RAPID FREEZE-DRYING METHOD

Abstract

 A method is described which allows the rapid and reproducible freeze-drying of compositions containing a liquid phase, in particular pharmaceutical compositions and wherein the liquid phase is water.

Description

FIELD OF THE INVENTION

[0001] The present invention refers to a method for obtaining rapid and uniform freeze-drying of solutions or suspensions, with particular regard to freeze-drying of pharmaceutical products.

BACKGROUND

[0002] Freeze-drying is a dehydration process that allows the removal by sublimation of a liquid phase, typically water, from a substance, such as a foodstuff, a pharmaceutical or biological product. The process is widely used in the industry because the removal of the solvent takes place at low temperatures, typically below 0 °C, thus avoiding the risk of thermal alteration of the present organic compounds that would occur with a classical evaporation.

[0003] Freeze-drying can be used in different sectors and different fields of application: from food to pharmaceuticals to diagnostics up to taxidermy, or to treat flowers, documents damaged by exposure to water (e.g. books or documents after floods), heat-sensitive chemicals and museum artefacts and/or archaeological finds.

[0004] This technique can therefore be applied to different products such as coffee, milk or fruit, up to meat; from the pharmaceuticals such as small molecules to biological products, more or less complex such as peptides, proteins and their derivatives (e.g. antibody-drug conjugates, known as ADCs). Other products that can be freeze-dried are cells, tissues, tissue replacement implants; so-called "scaffolds" up to gene therapy products (viral vectors and/or cells). Sugars and polysaccharides are also lyophilisable products that are commonly used, for various reasons.

[0005] In the case of the pharmaceutical and food sector, the finished product is also subject to specific aesthetic requirements.

[0006] Freeze-drying is used to preserve various perishable products as the resulting greatly reduced water content inhibits chemical degradation and/or the action of microorganisms and enzymes that would degrade the product. In addition, the process makes it more convenient to transport the product thanks to the reduction in weight and volume of the same. The freeze-dried products can be easily rehydrated or reconstituted at the time of use by adding water and/or solvents.

[0007] Given the importance of freeze-drying in the pharmaceutical industry, particular reference is made in the remainder of the description to applications in this field, but the invention has a more general character and can also be employed in other industrial sectors where freeze-drying is required.

[0008] Similarly, in the remainder of the description reference is made to water as solvent to be removed, but the invention also finds application with different solvents (by way of example, tert-butanol).

[0009] The freeze-drying process comprises three main steps:

• freezing the product to bring the water to be removed in solid phase;
• first step of removal of water at subatmospheric pressure and low temperature;
• second step of completion of the removal of the water at subatmospheric pressure and temperature higher than that of the previous step.

[0010] The process is carried out in systems known as freeze-dryers, also referred to as lyophilizers, consisting of pressure-tight chambers provided with cooling systems and connected to vacuum pumps. Typically, there are several shelves inside the freeze-dryers, commonly referred to as plates, on which the products to be freeze-dried are housed during the process. The size of a freeze-dryer and of the relative chamber is generally expressed in square metres relative to the total surface area given by the sum of the loading surface of the plates. Small laboratory and/or pilot machines have surfaces comprised between 0.1 and 1 square metre; the industrial machines generally have sizes from 2 square metres up to 50 square metres.

[0011] In the pharmaceutical industry, the products to be freeze-dried are typically contained in trays, bags, bottles or vials which are open during the process; in the rest of the description and in the claims, the term "container" will be used to refer to all these forms. Cooling takes place through control of the temperature of the shelves (also called plates) of the freeze-dryer, which can be made for example with cooling liquids.

[0012] During the initial cooling step, the system is brought to a temperature lower, even by 20-40 °C, and in some cases by 50 °C, than the thermodynamic one of water solidification, whereby an undercooled metastable liquid phase is obtained.

[0013] The freezing step is the most critical one for obtaining a homogeneous product, both within the single container and between the different containers.

[0014] In fact, the solidification of the liquid phase begins with the nucleation process, in which the first crystalline solid nuclei are formed inside the liquid from which, by growth, the complete solidification of the liquid is caused. Nucleation is a stochastic process that can be affected by minor variations in surrounding conditions, such as inhomogeneities of composition within the containers and especially of temperature in the freeze-drying chamber (e.g. inhomogeneity of temperature on the same shelf or plate and therefore in the same containers placed on the same plate). This leads to significant differences, even up to 10 °C, in the temperatures at which water freezes in the different containers, and also in the sizes of the ice crystals formed, with consequent inhomogeneities in the subsequent steps of the process (rate of water removal) and ultimately in the physical properties of the freeze-dried product, which can influence for example the rehydration characteristics. In addition, to adjust these differences in freezing temperatures, both the freezing step of the freeze-drying process and the sublimation step of ice crystals of very different sizes generally require very long times.

[0015] To overcome these problems, various methods have been studied in order to make the characteristics and the results of the freeze-drying process less variable.

[0016] A first method consists in adding additives (e.g. silver iodide, AgI) which act as nucleation centre for the ice crystals; the presence of these additives is however generally not desirable within pharmaceutical compositions.

[0017] One type of additive that does not pose problems is the ice crystallites used in the so-called "ice mist" method, in which a humid atmosphere is introduced into the freeze-drying chamber which is then cooled producing a suspension of small ice particles that, in contact with the product to be freeze-dried, act as crystallization nuclei. However, this method too is affected by the temperature inhomogeneities within the chamber, which lead to different sizes of the ice particles or different timings of formation thereof at different points in the chamber.

