TW202220639A - Lyophilisation process - Google Patents

Abstract

The invention relates to improved processes for lyophilising bacterial products which minimise the loss of viable cells during the lyophilisation process, enabling the products to be lyophilised in an efficient and economic manner. The lyophilisation process of the invention achieves improved viability of the lyophilised cells.

Description

Freeze drying method

The present invention relates to lyophilization methods of live biotherapeutic products that improve the viability of lyophilized live biotherapeutic products and the efficiency of making such products.

Lyophilization is a widely used method for formulating pharmaceutical, biotechnological and other types of products. It is an efficient way to prepare solid products, even if they are medicinal products that must be administered to patients in liquid form. Lyophilization is also a convenient way to produce formulations containing living organisms or chemically sensitive products obtained from organisms.

There are many commercial lyophilization methods; these methods typically involve three stages, namely i) freezing, ii) primary drying or sublimation and iii) secondary drying or desorption [1].

During the freezing stage and optionally also during the sublimation stage, the product is frozen by exposure to low temperatures typically between -20°C and -80°C. During this time, the fluid in the sample becomes a crystalline, amorphous or glassy solid body. The amorphous state occurs when the material is in a solid state but the molecules do not pack in a repeating long-range order. During one or more freezing steps, as the temperature decreases, so-called freeze-concentration continues until the solution reaches the glass transition temperature. This is the time point at which freeze-concentration is greatest and any further thermodynamically supported freeze-concentration is inhibited because the mobility of the remaining liquid in the sample is too low (glassy state) to allow crystallization to the ice interface. When the product has reached the target low temperature, the frozen material is placed under vacuum and heated gradually to deliver enough energy to sublime the ice. This is referred to as a preliminary drying step, which generally results in the removal of most of the ice present in the product. The preliminary drying step is usually not carried out at a temperature above the glass transition temperature, so that only sublimation of the water of crystallization occurs. The third stage (secondary drying or desorption step) begins when the ice has distilled off, so that the higher vacuum allows for the gradual extraction of bound water at temperatures above zero.

During lyophilization, products comprising living cells, such as probiotics, are exposed to various stresses, especially dehydration, which impair cell survival. Since water plays an important role in cellular integrity and stability, freezing is not only from a structural point of view considering ice crystal formation, but also due to increased solute concentrations in the non-frozen fraction (which can cause chemical and osmotic damage), freezing Both and drying can be detrimental to vitality. Many different strategies have been developed to enhance cell viability during freeze-drying, such as adding protective agents to the drying medium.

Known methods for lyophilizing bacterial cells (eg, in the manufacture of probiotic health supplements) achieve moderate levels of viability of live biotherapeutic products after lyophilization. While such loss of viability can be tolerated in the manufacture of probiotic products, these methods are not necessarily suitable for the manufacture of medicinal products (such as live biotherapeutic products or LBPs) that are subject to more stringent controls, including stringent requirements for maintaining cell viability . The inventors have determined that the viability of live biotherapeutic products can be significantly increased when an annealing step is incorporated into the lyophilization process.

In a first aspect, the present invention provides a method for preparing a lyophilized product comprising a viable bacterial population, the method comprising: (i) provide lyophilized media containing viable bacterial populations; (ii) subjecting the lyophilized medium to: a. One or more freezing steps; b. performing one or more annealing steps at a temperature different from the one or more freezing steps; and c. At least one preliminary drying step; and (iii) Collect the lyophilized product.

An annealing step is a step of changing the temperature to which the lyophilized medium is exposed, eg, increasing from the temperature of the freezing step to a temperature above the glass transition temperature of the lyophilized medium. Annealing can be performed during the initial cooling in the freezing step. Additionally or alternatively, annealing can be used as a post-freeze ramp and hold step followed by cooling (eg, to return the sample to below the glass transition temperature). Such annealing steps can dilute the remaining liquid in the glass state, allowing other processes, including ice crystal maturation, solute crystallization, and possible degradation reactions. The maturation of ice crystals increases the size of their crystals. When lyophilizing live biotherapeutic products, this ice crystal growth would be considered undesirable as it could disrupt bacterial cells leading to their inactivation, which would be highly unattractive as in the preparation of live biotherapeutics It is extremely important to maintain high bacterial viability. However, surprisingly and unexpectedly, performing an annealing step (as demonstrated in the Examples) has been shown to enhance the viability of bacterial cells in live biotherapeutic products.

In embodiments of the invention, one or more freezing steps are cycled with one or more annealing steps.

In certain embodiments, the water content of the lyophilized product is less than 5.0 wt%. In certain embodiments, the water content of the lyophilized product is between 3.0 wt% and 4.5 wt%. In certain preferred embodiments, the water content of the lyophilized product is between 3.3 wt% and 4.4 wt%. In certain embodiments, the water content of the lyophilized product is less than 4.5 wt%. In certain embodiments, the water content of the lyophilized product is less than 4.0 wt%. In certain embodiments, the water content of the lyophilized product is less than 3.5 wt%.

In certain embodiments, the viability (CFU/g, in dry weight) of the lyophilized product is equal to or less than 10 ³ CFU/g, equal to or less than the viability (CFU/ml) of the lyophilized medium prior to lyophilization 10 ² CFU/g or equal to or less than 10 CFU/g. Additionally or alternatively, the lyophilized product has about 1 x 10 ⁹ CFU/g or higher, about 5 x 10 ⁹ CFU/g or higher, about 1 x 10 ¹⁰ CFU/g or higher or 5 x 10 ¹⁰ CFU/g The vitality of g.

In certain embodiments, the annealing step is performed at a higher temperature than the freezing step preceding the annealing step. In certain embodiments, the annealing step is performed at a temperature higher than the glass transition temperature of the lyophilized product. Additionally or alternatively, the annealing step is performed at a temperature higher than the collapse temperature of the lyophilized product.

A "glass transition temperature" is the temperature at which an amorphous form, such as a lyophilized product, has a change in properties. In certain embodiments, the amorphous form will be brittle (ie, glassy) if the sample is stored below the glass transition temperature. If the sample is stored above the glass transition temperature, the amorphous form will become rubbery. At this temperature, the molecules in the glass exhibit significant changes in mobility.

The "collapse temperature" is the temperature at which the freeze-dried product collapses in structure.

Those skilled in the art will appreciate that determination of the glass transition temperature or collapse temperature of a lyophilized product is straightforward and can be accomplished using techniques and equipment with which they will be familiar. For the purposes of the present invention, where a heating/cooling step is performed with reference to the glass transition temperature or collapse temperature (eg where the annealing step is performed by heating the freeze-drying medium to or above the glass transition temperature) ), the lyophilized product would first need to be provided and its glass transition temperature or collapse temperature determined. Next, it will be possible to carry out the method according to the invention based on the glass transition temperature or collapse temperature of the lyophilized product. Those skilled in the art will of course recognize that such steps are routine and not onerous.

In embodiments of the present invention, the lyophilized product is a live biotherapeutic product. For the purposes of the present invention, a "live biotherapeutic product" ("LBP") is a composition comprising a population of live bacterial cells intended for therapeutic applications. The US FDA defines a live biotherapeutic product in a guide published in June 2016 (Early Clinical Trials with Live Biotherapeutic Products: Chemistry, Manufacturing, and Control Information) as a biological product that 1) contains a living organism, such as bacteria; 2) is suitable for the prevention, treatment or cure of a disease or disorder in humans; and 3) is not a vaccine. Therefore, LBP is a regulated product, which in order to be approved as a medical product (unlike conventional probiotics) must meet strict and legally responsible safety and efficacy requirements.

Unless otherwise stated, reference to "maintaining" relative to a temperature herein means that a particular temperature is achieved, regardless of the time spent at that particular temperature. The steps of the freeze-drying method

The lyophilization method includes at least the following steps: (i) provide lyophilized media containing viable bacterial populations; (ii) subjecting the lyophilized medium to: a. One or more freezing steps; b. Optionally at least one annealing step between the one or more freezing steps, which is performed at a different temperature than the one or more freezing steps; and c. At least one preliminary drying step; and (iii) Collect the lyophilized product.

In certain embodiments, the lyophilization method comprises at least the following steps: (i) provide lyophilized media containing viable bacterial populations; (ii) subjecting the lyophilized medium to: a. At least two freezing steps; b. at least one annealing step between the at least two freezing steps, which is performed at a different temperature than the two freezing steps; and c. At least one preliminary drying step; and (iii) Collect the lyophilized product.

In certain embodiments, the lyophilization method includes one or more additional steps, such as a pretreatment step and/or a secondary drying step. Freeze-dried medium available

Lyophilized media can be provided by preparing biomass comprising viable bacterial populations using conventional fermentation processes. In certain embodiments, the live biotherapeutic biomass can be concentrated. In certain embodiments, the biomass can be concentrated to ¹⁰⁸ bacterial cells per milliliter or gram. In certain embodiments, the biomass is concentrated to ¹⁰⁹ bacterial cells per milliliter or gram. In certain embodiments, the biomass is concentrated to ¹⁰¹⁰ bacterial cells per milliliter or gram. In certain embodiments, the biomass is concentrated to 10 ¹¹ bacterial cells per milliliter or gram. In certain embodiments, the biomass is concentrated to 10 ¹² cells per milliliter or gram.

The examples in this application demonstrate that the methods of the present invention can be used to prepare lyophilized material on a commercial scale. Therefore, in an embodiment of the present invention, the lyophilized medium contains at least about 1 g, at least about 2 g, at least about 5 g, at least about 10 g, at least about 20 g, at least about 50 g, at least about 100 g, at least about 200 g, at least about 500 g, Provided in an amount of at least about 1 kg or at least about 2 kg.

In certain embodiments, the lyophilized medium comprises a buffer. In certain embodiments, the buffer has a pH between 5.5 and 8.5.

Additionally or alternatively, the lyophilized medium contains excipients such as non-reducing sugars. In certain embodiments, the lyophilized medium comprises cryoprotectants such as sucrose, maltose, maltodextrin and/or glycerol. In certain embodiments, the lyophilized medium comprises sucrose. In certain embodiments, the lyophilized medium does not contain trehalose. In certain embodiments, the lyophilized medium comprises a compatibilizer, such as albumin, glycine, mannitol, hydroxyethyl starch, or dextran. In certain embodiments, the lyophilized medium comprises glycine. In certain embodiments, the lyophilized medium comprises a nonionic surfactant. In embodiments of the present invention, the lyophilized medium comprises antioxidants, such as cysteine. In a preferred embodiment, the lyophilization medium comprises a lyoprotectant formulation (at final concentration prior to lyophilization) containing the following components: 2% sucrose, 4% maltodextrin DE9, and 0.2% cysteine acid salt. In a preferred embodiment, the ratio of viable bacterial population to lyoprotectant is about 70:30. preprocessing step

In certain embodiments, the lyophilization method includes a pretreatment step. Such pretreatment steps may include diluting the viable bacterial population; modifying the formulation of the lyophilized medium, such as adding compounds to increase the stability of the viable bacterial population; reducing high vapor pressure solvents in the lyophilizing medium; and/or increasing the stability of the viable bacterial population. surface area.

In certain embodiments, the pretreatment step includes adding stabilizers and/or protectants to the lyophilized medium. Freezing step

In certain embodiments, the lyophilized medium is cooled to a temperature below the glass transition temperature of the lyophilized product during one or more freezing steps. Additionally or alternatively, during one or more freezing steps, the lyophilized medium is cooled to a temperature below the breakdown temperature of the lyophilized product. In certain embodiments, the temperature of one or more freezing steps is 0°C or less, about -5°C or less, about -10°C or less, about -15°C or less, about -20°C or less Low, about -25°C or lower or about -30°C or lower, eg, between -40°C and -80°C. In certain embodiments, the temperature of the one or more freezing steps is between -40°C and -60°C. In certain embodiments, the temperature of the one or more freezing steps is between -60°C and -80°C. In certain embodiments, the temperature of the one or more freezing steps is between -30°C and -40°C. In certain embodiments, the temperature of the one or more freezing steps is between -40°C and -50°C. In certain embodiments, the temperature of the one or more freezing steps is between -50°C and -60°C. In certain embodiments, the temperature of the one or more freezing steps is between -60°C and -70°C. In certain embodiments, the temperature of the one or more freezing steps is between -70°C and -80°C. In certain embodiments, the temperature of one or more freezing steps is -30°C or less. In certain embodiments, the temperature of one or more freezing steps is -35°C or lower. In certain embodiments, the temperature of one or more freezing steps is -40°C or less. In certain embodiments, the temperature of one or more freezing steps is -45°C or less. In certain embodiments, the temperature of one or more freezing steps is -50°C or less. In certain embodiments, the temperature of one or more freezing steps is -55°C or less. In certain embodiments, the temperature of one or more freezing steps is -60°C or lower. In certain embodiments, the temperature of one or more freezing steps is -65°C or less. In certain embodiments, the temperature of one or more freezing steps is -70°C or less. In certain embodiments, the temperature of one or more freezing steps is -75°C or lower. In certain embodiments, the temperature of one or more freezing steps is -80°C or lower. In a preferred embodiment, the temperature of the one or more freezing steps is between about -40°C and about -50°C. In a preferred embodiment, the temperature of one or more freezing steps is below -45°C. In another preferred embodiment, the temperature of one or more freezing steps is about -45°C.

In certain embodiments, the one or more freezing steps involve snap-freezing a lyophilized medium comprising a viable bacterial population. In certain embodiments, the flash freezing process involves the rapid cooling of the lyophilized medium, eg, using liquid nitrogen. In certain embodiments, the flash freezing process involves exposure to an environment (eg, a liquid nitrogen cooled freezer) at a temperature of about -50°C, about -70°C, about -90°C, or about -110°C for about 4 hours or less, about 3 hours or less, about 2 hours or less, or about 1 hour or less to cool the lyophilized medium.

In certain embodiments, the temperature during one or more freezing steps is not constant and may fluctuate between different temperature values. In embodiments in which more than one freezing step is performed, the temperature to which the lyophilized medium is cooled can be the same or different.

In certain embodiments, the lyophilized medium is maintained at any temperature between -40°C and -80°C during one or more freezing steps. In certain embodiments, the lyophilized medium is maintained at any temperature between -40°C and -60°C during one or more freezing steps. In certain embodiments, the lyophilized medium is maintained at any temperature between -60°C and -80°C during one or more freezing steps. In certain embodiments, the lyophilized medium is maintained at any temperature below the glass transition temperature of the lyophilized product during one or more freezing steps. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is between -30°C and -80°C. In certain embodiments, the lyophilized medium is maintained at any temperature between -40°C and -50°C during one or more freezing steps. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is between -50°C and -60°C. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is between -60°C and -70°C. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is between -70°C and -80°C. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -30°C or lower. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -35°C or less. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -40°C or lower. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -45°C or lower. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -50°C or lower. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -55°C or less. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -60°C or less. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -65°C or lower. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -70°C or less. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -75°C or lower. In certain embodiments, the temperature of the lyophilized medium during one or more freezing steps is -80°C or less. In a preferred embodiment, the temperature of the lyophilized medium during one or more freezing steps is below -45°C. In a preferred embodiment, the temperature of the lyophilized medium during one or more freezing steps is about -45°C.

In certain embodiments, the temperature used in the methods of the present invention refers to the temperature of a component of a lyophilization apparatus (eg, a lyophilization chamber, a shelf of a lyophilization apparatus, or a tray loaded into a lyophilization apparatus). In a preferred embodiment, the temperature used in the method of the present invention refers to the temperature of the shelf of the freeze-drying apparatus. Thus, in certain embodiments, the lyophilization chamber is at the temperatures disclosed herein during one or more freezing steps. In certain embodiments, the lyophilization chamber is maintained at any temperature between -40°C and 80°C during one or more freezing steps. In certain embodiments, the lyophilization chamber is at any temperature between -40°C and -60°C during one or more freezing steps. In certain embodiments, the lyophilization chamber is maintained at any temperature between -60°C and -80°C during one or more freezing steps. In certain embodiments, the lyophilization chamber is maintained at any temperature between -30°C and -40°C during one or more freezing steps. In certain embodiments, the lyophilization chamber is maintained at any temperature below the eutectic point of the lyophilization medium during one or more freezing steps. Thus, in certain embodiments, the shelves or trays of the lyophilization chamber are maintained at any temperature between -40°C and -80°C during one or more freezing steps. In certain embodiments, the shelves or trays of the lyophilization chamber are maintained at any temperature between -40°C and -60°C during one or more freezing steps. In certain embodiments, the shelves or trays of the lyophilization chamber are maintained at any temperature between -60°C and -80°C during one or more freezing steps. In certain embodiments, the shelves or trays of the lyophilization chamber are maintained at any temperature between -30°C and -40°C during one or more freezing steps. In certain embodiments, the shelves or trays of the lyophilization chamber are maintained at any temperature below the eutectic point of the lyophilization medium during one or more freezing steps. In certain preferred embodiments, the chamber is pre-cooled prior to placing the sample in the lyophilization chamber. In certain preferred embodiments, the shelves or trays of the lyophilization chamber are pre-cooled prior to placing the samples on the shelves of the lyophilization chamber.

