreatic tissue includes pancreatic tissue that has undergone minimal or no manipulation or alteration

- 6 045327 tion or change in such a way that, for example, it destroys active enzymes and/or converts proenzymes in tissues into their active form. For example, a tissue homogenate that has undergone significant chemical or enzymatic treatment to convert proenzymes to their active form is not intact tissue in the sense used here. As another example, tissue that has been crushed or minced under conditions in which active enzymes are destroyed and/or proenzymes are converted into the tissue into their active form is not intact tissue in the sense used herein.

In some embodiments, the animal tissue, preferably frozen animal tissue, is finely ground. In some embodiments, fine comminution can be achieved using a frozen block crusher, such as the Hydrauflake Chunker, available from General Machinery Corporation (Sheboygan, WI), which is designed to crush frozen treated tissue for subsequent processing.

In certain embodiments, the animal tissue is irradiated. In certain embodiments, the animal tissue is exposed to a sterilizing beam of accelerated electrons, such as an E-beam or electron beam radiation. In some embodiments, intact animal tissue is irradiated with an electron beam. In some embodiments, the whole pancreas or an intact portion thereof is irradiated with an electron beam. In some embodiments, exfoliated porcine pancreatic tissue is irradiated with an electron beam.

An electron beam is a form of ionizing energy that is typically characterized by shallow penetration and high doses. Bunch, i.e. a concentrated, highly charged stream of electrons is generated by the acceleration and conversion of electricity. Electrons are generated using equipment called accelerators here, which are capable of producing beams that are either pulsed or continuous. When the irradiated material passes under or in front of the electron beam, energy from the electrons is absorbed. This absorbed energy changes various chemical bonds and biological properties of the product/material. The energy that has been absorbed is called the absorbed dose. It is this energy absorption, or dosage, that destroys viruses and microorganisms, for example by destroying their DNA or RNA chains.

Irradiation can be performed in a conventional manner, such as placing the tissue in a suitable container and exposing the tissue to a beam of electrons. In certain embodiments, the container containing the tissue is placed on a conveyor that passes through the electron beam. In certain embodiments, the container containing the tissue does not contain any solvent. In certain embodiments, the container containing the tissue is substantially free of solvent. In certain embodiments, the container containing the tissue does not contain any flammable solvent. In certain embodiments, the container containing the tissue is substantially free of flammable solvent, such as alcohol. For example, the container may contain frozen intact tissue, such as a whole gland, a portion of a whole gland, or detached tissue.

In some embodiments, irradiation is administered with a dose sufficient to substantially inactivate resistant viruses and/or microbes in the tissue. In certain embodiments, irradiation is administered at a dose that prevents loss of biological activity, preferably enzyme activity, relative to a reference enzyme isolated from non-irradiated tissue.

The accelerated electron beam can be produced by an electron accelerator, such as one available from Iotron Industries USA, Inc. (Columbia City, IN). In certain embodiments, the electron accelerator operates at a power of 20 to 250 kW with electron energies of 5 to 18 MeV. In some embodiments, the electron accelerator is operated at 50 kW and 10 MeV. In some embodiments, the electron accelerator provides a beam energy of 10 MeV or higher.

The tissue may be irradiated with an electron beam for a time and dose sufficient to achieve inactivation of viruses and microbes without adversely affecting the biological activity of one or more enzymes that are subsequently extracted or isolated from the irradiated tissue. The dose of electron irradiation required to sterilize tissue may vary, for example, depending on the size of the tissue, the type of tissue, and the type and amount of viral or microbial contaminant suspected or present in the tissue sample. One of ordinary skill in the art will recognize and be able to determine the appropriate dose and time appropriate for a particular tissue and based on the characteristics of the tissue and the accelerator used. The selected dose of electron radiation is effective in inactivating infectious agents that are difficult to destroy by conventional methods without minimal loss of enzymatic activity.

The absorbed dose of radiation is expressed in kilograys (kGy), where 1 kGy = 1000 J of energy applied to 1 kg of material. In certain embodiments, the tissue is exposed to a beam of electrons until an amount of radiation that inactivates the infecting agent is absorbed. For example, tissue can be exposed to a beam of electrons up to

- 7 045327 dose intensification of approximately 10, approximately 15, approximately 20, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45 or approximately 50 kGy or more. As another example, tissue may be exposed to a beam of electrons to achieve a dose of about 5 to 50, about 10 to 40, about 15 to 35 kGy. In some embodiments, the tissue may be exposed to a beam of electrons to achieve a dose of approximately 30 kGy. In some embodiments, the porcine pancreas is irradiated with a dose of an electron beam sufficient to provide high log ₁₀ killing of infectious agents, preferably infectious agents that are difficult to inactivate, such as non-enveloped viruses, with minimal loss of enzymatic activity.

In some embodiments, the dose may be determined using films of radiochromic dyes. Such films can be calibrated to national reference standards.

In some embodiments, the tissue is irradiated with an electron beam for a time and at a dose sufficient to achieve at least three log ₁₀ , preferably at least four log ₁₀ , reduction in the magnitude of the viral load of the model virus relative to the reference sample. In one such embodiment, the tissue is irradiated with an electron beam for a time and dose sufficient to achieve at least three log ₁₀ , preferably at least four log ₁₀ , reduction in the magnitude of the viral load of the model virus relative to the reference sample. In some embodiments, the model virus is porcine parvovirus. In some embodiments, the tissue is intact tissue. In some of these embodiments, the intact tissue is a gland, such as a whole pancreas or a portion thereof, such as one or more pancreatic lobes. In another such embodiment, the intact tissue is frozen exfoliated tissue.

In some embodiments, exposure to the electron beam includes exposure from one side or the other. In some embodiments, the tissue is exposed to the electron beam on one side. In some embodiments, the tissue is exposed to an electron beam from multiple directions, such as a bidirectional electron beam. Thus, a dose of approximately 20 kGy may involve bilateral exposure of approximately 10 kGy/side, a dose of approximately 30 kGy may include bilateral exposure of approximately 15 kGy/side, a dose of approximately 40 kGy may include bilateral exposure of approximately 20 kGy/side, dose of approximately at 50 kGy may include bilateral exposure of approximately 25 kGy/side.

In some embodiments, methods for producing an enzyme preparation reduce the risk of viral or microbial contamination of a pharmaceutical composition containing the enzyme preparation.

In some embodiments, the infection rate of the irradiated pancreas is reduced by at least three log ₁₀ , preferably at least four log ₁₀ , compared to the infection rate of the gland before radiation treatment.

In some embodiments, the contamination of an enzyme preparation derived from or isolated from an irradiated pancreas is reduced by at least three log ₁₀ , preferably at least four log ₁₀ , compared to the contamination of the gland before radiation treatment. Alternatively, the contamination of the enzyme preparation is determined relative to the enzyme a preparation obtained or isolated from a non-irradiated pancreas.

In some embodiments, the reduced infection is a viral infection, particularly a non-enveloped virus such as porcine parvovirus (PVV). For example, the PVA infection of a pancreatin product isolated from an irradiated pancreas is reduced by at least three log ₁₀ , preferably by at least four log10 relative to the reference drug isolated from non-irradiated pancreas. As another example, porcine parvovirus contamination of a pancreatin product isolated from irradiated pancreas is reduced by at least three log ₁₀ , preferably by at least four log 10 relative to non-irradiated pancreas. In one such embodiment, porcine parvovirus infection of the enzyme preparation is further reduced by subsequent orthogonal viral inactivation steps performed after irradiation.