[0018] Other proposed methods are based on the physical destabilization of the undercooled metastable liquid phase. These methods may consist in subjecting the solutions to vibrations in the range 10-20 kHz or to frequencies in the field of the ultrasounds; or to relatively strong electric fields. These systems, however, require substantial modifications of the freeze-drying chambers with increased process costs; moreover, in the case of the use of electric fields, the method cannot be applied on conductive solutions (for example, isotonic solutions generally containing NaCl).

[0019] Finally, a further method proposed is that described in patent EP 1982133 B1. In the method of this document, the material to be freeze-dried is brought to a temperature lower, by a value comprised between 3 and 20 °C, than the thermodynamic one of solidification of the liquid phase, maintaining in the chamber a pressure higher than the atmospheric one: see in this regard paragraph [0043] of the patent text and the examples, in which in all cases it is started from an initial pressure of 197.8 kPa (1.978 bar), with the need to have to modify the machine having to operate under overpressure. Subsequently, when the interior of the chamber is balanced to the desired values of temperature and pressure, the pressure is rapidly decreased (over a few tens of seconds) to a value lower than the initial one, which can be lower or in turn higher than the atmospheric pressure (paragraph [0043]). Rapid depressurization leads to a localized decrease in the temperature within the liquid phase, which can be estimated by a few degrees at most, followed by a rapid rise in the same temperature (see figs. 2 and 3 of the patent); the effect obtained is the induction of nucleation almost simultaneously in all containers containing the liquid phase. While presenting some advantages over the previous ones, the method of this document requires pipes, valves, modifications to the software and regulation systems (e.g. pressure regulators and modulating valves) built specifically to be able to generate and then withstand the initial overpressure, like the freeze-dryer described in international patent application WO 2010/117508 A2 in the name of the same owner of EP 1982133 B1; the method is therefore not suitable for use in normal commercial freeze-drying chambers.

[0020] The need to have available a freeze-drying method that allows to overcome the disadvantages of known systems is therefore still felt in the field.

[0021] An object of the present invention is to provide an improved method for freeze-drying compositions comprising a liquid phase, and which allows in particular to obtain nucleation of the solid phase from said liquid phase rapidly and uniformly, both within the single container of composition to be freeze-dried, and on all the containers present in the chamber, without modifications to the equipment itself.

SUMMARY OF THE INVENTION

[0022] This and other objects are achieved with the present invention by a method comprising the steps of:

a) arranging one or more containers containing the composition to be freeze-dried on one or more temperature-controlled plates in a freeze-drying chamber;

b) by adjusting the temperature of the plates, bringing the temperature of the composition to a value comprised between 0 °C and -20 °C;

c) reducing, over a time comprised between 5 min and 60 minutes, the pressure in the chamber to a value comprised between 5.0 mbar and 0.01 mbar, and leaving the system under these conditions for a period comprised between 12 hours and 120 hours, causing the temperature of the product being freeze-dried to decrease below that of the plates;

d) by keeping the pressure at a value comprised between 5.0 mbar and 0.01 mbar, keeping the plates at a temperature equal to that set in point b) until the temperature of the product being freeze-dried reaches a temperature equal to or higher than that of the plates;

e) when the temperature of the product being freeze-dried has equalled the temperature of the plates, raising the temperature of the plates to a temperature comprised between +10 °C and +40 °C;

characterised in that during step b) the atmospheric pressure is maintained inside the chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 

• Fig. 1 is a schematic reproduction of the positioning of the temperature probe outside a container during experimental tests;
• Fig. 2 graphically represents the trend of the temperature and of the pressure in the chamber and of the temperature of a solution of mannitol in water during a test according to the invention;
• Fig. 3 shows a magnification of Fig. 2;
• Fig. 4 graphically represents the trend of the temperature and of the pressure in the chamber and of the temperature of a solution of trehalose in water during a test according to the invention;
• Fig. 5 shows a magnification of Fig. 4;
• Fig. 6 graphically represents the trend of the temperature and of the pressure in the chamber and of the temperature of a solution of mannitol in water during a test according to the invention, with the temperature probe immersed in the sample;
• Fig. 7 shows a magnification of Fig. 6;
• Fig. 8 graphically represents the trend of the temperature and of the pressure in the chamber and of the temperature of a solution of albumin in water during a test according to the invention
• Fig. 9 shows a magnification of Fig. 8;
• Fig. 10 graphically represents the trend of the temperature and of the pressure in the chamber and of the temperature of a solution of egg white in water during a test according to the invention; and
• Fig. 11 shows a magnification of Fig. 10.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The following definitions and abbreviations are used in the description and examples that follow:

• the term "solvent" means a liquid component selected from water, ethanol, tert-butanol, dimethyl sulfoxide, isopropanol, hexafluoroacetone, tetrachloromethane, 1,1,1-trichloro-2-methyl-2-propanol (also known as chlorobutanol), acetic acid, cyclohexane, acetonitrile and combinations thereof;
• PVP K12: Polyvinyl pyrrolidone K12
• BSA (Bovine Serum Albumin): Bovine serum albumin.

