WO2013135826A1

WO2013135826A1 - Improved lyophilization method

Info

Publication number: WO2013135826A1
Application number: PCT/EP2013/055260
Authority: WO
Inventors: Andreas Schuetz; Klaus Hellerbrand
Original assignee: Project Pharmaceutics Gmbh
Priority date: 2012-03-14
Filing date: 2013-03-14
Publication date: 2013-09-19

Abstract

The present invention is directed to an improved lyophilization process which comprises the thawing of a frozen composition and the subsequent refreezing of the thawed composition. The present invention is further directed to lyophilized cake and intermediate cake obtainable according to the process of the invention.

Description

Improved lyophilization method FIELD OF THE INVENTION

[0001] The present invention is directed to an improved method for lyophilizing compositions. The present invention is further directed to the lyophilizate obtainable according to the improved methods of the invention.

BACKGROUND OF THE INVENTION

[0002] Due to potential instability and degradation, food or pharmaceutically active ingredients, such as proteins, nucleic acids and viruses (for example, as components of vaccines) often have to be made into solid forms to achieve an acceptable shelf life. The most commonly used method for achieving a longer shelf life is the lyophilization.

[0003] Lyophilization traditionally consists of two major steps: (1 ) freezing of a solution, and (2) drying of the frozen solid under vacuum. The drying step is further divided into two phases: primary and secondary drying. The primary drying step attempts to remove the frozen water or solvent (sublimation) and the second drying step attempts to remove the non-frozen 'bound' water or solvent (desorption) from the solidified solution. The removal of water or other solvent by lyophilization stabilizes the preparation by greatly reducing the degradation rate of the ingredients. The process inhibits the degradation process by removing solvent components in the preparation to levels that no longer support chemical reactions or biological growth. Additionally, the removal of solvent reduces molecular mobility, reducing the potential for degradative reaction. The removal of solvent(s) is accomplished, first, by freezing the solution such that the freezing process separates the solvent or solvents from solutes and immobilizes any non-frozen solvent molecule in the interstitial regions between the frozen solvent crystals. The solvent is then removed by sublimation (primary drying) and next by desorption (secondary drying).

[0004] In the freezing step, as the solvent or water freezes, the dissolved components in the solution (formulation) remain in the residual liquid (freeze concentrate). At the point of maximal ice formation (=eutectic point or glass transition point) the freeze concentrate solidifies between the ice crystal that make up the lattice.

[0005] Lyophilization cycle development typically focuses on optimizing the primary drying step because this is the most time consuming of the three steps and because generally the primary drying parameters are easily adjustable. The parameters generally affect both the time involved and the quality of the resulting "cake". It is known that in primary drying two important parameters are the chamber pressure and the shelf temperature. They are usually adjusted to maximize the rate of heat transfer to each vial. An excessive speeding up of the process may however cause the cake collapse.

[0006] It is further known that with standard freezing procedure cakes are formed that have the upper layers with an increased density (lid). The lid contributes to create a resistance against the flow of the sublimate during the drying step which further slows the drying process. Moreover, a lid in the upper part of the cake facilitates the risk of collapse of the cake.

[0007] There is the need of an improved method to shorter the drying step in particular the primary drying step (sublimation of solvent) without affecting the quality of the lyophilized cake and at the same time avoiding the frozen cake to collapse during the drying step.

DESCRIPTION OF THE INVENTION [0008] The present invention provides a lyophilization method that allows to have a faster drying step while avoiding the collapse of the cake.

[0009] The present invention provides a method for lyophilizing a composition, the method comprising:

(a) freezing a composition to be lyophilized and obtaining a frozen composition a';

(b) thawing the frozen composition a' obtained in step (a) and obtaining a thawed composition b'; (c) re-freezing the composition b' obtained in step (b) and obtaining a frozen composition c'; and

(d) drying the frozen composition c' of step (c)

wherein the composition to be lyophilized comprises a solvent or a mixture of solvents.

[0010] In step (a) a partially frozen composition a" may be also obtained. In step b) the thawing of the partially frozen composition a" obtained in step (a) can be performed to obtain a thawed composition b'. [0011] Hence, in an aspect the present invention is directed to a method for lyophilizing a composition to be lyophilized comprising:

(a) freezing a composition to be lyophilized and obtaining a frozen composition a'or a partially frozen composition a";

(b) thawing the frozen composition a' or the partially frozen composition a" obtained in step (a) and obtaining a thawed composition b';

(c) re-freezing the composition b' obtained in step (b) and obtaining a frozen composition c'; and

(d) drying the frozen composition c' of step (c)

[0012] In an aspect, the present invention is directed to a lyophilized cake obtainable according to the above method. [0013] The present invention further provides a method for lyophilizing a composition, the method comprising:

(b) thawing the frozen composition a' obtained in step (a) and obtaining a thawed composition b';

(d') primary drying the frozen composition obtained in step (c); and optionally (d") secondary drying wherein the composition to be lyophilized comprises a solvent or a mixture of solvents.

[0014] In step (a) a partially frozen composition a" may be also obtained. In step b) the thawing of the partially frozen composition a" obtained in step (a) can be performed to obtain a thawed composition b'.

[0015] Hence, in an aspect, the present invention further provides a method for lyophilizing a composition, the method comprising:

(a) freezing a composition to be lyophilized and obtaining a frozen composition a or a partially frozen composition a'";

[0016] In an aspect, the present invention is directed to a lyophilized cake obtainable according to the above method.

[0017] The methods of the invention may further comprise an evacuation step (d°) performed directly before the drying step (d) or the primary drying step (d ) to reduce the vial pressure. The evacuation step (d°) is performed preferably, at the same shelf temperature of the re-freezing step (c). Alternatively, it can be performed at a temperature below the eutectic point. The evacuation step (d°) is preferably performed for a time necessary to reach the pressure at which the drying step or the primary drying step will be performed.

[0018] In the methods of the invention the thawing of step (b) is a complete or a partial thawing and the composition b' obtained in step (b) can be a completely thawed composition or a partially thawed composition. The partially thawed composition is a composition b' wherein at least the 10% (w/w) of the frozen composition or of the partially frozen composition obtained in step (a) is thawed, more preferably at least the 30% (w/w) or at least the 50% (w/w) of the frozen composition or of the partially frozen composition obtained in step (a) is thawed, even more preferably at least the 80 % (w/w) of the frozen composition obtained in step (a) is thawed. Even more preferably, the composition b' is completely thawed. In the partially frozen composition the thawing refers to the frozen part of the partially frozen composition. [0019] In an aspect, the present invention is directed to a lyophilized cake (lyophilizate) obtainable according to the above methods.

[0020] In the methods of the invention, the freezing in step (a) is conducted at any suitable pressure, preferably it is conducted at atmospheric pressure.

