Method for producing a pancreatin product

Abstract

The present invention relates to an enzyme preparation obtained from animal tissue irradiated with an electron beam, such as porcine pancreas. The present invention also relates to methods for producing such enzyme preparations, to pharmaceutical compositions comprising such enzyme preparations, and to methods for using such pharmaceutical compositions and enzyme preparations.

Description

METHOD FOR PRODUCING A PANCREATIN PRODUCT Technical field

[0001] The present invention relates to enzyme preparations derived from animal tissue, to pharmaceutical compositions comprising such enzyme preparations, and to methods for reducing the risk of viral or microbial contamination in such preparations and compositions. Exemplary enzyme compositions include pancreatic extracts suitable for therapeutic use, such as for the treatment and / or prophylaxis of maldigestion, and in particular maldigestion based on exocrine pancreatic insufficiency, in mammals and humans. History

[0002] Products derived from animal tissue may exhibit viral and / or microbial contamination. Certain biological contaminants such as bacteria or protozoa can be inactivated during manufacturing processes. However, other biological contaminants such as uncoated viruses and certain spore-forming bacteria (e.g., Bacillus cereus) are resistant to established methods for reducing or inactivating contaminants. A particular challenge is the inactivation or removal of viruses and spore-forming bacteria from enzyme compositions derived from animal tissue without destroying or altering the enzyme activity in the process.

[0003] Established methods for viral inactivation include, for example, pasteurization, dry heat, steam heat, solvent / detergent treatment, and low pH. The selection of methods to be employed for viral inactivation 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 Petition 870260070403, dated 07 / 15 / 2026, page 17 / 79 2 / 61 coated viruses, and thus it has been used for the inactivation of coated viruses. However, in general, many uncoated viruses are not inactivated by treatment with solvent or detergent. Similarly, many spore-forming bacteria are resistant to both heat and organic solvents.

[0004] Heat, particularly dry heat, is another established method for viral inactivation. Although dry heat treatment can inactivate even highly resistant viruses, such as non-coated viruses, the treatment requires long periods of time (several hours) and monitoring of moisture content. Furthermore, heat treatment can compromise the desired biological activity of the product, particularly where the product is an enzymatic composition.

[0005] Pancreatic enzyme products have long been used to treat exocrine pancreatic insufficiency, a condition associated with cystic fibrosis (CF), chronic pancreatitis, obstruction of the pancreas or common bile duct (as a neoplastic disease), surgical procedures such as pancreatectomy or gastrointestinal bypass surgery, as well as other diseases and disorders. The pancreas secretes digestive enzymes, including lipases, proteases, and amylases, into the proximal duodenal lumen, where they facilitate the hydrolysis of macronutrients. Amylases and proteases are secreted by organs other than the pancreas and contribute to the digestion of carbohydrates and proteins. However, there is relatively little lipase from sources other than the pancreas involved in lipid digestion.Thus, patients with untreated exocrine pancreatic insufficiency have difficulty digesting fat and may present with symptoms of maldigestion or malnutrition, or both, with deficiencies in essential fatty acids and fat-soluble vitamins, weight loss, cramps, flatulence, oily stools and foul odor, and excessive amounts of fat in the stool. Petition 870260070403, dated 07 / 15 / 2026, page 18 / 79 3 / 61 stools (steatorrhea). For patients with CF, inadequate treatment can have serious consequences, as good nutritional status is directly related to good lung function.

[0006] Pancreatic enzyme therapy treats and / or prevents malabsorption and facilitates growth and development in patients with exocrine pancreatic insufficiency. In patients with CF, mucus blocks the pancreatic duct in the pancreas as well as in the lungs. Pancreatic digestive enzymes are not secreted into the intestine, and therefore the digestion of starch, fat, and protein is impaired. The lack of digestion results in steatorrhea, abdominal pain, and weight loss, among other symptoms.

[0007] Poor digestion in mammals and humans is generally based on a deficiency of digestive enzymes, particularly a deficiency of endogenous lipase, but also of protease and / or amylase. If pancreatic insufficiency is pathological, it may be congenital or acquired. Acquired chronic pancreatic insufficiency can, for example, be attributed to alcoholism. Congenital pancreatic insufficiency can, for example, be attributed to congenital cystic fibrosis. The consequences of digestive enzyme deficiency can be severe symptoms of undernutrition and malnutrition, which may be accompanied by increased susceptibility to secondary diseases.

[0008] Replacement with exogenous digestive enzymes or digestive enzyme mixtures (i.e., pancreatic enzyme therapy) has proven to be an effective treatment for endogenous digestive enzyme deficiency. Most frequently, pharmaceutical preparations containing porcine pancreatin are used for pancreatic enzyme therapy (also known as “enzyme replacement therapy”). For these pharmaceutical preparations, the active ingredient evaluated in clinical trials is lipase, and dosage amounts for commercial products are given in lipase units. Petition 870260070403, dated 07 / 15 / 2026, page 19 / 79 4 / 61 However, these digestive enzyme mixtures obtained from pig pancreas comprise lipases, amylases, and proteases, and can be used effectively for pancreatic enzyme therapy in humans due to the great similarity of the enzymes and associated substances contained in human pancreatic juices. Pancreatic enzymes are generally administered orally in the form of solid preparations. Pancreases can be obtained from animals, such as pigs, raised and slaughtered for food. Government regulations generally require that pancreases be obtained from a single-species slaughterhouse (i.e., no other species are slaughtered and processed at that facility) and thus limit the availability of starting material. Widespread contamination of facilities with infectious agents can lead to production shutdowns and supply shortages.Current testing procedures can identify contaminated batches, and the disposal of such batches places further burdens on an already limited supply of starting materials.

[0009] Processes are available for obtaining pancreatic enzymes from mammalian pancreas. For example, processes are described in US 4,623,624 from which pancreatin is obtained by autolysis of a semi-fluid tissue paste containing aqueous isopropanol.

[0010] The presence of infectious agents, and in particular viruses and spore-forming bacteria, is recognized in the pancreases of pigs used to manufacture pancreatin. In fact, most pig herds have been infected with porcine parvovirus (PPV), which exhibits high resistance to inactivation. PPV has been detected in pancreatin as an infectious agent. Although PPV is not believed to be pathogenic to humans, it is desirable to obtain pancreatin with a reduced PPV load. Furthermore, PPV is a common model virus because it is difficult to inactivate by standardized methods, such as processing. Petition 870260070403, dated 07 / 15 / 2026, page 20 / 79 5 / 61 chemical or thermal. Similarly, contamination of the pancreatin drug substance with Bacillus cereus and / or Bacillus cereus enterotoxin has been reported.

[0011] US 2010 / 0119654 refers to the irradiation of an aqueous or alcoholic biological extract containing solids in the form of a suspension. The radiation employed in US 2010 / 0119654 is ultraviolet (UV) radiation, X-ray radiation, β radiation, or γ radiation. UV irradiation of a pancreatin intermediate dissolved in 40% isopropanol produced a reduction of up to 4 log10 in phage M2. Gamma irradiation of pancreatin (API) produced a decrease of approximately 40% in lipase activity at 27 kGy and a decrease of 13% in lipase activity at 5 kGy. Bacterial content was reduced by more than 2.5 log10, but virus inactivation was not reported. When pancreatin (API) was treated with β irradiation, it was reported that more than 85% of the enzymatic activity was maintained, but the “germ count” was reduced by only approximately 1.5log10.

[0012] WO 2003 / 020324 refers to the sterilization of digestive enzymes, such as trypsin, α-galactosidase, and iduronate-2-sulfatase, by irradiation. Liquid or lyophilized enzymes (trypsin, a glucosidase, or a sulfatase) were irradiated alone or in the presence of a stabilizer. γ-irradiation was performed using a 60Co source. No viral inactivation was reported.

[0013] WO 2007 / 014896 refers to the reduction of the concentration of one or more biological contaminants, in particular viral ones, in pancreatin by heating the pancreatin.

[0014] In US 2009 / 0233344, heat treatment of pancreatin at 80°C for 32 hours provided a reduction of approximately 2.5 log10 in PPV viral titer, but also a 20% loss in lipase activity. Heat treatment of pancreatin at 100°C for 8 hours provided a reduction greater than 3 log10 in Petition 870260070403, dated 07 / 15 / 2026, p. 21 / 79 6 / 61 viral titer of PPV, but approximately 50% of lipase activity was lost.

[0015] Thus, process steps that may be effective against viruses that are difficult to inactivate, such as PPV, have a high potential to change the nature of the pancreatin product by degrading or reducing pancreatic enzymes, particularly lipase, to unacceptable levels. Such changes in potency may reduce or alter the efficacy profile of the final product. Therefore, it is desirable to maintain enzymatic activity, particularly lipase activity, during the manufacturing process.

[0016] As each of the previously tested viral clearance processes, which demonstrated some efficacy against difficult-to-inactivate viruses (e.g., PPV), resulted in significant loss of enzymatic activity, including lipase, there was skepticism in the industry as to whether a robust level of viral inactivation / clearance could be achieved without compromising product quality. In particular, there was skepticism in the industry that a robust and adequate orthogonal viral clearance step could be developed without adversely impacting the chemical, physical, and pharmaceutical properties of pancreatin. See, for example, the letter from Scientific Protein Laboratories to the FDA dated June 22, 2004, in bulletin no. 2003D-0206. Summary of the invention

[0017] 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 animal tissue source, and retains at least one biological activity of the animal tissue source. In certain embodiments, the enzyme preparation is produced by subjecting the animal tissue source to radiation, preferably electron beam radiation, and subsequently isolating one or more enzymes from the irradiated tissue. In certain embodiments, the Petition 870260070403, dated 07 / 15 / 2026, p. 22 / 79 7 / 61 The source animal tissue is intact tissue. In certain incorporations, the irradiated tissue exhibits a reduction of at least three log10, preferably at least four log10, in viral load when compared with the source animal tissue. In certain incorporations, additional orthogonal viral reduction steps are employed (e.g., during the isolation step, one or more enzymes from the irradiated tissue). In certain incorporations, the enzyme preparation isolated from the irradiated tissue has a biological activity corresponding to at least 50%, preferably at least 90%, of the biological activity of a control enzyme preparation, such as an enzyme preparation isolated from a non-irradiated source animal tissue. In certain incorporations, the biological activity is lipase activity.In certain embodiments, the irradiated tissue exhibits a reduction of at least three log10, preferably at least four log10, in viral and / or microbial contaminants when compared to a non-irradiated source animal tissue, and the enzyme preparation isolated from the irradiated tissue has a biological activity corresponding to at least 50%, preferably at least 90%, of the biological activity of a control enzyme preparation, such as an enzyme preparation isolated from a non-irradiated source animal tissue.

[0018] Another aspect of the present invention relates to a method for producing an enzyme preparation derived from animal tissue, and the enzyme preparation has reduced viral and / or microbial contamination compared to the source animal tissue. The method includes sufficient treatment to produce a reduction of at least three log10, preferably at least four log10, in viral and / or microbial contaminants when compared with the source animal tissue. In certain embodiments, the treatment comprises subjecting an intact source animal tissue to radiation, Petition 870260070403, dated 07 / 15 / 2026, p. 23 / 79 8 / 61 preferably electron beam radiation, to produce irradiated animal tissue. In certain embodiments, treatment with electron beam radiation is sufficient to reduce viral and / or microbial contamination of the source animal tissue while simultaneously maintaining at least one biological activity of the source animal tissue. In certain embodiments, the biological activity is lipase activity. In certain embodiments, one or more enzymes and / or proenzymes are extracted from the irradiated animal tissue. In certain embodiments, one or more enzymes are isolated from the irradiated animal tissue. In certain embodiments, the method reduces the risk of infectious contamination of the animal-derived enzyme preparation or a pharmaceutical composition comprising the animal-derived enzyme preparation compared to an untreated control.

