MXPA06009231A

MXPA06009231A - Lyophilization method to improve excipient crystallization

Info

Publication number: MXPA06009231A
Application number: MXPA/A/2006/009231A
Authority: MX
Inventors: Juneau Jennifer; Knowles Susan
Original assignee: Juneau Jennifer; Knowles Susan; Wyeth
Priority date: 2004-03-04
Filing date: 2006-08-14
Publication date: 2007-04-10

Abstract

The present invention provides improved methods to lyophilize (freeze-dry) active ingredients such as proteins, nucleic acids and viruses. The present methods improve the degree of excipient crystallization during lyophilization over prior methods. The improvement in excipient crystallization is based, in part, on a high-temperature annealing step that is conducted prior to or at the same time as secondary drying. Importantly, the high-temperature annealing step does not destabilize active ingredients. Further, the high-temperature annealing step does not require sub-zero annealing steps prior to its enactment in order to provide complete excipient crystallization.

Description

METHOD OF IOFICIATION TO IMPROVE THE CRYSTALIZATION OF EXCIPIENT BACKGROUND OF THE INVENTION Due to potential instability and degradation, active ingredients such as proteins, nucleic acids and viruses (e.g., as components of vaccines) often need to be made into solid forms to obtain an acceptable shelf life as pharmaceuticals. The most commonly used method for preparing pharmaceutical substances as a solid protein is lyophilization. Lyophilization traditionally consists of two main stages: (1) freezing a protein solution and (2) drying the frozen solid, under vacuum. The drying stage is further divided into two phases: primary and secondary drying. The primary drying stage tries to eliminate the frozen water or the solvent (sublimation) and the second drying stage tries to eliminate the water or "frozen" non-frozen solvent (desorption). The separation of water or other solvents by lyophilization stabilizes the pharmaceutical formulations but greatly reduces the rate of degradation of the active ingredient. The process inhibits the degradation process by separating the solvent component in a formulation at levels that no longer support chemical reactions or biological growth. Additionally, REF: 174636 solvent separation reduces molecular mobility, reduces the potential for degradative reaction. The separation of the solvents is carried out, first, by freezing the formulation in such a way that the freezing process separates the solvent or the solvents from the solutes and immobilizes any non-frozen solvent molecule in the interstitial regions between the crystals. frozen solvent. The solvent is then separated by sublimation (primary drying) and then by desorption (secondary drying). The solutes in the solution before lyophilization comprise the protein or drug of interest (active ingredient) and the inactive ingredients (excipients). At the time of lyophilization, the excipients may remain in the same phase as the protein or the phases of that phase containing the protein (active ingredient) may be separated. The phase that contains the protein is typically an amorphous phase. When the excipients are phase separated from the phase containing the protein, they can form a crystalline phase or an amorphous phase. In addition, crystallizing excipients are commonly used in lyophilized products as bulking agents and sometimes as stabilizers. Commonly used crystallizing excipients include amino acids such as glycine, polyols such as mannitol and salts such as sodium chloride. It is usually desirable to crystallize the excipients so that they are completely crystalline after lyophilization. If the crystallizing excipients do not fully crystallize after lyophilization, they may remain in the same amorphous phase as the protein. This can destabilize protein by allowing greater molecular mobility. Complete crystallization improves drying and cake structure, thereby reducing final residual moisture levels. Full crystallization also prevents unwanted crystallization from occurring during storage. Since a higher degree of crystallization reduces the amount of amorphous material, a higher glass transition temperature is obtained. Typically, crystallization of these kinds of excipients is carried out through a low temperature annealing step carried out at temperatures below zero (degrees centigrade) before primary drying. However, this annealing process below zero is slow and does not always result in sufficient or complete crystallization. In particular, the mixing of glycine and sodium chloride inhibits the crystallization of any excipient and said annealing process at low temperature is ineffective in promoting crystallization, since multiple low temperature annealing steps are necessary to further crystallize the excipient.