In some embodiments, the methods may also include following the irradiation step the step of testing for the presence or quantitation of one or more microorganisms (eg, viruses, bacteria, protozoa) in the tissue or in an enzyme preparation isolated from the tissue. Methods for determining whether a sample contains a microorganism are known in the art and include, for example, a plaque formation assay or a colony formation assay. Effective sterilization can also be determined using conventional microbiological techniques, such as, for example, including suitable biological indicators in the irradiated batch or contacting tissue with culture medium and incubating the medium to determine sterility

- 8 045327 fabrics.

In certain embodiments, viral infection can be determined by titration endpoint followed by calculation of the tissue culture infecting dose (TCID ₅₀ ). Viral titers calculated in this way can be expressed as log ₁₀ TCID ₅₀ per ml with a 95% confidence interval.

In some embodiments, the reduction in viral contamination is reported in accordance with USP-NF general chapter <1050> as a logarithmic reduction factor, which is the difference in viral titer between the reference sample and the sample obtained from tissue irradiated with the electron beam after isolation. For example, a three log ₁₀ reduction could mean a 1,000-fold reduction in viral load, and a four-log ₁₀ reduction could mean a 10,000-fold reduction in viral load.

In some embodiments, methods for producing an enzyme preparation can reduce viral and/or microbial contamination of the enzyme preparation without significantly reducing its enzyme activity.

In some embodiments, the enzyme activity of the enzyme preparation isolated from the irradiated pancreas is preserved. For example, in some embodiments, the biological activity of the enzyme preparation is at least 50%, at least 60%, at least 70%, at least 80%, or preferably at least 90% of the biological activity of the comparator drug isolated from non-irradiated pancreas glands. In certain embodiments, the biological activity is an enzyme activity, such as protease activity, amylase activity, or preferably lipase activity.

After irradiation, the tissue can be further processed to obtain an enzyme composition. For example, in some embodiments, one or more enzymes and/or proenzymes may be extracted from the irradiated tissue. In some embodiments, one or more enzymes and/or proenzymes may be isolated from the irradiated tissue. Various methods are known for isolating enzymes from tissue samples. For example, US 4,623,624 provides methods for isolating pancreatin by autolysis of an aqueous, isopropanol-containing tissue suspension.

In some embodiments, the irradiated tissue may be subjected to autolysis and/or hydrolysis to convert the proenzymes to their active form. For example, irradiated tissue can be combined with a hydrolysis initiator to initiate autolysis. In some embodiments, the autolysis and/or hydrolysis is carried out at ambient temperature. In some embodiments, the reaction mixture is filtered at the end of the reaction, the filtrate is collected, the enzymes present in the filtrate are precipitated (eg, with isopropanol), the precipitate is filtered, washed with isopropanol and dried under reduced pressure.

It should be understood that the methods described above and illustrated in the Examples section are illustrative and should not be construed as limiting the scope of the invention as defined in the accompanying claims. All alternatives, modifications and equivalents of the methods and specific examples are included within the scope of the claims.

B. Compositions.

In at least one embodiment, the present invention includes compositions containing an enzyme preparation disclosed herein. In certain embodiments, the composition includes one or more enzymes and/or proenzymes extracted from irradiated animal tissue. In certain embodiments, the composition includes one or more enzymes isolated from irradiated animal tissue. In certain embodiments, the composition is a crude mixture containing one or more enzymes isolated from animal tissue.

In another aspect, a pharmaceutical composition is provided comprising the enzyme preparation disclosed herein, and further optionally containing one or more conventional pharmaceutically acceptable excipients such as those found in monographs such as Remington's Pharmaceutical Sciences, ^18th Ed. (Alfonso R. Gennaro, ed., Mack Publishing Company, Easton, PA, 1990), Remington the Science and Practice of Pharmacy, ^19th Ed. (Lippincott, Williams & Wilkins, 1995), Handbook of Pharmaceutical Excipients, ^3rd Ed. (Arthur H. Kibbe, ed., Amer Pharmaceutical Assoc, 1999), the Pharmaceutical Codex Principles and Practice of Pharmaceutics ^12th Ed. (Water Lund ed., Pharmaceutical Press, London, 1994), The United Kingdom Pharmacopeia The National Formulary (United States Pharmacopeial Convention), and Goodman and Gilman's the Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds, McGraw Hill, 1992), the contents of which are incorporated herein by reference.

Pharmaceutical compositions containing the enzyme preparation disclosed herein can be used to replenish digestive enzymes in the treatment and/or prevention of digestive disorders in animals, in particular digestive disorders due to chronic exocrine pancreatic insufficiency, such as in patients suffering from cystic fibrosis, chronic pancreatitis , or in patients who have undergone surgery on the upper gastrointestinal tract. Pharmaceutical compositions or dosage forms,

- 9 045327 disclosed in the description may preferably be oral dosage forms, which, in particular, can be administered to humans.

The oral dosage form containing the enzyme preparation may be, for example, in the form of capsules, granules, granulates, micropellets, microspheres, microtablets, pellets, pills, powders and/or tablets. For purposes of this specification, the prefix micro is used to describe an oral dosage form if the diameter of the oral dosage form or all of its dimensions (length, height, width) is equal to or less than about 5 mm.

In certain embodiments, the oral dosage form is a capsule. The capsule may contain from about 2000 to about 40,000 lipase units per capsule. In some embodiments, the oral dosage form is a capsule containing 3000, 6000, 12000, 24000, or 36000 lipase units per capsule. In some embodiments, the oral dosage form is a capsule containing 3000, 5000, 10000, 15000, 20000, 25000, or 40000 lipase units per capsule. In some embodiments, the oral dosage form is a capsule containing 2600, 4200, 10500, 16800, or 21000 lipase units per capsule. In some embodiments, the oral dosage form is a capsule containing 4000, 13800, 20700, or 23000 lipase units per capsule. In some embodiments, the oral dosage form is a capsule containing 4000, 8000, or 16000 lipase units per capsule. In some embodiments, the oral dosage form is a capsule containing 4000, 8000, or 16000 lipase units per capsule. In other embodiments, the oral dosage form is a capsule containing 3000, 4000, 6000, or 8000 lipase units per capsule. Dosage can be expressed in a variety of ways, which include USP units, European Pharmacopoeia units, or British Pharmacopoeia units.

In some embodiments, the oral dosage form is a tablet containing 10,440 or 20,880 lipase units per tablet.

A variety of pharmaceutical compositions and dosage forms containing pancreatin are known, such as delayed or immediate release formulations. For example, US 9198871 provides delayed release pancreatin compositions.

