WO2006029467A1 - Rapid freeze drying process - Google Patents

Rapid freeze drying process Download PDF

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Publication number
WO2006029467A1
WO2006029467A1 PCT/AU2005/001408 AU2005001408W WO2006029467A1 WO 2006029467 A1 WO2006029467 A1 WO 2006029467A1 AU 2005001408 W AU2005001408 W AU 2005001408W WO 2006029467 A1 WO2006029467 A1 WO 2006029467A1
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Prior art keywords
process
sample
freeze
temperature
drying
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PCT/AU2005/001408
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French (fr)
Inventor
Charlotte Ann Morgan
Nicholas Herman
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Btf Pty Ltd
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04Preserving or maintaining viable microorganisms
    • FMECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F26DRYING
    • F26BDRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
    • F26B5/00Drying solid materials or objects by processes not involving the application of heat
    • F26B5/04Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
    • F26B5/06Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing

Abstract

A process for rapid freeze drying a sample including freezing the sample followed by freeze drying the sample under temperature and vacuum conditions such that the sample is dried close to the collapse temperature of the sample. T collapse temperature of the sample has been defined as the temperature during freezing that results in the collapse of the structural integrity of the product. Further, 'close to the collapse temperature' means a temperature about 0.1 to 1O°C below the collapse temperature.

Description

RAPID FREEZE DRYING PROCESS

Technical Field

The invention relates to a process for freeze drying, particularly freeze drying samples containing biological material.

Background Art

The process of lyophilization, or freeze-drying involves removing water from a material by sublimation and desorption. Freeze drying is used widely in the food and pharmaceutical industries to preserve biological molecules as well as whole cells.

Freeze drying involves firstly freezing the material that is to be freeze dried and then placing the frozen material into a sealed vessel and applying a vacuum. The sealed vessel is normally comprised of two compartments, one which holds the material and a second that acts as a condenser. The condenser is usually a cylindrical chamber that can be cooled to below 0°C. When a vacuum is applied to the vessel the frozen water molecules within the material undergo sublimation and are collected on the cooled surface of the condenser. This process is continued until sufficient water has been ' removed from the material.

In many freeze dryers the material that is being freeze dried is held on shelves that can be heated. The application of heat to the material speeds up the drying process and provides, control over the rate of drying. The application of heat through a shelf or tray that is in contact with a large surface area of the material that is being freeze dried, has the added advantage that it reduces variation in the rate of drying across the material.

Sensitive biological material is normally freeze dried in glass vials. The vials are loaded into a tray that is placed onto a heat controlled shelf within the freeze dryer. The material may be frozen prior to loading into the freeze dryer or the materials loaded in a liquid form and then frozen by lowering the temperature of the shelf. The pressure in the chamber is then lowered and the drying process begins. The temperature of the shelves is gradually increased to help drive the water molecules from the frozen material. The drying process is normally complete within 24 to 72 hours depending on the amount of material loaded into the freeze dryer and the capabilities of the freeze dryer.

It is important to control the drying rate and the heating rate during the freeze drying process. If the product is heated too rapidly, it will melt or collapse. This may cause degradation of the product, and will certainly change the physical characteristics of the dried material, making it visually unappealing and harder to reconstitute. While frozen mobile water is present, the product must be held below the collapse temperature. It is difficult to accurately determine the collapse temperature for a particular material, therefore, heat is normally applied very gradually to ensure that the collapse temperature is not reached.

If heat is applied to the product at a rate that brings the product close to the collapse temperature, then variation between the vials occurs depending on the position of the vial on the shelf. Material in some vials will collapse whilst others will not. This is because there is variation in the temperature of the viais depending on the position of the vial. For example, vials on the outside of the shelf may warm up quicker than vials in the middle of the shelf because the ones in the middle are surrounded by a large mass of cold material of the neighbouring vials, whereas vials on the outside of the shelf are not surrounded by such a large mass of cold material. Sensitive biological materials such as cells are often irrevocably damaged during freeze drying. It is thought that changes occur in the physical state of lipids and structure changes occur in some proteins. These changes can cause lethal damage to biological membranes. It is well known that crystal growth during freezing causes damage to biological material. Furthermore, it is likely that crystal growth during the freeze drying process contributes to cell damage and cell death.

Additives used to exert a mechanism of protection to biological materials are termed cryoprotectants, if the material is frozen, and lyoprotectants if the material undergoes freeze drying. There are a wide range of protectants used in the field and include sulfoxides, alcohols and derivatives, saccharides, polysaccharides, amides, peptides, proteins, glycoproteins, complex compounds and cations.