[0025] The method of the present invention is analogous to that described in patent EP 1982133 B1, with the difference that the method of this prior art document necessarily required that at the beginning of the process the freeze-drying chamber be under overpressure, while in the method of the invention at the beginning of the procedure the chamber is at ambient pressure (about 1 bar) and without any modifications to the equipment. Although the phenomenon has not been studied extensively and the reasons for it have not been clarified, this initial pressure difference has the consequence that the material to be freeze-dried undergoes a treatment with a completely different thermal profile: in the method of EP 1982133 B1, as described above, following the lowering of the pressure the temperature undergoes a lowering by a few degrees or fractions of degree and subsequently, within less than a minute, a "bounce" with an increase by about 5 °C; in the method of the present invention, instead, in conjunction with the depressurization the temperature inside the sample undergoes a sudden decrease up to about 25 °C, and in some cases up to 30 °C, while keeping the temperature of the plates constant.

[0026] In step a) of the method of the invention, the composition to be freeze-dried is arranged in the freeze-drying chamber (freeze-dryer) inside one or more containers (e.g. vials) positioned on plates at controlled temperature; the containers are positioned on the plates side by side in such a way as to cover all the available surface for each plate. In the case of rigid containers (e.g. vials) these can be placed in direct contact with the plate or positioned on steel trays in turn in contact with the plate; the bags are instead placed in dedicated supports in contact with the plates.

[0027] As is well known in the sector, the containers are inserted pre-capped into the freeze-dryer, that is, with a closure element already provided and partially inserted into the opening of the container, but in such a way as to leave a way of communication between the inside and the outside of the container to allow the removal of the solvent; at the end of the process, before extracting the containers, they are sealed by completely inserting the closure element into the opening of the container.

[0028] The insertion of the cap takes place through the movement of the plates that slide on support guides and push the cap into the vial bringing it into the closed position.

[0029] The trays can be open, and are generally made of metal or plastic; the plastic trays can be covered with a breathable membrane, like in the case of Lyoguard^® trays produced by the company W. L. Gore & Associates (Gore^®).

[0030] The open trays (directly containing the solution or pre-filled vials) can be placed in breathable bags (e.g. Lyoprotect^®) that exploit the same technology as Gore^® systems.

[0031] The vials can be made of both blown glass and from a pipe or plastic material.

[0032] The bags may be of different flexible plastic material, with different design and various possibilities of hooks for tubings, and may have one or more compartments; bags suitable for the purposes of the present invention are described for example in patent application WO 2010/019217 A1 or in patent EP 3412272 B1. The plastic materials used for the bags may be breathable, consisting of a permeable membrane (as described in US patent 10377520 B2).

[0033] In step b), through the control of the temperature of the plates, the temperature of the composition is brought to a value comprised between 0 °C and -20 °C, preferably between - 5 °C and -15 °C.

[0034] The time required for the product to reach these temperatures varies depending on the load, the type of container and the composition of the solution.

[0035] Having the temperature of the product at temperatures between 0 °C and -2 °C, especially for solutions at concentrations greater than or equal to 5% w/w, leads to obtaining foaming phenomena that are useful especially when the appearance of the product is not the predominant condition in the choice of the process parameters (e.g. freeze-dried coffee or milk), and in particular in the processes especially in a tray (bulk).

[0036] The cooling of the plates generally takes place through liquids such as silicone oils, cooled by refrigeration units (or heated through electrical resistors in subsequent steps of the method). With the use of refrigerated silicone oils, it is possible to reach minimum temperatures of the plates of up to -50 °C. In some freeze-dryers it is possible to use liquid nitrogen, which allows reaching particularly low temperatures of the plates, up to -70 °C.

[0037] When the desired temperature in the range indicated in point b) has been reached, step c) is carried out in which, in a time comprised between 5 min and 60 min and preferably between 10 and 40 min, the pressure in the chamber of the freeze-dryer is brought to a value comprised between 5 mbar and 0.01 mbar, more preferably between 1 mbar and 0.01 mbar, and the system is left under these conditions for a period comprised between 12 hours and 120 hours, preferably between 12 hours and 40 hours. In this step the temperature of the plates is not changed, and is kept the same as the one set in step b).

[0038] The execution of step c), by operating under the conditions of the invention and in particular starting in step b) from the ambient pressure (about 1 bar), causes the rapid decrease of the temperature of the composition in the containers to a value of about 10-30 °C lower than that of the plates.

[0039] In step d) the temperature of the plates is still kept constant at the value set in step b) and the pressure at the value of step c), and the conditions of the system are allowed to evolve spontaneously until the temperature of the composition or product being freeze-dried reaches a temperature equal to or higher than that of the plates. This stage is known in the industry as "primary drying".

[0040] The moment at which the temperature of the composition equals the set temperature of the plates is an indication of the end of the sublimation of the solvent.

[0041] Finally, once the product has reached the temperature of the plates, it is possible to proceed with step e), which consists in raising the temperature of the plates themselves to further reduce the solvent content while keeping the pressure in the range indicated above for step c); this stage is known in the sector as "secondary drying". At this stage the working temperature of the plates is generally comprised between +10 °C and +40 °C.

[0042] The present invention allows to manage temperature decreases on products both in large volumes as is the case of products in bulk or in large vials (vials with volume greater than 100 mL, containing for example plasma, human or not, or albumin, human or not) and products where there is a limited heat exchange like in the case of products contained in bags, which are housed on supports in turn placed on the plates. In this case the use of supports strongly limits the heat exchange with the product and therefore a temperature lowering induced by the pressure lowering is particularly advantageous in terms of process economy.