In certain embodiments, the lyophilized medium is frozen with incremental temperature reductions. In certain embodiments, the temperature of the lyophilized medium is slowly lowered. In certain embodiments, one or more freezing steps are slow freezing, such as by lowering the lyophilization chamber and/or the shelves of the lyophilization chamber at a rate between 0.1°C/min and 0.3°C/min or the temperature of the tray. In certain embodiments, one or more freezing steps are slow freezing steps, such as by reducing the lyophilization chamber and/or the shelf or tray of the lyophilization chamber at a rate of 0.1°C/min or higher. temperature to proceed. In certain embodiments, one or more freezing steps are performed by reducing the temperature of the lyophilization chamber and/or shelves or trays of the lyophilization chamber at a rate of 0.5°C/min or higher. In certain embodiments, one or more freezing steps are performed by reducing the temperature of the lyophilization chamber and/or shelves or trays of the lyophilization chamber at a rate of 1°C/min or higher. In certain embodiments, one or more freezing steps are moderate-speed freezing steps, eg, by reducing the lyophilization chamber and/or the shelf or tray of the lyophilization chamber at a rate of 1.5°C/min or higher. temperature to proceed. In certain embodiments, the one or more freezing steps are performed by reducing the temperature of the lyophilization chamber and/or shelves or trays of the lyophilization chamber at a rate between 0.3°C/min and 1.5°C/min . In certain embodiments, one or more freezing steps are performed by reducing the temperature of the lyophilization chamber and/or the shelves or trays of the lyophilization chamber by 2°C or more per minute. In certain embodiments, one or more freezing steps are performed by reducing the temperature of the lyophilization chamber and/or the shelves or trays of the lyophilization chamber by 5°C or more per hour. In certain embodiments, the one or more freezing steps are performed by reducing the temperature of the lyophilization chamber and/or the shelves or trays of the lyophilization chamber by 30°C or more per hour. In certain embodiments, one or more freezing steps are performed by reducing the temperature of the lyophilization chamber and/or the shelves or trays of the lyophilization chamber by 60°C or more per hour. In certain embodiments, the one or more freezing steps are snap or flash freezing steps, eg, performed by submerging the lyophilized medium in liquid nitrogen. In certain embodiments, the lyophilized medium is incubated at a single freezing temperature or a range of freezing temperatures and maintained at a single freezing temperature or a range of freezing temperatures. In certain embodiments, the lyophilized medium is incubated in a pre-cooled lyophilization chamber.

In certain embodiments, the lyophilized medium is maintained at the temperature of one or more stages of the incremental freezing process. In certain embodiments, the lyophilized medium is maintained at that temperature for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In certain embodiments, the lyophilized medium is maintained at that temperature for less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes . In certain embodiments, the lyophilized medium is maintained at that temperature for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.

In certain embodiments, the lyophilized medium is maintained at freezing temperature for less than 1 hour. In certain embodiments, the lyophilized medium is maintained at freezing temperature for at least 1 hour. In certain embodiments, the lyophilized medium is maintained at the lowest freezing temperature for less than 1 minute. In certain embodiments, the lyophilized medium is maintained at the minimum freezing temperature for at least 1 minute. In certain embodiments, the lyophilized medium is maintained at the lowest freezing temperature for 1 minute to 1 hour. Thus, in certain embodiments, the lyophilized medium is maintained at the lowest freezing temperature for less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. Thus, in certain embodiments, the lyophilized medium is maintained at the minimum freezing temperature for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. Thus, in certain embodiments, the lyophilized medium is maintained at the minimum freezing temperature for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In certain embodiments, the lyophilized medium is maintained at the minimum freezing temperature for 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In certain embodiments, the lyophilized medium is maintained at the lowest freezing temperature for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In certain embodiments, the lyophilized medium is maintained at the minimum freezing temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In certain embodiments, the lyophilized medium is maintained at the minimum freezing temperature for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In certain embodiments, the lyophilized medium is maintained at the lowest freezing temperature for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In a preferred embodiment, the lyophilized medium is maintained at the lowest freezing temperature for at least 2 hours. In a preferred embodiment, the lyophilized medium is maintained at the lowest freezing temperature for about 2 hours. In certain embodiments wherein the lyophilization method comprises at least two freezing steps followed by an annealing step, the lyophilized medium is maintained at a minimum freezing temperature in the first freezing step for a first period of time, such as at least 2 hours, and maintained in the second freezing step for a second, optionally longer period of time (eg, at least 3 hours). In a preferred embodiment wherein the lyophilization method comprises at least two freezing steps followed by an annealing step, the lyophilized medium is maintained at the lowest freezing temperature for about 2 hours in the first freezing step and in the second freezing step for about 3 hours.

In certain embodiments, the freezing step is performed at atmospheric pressure. In certain embodiments, one or more freezing steps are performed under vacuum. In certain embodiments, the one or more freezing steps are performed at a pressure of about 10 microbars (µBar) or higher, about 20 microbars or higher, or about 50 microbars or higher. In certain embodiments, the one or more freezing steps are performed at a pressure of about 5000 microbars or less, about 2000 microbars or less, about 1000 microbars or less, or about 500 microbars or less. In certain embodiments, one or the freezing step is performed at a pressure of 50 to 300 microbars. In certain embodiments, the one or more freezing steps are performed at a pressure of 50 to 100 microbars. In certain embodiments, the one or more freezing steps are performed under anaerobic conditions consistent with the disclosure of European Application No. 19383175.7.

In certain preferred embodiments, the lyophilization chamber and/or shelves or trays of the lyophilization chamber are pre-cooled to a temperature of about -45°C, and the lyophilization medium is loaded into the lyophilization chamber to start Initiate one or more freezing steps. The lyophilization chamber and/or the shelves or trays of the lyophilization chamber can be maintained at about -45°C for at least about 2 hours during the first freezing step. The temperature of the lyophilization chamber and/or shelves or trays of the lyophilization chamber can then be increased to a temperature of about -15°C at a rate of about 1°C/min (60°C/h) for about 30 minutes. This temperature of the lyophilization chamber and/or the shelves or trays of the lyophilization chamber can be maintained for at least 5 hours during the annealing step. The temperature of the lyophilization chamber and/or the shelves or trays of the lyophilization chamber can then be lowered to a temperature of about -45°C at a rate of about 0.5°C/min (30°C/h) for 1 hour. The temperature of the lyophilization chamber and/or the shelves or trays of the lyophilization chamber can be maintained at about -45°C for at least about 3 hours during the second freezing step.

One or more freezing steps are followed by at least one preliminary drying step.

In certain embodiments, during one or more steps of freezing the lyophilized medium, the lyophilized medium can be exposed to about -50°C or less, about -70°C or less, or about -90°C or less the temperature. In certain embodiments, during one or more steps of freezing the lyophilized medium, the lyophilized medium can be exposed to about -130°C or higher, about -150°C or higher, or about -200°C or higher the temperature. For example, in certain embodiments, the lyophilized medium can be exposed to a temperature from about -50°C to about -200°C during one or more freezing steps. For example, in certain embodiments, the lyophilized medium can be exposed to a temperature of about -70°C to about -150°C during one or more freezing steps. For example, in certain embodiments, the lyophilized medium can be exposed to a temperature of about -90°C to about -130°C during one or more freezing steps. In certain embodiments, one or more freezing steps may last about 5 minutes or more, about 10 minutes or more, about 20 minutes or more, about 30 minutes or more, about 60 minutes or more. In certain embodiments, one or more freezing steps may last about 600 minutes or less, about 300 minutes or less, about 240 minutes or less, or about 180 minutes or less. For example, in certain embodiments, one or more freezing steps may last from about 5 minutes to about 600 minutes. For example, in certain embodiments, one or more freezing steps may last from about 10 minutes to about 300 minutes. For example, in certain embodiments, one or more freezing steps may last from about 20 minutes to about 240 minutes. For example, in certain embodiments, the one or more freezing steps may last from about 30 minutes to about 180 minutes. Such freezing steps can be carried out in a lyophilization facility or in a separate freezing facility.

In certain embodiments, the temperature to which the lyophilized medium is exposed during one or more freezing steps may vary. For example, the lyophilized medium can be exposed to a first temperature (eg, about 20°C, about 10°C, or about 0°C to about -20°C, about -30°C, about -40°C, or about -50°C) and at maintained at that temperature for a first period of time (eg, about 1 minute, about 5 minutes, or about 10 minutes to about 30 minutes, about 60 minutes, about 90 minutes, or about 120 minutes), and then cooled to a second temperature (eg, the previous paragraph) those temperatures suggested in) and maintained at that second temperature for a second period of time (eg, about 10 minutes, about 20 minutes, about 30 minutes, or about 60 minutes to about 120 minutes, 180 minutes, 240 minutes, 300 minutes, or longer). In such embodiments, the second period of time may be longer than the first period of time.

One or more freezing steps can be carried out at atmospheric pressure. Alternatively, subatmospheric or superatmospheric pressure may be employed. In embodiments of the present invention, atmospheric pressure or moderate sub-atmospheric or super-atmospheric pressure is preferred. For example, in certain embodiments, the pressure employed during one or more freezing steps ranges from about 10 kPa below atmospheric pressure to about 10 kPa above atmospheric pressure. In certain embodiments, the pressure employed during one or more freezing steps ranges from about 5 kPa below atmospheric pressure to about 5 kPa above atmospheric pressure. In certain embodiments, the pressure employed during one or more freezing steps ranges from about 2 kPa below atmospheric pressure to about 2 kPa above atmospheric pressure.

Any number of freezing steps may be performed during the method of the present invention. For example, 1, 2, 3, 4, 5 or more than 5 freezing steps can be performed. In embodiments in which multiple freezing steps are performed, one or more annealing steps may be performed between the freezing steps. Preliminary drying step background

During this method, ice formed during one or more freezing steps is removed by sublimation at low temperature under vacuum. Sublimation is the result of coupled heat transfer and mass transfer processes. The driving force for sublimation is the pressure difference related to the corresponding temperature difference between the product ice surface and the condenser ice surface. A larger temperature difference means a larger pressure difference, which allows for a faster drying process. The purpose of the vacuum is to speed up the process by removing air molecules to allow sample vapor molecules to more easily move from the sample through the lyophilization chamber and into the condenser of the lyophilization apparatus. Typically, this process produces a highly porous structure in the remaining amorphous solute where about 30% of the sample is water. Certain Examples of Preliminary Drying Steps

In certain embodiments, the preliminary drying step referred to herein is the only drying step. In certain embodiments, the preliminary drying steps referred to herein are one or more of the drying steps.

In certain embodiments, during the preliminary drying step, the temperature of the lyophilized medium is maintained at about 20°C or lower, about 10°C or lower, about 0°C or lower, or about -5°C or lower ( For example between -25°C and -5°C). In certain embodiments, the temperature during the preliminary drying step is not constant and may fluctuate between different temperature values. In certain embodiments, the temperature of the lyophilized medium is maintained at any temperature between -45°C and -5°C during the preliminary drying step. In certain preferred embodiments, the temperature of the lyophilized medium is maintained below the glass transition temperature of the lyophilized product during the preliminary drying step. In certain embodiments, during the preliminary drying step, the temperature of the lyophilized medium is maintained at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10°C below the glass transition temperature of the lyophilized product . In certain embodiments, during the preliminary drying step, the temperature of the lyophilized medium is maintained at least 1°C below the glass transition temperature of the lyophilized product.

In certain embodiments, during the preliminary drying step, the temperature of the lyophilized medium is adjusted (eg, increased) in temperature increments between the temperature of the freezing step and the glass transition temperature of the lyophilized product. In certain embodiments, the temperature of the lyophilization medium is adjusted according to the adjusted temperature of the shelf or tray of the lyophilization apparatus. In certain embodiments, the lyophilized medium is not adjusted to a temperature above the glass transition temperature of the lyophilized product during the preliminary drying step.

In certain embodiments, the temperature of the preliminary drying step is -5°C or lower. In certain embodiments, the temperature of the preliminary drying step is between about -5°C and -25°C. In certain embodiments, the temperature of the preliminary drying step is between about -5°C and -15°C. In certain embodiments, the temperature of the preliminary drying step is between about -15°C and -25°C. In a preferred embodiment, the temperature of the preliminary drying step is -9°C or lower. In certain embodiments, the temperature of the preliminary drying step is -20°C or lower. In another preferred embodiment, the temperature of the preliminary drying step is -21°C or lower. In certain embodiments, the temperature of the shelf or tray of the lyophilization apparatus is maintained at -5°C or lower during the preliminary drying step. In a preferred embodiment, the temperature of the shelf or tray of the lyophilization apparatus is maintained at -9°C or lower during the preliminary drying step. In certain embodiments, the temperature of the shelf or tray of the lyophilization apparatus is maintained at -20°C or lower during the preliminary drying step. In another preferred embodiment, the temperature of the shelf or tray of the lyophilization apparatus is maintained at -21°C or lower during the preliminary drying step.

In certain embodiments, the temperature of the lyophilized medium during the preliminary drying step is controlled by the temperature of the shelf or tray of the lyophilization apparatus. In certain embodiments, the temperature during the preliminary drying step (eg, shelf or tray temperature) is adjusted from -50°C to -5°C. In certain embodiments, at 1°C or more, 2°C or more, 3°C or more, 4°C or more, 5°C or more, 6°C or more, 7°C or more, The temperature (eg shelf or tray temperature) is adjusted in increments of 8°C or more, 9°C or more, or 10°C or more. In certain embodiments, the temperature (eg, shelf or tray temperature) is adjusted in 5°C increments. In certain embodiments, the temperature adjustment is performed at a rate of between 0.05°C/min and 1°C/min. In certain embodiments, the temperature adjustment is performed at a rate of at least 0.05°C/min. In certain embodiments, the temperature adjustment is performed at a rate of 0.1 °C/min. In certain embodiments, the temperature adjustment is performed at a rate of between 0.05°C/min and 0.1°C/min. In a preferred embodiment, the temperature adjustment is performed at a rate of about 0.08°C/min or greater (ie, about 5°C/h or greater). In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at an incremental temperature for a specified time. In certain embodiments, the increment is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10°C. In certain embodiments, the temperature of each increment is maintained for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In certain embodiments, the temperature of each increment is maintained for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In certain embodiments, the temperature of each increment is maintained for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In certain embodiments, the temperature of each increment is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In certain embodiments, the temperature of each increment is maintained for at least 10 hours. In a preferred embodiment, the temperature during the preliminary drying step (eg shelf or tray temperature) is adjusted from about -45°C to about -9°C for about 7 hours (ie at a rate of about 5°C/h) . In another preferred embodiment, the temperature during the preliminary drying step (eg, shelf or tray temperature) is adjusted from about -45°C to about -21°C for about 5 hours (ie, at a rate of about 5°C/h) °C.

In certain embodiments, the pressure is maintained at a constant value during the adjustment of the temperature during the preliminary drying step. In certain embodiments, during the adjustment of the temperature during the preliminary drying step, the pressure is changed according to the adjustment of the temperature.

In certain embodiments, the pressure is maintained at atmospheric pressure during the preliminary drying step. Alternatively, during the preliminary drying step, the maintenance pressure can be maintained at about 1 microbar or higher, about 5 microbar or higher, about 10 microbar or higher, about 20 microbar or higher, or about 50 microbar or higher. In certain embodiments, during the preliminary drying step, the pressure may be maintained at about 10000 microbars or less, about 5000 microbars or less, about 2000 microbars or less, about 1000 microbars or less, About 500 microbars or less. In embodiments of the present invention, the pressure may be maintained at about 1 to about 350 microbars during the preliminary drying step. In certain embodiments, the pressure is constant during the preliminary drying step. In certain embodiments, during the preliminary drying step, the pressure is changed to any value in the range of 1 to 350 microbars. In certain embodiments, the pressure is maintained at 30, 40, 50, 60 or 70 microbars during the preliminary drying step. In certain embodiments, the pressure is maintained at 40 microbars during the preliminary drying step. In certain embodiments, the pressure is maintained at 50 microbars during the preliminary drying step. In certain embodiments, the pressure is maintained at 260, 270, 280, 290, 300, 310, or 320 microbars during the preliminary drying step. In certain embodiments, the pressure is maintained at 300 microbars during the preliminary drying step. In a preferred embodiment, the pressure is maintained at about 50 microbars during the preliminary drying step. In another preferred embodiment, the pressure is maintained at about 275 microbars or higher, eg, about 276 microbars, during the preliminary drying step.

In certain embodiments, the preliminary drying step is performed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 20, 24, 30, or 36 hours. In certain embodiments, the preliminary drying step is performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 20, 24, 30, or 36 hours. In certain embodiments, the preliminary drying step is performed for at least 10 hours. In certain embodiments, the preliminary drying step is performed for about 10 hours. In a preferred embodiment, the preliminary drying step is performed for about 20 hours. In another preferred embodiment, the preliminary drying step is carried out for about 24 hours.

In certain embodiments, the lyophilization method includes two or more preliminary drying steps. In a preferred embodiment, the lyophilization method includes two preliminary drying steps. In certain embodiments, the first preliminary drying step of the two or more preliminary drying steps is performed under the conditions outlined above. In certain embodiments, the second preliminary drying step of the two or more preliminary drying steps is performed under the conditions outlined above.