There are three mechanisms of protection afforded by protectants according to their permeability. Additives such as glycerol, dimethylsulfoxide (Me2SO) and glucose are cell permeant and can therefore exert intracellular protection. Larger molecules such as mono- and disaccharides, amino acids and low molecular weight polymers that cannot pass through the cell membrane, but are small enough to cross the cell wall are termed semi-permeable protectants. The large molecules, for example polymers with high molecular weights, proteins, polysaccharides and dextran, that cannot pass either the cell wall or the cell membrane can only exert extracellular protection mechanisms. Permeant protectants are generally hydrophilic (water - loving), and possess chemical groups that enable strong hydrogen bonds with water molecules. This ability to bind intracellular water prevents excessive dehydration, reduces the build up of high salt concentrations and stops the formation of large ice crystals. Permeable protectants stimulate a fine crystalline ice structure and form a gel-type glass phase, helping cells to avoid hyperosmotic injury, as the salt is bound in the gel phase and not allowed to concentrate in solution as water crystals remove the water solvent.

Semi-permeable protectants can induce partial dehydration of cells prior to freezing, by concentrating as a buffer layer between the cell wall and the cell membrane and drawing water osmotically from the cell. They can also exert a mechanical support to the cell wall.

Non-penetrating protectants generally protect against extracellular ice crystal formation, they do not interact directly with the cells, but they form a viscous protective layer around cells. Like the semi-permeable protectants, they can cause an osmotic efflux of water from the cell, but more importantly they inhibit the growth of ice crystals by forming amorphous glass structures during drying, due to gradually increasing viscosity of the protectant solution.

The present inventors have now developed a process for freeze-drying which results in minimal loss of activity or viability of biological materials.

Summary of Invention

The present invention provides a process for rapid freeze drying samples which results in improved recovery of activity or viability of the freeze-dried samples compared with conventional freeze-drying processes for equivalent samples. In a first aspect, the present invention provides a process for freeze drying a sample comprising: freezing the sample; and freeze drying the sample under temperature and vacuum conditions such that the sample is dried close to the collapse temperature of the sample. In a preferred form, the process further includes: adding a penetrating cryoprotectant to the sample. Preferably, the penetrating cryoprotectant is selected from glycerol, 2-methyl-2,4- pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, dimethyl sulfoxide (DMSO), methanol, 1 ,2-propanediol, proline, and ethylene glycol.

Preferably, the cryoprotectant is glycerol. Preferably, one or more cryoprotectants are included in the sample medium, usually prior to freezing. In a preferred embodiment, at least about 0.1% cryoprotectant (v/v) is included, preferably at least about 0.2%. Typically, not more than about 10% cryoprotectant is added, preferably not more than about 5% cryoprotectant, more preferably not more than about 1 % cryoprotectant. In a particularly preferred embodiment, at least 0.1 % glycerol (v/v) is included, preferably at least 0.2%. Typically, not more than about 10% glycerol is added, preferably not more than about 5%, more preferably not more than about 1 % glycerol. It will be appreciated that other suitable cryoprotectants can also be used.

In a second aspect, the present invention provides a process for freeze drying a plurality of samples comprising: ' freezing a plurality of samples; placing the plurality of frozen samples into a plurality of containers; and freeze drying the plurality of samples under temperature and vacuum conditions such that the samples are dried close to the collapse temperature of the sample. In a preferred form, the process further includes: adding a penetrating cryoprotectant to the samples.

The present inventors have found that activity or viability of biological materials can be maintained in samples when freeze dried according to the present invention.

Biological materials include, but not limited to, microorganisms such as bacteria, yeasts, fungi, viruses; proteins such as antibodies, growth factors, hormones and the like; nucleic acids such as DNA and RNA. It will be appreciated, however, that the process of the present invention is suitable for freeze drying any suitable material.

The collapse temperature is the temperature during freeze drying that results in collapse of the structural integrity of a product. It will be appreciated therefore that the collapse temperature will vary between different sample types. By close to the collapse temperature, it is meant to be about 0.1 to 1O0C below the collapse temperature. It will be appreciated that a collapse temperature may be calculated by experiment or measured for each sample type. When a sample is freeze dried close to its collapse temperature, the present inventors have found that the product looks shrivelled and / or shrunken. Freeze drying further away from the collapse temperature produces a more smooth and well formed product. It is therefore preferred to form a shrivelled and shrunken product using the present invention. This is quite different from what has been done prior to the present invention as a smooth and well formed product was considered desirable.