[0021] In any of the methods of the invention, the freezing in step (a) is conducted to obtain a frozen composition a' (frozen cake) or partially frozen composition a" (a partially frozen cake). The shelf temperature is typically set below the eutectic melting temperature of the composition. The freezing step (a) may be conducted at a shelf temperature set at any temperature between -10 °C and -55 °C or between -10 °C and -45 °C. Preferably, the freezing in step (a) is conducted at a shelf temperature of less than -10 °C, more preferably of less than -45 °C, more preferably at -55 °C.

[0022] In the methods of the invention, the thawing step (b) which can be complete or partial is conducted at a shelf temperature set at any temperature between 0 °C and 25 °C above the melting point of the solvent. Preferably the shelf temperature is set at 20 °C above the melting point of the solvent. The temperature is set so as to allow to have a partial or a complete thawing of the frozen composition a' or of the partially frozen composition a". The degree of thawing from partial to complete may also depend on the time the frozen composition a' or the partially frozen composition a" is exposed to the mentioned shelf temperature of the invention. The frozen composition a' or the partially frozen composition a" is exposed to a suitable temperature and for a suitable period of time to reach a partial or a complete thawing. [0023] In the methods of the invention, the composition during the thawing step (b) reaches a temperature 1 to 30 °C, preferably from 5 to 30 °C above the eutectic melting point of the composition or from 5 to 20 °C above the eutectic melting point of the composition, more preferably from 9 to 20 °C above the eutectic melting point of the composition. In the complete thawing, the composition may reach the shelf temperature.

[0024] In the methods of the invention, the composition during the partial thawing step (b) may reach a temperature of 1 , 2, 3, 4, 5, 6 or 7 °C above the eutectic melting point of the composition or from 5 to 30 °C above the eutectic melting point of the composition, from 5 to 20 °C above the eutectic melting point of the composition to be lyophilized, from 9 to 20 °C above the eutectic melting point of the composition. The skilled person understands that the temperature of the composition is such that a partial thawing is reached. The temperature is such that the partially thawed composition is a composition wherein at least the 10% (w/w) or at least the 30% (w/w) of the frozen composition a' or the partially frozen composition a" is thawed, more preferably at least the 50% (w/w) of the frozen composition a' or the partially frozen composition a" is thawed, even more preferably at least the 80% (w/w) of the frozen composition a' or the partially frozen composition a" is thawed. In the partially frozen composition the thawing refers to the frozen part of the partially frozen composition..

[0025] In an aspect, the present invention is directed to a lyophilized cake (lyophilizate) obtainable according to the above methods.

[0026] In the methods of the invention, the freezing step (a), the thawing step (b) and the re-freezing step (c) are conducted at any suitable pressure for the purpose of each of step (a), (b) and (c), preferably they are conducted at atmospheric pressure.

[0027] In the methods of the invention, the freezing in step (c) is performed to refreeze the complete or partially thawed composition obtained in step (b). The shelf temperature is at least set below the eutectic melting temperature of the composition or the lowest eutectic melting temperature of the composition when the composition to be lyophilized has more than one eutectic point. [0028] The refreezing step (c) may be conducted at a shelf temperature set at any temperature between -10 °C and -55 °C or between -10 °C and -45 °C.

[0029] In the methods of the invention, the freezing in step (c) is preferably conducted at a shelf temperature of less than -10 °C.

[0030] In the methods of the invention, the freezing in step (c) is preferably conducted at a shelf temperature of less than -45 °C, preferably at -55 °C.

[0031] In the methods of the invention, the freezing step (c) is preferably conducted at a shelf temperature set at any temperature between -10 °C and -45 °C.

[0032] In the methods of the invention, the drying step (d) may comprise or consist of the primary drying step (d') and optionally of the secondary drying step (d"). [0033] In the methods of the invention, the drying in step (d) or the primary drying step (d') and the secondary drying step (d"), the latter when performed, is preferably conducted at any shelf temperature at or above 0 °C to 20 °C.

[0034] In the methods of the invention, the drying in step (d) or the primary drying step (d') and the secondary drying step (d"), the latter when performed, is preferably conducted at a shelf temperature set at any temperature between 20 °C and 40 °C.

[0035] In the methods of the invention, the starting composition to be lyophilized can be a solution or a suspension, for example the composition of the invention is a nanoparticle or a liposome suspension wherein the nanoparticles or the liposomes are suspended in a suitable solvent or mixture of solvents.

[0036] The composition to be lyophilized comprises a solvent or a mixture of solvents. The solvent is selected from water, DMSO, glacial acetic acid, anisole, benzylalcohol, tert-butanol and mixtures thereof. Preferably, the solvent is water. [0037] In the methods of the invention, the composition is a food composition or the composition is a pharmaceutical composition which comprises a pharmaceutically active ingredient or at least a pharmaceutically active ingredient. Preferably, the pharmaceutical active ingredient is selected from, but not limited to, proteins, peptides, virus, nucleic acids, antibodies, small molecules. The pharmaceutically active ingredient may be contained in liposomes or be in the form of nanoparticles.

[0038] In the methods of the invention, the composition to be lyophilized is preferably a pharmaceutical formulation.

[0039] In the methods of the invention, the composition to be lyophilized preferably comprises at least one of the following components: buffer, tonicity modifier, stabilizer, bulking agent and/or an excipient. It is clear that these components are present in composition a', composition a", composition b', composition c' and in the final lyophilized cake after the drying step whose purpose is to remove the solvent or the solvents from the composition to be lyophilized to obtain a lyophilized cake.

[0040] In the methods of the invention, the lyophilized product obtained according to the methods of the invention is preferably a pharmaceutical formulation.

[0041] In other embodiment, an object of the present invention is the lyophilized product obtainable (obtained) by the methods of the invention as disclosed in any of the embodiments or aspects disclosed above in connection with the methods of the invention.

[0042] Hence, the present invention is directed to a lyophilized product obtainable by:

(c) re-freezing the composition b' obtained in step (b) and obtaining a re- frozen composition c'; and (d) drying the frozen composition c' of step (c)

wherein the composition to be lyophilized comprises solvents or a mixture of solvents.

[0043] In step (a) a partially frozen composition a" may be also obtained. In step b) the thawing of the partially frozen composition a" obtained in step (a) can be performed to obtain a thawed composition b'.

[0044] Hence, the present invention is directed to a lyophilized product obtainable by:

(a) freezing a composition to be lyophilized and obtaining a frozen composition a' or a partially frozen composition a";

wherein the composition to be lyophilized comprises solvents or a mixture of solvents. I

[0045] The thawing of step (b) is a complete or a partial thawing and the composition b' obtained in step (b) can be a completely thawed composition or a partially thawed composition. The partially thawed composition is preferably a composition b' wherein at least the 10% (w/w) of the frozen composition or the partially frozen composition a" obtained in step (a) is thawed, more preferably at least the 30% (w/w) or at least the 50% (w/w) of the frozen composition or the partially frozen composition a" obtained in step (a) is thawed, even more preferably at least the 80% (w/w) of the frozen composition or of the partially frozen composition obtained in step (a) is thawed. Even more preferably the composition b' is a completely thawed composition. In the partially frozen composition the thawing refers to the frozen part of the partially frozen composition. [0046] In any embodiment of the invention, the drying step (d) may comprise

(d') primary drying the frozen composition obtained in step (c); and optionally (d") secondary drying. [0047] In other embodiment, an object of the present invention is a refrozen cake (composition c') obtainable by

(a) freezing the composition to be lyophilized and obtaining a frozen composition a';

(c) re-freezing the composition b' obtained in step (b) and obtaining a re-frozen composition c'

[0048] In step (a) a partially frozen composition a" may be also obtained. In step b) the thawing of the partially frozen composition a" obtained in step (a) can be performed to obtain a thawed composition b'.

