CN112566649A

CN112566649A - Microbiota Restoration Therapy (MRT) compositions and methods of manufacture

Info

Publication number: CN112566649A
Application number: CN201980053918.3A
Authority: CN
Inventors: 李·A·琼斯; 考特尼·R·琼斯; 贝丝·安妮-舒库德拉克·布朗; 约舒亚·埃里克森; 梅萨克·彻·内巴
Original assignee: Rebiotix Inc
Current assignee: Rebiotix Inc
Priority date: 2018-06-14
Filing date: 2019-06-13
Publication date: 2021-03-26
Also published as: EP3806874A1; KR20210021023A; WO2019241523A1

Abstract

Microbiota Restoration Therapy (MRT) compositions (e.g., oral MRT compositions) and methods for making MRT compositions are disclosed. An exemplary method for making an MRT composition may comprise: the method includes the steps of collecting a fecal sample, purifying the fecal sample to form a purified sample, stabilizing the purified sample to form a stabilized sample, converting the stabilized sample to a solid, adding one or more additives and/or adjuvants to the solid to form a therapeutic composition, and encapsulating the therapeutic composition.

Description

Microbiota Restoration Therapy (MRT) compositions and methods of manufacture

Technical Field

The present disclosure relates to compositions and methods for treating patients.

Background

A wide variety of compositions and methods have been developed for the treatment of diseases and/or conditions of the digestive tract. Each of the compositions and methods has certain advantages and disadvantages with respect to known compositions and methods. There is a continuing need for compositions and methods that provide alternatives for the treatment of diseases and/or conditions of the digestive tract.

Disclosure of Invention

The present disclosure provides design, materials, methods of manufacture, and alternatives to use of compositions and methods for treating patients. An exemplary method for manufacturing an oral Microbiota Restoration Therapy (MRT) composition is disclosed. The method comprises the following steps:

collecting a fecal sample;

purifying the fecal sample to form a purified sample;

stabilizing the purified sample to form a stabilized sample;

converting the stabilized sample into a solid;

adding one or more additives and/or adjuvants to the solid to form a therapeutic composition; and

the therapeutic composition is encapsulated.

An exemplary method for manufacturing an oral Microbiota Restoration Therapy (MRT) composition is disclosed. The method comprises the following steps:

collecting a fecal sample;

purifying the fecal sample to form a purified intermediate, wherein purifying the fecal sample comprises:

adding a diluent to the fecal sample;

mixing a fecal sample with a diluent to form a mixture;

filtering the mixture;

transferring the filtrate from the filtration step to a centrifuge tube; and

centrifuging the filtrate to obtain a purified intermediate;

lyophilizing the purified intermediate to form a plurality of lyophilized pellets; and

a plurality of the lyophilized pellets are encapsulated in one or more capsules.

Alternatively or additionally to any of the embodiments above, filtering the mixture comprises filtering the mixture to obtain a sample having particles in a range of 50 to 70 micrometers (μ ι η).

Alternatively or additionally to any of the embodiments above, centrifuging the filtrate comprises centrifuging the filtrate at a rotational speed such that the centrifugal force is in the range of about 8-12,000g for a time in the range of 15 to 45 minutes.

Alternatively or additionally to any of the embodiments above, lyophilizing the purified intermediate comprises the steps of:

mixing the purified intermediate with a lyophilization excipient to form a lyophilized intermediate;

placing the lyophilized intermediate in a plate having a plurality of wells;

reducing the temperature of the lyophilized intermediate to a temperature in the range of-40 to-45 ℃;

evacuating the lyophilized intermediate and raising the temperature of the lyophilized intermediate to about 0 ℃;

starting a secondary drying step and raising the temperature of the lyophilized intermediate to about 25 ℃;

releasing the vacuum; and

a plurality of lyophilized pellets were removed from the plate.

Alternatively or additionally to any of the embodiments above, the lyophilization excipient comprises at least 2.3% PEG3350, 1% glycerol, 10% trehalose, and 10% sucrose.

Alternatively or additionally to any of the embodiments above, the one or more capsules comprise hydroxypropyl methylcellulose capsules.

Alternatively or additionally to any of the embodiments above, further comprising sealing the capsule.

Alternatively or additionally to any of the embodiments above, the edge sealing material comprises hydroxypropyl methylcellulose (an anionic copolymer based on methacrylic acid and methyl methacrylate), hydroxypropyl methylcellulose phthalate, or hypromellose acetate succinate.

A method for manufacturing an oral Microbiota Restoration Therapy (MRT) composition is disclosed. The method comprises the following steps:

adding a diluent to a purified fecal sample comprising feces, and a solution of 2.3% cryoprotectant, and a 0.9% sodium chloride solution;

mixing a fecal sample with a diluent to form a mixture;

filtering the mixture;

transferring the filtrate from the filtration step to a centrifuge tube; and

centrifuging the filtrate to obtain a purified intermediate;

the multiple lyophilized pellets are encapsulated in one or more capsules.

Alternatively or additionally to any of the embodiments above, filtering the mixture includes filtering the mixture to obtain a sample having particles with a particle size in a range of 50 to 70 micrometers (μm).

placing the lyophilized intermediate in a plate having a plurality of wells;

releasing the vacuum; and

a plurality of lyophilized pellets were removed from the plate.

Alternatively or additionally to any of the embodiments above, further comprising packaging the encapsulated lyophilized pellets in sachets in a single administration dose.

Alternatively or additionally to any of the embodiments above, the pouch comprises a metallized polyester/polyethylene adhesive film.

Alternatively or additionally to any of the embodiments above, further comprising placing the pouch in one or more child-resistant containers.

The above summary of some embodiments is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The figures and the detailed description that follow more particularly exemplify these embodiments.

Drawings

The disclosure may be more completely understood in consideration of the following detailed description of various embodiments of the disclosure in connection with the accompanying drawings, in which:

FIG. 1 is a flow chart depicting the overall process for manufacturing a standardized FMT composition;

FIG. 2 is a flow chart depicting additional steps in an exemplary manufacturing process.

FIG. 3 is a flow chart depicting additional steps in another exemplary manufacturing process.

FIG. 4 is a flow chart depicting additional steps in another exemplary manufacturing process.

FIG. 5 is a flow chart depicting additional steps in another exemplary manufacturing process.

While the disclosure is amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the disclosure to the particular embodiments described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure.

Detailed Description

To the extent that the terms are defined below, these definitions should be used unless a different definition is given in the claims or elsewhere in this specification.

All numerical values herein are assumed to be modified by the term "about," whether or not explicitly indicated. The term "about" generally refers to a series of numbers that one of ordinary skill in the art would consider equivalent to the recited value (i.e., having the same function or result). In many cases, the term "about" may include numbers that are rounded to the nearest significant figure.

The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and/or" unless the context clearly dictates otherwise.

It should be noted that references in this specification to "one embodiment," "some embodiments," "other embodiments," and so forth, indicate that the embodiment described may include one or more particular features, structures, and/or characteristics. However, such recitations do not necessarily imply that all embodiments include the particular feature, structure, and/or characteristic. Further, when a particular feature, structure, and/or characteristic is described in connection with an embodiment, it is understood that such feature, structure, and/or characteristic may also be used in connection with other embodiments, whether or not explicitly described, unless explicitly stated to the contrary.

The following detailed description should be read with reference to the drawings, in which like elements in different drawings are numbered identically. The drawings, which are not necessarily to scale, depict illustrative embodiments and are not intended to limit the scope of the disclosure.

As used herein, "mammal" refers to any member of the class mammalia, including but not limited to: human and non-human primates, such as chimpanzees, and other apes and monkeys; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents (e.g., mice, rats, guinea pigs, and the like). The term does not refer to a particular age or gender. Thus, adult and infant subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

The term "cryopreservation" as used herein refers to a process of cooling and storing biological cells, tissues, or organs at very low temperatures to maintain their viability. As one non-limiting example, cryopreservation may be a technique that allows for high survival of cells when thawed, cooling and storing the cells at a temperature below freezing (e.g., 196K).

The term "cryoprotectant" as used herein refers to a substance used to protect biological cells or tissues from freezing.

The term "microbiota" as used herein may refer to a human microbial flora, a human microbiota or a human intestinal microbiota. The human microbial flora (or microbiota) is an aggregate of microorganisms present on the surface of the skin and in the deep layers of the skin, in saliva and the oral mucosa, in the conjunctiva, and in the gastrointestinal tract, urogenital tract, or vagina of humans. The human microbial flora is composed of bacteria, fungi, and archaea. Some of these organisms perform tasks that are useful to the human host, but the function of most of the organisms that make up the human microbial flora is unknown. Under normal circumstances, these microorganisms do not cause diseases in the human host, but are involved in maintaining health. Thus, the population of organisms is often referred to as the "normal flora".

The population of microorganisms living in the human gastrointestinal tract is commonly referred to as the "gut flora" or "gut microbiota". The human intestinal microflora includes a wide variety of microorganisms that contribute to digestion, vitamin synthesis, and the production of enzymes that the human body cannot produce.

As used herein, the phrase "microbiota restoration therapy" refers to a composition that may include, but is not limited to: human fecal material comprising a viable intestinal flora derived from a patient or donor, a diluent, and a cryoprotectant. Other compositions comprise equivalent freeze-dried and reconstituted feces or "synthetic" feces compositions. Human fecal material is screened for the presence of pathogenic microorganisms prior to its use in microbiota restoration therapy. Human fecal material is screened for the presence of clostridia (including clostridium difficile), norovirus, adenovirus, enteropathogen, giardia antigens, cryptosporidium and other pathogens (including acid-fast bacteria, enterococci including, but not limited to, vancomycin-resistant enterococci (VRE), methicillin-resistant staphylococcus aureus (MSRA)), as well as any ovalbumin or parasite, or spore-forming parasites (including, but not limited to, isospora, cyclosporine, and cryptosporidium).

The process of fecal bacteria treatment may include: a fecal sample of a healthy donor or a donor having one or more desired characteristics is introduced into the gastrointestinal tract of a patient to rejoin a healthy or desired intestinal microbiota. In certain instances, antibiotics can be used to destroy the intestinal microbiota of a patient prior to introduction of a fecal sample, such that a healthy or desired intestinal microbiota can readily colonize the intestine once introduced into the patient.

Optionally, the human fecal material is filtered before it is used in microbiota restoration therapy.

The present disclosure relates to compositions, methods of manufacture, and methods of treatment using Microbiota Restoration Therapy (MRT) for the treatment of Clostridium Difficile Infection (CDI). CDI is a common nosocomial infection and is often associated with significant morbidity and mortality, particularly in elderly patients. While CDI treatment is one example for the MRT compositions disclosed herein, this is not intended to be limiting. Other diseases and/or conditions are also contemplated. Partial conditions that are desirably affected by treatment with MRT compositions may include: cardiovascular and/or peripheral vascular disease, allergy, obesity, hypoglycemia, constipation, sprue (e.g., celiac disease), gastrointestinal cancer (e.g., gastrointestinal cancer is at least one of gastric cancer, esophageal cancer, colon cancer, gallbladder cancer, liver cancer, pancreatic cancer, colorectal cancer, anal cancer, and gastrointestinal stromal tumors), melanoma, non-squamous cell lung cancer, renal cell cancer, head and neck tumors, bladder cancer, hodgkin's lymphoma, non-hodgkin's lymphoma, gastric cancer, colorectal cancer, multiple myeloma, esophageal cancer. Breast cancer, glioblastoma, mediastinal B-cell lymphoma, other hematologic malignancies, testicular cancer, pancreatic cancer, lymphoma, cervical cancer, ovarian cancer, basal cell carcinoma, neuroblastoma, leukemia, sarcoma, other cancers, myoclonic-dystonia, sacroiliac arthritis, spondyloarthropathies, spondyloarthritis, proximal myotonic myopathy; autoimmune diseases nephritic syndrome, autism, traveler's diarrhea, small intestine bacterial overgrowth, chronic pancreatitis, pancreatic insufficiency, chronic fatigue syndrome, benign myalgic encephalomyelitis, chronic fatigue immune dysfunction syndrome, Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), neurodegenerative diseases, grand mal or petit mal seizures, dystrophic myotonia, chronic infectious mononucleosis, epidemic myalgic encephalomyelitis, Idiopathic Thrombocytopenic Purpura (ITP), acute or chronic allergic obesity, anorexia, irritable bowel syndrome (IBS or colonic spasm), crohn's disease, Irritable Bowel Disease (IBD), colitis, ulcerative colitis or regional colitis, chronic infectious mononucleosis, epidemic myalgic encephalomyelitis, chronic fatigue syndrome, Acute or chronic urticaria, lupus, Rheumatoid Arthritis (RA) or Juvenile Idiopathic Arthritis (JIA), pre-diabetic syndrome, Fibromyalgia (FM), type I or type II diabetes, acute or chronic insomnia, migraine, and attention deficit/hyperactivity disorder (ADHD).