Prior art freeze drying methods are carried out far from the collapse temperature of the sample to produce smooth and evenly formed products.

In a preferred form, the plurality of containers are arranged in the freeze dryer such that the containers are isolated from each other.

In a further preferred form, heat is applied to the plurality of containers such that each container is exposed to substantially an equivalent temperature at any given point during the freeze drying process.

Preferably, during the freeze drying the rate of heating and rate of vacuum pressure are controlled such that the drying process occurs as rapidly as possible without the sample collapsing.

Preferably, there is substantially no inactivation or loss of activity or viability of the samples during the process. The present inventors have surprisingly found that by freeze-drying samples of labile or living material close to the collapse temperature not only results in rapid freeze drying but retains the activity or viability of the sample content.

Preferably, there is no adverse affect to the appearance of the freeze-dried samples.

Preferably, the heating is rapid. Such rapid heating is defined as increasing the temperature by about 10C per minute. A starting temperature of around -4O0C and a finish temperature of around 250C are typically used for the present invention. It will be appreciated that the process may be started at other temperatures such as about -350C, -3O0C, -250C, or -2O0C, for example. The finishing temperature may be about 1O0C, 150C, 2O0C, or 3O0C, for example. A vacuum is typically applied during the heating process.

Preferably, the process takes about 3 to 5 hours depending on the volume of material within the freeze dryer.

The process may further include sealing the containers. The sample can be a compound, composition, or biological material. Preferably, the compound or composition is a pharmaceutical agent, an antibody, an enzyme, a protein, a peptide or a nucleic acid. Preferably, the biological sample is a microorganism, cell, cell component or a virus. The present invention is particularly suitable for freeze-drying bacteria in relatively small numbers without significant loss of viability.

The sample may further contain bulking agents, cryoprotectants, lyoprotectants, surfactants, buffering agents, buffers or excipients. It will be appreciated, however, that other components as required may also be added to the sample prior to processing. Preferably, the plurality of containers are placed in an rack made of an insulating material to isolate the containers from each other. Preferably, the rack is made of polystyrene, or other suitable insulating material, having holes adapted to receive the containers. The rack preferably does not provide insulation to the bottom of the containers such that the heating is provided from above and below the containers in a freeze drying apparatus.

The present inventors have found that if the temperature applied to each container during freeze-drying is relatively consistent, then it is possible to rapidly freeze dry samples with significantly less adverse affects on the characteristics of the sample. Instead of requiring several days to prepare samples by conventional freeze-drying, the present invention can be carried out over several hours. Not only does this result in an improved product, it also reduces processing time and thus production costs.

In a third aspect, the present invention provides a freeze-dried product produced by the process according to the first and second aspects of the present invention.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this specification. In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following drawings and examples.

Brief Description of the Drawings Figure 1 shows vacuum pressure and temperature profile of a preferred freeze drying cycle according to the present invention.

Figure 2 shows results of the. lowest freeze dried count of 5 replicates produced after E. coli 0157 BioBall™ had been stored for 1 week at 22°C.

Mode(s) for Carrying Out the Invention Definitions

Protein stability can be affected inter alia by such factors as ionic strength, pH, temperature, repeated cycles of freeze/thaw and exposures to shear forces. Active protein may be lost as a result of physical instabilities, including denaturation and aggregation (both soluble and insoluble aggregate formation), as well as chemical instabilities, including, for example, hydrolysis, deamidation, and oxidation. For a general review of stability of protein pharmaceuticals, see, for example, Manning, et al., Pharmaceutical Research 6:903-918 (1989).

"Collapse temperature" is the temperature during freeze drying that results in collapse of the structural integrity of a product. It will be appreciated therefore that the collapse temperature will vary between different sample types.

"Close to the collapse temperature" is meant to be about 0.1 to 100C below the collapse temperature. It will be appreciated that a collapse temperature may be calculated by experiment or measured for each sample type. "Storage-stable" means the product is stabilised for storage at temperatures between -200C to 250C and remains within product specifications for a suitable time period, often several months.

"Bulking agent" generally includes agents which provide good lyophilised cake properties, which form a pharmaceutically elegant product, which help the material overcome various stresses (shear/freezing for example) associated with the lyophilization process, and which help to maintain protein activity levels during the freeze-drying process and subsequent storage. Examples of often-used bulking agents include mannitol, glycine, sucrose, lactose, etc. The agents can also contribute to the tonicity of the formulations.