In certain embodiments, the second preliminary drying step of the two or more preliminary drying steps is at a temperature (eg, shelf or tray temperature) that is greater than the temperature of the first preliminary drying step (eg, about 20-30° C.) proceed below. In certain embodiments, the second preliminary drying step of the two or more preliminary drying steps is performed at a temperature (eg, shelf or tray temperature) of about 25°C. In certain embodiments, the temperature (eg, shelf or tray temperature) is increased from two or more preliminary drying steps at a rate of about 5°C/h or higher (ie, about 0.08°C/min or higher). The temperature of the first preliminary drying step of the two or more preliminary drying steps is adjusted to the temperature of the second preliminary drying step of the two or more preliminary drying steps. In a preferred embodiment, the temperature (eg, shelf or tray temperature) is maintained for about 7 hours (ie, at a rate of about 5°C/h (ie, about 0.08°C/min) from about -9°C (two (the temperature of the first drying step of the two or more drying steps) is adjusted to about 25°C (the temperature of the second drying step of the two or more drying steps). In another preferred embodiment, the temperature (eg shelf or tray temperature) for about 9 hours (ie at a rate of about 5°C/h (ie about 0.08°C/min)) from about -21°C (the first of two or more drying steps) The temperature of one drying step) is adjusted to about 25°C (the temperature of the second drying step of the two or more drying steps). In certain embodiments, the first drying step of the two or more preliminary drying steps The preliminary drying step is performed for about 24 hours. In certain embodiments, the second preliminary drying step of the two or more preliminary drying steps is performed for about 20 hours. In a preferred embodiment, the two or more The first preliminary drying step of the preliminary drying steps is performed for about 24 hours (eg, at a temperature of about -9°C or about -21°C) and the second preliminary drying step of the two or more preliminary drying steps is performed for about 20 hours. hours (eg at a temperature of 25°C).

In certain embodiments, the preliminary drying step requires the lyophilization apparatus to have a vacuum pump or alternative means for creating a vacuum. A vacuum pump is used to reduce the pressure of the environment around the lyophilized medium, eg by removing all non-condensable gases.

In certain embodiments, the preliminary drying step requires the freeze-drying apparatus to have a condensation system or collector. The collector condenses out all condensable gases such as water molecules from the sublimation process.

In certain embodiments, the preliminary drying step requires the application of heat to the lyophilized medium. Heat causes water to be removed from the lyophilized medium as water vapor. In certain embodiments, the application of heat is controlled to ensure that the temperature of the lyophilized medium does not exceed the glass transition temperature of the lyophilized product. In certain embodiments, heat is applied to the lyophilized medium directly through the thermal conductor shelf or tray of the lyophilization apparatus. In certain embodiments, the lyophilized medium is heated using, eg, manifold drying. In certain embodiments, heat enters the lyophilized medium by direct contact between the bottom of the container of the lyophilized medium and the shelf or tray of the lyophilized apparatus. In certain embodiments, heat enters the lyophilized medium by conduction of heat through the bottom of the container of the lyophilized medium followed by conduction through the frozen mass of the lyophilized medium to the sublimation interface. In certain embodiments, heat enters the lyophilization medium by gas convection between the lyophilization medium and residual gas molecules in the chamber of the lyophilization apparatus. In certain embodiments, heat is introduced into the lyophilized medium by radiation. In a preferred embodiment, heat is applied to the lyophilized medium by gas convection.

In certain preferred embodiments, during the preliminary drying step, the temperature (eg, shelf or tray temperature) is maintained at a pressure of about 50 to about 300 microbars at about -25 to about -5°C for about 24 hours . In certain embodiments, during the preliminary drying step, the temperature (eg, shelf or tray temperature) is maintained at a pressure of about 50 to about 100 microbars at about -15 to about -5°C for about 24 hours. In certain embodiments, during the preliminary drying step, the temperature (eg, shelf or tray temperature) is maintained at about -25 to about -15°C at a pressure of about 250 to about 300 microbars for about 24 hours. In a preferred embodiment, during the preliminary drying step, the temperature (eg, shelf or tray temperature) is maintained at about -9°C for about 24 hours at a pressure of about 50 microbars. In another preferred embodiment, during the preliminary drying step, the temperature (eg, shelf or tray temperature) is maintained at about -21°C for about 24 hours at a pressure of about 275 microbars. In certain embodiments, during the preliminary drying step, the temperature (eg, shelf or tray temperature) is incrementally increased from the temperature of the freezing step to the temperature of the preliminary drying step in increments of about 4-10°C/h. In a preferred embodiment, during the preliminary drying step, the temperature (eg shelf or tray temperature) is increased incrementally from the temperature of the freezing step to the temperature of the preliminary drying step in increments of about 5°C/h. In certain embodiments, temperature conditioning is performed for 4 to 8 hours during the preliminary drying step. In a preferred embodiment, the temperature conditioning is performed for about 5 or about 7 hours during the preliminary drying step.

In certain embodiments, the pressure is constant for the duration of the increment. In certain preferred embodiments, the conditions applied to the lyophilized medium are such that the lyophilized medium is as close to this temperature as possible without exceeding the glass transition temperature of the lyophilized product.

In certain embodiments, during the second preliminary drying step of the two or more preliminary drying steps, the temperature (eg, shelf or tray temperature) is maintained at about 50 to about 300 microbars of pressure 20 to about 30°C. In a preferred embodiment, the temperature (eg, shelf or tray temperature) is maintained at a pressure of about 50 or about 275 microbars during the second preliminary drying step of the two or more preliminary drying steps. about 25°C. In a preferred embodiment, during the second preliminary drying step of the two or more preliminary drying steps, the temperature (eg shelf or tray temperature) is increased at about 5°C/h from the two or more preliminary drying steps The temperature of the first preliminary drying step of the preliminary drying steps is adjusted to the temperature of the second preliminary drying step of the two or more preliminary drying steps. In certain preferred embodiments, the temperature (eg, shelf or tray temperature) is maintained for about 7 hours (ie, at 5°C/h) during the second of the two or more preliminary drying steps rate) from about -9°C (the temperature of the first preliminary drying step of the two or more preliminary drying steps) to about 25 °C (the second preliminary drying step of the two or more preliminary drying steps) temperature). In other preferred embodiments, the temperature (eg, shelf or tray temperature) is maintained for about 9 hours (ie, at a rate of 5°C/h) during the second of the two or more preliminary drying steps. rate) from about -21°C (the temperature of the first preliminary drying step of the two or more preliminary drying steps) to about 25°C (the temperature of the second preliminary drying step of the two or more preliminary drying steps) temperature).

In certain embodiments, during the preliminary drying step, the lyophilized medium can be exposed to a lyophilized medium containing about 100 ppm or more, about 200 ppm or more, about 500 ppm or more, about 1000 ppm or more, about 2000 ppm or more An atmosphere of high, about 5,000 ppm or more, about 10,000 ppm or more, about 20,000 ppm or more, or about 50,000 ppm or more oxygen, or ambient air. Such exposure of lyophilized medium can be produced by transferring an oxygen-permeable reservoir containing the lyophilized medium from anaerobic conditions to such an atmosphere. Alternatively, such exposure may be by opening a sealed oxygen-impermeable receptacle in such an atmosphere (eg, by removing a closure (eg, a fill hole) of the opening in the receptacle and/or removing the lining of the receptacle wall part) to produce.

Thus, in certain embodiments, the oxygen content of the environment to which the lyophilized medium is exposed during the preliminary drying step can be about 100 ppm or more, about 200 ppm or more, about 500 ppm or more, about 1000 ppm or more , about 2000 ppm or more, about 5000 ppm or more, about 10000 ppm or more, about 20000 ppm or more, or about 50000 ppm or more. In certain embodiments, the preliminary drying step may be performed in ambient air.

In certain embodiments, the temperature to which the lyophilized medium is exposed during the preliminary drying step can be about 50°C or less, about 30°C or less, or about 10°C or less. In certain embodiments, the temperature to which the lyophilized medium is exposed during the preliminary drying step can be about -30°C or higher, about -50°C or higher, about -70°C or higher, about -100°C or About -150°C or higher. In certain embodiments, the temperature to which the lyophilized medium is exposed during the preliminary drying step may be between about -150°C and about 50°C. In certain embodiments, the temperature to which the lyophilized medium is exposed during the preliminary drying step may be between about -100°C and about 30°C. In certain embodiments, the temperature to which the lyophilized medium is exposed during the preliminary drying step may be between about -70°C and about 10°C. In certain embodiments, the temperature to which the lyophilized medium is exposed during the preliminary drying step may be between about -50°C and about 10°C. In certain embodiments, the temperature to which the lyophilized medium is exposed during the preliminary drying step may be between about -30°C and about 10°C.

Additionally or alternatively, in certain embodiments, the preliminary drying step may be performed at a pressure of about 5000 microbars or less, about 2000 microbars or less, about 1000 microbars or less, or about 500 microbars or more Low. In certain embodiments, the pressure at which the preliminary drying step is performed may be about 50 microbars or higher, about 25 microbars or higher, about 10 microbars or higher, or about 0 microbars or higher. In certain embodiments, the pressure at which the preliminary drying step is performed may be between about 0 microbar and about 5000 microbar. In certain embodiments, the pressure at which the preliminary drying step is performed may be between about 10 microbars and about 2000 microbars. In certain embodiments, the pressure at which the preliminary drying step is performed may be between about 25 microbars and about 1000 microbars. In certain embodiments, the pressure at which the preliminary drying step is performed may be between about 50 microbars and about 500 microbars.

In embodiments wherein the lyophilized medium is provided in a receptacle, the methods of the present invention may include the step of exposing the lyophilized medium within the receptacle. Doing this may facilitate the primary drying step, the secondary drying step (if performed), or other steps of the lyophilization process. For example, a portion of the wall of the receptacle can be removed and/or a closure (eg, a filling hole) in the receptacle can be opened. This step of exposing the lyophilization medium can be performed before loading the reservoir into the lyophilization apparatus, or after loading the reservoir into the lyophilization apparatus and before or during the preliminary drying step.

The preliminary drying step can be carried out using any technique or equipment known to those skilled in the art of lyophilization. For example, the preliminary drying step can be carried out in a lyophilization apparatus. In embodiments of the present invention, the lyophilization apparatus can be of any size without so substantially affecting the viability of the cells present in the lyophilization medium. Thus, in certain embodiments, the preliminary drying step is performed ^on a pilot scale lyophilization facility (eg, having about 0.1 m or more, about 0.2 m or more, about ^0.5 m or about ^{2 m} or less, about 3m ² or less or about 4m ² or less of operating shelf area freeze-drying equipment). In certain embodiments of the invention, the preliminary drying step is performed on a commercial scale lyophilization facility (eg, having about ⁵ m or more, about ¹⁰ m or more, or about ²⁰ m or more or about 50 ^m or less, about 100 m ² or less, about 150 m ² or less, or about 200 m ² or less operating shelf area freeze-drying equipment). Secondary drying step background

This optional method step can be initiated after the product has reached a temperature above its glass transition point and used to remove any residual solvent from the product. Therefore, this step may be referred to as a desorption step. In certain embodiments, up to 7-8% bound moisture may still be present in the lyophilized product after the initial drying step during which the ice has sublimated. Therefore, the secondary drying step is conventionally performed at high temperature to reduce the residual moisture content of the lyophilized product. Certain Embodiments of Secondary Drying Steps

In certain embodiments, the lyophilization method of the present invention includes a secondary drying step. In certain other embodiments, the lyophilization method of the present invention does not include a secondary drying step.

In certain embodiments, the secondary drying step is at a temperature above about -20°C, above about -10°C, above about 0°C, above about 10°C, such as at ambient temperature (about 20°C) or higher proceed below. In certain embodiments, the temperature of the secondary drying step is compatible with the sensitivity of the lyophilized product. In certain embodiments, the secondary drying step is performed at a temperature higher than the glass transition temperature of the lyophilized biotherapeutic product. In certain embodiments, during the secondary drying step, the lyophilized product is maintained at a temperature between the temperature of the primary drying step and the ambient (eg, 20°C) temperature. In certain embodiments, the lyophilized product is maintained at a temperature between the glass transition temperature and the ambient (eg, 20°C) temperature during the secondary drying step. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at a temperature between -20°C and 37°C during the secondary drying step. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained below 37°C during the secondary drying step. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained below 42°C during the secondary drying step. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at a temperature between about 20°C and about 25°C during the secondary drying step. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at a temperature of about 25°C during the secondary drying step. In certain other embodiments, the lyophilized product is maintained at a temperature of about 22°C during the secondary drying step.

In certain embodiments, the secondary drying step temperature (eg, shelf or tray temperature) is between -20°C and 37°C. In certain embodiments, the secondary drying step temperature (eg, shelf or tray temperature) is below 37°C. In certain embodiments, the secondary drying step temperature (eg, shelf or tray temperature) is below 42°C. In certain embodiments, the secondary drying step temperature (eg, shelf or tray temperature) is about 25°C.

In certain embodiments, the temperature (eg, shelf or tray temperature) is adjusted in temperature increments between the temperature of the glass transition temperature and the ambient (eg, 20° C.) temperature during the secondary drying step. In certain embodiments, the temperature (eg, shelf or tray temperature) is adjusted according to the temperature of the shelf or tray of the conditioned lyophilization apparatus. In certain embodiments, the temperature (eg, shelf or tray temperature) is not adjusted to a temperature above the glass transition temperature during the secondary drying process.

In certain embodiments, the temperature of the lyophilized product during the secondary drying step is controlled by the shelf or tray temperature of the lyophilization apparatus. In certain embodiments, the temperature during the secondary drying step (eg, shelf or tray temperature) is adjusted from the temperature of one or more primary drying steps to the temperature of the secondary drying step. In certain embodiments, the temperature during the secondary drying step (eg, shelf or tray temperature) does not exceed 42°C.

In certain embodiments, at 1°C or higher, 2°C or higher, 3°C or higher, 4°C or higher, 5°C or higher, 6°C or higher, 7°C or higher, The temperature (eg shelf or tray temperature) is adjusted in increments of 8°C or higher, 9°C or higher, or 10°C. In certain embodiments, the temperature (eg, shelf or tray temperature) is adjusted in increments of 5°C or higher. In certain embodiments, the adjustment is performed at a rate between 0.05°C/min and 1°C/min. In certain embodiments, the conditioning is performed at a rate of at least 0.05°C/min. In certain embodiments, the conditioning is performed at a rate of 1.5°C/min. In certain preferred embodiments, the conditioning is performed at a rate of at least 0.08°C/min (ie, 5°C/h). In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at an incremental temperature for a specified time. In certain embodiments, the increment is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10°C. In certain embodiments, the temperature of each increment is maintained for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In certain embodiments, each incremental temperature is maintained at that temperature (eg, shelf or tray temperature) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours . In certain embodiments, the temperature of each increment is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In certain embodiments, the temperature of each increment is maintained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certain embodiments, the final shelf temperature is such that the lyophilized product is maintained at 25°C. In certain embodiments, the final shelf temperature is such that the lyophilized product is maintained at a temperature of 22°C. In certain embodiments, the final shelf temperature is such that the lyophilized product is maintained at a temperature of 22°C for at least 10 hours.

In certain embodiments, the temperature during the secondary drying step (eg, shelf or tray temperature) is maintained at the final temperature after temperature conditioning is completed. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at the final temperature for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 , 55 or 60 minutes. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 at the final temperature , 55 or 60 minutes. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at the final temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 24 or 36 hours. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 at the final temperature , 15, 16, 17, 18, 19, 20, 24 or 36 hours. In certain embodiments, the temperature (eg shelf or tray temperature) is maintained at the final temperature for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24 or 36 hours. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at the final temperature for at least 10 hours. In a preferred embodiment, the temperature (eg shelf or tray temperature) is maintained at 25°C for at least 10 hours. In another preferred embodiment, the secondary drying step is completed after the lyophilized product has been maintained at a temperature of 22°C for at least 10 hours. In certain embodiments, the temperature during the secondary drying step (eg, shelf or tray temperature) is maintained at 25°C for at least 25, 30, or 35°C as the lyophilized product takes time to reach a final target temperature of 22°C Hour. In a preferred embodiment, the temperature during the secondary drying step (eg, shelf or tray temperature) is maintained at 25°C for about 35 hours.

In certain embodiments, the secondary drying step is one third or half the time of the primary drying step. In certain embodiments, the secondary drying step is one and a half or twice the time of the primary drying step. In certain embodiments, the secondary drying step is approximately the same time as the one or more primary drying steps. In certain embodiments, the secondary drying step is approximately the same time as all primary drying steps.

In certain embodiments, the pressure is maintained at a constant value during the adjustment of the temperature during the secondary drying step. In certain embodiments, during the adjustment of the temperature during the secondary drying step, the pressure is changed according to the temperature adjustment.

In certain embodiments, the pressure to which the lyophilized medium is exposed during the secondary drying step is atmospheric pressure. Alternatively, during the secondary drying step, the pressure to which the lyophilized medium is exposed can be about 1 microbar or higher, about 2 microbar or higher, about 5 microbar or higher, about 10 microbar or higher, About 20 microbars or higher or about 50 microbars or higher. In particular embodiments, the pressure to which the lyophilized medium is exposed during the secondary drying step can be about 10,000 microbars or less, about 5,000 microbars or less, about 2000 microbars or less, about 1000 microbars or less or about 500 microbars or less, for example between 50 and 350 microbars. In certain embodiments, the pressure to which the lyophilized medium is exposed is 50 microbars. In certain embodiments, the pressure to which the lyophilized medium is exposed is 275 microbars.