In some embodiments, the oral dosage form is a pancreatin micropellet or pancreatin microsphere. In some embodiments, the pancreatin micropellet or pancreatin microsphere is coated, for example, with an enteric coating. In some embodiments, the pancreatin micropellet or pancreatin microsphere, regardless of the presence of any such coating, contains from about 10 to about 95% by weight pancreatin, from about 5 to about 90% by weight of at least one pharmaceutically acceptable binding agent, and between 0 and about 10% by weight of at least one pharmaceutically acceptable excipient. More specifically, the pancreatin micropellet or pancreatin microsphere contains from about 70 to about 90% by weight pancreatin, from about 10 to about 30% by weight of at least one pharmaceutically acceptable binding agent, and between 0 and about 5% by weight of at least one a pharmaceutically acceptable excipient. In some embodiments, the pancreatin micropellet or pancreatin microsphere contains from about 70 to about 90% by weight pancreatin, from about 10 to about 30% by weight of at least one pharmaceutically acceptable binding agent. In some embodiments, the pancreatin micropellet or pancreatin microsphere is approximately spherical and has a diameter of from about 0.5 to about 2.0 mm. In some embodiments, the pancreatin micropellet or pancreatin microsphere has a first dimension of about 0.5 to about 2.0 mm and a second dimension of about 0.5 to about 2.0 mm. In some embodiments, the pancreatin micropellet or pancreatin microsphere has a first dimension of about 0.8 to about 1.0 mm and a second dimension of about 0.5 to about 2.0 mm.

Examples of pharmaceutically acceptable coupling agents include polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, hydroxypropyl methylcellulose, polyoxyethylene, polyoxyethylene-pol copolymers ioxypropylene and mixtures of these organic polymers. The preceding list of pharmaceutically acceptable binding agents is not to be understood as exhaustive, but merely illustrative, as one of ordinary skill in the art will appreciate that a variety of other pharmaceutically acceptable binding agents or combinations of binding agents may be used. Polyethylene glycol 4000 is the preferred pharmaceutically acceptable binding agent.

Examples of suitable pharmaceutically acceptable excipients include glidants such as magnesium stearate or calcium stearate, stearic acid, talc and/or starch; fillers such as calcium phosphate, corn starch, dextrans, dextrin, hydrated silica, microcrystalline cellulose, kaolin, lactose, mannitol, polyvinylpyrrolidone, precipitated calcium carbonate, sorbitol and/or talc; disintegrating agents such as aerosil (silicic acid), alginic acid, amylose, calcium alginate, calcium carbonate, formaldehyde gelatin, pectin carbonate, sago starch, sodium bicarbonate and/or starch; and/or wetting agents such as glycerin and/or starch. The foregoing list of pharmaceutically acceptable excipients is not to be understood as exhaustive, but merely as illustrative, as one of ordinary skill in the art will appreciate that many other pharmaceutically acceptable excipients or combinations of excipients may be used. For the purposes of this description, synthetic oils and monomeric phthalate esters should not be considered suitable pharmaceutically acceptable excipients. In some embodiments, pancreatin micropellets or pancreatin microspheres do not contain pharmaceutically acceptable excipients, but may optionally contain increased amounts of pancreatin.

In another embodiment, an oral dosage form of pancreatin, such as an enteric-coated oral dosage form, is provided for the manufacture of a medicament for the treatment of medical conditions such as digestive disorders, exocrine pancreatic insufficiency, pancreatitis, cystic fibrosis, type I diabetes and/or diabetes mellitus II.

In some embodiments, the pharmaceutical composition is a controlled release pharmaceutical composition, for example, the controlled release pharmaceutical composition can be prepared by applying an enteric coating to an oral dosage form. In some embodiments, the enteric coating includes a film-forming agent, a plasticizer, and optionally anti-tack additives.

Suitable film-forming agents include agar, carbomer homopolymer and copolymers (i.e., high molecular weight acrylic acid cross-linked polymers), carboxymethyl cellulose, carboxymethyl ethyl cellulose, carrageen, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate trimelliate, chitin, protein extract cereals, ethyl cellulose, gum arabic, hydroxypropyl cellulose, hydroxypropyl methyl acetate succinate, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, methacrylic acid-ethyl methacrylate copolymer, methyl cellulose, pectin, polyvinyl acetate phthalate, polyvinyl alcohol, shellac, sodium alginate, starch acetate phthalate and /or styrene/maleic acid copolymer or mixtures of said film-forming polymers. Preferred film-forming agents are cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate and methacrylic acid-ethyl methacrylate copolymer. Most preferred is a hydroxypropyl methylcellulose phthalate such as HP 55 or HPMCP HP-50. Synthetic oils should not be considered the preferred film-forming agents. The foregoing list of film-forming agents should not be understood as exhaustive, but merely as illustrative, as one of ordinary skill in the art will appreciate that a variety of other film-forming agents or a combination of film-forming agents may be used.

The plasticizer(s) typically may be present in an amount of greater than about 1.5% and typically in an amount of from about 2 to about 20% by weight of the film-forming agent. The plasticizer may contain saturated, unbranched monohydric alcohols having from 12 to 30 carbon atoms. More specifically, suitable plasticizers include lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, heptdecyl alcohol, stearyl alcohol, nonadecyl alcohol, arachidonic alcohol, behenyl alcohol, carnaubyl alcohol, ceryl alcohol, corianyl alcohol, melissyl alcohol, acetyltributyl citrate, dibutyl sebacate, glycerol fatty acid esters, glycerin, polyethylene glycol, propylene glycol, sorbitol fatty acids, triacetin, triethyl citrate and mixtures of these plasticizers. Preferred plasticizers are cetyl alcohol, stearyl alcohol, triethyl citrate, and mixtures thereof. When cetyl alcohol is used as the sole plasticizer, it may be present in an amount of greater than about 1.5%, typically in an amount of from about 2 to about 15%, preferably from about 2 to about 10%, by weight of the film-forming agent. When triethyl citrate is used as the sole plasticizer, it may be present in an amount of from about 5 to about 20%, preferably from about 12 to about 15%, by weight, based on the film-forming agent. Synthetic oils and phthalic acid monomer esters should not be considered suitable plasticizers. The foregoing list of plasticizers should not be understood as exhaustive, but merely as illustrative, as one of ordinary skill in the art will appreciate that many other plasticizers or a combination of plasticizers may be used so long as they are substantially free of both synthetic oils and monomeric phthalic acid esters.

In certain embodiments, the plasticizer consists of cetyl alcohol and triethyl citrate, which together are present in an amount greater than about 3%, typically in an amount

- 11 045327 from about 4 to about 20%, in particular between about 6 and about 15%, more particularly between about 7 and about 10% by weight, based on the film-forming agent. The weight ratio of cetyl alcohol and triethyl citrate when present together may be from about 0.05:1 to about 1:1, for example 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1 or 0.9:1. Specifically, the ratio of cetyl alcohol to triethyl citrate may be from about 0.25:1 to about 0.5:1, preferably from about 0.3:1 to about 0.45:1, more preferably from about 0.35:1 to about 0.4:1, and even more preferably from about 0.38:1 to about 0.4:1 (w/w).

The enteric coating optionally contains a detack additive. Suitable detackifiers include dimethicone and castor oil. Dimethicone, particularly dimethicone 1000, is a preferred anti-stick additive. The amount of tack reducing agent (if present) in the enteric coating is from about 1.5 to about 3% by weight based on the film-forming agent. Synthetic oils should not be considered the preferred anti-stick additives. The foregoing list of detackifiers is not to be understood as exhaustive, but merely illustrative, as one of ordinary skill in the art will appreciate that a variety of other detackifiers, or a combination of detackifiers, may be used.