"Cryoprotectants" generally includes agents which provide stability to the material to be freeze-dried from freezing-induced stresses. The term also includes agents that provide stability, e.g. to bulk drug formulations during storage from non-freezing-induced stresses. Examples of cryoprotectants include solvents such as glycerol and dimethyl sulfoxide (DMSO), polyols such as, for example, mannitol, and include saccharides such as, for example, sucrose, as well as including surfactants such as, for example, polysorbate, poloxamer or polyethylene glycol, and the like. Cryoprotectants can also contribute to the tonicity of the formulations.

The term "lyoprotectant" includes agents that provide stability to the material during water removal from the system during the drying process, presumably by maintaining the proper conformation of the protein through hydrogen bonding. Examples of lyoprotectants include saccharides and di- or trisaccharides and complex mixtures such as serum. Cryoprotectants may also have lyoprotectant effects. While preferred concentrations range from about 0.5 to 2%, relatively higher concentrations (for example 75%) of serum is often used.

The term "surfactants" generally include those agents, which protect the material from air/solution interface-induced stresses and solution/surface induced-stresses (e.g. resulting in protein aggregation), and may include detergents such as polysorbate, poloxamer or polyethylene glycol, and the like. Optionally, concentrations from about 0.01% to about 1 % (w/w) are suitable for maintaining protein stability, however, the levels used in actual practice are customarily limited by clinical practice.

The term "buffering agent" or "buffer" encompasses those agents which maintain the solution pH in an acceptable range prior to and during lyophilization and may include histidine, phosphate, citrate, tris, diethanolamine, and the like. The upper concentration limits are generally higher for "bulk" materials than for "dosage" material forms as is readily appreciated by one skilled in the art. For example, while buffer concentrations can range from several millimolar up to the upper limit of their solubility (e.g., histidine could be as high as 200 mM), one skilled in the art would also take into consideration achieving/maintaining an appropriate physiologically suitable concentration. Percentages are weight/weight when referring to solids and weight/volume when referring, to liquids.

The term "isotonic", 300+-.50 mOsM, is meant to be a measure of osmolality of the material solution prior to lyophilization; reconstitution is typically with water for injection (WFI). Maintaining physiological osmolality can be important for the dosage formulations. However for bulk formulations, much higher concentrations can be effectively utilised as long as the solution is made isotonic prior to use.

. The term "excipients" includes pharmaceutical acceptable reagents to provide good lyophilised cake properties (bulking agents) as well as provide lyoprotection and cryoprotection of the protein, maintenance of pH, and proper conformation of the material during storage so that substantial retention of biological activity and protein stability is maintained.

The term "viability" refers to the ability of a culture of microorganisms to grow. For example, a "viable" sample is comprised of live organisms that are capable of metabolism and growth. Consequently, a freeze-dried sample is viable if it can be revived, such as by rehydration, and grown in a suitable median. Preferably, the sample remains viable for at least one month, preferably at least two, three or six months, more preferably at least a year. The term "activity" refers to the ability of a biological material to react in a specific manner. For example, an "active" antibody is capable of binding to an antigen. Consequently, a freeze-dried sample is active if it maintains activity after rehydration. Preferably, the sample remains active for at least one month, preferably at least two, three or six months, more preferably at least a year.

Microorganisms

The term "microorganism" includes prokaryotes and eucaryotes such as yeast, fungi and animal and plant cells capable of being propagated in culture, such as immortalised cell lines. Examples of. prokaryotes include eubacteria and archaebacteria. Eubacteria include gram negative bacteria such as Escherichia sp. (e.g. E. coli),

Neisseria sp, Campylobacter sp, Haemophilus sp, Streptococcus sp and Staphylococcus sp and gram positive bacteria such as Bacillus sp (e.g. B. subtilis).

Table 1 contains a list of bacteria that can be preserved or freeze-dried according to the present invention. It will be appreciated, however, that this list is not exhaustive. . Table 1

Figure imgf000011_0001
Table 1 continued

Figure imgf000012_0001

Microorganisms are typically grown prior to freeze-drying in standard culture medium appropriate to that specific type of microorganism, such as nutrient broth for E. coli. In a preferred embodiment of the present invention, the microorganisms are grown in the presence of a high concentration of a carbohydrate at a desired pH for sufficient time so as to cause an accumulation of the carbohydrate in the cell.