[0043] The process described has proven effective even with excipients traditionally difficult to manage eliminating the annealing step: after a year of evaluation of the samples, no breakages of the vials can be highlighted, using mannitol as an emblematic excipient.

[0044] With the method of the present invention it is possible to treat active pharmaceutical ingredients, freeze-dried as such in a tray (or "bulk") or formulated as finished products. The following products as such and/or salts and derivatives thereof are mentioned: Abatacept, Aciclovir, Clavulanic acid, Hyaluronic acid, Albumin (human, ovine, porcine) Allopurinol, Alprazolam, Alprostadil, Amphetamine, Hydroxyethyl starch (HES), Amifostine, Amikacin, Amoxocillin, Annamycin, Antitrypsin, Aplidin, Aripiprazole Azacitidine, Bacitracin, Belimumab, Bendamustine, Bleomycin, Bortezomib, Brentuximab, Busulfan, Coffee, Carbidopa, Carboplatin, Casein, Carfilzomib, Carmustine, Caspofungin, Cefacetril, 0020Cefadroxil, Cephalexin, Cefazaflur, Cephalotin, Cefazolin, Cefepime, Ceftaroline, Ceftobiprole, Ceftriaxone, Cetrorelix, Cyclophosphamide, Cilastatin, Cilastatine, Cisapride, cis-platinum, Cytarabine, Citalopram, Cladribine, Clonazepam, Clozapine, Cloxacillin, Cholestyramine, Copanlisib, Daptomycin, Daunorubicin, Decitabine, Degarelix, Desloratadine, Dexrazosan, Diclofenac, Donepezil, Doxorubicin, Ebastine, Elastin, Haemoglobin (equine, swine, human), Epicillin, Epirubicin, Erythromycin, Esmolol, Esomeprazole, Insulin-like growth factor type I (IGF-1), Insulin-like growth factor type II (IGF-2), Coagulation factor VII, Coagulation factor VIII, Coagulation factor IX, Phenytoin, Phentermine, Phenobarbital, Phentolamine, Fingolimod lauryl sulfate, Flucloxacillin, Fludarabine, Fosaprepitant Dimeglumine, Fotemustine, Gabexate, Gallium Ga-68, Ganciclovir, Gemcitabine, Gentamicin, Gozetotide, Glycopyrronium (or glycopyrrolate) Glucagon, Glutathione, Ibuprofen, Ifosfamide, Hygromycin B, Imipenem, Indomethacin, Infliximab, Trypsin inhibitor (pancreatic) (from bovine), Human Insulin (recombinant), Insulin glargine, Insulin lispro, Insulin aspart, Insulin detemir, Irinotecan, Lamotrigine, Lansoprazole, Leptin, Leuprolide, Levodopa, Levoleucovorin, Levothyroxine, Loratadine, Melphalan, Meropenem, Methadone, Methicillin, Methylpheniudate, Micafungin, Mifamurtide, Myoglobin (equine, swine, human), Mirtanzapine, Mitomycin, Motixafortide, Sodium nitroprusside, Nesiritide, Omeprazole, Omacetaxins, Olanzapine, Ondasetron, Oritavancin, Oxacillin, Oxytocin, Paclitaxel, Palonosetron, Pantoprazole, Paracetamol, Pemetrexed, Penicillin F, Penicillin G, Penicillin X, Penicillin K, Penicillin V, Penicillin O, Penicillin N, c-Myc Tag peptide, Pyraubidine, Pivampicillin, Pixantrone, Plasma (human, sheep, swine, bovine, equine), Plasminogen, Polysaccharose 400 (Ficoll^® 400), Prednisolone, Progesterone, Prostaglandin E1, Rifampicin, Rimegepant, Remdesivir, Remimazolam, Risperidone, Rizatriptan, Romidepsin, Secretin, Somatorelin, Selegiline, Selexipag, Syncalide, Sinvastatin, Sirolimus, Sulbactam, Tasonermin, Technetium Tc-99m, Teduglutide, Telavancin, Temozolomide, Tigecycline, Thiotepa, Tobramycin, Botulinum toxin type A of Clostridium botulinum (onabotulinumtoxin A), Trabectedin, Trastuzumab, Trilaciclib, Vaborbactam, Vancomycin, Vancuronium, Vardanafil, Verteporfin, Vinblastine, Vincristine, Voriconazole, Vosoritide and Ziprasidone, Zolmitriptan, their salts and derivatives.

[0045] The invention will be further described by means of the following examples.

[0046] The following instruments and materials were used in the experimental tests:

• the residual humidity measurements at the end of the tests of the examples were performed with Karl Fischer method with Metrohm instrument, 684KF coulometer.
• infusion bags (30 ml bags obtained from double chamber bags HAEMOFARM^™, 500 ml bags HAEMOFARM ^™), trays (e.g. GORE^® LYOGUARD ^® Freeze drying trays LGT2000 3/4" - 19.0 mm) and vials of various sizes (20 and 30 R-sized vials Nuova Ompi/Stevanato; "500 ml infusion" vials from Bormioli code HP1001/500), 20 mm bromobutyl caps code WEST S-87-J 4416/50 for 20 and 30 R vials from West Pharmaceuticals and "32 mm" freeze-drying caps for 500 ml vials Datwyler code V9262);
• Mannitol PEARLITOL^® PF, Roquette Italia S.p.A. Cassano Spinola (AL);
• Sucrose "white sugar", Eridania, Bologna;
• Trehalose dihydrate, purity ≥ 98%, Biospectra, Pennsylvania, USA;
• Galactose, purity ≥ 98%, Biospectra, Pennsylvania, USA;
• PVP K12 Kollidon^®, BASF, Ludwigshafen (Germany);
• Bovine serum albumin (BSA), Sigma Aldrich, purity ≥ 98%;
• Soluble dehydrated coffee, freeze-dried Carrefour/Interdis brand;
• Pasteurized liquid egg white "Le naturelle", Eurovo srl, Santa Maria In Fabriago (RA).