SUMMARY OF THE INVENTION The present invention provides improved methods for lyophilizing (drying and freezing) active ingredients including proteins, nucleic acids and viruses. The present methods improve the degree of crystallization of excipient during lyophilization with respect to the above methods. The improvement in the crystallization of excipients is based, in part, on the introduction of an annealing step before secondary drying. This annealing step occurs at high temperature (higher than 0aC) and does not require multiple annealing steps below zero before its action. Although the active ingredients, such as proteins, viruses and nucleic acids are technically inherently stable such as at high temperature exposure which causes degradation, the present invention provides an unexpected finding that high temperature annealing does not cause degradation or instability of the active ingredients. In addition, the present invention provides lyophilized products made by the present lyophilization methods, wherein the products comprise glycine and sodium chloride and wherein the glycine is substantially completely crystallized or more crystallized (or is more crystalline) with respect to to the above methods so that it does not include high temperature annealing. In one aspect, the present invention provides a method for lyophilizing an aqueous pharmaceutical formulation, the method comprising: (a) freezing the aqueous pharmaceutical formulation at a temperature of less than -10 SC; (b) drying the pharmaceutical formulation of step (c) at a temperature between about -35 ° C of about 20 ° C; (c) annealing the pharmaceutical formulation of step (b) at a temperature greater than about 252C; and (d) drying the pharmaceutical formulation of step (c) at a temperature lower than the temperature used in step (c). In one aspect of the present invention, the temperature and step (a) is less than -35 ° C and the freezing is carried out for a duration of more than 1 hour. In another aspect, the temperature in step (b) is between about -30 ° C and about 20 ° C or between about -25 ° C and about 10 ° C, or about 0 ° C. In another aspect, the temperature in step (c) is between about 25 ° C and about 75 ° C, or between about 35 ° C and about 60 ° C, or at about 50 ° C. In another aspect, the temperature in step (d) is between about 25 ° C and about 35 ° C; in one aspect, the temperature in step (d) is about 25 ° C. The aqueous pharmaceutical formulation which is lyophilized by the present methods can contain essentially any active ingredient, which instructs, but is not limited to proteins, peptides, nucleic acids and viruses.

In another aspect, the present invention provides a method for lyophilizing an aqueous pharmaceutical formulation, the method comprising: (a) freezing the aqueous pharmaceutical formulation at a temperature of -10 ° C; (b) annealing the pharmaceutical formulation of step (a) at a temperature between about -35 ° C and about 0 ° C; (c) drying the pharmaceutical formulation of step (b) at a temperature between about -35 ° C and about 10 ° C; (d) annealing the pharmaceutical formulation of step (c) at a temperature between about 25 ° C and about 75 ° C; and (e) drying the pharmaceutical formulation of step (d) at a temperature lower than the temperature used in step (d). In another aspect, the temperature in step (b) is between about -25 ° C and -10 ° C, or between about -20 ° C and -10 ° C, or at about -15 ° C. In another aspect, the temperature in step (c) is between about -30 ° C and 5 ° C or between about -25 ° C and 10 ° C, or between about -20 ° C and 0 ° C, or between about -20 ° C and -10 ° C, or at approximately 0 ° C. In another aspect, the temperature in step (d) is between about 35 ° C and 60 ° C or at about 50 ° C. In another aspect, the temperature in stage (e) is approximately 25 ° C. In still another aspect, the methods may further comprise a freezing step which is carried out after step (b) and before step (c), wherein the freezing step comprises freezing the formulation at a temperature of - 35 ° C, or at about -40 ° C to about -50 ° C. In another aspect, the present invention provides lyophilization methods wherein the aqueous pharmaceutical formulation comprises at least one crystallization excipient. One or more crystallization excipients may be selected from the group consisting of an amino acid, a salt and a polyol. In one aspect, the amino acid is glycine or histidine. In another aspect, the salt is sodium chloride. In another aspect, the polyol is mannitol. In one aspect, the aqueous pharmaceutical formulation comprises a combination of crystallization excipients, wherein the combination is a salt and an amino acid. In one aspect, the salt in the combination is sodium chloride, wherein the sodium chloride is present in the formulation in a concentration greater than about 25 M, or at a concentration between about 25 mM and 200 mM, 30 mM and 100 mM or 40 mM and 60 mM, or at a concentration of approximately 50 mM. In another aspect, the amino acid in the combination is present in the formulation in a concentration between about 1% to about 10%, 1.5% to 5%, 1.5% to 3% or to about 2%. In another aspect, the amino acid in the combination is glycine. In one aspect, the present invention provides a method for lyophilizing an aqueous pharmaceutical formulation, wherein the method comprises: (a) freezing the aqueous pharmaceutical formulation at a temperature of -35 ° C; (b) optionally annealing the pharmaceutical formulation of the stage (a) at a temperature between about -20 ° C and about -10 ° C; (c) drying the phceutical formulation of step (b) at a temperature between about -10 ° C and about 10 ° C; (d) annealing the phceutical formulation of step (c) at a temperature between about 35 ° C and about 60 ° C or between about 35 ° C and about 50 ° C; and (e) drying the phceutical formulation of step (d) at a temperature lower than the temperature used in step (d). In one aspect, the present invention provides a method for lyophilizing an aqueous phceutical formulation, comprising sodium chloride and glycine, wherein the method comprises: (a) freezing the aqueous phceutical formulation at a temperature of -35 ° C; (b) optionally annealing the phceutical formulation of step (a) at a temperature between about -20 ° C and about -10 ° C; (c) drying the phceutical formulation of step (b) at a temperature between about -10 ° C and about 10 ° C; (d) annealing the phceutical formulation of step (c) at a temperature between about 35 ° C and about 50 ° C; and (e) drying the phceutical formulation of step (d) at a temperature lower than the temperature used in step (d).

