WO2022029303A1

WO2022029303A1 - Lyophilisation process

Info

Publication number: WO2022029303A1
Application number: PCT/EP2021/072033
Authority: WO
Inventors: Christophe Carite; Sophie Declomesnil; Pierre PEYNICHOU
Original assignee: 4D Pharma Plc
Priority date: 2020-08-06
Filing date: 2021-08-06
Publication date: 2022-02-10
Also published as: TW202220639A

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

LYOPHILISATION PROCESS

FIELD OF THE INVENTION

The invention relates to processes of lyophilisation of live biotherapeutic products which improve the viability of the lyophilised live biotherapeutic product as well as the efficiency of production of such products.

BACKGROUND TO THE INVENTION

Lyophilisation is a widely used process for formulating pharmaceutical, biotechnological and other types of products. It is an effective way to prepare solid products, even if those products are pharmaceutical products which are destined to be administered in liquid form to patients. Lyophilisation is also a convenient way to produce preparations containing live organisms, or chemically sensitive products obtained from organisms.

There are many commercially operated lyophilisation processes; these typically involve three phases, namely i) freezing, ii) primary drying or sublimation and iii) secondary drying or desorption [1],

During the freezing phase and optionally also during the sublimation phase, the product is frozen by exposure to low temperatures, typically between -20°C and -80°C. During this period, the fluids in the sample become solid bodies, either crystalline, amorphous or glass. An amorphous state occurs when a material is in a solid state but the molecules are not packed in a repeating long-range ordered fashion. As the temperature decreases during the one or more freezing steps, the so-called freeze-concentration continues until the solution reaches the glass transition temperature. This is the point at which the freeze-concentration is at a maximum, and any further thermodynamically favoured freeze-concentration is arrested because the mobility of the remaining liquid in the sample is too low (glassy state) to permit crystallisation to the ice interface. Once the product has attained the target reduced temperature, the frozen material is placed under vacuum and is progressively heated to deliver enough energy for the ice to sublimate. This is known as the primary drying step, which generally results in much of the ice present in the product being removed. The primary drying step usually does not take place at a temperature above the glass transition temperature such that only sublimation of crystalline water occurs. The third phase, the secondary drying or desorption step, starts when ice has been distilled away such that a higher vacuum allows the progressive extraction of bound water at above-zero temperatures.

During lyophilisation, products comprising live cells, such as probiotic bacteria, are exposed to various stresses, in particular dehydration, compromising cell survival. As water plays an important role in cell integrity and stability, both freezing and drying may be detrimental to viability not least from a structural perspective in light of the formation of ice crystals, but also due to the increased concentration of solutes in the unfrozen fraction, which can cause chemical and osmotic damage. A number of different strategies have been developed to enhance cellular viability during freeze-drying, for example, the addition of protective agents to the drying medium.

Known processes for lyophilising bacterial cells (e.g. in the manufacture of probiotic health supplements) achieve modest levels of live biotherapeutic product viability post-lyophilisation. While such losses in viability may be tolerated in the manufacture of probiotic products, these processes are not necessarily appropriate for the preparation of pharmaceutical products (e.g. live biotherapeutic products or LBPs) which are subject to more stringent regulation including strict requirements regarding the maintenance of cellular viability. The present inventors have identified that the viability of a live biotherapeutic product can be significantly increased with the incorporation of an annealing step into the lyophilisation process.

BRIEF DESCRIPTION OF THE INVENTION

In a first aspect, the invention provides a process for preparing a lyophilised product comprising a viable bacterial population, the process comprising:

(i) providing a lyophilisation medium comprising a viable bacterial population;

(ii) subjecting the lyophilisation medium to: a. one or more freezing steps; b. one or more annealing steps conducted at a different temperature to the one or more freezing steps; and c. at least one primary drying step; and

(iii) collecting a lyophilised product.

The annealing step is one in which the temperature to which the lyophilisation medium is exposed is altered, e.g. raised from the temperature of the freezing step to a temperature above the glass transition temperature of the lyophilisation medium. Annealing can be carried out during the initial cooling in the freezing step. Additionally or alternatively, annealing can be used as a post-freezing warming and hold step, followed by cooling (e.g. returning the sample to below the glass transition temperature). Such an annealing step may dilute the glassy state of the remaining liquid allowing further processes to take place, including ice crystal maturation, the crystallisation of solutes and possibly degradative reactions. Maturation of ice crystals results in those crystals increasing in size. This growth of ice crystals would not be considered to be desirable when lyophilising live biotherapeutic products as this could result in the crushing of bacterial cells resulting in their inactivation which would be highly unattractive as maintenance of high bacterial viability is of critical importance in the production of live biotherapeutics. Surprisingly and unexpectedly, however, the performance of an annealing step (as demonstrated in the examples) has been shown to enhance the viability of bacterial cells in a live biotherapeutic product.

In embodiments of the invention, the one or more freezing steps are cycled with the one or more annealing steps. In certain embodiments, the water content of the lyophilised product is less than 5.0 wt%. In certain embodiments, the water content of the lyophilised product is between 3.0 and 4.5 wt%. In certain preferred embodiments, the water content of the lyophilised product is between 3.3 and 4.4 wt%. In certain embodiments, the water content of the lyophilised product is less than 4.5 wt%. In certain embodiments, the water content of the lyophilised product is less than 4.0 wt%. In certain embodiments, the water content of the lyophilised product is less than 3.5 wt%.

In certain embodiments, the viability (CFU/g as dry weight) of the lyophilised product is equal to or less than 10³ CFU/g, equal to or less than 10² CFU/g, or equal to or less than 10 CFU/g lower than the viability of the lyophilisation medium prior to lyophilisation (CFU/ml). Additionally or alternatively, the lyophilised product has a viability of about 1 x 10⁹ CFU/g or higher, about 5 x 10⁹ CFU/g or higher, about 1 x 10¹° CFU/g or higher or 5 x 10¹° CFU/g.

In certain embodiments, the annealing step occurs at a temperature higher than the temperature of the freezing step which precedes the annealing step. In certain embodiments, the annealing step occurs at a temperature higher than the glass transition temperature of the lyophilised product. Additionally or alternatively, the annealing step occurs at a temperature higher than the collapse temperature of the lyophilised product.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Macroscopic aspects of the lyophilised products obtained from the process of Example 1 (A - with an annealing step; B - without an annealing step). The arrows indicate the position at which the thickness of the bands of distinct macroscopic crystal organization were measured.

Figure 2: Macroscopic aspects of the lyophilised products obtained from the process of Example 2 (A-B - with an annealing step; C-F - without an annealing step). The arrows indicate the position at which the thickness of the bands of distinct macroscopic crystal organization were measured.

Figure 3: Microscopic aspects of the lyophilised products obtained from the process of Example 2 (A-D - left-hand images show product obtained without an annealing step; right-hand images show product obtained with an annealing step).

DETAILED DESCRIPTION OF THE INVENTION

The ‘glass transition temperature’ is the temperature at which amorphous forms such as lyophilised products have a change in properties. In certain embodiments, if the sample is stored below the glass transition temperature, the amorphous form will be brittle (/.e. in a glassy state). If the sample is stored above its glass transition temperature, the amorphous form will become rubbery. At this temperature, the molecules in the glass exhibit a major change in mobility.

The ‘collapse temperature’ is the temperature at which lyophilised products structurally collapse. Those skilled in the art will appreciate that the determination of the glass transition temperature or collapse temperature of lyophilised products are straightforward and can be achieved using techniques and apparatus with which they will be familiar. For the purposes of the present invention, in embodiments in which heating I cooling steps are conducted with reference to the glass transition temperature or collapse temperature (e.g. in which an annealing step is carried out by heating a frozen lyophilisation medium to or in excess of the glass transition temperature), it will firstly be necessary to provide a lyophilised product and determine its glass transition temperature or collapse temperature. Then it would be possible to perform the process in accordance with the invention based on the glass transition temperature or collapse temperature of the lyophilised product. Those skilled in the art will of course recognise that such steps are routine and not burdensome.

In embodiments of the invention, the lyophilised 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 viable bacterial cells intended for therapeutic application. The US FDA, in guidance published in June 2016 (Early Clinical Trials with Live Biotherapeutic Products: Chemistry, Manufacturing, and Control Information), defined live biotherapeutic products as a biological product that 1) contains live organisms, such as bacteria; 2) is applicable to the prevention, treatment, or cure of a disease or condition of human beings; and 3) is not a vaccine. Thus, LBPs are regulated products which, in order to be approved as medicinal products (unlike conventional probiotics) must meet strict and onerous requirements of safety and efficacy.

Unless stated otherwise, reference to ‘maintained’ with respect to temperature herein means the achieving of a particular temperature irrespective of the time spent at that particular temperature.

Steps of the lyophilisation process

The lyophilisation process comprises at least the following steps:

(i) providing a lyophilisation medium comprising a viable bacterial population;

(ii) subjecting the lyophilisation medium to: a. one or more freezing steps; b. at least one annealing step, optionally between the one or more freezing steps, occurring at a different temperature to the one or more freezing steps; and c. at least one primary drying step; and

(iii) collecting a lyophilised product.

In certain embodiments, the lyophilisation process comprises at least the following steps:

(i) providing a lyophilisation medium comprising a viable bacterial population;

(ii) subjecting the lyophilisation medium to: a. at least two freezing steps; b. at least one annealing step, between the at least two freezing steps, occurring at a different temperature to the two freezing steps; and c. at least one primary drying step; and (iii) collecting a lyophilised product.

In certain embodiments, the lyophilisation process comprises one or more additional steps, for example a pre-treatment step and/or a secondary drying step.

Provision of the lyophilisation medium

The lyophilisation medium may be provided by preparing a biomass comprising the viable bacterial population using conventional fermentation processes. In certain embodiments, the live biotherapeutic biomass may be concentrated. In certain embodiments, the biomass may be concentrated to 10⁸ bacterial cells/ml or /g. In certain embodiments, the biomass is concentrated to 10⁹ bacterial cells/ml or /g. In certain embodiments, the biomass is concentrated to 10¹° bacterial cells/ml or /g. In certain embodiments, the biomass is concentrated to 10¹¹ bacterial cells/ml or /g. In certain embodiments, the biomass is concentrated to 10¹² cells/ml or /g.

The examples of the present application demonstrate that the process of the present invention can be used to produce lyophilised material on a commercial scale. Thus, in embodiments of the invention, the lyophilisation medium is provided in an amount of at least about 1g, at least about 2g, at least about 5g, at least about 10g, at least about 20g, at least about 50g, at least about 100g, at least about 200g, at least about 500g, at least about 1 kg or at least about 2kg.

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

Additionally or alternatively, the lyophilisation medium comprises excipients such as non-reducing sugars. In certain embodiments, the lyophilisation medium comprises cryopreservatives, for example sucrose, maltose, maltodextrin, and/or glycerol. In certain embodiments, the lyophilisation medium comprises sucrose. In certain embodiments, the lyophilisation medium does not comprise trehalose. In certain embodiments, the lyophilisation medium comprises bulking agents, such as albumin, glycine, mannitol, hydroxyethyl starch or dextran. In certain embodiments, the lyophilisation medium comprises glycine. In certain embodiments, the lyophilisation medium comprises a non-ionic surfactant. In embodiments of the invention, the lyophilisation medium comprises an antioxidant, for example cysteine. In a preferred embodiment, the lyophilisation medium comprises a lyoprotectant formula comprising (at a final concentration priorto lyophilisation): 2% sucrose, 4% maltodextrine DE9 and 0.2% cysteine HCI. In a preferred embodiment, the ratio of the viable bacterial population to lyoprotectant is about 70:30.

The pre-treatment step

In certain embodiments, the lyophilisation process comprises a pre-treatment step. Such a pretreatment step can include diluting the viable bacterial population; revision of the formulation of the lyophilisation medium, for example the addition of compounds to increase the stability of the viable bacterial population; decreasing a high vapour pressure solvent in the lyophilisation medium; and/or increasing the surface area of the viable bacterial population.

In certain embodiments, the pre-treatment step comprises the addition of stabilising agents and/or protective agents to the lyophilisation medium.

The freezing step

In certain embodiments, during the one or more freezing steps, the lyophilisation medium is cooled to a temperature below the glass transition temperature of the lyophilised product. Additionally or alternatively, during the one or more freezing steps, the lyophilisation medium is cooled to a temperature below the collapse temperature of the lyophilised product. In certain embodiments, the temperature of the one or more freezing steps is below 0°C, about -5°C or lower, about -10°C or lower, about -15°C or lower, about -20°C or lower, about -25°C or lower or about -30°C or lower, for example between -40 and -80°C. In certain embodiments, the temperature of the one or more freezing steps is between -40 and -60°C. In certain embodiments, the temperature of the one or more freezing steps is between -60 and -80°C. In certain embodiments, the temperature of the one or more freezing steps is between -30 and -40°C. In certain embodiments, the temperature of the one or more freezing steps is between -40 and -50°C. In certain embodiments, the temperature of the one or more freezing steps is between -50 and -60°C. In certain embodiments, the temperature of the one or more freezing steps is between -60 and -70°C. In certain embodiments, the temperature of the one or more freezing steps is between -70 and -80°C. In certain embodiments, the temperature of the one or more freezing steps is -30°C or lower. In certain embodiments, the temperature of the one or more freezing steps is -35°C or lower. In certain embodiments, the temperature of the one or more freezing steps is -40°C or lower. In certain embodiments, the temperature of the one or more freezing steps is -45°C or lower. In certain embodiments, the temperature of the one or more freezing steps is -50°C or lower. In certain embodiments, the temperature of the one or more freezing steps

-55°C or lower. In certain embodiments, the temperature of the one or more freezing steps

-60°C or lower. In certain embodiments, the temperature of the one or more freezing steps is -65°C or lower. In certain embodiments, the temperature of the one or more freezing steps is -70°C or lower. In certain embodiments, the temperature of the one or more freezing steps is -75°C or lower. In certain embodiments, the temperature of the 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 and about -50°C. In a preferred embodiment, the temperature of the one or more freezing steps is below -45°C. In another preferred embodiment, the temperature of the one or more freezing steps is about -45°C.