[0047] In the first tests the vials are in direct contact with the plates. They are then loaded onto trays and are placed on the plates.

[0048] All are pre-capped with freeze-drying caps.

[0049] The effect of simultaneous nucleation is most marked (fast) when the product is placed on the plates without a tray.

EXAMPLE 1

[0050] 20R vials (20 mL capacity) are filled with 5 millilitres of 2% w/w mannitol solution in water.

[0051] The vials containing the solution are subsequently loaded onto the tray and pre-capped.

[0052] The tray on which 120 vials prepared as described above are present is positioned on one of the three plates of a 0.45 m² pilot freeze-dryer.

[0053] Three vials are each externally probed with a temperature probe (heat resistance) in order to monitor the temperature trend of the product during the execution of the cycle; the fixing of the temperature probe outside the vials is shown in Fig. 1, showing the probe, 10, fixed to the vial, 11, by adhesive tape.

[0054] Fig. 2 represents the trend, during the test, of the temperature of the tray (continuous curve), of the temperature of the samples (dotted curve) and of the pressure in the chamber (dashed curve). The temperature values are read on the vertical scale to the left of the figure, while the pressure values are read on the vertical scale to the right of the figure. As can be seen in the figure, the temperature of the plate, in step b) of the method of the invention, is set at -10 °C; when the temperature of the sample reaches a value of about -5 °C the pressure is brought rapidly to a value of about 0.1 mbar (step c)); in conjunction with the pressure decrease, the temperature of the sample undergoes a very rapid decrease (less than one hour) of about 25 °C. Fig. 3 shows a magnification relating to the first 11 hours (approx.) of the test of Fig. 2, in which the temperature trend of the samples is better highlighted (dotted curve).

[0055] The maximum verified time to freeze all vials in this example is 58 seconds.

[0056] The primary drying step takes approximately 21 hours, while the secondary drying takes approximately an additional 8 hours. The whole process takes less than a day and a half, against the durations of even 5-6 days of known processes.

[0057] The product is thus ready to be reconstituted with the quantities of liquid suitable to obtain the desired final concentration of the solution.

[0058] The product characterization results obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Compact and homogeneous lyophile. Perfectly adhering to the vial
Humidity (average ± std dev)	0.63 ± 0.02% w/w
Number of Broken Vials	0/120

[0059] The standard deviation (std dev) expressed is by sample with three samples tested by Karl Fisher method.

[0060] No breakage of vials is reported after 14 months, storing them at room temperature.

EXAMPLE 2

[0061] 20R vials are filled with 5 millilitres of 2% w/w mannitol solution in water.

[0062] The vials containing the solution are subsequently loaded onto a tray and pre-capped.

[0063] This example is done on industrial freeze-dryers (4 m²) where six trays containing 120 vials each are thus positioned on two of the eight plates of the freeze-dryers; three trays for each shelf.

[0064] Three vials are probed by immersing the probe in the solution each with a temperature probe (heat resistance) in order to monitor the temperature trend of the product during the execution of the cycle.

[0065] In conjunction with the pressure decrease, the temperature of the sample undergoes a very rapid decrease (less than one hour) by about 25 °C.

[0066] The maximum time required to freeze all vials (on a shelving, visible from the freeze-dryer port) in this example is 90 seconds.

[0067] The product characterization results obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Compact and homogeneous lyophile. Perfectly adhering to the vial
Humidity (average ± std dev)	0.74 ± 0.04% w/w
Number of Broken Vials	0/720

[0068] The standard deviation expressed is by sample with three samples tested by Karl Fisher method.

[0069] It should be noted that with a clearly problematic excipient there are no breakages of any vial, even after 6 months of storage at room temperature.

EXAMPLE 3

[0070] 20R vials are filled with 5 millilitres of 3% w/w sucrose solution in water.

[0071] The vials containing the solution are subsequently loaded onto a tray and pre-capped.

[0072] This example is done on a pilot freeze-dryer (0.45 m²) where a tray containing 120 vials is positioned on one of the three plates of the freeze-dryer.

[0073] Three vials are probed internally each with a temperature probe (heat resistance) in order to monitor the temperature trend of the product during the execution of the cycle.

[0074] The product characterization results (N = 3) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Compact and homogeneous lyophile. Slightly rough surface
Humidity (average ± std dev)	2.70 ± 0.08% w/w
Number of Broken Vials	0/120

EXAMPLE 4

[0075] 30R vials are filled with 5 millilitres of 2% w/w PVP K12 solution in water.

[0076] The vials containing the solution are subsequently loaded onto a tray and pre-capped.