In some embodiments, the enteric coating comprises from about 5 to about 30% by weight, more preferably from about 7 to about 20%, even more preferably from about 10 to about 15% by weight of the total composition of the enteric-coated oral dosage form or pharmaceutical controlled release compositions. In some embodiments, the enteric coating comprises from about 20 to about 30% by weight, more preferably from about 22 to about 26% by weight, even more preferably from about 22.5 to about 25% by weight of the total composition of the oral dosage form with enteric coating or controlled release pharmaceutical composition.

In some embodiments, the pharmaceutical composition is a controlled release capsule for oral administration. The capsule may contain enteric-coated pellets including lipase, protease and amylase. The enteric coated pellets may have a first dimension of about 0.5 to about 2 mm and, optionally, a second dimension of about 0.5 to about 2 mm. For example, enteric-coated pellets may be approximately spherical and have a diameter of from about 0.71 to about 1.60 mm. As another example, enteric-coated pellets may be filamentous and have a diameter of from about 0.5 to about 2.0 mm and a length of from about 0.5 to about 2.0 mm. The composition may further include inactive ingredients disclosed herein, such as cetyl alcohol, dimethicone, hypromellose phthalate, polyethylene glycol and triethyl citrate.

In some other embodiments, the pharmaceutical composition is an immediate release pharmaceutical composition. For example, an immediate release pharmaceutical composition may not have an enteric coating.

D. Methods of application.

In at least one aspect, the present invention includes a method of treating exocrine pancreatic insufficiency in a subject, particularly in a human subject for the purpose of such treatment. The method includes administering to a subject an enzyme preparation or a pharmaceutical composition containing an enzyme preparation. In some embodiments, exocrine pancreatic insufficiency is a consequence of cystic fibrosis, chronic pancreatitis, pancreatectomy, or other conditions.

In another aspect, the present invention includes a method of treating or preventing a digestive disorder in a subject, particularly in a human subject for the purposes of such treatment or prevention. In some embodiments, the digestive disorder is a consequence of chronic exocrine pancreatic insufficiency, such as in patients suffering from cystic fibrosis, chronic pancreatitis, or in patients undergoing upper gastrointestinal surgery.

In another aspect, the present invention includes the use of an enzyme preparation or a pharmaceutical composition containing an enzyme preparation for the treatment of exocrine pancreatic insufficiency in a subject for the purpose of such treatment.

In another aspect, the present invention includes the use of an enzyme preparation or a pharmaceutical composition containing an enzyme preparation for the treatment or prevention of a digestive disorder in a subject for purposes of such treatment or prevention.

In some embodiments related to the methods and uses mentioned above, the enzyme preparation contains pancreatin. In some embodiments, the appropriate dosage of pancreatin may be based on lipase units. The Cystic Fibrosis Committee (CFF) has published Consensus Guidelines that contain recommended total daily doses

- 12 045327 lipase units.

In some embodiments, infants under 12 months of age are administered 2,000 to

4000 lipase units, preferably 3000 lipase units per 120 ml of nutritional composition or per breastfeeding.

In some embodiments, patients 1 to 4 years of age are administered 1000 to 2500 lipase units per kg of body weight per meal. In some embodiments, 500 to 2500 lipase units per kg of body weight are administered per meal at at least 4 years of age.

In some embodiments, the maximum daily dosage does not exceed 10,000 lipase units per kg of body weight. In some embodiments, the maximum daily dosage does not exceed 4000 lipase units per gram of fat absorbed.

In some embodiments, the enzyme preparation or pharmaceutical composition containing the enzyme preparation is administered immediately before a meal. In some embodiments, the enzyme preparation or pharmaceutical composition containing the enzyme preparation is administered with a meal or snack.

In yet another embodiment, a method is provided for treating a medical condition, such as digestive disorders, exocrine pancreatic insufficiency, pancreatitis, cystic fibrosis, type I and/or II diabetes, by administering to a person a therapeutically effective amount of an enzyme preparation or a pharmaceutical composition containing an enzyme preparation for the purpose of such treatment.

In at least one aspect, the present invention includes a method for protein degradation. The method involves contacting the protein to be digested with an enzyme composition under conditions sufficient to break down the protein.

In some embodiments, the enzyme preparation includes one or more enzymes and/or proenzymes extracted from animal tissue irradiated with an electron beam. In some embodiments, the enzyme preparation includes one or more enzymes and/or proenzymes isolated from animal tissue irradiated with an electron beam. In some embodiments, the enzyme preparation is in a crude form.

In some embodiments, the contacting step occurs in vivo. In some embodiments, the contacting step occurs in vitro.

In some embodiments, the digestible protein is used to prepare a hydrolyzed protein product.

The enzyme preparations, compositions, methods and methods of use disclosed herein will be better understood with reference to the following embodiments and examples, which are included herein by way of illustration and are not intended to limit the scope of the invention.

D. Embodiments.

One aspect of the present invention includes an enzyme preparation obtained by a method comprising the steps of:

(a) obtaining pancreatic tissue from an animal;

(b) irradiating pancreatic tissue with an electron beam to produce irradiated pancreatic tissue, wherein irradiation with the electron beam is sufficient to achieve a reduction in microbial and/or viral load; and (c) release of pancreatin from irradiated pancreatic tissue.

In some embodiments, the isolation step eliminates the initiation of hydrolysis or autolysis of pancreatic tissue. In some embodiments, the isolation step involves mixing the irradiated pancreatic tissue with water. In some embodiments, step (c) involves activating the proenzyme from the irradiated pancreatic tissue. In some embodiments, the reduction in microbial and/or viral load is at least three log ₁₀ , preferably at least four log ₁₀ relative to a reference sample obtained from non-irradiated tissue. For example, the reduction in microbial and/or viral load of porcine parvovirus (PPV) is at least three log ₁₀ , preferably at least four log ₁₀ relative to a reference sample obtained from non-irradiated tissue. In some embodiments, the biological activity of the enzyme preparation obtained in step (c) corresponds to at least 50%, preferably at least 90% of the biological activity of the reference enzyme preparation obtained from non-irradiated pancreatic tissue. In some embodiments, the biological activity is a lipase activity. In some embodiments, the biological activity is a protease activity or an amylase activity. In some embodiments, the method further includes one or more additional steps that provide further reduction in microbial and/or viral load.

Another aspect of the present invention includes an enzyme preparation containing one or more enzymes isolated from tissue of a mammal that has been subjected to processing sufficient to achieve a reduction in viral load of at least three log ₁₀ , preferably at least

- 13 045327 by four log1o. For example, the treatment is sufficient to achieve a reduction in PVA viral load of at least three log ₁₀ , preferably at least four log ₁₀ relative to an unirradiated control sample.