In the case of humans, the present disclosure includes methods of treating chronic disorders associated with the presence of abnormal gut microbiota. Such disorders include, but are not limited to, conditions belonging to the following types: gastrointestinal disorders (including irritable bowel syndrome or colonic spasm), Functional Bowel Disorders (FBD) (including constipated FBD, FBD with pain as the main manifestation, epigastric FBD), non-ulcerative dyspepsia (NUD), gastroesophageal reflux, inflammatory bowel disorders (including crohn's disease), ulcerative colitis, indeterminate colitis, collagenous colitis, microscopic colitis, chronic clostridium difficile infection, pseudomembranous colitis, mucous colitis, antibiotic-associated colitis, idiopathic or simple constipation, diverticulosis, aids enteropathy, small intestinal bacterial overgrowth, celiac disease, colonic polyposis, colon, chronic idiopathic pseudoobstructive syndrome; chronic intestinal infections with specific pathogens (including bacteria, viruses, fungi, and protozoa); viral gastrointestinal disorders including viral gastroenteritis, norwalk viral gastroenteritis, rotavirus gastroenteritis, aids-related gastroenteritis; liver diseases, such as primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver, or cryptogenic cirrhosis; rheumatism such as rheumatoid arthritis, non-rheumatoid factor positive arthritis, ankylosing spondylitis, lyme disease, and reiter's syndrome; immune-mediated diseases such as glomerulonephritis, hemolytic uremic syndrome, juvenile diabetes, mixed cryoglobulinemia, polyarteritis, familial mediterranean fever, amyloidosis, scleroderma, systemic lupus erythematosus, and Behcet's syndrome; autoimmune diseases, including systemic lupus, psoriasis, idiopathic thrombocytopenic purpura, sjogren's syndrome, hemolytic uremic syndrome, or scleroderma; neurological syndromes such as chronic fatigue syndrome, migraine, multiple sclerosis, amyotrophic lateral sclerosis, myasthenia gravis, Barger's syndrome, Parkinson's disease, Alzheimer's disease, chronic inflammatory demyelinating polyneuropathy, and other degenerative diseases; psychiatric disorders including chronic depression, schizophrenia, psychosis, manic depression; degenerative disorders including asperger's syndrome, rett's syndrome, Attention Deficit Hyperactivity Disorder (ADHD), and Attention Deficit Disorder (ADD); degenerative disorders, autism; sudden Infant Death Syndrome (SIDS), anorexia nervosa; skin diseases such as chronic urticaria, acne, dermatitis herpetiformis, and vasculitis; and cardiovascular and/or vascular disorders and diseases.

The increase in the spread of drug resistant organisms has created many challenges for clinicians that pose public health risks throughout the world. Infection by resistant organisms (e.g., vancomycin-resistant enterococci (VRE)) and clostridium difficile share similar risk factors. VRE is an in-hospital pathogen that can cause complications in transplant and immunodeficiency patients. VRE vectors can also be at increased risk of infection due to VRE and are also a potential source of transmission of VRE to others. VRE shedding in feces is increased by antimicrobial contact and decreased by normalization of the intestinal microbiota after the antimicrobial is discontinued. Thus, normalization of the gut microbiota may not only be useful in the treatment of clostridium difficile infections (including chronic infections), but these treatments may also be useful in the treatment of infections caused by resistant microorganisms (e.g., VRE and/or other resistant organisms, including those disclosed herein).

In some cases, the microbiota restoration therapy compositions (and/or fecal bacteria therapy compositions) disclosed herein can be used to treat patients infected with drug-resistant microorganisms and/or multi-drug resistant Microorganisms (MDRO). Resistant organisms develop resistance to antimicrobial agents (e.g., antibiotics, antivirals, antifungals, antiparasitics, other drugs, combinations thereof, and the like) and may include resistant microorganisms such as bacteria, viruses, fungi, parasites, and the like. Infections that can be treated with the microbiota restoration therapeutic compositions disclosed herein can be along the alimentary canal or along other systems of the patient.

Microbiota restoration therapy compositions are useful for treating infections caused by a variety of resistant organisms, such as vancomycin-resistant enterococci (VRE), methicillin-resistant staphylococcus aureus (MRSA), ultra-broad spectrum beta-lactamase-producing gram-negative bacteria, klebsiella pneumoniae, carbapenemase-producing gram-negative bacteria, multiple-resistant gram-negative bacteria (e.g., enterobacter, escherichia coli, klebsiella pneumoniae, acinetobacter baumannii, and pseudomonas aeruginosa), resistant enterobacter, multiple-resistant mycobacterium tuberculosis (e.g., mycobacterium tuberculosis), resistant staphylococcus, resistant enterococcus, resistant gonococcus, resistant streptococcus (e.g., including streptococcus pneumoniae), resistant salmonella, resistant gram-negative bacteria, resistant candida, resistant HIV, resistant influenza virus, resistant cytomegalovirus, resistant herpes simplex virus, Drug-resistant malaria, drug-resistant plasmodium vivax, drug-resistant plasmodium falciparum, drug-resistant toxoplasma, and the like, and/or other drug-resistant organisms. These are only examples.

The microbiota restoration therapy composition may be used to treat other infections, including urinary tract infections.

Treatment of an infection by a drug resistant organism with a microbiota restoration therapeutic composition disclosed herein may include: patients without a prior history of infection are treated with resistant organisms: treating a patient having a prior infection with a drug-resistant organism; treating a patient having more than 2 (e.g., 2, 3, 4, 5,6, or more) prior infections with a drug-resistant organism, and the like. In some cases, the microbiota restoration therapeutic composition may be used to treat patients with 3 previous infections with drug resistant organisms. In other cases, the microbiota restoration therapy composition may be used to treat patients with 2 previous infections utilizing resistant organisms if the previous infection resulted in hospitalization, if the previous or current infection required treatment with a toxic drug, or if the previous infections were all derived from the same organism.

In some cases, the MRT composition may be administered to the patient using enema or other suitable techniques. However, it is desirable to administer the MRT composition orally. In order to prepare the MRT composition in a dosage form suitable for oral administration, several steps may be performed. Generally, these steps may include: collecting a fecal sample; treating a fecal sample; lyophilizing or "freeze-drying" (or converting a treated fecal sample from a liquid to a solid) a treated fecal sample; adding one or more additives and/or auxiliary materials; and oral formulations (e.g., tablets, capsules, liquid formulations, etc.) of the MRT compositions formed from the lyophilized material and additives. Some additional details regarding at least some of these steps are disclosed herein.

FIG. 1 is a flow chart depicting a portion of an exemplary MRT production process. This is just one example. Other examples of screening donors, obtaining human stool samples, and processing the stool samples into MRT products are disclosed in commonly assigned U.S. patent publication 2014/0363398, the contents of which are incorporated herein by reference. More specifically, fig. 1 schematically depicts a process for collecting and examining a donor stool sample. As a first step in the collection/inspection process, potential fecal donors are screened. Screening/pre-screening is described in more detail herein. Once the donor passes the screening, step 2 may comprise collecting the donor's stool, whether at home or at a collection facility, using a human stool collection kit as defined herein. The kit may include, but is not limited to: a clean lidded human fecal collection container, a large closable/sealable pouch, a donation form, and a human fecal collection instruction chart. The time and data of collection, as well as the donor identity and method of transport, may be recorded to track the time from collection to processing, and the status of the transport. As one non-limiting example, the collection container may include an indicator of the minimum and maximum temperatures to which the sample is exposed. As another non-limiting example, more than one temperature-sensitive sticker that changes color at temperatures below about 4 ℃ and at temperatures above about room temperature (about 22-29 ℃) may be adhered to the container.

Step 3 may include transporting the sample to a processing facility. It will be appreciated that if the sample is taken at a processing facility, then transport of the sample is not necessary. In some cases, it may be desirable to collect the sample at the processing device to more specifically establish a chain of custody of the sample. When the first fecal donation is received for any individual, a profile will be established for each donor. Subsequent stool samples may undergo human stool examination that is used to match and confirm the identity of the donor providing the donation. Based on previously collected samples, a human stool profile for the donor is generated and can be maintained or enhanced in repeated donations. Any new sample will be compared to this profile to confirm that it belongs to the same donor. Identification to confirm donor identity can be done based on the characterization of bacteroides in human feces. In one non-limiting example, a base set of stool samples used to build an archive is collected at a processing device to confirm donor identity in the archived samples. In another non-limiting example, the base set of stool samples used to build the profile may be collected at a location different from the processing facility, provided with a donor identity assurance scheme appropriate to the situation or location.

Step 4 of the method may comprise: the donations are labeled "quarantine" and are kept at room temperature or at a temperature below room temperature for no more than a period in the range of 24 hours to 5 days prior to treatment in quarantine. In the case where the temperature indicator has been activated or the time between donation and receipt exceeds 24 hours, the donation may be rejected. In addition, where applicable, human stool examination results must be matched to the donor profile. If the human stool examination does not match the donor profile, the donations collected this day will be abandoned and the donor will be disqualified.

In one method of the present disclosure, a human stool sample is processed within about 24 hours of collection. In another method of the present application, the time of collection is recorded when the fecal sample reaches the processing device. Step 6 may include examination of the fecal donation. When the fecal sample reaches the processing equipment, a visual inspection can be completed. In the event that a human stool sample is loose, unformed, does not have sufficient weight (e.g., less than about 50g), or for any other reason, including but not limited to evidence that the sample quality or factors related to donor health are poor, the sample may be rejected, flagged as "check-reject" and the donation discarded. In addition, answers to questions on the human stool collection form may be reviewed by trained personnel. Some answers in the collection form may require sufficient rejection. If a sample is received, it may be marked as "check-receive" and may be transferred to the manufacturing process.

Fig. 2 is a flow chart depicting a portion of a general illustrative method for preparing stool samples for MRT as oral dosage forms. It is conceivable that the intermediate product in the method for preparing a stool sample for MRT as an oral dosage form may be suitable for MRT via enema or nasogastric tube. The fecal sample may first be collected and screened 100, such as in the method described with reference to fig. 1. Once the sample has been received, the sample may be purified and concentrated 102. The sample may be purified by centrifugation, membrane filtration, or a combination thereof to remove fecal material above a certain particle size. It is conceivable that the sample may be treated to remove particles larger than 50-70 μm, since the majority of the bacteria of interest is in the range of 0.3 micrometers (μm) to 30 μm. The sample may be treated to obtain a concentration of bacteria of 75% to 90%. This may increase the flexibility of the formulation adjuvant to bacteria ratio for further processing.

The sample may be membrane filtered in a number of different ways, including but not limited to the use of filter bags, pressure filters, and/or vacuum filters. In some cases, the sample may be filtered multiple times, with each subsequent filtration being performed with a smaller filter membrane. In some cases, saline may be added as a diluent at a ratio of 1:3 (feces to saline), although this is not required. In other cases, a mixture of saline and a cryoprotectant (e.g., polyethylene glycol (PEG)3350) may be used as a diluent. The PEG concentration of the diluent may be about 30-90 g/l (or about 10-90 g/l). The PEG concentration of the diluent may also be between about 25-75 g/l. In one example, the ratio of saline/PEG mixture to fecal sample is 2:1, or 2mL of saline/PEG mixture to 1 gram of human feces. As a non-limiting example, approximately 100mL of the saline/PEG mixture can be used for 50g of human feces. In another example, the ratio of saline/PEG mixture to fecal sample is 3:1, or 3mL of saline/PEG mixture to 1 gram of human feces. As a non-limiting example, approximately 150mL of the saline/PEG mixture can be used for 50g of human feces. While saline/PEG may be suitable for use as a diluent (and/or cryoprotectant), this is not intended to be limiting. Other cryoprotectants may also be used. For example, dextrose, betaine, glycine, sucrose, polyvinyl alcohol, pluronic F-127, mannitol, Tween 80, ethylene glycol, 1, 3-propylene glycol, hydroxypropyl cellulose, glycerol, PEG/glycerol mixtures, propylene glycol, or combinations thereof can be used as cryoprotectants. These may be used alone or in combination with a solvent (e.g., brine).

