Methods for rapid preparation of microbes for microbiota transplantation therapy

Abstract

Provided herein are rapid methods for obtaining an extract of human feces, and methods for using such compositions, including methods for microbiota transplantation therapy, including replacing or supplementing or modifying a subject's colon microbiota, and methods for treating a disease and / or condition.

Description

METHODS FOR RAPID PREPARATION OF MICROBES FOR MICROBIOTATRANSPLANTATION THERAPY

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 650,537, filed May 22, 2024, which is incorporated by reference herein in its entirety.

[0003] BACKGROUND

[0004] Over the last decade, fecal microbiota transplantation (FMT) has emerged as a highly effective treatment for recurrent Clostridioides difficile infections (rCDI) that fail traditional treatment with antibiotics alone. As initially envisioned and practiced, FMT [now referred to as microbiota transplantation therapy (MTT)] involved administration via nasogastric tube or enema (Brandt and Aroniadis, Gastrointestinal endoscopy, 2013;78:240-249; Aas et al., Clinical infectious diseases. 2003;36:580-585) of a fecal slurry prepared from stool of a healthy donor typically identified by the patient 2013;78:240-249; Aas et al., Clinical infectious diseases. 2003;36:580-585). These treatments, however, lacked standardization and presented formidable logistical difficulties for routine implementation into clinical practice. These problems were solved, in large part, by the development of formal criteria for use of standardized stool donors, protocols for separating and purifying microbiota from stool, cryopreservation using glycerol as a cryoprotectant, and dose quantification in terms of viable bacterial counts (Hamilton et al, The American journal of gastroenterology. 2012;107:761-767). While colonoscopy was initially one of the most common methods for administering the purified liquid suspension of fecal microbiota, an encapsulated preparation of purified and freeze-dried fecal microbiota was more recently introduced and shown to have comparable efficacy in treatment of rCDI, relative to freshly prepared materials (Staley et al., The American journal of gastroenterology. 2017;112:940-947; Vaughn et al., Clin Gastroenterol Hepatol. 2023 May;21(5): 1330-1337. e2). The use of oral capsules has helped to reduce the burden, costs, and intrinsic risks associated with colonoscopy, and enabled more mainstream practice of MTT for C. difficile, as well as for other indications that require repeateddosing (Kang et al., Microbiome. 2017; : 10; Haifer et al., Lancet Gastroenterol Hepatol. 2022;7:141-151).

[0005] SUMMARY OF THE APPLICATION

[0006] Described herein are methods to rapidly extract and purify microbiota from donor stool for use in Microbiota Transplantation Therapy (MTT). The microbial preparation includes a flowable, suspendable, wettable powder that can be easily administered to the patient by various methods including, but not limited to: (i) a liquid buffered suspension introduced via endoscopy, (ii) encapsulated freeze-dried microbiota taken by capsules via swallowing, or (iii) via an oral drinkable suspension using flavored freeze-dried microbiota dispersed in a liquid. Advantages of the methods disclosed herein include ease of maintaining anaerobiosis during the extraction, which is expected to result in better preservation of strict anaerobes, thus providing greater anaerobe viability, greater microbe diversity, or the combination thereof, compared to currently available methods of obtaining microbiota from donor stool for use in MTT. Other advantages of the methods disclosed herein include an expected increase in ease of engraftment compared to that obtained from donor stool using currently available methods, and improved efficiency of purification, resulting in reduced time and costs for obtaining microbiota from donor stool for use in MTT.

[0007] In one embodiment, a method of the present disclosure includes homogenizing a fecal sample in a cell extraction buffer to result in a slurry, and filtering the slurry with a hand pressurized filter press that has a filter medium, where the filter medium includes at least one filter screen to result in a filtrate comprising biological material and, in some embodiments, a small amount of non-biological and unfiltered materials. The unfiltered material can be resuspended in a solution to result in resuspended material. The method can further include filtering the resuspended material with a filter medium, where the filter medium comprises at least one filter. A force, such as centrifugal force, can be applied to the combined filtrate to form a pellet that includes microbial cells, and the pellet resuspended in a reduced volume compared to the combined filtrate before application of the force. The application of the force and the resuspending of can be repeated at least once to result in a processed filtrate.

[0008] In another embodiment, a method of the present disclosure includes homogenizing a fecal sample using a fdtration device that includes a container. In one embodiment, the container can include a solid materials region, a fdtrate region, and a filter separating the solid materials region and the filtrate region, where the homogenized fecal sample is present in the solid materials region. The fecal sample is filtered, where the filtering can include passage of liquid present in the fecal sample through the filter into the filtrate region. In some embodiments, the material passes back and forth through the filters multiple times, to result in a filtered liquid material that includes mostly a biological component, a small amount of non-biological material, and unfiltered material retained on the filter surface. The suspended filtered material is removed from the container and retained. Additional solution, equal to the first amount added to the bag, is then added to the remaining unfiltered material in the solid materials region, and re-homogenized. The resulting second filtered liquid material contains additional biological material. The second filtered material is removed from the container and retained, while the remaining unfiltered solid material is safely discarded. The first and second filtered material are combined, and a force, such as a centrifugal force, is applied to the combined filtrate to form a pellet. The pellet can be resuspended in a reduced volume of buffer compared to the combined filtrate before application of the force. The application of the force and the resuspending can be repeated multiple timer to result in a partially purified preparation containing mostly microbiota.

[0009] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein, and their meanings, are set forth below.

[0010] As used herein, the term “filtrate” refers to the material that has moved through a filter and is separated from unfiltered material. Filtrate typically includes biological material, nonliving material, and a liquid component. For instance, applying force to a fecal material causes biological material, liquid, and some non-living material present in the fecal material to pass through a filter, while most non-living material does not pass through the filter. The biological material, liquid, and some non-living material present in the fecalmaterial that pass through the filter is the filtrate, and the non-living material that does not pass through the filter is the unfiltered material.

[0011] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0012] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all the listed elements or a combination of any two or more of the listed elements. The use of "and / or" in some instances does not imply that the use of "or" in other instances may not mean "and / or."

[0013] The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.

[0014] As used herein, "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" or the like are used in their open ended inclusive sense, and generally mean "include, but not limited to," "includes, but not limited to," or "including, but not limited to."

[0015] It is understood that wherever embodiments are described herein with the language "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" and the like, otherwise analogous embodiments described in terms of "consisting of' and / or "consisting essentially of' are also provided. The term "consisting of' means including, and limited to, whatever follows the phrase "consisting of." That is, "consisting of1indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of' indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0016] Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0017] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0018] In the description herein particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

[0019] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0020] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The description thatfollows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0021] BRIEF DESCRIPTION OF THE FIGURES

[0022] The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following drawings.

[0023] FIG. 1A shows an elevation view of a container according to the present disclosure.

[0024] FIG. IB shows a top plan view taken along line 1-1 of FIG. 1A.

[0025] FIG. 1C shows an enlarged view of a portion of FIG. IB.

[0026] FIG. 2 shows a flowchart illustrating an example of a method for processing a fecal sample according to various aspects of the disclosure presented herein.

[0027] Relative terms such as “left,” “right,” “front,” “back,” “top,” “bottom,” “side,” “upper,” “lower,” “above,” “below,” and the like may be used herein and, if so, are from the perspective shown in the particular figure. These terms are used only to simplify the description, however, and not to limit the interpretation of any embodiment described. In a similar manner, terms such as “first” and “second” may be used herein to describe various elements. However, such terms are provided merely to simplify identification of the element(s) or step(s). Accordingly, if an element is described as “first,” there may or may not be any other subsequent elements or steps - that is, a “second” element or step is not necessarily present.

[0028] DETAILED DESCRIPTION

[0029] Provided herein are compositions and methods for making compositions that include microbes (microbiota) recovered from feces of human donors. As used herein, the term “fecal microbes” refers to microorganisms (microbiota) that are present in the gut, intestine, colon, or feces of a normal healthy adult human. Such a composition may beprepared by processing fecal material. As used herein, the term “fecal material” refers to human stool. Unprocessed fecal material contains non-living material and biological material. The “non-living material” refers to the non-living material in fecal material, and may include, but is not limited to, dead bacteria, shed host cells, proteins, carbohydrates, fats, minerals, mucus, bile, undigested fiber and other foods, and other compounds resulting from food and metabolic ingestion and waste products and partial or complete digestion of food materials. “Non-living material” does not include an excipient, e.g., a pharmaceutically inactive substance, such as a cryoprotectant or lyoprotectant, added to a processed fecal material. “Biological material” refers to the living material, in fecal material (e.g., all possible microorganisms present in a human gut), and includes microbes including prokaryotic cells, such as bacteria and archaea (e.g., living prokaryotic cells and spores that can sporulate to become living prokaryotic cells), viruses, and eukaryotic cells such as protozoa and fungi. In one embodiment, "biological material” refers to all living material, e.g., the microbes, eukaryotic cells, fungi, and viruses, which are present in the colon of a normal healthy human.

[0030] Prokaryotic cells that may be present in a composition produced as described herein include the normal members of the intestinal microbiota present in any healthy human. In one embodiment, prokaryotic cells in a composition of the present disclosure include all genus members of the families indicated herein. Examples of prokaryotic cells that may be present cells include those that are members of the class Actinobacteria, such as the subclass Actinobacteridae and subclass Coriobacteridae. Examples of the subclass Actinobacteridae include members of the order Actinomycetales, and members of the order Bifidobacteriales. Members of the order Bifidobacteriales include members of the family Bifidobacteriaceae. Examples of the subclass Coriobacteridae include members of the order Coriobacteriales. Members of the order Coriobacteriales include members of the family Coriobacteriaceae.

[0031] Other examples of prokaryotic cells include members of the phylum Bacteroidetes, such as class Bacteroidia. Members of class Bacteroidia include order Bacteroidales. Members of order Bacteroidales include members of the family Bacteroidaceae, members of the familyPorphyromonadaceae, members of the family Prevotellaceae (such as members of the genus Prevotelid), and members of the family Rikenellaceae.