Another aspect of the present invention includes an enzyme preparation containing one or more enzymes isolated from tissue of a mammal, wherein prior to isolating the enzyme, the tissue of the mammal is subjected to a treatment sufficient to achieve a reduction in viral load of at least three log ₁₀ , preferably at least four log ₁₀ . For example, the treatment is sufficient to achieve a reduction in PVA viral load of at least three log ₁₀ , preferably at least four log ₁₀ relative to the untreated control sample. In some embodiments, the mammalian tissue is porcine pancreatic tissue. In some embodiments, the porcine pancreatic tissue is dissected. In some embodiments, the porcine pancreatic tissue is in the form of a frozen block. In some embodiments, the porcine pancreatic tissue is a complete pancreas or a portion thereof, such as one or more lobes. In some embodiments, the one or more enzymes include pancreatin. In some embodiments, the treatment includes irradiation with an electron beam. In some embodiments, the electron beam irradiation has a dose of from about 5 to about 50 kGy, preferably from about 10 to about 40 kGy. In some embodiments, the biological activity of the enzyme preparation corresponds to at least 50%, preferably at least 90% of the biological activity of the comparator obtained from non-irradiated mammalian tissue. In some embodiments, the biological activity is a lipase activity. In some embodiments, the biological activity is a protease activity or an amylase activity. In some embodiments, the reduction in viral load is an orthogonal reduction.

Another aspect of the present invention includes a method of reducing the risk of contamination of a pancreatin product with an infectious agent, comprising the steps of:

(a) providing pancreatic tissue to a mammal;

(b) exposing the pancreatic tissue to an electron beam to produce irradiated pancreatic tissue, wherein the electron beam irradiation is sufficient to achieve a reduction in the risk of contamination of the pancreatin product with an infectious agent; and (c) isolating pancreatin from the irradiated pancreatic tissue, thereby obtaining a pancreatin product with a reduced risk of contamination by an infectious agent relative to the mammalian pancreatic tissue obtained in step (a) or the pancreatin sample isolated from non-irradiated pancreatic tissue.

In some embodiments, the infectious agent is porcine parvovirus (PVV). In some embodiments, the method provides a reduction of at least three log ₁₀ , preferably at least four log ₁₀ , in a measure of the activity level of a non-enveloped virus, such as porcine parvovirus (PVV). In some embodiments, the measure of the level of activity of the non-enveloped virus is the viral load.

Another aspect of the present invention includes an enzyme preparation containing one or more enzymes isolated from mammalian tissue and a substantially inactivated non-enveloped virus, wherein the preparation has a biological activity that corresponds to at least 50%, preferably at least 90%, of the biological activity of the comparator drug . In some embodiments, the comparator drug has not been processed sufficiently to inactivate the non-enveloped virus. In some embodiments, the substantially inactivated non-enveloped virus is porcine parvovirus (PVV). In some embodiments, the one or more enzymes include pancreatin. In some embodiments, one or more enzymes are isolated from mammalian tissue. In some embodiments, the mammalian tissue is mammalian tissue irradiated with an electron beam.

Another aspect of the present invention includes a pharmaceutical composition containing one or more enzymes isolated from mammalian tissue that has been processed to reduce the risk of viral or microbial infection, wherein the composition has a biological activity corresponding to at least 50%, preferably at least 90% biological activity of the comparison composition. In some embodiments, the one or more enzymes include a lipase. In some embodiments, the comparison composition comprises an untreated sample of mammalian tissue corresponding to mammalian tissue that has been treated to reduce the risk of viral or microbial infection. In some embodiments, the treatment includes irradiation with an electron beam. In some embodiments, the electron beam irradiation has a dose of from about 5 to about 50 kGy, preferably from about 10 to about 40 kGy.

Another aspect of the present invention includes a method for producing a pancreatin product,

- 14 045327 including stages:

(a) obtaining pancreatic tissue from a mammal;

(b) irradiation of pancreatic tissue with an electron beam to obtain irradiated pancreatic tissue:

(c) isolating pancreatin from irradiated pancreatic tissue to obtain a pancreatin product.

In some embodiments, the biological activity of the pancreatin product obtained in step (c) corresponds to at least 50%, preferably at least 90% of the biological activity of the reference pancreatin product. In some embodiments, the pancreatin reference product is derived from non-irradiated pancreatic tissue. In some embodiments, electron beam irradiation is sufficient to achieve a reduction in viral load of at least three log ₁₀ , preferably at least four log ₁₀ . For example, the treatment is sufficient to achieve a reduction in PVA viral load of at least three log ₁₀ , preferably at least four log ₁₀ relative to an unirradiated reference sample. In some embodiments, the electron beam irradiation has a dose of from about 5 to about 50 kGy, preferably from about 10 to about 40 kGy. In some embodiments, the biological activity is lipase activity. In some embodiments, the biological activity is protease activity or amylase activity.

Another aspect of the present invention includes a method for digesting protein, comprising the steps of (a) providing an enzyme or proenzyme preparation isolated from animal tissue irradiated with an electron beam; and (b) contacting the protein with an enzyme or proenzyme preparation under conditions sufficient to digest the protein. In some embodiments, the contacting step occurs in vivo. In some embodiments, the contacting step occurs in vitro. In some embodiments, the animal tissue is porcine pancreas. In some embodiments, the digestible protein is used to prepare a hydrolyzed protein product. In some embodiments, an enzyme or proenzyme preparation isolated from animal tissue irradiated with an electron beam exhibits a reduction in viral load of at least three log ₁₀ , preferably at least four log ₁₀ relative to the enzyme or proenzyme preparation isolated from non-irradiated animal tissue. In some embodiments, an enzyme or proenzyme preparation isolated from animal tissue irradiated with an electron beam exhibits biological activity corresponding to at least 50%, preferably at least 90% of the biological activity of a comparator enzyme or proenzyme preparation isolated from non-irradiated animal tissue.

Another aspect of the present invention includes an enzyme preparation containing one or more enzymes isolated from pancreatic tissue irradiated with an electron beam. In some embodiments, the pancreatic tissue includes porcine pancreas. In some embodiments, prior to irradiation, the porcine pancreas is frozen and mechanically processed into flakes or blocks. Thus, in some such embodiments, the pancreatic tissue irradiated with the electron beam is dissected frozen pancreatic tissue or a frozen block of pancreatic tissue. In some embodiments, the porcine pancreas is a complete pancreas or a portion thereof, such as one or more lobes. In some embodiments, the one or more enzymes include pancreatin. In some embodiments, the enzyme preparation exhibits a reduction in viral load of at least three log ₁₀ , preferably at least four log ₁₀ , relative to the comparator enzyme preparation. For example, the enzyme preparation exhibits a reduction in PVA viral load of at least three log ₁₀ , preferably at least four log ₁₀ , relative to the comparator enzyme preparation. In some embodiments, the biological activity of the enzyme preparation corresponds to at least 50%, preferably at least 90% of the biological activity of the reference enzyme preparation. In some embodiments, the biological activity is a lipase activity. In some embodiments, the biological activity is a protease activity or an amylase activity. In some embodiments, the comparator enzyme preparation is derived from non-irradiated tissue. In some embodiments, the reduction in viral load is an orthogonal reduction.

Another aspect of the present invention includes a method of treating exocrine pancreatic insufficiency, comprising administering to a patient a dose of any of the above enzyme preparations and/or pharmaceutical compositions. In some embodiments, exocrine pancreatic insufficiency is a consequence of cystic fibrosis or chronic pancreatitis.

E. Examples.

Example 1. Electron beam irradiation of porcine parvovirus (PVV), pancreatin API and porcine pancreas.

Materials and methods.

Porcine parvovirus in cell culture fluid in vials.