Prior to freeze drying the microorganisms are mixed with a lyoprotectant to provide a sample with a suitable glass transition point (Tg), as discussed below.

Freeze-drying

Freeze-drying involves the removal of water or other solvent from a frozen product by a process called sublimation. Sublimation occurs when a frozen liquid goes directly to the gaseous state without passing through the liquid phase. Freeze-drying is a routine technique used in the art and suitable equipment is available from commercial sources. However, for completeness, a brief description of the freeze-drying process is set out below, some of which is based on information provided in "A Guide to Freeze- drying for the Laboratory"~an industry service publication by Labconco, 1998. See also Franks, F. (1994), Effective freeze-drying: a combination of physics, chemistry, engineering and economics. Proc. Inst. Refrigeration 91 : 32-39.

The freeze-drying process consists of three stages: pre-freezing, primary drying and secondary drying.

Pre-freezing: the material to be freeze-dried is adequately prefrozen. In traditional freeze-drying it is usual to pre-freeze the product below the glass transition point (Tg). As the temperature of the sample is lowered, the water in the sample freezes, increasing the concentration of solutes in the sample and increasing the viscosity of the mixture. As the viscosity increases steeply, the rate of freezing reduces, eventually stopping altogether. The residual mixture, including the ice, undergoes a glass transition, where the viscosity increases by many orders of magnitude over a narrow temperature interval. Structurally, the sample is then in the form of an amorphous solid and is described as a glass. This transition from a super-saturated, frozen aqueous solution of very high Viscosity to a brittle solid is operationally characterized by a glass transition temperature (Tg). The Tg for a sample can be determined by methods known in the art, such as differential scanning calorimetry or thermomechanical analysis.

However the Tg for samples of microorganisms in standard culture medium is often too low for practical purposes and must be raised by the addition of excipients. Suitable excipients include skimmed milk powder, glucose, sucrose, maltose, lactose, trehalose, raffinose, maltotriose, stachyose and dextran. Other suitable excipients are known in the art. Preferably the excipient is added in such an amount as to raise the Tg to about from -4O0C to -2O0C. The amounts may vary between types of sample but may readily be determined by the person skilled in the art. A suitable amount is typically from 5 to 15% w/v, such as from 7.5 to 12.5% w/v.

Primary drying: after pre-freezing the sample, conditions are established in which ice can be removed from the frozen product by sublimation. Samples are typically pre- frozen to well below their Tg and then the temperature is raised to just below the Tg. The samples are then subjected to reduced pressure in a vacuum chamber. At this point the freeze-drying process begins.

Secondary drying: after primary freezing is completed, all the frozen water has sublimed. However, non-frozen bound moisture is still present in the product. Continued drying at a higher temperature is often required to drive off the residual moisture by evaporation.

Several types of freeze-drying methods are currently in use including the manifold method, the batch method and the bulk method. It is preferred according to the present invention to use an apparatus suited to the batch method, such as a tray dryer.

Samples of microorganisms may be grown in the container prior to freeze-drying or grown in a separate vessel and dispensed into containers.

If a suitable excipient has not yet been added then it is added to the material prior to freezing. Once the excipient has been added, if not already added, the container may be frozen at a temperature below the Tg (e.g. in a -8O0C freezer or by a cryogenic liquid such as liquid nitrogen) and maintained at that temperature until it is freeze-dried, if it cannot be freeze-dried immediately. The container is then placed in the freeze-drying apparatus and freeze-dried according to the present invention. Once freeze-drying is complete, the container is typically sealed. The container may then be kept at room temperature, or below room temperature, as required, such that the viability of the microorganisms is maintained.

In general, cryoprotectants such as DMSO and glycerol, which are often used to protect the cells from damage during the freezing process, are not required in the method of the invention, but may be added if necessary. Preferably cryoprotectants are included in the sample medium, more preferably glycerol is included in the sample medium, prior to freezing. In a particularly preferred embodiment, at least 0.1 % glycerol v/v is included, preferably at least 0.2%. Typically, not more than 10% glycerol is added, preferably not more than 5 or 1% glycerol.