In certain preferred embodiments, the temperature (eg, shelf or tray temperature) is maintained at about 25°C for at least about 20 h at a pressure of about 50 microbars during the secondary drying step. In certain preferred embodiments, during the secondary drying step, the temperature (eg, shelf or tray temperature) is maintained at about 25°C for at least about 20 h at a pressure of about 275 microbars. In certain preferred embodiments, the temperature (eg, shelf or tray temperature) is maintained at about 25°C during the secondary drying step until the temperature of the lyophilized product is maintained at 22°C for at least 10 hours.

As will be recognized by those skilled in the art, conventional lyophilization methods typically include a plurality of drying steps, such as a primary drying (sublimation) step and a secondary drying (desorption) step. Accordingly, in certain embodiments, the lyophilization method includes one or more secondary drying steps.

For the preliminary drying step, the oxygen content of the environment in which the desorption step is performed may be about 100 ppm or more, about 200 ppm or more, about 500 ppm or more, about 1000 ppm or more, about 2000 ppm or more, about 5000 ppm or more, about 10000 ppm or more, about 20000 ppm or more, or about 50000 ppm or more. In certain embodiments, the secondary drying step may be performed in ambient air.

In certain embodiments, the lyophilized medium can be exposed to a temperature of about 70°C or lower, about 50°C or lower, or about 40°C or lower during the secondary drying step, if performed. In certain embodiments, the lyophilized medium can be exposed to about 10°C or higher, about 0°C or higher, about -10°C or higher, or about -20°C or higher during the secondary drying step the temperature. In certain embodiments, the lyophilized medium can be exposed to a temperature between about -20°C and about 70°C during the secondary drying step. In certain embodiments, the lyophilized medium can be exposed to a temperature between about -10°C and about 50°C during the secondary drying step. In certain embodiments, the lyophilized medium can be exposed to a temperature between about 0°C and about 40°C during the secondary drying step. In certain embodiments, the lyophilized medium can be exposed to a temperature between about 10°C and about 40°C during the secondary drying step.

Additionally or alternatively, in certain embodiments, during the secondary drying step (if performed), the lyophilized medium may be exposed to about 2000 microbars or less, about 1000 microbars or less, about 500 microbars or A pressure of lower or about 300 microbars or lower. In certain embodiments, during the secondary drying step, the lyophilized medium can be exposed to about 50 microbars or higher, about 25 microbars or higher, about 10 microbars or higher, or about 0 microbars or higher pressure. In certain embodiments, the lyophilized medium can be exposed to a pressure between about 0 microbar and about 2000 microbar during the secondary drying step. In certain embodiments, the lyophilized medium can be exposed to a pressure between about 10 microbars and about 1000 microbars during the secondary drying step. In certain embodiments, the lyophilized medium can be exposed to a pressure between about 25 microbars and about 500 microbars during the secondary drying step. In certain embodiments, the lyophilized medium can be exposed to a pressure between about 50 microbars and about 300 microbars during the secondary drying step.

Preferably, the secondary drying step is performed in the same equipment as the primary drying step.

In certain embodiments, the water content of the lyophilized product after the primary drying step and the secondary drying step, if performed, and/or after the lyophilized product is collected, is less than about 5%. In certain embodiments, the water content of the lyophilized product after the primary drying step and the secondary drying step (if performed) and/or after the lyophilized product is collected is between 5% and 30%. In certain embodiments, the water content of the lyophilized product is less than about 5%, less than about 10%, less than about 15% after the primary drying step and the secondary drying step (if performed) and/or after the lyophilized product is collected , less than about 20%, less than about 25%, or less than about 30%. In certain embodiments, the water content of the lyophilized product after the primary drying step and the secondary drying step, if performed, and/or after the lyophilized product is collected, is less than about 4%. In certain embodiments, the water content of the lyophilized product after the primary drying step and the secondary drying step, if performed, and/or after the lyophilized product is collected, is less than about 3%. In certain embodiments, the water content of the lyophilized product after the primary drying step and the secondary drying step, if performed, and/or after the lyophilized product is collected, is less than about 2%. In certain embodiments, the water content of the lyophilized product after the primary drying step and the secondary drying step (if performed) and/or after the lyophilized product is collected is less than about 1%. Annealing step

In an embodiment of the present invention, the lyophilization method includes at least one annealing step. The annealing step can be performed between one or more freezing steps. Additionally or alternatively, the (or another) annealing step may be performed after one or more freezing steps and before the preliminary drying step. As shown in the examples herein, the inventors determined that incorporating an annealing step into a lyophilization process achieves a significant increase in the viability of the lyophilized product compared to a lyophilization process without a corresponding annealing step.

In certain embodiments, the lyophilized medium is maintained at a temperature above the glass transition temperature of the lyophilized product during the annealing step. In certain embodiments, during the annealing step, the lyophilized medium is maintained at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 above the glass transition temperature of the lyophilized product , 13, 14, 15, 16, 17, 18, 19 or 20°C. In certain embodiments, during the annealing step, the lyophilized medium is maintained less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 above the glass transition temperature of the lyophilized product , 13, 14, 15, 16, 17, 18, 19 or 20°C. In certain embodiments, during the annealing step, the lyophilized medium is maintained at any temperature between the glass transition temperature of the lyophilized product and -20°C. In certain embodiments, during the annealing step, the lyophilized medium is maintained at any temperature between the glass transition temperature of the lyophilized product and -15°C. In certain embodiments, during the annealing step, the lyophilized medium is maintained at any temperature between the glass transition temperature of the lyophilized product and -10°C. In certain embodiments, during the annealing step, the lyophilized medium is maintained at any temperature between the glass transition temperature of the lyophilized product and -5°C. In certain embodiments, during the annealing step, the lyophilized medium is maintained at any temperature between the glass transition temperature of the lyophilized product and 0°C.

In certain embodiments, during the annealing step, the lyophilized medium is maintained at least 1°C above the glass transition temperature of the lyophilized product. In certain embodiments, during the annealing step, the lyophilized medium is maintained at least 2°C above the glass transition temperature of the lyophilized product. In certain embodiments, the lyophilized medium is maintained at least 3°C above the glass transition temperature of the lyophilized product during the annealing step. In certain embodiments, during the annealing step, the lyophilized medium is maintained at least 4°C above the glass transition temperature of the lyophilized product. In certain embodiments, during the annealing step, the lyophilized medium is maintained at least 5°C above the glass transition temperature of the lyophilized product. In certain embodiments, the lyophilized medium is maintained less than 1°C above the glass transition temperature of the lyophilized product during the annealing step. In certain embodiments, the lyophilized medium is maintained less than 2°C above the glass transition temperature of the lyophilized product during the annealing step. In certain embodiments, the lyophilized medium is maintained at less than 3°C above the glass transition temperature of the lyophilized product during the annealing step. In certain embodiments, the lyophilized medium is maintained at less than 4°C above the glass transition temperature of the lyophilized product during the annealing step. In certain embodiments, the lyophilized medium is maintained less than 5°C above the glass transition temperature of the lyophilized product during the annealing step.

In certain embodiments, the annealing temperature is at least 1°C above the glass transition temperature of the lyophilized product. In certain embodiments, the annealing temperature is at least 2°C above the glass transition temperature of the lyophilized product. In certain embodiments, the annealing temperature is at least 3°C above the glass transition temperature of the lyophilized product. In certain embodiments, the annealing temperature is at least 4°C above the glass transition temperature of the lyophilized product. In certain embodiments, the annealing temperature is at least 5°C above the glass transition temperature of the lyophilized product. In certain embodiments, the annealing temperature is less than 1°C above the glass transition temperature of the lyophilized product. In certain embodiments, the annealing temperature is less than 2°C above the glass transition temperature of the lyophilized product. In certain embodiments, the annealing temperature is less than 3°C above the glass transition temperature of the lyophilized product. In certain embodiments, the annealing temperature is less than 4°C above the glass transition temperature of the lyophilized product. In certain embodiments, the annealing temperature is less than 5°C above the glass transition temperature of the lyophilized product.

In certain embodiments, the annealing temperature is about -15°C or higher, -20°C or higher, -25°C or higher, -30°C or higher, -35°C or higher, -40°C or higher, -45°C or higher, -50°C or higher, or -55°C or higher. In certain embodiments, the annealing temperature is an integer value between about -15°C and about -55°C. In certain embodiments, the annealing temperature is about -35°C. In certain embodiments, the annealing temperature is -33°C. In certain preferred embodiments, the annealing temperature is between about -10°C and about -20°C. In certain preferred embodiments, the annealing temperature is about -15°C.

In certain embodiments, the temperature (eg, shelf or tray temperature) is adjusted in increments of at least about 1, 2, 3, 4, or 5°C during the annealing step. In certain embodiments, the adjustment is performed at a rate between about 0.01°C/min and 1°C/min. In certain embodiments, the conditioning is performed at a rate of at least about 0.1°C/min. In a preferred embodiment, the conditioning is performed at a rate of about 0.5°C/min (ie, 30°C/h) or higher. In another preferred embodiment, the conditioning is performed at a rate of about 1°C/min (ie, 60°C/h) or higher. In certain embodiments, the temperature of the shelves of the lyophilization apparatus is adjusted in increments of at least about 1, 2, 3, 4, or 5°C during the annealing step.

In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at less than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 at the annealing temperature , 50, 55 or 60 minutes. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 at the annealing temperature , 55 or 60 minutes. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at the annealing temperature for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at the annealing temperature for less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at the annealing temperature for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In a preferred embodiment, the temperature (eg, shelf or tray temperature) is maintained at the annealing temperature for at least about 5 hours. In certain embodiments, the temperature (eg, shelf or tray temperature) is maintained at the annealing temperature for about 5 hours. In a preferred embodiment, during the annealing step, the temperature (eg, shelf or tray temperature) is maintained at about -15°C for about 5 hours.

In certain embodiments, at the end of the one or more freezing steps, the lyophilized medium is adjusted from the temperature of the one or more freezing steps to above the glass transition temperature of the lyophilized product and maintained at this temperature, and then cooled to The temperature below the glass transition temperature of the lyophilized product. In certain embodiments, at the conclusion of the one or more freezing steps, the lyophilized medium is adjusted from the temperature of the one or more freezing steps to the annealing temperature and maintained at this temperature, and then cooled to the freezing step temperature. In certain embodiments, at the conclusion of the one or more freezing steps, the shelf of the lyophilization apparatus is adjusted from the temperature of the one or more freezing steps to above the glass transition temperature of the lyophilized product and maintained at this temperature, It is then cooled to below the glass transition temperature of the lyophilized product. In certain embodiments, at the conclusion of one or more freezing steps, the shelves of the lyophilization apparatus are adjusted from the temperature of the freezing step to an annealing temperature and maintained at this temperature, and then cooled to the freezing step temperature.

In certain embodiments, the annealing temperature is adjusted at a rate between about 0.1°C/min and 1°C/min. In certain embodiments, the annealing temperature is adjusted at a rate of at least about 0.5°C/min. In certain embodiments, the annealing temperature is adjusted at a rate of about 1°C/min. In certain embodiments, the annealing temperature is adjusted at a rate of about 5°C/min. In a preferred embodiment, the annealing temperature is adjusted at a rate of about 1°C/min (ie, 60°C/h). In certain embodiments, the back freezing step temperature is adjusted at a rate between about -0.1°C/min and -1°C/min. In certain embodiments, the back-freezing step temperature is adjusted at a rate of at least about -0.5°C/min. In certain embodiments, the back freezing step temperature is adjusted at a rate of about -1°C/min. In certain embodiments, the back freezing step temperature is adjusted at a rate of about -5°C/min. In a preferred embodiment, the back freezing step temperature is adjusted at a rate of about 0.5°C/min (ie about 30°C/h).

In certain embodiments, the pressure to which the lyophilized medium is exposed during the annealing step is atmospheric pressure. Alternatively, during the annealing step, the pressure to which the lyophilized medium is exposed can be about 1 microbar or higher, about 2 microbar or higher, about 5 microbar or higher, about 10 microbar or higher, about 20 microbar or higher Microbars or higher or about 50 microbars or higher. In particular embodiments, the pressure to which the lyophilized medium is exposed during the annealing step can be about 10,000 microbars or less, about 5,000 microbars or less, about 2000 microbars or less, about 1000 microbars or more Low or about 500 microbars or less, such as between 50 and 350 microbars or between 50 and 100 microbars. In certain embodiments, the pressure to which the lyophilized medium is exposed is constant during the annealing step. In certain embodiments, during the annealing step, the pressure to which the lyophilized medium is exposed is optionally varied according to temperature.

In certain embodiments, at the conclusion of one or more freezing steps, the temperature (eg, shelf or tray temperature) is adjusted from a freezing step temperature of between about -40°C and about -50°C to about -10°C and An annealing temperature between -20°C and held at the annealing temperature for about 4-8 hours, then reduced to a freezing temperature between about -40°C and about -50°C. In certain embodiments, the temperature of the first freezing step is maintained for about 1-3 hours. In certain embodiments, the temperature of the second freezing step is maintained for about 2-4 hours. In a preferred embodiment, at the end of one or more freezing steps, the temperature (eg, shelf or tray temperature) is adjusted from a freezing step temperature of about -45°C to an annealing temperature of about -15°C, and at the end of the annealing step The temperature is maintained for at least about 5 hours and then lowered to a freezing temperature of about -45°C. In another preferred embodiment, the temperature (eg shelf or tray temperature) is maintained for 30 minutes (i.e. at 60°C/ The rate of h) was adjusted from a freezing step temperature of about -45°C to an annealing temperature of about -15°C, and held at the annealing temperature for at least about 5 hours, then decreased for 60 minutes (ie, at a rate of 30°C/h) to a freezing temperature of about -45°C and maintained at this temperature for at least about 3 hours.

In certain embodiments, the lyophilization method of the present invention includes at least 1, 2, 3, 4, or 5 annealing steps. In a preferred embodiment, one or more annealing steps are followed by a freezing step. Thus, in certain embodiments, the lyophilization methods of the present invention comprise at least 2, 3, 4, 5, 6 or 7 freezing steps. In certain embodiments, the lyophilization methods of the present invention comprise less than 2, 3, 4 or 5 annealing steps. In certain embodiments, the lyophilization methods of the present invention comprise less than 2, 3, 4, 5, 6 or 7 freezing steps. In a preferred embodiment, the lyophilization method includes two freezing steps followed by an annealing step. Glass transition temperature Determination of glass transition temperature

In certain embodiments, the glass transition temperature of the lyophilized product is determined using differential scanning calorimetry (DSC) (see [2]). Such techniques will be routine to the skilled artisan. In a preferred embodiment, the glass transition temperature is determined using DSC according to ASTM E1356-08 as exemplified by Drake et al., Plos One, Jan. 5, 2018. glass transition temperature

In certain embodiments, the glass transition temperature of the lyophilized product is between -15°C and -55°C. In certain embodiments, the glass transition temperature of the lyophilized product is between -10°C and -26°C. In certain embodiments, the glass transition temperature of the lyophilized product is between -10°C and -55°C. In certain embodiments, the glass transition temperature of the lyophilized product is between -26°C and -55°C. In certain embodiments, the glass transition temperature of the lyophilized product is between -15°C and -25°C. In certain embodiments, the glass transition temperature of the lyophilized product is less than -30°C. In a preferred embodiment, the glass transition temperature of the lyophilized product is about -33°C. In a preferred embodiment, the glass transition temperature of the lyophilized product is less than -15°C. Lyophilization Buffer Preparation

In certain embodiments, the lyophilization medium comprises a lyophilization buffer. In certain embodiments, the lyophilization buffer is formulated according to regulatory requirements and the route of administration of the lyophilized product, as known to those skilled in the art. In certain embodiments, the lyophilization buffer may contain one or more excipients. In certain embodiments, the excipients are buffers, pH adjusters, compatibilizers, stabilizers and/or tonicity adjusters.

In certain embodiments, the lyophilized medium comprises a buffer. In certain embodiments, the buffer stabilizes the pH of the lyophilization buffer. In certain embodiments, the buffer is one that undergoes minimal pH change during freezing, such as a citrate and/or histidine buffer. Thus, in certain embodiments, the buffer is a citrate buffer. In certain embodiments, the buffer is a histidine buffer. In certain embodiments, the lyophilization buffer has a pH between 5.5 and 8.5.

In certain embodiments, the lyophilization buffer includes a compatibilizer. In certain embodiments, the compatibilizer is a crystalline compatibilizer. In certain embodiments, the compatibilizer is a disaccharide. In certain embodiments, the compatibilizer is mannitol. In certain embodiments, the compatibilizer is sucrose. In certain embodiments, the compatibilizer is albumin, glycine, hydroxyethyl starch, or dextran.

In certain embodiments, the lyophilization buffer contains stabilizers. In certain embodiments, the stabilizer comprises a compound that can form an amorphous glass structure, such as one or more disaccharides. In certain embodiments, the stabilizer is glucose, lactose and/or maltose. In certain embodiments, the stabilizer is glucose. In certain embodiments, the stabilizer is lactose. In certain embodiments, the stabilizer is maltose. In certain embodiments, the stabilizer is sucrose. In certain embodiments, the stabilizer is trehalose. In certain embodiments, the stabilizer is not trehalose.

In certain embodiments, the lyophilization buffer includes a tonicity adjusting agent. In certain embodiments, the tonicity adjusting agent is an excipient such as mannitol, sucrose, glycine, glycerol, or sodium chloride. Thus, in certain embodiments, the lyophilization buffer comprises mannitol, sucrose, glycine, glycerol, and/or sodium chloride.