In another aspect, the present invention provides a method for lyophilizing an aqueous phceutical formulation, comprising sodium chloride in a concentration greater than 35 mM and glycine in a concentration between approximately 250 mM and approximately 300 mM, or glycine in a concentration of approximately 250 mM and approximately 270 mM, wherein the method comprises: (a) freezing the aqueous phceutical formulation at a temperature of -35 ° C; (b) annealing the phceutical formulation of step (a) to about -15 ° C; (c) drying the phceutical formulation of step (b) at about 0 ° C; (d) annealing the phceutical formulation of step (c) to about 50 ° C and (e) drying the phceutical formulation of step (d) at about 25 ° C. This method may further comprise a refreezing step after step (b) and before step (c) wherein the refreezing step comprises freezing the phceutical formulation of step (b) at about -40 ° C to about - 50 ° C. In one aspect, the present invention provides a method for increasing the crystallization of excipient during lyophilization, comprising: (a) providing an aqueous phceutical formulation comprising sodium chloride and another bulking agent such as glycine; (b) freezing the aqueous phceutical formulation; (c) optionally annealing the phceutical formulation of step (b) at a temperature between about -35 ° C and about 0 ° C, or between about 20 ° C and about -10 ° C; (d) drying the phceutical formulation of step (b) or step (c) at a temperature between about -35 ° C and about 10 ° C or between about -5 ° C and about 5 ° C; (e) annealing the phceutical formulation of step (d) at a temperature between about 25 ° C and about 75 ° C, so that the bulking agent and / or the sodium chloride is more crystallized after the step ( e) before stage (e); and (f) drying the phceutical formulation of step (e) at a temperature that is the same or lower than the temperature used in the step (e), so that the crystallization of excipient is increased.