[0049] Hence, in other embodiment, an object of the present invention is a refrozen cake (composition c') obtainable by

(a) freezing the composition to be lyophilized and obtaining a frozen composition a' or a partially frozen composition a";

[0050] The thawing of step (b) is a complete or a partial thawing and the composition b' obtained in step (b) can be a completely thawed composition or a partially thawed composition. The partially thawed composition is preferably a composition b' wherein at least the 10% (w/w) of the frozen composition or the partially frozen composition a" obtained in step (a) is thawed, more preferably at least the 30% (w/w) or at least the 50% (w/w) of the frozen or partially frozen composition obtained in step (a) is thawed, even more preferably at least the 80 %(w/w) of the frozen composition or the partially frozen composition a" obtained in step (a) is thawed. Even more preferably the composition b' is a completely thawed composition. In the partially frozen composition the thawing refers to the frozen part of the partially frozen composition. [0051] In the methods of the invention, the composition to be lyophilized contains up to 30% (w/w) of solute, preferably up to 20% (w/w) of solute. Preferably, the excipients constitute the 1 % to 10% (w/w) of the composition. The percentages are referred to the starting composition i.e. to the composition to be lyophilized. DETAILED DESCRIPTION OF THE INVENTION

[0052] The present inventors have found that by varying the freezing conditions of a lyophilization (freeze-drying) process, a (re)frozen cake which easily undergoes the drying step, in particular, the sublimation (primary drying step), is prepared. The present inventors have found that by performing a freezing/thawing/freezing (FTF) process according to steps a) to c) of the methods of the invention an altered structure of the re- frozen cake is obtained that is able to positively influence the drying step of the lyophilization process. In particular, it is able to positively influence the sublimation of the solvent in the drying step. More in particular, the structure of the re-frozen cake is such that it allows the shortening of the drying step, in particular, the sublimation phase while avoiding the re-frozen cake collapse.

[0053] The present inventors have found that when the freezing step (a) is followed by a thawing step (b) wherein the frozen composition (cake) is complete or partially thawed and then re-frozen according to step (c), the re-frozen composition (cake) obtained can easily undergo sublimation (primary drying step). It is in fact been observed that the sublimation is shorter in time and no cake-collapse occurs. On the contrary, standard frozen cakes (i.e. one freezing step) generally collapse under the very same primary drying conditions.

[0054] The present inventors have further surprisingly found that the thawing (complete or partial) of the frozen composition (cake) affects the concentration gradient in the re- frozen composition (cake). It has been indeed observed by the present inventors that the process of freezing/thawing/freezing (FTF) according to steps (a) to (c) of the present invention yields to less concentrate layers on the top of the re-frozen cake (Fig. 1 ). In other words, the FTF process of the invention avoids the formation of a "lid" on the top of the re-frozen cake. This is clearly an advantage because concentrate top layers (lid) on the top of a frozen cake form a barrier for water/solvent transport during the drying step (sublimation of the solvent). On the contrary, in the presence of less concentrate layers at the top of the cake, the drying step is facilitated because the sublimate stream is facilitated. Thereby, the sublimation time is strongly reduced and the lyophilized cake reaches the shelf temperature faster. Moreover, the lack of hyper-concentration on the top of the re-frozen cake obtained according to the present invention is such that cake collapse is absent or limited. Accordingly, vials having a higher fill height can be prepared with the methods of the invention. For example, filling heights above 20 mm or above 26 mm or above 30 mm have been dried for the first time with technical relevance and without observing the cake collapse. [0055] Additionally, an increase in the ice crystal size caused by the thawing and the re- freezing steps has been observed. An increased ice crystal size contributes to a faster primary drying, as well, as it contributes to decrease the product resistance to the flow of the solvent vapor. [0056] The present invention is even more surprising in case of complete thawing. It was in fact expected that a complete thawing of the frozen composition a' would have led to a composition (composition b') identical (e.g. in terms of structure, concentration gradient, etc.) to the starting composition and therefore that the re-frozen cake/composition c' would have had the very same structure and features of frozen composition a'. Surprisingly, the present inventors have found that the complete thawing/freezing of a frozen cake (composition a') or partially frozen cake (composition a") leads to a re-frozen cake-composition c' that differs from the frozen cake-composition a' in that no lid and an increased ice/ solvent crystal size are observed thereby resulting in a faster drying step. [0057] The composition to be lyophilized of the invention may have one or more eutectic melting points. When the composition has more than one eutectic melting point, the skilled person understands to which melting point reference is made. For example, in the freezing step (a) and the re-freezing step (c) the shelf temperature is set at least below the lowest eutectic melting point to reach a frozen composition. In the thawing step, the shelf temperature is at least above the highest eutectic point to reach thawing. To achieve partial thawing the shelf temperature may be set above the lowest eutectic temperature and below the melting temperature of the composition. The shelf temperatures may be also set above the melting temperature of the composition. In this case however, the time for thawing will be limited so as to avoid complete thawing.

[0058] A complete thawing is obtained by allowing the frozen composition a' or the partially frozen composition a" obtained in freezing step (a) of the invention step to reach a temperature at least above the eutectic point of the composition and at least above the melting point of the component of the composition having the highest melting point so as to assure a complete thawing. Generally, the component having the highest melting point is the solvent. Generally, if the composition is an aqueous solution, the completely thawed composition will reach, during the thawing step, a temperature above the melting point of the water i.e. 0°C. In order to achieve a complete thawing, the shelf temperature is set at least above the melting point of the component of the composition having the highest melting point. Preferably, the shelf temperature during the thawing step is set 0°C, 10°C, 15°C, 20°C or 45°C above the melting point of the solvent (it is intended the melting point of the solvent alone not in the composition (e.g. the melting point of the water is 0°C)). Preferably, the shelf temperature is set at any temperature between 10°C and 45°C above the melting point of the solvent, more preferably is set at any temperature between 10°C and 35°C above the melting point of the solvent, even more preferably is set at any temperature between 10°C and 20°C above the melting point of the solvent. The preferred shelf temperature is 20°C. Step (b) is preferably performed for a period of time necessary to obtain a complete thawing of the frozen composition or the partially frozen composition a" of step (a). The time necessary for achieving the thawing may depends also on the chosen shelf temperature.