Because porcine pancreatic tissue itself may have some inactivating effect on viruses, virus samples used in these studies are initially prepared from infected cell cultures. Porcine parvovirus (PVV) strain NADL-2 (ATCC® VR-742™) and porcine seminal cells (ATCC® CRL-1746™) were purchased from the American Type Culture Collection (ATCC). PVA is propagated, cultured and stored according to ATCC guidelines. PVS are propagated to approximately a titer of 10 ⁸ viral infectious units (IU)/ml. The virus is collected from lysed cells in a cell culture medium consisting of minimal essential medium (MPM), 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin.

Before delivery, PVA is packaged in vials and then placed in double bags. The vials are Nalgene cryogenic vials, i.e. polypropylene with screw-on external high-density polyethylene (HDPE) caps with a leak-proof ring, having a length of 1.87 inches, a diameter of 0.5 inches, a capacity of 2.0 ml and a fill volume of 1.0 ml. Each individual bottle is placed in a Food Saver bag (polyethylene with a nylon outer layer) and vacuum sealed. The bags are cut so that they fit the bottles exactly. The four individual bottles are then placed inside another Food Saver bag (approximately 11x14 inches) and vacuum sealed.

Frozen dissected porcine pancreas.

Pancreas obtained from a slaughter hog (Animal Technology, Tyler, TX) was stored on dry ice.

Frozen dissected porcine pancreas is placed in a 12 x 12 x ¹ 3/4 inch container (clear polypropylene) which is vacuum sealed in a 3 mil polypropylene/nylon bag. The bag is heat sealed. Pancreases are stored on dry ice to ensure temperatures below 20°C. The mass of the frozen dissected porcine pancreas sample is 1.05 kg plus the packaging mass of 270 g (0.27 kg). The lid weighs 100 g. Surface density 1.3 g/cm ² . For each dose of radiation, two packages are prepared, including a control without irradiation, one unopened package is used for the isolation of the enzyme preparation after delivery back to the site of isolation with the other package constituting a supply.

Pancreatin (API).

Two types of pancreatin APIs were used: pancreatin N and pancreatin S. Both APIs were stored under ambient conditions. Pancreatin N (material number 1030828, Abbott Laboratories) is a yellowish/gray to off-white powder. Pancreatin N is derived from the pancreas of the slaughter hog. Pancreatin S (material number 1030829, Abbott Laboratories) is a yellowish/gray to off-white powder. Pancreatin S from the cow's pancreas was used.

Pancreatin API was placed in a 2 ^1/2 x ³ 1/2 x 1 ^5/8 inch container (clear polypropylene) _which was vacuum sealed in a 3 mil polypropylene/nylon bag. The bag was heat sealed. The weight of the pancreatin API sample is 80 g plus the package weight is 26 g. Surface density is 1.4 g/cm ² . Pancreatin N samples were irradiated with an electron beam at the indicated dose. The control was not irradiated with an electron beam.

Pancreatin N activity was determined after returning to the study site. Pancreatin N from the pancreas of a slaughter hog was used. All packages of Pancreatin N arrive unopened from the irradiation site.

Irradiation with an electron beam.

The electron beam source is an Industrial Materials Processing Linear Accelerator (IMPELA®, Iotron Industries, Inc Columbia City, IN), 10 MeV, scan width 80 cm.

Frozen dissected porcine pancreas and porcine parvovirus samples are stored below -20°C on dry ice on an aluminum tray. Dosimeters are placed on the top surface of each sample. Samples of packaged pancreatin API, at ambient temperature, are placed on a stretched sheet of polystyrene foam, with dosimeters placed on the top surface. The packages are directed onto a conveyor belt under the cathode ray tube. After one irradiation pass, the dosimeters are removed. The packages are directed to a second pass under the emitting tube after the package has been inverted (the top side is now facing down) and a new dosimeter is placed on the top surface. After the second round of irradiation, the packages and dosimeters are taken away. Packages of frozen dissected porcine pancreas and porcine parvovirus are picked up and placed on dry ice for transport. Pancreatin API packages are picked up and stored during transport at ambient temperature. Frozen dissected porcine pancreas and porcine parvovirus control packages placed on dry ice for return shipment on dry ice are prepared without exposure to radiation. Pancreatin API control packages prepared for return shipment are stored at ambient temperature and prepared without exposure to radiation. The electron beam dose is calculated from the dosimeter readings using a calibration curve.

Study of viral infection.

Viral contamination was determined by titrating tenfold dilutions in 96-well microplates using appropriate controls in triplicate for each energy dose. Viral titers are calculated using the Reed and Muench method described in Reed LJ, Muench H. (1938), A simple method of estimating fifty percent endpoints, The American Journal of Hygiene, 27:493-493, and expressed as 50% TCID ₅₀ per 1 ml.

Isolation and study of pancreatin.

The method used to isolate pancreatin is substantially similar to that disclosed in US 4,623,624 and involves hydrolysis and/or autolysis followed by fiber screening, enzyme precipitation, separation of the precipitate by filtration and/or centrifugation, washing of the filter cake, drying and particle size reduction. Hydrolysis is carried out using one package corresponding to each dose of radiation, including a non-irradiated control. For each isolation experiment, select packaging without damage (such as large cracks). Due to precautions taken during return shipment to the study site, all packages arrive undamaged and one package for each radiation dose, including controls, is randomly selected for isolation. To begin hydrolysis, pancreatin is used as a source of protease from an earlier release. Calcium hydroxide is used to supply calcium ions necessary to activate proteases. Sodium bicarbonate is used as a buffering agent for hydrolysis. Simethicone is used as an antifoaming agent. Hydrolysis of the pancreas is carried out at a temperature close to ambient temperature. The completion of hydrolysis is determined by centrifuging the hydrolyzed mixture after adding isopropanol as described below. After hydrolysis is completed, additional isopropanol is added to reduce the rate of hydrolysis, the mixture is cooled, the mixture is mixed, and the fibers are separated using a 0.425 inch sieve. The fibers are washed with isopropanol and squeezed to force out the retained liquid. The washing liquid is combined with the previously obtained filtrate. To precipitate the enzymes, additional isopropanol is added to the filtrate. The suspension is filtered through a 15 µm filter cloth and washed with increasing concentrations of isopropanol and absolute isopropanol as a final rinse. The cake is dried on a filter cloth under a stream of nitrogen, creating a vacuum under the filter cloth until the cake appears visually dry (the color of the cake becomes lighter once the isopropanol and water are removed). The cake is removed from the filter cloth and dried under vacuum and a stream of nitrogen at a temperature below about 50°C to a Karl Fischer water content of 3.5% or less. The yield of pancreatin ranges from 80 to 100 g per 1 kg of frozen dissected pancreas of a slaughter hog.

Centrifuge test to determine the completion of hydrolysis.

Three scoops of approximately 10 ml each are taken from the hydrolysis vessel and passed through a 0.425 mm sieve, the filtrate is collected in a plastic glass (the filtrate is also scraped out into the plastic glass), and the fibers that are retained on the sieve are discarded. Using a pipette, add 10 g of solution from the reactor to a 50 ml centrifuge tube, add 5.5 ml of 85% IPA, mix for 1 min with a spatula. In a 50 ml centrifuge tube, add 20 ml of 85% IPA to the filtrate and mix for 1 min with a spatula. Centrifuge the suspension at approximately 90xg for 2 minutes.