In this method, the bulking agent may comprise glycine, alanine or mannitol (in addition to sodium chloride) for example. In one aspect, the bulking agent is glycine. In another aspect, this method for increasing the crystallization of excipient during lyophilization further comprises a refreezing step carried out after step (c) and before step (d) wherein the refreezing step comprises freezing the formulation of step (c) at a temperature between about -40 ° C and -50 ° C or at a temperature of about -50 ° C. In another aspect, the present invention provides a lyophilized product produced by a process comprising: (a) providing a formulation comprising glycine and sodium chloride; (b) freeze the formulation; (c) optionally annealing the formulation to step (b) - at a temperature between about -35 ° C and about 0 ° C; (d) drying the formulation of step (c) at a temperature between about -35 ° C and about 10 ° C; (e) annealing the formulation of step (d) at a temperature between about 25 ° C and about 70 ° C: and (f) drying the formulation of step (e) at a temperature that is the same or less than the temperature used in step (e) and in this way the lyophilized product is provided. The active ingredient in the formulation that is lyophilized by this method may comprise a protein, a nucleic acid or a virus. In addition, the glycine in the lyophilized product after step (f) may be more crystalline than before step (e). In one aspect, the present invention provides a lyophilized product produced by the process comprising: (a) providing a formulation comprising glycine and sodium chloride; (b) freeze the formulation; (c) optionally annealing the formulation of step (b) at a temperature between about -20 ° C and about -10 ° C; (d) drying the formulation of step (c) at a temperature between about -5 ° C and about 5 ° C; (e) annealing the formulation of step (d) at a temperature between about 35 ° C and about 60 ° C or between about 35 ° C and about 50 ° C; and (f) drying the formulation of step (e) at a temperature that is the same or lower than the temperature used in step (e) and thus a lyophilized product is provided. In another aspect, the glycine in the lyophilized product after step (f) is substantially completely crystallized or is more crystalline than without step (e) (or more crystalline with respect to a lyophilization method not comprising the step of annealing at high temperature before secondary drying). In another aspect, the lyophilized product is substantially stable for extended periods of time at high storage temperatures or accelerators. Prolonged periods of time may be, for example, at least 1 month, 3 months, 6 months, 1 year or longer. The high or accelerated storage temperatures can be, for example, between about 25 ° C and about 50 ° C. The stability can be tested, for example, by the percentage of HMW entities present in a lyophilized product, the concentration of the active ingredient and the activity of the active ingredient. BRIEF DESCRIPTION OF THE FIGURES Figure 1A shows a first exploration) and Figure IB shows a second differential scanning calorimetry (DSC) scan representative of a solid cake of formulation 1 lyophilized according to the cycles Lyo G, H ol (see table 2 for contents of formulations 1, 2 and 3, see tables 3, 4 and 5 for stages of cycles of Lyo G, H and I, see example 1 for description of experiments). A crystallization event is observed in the first explorations of the solid cakes of the formulation 1 lyophilized according to the cycles Lyo G, H and I, which indicates a lack of complete or substantial crystallization in the cakes. As stated herein,. when a - first DSC scan shows crystallization, this indicates that complete or substantially complete crystallization does not occur in a lyophilized cake. In this way, the first exploration shows that a crystallization event occurred and the second exploration conforms that the exothermic event was recrystallization. Figure 2A shows a first scan and Figure 2B shows a second DSC scan representative of a solid cake of formulation 2 lyophilized according to the cycles Lyo G, H or I (see table 2 for contents of formulations 1, 2 and 3; see tables 3, 4 and 5 for cycle stages of Lyo G, H and I, see example 1 for description of experiments). A crystallization event is observed in the first explorations of the solid cakes of formulation 2 lyophilized according to the cycles Lyo G, H and I, which indicates a. lack of complete or substantial crystallization in the cakes. The second explorations confirm that the exothermic event observed in the first explorations is crystallization. In addition, the second scans do not show a Tg transition. Figure 3A shows a first scan and Figure 3B shows a second DSC scan representative of a solid cake of formulation 3 lyophilized according to the cycles Lyo G, H or I (see table 2 for contents of formulations 1, 2 and 3; see tables 3, 4 and 5 for cycle stages of Lyo G, H and I, see example 1 for description of experiments). A crystallization event is observed in the first explorations of the solid cakes of the formulation 3 lyophilized according to the cycles Lyo G, H and I, which indicates a lack of complete or substantial crystallization in the cakes. The second explorations confirm that the exothermic event observed in the first explorations is crystallization. In addition, the second scans do not show a Tg transition. Figure 4A shows a