In certain embodiments, the one or more freezing step involves the snap freezing of the lyophilisation medium comprising the viable bacterial population. In certain embodiments, the snap freezing process involves the rapid cooling of the lyophilisation medium, e.g. using liquid nitrogen. In certain embodiments, the snap freezing process involves the cooling of the lyophilisation medium by exposure to an environment (e.g. 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 of less.

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

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

In certain embodiments, the temperatures used in the process of the invention refer to the temperature of a component of the lyophilisation apparatus, for example, the lyophilisation chamber, the shelf of the lyophilisation apparatus or a tray loaded into the lyophilisation apparatus. In a preferred embodiment, the temperatures used in the process of the invention refer to the temperature of the shelf of the lyophilisation apparatus. Accordingly, in certain embodiments, during the one or more freezing steps, the lyophilisation chamber is at a temperature disclosed herein. In certain embodiments, during the one or more freezing steps, the lyophilisation chamber is maintained at any temperature between -40 and - 80°C. In certain embodiments, during the one or more freezing steps, the lyophilisation chamber is at any temperature between -40 and -60°C. In certain embodiments, during the one or more freezing steps, the lyophilisation chamber is maintained at any temperature between -60 and -80°C. In certain embodiments, during the one or more freezing steps, the lyophilisation chamber is maintained at any temperature between -30°C and -40°C. In certain embodiments, during the one or more freezing steps, the lyophilisation chamber is maintained at any temperature below the eutectic point of the lyophilisation medium. Accordingly, in certain embodiments, during the one or more freezing steps, the shelf or tray of the lyophilisation chamber is maintained at any temperature between -40 and -80°C. In certain embodiments, during the one or more freezing steps, the shelf or tray of the lyophilisation chamber is maintained at any temperature between -40 and -60°C. In certain embodiments, during the one or more freezing steps, the shelf or tray of the lyophilisation chamber is maintained at any temperature between -60 and -80°C. In certain embodiments, during the one or more freezing steps, the shelf or tray of the lyophilisation chamber is maintained at any temperature between -30°C and -40°C. In certain embodiments, during the one or more freezing steps, the shelf or tray of the lyophilisation chamber is maintained at any temperature below the eutectic point of the lyophilisation medium. In certain preferred embodiments, the lyophilisation chamber is pre-cooled before placing the sample in the chamber. In certain preferred embodiments, the shelf or tray of the lyophilisation chamber is pre-cooled before placing the sample on the shelf of the lyophilisation chamber.

In certain embodiments, the lyophilisation medium is frozen in incremental decreases in temperature. In certain embodiments, the temperature of the lyophilisation medium is slowly reduced. In certain embodiments, the one or more freezing step is slow freezing, for example by decreasing the temperature of lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber at a rate of between 0.1 and 0.3°C/min. In certain embodiments, the one or more freezing step is a slow freezing step, for example by decreasing the temperature of lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber at a rate of 0.1 °C/min or higher. In certain embodiments, the one or more freezing step occurs by decreasing the temperature of lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber at a rate of 0.5°C/min or higher. In certain embodiments, the one or more freezing step occurs by decreasing the temperature of lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber at a rate of 1 °C/min or higher. In certain embodiments, the one or more freezing step is a moderate freezing step, for example by decreasing the temperature of lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber at a rate of 1 ,5°C/min or higher. In certain embodiments, the one or more freezing step occurs by decreasing the temperature of lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber at a rate of between 0.3 and 1 ,5°C/min. In certain embodiments, the one or more freezing step occurs by decreasing the temperature of the lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber by 2°C/min or higher. In certain embodiments, the one or more freezing step occurs by decreasing the temperature of the lyophilisation chamber and/orthe shelf ortray ofthe lyophilisation chamber by 5°C/h or higher. In certain embodiments, the one or more freezing step occurs by decreasing the temperature of the lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber by 30°C/h or higher. In certain embodiments, the one or more freezing step occurs by decreasing the temperature of the lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber by 60°C/h or higher. In certain embodiments, the one or more freezing step is a fast or snap freezing step, for example by immersing the lyophilisation medium in liquid nitrogen. In certain embodiments, the lyophilisation medium is incubated and maintained at a single freezing temperature or within a range of freezing temperatures. In certain embodiments, the lyophilisation medium is incubated in a pre-cooled lyophilisation chamber.

In certain embodiments, the lyophilisation medium is maintained at the temperature of one or more of the stages of the incremental freezing process. In certain embodiments, the lyophilisation 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 lyophilisation 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 lyophilisation 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 lyophilisation medium is retained at a freezing temperature for less than 1 hour. In certain embodiments, the lyophilisation medium is retained at a freezing temperature for at least 1 hour. In certain embodiments, the lyophilisation medium is retained at a minimum freezing temperature for less than 1 minute. In certain embodiments, the lyophilisation medium is retained at a minimum freezing temperature for at least 1 minute. In certain embodiments, the lyophilisation medium is retained at a minimum freezing temperature for between 1 minute and 1 hour. Accordingly, in certain embodiments, the lyophilisation medium is retained at a minimum freezing temperature for less than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. Accordingly, in certain embodiments, the lyophilisation medium is retained at a minimum freezing temperature for at least 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. Accordingly, in certain embodiments, the lyophilisation medium is retained at a 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 lyophilisation medium is retained at a 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 lyophilisation medium is retained at a minimum freezing temperature for less than 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. In certain embodiments, the lyophilisation medium is retained at a minimum freezing temperature for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. In certain embodiments, the lyophilisation medium is retained at a minimum freezing temperature for about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. In certain embodiments, the lyophilisation medium is retained at a minimum freezing temperature for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. In a preferred embodiment, the lyophilisation medium is retained at a minimum freezing temperature for at least 2 hours. In a preferred embodiment, the lyophilisation medium is retained at a minimum freezing temperature for about 2 hours. In certain embodiments, wherein the lyophilisation process comprises at least two freezing steps, flanking an annealing step, the lyophilisation medium is retained at the minimum freezing temperature for a first time period, e.g. at least 2 hours in the first freezing step and for a second, optionally longer, time period (e.g. at least 3 hours) in the second freezing step. In a preferred embodiment, wherein the lyophilisation process comprises at least two freezing steps, flanking an annealing step, the lyophilisation medium is retained at the minimum freezing temperature for about 2 hours in the first freezing step and for about 3 hours in the second freezing step.

In certain embodiments, the freezing step takes place at atmospheric pressure. In certain embodiments, the one or more freezing step takes place under vacuum. In certain embodiments, the one or more freezing step takes place at a pressure of about 10 pBar or higher, about 20 pBar or higher or about 50 pBar or higher. In certain embodiments, the one or more freezing step takes place at a pressure of about 5000 pBar or lower, about 2000 pBar or lower, about 1000 pBar or lower or about 500 pBar or lower. In certain embodiments, the one or freezing step takes place at a pressure of 50 to 300 pBar. In certain embodiments, the one or more freezing step takes place at a pressure of 50 to 100 pBar. In certain embodiments, the one or more freezing step takes place under anaerobic conditions in line with the disclosure of EP Application No. 19383175.7.

In certain preferred embodiments, the lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber is pre-cooled to a temperature of about -45°C, and the lyophilisation medium is loaded into the lyophilisation chamber to initiate the one or more freezing steps. The lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber may be retained at about -45°C for at least about 2 hours during the first freezing step. The temperature of lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber may then be increased at a rate of about 1 °C/min (60°C/h) for about 30 minutes to a temperature of about -15°C. This temperature of the lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber may be retained for at least 5 hours during the annealing step. The temperature of the lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber may then be decreased at a rate of about 0.5°C/min (30°C/h) for 1 hour to a temperature of about -45°C. The temperature of the lyophilisation chamber and/or the shelf or tray of the lyophilisation chamber may be retained at about -45°C for at least about 3 hours during the second freezing step.

The one or more freezing steps is followed by at least one primary drying step.

In certain embodiments, during the one or more step of freezing the lyophilisation medium, the lyophilisation medium may be exposed to a temperature of about -50°C or lower, about -70°C or lower, or about -90°C or lower. In certain embodiments, during the one or more step of freezing the lyophilisation medium, the lyophilisation medium may be exposed to a temperature of about -130°C or higher, about -150°C or higher, or about -200°C or higher. For example, in certain embodiments, during the one or more freezing steps, the lyophilisation medium may be exposed to a temperature of about -50°C to about -200°C. For example, in certain embodiments, during the one or more freezing steps, the lyophilisation medium may be exposed to a temperature of about -70°C to about -150°C. For example, in certain embodiments, during the one or more freezing steps, the lyophilisation medium may be exposed to a temperature of about -90°C to about -130°C. In certain embodiments, the one or more freezing step may last for 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, the one or more freezing step may last for 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, the one or more freezing step may last for about 5 minutes to about 600 minutes. For example, in certain embodiments, the one or more freezing step may last for about 10 minutes to about 300 minutes. For example, in certain embodiments, the one or more freezing step may last for about 20 minutes to about 240 minutes. For example, in certain embodiments, the one or more freezing step may last for about 30 minutes to about 180 minutes. Such a freezing step may be conducted in lyophilisation apparatus or in separate freezing apparatus.

In certain embodiments, the temperature to which the lyophilisation medium is exposed may be varied during the one or more freezing steps. For example, the lyophilisation medium may be exposed to a first temperature (e.g. 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 maintained at that temperature for a first period (for example 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 (for example those proposed in the preceding paragraph) and maintained at that second temperature for a second period (e.g. 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 may be longer than the first period.

The one or more freezing step may be carried out at atmospheric pressure. Alternatively, sub- atmospheric or supra-atmospheric pressure may be employed. In embodiments of the invention, atmospheric, or mildly sub- or supra-atmospheric pressures are preferred. For example, in certain embodiments, the pressure employed during the one or more freezing steps is about 10kPa below atmospheric pressure to about 10kPa above atmospheric pressure. In certain embodiments, the pressure employed during the one or more freezing steps is about 5kPa below atmospheric pressure to about 5kPa above atmospheric pressure. In certain embodiments, the pressure employed during the one or more freezing steps is about 2kPa below atmospheric pressure to about 2kPa above atmospheric pressure.

Any number of freezing steps may be conducted during the process of the present invention. For example, 1 , 2, 3, 4, 5 or more than 5 freezing steps may be carried out. In embodiments in which a plurality of freezing steps are conducted, one or more annealing steps may be carried out in between these.

The primary drying step

Background During this process, the ice formed during the one or more freezing steps is removed by sublimation under vacuum at low temperatures. Sublimation is the result of coupled heat-transfer and mass-transfer processes. The driving force of sublimation is the pressure difference related to the corresponding temperature difference between the product ice surface and the condenser ice surface. Larger temperature differences mean larger pressure differences, which allow for a faster drying process. The purpose of the vacuum is to speed up the process by removing air molecules to allow sample vapour molecules to move more easily from the sample, through the lyophilisation chamber and into the condenser of the lyophilisation apparatus. Typically, this process results in a highly porous structure in the remaining amorphous solute of the sample that is around 30% water.

Certain embodiments of the primary drying step

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

In certain embodiments, during the primary drying step, the temperature of the lyophilisation medium is maintained at a temperature of 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 and -5°C. In certain embodiments, the temperature during the primary drying step is not constant and can fluctuate between different temperature values. In certain embodiments, during the primary drying step, the temperature of the lyophilisation medium is maintained at any temperature between -45 and -5°C. In certain preferred embodiments, during the primary drying step, the temperature of the lyophilisation medium is maintained below the glass transition temperature of the lyophilised product. In certain embodiments, during the primary drying step, the temperature of the lyophilisation medium is maintained at least 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10°C below the glass transition temperature of the lyophilised product. In certain embodiments, during the primary drying step, the temperature of the lyophilisation medium is maintained at least 1 °C below the glass transition temperature of the lyophilised product.

In certain embodiments, during the primary drying step, the temperature of the lyophilisation medium is adjusted (e.g. increased) in temperature increments between the temperature of the freezing step and the glass transition temperature of the lyophilised product. In certain embodiments, the temperature of the lyophilisation medium is adjusted according to the adjusted temperature of the shelf or tray of the lyophilisation apparatus. In certain embodiments, the lyophilisation medium is not adjusted to a temperature above the glass transition temperature of the lyophilised product during the primary drying step.