The first sample can be taken 2 hours after the start of hydrolysis or when the color changes from pink to brownish and the suspension becomes thinner (approximately 2 hours).

The next sample is taken when the color turns brown without a pink tint, at this point approximately 25% sediment is expected, the sediment will be less solid and residue may be present in the clear top layer. Sampling can then be carried out at half-hourly intervals if possible.

Hydrolysis is stopped when two consecutive measurements show less than 20% precipitation. At this point the sediment will be solid with no residue in the clear top layer.

Bilateral irradiation of PVA in vials with an electron beam is carried out in three repetitions. Data on the viral load and logarithmic reduction of PVA exposed to electron beam irradiation are given in Table. 1.

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Table 1

Dose 0 9.5 19.25 38.45 0 9.5 19.25 38.45 (kGy) Viral Viral Viral Viral Logarith- Logarith- Logarith- Logarith- load load load load mystical mystical mystical mystical reduction reduction reduction reduction Virus, Virus, Virus, Virus, titer/ml titer/ml titer/ml titer/ml Test A 1.50E+08 2.50E+06 1.50E+05 0.00E+00 1.78E+00 3.00E+00 Test B 1.50E+08 6.34E+06 6.34E+04 0 1.37E+00 3.37E+00 Test C 4.00E+08 1.26E+06 4.00E+05 1.50E+02 0 2.50+00 3.00E+00 6.43E+00 Average 0 1.88E+00 ZD2E+00 6.43E+00

The electron beam dose is determined by estimating the dose absorbed by each sample from the surface dose indicated by a dosimeter mounted on the sample container. In table 1 shows that exposure of the PVA to approximately 40 kGy provides approximately 6.5 log ₁₀ log reduction, while exposure to approximately 20 kGy provides approximately 3 log ₁₀ log reduction. Based on these data, exposure of approximately 30 kGy is expected to provide approximately 4 log ₁₀ log reduction.

Exposure of PVA to approximately 60 kGy, approximately 80 kGy, or approximately 100 kGy results in final viral titers below the study detection limit.

Pancreatin is tested by validated methods for free protease, amylase, and lipase activities as described in the United States Pharmacopoeia. Pancreatin is tested by validated methods for total protease, as described in the European Pharmacopoeia (EP).

Performing bilateral irradiation of pancreatin N with an electron beam.

All pancreatin N containers at the study site are obtained intact and used for the study.

Data on the enzyme activity of pancreatin API irradiated with an electron beam are given in Table. 2.

table 2

Dose of electronic Activity Activity General Activity beam lipases amylase activity free proteases proteases kGy units units units units American American American American pharmacopeia/g pharmacopeia/g pharmacopeia/g pharmacopeia/g Control (0 kGy) 89106 564390 383192 314093 18.6 kGy 63328 398752 321567 266540 37.45 kGy 58631 386628 302341 244531 56.5 kGy 47755 282378 272499 232087 76.35 kGy 40583 272760 259117 214914 99.4 kGy 37799 286652 247005 196356

In table Table 2 shows that exposing the pancreatin API to approximately 20 kGy results in a loss of approximately 30% of lipase activity, exposing the pancreatin API to approximately 40 kGy results in a loss of approximately 35% of lipase activity, and exposing the pancreatin API to approximately 60 kGy results in a loss of approximately 46% of lipase activity. activity, exposure of the pancreatin API to approximately 80 kGy results in a loss of approximately 54% of lipase activity, exposure of the pancreatin API to approximately 100 kGy results in a loss of approximately 58% of lipase activity. Based on these data, exposure to approximately 30 kGy is expected to result in a loss of approximately 30% of lipase activity.

Performing bilateral irradiation of the pancreas of a slaughter hog with an electron beam. Data on the enzymatic activity of pancreatin obtained from the frozen dissected pancreas of a slaughter hog, subjected to irradiation with an electron beam, are given in table. 3.

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Table 3

Dose of electronic Activity Activity General activity Activity beam lipases amylase proteases free proteases kGy units units units units American American American American pharmacopeia/g pharmacopeia/g pharmacopeia/g pharmacopeia/g Control (0 kg r) 74986 423487 243421 151365 18.75 kGy 74741 449569 337594 128749 35.5 kg r 65348 349816 360407 128506 56.55 kg p 57976 398281 268297 119727 77.4 kGy 34806 251691 175994 109158 100.4 kGy 36362 276118 206217 100342

In table 3 shows that exposure of frozen dissected slaughter hog pancreas to approximately 20 kGy results in a loss of approximately 1% lipase activity, exposure of frozen dissected slaughter hog pancreas to approximately 40 kGy results in loss of approximately 13% lipase activity, exposure of frozen dissected slaughter hog pancreas to approximately 13% loss of lipase activity. exposure of a slaughter hog to approximately 60 kGy results in a loss of approximately 23% of lipase activity, exposure of a frozen dissected slaughter hog pancreas to approximately 80 kGy results in a loss of approximately 54% of lipase activity, and exposure of a frozen dissected slaughter hog pancreas to approximately 100 kGy results in a loss of approximately 52 % lipase activity. Based on these data, exposure to approximately 30 kGy is expected to result in a loss of approximately 10% of lipase activity.

As shown in table. 2 and 3, irradiation of pancreatin API with an electron beam leads to less loss of enzyme activity compared to irradiation of pancreatic tissue with an electron beam before isolation. Without wishing to be bound by theory, the resistance of intact tissue to electron beam irradiation may be related to the conformation of the enzyme (ie, proenzyme form) in the source tissue and/or concomitant coenzymes in the source tissue providing structure protection.

Example 2. Electron beam irradiation of a whole pig pancreas.

Further research is performed using whole porcine pancreas. Approximately 3.6 kg of thawed porcine pancreas is placed in waxed cardboard boxes measuring approximately 10 x 15 x 1.5 inches. Boxes are frozen at -20°C and stored until used for electron beam irradiation. The boxes are transported for electron beam irradiation in a refrigerated truck (-20°C). The boxes are removed from the refrigerated truck and then subjected to double-sided electron beam irradiation, with the unirradiated boxes serving as controls. Five boxes are used for each nominal radiation dose of 0, 15, 20 and 25 kGy and sent back for evaluation in a refrigerated truck (-20°C), along with unirradiated boxes, and then stored at -20°C.

Frozen pancreas samples are selected from each box in a random order within the box for further pancreatin isolation. The isolation is carried out as described in this description of the invention. Pancreatin, isolated from whole pancreas irradiated with an electron beam, is then tested for enzyme activity according to a colorimetric assay.

Pancreatin API samples are tested using a microplate reader colorimetric kinetic assay using substrates structurally similar to those used in USP 39 <Pancrelipase>. Enzyme activities are determined by measuring the rate of product formation relative to a standard pancrelipase sample. Analytical comparability between registered methods from the American Pharmacopoeia monograph and alternative methods using microplate reader methods is demonstrated.

The sample preparation, isolation and evaluation procedures are repeated four times for each dose to obtain a total of five measurements for each dose. The average values of five measurements are given in table. 4.