first scan and Figure 4B shows a second DSC scan of a solid cake of formulation 4 ("fix9271yoJ.001"; the line with. a graph of long dashes and dots), formulation 5 ("fix502501yoja.001", line without dashes) and formulation 6 ("fix502701yoja.001", dashed line), lyophilized according to the Lyo J cycle (see table 7 for contents of formulations 4, 5 and 6, see tables 8, 9 and 10 for the stages of the cycles of Lyo J, K and L, see example 2 for description of experiments). Table 11 summarizes the data from the first and second DSC scans in Example 2, where formulations 5 and 6 are lyophilized according to Lyo J which shows crystallization on the first scan and no Tg transition on the second scan. Figure 5A shows a first scan and Figure 5B shows a second DSC scan of a solid cake of formulation 4 ("fix927yoK.002" the line with long dashes and dots), formulation 5 ("fix50250yok.001"; dashes) and formulation 6 ("fix50270yok.001", dashed line), lyophilized according to the Lyo K cycle (see table 7 for contents of formulations 4, 5 and 6, see tables 8, 9 and 10). for the stages of the cycles of Lyo J, K and L, see example 2 for description of experiments). Figure 5C represents data from another second exploration of the solid cake of formulation 4 ("fix 927 lyo K") lyophilized by cycle Lyo K. Table 11 summarizes the data of the first and second DSC scans in example 2, wherein formulations 4, 5 and 6 are lyophilized according to Lyo K do not show crystallization on the first scan and have a Tg transition on the second scan. Figure 6A shows a first scan and Figure 6B shows a second DSC scan of a solid cake of formulation 4 ("fix9271yol .002" the dashed line), formulation 5 ("fix502501yoL.001", line without dashes) and formulation 6 ("fix502701yoL.001", dashed line), lyophilized according to the Lyo L cycle (see table 7 for contents of formulations 4, 5 and 6, see tables 8, 9 and 10 for the stages of the Lyo J, K and L cycles, see example 2 for description of experiments). Figure 6C depicts data from another second exploration of the solid cake of formulation 4 ("fix 927 lyo L") lyophilized by cycle Lyo L. Table 11 summarizes the first and second DSC scan data in example 2, in where formulations 4, 5 and 6 are lyophilized according to Lyo L do not show crystallization on the first scan and have a Tg transition on the second scan. Figure 7 shows the percentage of high molecular weight present in the pre-lyophilized formulations and in the post-lyophilized cakes of the formulations 4, 5 and 6 lyophilized according to Lyo J, K and L (see example 2). For each formulation, 10 bottles are tested and assays are analyzed in triplicate. The term "PCTRL" indicates formulation 4 prior to lyophilization; the term "CTRL" indicates formulation 4 after lyophilization. The term "P50 / 250" indicates the formulation 5 before lyophilization; the term "50/250" indicates the formulation '5 after lyophilization. The term "P50 / 270" indicates formulation 6 prior to lyophilization; the term "50/270" indicates the formulation, 6 after lyophilization. Figure 8A represents the coagulation activity and Figure 8B shows the recovery percentage of the coagulation activity of the factor IX protein in the pre-lyophilized formulations and the post-lyophilized cakes of the formulations 4, 5 and 6, lyophilized in accordance with Lyo J, K and L (see example 2). For each formulation 8 flasks are tested by post-lyophilization formulation. Due to a 4 ml fill and a 5 ml reconstitution dilution, 80% recovery is the highest recovery value expected in Figure 8B. Figure 9 represents the recovery rate of specific activity of factor IX protein in pre-lyophilized formulations and post-lyophilized cakes of formulations 4, 5 and 6, lyophilized according to Lyo, J, K and L (see example 2). Figure 10A depicts X-ray diffraction patterns (XRD) on the cake of the lyophilized formulation 2 according to Lyo G and Figure 10B shows on the cake of. the formulation 6 lyophilized according to Lyo L. The patterns of XRD indicate that crystalline glycine is present in the lyophilized samples. In addition, the XRD standards show a qualitative pattern such that a glycine / sodium chloride formulation lyophilized by Lyo L has more crystalline material than a lyophilized glycine / sodium chloride formulation by Lyo G. The maximum XRD heights are relative to the crystallinity, where the highest peaks reflect the presence of more crystalline material. Figure 11 shows a XRD pattern on the cake of the lyophilized formulation 7 according to Lyo Q (see example 3). The XRD pattern for Lyo Q is compared to an XRD pattern for Lyo G. Lyo G is used for comparison because it includes the same lyophilization cycle parameters as Lyo Q, except that Lyo G does not have a heat treatment at 50 ° C. The comparison of the XRD pattern shows that lyophilization by Lyo Q results in an increase in the crystallization of glycine, as can be observed by the intensity of the peak at 17.5 °. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved process for lyophilizing pharmaceutical formulations. Lyophilization is important, in part, because it helps stabilize active ingredients (such as proteins, nucleic acids and viruses) or by decreasing or preventing degradation. The present methods provide improved crystallization of excipient during the lyophilization steps so that in this way the stability and efficacy of the lyophilized products is improved.