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

In certain embodiments, the temperature of the lyophilisation medium during the primary drying step is controlled by the shelf or tray temperature of the lyophilisation apparatus. In certain embodiments, the temperatures (for example, the shelf or tray temperature) during the primary drying step are adjusted from -50 to -5°C. In certain embodiments, the temperature (for example, the shelf or tray temperature) is adjusted by increments of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10°C or more. In certain embodiments, the temperature (for example, the shelf or tray temperature) is adjusted by increments of 5°C. In certain embodiments, the temperature adjustment occurs at a rate of between 0.05 and 1 °C/min. In certain embodiments, the temperature adjustment occurs at a rate of at least 0.05°C/min. In certain embodiments, the temperature adjustment occurs at a rate of 0.1 °C/min. In certain embodiments, the temperature adjustment occurs at a rate of between 0.05 and 0.1 °C/min. In a preferred embodiment, the temperature adjustment occurs at a rate of about 0.08°C/min or more (/.e. about 5°C/h or more). In certain embodiments, the temperature (for example, the shelf or tray temperature) is maintained at the temperature of an increment for a specific 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 of the increments 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 of the increments 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 of the increments is maintained for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. In certain embodiments, the temperature of each of the increments is maintained for about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. In certain embodiments, the temperature of each of the increments is maintained for at least 10 hours. In a preferred embodiment, the temperature (for example, the shelf or tray temperature) during the primary drying step is adjusted from about -45°C to about -9°C for about 7 hours (/.e. at a rate of about 5°C/h). In another preferred embodiment, the temperature (for example, the shelf or tray temperature) during the primary drying step is adjusted from about -45°C to about -21 °C for about 5 hours (/.e. at a rate of about 5°C/h).

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

In certain embodiments, during the primary drying step, the pressure is maintained at atmospheric pressure. Alternatively, during the primary drying step, the pressure may be maintained at about 1 pBar or higher, about 5 pBar or higher, about 10 pBar or higher, about 20 pBar or higher or about 50 pBar or higher. In certain embodiments, during the primary drying step, the pressure may be maintained at about 10000 pBar or lower, about 5000 pBar or lower, about 2000 pBar or lower, about 1000 pBar or lower, about 500 pBar or lower. In embodiments of the invention, during the primary drying step, the pressure may be maintained at about 1 to about 350 pBar. In certain embodiments, during the primary drying step, the pressure is constant. In certain embodiments, during the primary drying step, the pressure is varied to any value within the range of 1 to 350 pBar. In certain embodiments, during the primary drying step, the pressure is maintained at 30, 40, 50, 60 or 70 pBar. In certain embodiments, during the primary drying step, the pressure is maintained at 40 pBar. In certain embodiments, during the primary drying step, the pressure is maintained at 50 pBar. In certain embodiments, during the primary drying step, the pressure is maintained at 260, 270, 280, 290, 300, 310 or 320 pBar. In certain embodiments, during the primary drying step, the pressure is maintained at 300 pBar. In a preferred embodiment, during the primary drying step, the pressure is maintained at about 50 pBar. In another preferred embodiment, during the primary drying step, the pressure is maintained at about 275 pBar or higher, for example at about 276 pBar.

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

In certain embodiments, the lyophilisation process comprises two or more primary drying steps. In a preferred embodiment, the lyophilisation process comprises two primary drying steps. In certain embodiments, the first of the two or more primary drying steps occurs at the conditions outlined above. In certain embodiments, the second of the two or more primary drying steps occurs at the conditions outlined above.

In certain embodiments, the second of the two or more primary drying steps occurs at a temperature (for example, the shelf or tray temperature) greater than the temperature of the first primary drying step, for example, about 20-30°C. In certain embodiments, the second of the two or more primary drying steps occurs at a temperature (for example, the shelf or tray temperature) of about 25°C. In certain embodiments, the temperature (for example, the shelf or tray temperature) is adjusted from the temperature of the first of the two or more primary drying steps to the temperature of the second of the two or more primary drying steps at a rate of about 5°C/h or higher (/.e. about 0.08°C/min or higher). In a preferred embodiment, the temperature (for example, the shelf or tray temperature) is adjusted from about -9°C (the temperature of the first of the two or more drying steps) to about 25°C (the temperature of the second of the two or more drying steps) for about 7 hours (/.e. at a rate of about 5°C/h (/.e. about 0.08°C/min). In another preferred embodiment, the temperature (for example, the shelf or tray temperature) is adjusted from about -21 °C (the temperature of the first of the two or more drying steps) to about 25°C (the temperature of the second of the two or more drying steps) for about 9 hours (/.e. at a rate of about 5°C/h (/.e. about 0.08°C/min)). In certain embodiments, the first of the two or more primary drying steps occurs for about 24 hours. In certain embodiments, the second of the two or more primary drying steps occurs for about 20 hours. In a preferred embodiment, the first of the two or more primary drying steps occurs for about 24 hours (for example at a temperature of about -9°C or about -21 °C) and the second of the two of more primary drying steps occurs for about 20 hours (for example at a temperature of 25°C).

In certain embodiments, the primary drying step requires the lyophilisation apparatus to have a vacuum pump or an alternative device for creating a vacuum. The vacuum pump serves to lower the pressure of the environment around the lyophilisation medium, for example by removing all non-condensable gases.

In certain embodiments, the primary drying step requires the lyophilisation apparatus to have a condensing system or collector. The collector condenses out all condensable gases, for example the water molecules from the sublimation process.

In certain embodiments, the primary drying step requires the application of heat to the lyophilisation medium. Heat encourages the removal of water in the form of water vapour from the lyophilisation medium. In certain embodiments, the application of heat is controlled to ensure the temperature of the lyophilisation medium does not exceed the glass transition temperature of the lyophilised product. In certain embodiments, heat is applied to the lyophilisation medium directly through a thermal conductor shelf or tray of the lyophilisation apparatus. In certain embodiments, the lyophilisation medium is heated using, for example manifold drying. In certain embodiments, the heat enters the lyophilisation medium by direct contact between the base of the container of the lyophilisation medium and the shelf or tray of the lyophilisation apparatus. In certain embodiments, the heat enters the lyophilisation medium by conduction of heat across the base of the container of the lyophilisation medium followed by conduction through the frozen mass of the lyophilisation medium to the sublimation interface. In certain embodiments, the heat enters the lyophilisation medium by gaseous convection between the lyophilisation medium and the residual gas molecules in the chamber of the lyophilisation apparatus. In certain embodiments, the heat enters the lyophilisation medium by radiation. In a preferred embodiment, heat is applied to the lyophilisation medium by gaseous convection.

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

In certain embodiments, the pressure is constant for the duration of the increments. In certain preferred embodiments, the conditions applied to the lyophilisation medium bring the lyophilisation medium as close as possible to the glass transition temperature of the lyophilised product, without exceeding this temperature.

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

In certain embodiments during the primary drying step, the lyophilisation medium can be exposed to an atmosphere comprising oxygen at a level of about 100 ppm or higher, about 200ppm or higher, about 500ppm or higher, about 10OOppm or higher, about 2000ppm or higher, about 5000ppm or higher, about l OOOOppm or higher, about 20000ppm or higher or about 50000ppm or higher, or ambient air. Such exposure of the lyophilisation medium could arise through an oxygen permeable receptacle containing the lyophilisation medium being transferred from anaerobic conditions to such an atmosphere. Alternatively, such exposure could arise through a sealed oxygen impermeable receptacle being opened (e.g. by removal of a closure of an opening (e.g. a filling port) in the receptacle and / or removal of a portion of the wall of the receptacle) in such an atmosphere.

Accordingly, in certain embodiments, the oxygen level of the environment to which the lyophilisation medium is exposed during the primary drying step may be about 100 ppm or higher, about 200ppm or higher, about 500ppm or higher, about 10OOppm or higher, about 2000ppm or higher, about 5000ppm or higher, about l OOOOppm or higher, about 20000ppm or higher or about 50000ppm or higher. In certain embodiments, the primary drying step may be carried out in ambient air.

In certain embodiments, the temperature to which the lyophilisation medium is exposed during the primary drying step may be about 50°C or lower, about 30°C or lower, or about 10°C or lower. In certain embodiments, the temperature to which the lyophilisation medium is exposed during the primary drying step may 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 lyophilisation medium is exposed during the primary drying step may be between about -150°C to about 50°C. In certain embodiments, the temperature to which the lyophilisation medium is exposed during the primary drying step may be between about -100°C to about 30°C. In certain embodiments, the temperature to which the lyophilisation medium is exposed during the primary drying step may be between about -70°C to about 10°C. In certain embodiments, the temperature to which the lyophilisation medium is exposed during the primary drying step may be between about -50°C to about 10°C. In certain embodiments, the temperature to which the lyophilisation medium is exposed during the primary drying step may be between about -30°C to about 10°C.

Additionally or alternatively, in certain embodiments, the pressure at which the primary drying step is carried out may be about 5000pBar or lower, about 2000pBar or lower, about 1000pBar or lower or about 500pBar or lower. In certain embodiments, the pressure at which the primary drying step is carried out may be about 50pBar or higher, about 25pBar or higher, about 10pBar or higher, or about OpBar or higher. In certain embodiments, the pressure at which the primary drying step is carried out may be between about OpBar to about 5000pBar. In certain embodiments, the pressure at which the primary drying step is carried out may be between about 10pBar to about 2000pBar. In certain embodiments, the pressure at which the primary drying step is carried out may be between about 25pBar to about WOOpBar. In certain embodiments, the pressure at which the primary drying step is carried out may be between about 50pBar to about 500pBar.

In embodiments in which the lyophilisation medium is provided in a receptacle, the process of the invention may comprise the step of exposing the lyophilisation medium within the receptacle. This may be done to facilitate the primary drying step, secondary drying step (if performed) or other steps of the lyophilisation process. For example, a portion of the wall of the receptacle could be removed and / or a closure (e.g. a filling port) in the receptacle could be opened. This step of exposing the lyophilisation medium may be performed prior to loading the receptacle into the lyophilisation apparatus, or following loading of the receptacle into the lyophilisation apparatus and prior to or during the primary drying step. The primary drying step can be conducted using any technique or apparatus known to those skilled in the art of lyophilisation. For example, the primary drying step may be conducted in lyophilisation apparatus. In embodiments of the invention, the lyophilisation apparatus may be of any size without this impacting substantially on the viability of the cells present in the lyophilisation medium. Thus, in certain embodiments, the primary drying step is carried out in pilot scale lyophilisation apparatus (e.g. freeze drying apparatus having an operating shelf area of about 0.1 m² or higher, about 0.2m² or higher, about 0.5m² or about 2m² or lower, about 3m² or lower or about 4m² or lower). In certain embodiments of the invention, the primary drying step is carried out in commercial scale lyophilisation apparatus (e.g. freeze drying apparatus having an operating shelf area of about 5m² or higher, about 10m² or higher, or about 20m² or higher, or about 50m² or lower, about 100m² or lower, about 150m² or lower, or about 200m² or lower).

The secondary drying step

Background

This optional process step may be initiated after the product has reached a temperature above its glass transition point and is used to remove any remaining solvent from the product. Accordingly, this step may be referred to as the desorption step. In certain embodiments, after the primary drying step during which the ice has sublimed, bound moisture, as much as 7-8% may still be present in the lyophilised product. Accordingly, the secondary drying step conventionally occurs at a high temperature in order to reduce the residual moisture content of the lyophilised product.

Certain embodiments of the secondary drying step

In certain embodiments, the lyophilisation process of the invention comprises a secondary drying step. In certain other embodiments, the lyophilisation process of the invention does not comprise a secondary drying step.

In certain embodiments, the secondary drying step occurs at a temperature above about -20°C, above about -10°C, above about 0°C, above about 10°C, for example at ambient temperature (about 20°C) or higher. In certain embodiments, the temperature of the secondary drying step corresponds with the sensitivity of the lyophilised product. In certain embodiments, the secondary drying step occurs at a temperature higher than the glass transition temperature of the lyophilised biotherapeutic product. In certain embodiments, during the secondary drying step, the lyophilised product is maintained at a temperature between the temperature of the primary drying step and ambient (e.g. 20°C) temperature. In certain embodiments, during the secondary drying step, the lyophilised product is maintained at a temperature between the glass transition temperature and ambient (e.g. 20°C) temperature. In certain embodiments, during the secondary drying step, the temperature (for example, the shelf or tray temperature) is maintained at a temperature between -20 and 37°C. In certain embodiments, during the secondary drying step, the temperature (for example, the shelf or tray temperature) is maintained at a temperature below 37°C. In certain embodiments, during the secondary drying step, the temperature (for example, the shelf or tray temperature) is maintained at a temperature below 42°C. In certain embodiments, during the secondary drying step, the temperature (for example, the shelf or tray temperature) is maintained at a temperature between about 20°C and about 25°C. In certain embodiments, during the secondary drying step, the temperature (for example, the shelf or tray temperature) is maintained at a temperature of about 25°C. In certain other embodiments, during the secondary drying step, the lyophilised product is maintained at a temperature of about 22°C.

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

In certain embodiments, during the secondary drying step, the temperature (for example, the shelf or tray temperature) is adjusted in temperature increments between the temperature of the glass transition temperature and ambient (e.g. 20°C) temperature. In certain embodiments, the temperature (for example, the shelf or tray temperature) is adjusted according to the adjusted temperature of the shelf or tray of the lyophilisation apparatus. In certain embodiments, the temperature (for example, the 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 lyophilised product during the secondary drying step is controlled by the shelf or tray temperature of the lyophilisation apparatus. In certain embodiments, the temperature (for example, the shelf or tray temperature) during the secondary drying step is adjusted from the temperature of the one or more primary drying steps to the temperature of the secondary drying step. In certain embodiments, the temperature (for example, the shelf or tray temperature) during the secondary drying step do not exceed 42°C.