[0019] Another aspect of the present invention relates to a pharmaceutical composition comprising the enzyme preparations described herein. The pharmaceutical composition may be in an oral pharmaceutical dosage form. In certain 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 for treating or preventing exocrine pancreatic insufficiency comprising administering to an individual in need thereof a dose of an enzyme preparation or pharmaceutical composition described herein.

[0020] Another aspect of the present invention relates to a kit comprising the enzyme preparations or pharmaceutical compositions described herein.

[0021] These and other objectives are described in the following paragraphs. These objectives should not be considered as limiting the scope of Petition 870260070403, dated 07 / 15 / 2026, page 24 / 79 9 / 61 invention. Detailed description of the invention

[0022] This detailed description is intended only to familiarize other skilled individuals in the art with the present invention, its principles, and its practical application, so that other skilled individuals in the art may adapt and apply the invention in its numerous forms as may be more suitable for the requirements of a particular use. This description and its specific examples are for illustrative purposes only. Therefore, this invention is not limited to the embodiments described in this patent application and may be modified in various ways. A. Definitions

[0023] As used in the descriptive report and accompanying claims, unless otherwise specified, the following terms have the meaning indicated:

[0024] When used herein, the term API means “active pharmaceutical ingredient.” The preferred API disclosed herein is pancreatin, in particular porcine pancreatin when generally used for therapeutic purposes, i.e., pancreatin according to the requirements of standard pharmacopoeias, e.g., Ph.Eur. and / or USP, and suitable for oral administration in the treatment and prophylaxis of maldigestion in mammals, in particular in humans, and in particular maldigestion due to chronic exocrine pancreatic insufficiency such as in patients suffering from cystic fibrosis, chronic pancreatitis, or patients who have undergone upper gastrointestinal surgery.

[0025] When used herein, the term “crude” refers to an unpurified preparation or mixture containing enzymes and / or proenzymes as well as additional components derived from the source tissue. A crude preparation or mixture includes, but is not limited to, the animal tissue itself. Petition 870260070403, dated 07 / 15 / 2026, page 25 / 79 10 / 61

[0026] The term “enzyme preparation” refers to any composition of matter containing one or more enzymes, either in the active or inactive form (i.e., proenzymes or zymogens). The term includes cell or tissue extracts as well as crude preparations derived from animal tissue or other cellular material. An example of an enzyme preparation is pancreatin, pancrelipase, an extract derived from mammals, preferably porcine pancreas.

[0027] The term “extract”, as it relates to the present enzyme preparations, refers to one or more enzymes and / or proenzymes that have been separated from at least one component of the tissue from which they were derived. The extracted components may be in the form of an active enzyme or a proenzyme (zymogen) that requires subsequent conversion to the active form.

[0028] The term “isolate,” as it relates to the present enzyme preparations, refers to one or more active enzymes that have been separated from at least one component of the tissue from which they are derived. Thus, in certain embodiments, an “isolated enzyme” or an “isolated enzyme preparation” includes one or more active enzymes that have been converted from the corresponding proenzyme form via hydrolysis and / or autolysis. Hydrolysis and / or autolysis to convert the proenzyme into an active enzyme may occur before, during, or after extraction.

[0029] When used herein, the terms “pancreatic enzymes”, “pancreatin” and “pancrelipase” refer to enzyme mixtures derived from mammalian pancreases comprising digestive enzymes such as lipase, protease and amylase as principal components. In particular, the terms “pancreatic enzymes”, “pancreatin” and “pancrelipase” may be used herein synonymously and refer to pancreatic extracts suitable for therapeutic use, according to standard pharmacopoeias, containing various enzymes. Petition 870260070403, dated 07 / 15 / 2026, page 26 / 79 11 / 61 digestive enzymes whose properties are defined by standard monographs explained above. Due to standardized manufacturing processes, “pancreatic enzymes”, “pancreatin”, and “pancrelipase” are usually supplied in powder form as “pancreatin powder”, sometimes also referred to as “pancreas powder”. Pancreatic enzymes, pancreatin, and pancrelipase can also be, and preferably are, APIs (Indicated Polycyclic Aromatic Formulas). Pancreatin for pharmaceutical use is typically of bovine or porcine origin. Porcine pancreatin is preferred. Pancrelipase has been described in some references as an enzymatic preparation with increased (lipase) activity compared to pancreatin.

[0030] When used herein, the term “pharmaceutical composition” means a composition comprising an enzyme preparation as described herein and optionally one or more pharmaceutically acceptable excipients.

[0031] The term “pharmaceutically acceptable” is used adjectivally to mean that the modified noun is suitable for use as a pharmaceutical product or as part of a pharmaceutical product.

[0032] An “orthogonal” microbial and / or viral reduction step refers to a distinct method for reducing microbes and / or viruses that may be present in a sample. A microbial and / or viral reduction step can be orthogonal as long as there is one or more additional microbial and / or viral reduction steps in the process. In certain embodiments, an “orthogonal” microbial and / or viral reduction step has a mechanism sufficiently distinct from all other microbial and / or viral reduction steps used in the process such that the log10 mortality achieved by the “orthogonal” step becomes additive with the cumulative log10 mortality obtained from all other microbial and / or viral reduction steps that are used to obtain the enzyme preparation.

[0033] The terms “prevent”, “preventing” and “prevention” refer to a Petition 870260070403, dated 07 / 15 / 2026, p. 27 / 79 12 / 61 method to prevent the onset of a condition, disorder, or disease and / or associated symptoms and to prevent an individual from acquiring a condition, disorder, or disease. When used herein, the terms prevent, deter, and prevention also include delaying the onset of a condition, disorder, or disease and / or associated symptoms and reducing an individual's risk of acquiring a condition, disorder, or disease.

[0034] The term individual includes human beings and other primates as well as domestic and semi-domestic animals including, but not limited to, domestic fowl, bees, cows, sheep, goats, pigs, horses, dogs, cats, rabbits, rats, mice and the like. The term domestic fowl encompasses all types of domestic birds, including, but not limited to, chickens, turkeys, ducks, geese, emus, ostriches and game birds. In certain embodiments, the individual is a human being.

[0035] The term therapeutically effective amount means a sufficient quantity of the enzyme preparation or pharmaceutical composition to treat a condition, disorder or disease, in a reasonable benefit / risk ratio applicable to any medical treatment. When used in a medical treatment, a therapeutically effective amount of one of the enzyme preparations may be employed as an extract or in crude form. Alternatively, the enzyme composition may be administered as a pharmaceutical composition containing the enzyme composition of interest in combination with one or more pharmaceutically acceptable carriers.

[0036] The terms treat, treat and treatment refer to a method for relieving or eliminating a condition, disorder or disease and / or its associated symptoms. B. Enzyme preparations and manufacturing methods

[0037] In one aspect, the present invention includes a preparation of Petition 870260070403, dated 07 / 15 / 2026, page 28 / 79 13 / 61 enzyme comprising one or more enzymes and / or proenzymes derived from animal tissue, preferably mammalian. In certain embodiments, the enzyme preparation comprises a mixture of digestive enzymes and / or proenzymes. In certain embodiments, the enzyme preparation comprises a lipase. In certain embodiments, the enzyme preparation comprises an amylase. In certain embodiments, the enzyme preparation comprises pancreatin. In certain embodiments, the enzyme preparation comprises a proenzyme, such as a prolipase or a trypsinogen. In certain embodiments, the enzyme preparation is in crude form. In certain embodiments, the enzyme preparation comprises one or more enzymes and / or proenzymes that have been extracted from an animal tissue. In certain embodiments, the enzyme preparation comprises one or more enzymes that have been isolated from an animal tissue.

[0038] In certain respects, an isolated enzyme preparation has the same or substantially the same biological activity, but less infectivity, as the tissue from which it was isolated. In certain embodiments, the isolated enzyme preparation has the same or substantially the same biological activity as a control enzyme preparation. In certain embodiments, the infectivity of the isolated enzyme preparation is reduced by at least three log10, preferably by at least four log10, relative to the infectivity of the tissue from which it was isolated. In certain embodiments, the infectivity that is reduced is viral infectivity, particularly uncoated viral infectivity and / or coated viral infectivity. 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 a Petition 870260070403, dated 07 / 15 / 2026, page 29 / 79 14 / 61 control enzyme preparation. In certain incorporations, the biological activity is enzymatic activity, such as protease activity, amylase activity or, preferably, lipase activity.

[0039] In another aspect, the enzyme preparation is isolated from a pre-treated tissue source and has the same or substantially the same biological activity, but less infectivity, than a control preparation isolated from an untreated tissue source. In certain embodiments, the infectivity of the enzyme preparation is reduced by at least three log10, preferably by at least four log10, compared to the control preparation. In certain embodiments, the infectivity of the pre-treated tissue source is reduced by at least three log10, preferably by at least four log10, compared to the untreated tissue source. In certain embodiments, the infectivity that is reduced is viral infectivity, particularly uncoated viral infectivity.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 a control preparation isolated from an untreated tissue source. In certain embodiments, biological activity is enzymatic activity, such as protease activity, amylase activity, or preferably lipase activity.

[0040] In another aspect, the enzyme preparation is derived from electron beam-irradiated tissue. In certain embodiments, the electron beam-irradiated tissue is preferably mammalian tissue, preferably porcine tissue. In certain embodiments, the enzyme preparation derived from electron beam-irradiated tissue comprises pancreatin. In certain embodiments, the enzyme preparation derived from electron beam-irradiated tissue comprises a Petition 870260070403, dated 07 / 15 / 2026, p. 30 / 79 15 / 61 proenzyme, such as prolipase or trypsinogen. In certain embodiments, the enzyme preparation derived from electron beam-irradiated tissue is in crude form. In certain embodiments, the enzyme preparation derived from electron beam-irradiated tissue comprises one or more enzymes and / or proenzymes that have been extracted from irradiated tissue. In certain embodiments, the enzyme preparation derived from electron beam-irradiated tissue comprises one or more enzymes that have been isolated from irradiated tissue.

[0041] In another aspect, the enzyme preparation is isolated from electron beam-irradiated tissue and has the same or substantially the same biological activity, but less infectivity, than a control preparation isolated from a non-irradiated tissue source. In certain incorporations, the irradiated tissue is pancreatic tissue. In certain incorporations, the irradiated tissue is a laminated pancreatic tissue, a whole pancreas, or a portion of a whole pancreas. In certain incorporations, the non-irradiated tissue source is pancreatic tissue. In certain incorporations, the infectivity of the enzyme preparation is reduced by at least three log10, preferably by at least four log10, compared to the control preparation. In certain incorporations, the infectivity that is reduced is viral infectivity, particularly coated or uncoated viral infectivity. In certain incorporations, the infectivity that is reduced is PPV infectivity.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 control preparation. In certain embodiments, biological activity is enzymatic activity, such as protease activity, amylase activity, or preferably lipase activity. Petition 870260070403, dated 07 / 15 / 2026, page 31 / 79 16 / 61

[0042] In another aspect, the enzyme preparation comprises proenzymes irradiated with an electron beam. In certain incorporations, the enzyme preparation is further processed, such as converting the irradiated proenzymes into their active form (e.g., by autolysis and / or hydrolysis).