It is usually desirable that the crystallization excipients be completely crystalline after lyophilization because if the crystallization excipients are left in an amorphous phase containing protein or medicament, the excipients reduce the vitreous transition temperature (of that phase). An amorphous phase with a reduced Tg may increase molecular mobility. An increased molecular mobility can allow increased rates of degradation reactions. Thus, for a drug that is expected to be in the -amorpha phase, the increase in vitreous transition temperature of that phase will result in improved stability. Conversely, a formulation with a decreased vitreous transition temperature due to a non-crystallized excipient still remaining in the amorphous phase due to poor lyophilization methods, can be expected to have poorer stability. The above methods crystallized excipients through one or more annealing steps carried out at temperatures below zero centigrade before primary drying. However, this annealing process at temperatures below zero is slow and does not always result in complete crystallization. In particular, the mixture of glycine and sodium chloride inhibits the crystallization of any excipient, and such an annealing process at low temperature is not effective to improve the crystallization of excipient since longer cycles are required. In contrast, the present invention provides methods that introduce a high temperature annealing step prior to secondary drying so that multiple de-annealing steps below zero are not required for enhanced crystallization of excipients. In addition, although active ingredients such as proteins, viruses and nucleic acids are inherently thermally unstable so that exposure to high temperature causes degradation, the present invention provides the unexpected finding that high temperature annealing does not cause degradation or instability of the active ingredients. In this manner, the present invention provides effective methods for improving the crystallization of excipient during lyophilization, including improving the crystallization of excipients of the formulations comprising both glycine and sodium chloride. As used herein, the terms "lyophilization", "lyophilization" and "freeze-drying" relate to a process such as "freezing" a solution followed by "drying". In general, the lyophilization methods of the present invention comprise the following steps: (1) freezing, (2) primary drying, (3) annealing at high temperature, at a temperature greater than about 25 ° C, and (4) drying secondary to a temperature that is the same or lower than the temperature in the high temperature annealing stage. Prior to primary drying one or more optional low temperature annealing steps may be carried out and an optional refreezing step may be carried out after the low temperature annealing step. The present invention provides the unexpected finding that the high temperature annealing step prior to secondary drying does not destabilize the active ingredient and therefore the present methods are capable of improving the crystallization of excipient and at the same time provide a freeze-drying protocol more effective, practical or robust. The present lyophilization methods allow an increased degree of crystalline bulking agents with respect to the above methods, and at the same time maintain the stability and activity of the active ingredient. The present invention is sometimes referred to as the objective of complete crystallization of excipient and a person skilled in the art understands that "complete crystallization" is difficult to verify, because the current sensitivity of the technology can not inform us with absolute certainty of that the excipient has been crystallized 100%. Thus, in practical terms, the invention provides lyophilization methods that improve the crystallization of excipient with respect to the above methods. Accordingly, as used herein, the term "complete crystallization" of lyophilized products can be determined, for example, by differential scanning calorimetry (DSC), where a person skilled in the art recognizes that a non-reversible exothermic event. in a first scan it represents a crystallization event, which indicates that the crystallization excipient does not completely crystallize during lyophilization (see examples). Lyophilization Procedures Lyophilization cycles traditionally include three phases: freezing (heat treatment), primary drying (sublimation) and secondary drying (desorption). In various embodiments, the present invention improves traditional lyophilization processes by introducing one or more annealing steps prior to secondary drying, wherein the annealing and drying step occurs at specific temperature ranges. In the present invention, the specific temperatures and temperature ranges of a lyophilization process refer to the shelf temperature of the lyophilizing equipment, unless otherwise indicated. The shelf temperature refers to the control temperature for a refrigerant flowing through the freeze dryer shelves, which is the one that controls in terms of temperature during lyophilization. The temperature of the sample (the temperature of the product) depends on the shelf temperature, the chamber pressure and the evaporation / sublimation rate during primary drying (an evaporative cooling generates product temperatures less than the shelf temperature). The present invention provides an improved lyophilization process in order to provide, for example, a more consistent, stable and aesthetically acceptable product. In the present invention, the percentages are weight / weight when referring to solids and weight / volume when referring to liquids. Terms of Formulas: The formation of ice (solvent crystal) during the cooling of a pharmaceutical formulation concentrates all the solutes. The solute concentration finally changes the solution from a liquid to a glass. The temperature of this reversible transition of a frozen-concentrated solution is called the vitreous transition temperature, Tg ', of the frozen solution-maximally concentrated. This temperature is also called the vitreous transformation temperature Tg 'which is used to differentiate this transition from the softening point of a true vitreous transition Tg of a pure polymer. The collapse temperature, Tco ?, is the temperature at which the interstitial water in the frozen matrix becomes significantly mobile.