[0032] Other examples of prokaryotic cells include members of the phylum Firmicutes, such as class Bacilli, Clostridia, Erysipelotrichi, and Negativicutes. Examples of the class Bacilli include members of the order Bacillales (including members of the family Paenibacillaceae and members of the family Planococcaceae) and the order Lactobacillales (including members of the family Aerococcaceae, Enterococcaceae, Lactobacillaceae, and Streptococcaceae). Examples of the class Clostridia include members of the order Clostridiales, and examples of the order Colstridiales include the family Catabacteriaceae, Peptococcaceae, Peptostreptococcaceae, Ruminococcaceae, Clostridiaceae, Eubacteriaceae, and Lachnospiraceae. Examples of the class Erysipelotrichi include members of the family Erysipelotrichaceae. Examples of the class Negativicutes include members of the family Veillonellaceae. Other examples of the order Bacillales include Bacillales Family XI. Incertae Sedis, and Bacillaceae 1. Other examples of the order Clostridiales include Clostridiales Family XI. Incertae Sedis, Clostridiales Family XIII. Incertae Sedis, and Clostridiaceae.

[0033] Other examples of prokaryotic cells include members of the phylum Proteobacteria, such as class Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, and Gammaproteobacteria. Examples of the class Alphaproteobacteria include members of the order Rhizobiales, and examples of members of the order Rhizobiales includes members of the family Rhodobiaceae, members of the family Brucellaceae, and members of the family Hyphomicrobiaceae. Examples of the class Betaproteobacteria include members of the order Burkholderiales, and examples of members of the order Burkholderiales include members of the family Alcaligenaceae, members of the family Burkholderiaceae, and members of the family Sutterellaceae. Examples of the class Deltaproteobacteria include members of the order Desulfovibrionales, and examples of members of this order include members of the family Desulfovibrionaceae and Desulfomicrobiaceae. Examples of the class Epsilonproteobacteria include members of the order Desulfobacterales, and examples of members of this order include members of the family Desulfobacteraceae. Examples of the class Gammaproteobacteria includes membersof the order Alteromonadales and Enterobacteriales. Examples of members of the order Alteromonadales include members of the family Shewanellaceae, and examples of members of the order Enterobacteriales include members of the family Enterob act eriaceae.

[0034] Other examples of prokaryotic cells include members of the phylum Mycoplasmatota, formerly Tenericutes. Examples of members of the phylum Mycoplasmatota include members of the class Mollicutes. Examples of the class Mollicutes include members of the order Entomoplasmatales, and members of the order Entomoplasmatales include members of the family Spiroplasmataceae.

[0035] Other examples of prokaryotic cells include members of the class Verrucomicrobiae. Examples of members of the class Verrucomicrobiae include members of the order Verrucomicrobiales, and examples of members of the order Verrucomicrobiales includes members of the family Verrucomicrobiaceae. Other examples of prokaryotic cells include members of the family Fusobacteriaceae.

[0036] In one embodiment, a composition produced as described herein may include prokaryotic bacteria that are members of at least 1 phylum, at least 2 phyla, at least 3 phyla, at least 4 phyla, at least 5 phyla, at least 6 phyla, at least 7 phyla, at least 8 phyla, at least 9 phyla, or at least 10 phyla. In one embodiment a composition produced as described herein may include prokaryotic bacteria that are members of at least 1 class, at least 2 classes, at least 3 classes, at least 4 classes, at least 5 classes, at least 6 classes, or at least 7 classes. In one embodiment a composition of the present disclosure may include prokaryotic bacteria that are members of at least 1 order, at least 2 orders, at least 3 orders, at least 4 orders, at least 5 orders, at least 6 orders, or at least 7 orders. In one embodiment a composition produced as described herein may include prokaryotic bacteria that are members of at least 1 family, at least 2 families, at least 3 families, at least 4 families, at least 5 families, at least 6 families, at least 7 families. In one embodiment a composition produced as described herein may include at least 5, at least 10, at least 20, or at least 30 different genera of prokaryotic bacteria. In one embodiment a composition produced as described herein may include at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least500, at least 600, at least 700, at least 800, at least 900, or at least 1000 different species of prokaryotic bacteria.

[0037] Methods of making

[0038] The methods described herein use a fecal sample, also referred to herein as a stool sample. Typically, the fecal sample does not include pathogenic biological material. Accordingly, the compositions described herein preferably do not include pathogenic biological material. In one embodiment, fecal material is from a person that has undergone a rigorous clinical evaluation, including medical history, a physical examination, and laboratory testing. The clinical evaluation may include, but is not limited to, risk of infectious agents, carriage of multi-drug resistant organisms, co-morbidities, use of pharmaceuticals, factors that can or do affect the composition of the intestinal microbiota, and systemic medical conditions. Exclusion criteria for stool donors include, but are not limited to, risk of infectious agents; gastrointestinal comorbidities; use of pharmaceuticals, factors that can, or do affect, the composition of the intestinal microbiota: systemic medical conditions; and physical status. Guidelines related to examination of these exclusion criteria can be found in International Patent Publication No. WO 2014 / 152484 and US Published Patent Application No. 2018 / 0110810.

[0039] Exclusion criteria regarding risk of infectious agents may include, but are not limited to, known bacterial infections, known fungal infections, known parasitic infections, and known viral infections with Hepatitis B, C, SARS-CoV-2, or HIV; known exposure to HIV or viral hepatitis at any time; high risk behaviors including sex for drugs or money, any past use of intravenous drugs or intranasal cocaine, history of incarceration; tattoo or body piercing within 12 months; travel to areas of the world where risk of traveler's diarrhea is higher than the US; and current communicable disease, e.g., upper respiratory viral infection.

[0040] Exclusion criteria regarding gastrointestinal comorbidities include, but are not limited to, history of irritable bowel syndrome, wherein specific symptoms may include frequent abdominal cramps, excessive gas, bloating, abdominal distension, fecal urgency, diarrhea, constipation; history of inflammatory bowel disease such as Crohn's disease, ulcerativecolitis, microscopic colitis, chronic diarrhea; chronic constipation or use of laxatives; history of gastrointestinal malignancy or known colon polyposis; history of any abdominal surgery, e.g., gastric bypass, intestinal resection, appendectomy, cholecystectomy, and the like; use of probiotics or any other over the counter aids used by the potential donor for purpose of regulating digestion, but yogurt and kefir products may be allowed if taken merely as food rather than nutritional supplements.

[0041] Exclusion criteria regarding factors that can, or do affect, the composition of the intestinal microbiota include, but are not limited to, use of antibiotics for any indication within the preceding 6 months, and any prescribed medications, including but not limited to immunosuppressive or anti-neoplastic medications.

[0042] Exclusion criteria regarding systemic medical conditions include, but are not limited to, established or emerging metabolic syndrome, where criteria used for definition here are stricter than established criteria, including history of increased blood pressure, history of diabetes or glucose intolerance; known systemic autoimmunity, e.g., connective tissue disease, multiple sclerosis; known atopic diseases including asthma or eczema, chronic pain syndromes including fibromyalgia, chronic fatigue syndrome; ongoing (even if intermittent) use of any prescribed medications, including inhalers or topical creams and ointments; neurologic, neurodevel opmental, and neurodegenerative disorders including autism, Parkinson's disease.

[0043] Exclusion criteria on physical examination may include, but are not limited to, general, such as body mass index < 30 kg / m2, central obesity defined by waist : hip ratio > 0. 90 (male) and > 0. 85 (female); blood pressure > 135 mmHg systolic and > 85 mmHg diastolic; skin — presence of a rash, tattoos or body piercing placed within a year, jaundice; enlarged lymph nodes; wheezing on auscultation; hepatomegaly or stigmata of liver disease; swollen or tender joints; muscle weakness; abnormal neurologic examination.

[0044] Exclusion criteria on laboratory testing may include, but is not limited to, detection of Clostridium difficile toxin B, Escherichia coli O157:H7, Shiga toxins, Enteropathogenic E. coli (EPEC), Salmonella, Shigella, Yersinia, Campylobacter, Pleisomonas, and Vibrio, Giardia, Cryptosporidium, Cyclospora, and Cystoisospora (previously Isospora)', Rotavirus,Norovirus I and II, and adenovirus by PCR; detection of multi-drug resistant organisms, including, but not limited to, MRS A, ESBL, CRE, VRE using PCR or culture in selective media; positive screening for any circulating viral pathogens, including HIV 1 and 2, Hepatitis A, Hepatitis B, Hepatitis C, Cytomegalovirus, SARS-CoV-2, Epstein-Barr Virus; any abnormal liver function tests including alkaline phosphatase, aspartate aminotransaminase, alanine aminotransferase; raised serum triglycerides > 150 mg / dL; HDL cholesterol < 40 mg / dL (males) and < 50 mg / dL (females); high sensitivity CRP > 2. 4 mg / L; raised fasting plasma glucose (> 100 mg / dL).

[0045] Methods disclosed herein for making a fecal extract include providing a fecal sample in a container subjecting a fecal sample to homogenization. As used herein, a “fecal extract” is a composition derived from a fecal sample, but includes predominantly biological material, including bacteria, and little non-living material, e.g., undigested fiber and other foods. As used herein, “homogenization” refers to conditions that promote the break-up or dispersion of non-living and living material present in a fecal sample and increase the release of living biological material from the non-living material. A fecal sample can be present in a container in which homogenization can occur. An example of such a container is a flexible sterile plastic bag.

[0046] To increase the ability to prepare a fecal extract that includes a population of microbes, eukaryotic cells, and viruses that is essentially identical to a population of microbes, eukaryotic cells, and viruses present in the colon or feces of a normal healthy human, e.g., the donor from whom a fecal sample was obtained, in one embodiment conditions for preparing a composition of the present disclosure include the use of temperatures that decrease the replication of the microbes and eukaryotic cells. In one embodiment, the conditions used for preparation are maintained below 37 degrees Celsius. For instance, the conditions used for preparation are maintained at a temperature of no greater than 30 degrees Celsius, no greater than 20 degrees Celsius, no greater than 10 degrees Celsius, or no greater than 5 degrees Celsius. In one embodiment, conditions are used such that replication of the microbes and eukaryotic cells is undetectable and preferably does not occur. In one embodiment, the conditions used for preparing a composition of the present disclosure include maintaining anaerobiosis during practice of the methods describedherein to reduce exposure to oxygen and can include using nitrogen gas (or other inert gases or gas mixtures) to replace any air that may be present in the flexible sterile plastic bag containing stool and removing oxygen from aqueous solutions.