In one example, the sample can be placed in a 500 μm filter bag with or without diluent, and stirred for about 2 minutes at 230rpm using, for example, a Stomacher stirrer, to obtain a filtrate having a particle size below about 500 μm. This filtrate can then be placed in a filter bag having a pore size of less than 500 μm, for example 280 μm. The sample can be again stirred at 230rpm for about 4 minutes, e.g., using a Stomacher stirrer, with or without diluent, to obtain a filtrate having a particle size below about 280 μm. The filtrate may be placed in another filter bag having a pore size of less than, for example, 280 μm, such as, but not limited to, 60 μm. The sample can be again stirred, for example, with a Stomacher stirrer at 230rpm for about 4 minutes, with or without diluent, to form a filtrate having a particle size of about 50-70 μm or less.

In another example, the sample can be placed in a 500 μm filter bag with or without diluent and stirred using, for example, a Stomacher stirrer to obtain a filtrate having a particle size below about 500 μm. The filtrate can then be processed with a pressure filter having a pore size of about 160 μm, and the resulting filtrate can be processed with a pressure filter having a pore size of about 60 μm. In some cases, it may be necessary to perform a secondary treatment of the sample with a bag filter having a pore size between 160 μm and 500 μm before using the pressure filter.

In another example, the sample can be placed in a 500 μm filter bag with or without diluent and stirred using, for example, a Stomacher stirrer to obtain a filtrate having a particle size below about 500 μm. The filtrate can then be processed with a vacuum filter having a pore size of about 160 μm, and the resulting filtrate can be processed with a vacuum filter having a pore size of about 60 μm. In some cases, it may be necessary to perform a second treatment of the sample with a bag filter having a pore size between 160 μm and 500 μm before using the pressure filter.

Once the sample has been processed to have a particle size below about 60 μm, the sample can then be washed and further concentrated using centrifugation. In some cases, the centrifuge tube may have a volume in the range of 50 to 500mL or more. Fill the filtered suspension to about 20 to 80% of the centrifuge tube volume. In one example, the sample may be centrifuged at 1100 to 3600 revolutions per minute (rpm) for a period of 10 to 15 minutes. In another example, the sample can be centrifuged for 15-45 minutes or 20-30 at a number of revolutions such that the centrifugal force is in the range of about 8-12,000g (e.g., about 10,000g)And (3) minutes. The centrifugal force may be increased or gradually accelerated to a speed required to generate a centrifugal force in the range of about 8-12,000g (e.g., about 10,000 g). It is also contemplated that the centrifugation may also be slowed or slowed slowly as the centrifugation process is completed. In some cases, it is desirable to decelerate the centrifugation as slowly as possible so that the return to atmospheric pressure is slow to prevent possible rupture of the bacterial cells. The supernatant was removed and the remaining material in the tube was the purified intermediate MRT composition. This results in a product that has been concentrated up to about 60%. In some cases, the centrifugation process may be a 2-stage process. For example, the product may first undergo a "pre-spin" (e.g., about 300-. For example, after "pre-spinning", the supernatant can be transferred to a new centrifuge tube/vial and then spun at a higher speed (e.g., about 5,000 Xg or about 10,000Xg for 30-60 minutes or for about 45 minutes). After high speed rotation, the supernatant can be discarded and the recovered microbiota can be further processed. It is also contemplated that volumes of up to 300mL may be centrifuged and that no drop in the amount of concentration is formed. The resulting MRT composition is of a particle size of 70 μm or less and about 1X10¹⁰Bacterial suspension at CFU/g bacterial concentration. The resulting MRT composition may also be stable under refrigeration conditions for up to 3 weeks.

In some embodiments, multiple centrifugations can be used alone for purification and concentration. However, the particle size of the bacterial suspension will still be in the range of clogged pipette tips (e.g. greater than 60 μm). However, in some cases, a wide pipette tip may be used. Whether this is successful depends on the input fecal material, which is variable. It is also conceivable that a system of separators and decanters could be used if the batch size is in the range of tens of litres or more. However, this may not be necessary if the starting product has been previously treated.

In other embodiments, density gradient centrifugation may be used for purification and concentration of fecal samples. Density gradient centrifugation can be combined with the above-described processFiltering techniques, or used separately. Density gradient centrifugation can be used strictly for separation by density, while differential centrifugation can be used for separation by particle size and density. To perform density gradient centrifugation, a density gradient medium can be added to a sample (e.g., a diluted native or diluted filtered sample). The density gradient media may be a solution with varying concentrations (e.g., sucrose with varying concentrations). For example, a density gradient medium can be formed by superimposing a lower solution concentration on a higher solution concentration in a centrifuge tube. The sample may be placed on top of the density gradient medium and subsequently centrifuged. Particles in the sample may travel through the density gradient medium until they reach a point in the gradient where their density matches that of the surrounding solution. For example, due to the density of the bacteria and the density gradient medium, the object (e.g., bacteria) may settle in the middle of the centrifuge tube. A wide variety of density gradient media may be used for centrifugation, including but not limited to: polyhydric (sugar) alcohols, polysaccharides, inorganic salts, iodine compounds, silica gel, and the like. Other density gradient materials may include iohexol (e.g., iodine, or iodine-containing compounds)

Manufactured by Axis-Shield), iodixanol solution (e.g., OptiPrep)^TM(manufactured by Axis Shield)), and/or PEGs of various molecular weights. It is contemplated that concentrations in the range of 40% to 80% weight/volume (w/v) may be used

The medium may be pharmaceutical grade, biologically inert, and/or isotonic. In some cases, density gradient centrifugation can more efficiently purify bacteria from feces than differential centrifugation.

In some embodiments, tangential flow filtration (or cross-flow filtration) may be used in conjunction with density gradient centrifugation to further remove any undesirable soluble species. In tangential flow filtration, a substantial portion of the feed stream may flow tangentially across the surface of the filter rather than into the interior of the filter. Tangential flow filtration of the target (e.g., bacteria) can further remove soluble impurities from the target. During tangential flow filtration, additional fibrous material can be pushed out as the bacterial suspension (obtained by conventional centrifugation and/or density gradient centrifugation) is passed over the surface of the filter. In some cases, a buffer is followed after each pass of the bacterial suspension through the tangential flow filtration system. It is contemplated that a larger volume (e.g., up to about 10L) of bacterial suspension may be treated once through the tangential flow filtration system. In some cases, the filtrate from the tangential flow filtration process can be used as a purified intermediate stool sample. It is conceivable that the filtered suspension (e.g. filtrate) may be diluted with saline and/or Phosphate Buffered Saline (PBS). In other cases, the filtrate from the tangential flow filtration process can be further processed using, for example, but not limited to, differential centrifugation and/or dead-end filtration.

In some embodiments, it is desirable to stabilize the treated sample in suspension under refrigerated conditions for a period 104 in the range of 1 to 2 weeks. In some cases, removal of fecal material and replacement with a carrier or adjuvant that is soluble in aqueous solution may allow the bacteria to be suspended in the liquid and further processed without stability problems. Considerations for these adjuvant solutions may be pH, concentration, and isotonicity or isotonicity. Adjuvants may be selected based on protein and monoclonal antibody preparations and their recommended use in stabilizing biologics. Some exemplary excipients that may be used to provide liquid stabilization 104 of a sample may include, but are not limited to: salt (NaCl), sucrose, trehalose, L-arginine hydrochloride, and/or PEG3350, as summarized in table 1 below. A list of other potential excipients can be found in tables I and III, in the tables of Seong Hoon Jeong, Arch Pharm Res, Vol. 35, No. 11, p.1871-1886, 2012, and in the tables of Pharma Times, Vol.45, No. 3, p.2013, Pramanick et al.

Table 1: summary of illustrative adjuvants

In some cases, the excipient may comprise 2-20% sucrose, 0.1-5% L-arginine hydrochloride, 0.5-20% PEG3550, or a combination thereof.

Combinations of adjuvants may be used to protect biological cells or tissues from freezing and/or to provide stability to the product during storage (e.g., to minimize cell death). Some exemplary adjuvant formulations that can provide cryoprotection and stability during storage are shown in table 2 below. However, the formulations listed in table 2 are not intended to be limiting. Other combinations and/or amounts of adjuvants may also be employed.

Table 2: adjuvant solution composition prior to addition to a drug

In certain instances, adjuvants (e.g., lyophilization adjuvants) can include polyethylene glycol (e.g., about 1-5%, or about 2-3%, or about 2.3%), trehalose (e.g., about 1-25%, or about 5-15%, or about 10%), sucrose (e.g., about 1-25%, or about 5-15%, or about 10%), and glycerol (e.g., about 0.1-5%, or about 0.5-2%, or about 1%) in purified water.

It is envisaged that the above adjuvant formulations, when added to a medicament (e.g. a faecal sample or a treated faecal sample), may provide freeze protection to biological cells in liquid and/or solid formulations and stability during storage. In some cases, the adjuvant formulation may be added to the drug in a 1:1 ratio. This is just one example. Other ratios of excipients to drug are also contemplated, such as, but not limited to, 0.25:1, 0.5:1, 1.5:1, 2:1, and the like.

In some of these cases and in other cases, the adjuvant formulation may comprise:

(a) 0.5-20% PEG, (b) 0.1-5% glycerol, (c) 10-30% PVP, (d) 40-80% trehalose, (e) 40-80% sucrose, (f) 10-30% phosphate buffer solution, or (g) combinations thereof. Other formulations are also conceivable.

It is conceivable that similar excipients can also be used to protect the bacteria during membrane filtration. For example, Farber and Sharpe in Applied and Environmental Microbiology, 8 1984, page 441-443 states that bacterial recovery is improved in the presence of certain food fragments (carrots, cheese, peaches, tuna), pH values can be important: pH5.88 to 6.40 (carrot), pH4.75 to 5.02 (cheese), pH5.9 to 6.2 (tuna), pH3.3 to 4.05 (peach). The presence of sugars, carbohydrates, or proteins can be important, and the properties of these foods that coat the bacteria, support bacterial growth (pro-probiotic activity), or support the bacterial cell wall during filtration can be important.

Suitable carriers may vary with the desired dosage form and mode of administration of the composition. For example, they may contain diluents or adjuvants such as fillers, binders, wetting agents, disintegrants, surfactants, glidants, lubricants, and the like. Generally, the carrier can be a solid (including a powder), a liquid, or a combination thereof. Preferably, each carrier is "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. The carrier can be biologically acceptable and inert (e.g., it allows the composition to maintain viability of the biological material until delivered to the appropriate site).

Oral compositions may comprise an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compounds can be combined with adjuvants and used in the form of tablets, dragees, or capsules, such as gelatin capsules. Oral compositions can also be prepared by mixing the compositions of the present disclosure with food. In one embodiment, the food for administration is chilled (e.g., ice cream). Pharmaceutically compatible binders, and/or adjuvants may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients, or compounds of similar properties: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; adjuvants, such as starch or lactose, disintegrating agents, such as alginic acid, sodium starch glycolate, or corn starch; lubricants, such as magnesium stearate or hydrogenated vegetable oil; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, orange flavoring, or other suitable flavoring. These are for purposes of example only and are not intended to be limiting.

Once the purified sample has been purified and stabilized into an aqueous suspension suitable for delivery by nasogastric tube or enema, the sample can be further processed into a dosage form suitable for oral administration, such as in the form of a tablet, lozenge, or capsule. For example, the aqueous solution may be converted to a solid 106. A series of bacterial treatment techniques can be found in Innovative Food Science and engineering Technologies, 27(2015)15-25 by Martin et al.

In some cases, lyophilization or freeze-drying can be used to convert a sample from a liquid to a solid. The sample may have a cryoprotectant, such as, but not limited to, PEG, skim milk, activated carbon, ascorbic acid, or combinations thereof, to protect the bacteria from freezing. The sample may also have a lyoprotectant, such as, but not limited to, sucrose, inositol, trehalose, glycerol, or a combination thereof. In some cases, the sample may also have an enrichment material that can provide acid buffering. Alternatively or in addition, the enrichment material may also keep the bacteria more viable, which may facilitate analytical testing. Some exemplary enrichment materials may include, but are not limited to: skim milk, activated carbon, gelatin, ascorbic acid, GI medium, or combinations thereof. Alternatively or additionally, an oxygen scavenger may be added to the sample before and/or after lyophilization. While not wishing to be bound by theory, it is believed that oxygen scavengers may improve the stability and/or viability of the sample. It is contemplated that the lyophilization tube may include an insert that may be used to expel the lyophilized pellets from the lyophilization tube after lyophilization. The width of the freeze-dried tube may be less than the width of a capsule shell for oral treatment. This allows a tray of pellets to be transferred directly into the capsule shell. It is contemplated that this may reduce or eliminate particle size measurement of the formulation or further mix 108 it to improve flow characteristics into the capsuleAnd (4) sex requirements. The dosage may also be determined by the pellet size. In some cases, the pellets produced during the lyophilization process may comprise about 4.5X 10⁸CFU (CDC). Size 0 capsules can contain 3 pellets. Thus, the capsule may contain about 6.7 x10⁹CFU (CDC). Eight capsules would need to be taken twice a day, which corresponds to one enema dose. Furthermore, there is no need to check the homogeneity of the batch of pellets that are mixed together before filling the capsule. In some cases, compaction may allow for a greater concentration or number of pellets inside each capsule. For example, compaction of the pellets inside the capsule may allow for about 2-4 times (e.g., about 2.5 times) the number of pellets in each capsule (e.g., each capsule may hold 2-4 or about 3 pellets without compaction, and each capsule may hold about 7-10 or about 8 pellets with compaction). This may help to reduce the number of capsules that a patient needs to take in order to reach a desired dose. In some cases, the pellets may be ground prior to compaction into a capsule. If the pellets are ground, it is desirable that the powder have a Carl index value in the range of 15 to 30 to facilitate capsule filling. Alternatively, the pellets may be ground and compressed into tablets. The enteric powder can then be compressed over the tablet to form an oral formulation that is stable in the acid environment of the stomach but dissolves in the intestinal tract.