For reference, table 1 includes a list of some commonly used excipients and buffers (as well as proteins, when these proteins are not the active ingredient of a formulation but rather additional elements to a formulation). Table 1: List of buffers, excipients and proteins Compound Buffering agents: citric acid; Hepes, histidine; potassium acetate, potassium citrate; Potassium Phosphate; sodium acetate; sodium bicarbonate; sodium citrate; Tris (tromethamine) -base; Tris-HCl. Excipients of low molecular weight: β-alanine; arabinose; arginine; cellobiose; fruitful fucose; galactose; glucose; glutamic acid; glycerol; glycine; histidine; lactose; lysine; maltose; maltotriose; mannitol; crafty; melibious octulose; raffinose; ribose; sodium chloride; sorbitol (glucito); saccharose; trehalose; Water; xylitol; xylose (a person skilled in the art will understand that some of these excipients are crystallization excipients, such as glycine, mannitol and sodium chloride, and some of these excipients generate amorphous phases) Excipients with high molecular weight: cellulose; β-cyclodextrin; dextran, Ficoll, gelatin, hydroxypropylmethylcellulose, hydroxyethyl starch, maltodextrin 860, metocel, PEG, polydextrose, PVP, Sephadex, protein, BSA, a-casein, globulins, HSA, a-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, ribonuclease, TO.