In certain embodiments, the lyophilization buffer includes a cryoprotectant. In certain embodiments, cryoprotectants are water-soluble substances that lower the melting point of water and/or increase the non-frozen portion of the lyophilized medium. In certain embodiments, the cryoprotectant is a nontoxic low molecular weight solute. In certain embodiments, the cryoprotectant is a monosaccharide or a disaccharide. In certain embodiments, the cryoprotectant is trehalose, sucrose, glucose and/or lactose. In certain embodiments, the cryoprotectant is a sugar alcohol, such as glycerol and/or sorbitol. In certain embodiments, the cryoprotectant is a polymer. In certain embodiments, the cryoprotectant is polyethylene glycol, polyvinylpyrrolidone, and/or dextran. In certain embodiments, the cryoprotectant is skim milk, egg whites and/or various amino acids and derivatives.

In certain embodiments, the lyophilization buffer comprises sucrose. In certain embodiments, the lyophilization buffer comprises glycerol. In certain embodiments, the lyophilization buffer comprises DMSO. In certain embodiments, the lyophilization buffer comprises sucrose, glycerol and/or DMSO. In certain embodiments, the lyophilization buffer does not contain trehalose. In certain embodiments, the lyophilization buffer does not contain trehalose.

In certain embodiments, a live bacterial population may comprise more than one bacterial strain (such as a consortium of different bacterial strains). In such embodiments, the population of live bacteria can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 Or at least 15 different bacterial strains. Additionally or alternatively, the viable bacterial population may comprise 50 or less, 40 or less, 30 or less, or 20 or less different bacterial strains.

Those skilled in the art will be familiar with typical compositions of lyophilized media and how they can be prepared. In certain embodiments, the lyophilized medium comprises a viable bacterial population in the form of concentrated biomass. In certain embodiments, biomass can be stored under anaerobic conditions.

In certain embodiments, the lyophilisation medium may comprise a lyophilisation buffer/lyobuffer. The lyophilization buffer may contain excipients known to those skilled in the art, such as: Cryoprotectants (eg polyols such as ethylene glycol, sorbitol, propylene glycol and/or glycerol; DMSO; skim milk; yeast extract; bovine serum albumin (BSA); starch hydrolysates; sugars (including monosaccharides, disaccharides and/or polysaccharides) such as glucose, maltose, maltotriose, trehalose, mannitol, dextran, maltodextrin, lactose and/or sucrose; and/or amino acids such as cysteine , glutamic acid (in salt form as appropriate, such as sodium glutamate), arginine and/or glycine); Antioxidants (eg cysteine, arginine, ascorbic acid (and salts and esters thereof, eg ascorbyl palmitate, sodium ascorbate)), butylating agents (eg butylated hydroxyanisole or butylated hydroxytoluene) ), citric acid, erythronic acid, fumaric acid, glutamic acid, glutathione, malic acid, methionine, monothioglycerol, pentetic acid, metabisulfites (such as sodium metabisulfite) , potassium metabisulfite), propionic acid, propyl gallate, uric acid, sodium formaldehyde sulfoxylate, sulfites (e.g. sodium sulfite), sodium thiosulfate, sulfur dioxide, thymol, tocopherol (free or ester sulfonic acid), uric acid (and its salts) and its salts and/or esters); Compatibilizers (eg mannitol, maltodextrin and/or glycine); Buffers (eg phosphate, citrate, tris and/or Hepes); and/or Surfactants such as polysorbates (such as those commercialized under the trademark Tween) and/or sorbitans (such as those commercialized under the trademark Span).

In a preferred embodiment, the lyophilization buffer comprises a lyoprotectant formulation (at final concentration prior to lyophilization) containing the following components: 2% sucrose, 4% maltodextrin DE9, and 0.2% cysteine Hydrochloride. In a preferred embodiment, the ratio of biomass to lyoprotectant is about 70:30. In a preferred embodiment, the lyophilization buffer does not contain trehalose. In certain embodiments, the lyophilization buffer does not contain maltodextrin. In certain embodiments, the lyophilization buffer does not contain maltodextrin DE9. In certain embodiments, the lyophilization buffer comprises (at final concentration prior to lyophilization): 2% sucrose and 0.2% cysteine hydrochloride.

In an embodiment of the present invention, the lyophilized medium is not prepared by at least partially solidifying a liquid or paste-like mixture of substances between two surfaces having different temperatures prior to lyophilization. Additionally or alternatively, the lyophilized product does not contain a sponge. Freeze -drying equipment Components of freeze-drying equipment

In certain embodiments, the lyophilization apparatus is a manifold freeze dryer, a rotary freeze dryer, and/or a tray freeze dryer. In certain embodiments, the freeze-drying apparatus is a tray-type freeze dryer. In an embodiment of the present invention, the freeze-drying apparatus includes a shelf. In certain embodiments, the methods of the present invention include the step of filling the lyophilized medium into a receptacle for lyophilization. In such embodiments, the lyophilized medium is filled into vials. In an alternative embodiment, the lyophilization medium is filled into receptacles for bulk lyophilization (eg, lyophilization trays (eg, conventional open trays or specialty trays, such as those commercialized by ^Gore® under the trademark ^Lyogard® ) or lyophilized bags such as those disclosed in International Patent Application No. PCT/IB2018/055246 (the contents of which are incorporated herein by reference)).

In an embodiment of the present invention, the lyophilized medium can be prepared at least about 1 g, at least about 2 g, at least about 5 g, at least about 10 g, at least about 20 g, at least about 50 g, at least about 100 g, at least about 200 g, at least about 500 g, An amount of at least about 1 kg or at least about 2 kg is filled into the receptacle for freeze drying.

In an embodiment of the invention, the lyophilization reservoir and/or the lyophilization apparatus does not contain a sample array.

In an embodiment, the step of filling the lyophilized medium into the receptacle may be performed before one or more freezing steps and/or before the preliminary drying step. After filling, the filled receptacles can be placed inside the lyophilization apparatus (eg, where the lyophilization apparatus includes shelves, the filled receptacles are placed on one or more of the shelves).

In certain embodiments, the lyophilization device can be of any size without so affecting the viability of cells present in the lyophilization medium. Thus, in certain embodiments, the lyophilization method is performed ^on a pilot scale lyophilization facility (eg, having about 0.1 m or more, about 0.2 m or more, about ^0.5 m or about ^{2 m} or less, about 3m ² or less or about 4m ² or less of operating shelf area freeze-drying equipment). In certain embodiments of the invention, the sublimation step is performed on a commercial scale lyophilization facility (eg, having about ⁵ m or more, about ¹⁰ m or more, or about ²⁰ m or more or about 50 ^m or less, about 100m ² or less, about 150m ² or less, or about 200m ² or less of operating shelf area freeze-drying equipment).

In certain embodiments, the lyophilization apparatus includes a lyophilization chamber, a condenser, and/or a vacuum pump. In certain embodiments, the lyophilization chamber is adapted to use product vials. In certain embodiments, the lyophilization chamber is adapted to use trays.

In certain embodiments, the lyophilization apparatus includes a lyophilization chamber. In certain embodiments, the lyophilization chamber contains one or more shelves. In certain embodiments, the shelf acts as a heat exchanger to help remove energy from the lyophilized medium during freezing and/or supply energy to the lyophilized medium during the drying step. In certain embodiments, the shelves are connected to a fluid system that enables a fluid of a particular temperature to circulate through the shelves. In certain embodiments, the circulating fluid is silicone oil. In certain embodiments, the temperature of the circulating fluid is set in an external heat exchange system comprising at least one cooling heat exchanger and at least one electric heater.

In certain embodiments, the lyophilization apparatus includes a vacuum pump. In some embodiments, the vacuum pump can achieve a vacuum of 50 to 100 microbars. In certain embodiments, the vacuum pump is a two-stage rotary pump. In some embodiments, two or more vacuum pumps may be used.

In certain embodiments, the lyophilization apparatus includes a control system. In certain embodiments, the control system controls shelf temperature and/or pressure within the lyophilization chamber. In certain embodiments, the control system controls how long the lyophilized medium is maintained at a particular temperature and/or pressure.

In certain embodiments, the control system can monitor the temperature of the lyophilized medium. In certain embodiments, a temperature sensor is placed with the sample to measure the core temperature of the lyophilization medium during the lyophilization process. In some embodiments, a temperature sensor is used to measure the temperature of the shelves within the freeze-drying chamber. In certain embodiments, a temperature sensor allows a comparison between the shelf temperature and the temperature of the lyophilized medium.

In certain embodiments, the reservoir may be oxygen impermeable to help maintain anaerobic conditions therein. As used herein, the term "oxygen impermeable" is used to identify a receptacle as having about 10 cc/m ² /24h or less, about 5 cc/m ² / 24h or less, about 1 cc/m ² /24h or less, about 0.5 cc/m ² /24h or less, about 0.1 cc/m ² /24h or less, about 0.05 cc/m ² /24h or An oxygen transmission rate (OTR) of less, about 0.01 cc/m ² /24h or less, about 0.005 cc/m ² /24h or less, or about 0.001 cc/m ² /24h or less. In certain embodiments, the reservoir has an oxygen transmission rate (OTR) of about 1 cc/m ² /24h or less. In certain embodiments, the reservoir has an oxygen transmission rate (OTR) of about 0.1 cc/m ² /24h or less. In certain embodiments, the reservoir has an oxygen transmission rate (OTR) of about 0.01 cc/m ² /24h or less. Conditions in the freeze-drying chamber

As outlined above, the conditions in the lyophilization chamber depend on the lyophilization method. In certain embodiments, the temperature and pressure of the lyophilization chamber are controlled during the lyophilization process. In certain embodiments, the temperature and/or pressure of the lyophilization chamber is changed during the lyophilization process. In certain embodiments, the temperature and/or pressure of the lyophilization chamber is constant for a specified period of time during the lyophilization method. In certain embodiments, the conditions in the lyophilization chamber are those for the lyophilization medium. In certain embodiments, the conditions within the lyophilization chamber correspond to the conditions disclosed herein for each of the steps of the lyophilization method.

In certain embodiments, the temperature of the lyophilization chamber is between -80°C and 40°C. In certain embodiments, the temperature of the lyophilization chamber is about -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25 , -20, -15, -10, -5, 0, 5, 10, 15, 20, 15, 30, 35 or 40°C. In certain embodiments, the temperature of the lyophilization chamber is at least -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25 , -20, -15, -10, -5, 0, 5, 10, 15, 20, 15, 30, 35 or 40°C. In certain embodiments, the temperature of the lyophilization chamber is less than -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25, -20, -15, -10, -5, 0, 5, 10, 15, 20, 15, 30, 35 or 40°C. In certain embodiments, the temperature of the lyophilization chamber is between -40°C and -50°C during one or more freezing steps. In a preferred embodiment, the temperature of the lyophilization chamber during one or more freezing steps is about -45°C. In certain embodiments, the temperature of the lyophilization chamber during the annealing step is between -10°C and -20°C. In a preferred embodiment, the temperature of the lyophilization chamber during the annealing step is about -15°C. In certain embodiments, the temperature of the lyophilization chamber during the preliminary drying step is between -5°C and -25°C. In preferred embodiments, the temperature of the lyophilization chamber during the preliminary drying step is about -9°C or about -21°C. In certain embodiments, the lyophilization chamber is between 15°C and 25°C during another preliminary drying step. In certain embodiments, the lyophilization chamber is about 20°C during another preliminary drying step.

In certain embodiments, the temperature of the lyophilization chamber can be varied. In certain embodiments, the temperature of the lyophilization chamber is increased at a rate between 0.05 and 5.0°C/min. In certain embodiments, at approximately , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to increase the temperature of the lyophilization chamber. In certain embodiments, at least 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to increase the temperature of the lyophilization chamber. In certain embodiments, at less than 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to increase the temperature of the lyophilization chamber. In certain embodiments, the temperature of the lyophilization chamber is lowered at a rate between 0.05 and 5.0°C/min. In certain embodiments, at approximately , 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to reduce the temperature of the lyophilization chamber. In certain embodiments, at least 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to reduce the temperature of the lyophilization chamber. In certain embodiments, at less than 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to reduce the temperature of the lyophilization chamber. In a preferred embodiment, the temperature of the lyophilization chamber is increased at a rate of 0.08°C/min (ie 5°C/h). In a preferred embodiment, the temperature of the lyophilization chamber is lowered at a rate of 0.5°C/min (ie 30°C/h). In another preferred embodiment, the temperature of the lyophilization chamber is increased at a rate of 1°C/min (ie 60°C/h).

In certain embodiments, the temperature of the lyophilization chamber is the temperature of the shelf or tray of the lyophilization chamber. In certain embodiments, the temperature of the shelf or tray of the lyophilization chamber is between -80°C and 40°C. In certain embodiments, the temperature of the shelf or tray of the lyophilization chamber is about -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25, -20, -15, -10, -5, 0, 5, 10, 15, 20, 15, 30, 35 or 40°C. In certain embodiments, the temperature of the shelf or tray of the lyophilization chamber is at least -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25, -20, -15, -10, -5, 0, 5, 10, 15, 20, 15, 30, 35 or 40°C. In certain embodiments, the temperature of the shelf or tray of the lyophilization chamber is less than -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, - 30, -25, -20, -15, -10, -5, 0, 5, 10, 15, 20, 15, 30, 35 or 40°C. In certain embodiments, the temperature of the shelves or trays of the lyophilization chamber is between -40°C and -50°C during one or more freezing steps. In a preferred embodiment, the temperature of the shelves or trays of the lyophilization chamber during one or more freezing steps is about -45°C. In certain embodiments, the temperature of the shelf or tray of the lyophilization chamber during the annealing step is between -10°C and -20°C. In a preferred embodiment, the temperature of the shelf or tray of the lyophilization chamber during the annealing step is about -15°C. In certain embodiments, the temperature of the shelves or trays of the lyophilization chamber during the preliminary drying step is between -5°C and -25°C. In a preferred embodiment, the temperature of the shelf or tray of the lyophilization chamber during the preliminary drying step is about -9 or about -21°C. In certain embodiments, the shelves or trays of the lyophilization chamber are between 15°C and 25°C during another preliminary drying step. In certain embodiments, the shelf or tray of the lyophilization chamber is about 20°C during another preliminary drying step.

In certain embodiments, the temperature of the shelves or trays of the lyophilization chamber can be varied. In certain embodiments, the temperature of the shelf or tray of the lyophilization chamber is increased at a rate between 0.05 and 5.0°C/min. In certain embodiments, at approximately , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to increase the temperature of the shelf of the freeze-drying chamber. In certain embodiments, at least 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to increase the temperature of the shelf or tray of the freeze-drying chamber. In certain embodiments, at less than 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to increase the temperature of the shelf or tray of the freeze-drying chamber. In certain embodiments, the temperature of the shelves or trays of the lyophilization chamber is lowered at a rate between 0.05 and 5.0°C/min. In certain embodiments, at approximately , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to reduce the temperature of the shelf or tray of the freeze-drying chamber. In certain embodiments, at least 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to reduce the temperature of the shelf or tray of the freeze-drying chamber. In certain embodiments, at less than 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 , 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0°C/min to reduce the temperature of the shelf or tray of the freeze-drying chamber. In a preferred embodiment, the temperature of the shelf or tray of the lyophilization chamber is increased at a rate of 0.08°C/min (ie 5°C/h). In a preferred embodiment, the temperature of the shelf or tray of the lyophilization chamber is lowered at a rate of 0.5°C/min (ie 30°C/h). In another preferred embodiment, the temperature of the shelf or tray of the freeze-drying chamber is increased at a rate of 1°C/min (ie 60°C/h).

In certain embodiments, the pressure in the lyophilization chamber is between 1 and 350 microbars. In certain embodiments, the pressure in the lyophilization chamber is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225 , 250, 275, 300, 325 or 350 microbars. In certain embodiments, the pressure in the lyophilization chamber is at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225 , 250, 275, 300, 325 or 350 microbars. In certain embodiments, the pressure in the lyophilization chamber is less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 or 350 microbars. In certain embodiments, the pressure in the lyophilization chamber is between 50 and 100 microbars. In a preferred embodiment, the pressure in the lyophilization chamber is about 50 microbars. In another preferred embodiment, the pressure in the lyophilization chamber is about 275 microbars.