[0043] The present enzyme preparations can be better understood together with the following methods which illustrate exemplary techniques by which the enzyme preparations can be obtained.

[0044] In one aspect, the present invention includes a method for manufacturing an enzymatic composition comprising subjecting a proenzyme source to radiation, preferably electron beam radiation. In certain embodiments, the proenzyme source is a cell population. In certain embodiments, the cell population is intact tissue obtained from a mammalian gland or a portion thereof. In certain embodiments, the cell population is an entire gland obtained from a mammal. In certain embodiments, the cell population is a portion of a gland obtained from a mammal. In certain embodiments, the cell population is a frozen tissue block. In certain embodiments, the cell population is rolled or minced animal tissue.

[0045] In another aspect, the present invention includes a method for manufacturing an enzyme preparation. The method comprises subjecting an animal tissue to radiation, preferably electron beam radiation. In certain embodiments, the animal tissue is an intact tissue. In certain embodiments, the intact animal tissue is a frozen tissue block. In certain embodiments, the intact animal tissue is a rolled or minced animal tissue.

[0046] In certain incorporations, the method begins with tissue Petition 870260070403, dated 07 / 15 / 2026, p. 32 / 79 17 / 61 animal, preferably intact animal tissue. In certain incorporations, the animal tissue is mammalian pancreas, preferably porcine. In certain incorporations, porcine pancreas is sought from an approved slaughterhouse, preferably a single-species slaughterhouse.

[0047] In certain embeddings, intact animal tissue includes intact pancreatic tissue. In certain embeddings, intact pancreatic tissue includes an entire pancreas or a portion thereof, such as one or more lobules. In certain embeddings, pancreatic tissue includes laminated frozen tissue. In certain embeddings, intact pancreatic tissue includes a block of frozen tissue, which may have been mechanically processed. In certain embeddings, intact pancreatic tissue includes pancreatic tissue that has been minimally manipulated or altered, or preferably not manipulated or altered, so as to, for example, destroy active enzymes and / or convert proenzymes in the tissue to their active form. For example, a homogenized tissue that has undergone significant chemical or enzymatic processing to convert proenzymes to their active form is not “intact tissue” as the term is used herein.

[0048] Chopping typically involves processing the source tissue in a grinder into pieces smaller than or equal to 0.5 cm3, smaller than or equal to 0.4 cm3, smaller than or equal to 0.3 cm3, smaller than or equal to 0.2 cm3, or smaller than or equal to 0.1 cm3. Homogenization typically involves mixing the chopped material with water to form a semi-fluid suspension or paste as known in the art. The tissue may also be mixed with other solutions and / or stabilizers to enable or facilitate mechanical processing, provided that mixing with other solutions and / or stabilizers does not destroy active enzymes and / or convert proenzymes in the tissue to their active form, for example, by maintaining the temperature of the homogenate, suspension, or semi-fluid paste at Petition 870260070403, dated 07 / 15 / 2026, p. 33 / 79 18 / 61 10°C + 5°C, preferably below 10°C.

[0049] Thus, the mechanical processing of fabric explicitly includes the addition of or mixing with water and / or a lower alcohol (e.g., isopropyl alcohol) in sufficient quantity to allow or facilitate mechanical processing such as chopping or homogenizing, provided that the mixing / combination is not accompanied by concomitant autolysis or hydrolysis. Therefore, mechanical processing according to the methods of the invention can be carried out to maintain the fabric at 10°C ± 5°C, preferably below 10°C (including freezing), during processing to minimize or stop autolysis or hydrolysis. Consequently, for example, a homogenized fabric can be a mixture of fabric and water that has been maintained at 10°C ± 5°C, preferably below 10°C during processing so as to prevent the fabric from undergoing hydrolysis and / or autolysis.

[0050] On the other hand, “process intermediate” (and analogous terms) refers to tissues that, in addition to optional mechanical alteration from the source tissue, have also been subjected to one or more steps of chemical and / or enzymatic processing, including hydrolysis and autolysis. In particular, one or more steps of chemical and / or enzymatic processing alter the biomolecular population and / or the biomolecular profile, for example, by destroying active enzymes and / or converting proenzymes in the tissue to their active form. Thus, a process intermediate may also have an altered ratio of proenzymes to active enzymes.

[0051] In certain embodiments, animal tissue, preferably frozen animal tissue, is ground. In certain embodiments, grinding may be achieved using a cast block grinder, such as a Hydrauflake Chunker supplied by General Machinery Corporation (Sheboygan, WI), which is designed to cut thick pieces of tissue. Petition 870260070403, dated 07 / 15 / 2026, page 34 / 79 19 / 61 frozen in preparation for further processing.

[0052] In certain embeddings, animal tissue is irradiated. In certain embeddings, animal tissue is exposed to a sterilizing beam of accelerated electrons, i.e., an electron beam or electron beam radiation. In certain embeddings, intact animal tissue is exposed to electron beam irradiation. In certain embeddings, an entire pancreas or an intact portion thereof is exposed to electron beam irradiation. In certain embeddings, rolled porcine pancreatic tissue is exposed to electron beam irradiation.

[0053] Electron beam radiation is a form of ionizing energy that is generally characterized by its low penetration and high dosage rates. The beam, a highly charged and concentrated stream of electrons, is generated by the acceleration and conversion of electricity. Electrons are generated by equipment referred to as accelerators, which are capable of producing beams that are either pulsed or continuous. When the material being irradiated passes under or in front of the electron beam, the energy of the electrons is absorbed. This energy absorption alters various chemical bonds and biological properties within the product / material. The energy that is absorbed is referred to as the absorbed dose. It is this energy absorption that destroys viruses and microorganisms, for example, by destroying their DNA or RNA chains.

[0054] Irradiation can be performed conventionally, such as by placing the tissue in a suitable container and exposing the tissue to an electron beam. In certain embodiments, the container holding the tissue is placed on a carrier that then passes through the electron beam. In certain embodiments, the container holding the tissue does not contain any solvent. In certain embodiments, the container holding the tissue is substantially solvent-free. In certain embodiments, Petition 870260070403, dated 07 / 15 / 2026, page 35 / 79 20 / 61 The container holding the tissue does not contain any flammable solvent. In certain embodiments, the container holding the tissue is substantially free of a flammable solvent, such as alcohol. For example, the container may contain intact frozen tissue such as a whole gland, a portion of a whole gland, or tissue in sheets.

[0055] In certain embodiments, radiation is delivered at a dose sufficient to substantially inactivate resistant viruses and / or microbes in the tissue. In certain embodiments, the radiation is at a dose that prevents loss of biological activity, preferably enzymatic activity, compared to an isolated enzymatic control preparation from non-irradiated tissue.

[0056] The accelerated electron beam may be provided by an electron accelerator, such as an electron accelerator provided by Iotron Industries USA, Inc. (Columbia City, IN). In certain embodiments, the electron accelerator operates at power ratings of 20 to 250 kW and beam energies of 5 to 18 megaelectron-volts (MeV). In certain embodiments, the electron accelerator operates at 60 kW and 10 MeV. In certain embodiments, the electron accelerator provides a beam energy greater than or equal to 10 MeV.

[0057] A tissue exposed to electron beam irradiation for a time interval and in a quantity sufficient to achieve viral or microbial inactivation without compromising the biological activity of one or more enzymes subsequently extracted or isolated from the irradiated tissue. The dosage of electron beam irradiation required to sterilize a tissue may vary based, for example, on the size of the tissue, the type of tissue, and the type and quantity of viral or microbial contaminant in, or suspected of being present in, the tissue sample. A specialist in the technique will recognize and be Petition 870260070403, dated 07 / 15 / 2026, p. 36 / 79 21 / 61 capable of determining an appropriate dose and time interval for a particular tissue based on tissue characteristics and the accelerator being used. The selected electron beam dose is effective for inactivating infectious agents that are difficult to destroy by conventional processes, while causing minimal loss in enzymatic activity.

[0058] An “absorbed dose” of radiation is expressed in terms of kilograys (kGy) with one kilogray equal to one thousand Joules of energy deposited per kilogram of material. In certain embodies, the tissue is exposed to the electron beam until an inactivating amount of infectious agent is absorbed. For example, the tissue may be exposed to the electron beam until it reaches a dose of about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 kGy or more. As another example, the tissue may be exposed to the electron beam until it reaches a dose of about 5 to about 50, about 10 to about 40, about 15 to about 35 kGy. In certain embeddings, the tissue is exposed to the electron beam until it absorbs a dose of approximately 30 kGy.In certain incorporations, porcine pancreas is irradiated with an electron beam dose sufficient to provide high log10 mortality for infectious agents, preferably infectious agents that are difficult to inactivate such as uncoated viruses, while causing minimal loss in enzymatic activity.

[0059] In certain incorporations, the dosage can be determined using radiochromic dye films. Such films can be calibrated by reference to a national standard.

[0060] In certain embeddings, the tissue is exposed to electron beam radiation for a time and in a quantity sufficient to produce Petition 870260070403, dated 07 / 15 / 2026, page 37 / 79 22 / 61 A reduction of at least three log10, preferably at least four log10, in the viral load of a model virus when compared with a control sample. In some such embeddings, the tissue is exposed to electron beam radiation for a time and in a quantity sufficient to produce a reduction of between about three log10 and about five log10 in the viral load of a model virus when compared with a control sample. In some such embeddings, the tissue is exposed to electron beam radiation for a time and in a quantity sufficient to produce a reduction of about four log10 in the viral load of a model virus when compared with a control sample. In certain embeddings, the model virus is PPV. In certain embeddings, the tissue is intact tissue. In some such embeddings, the intact tissue is a gland, such as an entire pancreas or a portion thereof, such as one or more lobes of a pancreas.In other such incorporations, the intact tissue is frozen laminated tissue.

[0061] In certain embodiments, electron beam exposure includes single-sided or multi-sided exposure. In certain embodiments, the tissue is subjected to single-sided electron beam exposure. In certain embodiments, the tissue is subjected to multi-sided exposure, for example, double-sided electron beam exposure. Thus, a dose of about 20 kGy may comprise double-sided exposure of about 10 kGy / side; a dose of about 30 kGy may comprise double-sided exposure of about 15 kGy / side; a dose of about 40 kGy may comprise double-sided exposure of about 20 kGy / side; and a dose of about 50 kGy may comprise double-sided exposure of about 25 kGy / side.

[0062] In certain embodiments, the methods for manufacturing the enzyme preparation reduce the risk of viral or microbial infectivity of Petition 870260070403, dated 07 / 15 / 2026, page 38 / 79 23 / 61 a pharmaceutical composition comprising an enzymatic preparation.

[0063] In certain embodiments, the infectivity of an irradiated pancreas is reduced by at least three log10, preferably by at least four log10, relative to the infectivity of the gland before radiation treatment. In certain embodiments, the infectivity of an enzyme preparation derived from or isolated from an irradiated pancreas is reduced by at least three log10, preferably by at least four log10, relative to the infectivity of the gland before radiation treatment. Alternatively, the infectivity of the enzyme preparation is determined relative to a control enzyme preparation derived from or isolated from a non-irradiated pancreas.