In other cases, it may be desirable to preserve the sample by evaporative foam drying. It is contemplated that conventional adjuvants and equipment may be used in the process. Higher adjuvant concentrations and optimized process parameters for generating foam during processing can result in formulations with low water content. The lower the water content, the greater the likelihood of being stable at room temperature. Once the sample has been dried 106, the sample may be further processed to obtain a desired particle size and/or mixed 108 to prepare the sample for oral product processing.

In other embodiments, the liquid sample may be microencapsulated using liposomes to isolate the liquid sample from bile, alginate, and/or polymers. Once the sample has been embedded, the sample may be further processed to obtain a desired particle size and/or mixed 108 to prepare the sample for oral product processing.

After the sample has been processed to a desired particle size and/or mixed 108 to prepare the sample for oral product processing, the sample may be packaged 110. It is contemplated that the encapsulation process may provide low pH protection 112. For example, the encapsulation process can prevent or substantially prevent the capsule shell, tablet, and/or lozenge from disintegrating in the acidic environment of the stomach such that the MRT composition is released in the desired portion of the intestinal tract. It is conceivable that an enteric coated capsule would be required to provide protection in the stomach and have disintegration of the capsule in the small and large intestines. In some cases, the capsules may be pan-coated with an enteric coating. Enteric coating materials may include fatty acids, waxes, shellac, plastics, and vegetable fibers. Pan coating of hydroxypropyl methylcellulose (HPMC), otherwise known as hydroxypropyl methylcellulose capsules, will protect at low pH and also help protect against moisture. Some suitable capsules may include capsules made from

DRcaps obtained^TMAnd Vcaps^TM. Likewise, AR capsules with a composition of 60% HPMC and 40% HPMCP (hydroxypropylmethylcellulose phthalate) may have the same performance. Non-gelatin capsule types may contain less water (gelatin capsules typically contain 10 to 12% water, while other polymer capsules contain less than 3-4% water). It may be desirable to edge seal the capsule with a polymer that is insoluble in low pH environments, as will be discussed in more detail below. In other cases, the capsules may be stacked such that more than 2 capsules are used to encapsulate the sample. For example, a sample may be placed in a capsule and then the capsule placed in another larger capsule. Stacked (e.g., more than 2 capsules) and/or edge-sealed capsules can be present in an acidic environment (e.g., stomach) for at least 2 hours or more and dissolve in a more neutral intestinal tract.

In some cases, capsules that have been edge sealed with a low pH resistant polymer may not completely disintegrate and/or release the product for more than 5 hours. This may allow the capsule to pass intact through the stomach and allow the product to be released into the intestine, which is ideal for bacteria. It is also conceivable that releasing the MRT composition into the more neutral environment of the intestine may allow more bacteria to survive, as opposed to the acidic environment of the stomach (in the pH range of 1.2). Sealing the capsule may include placing a band of low pH resistant polymer over an area where the first capsule portion overlaps the second capsule portion. In other cases, two capsules (e.g., double encapsulation), one within the other, may allow the capsule to pass entirely through the stomach and/or allow a desired number of viable bacteria to reach the target area.

In some embodiments, super-disintegrants may be used to swell a dosage form (e.g., a capsule or tablet) to increase the likelihood of bacteria coming into contact with the intestinal wall. For example, crosslinked cellulose swells 4 to 8 times in 10 seconds, crosslinked starch swells 7 to 12 times in less than 30 seconds, and crosslinked alginic acid undergoes rapid swelling or capillary action in an aqueous medium.

The presence of prebiotics may be desirable to ensure bacterial growth at the site of action in the intestine. These are substances that can be added to the capsule formulation or administered separately at the same time of administration. Some suitable additives include: galactooligosaccharides, inulin derivatives (e.g., fructooligosaccharides), celluloses, dextrins, chitins, gums, beta-glucans, waxes, lignins, phytochemicals (bioactive non-nutritive plant compounds present in fruits, vegetables, grains, and other plant foods), carotenoids, phenolics, alkaloids, nitrogenous compounds, and organosulfur compounds. It is contemplated that in certain concentration ranges, the L-arginine and PEG excipients may produce water and electrolyte secretions when delivering the drug. This may enhance the ability of the bacteria to attach to and grow in the intestine. Other adjuvants which produce this effect may also improve the therapeutic effect.

An oral product can be packaged in several different ways, including but not limited to: blister packs or bottles. In some cases, oxygen scavengers and/or desiccants may be placed in the bottle and/or blister. The blister pack and/or bottle may include features for rendering the pack child-resistant. For example, bottles may be provided with child-resistant caps, and blister packages may be provided with child-resistant sleeves. In some cases, the blister package may include instructions designed to instruct the patient how to use the package. For example, a blister pack may provide guidance as to how many pills to take on a given day and/or when to take pills. The package may include monitoring means for monitoring the transport condition. As a non-limiting example, the packaging container may include indicators of the minimum and maximum temperatures to which the product is exposed. As another non-limiting example, one or more temperature sensitive decals that change color at temperatures below about 4 ℃ and temperatures greater than about room temperature (about 22-29 ℃) may be affixed to the container.

Fig. 3 and 4 are flow charts depicting two

illustrative methods

201, 300 for preparing stool samples for MRT as an oral formulation. In some embodiments, oral dosage forms may be prepared from fresh stool samples (fig. 3), in other embodiments oral dosage forms may be prepared from materials that have been processed (fig. 4). As used herein, a fresh stool sample will be referred to as drug a and a previously processed sample will be referred to as drug B. Other drugs are conceivable, including substances obtained from cultures of fecal microbiota. Referring first to fig. 3, a stool sample may first be collected and screened, such as in the method described with reference to fig. 1. Once the sample has been received, the sample may be weighed and loaded into a filter bag, as shown at step 200. It is contemplated that multiple collection containers (e.g., the same or different donors and collected at various times) within their expiration dates may be used (e.g., pooled together). The sample may be purified by centrifugation, membrane filtration, or a combination thereof to remove fecal material above a certain particle size. It is conceivable that the sample may be treated to remove particles larger than in the 50-70 μm range, since the majority of the bacteria of interest is in the 0.3 micrometer (μm) to 30 μm range. The sample may be treated to obtain a concentration of about 60% bacteria. This may allow for increased flexibility in formulation adjuvant to bacteria ratio for further processing.

The filtration solution or diluent may be added to the filter bag, as shown at step 202. In some cases, saline may be used as a diluent. For example, a solution of 0.9% sodium chloride (NaCl) can be added to the filter bag at a rate of about 3 milliliters (mL) per gram of drug a. In other cases, phosphate buffered saline may be added to the filter bag at a ratio of 1:1 (by weight). It is contemplated that other diluents, other diluent concentrations, and dilution ratios may be used, as desired. For example, a mixture of saline and a cryoprotectant (e.g., polyethylene glycol (PEG)3350) may be used as a diluent. The PEG concentration of the diluent may be about 30-90 g/l (or about 10-90 g/l). The PEG concentration of the diluent may also be between about 25-75 g/l. In one example, the ratio of saline/PEG mixture to fecal sample is 2:1, or 2mL saline/PEG mixture to 1 gram of human feces. However, in some cases, such as when drug a is specifically treated to facilitate lyophilization, the diluent may not comprise a cryoprotectant. The sample may then be subjected to membrane filtration in a number of different ways, including but not limited to the use of a filter bag, a pressure filter, and/or a vacuum filter, as shown at step 204. In some cases, the sample may be filtered multiple times, with each subsequent filtration being performed with a smaller filter membrane. In one example, the sample can be placed in a 500 μm filter bag and stirred at 230rpm for about 2 minutes using, for example, a Stomacher stirrer to obtain a filtrate having a particle size below about 500 μm. The filtrate can then be placed in a filter bag having a pore size of less than 500 μm, for example 280 μm. The sample can be again stirred at 230rpm, for example, using a Stomacher stirrer, with or without a diluent, for a period of about 4 minutes, to obtain a filtrate having a particle size below about 280 μm. The filtrate may be placed in another filter bag having a pore size of less than, for example, 280 μm, such as, but not limited to, 50-70 μm. The sample can be again stirred, for example, with a Stomacher stirrer at 230rpm for a period of about 4 minutes, with or without diluent, to form a filtrate having a particle size of about 50-70 μm or less.

In another example, the sample can be placed in a 500 μm filter bag with or without diluent and stirred using, for example, a Stomacher stirrer to obtain a filtrate having a particle size below about 500 μm. The filtrate can then be processed with a pressure filter having a pore size of about 160 μm, and the resulting filtrate can be processed with a pressure filter having a pore size of about 60 μm. In some cases, it may be necessary to perform a second treatment of the sample with a bag filter having a pore size between 160 μm and 500 μm before using the pressure filter.

In another example, the sample can be placed in a 500 μm filter bag with or without diluent and stirred using, for example, a Stomacher stirrer to obtain a filtrate having a particle size below about 500 μm. The filtrate can then be processed with a vacuum filter having a pore size of about 160 μm, and the resulting filtrate can be processed with a vacuum filter having a pore size of about 60 μm. In some cases, the sample may need to be treated a second time with a bag filter having a pore size between 160 μm and 500 μm before the pressure filter is used.

Once the sample has been processed to have a particle size of about 50-70 μm or less, the sample may be placed in an intermediate storage container, as shown at step 206. One example of an acceptable intermediate storage container is a 250mL capped sterile plastic container. In some cases, the filtered suspension may be stored in a refrigerator at a temperature of 5 ± 3 ℃ for up to 5 days, although this is not essential. The filtered suspensions may be combined and mixed into a larger container, as shown at step 208. One example of an acceptable intermediate storage container is a multi-liter sterile plastic container with a lid.

An aliquot of the mixed filtered suspension can then be placed into a centrifuge tube having a volume of 50 to 500mL, as shown at step 210. The filtered suspension is filled to approximately 20 to 80% of the volume of the centrifuge tube. In some cases, centrifuge tubes having a volume greater than 500mL may be used. The filtered suspension may then be washed and further concentrated using centrifugation, as shown at step 212. In one example, the sample may be centrifuged at 1100 to 3600 revolutions per minute (rpm) for a period of 10 to 15 minutes. In another example, each sample may be centrifuged for 15-45 minutes or 20-30 minutes at a speed such that the centrifugal force is in the range of about 8-12,000g (e.g., about 10,000 g). The centrifugation can be accelerated or gradually accelerated to a speed required to generate a centrifugal force in the range of about 8-12,000g (e.g., about 10,000 g). It is also conceivable that the centrifugation can also be slowed or slowed down slowly when the centrifugation process is completed. In some cases, it is desirable to decelerate the centrifugation as slowly as possible so that the rate of return to atmospheric pressure is slow to prevent possible rupture of the bacterial cells. The supernatant was removed and the remaining material in the tube was the purified intermediate MRT composition. This can result in a product that has been concentrated up to about 60%.

In some cases, the centrifugation process may be a 2-stage process. For example, the product may first undergo a "pre-spin" (e.g., about 300-. For example, after "pre-spinning", the supernatant can be transferred to a new centrifuge tube/vial and then spun at a higher speed (e.g., about 5,000-. After high speed rotation, the supernatant can be discarded and the recovered microbiota can be further processed. It is also contemplated that volumes of up to 300mL may be centrifuged without causing a decrease in the amount of concentration. In some cases, a volume greater than 300mL may be centrifuged. For example, as described above, the centrifugation volume may be selected as a percentage of the volume of the container (e.g., in the range of 60%). The resulting MRT composition is of a particle size of 70 μm or less and about 1X10¹⁰Bacterial suspension at CFU/g bacterial concentration. Purified intermediate bacterial survival can be measured using the quantitative polymerase chain reaction (qPCR) method using propidium monoazide bromide (PMA). The resulting MRT composition may also be stable under refrigerated conditions for up to 3 weeks.