[0047] Diluents useful in the methods of the present disclosure include aqueous solutions that are routinely used for manipulating microbes, eukaryotic cells, and / or viruses. Diluents can be freed of contaminating oxygen prior to use by autoclaving at 15 PSI for 15 min and purging with an inert gas or gas mixture. Useful diluents may include constituents to maintain physiological buffer, osmolarity, and the like. The diluent is preferably sterile and / or non- allergenic. Examples of diluents include, but are not limited to, phosphate buffered saline, at pH 7, and physiological (normal) saline (0.9% w / v NaCl). In one embodiment, the oxygen in a container may be decreased or removed by purging with an inert gas such as nitrogen or argon prior to homogenization. Such anaerobic conditions may be useful to maintain viability of strict anaerobic bacteria present in a colon. The sample may be homogenized multiple times and / or additional diluent may be added until a consistency is achieved that will permit the following steps to occur. After homogenization, the solutions used for washing and resuspension can be treated to remove oxygen. Manipulation of the microbiota may occur in an oxygen free cabinet or anaerobic glove box chamber.

[0048] Two methods of processing a fecal sample described herein include the use of filters. Typical filtration methods use filters having a pore size of about 0.25 millimeters (mm). Larger sized pores are not used because of the expectation that the resulting filtrate containing the desired biological material will include non-biological material, such as undigested fiber and food, at too high a level. Increased non-biological material can lead to difficulties in resuspending a freeze-dried composition. In addition, it is difficult to count cells in the presence of non-biological material, such as undigested fiber and food, at too high a level, and such a composition can clog a syringe used during colonoscopy. The inventors have surprisingly found that filters with a pore size of 0.5 mm to 0.7 mm, such as 0.5 mm to 0.65 mm, and in some embodiments 0.6 mm, yielded compositions with sufficiently low amounts of non-biological material. The use of filters with higher pore sizes results in the advantage of easier maintenance of anaerobic conditions; the filters do not clog easily, and the filtration steps are faster, thereby leaving less time for oxygen tonegatively impact viability of some microbes and eukaryotic cells during stool processing to produce a fecal extract. Filters useful herein can include any suitable material, including but not limited to stainless steel, polymer, nylon, rayon, plastic, and the like.

[0049] Pressurized fdter press method

[0050] In one embodiment, a method disclosed herein can use a container such as a flexible sterile plastic bag that is impervious to liquid. Examples of such containers are described in, but not limited to, US 5,180,229, US 2024 / 0009908, and US 2024 / 0017888, and those available from WHIRL-PAK® Filtration Group (Pleasant Prairie, WI). Care should be taken during the methods described herein to reduce exposure to oxygen, and the transfer can include using nitrogen gas to replace any air that may be present.

[0051] A cell extraction buffer is added to the fecal sample. The inventors observed that the use of gelatin in combination with ammonium phosphate aided in the release of biological material from the non-living material present in a fecal sample, accordingly, in some embodiments, a cell extraction buffer includes gelatin and ammonium phosphate.

[0052] In one embodiment a cell extraction buffer includes, but is not limited to a buffer, or both a buffer and a gelatin. In one embodiment, 1 part donor feces may be combined with 5 parts cell extraction buffer (e.g., 50 grams of donor feces may be combined with 250 ml cell extraction buffer) and homogenized. An example of a buffer includes, but is not limited to, (NH4)2HPO4. The buffer, for instance (NH^HPCU, can be present at a concentration of at least 0.01 M, at least 0.05 M, or at least 0.1 M, and at a concentration of no greater than 1 M, no greater than 0.5 M, or no greater than 0.1 M. Examples of ranges of a buffer include, but are not limited to, 0.01 M to 1 M, and 0.05 M to 0.5 M. Gelatin is typically a mixture of peptides and proteins commonly derived from collagen. In one embodiment, the gelatin can be partially hydrolyzed. In one embodiment, the gelatin can be USP grade. Gelatin can be present at a concentration of at least 0.01% volume / volume (v / v), at least 0.05% v / v, or at least 0.1% v / v, and at a concentration of no greater than 1% v / v, no greater than 0.5% v / v, or no greater than 0.1% v / v. In one embodiment, a 10% v / v gelatin solution in water is adjusted to pH 10.0 and is partially hydrolyzed by autoclaving for 10 min at 15 PSI (Kingsley and Bohlool , Appl Environ Microbiol. 1981 Aug;42(2):241-8. doi:10.1128 / aem.42.2.241-248.1981). Examples of ranges of gelatin in a cell extraction buffer include, but are not limited to, 0.01% (v / v) to 1% (v / v), and 0.05% to 0.5% (v / v).

[0053] A cell extraction buffer can also include other components, including but not limited to a nonionic surfactant and emulsifier. Examples of a nonionic surfactant and emulsifier include, but are not limited to, polysorbate 80 (available under the trade name TWEEN- 80®). A polysorbate 80 can be present at a concentration of at least 0.001%, at least 0.005%, or at least 0.01%, and at a concentration of no greater than 0.1%, no greater than 0.05%, or no greater than 0.01%.

[0054] The fecal sample in the container is subjected to conditions that homogenize the sample. In one embodiment, such conditions can be obtained through the use of a paddle homogenizer, also known in the art as a stomacher. Typically, the sample is homogenized until it is a slurry, e.g., a mixture of solids suspended in a liquid. The slurry is then filtered with a pressurized filter press. An example of a pressurized filter press useful in the method includes a cylinder, a lid, and a plunger. The side of the plunger in contact with the surface of the cylinder fits tightly to prevent seepage of the slurry, and the surface of the plunger in contact with the slurry includes at least one filter medium. An example of a pressurized filter press is a coffee press, modified to include a filter and plunging rod as described herein.

[0055] The filter medium present at the surface of the plunger in contact with the slurry includes at least one, at least two, at least three, or at least four filters. In one embodiment, the surface of the plunger in contact with the slurry includes no greater than six filters. The one or more filters can be described by sieve size, also referred to as pore size, in millimeters (mm) or sieve size based on the US Standard sieve sizes. The sieve size (e.g. porosity or pore size) of one or more of the filters can be no greater than 2 mm, no greater than 1 mm, no greater than 0.7 mm, no greater than 0.65 mm, no greater than 0.6 mm, no greater than 0.55 mm, no greater than 0.5 mm, no greater than 0.25 mm, no greater than 0.212 mm, no greater than 0. 18 mm, no greater than 0. 15 mm, no greater than 0. 125 mm, no greater than 0. 106 mm, no greater than 0. 09 mm, no greater than 0. 075 mm, no greater than 0. 063 mm, no greater than 0. 053 mm, no greater than 0. 045 mm, no greater than 0. 038 mm,no greater than 0. 033 mm, no greater than 0. 025 mm, no greater than 0. 02 mm, or no greater than 0. 01 mm. The sieve size of one or more filters can be no less than 0.212 mm, no less than 0.25 mm, no less than 0.3 mm, no less than 0.400 mm, no less than 0.5 mm, no less than 0.55 mm, no less than 0.6 mm, no less than 0.65 mm, no less than 0.7 mm, or no less than 0. mm. Sieve size of one or more of the filters can be, when described as US Standard sieve sizes, no greater than 400, no greater than 325, no greater than 270, no greater than 230, no greater than 200, no greater than 170, no greater than 140, no greater than 120, no greater than 100, no greater than 80, no greater than 70, no greater than 60, no greater than 35, no greater than 18, or no greater than 10. In one embodiment, the filter medium present at the surface of the plunger in contact with the slurry includes at least one, at least two, at least three, or at least four filters, each of which has a sieve size of no greater than 0.15 mm or no greater than US Standard sieve size 100. In one embodiment, the filter medium present at surface of the plunger in contact with the slurry includes at least four filters, each of which has a sieve size of 0.15 mm or US Standard sieve size 100.

[0056] Pressure can be applied manually to push the liquid component and filterable biological material through the filter while preventing passage of the non-living material and biological material that exceeds the pore size of the smallest filter. The liquid component containing biological material is removed (the filtrate) and the solids (unfiltered material) resuspended in a diluent, such as a saline solution. Saline solutions useful in maintaining the viability of biological material present in a fecal sample are known, and in one embodiment includes phosphate buffered saline at pH 7.0. This suspension is then filtered with the pressurized filter press to yield a second liquid component (second filtrate) containing biological material. The first and second liquid components are combined in a container suitable for centrifugation. The container can be purged with an inert gas or gas mixture, such as nitrogen gas, to maintain conditions for anaerobes. The force of a centrifugation step can be at any speed that causes the separation of biological material from liquid but does not reduce ease of resuspension in a suitable buffer, and does not cause loss of therapeutic efficacy of the composition in MTT. Care should be taken as damage of the fecal microbes can be increased at greater speeds, and the fecal microbes can be more difficult to resuspend. Examples of suitable conditions include speeds resulting in at least 3,000 xg, at least 4,000 xg, or at least 5,000 xg. Other examples of suitableconditions include speeds resulting in no greater than 9,000 xg, no greater than 8,000 xg, or no greater than 7,000 xg. Suitable ranges include at least 3,000 xg to no greater than 9,000 xg, at least 4,000 xg to no greater than 8,000 xg, and at least 5,000 xg to no greater than 7,000 xg. Typically, the volume of diluent added back to the biological material after removal of the supernatant is reduced to cause concentration of the material. For instance, the diluent added back to the material can be one half the volume present before the separation step. The washing procedure of centrifugation followed by removal of the supernatant and resuspending the pellet in a reduced volume of diluent is repeated at least once for a total of four washes. The final resuspended pellet can be referred to herein as fecal microbiota or fecal extract.

[0057] Linear filtration method

[0058] In another embodiment, a method disclosed herein can use a container such as a flexible sterile plastic bag that is impervious to aqueous liquids, and has been further modified to include one or more linear filters. In one embodiment, a linear filter in a container is sealed to both sides and bottom of the container, resulting in two compartments that share one wall that is the filter. The container can include a mechanism for closing. For instance, a container such as those described in, but not limited to, US 5,180,229, US 2024 / 0009908, and US 2024 / 0017888, can be modified to include one or more filters. Useful containers containing a filter are available from WHIRL-PAK® Filtration Group (e g., WHIRL- PAK® Filter Sterilized Bags). A fecal sample can be placed on either side, and the liquid component with biological material will transfer through the filter, while the non-living materials remain separate and contained on the filter surface.