[0059] In another embodiment of the invention, the thawing can be a partial thawing and the composition obtained in step (b) can be a partially thawed composition. The partially thawed composition is preferably a composition wherein at least the 10% (w/w) or the 30% (w/w) of the frozen composition a' or the partially frozen composition a" is thawed, more preferably at least the 50% (w/w) of the frozen composition a' or the partially frozen composition a" is thawed, even more preferably at least the 80% (w/w) of the frozen composition a' or the partially frozen composition a" is thawed. A partial thawing of the frozen composition or partially frozen composition obtained in step (a) occurs because, after the first freezing step (a), the shelf temperature is raised to allow the frozen or partially frozen composition to reach a temperature which is above the eutectic point or the highest eutectic point of the composition but not above the melting point of a component (e.g. the solvent) of the composition, preferably not above the melting point of the component of the composition having the highest melting point (e.g. the solvent). Generally, the component having the highest melting point is the solvent. Generally, the composition should not be allowed to reach a temperature above the melting point of the component of the composition having the highest melting point so as to assure that a solid frozen phase is still present. Generally, the component having the highest melting point is the solvent. It is clear that within this interval of temperatures of the composition both a liquid and a solid (frozen) state coexist. In order to achieve a partial thawing, the shelf temperature depends on the composition considered. The shelf temperature may be set at a temperature at least above the lowest eutectic point and not above the melting point of the component of the composition having the highest melting point. The shelf temperature may also be set at or above this temperature. For example for aqueous solutions the shelf temperature may be set at or above 0 °C, or at or above 10°C or at or above 20°C. The temperature of the sample can be appropriately measured and monitored to check whether a partial thawing has been achieved (for example, when the composition temperature is between at least a eutectic point and the melting point of the solvent). E.g., to achieve a partial thawing the shelf temperature is set at least above the melting point of the component of the composition having the highest melting point. Generally this component is the solvent. When the composition to be lyophilized comprises a mixture of solvents, the shelf temperature may be set above 0 °C, or above 10°C or above 20°C the melting point of the solvent of the mixture of solvents that has the highest melting point. Preferably, the shelf temperature during the thawing step is set above -10 °C, 0 °C, 10 °C, 15 °C, 20 °C or 45 °C, preferably is set at any temperature between 10 °C and 45 °C, more preferably is set at any temperature between 10 °C and 35 °C, even more preferably is set at any temperature between 10 °C and 20 °C. The preferred shelf temperature is 20 °C. Step (b) is preferably performed for a period of time necessary to obtain a partial thawing. The composition may be not allowed to reach a temperature above the melting point of the component of the composition having the highest melting point so as to assure that a solid frozen phase is still present. The partial thawing is achieved by bringing the frozen composition or the partially frozen composition a" obtained in step (a) above the eutectic point of the composition.

[0060] Preferably, in the methods of the present invention the thawing is complete.

[0061] In another aspect, step (b) is conducted at any suitable pressure to obtain a thawing or a partial thawing as disclosed above. Preferably, step (b) is conducted at atmospheric pressure. [0062] The freezing step (a) is performed to obtain a completely frozen composition. Hence, the freezing step is performed at a shelf temperature that allows obtaining a frozen composition. Alternatively, the freezing step (a) is performed to obtain a partially frozen composition (a"). Hence, the freezing step is performed at a shelf temperature that allows obtaining a partially frozen composition. In the methods of the invention, the freezing in step (a) is conducted at the shelf temperature set below the eutectic melting temperature of the composition. Compositions having more than a eutectic point may exist. In this case, the shelf temperature is set below the lowest eutectic point. Hence, in an aspect, the freezing step (a) is performed at a shelf temperature of less than -10 °C, of less than -20 °C, of less than -30 °C, of less than -40 °C or of less than -45 °C or of less than -55 °C. Preferably, the shelf temperature is set at -45 °C or at -55 °C. The freezing step (a) is conducted for the time necessary to obtain a frozen composition or a partially frozen composition (a"). The freezing step (a) is conducted for a total time of at least 1 hour. Preferably, the freezing of step (a) is conducted for a total time of at least 2 hours, at least 3 hours, at least 4 hours. The freezing of step (a) is conducted at any suitable pressure to obtain a frozen cake or partially frozen composition a". Preferably, freezing step (a) is conducted at atmospheric pressure.

[0063] The re-freezing step (c) is performed to obtain a completely frozen composition. In the methods of the invention, the freezing in step (c) is conducted at the shelf temperature set below the eutectic melting temperature of the composition. Compositions having more than a eutectic point may exist. In this case, the shelf temperature is set below the lowest eutectic point. Hence, in an aspect, the re-freezing step (c) is performed at a shelf temperature of less than -10 °C, of less than -20 °C, of less than -30 °C, of less than -40 °C or of less than -45 °C or of less than -55 °C . Preferably, the shelf temperature is set at -45 °C or at -55 °C. The re-freezing step is preferably performed at a shelf temperature set below the eutectic point or the lowest eutectic point. The re-freezing step (c) is conducted for the time necessary to obtain a re- frozen composition. The re-freezing step (c) is conducted for a total time of at least 1 hour. Preferably, the freezing of step (c) is conducted for a duration of greater than 2 hours, 3 hours, 4 hours.

[0064] Preferably, the re-freezing of step (c) is conducted at atmospheric pressure. [0065] The re-freezing step (c) may be performed as a freezing step usually performed in lyophilization process.

[0066] The methods of the invention comprise a drying phase or drying step to form the lyophilized composition. The drying phase or drying step can comprise or consist of a primary drying step and optionally a secondary drying step.

[0067] A primary drying step (d') of the frozen composition obtained in step (c) may be performed.

[0068] The primary drying has the purpose of sublimating the solid frozen solvent or mixture of solvents contained in the re-frozen cake obtained in step (c). The pressure and the temperature of the primary drying step are set so as to obtain the sublimation of the solvent(s) from the frozen composition c'. The primary drying is preferably performed at reduced atmospheric pressure. The pressure is preferably set below 1 mbar. Preferably, the pressure is set at or below 200 μbar, preferably in a range between 200 μbar and 20 μbar. In one embodiment, the primary drying is preferably conducted at a shelf temperature of 10 °C to 40 °C, preferably of 5 °C to 30 °C, more preferably at 25°C. In one aspect, the primary drying is preferably conducted for a time sufficient to ensure that the frozen solvent or the frozen water, when water is the solvent, is removed from the refrozen composition. One skilled in the art understands that the primary drying time varies in dependence of different parameters. The duration of the primary drying may depend on the fill volume, surface area of the cake, concentration etc.. For example, the primary drying is of at least 1 hour, of at least 3 hours to 10 hours, of at least 10 to 30 hours. [0069] The primary drying can be monitored by a number of methods. E.g. it may be monitored by measuring the changes in the product temperature. The end of the primary drying can be determined to be when the product temperature approaches the shelf temperature. For example, this can be noted by the change in slope of the product temperature trace due to a reduced sublimation rate: when evaporation ends the cooling due to the evaporation ends as well (Figs. 2, 5 and 10). Other methods to monitor the primary drying include the monitoring of the pressure rise or the measurement of the heat transfer rate. [0070] In one aspect, the present invention provides a method for lyophilizing a solution as disclosed above which further comprises an evacuation step (d°) performed directly before the drying step (d) or the primary drying step (d ) to reduce the pressure. The evacuation step (d°) is performed preferably at the same shelf temperature of re-freezing step (c). Alternatively, it can be performed at a temperature below the eutectic point. The evacuation step (d°) is preferably performed for a time necessary to reach the pressure at which the primary drying step will be performed.