[0064] In certain embodiments, the infectivity that is reduced is viral infectivity, particularly the infectivity of an uncoated virus such as PPV. For example, the PPV infectivity of a pancreatin product isolated from an irradiated pancreas is reduced by at least three log10, preferably by at least four log10, compared to a control enzyme preparation isolated from a non-irradiated pancreas. As another example, the PPV infectivity of an irradiated pancreas is reduced by at least three log10, preferably by at least four log10, compared to a non-irradiated pancreas. In some such embodiments, the PPV infectivity of an enzyme preparation is further reduced by subsequent orthogonal viral inactivation steps performed after irradiation.

[0065] In certain embodiments, the methods may also include the step of, after the irradiation step, testing for the presence or quantity of one or more microorganisms (e.g., viruses, bacteria, or protozoa) in the tissue or in the tissue-derived enzyme preparation. Methods for determining whether a sample contains a microorganism are known in Petition 870260070403, dated 07 / 15 / 2026, page 39 / 79 24 / 61 techniques and include, for example, plaque assays or colony formation tests. Effective sterilization can also be determined using conventional microbiological techniques, such as, for example, including appropriate biological indicators in a batch of radiation or contacting the tissue with a culture medium and incubating the medium to determine the sterility of the tissue.

[0066] In certain incorporations, viral infectivity can be calculated by endpoint titration and subsequent calculation of half the tissue culture infectious dose (TCID50). Virus titers calculated in this way can be given as log10 TCID50 per mL with 95% confidence intervals.

[0067] In certain incorporations, a reduction in viral contamination is given in accordance with USP-NF General Chapter 1050, as a logarithmic reduction factor which is the difference in viral titer between a control sample and the sample derived from electron beam irradiated tissue after isolation. For example, a 3log10 reduction might indicate a reduction in viral load by a factor of 1,000 and a 4log10 reduction might indicate a reduction in viral load by a factor of 10,000.

[0068] In certain embodiments, the methods for manufacturing the enzyme preparation allow for the reduction of viral and / or microbial contamination of the enzyme preparation without a substantial reduction in its enzymatic activity.

[0069] In certain embodiments, the enzymatic activity of an enzyme preparation isolated from an irradiated pancreas is maintained. For example, 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 a control preparation isolated from a non-irradiated pancreas. In certain embodiments, Petition 870260070403, dated 07 / 15 / 2026, page 40 / 79 25 / 61 Biological activity is enzymatic activity, such as protease activity, amylase activity or, preferably, lipase activity.

[0070] After irradiation, the tissue can be further processed to provide the enzyme composition. For example, in certain embeddings, one or more enzymes and / or proenzymes are extracted from the irradiated tissue. In certain embeddings, one or more enzymes are isolated from the irradiated tissue. Several 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 semi-fluid tissue paste containing isopropanol.

[0071] In certain embeddings, the irradiated tissue may be subjected to autolysis and / or hydrolysis to convert proenzymes to their active form. For example, the irradiated tissue may be combined with a hydrolysis starter to initiate autolysis. In certain embeddings, autolysis and / or hydrolysis is performed at room temperature. In certain embeddings, the reactant mixture is filtered after the reaction is complete; the filtrate is collected; the enzymes present in the filtrate are precipitated (e.g., with isopropanol); and the precipitate is filtered, washed with isopropanol, and vacuum dried.

[0072] The methods described above and illustrated in the Examples section may be considered illustrative and should not be read as limiting the scope of the invention as defined in the appended claims. All alternatives, modifications and equivalents of the specific methods and examples are included within the scope of the claims. C. Compositions

[0073] In at least one aspect, the present invention includes compositions comprising an enzyme composition described herein. In certain embodiments, the composition comprises one or more enzymes and / or Petition 870260070403, dated 07 / 15 / 2026, page 41 / 79 26 / 61 proenzymes extracted from irradiated animal tissue. In certain embodiments, the composition comprises one or more enzymes isolated from irradiated animal tissue. In certain embodiments, the composition is a crude mixture containing one or more enzymes derived from animal tissue.

[0074] In another aspect, a pharmaceutical composition is provided comprising an enzyme preparation described herein, optionally further comprising one or more conventional pharmaceutically acceptable excipients, such as those found in compendia 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 195th Ed. (Lippincott, Williams & Wilkins, 1995); Handbook of Pharmaceutical Excipients, 35th Ed. (Arthur H. Kibbe, ed.; Amer. Pharmaceutical Assoc., 1999); Pharmaceutical Codex: Principles and Practice of Pharmaceutics 125th Ed. (Walter Lund ed.; Pharmaceutical Press, London, 1994); The United States 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), whose publications are incorporated herein by reference.

[0075] Pharmaceutical compositions comprising an enzyme preparation described herein may be used to supplement digestive enzymes in the treatment and / or prophylaxis of maldigestion in mammals, in particular maldigestion due to chronic exocrine pancreatic insufficiency such as in patients suffering from cystic fibrosis, chronic pancreatitis or patients who have undergone upper gastrointestinal surgery. The pharmaceutical compositions or dosage forms described herein may preferably be oral dosage forms that may, in particular, be administered to humans.

[0076] An oral dosage form containing an enzyme preparation Petition 870260070403, dated 07 / 15 / 2026, page 42 / 79 27 / 61 may be, for example, in the form of capsules, granules, pellets, micropellets, microspheres, microtablets, pills, powders and / or tablets. For the purposes of this description, the prefix micro is used to describe an oral dosage form if the diameter of the oral dosage form or all its dimensions (length, height, width) is less than or equal to about 5 mm.

[0077] In certain embodiments, the oral dosage form is a capsule. The capsule may comprise between about 2,000 and about 40,000 lipase units per capsule. In certain embodiments, the dosage form is a capsule comprising 3,000, 6,000, 12,000, 24,000 or 36,000 lipase units per capsule. In certain embodiments, the oral dosage form is a capsule comprising 3,000, 5,000, 10,000, 15,000, 20,000, 25,000 or 40,000 lipase units per capsule. In certain embodiments, the oral dosage form is a capsule comprising 2,600, 4,200, 10,500, 16,800 or 21,000 lipase units per capsule. In certain embodiments, the oral dosage form is a capsule comprising 4,000, 13,800, 20,700 or 23,000 lipase units per capsule. In certain embodiments, the oral dosage form is a capsule comprising 4,000, 8,000 or 16,000 lipase units per capsule.In other embodiments, the oral dosage form is a capsule comprising 3,000, 4,000, 6,000, or 8,000 lipase units per capsule. Dosage strength may be expressed in various ways, including USP units, Ph.Eur units, or BP units.

[0078] In certain embodiments, the oral dosage form is a tablet comprising 10,440 or 20,880 lipase units per tablet.

[0079] Several pharmaceutical compositions and forms are known Petition 870260070403, dated 07 / 15 / 2026, page 43 / 79 28 / 61 dosage containing pancreatin, such as immediate-release and delayed-release compositions. For example, US 9,198,871 provides delayed-release pancreatin compositions.

[0080] In certain embodiments, the oral dosage form is a pancreatin micropellet or a pancreatin microsphere. In certain embodiments, the pancreatin micropellet or pancreatin microsphere is coated, for example, with an enteric coating. In certain embodiments, the pancreatin micropellet or pancreatin microsphere – regardless of any such coating – comprises between about 10% and about 95% by weight of pancreatin, between about 5% and about 90% by weight of at least one pharmaceutically acceptable binding agent, and between about 0% and about 10% by weight of at least one pharmaceutically acceptable excipient.More specifically, the pancreatin micropellet or pancreatin microsphere comprises between about 70% and about 90% by weight of pancreatin, between about 10% and about 30% by weight of at least one pharmaceutically acceptable binder, and between about 0% and about 5% by weight of at least one pharmaceutically acceptable excipient. In certain embodiments, the pancreatin micropellet or pancreatin microsphere comprises between about 70% and about 90% by weight of pancreatin and between about 10% and about 30% by weight of at least one pharmaceutically acceptable binder. In certain embodiments, the pancreatin micropellet or pancreatin microsphere is approximately spherical and has a diameter between about 0.5 mm and about 2.0 mm.In certain embodiments, pancreatin micropellets or pancreatin microspheres have a first dimension between about 0.5 mm and about 2.0 mm and a second dimension between about 0.5 mm and about 2.0 mm. In certain embodiments, micropellets of... Petition 870260070403, dated 07 / 15 / 2026, page 44 / 79 29 / 61 pancreatin or pancreatin microsphere has a first dimension between about 0.8 mm and about 1.0 mm and a second dimension between about 0.5 mm and about 2.0 mm.

[0081] Examples of pharmaceutically acceptable binding agents include polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, hydroxypropyl methyl cellulose, polyoxyethylene, polyoxyethylene-polyoxypropylene copolymers and mixtures of said organic polymers. The foregoing list of pharmaceutically acceptable binding agents is not intended to be exhaustive, but merely illustrative, as a person skilled in the art will understand that many other pharmaceutically acceptable binding agents may also be used. Polyethylene glycol 4000 is a preferred pharmaceutically acceptable binding agent.

[0082] 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 silicon dioxide, microcrystalline cellulose, kaolin, lactose, mannitol, polyvinylpyrrolidone, precipitated calcium carbonate, sorbitol and / or talc; disintegrants such as Aerosil (silicic acid), alginic acid, amylose, calcium alginate, calcium carbonate, formaldehyde gelatin, pectic carbonate, sago starch, sodium bicarbonate and / or starch; and / or moisturizers such as glycerol and / or starch. The above list of pharmaceutically acceptable excipients is not intended to be exhaustive, but merely illustrative, as a person skilled in the art will understand that many other pharmaceutically acceptable excipients or combinations of excipients may also be used.For the purposes of this disclosure, synthetic oils and monomeric phthalic acid esters should not be considered. Petition 870260070403, dated 07 / 15 / 2026, p. 45 / 79 30 / 61 as appropriate pharmaceutically acceptable excipients. In certain embodiments, pancreatin micropellets or pancreatin microspheres do not contain any pharmaceutically acceptable excipients, but may optionally contain a higher amount of pancreatin.

[0083] In another embodiment, an oral dosage form, such as an enteric-coated oral dosage form, of pancreatin 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 type II diabetes.

[0084] In certain embodiments, the pharmaceutical composition is a controlled-release pharmaceutical composition. For example, a controlled-release pharmaceutical composition may be obtained by applying an enteric coating to an oral dosage form. In certain embodiments, the enteric coating comprises a film-forming agent, a plasticizer and, optionally, an anti-adherent agent.