[0059] FIG. 1A, FIG. IB, and FIG. 1C illustrate a container 10 in elevation view, top plan view, and enlarged view, respectively, formed by a front wall 15 and a rear wall 16. Walls 15 and 16 are a flexible material, such as a plastic, that is impervious to aqueous liquids. Two linear filters 17 are present between front wall 15 and rear wall 16. Walls 15 and 16 and linear filters 17 are bonded together along a pair of side seams 12 and 13, with a bottom seam 14 extending between the side seams 12 and 13 at their lower ends. Referring to FIG. IB and 1C, front wall 15 and linear filters 17 form a first compartment 18 (e.g., a solidmaterials region), and rear wall 16 and linear filters 17 form a second compartment 19 (e g., a filtrate region). The top 11 of the container is open to allow for placement of fecal material into one of the compartments 18 or 19.

[0060] In another embodiment, a fecal sample can be placed in a bag that includes one or more walls that are made of a linear filter. The bag can be sealed and then placed in a container such as a flexible sterile plastic bag that is impervious to liquid. The container can include a mechanism for closing. For instance, a container such as those described in, but not limited to, US 5,180,229, US 2024 / 0009908, and US 2024 / 0017888. Useful containers containing a filter are available from WHIRL-PAK® Filtration Group (e.g., WHIRL -PAK® Filter Sterilized Bags). The liquid component of the fecal sample with biological material will transfer through the filter, while the non-living materials remain separate and contained. The inventors have found that the method of using this double filtration system resulted in surprisingly efficient production of a homogenized sample that can be further processed to result in a fecal extract. Without intending to be limited by theory, it is possible the homogenization step causes the fecal sample to pass through the filter multiple times.

[0061] The linear filter can be described by sieve size, also referred to as pore size, in millimeters (mm) or sieve size based on the US Standard sieve sizes. The sieve size of the linear filter can be no greater than 2 mm, no greater than 1 mm, no greater than 0.7 mm, no greater than 0.65 mm, no greater than 0.6 mm, no greater than 0.55 mm, no greater than 0.5 mm, no greater than 0.33 mm, no greater than 0.25 mm, no greater than 0.212 mm, no greater than 0. 18 mm, no greater than 0. 15 mm, no greater than 0. 125 mm, no greater than 0. 106 mm, no greater than 0. 09 mm, no greater than 0. 075 mm, no greater than 0. 063 mm, no greater than 0. 053 mm, no greater than 0. 045 mm, no greater than 0. 038 mm, no greater than 0. 032 mm, no greater than 0. 025 mm, no greater than 0. 02 mm, or no greater than 0. 01 mm. The sieve size of one or more filters can be no less than 0.212 mm, no less than 0.25 mm, no less than 0.3 mm, no less than 0.400 mm, no less than 0.5 mm, no less than 0.55 mm, no less than 0.6 mm, no less than 0.65 mm, no less than 0.7 mm, or no less than 0. mm. Sieve size of one or more of the filters can be, when described as US Standard sieve sizes, no greater than 400, no greater than 325, no greater than 270, no greater than 230, no greater than 200, no greater than 170, no greater than 140, no greater than 120, no greaterthan 100, no greater than 80, no greater than 70, no greater than 60, no greater than 35, no greater than 18, or no greater than 10. In one embodiment, the pores can be distributed at 200 to 300 pores per cm2.

[0062] A cell extraction buffer is added to the fecal sample. The inventors observed that the use of gelatin in combination with ammonium phosphate aided in the release of biological material from the non-living material present in a fecal sample, accordingly, in some embodiments, a cell extraction buffer includes gelatin, ammonium phosphate, or both gelatin and ammonium phosphate.

[0063] In one embodiment a cell extraction buffer includes, but is not limited to a buffer, or both a buffer and a gelatin. In one embodiment, 1 part donor feces may be combined with 5 parts cell extraction buffer (e.g., 50 grams of donor feces may be combined with 250 ml cell extraction buffer) and homogenized. An example of a buffer includes, but is not limited to, (NH4)2HPO4. The buffer, for instance, phosphate buffered saline (monosodium phosphate (NaffcPC ) and disodium phosphate (TfeHPC ), pH 7.0 or (NH^HPC , can be present at a concentration of at least 0.01 M, at least 0.05 M, or at least 0.1 M, and at a concentration of no greater than 1 M, no greater than 0.5 M, or no greater than 0.1 M. Examples of ranges of a buffer include, but are not limited to, 0.01 M to 1 M, and 0.05 M to 0.5 M. The gelatin is typically a mixture of peptides and proteins commonly derived from collagen. In one embodiment, the gelatin can be partially hydrolyzed. In one embodiment, the gelatin can be USP grade. The gelatin can be present at a concentration of at least 0.01% volume / volume (v / v), at least 0.05% v / v, or at least 0.1% v / v, and at a concentration of no greater than 1% v / v, no greater than 0.5% v / v, or no greater than 0.1% v / v. In one embodiment, a 10% v / v gelatin solution in water is adjusted to pH 10.0 and is partially hydrolyzed by autoclaving for 10 min at 15 PSI (Kingsley and Bohlool , Appl Environ Microbiol. 1981 Aug;42(2):241-8. doi: 10.1128 / aem.42.2.241-248.1981). Examples of ranges of gelatin in a cell extraction buffer include, but are not limited to, 0.01% v / v to 1% v / v, and 0.05% v / v to 0.5% v / v.

[0064] A cell extraction buffer can also include other components, including but not limited to a nonionic surfactant and emulsifier. Examples of a nonionic surfactant and emulsifierinclude, but are not limited to, polysorbate 80 (available under the trade name TWEEN- 80®), Tween 60, and Nonident P40 . A polysorbate 80 can be present at a concentration of at least 0.001%, at least 0.005%, or at least 0.01%, and at a concentration of no greater than 0.1%, no greater than 0.05%, or no greater than 0.01%.

[0065] The fecal sample in the container is subjected to conditions that homogenize the sample. In one embodiment, such conditions can be obtained through the use of a paddle blender, also known in the art as a stomacher. Typically, the sample is homogenized until it is a slurry of dispersed non-living and living material. Force can then be applied to the slurry to push the liquid component and biological material through the linear filter while preventing passage of the non-living material. Movement of the liquid component and biological material through the linear filter typically occurs during the homogenization. The liquid component (the filtrate) containing biological material is removed and the non-living material (the unfiltered material) resuspended in a diluent, such as a saline solution. Saline containing solutions useful in maintaining the viability of biological material present in a fecal sample are known, and in one embodiment includes phosphate buffered saline at pH 7.0. This can be subjected to conditions that homogenize the sample, and force applied to the resulting slurry to push the liquid component and biological material through the linear filter while preventing passage of the non-living material. The second liquid component containing biological material is removed and combined with the first filtrate in a container suitable for centrifugation. The container can be purged with nitrogen gas to maintain conditions for anaerobes. The force of a centrifugation step can be at any speed that causes the separation of biological material from liquid but does not cause loss of therapeutic efficacy of the composition in MTT. Care should be taken as damage of the fecal microbes can be increased at greater speeds, and the fecal microbes can be more difficult to resuspend. Examples of suitable conditions include speeds resulting in at least 3,000 xg, at least 4,000 xg, or at least 5,000 xg. Other examples of suitable conditions include speeds resulting in no greater than 9,000 xg, no greater than 8,000 xg, or no greater than 7,000 xg. Suitable ranges include at least 3,000 xg to no greater than 9,000 xg, at least 4,000 xg to no greater than 8,000 xg, and at least 5,000 xg to no greater than 7,000 xg. Typically, the volume of diluent added back to the biological material after removal of the supernatant is reduced to cause concentration of the material. For instance, the diluent added back to thematerial can be one half the volume present before the separation step. The washing procedure of centrifugation followed by removal of the supernatant and resuspending the pellet in a reduced volume of diluent is repeated at least twice for a total of four washes.

[0066] An example of a linear filtration method is illustrated in FIG. 2. In this embodiment, the method includes providing a container that includes a fecal sample (FIG. 2, block 20). As described herein, the container can include one or more compartments separated by one or more filters, and the fecal sample can be present in one of the compartments. The fecal sample is subjected to a homogenization (FIG. 2, block 21) that disperses or breaks-up the fecal sample. Liquid present in the fecal sample is transferred across the one or more filters, and in some embodiments the transfer across the one or more filters can occur during the homogenizing. The result is two compartments. One compartment includes a liquid component (a filtrate, FIG. 2, block 22) containing biological material from the fecal sample, and the liquid component can be removed from the container. The second compartment is one that includes a slurry of dispersed non-living material (filtered material, FIG. 2, block 23). A suitable diluent can be added to the second compartment and subjected to a second homogenization (FIG. 2, block 24) that further disperses or breaks-up the slurry of dispersed non-living material to obtain more living material, and results in another liquid component (a filtrate, FIG. 2, block 25) containing biological material from the dispersed non-living material (FIG. 2, block 24). The two liquid components can be combined (FIG. 2, block 26) and then used as described herein.

[0067] The final pellet resulting from either the pressurized filter press method or the linear filtration method is resuspended in a diluent. The diluent used can be a volume that is no greater than 50% of the starting volume (e.g., the volume present when the fecal sample and cell extraction buffer are combined), no greater than 40% of the starting volume, no greater than 30% of the starting volume, no greater than 20% of the starting volume, or no greater than 10% of the starting volume. The final volume is typically dictated by the final numbers of bacteria desired after processing as described herein (e.g., adding a cryoprotectant or freeze drying). Typically, resuspending the final pellet in an aqueous composition results in a final slurry. The final slurry can be referred to herein as a fecal extract.