[0071] The drying step may optionally comprise one or more secondary drying step. The secondary drying step is performed to reduce the moisture of the primary dried cake. Preferably, the secondary drying is conducted at shelf temperature close to the ambient temperature. The secondary drying step is preferably conducted at a temperature of at least 20°C. Preferably the secondary drying is conducted at a temperature from 20 to 40 °C. The pressure, preferably, is the same pressure of the primary step or a lower pressure as applied during the primary step. The secondary drying step is conducted for a time sufficient to reduce or remove the residual moisture level in the lyophilized product. The secondary drying step can be performed for a total time of at least 1 hour or at least 2 hours or more.

[0072] The composition to be lyophilized can be a solution or a suspension (e.g. a nanoparticles or a liposome suspension). Preferably, it is a solution or a suspension. For example, compositions of the invention are nanoparticle or liposome suspensions wherein the nanoparticles or the liposomes are suspended in a suitable solvent or mixture of solvents. The solution or suspension of the invention may contain additional components such as excipient, bulking agent, etc. which are generally used in a lyophilization process. The composition to be lyophilized according to the method of the invention comprises one or more solvents. Preferably, the solvent is one solvent.

[0073] The solvent of the composition to be lyophilized can be any solvent or any mixture of solvents suitable for lyophilization. The solvent as a frozen liquid should be able to sublime, preferably it should be able to sublime under reduced pressure. Solvents used according to the present invention are selected from water, DMSO, glacial acetic acid, anisole, benzylalcohol, tert-butanol, and mixtures thereof. Preferably, the solvent is water.

[0074] The composition to be lyophilized comprises one or more components selected from food or a pharmaceutically active ingredient, or polymers, or lipids or liposomes or nanoparticles. The pharmaceutical active ingredient may have previously been formulated in liposomes or nanoparticle. The liposomes or nanoparticle suspensions used in the methods of the invention may contain one or more pharmaceutically active ingredients. The composition to be lyophilized can be a pharmaceutical formulation, preferably an aqueous pharmaceutical formulation that is lyophilized with the present methods. The pharmaceutical formulation to be lyophilized may contain any active ingredient that can be lyophilized. Active ingredients according to the present invention include, but are not limited to, proteins, peptides, nucleic acids, viruses, viruses, liposomes or small molecules. Preferably, the compositions of the invention are aqueous pharmaceutical formulations comprising as pharmaceutically active ingredients an active ingredient selected from proteins, peptides, nucleic acids and viruses liposomes or small molecules. Any active ingredient may be in the form of nanoparticles or liposomes.

[0075] In the methods of the invention, the composition to be lyophilized may comprise at least one excipient. Non limiting examples of excipients include: buffering agent, tonicity modifier, stabilizer, bulking agent, and surfactants. Preferred excipients are sucrose, mannitol, trehalose or glycine.

[0076] The starting composition of the methods of the invention has a concentration below 30% (w/w) of solute (solute is the sum of components which are not the solvent or solvents) which has been dissolved in the solvent or mixture of solvents. Preferably, the starting composition has a concentration of solute at or below the 20% (w/w). [0077] Excipients (e.g. sugars) are present in a concentration ranging from 1 % to 10% (w/w). Preferably sucrose, mannitol, trehalose or glycine is present in the starting composition in a concentration of about 1 % to about 10% (w/w).

[0078] In another aspect, the present invention is directed to a lyophilized product obtainable with any of the method of the invention as disclosed above.

[0079] Hence, one aspect of the invention is directed the lyophilized cake obtainable by (a) freezing a composition to be lyophilized and obtaining a frozen composition a';

(c) re-freezing the composition b' obtained in step (b) and obtaining a frozen composition c' and

(d) drying the frozen composition c' of step (c) wherein the composition to be lyophilized comprises a solvent or a mixture of solvents.

[0080] In step (a) a partially frozen composition a" may be also obtained. In step b) the thawing of the partially frozen composition a" obtained in step (a) can be performed to obtain a thawed composition b'.

[0081] Hence, in a further aspect of the invention is directed the lyophilized cake obtainable by

(a) drying the frozen composition c' of step (c) wherein the composition to be lyophilized comprises a solvent or a mixture of solvents. [0082] The drying step may comprise a primary drying step or a primary drying step and a secondary drying step.

[0083] The thawing of step (b) is a complete or a partial thawing and the composition b' obtained in step (b) can be a completely thawed composition or a partially thawed composition. The partially thawed composition is a composition b' wherein preferably at least the 10% (w/w) of the frozen composition or the partially frozen composition a" obtained in step (a) is thawed, more preferably at least the 30% (w/w) or at least the 50% (w/w) of the frozen composition or partially frozen composition (a") obtained in step (a) is thawed, even more preferably at least the 80%(w/w) in weight of the frozen composition or the partially frozen composition a" obtained in step (a) is thawed. Even more preferably the composition b' is completely thawed. In the partially frozen composition the thawing refers to the frozen part of the partially frozen composition. [0084] Steps (a), (b), (c) (d) including d°, d' and d" are performed as disclosed for the methods of the invention.

[0085] A further aspect the invention is directed the an intermediate cake obtainable by (a) freezing a composition to be lyophilized and obtaining a frozen composition a';

(c) re-freezing the composition b' obtained in step (b) and obtaining a frozen composition c'

[0086] In step (a) a partially frozen composition a" may be also obtained. In step b) the thawing of the partially frozen composition a" obtained in step (a) can be performed to obtain a thawed composition b'.

[0087] Hence, a further aspect the invention is directed the an intermediate cake obtainable by (a) freezing a composition to be lyophilized and obtaining a frozen composition a' or a partially frozen composition a"

[0088] The features of steps (a), (b), (c) are as disclosed above for the methods of the invention.

[0089] The thawing of step (b) is a complete or a partial thawing and the composition b' obtained in step (b) can be a completely thawed composition or a partially thawed composition. The partially thawed composition is a composition b' wherein preferably at least the 10% (w/w) of the frozen composition or the partially frozen composition a" obtained in step (a) is thawed, more preferably at least the 30% (w/w) or 50% (w/w) of the frozen composition or the partially frozen composition a" obtained in step (a) is thawed, even more preferably at least the 80%(w/w) in weight of the frozen composition or the partially frozen composition a" obtained in step (a) is thawed. Even more preferably, the composition b' is completely thawed. In the partially frozen composition the thawing refers to the frozen part of the partially frozen composition.

[0090] As already disclosed above, the lyophilized cake or the intermediate cake obtainable with the methods of the invention do not present a lid or an hyper- concentration on the top. Moreover, the solvent crystal has a bigger size with respect to the lyophilized cake obtained without the thawing and refreezing steps of the invention.