[0085] Suitable film-forming agents include agar, carbomer homopolymers and copolymers (i.e., high molecular weight crosslinked acrylic acid-based polymers), carboxymethyl cellulose, carboxymethyl ethyl cellulose, carrageenan, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate trimellitate, chitin, corn protein extract, ethyl cellulose, gum arabic, hydroxypropyl cellulose, hydroxypropylmethyl acetate succinate, hydroxypropylmethyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methacrylic acid-ethyl methacrylate copolymer, methyl cellulose, pectin, poly(vinyl acetate phthalate), poly(vinyl alcohol), shellac, sodium alginate, starch acetate phthalate and / or styrene / maleic acid copolymer or mixtures of said film-forming polymers. of films. Cellulose acetate phthalate, hydroxypropylmethylcellulose acetate succinate Petition 870260070403, dated 07 / 15 / 2026, page 46 / 79 31 / 61 and / or methacrylic acid-methyl methacrylate copolymer are preferred film-forming agents. Hydroxypropyl methyl cellulose phthalate is highly preferred, such as HP 55 or HPMCP HP-50. Synthetic oils are not considered preferred film-forming agents. The preceding list of film-forming agents is not exhaustive but merely illustrative, as a person skilled in the art will understand that many other film-forming agents or combinations of film-forming agents may also be used.

[0086] In general, plasticizers may be present in an amount greater than about 1.5% and typically in an amount between about 2% and about 20% by weight, relative to the film-forming agent. The plasticizer may contain saturated linear monohydric alcohols having from 12 to 30 carbon atoms. More specifically, acceptable plasticizers include lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, heptadecyl alcohol, stearyl alcohol, nonadecyl alcohol, arachidic alcohol, behenyl alcohol, carnaubyl alcohol, ceryl alcohol, corianyl alcohol, melissyl alcohol, tributyl acetyl citrate, dibutyl sebaceate, glycerol fatty acid esters, glycerol, polyethylene glycol, sorbitan fatty acids, triacetyl, triethyl citrate, and mixtures thereof. 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 greater than about 1.5%, typically in an amount of about 2% to about 15%, preferably from about 2% to about 10% by weight relative to the film-forming agent. When triethyl citrate is used as the sole plasticizer, it may be present in an amount between about 5% and about 20%, preferably between about 12% and about 15% by weight relative to the film-forming agent. Oils. Petition 870260070403, dated 07 / 15 / 2026, p. 47 / 79 32 / 61 Synthetic and monomeric phthalic acid esters are not considered suitable plasticizers. The preceding list of plasticizers is not intended to be exhaustive, but merely illustrative, as a person skilled in the art will understand that many other plasticizers or combinations of plasticizers may also be used, provided they are substantially free of synthetic oils and monomeric phthalic acid esters.

[0087] In certain embodiments, the plasticizer comprises cetyl alcohol and triethyl citrate which are collectively present in an amount greater than about 3%, typically in an amount of about 4% to about 20%, in particular between about 6% and about 15%, more particularly between about 7% and about 10% by weight relative to the film-forming agent. The weight ratio of cetyl alcohol to triethyl citrate when both are present 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. In particular, the ratio of cetyl alcohol to triethyl citrate can 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 (weight / weight).

[0088] Optionally, the enteric coating comprises an anti-adherent agent. Suitable anti-adherent agents include dimethicone and castor oil. Dimethicone, in particular dimethicone 1000, is the preferred anti-adherent agent. The amount of anti-adherent agent (if present) in the enteric coating is between about 1.5% and about 3% by weight relative to the film-forming agent. Synthetic oils are not considered preferred anti-adherent agents. The preceding list of anti-adherent agents is not intended to be exhaustive, but merely illustrative, as a person skilled in the art will understand that many other anti-adherent agents or combinations of agents may exist. Petition 870260070403, dated 07 / 15 / 2026, page 48 / 79 33 / 61 non-stick coatings can also be used.

[0089] In certain embodiments, the enteric coating comprises between about 5% and about 30% by weight, more preferably between about 7% and about 20% by weight, even more preferably between about 10% and about 15% by weight of the total composition of the enteric-coated oral dosage form or controlled-release pharmaceutical composition. In certain embodiments, the enteric coating comprises between about 20% and about 30% by weight, more preferably between about 22% and about 26% by weight, even more preferably between about 22.5% and about 25% by weight of the total composition of the enteric-coated oral dosage form or controlled-release pharmaceutical composition.

[0090] In certain embodiments, the pharmaceutical composition is a controlled-release capsule for oral administration. The capsule may contain enteric-coated pellets comprising lipase, protease, and amylase. The enteric-coated pellets may have a first dimension between about 0.5 mm and about 2.0 mm and, optionally, a second dimension between about 0.5 mm and about 2.0 mm. For example, the enteric-coated pellets may be spun and have a diameter between about 0.5 mm and about 2.0 mm and a length between about 0.5 mm and about 2.0 mm. The composition may further include inactive ingredients described herein, such as cetyl alcohol, dimethicone, hypromellose phthalate, polyethylene glycol, and triethyl citrate.

[0091] In certain embodiments, the pharmaceutical composition is an immediate-release pharmaceutical composition. For example, the immediate-release pharmaceutical composition may not have an enteric coating. D. Methods of use

[0092] In at least one aspect, the present invention includes a method for treating exocrine pancreatic insufficiency in an individual, in particular a Petition 870260070403, dated 07 / 15 / 2026, page 49 / 79 34 / 61 human individual requiring such treatment. The method comprises administering an enzyme preparation or a pharmaceutical composition containing an enzyme preparation to the individual. In certain embodiments, exocrine pancreatic insufficiency is due to cystic fibrosis, chronic pancreatitis, pancreatectomy, or other conditions.

[0093] In another aspect, the present invention includes a method for treating or preventing indigestion in an individual, in particular a human individual, requiring such treatment or prevention. In certain embodiments, indigestion is due to exocrine pancreatic insufficiency, such as in individuals suffering from cystic fibrosis, chronic pancreatitis, or patients undergoing upper gastrointestinal surgery.

[0094] In another aspect, the present invention includes the use of an enzyme preparation or a pharmaceutical composition containing an enzyme preparation to treat exocrine pancreatic insufficiency in an individual requiring such treatment.

[0095] 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 prophylaxis of maldigestion in an individual requiring such treatment or prophylaxis.

[0096] In certain embodiments related to the methods and uses mentioned above, the enzyme preparation comprises pancreatin. In certain embodiments, an appropriate dosage of pancreatin may be based on lipase units. The Cystic Fibrosis Foundation (CFF) has published consensus guidelines containing recommended total daily doses of lipase units.

[0097] In certain formulations, 2,000 to 4,000 lipase units are administered, preferably 3,000 lipase units, per 120 mL of Petition 870260070403, dated 07 / 15 / 2026, pp. 50 / 79 35 / 61 formula or by breastfeeding to a child up to 12 months of age.

[0098] In certain incorporations, 1,000 to 2,500 units per kg of body weight are administered per meal to an individual aged one (1) to four (4) years. In certain incorporations, 500 to 2,500 units per kg of body weight are administered per meal to an individual aged at least four (4) years.

[0099] In certain formulations, the maximum daily dosage does not exceed 10,000 lipase units per kg of body weight. In certain formulations, the maximum daily dosage does not exceed 4,000 lipase units per g of fat ingested.

[0100] In certain embodiments, the enzyme preparation or the pharmaceutical composition containing the enzyme preparation is administered immediately before a meal. In certain embodiments, the enzyme preparation or the pharmaceutical composition containing the enzyme preparation is administered during a meal or a snack.

[0101] In another embodiment, a method is provided for treating a medical condition such as digestive disorders, exocrine pancreatic insufficiency, pancreatitis, cystic fibrosis, type I diabetes and / or type II diabetes by administering a therapeutically effective amount of an enzyme preparation or a pharmaceutical composition containing the enzyme preparation to a person in need of such treatment.

[0102] In at least one aspect, the present invention includes a method of digesting a protein. The method comprises contacting the protein to be digested with an enzyme preparation under conditions sufficient to digest the protein.

[0103] In certain embodiments, the enzyme preparation comprises one or more enzymes and / or proenzymes extracted from animal tissue. Petition 870260070403, dated 07 / 15 / 2026, p. 51 / 79 36 / 61 irradiated with an electron beam. In certain embodiments, the enzyme preparation comprises one or more enzymes isolated from an animal tissue irradiated with an electron beam. In certain embodiments, the enzyme preparation is in crude form.

[0104] In certain incorporations, the contact step occurs in vivo. In certain incorporations, the contact step occurs in vitro.

[0105] In certain incorporations, the digested protein is used to prepare a protein hydrolysate.

[0106] The enzyme preparations, compositions, methods and uses described herein will be better understood by reference to the following exemplary embodiments and examples, which are included by way of illustration and not to limit the scope of the invention. E. Exemplary Incorporations

[0107] One aspect of the present invention includes an enzyme preparation produced by a method comprising the steps of: (a) providing mammalian pancreatic tissue; (b) subjecting the pancreatic tissue to electron beam radiation to produce irradiated pancreatic tissue, wherein the electron beam radiation is sufficient to produce a reduction in microbial and / or viral load; and (c) isolating pancreatin from the irradiated pancreatic tissue. In certain embodiments, the isolation step comprises mixing the irradiated pancreatic tissue with water. In certain embodiments, step (c) comprises activating a proenzyme from the irradiated pancreatic tissue. In certain embodiments, the reduction in microbial and / or viral load is at least three log10, preferably a reduction of at least four log10 relative to a control sample obtained from non-irradiated tissue.For example, a reduction in microbial and / or viral load is a reduction of at least three log10, preferably a reduction of at least four log10 in viral load. Petition 870260070403, dated 07 / 15 / 2026, page 52 / 79 37 / 61 of PPV in relation to a control sample obtained from non-irradiated tissue. In certain 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 a control enzyme preparation obtained from non-irradiated pancreatic tissue. In certain embodiments, the biological activity is lipase activity. In certain embodiments, the biological activity is protease activity or amylase activity. In certain embodiments, the method further comprises one or more additional steps that provide a further reduction in microbial and / or viral load.

[0108] Another aspect of the present invention includes an enzymatic preparation comprising one or more enzymes isolated from a mammalian tissue subjected to treatment sufficient to produce a reduction of at least three log10, preferably a reduction of at least four lo10 in the viral load. For example, the treatment is sufficient to produce a reduction of at least three log10, preferably a reduction of at least four lo10 in the PPV viral load relative to a non-irradiated control sample.

[0109] Another aspect of the present invention includes an enzymatic preparation comprising one or more enzymes isolated from mammalian tissue, wherein, prior to isolating the enzyme, the mammalian tissue has been subjected to treatment sufficient to produce a reduction of at least three log10, preferably a reduction of at least four lo10, in the viral load. For example, the treatment is sufficient to produce a reduction of at least three log10, preferably a reduction of at least four lo10, in the PPV viral load relative to an untreated control sample. In certain embodiments, the mammalian tissue is a pancreatic gland. Petition 870260070403, dated 07 / 15 / 2026, page 53 / 79 38 / 61 porcine. In certain embodiments, the porcine pancreatic gland is laminated. In certain embodiments, the porcine pancreatic gland is in a frozen block. In certain embodiments, the porcine pancreatic gland is a whole gland or a portion thereof, such as one or more lobules. In certain embodiments, one or more enzymes comprise pancreatin. In certain embodiments, the treatment comprises electron beam radiation. In certain embodiments, the electron beam radiation has a dose of about 5 to about 50 kGy, preferably about 10 to about 40 kGy. In certain embodiments, the biological activity of the enzyme preparation corresponds to at least 50%, preferably at least 90%, of the biological activity of a control enzyme preparation obtained from untreated mammalian tissue. In certain embodiments, the biological activity is lipase activity.In certain incorporations, the biological activity is protease activity or amylase activity. In certain incorporations, the reduction in viral load is an orthogonal reduction.