In certain embodiments, the temperature (for example, the shelf or tray temperature) is adjusted by increments of 1 or higher, 2 or higher, 3 or higher, 4 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher or 10°C. In certain embodiments, the temperature (for example, the shelf or tray temperature) is adjusted by increments of 5°C or higher. In certain embodiments, the adjustment occurs at a rate of between 0.05 and 1 °C/min. In certain embodiments, the adjustment occurs at a rate of at least 0.05°C/min. In certain embodiments, the adjustment occurs at a rate of 1.5°C/min. In certain preferred embodiments, the adjustment occurs at a rate of at least 0.08°C/min (/.e. 5°C/h). In certain embodiments, the temperature (for example, the shelf or tray temperature) is maintained at the temperature of an increment for a specific 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 of the increments 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 of the increments is maintained at the temperature (for example, the shelf or tray temperature) at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certain embodiments, the temperature of each of the increments is maintained for about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 hours. In certain embodiments, the temperature of each of the increments 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 lyophilised product is maintained at a temperature of 25°C. In certain embodiments, the final shelf temperature is such that the lyophilised product is maintained at a temperature of 22°C. In certain embodiments, the final shelf temperature is such that the lyophilised product is maintained at a temperature of 22°C for at least 10 hours.

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

In certain embodiments, the secondary drying step is a 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 about the same time as the one or more primary drying steps. In certain embodiments, the secondary drying step is about the same time as all of the primary drying steps.

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

In certain embodiments, during the secondary drying step, the pressure to which the lyophilisation medium is exposed is atmospheric. Alternatively, during the secondary drying step, the pressure to which the lyophilisation medium is exposed may be about 1 pBar or higher, about 2 pBar or higher, about 5 pBar or higher, about 10 pBar or higher, about 20 pBar or higher, or about 50 pBar or higher.ln specific embodiments, during the secondary drying step, the pressure to which the lyophilisation medium is exposed may be about 10000 pBar or lower, about 5000 pBar or lower, about 2000 pBar or lower, about 1000 pBar or lower or about 500 pBar or lower, for example between 50 and 350 pBar. In certain embodiments, the pressure to which the lyophilisation medium is exposed is 50 pBar. In certain embodiments, the pressure to which the lyophilisation medium is exposed is 275 pBar.

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

As those skilled in the art will recognise, conventional lyophilisation processes typically include a plurality of drying steps, for example, a primary drying (sublimation) step and a secondary drying (desorption) step. Thus, in certain embodiments, the lyophilisation process comprises one or more secondary drying steps.

As with the primary drying step, the oxygen level of the environment in which the desorption step is carried out may be about 100 ppm or higher, about 200ppm or higher, about 500ppm or higher, about OOppm or higher, about 2000ppm or higher, about 5000ppm or higher, about l OOOOppm or higher, about 20000ppm or higher or about 50000ppm or higher. In certain embodiments, the secondary drying step may be carried out in ambient air.

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

Additionally or alternatively, in certain embodiments, during the secondary drying step, if carried out, the lyophilisation medium may be exposed to a pressure of about 2000pBar or lower, about 10OOpBar or lower, about 500pBar or lower, or about 300pBar or lower. In certain embodiments, during the secondary drying step, the lyophilisation medium may be exposed to a pressure of about 50pBar or higher, about 25pBar or higher, about 10pBar or higher, or about OpBar or higher. In certain embodiments, during the secondary drying step, the lyophilisation medium may be exposed to a pressure of between about OpBar to about 2000pBar. In certain embodiments, during the secondary drying step, the lyophilisation medium may be exposed to a pressure of between about 10pBar to about WOOpBar. In certain embodiments, during the secondary drying step, the lyophilisation medium may be exposed to a pressure of between about 25pBar to about 500pBar. In certain embodiments, during the secondary drying step, the lyophilisation medium may be exposed to a pressure of between about 50pBar to about 300pBar.

Preferably, the secondary drying step is carried out in the same apparatus as the primary drying step.

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

The annealing step

In embodiments of the invention, the lyophilisation process comprises at least one annealing step. The annealing step may be performed between the one or more freezing steps. Additionally or alternatively, the (or a further) annealing step may be performed following the one or more freezing steps and prior to the primary drying step. As shown in the Examples herein, the inventors identified that the incorporation of an annealing step in the lyophilisation process achieves significant increases in viability of the lyophilised product compared to lyophilisation processes without a corresponding annealing step.

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

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

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

In certain embodiments, during the annealing step, the temperature (for example, the shelf or tray temperature) is adjusted by increments of at least about 1 , 2, 3, 4 or 5°C. In certain embodiments, the adjustment occurs at a rate of between about 0.01 and 1 °C/min. In certain embodiments, the adjustment occurs at a rate of at least about 0.1 °C/min. In a preferred embodiment, the adjustment occurs at a rate of about 0.5°C/min (/.e. 30°C/h) or higher. In another preferred embodiment, the adjustment occurs at a rate of about 1 °C/min (/.e. 60°C/h) or higher. In certain embodiments, during the annealing step, the temperature of the shelf of the lyophilisation apparatus is adjusted by increments of at least about 1 , 2, 3, 4 or 5°C.

In certain embodiments, the temperature (for example, the shelf or tray temperature) is maintained at the annealing temperature for 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 (for example, the shelf or tray temperature) is maintained at the annealing temperature for less than about 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In certain embodiments, the temperature (for example, the shelf or tray temperature) is maintained at the annealing 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 temperature (for example, the 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 (for example, the 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 (for example, the 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 (for example, the shelf or tray temperature) is maintained at the annealing temperature for at least about 5 hours. In certain embodiments, the temperature (for example, the shelf or tray temperature) is maintained at the annealing temperature for about 5 hours. In a preferred embodiment, during the annealing step, the temperature (for example, the 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 lyophilisation medium is adjusted from the temperature of the one or more freezing steps to above the glass transition temperature of the lyophilised product and held at this temperature, before being cooled to a temperature below the glass transition temperature of the lyophilised product. In certain embodiments, at the end of the one or more freezing steps, the lyophilisation medium is adjusted from the temperature of the one or more freezing step to the annealing temperature and held at this temperature, before being cooled to the freezing step temperature. In certain embodiments, at the end of the one or more freezing steps, the shelf of the lyophilisation apparatus is adjusted from the temperature of the one or more freezing steps to above the glass transition temperature of the lyophilised product and held at this temperature, before being cooled to below the glass transition temperature of the lyophilised product. In certain embodiments, at the end of the one or more freezing steps, the shelf of the lyophilisation apparatus is adjusted from the temperature of the freezing step to the annealing temperature and held at this temperature, before being cooled to the freezing step temperature.

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

In certain embodiments, during the annealing step, the pressure to which the lyophilisation medium is exposed is atmospheric. Alternatively, during the annealing step, the pressure to which the lyophilisation medium is exposed may be about 1 pBar or higher, about 2 pBar or higher, about 5 pBar or higher, about 10 pBar or higher, about 20 pBar or higher, or about 50 pBar or higher.ln specific embodiments, during the annealing step, the pressure to which the lyophilisation medium is exposed may be about 10000 pBar or lower, about 5000 pBar or lower, about 2000 pBar or lower, about 1000 pBar or lower or about 500 pBar or lower, for example between 50 and 350 pBar or between 50 and 100 pBar. In certain embodiments, during the annealing step, the pressure to which the lyophilisation medium is exposed is constant. In certain embodiments, during the annealing step, the pressure to which the lyophilisation medium is exposed is varied optionally according to the temperature.

In certain embodiments, at the end of the one or more freezing steps, the temperature (for example, the shelf or tray temperature) is adjusted from the freezing step temperature of between about -40 and about -50°C to the annealing temperature of between about -10 and -20°C, and held at the annealing temperature for between about 4-8 hours, before being decreased to the freezing temperature of between about -40 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 the one or more freezing steps, the temperature (for example, the shelf or tray temperature) is adjusted from the freezing step temperature of about -45°C to the annealing temperature of about -15°C, and held at the annealing temperature for at least about 5 hours, before being decreased to the freezing temperature of about -45°C. In another preferred embodiment, at the end of the first of the one or more freezing steps of at least about 2 hours, the temperature (for example, the shelf or tray temperature) is adjusted for 30 minutes (/.e. at a rate of 60°C/h) from the freezing step temperature of about -45°C to the annealing temperature of about -15°C, and held at the annealing temperature for at least about 5 hours, before being decreased for 60 minutes (/.e. at a rate of 30°C/h) to the freezing temperature of about -45°C and maintained at this temperature for at least about 3 hours.

In certain embodiments, the lyophilisation process of the present invention comprises at least 1 , 2, 3, 4 or 5 annealing steps. In preferred embodiments, the one or more annealing step is flanked by freezing steps. Accordingly, in certain embodiments, the lyophilisation process if the present invention comprises at least 2, 3, 4, 5, 6 or 7 freezing steps. In certain embodiments, the lyophilisation process of the present invention comprises less than 2, 3, 4 or 5 annealing steps. In certain embodiments the lyophilisation process of the present invention comprises less than 2, 3, 4, 5, 6 or 7 freezing steps. In a preferred embodiment, the lyophilisation process comprises two freezing steps flanking one annealing step.

The glass transition temperature

Determination of the glass transition temperature

In certain embodiments, the glass transition temperature of the lyophilised product is determined using differential scanning calorimetry (DSC) (see [2]). Such a technique would be routine for the skilled person. In preferred embodiments, the glass transition temperature is determined using DSC in accordance with ASTM E1356-08 as exemplified by Drake et al., Pios One, 5 January 2018 G/ass transition temperatures

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

Lyophilisation buffer formulations

In certain embodiments, the lyophilisation medium comprises a lyophilisation buffer. In certain embodiments, the lyophilisation buffer is formulated according to regulatory requirements and route of administration of the lyophilised product, as known to those of skill in the art. In certain embodiments, the lyophilisation buffer may comprise one or more excipients. In certain embodiments, the excipients are buffers, pH adjusters, bulking agents, stabilisers and/or tonicity modifiers.

In certain embodiments, the lyophilisation medium comprises a buffering agent. In certain embodiments, the buffer stabilises the pH of the lyophilisation buffer. In certain embodiments, the buffer is a buffer that undergoes minimal changes in pH during freezing, for example citrate and/or histidine buffers. Accordingly, in certain embodiments, the buffer is a citrate buffer. In certain embodiments, the buffer is a histidine buffer. In certain embodiments, the lyophilisation buffer has a pH of between 5.5 and 8.5.

In certain embodiments, the lyophilisation buffer comprises a bulking agent. In certain embodiments, the bulking agent is a crystalline bulking agent. In certain embodiments, the bulking agent is a disaccharide. In certain embodiments, the bulking agent is mannitol. In certain embodiments, the bulking agent is sucrose. In certain embodiments, the bulking agent is albumin, glycine, hydroxyethyl starch or dextrane.

In certain embodiments, the lyophilisation buffer comprises a stabilising agent. In certain embodiments, the stabiliser comprises a compound that can form an amorphous glass structure, for example one or more disaccharides. In certain embodiments, the stabilising agent is glucose, lactose and/or maltose. In certain embodiments, the stabilising agent is glucose. In certain embodiments, the stabilising agent is lactose. In certain embodiments, the stabilising agent is maltose. In certain embodiments, the stabilising agent is sucrose. In certain embodiments, the stabilising agent is trehalose. In certain embodiments, the stabilising agent is not trehalose. In certain embodiments, the lyophilisation buffer comprises a tonicity adjuster. In certain embodiments, the tonicity adjuster is an excipient such as mannitol, sucrose, glycine, glycerol or sodium chloride. Accordingly, in certain embodiments, the lyophilisation buffer comprises mannitol, sucrose, glycine, glycerol and/or sodium chloride.

In certain embodiments, the lyophilisation buffer comprises cryoprotective agents. In certain embodiments, cryoprotective agents are water soluble substances that lower the melting point of water and/or increase the unfrozen part of the lyophilisation medium. In certain embodiments, the cryoprotective agent is a non-toxic, low molecular weight solute. In certain embodiments, the cryoprotectant is a monosaccharide or disaccharide. In certain embodiments, the cryoprotectant is trehalose, sucrose, glucose and/or lactose. In certain embodiments, the cryoprotectant is a sugar alcohol, for example glycerol and/or sorbitol. In certain embodiments, the cryoprotectant is a polymer. In certain embodiments, the cryoprotective agent is polyethylene glycol, polyvinylpyrrolidone and/or dextrane. In certain embodiments, the cryoprotectant is skim milk, peptones and/or different amino acids and derivatives.

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

In certain embodiments, the viable bacterial population may comprise more than one bacterial strain (such as a consortium of different bacterial strains). In such embodiments, the viable bacterial population may 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 the typical compositions of lyophilisation media and how they may be prepared. In certain embodiments, the lyophilisation medium comprises the viable bacterial population in the form of a concentrated biomass. In certain embodiments, the biomass may be stored under anaerobic conditions.

In certain embodiments, the lyophilisation medium may comprise a lyophilisation buffer or lyobuffer. The lyobuffer may comprise excipients known to those of skill in the art, for example: cryoprotectants (e.g. polyol such as ethylene glycol, sorbitol, propylene glycol, and/or glycerol; DMSO; skim milk; yeast extract; bovine serum albumin (BSA); starch hydrolysates; saccharides (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 (optionally in the form of a salt, such as sodium glutamate), arginine and/or glycine); antioxidants (e.g cysteine, arginine, ascorbic acid (and salts and esters thereof e.g. ascorbyl palmitate, sodium ascorbate)), butylated agents such as butylated hydroxyanisole or butylated hydroxytoluene, citric acid, erythorbic acid, fumaric acid, glutamic acid, glutathione, malic acid, methionine, monothioglycerol, pentetic acid, metabisulfite (such as sodium metabisulfite, potassium metabisulfite), propionic acid, propyl gallate, uric acid, sodium formaldehyde sulfoxylate, sulphite (e.g. sodium sulphite), sodium thiosulfate, sulphur dioxide, thymol, tocopherol (free or esterified), uric acid (and salts thereof) and salts and/or esters thereof); bulking agents (e.g. mannitol, maltodextrin and/or glycine); buffers (e.g. phosphate, citrate, tris and/or Hepes); and/or surfactants (e.g. polysorbate (such as those commercialised under the trade mark Tween)) and/or sorbitan (such as those commercialised under the trade mark Span).