[0091] In still a further aspect also it is provided kit comprising a) the lyophilized composition obtained according to the methods of the invention and b) a liquid component. The liquid component may be used as diluent to reconstitute the lyophilized composition. The lyophilized composition and the liquid component can be present in separate containers. Additional reagent may be present. The kits may further include the instructions for using the components of the kit e.g. instruction for reconstituting the lyophilized composition. [0092] In the partially frozen composition a", any percentage of frozen volume is suitable for the purpose of the invention. For example the partially frozen composition a" according of the invention is a composition wherein at least 10% of the volume of the whole composition is frozen, at least 20% of the volume of the whole composition is frozen, at least 30% of the volume of the whole composition is frozen, at least 50%, 60%, 70%, 80%, 90% or more of the volume of the whole composition is frozen.

DEFINITIONS

[0093] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0094] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".

[0095] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of may be replaced with either of the other two terms.

[0096] As described herein, "preferred embodiment" or "preferred aspect" means "preferred embodiment of the present invention" or "preferred aspect of the present invention". Likewise, as described herein, "various embodiments" and "another embodiment" means "various embodiments of the present invention" and "various embodiment of the present invention", respectively. [0097] "Eutectic" or "eutectic point" or "eutectic melting point" or "eutectic temperature" means the temperature at which only solid phase exists, representing the minimum melting temperature of the solution.

[0098] The term "melting point" refers to the temperature at which the substance changes its state from solid to liquid. Generally, it refers to the melting point of a component of a composition as distinct from the eutectic temperature or eutectic melting point (melting point of the eutectic mixture). The melting point of the solvent is the melting point of the solvent per se not in the composition. For example, the melting point of the water is understood to be 0°C.

[0099] "Freezing": the freezing is the step in which the composition to be lyophilized is brought from a liquid state to a solid state. During the freezing step nucleation and formation of ice/ solvent crystals occurs at a certain temperature throughout the composition. Both the shelf and the composition temperatures are set to be below the lowest eutectic temperature. Freezing according to the present invention may lead to a partially frozen composition such as the partially composition a". Hence in this sense freezing encompass freezing of a part of the volume of a composition. - [0100] "Re-Freezing" is the step in which the partially or completely thawed solution is brought to a solid state. As in the freezing step, both the final shelf and solution temperature are set to be below the eutectic temperature or the lowest eutectic temperature.

[0101] "Primary drying": the frozen solution sublimes and the drying front gradually retracts the frozen core leaving a dry cake. The formed solvent vapor passes through the dried cake to the surface and then through the chamber to the condenser. Heat is transferred from the shelf and the chamber walls to the drying front mainly by conduction and radiation.

[0102] "Secondary drying" involves the removal of the solvent (water) that does not separate out as solid (ice) during the freezing, and hence does not sublimate off. [0103] "Shelf temperature" is temperature at which the lyophilizer is set. It does not necessarily correspond with the temperature of the composition during each step of the lyophilization process. [0104] "Sublimation" is the transition from the solid state to a gas phase with no intermediate liquid phase. It occurs in the primary drying. After the composition is completely re-frozen, the pressure of the freeze dryer is reduced, and heat is applied to initiate sublimation of the ice or of the solvent crystal. [0105] "Desorption": it occurs in the secondary drying. It involves the removal of the excess moisture (e.g. bound water/solvent) by increasing the temperature of the product and reducing the partial pressure of water (solvent) vapor in the container.

[0106] The term "thawing" or/and "melting" are used interchangeably. They mean the passage from a solid state to a liquid state. The thawing or the melting can be complete indicating a complete passage from the solid to the liquid phase or partial indicating the coexistence of a solid frozen and a liquid phase.

[0107] With the term "cake" is intended the porous and spongy like material resulting from the freezing/thawing/freezing (FTF) process or from the entire lyophilization process (including drying). Sometime it is referred also to the solid product obtained after the first step (a) of the invention or the product obtained after the primary drying step.

[0108] With the term "collapse" or "collapsing" is intended the point at which the product softens to the extent that it can no longer support its structure.

[0109] "Buffers" are required in pharmaceutical formulations to stabilize pH. In the development of lyophilized formulations, the choice of buffer can be critical. Examples of buffer components according to the present invention are phosphate, citrate, Tris, histidine, acetic acid, glutamic acid, fumaric acid, succinic acid.

[0110] The purpose of the "bulking agent" is to provide bulk of the formulation. This is important in cases in which very low concentrations of the active ingredient are used. Bulking agents according to the present invention are for example, sucrose, trehalose or other mono- or disaccharides, sugar alcohols like mannitol, amino acids and inorganic salts.

[0111] "Tonicity adjusters": in several cases, an isotonic formulation might be required. The need for such a formulation may be dictated by either the stability requirements of the bulk solution or those for the route of administration. Excipients such as, sucrose, trehalose, glycine, mannitol and sodium chloride are good tonicity adjusters that can be used according to the present invention. Tonicity modifiers also can be included in the diluent that reconstitutes the lyophilized product rather than in the lyophilized.

[0112] With the term "small molecule" is meant any molecule, preferably a pharmaceutically active ingredient, which is not a protein, a peptide, nucleic acids or viruses and has a molecular weight of less than 1200 Da (Dalton), less than 1000 Da, less than 800Da or less than 600 Da preferably less than 800Da.

FIGURES

[0113] The figures show: [0114] Figure 1 shows the concentration gradient in the frozen cake after the freezing step c) of the invention (FTF lyophilization FTF-1 and FTF-2) and after a standard freezing (prior art) according to Example 1.

[0115] Figure 2 shows the plot of the temperature vs. the time of the FTF-lyophilization process of the invention (FTF-1 and FTF-2) and of the standard process according to Example 2.

[0116] Figure 3 shows the photos of the lyophilized cakes of Example 2. [0117] Figure 4 shows the distributions of the vials of Example 3 in the plate of the lyophilizer. The internal temperature of the solution was measured in the dark color vials.

[0118] Figure 5 shows the plot of the temperature vs. the time of the FTF-lyophilization process of the invention and of the standard process according to Example 3.

[0119] Figure 6 shows the photos of the lyophilized cakes containing arginine, sucrose and trehalose of Example 3.

[0120] Figure 7 shows the photos of the lyophilized cakes containing glycine of Example 3.

[0121] Figure 8 shows the photos of the vertical section of the lyophilized cake of Example 3. [0122] Figure 9 shows the distributions of the vials of Example 4 in the plate of the lyophilizer. The internal temperature of the composition was measured in the dark color vials. [0123] Figure 10 shows the plot of the temperature vs. the time of the FTF-lyophilization process of the invention and of the standard process according to Example 4.

[0124] Figure 11 shows the photos of the lyophilized cakes in the vials of Example 4.

[0125] Figure 12 shows the photos of the surface of lyophilized cake containing sucrose and trehalose of Example 4.

[0126] Figure 13 shows the different drying profiles of the two variants: the freeze-thaw- Freez (FTF) and standard procedures.