Conventionally employed freeze-drying apparatus have a construction in which a plurality of heating shelves in a form of a multistage unit are positioned in a sealable vessel provided with an exhaust manifold. The frozen material to be freeze-dried is placed on each shelf and the shelves holding the frozen material are placed in the vessel. The vessel is closed, sealed and evacuated to generate a vacuum. The shelves are then heated to effect vacuum drying of the frozen material on the shelves, while the gases and moisture given off under sublimation from the frozen material are removed. By such a conventional freeze-drying apparatus, a large scale processing of materials can be difficult due to restrictions in the amount and the size of the material to be freeze-dried. It can often take several days to ensure complete freeze drying of the samples therefore increasing the likelihood of inconsistency between the products. A further problem is that irregular heat transfer on heating the material to be freeze-dried may occur due to confinement of the material within a narrow interstitial spaces between the shelves, resulting in potential denaturation or inactivation of the material caused by local heat accumulation in different parts of the freeze-dryer.

Figure 1 shows vacuum pressure and temperature profile of a preferred freeze drying cycle according to the present invention.

EXAMPLE 1

This example describes a process for freeze drying droplets containing Escherichia coli cells. Culture conditions

E. co// ATCC 11775 was grown in 2xYT media (tryptone-yeast extract rich medium) (Sun, 1991) supplemented with 250 mM HEPES buffer pH 7.2 and 20% w/v sucrose in 250 ml erlenmeyer flasks. A standard aliquot containing 105 cells was used to inoculate the media before incubating for 24 hrs in a 370C shaker at 300 rpm.

Preparation of droplets containing E. coli cells

Diluted culture was then mixed 1 :1 with filter sterilized sheep serum. A Pasteur pipette was used to dispense drops from this mixture into liquid nitrogen. The sample was then formed into frozen spherical pellets after freezing in liquid nitrogen for 20 seconds. The sample pellets were then transferred individually into labelled 2 ml freeze drying vials and partially capped with a freeze drying bung. The capped vials were held in liquid nitrogen. until loaded into the freeze dryer.

To obtain a control plate to represent the number of cells contained in a drop prior to freeze-drying, drops were spread plated onto nutrient agar plates for each sample. The process of pre-freezing and control plating for each sample tested was repeated at least three times. The control plates were then incubated at 370C overnight until growth was achieved.

Freeze drying

Two different freeze drying cycles were tested. The cycles differed in the temperature that the shelves were set during various stages of the drying cycle. The first cycle involved freeze drying slowly and maintaining the shelves at a temperature well below the collapse temperature. The second cycle involved starting the shelves at a highertemperature and rapid increasing the shelf temperature to 250C so that the product was freeze dried close to the collapse temperature. Details of the freeze drying cycles are given in Table 2. Table 2.

Figure imgf000016_0001

A Telstar Lyobeta freeze dryer (Telstar, Spain) was prepared by setting the shelf temperature to the start temperature and setting the condenser temperature to -6O0C.

The viais containing the frozen droplets were placed directly onto the shelves of the freeze dryer. Alternatively, the vials were loaded into a polystyrene test tube rack that had had the bottom cut off so that the holes passed completely through the rack. The polystyrene rack was held on a stainless steel tray. The tray and rack were placed onto a shelf within the freeze dryer. The tray was slid out from the rack leaving the vials in contact with the shelf.

The vacuum pump was started and the heater for the shelf was adjusted as detailed in Table 2. At the end of the cycle the vials were capped and the freeze dryer turned off. The vials were removed from the freeze dryer and analysed immediately. Vials were examined to determine if the freeze dried product had formed a uniform ball shape or if the product had melted.

Determination of cell viability

To rehydrate the freeze-dried sample pellets, the vials containing the sample were opened and the freeze-dried pellet was tipped onto a nutrient agar plate. Some 100 μl of sterile 0.9% saline was then dispensed over the pellet on the plate and a spreader was then used to dispense the sample. The plates were then incubated at 370C over night.

Control counts from samples plated prior to freeze-drying experiments were compared to counts obtained from samples re-hydrated after freeze-drying. From the data a mean with its corresponding standard deviation was obtained for every control and freeze-dried sample. Percentage survival was obtained from this data by dividing the mean of the freeze-dried sample by the mean of the control sample, then multiplying by one hundred.

RESULTS

Freeze drying cycle number one resulted in uniform and well shaped freeze dried balls, however, the recovery of viable E. coli was low (Table 3). Freeze dryer cycle number 2 resulted in a collapsed product in approximately half of the vials and a variably shaped product in all the vials. Many vials contained product that stuck to the side of the vial.

The recovery of viable E. coli was high for freeze drying cycle 2 (Table 3). The use of freeze drying cycle number 2 with the vials insulated by placing in polystyrene racks resulted in uniform and weil shaped freeze dried balls and high recovery of £. coli cells (Table 3).

Table 3.