In a preferred embodiment, the lyophilisation buffer comprises a lyoprotectant formula comprising (at a final concentration prior to lyophilisation): 2% sucrose, 4% maltodextrine DE9 and 0.2% cysteine HCI. In a preferred embodiment, the ratio of biomass to lyoprotectant is about 70:30. In a preferred embodiment, the lyophilisation buffer does not comprise trehalose. In certain embodiments, the lyophilisation buffer does not comprise maltodextrine. In certain embodiments, the lyophilisation buffer does not comprise maltodextrine DE9. In certain embodiments, the lyophilisation buffer comprises (at a final concentration prior to lyophilisation): 2% sucrose and 0.2% cysteine HCI.

In embodiments of the invention, the lyophilisation medium is not prepared by bringing a liquid or pasty mixture of substances at least partially to solidification between two surfaces with different temperatures prior to freeze drying. Additionally or alternatively, the lyophilised product does not comprise a sponge.

Lyophilisation apparatus

Components of the lyophilisation apparatus

In certain embodiments, the lyophilisation apparatus is a manifold freeze-dryer, a rotary freeze-dryer and/or a tray-style freeze-dryer. In certain embodiments, the lyophilisation apparatus is a tray-style freeze-dryer. In embodiments of the invention, the lyophilisation apparatus comprises shelves. In certain embodiments, the process of the invention includes the step of filling the lyophilisation medium into receptacles for freeze drying. In such embodiments, the lyophilisation medium is filled into vials. In alternative embodiments, the lyophilisation medium is filled into receptacles for bulk lyophilisation, e.g. lyophilisation trays (e.g. a conventional open tray or a specialist tray such as those commercialised by Gore® under the trade mark Lyogard®) or a lyophilisation bag (e.g. a bag such as that disclosed in International Patent Application No. PCT/IB2018/055246 (the contents of which are incorporated by reference herein)).

In embodiments of the invention, the lyophilisation medium may be filled into receptacles for freeze drying in an amount of at least about 1g, at least about 2g, at least about 5g, at least about 10g, at least about 20g, at least about 50g, at least about 100g, at least about 200g, at least about 500g, at least about 1 kg or at least about 2kg.

In embodiments of the invention, the lyophilisation receptacle and / or the lyophilisation apparatus does not comprise an array of samples.

In embodiments, the step of filling the lyophilisation medium into receptacles may take place prior to the one or more freezing steps and I or before the primary drying step. Once filled, the filled receptacles may be placed inside the lyophilisation apparatus (e.g. where the lyophilisation apparatus comprises shelves, placing the filled receptacles on one or more of the shelves).

In certain embodiments, the lyophilisation apparatus may be of any size without this impacting on the viability of the cells present in the lyophilisation medium. Thus, in certain embodiments, the lyophilisation process is carried out in pilot scale lyophilisation apparatus (e.g. freeze drying apparatus having an operating shelf area of about 0.1 m² or higher, about 0.2m² or higher, about 0.5m² or about 2m² or lower, about 3m² or lower or about 4m² or lower). In certain embodiments of the invention, the sublimation step is carried out in commercial scale lyophilisation apparatus (e.g. freeze drying apparatus having an operating shelf area of about 5m² or higher, about 10m² or higher, or about 20m² or higher, or about 50m² or lower, about 100m² or lower, about 150m² or lower, or about 200m² or lower).

In certain embodiments, the lyophilisation apparatus comprises a lyophilisation chamber, a condenser and/or a vacuum pump. In certain embodiments, the lyophilisation chamber is adapted for the use of product vials. In certain embodiments, the lyophilisation chamber is adapted for the use of trays.

In certain embodiments, the lyophilisation apparatus comprises a lyophilisation chamber. In certain embodiments, the lyophilisation chamber contains one or more shelves. In certain embodiments, a shelf acts as a heat exchanger so as to assist in the removal of energy from the lyophilisation medium during freezing and/or the supplying of energy to the lyophilisation medium during the drying steps. In certain embodiment, the shelf is connected to a fluid system that enables the circulation of fluid at a particular temperature through the shelf. 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 electrical heater.

In certain embodiments, the lyophilisation apparatus comprises a vacuum pump. In certain embodiments, the vacuum pump can achieve a vacuum level of 50 to 100 pBar. In certain embodiments, the vacuum pump is a two stage rotary pump. In certain embodiments, two or more vacuum pumps may be used.

In certain embodiments, the lyophilisation apparatus comprises a control system. In certain embodiments, the control system controls the shelf temperature and/or the pressure within the lyophilisation chamber. In certain embodiments, the control system controls the time for which the lyophilisation medium is retained at a particular temperature and/or pressure.

In certain embodiments, the control system can monitor the temperature of the lyophilisation medium. In certain embodiments, a temperature sensor is placed with the sample to measure the core temperature of the lyophilisation medium during the lyophilisation process. In certain embodiments, a temperature sensor is used to measure the temperature of the shelf within the lyophilisation chamber. In certain embodiments, the temperature sensors allow for a comparison between the shelf temperature and the temperature of the lyophilisation medium.

In certain embodiments, the receptacle may be oxygen impermeable to facilitate the maintenance of anaerobic conditions therein. As used herein, the term ‘oxygen impermeable’ is used to identify a receptacle as having an oxygen transmission rate (OTR) of about 10 cc/m²/24hrs or less, about 5 cc/m²/24hrs or less, about 1 cc/m²/24hrs or less, about 0.5 cc/m²/24hrs or less, about 0.1 cc/m²/24hrs or less, about 0.05 cc/m²/24hrs or less, about 0.01 cc/m²/24hrs or less, about 0.005 cc/m²/24hrs or less or about 0.001 cc/m²/24hrs or less as measured using a coulometric sensor operated in accordance with ASTM D3985. In certain embodiments, the receptacle has an oxygen transmission rate (OTR) of about 1 cc/m²/24hrs or less. In certain embodiments, the receptacle has an oxygen transmission rate (OTR) of about 0.1 cc/m²/24hrs or less. In certain embodiments, the receptacle has an oxygen transmission rate (OTR) of about 0.01 cc/m²/24hrs or less.

Conditions within the lyophilisation chamber

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

In certain embodiments, the temperature of the lyophilisation chamber is between -80 and 40°C. In certain embodiments, the temperature of the lyophilisation 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 lyophilisation 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 lyophilisation 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 lyophilisation chamber during the one or more freezing steps is between -40 and -50°C. In preferred embodiments, the temperature of the lyophilisation chamber during the one or more freezing steps is about -45°C. In certain embodiments, the temperature of the lyophilisation chamber during the annealing step is between -10 and -20°C. In preferred embodiments, the temperature of the lyophilisation chamber during the annealing step is about -15°C. In certain embodiments, the temperature of the lyophilisation chamber during the primary drying step is between -5 and -25°C. In preferred embodiments, the temperature of the lyophilisation chamber during the primary drying step is about -9 or about -21 °C. In certain embodiments, the lyophilisation chamber during a further primary drying step is between 15 and 25°C. In certain embodiments, the lyophilisation chamber during a further primary drying step is about 20°C.

In certain embodiments, the temperature of the lyophilisation chamber can be altered. In certain embodiments, the temperature of the lyophilisation chamber is increased at a rate of between 0.05 to 5.0°C/min. In certain embodiments, the temperature of the lyophilisation chamber is increased at a rate of about 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. In certain embodiments, the temperature of the lyophilisation chamber is increased at a rate of 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. In certain embodiments, the temperature of the lyophilisation chamber is increased at a rate of 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. In certain embodiments, the temperature of the lyophilisation chamber is decreased at a rate of between 0.05 to 5.0°C/min. In certain embodiments, the temperature of the lyophilisation chamber is decreased at a rate of about 0.0.05, 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. In certain embodiments, the temperature of the lyophilisation chamber is decreased at a rate of 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. In certain embodiments, the temperature of the lyophilisation chamber is decreased at a rate of 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. In preferred embodiments, the temperature of the lyophilisation chamber is increased at a rate of is 0.08°C/min (/.e. 5°C/h). In preferred embodiments, the temperature of the lyophilisation chamber is decreased at a rate of is 0.5°C/min (/.e. 30°C/h). In another preferred embodiment, the temperature of the lyophilisation chamber is increased at a rate of 1 °C/min (/.e. 60°C/h).

In certain embodiments, the temperature of the lyophilisation chamber is the temperature of the shelf or tray of the lyophilisation chamber. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber is between -80 and 40°C. In certain embodiments, the temperature of the shelf or tray of the lyophilisation 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 lyophilisation 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 lyophilisation 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 shelf or tray of the lyophilisation chamber during the one or more freezing steps is between -40 and -50°C. In preferred embodiments, the temperature of the shelf or tray of the lyophilisation chamber during the one or more freezing steps is about -45°C. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber during the annealing step is between -10 and -20°C. In preferred embodiments, the temperature of the shelf or tray of the lyophilisation chamber during the annealing step is about -15°C. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber during the primary drying step is between -5 and -25°C. In preferred embodiments, the temperature of the shelf or tray of the lyophilisation chamber during the primary drying step is about -9 or about -21 °C. In certain embodiments, the shelf or tray of the lyophilisation chamber during a further primary drying step is between 15 and 25°C. In certain embodiments, the shelf or tray of the lyophilisation chamber during a further primary drying step is about 20°C.

In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber can be altered. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber is increased at a rate of between 0.05 to 5.0°C/min. In certain embodiments, the temperature of the shelf of the lyophilisation chamber is increased at a rate of about 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. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber is increased at a rate of 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. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber is increased at a rate of 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. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber is decreased at a rate of between 0.05 to 5.0°C/min. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber is decreased at a rate of about 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. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber is decreased at a rate of 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. In certain embodiments, the temperature of the shelf or tray of the lyophilisation chamber is decreased at a rate of 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. In preferred embodiments, the temperature of the shelf or tray of the lyophilisation chamber is increased at a rate of is 0.08°C/min (/.e. 5°C/h). In preferred embodiments, the temperature of the shelf or tray of the lyophilisation chamber is decreased at a rate of is 0.5°C/min (/.e. 30°C/h). In another preferred embodiment, the temperature of the shelf or tray of the lyophilisation chamber is increased at a rate of 1 °C/min (/.e. 60°C/h).

In certain embodiments, the pressure of the lyophilisation chamber is between 1 and 350 pBar. In certain embodiments, the pressure of the lyophilisation 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 pBar. In certain embodiments, the pressure of the lyophilisation 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 pBar. In certain embodiments, the pressure of the lyophilisation 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 pBar. In certain embodiments, the pressure of the lyophilisation chamber is between 50 and 100 pBar. In a preferred embodiment, the pressure of the lyophilisation chamber is about 50 pBar. In another preferred embodiment, the pressure of the lyophilisation chamber is about 275 pBar.

In certain embodiments, the temperature and/or pressure of the lyophilisation chamber is constant for between 1 and 60 minutes. In certain embodiments, the temperature and/or pressure of the lyophilisation 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 lyophilisation 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 lyophilisation 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 lyophilisation chamber is constant for between 1 and 36 hours. In certain embodiments, the temperature and/or pressure of the lyophilisation 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 lyophilisation 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 lyophilisation 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, during the one or more freezing steps, the temperature and/or pressure of the lyophilisation chamber is constant for at least 2 hours, for example 3 hours. In a preferred embodiment, during the annealing step, the temperature and/or pressure of the lyophilisation chamber is constant for about 5 hours. In a preferred embodiment, during the primary drying step, the temperature and/or pressure of the lyophilisation chamber is constant for about 24 hours. In a preferred embodiment, during a further primary drying step (if included), the temperature and/or pressure of the lyophilisation chamber is constant for about 20 hours. In a preferred embodiment, during the secondary drying step, the temperature and/or pressure of the lyophilisation chamber is constant for at least 10 hours or until the lyophilised product is maintained at a temperature of at least 22°C for at least 10 hours.

Viable bacterial populations for lyophilisation

In certain embodiments, the genera of the bacteria of the viable bacterial population for lyophilisation is selected from the group consisting of: Enterococcus (e.g. Enterococcus gallinarum, Enterococcus caselliflavus, Enterococcus durans, Enterococcus faecalis, or Enterococcus faecium), Blautia (e.g. Blautia hydrogenotrophica, Blautia stercoris, Blautia wexlerae, Blautia coccoides or Blautia producta), Bacteroides (e.g. Bacteroides thetaiotaomicron, Bacteroides massiliensis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides dorei, or Bacteroides copricola), Faecalibacterium (e.g. Faecalibacterium prausnitzii), Bariatricus (e.g. Bariatricus massiliensis), Bifidobacterium (e.g. Bifidobacterium breve, Bifidobacterium adolescentis or Bifidobacterium longum), Roseburia (e.g. Roseburia hominis, Roseburia intestinalis or Roseburia inulinivorans), Flavonifractor (e.g. Flavonifractor plautii), 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), Pseudoflavonifractor (e.g. Pseudoflavonifractor capillosus), Clostridium (e.g. Clostridium nexile, Clostridium hylemonae, Clostridium butyricum, Clostridium tertium, Clostridium disporicum, Clostridium bifermentans, Clostridium inocuum, Clostridium mayombei, Clostridium bolteae, Clostridium bartletti, Clostridium symbiosum or Clostridium orbiscindens), or Coprococcus (e.g. Coprococcus comes, or Coprococcus cattus), Bifidobacterium (e.g. Bifidobacterium breve, Bifidobacterium adolescentis or Bifidobacterium longum), Acetivibrio (e.g. Acetovibrio ethanolgignens), Dorea (e.g. Dorea longicatena), Anaerostipes (e.g. Anaerostipes hadrus or Anaerostipes caccae), Fusicatenibacter (e.g. Fusicatenibacter saccharivorans), Lachnospiraceae or Clostridiaceae. Examples of such organisms include those disclosed in European Patent Nos. 1280541 , 1448995, and 3209310, European Patent Publication No. 3206700, 2763685 and UK Patent Application No. 1423084.1 , the contents of which are all incorporated herein by reference. Further examples of organisms that can be formulated according to the present invention include those disclosed in UK Patent Application Nos. 1510470.6, 1510468.0, 1510469.8, 1510466.4 and 1510467.2, the contents of which are all incorporated herein by reference.