[0077] This example is done on a pilot freeze-dryer (0.45 m²) where a tray containing 120 vials is positioned on one of the three plates of the freeze-dryer.

[0078] Three vials are probed internally each with a temperature probe (heat resistance) in order to monitor the temperature trend of the product during the execution of the cycle.

[0079] The product characterization results (N = 3) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Compact and uniform lyophile
Humidity (average ± std dev)	2.35 ± 0.03% w/w
Number of Broken Vials	0/120

EXAMPLE 5

[0080] 30R vials are filled with 5 millilitres of 2.2% w/w trehalose dihydrate solution in water.

[0081] The vials containing the solution are subsequently loaded onto a tray and pre-capped.

[0082] Three vials are probed internally each with a temperature probe (heat resistance immersed in the solution to be freeze-dried) in order to monitor the temperature trend of the product during the execution of the cycle.

[0083] The product characterization results (N = 3) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Compact and uniform lyophile
Humidity (average ± std dev)	3.23 ± 0.09% w/w
Number of Broken Vials	0/120

[0084] This example was carried out in duplicate. The same identical load and cycle with the same compound, led in the first case to a literally simultaneous freezing in all vials (less than 2 seconds).

[0085] In the replicate the effect was obtained in 150 seconds, obtaining similar results in terms of appearance of the lyophile and in the number of intact vials (0/120).

[0086] Fig. 4 represents the trend, during the test, of the temperature of the tray (continuous curve), of the temperature of the samples (dotted curve) and of the pressure in the chamber (dashed curve). As can be seen in the figure, the temperature of the plate, in step b) of the method of the invention, is set at -15 °C; when the temperature of the sample reaches a value of about -10 °C, the pressure in the chamber is brought rapidly to a value of about 0.1 mbar (step c)); in conjunction with the pressure decrease, the temperature of the sample undergoes a very rapid decrease (less than one hour) of about 25 °C. Fig. 5 shows a magnification relating to the first 19 hours of the test of Fig. 4, in which the temperature trend of the samples is better highlighted (dotted curve).

EXAMPLE 6

[0087] 20R vials are filled with 5 millilitres of 2% w/w galactose solution in water.

[0088] The vials containing the solution are subsequently loaded onto a tray and pre-capped.

[0089] This example is done on a pilot freeze-dryer (0.45 m²) where a tray containing 120 vials is positioned on one of the three plates of the freeze-dryer.

[0090] Three vials are probed internally each with a temperature probe (heat resistance) in order to monitor the temperature trend of the product during the execution of the cycle.

[0091] The product characterization results (N = 6) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Perfectly compact and uniform lyophile at the time of unloading. First signs of collapse after 24 h. Product collapsed after 72 h
Humidity (average ± std dev)	1.00 ± 0.08% w/w
Number of Broken Vials	0/120

[0092] The collapse is attributed to the low glass transition of galactose (30-37 °C) in which water, acting as a plasticizer, further lowers its value, leading to the collapse of the lyophile and de facto making it unsuitable for storage at room temperature (with these humidity values in the lyophile).

EXAMPLE 7

[0093] Eight 30 ml bags are filled with 15 millilitres of 2% w/w mannitol solution in water.

[0094] The bags are housed on the relative dedicated support.

[0095] In conjunction with the pressure decrease, the temperature of the sample undergoes a very rapid decrease (less than an hour) and the simultaneous freezing of the solutions in the bags.

[0096] The maximum time required to freeze all unprobed bags in this example is 120 seconds.

[0097] The product characterization results (N=3) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Compact and satisfactory lyophile. Different lyophile thickness in some areas of the pouch.
Humidity (average ± std dev)	0.35 ± 0.07% w/w

[0098] The uneven appearance of this test is related to the formation of gas pockets within the frozen matrix which are then reflected in the dried product once the process is concluded.

[0099] Fig. 6 represents the trend, during the test, of the temperature of the tray (continuous curve), of the temperature of the samples (dotted curve) and of the pressure in the chamber (dashed curve). As can be seen in the figure, the temperature of the plate, in step b) of the method of the invention, is set at -15 °C; when the temperature of the sample reaches a value of about -6 °C, the pressure in the chamber is brought rapidly to a value of about 0.1 mbar (step c)); in conjunction with the pressure decrease, the temperature of the sample undergoes a very rapid decrease (less than one hour) of about 20 °C. Fig. 7 shows a magnification relating to the first 24 hours (approx.) of the test of Fig. 6, in which the temperature trend of the samples is better highlighted (dotted curve).

EXAMPLE 8

[0100] Trays for freeze-drying in bulk are filled with 1.5 kilograms of solution in water composed of 4% w/w mannitol.

[0101] The tray containing the solution to be freeze-dried (in the specific example Lyoguard^®) is positioned on the plate of the freeze-dryer and the cycle started.

[0102] The product characterization results (N = 2, sampling at two different points of the lyophile) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Compact and uniform lyophile
Humidity (average ± std dev)	0.22 ± 0.03% w/w

EXAMPLE 9

[0103] 20R vials are filled with 5 millilitres of solution composed of 5% w/w albumin in water.

[0104] The vials containing the solution are subsequently loaded onto a tray and pre-capped.

[0105] This example is done on a pilot freeze-dryer (0.45 m²) where a tray containing 120 vials is positioned on one of the three plates of the freeze-dryer.

[0106] Three vials are each probed with a temperature probe (heat resistance) in order to monitor the temperature trend of the product during the execution of the cycle.