[0127] Figure Mshows on the left the pattern completely dry and free of defects FTF variant and on the right the standard version with melted ice core. All 44 standard versions after discharge showed complete meltdown by melting residual ice. In contrast, all 44 FTF variants were defect-free.

EXAMPLES In the following examples the primary and the secondary drying have been performed in one step. The primary drying is finished when the sublimation is finished. This is indicated by converging of product temperature to the shelf temperature.

[0128] Example 1 : Sucrose concentration gradient in FTF treatment vs. standard treatment 20RVials.

[0129] 20Rvials (FTF1 and FTF 2) were charged with a 15 ml of a sterile 5% sucrose solution. The resulting fill height was of ca. 26mm. They were placed in a lyophilizer and processed according to the following schedule (Table 1 ): Table 1

[0130] After the thawing of the first 2 vials, 2 additional vials (Standard 1 and standard 2) with the same quantity of sucrose solution were charged in the lyophilizer and frozen (i.e. the latter two vials underwent the "standard" freezing step).

[0131] The 4 vials were then removed from the lyophilizer, the concentration gradient of each cake was analyzed. In particular, each frozen cake was divided in an upper half cake and in a lower half cake. The two parts were left to melt and the concentration of each melted solution was measured with respect to the concentration. The results are reported in Fig 1.

[0132] The upper halves of the cakes obtained with the FTF freezing steps show a far lower concentration with respect to the lower halves of the cake. This difference is less remarkable in the cake obtained with the standard freezing procedure.

[0133] Example 2: Lyophilization with FTF treatment [0134] 6 20Rvials (FTF1 to FTF 6) were charged with a 15 ml of a sterile 5% sucrose solution. The resulting fill height was of ca. 26mm. They were placed in a lyophilizer and processed according to the following schedule (Table 2): Table 2

1 ) A longer freezing step before the evacuation of the lyophilizer has been done.

5 [0135] After the thawing of the first 6 vials, 6 additional vials (Standard 1 to standard 6) with the same quantity of sucrose solution were charged in the lyophilizer and frozen (i.e. the latter 6 vials undergo the "standard" freezing step).

[0136] The process is ended after 41 h of drying.

10

[0137] As it is shown in Fig. 2 (only FTF1 and FTF 2 reported) a complete thawing has been performed. This is indicated by the fact that the solutions FTF1 and FTF2 reach the shelf temperature. The drying of the FTF Vials was stopped 41 h the setting of the shelf temperature of 30°C. As can be seen from the plot, the vials which have undergone the 15 FTF treatment have already reached the shelf temperature of 30°C at the 41 h. This is indicative of a complete sublimation. On the contrary, the vials (F) that have undergone a standard treatment have not yet reached the shelf temperature after 41 h. In other worlds they have not yet completed the sublimation process.

20 [0138] The results indicate that the primary drying step is shortened in the vials which have undergone the FTF treatment.

[0139] Subsequently, the vials were brought at atmosphere pressure. As the pressure raises in the standard vials, the ice core started melting and the lyophilized collapsed. 25 On the contrary, in the FTF vials the lyophilized cakes remained intact. (Fig. 3). [0140] Example 3

[0141] 20R vials were charged with 18 ml of a solution as disclosed in Table 3. The resulting fill height was of ca. 31 mm.

Table 3

[0142] The arginine-phosphate solution was prepared by dissolving L-arginine in water for injection and the pH was adjusted with phosphoric acid at pH 7.2.

The solutions were sterilized and the vials were placed in a lyophilizer and processed according to the following schedule (Table 4):

[0143] After the thawing of the first vials, additional vials (Standard vials) with the same quantity of solution were charged in the lyophilizer and frozen (i.e. the latter vials undergo the "standard" freezing step). [0144] In Fig. 4 it is shown how the vials plate was charged in the lyophilizer. From the plot in Fig. 5, it can be seen that a complete thawing was performed. In the FTF-vials the drying step (sublimation) occurs faster than in the standard vials. As shown in the plot, the standard-vials reach the shelf temperature more slowly than the FTF-vials. Moreover, not all the standard vials reached the shelf temperature (i.e. a complete sublimation) when the process was stopped (57.06 h). This is indicative of a slower sublimation due to a denser structure of the standard vials. The compacter structure especially in upper part of the standard lyophilized cake can be also seen in Fig. 8 wherein a section of the structure the lyophilized cake of example 3 (vertical cut) is shown. It was not possible to obtain a vertical cut of the standard sucrose lyophilized cake due to the high content of residual water still present in the sample.

[0145] Example 4

As in example 3, 20R vials were charged with 18 ml of a solution at 5% sucrose or at 5% trehalose. The resulting fill height was of ca. 31 mm. The solutions were 0.22 μηι filtered and processed with the same parameters of example 3. The drying step was however longer with respect to the drying step of example 3 so has to obtain the complete sublimation of all the vials (FTF and standard). The process was manually stopped after 90h when all the vials reached the niveau of the shelf temperature. In Fig. 9 it is shown how the vials plate was charged in the lyophilizer.

[0146] From the plot in Fig. 10, it can be seen that in the FTF-vials the drying step (sublimation) occurs faster than in the standard vials. As shown in the plot the standard- vials reach the shelf temperature more slowly than the FTF-vials. The FTF-vials reaches the shelf temperature with a steeper temperature rise. This is indicative of a faster sublimation due to a lower density of the lyophilized cakes especially in the upper part of the cakes of the FTF vials. The sublimation pattern of the standard vials shows, on the contrary, a non-homogeneous pattern with different times and gradient. [0147] The macroscopic structure of the lyophilized cake obtained it has been studied. With the purpose of identifying defects in the cake the vials were photographed from the bottom. Approximately, the half of the sucrose cakes obtained with the standard method show evident defects (Fig. 1 1 right side). These defects can derive by the compact surface layer when the sublimation step does not occur sufficiently fast and a local increased of pressure leads to an overriding of the glass transition temperature. The FTF-cake do not show defect. In addition one third of the standard vials show a melting which was not present in the FTF-vials. [0148] Further on the surface of the cakes obtained in example 4 was observed with the Scanning Electron Microscopy (SEM). The upper site of the standard sucrose cake does not show any abnormalities. The surface layer of the FTF cakes was sometime very thin and fragile that could not be prepared for SEM analysis. The FTF cakes show big portions with extensive portion with plate-like structure, while the standard cakes show big portions with a dick structure.

[0149] The improved sublimation process has been achieved in the FTF-vials due to a thinner surface layer of the cakes and a bigger ice-crystal structure with respect to the standard vial. The lack of a super concentration on the top of the cake obtained with the FTF process improved the diffusion of the sublimate. In particular, the sucrose lyophilized cakes prepared with the standard process often have defects and melt, while these phenomena are not observed in the cake prepared with the FTF process. The dicker upper layers of the standard cakes prevent a sublimate stream and therefore the drying of the cake. As a consequence a local improved pressure internal to the cake can occur with a consequent improve in the temperature. When temperature is higher than the collapse temperature defect and/or collapse of the standard cake is observed. On the contrary the FTF cake has a structure that assures an optimal sublimate stream and therefore and improved sublimation. Since the FTF cakes do not collapse the vial can have a higher fill height.