In some embodiments, a single use may be centrifuged multiple times to purify and concentrate. However, the particle size of the bacterial suspension may still be in the range of clogging the pipette tip (e.g. greater than 60 μm). Whether this is successful depends on the variable input fecal material. It is also conceivable that a system of separators and decanters could be used if the batch size is in the range of tens of litres or more.

The intermediate MRT composition may optionally be transferred to an intermediate tube and, if necessary, shipped to a lyophilization apparatus, as shown at step 214. If necessary, the purified intermediate can be shipped to a contract lyophilizer for lyophilization in a pre-approved transporter for refrigerated conditions of 5 ± 3 ℃.

The purified intermediate may be mixed with a lyophilization excipient solution in a 1:1 ratio, as shown at step 216. The lyophilized adjuvant solution may be composed of 2.3% PEG3350, 1% glycerol, 10% trehalose, and 10% sucrose. However, other lyophilization excipients may be used. The lyophilized excipient solution (without glycerol) was filtered through a 0.2 μm filter before the excipient solution was added to the purified intermediate. Glycerol was autoclaved at 121 ℃ for a minimum of 15 minutes and added aseptically. Once the lyophilization excipient has been mixed with the purified intermediate (lyophilization suspension), a single 200 microliter (200 μ Ι) aliquot of the lyophilization suspension is placed into each well of a 96-well plate, as shown at step 218, and lyophilized, as shown at step 220.

The lyophilization process will be described further below with reference to fig. 5, which shows a flow diagram of an illustrative lyophilization process 220. To perform lyophilization, the 96-well plate, once filled, may be shielded in a sterile biological protective cover, as shown at step 402. Other plate sizes are also conceivable. After all plates are masked, they can be immediately transported and loaded into a lyophilizer, as shown at step 404. The lyophilizer can be sealed and the lyophilization cycle started. The product is frozen by reducing the product shelf temperature to a range of about-40 ℃ to-45 ℃, as shown at step 406. After the product is frozen, primary drying (sublimation) is performed by drawing a vacuum and raising the shelf temperature up to 0 ℃, as shown at step 408. A secondary drying step is initiated to further reduce the moisture content and bring the product to ambient temperature (about 25 c), as shown at step 410. At the end of the secondary drying step, the vacuum is released and the product is removed from the lyophilizer, as shown at step 412. The product may be placed inside the anaerobic chamber for collection of the lyophilized aliquot. The lyophilized aliquot may be in pellet form and transferred to a package containing a desiccant, as shown at step 414. The filled package may be purged with nitrogen and heat sealed as shown at step 416. Returning now to fig. 3, if the intermediate MRT composition has been shipped off-site for lyophilization, the lyophilized pellets can be shipped back to the MRT composition manufacturer in a pre-approved transporter for refrigerated conditions, as shown at step 222.

In some cases, it is desirable that the lyophilized material or pellet has a glass transition temperature (T) above 30 ℃_g). In some examples, the glass transition temperature may be in the range of 30-75 ℃. This results in a final product that is stable at room temperature. The glass transition temperature may also be used as a means to screen the product received from the lyophilization process and/or to verify the stability of the final product. E.g. T_gCan be used to predict the stability of the product during storage. In some cases, T of 50 ℃ over the storage temperature_gThe lyophilized intermediate and/or final oral drug product may be allowed to be stored for a period of time without significant loss of bacteria.

When the lyophilized intermediate is received, it may be removed from the package and filled into a capsule, as shown at step 224. Lyophilized intermediates can also be sampled and the overall survival measured using the PMA-qPCR method. Encapsulation may be performed in a nitrogen purge zone at ambient temperature to minimize contact of the lyophilized intermediate with oxygen. The lyophilized intermediate was encapsulated in hydroxypropyl methylcellulose capsules. Based on the capsule size (e.g., 1,0, or 00 size), multiple lyophilized intermediates can be loaded into a hydroxypropyl methylcellulose capsule.

The capsule may then be edge sealed as shown at step 226. In some cases, the capsules can be edge sealed with hydroxypropyl methylcellulose. In some cases, the edge sealing material may be an anionic copolymer based on methacrylic acid and methyl methacrylate, such as, but not limited to

L100. In other cases, the edge banding material can be hydroxypropylmethyl cellulose ortho-cellulosePhthalate or hypromellose acetate succinate. These are only examples. The edge sealing material may be any material that is resistant to degradation in low pH environments (e.g. stomach) and in high pH environments (e.g. intestine). Each capsule was given a consistent edge seal thickness so the disintegration performance met the acceptance limit. Additionally, the capsule may not be edge-sealed and/or otherwise be free of edge-sealing material. The capsules were stored under refrigeration at 5 ± 3 ℃ in nitrogen purged bulk plastic containers or packages containing desiccant. The packaged and edge sealed pharmaceutical product may be packaged in a package containing a desiccant and heat sealed, as shown at step 228. In some cases, the packaged and edge sealed pharmaceutical product may be packaged in individual doses of medication in a metallized polyester/polyethylene adhesive film. This may minimize exposure of the drug to oxygen and/or moisture which may lead to product degradation. The metallized polyester/polyethylene adhesive film may have a thickness of 0.02gr/100in²(ii) a moisture vapor transmission rate of 0.0402/mL/100in²Oxygen permeability (within 24 hours). The adhesive film pouch may be provided to a patient in a child-resistant container to meet the need for a child-resistant clinical supply package. The child-resistant container may be a 40 gram (2.5 ounce) green vial with a child-resistant cap. The vial may be made of translucent, light-resistant polypropylene. Low Density Polyethylene (LDPE) child-resistant closures help prevent unauthorized access by requiring a user to depress the closure and rotate the closure to open the container.

Referring now to fig. 4, an illustrative method 300 for preparing a previously purified fecal sample (drug B) for MRT as an oral dosage form is shown. Drug B can be a fecal microbiota frozen preparation prepared in an enema form comprising human feces enemas performed at a ratio of 1g of feces to 3mL of solution and 2.3% polyethylene glycol 3350 (or its cryoprotectant) solution and 0.9% sodium chloride solution. For example, drug B has been treated in a manner similar to steps 200 to 212 described above, and a cryoprotectant is added at step 202. Following the centrifugation process outlined in step 212, the purified intermediate (e.g., now drug B) may be refrigerated, frozen, or otherwise used for treatment.

Beginning at step 302, the frozen preparation can be thawed, if necessary, and placed in a filter bag. It is contemplated that multiple collection containers (e.g., the same or different donors and collected at various times) within their life data may be used. The sample may be purified by centrifugation, membrane filtration, or a combination thereof to remove fecal material above a certain particle size. It is conceivable that the sample may be treated to remove particles larger than in the 50-70 μm range, since the majority of the bacteria of interest is in the 0.3 micrometer (μm) to 30 μm range. The sample may be treated to obtain a concentration of about 60% bacteria. This may allow for increased flexibility in the ratio of formulation adjuvants to bacteria for further processing.

The filtration solution or diluent may be added to the filter bag, as shown at step 304. In some cases, saline may be used as a diluent. For example, a 0.9% sodium chloride (NaCl) solution can be added to the filter bag at a rate of about 3 milliliters (mL) per gram of drug B. It is contemplated that other diluents, other diluent concentrations, and dilution ratios may be used, as desired. The sample may then be subjected to membrane filtration in a number of different ways, including but not limited to the use of a filter bag, a pressure filter, and/or a vacuum filter, as shown at step 306. In some cases, the sample may be filtered multiple times with smaller filter membranes, each followed by subsequent filtration. In one example, the sample can be placed in a 500 μm filter bag and stirred at 230rpm for about 2 minutes using, for example, a Stomacher stirrer to obtain a filtrate having a particle size below about 500 μm. The filtrate can then be placed in a filter bag having a pore size of less than 500 μm, for example 280 μm. The sample can be again stirred at 230rpm for about 4 minutes, e.g., using a Stomacher stirrer, with or without diluent, to obtain a filtrate having a particle size below about 280 μm. The filtrate can be placed in another filter bag having a pore size of less than, for example, 280 μm, such as, but not limited to, 50-70 μm. The sample can be stirred at 230rpm for about 4 minutes, again using, for example, a Stomacher stirrer, with or without a diluent, to form a filtrate having a particle size below about 50-70 μm.

In another example, the sample can be placed in a 500 μm filter bag with or without diluent and stirred using, for example, a Stomacher stirrer to obtain a filtrate having a particle size below about 500 μm. The filtrate was then treated with a pressure filter having a pore size of about 160 μm, and the resulting filtrate was then treated with a pressure filter having a pore size of about 60 μm. In some cases, it may be necessary to perform a second treatment of the sample with a bag filter having a pore size between 160 μm and 500 μm before using the pressure filter.

Once the sample has been processed to have a particle size of about 50-70 μm or less, the sample may be placed in an intermediate storage container, as shown at step 308. An example of an acceptable intermediate storage container is a 250mL lidded sterile plastic container. In some cases, the filtered suspension may be stored in a refrigerator at a temperature of 5 ± 3 ℃ for up to 5 days, although this is not essential. The filtered suspensions may be combined and mixed into a larger container, as shown at step 310. One example of an acceptable intermediate storage container is a multi-liter sterile plastic container with a lid.

An aliquot of the mixed filtered suspension may then be placed into a centrifuge tube having a volume of 50 to 500mL, as shown at step 312. The filtered suspension is filled to approximately 20 to 80% of the volume of the centrifuge tube. In some cases, centrifuge tubes having a volume greater than 500mL may be used. The filtered suspension may then be washed and further concentrated using centrifugation, as shown at step 314. In one example, the sample may be centrifuged at 1100 to 3600 revolutions per minute (rpm) for a period of 10 to 15 minutes. In another example, the sample may be centrifuged for 15-45 minutes or 20-30 minutes at a rotational speed such that the centrifugal force is in the range of about 8-12,000g (e.g., about 10,000 g). The centrifugation can be accelerated or gradually accelerated to a speed required to establish a centrifugal force in the range of about 8-12,000g (e.g., about 10,000 g). It is also conceivable that the centrifugation can also be slowed or slowed down slowly when the centrifugation process is completed. In some cases, it is desirable to decelerate the centrifugation as slowly as possible so that the process of returning to atmospheric pressure is slow, thereby preventing possible rupture of the bacterial cells. The supernatant was removed and the remaining material in the tube was the purified intermediate MRT composition. This may form a product that has been concentrated up to about 60%.

In some cases, the centrifugation process may be a 2-stage process. For example, the product may first undergo a "pre-spin" (e.g., about 300-. For example, after "pre-spinning", the supernatant can be transferred to a new centrifuge tube/vial and then spun at a higher speed (e.g., about 5,000-. After high speed rotation, the supernatant can be discarded and the recovered microbiota can be further processed. It is also contemplated that volumes of up to 300mL may be centrifuged without causing a decrease in the amount of concentration. In some cases, a volume greater than 300mL may be centrifuged. For example, as described above, the centrifugation volume may be selected as a percentage of the volume of the container (e.g., in the range of 60%). The resulting MRT composition is of a particle size of 70 μm or less and about 1X10¹⁰Bacterial suspensions at bacterial concentrations of the order of CFU/g. Bacterial survival of the purified intermediates can be measured using the quantitative polymerase chain reaction (qPCR) method with propidium monoazide bromide (PMA). The resulting MRT composition was also stable for up to 3 weeks in the frozen state.

In some embodiments, multiple centrifugations may be performed separately for purification and concentration. However, the particle size of the bacterial suspension may still be in the range of clogging the pipette tip (e.g. greater than 60 μm). Whether this is successful depends on the variable input fecal material. It is also conceivable that a system of separators and decanters could be used if the batch size is in the range of tens of litres or more.

The intermediate MRT composition may optionally be transferred to an intermediate vial and, if necessary, shipped to a lyophilization apparatus, as shown at step 316. If necessary, the purified intermediate can be shipped to a contract lyophilizer in a pre-approved transporter for 5 ± 3 ℃ refrigerated conditions for lyophilization.