In certain embodiments, the viable bacterial population does not comprise conventional probiotic bacteria, e.g. they do not belong to the genera Lactobacillus, Bifidobacterium and/or are not lactic acid bacteria. In certain embodiments, the viable bacterial population does not comprise a lactic acid bacteria. In certain embodiments, the viable bacterial population is not a bacterial strain from the genus Lactobacillus or does not comprise a strain from that genus.

In certain embodiments, the viable bacterial population comprises or consists of non-sporulating bacteria.

Properties of the lyophilised product

Viability post-lyophilisation

Those skilled in the art will be familiar with methods for conducting viable cell counts, for example plate counts using a spiral plater, e.g. that commercialised under the trademark easySpiral® Pro. The plate count can be performed under an anaerobic hood.

In certain embodiments, the viable cell count (in CFU/g) in the lyophilised product is no more than 10³ CFU/g, 10² CFU/g or 10 CFU/g lower than the viable cell count (in CFU/g) of the lyophilisation medium prior to freezing (excluding moisture). In certain embodiments, the viability (CFU/g as dry weight) of the lyophilised product is equal to or less than 10³ CFU/g, equal to or less than 10² CFU/g, or equal to or less than 10 CFU/g lower than the viability of the lyophilisation medium prior to lyophilisation (CFU/ml). As shown in the examples, the process of the invention is particularly suitable for maintaining viable cell count in the lyophilised product compared to the viable cell count in the lyophilisation medium prior to freezing.

In certain embodiments of the invention, the viable cell count (in CFU/g) in the lyophilised product is no more than a factor of 5 CFU/g, no more than a factor of 4 CFU/g, no more than a factor of 3 CFU/g or no more than a factor of 2 CFU/g lower than the viable cell count (in CFU/g) of the lyophilisation medium prior to freezing (excluding moisture).

In certain embodiments, the viable cell count (in CFU/g as dry weight) of the lyophilised product is equal to or less than a factor of 5 CFU/g, equal to or less than a factor of 4 CFU/g, equal to or less than a factor of 3 CFU/g, or equal to or less than a factor of 2 CFU/g lower than the viability of the lyophilisation medium prior to lyophilisation (CFU/ml).

In certain embodiments, the reduction in viable cell count in the lyophilised product compared to the viable cell count in the lyophilisation medium prior to freezing (excluding moisture) is 0.4 log or less or 0.3 log or less, 0.2 log or less or 0.1 log or less.

In order to accurately assess the reduction (e.g. log loss) in viable cell count in the lyophilised product (/.e. in CFU/g) compared to the viable cell count in the lyophilisation medium (/.e. in CFU/ml), the skilled person would appreciate that it is necessary to account for the increased concentration of potentially viable cells in the /g of lyophilised product compared to in the /ml of lyophilisation medium (/.e. caused by the removal of moisture during the process of lyophilisation). The increased concentration of cells in the lyophilised product can be accounted for by determining a so-called theoretical viable cell count (in CFU/g) for the lyophilised product which is calculated as a function of the moisture content and the viable cell count of the lyophilisation medium. The theoretical viable cell count assumes that no loss of viability occurs upon the removal of moisture and therefore corresponds to the maximum possible viable cell count in the lyophilised product after lyophilisation (in CFU/g). By comparing the theoretical viable cell count (in CFU/g) to the real viable cell count (in CFU/g) measured after lyophilisation, it is possible to accurately determine the reduction (e.g. log loss) in viable cell count in the lyophilised product, accounting for the increase in concentration of bacterial cells occurring upon removal of moisture during lyophilisation.

This process of accounting for the loss of moisture between the lyophilisation medium and lyophilised product (/.e. before and after lyophilisation) can be explained with the following illustration. Three lyophilisation media have the same viable cell count (in CFU/ml) priorto lyophilisation, but have different % water contents. In a lyophilisation process in which, for the purposes of this illustration, half of the bacteria perish, the overall reduction (e.g. log loss) in viability should be identical for each lyophilisation media irrespective of their original water content. This is possible, as demonstrated in Table 1 below, by calculating the theoretical viable cell count (on the basis of moisture content and viable cell count of the lyophilisation medium) for each of the respective lyophilised products (in CFU/g), and comparing this theoretical viable cell count with the ‘real’ viable cell count of the corresponding lyophilised product (in CFU/g) measured after lyophilisation. Accordingly, the reduction (e.g. log loss) in viability for each of the lyophilisation products is identical irrespective of the original water content. Therefore, the inherent increase in concentration of bacterial cells upon lyophilisation and/or any differences in the moisture content of different lyophilisation media prior to lyophilisation are accounted for in the calculation. This factoring ensures at least that (I) the reduction (e.g. log loss) in viable cell count determined when comparing the viable cell count before lyophilisation (in CFU/ml) and after lyophilisation (in CFU/g) accurately represents the loss of bacterial cell viability during the lyophilisation process and (ii) the reduction (e.g. log loss) in viable cell count determined for lyophilised products from lyophilisation media having different moisture contents can be directly compared.

Table 1 - Determination of reduction (e.g. log loss) in viable cell count

* As outlined above, the ‘real’ viable cell count for the purposes of this example assumes that half of the bacteria perish during the lyophilisation process.

In line with the calculations outlined above, the viability before lyophilisation (in CFU/ml) and after lyophilisation (in CFU/g) can be directly compared.

In certain embodiments, the loss of viability of the lyophilised product after 1 month of storage at 2 to 8°C when packed in moisture impermeable packaging (e.g. alu/alu blister packaging) is equal to or less than 10³ CFU/g, equal to or less than 10² CFU/g or equal to or less than 10 CFU/g.

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

Surprisingly, the incorporation of an annealing step in the lyophilisation process resulted in a striking improvement in the sublimation of lyophilisation media and/or viability of the bacterial population post- lyophilisation. Without wishing to be bound by theory, the incorporation of an annealing step results in the generation of larger, more organised, water crystals. These water crystals allow faster and I or less intense sublimation procedures. Accordingly, the lyophilised products have an improved ease of sublimation and/or viability.

In certain embodiments, the viability of the bacterial population in the lyophilised product is determined by culturing the lyophilised product in appropriate culture medium. The lyophilised product is resuspended in culture medium prior to culturing. For example, 0.1g of the lyophilised product may be resuspended in 25ml of culture medium. The resuspended culture is incubated at room temperature for 30 minutes. The culture is then diluted (for example, 100-fold) and the cells are plated on solid medium using an appropriate pour plate technique (with a number of repeats). The plates are incubated at an appropriate temperature, for example 37°C, until colonies are formed. The colonies can then be counted and viability compared to the pre-lyophilised bacterial population can be calculated. In certain embodiments, a spiral plater (e.g. that commercialised under the trademark easySpiral® Pro) can be employed. The plate count can be performed under an anaerobic hood.

Residual moisture content and water activity post-lyophilisation

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

In embodiments of the invention, the water activity of the lyophilised product is less than 1. In certain embodiments, the water activity of the lyophilised product is between 0.01 and 0.5. In certain embodiments, the water activity of the lyophilised product is 0.01 to 0.1 , for example 0.01 to 0.05. As shown in the examples, the process of the invention achieves a water activity of less than 0.05.

In certain embodiments, the water content is determined using a Karl Fischer coulometer, for example one commercialised by Mettler Toledo. In certain embodiments, the water activity is determined using a mirror dew point system, for example one commercialised by AquaLab.

Formulations and dosage forms of the lyophilised product

The lyophilised product can be blended with one or more excipients before being provided in dosage forms. Such excipients can comprise diluents, stabilisers, growth stimulators, fillers, lubricants, glidants and the like. Examples of such suitable excipients can be found in the Handbook of Pharmaceutical Excipients. Acceptable excipients for therapeutic use are well known in the pharmaceutical art. Exemplary pharmaceutically acceptable excipients which may be blended with the lyophilised product include, but are not limited to, binders, disintegrants, superdisintegrants, lubricants, diluents, fillers, flavours, glidants, sorbents, solubilizers, chelating agents, emulsifiers, thickening agents, dispersants, stabilizers, suspending agents, adsorbents, granulating agents, preservatives, buffers, colouring agents and sweeteners or combinations thereof. Examples of binders include microcrystalline cellulose, hydroxypropyl methylcellulose, carboxyvinyl polymer, polyvinylpyrrolidone, polyvinylpolypyrrolidone, carboxymethylcellulose calcium, carboxymethylcellulose sodium, ceratonia, chitosan, cottonseed oil, dextrates, dextrin, ethylcellulose, gelatin, glucose, glyceryl behenate, galactomannan polysaccharide, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, hypromellose, inulin, lactose, magnesium aluminium silicate, maltodextrin, methylcellulose, poloxamer, polycarbophil, polydextrose, polyethylene glycol, polyethylene oxide, polymethacrylates, sodium alginate, sorbitol, starch, sucrose, sunflower oil, vegetable oil, tocofersolan, zein, or combinations thereof. Examples of disintegrants include hydroxypropyl methylcellulose (HPMC), low substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium, sodium starch glycolate, lactose, magnesium aluminum silicate, methylcellulose, polacrilin potassium, sodium alginate, starch, or combinations thereof. Examples of a lubricant include stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, glycerin monostearate, glyceryl palmitostearate, magnesium lauryl sulphate, mineral oil, palmitic acid, myristic acid, poloxamer, polyethylene glycol, sodium benzoate, sodium chloride, sodium lauryl sulphate, 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 sulphate, cellulose, cellulose acetate, corn starch, dextrates, dextrin, dextrose, erythritol, ethylcellulose, fructose, fumaric acid, glyceryl palmitostearate, isomalt, kaolin, lactitol, lactose, magnesium carbonate, magnesium oxide, maltodextrin, maltose, mannitol, microcrystalline cellulose, polydextrose, polymethacrylates, simethicone, sodium alginate, sodium chloride, sorbitol, starch, sucrose, sulfobutylether p-cyclodextrin, tragacanth, trehalose, xylitol, or combinations thereof.

Various useful fillers or diluents include, but are not limited to calcium phosphate, dibasic anhydrous, calcium phosphate, dibasic dihydrate, calcium phosphate tribasic, calcium sulphate, cellulose powdered, silicified microcrystalline cellulose, cellulose acetate, compressible sugar, confectioner's sugar, dextrates, dextrin, dextrose, fructose, kaolin, lactitol, lactose, lactose monohydrate, magnesium carbonate, magnesium oxide, maltodextrin, maltose, mannitol, microcrystalline cellulose, polydextrose, 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, sodium lauryl sulphate, magnesium lauryl sulphate, sodium oleate, sodium stearyl fumarate, DL- leucine, colloidal silica, and others as known in the art. In certain embodiments, a lubricant is magnesium stearate. Various useful glidants include, but are not limited to, tribasic calcium phosphate, calcium silicate, cellulose, powdered, colloidal silicon dioxide, magnesium silicate, magnesium trisilicate, starch and talc, or mixtures thereof.

Pharmaceutically acceptable surfactants include, but are limited to both non-ionic and ionic surfactants suitable for use in pharmaceutical dosage forms. Ionic surfactants can include one or more of anionic, cationic or zwitterionic surfactants. Various useful surfactants include, but are not limited to, sodium lauryl sulphate, monooleate, monolaurate, monopalmitate, monostearate or another ester of olyoxyethylene sorbitan, sodium dioctylsulfosuccinate (DOSS), lecithin, stearic alcohol, cetostearylic alcohol, cholesterol, polyoxyethylene ricin oil, polyoxyethylene fatty acid glycerides, and poloxamer.

Excipients which may be blended with the lyophilised product can comprise a prebiotic. The term "prebiotic" means a non-digestible ingredient that beneficially affects the 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 embodiments of the invention, the process comprises the step of preparing a dosage form comprising the lyophilised product. Dosage forms comprising the lyophilised product can be prepared by punching or pressing tablet cores comprising the lyophilised product. In certain embodiments, tablet cores are coated (e.g. enteric coating) to provide tablets. In certain embodiments, the lyophilised product is encapsulated into capsule shells to provide capsules. In certain embodiments, the lyophilised product is provided in sachets and sealing the sachets.

In certain embodiments, the viable bacterial population comprised in dosage forms of the lyophilised product comprise one or more bacterial strains of a specific genus and do not contain bacteria from any other genera, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another genera.

In certain embodiments, the viable bacterial population comprised in dosage forms of the lyophilised product comprise one or more bacterial strains of a specific species and do not contain bacteria from any other species, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another species.

In certain embodiments, the viable bacterial population comprised in dosage forms of the lyophilised product contain a single bacterial strain or species and do not contain any other bacterial strains or species. Such viable bacterial populations can comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species.