In certain embodiments, the temperature and/or pressure of the lyophilization chamber is constant for 1 to 60 minutes. In certain embodiments, the temperature and/or pressure of the lyophilization chamber is constant for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In certain embodiments, the temperature and/or pressure of the lyophilization chamber is constant for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In certain embodiments, the temperature and/or pressure of the lyophilization chamber is constant for less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In certain embodiments, the temperature and/or pressure of the lyophilization chamber is constant for 1 to 36 hours. In certain embodiments, the temperature and/or pressure of the lyophilization chamber is constant for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24 or 36 hours. In certain embodiments, the temperature and/or pressure of the lyophilization chamber is constant for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24 or 36 hours. In certain embodiments, the temperature and/or pressure of the lyophilization chamber is constant for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 24 or 36 hours. In a preferred embodiment, the temperature and/or pressure of the lyophilization chamber is constant for at least 2 hours, eg, 3 hours, during one or more freezing steps. In a preferred embodiment, the temperature and/or pressure of the lyophilization chamber is constant for about 5 hours during the annealing step. In a preferred embodiment, the temperature and/or pressure of the lyophilization chamber is constant for about 24 hours during the preliminary drying step. In a preferred embodiment, the temperature and/or pressure of the lyophilization chamber is constant for about 20 hours during another preliminary drying step (if included). In a preferred embodiment, during the secondary drying step, the temperature and/or pressure of the lyophilization chamber is constant for at least 10 hours or until the lyophilized product is maintained at a temperature of at least 22°C for at least 10 hours. Live bacterial population for lyophilization

In certain embodiments, the bacterial genus of the live bacterial population used for lyophilization is selected from the group consisting of Enterococcus ( eg , Enterococcus gallinarum ), Enterococcus caselliflavus , Enterococcus durans , Enterococcus faecalis or Enterococcus faecium ), Blautia ( e.g. Blautia hydrogenotrophica ), fecal cloth Blautia stercoris , Blautia wexlerae , Blautia coccoides or Blautia producta ), Bacteroides ( Bacteroides) ( e.g. Bacteroides thetaiotaomicron , Bacteroides massiliensis , Bacteroides fragilis , Bacteroides ovatus , Bacteroides vulgatus , Bacteroides dorea ( Bacteroides dorei or copricola ), faecalibacterium ( e.g. Faecalibacterium prausnitzii ), Bariatricus ( e.g. Marseille Bariatricus ), Bifidobacterium ( e.g. Bifidobacterium breve ) breve ), Bifidobacterium adolescentis or Bifidobacterium longum ), Roseburia ( e.g. Roseburia hominis ), Roseburia intestinalis or Glucophagus Roseburia inulinivorans ), Flavonifractor ( eg Flavonifractor plautii ), anaerobic Anaerotruncus ( e.g. Anaerotruncus colihominis ), Parabacteroides ( e.g. Parabacteroides distasonis ), Parabacteroides goldsteinii , Parabacteroides merdae or Parabacteroides johnsonii ), Erysipelatoclostridium ( e.g. Erysipelatoclostridium ramosum ), Megasphaera ) ( e.g. Megasphaera massiliensis , Megasphaera elsdenii ), Pediococcus ( e.g. Pediococcus acidilacticii ), Eubacterium ( e.g. Eubacterium contortum , fissicatena , Eubacterium eligens , Eubacterium hadrum , Eubacterium hallii , Eubacterium callenderi or Eubacterium rectale ), Ruminococcus ( e.g. Ruminococcus torques , Ruminococcus gnavus or Ruminococcus bromii ), Pseudolytic flavonoids ( Pseudoflavonifractor ( eg Pseudoflavonifractor capillosus ), Clostridium ( eg Clostridium nexile , Clostridium hylemonae , Clostridium butyricum ) , Clostridium tertium ), Clostridium disporicum , Clostridium bifermentans , Clostridium inocuum , Clostridium mayombei , Clostridium bolteae, Pasteurella Clostridium bartleetti , Clostridium symbiosum or Clostridium orbiscindens ) or Coprococcus ( e.g. Coprococcus comes or Coprococcus cattus ), Bifidobacterium ( eg Bifidobacterium breve , Bifidobacterium adolescentis or Bifidobacterium longum ), Acetivibrio ( eg ethanologenic Arc Acetovibrio ethanolgignens ), Dorea ( e.g. Dorea longicatena) , Anaerostipes ( e.g. Anaerostipes hadrus) or Anaerostipes caccae) , Fusicatenibacter ( eg Fusicatenibacter saccharivorans , Lachnospiraceae or Clostridiaceae ). Examples of such organisms include those disclosed in European Patent Nos. 1280541, 1448995 and 3209310, European Patent Publication Nos. 3206700, 2763685 and UK Patent Application No. 1423084.1, the The contents are incorporated herein by reference in their entirety. Other examples of organisms that may be formulated according to the present invention include those disclosed in UK Patent Applications Nos. 1510470.6, 1510468.0, 1510469.8, 1510466.4 and 1510467.2, all of which refer to Incorporated herein by reference.

In certain embodiments, the live bacterial population does not comprise conventional probiotics, e.g., it does not belong to the genus Lactobacillus , Bifidobacterium, and/or is not a lactobacillus. In certain embodiments, the viable bacterial population does not contain lactic acid bacteria. In certain embodiments, the viable bacterial population is not or does not comprise a bacterial strain from the genus Lactobacillus.

In certain embodiments, the viable bacterial population comprises or consists of non-spore-forming bacteria. Characteristics of lyophilized products Viability after lyophilization

Those skilled in the art will be familiar with methods for performing viable cell counts, such as plate counting using a spiral seeder such as that commercialized under the trademark ^easySpiral® Pro. Plate counting can be performed under an anaerobic fume hood.

In certain embodiments, the viable cell count (CFU/g) in the lyophilized product is no more than 10 ³ CFU/g, 10 ² CFU/g or 10 CFU/g.

In certain embodiments, the viability (CFU/g, in dry weight) of the lyophilized product is equal to or less than 10 ³ CFU/g, equal to or less than the viability (CFU/ml) of the lyophilized medium prior to lyophilization 10 ² CFU/g or equal to or less than 10 CFU/g. As shown in the Examples, the methods of the present invention are particularly suitable for maintaining viable cell counts in lyophilized products compared to the viable cell counts in lyophilized media prior to freezing.

In certain embodiments of the invention, the viable cell count (CFU/g) in the lyophilized product is no more than 5 CFU/g lower than the viable cell count (CFU/g) in the lyophilized medium prior to freezing (water excluded) , no more than 4 CFU/g, no more than 3 CFU/g, or no more than 2 CFU/g.

In certain embodiments, the viable cell count (CFU/g, in dry weight) of the lyophilized product is equal to or less than 5 CFU/g, equal to or less than the viability (CFU/ml) of the lyophilized medium prior to lyophilization Less than 4 CFU/g, equal to or less than 3 CFU/g, or equal to or less than 2 CFU/g.

In certain embodiments, the viable cell count in the lyophilized product is reduced by 0.4 log or less, or 0.3 log or less, 0.2 log or more, as compared to the viable cell count in the lyophilized medium prior to freezing (moisture exclusion) less or 0.1 log or less.

In order to accurately assess the reduction (eg, log loss) in cell counts (ie, CFU/g) in live lyophilized product compared to viable cell counts (ie, CFU/ml) in lyophilized media, the skilled artisan will understand , the increase in potential viable cell concentration per gram of lyophilized product compared to per milligram of lyophilized medium (ie caused by removal of water during the lyophilization process) must be accounted for. The increased cell concentration in the lyophilized product can be explained by determining the so-called theoretical viable cell count (CFU/g) of the lyophilized product, which is calculated as a function of the moisture content of the lyophilized medium and the viable cell count. The theoretical viable cell count assumes no loss of viability after removal of moisture and therefore corresponds to the maximum possible viable cell count (CFU/g) in the lyophilized product after lyophilization. By comparing the theoretical viable cell count (CFU/g) with the actual viable cell count (CFU/g) measured after lyophilization, the reduction in viable cell count (e.g., log loss) in the lyophilized product can be accurately determined. ), thereby explaining the increase in bacterial cell concentration that occurs after removal of moisture during lyophilization.

This method of explaining the water loss between the lyophilized medium and the lyophilized product (ie, before and after lyophilization) can be explained in the context of the following description. The three lyophilized media had the same viable cell count (CFU/ml) before lyophilization, but had different % water content. In a lyophilization process in which half of the bacteria die for the purposes of this illustration, the overall viability reduction (eg, log loss) should be the same for each lyophilization medium, regardless of the original water content. As shown in Table 1 below, by calculating the theoretical viable cell count (CFU/g) (based on the moisture content of the lyophilized medium and the viable cell count) for each of the corresponding lyophilized products, and by calculating this theoretical viable cell count This is possible compared to the "actual" viable cell count (CFU/g) of the corresponding lyophilized product measured after lyophilization. Thus, the reduction in viability (eg, log loss) was the same for each of the lyophilized products, regardless of the original water content. Therefore, the calculation accounts for the increase in resident bacterial cell concentration after lyophilization and/or any differences in the moisture content of the different lyophilization media prior to lyophilization. This factoring ensures at least (i) that the determined reduction in viable cell count (eg, log loss) when compared to the viable cell count before lyophilization (CFU/ml) and after lyophilization (CFU/g) is accurately represented The loss of bacterial cell viability during the lyophilization process, and (ii) the reduction in viable cell count (eg, log loss) determined for lyophilized products from lyophilized media with different moisture contents, can be directly compared. Table 1 - Determination of Viable Cell Count Reduction ( eg Log Loss ) Freeze-dried medium Viable cell count before lyophilization (CFU/ml) Water content (%) Concentration factor Theoretical viable cell count after lyophilization (CFU/g) Logarithm of theoretical value "Actual" viable cell count after lyophilization (CFU/g)* logarithm of the "actual" value log of loss A 1.00 x 10 ¹⁰ 20 1.25 1.25 x 10 ¹⁰ 10.10 6.25 x ¹⁰⁹ 9.80 0.30 B 1.00 x 10 ¹⁰ 50 2 2.00 x 10 ¹⁰ 10.30 1.00 x 10 ¹⁰ 10.00 0.30 C 1.00 x 10 ¹⁰ 80 5 5.00 x 10 ¹⁰ 10.70 2.50 x 10 ¹⁰ 10.40 0.30 *As outlined above, "actual" viable cell counts for the purposes of this example assume that half of the bacteria die during the lyophilization process.

Consistent with the calculations outlined above, viability before lyophilization (CFU/ml) and after lyophilization (CFU/g) can be directly compared.

In certain embodiments, the lyophilized product has a viability loss equal to or less than 10 ³ CFU/g, after 1 month storage at 2 to 8° C. when packaged in a water impermeable package (eg, alu/alu blister pack), Equal to or less than 10 ² CFU/g or equal to or less than 10 CFU/g.

In certain embodiments, the loss of viability of the lyophilized product is determined after long-term storage at a temperature of 2 to 8°C when packaged in a water impermeable package (eg, alu/alu blister pack). In certain embodiments, the lyophilized product is stored for 1, 2, 3, 4, 5, 6, 12, 24, or 36 months. In certain embodiments, the lyophilized product is stored for less than 1, 2, 3, 4, 5, 6, 12, 24, or 36 months. In certain embodiments, the lyophilized product is stored for at least 1, 2, 3, 4, 5, 6, 12, 24, or 36 months.

Surprisingly, incorporating the annealing step into the lyophilization process resulted in a significant improvement in the viability of the bacterial population after sublimation of the lyophilization medium and/or lyophilization. Without wishing to be bound by theory, incorporating an annealing step results in larger, more organized crystals. These crystals allow for a faster and/or less intense sublimation process. Thus, the lyophilized product has improved ease of sublimation and/or viability.

In certain embodiments, the viability of the bacterial population in the lyophilized product is determined by culturing the lyophilized product in an appropriate medium. The lyophilized product was resuspended in culture medium prior to cultivation. For example, 0.1 g of lyophilized product can be resuspended in 25 ml of medium. The resuspended cultures were incubated at room temperature for 30 minutes. The culture is then diluted (eg, 100-fold) and the cells are plated on solid medium using appropriate pour plate techniques (multiple replicates are performed). The plate is incubated at an appropriate temperature (eg, 37°C) until colonies form. Colonies can then be counted and viability can be calculated compared to the bacterial population prior to lyophilization. In certain embodiments, a spiral seeder (eg, the one commercialized under the trademark ^easySpiral® Pro) can be used. Plate counting can be performed under an anaerobic fume hood. Residual water content and water activity after lyophilization

In certain embodiments, the water content of the lyophilized product collected from the methods of the present invention is less than 5.0 wt%. In certain embodiments, the water content of the lyophilized product is between 3.0 wt% and 4.5 wt%. In certain embodiments, the water content of the lyophilized product is 3.0, 3.5, 4.0 or 4.5 wt%. In certain preferred embodiments, the water content of the lyophilized product is between 3.3 wt% and 4.4 wt%. In certain embodiments, the water content of the lyophilized product is less than 4.5 wt%. In certain embodiments, the water content of the lyophilized product is less than 4.0 wt%. In certain embodiments, the water content of the lyophilized product is less than 3.5 wt%.

In the embodiment of the present invention, the water activity of the lyophilized product is less than 1. In certain embodiments, the water activity of the lyophilized product is between 0.01 and 0.5. In certain embodiments, the water activity of the lyophilized product is 0.01 to 0.1, eg, 0.01 to 0.05. As shown in the examples, the methods of the present invention achieve a water activity of less than 0.05.

In certain embodiments, the water content is determined using a Karl Fischer coulomb counter (eg, the Karl Fischer coulomb counter commercialized by Mettler Toledo). In certain embodiments, water activity is determined using a mirror dew point system (eg, the mirror dew point system commercialized by AquaLab). Preparation and dosage form of lyophilized product

The lyophilized product can be admixed with one or more excipients before being provided in the dosage form. Such excipients may include diluents, stabilizers, growth stimulators, fillers, lubricants, glidants and the like. Examples of such suitable excipients can be found in the Handbook of Pharmaceutical Excipients. Excipients acceptable for therapeutic use are well known in the pharmaceutical art.

Exemplary pharmaceutically acceptable excipients that can be blended with the lyophilized product include, but are not limited to, binders, disintegrants, superdisintegrants, lubricants, diluents, fillers, flavors, glidants, adsorbents agents, solubilizers, chelating agents, emulsifiers, thickening agents, dispersing agents, stabilizers, suspending agents, adsorbents, granulating agents, preservatives, buffers, colorants and sweeteners or combinations thereof. Examples of binding agents include microcrystalline cellulose, hydroxypropyl methylcellulose, carboxyvinyl polymers, polyvinylpyrrolidone, polyvinylpolypyrrolidone, calcium carboxymethylcellulose, sodium carboxymethylcellulose, Carob Gum, Chitosan, Cottonseed Oil, Dextrose Binding Agent, Dextrin, Ethylcellulose, Gelatin, Dextrose, Glyceryl Behenate, Galactomannan Polysaccharide, Hydroxyethyl Cellulose, Hydroxyethyl Methyl cellulose, hydroxypropyl cellulose, hypromellose, inulin, lactose, magnesium aluminum silicate, maltodextrin, methylcellulose, poloxamer, polycarbophil ), polydextrin, polyethylene glycol, polyethylene oxide, polymethacrylate, sodium alginate, sorbitol, starch, sucrose, sunflower oil, vegetable oil, tocofersolan, zein or its combination. Examples of disintegrants include hydroxypropylmethylcellulose (HPMC), low-substituted hydroxypropylcellulose (L-HPC), croscarmellose sodium, sodium starch glycolate, lactose, magnesium aluminum silicate , methylcellulose, polacrilin potassium, sodium alginate, starch, or a combination thereof. Examples of lubricants include stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, glyceryl monostearate, glyceryl palmitostearate, magnesium lauryl sulfate, Mineral oil, palmitic acid, myristic acid, poloxamer, polyethylene glycol, sodium benzoate, sodium chloride, sodium lauryl sulfate, talc, zinc stearate, potassium benzoate, magnesium stearate, or combinations thereof . Examples of diluents include talc, ammonium alginate, calcium carbonate, calcium lactate, calcium phosphate, calcium silicate, calcium sulfate, cellulose, cellulose acetate, corn starch, glucose binders, dextrin, dextrose, erythromycin Sugar alcohol, ethyl cellulose, fructose, fumaric acid, glyceryl palmitostearate, isomalt, kaolin, lactitol, lactose, magnesium carbonate, magnesium oxide, maltodextrin, maltose, mannose Alcohol, microcrystalline cellulose, polydextrin, polymethacrylate, simethicone, sodium alginate, sodium chloride, sorbitol, starch, sucrose, sulfobutyl ether beta-cyclodextrin, yellow Gum yarrow, trehalose, xylitol, or a combination thereof.

Various suitable fillers or diluents include, but are not limited to, anhydrous calcium hydrogen phosphate, calcium hydrogen phosphate dihydrate, tricalcium diphosphate, calcium sulfate, cellulose powder, silicified microcrystalline cellulose, cellulose acetate, compressible sugar, confectionery Store's sugar, glucose binder, dextrin, dextrose, fructose, kaolin, lactitol, lactose, lactose monohydrate, magnesium carbonate, magnesium oxide, maltodextrin, maltose, mannitol, microcrystalline cellulose, Polydextrin, simethicone, sodium alginate, sodium chloride, sorbitol, starch, pregelatinized starch, sucrose, trehalose and xylitol or mixtures thereof.

Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, zinc stearate, stearic acid, talc, glyceryl behenate, polyethylene glycol, polyethylene oxide polymers, lauryl Sodium sulfate, magnesium lauryl sulfate, sodium oleate, sodium stearyl fumarate, DL-leucine, colloidal silica and others known in the art. In certain embodiments, the lubricant is magnesium stearate.

Various suitable glidants include, but are not limited to, tricalcium phosphate, calcium silicate, cellulose powder, colloidal silica, magnesium silicate, magnesium trisilicate, starch and talc or mixtures thereof.