The purified intermediate may be mixed with a lyophilization excipient solution in a 1:1 ratio, as shown at step 318. The lyophilization excipient solution may include 2.3% PEG3350, 1% glycerol, 10% trehalose, and 10% sucrose. However, other lyophilization excipients may also be used. The lyophilized excipient solution (without glycerol) was filtered through a 0.2 μm filter before the excipient solution was added to the purified intermediate. Glycerol was autoclaved at 121 ℃ for a minimum of 15 minutes and added aseptically. Once the lyophilization excipient and purified intermediate have been mixed (lyophilization suspension), a single 200 microliter (200 μ Ι _ L) aliquot of the lyophilization suspension is placed into each well of a 96-well plate, as shown at step 320, and lyophilized, as shown at step 322.

The purified intermediate can be mixed with the lyophilization excipient solution in a suitable ratio (e.g., a ratio of 1: 1), as shown at step 318. In some cases, the lyophilization excipient may include polyethylene glycol (e.g., about 1-5%, or about 2-3%, or about 2.3%), trehalose (e.g., about 1-25%, or about 5-15%, or about 10%), sucrose (e.g., about 1-25%, or about 5-15%, or about 10%), and glycerol (e.g., about 0.1-5%, or about 0.5-2%, or about 1%) in purified water. For example, a lyophilization excipient solution may include 2.3% PEG3350, 1% glycerol, 10% trehalose, and 10% sucrose. However, other lyophilization excipients may be used. The lyophilized excipient solution (without glycerol) was filtered through a 0.2 μm filter before the excipient solution was added to the purified intermediate. Glycerol was autoclaved at 121 ℃ for a minimum of 15 minutes and added aseptically. Once the lyophilization excipient and purified intermediate have been mixed (lyophilization suspension), a single 200 microliter (200 μ Ι _ L) aliquot of the lyophilization suspension is placed into each well of a 96-well plate, as shown at step 320, and lyophilized, as shown at step 322.

Another exemplary method may include manufacturing an MRT composition suitable for use as a medicament for administration by enema and/or suitable for use as a raw material for manufacturing a medicament for oral administration. The process may include collecting a fresh human stool sample from a pre-screened donor. Such processes may be similar to those disclosed herein and/or similar to those disclosed in U.S. patent nos. 9,675,648 and 9,629,881, which are hereby incorporated by reference. In some cases, multiple samples from the same donor will be collected and pooled. Pooled samples (also referred to as drugs) can be stored in sterile microbiological containers at 5 ℃ ± 3 ℃. One or more other samples and/or partially pooled samples may be stored as stock samples in sterile microbiological containers in a-80 ℃ freezer.

A portion of the pooled sample/drug (e.g., 50 ± 10g) may be removed from the pooled sample and placed in a filter bag assembly. The filter bag assembly may include a filter bag within an outer closed bag. An adjuvant solution (e.g., also understood as a diluent, cryoprotectant, or other solution) may be added to the drug. The adjuvant solution may comprise 10-90g/L, or about 20-50g/L or about 30g/L polyethylene glycol (e.g., polyethylene glycol 3350 powder) in 0.9% sodium chloride. The excipient solution may be added at a suitable rate, such as 1-5mL per 1g of drug (e.g., 3mL of excipient solution per 1g of drug). The process of placing the pooled sample/portion of the drug/sample in the filter bag assembly and then adding the excipient solution may be repeated until the pooled sample/drug is sufficiently depleted. For example, if the remaining pooled sample/drug weighs 50 ± 10g or more, another pooled sample/drug sample may be added to another filter bag assembly. Once the weight of the remainder of the pooled sample is less than 50 ± 10g, the remainder of the pooled sample/drug may be discarded and the filter bag assembly may be closed.

The closed filter bag assemblies containing the drug and excipient solutions can be mixed one at a time. For example, the filter bag assembly may be placed into a paddle mixer and processed (e.g., at a speed of about 230RPM for about 2 minutes). The operating time and speed of the paddle mixer can be controlled electronically and the settings can be verified before each batch of product is manufactured.

The first filter bag assembly processed in the paddle mixer may be opened, and the filtrate may be taken out and filled into the freezing pipe. The cryostraw can be Quality Controlled (QC) and stored in a freezer at-80 ℃. Samples of quality control drug release can be tested in QC laboratories. The stock samples may be stored in a freezer at-80 ℃.

The fill tube cap of an Ethylene Vinyl Acetate (EVA) enema bag may be removed, and 150 ± 30g of the microbial suspension (e.g., filtered drug and adjuvant) is extracted from the filter bag assembly and filled into the EVA bag through the fill port. After filling is complete, the fill tube cap is replaced and the EVA bag is sealed before removal from the biohazard cabinet. The fill tube on the EVA bag can be sealed between the bag body and the fill cap using a tube sealer to prevent inadvertent opening of the container seal. Each EVA enema bag can be pasted with a label marked with a medicine batch number and a quarantine batch state sticker.

The drug product under treatment may be refrigerated at 5 ℃. + -. 3 ℃ before being frozen in a freezer at-80 ℃. The drug product may be held at 5 ℃. + -. 3 ℃ for up to 24 hours before being frozen in a freezer at-80 ℃. The drug (e.g., drug contained in a sealed enema bag) can be transferred from refrigerated storage to a designated-80 ℃ drug quarantine freezer. The quarantine medication is left in this position until QC treatment. If all donors and QC test results are acceptable, the batch will be processed as a release. If the result is not acceptable, the batch will be considered rejected and discarded. The drug disposed of as accepted will be removed from the-80 ℃ quarantine freezer, labeled with an "accept" label, and transferred to the designated-80 ℃ freezer where the drug is released. The accepted, released medication, which may be similar to medication B, may be thawed and administered to the patient (e.g., by enema). A released drug product is also understood to be a suspended intermediate suitable for use in the manufacture of an oral MRT composition as described below.

Batch manufacturing of oral MRT compositions begins with the selection of multiple bags of a released pharmaceutical product (e.g., where each bag of released pharmaceutical product comprises a frozen suspension intermediate manufactured as described above). Each bag of released medication may be from the same donor. The selected bag may be thawed at a suitable temperature (e.g., room temperature) for a suitable time (e.g., about 2 hours), and a quantity (e.g., 500 grams) of the suspended intermediate may be transferred to one or more 1 liter centrifuge bottles. A diluent may be added to the suspension intermediate. The diluent may be Phosphate Buffered Saline (PBS) or other suitable diluent. The diluent may be added to the suspended intermediate in an appropriate ratio (e.g., 1:1 by weight), mixed with intermittent gentle shaking, and held at 4 ℃ for 30 minutes. At this point, the sample may be referred to as a diluted intermediate.

The diluted intermediate may be subjected to differential centrifugation. For example, the diluted intermediate may be centrifuged at a relatively low speed. For example, the diluted intermediate may be centrifuged at about 500-2,000Xg, or about 1,000-1,500Xg, or about 1,400Xg for about 1-5 minutes (e.g., 2 minutes). The low speed centrifugation can be performed at 4 ℃. + -. 3 ℃. The supernatant from this low speed rotation can be transferred to one or more new 1L centrifuge bottles (e.g., containing bottle liners) for further processing, and the pellet material can be discarded. The bottle containing the collected supernatant may then be centrifuged at a relatively high speed. For example, the collected supernatant may be centrifuged at about 5,000-. High speed centrifugation can be carried out at 4 ℃. + -. 3 ℃. After the high speed rotation, the supernatant may be discarded and the remaining pellets (e.g., pellets if multiple vials are centrifuged) retained for further processing. The pellets may be referred to as a recovered microbiota.

The lyophilization excipient/cryoprotectant may be added to the recovered microbiota in an appropriate ratio. For example, lyophilization adjuvants may be added to the recovered microbial flora at a ratio of 1:1 (w/w). In some cases, the lyophilization excipient may include polyethylene glycol (e.g., about 1-5%, or about 2-3%, or about 2.3%), trehalose (e.g., about 1-25%, or about 5-15%, or about 10%), sucrose (e.g., about 1-25%, or about 5-15%, or about 10%), and glycerol (e.g., about 0.1-5%, or about 0.5-2%, or about 1%) in purified water. For example, a lyophilization excipient solution may comprise 2.3% PEG3350, 1% glycerol, 10% trehalose, and 10% sucrose. The mixture loaded in the bottle liner can be mixed using a paddle mixer to form a homogeneous suspension. The resuspended microbial colony solution can be aliquoted into 96-well plates (e.g., 200 microliters per well) and lyophilized. Other plate types and/or single well dishes may be utilized.

The lyophilization process will be described further below with reference to fig. 5, which shows a flow diagram of an exemplary lyophilization process 220/322. To perform lyophilization, once filled, the 96-well plate can be masked in a sterile bioprotective shield, as shown at step 402. Other plate sizes are also conceivable. In some embodiments, trays with zero holes may also be used. This may maximize the volume available for receiving the lyophilized suspension, which may improve the efficiency of the lyophilization process. After all plates are masked, they can be immediately transported and loaded into a lyophilizer, as shown at step 404. The lyophilizer can be sealed and the lyophilization cycle started. The product is frozen by reducing the product shelf temperature to a range of about-40 ℃ to-45 ℃, as shown at step 406. After the product is frozen, primary drying (sublimation) is performed by drawing a vacuum and raising the shelf temperature up to 0 ℃, as shown at step 408. A secondary drying step is initiated to further reduce the moisture content and bring the product to ambient temperature (about 25 c), as shown at step 410. At the end of the secondary drying step, the vacuum is released and the product is removed from the lyophilizer, as shown at step 412. The product may be placed inside the anaerobic chamber for collection of the lyophilized aliquot. The lyophilized aliquot may be in the form of pellets and transferred to a package containing a desiccant, as shown at step 414. The filled package may be purged with nitrogen and heat sealed as shown at step 416. If the intermediate MRT composition has been shipped off-site for lyophilization, the lyophilized pellets can then be shipped back to the MRT composition manufacturer in a pre-approved transporter for refrigerated conditions, as shown at step 324. In some cases, the lyophilized pellet may be referred to as a lyophilized intermediate.

In some cases, the composition (% w/w) of the lyophilized intermediate may include a post-treatment microbiota (e.g., about 10-75%, or about 40-60%, or about 45-50%, or about 47.9%), polyethylene glycol (e.g., about.1-10%, or about 3-8%, or about 5.2%), glycerol (e.g., about 0.5-5%, or about 1-4%, or about 2.2%), trehalose (e.g., about 10-40%, or about 20-30%, or about 22.4%), and sucrose (e.g., about 10-40%, or about 20-30%, or about 22.4%). The treated microbial population may comprise about 1X10⁵To 1X10¹²Live bacteria, or about 1X10⁶To 1X10¹¹Individual living bacteria, or about 1X10⁷To 1X10¹⁰Individual live bacteria. It will be appreciated that the composition of the lyophilized intermediate may be mechanically processed prior to being placed in a capsule, representing the composition of the active ingredient of the oral MRT composition. The disclosed compositions are obtained when the manufacturing process utilizes a lyophilization excipient comprising polyethylene glycol (e.g., about 1-5%, or about 2-3%, or about 2.3%), trehalose (e.g., about 1-25%, or about 5-15%, or about 10%), sucrose (e.g., about 1-25%, or about 5-15%, or about 10%), and glycerol (e.g., about 0.1-5%, or about 0.5-2%, or about 1%) in purified water.

In some cases, it is desirable for the lyophilized pellets to have a glass transition temperature (T) greater than 30 ℃_g). In some examples, the glass transition temperature may be in the range of 30-75 ℃. This results in a final product that is stable at room temperature. The glass transition temperature may also be used as a tool for screening received products from a lyophilization process and/or for verifying the stability of the final product. E.g. T_gCan be used to predict the stability of the product during storage. In some cases, T of 50 ℃ over the storage temperature_gThe lyophilized intermediate and/or final oral drug product may be allowed to be stored for a period of time without significant loss of bacteria.

Upon receiving the lyophilized intermediate, it may be removed from the package, milled or otherwise broken into a smaller granular and/or powdered consistency, and the milled material may be filled into capsules, as shown at step 326. The lyophilized intermediates can also be sampled and the overall survival measured using the PMA-qPCR method (an exemplary method is disclosed in, for example, U.S. patent application publication No. US2017/0327862, which is incorporated herein by reference). Encapsulation was performed at ambient temperature in a nitrogen purge zone to minimize contact of the lyophilized intermediate with oxygen. The lyophilized intermediate is encapsulated in one or more hydroxypropyl methylcellulose capsules. Multiple lyophilized intermediates (e.g., multiple pellets) can be loaded into a hydroxypropyl methylcellulose capsule based on capsule size (e.g., 1,0, or 00 size). For example, the ground drug product may be enclosed in a size 0 capsule using a manual capsule filling apparatus within a NF grade nitrogen purged glove box. The filled capsules may be tested for inter-capsule content uniformity. These capsules can then be filled into size 00 capsules. In other words, the drug product may be double-packaged.