Figure imgf000017_0001

In the present inventors' experience with bacteria, over 90% of the cells die during the freezing and freeze drying process that are normally employed to preserve bacteria. However, the present inventors have found that when biological material is freeze dried at a rapid rate so that the freeze dried material is close to its collapse temperature then less damage to the material occurs. In fact the present inventors have successfully freeze dried cells in this manner without causing death to any of the cells. This has been performed by freeze drying frozen droplets of fluid that contain bacteria. The frozen droplets are placed into vials and then freeze dried rapidly just below the collapse temperature of the droplets. Whilst the viability of the bacteria was maintained, the physical appearance of the freeze dried droplets varied between the vials depending on the position of the vial within the freeze dryer. Some of the droplets collapsed resulting in a freeze dried product with a poor appearance and which was often stuck to the bottom of the vial.

This effect was overcome by insulating each vial in a polystyrene foam rack that consisted of a polystyrene tray with holes, slightly larger than the viais. The holes pass all the way through the rack so that the vials are insulated from each other but the vials touch the heated shelf.

EXAMPLE 2

This example describes a process for freeze drying droplets containing a fluorescent monoclonal antibody.

Monoclonal antibody

A monoclonal antibody specific to the surface of Cryptosporidium oocysts was conjugated with fluorescein isothiocyanate (FITC) as previously described (Weir, C, Vesey, G, Slade, M, Ferrari, B, Veal, DA, and Williams, K. 2000. An immunoglobulin G1 monoclonal antibody highly specific to the wall of Cryptosporidium oocysts. Clinical & Diagnostic Laboratory Immunology. 7(5):745-750). The conjugated antibody was diluted in sterile sheep serum to a concentration of 20 μg per ml.

Freezing and freeze drying A Pastuer pipette was used to dispense drops of the antibody solution into liquid nitrogen. The frozen spherical pellets were then transferred individually into labelled 2 ml freeze drying vials and partially capped with a freeze drying bung. The capped vials were held in liquid nitrogen until loaded into the freeze dryer.

Additional samples were prepared by pipetting 750 μl of antibody solution directly into freeze drying vials and then freezing the samples by placing the vials into liquid nitrogen. A frozen cake of antibody was obtained. The two different freeze drying cycles described in Example 1 were used. Vials containing frozen spheres were freeze dried using cycle number 2. Vials containing 750 μl of frozen antibody were freeze dried using cycle 1.

After completion of the freeze drying cycle the vials were removed from the freeze dryer, crimped and stored at -2O0C for 8 weeks prior to analysis.

Flow cytometric analysis of antibodies

The vials of antibody were reconstituted by adding a volume of water equivalent to the volume of antibody prior to freeze drying. The vials were then vortexed for 2 minutes.

A 20 μl aliquot of antibody was mixed in a flow cytometry test tube with 200 μl of saline that contained approximately 50,000 Cryptosporidium oocysts. The samples were vortexed for 10 seconds and then incubated at room temperature for 30 minutes.

The samples were then analysed using a FACScalibur flow cytometer (Becton Dickinson, CA, USA) and the intensity of the green fluorescence (525 nm) of 2000 oocysts was recorded.

RESULTS

The fluorescence intensities of oocysts stained with the freeze dried fluorescent antibodies are presented in Table 4.

Table 4

Figure imgf000019_0001

The antibody that was freeze dried as a frozen sphere using the rapid freeze drying cycle produced significantly brighter oocysts than the antibody freeze dried as a cake using the slower freeze drying cycle. Freeze drying sensitive biological material in this manner resulted in preservation of the material without damage and results in a freeze dried material that is not stuck to the vial and has an acceptable visual appearance.

EXAMPLE 3

This example describes the difference when glycerol is used in protectant for freeze drying E. coli cells.

Culture Conditions The same culture conditions as described in Example 1 above were used.

Preparation of E. coli cells for freeze drying

An aliquot of 1 μl of E. coli cell culture was diluted into 1 ml PBS. This cell dilution was loaded onto a FACScalibur flow cytometer (Becton Dickinson). The cells were sorted by. the cytometer and released as a single droplet from the outlet nozzle. An equal flow rate of a lyoprotectant was pumped into the nozzle, so that the resulting droplet consisted of 50% cytometer fluids (PBS) and sorted cells and 50% lyoprotectant.

Two lyoprotectants were tested: A. Lyoprotectant + 4% glycerol; B. Lyoprotectant

Freeze drying

The droplets dispensed by the cytometer were collected in cups of liquid nitrogen and allowed to freeze. Once frozen, the droplets were placed into vials and loaded onto the freeze dryer, with the shelves chilled to -300C.