[0107] The product is thus ready to be reconstituted with the quantities of liquid suitable to obtain the desired final concentration of the solution.

[0108] The product characterization results (N = 3) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Lyophile non-uniform in appearance from vial to vial. The lyophile is spongy in some cases due to partial foaming effect during the freezing step
Humidity (average ± std dev)	1.85 ± 0.12% w/w
Number of Broken Vials	0/20

[0109] Fig. 8 represents the trend, during the test, of the temperature of the tray (continuous curve), of the temperature of the samples (dotted curve) and of the pressure in the chamber (dashed curve). As can be seen in the figure, the temperature of the plate, in step b) of the method of the invention, is set at -5 °C; when the temperature of the sample reaches a value of about -2 °C, the pressure in the chamber is brought rapidly to a value of about 0.1 mbar (step c)); in conjunction with the pressure decrease, the temperature of the sample undergoes a very rapid decrease (less than one hour) of almost 30 °C. Fig. 9 shows a magnification relating to the first 14 hours of the test of Fig. 8, in which the temperature trend of the samples is better highlighted (dotted curve).

EXAMPLE 10

[0110] 30R vials are filled with 5 millilitres of solution composed of 10% w/w albumin in water.

[0111] The product characterization results (N = 3) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Non-uniform lyophile in appearance. The lyophile is spongy due to foaming during freezing step in almost all vials
Humidity (average ± std dev)	2.04 ± 0.16% w/w
Number of Broken Vials	0/20

EXAMPLE 11

[0112] A 500 ml bag is filled with 200 ml of 2% w/w mannitol solution in water.

[0113] The bag is housed on the relative and dedicated support, which is thus positioned on one of the three plates of the freeze-dryer.

[0114] The bag is probed (by immersing the probe in the liquid) with one of the three available probes. The other two probes are used to monitor the temperatures at different points of the support.

EXAMPLE 12

[0115] Trays for freeze-drying in bulk are filled with 1.0 kg of solution in water composed of 5% w/w soluble coffee (soluble dehydrated coffee, freeze-dried, Carrefour / Interdis brand).

[0116] The tray containing the solution to be freeze-dried (in the specific example Lyoguard^®) is positioned on the plate of the freeze-dryer and the cycle started.

[0117] The product characterization results (N = 2, sampling at two different points of the lyophile) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Compact and uniform lyophile
Humidity (average ± std dev)	3.72 ± 0.12% w/w

EXAMPLE 13

[0118] A cycle with egg white is tested as it is wished to test a human plasma surrogate having a similar composition.

[0119] 500 ml vials are filled with 150 ml of pasteurized liquid egg white.

[0120] Below is the generic composition as per label of the tested liquid.

Material	Solution unit composition (g/vial)	Composition % (w/w)
Protein	15.3	10.2
Carbohydrates	1.8	1.2
NaCl	0.6	0.4
Water	132.3	88.2

[0121] The vials containing the solution are subsequently loaded onto a tray and pre-capped with special freeze-drying caps.

[0122] This example is done on a pilot freeze-dryers (0.45 m²) where five vials on a tray are positioned on one of the three plates of the freeze-dryers.

[0123] Three vials are probed externally each with a temperature probe (heat resistance) in order to monitor the evolution of the temperature of the product during the execution of the cycle.

[0124] In conjunction with the pressure decrease, the temperature of the sample undergoes a very rapid decrease also in this case (less than an hour).

[0125] The maximum time required to freeze all vials in this example is 80 seconds.

[0126] The product characterization results (N = 2) obtained by applying the parameters as described are reported:

Characteristic	Results
Appearance	Non-uniform lyophile in appearance. Areas on top of the lyophile that have a very spongy solid due to foaming.
Humidity (average ± std dev)	4.96 ± 0.08% w/w
Number of Broken Vials	0/5

[0127] Moisture was evaluated on the most compact lyophile portion where a possible higher content is expected compared to the most porous area.

[0128] Fig. 10 represents the trend, during the test, of the temperature of the tray (continuous curve), of the temperature of the samples (dotted curve) and of the pressure in the chamber (dashed curve). As can be seen in the figure, the temperature of the plate, in step b) of the method of the invention, is set at -12 °C; when the temperature of the sample reaches a value of about -2 °C, the pressure in the chamber is brought rapidly to a value of about 0.1 mbar (step c)); in conjunction with the pressure decrease, the temperature of the sample undergoes a very rapid decrease (less than one hour) of about 23 °C. Fig. 11 shows a magnification relating to the first 15 hours approximately of the test of Fig. 10, in which the temperature trend of the samples is better highlighted (dotted curve).

Claims

1. Method for freeze-drying a composition comprising a liquid phase, which comprises the steps of:

a) arranging one or more containers containing the composition to be freeze-dried on one or more temperature-controlled plates in a freeze-drying chamber;

b) by adjusting the temperature of the plates, bringing the temperature of the composition to a value comprised between 0 °C and -20 °C;

c) reducing, over a time comprised between 5 min and 60 minutes, the pressure in the freeze-drying chamber to a value comprised between 5.0 mbar and 0.01 mbar, and leaving the system under these conditions for a period comprised between 12 hours and 120 hours, causing the temperature of the product being freeze-dried to decrease below that of the plates;

d) by keeping the pressure at a value comprised between 5.0 mbar and 0.01 mbar, keeping the plates at a temperature equal to that set in point b) until the temperature of the product being freeze-dried reaches a temperature equal to or higher than that of the plates;

e) when the temperature of the product being freeze-dried has equalled the temperature of the plates, raising the temperature of the plates to a temperature comprised between +10 °C and +40 °C;

characterised in that during step b) the atmospheric pressure is maintained inside the freeze-drying chamber.