[0150] Example 5 Drying of 18 mi of sucrose solution dyed in 20R Vial

[0151] Figure 13 shows the different drying profiles of the two variants: the freeze-thaw-

Freez (FTF) and standard procedures.

[0152] The delay of the freezing and thawing as a whole was about 5 to 6 hours. In contrast, a much shorter drying time, compared to the standard version is obtained with FTF. This is confirmed by at the product temperature probe of FTF variant, the temperature of which rises earlierto the level of the plate temperature (at 55 C in the graph).

[0153] The much stronger counter-cooling at the start of drying indicates a more efficient sublimation compared to the standard version,

[0154] The cycle was approximately 10 h after the end of the sublimation of the FTF variant The variations with the standard process at this stage were still far from the end of the drying and still had a high proportion of non-sublimated ice.

[0155] The much more homogeneous drying process of the FTF is really remarkable while the scattering of the temperature profiles of the standard variation is, at the end of the primary drying, significantly greater. [0156] Figure 14 shows on the left the pattern completely dry and free of defects FTF variant and on the right the standard version with melted ice core. All 44 standard versions after discharge showed complete meltdown by melting residual ice. In contrast, all 44 FTF variants were defect-free. [0157] In summary, in this experiment, in particular, by comparing the temperatures of the product the difference in the process duration of FTF and standard variant was studied and identified:

[0158] It has been observed that an additional time of 5-6 h at the begin of the process for FTF variant means 15 hours or more of time gained at the end of drying, i.e., a net gain of time of at least 10 h. An additional advantage is the uniform drying process of the FTF version. The standard models equipped with temperature sensors had a much stronger scattering at the end of primary drying.

Claims

1. A method for lyophilizing a composition to be lyophilized comprising:

(d) drying the frozen composition c' of step (c)

2. The method of claim 1 wherein the drying step (d) comprises a primary drying step (d ) and optionally further comprises a secondary drying step (d ).

3. The method of claim 1 or 2 wherein the thawing of step (b) is a partial thawing and the composition b' is a partially thawed composition.

4. The method of claims 1 to 3 wherein the composition b' is a composition obtained by thawing at least the 10% (w/w) or at least the 30% (w/w) or at least the 50% (w/w) or at least the 80% (w/w) of the frozen composition a' or of the partially frozen composition a" obtained in step (a).

5. The method of claims 1 to 4 wherein the partial thawing occurs at a shelf temperature above the eutectic melting temperature.

6. The method of claim 1 or 2 wherein the thawing of step (b) is a complete thawing and the composition b' is a completely thawed composition.

7. The method of claims 1 to 6, wherein the freezing in step (a) is conducted at atmospheric pressure.

8. The method of any one of the preceding claims, wherein the freezing in step (a) is conducted at the shelf temperature set at least below the eutectic melting temperature or the lowest eutectic melting temperature of the composition to be lyophilized.

9. The method of any one of the preceding claims, wherein the freezing in step (a) is conducted at a shelf temperature of less than -10 °C.

10. The method of any one of the preceding claims, wherein the freezing in step (a) is conducted at a temperature of less than -45 °C, preferably less than -55 °C.

11. The method of any one of the preceding claims, wherein the freezing step (a) is conducted at a shelf temperature set at any temperature between -10 °C and -45 °C.

12. The method of any one of the preceding claims, wherein the thawing in step (b) is conducted at a shelf temperature set at any temperature between 0 °C and 25 °C above the melting point of the solvent or wherein, when the composition to be lyophilized comprises a mixture of solvents, the thawing in step (b) is conducted at a shelf temperature set at any temperature between 0 °C and 25 °C above the melting point of the solvent having the highest melting point.

13. The method of claim 12 wherein the shelf temperature is set at 20 °C above the melting point of the solvent or above the melting point of the solvent having the highest melting point.

14. The method of any proceeding claims wherein the composition during the thawing step (b) reaches a temperature from 5 °C to 20 °C above the eutectic melting point.

15. The method of claims 1 to 5 and 7 to 14 wherein the composition during the partial thawing step (b) reaches a temperature above the eutectic melting point.

16. The method of any one of the preceding claims, wherein the freezing in step (c) is conducted at atmospheric pressure.

17. The method of any one of the preceding claims, wherein in the re-freezing step (c) the shelf temperature is set at least below the eutectic melting temperature of the composition or below the lowest eutectic melting temperature of the composition.

18. The method of any one of the preceding claims, wherein the freezing in step (c) is conducted at a shelf temperature of less than -10 °C.

19. The method of any one of the preceding claims, wherein the freezing in step (c) is conducted at a shelf temperature of less than -45 °C.

20. The method of any one of the preceding claims, wherein the freezing step (c) is conducted at a shelf temperature set at any temperature between -10 °C and -55 °C.

21. The method of any one of the preceding claims, wherein the drying in step (d) or the primary drying step (d') is conducted at a shelf temperature between -30 °C to +30 °C.

22. The method of any one of the preceding claims, wherein the drying in step (d) or the primary drying step (d') is conducted at a shelf temperature set at any temperature between 20 °C and 40 °C.

23. The method of any preceding claims wherein the composition comprises a solvent or a mixture of solvents selected from water, DMSO, glacial acetic acid, anisole, benzylalcohol, tert-butanol.

24. The method of any preceeding claims wherein the composition to be lyophilized comprises a food product or wherein the composition to be lyophilized comprises a pharmaceutically active ingredient.

25. The method of claim 24, wherein the pharmaceutical active ingredient is selected from proteins, peptides, virus, nucleic acids, antibodies, small molecules.

26. The method of claim 24 or 25, wherein the composition to be lyophilized, preferably when it contains a pharmaceutically active ingredient, comprises at least one of the following components: buffer, tonicity modifier, stabilizer, bulking agent and/or an excipient.

27. The method of any of the preceding claims wherein the lyophilized composition is a pharmaceutical formulation.

28. The lyophilized composition obtainable by the method of any one of claims 1 to 27.

29. An intermediate re-frozen composition obtainable by

(c) re-freezing the composition b' obtained in step (b) and obtaining a frozen composition c'.

30. The intermediate re-frozen composition of claim 29 wherein the thawing step (b) is a complete or a partial thawing.

31. The composition of claims 28, 29 or 30 wherein the thawing of step (b) is a partial thawing and the composition b' is a partially thawed composition, preferably the composition b' is a composition obtained by thawing at least the 10% (w/w) or at least the 30% (w/w) or at least the 50%(w/w) or at least the 80% (w/w) of the composition a' or of the partially frozen composition a" obtained in step (a).

32. The composition of claims 28 to 31 wherein the partial thawing occurs at a temperature above the eutectic melting temperature.

33. The methods and the composition of any of the preceding claims wherein the composition is a liquid composition and contains up to 30% in weight of solute.