Optionally, the capsule may be edge sealed, as shown at step 328. In some cases, the capsules were edge sealed with hydroxypropyl methylcellulose. In some cases, the edge banding material may be Eudragit L100, hydroxypropyl methylcellulose phthalate, or hydroxypropyl methylcellulose acetate/succinate. These are only examples. The edge sealing material may be any material that is resistant to degradation in low pH environments (e.g. stomach) and in high pH environments (e.g. intestine). Each capsule is given a consistent edge seal thickness, so the disintegration properties meet the acceptance limits. The capsules were stored under refrigeration at 5 ± 3 ℃ in nitrogen purged bulk plastic containers or packages containing desiccant. The capsules may be stored in a package containing a desiccant and heat sealed, as shown at step 330. In some cases, the capsules may be packaged in individual doses of medication in metallized polyester/polyethylene adhesive films. This may minimize exposure of the drug product to oxygen and/or moisture, which may lead to product degradation. The metallized polyester/polyethylene adhesive film may have a thickness of 0.02gr/100in²(ii) a moisture vapor transmission rate of 0.0402/mL/100in²Oxygen permeability (within 24 hours). The adhesive film pouch may be provided to the patient in a child-resistant container to satisfy child-resistanceThe requirement for clinical supply packaging. The child-resistant container may be a 40 gram (2.5 ounce) green medicinal vial with a child-resistant cap. The vial may be made of translucent, light-resistant polypropylene. The Low Density Polyethylene (LDPE) child-resistant cap helps prevent unauthorized access by requiring a user to depress the cap and rotate the cap to open the container.

In at least some examples, the moisture content of the packaged pharmaceutical product (e.g., including a dual packaged pharmaceutical product) is less than or equal to about 10%, or less than or equal to about 8%, or less than or equal to about 6%. In at least some examples, the packaged pharmaceutical product is packaged to limit/minimize any further moisture renewal.

In at least some examples, the pharmaceutical product can include about 10% or more of bacteria from the bacteroides class, or about 15% or more of bacteria from the bacteroides class, or about 20% or more of bacteria from the bacteroides class.

Examples

The disclosure is further illustrated by reference to the following examples, which are intended to be illustrative of some embodiments and not limiting of the invention.

Example 1: determination of the collapse temperature of MRT sample formulations

The collapse temperature results for the 12 sample microbiota recovery therapeutic formulations were determined. The collapse temperature can be used to help develop optimized formulation and lyophilization cycle parameters to freeze-dry this type of product without affecting its physical or chemical integrity in a reasonable amount of time. Standard lyophilization cycles were performed on these formulations, and the anaerobic microbial cell suspensions contained.

Example 2: materials and methods for freeze-drying microscopy

12 preparations were used for examination. Each matrix contains 10% of skimmed milk, 1% of ascorbic acid, 1.4% of gelatin and 0.3% of activated carbon. The ingredients are food grade, USP (united states pharmacopeia) or NF grade chemicals. The matrix was then supplemented with each of the following additives:

trehalose 10% and sucrose 10%

Sucrose 10% and inositol 5%

Trehalose 10% and glycerol 1%

Raffinose 10% and inositol 5%

Raffinose 10% and Glycerol 1%

Glucose 5% and inositol 5%

PEG 1% and sucrose 10%

PEG 1% and Glycerol 1%

Trehalose 10%, sucrose 10% and glycerol 1%

Sucrose 10% and lactose 8%

Trehalose 10% and inositol 5%

PEG 1% and lactose 8%

Each formulation was prepared. The freeze-dried microscopy instrument consisted of an Olympus BX53 polarizing microscope with Linkam FDCS196 hot stage, a T95 system controller, an LNP liquid nitrogen pump, and an Edwards E2M1.5 vacuum pump.

A 20 microliter (μ L) aliquot of a 100 milliliter (ml) sample was placed on a glass slide that had been placed on a hot stage after a small drop of silicone oil was added. A small cover slip was placed over the sample and the chamber was sealed. The sample was then cooled to-45 degrees Celsius (C.) at 10 deg.C/min. The temperature at which the material became frozen during the cooling phase was recorded. Once the temperature had dropped to-45 deg.C, vacuum was started. The product samples were then warmed at 1 deg.C/minute. The product samples were continuously monitored during this period to observe the drying and sublimation fronts. Once evidence of collapse was observed, the temperature was recorded. Table 3 is a summary of the freezing and collapse temperatures for each formulation.

TABLE 3 freezing and collapse temperatures recorded for each formulation

Preparation	Freezing temperature	Collapse temperature
			Trehalose and sucrose	-20℃	-24℃
Sucrose and inositol	-15℃	-20℃
			Trehalose and glycerol	-16℃	-24℃
Raffinose and inositol	-22℃	-22℃
			Raffinose and glycerol	-18℃	-26℃
Glucose and inositol	-13℃	-23℃
			PEG and sucrose	-11℃	-23℃
PEG and Glycerol	-12℃	-22℃
			Trehalose, sucrose and glycerol	-16℃	-26℃
Sucrose and lactose	-17℃	-25℃
			Trehalose and inositol	-16℃	-25℃
PEG and lactose	-17℃	-20℃

The lyophilization cycle is affected by a number of factors, including the percent solids in the formulation, vial size and diameter, collapse temperature, chamber pressure, shelf temperature, product resistance, and the like. The chamber pressure and shelf temperature necessary to complete the primary drying process are determined by the thermal properties of the formulation, primarily the collapse temperature. The primary drying temperature is lower than the collapse temperature to compensate for the product warming due to the increased resistance of the growing drying layer. Three cycles are designed based on a combination of the above factors. All cycle times were completed in less than 48 hours. Table 4 is a summary of the drying temperature and chamber pressure for the lyophilization cycle based on the critical collapse temperature.

TABLE 4 Primary drying temperature and Chamber pressure for Freeze drying cycle based on Critical collapse temperature

Critical temperature	Primary drying temperature	Room pressure
			-20 ℃ to-22 DEG C	-30℃	120 mTorr
-23 ℃ to-24 DEG C	-33℃	95 mTorr
			-25 ℃ to-26 DEG C	-35℃	75 mTorr

The lyophilization cycle was designed based on data collected during the freeze-drying microscopy study. A pilot lyophilization cycle was performed for each formulation to examine the cake structure and viability of the bacterial cell mixture. Harvesting of cells and dispersion of suspensions was done based on the protocol established by Gibson Bioscience to obtain aliquots of mixture per 100 microliters of colony forming units in the range of microorganisms from 10e7 to 10e 8. The microbial population used was selected from the first stage of the study and included the following anaerobes: bacteroides monomorphus Bacteroides ATCC 8492^TM、Alistipes putredinis ATCC 29800^TMRuminococcus gnavus ATCC 29149, Ruminococcus gnavus^TMAnd Bacteroides ovorans ATCC 8484^TM。

Viable cell number (CFU) before and after lyophilization was determined using serial dilution. Each dilution included the following levels: 10e3, 10e5, 10e7, and 10e 9. Pellet samples were rehydrated in 1mL of phosphate buffered saline. All samples were plated in duplicate onto pre-reduced CDC anaerobic blood agar plates and selective bacteroides bile esculin agar plates. Agar plates were incubated in an anaerobic environment at 35-37 ℃ for up to 48 hours.

For all formulations, the lyophilization cycle formed a good quality cake-like structure. Each pellet was solid and had a uniform appearance. Each lyophilized pellet dissolved within 30 seconds when rehydrated in 1.0mL of phosphate buffered saline. Viability was calculated as the percentage of the total number of bacterial colony forming units after freeze-drying divided by the total number of colony forming units before freeze-drying. Colony forming units are based on a mixture of 4 organisms. The stock and percent survival of all colony forming units for each formulation are summarized in tables 5 and 6.

TABLE 5 stock and percent survival of all colony forming units of each formulation inoculated directly onto CDC anaerobic blood agar

Data conversion based on logarithm

TABLE 6 stock and percent survival of total colony forming units for each formulation inoculated directly onto Bacteroides selectivatus bile esculin agar

Data conversion based on logarithm

Based on the data collected, the selected anaerobes showed the highest survival when trehalose and sucrose or a combination of sucrose and inositol was used for the base preparation. This was true for recovery on both CDC anaerobic blood agar and selective bacteroides bile esculin agar. These results indicate that the combination of trehalose with sucrose or sucrose with inositol provides the best protection for bacteroides during lyophilization.

Example 3: bacterial stability of solid products during storage

Studies were conducted to determine the stability of packaged encapsulated capsules after manufacture and upon storage. The active components (bacteria) present in the solid drug product were characterized using standard microbial plate counting methods, molecular non-cultured PMA-qPCR methods, and 16s rRNA gene sequencing of both PMA and non-PMA treated samples. Plate count and total survival stability data indicate: the lyophilized packaged product (using the first lyophilization process) is more stable under colder storage conditions (5 + -3 deg.C) than under higher storage temperatures and relative humidities (25 + -2 deg.C/60% + -5% RH and 30 + -2 deg.C/65% + -5% RH). Plate count and total survival stability data for lyophilized packaged products (using the second lyophilization process) indicate that the packaged products are stable at storage temperatures of about 5 ± 3 ℃ and 25 ± 2 ℃.

Example 4: manufacture of a medicament suitable for administration by enema and/or manufacture of a starting material for oral administration Exemplary procedure for MRT composition of Material

Note: steps 1-3 can be performed in a class II B2 biosafety cabinet. All instruments used for handling drugs, adjuvant solutions or drug formulations (e.g. tongue depressors, serological pipettors) are single-use instruments supplied pre-sterilized by the manufacturer. Filter bags, seal bags and EVA bags are also supplied by the manufacturer for pre-sterilization.

Step 1-Collection of Donor pooled drug test samples and drug stock samples

Fresh human stool samples can be collected from pre-screened donors. In some cases, multiple samples may be taken from the same donor and pooled. Pooled samples can be stored in sterile microbiological containers at 5 ℃ ± 3 ℃. One or more additional samples and/or a portion of the pooled sample may be stored as a stock sample in a sterile microbiological container at-80 ℃.

Step 2-dispensing the medicament

50 + -10 g of a sample (e.g., a drug) can be drawn from the pooled samples and placed in a filter bag assembly. The filter bag assembly may include a filter bag within an outer closed bag.

Step 3-dispensing of the adjuvant solution

An adjuvant solution (e.g., also understood as a diluent, cryoprotectant, or other solution) may be added to the drug substance. The adjuvant solution may include a solution of 30g/L polyethylene glycol (e.g., polyethylene glycol 3350 powder) in 0.9% sodium chloride. Can be added in a proportion of 3mL of the adjuvant solution per 1g of the drug. If the remaining weight of the pooled samples is 50 + -10 g or more, steps 2 and 3 may be repeated. If the remaining weight of the pooled sample is less than 50 ± 10g, the remaining portion of the pooled sample may be discarded and the filter bag assembly closed.

Step 4-mixing and filtration

The closed filter bag assembly with drug and excipient solutions was placed in a paddle mixer and processed at 230rpm for 2 minutes each time. The paddle mixer run time and speed are electronically controlled and settings are verified before each batch of product is manufactured.

Note: steps 5-6 were performed in a class II B2 biosafety cabinet.

Step 5-collecting the drug quality control and storage samples

The first filter bag treated in the paddle mixer is open and filtrate can be withdrawn and filled into the freeze tube. The frozen bottles may be submitted for Quality Control (QC) and stored in a freezer at-80 ℃. QC samples were taken from only the first dose produced in each batch.

Step 5A-drug quality control Release test

Samples of quality control drug release were tested in a QC laboratory. The stock samples may be stored in a freezer at-80 ℃.

Step 6-filling

The fill tube cap of an Ethylene Vinyl Acetate (EVA) enema bag may be removed, and 150 ± 30g of the microbial suspension (e.g., filtered drug and adjuvant) is extracted from the filter bag assembly and filled into the EVA bag through the fill port. And after filling, replacing the filling pipe cover, sealing the EVA bag and taking out the EVA bag from the biological safety cabinet.

Step 7-sealing the EVA bag

The fill tube on the EVA bag can be sealed between the bag body and the fill cap using a tube sealer to prevent inadvertent opening of the container seal.

Step 8, attaching batch identification label and quarantine label

Each EVA enema bag is pasted with a label marked with a medicine batch number and a quarantine batch state sticker.

Step 9-refrigerating the medicine at 5 +/-3 DEG C

The drug product under treatment was refrigerated at 5 ℃. + -. 3 ℃ before being frozen in a-80 ℃ freezer. The medicine can be stored at 5 + -3 deg.C for 24 hr before being frozen in a freezing chamber at-80 deg.C.