In certain embodiments, the viable bacterial population comprised in dosage forms of the lyophilised product comprise more than one bacterial strain. For example, in certain embodiments, the viable bacterial population comprised in dosage forms of the lyophilised product comprise more than one strain from within the same species (e.g. more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 45 strains), and, optionally, do not contain bacteria from any other species.

In certain embodiments, the viable bacterial population comprised in dosage forms of the lyophilised product comprise less than 50 strains from within the same species (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 strains), and, optionally, do not contain bacteria from any other species. In certain embodiments, the viable bacterial population comprised in dosage forms of the lyophilised product comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 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 from within the same species and, optionally, do not contain bacteria from any other species. Certain embodiments comprise any combination of the foregoing.

In certain embodiments, the viable bacterial population comprised in the dosage form comprises a microbial consortium. For example, in certain embodiments, the viable bacterial population comprised in the dosage form comprises a specific bacterial strain as part of a microbial consortium. For example, in certain embodiments, the viable bacterial population comprised in the dosage form comprises a bacterial strain which is present in combination with one or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterial strains from other genera with which it can live symbiotically in vivo in the intestine.

For example, in certain embodiments, the viable bacterial population comprised in the dosage form comprises a specific bacterial strain in combination with a bacterial strain from a different genus. In certain embodiments, the microbial consortium comprises two or more bacterial strains obtained from a faeces sample of a single organism, e.g. a human. In certain embodiments, the microbial consortium is not found together in nature. For example, in certain embodiments, the microbial consortium comprises bacterial strains obtained from faeces samples of at least two different organisms. In certain embodiments, the two different organisms are from the same species, e.g. two different humans. In certain embodiments, the two different organisms are an infant human and an adult human. In certain embodiments, the two different organisms are a human and a non-human mammal.

In certain embodiments, the lyophilised product is in a pharmaceutical dosage form. In certain embodiments, the amount of the bacterial strain in the viable bacterial population comprised in the pharmaceutical dosage form is from about 1 x 10³ to about 1 x 10¹¹ colony forming units per gram with respect to a weight of the dosage form (excluding the capsule body (if present) and any enteric coating (if present).

In certain embodiments, the lyophilised product is in a pharmaceutical composition. In certain embodiments, the pharmaceutical composition is stored in a moisture tight container at 2°C to 8°C, the loss of viable bacteria in the lyophilised product as measured in colony forming units (CFU) per gram is no greater than 3 log, no greater than 2 log or no greater than 1 log after a period of at least about 1 year, 1 .5 years, 2 years, 2.5 years or 3 years. Additionally or alternatively, the lyophilised product, when stored in a moisture tight container at 5°C and the container is placed in an atmosphere having 50% relative humidity, the loss of viable bacteria in the lyophilised product strain as measured in colony forming units (CFU) per gram is no greater than 3 log, no greater than 2 log, no greater than 1 log or no greater than 0.5 log after a period of 6 months.

In certain embodiments, the dosage form contains the lyophilised product in an amount of from about 1 x 10³to about 1 x 10¹³ CFU/g, respect to the weight of the dosage form (excluding the capsule body (if present) and any enteric coating (if present), for example, from about 1 x 10⁴ to about 1 x 10¹² CFU/g, from about 1 x 10⁶ to about 1 x 10¹¹ CFU/g, from about 1 x 10⁸ to about 1 x 10¹² , or from about 1 x 10⁸ to about 1 x 10¹° CFU/g. In certain embodiments, the dosage form can comprise at least 1 x 10¹° CFU/g, at least 1 x 10⁹ CFU/g, at least 1 x 10⁸ CFU/g, at least 1 x 10⁷ CFU/g, or at least 1 x 10⁶ CFU/g.

The viable bacterial population present in the dosage form can be commensal, i.e. it is obtained from a donor (e.g. a human infant, child, adolescent or adult).

In certain embodiments, the dosage form comprises a biologically pure single strain of bacteria. As used herein the term "biologically pure" refers to a culture that comprises de minimis or biologically irrelevant levels of other strains of bacteria. 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 % as a proportion of the total number of bacterial cells of other bacterial species.

In alternative embodiments, the dosage form can comprise a plurality, e.g. 2, 3, 4, 5 or more than 5 strains of bacteria.

In addition to the lyophilised product, the pharmaceutical products can comprise one or more excipients including, for example diluents, stabilisers, growth stimulators, fillers, lubricants, glidants and the like.

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

Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, 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 regard to standard pharmaceutical practice. The dosage forms can comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatine, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, 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 flavouring agents can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid. Suspending agents can be also used.

In certain embodiments, the lyophilised product is not microencapsulated.

In certain embodiments, the lyophilised product can comprise a sugar for example a monosaccharide or disaccharide. Additionally or alternatively, the sugar can be a reducing sugar or non-reducing sugar. In certain embodiments, where the products comprise a reducing sugar, non-reducing sugars can be excluded from the product, and vice versa. Examples of specific sugars that can be employed as excipients include sucrose and trehalose.

The pharmaceutical products can further comprise a prebiotic. The term "prebiotic" means a non-digestible ingredient that beneficially affects the lyophilised 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 lyophilised product can be prepared into an orally administrable enteric dosage form, i.e. one which is capable of dissolution only in selective media (/.e. the intestinal environment) thus preventing release of its contents in the stomach. Such a gastroprotective dosage form can comprise an effective amount of a viable bacterial population and an antioxidant, where the effective amount of the viable bacterial population in colony forming units (CFU) decreases by no more than 1 log in a simulated gastric environment. Gastroprotective properties can be determined by, for example: (a) exposing a dosage form described herein to an acid media at pH 1 .2 for 30 minutes, (b) exposing the dosage form to an intestinal medium at pH 6.8 for 45 minutes, and (c) comparing the CFU after the exposing relative to prior to the exposing.

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

The skilled person will also be familiar with techniques for rendering dosage forms enteric. This is typically achieved by the application of enteric coatings to dosage forms, such as tablets or capsules. Useful enteric coating materials include polymers which dissolve at pH 5.5 or above (e.g. the Eudragit L 30 D-55 and L 100-55 grades), those which dissolve at pH 6.0 or above (for the Eudragit L 100 and L 12,5 grades) and/or those which dissolve at above pH 7.0 (for the Eudragit S 100, S 12,5 and FS 30 D grades). Additionally or alternatively, where the dosage form comprises a capsule, in addition to being enterically coated, the capsule can also be banded to prevent the ingress of gastric medium at the join between the two capsule halves.

In alternative embodiments, the dosage form can be an intrinsically enteric dosage form. As used herein, the term 'intrinsically enteric capsule' is used to refer to a capsule which is formed (either partially or totally) from material which dissolves when exposed to medium having a mildly acidic, neutral or basic pH, thus releasing the contents of the capsule into the medium. In certain embodiments, the intrinsically enteric capsule releases its contents when exposed to media having a pH of about 4.0 or above, about 4.5 or above, about 5.0 or above, about 5.5 or above, about 6.0 or above, about 6.5 or above or about 7.0 or above. Likewise, the term 'enteric' is used to refer to a material which dissolves upon exposure to media having a pH of about 4.0 or above, about 4.5 or above, about 5.0 or above, about 5.5 or above, about 6.0 or above, about 6.5 or above or about 7.0 or above.

Owing to its intrinsic enteric properties, the capsule does not require post-fill processing that could otherwise be potentially damaging to the viable bacterial population, for example, coating, drying and/or banding. Thus, the intrinsically enteric capsule does not comprise a continuous coating (/.e. one that covers the entirety of the capsule) and/or is unbanded.

The intrinsically enteric capsule can be single layered or multi-layered and I or be wholly or partly formed of gastrointestinal material which dissolves at the specific pH. In multilayered embodiments, one or more of the layers can be formed of enteric material which dissolves at the specific pH.

The intrinsically enteric capsule can be formed of any material/s which permit the total or partial dissolution of the capsule when exposed to medium having a mildly acidic, neutral or basic pH. In embodiments of the invention, the intrinsically enteric capsule can be formed partially or totally from fatty acids, waxes, shellac, plastics, plant fibers, enteric polymers or mixtures thereof. Enteric materials which can be employed in the present invention (either to produce intrinsically enteric dosage forms, or in alternative embodiments, enteric coatings) include, but are not limited to methacrylate polymers, methyl acrylate-methacrylic acid copolymers, methacrylic acid-methyl methacrylate copolymers, polyvinyl acetate phthalate, shellac, sodium alginate, zein, dextrins, amylose starch and starch derivatives, and cellulose and cellulose derivatives including hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate, cellulose acetate succinate, cellulose acetate trimellitate, cellulose acetate phthalate, or mixtures thereof. Plasticisers can also be comprised in the material from which the intrinsically enteric capsule is formed. Examples of materials that can be used in the production of intrinsically enteric capsules as well as methods for preparing such capsules are provided in European Patent No. 2722104, the contents of which are incorporated herein by reference. An example of an intrinsically enteric capsule is provided by Capsugel underthe trade names enTRinsic DDT or ECDDT.

The capsules on can take any shape, form or construction provided that they can be closed to provide an enteric seal around the LBP comprised therein. For example, the capsules can be hard or soft. In certain embodiments, the capsule is a two part capsule or a multi part capsule (/.e. a capsule closed by coupling more than two parts).

For two part capsules or multi part capsules, the capsule parts can be closed by mechanically coupling the two or more parts of the capsule. Any form of mechanical interaction which results in the formation of a seal around the LBP can be employed. Examples of mechanical interaction that are envisaged include push-fit coupling, friction coupling and/or threaded coupling.

The invention is exemplified in the following Examples. The Examples are not intended to limit the scope of the invention and are intended for exemplification only.

EXAMPLES

Example 1 - An annealing step improves post-lyophilisation viability

Materials and methods

A bacterial population of Parabacteroides distasonis (strain MRx0005, deposited under accession number NCIMB 42382) was grown to stationary phase using conventional fermentation techniques. The bacterial population was then harvested from the fermenter and subjected to concentration (27X). The harvested biomass was mixed with a lyophilisation buffer comprising sucrose, maltodextrine DE9 and cysteine HCI to provide a lyophilisation medium. The ratio of biomass to lyoprotectant prior to lyophilisation was 69.4:30.6, and the components of the lyophilisation buffer were at a final concentration prior to lyophilisation of 2% sucrose, 4% maltodextrin and 0.2% cysteine HCI.

The lyophilisation medium was split into two batches prior to lyophilisation, each of 1 kg. Both batches were subjected to a freeze drying cycle, which, aside from the performance of an annealing step, were essentially identical.

More specifically, the lyophilisation apparatus was cooled to -45°C before the batches were loaded. The two 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 primary drying steps at a shelf temperature of (i) -9°C for 24 hours and (ii) 25°C for 20 hours, respectively, both at a pressure of 50 pBar, and a secondary drying step at a shelf temperature of 25°C for at least 10 hours. During the primary drying steps, the shelf temperature was increased by about 5°C/hour. One of the batches was subjected to an annealing step during the one or more freezing steps, in which the bacteria, after 2 hours at a shelf temperature of -45°C, were heated to an annealing temperature of -15°C, at which they were maintained for 5 hours, before being cooled to a shelftemperature of -45°C, at which they were incubated for a further 3 hours. The second batch was not subjected to an annealing step. The lyophilised biomass from each batch was then resuspended in media and tested for viability.

The conditions of the lyophilisation process for both batches (/.e. with and without an annealing step) are outlined in Table 2, below.

Table 2

The temperatures listed in Table 2 refer to the temperature of the shelf of the lyophilisation apparatus. The secondary drying step in Table 2 continues until the product temperature has been at least 22°C for 10 hours.

The lyophilisation apparatus a Lyovac GT41 freeze dryer, with a capacity of 0.5m². The water content was determined using a Karl Fischer coulometric device (Mettler Toledo) with the following analysis parameters (speed: 40%; mix: 600s and set point temperature: 120°C). The water activity of the sample was determined using an Aqualab water activity monitor using the chilled mirror dew point technique at 20°C. Results

The results are shown in Figure 1 , and Table 3.

Figure 1A and B display the structural integrity of the lyophilised biomass obtained from the lyophilisation process with an annealing step (Figure 1A) and without an annealing step (Figure 1 B). These figures demonstrate that the lyophilised biomass obtained from the lyophilisation process using an annealing step displays a greater level of macroscopic crystal organization. The product obtained without an annealing step has a layer, about 30% of the depth of the product (measured at the point marked with the arrow), with crystals organized in a vertical manner. However, this layer in the product obtained with an annealing step is about 55% of the depth of the product (measured at the point marked with the arrow). Accordingly, the inclusion of an annealing step improves the crystal organization of the lyophilisation medium during the lyophilisation procedure in turn facilitating the sublimation of moisture therefrom, thus improving lyophilisation efficiency.

Table 3 demonstrates the effect of an annealing step on the viability of the lyophilised biomass with and without an annealing step. The viability of the biomass (originally from a culture having a viability of 1.6x10¹¹ CFU/ml) obtained with an annealing step was 1.3x1 O¹⁰ CFU/g, while the biomass obtained without an annealing step was 5.1x10⁹ CFU/g. As outlined in the detailed description, it is appropriate to compare the viability before lyophilisation (CFU/ml) and after lyophilisation (CFU/g) by accounting for the loss of moisture content and thus the increase in concentration of bacterial cells that inherently occurs during the process of lyophilisation.

In addition, the lyophilisation process including an annealing step resulted in a slightly lower % moisture content compared to the same process without an annealing step.