[0110] Another aspect of the present invention includes a method for reducing the risk of contamination of a pancreatin product by an infectious agent comprising the steps of: (a) providing mammalian pancreatic tissue; (b) subjecting the pancreatic tissue to electron beam radiation to produce irradiated pancreatic tissue, wherein the electron beam radiation is sufficient to reduce the risk of contamination by an infectious agent; and (c) isolating the pancreatin from the irradiated pancreatic tissue, thereby obtaining a pancreatin product with a reduced risk of contamination by the infectious agent compared to mammalian pancreatic tissue provided in step (a) or a pancreatin sample derived from non-irradiated pancreatic tissue. In certain embodiments, the infectious agent is parvovirus. Petition 870260070403, dated 07 / 15 / 2026, pp. 54 / 79 39 / 61 porcine parvovirus (PPV). In certain incorporations, the method provides a reduction of at least three log10, preferably a reduction of at least four l0 l0 l0 in a measure indicative of the level or activity of an uncoated virus, such as porcine parvovirus (PPV). In certain incorporations, the measure indicative of the level or activity of the uncoated virus is the viral load.

[0111] Another aspect of the present invention includes an enzyme preparation comprising one or more enzymes isolated from mammalian tissue and a substantially inactivated uncoated virus, wherein the preparation has a biological activity corresponding to at least 50%, preferably at least 90%, of the biological activity of a control preparation. In certain embodiments, the control preparation has not been subjected to sufficient treatment to inactivate the uncoated virus. In certain embodiments, the substantially inactivated uncoated virus is porcine parvovirus (PPV). In certain embodiments, one or more enzymes comprise pancreatin. In certain embodiments, one or more enzymes are isolated from mammalian tissue. In certain embodiments, the mammalian tissue is electron beam irradiated mammalian tissue.

[0112] Another aspect of the present invention includes a pharmaceutical composition comprising one or more enzymes isolated from mammalian tissue that has been treated to reduce the risk of viral and microbial infection, the composition having a biological activity that corresponds to at least 50%, preferably at least 90%, of the biological activity of a control composition. In certain embodiments, one or more enzymes comprise lipase. In certain embodiments, the control composition comprises an untreated sample of mammalian tissue corresponding to the mammalian tissue that was Petition 870260070403, dated 07 / 15 / 2026, pp. 55 / 79 40 / 61 undergoing treatment to reduce the risk of viral and microbial infection. In certain embodiments, the treatment comprises electron beam radiation. In certain embodiments, the electron beam radiation has a dose of about 5 to about 50 kGy, preferably about 10 to about 40 kGy.

[0113] Another aspect of the present invention includes a method for producing a pancreatin product comprising the steps of: (a) providing mammalian pancreatic tissue; (b) subjecting the pancreatic tissue to electron beam radiation to produce irradiated pancreatic tissue; and (c) isolating pancreatin from the irradiated pancreatic tissue to obtain the pancreatin product. In certain 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 a control pancreatin product. In certain embodiments, the control pancreatin product is obtained from non-irradiated pancreatic tissue. In certain embodiments, the electron beam radiation is sufficient to produce a reduction of at least three log10, preferably a reduction of at least four lo10, in the PPV viral load relative to a non-irradiated control sample.In certain incorporations, the electron beam radiation has a dosage of about 5 to about 50 kGy, preferably about 10 to about 40 kGy. In certain incorporations, the biological activity is lipase activity. In certain incorporations, the biological activity is protease activity or amylase activity.

[0114] Another aspect of the present invention includes a method for digesting a protein comprising the steps of: (a) providing an enzyme or proenzyme preparation isolated from an animal tissue irradiated with an electron beam; and (b) contacting the protein with the enzyme or proenzyme preparation. Petition 870260070403, dated 07 / 15 / 2026, pp. 56 / 79 41 / 61 under conditions sufficient to digest the protein. In certain incorporations, the contact step occurs in vivo. In certain incorporations, the contact step occurs in vitro. In certain incorporations, the animal tissue is porcine pancreas. In certain In certain embodiments, the enzyme or proenzyme preparation derived from an electron beam-irradiated animal tissue exhibits at least a reduction of at least three log10, preferably a reduction of at least four log10, in viral load when compared with an enzyme or proenzyme preparation derived from non-irradiated animal tissue. In certain embodiments, the enzyme or proenzyme preparation exhibits a biological activity corresponding to at least 50%, preferably at least 90%, of the biological activity of an enzyme or proenzyme preparation derived from non-irradiated animal tissue.

[0115] Another aspect of the present invention includes an enzyme preparation comprising one or more enzymes isolated from pancreatic tissue irradiated with an electron beam. In certain embodiments, the pancreatic tissue comprises a porcine pancreatic gland. In certain embodiments, the porcine pancreatic gland is frozen and mechanically processed into flakes or blocks before irradiation. Thus, in some such embodiments, the pancreatic tissue subjected to electron beam irradiation is frozen pancreatic tissue in flakes or a frozen block of pancreatic tissue. In certain embodiments, the porcine pancreatic gland is a whole gland or a portion thereof, such as one or more lobules. In certain embodiments, one or more enzymes comprise pancreatin.In certain incorporations, the enzyme preparation exhibits a reduction of at least three log10, preferably a reduction of at least four log10, in viral load when compared to a preparation of... Petition 870260070403, dated 07 / 15 / 2026, pp. 57 / 79 42 / 61 control enzyme. For example, the enzyme preparation exhibits a reduction of at least three log10, preferably a reduction of at least four log10 in PPV viral load relative to a control enzyme preparation. In certain incorporations, the biological activity of the enzyme preparation corresponds to at least 50%, preferably at least 90%, of the biological activity of a control enzyme preparation. In certain incorporations, the biological activity is lipase activity. In certain incorporations, the biological activity is protease activity or amylase activity. In certain incorporations, the control enzyme preparation is obtained from non-irradiated tissue. In certain incorporations, the reduction in viral load is an orthogonal reduction.

[0116] Another aspect of the present invention includes a method for treating exocrine pancreatic insufficiency comprising: administering a dose of the aforementioned enzymatic preparations and / or pharmaceutical compositions to an individual in need thereof. In certain embodiments, the exocrine pancreatic insufficiency is due to cystic fibrosis or chronic pancreatitis. F. Examples Example 1. Electron beam irradiation of supine parvovirus (PPV), pancreatin API and porcine pancreas Materials and methods

[0117] Porcine parvovirus in a vial for injection in cell culture fluid. Because porcine gland tissue itself may have some inactivating effects on viruses, initially, the virus samples used for these studies were prepared from infected cell cultures. Parvovirus (PPV) cells, NADL-2 cell line (ATCC® VR-742™) and pig testes (ST) cells (ATCC® CRL-1746™) were acquired from American Type Culture. Petition 870260070403, dated 07 / 15 / 2026, pp. 58 / 79 43 / 61 Collection (ATCC). PPV was propagated, cultured, and maintained according to ATCC recommendations. PPV was propagated to an approximate titer of 10⁸ infectious units (IU) of virus / mL. The virus was harvested from lysed cells in cell culture medium consisting of minimum essential medium (MEM), 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units / mL of penicillin, and 100 mg / mL of streptomycin.

[0118] The PPV was packaged in vials and then double-bagged before shipment. The vials were Nalgene cryogenic vials (i.e., polypropylene with a high-density polyethylene (HDPE) screw cap with a leak-proof sealing ring having a length of 1.87 inches; diameter of 0.5 inches; a capacity of 2.0 mL; and a usable volume of 1.0 mL). Each vial was placed in a Food Saver bag (polyethylene with an outer layer of nylon) and vacuum-sealed. The bags were cut to fit the vial exactly. Four individually sealed vials were then placed inside another Food Saver bag (approximately 11 x 14”) and vacuum-sealed.

[0119] Frozen porcine pancreas flakes. Pancreases obtained from butcher pigs (Animal Technologies, Tyler, TX) were kept on dry ice.

[0120] Frozen porcine pancreas flakes were placed in a 12" x 12" x 1" transparent polypropylene container, which was vacuum-sealed inside a 3-milliinch transparent poly / nylon bag. The bag was then thermally sealed. The pancreases were kept on dry ice to ensure temperatures below -20°C. The weight of the frozen porcine pancreas flake sample was 1.05 kg plus the weight of the packaging of 270 grams (0.27 kg). The lid weighed 100 g. The surface density was 1.3 g / cm². Two packages were made for each radiation dose, including the non-radiation control; one intact package was used for the isolation of the enzymatic preparation. Petition 870260070403, dated 07 / 15 / 2026, pp. 59 / 79 44 / 61 after transit back to the location where the isolation was conducted, the other package being a reserve.

[0121] Pancreatin (API). Two types of API pancreatin were used: pancreatin N and pancreatin S. Both APIs were kept at ambient conditions. Pancreatin N (Material #1030828, Abbott Laboratories) is a light yellow / gray to ivory-colored powder. Pancreatin S (Material #1030829, Abbott Laboratories) is a light yellow / gray to ivory-colored powder. Pancreatin S is derived from the pancreas of sows.

[0122] API pancreatin was placed in a 2" x 3" x 1 5 / 8" (transparent polypropylene) container, which was vacuum-sealed inside a 3-milliinch transparent poly / nylon bag. The bag was heat-sealed. The weight of the API pancreatin sample was 80 grams plus a packaging weight of 26 grams. The surface density was 1.4 g / cm2. The pancreatin samples were exposed to electron beam radiation at the indicated dose. The control was not exposed to electron beam radiation. The pancreatin was tested for activity after transit back to the experimental site. The pancreatin was derived from butcher's pig. All pancreatin packages arrived intact from the irradiation site. Electron beam irradiation

[0123] The electron beam source was an industrial materials processing electron linear accelerator (IMPELA®; Iontron Industries, Inc., Columbia City, IN), 10 MeV, 80 cm sweep amplitude.