[0068] In one embodiment, the fecal material present in a fecal extract described herein does not include particles (e.g., particles of non-living material and / or particles of biological material) having a size of greater than 2.0 millimeters (mm), greater than 1.0 mm, greater than 0.7 mm, greater than 0.65 mm, greater than 0.6 mm, greater than 0.55 mm, greater than 0.5 mm, greater than 0.4 mm, greater than 0.3 mm, greater than 0.25 mm, greater than 0.212 mm, greater than 0.180 mm, greater than 0.150 mm, greater than 0.125 mm, greater than 0.106 mm, greater than 0.090 mm, greater than 0.075 mm, greater than 0.063 mm, greater than 0.053 mm, greater than 0.045 mm, greater than 0.038 mm, greater than 0.032 mm, greater than 0.025 mm, greater than 0.020 mm, or greater than 0.01 mm. In one embodiment, the fecal material present in a fecal extract of the present disclosure includes, consists essentially of, or consists of, particles of non-living material and / or biological material having a size that will pass through a sieve having a sieve size of 2.0 mm, 1.0 mm, 0. 7 mm, 0.65 mm, 0.6 mm, 0.55 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.25 mm, 0.212 mm, 0.180 mm, 0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053 mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, or 0.01 mm. Thus, in such an embodiment, the fecal material present in a fecal extract has a size that is less than or equal to 2.0 mm, less than or equal to 1.0 mm, less than or equal to 0. 7 mm, less than or equal to 0.65 mm, less than or equal to 0.6 mm, less than or equal to 0.55 mm, less than or equal to 0.5 mm, less than or equal to 0.4 mm, less than or equal to 0.3 mm, less than or equal to 0.25 mm, less than or equal to 0.212 mm, less than or equal to 0.180 mm, less than or equal to 0.150 mm, less than or equal to 0.125 mm, less than or equal to 0.106 mm, less than or equal to 0.090 mm, less than or equal to 0.075 mm, less than or equal to 0.063 mm, less than or equal to 0.053 mm, less than or equal to 0.045 mm, less than or equal to 0.038 mm, less than or equal to 0.032 mm, less than or equal to 0.025 mm, less than or equal to 0.020 mm, or less than or equal to 0.01 mm.

[0069] Cell numbers in a fecal extract can be determined. Methods for determining cell numbers include any combination of the Bacteria Counting Kit (ThermoFisher, Waltham, MA), an epifluorescence microscope, equipped with a 470-490 nm excitation filter and 500 nm cut- on long pass emission filter, and a Petroff Hauser counting chamber. A membrane integrity assay can be used to directly enumerate microbes present in a diluted sample and to calculate the percentage of microbes with intact membranes, thereby indicating potentialviability. An example of a membrane assay is the BacLight live / dead membrane integrity assay (ThermoFisher, Waltham, MA) and a microscope and counting chamber.

[0070] The number of cells in a fecal extract can be in at least 5xlO10cells, at least 5.5xlO10cells, at least 6xlO10cells, at least 6.5xlO10cells, at least 7xlO10cells, at least 7.5xlO10cells, at least 8xlO10cells, at least 8.5xlO10cells, at least 9xlO10cells, at least 9.5xlO10cells, at least IxlO11cells, at least 2.5xlOncells, at least 3xlOncells, at least 3.5xlOncells, at least4xlOncells, at least 4.5xlOucells, at least 5xlOncells, at least 5.5xlOncells, at least6xlOncells, at least 6.5xlOncells, at least 7xlOncells, at least 7.5xlOncells, at least8xlOncells, at least 8.5xlOncells, at least 9xlOncells, at least 9.5xlOncells, at leastIxlO12cells, at least 1.5xl012cells, at least 2xl012cells, at least 2.5xl012cells, at least3xl012cells, at least 3.5xl012cells, at least 4xl012cells, at least 4.5xl012cells, at least5xl012cells, at least 5.5xl012cells, at least 6xl012cells, at least 6.5xl012cells, at least7xl012cells, at least 7.5xl012cells, or at least 8xl012cells. In one embodiment, the material is concentrated to result in no greater than 9xl012cells in a volume that is subsequently freeze-dried. The number of cells in a fecal extract can be adjusted, if necessary, by addition of a diluent.

[0071] In some embodiments, where the fecal extract is to be freeze-dried, a cryoprotectant, a lyoprotectant, or both a cryoprotectant and a lyoprotectant can be added. A cryoprotectant useful herein maintains the viability of fecal microbes when subjected to freeze-drying conditions, and / or when stored as a freeze-dried composition. A cryoprotectant and / or lyoprotectant useful herein results in a freeze-dried composition that is friable. As used herein, a "friable” composition refers to a composition that can be easily milled to result in a fine powder. Milling, also referred to as grinding, is a process that physically changes a material into smaller particles. Methods for milling freeze-dried compositions are known to the skilled person, and can occur at various temperatures, e.g., at or below 0 degrees Celsius, or above 0 degrees Celsius. In one embodiment, a freeze-dried composition described herein that is friable is one that results in a powder that can be subsequently used to produce an oral liquid composition. In one embodiment, a freeze-dried composition described herein that is friable is one that results in a powder that can be subsequently usedto produce a capsule. In one embodiment, a useful powder may have size, density, flow, and compression characteristics suitable for, but not limited to, encapsulation.

[0072] Examples of cryoprotectants and lyoprotectants include, but are not limited to, amino acids such as alanine, glycine, proline; simple sugars such as sucrose, glucose, lactose, ribose, mannitol, and trehalose; and other compounds such as dimethyl sulfoxide (DMSO), and glycerol. The total cryoprotectant and / or lyoprotectant used to produce a freeze-dried composition may be 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 21 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, or 30 % (vol / vol) of the final concentration of a fecal extract and the cryoprotectant and / or lyoprotectant before freeze drying the composition. For instance, to produce a composition having a cryoprotectant and / or lyoprotectant at a final concentration of 10 %, equal volumes of a 20 % solution of the cryoprotectant and / or lyoprotectant and the fecal extract can be combined and mixed, and then freeze-dried. A composition that includes a fecal extract, e.g., a fecal extract supplemented with a cryoprotectant and / or lyoprotectant, can be referred to herein as a fecal extract composition.

[0073] In one embodiment, trehalose is added to a final concentration (w / v) of 8%. The combined slurry and cryoprotectant and / or lyoprotectant can be freeze-dried immediately or stored at -80 degrees Celsius until ready for freeze-drying.

[0074] Freeze-drying

[0075] In one embodiment, a fecal extract composition is freeze-dried to form solid dried powder. A fecal extract composition is subjected to conditions that result in freeze-drying. In those embodiments where the freeze-dried composition will be used to make a liquid solution for oral consumption, a composition of fecal microbes can be mixed with a flavor agent, and in other embodiments a flavor agent is added when the freeze-dried composition is reconstituted. Conditions that result in freeze-drying typically include freezing the sample, and reducing the pressure surrounding the frozen sample, for instance to under 100 mT vacuum or less, to remove water from the sample. Once freeze-dried, the composition may be further processed by subjecting the dried material to force sufficient to break up the material into a powder that can be easily stored until used. In one embodiment, the powdermay be used to form granules, pills, capsules, wafers, and the like. In one embodiment, the freeze-dried material can be formulated such that it is released from a capsule into the small or large intestine or the colon, and not the stomach.

[0076] In one embodiment, a freeze-dried composition described herein is reconstituted to result in a liquid suspension. A freeze-dried composition is mixed with an aqueous diluent. The freeze-dried composition can include a flavor agent and / or a gut-transit protectant, or a flavor agent and / or a gut-transit protectant can be added at the time of reconstitution. In one embodiment, the aqueous diluent includes a dairy product such yogurt or a milk product. Examples of milk include whole milk or milk having reduced fat, such as 1% or 2% milk fat, and the milk can be regular or chocolate milk, or vegetable-based milk products. Typically, the reconstitution occurs using conditions that maintain the efficacy of the composition, e.g., conditions that reduce inactivating the microbes present. Appropriate conditions can include gentle mixing to dissolve or suspend the freeze-dried composition in the aqueous diluent.

[0077] Pharmaceutically acceptable formulations

[0078] The compositions of the present disclosure may be included in a diversity of pharmaceutically acceptable formulations. In one embodiment, a formulation may be a liquid composition. Liquid compositions include, but are not limited to, solutions, suspensions, dispersions, and the like. In one embodiment, a formulation may be a solid composition. Solid compositions include, but are not limited to, powder (e.g., a friable freeze-dried powder), granule, pill, capsule, chewing gum, microsphere, wafer, and the like. Those formulations may include a pharmaceutically acceptable carrier to render the composition appropriate for administration to a subject. As used herein “pharmaceutically acceptable carrier” includes pharmacologically inactive compounds compatible with pharmaceutical administration. A pharmaceutically acceptable carrier can be, and typically is, United States Pharmacopeia (USP) grade. Examples of pharmaceutically acceptable carriers include flavor agents and gut-transit protectants. The compositions of the present disclosure may be formulated to be compatible with its intended route of administration.

[0079] A composition of the present disclosure may be administered by any method suitable for depositing in the gastrointestinal tract, such as the mouth, stomach, colon, or rectum, of a subject. Examples of routes of administration include rectal administration (e.g., by suppository, enema, upper endoscopy, upper push enteroscopy, flexible sigmoidoscopy, or colonoscopy), intubation through the nose or the mouth (e.g., by nasogastric tube, nasoenteric tube, or nasal jejunal tube), or oral administration (e.g., by a solid such as a pill or capsule, or by liquid). In embodiments where a liquid form of the composition is delivered to a subject, the freeze-dried composition can be reconstituted with an aqueous solution, such as by adding water, saline, milk, or plant-based milk, a flavor agent, and / or a gut-transit protectant, or by exposing the freeze-dried composition to a body fluid. In one embodiment, a composition is formulated for oral administration as a liquid that is swallowed by the subject. Such a composition is also referred to herein as an oral liquid preparation.

[0080] For therapeutic use in the method of the present disclosure, a composition described herein may be conveniently administered in a form containing one or more pharmaceutically acceptable carriers. Suitable carriers are well known in the art and vary with the desired form and mode of administration of the composition. For example, they may include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, glidants, lubricants, and the like. Typically, the carrier may be a solid (including powder), liquid, or combinations thereof. Each carrier is preferably "acceptable” in the sense of being compatible with the other ingredients in the composition and not injurious to the subject. The carrier is preferably biologically acceptable and inert, i.e., it permits the composition to maintain viability of the biological material until delivered to the appropriate site.