The freeze drying cycle, ramped the shelf temperature from -300C to 25°C in 45 minutes, and then remained at 25°C for a further 2.25 hours, until the chamber pressure level reached 0.06 mbar. Table 5

Figure imgf000021_0001

There was no significant difference observed between the two different lyoprotectants immediately after freeze drying. However, there was a significant difference between the E. coli cell recoveries after a 1 week period of storage at 22°C. The addition of glycerol to the lyoprotectant enables E. coli to survive for longer periods of time at elevated temperatures.

EXAMPLE 4 E. coli 0157

Figure 2 shows results of the lowest freeze dried count of 5 replicates produced after E. coli 0157 BioBall™ had been stored for 1 week at 22°C and then plated onto non-selective agar. The limit pass level for these batches was all 5 counts to be above 24 cfu. The light shaded bars show batches that were freeze dried using a slow cycle and failed to meet the pass specification of 24 cfu whereas, the black shaded bars are batches that used a rapid freeze drying cycle and passed the limit of 24 cfu.

The rapid freeze drying cycle had a shelf temperature ramp to 25°C of 20 minutes, whereas the slow freeze drying cycle had a shelf temperature ramp to 25° C of 45 minutes. The shelf temperature ramp rate was the controlling force behind the speed of the drying process. Both the mentioned rates were significantly faster than traditional freeze drying methodologies. However, the 25 minute increase in rate resulted in differences between a freeze dried E. coli 0157 being stable at 22°C for 1 week and producing significantly lower counts after 1 week storage. Table 6

Figure imgf000022_0001

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

Claims:
1. A process for rapid freeze drying a sample comprising: freezing the sample; and freeze drying the sample under temperature and vacuum conditions such that the sample is dried close to the collapse temperature of the sample.
2. The process of claim 1 wherein close to the collapse temperature is about 0.10C to 1O0C below the collapse temperature of the sample.
3. The process of claim 1 or 2 wherein the sample is in a plurality of containers.
4. The process of claim 3 wherein the plurality of containers are arranged in a freeze dryer such that the containers are isolated from each other.
5. The process of claim 4 wherein the plurality of containers are isolated from each other in a rack having holes adapted to receive the containers.
6. The process of claim 5 wherein the rack is made of an insulating material.
7. The process of claim 6 wherein the insulating material is polystyrene.
8. The process of any one of claims 3 to 7 wherein the sample is heated such that each container is exposed to substantially an equivalent temperature at any given point during the freeze drying process.
9. The process of any one of claims 1 to 8 wherein the freeze drying is started at a temperature of about -4O0C and finished at a temperature of about 250C.
10. The process of claim 10 wherein the sample is heated at a rate of about 10C per minute.
1 1. The process of any one of claims 1 to 10 further comprising adding a penetrating cryoprotectant to the sample.
12. The process of any one of claims 1 to 11 wherein the penetrating cryoprotectant is selected from the group consisting of glycerol, 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, dimethyl sulfoxide (DMSO), methanol, 1 ,2-propanediol, proline, and ethylene glycol.
13. The process of claim 12 wherein the cryoprotectant is glycerol.
14. The process of any one of claims 11 to 13 wherein at least about 0.1% (v/v) cryoprotectant is added.
15. The process of claim 14 wherein at least about 0.2% (v/v) cryoprotectant is added.
16. The process of claim 15 wherein not more than about 10% cryoprotectant is added.
17. The process of claim 16 wherein not more than about 5% cryoprotectant is added.
18. The process of claim 17 wherein not more than about 1% cryoprotectant is added.
19. The process of any one of claims 1 to 18 further comprising adding bulking agents, cryoprotectants, lyoprotectants, surfactants, buffering agents, buffers or excipients to the sample.
20. The process of any one of claims 1 to 19 wherein the sample is a compound, composition, or biological material.
21. The process of claim 20 wherein the compound or composition is a pharmaceutical agent, an antibody, an enzyme, a protein, a peptide or a nucleic acid.
22. The process of claim 20 wherein the biological material is a microorganism, cell, cell component or a virus.
23. A freeze-dried product produced by the process of any one of claims 1 to 22.
24. The product according to claim 23 having shrivelled or shrunken appearance.
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WO2015148444A1 (en) * 2014-03-24 2015-10-01 Biogen Ma Inc. Lyophilized factor ix formulations

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