[0124] Packaged frozen porcine parvovirus and supine pancreas flakes samples were kept below -20°C by placing them on dry ice contained in an aluminum tray. Dosimeters were placed on the top surface of each sample. Packaged API pancreatin samples kept at room temperature were placed on a foam sheet. Petition 870260070403, dated 07 / 15 / 2026, pp. 60 / 79 45 / 61 expanded polystyrene and dosimeters were placed on the top surface. The packages were sent on a conveyor belt under the electron beam scanning horn. After one radiation pass, the dosimeters were retrieved. The packages were sent under the radiation horn for a second pass after inverting the package (the top side is now facing down) and a new dosimeter was placed on the top surface. After the second radiation cycle, the packages and dosimeters were retrieved. Packages of porcine parvovirus and frozen supine pancreas flakes were retrieved and placed on dry ice for shipment. Packages of API pancreatin were retrieved and kept at room temperature for shipment. Control packages of porcine parvovirus and frozen supine pancreas flakes placed on dry ice were prepared for reshipment on dry ice without undergoing radiation.The API pancreatin control packages, kept at room temperature, were prepared for reshipment in a radiation-free environment. The electron beam dosage was calculated from the dosimeter exposure using a calibration curve. Viral infectivity test

[0125] Viral infectivity was determined by tenfold titration in 96-well microplates using appropriate controls for triplicate samples at each energy dose. Virus titers were 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: 493-497 and expressed as 50% of TCID50 per mL. Pancreatin isolation and testing

[0126] The method used for pancreatin isolation is substantially similar to that described in US 4,623,624, comprising hydrolysis and / or autolysis, followed by fiber sieving, enzyme precipitation, separation Petition 870260070403, dated 07 / 15 / 2026, pp. 61 / 79 46 / 61 of precipitate by filtration and / or centrifugation, cake washing, drying, and particle size reduction. Hydrolysis was performed using a package corresponding to each radiation dosage, including the non-irradiated control. A package without packaging damage (such as large cracks) was selected for each isolation experiment. Due to the care taken during transport back to the experimental site, all packages arrived intact, and one package was randomly selected for each radiation dosage, including the control for isolation. Pancreatin from a previous isolation was used as a protease source to initiate hydrolysis. Calcium hydroxide was used to provide calcium ions necessary for protease activation. Sodium bicarbonate was used as a buffering agent for hydrolysis. Simethicone was used as an antifoaming agent. Pancreatic hydrolysis was performed near room temperature.The completion of hydrolysis was checked by centrifugation of the hydrolyzed mixture after the addition of isopropanol as described below. After the completion of hydrolysis, more isopropanol was added to reduce the rate of hydrolysis, the mixture was cooled, the mixture was stirred, and the fibers were separated using a 0.425-inch mesh. The fibers were washed with isopropanol and compressed to expel the liquid retained in them. The wash was combined with the previously obtained filtrate. More isopropanol was added to the filtrate to cause the precipitation of the enzymes. The suspension was filtered through a cloth filter with 15-micron openings and washed with increasing concentrations of isopropanol with absolute isopropanol as the final wash. The cake was dried on the cloth filter under a stream of nitrogen while vacuum pulled down the cloth filter until the cake appeared visually dry (the color of the cake becomes lighter after the removal of isopropanol and water).The cake was removed from the cloth filter and dried under vacuum and nitrogen flow at a temperature below approximately 50°C until a water content was reached. Petition 870260070403, dated 07 / 15 / 2026, pp. 62 / 79 47 / 61 by Karl Fischer less than or equal to 3.5%. The yield of dry pancreatin is 80 to 100 g from 1 kg of frozen porcine pancreas flakes from butcher's pig.

[0127] Centrifugation test to complete hydrolysis. Three 10 mL sample spoonfuls from the hydrolysis vessel are passed through a 0.425 mm sieve, the filtrate is collected in a plastic beaker (the filtrate is also scraped off in the plastic beaker), and any fibers retained in the mesh are discarded. Using a pipette, 10 g of reactor solution are added to the 50 mL centrifuge tube, 5.5 mL of 85% IPA are added, and the mixture is stirred for 1 minute with a spatula. 20 mL of 85% IPA are added to the filtrate in the 50 mL centrifuge tube. The mixture is stirred for one minute with a spatula. The suspension is centrifuged at approximately 90X for two minutes.

[0128] The first sample can be collected two hours after the start of hydrolysis or when the color is changing from pink to brown and the suspension becomes thinner (about 2 hours).

[0129] The next sample is taken when the color is brown without pink tones, at this point the sediment is expected to be about 25%, the sediment will be less firm and retarders may be present in the clear layer above. After that, sampling can be done at every possible half-hour interval.

[0130] Hydrolysis will stop if two subsequent measurements show less than 20% sediment. At this point, the sediment will be firm and without retarders in the clear layer above.

[0131] Electron beam irradiation of the front and back of the PPV in a vial was performed in triplicate. Data for viral load reduction and the log mortality of PPV exposed to electron beam radiation are shown in Table 1: Petition 870260070403, dated 07 / 15 / 2026, pp. 63 / 79 Table 1 Dosage (KGy) 0 9.5 19.25 38.45 Viral load Viral load Viral load Viral load 0 9.5 19.25 38.45 Log of Log of Log of Log of mortality mortality mortality Title of Title of Title of Virus / mL Virus / mL Virus / mL Virus / mL Test A 1.50E+08 2.50E+06 1.50E+05 Test B Test C Average 1.50E+08 4.00E+08 6.34E+06 6.34E+04 1.26E+06 4.00E+05 1.50E+02 0.00E+00 1.78E+00 3.00E+00 0 1.37E+00 3.37E+00 0 2.50E+00 3.00E+00 6.43E+00 0 1.88E+00 3.12E+00 6.43E+00 48 / 61 Petition 870260070403, dated 07 / 15 / 2026, pp. 64 / 79 49 / 61

[0132] The electron beam dosage was determined by estimating the absorbed dose for each sample from the surface dose indicated by the dosimeter attached to the sample container.

[0133] Table 1 shows that PPV exposure to approximately 40 kGy provides about 6.5 log10 mortality, while exposure to approximately 20 kGy provides about 3 log10 mortality. Based on these data, exposure to approximately 30 kGy is expected to provide at least 4 log10 mortality.

[0134] PPV exposure to approximately 60 kGy, approximately 80 kGy, or approximately 100 kGy resulted in viral titers below the assay's detection limit.

[0135] Pancreatin was tested for free protease, amylase and lipase activity as described in the USP by validated methods. Pancreatin was tested for total protease as described in the European Pharmacopoeia (EP) by validated methods.

[0136] Bilateral electron beam irradiation of pancreatin N was performed. All pancreatin N containers were received intact at the experimental site and were used for assay. Table 2 shows the data for enzymatic activity of pancreatin API exposed to electron beam radiation: Table 2 Electron beam dose Lipase activity Amylase activity Total protease activity Free protease activity kGy USP units / gram USP units / gram USP units / gram USP units / gram Control (0 kGy) 89106 564390 383192 314093 Petition 870260070403, dated 07 / 15 / 2026, pp. 65 / 79 50 / 61 18.6kGy 40583 272760 259117 214914 99.4 kGy 37799 286652 247005 196356

[0137] Table 2 shows that exposure to pancreatin API at approximately 20 kGy provides approximately 30% loss in lipase activity, exposure to pancreatin API at approximately 40 kGy provides approximately 35% loss in lipase activity, exposure to pancreatin API at approximately 60 kGy provides approximately 46% loss in lipase activity, exposure to pancreatin API at approximately 80 kGy provides approximately 54% loss in lipase activity, and exposure to pancreatin API at approximately 100 kGy provides approximately 58% loss in lipase activity. Based on these data, it is expected that exposure to pancreatin API at approximately 30 kGy will provide approximately 30% loss in lipase activity.

[0138] Bilateral electron beam irradiation of butcher pig pancreas was performed. Table 3 shows the data for pancreatin enzymatic activity derived from frozen porcine pancreas flakes exposed to electron beam radiation: Table 3 Electron beam dose Lipase activity Amylase activity Total protease activity Free protease activity kGy USP USP USP USP units / gram units / gram units / gram units / gram Petition 870260070403, dated 07 / 15 / 2026, pp. 66 / 79 51 / 61 Control (0 kGy) 74986 423487 243421 151365 18.75 kGy 74741 449569 337594 128749 35.5 kGy 65348 349816 360407 128506 56.55 kGy 57976 398281 268297 119727 77.4 kGy 34806 251691 175994 109158 100.4 kGy 36362 276118 206217 100342

[0139] Table 3 shows that exposure of frozen porcine pancreas flakes to approximately 20 kGy provides a loss of approximately 1% in lipase activity; exposure of frozen porcine pancreas flakes to approximately 40 kGy provides a loss of approximately 13% in lipase activity; exposure of frozen porcine pancreas flakes to approximately 60 kGy provides a loss of approximately 23% in lipase activity; exposure of frozen porcine pancreas flakes to approximately 80 kGy provides a loss of approximately 54% in lipase activity; and exposure of frozen porcine pancreas flakes to approximately 100 kGy provides a loss of approximately 52% in lipase activity. Based on these data, exposure of frozen porcine pancreas flakes to approximately 30 kGy is expected to result in a loss of about 10% in lipase activity.

[0140] As shown in Tables 2 and 3, electron beam irradiation of pancreatin API produces more loss of enzymatic activity when compared to electron beam irradiation of pancreatic tissue before isolation. Without wanting to be bound by theory, the resistance of intact tissue to electron beam irradiation may be due to the conformation of the enzyme (e.g., as a proenzyme) in the source tissue and / or cofactors in the source tissue providing structural protection. Example 2. Electron beam irradiation of whole pig pancreases.

[0141] An additional study was performed using whole porcine pancreas. Petition 870260070403, dated 07 / 15 / 2026, pp. 67 / 79 52 / 61 Approximately 3.6 kg of thawed porcine pancreas were packed into wax-lined cardboard boxes approximately 10 x 15 x 1.5 inches thick. The boxes were frozen at -20°C and kept until electron beam irradiation. The boxes were shipped for electron beam irradiation by refrigerated truck (-20°C). The boxes were removed from the refrigerated truck and then exposed to bilateral electron beam irradiation – non-irradiated boxes served as controls. Five (5) boxes were used for each nominal radiation dose: 0, 15, 20 and 25 kGy and shipped back for evaluation by refrigerated truck (-20°C), including the non-irradiated boxes, and then kept at -20°C.

[0142] Frozen pancreas samples were selected from each box at random locations within the box for subsequent isolation of pancreatin. Isolation was performed as described here. Pancreatin isolated from whole pancreas irradiated with an electron beam was then tested for enzymatic activity according to a colorimetric assay.

[0143] Samples of pancreatin (API) were tested using colorimetric kinetic analysis by a microplate reader with the use of substrates structurally similar to those used in <pancrelipase>39 of USP. Enzymatic activities were determined by measuring the rate of product production relative to the pancrelipase reference standard. Analytical comparability was demonstrated between the USP monographic methods and alternative microplate reading methods.

[0144] The sampling, isolation, and analysis procedure was repeated four times for each dose to obtain a total of five measurements for each dose. The average values ​​of five measurements are presented in Table 4. Table 4: API enzyme activity after whole pancreas irradiation Petition 870260070403, dated 07 / 15 / 2026, pp. 68 / 79 53 / 61 Electron beam dose* Average lipase activity Average amylase activity Average free protease activity Average total protease activity kGy Units Units Units Units USP / mg USP / mg USP / mg USP / 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 104 395 183 301 *(minimum dose + maximum dose) / 2

[0145] Table 4 shows that pancreatin isolated from a whole pancreas exposed to an electron beam irradiation dose of up to about 25 kGy has the same or substantially the same lipase activity as pancreatin isolated from a non-irradiated control. Example 3. Low-dose electron beam irradiation of intact porcine pancreatic tissue.

[0146] Further studies were performed enriching porcine pancreas tissue with live viruses and then subjecting the virus-enriched tissue to lower dose electron beam irradiation (approximately 12.3 kGy) to allow for virus recovery and evaluation. This additional work was carried out to demonstrate the effective inactivation of several related viruses at a low dose, which would allow for effective enumeration of the impact of electron beam irradiation.