Step 10, putting the medicine into a freezing chamber with the temperature of 80 ℃ below zero for freezing

The medicines (e.g., the medicines contained in the sealed enema bag) can be transferred from the refrigerated storage to a prescribed-80 ℃ medicine quarantine freezing chamber. The drug being quarantined is left in this position until QC treatment.

Step 11-treatment of the drug

If all donors and QC test results are acceptable, the batch will be processed as a release. If the result is not acceptable, the batch will be considered rejected and discarded.

Step 12-Release of drug

The drug disposed of as accepted will be removed from the-80 ℃ quarantine freezer, labeled with an "accept" label, and transferred to the designated-80 ℃ freezer where the drug is released. The accepted, released medication may be thawed and administered to the patient (e.g., via an enema).

Example 5: exemplary Process for manufacturing oral MRT compositions from released pharmaceutical products

Step 1-thawing of suspended intermediates

Batch manufacturing of oral MRT compositions begins with the selection of multiple bags of a released pharmaceutical product (e.g., where each bag of released pharmaceutical product comprises a freeze-suspended intermediate manufactured as described in example 4). Each bag of released medication is from the same donor. The selected bags were thawed at room temperature for at least 2 hours and 500g of the suspension intermediate could be transferred to a 1 liter centrifuge bottle.

Step 2-addition of Diluent

Phosphate Buffered Saline (PBS) may be added to the suspended intermediate in a 1:1 weight ratio, mixed with intermittent gentle shaking, and held at 4 ℃ for 30 minutes.

Step 3-differential centrifugation

The diluted intermediate was centrifuged at 1,400Xg and 4 ℃. + -. 3 ℃ for 2 minutes. The low speed spun supernatant can be transferred to a new 1L centrifuge bottle containing a bottle liner for further processing and the pellet material can be discarded. The bottle containing the collection supernatant may then be centrifuged at 10,000Xg and 4 ℃. + -. 3 ℃ for 45 minutes. After high speed rotation, the supernatant can be discarded and the recovered microbiota retained for further processing.

Step 4-resuspending the recovered microbiota with lyophilized adjuvant solution

The lyophilization excipient/cryoprotectant may be added to the recovered microbial flora at a ratio of 1:1 (w/w). The lyophilization excipient may comprise 2.3% polyethylene glycol (e.g., PEG3350), 10% trehalose, 10% sucrose, and 1% glycerol in purified water. The mixture may be mixed using a paddle mixer to form a homogeneous suspension.

Step 5-Freeze drying

The resuspended microflora solution is aliquoted into 96-well plates (e.g., 200 microliters per well) and placed into a qualified lyophilizer. The lyophilization steps are shown in the following table.

Table 7: details of the lyophilization cycle

Step 6-grinding operation

In the NF grade nitrogen purged glove box workstation, the lyophilized drug particles were milled through a low energy mill.

Step 7-double encapsulation

The ground drug was encapsulated in a No. 0 capsule using a manual capsule filling device inside an NF grade nitrogen purged glove box. The filled capsules may be tested for inter-capsule content uniformity. These capsules can then be filled into size 00 capsules.

Step 7A-fill uniformity test (g/g)

A representative set of fill No. 0 capsule samples was weighed to ensure uniformity of fill.

Step 8-bulk packaging and labeling

The capsules are individually contained in an aluminium blister pack and provided with a child resistant lid foil. The blister packs were labeled and loaded into dose cartons and then stored at 5 ℃ ± 3 ℃.

Step 9-Collection of final drug QC, stability and stock samples

A representative sample test was performed on the filled size 00 capsules for product release.

Step 9A: release test of the final drug product

The drug may be tested for release.

Step 10-quarantine withholding

The batches of capsules are placed in designated quarantine medication coolers prior to packaging.

Step 11-end product treatment

If all QC test results and batch record review requirements are acceptable, the batch will be processed as accepted. If the result is not acceptable, the batch will be disposed of as rejected and discarded. Quality assurance will dispose of the final product batch as accepted or rejected.

Step 12-Final Package and Label

Step 13-Release

The product batch which is treated as accepted is taken out from the quarantine drug library, is pasted with an acceptance label and is transferred into a specified release drug library with the temperature of 5 +/-3 ℃.

Treatment holding time: the hold times for each intermediate step are shown in table 8.

TABLE 8 treatment hold times

Example 6 batch formulation

TABLE 9 batch formulation before encapsulation of microbial flora capsules

NA-not applicable

^aThe components are removed during the treatment; traces of sodium chloride and phosphate may remain after purification

^bComponents removed during lyophilization

As described in example 4, each batch of starting material was 60 released doses of the released drug, corresponding to approximately 9000 grams of starting material, and resulted in the recovery of approximately 1700 grams of the microbiota resuspended in the lyophilization medium. The material was lyophilized in a 96-well plate to give about 7700 lyophilized pellets. After milling of the freeze-dried pellets, the powder yield was about 300 g, the fill weight per capsule was 235 mg and the theoretical yield of capsules was about 1200 capsules per batch. Despite the donor-to-donor differences in starting materials, the estimated number of bacteria per mg is similar between donors and batches produced to date. The product is controlled by the fill weight.

Example 6 pharmaceutical product description and Components

The drug is a concentrated, lyophilized microbial powder encapsulated in an enteric capsule. The medicine comprises the following components (see tables 10-12).

TABLE 10 pharmaceutical compositions

NA-not applicable

^bReferred to in this document as trehalose, but formally named trehalose dihydrate.

^cComponents removed during lyophilization

^dRefer to

Capsule part 3.2.P.4.1

The milled lyophilized intermediate powder was filled and tamped into size 0 capsules without any excipients and over-encapsulated with size 00 capsules. The expected formulation percentage concentration is the same as the pre-encapsulated material.

TABLE 11 composition of lyophilized intermediate in each capsule

Table 12 describes the introduction of the drug.

TABLE 12 introduction of drugs

It should be understood that this disclosure is, in many respects, only illustrative. Changes may be made in details, particularly in matters of shape, size, and arrangement of steps without exceeding the scope of the disclosure. The scope of the invention is, of course, defined in the language in which the appended claims are expressed.

Claims

1. An oral microbiota restoration therapeutic composition comprising:

a capsule;

a lyophilized intermediate disposed within the capsule, the lyophilized intermediate comprising a fecal-derived microbiota, polyethylene glycol, trehalose, sucrose, and glycerol; and

wherein the lyophilized intermediate comprises 10-40% (w/w) sucrose.

2. An oral microbiota restoration therapeutic composition according to any one of claims 1 or 35 to 37 wherein the lyophilised intermediate comprises 40-60% (w/w) of a fecal-derived microbiota.

3. An oral microbiota restoration therapeutic composition according to any one of claims 1 or 35 to 37 wherein the lyophilised intermediate comprises 40-50% (w/w) of a fecal-derived microbiota.

4. The oral microbiota restoration therapeutic composition of any one of claims 1-3, 34, or 36-37, wherein the lyophilized intermediate comprises 1-10% (w/w) polyethylene glycol.

5. The oral microbiota restoration therapeutic composition of any one of claims 1-3, 34, or 36-37, wherein the lyophilized intermediate comprises 3-8% (w/w) polyethylene glycol.

6. The oral microbiota restoration therapeutic composition of any one of claims 1-5, 34-35, or 37, wherein the lyophilized intermediate comprises 0.5-5% (w/w) glycerol.

7. The oral microbiota restoration therapeutic composition of any one of claims 1-5, 34-35, or 37, wherein the lyophilized intermediate comprises 1-4% (w/w) glycerol.

8. An oral microbiota restoration therapeutic composition according to any one of claims 1 to 7 or 34 to 36 wherein the lyophilised intermediate comprises 10-40% (w/w) trehalose.

9. An oral microbiota restoration therapeutic composition according to any one of claims 1 to 7 or 34 to 36 wherein the lyophilised intermediate comprises 20-30% (w/w) trehalose.

10. The oral microbiota restoration therapeutic composition of any one of claims 1-9 or 34-37, wherein the lyophilized intermediate comprises 20-30% (w/w) sucrose.

11. An oral microbiota restoration combination therapeutic composition according to any one of claims 1 to 10 or 34 to 37 wherein the fecal derived microbiota is obtained from a fresh human fecal sample from a donor.

12. The oral microbiota restoration combination therapy composition of any one of claims 1-10 or 34-37, wherein the fecal derived microbiota is obtained from a plurality of pooled fresh human fecal samples from a single donor.

13. The oral microbiota restoration combination therapeutic composition of any one of claims 1-12 or 34-37, wherein the capsule comprises a bilayer capsule.

14. The oral microbiota restoration combination therapeutic composition of claim 13, wherein the bi-layer capsule comprises one capsule disposed within another capsule.

15. The oral microbiota restoration combination therapeutic composition of any one of claims 1-14 or 34-37, wherein the capsule is devoid of an edge seal.

16. An oral microbiota restoration combination therapeutic composition according to any one of claims 1 to 15 or 34 to 37 wherein the capsule is provided within an aluminium blister pack.

17. The oral microbiota restoration combination therapeutic composition of any one of claims 1-16 or 34-37, wherein the lyophilized intermediate comprises 22.4% (w/w) sucrose.

18. The oral microbiota restoration combination therapeutic composition of claim 17, wherein the lyophilized intermediate comprises 5.2% (w/w) polyethylene glycol.

19. The oral microbiota restoration combination therapeutic composition of claim 18, wherein the lyophilized intermediate comprises 2.2% (w/w) glycerol.

20. The oral microbiota restoration combination therapeutic composition of claim 19, wherein the lyophilized intermediate comprises 22.4% (w/w) trehalose.

21. An oral microbiota restoration combination therapeutic composition comprising:

a double-layer capsule; and

a lyophilized intermediate disposed within the bilayer capsule, the lyophilized intermediate comprising a fecal-derived microbiota, polyethylene glycol, trehalose, sucrose, and glycerol.

22. The oral microbiota restoration combination therapeutic composition of claim 21, wherein the bi-layer capsule comprises one capsule disposed within another capsule.

23. An oral microbiota restoration combination therapeutic composition according to any one of claims 12 to 22, wherein the bi-layer capsule is devoid of edge seals.

24. A package comprising an oral microbiota restoration therapy composition, the package comprising:

packaging the aluminum-aluminum bubble cap;

a plurality of capsules, each enclosed within the aluminum blister pack;

wherein each of the capsules comprises a lyophilized intermediate comprising fecal-derived microbiota, polyethylene glycol, trehalose, sucrose, and glycerol.

25. An oral microbiota restoration therapeutic composition comprising:

a capsule;

wherein the moisture content within the capsule is less than or equal to about 10%.

26. The oral microbiota restoration therapy composition of claim 25, wherein the moisture content within the capsule is less than or equal to about 8%.

27. The oral microbiota restoration therapy composition of claim 25, wherein the moisture content within the capsule is less than or equal to about 6%.

28. An oral microbiota restoration therapeutic composition comprising:

a capsule;

wherein the fecal-derived microbiota comprises 10% or more bacteria from the Bacteroides class.

29. The oral microbiota restoration therapy composition of claim 28, wherein the fecal-derived microbiota comprises 15% or more bacteria from the bacteroides class.

30. The oral microbiota restoration therapy composition of claim 28, wherein the fecal-derived microbiota comprises 20% or more of bacteria from the bacteroides class.

31. An oral microbiota restoration therapeutic composition comprising:

a capsule;

wherein the lyophilized intermediate comprises 1x10⁵To 1x10¹²And (4) living bacteria.

32. The oral microbiota restoration therapy composition of claim 31, wherein the lyophilized intermediate comprises 1x10⁶To 1x10¹¹And (4) living bacteria.

33. The oral microbiota restoration therapy composition of claim 31, wherein the lyophilized intermediate comprises 1x10⁷To 1x10¹⁰And (4) living bacteria.

34. An oral microbiota restoration therapeutic composition comprising:

a capsule;

wherein the lyophilized intermediate comprises 40-60% (w/w) of a fecal-derived microbiota.

35. An oral microbiota restoration therapeutic composition comprising:

a capsule;

wherein the lyophilized intermediate comprises 1-10% (w/w) polyethylene glycol.

36. An oral microbiota restoration therapeutic composition comprising:

a capsule;

wherein the lyophilized intermediate comprises 0.5-5% (w/w) glycerol.

37. An oral microbiota restoration therapeutic composition comprising:

a capsule;

wherein the lyophilized intermediate comprises 10-40% (w/w) trehalose.