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Table 4

Enzyme activity of API after irradiation of whole pancreas

Dose of electronic Average Average Average Average total bundle* lipase amylase activity activity activity activity free protease proteases kGy units units units units American American American American pharmacopoeias/mg pharmacopoeias/mg pharmacopoeias/mg pharmacopoeias/mg Control (0 kGy) 104 456 197 344 14.9 kGy 99 435 201 291 19.9 kGy 106 411 200 316 24.9 kGy 104 395 * (minimum dose + maximum dose)/2. 183 301

In table 4 shows that pancreatin isolated from a whole pancreas irradiated with an electron beam up to about 25 kGy has the same or substantially the same lipase activity as pancreatin isolated from an unirradiated control sample.

Example 3. Electron beam irradiation of intact porcine pancreatic tissue with low doses.

Further studies are performed by injecting porcine pancreatic tissue with live virus and then exposing the virus-containing tissue to low-dose electron beam radiation (approximately 12.3 kGy) to allow virus isolation and evaluation. This additional study is being conducted to demonstrate the effective inactivation of several related viruses at low doses to provide effective quantification of the effects of electron beam irradiation.

Selected viruses are deliberately injected into tissue samples and the level of virus elimination is determined by comparing the amount of virus injected with the amount of virus remaining after treatment with an electron beam. The viruses selected for this study are listed in Table. 5.

Table 5

Viruses selected for viral clearance studies

Virus Family Genome Shell Size(nm) Physico- Reovirus type 3 Reo RNA No 60-80 chemical resistance Average REOD Parvovirus Parvo DNA No 18-24 High pigs pvs Feline Calici RNA No 35-39 Average

calicivirus

KKV.

PEO3 has a double-stranded RNA, segmented genome and belongs to the Reoviridae family of viruses, which also includes rotavirus. Thus, PEO3 may serve as a model for rotavirus. KCV is used as a model virus to test the validity of inactivation methods in blood products and, in particular, as a model virus for hepatitis E virus (HEV).

The pancreas of pigs is cut and crushed. The tissue is then added to a petri dish and a specific virus is injected into it. Standard viral materials have certified titers of at least 1 x 10 ⁷ PFU/ml. The sample with the additive is kept at room temperature for at least 60 minutes (until the fabric returns to its original dryness). Following aging, additional porcine pancreatic tissue is added per plate. The dish is then sealed and placed on dry ice for transport to the electron beam irradiation facility. Each dish contains approximately 13 g of tissue, with tissue density similar to that of a whole gland.

For each virus, there are also processed recovery samples (not transported) and non-irradiated transported control samples (transported to the electron beam irradiation facility, but not irradiated).

Irradiation with an electron beam is carried out in an electron beam irradiation installation. After electron beam radiation treatment is completed, irradiated samples and unirradiated transported control samples are transported back for viral load determination. According to the method, samples are stored at a temperature of more than -60°C before testing.

For each sample, 50 ml of culture medium is added to a sterile bottle. The tissue is extracted by soaking three times for approximately 5-10 minutes at room temperature followed by vortexing for 15-30 seconds. The samples are then centrifuged at 3000 rpm for 10 min. The supernatant from the extraction is used to study the virus.

Viral titers are determined using a standard plaque test. Vero, PT-1 and CRFK are used as indicator cell types for PEO3, PVA and KKB, respectively. Viral titers and viral clearance factors were calculated according to standard procedures. The virus elimination factor (VEF) is calculated as follows:

FEV-log Volume * titer before treatment

Volume * titer after treatment

Data on the elimination of the virus caused by irradiation of a tissue sample containing the virus with an electron beam are presented in table. 6.

Table 6

Elimination of the virus after irradiation of the pancreas with the addition of virus

General virus log Factor 95% Model virus Before processing After processing elimination trusting virus interval REOD 6.5 <2.4 >4.1 0.08 KKV 6.7 4.9 1.8 0.05 PVS 5.8 3.6 2.2 0.29

These studies confirm that sufficient inactivation of viruses, including feline calicivirus (FCV) and reovirus type 3 (PEO3), can be achieved by irradiating intact tissue with a low-dose electron beam. Moreover, irradiation of the pancreas followed by isolation of an enzyme preparation (eg, pancreatin API) from the irradiated gland shows similar results of loss of enzyme activity relative to the non-irradiated control sample, as shown in Table. 3, where dissected porcine pancreas was used.

The introduction of a decontamination step prior to processing porcine pancreas provides effective control of known infectious agents, both with respect to operator safety during enzyme isolation and patient safety. Thus, the use of electron beam treatment, which is effective in inactivating a wide range of microbes and viruses, including viruses that are difficult to inactivate (eg, porcine parvovirus), with minimal loss of enzymatic activity, provides an advantage over other methods.

As disclosed herein, the electron beam irradiation step can be considered an orthogonal virus inactivation step. The reduction in microbial and/or viral load obtained in pancreatic tissue irradiated with an electron beam is additively transferred to the pancreatin released from it, in relation to the reduction achieved at other stages of reducing the microbial and/or viral load. The total logarithmic (log ₁₀ ) reduction achieved at all stages, including electron beam irradiation, is the cumulative logarithmic (log ₁₀ ) reduction achieved at all stages of microbial and/or viral inactivation performed on pancreatic tissue.

It is to be understood that the foregoing detailed description and the accompanying examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention, which is defined solely by the appended claims and their equivalents. Various substitutions and modifications to the disclosed embodiments will be apparent to one skilled in the art. Such substitutions and modifications, including, without limitation, those relating to chemical structures, substituents, derivatives, intermediates, syntheses, compositions or methods, or any combination of such substitutions and modifications in the application of the invention may be made without departing from the intent and scope of protection of the invention.

All sources (from patent and non-patent literature) cited above are incorporated herein by reference. The discussion of such sources is intended solely to summarize the statements made by their authors. It is not intended that any of these sources

Claims (8)

kov (or part of any source) is relevant from the prior art (and is generally included in the prior art). The applicant reserves the right to express doubts about the accuracy and appropriateness of cited sources. CLAIM 1. An enzyme preparation for the treatment of exocrine pancreatic insufficiency, obtained by a method including the stages: (a) irradiating intact mammalian pancreatic tissue with an electron beam to produce irradiated pancreatic tissue, wherein the electron beam irradiation has a dose of from about 5 to about 50 kGy and is sufficient to achieve a reduction in the viral load of the model virus by at least three logios relative to comparison sample; and (b) release of pancreatin from irradiated pancreatic tissue. 2. An enzyme preparation according to claim 1, where irradiation with an electron beam is sufficient to achieve a reduction in the viral load of the model virus by at least four logins relative to the comparison sample. 3. An enzyme preparation according to claim 1, where the model virus is porcine parvovirus (PVV). 4. The enzyme preparation of claim 1, wherein the intact mammalian pancreatic tissue is dissected tissue, a whole gland, or a portion of a whole gland. 5. An enzyme preparation according to claim 1, where step (b) includes initiating hydrolysis or autolysis of irradiated pancreatic tissue or activating a proenzyme from irradiated pancreatic tissue. 6. An enzyme preparation according to any one of claims 1 to 5, wherein the biological activity of the enzyme preparation obtained in step (b) corresponds to at least 50%, preferably at least 90% of the biological activity of the reference enzyme preparation. 7. An enzyme preparation according to claim 6, wherein the biological activity is lipase activity. 8. An enzyme preparation according to any one of claims 1 to 7, wherein the electron beam irradiation has a dose of from about 10 to about 40 kGy. ^gj) Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2