[0081] Compositions formulated for oral administration may include an inert diluent or an edible carrier. For purposes of oral therapeutic administration, in one embodiment a liquid composition, e.g., an oral liquid preparation, can include a flavor agent, a gut-transit protectant, or both a flavor agent and a gut-transit protectant. A flavor agent and / or a guttransit protectant can be present in a freeze-dried composition that is reconstituted to result in a liquid composition, or a flavor agent and / or a gut-transit protectant can be added to thecomposition at the time of reconstitution. A gut-transit protectant is a compound or composition that protects fecal microbes during gut transit, e.g., transit through the high acidity and digestive enzymes present in the stomach and / or intestine. Examples of guttransit protectants include, but are not limited to, a dairy product such yogurt, a milk, or a reconstituted powdered milk. Examples of milk include regular or chocolate milk, and whole milk or milk having reduced fat, such as 1% or 2% milk fat. Other examples of milk include a plant-based milk such as, but not limited to. almond, oat, and soy. Examples of powdered milk, also referred to as milk powder, dried milk, and dry milk, include but are not limited to nonfat skimmed milk, whole milk, buttermilk, and whey. Gut-transit protectants can be added to a liquid composition to a final concentration of at least 1 % w / w, at least 5 % w / w, at least 10 % w / w, or at least 15 % w / w, and no greater than 20 % w / w, no greater than 15 % w / w, no greater than 10 % w / w, or no greater than 5 % w / w. Exemplary ranges include but are not limited to at least 1 % w / w to no greater than 20 % w / w, or at least 5 % w / w to no greater than 15 % w / w. In one embodiment, for the purpose of oral therapeutic administration a freeze-dried composition can be incorporated with excipients and used in the form of, but not limited to, capsules, e.g., gelatin or hypromellose capsules. Oral compositions can also be prepared by combining a composition of the present disclosure with a food. In one embodiment a food used for administration is chilled, for instance, ice cream or milk. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The pills, capsules, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; or a sweetening agent such as sucrose or saccharin. Other ingredients may be added to a formulation to provide desired characteristics such as flow, compression, hardness, and taste.

[0082] In one embodiment, the freeze-dried composition may be present in a formulation that permits passage to the small intestine or colon. For instance, when the composition is to be administered orally using capsules, the dosage form may be formulated so the composition is not exposed to conditions prevalent in the gastrointestinal tract before the small intestineor colon, e.g., high acidity and digestive enzymes present in the stomach and / or intestine.In one embodiment, the dosage form may be formulated so the composition passes through the stomach and into the intestines and is released in conditions that include a pH of greater than 5. 5, greater than 6, greater than 6. 5, or greater than 7. In one embodiment an enteric coating is acid-resistant to protect the composition from the low pH of the stomach and break down when exposed to a pH greater than present in the stomach. The encapsulation, such as double encapsulation, of compositions in an enteric coating for therapeutic use is routine in the art. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers. Examples include, but are not limited to, methyl acrylatemethacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, enteric coatings (hydroxypropyl methylcellulose (HPMC), hydroxy propyl methyl cellulose acetate succinatelate (PVAP), methyl methacrylate-methacrylic acid copolymers, cellulose acetate trimellitate, and sodium alginate. Encapsulation, such as double encapsulation, may include hard-shelled capsules, which may be used for dry, powdered ingredients, or soft-shelled capsules. Capsules may be made from aqueous solutions of gelling agents such as animal protein (e.g., gelatin), plant polysaccharides or derivatives like carrageenans and modified forms of starch and cellulose. Other ingredients may be added to a gelling agent solution such as plasticizers (e.g., glycerin and or sorbitol), coloring agents, preservatives, disintegrants, lubricants, and surface treatment. Enteric coated capsules can be co-combined to provide for release of the freeze-dried composition within the large bowel or colon.

[0083] Methods of use

[0084] The present disclosure is further directed to methods of using the compositions described herein. One method includes administering to a subject in need thereof an effective amount of a composition described herein. The subject can be anyone in need thereof, including but not limited to a person having a disease or condition, or at risk of developing a disease or condition. A subject can be an infant, child, adolescent, or adult. The administering is done under conditions suitable for deposition of the composition in a region of the large or small intestine such that the biological material in the composition colonizes the small intestine and colon. For instance, administration may be into upper gastrointestinal tract, aswell as lower gastrointestinal tract, e.g., the terminal ileum, cecum, colonic areas containing diverticulosis, and rectum. In one embodiment the administering may be oral, such as by tablet. In one embodiment the administering may be by intubation, such as by nasogastric tube, of a freeze-dried composition that has been reconstituted. In one embodiment the administering may be rectal, for instance, via delivery using a colonoscope, enema, or suppository. Conditions that are "suitable” for an event to occur, or " suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and / or are conducive to the event. As used herein, an “effective amount” relates to a sufficient amount of a composition described herein, to provide the desired effect. For instance, in one embodiment an “effective amount” is an amount effective to alleviate one or more symptoms and / or signs of a disease or a condition as described herein. In some embodiments, an effective amount is an amount that is sufficient to affect a reduction in a symptom and / or sign associated with a disease or condition, such as diarrhea or C. difficile. A reduction in a symptom and / or a sign is, for instance, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, or at least 100 % in a measured sign as compared to a control, a non-treated subject, or the subject prior to administration of the composition. In one embodiment, an effective amount is an amount sufficient to result in at least 5xl010cells, at least 5.5xl010cells, at least 6xlO10cells, at least 6.5xl010cells, at least 7xlO10cells, at least 7.5xlO10cells, at least 8xl010cells, at least 8.5xl010cells, at least9xlO10cells, at least 9.5xlO10cells, at least IxlO11cells, at least 2.5xlOncells, at least3xl0ncells, at least 3.5xl0ncells, at least 4xlOncells, at least 4.5xlOncells, at least5xl0ncells, at least 5.5xl0ncells, at least 6xlOncells, at least 6.5xlOncells, at least7xlOncells, at least 7.5xlOucells, at least 8xl0ncells, at least 8.5xl0ncells, at least9xlOncells, at least 9.5xlOncells, at least IxlO12cells, at least 1.5xl012cells, at least2xl012cells, at least 2.5xl012cells, at least 3xl012cells, at least 3.5xl012cells, at least4xl012cells, at least 4.5xl012cells, at least 5xl012cells, at least 5.5xl012cells, at least6xl012cells, at least 6.5xl012cells, at least 7xl012cells, at least 7.5xl012cells, or at least8xl012cells delivered to the desired location in the gastrointestinal tract. In some embodiments, an initial high dosage is followed by one or more lower maintenance doses. An initial high dosage can include an amount sufficient to result in at least 5xl010cells, atleast 5.5x1010cells, at least 6x1010cells, at least 6.5x1010cells, at least 7x1010cells, at least 7.5xlO10cells, at least 8xlO10cells, at least 8.5xlO10cells, at least 9xlO10cells, at least9.5xlO10cells, at least IxlO11cells, at least 2.5xlOncells, at least 3xlOncells, at least3.5xlOncells, at least 4xlOncells, at least 4.5xlOncells, at least 5xlOncells, at least5.5xlOncells, at least 6xlOucells, at least 6.5xlOncells, at least 7xlOncells, at least7.5xlOncells, at least 8xlOncells, at least 8.5xlOncells, at least 9xlOncells, at least9.5xlOncells, at least IxlO12cells, at least 1.5xlO12cells, at least 2xl012cells, at least2.5xl012cells, at least 3xl012cells, at least 3.5xlO12cells, at least 4xl012cells, at least4.5xl012cells, at least 5xl012cells, at least 5.5xlO12cells, at least 6xl012cells, at least6.5xl012cells, at least 7xl012cells, at least 7.5xl012cells, or at least 8xl012cells delivered to the desired location in the gastrointestinal tract. In some embodiments, a patient can be administered a “maintenance dose.” Examples of a maintenance dose can include 2xl08, 2xl09, 2xlO10, or 2 xlO10cells, for instance from 2xl09to 2xlO10cells. It will be understood, however, that the total dosage of the compositions as disclosed herein will be decided by the attending professional, e.g., physician, within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type and extent of disease or condition being treated.

[0085] In one embodiment, a method of the present disclosure includes treating certain diseases in a subject in need of treatment. The subject may be a mammal, such as a human. In some embodiments animal models may be used, such as a mammal, including a rat, a mouse, a hamster, a gerbil, or a primate. As used herein, the term "disease” refers to any deviation from or interruption of the normal structure or function of a part, organ, or system, or combination thereof, of a subject that is manifested by a characteristic symptom or clinical sign. Diseases include those characterized by dysfunctional composition of intestinal microbiota. Such diseases include, but are not limited to, colitis, including autoimmune colitis (e.g., inflammatory bowel disease, ulcerative colitis, Crohn's disease, microscopic colitis), irritable bowel syndrome, and infectious colitis. Examples of infectious colitis include, but are not limited to, Clostridioides difficile colitis (e.g., acute C. difficde colitis, relapsing C. difficile colitis, or severe / fulminant C. difficile colitis) and enterohemorrhagic colitis (e.g., a colitis caused by Shigella spp. or E. coli). Other examples of diseases include, but are not limited to, chronic diarrhea; chronic constipation; metabolic syndromeand obesity; atopic diseases including asthma, eczema, food allergies, eosinophilic disorders of the GI tract; systemic autoimmunity including rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis, etc.; chronic pain disorders such fibromyalgia, chronic fatigue syndrome, neurodegenerative disorders, eating disorders, and malnutrition. As used herein, the term "condition” refers to an illness or other medical problem. Examples of conditions include but are not limited to Autism Spectrum disorder, Pitt- Hopkins Syndrome, Parkinson's disease, alcohol use disorder, obesity, Type II diabetes, and presence of multi-drug resistant organisms. The term "condition” also refers cancers that can be treated using immune-oncology, such as optimization of anti-cancer checkpoint immunotherapy, treatment of checkpoint inhibitor colitis, and treatment or prevention of complications of hematopoietic stem cell transplantation, such as graft-versus-host disease.

[0086] As used herein, the term “symptom” refers to subjective evidence of disease or condition experienced by the patient and caused by disease. As used herein, the term “clinical sign, " or simply “sign,” refers to objective evidence of a disease present in a subject. Symptoms and / or signs associated with diseases or conditions referred to herein and the evaluation of such signs are routine and known in the art. Typically, whether a subject has a disease or condition, and whether a subject is responding to treatment, may be determined by evaluation of signs associated with the disease or condition.