Table 3

Conclusion

The incorporation of an annealing step within the lyophilisation process maintains the viability of the bacterial population after lyophilisation. In addition, the annealing step appears to result in an improved crystal arrangement in the lyophilised biomass. Example 2 - An annealing step improves post-lyophilisation viability

Materials and methods

A Parabacteroides distasonis strain (MRX0005) deposited under accession number NCIMB 42382 was grown to stationary phase to a density of 1.6x10¹¹ CFU/ml before being collected and resuspended in lyophilisation buffer. The buffer comprised a lyoprotectant formula comprising (at a final concentration prior to lyophilisation): 2% sucrose, 4% maltodextrine DE9 and 0.2% cysteine HCI. The ratio of biomass to lyoprotectant was 69.4:30.6.

The lyophilisation medium was split into five batches prior to lyophilisation, each of 1 kg. All batches were subjected to a freeze drying cycle, which, aside from the performance of an annealing step, were essentially identical.

More specifically, the lyophilisation apparatus was cooled to -45°C before the batches were loaded. The 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 primary drying steps at a shelf temperature of (i) -21 °C for 24 hours and (ii) 25°C for 20 hours, respectively, both at a pressure of 276 pBar, and a secondary drying step at a shelf temperature of 25°C for at least 10 hours. During the primary drying steps, the shelf temperature was increased by about 5°C/hour. Two of the batches were subjected to an annealing step during the one or more freezing steps, in which the bacteria, after 2 hours at a shelf temperature of -45°C, were heated to an annealing temperature of -15°C, at which they were maintained for 5 hours, before being cooled to a shelftemperature of -45°C, at which they were incubated for a further 3 hours. The remaining three batches were not subjected to an annealing step. The lyophilised biomass from each batch was then resuspended in media and tested for viability.

The conditions of the lyophilisation process for the batches with and without an annealing step are outlined in Table 4, below.

Table 4

The temperatures listed in Table 4 refer to the temperature of the shelf of the lyophilisation apparatus. The secondary drying step in Table 4 continues until the product temperature has been at least 22°C for 10 hours.

The results are shown in Figure 2 and Table 5.

Figure 2A-F display the structural integrity of the lyophilised biomass obtained from the lyophilisation process with an annealing step (Figure 2A and B) and without an annealing step (Figure 2C-F). These figures demonstrate that the lyophilised biomass obtained from the lyophilisation process using an annealing step displays a greater level of macroscopic crystal organization. The product obtained without an annealing step has a layer, about 25-35% of the depth of the product (measured at the points marked with the arrow), with crystals organized in a vertical manner. However, this layer in the product obtained with an annealing step is about 50-55% of the depth of the product (measured at the points marked with the arrow). Accordingly, the inclusion of an annealing step improves the crystal organization of the lyophilised biomass during the lyophilisation procedure.

Table 5 demonstrates the effect of an annealing step on the viability of the lyophilised biomass with and without an annealing step. The viability of the biomass obtained from the batches with an annealing step was 9.4x10¹⁰ CFU/g and 1 .2x10¹¹ CFU/g. The viability of the biomass obtained from the batches without an annealing step was between 5.5x10¹⁰ CFU/g and 7.2x10¹⁰ CFU/g. As outlined in the detailed description, it is appropriate to compare the viability before lyophilisation (CFU/ml) and after lyophilisation (CFU/g) by accounting for the loss of moisture content and thus the increase in concentration of bacterial cells that inherently occurs during the process of lyophilisation.

In addition, in line with the observations above, the lyophilisation process including an annealing step resulted in a lower % moisture content compared to the same process without an annealing step.

Table 5

Conclusion

The incorporation of an annealing step within the lyophilisation process significantly increases the viability of the bacterial population after lyophilisation. In addition, the annealing step appears to result in an improved crystal arrangement in the lyophilised biomass. Therefore, the annealing step represents an advantageous additional step in the lyophilisation process.

Example 3 - Assessing the pore structure of the lyophilised product, generated using a lyophilisation process with and without an annealing step, using scanning electron microscopy (SEM)

Materials and methods

Samples of the lyophilised products obtained from Example 2 above were analysed using SEM. Fragments of the lyophilised product were attached to aluminium stubs using Electrodag 502 then coated in a layer of gold palladium. The prepared lyophilised products were then imaged using a Zeiss EVO MA10 Scanning Electron Microscope at various depths of field using the secondary electron detector.

Results and conclusion

The results of the SEM analysis are displayed in Figure 3 (A-D). The left-hand image in each of Figures 3A-D displays the microscopic structure of the lyophilised product obtained from the lyophilisation process in Example 2 without annealing. The right-hand image in each of Figures 3A-D displays the microscopic structure of the lyophilised product obtained from the lyophilisation process in Example 2 with annealing. Figures 3A and B show the crystal organisation under reduced magnification, indicating that the presence of an annealing step enables the crystals to form ordered layers more effectively. Figure 3C studies cake structure at a higher magnification, and supports the findings that the presence of an annealing step enables crystals to better align into layers. Finally, Figure 3D shows the crystals at the highest magnification, and indicates that the presence of an annealing step serves to maximize the consistency of crystal formation and reduces crystalline debris not present within the major layers.

Therefore, the use of the annealing step ensures a greater level and consistency of crystal organisation at all levels throughout the lyophilised product. BIBLIOGRAPHY Louis Rey and Joan May, Drugs and the Pharmaceutical Sciences, Third Edition Ablett et al. J Chem Soc Faraday Trans. (1992); 88:789-794

NUMBERED EMBODIMENTS

The present disclosure also provides the following numbered embodiments:

1. A process for preparing a lyophilised product comprising a viable bacterial population, the process comprising:

(i) providing a lyophilisation medium comprising a viable bacterial population;

(iii) collecting a lyophilised product.

2. The process of embodiment 1 , wherein the one or more freezing steps are cycled with the one or more annealing steps.

3. The process according to embodiment 1 , wherein the viability (CFU/g as dry weight) of the lyophilised product is equal to or less than 10³ CFU/g, equal to or less than 10² CFU/g, or equal to or less than 10 CFU/g lower than the viability of the lyophilisation medium prior to lyophilisation (CFU/ml).

4. The process according to embodiment 3, wherein the viability (CFU/g as dry weight) of the lyophilised product is equal to or less than 10 CFU/g lower than the viability of the lyophilisation medium prior to lyophilisation (CFU/ml).

5. The process according to any preceding embodiment, wherein the water content of the lyophilised product is less than 5.0, 4.5, 4.0 or 3.5 wt%.

6. The process according to any preceding embodiment, wherein the water content of the lyophilised product is between 3.0 and 4.5 wt%, optionally wherein the water content of the lyophilised product is between 3.3 and 4.4 wt%.

7. The process according to any preceding embodiment, wherein the one or more annealing steps occur at a higher temperature than the one or more freezing steps.

8. The process according to any preceding embodiment, wherein the one or more freezing steps occur at a temperature of between about -40 and -50°C, optionally wherein the one or more freezing steps occurs at a temperature of about -45°C.

9. The process according to any preceding embodiment, wherein the at least one primary drying step occurs at a temperature of between about -5 and -25°C, optionally wherein the at least one primary drying step occurs at a pressure of between about 50 and 300 pbar, optionally wherein the at least one primary drying step occurs at:

(i) a temperature of about -9°C and a pressure of about 50 pbar; or

(ii) a temperature of about -21 °C and a pressure of about 275 pbar. The process according to any preceding embodiment, wherein the one or more annealing steps occur at a temperature of between about -10 and -20°C, optionally wherein the one or more annealing steps occur at a temperature of about -15°C. The process according to any preceding embodiment, wherein the process further comprises a second primary drying step, wherein the second primary drying step occurs at a temperature of about 25°C and a pressure of between about 50 and 300 pbar. The process according to any preceding embodiment, wherein the viable bacterial population is not a lactic acid bacteria or does not comprise a lactic acid bacteria. The process according to any preceding embodiment, wherein the viable bacterial population is lyophilised in a lyophilisation medium, optionally wherein the lyophilisation medium comprises a lyophilisation buffer, for example wherein the lyophilisation buffer comprises a lyoprotectant formula comprising (at a final concentration prior to lyophilisation): 2% sucrose, 4% maltodextrine DE9 and 0.2% cysteine HCI, and optionally wherein the lyophilisation buffer does not comprise trehalose. The process according to any preceding embodiment, wherein the process comprises a first freezing step at a temperature of about -45°C. The process according to any preceding embodiment, wherein the process comprises an annealing step at a temperature of about -15°C. The process according to any preceding embodiment, wherein the process comprises a second freezing step at a temperature of about -45°C. The process according to any preceding embodiment, wherein the process comprises a primary drying step at a temperature of about -9°C and a pressure of about 50 pBar. The process according to any one of embodiment 1-16, wherein the process comprises a primary drying step at a temperature of about -21 °C and a pressure of about 275 pBar. The process according to any preceding embodiment, wherein the process comprises:

(i) providing a lyophilisation medium comprising a viable bacterial population;

(ii) lowering the temperature (for example, the shelf or tray temperature) to a temperature of between about -40 and -50°C;

(iii) maintaining the temperature of between about -40 and -50°C for about 1 to 3 hours; (iv) increasing the temperature (for example, the shelf or tray temperature) to a temperature of between about -10 and -20°C;

(v) maintaining the temperature of between about -10 and -20°C for about 4 to 8 hours;

(vi) lowering the temperature (for example, the shelf or tray temperature) to a temperature of between about -40 and -50°C;

(vii) maintaining the temperature of between about -40 and -50°C for about 2 to 4 hours;

(viii) drying the lyophilisation medium at a temperature of between about -5 and -25°C and a pressure of between about 50 and about 300 pBar;

(ix) maintaining the drying temperature for about 18 to 30 hours and

(x) collecting a lyophilised product. A lyophilised product produced by the process of any preceding embodiment, optionally wherein the lyophilised product is formulated in a pharmaceutical composition, for example wherein the lyophilised product is in a pharmaceutical dosage form. he process of any one of embodiments 1-19 or the product of embodiment 20, wherein the lyophilised product is a live biotherapeutic product.

(Original in Electronic Form)

(This sheet is not part of and does not count as a sheet of the international application)

FOR RECEIVING OFFICE USE ONLY

FOR INTERNATIONAL BUREAU USE ONLY

Claims

(i) providing a lyophilisation medium comprising a viable bacterial population;

(iii) collecting a lyophilised product.

2. The process of claim 1 , wherein the lyophilisation medium is provided in an amount of at least about 1g.

3. The process of claim 1 or 2, wherein the one or more freezing steps are cycled with the one or more annealing steps.

4. The process according to any preceding claim, wherein the viability (CFU/g as dry weight) of the lyophilised product is equal to or less than 10³ CFU/g, equal to or less than 10² CFU/g, or equal to or less than 10 CFU/g lower than the viability of the lyophilisation medium prior to lyophilisation (CFU/ml), optionally wherein the viability (CFU/g as dry weight) of the lyophilised product is equal to or less than 10 CFU/g lower than the viability of the lyophilisation medium prior to lyophilisation (CFU/ml).

5. The process according to any preceding claim, wherein the water content of the lyophilised product is:

(i) less than 5.0, 4.5, 4.0 or 3.5 wt%;

(ii) between 3.0 and 4.5 wt%; or

(iii) between 3.3 and 4.4 wt%.

6. The process according to any preceding claim, wherein the one or more annealing steps occur at a higher temperature than the one or more freezing steps.

7. The process according to any preceding claim, wherein the one or more freezing steps occur at a temperature of between about -40 and -50°C, optionally wherein the one or more freezing steps occurs at a temperature of about -45°C.

8. The process according to any preceding claim, wherein the at least one primary drying step occurs at a temperature of between about -5 and -25°C, optionally wherein the at least one primary drying step occurs at a pressure of between about 50 and 300 pbar, optionally wherein the at least one primary drying step occurs at:

(i) a temperature of about -9°C and a pressure of about 50 pbar; or

57 (ii) a temperature of about -21 °C and a pressure of about 275 pbar. The process according to any preceding claim, wherein the one or more annealing steps occur at a temperature of between about -10 and -20°C, optionally wherein the one or more annealing steps occur at a temperature of about -15°C. The process according to any preceding claim, wherein the process further comprises a second primary drying step, wherein the second primary drying step occurs at a temperature of about 25°C and a pressure of between about 50 and 300 pbar. The process according to any preceding claim, wherein the viable bacterial population is not a lactic acid bacteria or does not comprise a lactic acid bacteria. The process according to any preceding claim, wherein the viable bacterial population is lyophilised in a lyophilisation medium, optionally wherein the lyophilisation medium comprises a lyophilisation buffer, for example wherein the lyophilisation buffer comprises a lyoprotectant formula comprising (at a final concentration prior to lyophilisation): 2% sucrose, 4% maltodextrine DE9 and 0.2% cysteine HCI, and optionally wherein the lyophilisation buffer does not comprise trehalose. The process according to any preceding claim, wherein the process 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) a primary drying step: a. at a temperature of about -9°C and a pressure of about 50 pBar; or b. at a temperature of about -21 °C and a pressure of about 275 pBar. The process according to any preceding claim, wherein the process comprises:

(i) providing a lyophilisation medium comprising a viable bacterial population;

(iii) maintaining the temperature of between about -40 and -50°C for about 1 to 3 hours;

(iv) increasing the temperature (for example, the shelf or tray temperature) to a temperature of between about -10 and -20°C;

(ix) maintaining the drying temperature for about 18 to 30 hours and

58 (x) collecting a lyophilised product. A lyophilised product produced by the process of any preceding claim, optionally wherein the lyophilised product is formulated in a pharmaceutical composition, for example wherein the lyophilised product is in a pharmaceutical dosage form. he process of any one of claims 1-14 or the product of claim 15, wherein the lyophilised product is a live biotherapeutic product.

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