2. Method according to claim 1, wherein when the containers are rigid they are positioned on the plates in direct contact with them or on steel trays in turn in contact with the plates, while containers in the form of bags are arranged in dedicated supports in contact with the plates.

3. Method according to any one of claims 1 or 2, wherein in step b) the temperature of the composition is brought to a value comprised between -5 °C and -15 °C.

4. Method according to any one of claims 1 or 2 wherein, when the composition has a solids concentration equal to or greater than 5% by weight, in step b) the temperature of the composition is brought to a value comprised between 0 and -2°C.

5. Method according to any one of the preceding claims, wherein in step c) the pressure in the freeze-drying chamber is brought over a time comprised between 10 and 40 minutes to a value comprised between 1 mbar and 0.01 mbar, and the system is left under these conditions for a period comprised between 12 hours and 40 hours.

6. Method according to any one of the preceding claims, wherein the composition to be freeze-dried comprises as solid phase a compound selected from Abatacept, Aciclovir, Clavulanic acid, Hyaluronic acid, Albumin (human, ovine, porcine) Allopurinol, Alprazolam, Alprostadil, Amphetamine, Hydroxyethyl starch (HES), Amifostine, Amikacin, Amoxocillin, Annamycin, Antitrypsin, Aplidin, Aripiprazole Azacitidine, Bacitracin, Belimumab, Bendamustine, Bleomycin, Bortezomib, Brentuximab, Busulfan, Coffee, Carbidopa, Carboplatin, Casein, Carfilzomib, Carmustine, Caspofungin, Cefacetril, Cefadroxil, Cephalexin, Cefazaflur, Cephalotin, Cefazolin, Cefepime, Ceftaroline, Ceftobiprole, Ceftriaxone, Cetrorelix, Cyclophosphamide, Cilastatin, Cilastatine, Cisapride, cis-platinum, Cytarabine, Citalopram, Cladribine, Clonazepam, Clozapine, Cloxacillin, Cholestyramine, Copanlisib, Daptomycin, Daunorubicin, Decitabine, Degarelix, Desloratadine, Dexrazosan, Diclofenac, Donepezil, Doxorubicin, Ebastine, Elastin, Haemoglobin (equine, swine, human), Epicillin, Epirubicin, Erythromycin, Esmolol, Esomeprazole, Insulin-like growth factor type I (IGF-1), Insulin-like growth factor type II (IGF-2), Coagulation factor VII, Coagulation factor VIII, Coagulation factor IX, Phenytoin, Phentermine, Phenobarbital, Phentolamine, Fingolimod lauryl sulfate, Flucloxacillin, Fludarabine, Fosaprepitant Dimeglumine, Fotemustine, Gabexate, Gallium Ga-68, Ganciclovir, Gemcitabine, Gentamicin, Gozetotide, Glycopyrronium (or glycopyrrolate) Glucagon, Glutathione, Ibuprofen, Ifosfamide, Hygromycin B, Imipenem, Indomethacin, Infliximab, Trypsin inhibitor (pancreatic) (from bovine), Human Insulin (recombinant), Insulin glargine, Insulin lispro, Insulin aspart, Insulin detemir, Irinotecan, Lamotrigine, Lansoprazole, Leptin, Leuprolide, Levodopa, Levoleucovorin, Levothyroxine, Loratadine, Melphalan, Meropenem, Methadone, Methicillin, Methylpheniudate, Micafungin, Mifamurtide, Myoglobin (equine, swine, human), Mirtanzapine, Mitomycin, Motixafortide, Sodium nitroprusside, Nesiritide, Omeprazole, Omacetaxins, Olanzapine, Ondasetron, Oritavancin, Oxacillin, Oxytocin, Paclitaxel, Palonosetron, Pantoprazole, Paracetamol, Pemetrexed, Penicillin F, Penicillin G, Penicillin X, Penicillin K, Penicillin V, Penicillin O, Penicillin N, c-Myc Tag peptide, Pyraubidine, Pivampicillin, Pixantrone, Human plasma, Sheep plasma, Swine plasma, Bovine plasma, Equine plasma, Plasminogen, Polysaccharose 400, Prednisolone, Progesterone, Prostaglandin E1, Rifampicin, Rimegepant, Remdesivir, Remimazolam, Risperidone, Rizatriptan, Romidepsin, Secretin, Somatorelin, Selegiline, Selexipag, Syncalide, Sinvastatin, Sirolimus, Sulbactam, Tasonermin, Technetium Tc-99m, Teduglutide, Telavancin, Temozolomide, Tigecycline, Thiotepa, Tobramycin, Botulinum toxin type A of Clostridium botulinum (onabotulinumtoxin A), Trabectedin, Trastuzumab, Trilaciclib, Vaborbactam, Vancomycin, Vancuronium, Vardanafil, Verteporfin, Vinblastine, Vincristine, Voriconazole, Vosoritide and Ziprasidone, Zolmitriptan, their salts and derivatives.

Drawing