Pharmaceutically acceptable surfactants include, but are limited to, both nonionic and ionic surfactants suitable for use in pharmaceutical dosage forms. Ionic surfactants may include one or more of anionic, cationic or zwitterionic surfactants. Various suitable surfactants include, but are not limited to, sodium lauryl sulfate, monooleate, monolaurate, monopalmitate, monostearate, or another ester of polyoxyethylene sorbitan, dioctyl sulfonate Sodium succinate (DOSS), lecithin, stearyl alcohol, cetearyl alcohol, cholesterol, polyoxyethylene grate oil, polyoxyethylene fatty acid glycerides, and poloxamers.

Excipients that can be blended with the lyophilized product can include prebiotics. The term "prebiotic" means a non-digestible component that beneficially affects a bacterial population by selectively stimulating the growth and/or activity of one or a limited number of bacteria. Examples of prebiotics include oligosaccharides, fructooligosaccharides, and galactooligosaccharides.

In an embodiment of the invention, the method includes the step of preparing a dosage form comprising the lyophilized product. Dosage forms containing the lyophilized product can be prepared by punching or compressing dragee cores containing the lyophilized product. In certain embodiments, the dragee core is coated (eg, enteric coated) to provide a dragee. In certain embodiments, the lyophilized product is encapsulated into a capsule shell to provide capsules. In certain embodiments, the lyophilized product is provided in a sachet and the sachet is sealed.

In certain embodiments, the population of live bacteria included in the dosage form of the lyophilized product comprises one or more bacterial strains of a particular genera and does not contain bacteria from any other genera, or it comprises only minor or biologically irrelevant amounts from another genus of bacteria.

In certain embodiments, the population of live bacteria included in the dosage form of the lyophilized product comprises one or more bacterial strains of a particular species and does not contain bacteria from any other species, or it comprises only minor or biologically irrelevant amounts It comes from another kind of bacteria.

In certain embodiments, the live bacterial population included in the dosage form of the lyophilized product contains a single bacterial strain or species and is free of any other bacterial strain or species. Such viable bacterial populations may contain only minor or biologically irrelevant amounts of other bacterial strains or species.

In certain embodiments, the live bacterial population included in the dosage form of the lyophilized product comprises more than one bacterial strain. For example, in certain embodiments, the population of live bacteria included in the dosage form of the lyophilized product comprises more than one strain (eg, more than 1, 2, 3, 4, 5, 6, 7, 8) from within the same species , 9, 10, 15, 20, 25, 30, 35, 40 or 45 strains), as appropriate, without bacteria from any other species.

In certain embodiments, the population of live bacteria included in the dosage form of the lyophilized product comprises less than 50 strains from within the same species (eg, less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 strains), as appropriate, without bacteria from any other species. In certain embodiments, the live bacterial population included in the dosage form of the lyophilized product comprises 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1 from within the same species -10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20 , 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 strains, and as appropriate, without bacteria from any other species. Certain embodiments include any combination of the foregoing.

In certain embodiments, the viable bacterial population included in the dosage form comprises a microbial consortium. For example, in certain embodiments, the population of live bacteria included in the dosage form comprises a particular bacterial strain that is part of a microbial consortium. For example, in certain embodiments, the population of live bacteria included in the dosage form comprises one or more (eg, at least 2, 3, 4, 5, 10, 15 or 20) bacterial strains present in combination with other bacterial strains.

For example, in certain embodiments, the population of live bacteria included in the dosage form comprises a combination of a particular bacterial strain and bacterial strains from different genera. In certain embodiments, a microbial consortium comprises two or more bacterial strains obtained from a fecal sample of a single organism (eg, a human). In certain embodiments, microbial consortia do not exist together in nature. For example, in certain embodiments, a microbial consortium comprises bacterial strains obtained from fecal samples of at least two different organisms. In certain embodiments, two different organisms are from the same species, eg, two different humans. In certain embodiments, the two different organisms are a human infant and an adult human. In certain embodiments, the two different organisms are humans and non-human mammals.

In certain embodiments, the lyophilized product is in a pharmaceutical dosage form. In certain embodiments, the amount of bacterial strains in the viable bacterial population included in the pharmaceutical dosage form is about 1 x 10 relative to the weight of the dosage form (excluding the capsule body (if present) and any enteric coating (if present) ³ to about 1 x 10 ¹¹ colony forming units/gram.

In certain embodiments, the lyophilized product is in a pharmaceutical composition. In certain embodiments, the pharmaceutical composition is stored in a moisture-proof container at 2°C to 8°C, and the activity in the lyophilized product after a period of at least about 1 year, 1.5 years, 2 years, 2.5 years, or 3 years The loss of bacteria, as colony forming units (CFU)/gram, is no greater than 3 log, no greater than 2 log, or no greater than 1 log. Additionally or alternatively, loss of viable bacteria in the lyophilized product strain after a period of 6 months when the lyophilized product is stored in a moisture-proof container at 5°C and the container is placed in an atmosphere with 50% relative humidity Such as colony forming unit (CFU)/gram, it is not more than 3 log, not more than 2 log, not more than 1 log or not more than 0.5 log.

In certain embodiments, the dosage form contains an amount of cryogel in an amount from about 1 x 10 ³ to about 1 x 10 ¹³ CFU/g relative to the weight of the dosage form (excluding the capsule body (if present) and any enteric coating (if present) Dry product, such as about 1 x ¹⁰ to about 1 x ¹⁰ CFU/g, about 1 x ¹⁰ to about 1 x ¹⁰ CFU/g, about 1 x ¹⁰ to about 1 x 10 ¹² or about 1 x 10 ⁸ to about 1 x 10 ¹⁰ CFU/g. In certain embodiments, the dosage form may comprise at least 1 x 10 ¹⁰ CFU/g, at least 1 x 10 ⁹ CFU/g, at least 1 x 10 ⁸ CFU/g, at least 1 x 10 ⁷ CFU/g or at least 1 x 10 ⁶ CFU/g.

The live bacterial population present in the dosage form can be symbiotic, ie it is obtained from a donor (eg, a human infant, child, youth or adult).

In certain embodiments, the dosage form comprises a biologically pure single bacterial strain. As used herein, the term "biologically pure" means that the culture contains traces or biologically irrelevant levels of other bacterial strains. In certain embodiments, the dosage form comprises less than about 1%, less than about 0.5%, less than about 0.2%, less than about 0.1%, less than about 0.05%, less than about 0.02%, or less than about 0.01% of other bacterial species The total number of bacterial cells.

In alternative embodiments, the dosage form may contain a plurality (eg, 2, 3, 4, 5, or more than 5) bacterial strains.

In addition to the lyophilized product, the medicinal product may contain one or more excipients including, for example, diluents, stabilizers, growth stimulators, fillers, lubricants, glidants, and the like.

In addition to the lyophilized product, the dosage form may contain one or more pharmaceutically acceptable excipients. Examples of such suitable excipients can be found in the Handbook of Pharmaceutical Excipients. Excipients acceptable for therapeutic use are well known in the pharmaceutical art.

Examples of suitable carriers include lactose, starch, dextrose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with respect to standard pharmaceutical practice. The dosage form may contain any suitable binders, lubricants, suspending agents, coatings, solubilizers. Examples of suitable binding agents include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or seaweed sodium, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Preservatives, stabilizers, dyes and even flavoring agents can also be provided in the pharmaceutical compositions. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Suspending agents may also be used.

In certain embodiments, the lyophilized product is not microencapsulated.

In certain embodiments, the lyophilized product may comprise sugars, such as monosaccharides or disaccharides. Additionally or alternatively, the sugar can be a reducing sugar or a non-reducing sugar. In certain embodiments where the product comprises reducing sugars, non-reducing sugars can be excluded from the product, and vice versa. Examples of specific sugars that can be used as excipients include sucrose and trehalose.

The medicinal product may further comprise prebiotics. The term "prebiotic" means beneficially affecting the non-digestible components of a lyophilized product by selectively stimulating the growth and/or activity of one or a limited number of bacteria. Examples of prebiotics include oligosaccharides, fructooligosaccharides, and galactooligosaccharides.

In certain embodiments, the lyophilized product can be made into an orally administrable enteric form, that is, it is only capable of solubilizing in a selective medium (ie, the intestinal environment) thus preventing the release of its contents in the stomach. Enteric type. Such gastroprotective dosage forms may comprise an effective amount of a viable bacterial population and antioxidants, wherein the effective amount of the viable bacterial population (colony forming units (CFU)) is reduced by no more than 1 log in a simulated gastric environment. Gastroprotective properties can be determined by (a) exposing the dosage forms described herein to acidic media at pH 1.2 for 30 minutes, (b) exposing the dosage forms to enteric media at pH 6.8 for 45 minutes, and (c) comparing the relative exposure CFU before after exposure.

Those skilled in the art will be familiar with orally administrable enteric formulations, and such dosage forms include lozenges, capsules, granules, and other micro- or nano-formulations, such as alginate-encapsulated particles, and in the context of the present invention. In embodiments, the dosage form can be any of these dosage forms. Enteric-coated lozenges or capsules are especially preferred in some cases.

Skilled artisans will also be familiar with techniques for imparting enteric properties to dosage forms. This is typically achieved by applying an enteric coating to dosage forms such as lozenges or capsules. Suitable enteric coating materials include polymers that dissolve at a pH of 5.5 or above (eg Eudragit grades L 30 D-55 and L 100-55), those that dissolve at a pH of 6.0 or above ( For Eudragit L 100 and L 12, grades 5) and/or those materials that dissolve at pH values above 7.0 (for Eudragit S 100, S 12,5 and FS 30 grades D). Additionally or alternatively, where the dosage form includes a capsule in addition to the enteric coating, the capsule may also be bound at the junction between the two capsule halves to prevent the entry of gastric media.

In alternative embodiments, the dosage form may be inherently enteric. As used herein, the term "inherently enteric capsule" is used to refer to a material formed (partially or completely) that dissolves when exposed to a medium having a moderately acidic, neutral or basic pH value, thereby releasing the contents of the capsule into the medium ) capsules. In certain embodiments, the inherently enteric capsules when exposed to have about 4.0 or more, about 4.5 or more, about 5.0 or more, about 5.5 or more, about 6.0 or more, about 6.5 or more, or about 6.5 or more Or release its contents in a medium with a pH of about 7.0 or above. Likewise, the term "enteric-coated" is used to refer to a material that is Dissolves in media with a pH above 6.5 or about 7.0 or above.

Due to their inherent enteric properties, the capsules do not require post-fill processing, such as coating, drying and/or banding, that would otherwise potentially damage the viable bacterial population. Therefore, inherently enteric capsules do not contain a continuous coating (ie, a coating that covers the entire capsule) and/or are not bound.

Inherently enteric capsules may be single-layered or multi-layered and/or formed entirely or in part from gastrointestinal materials that dissolve at a particular pH. In multi-layer embodiments, one or more of the layers may be formed from enteric materials that dissolve at specific pH values.

Inherently enteric capsules can be formed from any material that allows the capsules to dissolve in whole or in part when exposed to a medium having a moderately acidic, neutral or basic pH. In embodiments of the present invention, inherently enteric capsules may be partially or completely formed from fatty acids, waxes, shellac, plastics, vegetable fibers, enteric polymers, or mixtures thereof. Enteric materials useful in the present invention (for creating inherently enteric forms, or in alternative embodiments, enteric coatings) include, but are not limited to, methacrylate polymers, methyl acrylate-methacrylic acid copolymers, Methacrylic acid-methyl methacrylate copolymer, polyvinyl acetate phthalate, shellac, sodium alginate, zein, dextrin, amylose and starch derivatives and cellulose and cellulose derivatives , including hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, cellulose acetate succinate, cellulose acetate trimellitate, cellulose acetate phthalate or its mixture. Plasticizers may also be included in the materials forming the inherently enteric capsules. Examples of materials that can be used to prepare inherently enteric capsules and methods for preparing such capsules are provided in European Patent No. 2722104, the contents of which are incorporated herein by reference. Examples of inherent enteric capsules are provided by Capsugel under the trade names enTRinsic DDT or ECDDT.

Capsules can be in any shape, form or configuration provided that they can be sealed to provide an enteric seal around the LBP contained therein. For example, capsules can be hard or soft. In certain embodiments, the capsule is a two-part capsule or a multi-part capsule (ie, the capsule is sealed by coupling more than two parts).

For two-part capsules or multi-part capsules, the capsule parts can be sealed by mechanically coupling two or more parts of the capsule. Any form of mechanical interaction that results in a seal around the LBP may be employed. Examples of contemplated mechanical interactions include push-in couplings, friction couplings, and/or thread couplings.

The invention is exemplified in the following examples. These examples are not intended to limit the scope of the invention and are intended to be illustrative only. EXAMPLES Example 1 - Annealing Step Improves Viability After Lyophilization Materials and Methods

Bacterial populations of Parabacteroides dineri (strain MRx0005, deposited under accession number NCIMB 42382) were grown to stationary phase using conventional fermentation techniques. The bacterial population was then harvested from the fermenter and concentrated (27X). The harvested biomass was mixed with a lyophilization buffer containing sucrose, maltodextrin DE9 and cysteine hydrochloride to provide a lyophilization medium. The ratio of biomass to lyoprotectant prior to lyophilization was 69.4:30.6 and the composition of the lyophilization buffer prior to lyophilization was 2% sucrose, 4% maltodextrin and 0.2% cysteine hydrochloride the final concentration.

The lyophilized medium was divided into two batches of 1 kg each prior to lyophilization. Freeze drying cycles were performed on both batches, which were essentially identical except for the performance of the annealing step.

More specifically, the lyophilization equipment was cooled to -45°C prior to loading the batches. Both batches of bacteria were frozen at a shelf temperature of -45°C for at least 2 hours (this time varied depending on the presence of an annealing step (see Table 2 below)), followed by: two preliminary drying steps at shelf temperature, (i) -9°C for 24 hours and (ii) 25°C for 20 hours, both at a pressure of 50 microbars, and a secondary drying step at a shelf temperature of 25°C for at least 10 hours . During the preliminary drying step, the shelf temperature was increased by about 5°C/hour. One of these batches was subjected to an annealing step during one or more freezing steps, wherein after 2 hours at a shelf temperature of -45°C, the bacteria were heated to an annealing temperature of -15°C, where The temperature was maintained for 5 hours, then cooled to a shelf temperature of -45°C, where it was incubated for an additional 3 hours. The second batch was not subjected to the annealing step. Lyophilized biomass from each batch was then resuspended in medium and tested for viability.

Conditions for the lyophilization process for both batches (ie, with and without the annealing step) are summarized in Table 2 below. Table 2 Steps of Freeze-Drying Method (A) Conditions for the batch used to perform the annealing step Conditions for batches without an annealing step pre-cooling Pre-cool lyophilization equipment to -45°C Freezing step Load batches into lyophilization equipment -45°C for 2 hours -45°C for 2 hours Annealing step Increase shelf temperature by approx. 60°C/h for 30 minutes Not applicable -15°C for 5 hours Decrease shelf temperature at approx. 30°C/h for 1 hour another freezing step -45°C for 3 hours Not applicable Preliminary drying step (1) Increase shelf temperature by approx. 5°C/h for 7 hours -9°C for 24 hours at 50 microbars Preliminary drying step (2) Increase shelf temperature by approx. 5°C/h for 7 hours 20 hours at 25°C at 50 microbars Secondary drying step 25°C for at least 10 hours at 50 microbars store 5℃

The temperatures listed in Table 2 refer to the shelf temperatures of the freeze-drying equipment. The secondary drying step in Table 2 continued until the product temperature had been at least 22°C for 10 hours.

The freeze-drying equipment was a Lyovac GT41 freeze-dryer with a capacity of 0.5 m ² . The water content was determined using a Karl Fischer Coulomb apparatus (Mettler Toledo) using the following analytical parameters (speed: 40%; mixing: 600 s and set point temperature: 120°C). The water activity of the samples was determined using an Aqualab water activity monitor at 20°C using the frozen mirror dew point technique. result

The results are shown in Figure 1 and Table 3.

1A and B show the structural integrity of lyophilized biomass obtained from a lyophilization process with and without an annealing step (FIG. 1A) and without an annealing step (FIG. IB). These figures demonstrate that the lyophilized biomass obtained from the lyophilization method using the annealing step exhibits a greater level of macroscopic crystal structure. The product obtained without the annealing step had a layer of about 30% of the product depth (measured at the point marked with the arrow) and the crystals were organized in a vertical manner. However, this layer in the product obtained with the annealing step is about 55% of the product depth (measured at the point marked with the arrow). Therefore, the inclusion of an annealing step improves the crystal structure of the lyophilized medium during the lyophilization process, which in turn facilitates the sublimation of moisture therefrom, thus improving lyophilization efficiency.

Table 3 shows the effect of the annealing step on the viability of lyophilized biomass with and without the annealing step. The viability of the biomass obtained without the annealing step (originally from a culture with a viability of ^1.6x1011 CFU/ml) was ^1.3x1010 CFU/g, while the biomass obtained without the annealing step Then 5.1x10 ⁹ CFU/g. As outlined in the embodiments, viability before (CFU/ml) and after lyophilization (CFU/g) was compared by explaining the loss of moisture content and thus the increase in bacterial cell concentration that occurs inherently during the lyophilization process as appropriate.