[0087] Treatment of a disease or condition can be prophylactic or, alternatively, can be initiated after the development of a disease or condition. Treatment that is prophylactic, for instance, initiated before a subject manifests signs of a disease or condition, is referred to herein as treatment of a subject that is at risk” of developing a disease or condition. An example of a subject that is at risk of developing a disease or condition is a person having a risk factor. An example of a risk factor for C. difficile colitis is antibiotic therapy of the gastrointestinal tract. Treatment can be performed before, during, or after the occurrence of the diseases described herein. Treatment initiated after the development of a disease may result in decreasing the severity of the signs of the disease, or completely removing the signs.

[0088] In one embodiment, a method of the present disclosure includes transplanting microbiota from a donor to a recipient.

[0089] In one embodiment, a method of the present disclosure includes increasing the relative abundance of members of the phylum Firmicutes, such as a non-pathogenic member of the class Clostridia, and / or members of the phylum Bacteroidetes, in a recipient's colon. In one embodiment, a method of the present disclosure includes increasing the relative abundance of members of the family Prevotellaceae, such as members of the genus Prevotella. The phrase "relative abundance" refers to the number of members of a phylum or class compared to the number of members of all other taxa in a recipient's colon. Such a comparison can be expressed as a percent. In one embodiment, the relative abundance of non-pathogenic members of the class Clostridia in a recipient's colon after the administration may be increased by at least 5 %, at least 10 %, at least 20 %, or at least 50 %, compared to the recipient's colon before the administration. In one embodiment, the relative abundance of members of the phylum Firmicutes in a recipient's colon after the administration may be increased by at least 5 %, at least 10 %, at least 20 %, or at least 50 % compared to the recipient's colon before the administration. The change in the abundance may be determined at, for instance, 3 days, 10 days, 15 days, or 25 days after the administration of fecal microbiota.

[0090] In one embodiment, a method of the present disclosure includes decreasing the relative abundance of members of the phylum Proteobacteria in a recipient's colon. In one embodiment, the relative abundance of members of the phylum Proteobacteria in a recipient's colon after the administration may be decreased by at least 10 %, at least 20 %, at least 30 %, or at least 40 % compared to the recipient's colon before the administration. The change in the abundance of members of the phylum Proteobacteria may be determined at, for instance, 3 days, 10 days, 15 days, or 25 days after the administration.

[0091] In one embodiment, the existing microbiota does not need to be cleared prior to administration of a composition of the present disclosure. In other embodiments clearance of the microbiota may be necessary. Methods for clearance of existing microbiota are known and routine. In one example, clearance can be accomplished by administering oneor a cocktail of antibiotics for one week until a day prior to transplant. An example of a useful cocktail is Metronidazole (1000 mg twice daily), Rifaximin (550 mg twice daily), Vancomycin (500 mg twice daily), and Neomycin (1000 mg twice daily).

[0092] The invention is defined in the claims. However, below there is provided a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0093] Exemplary Aspects

[0094] Aspect 1. A method for making a composition comprising a fecal extract, the method comprising: (A) homogenizing a fecal sample present in a filtration device, wherein the filtration device comprises a container comprising a solid materials region, a filtrate region, and at least one filter separating the solid materials region and the filtrate region, wherein the fecal sample is present in the solid materials region; (B) filtering the fecal sample, wherein the filtering comprises passage of liquid present in the fecal sample through the filter into the filtrate region and results in a first filtrate comprising biological material present in the filtrate region and a first unfiltered material present in the solid materials region; (C) adding a solution to the first unfiltered material in the solid materials region; (D) homogenizing the first unfiltered material present in the solid materials region; (E) filtering the homogenized first unfiltered material resulting from step (D), wherein the filtering comprises passage of liquid present in the solid materials region through the at least one filter into the filtrate region and results in a second filtrate comprising biological material present in the filtrate region and a second unfiltered material present in the solid materials region; and (F) combining the first and second filtrates to result in a combined filtrate.

[0095] Aspect 2. The method of any of Aspects 1 or 3-15, further comprising applying a centrifugal force to the combined filtrate to form a pellet and resuspending the pellet in a reduced volume compared to the combined filtrate before application of the centrifugal force.

[0096] Aspect 3. The method of any of Aspects 1-2 or 4-15, further comprising repeating the applying and the resuspending at least once to result in a first processed filtrate.

[0097] Aspect 4. The method of any of Aspects 1-3 or -15, wherein (i) the homogenizing of step (A) and the filtering of step (B), (ii) the homogenizing of step (D) and the filtering of step (E), or both (i) and (ii), occur simultaneously.

[0098] Aspect 5. The method of any of Aspects 1-4 or 5-15, wherein the at least one filter comprises a sieve size of no greater than 0.33 millimeters (mm).

[0099] Aspect 6. The method of any of Aspects 1-5 or 6-15, wherein the at least one filter comprises a sieve size of no greater than 0.6 mm.

[0100] Aspect 7. The method of any of Aspects 1-6 or 7-15, wherein the at least one filter comprises pores distributed at 200 to 300 pores per cm2.

[0101] Aspect 8. The method of any of Aspects 1-7 or 8-15, wherein the homogenizing comprises a stomacher.

[0102] Aspect 9. The method of any of Aspects 1-8 or 9-15, wherein the cell extraction buffer comprises a buffer and a gelatin.

[0103] Aspect 10. The method of any of Aspects 1-9 or 10-15, wherein the buffer comprises (NH4)2HPO4.

[0104] Aspect 11. The method of any of Aspects 1-10 or 11-15, wherein the (NH^HPCh is present at a concentration of 0.01 M to 0.1 M.

[0105] Aspect 12. The method of any of Aspects 1-11 or 12-15, wherein the gelatin comprises USP grade gelatin.

[0106] Aspect 13. The method of any of Aspects 1-12 or 13-15, wherein the gelatin is present at a concentration of 0.01% to 1%.

[0107] Aspect 14. The method of any of Aspects 1-13 or 15, further comprising freeze-drying the first processed filtrate.

[0108] Aspect 15. The method of any of Aspects 1-14, wherein trehalose is added to the first processed filtrate before the freeze-drying.

[0109] Aspect 16. A method for making a composition comprising a fecal extract, the method comprising: (A) homogenizing a fecal sample in a cell extraction buffer to result in a slurry; (B) filtering the slurry with a pressurized filter press comprising a filter medium, wherein the filter medium comprises at least one filter, to result in a first filtrate comprising biological material and an unfiltered material; (C) resuspending the unfiltered material in a solution to result in resuspended material; (D) filtering the resuspended material with a filter medium, wherein the filter medium comprises at least one filter, to result in a second filtrate; (E) combining the first and second filtrates to result in a combined filtrate; (F) applying a centrifugal force to the combined filtrate to form a pellet and resuspending the pellet in a reduced volume compared to the combined filtrate before application of the centrifugal force; (G) repeating the applying and the resuspending of step (F) at least once to result in a first processed filtrate.

[0110] Aspect 17. The method of any of Aspects 16 or 18-28, wherein the pressurized filter press is a manually pressurized filter press.

[0111] Aspect 18. The method of any of Aspects 16-17 or 19-28, wherein the homogenizing comprises a stomacher.

[0112] Aspect 19. The method of any of Aspects 16-18 or 20-28, wherein the at least one filter of step (B), step (D), or both steps (B) and (D) comprises a sieve size of no greater than 100 mesh (149 micrometers).

[0113] Aspect 20. The method of any of Aspects 16-19 or 21-28, wherein the filter medium of step (B), step (D), or both steps (B) and (D) comprises at least four filters.

[0114] Aspect 21. The method of any of Aspects 16-20 or 22-28, wherein the filtering of step (B), step (D), or both steps (B) and (D) comprises a pressurized filter press with a plunger.

[0115] Aspect 22. The method of any of Aspects 16- or 23-28, wherein the cell extraction buffer comprises a buffer and a gelatin.

[0116] Aspect 23. The method of any of Aspects 16-22 or 24-28, wherein the buffer comprises (NH4)2HPO4.

[0117] Aspect 24. The method of any of Aspects 16-23 or 25-28, wherein the (NT ^HPC is present at a concentration of 0.01 M to 0.1 M.

[0118] Aspect 25. The method of any of Aspects 16-24 or 26-28, wherein the gelatin comprises USP grade gelatin.

[0119] Aspect 26. The method of any of Aspects 16-25 or 27-28, wherein the gelatin is present at a concentration of 0.01% to 1%.

[0120] Aspect 27. The method of any of Aspects 16-26 or 28, further comprising freeze-drying the first processed filtrate.

[0121] Aspect 28. The method of any of Aspects 16-27, wherein trehalose is added to the first processed filtrate before the freeze-drying.

[0122] Aspect 29. A composition produced by the method of any of Aspects 1 or 16, wherein composition comprises particles of biological material, and wherein the particles of biological material are no greater than 0.6 millimeters (mm).

[0123] Aspect 30. A method of administering a fecal extract to a subject, comprising administering to a subject in need thereof an effective amount of the composition of any of Aspects 29.

[0124] EXAMPLES

[0125] The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.

[0126] Example 1

[0127] Production of microbiota by pressurized filter press method

[0128] Stool (50 grams) from a healthy donor (who is part of the UMN Donor Program (microbiota-therapeutics.umn.edu / donor-overview) was suspended in 250 of cell extraction buffer (0.1 M (NH^HPCh, pH 7.2, 0.1 % USP Gelatin dissolved in water, and 0.01% Tween 80 (Kingsley and Bohlool, Applied and Environ. Microbiol., 1980, 42(2):241-248)), in a 500ml Whirl-Pak bag (Whirl-Pak Filtration Group, Pleasant Prairie, WI). The head space of the bag was evacuated and fdled with Nitrogen gas for 3 min using a bag affixing device and associated purging tubes.

[0129] The contents of the bag were homogenized for 10 min at room temperature in a Stomacher® 3500 Jumbo Lab Blender (Seward Company), and the resulting slurry was transferred to a modified sterile 304 Grade Stainless Steel Coffee Press, containing four 100 mesh (149 um) filters. The coffee press was modified to include a longer plunging rod so that the filter press plate rested on the bottom of the glass cylinder and two barbed gas ports were added to the cover of the coffee press. The ports facilitate purging of the head space using nitrogen (N2) gas.