[0147] Selected viruses were deliberately enriched on the tissue sample and the degree of virus clearance was evaluated by comparing the amount of virus input with the amount of virus remaining after electron beam treatment. The viruses selected for this study are Petition 870260070403, dated 07 / 15 / 2026, pp. 69 / 79 54 / 61 identified in Table 5. Table 5: Viruses selected for viral clearance study. Virus Family Genome Coating Size (nm) Physicochemical Resistance Reovirus type 3 (REO3) Reo RNA None 60-80 Medium Porcine parvovirus (PPV) Parvo DNA None 18-24 High Feline calicivirus (FCV) Calici RNA None 35-39 Medium

[0148] REO3 has a double-stranded RNA, segmented genome and belongs to the Reoviridae family of viruses, which also includes Rotavirus. Thus, REO3 can serve as a model virus for the validation of inactivation methods in blood products and, in particular, as a model for the hepatitis E virus (HEV).

[0149] Porcine pancreases were cut and ground. The tissue was then added to a Petri dish and spiked with the indicated virus. Standard virus stocks had titers of at least 1 x 107 pfu / mL. The enriched sample was incubated at room temperature for a minimum of 60 minutes (until the tissue returned to its original dryness). After incubation, more porcine pancreatic tissue was added to the Petri dish. The Petri dish was sealed and placed on dry ice for shipment to the electron beam facility. Each Petri dish contained approximately 13 grams of tissue, and the tissue density was similar to that of a whole gland.

[0150] For each virus, enriched recovery samples (not sent) and a non-irradiated shipping control (sent to) were included. Petition 870260070403, dated 07 / 15 / 2026, pp. 70 / 79 55 / 61 the installation of an electron beam, but not irradiated).

[0151] Electron beam irradiation was performed in the electron beam facility. After completion of electron beam radiation exposure, irradiated samples and non-irradiated shipping controls were sent back for viral load assessment. Upon receipt, samples were stored at a temperature of -60°C or lower until testing.

[0152] For each sample, 50 mL of culture medium was added to a sterile bottle. Tissue was extracted by performing 3 immersion cycles for approximately 5-10 minutes at room temperature followed by a vortex of 15-30 seconds. Samples were then centrifuged at 3,000 rpm for 10 minutes. The extraction supernatant was used for viral testing.

[0153] Virus titers were determined by standard plate assays. The indicator cell types for REO3, PPV, and FCV were Vero, PT-1, and CRFK, respectively. Virus titers and viral clearance factors were calculated according to standard procedures. The virus clearance factor (VCF) was calculated as follows: VCF = log10[ Volume*title before processing Volume*title after processing

[0154] Table 6 shows data for viral clearance produced by exposure of enriched tissue to electron beam radiation. Table 6: Viral clearance after irradiation of virus-enriched pancreases Virus template, Total Virus Log (VCF), 95% confidence limit, Before processing, After processing Petition 870260070403, dated 07 / 15 / 2026, pp. 71 / 79 56 / 61 REO3 6.5 <2.4 >4.1 0.08 FCV 6.7 4.9 1.8 0.05 PPV 5.8 3.6 2.2 0.29

[0155] These studies confirmed that sufficient inactivation of viruses, including feline calicivirus (FCV) and reovirus 3 (REO3), can be achieved with electron beam irradiation of intact tissue. Furthermore, irradiation of a pancreas followed by isolation of an enzymatic preparation (e.g., pancreatin API) from the irradiated gland showed similar results with respect to the loss in enzymatic activity compared to a non-irradiated control to those shown in Table 3 where porcine pancreas flakes were used. Example 4. Electron beam irradiation of chopped tissue and homogenized tissue.

[0156] Minced pancreas: frozen porcine pancreas were ground in a mincer and subsequently frozen for irradiation as described herein.

[0157] Homogenized pancreas: frozen porcine pancreases were ground in a grinder as described above and agitated in water at a temperature of 10°C ± 5°C until a homogeneous mixture was obtained (approximately 75 minutes). The samples were subsequently frozen for irradiation as described herein.

[0158] As a final control product, pancreatin was used as a therapeutic-grade powder according to the Ph.Eur specification. The pancreatin powder was obtained directly from the manufacturer (Abbott Laboratories GmbH, Neustadt, Germany) and sampled from the manufacturing facility. Pancreatin for therapeutic use (according to Ph.Eur.) is also commercially available, for example, from Nordmark, Uetersen, Germany or from BIOZYM Gesellschaft für Enzymtechnologie mbH, Hamburg, Germany, and / or can be produced from Petition 870260070403, dated 07 / 15 / 2026, pp. 72 / 79 57 / 61 in accordance with known methods (see, for example, EP-A2 115023).

[0159] For irradiation treatment, samples were removed from the freezer or refrigerator and placed in cooling accumulators to maintain the temperature as described below. Since the use of a thermometer is not possible with frozen samples, a thermal imaging camera (Testo 880-3, Testo AG Lenzkirch, Germany) was used where necessary to determine surface temperatures.

[0160] Samples of homogenized pancreatic tissue were thawed and irradiated at a temperature above 0°C (T > 0°C), but below 20°C. Pancreatin samples were removed from the refrigerator and irradiated at T > 0°C.

[0161] Samples were exposed to electron beam radiation using a 10 MeV, 35 kW TT-100 Rhodotron electron beam accelerator (IBA Industrial, Louvain-La-Neuve, Belgium). The irradiation process was performed in stages. Sample boxes passed through the accelerator in 5 or 10 kGy steps until the required dose was reached. For example, samples planned for lower doses were removed in earlier steps, and samples planned for higher doses were re-exposed and irradiated again.

[0162] The enzymatic activities (i.e., lipolytic, proteolytic, amylolytic activity) of test samples before irradiation ("pretreatment samples") were determined according to Ph.Eur., relative to the relevant reference standards and taken as initial enzymatic activities (lipolytic, proteolytic, amylolytic, when applicable) before irradiation treatment. Subsequently, the test samples were irradiated as described herein. The enzymatic activities of the test samples after irradiation were determined ("post-treatment samples") in the same manner as for the pretreatment samples. The enzymatic activities retained in Petition 870260070403, dated 07 / 15 / 2026, pp. 73 / 79 58 / 61 post-treatment samples were determined and are reported as the relative percentage of the same enzymatic activity determined in the pre-treatment sample.

[0163] Lipase activity was determined as follows, as described in Ph.Eur.: the required amount of the test sample (depending on the expected activity, approximately 2.5 g) was triturated (e.g., crushed or ground depending on the state / consistency of the test sample) and extracted directly with buffer solution. Lipase hydrolytic activity is determined using an olive oil emulsion as a substrate. The free fatty acids cleaved from the olive oil triglycerides are titrated with sodium hydroxide solution at a constant pH of 9.0. The lipase activity of the sample is determined by comparing the rate at which a suspension of the sample hydrolyzes an olive oil emulsion substrate with the rate at which a suspension of a standard pancreas reference powder (Ph.Eur. reference standard, described in the monograph, "pancreas powder (lipase) BRP") hydrolyzes the same substrate under the same conditions.

[0164] Lipase activity loss values ​​are reported in Table 7. Table 7: Enzymatic activity after irradiation of chopped tissue and tissue homogenate. Electron beam dose (kGy) Lipase activity loss (%) Pancreatin (end product) Chopped pancreatic tissue Tissue homogenate 10 24.0 nt 7.8 15 30.3 13.1 11.3 20 35.0 10.9 11.4 25 nt 19.8 nt Petition 870260070403, dated 07 / 15 / 2026, pp. 74 / 79 59 / 61 30 42.5 18.5 16.9 40 48.9 nt 24.3 50 53.6 nt 29.7 Not tested.

[0165] Irradiation of chopped tissue or tissue homogenate (i.e., prior to chemical or enzymatic processing of the proenzyme population to active form) consistently resulted in improved enzymatic activity retention compared to final product irradiation. Irradiation between 20 and 30 kGy resulted in activity losses of less than 20%, lower than that achieved by standard protocols. Example 5. Irradiation of chopped tissue enriched with B. cereus

[0166] Pancreas frozen after chopping was thawed, packed in sterile 50 x 9 mm polystyrene Petri dishes (Ted Pella, Inc., Redding, CA, item 14014), enriched with approximately 108 cfu of B. cereus endospores from a commercial pre-titrated suspension (B. cereus ATCC 14579, Mesa Labs, Bozeman, MT), vacuum sealed in a bag (seal-a-meal) and then frozen.

[0167] An electron beam dosage range of 0 to 20 kGy in 5 kGy increments was selected in the present experiment with chopped pancreas. Bags were sent for electron beam irradiation in dry ice. Bags were exposed to electron beam irradiation - bags not sent and non-irradiated bags served as controls.

[0168] The samples were returned on dry ice. All samples were then diluted and placed on trypticase soy agar plates (Biomerieux item M1040) for CFU enumeration. The plates were incubated at 30-35°C for 1-2 days until the colonies were of countable size. CFU counts were recorded. Petition 870260070403, dated 07 / 15 / 2026, pp. 75 / 79 60 / 61

[0169] Introducing a decontamination step before processing porcine pancreas provides effective control of known infectious agents, both in terms of operator safety during enzyme isolation and patient safety. Thus, the use of an electron beam treatment that is effective in inactivating a broad spectrum of microbes and viruses, including difficult-to-inactivate viruses (e.g., porcine parvovirus) and spore-forming bacteria, with minimal loss in enzymatic activity, is advantageous compared to other methods.

[0170] When used herein, the electron beam irradiation step can be considered an orthogonal viral inactivation step. The reduction in microbial and / or viral load obtained in pancreatic tissue irradiated with electron beam transfers to the pancreatin that is isolated from it in an additive manner with respect to the reduction obtained by other microbial and / or viral reduction steps. The log10 of total mortality achieved by all steps, including electron beam irradiation, is the log10 of cumulative mortality achieved by all microbial and / or viral inactivation steps performed on pancreatic tissue.

[0171] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be considered as limiting the scope of the invention, which is defined only by the appended claims and their equivalents. Various changes and modifications of the disclosed embodiments will become apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to chemical structures, substituents, derivatives, intermediates, syntheses, formulations or methods, or any combination of such changes and modifications of use of the invention, may be made without departing from the spirit and scope of the invention.

[0172] All references (patents and non-patents) cited above are Petition 870260070403, dated 07 / 15 / 2026, pp. 76 / 79 61 / 61 are incorporated by reference in this patent application. The discussion of those references is intended merely to summarize the assertions made by their authors. No reference (or part of any reference) is admitted to be relevant prior art. The applicant reserves the right to contest the accuracy and relevance of the references cited.< / pancrelipase>

Claims

1. A method for producing a pancreatin product, characterized in that it comprises the steps of: (a) subjecting homogenized or chopped mammalian pancreatic tissue to electron beam radiation to produce irradiated pancreatic tissue; and (b) isolating pancreatin from the irradiated pancreatic tissue.

2. Method according to claim 1, characterized in that the biological activity of the pancreatin product obtained in step (b) corresponds to at least 80% of the biological activity of a control enzyme preparation, wherein the biological activity is a lipase activity.

3. Method according to claim 1 or 2, characterized in that electron beam radiation is sufficient to produce at least a three log10 reduction in a viral and / or microbial contaminant compared to a control sample, wherein the viral contaminant is a model virus that is porcine parvovirus (PPV), and wherein the microbial contaminant is a model bacterium that is Bacillus cereus.

4. Method according to claim 1 or 2, characterized in that the electron beam radiation has a dosage of 5 to 50 kGy.

5. Method according to any one of claims 1 to 4, characterized in that step (a) comprises maintaining the irradiated pancreatic tissue at a temperature of 0°C to 20°C.