Furthermore, the lyophilization method including the annealing step resulted in a slightly lower % moisture content compared to the same method without the annealing step. Table 3 Freeze-drying method (A) Moisture content (%) water activity Viability before lyophilization (CFU/ml) Viability after lyophilization (CFU/g) annealing 3.2 0.101 1.6x10 ¹¹ 1.3x10 ¹⁰ not annealed 3.3 0.028 5.1x10 ⁹ in conclusion

Incorporating the annealing step into the lyophilization process maintains the viability of the bacterial population after lyophilization. Furthermore, the annealing step appears to result in improved crystallographic alignment in the lyophilized biomass. Example 2 - Annealing step improves viability after lyophilization Materials and Methods

The Parabacteroides dineri strain (MRX0005) deposited under accession number NCIMB 42382 was grown to stationary phase to a density of ^1.6x1011 CFU/ml before harvesting and resuspended in lyophilization buffer. The buffer contains a lyoprotectant formulation (at final concentration prior to lyophilization) containing the following components: 2% sucrose, 4% maltodextrin DE9 and 0.2% cysteine hydrochloride. The ratio of biomass to lyoprotectant was 69.4:30.6.

The lyophilized medium was divided into five batches of 1 kg each prior to lyophilization. Freeze drying cycles were performed on all batches, which were essentially the same except for the performance of the annealing step.

More specifically, the lyophilization equipment was cooled to -45°C prior to loading the batches. Five batches of bacteria were frozen at a shelf temperature of -45°C for at least 2 hours (this time varied depending on the presence of an annealing step (see Table 4 below)), followed by: two preliminary drying steps at shelf temperature, (i) -21°C for 24 hours and (ii) 25°C for 20 hours, both at a pressure of 276 microbars, and a secondary drying at a shelf temperature of 25°C for at least 10 hours step. During the preliminary drying step, the shelf temperature was increased by about 5°C/hour. Both of these batches were subjected to an annealing step during one or more freezing steps, wherein after 2 hours at a shelf temperature of -45°C, the bacteria were heated to an annealing temperature of -15°C, where they were The temperature was maintained for 5 hours, then cooled to a shelf temperature of -45°C, where it was incubated for an additional 3 hours. The remaining three batches were not subjected to the annealing step. Lyophilized biomass from each batch was then resuspended in medium and tested for viability.

Conditions for the lyophilization process for batches with and without the annealing step are summarized in Table 4 below. Table 4 Steps of Freeze-Drying Method (B) Conditions for the batch used to perform the annealing step Conditions for batches without an annealing step pre-cooling Pre-cool lyophilization equipment to -45°C Freezing step Load batches into lyophilization equipment -45°C for 2 hours -45°C for 2 hours Annealing step Increase shelf temperature by approx. 60°C/h for 30 minutes Not applicable -15°C for 5 hours Decrease shelf temperature at approx. 30°C/h for 1 hour another freezing step -45°C for 3 hours Not applicable Preliminary drying step (1) Increase shelf temperature by approx. 5°C/h for 5 hours -21°C for 24 hours at 276 microbars Preliminary drying step (2) Increase shelf temperature by approx. 5°C/h for 9 hours 20 hours at 25°C at 276 microbars Secondary drying step 25°C for at least 10 hours at 276 microbars store 5℃

The temperatures listed in Table 4 refer to the shelf temperatures of the freeze-drying equipment. The secondary drying step in Table 4 continued until the product temperature had been at least 22°C for 10 hours.

The freeze-drying equipment was a Lyovac GT41 freeze-dryer with a capacity of 0.5 m ² . The water content was determined using a Karl Fischer Coulomb apparatus (Mettler Toledo) using the following analytical parameters (speed: 40%; mixing: 600 s and set point temperature: 120°C). The water activity of the samples was determined using an Aqualab water activity monitor at 20°C using the frozen mirror dew point technique. result

The results are shown in Figure 2 and Table 5.

2A-F show the structural integrity of lyophilized biomass obtained from a lyophilization process with and without an annealing step (FIGS. 2A and B) and without an annealing step (FIG. 2C-F). These figures demonstrate that the lyophilized biomass obtained from the lyophilization method using the annealing step exhibits a greater level of macroscopic crystal structure. The product obtained without the annealing step had a layer of about 25-35% of the product depth (measured at the point marked with the arrow) and the crystals were organized in a vertical manner. However, this layer in the product obtained with the annealing step is about 50-55% of the product depth (measured at the point marked with the arrow). Therefore, the inclusion of an annealing step improves the crystal structure of the lyophilized biomass during the lyophilization procedure.

Table 5 shows the effect of the annealing step on the viability of the lyophilized biomass with and without the annealing step. The viability of biomass obtained from the batches subjected to the annealing step was ^9.4x1010 CFU/g and ^1.2x1011 CFU/g. The viability of biomass obtained from batches without the annealing step was between ^5.5x1010 CFU/g and ^7.2x1010 CFU/g. As outlined in the embodiments, viability before (CFU/ml) and after lyophilization (CFU/g) was compared by explaining the loss of moisture content and thus the increase in bacterial cell concentration that occurs inherently during the lyophilization process as appropriate.

Furthermore, consistent with the observations above, the lyophilization method including the annealing step resulted in a lower % moisture content compared to the same method without the annealing step. Table 5 Freeze-drying method (B) Moisture content (%) water activity Viability before lyophilization (CFU/ml) Viability after lyophilization (CFU/g) annealing 3.3 0.04 1.6x10 ¹¹ 9.4x10 ¹⁰ annealing - 0.03 1.2x10 ¹¹ not annealed 3.5 0.08 7.1x10 ¹⁰ not annealed 3.6 0.08 7.2x10 ¹⁰ not annealed 3.6 0.07 5.5x10 ¹⁰ in conclusion

Incorporating the annealing step into the lyophilization process significantly increases the viability of the bacterial population after lyophilization. Furthermore, the annealing step appears to result in improved crystallographic alignment in the lyophilized biomass. Therefore, the annealing step represents an advantageous additional step in the lyophilization process. Example 3 - Using Scanning Electron Microscopy (SEM) to Assess Pore Structure of Lyophilized Products Produced Using a Lyophilization Process with and Without an Annealing Step Materials and Methods

A sample of the lyophilized product obtained from Example 2 above was analyzed using SEM. Fragments of the lyophilized product were attached to aluminum stubs using Electrodag 502, followed by coating with a layer of gold palladium. The prepared lyophilized product was then imaged using a Zeiss EVO MA10 scanning electron microscope at various depths of field using a secondary electron detector. Results and Conclusions

The results of the SEM analysis are shown in Figure 3 (A-D). The left image in each of FIGS. 3A-D shows the microstructure of the lyophilized product obtained from the lyophilization method in Example 2 without annealing. The right images in each of FIGS. 3A-D show the microstructure of the lyophilized product obtained from the lyophilization method annealed in Example 2. Figures 3A and B show the crystal structure at reduced magnification, indicating that the presence of an annealing step enables the crystal to form an ordered layer more efficiently. Figure 3C studies the pie structure at higher magnification and supports the result that there is an annealing step that allows the crystals to better align into layers. Finally, Figure 3D shows the crystal at the highest magnification and shows that the presence of an annealing step maximizes the uniformity of crystal formation and reduces crystalline debris that is not present in the main layers.

Therefore, the use of an annealing step ensures a greater level and consistency of crystal structure at all levels in the lyophilized product. Bibliography 1 Louis Rey and Joan May, Drugs and the Pharmaceutical Sciences, Third Edition 2 Ablett et al. J Chem Soc Faraday Trans. (1992); 88:789-794 Numbered Examples

The present invention also provides the following numbered examples: 1. A method for preparing a lyophilized product comprising a viable bacterial population, the method comprising: (i) providing a lyophilized medium comprising a viable bacterial population; (ii) making the The lyophilized medium is subjected to: a. one or more freezing steps; b. one or more annealing steps at a temperature different from the one or more freezing steps; and c. at least one preliminary drying step; and (iii) ) to collect the lyophilized product. 2. The method of embodiment 1, wherein the one or more freezing steps are cycled with the one or more annealing steps. 3. The method of embodiment 1, wherein the viability (CFU/g, in dry weight form) of the lyophilized product is lower than the viability (CFU/ml) of the lyophilized medium prior to lyophilization by equal to or less than 10 ³ CFU/ml. g, equal to or less than 10 ² CFU/g or equal to or less than 10 CFU/g. 4. The method of embodiment 3, wherein the viability (CFU/g, in dry weight form) of the lyophilized product is equal to or less than 10 CFU/g lower than the viability (CFU/ml) of the lyophilized medium prior to lyophilization . 5. The method of any one of the preceding embodiments, wherein the water content of the lyophilized product is less than 5.0, 4.5, 4.0 or 3.5 wt%. 6. The method of any one of the preceding embodiments, wherein the water content of the lyophilized product is between 3.0 wt% and 4.5 wt%, and wherein the water content of the lyophilized product is between 3.3 wt% and 4.4 wt% between wt%. 7. The method of any preceding embodiment, wherein the one or more annealing steps are performed at a higher temperature than the one or more freezing steps. 8. The method of any one of the preceding embodiments, wherein the one or more freezing steps are performed at a temperature between about -40°C and -50°C, optionally wherein the one or more freezing steps are performed at about- carried out at a temperature of 45°C. 9. The method of any one of the preceding embodiments, wherein the at least one preliminary drying step is performed at a temperature between about -5°C and -25°C, optionally wherein the at least one preliminary drying step is performed at a temperature between about 50 and 300°C. at a pressure of between microbars, optionally wherein the at least one preliminary drying step is performed under the following conditions: (i) a temperature of about -9°C and a pressure of about 50 microbars; or (ii) a temperature of about -21°C temperature and pressure of about 275 microbars. 10. The method of any one of the preceding embodiments, wherein the one or more annealing steps are performed at a temperature between about -10°C and -20°C, optionally wherein the one or more annealing steps are performed at about- carried out at a temperature of 15°C. 11. The method of any one of the preceding embodiments, wherein the method further comprises a second preliminary drying step, wherein the second preliminary drying step is performed at a temperature of about 25°C and a pressure between about 50 and 300 microbars . 12. The method of any of the preceding embodiments, wherein the population of viable bacteria is not or does not contain lactic acid bacteria. 13. The method of any one of the preceding embodiments, wherein the live bacterial colony is lyophilized in a freeze-dried medium, wherein the freeze-dried medium comprises a freeze-dried buffer as the case may be, such as wherein the freeze-dried buffer comprises and contains the following components The lyoprotectant formulation (at final concentration before lyophilization): 2% sucrose, 4% maltodextrin DE9 and 0.2% cysteine hydrochloride, and where the lyophilization buffer does not contain seaweed as appropriate sugar. 14. The method of any preceding embodiment, wherein the method comprises a first freezing step at a temperature of about -45°C. 15. The method of any preceding embodiment, wherein the method comprises an annealing step at a temperature of about -15°C. 16. The method of any preceding embodiment, wherein the method comprises a second freezing step at a temperature of about -45°C. 17. The method of any preceding embodiment, wherein the method comprises a preliminary drying step at a temperature of about -9°C and a pressure of about 50 microbars. 18. The method of any one of embodiments 1-16, wherein the method comprises a preliminary drying step at a temperature of about -21°C and a pressure of about 275 microbars. 19. The method of any one of the preceding embodiments, wherein the method comprises: (i) providing a lyophilized medium comprising a viable bacterial population; (ii) reducing the temperature (eg, shelf temperature or tray temperature) to about -40 a temperature between °C and -50 °C; (iii) maintaining the temperature between about -40 °C and -50 °C for about 1 to 3 hours; (iv) increasing the temperature (eg shelf or tray temperature) to a temperature between about -10°C and -20°C; (v) maintaining the temperature between about -10°C and -20°C for about 4 to 8 hours; (vi) maintaining the temperature (eg shelf temperature or tray temperature) temperature) to a temperature between about -40°C and -50°C; (vii) maintaining the temperature between about -40°C and -50°C for about 2 to 4 hours; (viii) placing the lyophilized medium in a drying at a temperature between about -5°C and -25°C and a pressure between about 50 and about 300 microbars; (ix) maintaining the drying temperature for about 18 to 30 hours, and (x) collecting the lyophilized product. 20. A lyophilized product produced by the method of any of the preceding embodiments, optionally wherein the lyophilized product is formulated into a pharmaceutical composition, eg, wherein the lyophilized product is in a pharmaceutical dosage form. 21. The method of any one of embodiments 1-19 or the product of embodiment 20, wherein the lyophilized product is a live biotherapeutic product.

Figure 1 : Macroscopic aspect of the lyophilized product obtained from the method of Example 1 ( A - with annealing step; B - without annealing step). Arrows indicate locations where the thickness of the strips of unique macroscopic crystal structure was measured. Figure 2 : Macroscopic view of the lyophilized product obtained from the method of Example 2 ( AB - with annealing step; CF - without annealing step). Arrows indicate locations where the thickness of the strips of unique macroscopic crystal structure was measured. Figure 3 : Microscopic aspect of the lyophilized product obtained from the method of Example 2 ( AD - left image shows the product obtained without the annealing step; right image shows the product obtained with the annealing step product).

Claims (16)

A method for preparing a lyophilized product comprising a viable bacterial population, the method comprising: (i) providing lyophilized media containing viable bacterial populations; (ii) subjecting the lyophilized medium to: a. one or more freezing steps; b. conducting one or more annealing steps at a temperature different from the one or more freezing steps; and c. at least one preliminary drying step; and (iii) Collect the lyophilized product. The method of claim 1, wherein the lyophilized medium is provided in an amount of at least about 1 g. The method of claim 1 or 2, wherein the one or more freezing steps are cyclically performed with the one or more annealing steps. The method of any one of the preceding claims, wherein the viability (CFU/g, in dry weight) of the lyophilized product is equal to or less than 10 ³ lower than the viability (CFU/ml) of the lyophilized medium prior to lyophilization CFU/g, equal to or less than 10 ² CFU/g, or equal to or less than 10 CFU/g, as the case may be wherein the viability of the lyophilized product (CFU/g, in dry weight) is greater than that of the lyophilized medium prior to lyophilization Viability (CFU/ml) was low equal to or less than 10 CFU/g. The method of any one of the preceding claims, wherein the water content of the freeze-dried product is: (i) less than 5.0, 4.5, 4.0 or 3.5 wt%; (ii) between 3.0 wt% and 4.5 wt%; or (iii) between 3.3 wt% and 4.4 wt%. The method of any of the preceding claims, wherein the one or more annealing steps are performed at a higher temperature than the one or more freezing steps. The method of any one of the preceding claims, wherein the one or more freezing steps are performed at a temperature between about -40°C and -50°C, optionally wherein the one or more freezing steps are at about -45°C at the temperature. The method of any of the preceding claims, wherein the at least one preliminary drying step is performed at a temperature between about -5°C and -25°C, optionally wherein the at least one preliminary drying step is at about 50 and 300 microbars under pressure between, as the case may be wherein the at least one preliminary drying step is carried out under the following conditions: (i) a temperature of about -9°C and a pressure of about 50 microbars; or (ii) a temperature of about -21°C and a pressure of about 275 microbars. The method of any of the preceding claims, wherein the one or more annealing steps are performed at a temperature between about -10°C and -20°C, optionally wherein the one or more annealing steps are at about -15°C at the temperature. The method of any of the preceding claims, wherein the method further comprises a second preliminary drying step, wherein the second preliminary drying step is performed at a temperature of about 25°C and a pressure between about 50 and 300 microbars. The method of any of the preceding claims, wherein the population of viable bacteria is not or does not contain lactic acid bacteria. The method of any one of the preceding claims, wherein the viable bacterial population is lyophilized in a lyophilization medium, optionally wherein the lyophilization medium comprises a lyophilization buffer, eg, wherein the lyophilization buffer comprises a lyophilization buffer comprising Dry protectant formulation (at final concentration prior to lyophilization): 2% sucrose, 4% maltodextrin DE9 and 0.2% cysteine hydrochloride, and optionally wherein the lyophilization buffer does not contain trehalose. The method of any of the preceding claims, wherein the method comprises: (i) a first freezing step at a temperature of about -45°C; (ii) an annealing step at a temperature of about -15°C; (iii) a second freezing step at a temperature of about -45°C; and/or (iv) Preliminary drying step: a. At a temperature of about -9°C and a pressure of about 50 microbars; or b. At a temperature of about -21°C and a pressure of about 275 microbars. The method of any of the preceding claims, wherein the method comprises: (i) providing lyophilized media containing viable bacterial populations; (ii) reducing the temperature (eg shelf temperature or tray temperature) to a temperature between about -40°C and -50°C; (iii) maintaining the temperature between about -40°C and -50°C for about 1 to 3 hours; (iv) increasing the temperature (eg shelf or tray temperature) to a temperature between about -10°C and -20°C; (v) maintaining the temperature between about -10°C and -20°C for about 4 to 8 hours; (vi) reducing the temperature (eg shelf temperature or tray temperature) to a temperature between about -40°C and -50°C; (vii) maintaining the temperature between about -40°C and -50°C for about 2 to 4 hours; (viii) drying the lyophilized medium at a temperature between about -5°C and -25°C and a pressure between about 50 and about 300 microbars; (ix) maintaining the drying temperature for about 18 to 30 hours, and (x) Collection of lyophilized product. A lyophilized product produced by the method of any of the preceding claims, optionally wherein the lyophilized product is formulated into a pharmaceutical composition, eg, wherein the lyophilized product is in a pharmaceutical dosage form. The method of any one of claims 1-14 or the product of claim 15, wherein the lyophilized product is a live biotherapeutic product.

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