[0130] The slurry was compressed by hand and the resulting liquid in the upper phase was removed. The remaining material was resuspended in normal saline to the original volume, mixed to resuspend, and is re-pressed to remove remaining particulate material.

[0131] The resulting second liquid suspension was combined with liquid from the first filtration, purged with N2 gas using centrifuge bottles modified to include ports to facilitate purging, and centrifuged at 6,000 * g for 15 min in a Sorvall SS-34 rotor. The upper supernatant layers after centrifugation were discarded, and the resulting pellet was resuspended to one- half the original volume in non-bacteriostatic normal sterile saline. The washing procedure of centrifugation and resuspension was repeated for a total of four times.

[0132] The final pellet was resuspended in normal saline and trehalose was added to a final concentration of 8% (w / v). This intermediate slurry suspension was stored in stainless steel trays at -80 degree C until ready for freeze-drying. The trays of frozen intermediate slurry suspension were freeze-dried using a lyophilizer (LyoStar II, Stone Ridge, NY or equivalent) with a shelf temperature of -70 degrees C for 24 hours followed by 6 hours at31+30 degrees C. All steps are done under 100 mT vacuum or less, and the final product was held at 20 degrees C until the trays were unloaded.

[0133] Cell counts of the intermediate slurry suspension and final lyophilized powder were determined by using the Bacteria Counting Kit (ThermoFisher, Waltham, MA), a Zeiss epifluorescence microscope equipped with a 470-490 nm excitation filter, a 500 nm cut-on long pass emission filter and a Petroff-Hauser counting chamber. Membrane integrity procedure done using BacLight live / dead membrane integrity assay (ThermoFisher, Waltham, MA) and a microscope and counting chamber, was used to directly enumerate intact microbes present in samples.

[0134] The final freeze-dried powder was suitable for (1) double-encapsulation in size 0 Hypromellose capsules (e.g., Capsugel® DRcaps®) packaged inside a size 00 capsule, (2) packaging in a sachet for suspension in bovine milk, non-dairy liquid media (e.g., plant based milks), or dairy or non-dairy yogurt or pudding for application via swallowing, or (3) rehydration in sterile saline buffer and applied to the distal intestine via the biopsy channel of a colonoscope.

[0135] Example 2

[0136] Production of microbiota by linear filtration method

[0137] Stool (50 grams) from a healthy donor (who is part of the UMN Donor Program (microbiota-therapeutics.umn.edu / donor-overview) were suspended in 250 of cell extraction buffer (0.1 M (NH^HPCL, pH 7.2, 0.1 % USP Gelatin dissolved in water, and 0.01% Tween 80 (Kingsley and Bohlool 1980)), in a 25.4 cm x 38.1 cm Whirl-Pak bag (Whirl-Pak Filtration Group, Pleasant Prairie, WI) containing a linear filter with a pore size of 0.33 mm (330 pm) distributed at 285 pores per cm2 (1,840 per square inch). Two linear filters with a pore size of 0.6 mm (600 pm) have also been successfully used. The head space of the bag was evacuated and filled with Nitrogen gas for 3 min using a bag affixing device and associated purging tubes.

[0138] The contents of the bag were homogenized for 10 min at room temperature in a Stomacher® 3500 Jumbo Lab Blender (Seward Company), and the resulting slurry wasmanually forced, by hand, through the linear mesh several times. The resulting fdtrate was decanted into a centrifuge bottle. A 50 ml aliquot of phosphate buffered saline (pH 7.0) was added to the bag and the residue remaining in the bag was homogenized for 5 min in the stomacher. The residue remaining in the was manually forced, by hand, through the linear mesh several times, and resulting fdtrate was decanted and combined with the original material in the centrifuge bottle. The procedure was repeated for a total of two times.

[0139] The combined liquid suspension was centrifuged at 6,000 x g for 15 min in a Sorvall SS-34 rotor, using N2 gas-purged centrifuge bottles. The upper layer was discarded, and the resulting pellet was resuspended to one-half the original volume in non-bacteriostatic normal sterile saline. The washing procedure was repeated for a total of three times.

[0140] The final pellet was resuspended in normal saline and trehalose was added to a final concentration of 10% (w / v). This intermediate slurry suspension was stored in stainless steel trays at -80 degree C until ready for freeze-drying. The trays of frozen intermediate slurry suspension were freeze-dried using a lyophilizer (LyoStar II, Stone Ridge, NY or equivalent) with a shelf temperature of -70 degrees C for 24 hours followed by 6 hours at +30 degrees C. All steps are done under 100 mT vacuum or less, and the final product was held at 20 degrees C until the trays were unloaded.

[0141] Cell counts of the intermediate slurry suspension and final lyophilized powder were determined by using the Bacteria Counting Kit (ThermoFisher, Waltham, MA), a Zeiss epifluorescence microscope equipped with a 470-490 nm excitation filter, a 500 nm cut-on long pass emission filter and a Petroff-Hauser counting chamber. Membrane integrity procedure was used to directly enumerate intact microbes present in samples.

[0142] The final freeze-dried powder was suitable for (1) double-encapsulation in size 0 Hypromellose capsules (e.g., Capsugel® DRcaps®) packaged inside a size 00 capsule, (2) packaging in a sachet for suspension in bovine milk, non-dairy liquid media (e.g., plant based milks), or dairy or non-dairy yogurt or pudding for application via swallowing, or (3) rehydration in sterile saline buffer and applied to the distal intestine via the biopsy channel of a colonoscope.

[0143] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and / or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.

[0144] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0145] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0146] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

CLAIMS1. A method for making a composition comprising a fecal extract, the method comprising:(A) homogenizing a fecal sample present in a filtration device, wherein the filtration device comprises a container comprising a solid materials region, a filtrate region, and at least one filter separating the solid materials region and the filtrate region, wherein the fecal sample is present in the solid materials region;(B) filtering the fecal sample, wherein the filtering comprises passage of liquid present in the fecal sample through the filter into the filtrate region and results in a first filtrate comprising biological material present in the filtrate region and a first unfiltered material present in the solid materials region;(C) adding a solution to the first unfiltered material in the solid materials region;(D) homogenizing the first unfiltered material present in the solid materials region;(E) filtering the homogenized first unfiltered material resulting from step (D), wherein the filtering comprises passage of liquid present in the solid materials region through the at least one filter into the filtrate region and results in a second filtrate comprising biological material present in the filtrate region and a second unfiltered material present in the solid materials region; and(F) combining the first and second filtrates to result in a combined filtrate.2 The method of claim 1, further comprising applying a centrifugal force to the combined filtrate to form a pellet and resuspending the pellet in a reduced volume compared to the combined filtrate before application of the centrifugal force.

3. The method of claim 2, further comprising repeating the applying and the resuspending at least once to result in a first processed filtrate.

4. The method of claim 1, wherein(i) the homogenizing of step (A) and the filtering of step (B),(ii) the homogenizing of step (D) and the filtering of step (E), orboth (i) and (ii), occur simultaneously.

5. The method of claim 1, wherein the at least one filter comprises a sieve size of no greater than 0.33 millimeters (mm).

6. The method of claim 1, wherein the at least one filter comprises a sieve size of no greater than 0.6 mm.

7. The method of claim 1, wherein the at least one filter comprises pores distributed at 200 to 300 pores per cm2.

8. The method of claim 1, wherein the homogenizing comprises a stomacher.

9. The method of claim 1, wherein the cell extraction buffer comprises a buffer and a gelatin.

10. The method of claim 9, wherein the buffer comprises (NH^HPCM.

11. The method of claim 10, wherein the (NHT^HPCh is present at a concentration of0.01 M to 0.1 M.

12. The method of claim 9, wherein the gelatin comprises USP grade gelatin.

13. The method of claim 12, wherein the gelatin is present at a concentration of 0.01% to 1%.

14. The method of claim 3, further comprising freeze-drying the first processed filtrate.

15. The method of claim 3, wherein trehalose is added to the first processed filtrate before the freeze-drying.

16. A method for making a composition comprising a fecal extract, the method comprising:(A) homogenizing a fecal sample in a cell extraction buffer to result in a slurry;(B) filtering the slurry with a pressurized filter press comprising a filter medium, wherein the filter medium comprises at least one filter, to result in a first filtrate comprising biological material and an unfiltered material;(C) resuspending the unfiltered material in a solution to result in resuspended material;(D) filtering the resuspended material with a filter medium, wherein the filter medium comprises at least one filter, to result in a second filtrate;(E) combining the first and second filtrates to result in a combined filtrate;(F) applying a centrifugal force to the combined filtrate to form a pellet and resuspending the pellet in a reduced volume compared to the combined filtrate before application of the centrifugal force;(G) repeating the applying and the resuspending of step (F) at least once to result in a first processed filtrate.

17. The method of claim 16, wherein the pressurized filter press is a manually pressurized filter press.

18. The method of claim 16, wherein the homogenizing comprises a stomacher.

19. The method of claim 16, wherein the at least one filter of step (B), step (D), or both steps (B) and (D) comprises a sieve size of no greater than 100 mesh (149 micrometers).

20. The method of claim 19, wherein the filter medium of step (B), step (D), or both steps (B) and (D) comprises at least four filters.

21. The method of claim 16, wherein the filtering of step (B), step (D), or both steps (B) and (D) comprises a pressurized filter press with a plunger.

22. The method of claim 16, wherein the cell extraction buffer comprises a buffer and a gelatin.

23. The method of claim 22, wherein the buffer comprises (NHa^HPC .

24. The method of claim 23, wherein the (NT ^HPCh is present at a concentration of0.01 M to O.l M.

25. The method of claim 22, wherein the gelatin comprises USP grade gelatin.

26. The method of claim 25, wherein the gelatin is present at a concentration of 0.01% to 1%.

27. The method of claim 16, further comprising freeze-drying the first processed filtrate.

28. The method of claim 27, wherein trehalose is added to the first processed filtrate before the freeze-drying.

29. A composition produced by the method of claim 1 or 16, wherein composition comprises particles of biological material, and wherein the particles of biological material are no greater than 0.6 millimeters (mm).

30. A method of administering a fecal extract to a subject, comprising: administering to a subject in need thereof an effective amount of the composition of claim 29.

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