When the temperature of an aqueous formulation falls below 0 ° C, the water is usually separated by crystallization first. Then, depending on the freezing rate, the crystallizable components having the lowest solubility in the formulation can then crystallize. This temperature (the temperature at which crystallizable components crystallize in a formulation) is called the crystallization temperature. When the temperature of an aqueous formulation further decreases after crystallization of the less soluble component, one or more of the crystallizable components and the water are separated by crystallization at the same time, as a mixture. This temperature is called the temperature of eutectic crystallization / fusion 7eut- Due to the - interactions with the excipient, some formulations of multiple components do not - present Teut. Freezing: The first stage in lyophilization is the freezing stage. The formulation or sample is frozen solid, which converts the water content of the material into ice.

In one embodiment, the freezing of an aqueous pharmaceutical formulation can be carried out at a temperature below -10 ° C. In another embodiment, the freezing can be carried out at or below -35 or -50 ° C. In the present invention, once the freezing temperature (as in the shelf temperature) reaches a target temperature of between about -35 ° C to about -50 ° C, for example, then the freezing temperature is maintained (a "freeze maintenance" stage) until the sample is frozen, or for about 1 hour to about 24 hours, for about 3 hours to about 12 hours, for about 5 hours to about 10 hours, or for about 5 hours . The freezing time depends on factors such as the volume of the solution per bottle, regardless of the composition of the formulation. Annealing at low temperature (one optional step): In the present invention, the step of annealing at low temperature is optional. The above methods were used - one or more steps of low temperature annealing prior to drying because the crystallizable component may not have crystallized "fully or sufficiently." However, these two previous methods are not effective as their stages of annealing at low temperature require prolonged or multiple cycles, and these prior methods are insufficient to promote sufficient or complete crystallization.The complete crystallization of a formulation component can provide a necessary cake structure or an active ingredient can be more stable in the formulation with complete crystallization The separation of the amorphous phase from a crystalline component such as glycine can increase the Tg 'of the amorphous phase.This increased Tg' can allow a more efficient primary drying at a higher temperature., a more complete crystallization of an excipient can also increase the Tg after lyophilization, which is critical for the stability of the active ingredient. In the present invention, an annealing step can be carried out at low temperature, at a temperature of about -35 ° C to about 0 ° C, or between about -25 ° C and -10 ° C, or between about - 20 ° C and -10 ° C. In one embodiment, the low temperature annealing step is carried out at a temperature of about -15 ° C. The temperature of the low temperature annealing step is obtained by increasing the temperature from the freezing step (also referred to as "freezing maintenance"). This method of regulating the temperature increase from the freezing temperature to the annealing temperature at low temperature is called the step of "gradual annealing change" which is optional in the present invention. The gradual annealing change step can be carried out at different speeds, for example at about 0.1 ° C to about 5 ° C per minute. Re-freezing (an optional step): The lyophilization methods of the present invention also encompass the option of including a refreezing step after the low temperature annealing step. The refreezing step can be carried out at a freezing temperature (freezing holding temperature) or between about -35 ° C to about -50 ° C for about 1-10 hours, 3-7 hours or 5 hours. The stage of gradual change of refreezing can be carried out at a rate of about -0.5 ° C to 5 ° C per minute, for example. Start of vacuum: Directly before primary drying, the formulation is placed under vacuum at the temperature of the stage directly prior to primary drying. This stage is called "vacuum start". Thus, for example, the refreezing stage is prior to the primary drying, so that the onset of vacuum occurs at the temperature of the refreezing stage. The vacuum may be at a level of between about 20 to about 300 microns. Once the vacuum is started, the vacuum is present during the remainder of the lyophilization process, although the level of vacuum may change. Primary drying: Drying is divided into two phases: primary and secondary drying. Primary drying eliminates frozen water (sublimation of ice) and secondary drying removes water "bound" not frozen (desorption of water). In primary drying, the objective is to separate unbound ice, which can be easily removed from the sample. Unbound water at the beginning of the primary drying stage must be in the form of free ice, which is separated by converting it directly from solid to steam, where the conversion process is called sublimation. In the present invention, the primary drying step can be carried at a temperature between about -35 ° C and about 20 ° C, or between about -25 ° C and 10 ° C, or between about -20 ° C and 0 ° C. In one embodiment, the primary drying step is carried out at 0 ° C. The regulation of the temperature increase from the previous stage to the primary drying to the primary drying temperature is called the "gradual drying primary change" stage, which is an optional step. The step of gradual change of primary drying can be carried out at a rate of about 0.1 ° C to about 5 ° C per minute. The primary drying step can be carried out for a sufficient time to ensure that substantially all of the frozen water is separated from the sample. A person skilled in the art will understand that the primary drying time varies with the configuration and that the duration of the primary drying depends on the filling volume and the geometry (surface area of the cake-strength / flow). In one embodiment, the duration of the primary drying is greater than 10 hours, in another embodiment it is from approximately 10 to approximately 100 hours. In another embodiment, the duration of the primary drying is about 30 to 50 hours. In another embodiment, the duration of the primary drying is 38 hours. Several methods can be used to verify that the primary drying stage has been completed. One method is to observe changes in product temperature during freeze-drying (lyophilization). Another method is to observe changes in chamber pressure, where, when sublimation ends, there are no more water molecules in the chamber that contribute to changes in pressure. The end of the primary drying stage occurs when the temperature of the product (sample) approaches the shelf temperature, which becomes evident by a significant change in the slope of the product temperature trace due to a reduced speed of the product. sublimation; When the sublimation ends, the evaporative cooling ends. To prevent premature completion, additional 2 to 3 hours of primary drying can be added to the duration. Another method to determine that the primary drying has finished is the temperature increase test. When disconnecting the vacuum source, the chamber pressure should be increased at a rate that depends on the amount of moisture in the product. The end of the primary drying process can be established when the rate of pressure increase is less than a specified value. Another method for determining the completion of the primary drying step is the measurement of the heat transfer rate (Jennings, T.A., Duan, N. (1995), J-Parent, Sci, Technol, 49, 272-282). High temperature annealing: The methods of the present invention include one or more stages of annealing (or heat treatment) at high temperature before secondary drying. The above methods report that annealing steps less than zero are necessary before primary drying when the secondary drying is carried out at high temperatures. However, the present invention discloses methods that do not require annealing steps below zero in order to perform secondary drying at high temperature. The present invention has determined that annealing steps below zero, before drying at high temperature are not necessary if a high temperature annealing step is carried out during or directly before secondary drying. The invention has determined that the high temperature annealing step improves the crystallization of the excipient, which includes crystallization of glycine and at the same time maintains stability of the active ingredient. Thus, the present invention provides a step of high temperature annealing before secondary drying, wherein the step of annealing at a high temperature is carried out at a temperature greater than about 25 ° C. In one embodiment, the temperature of the high temperature annealing step is from about 25 ° C to about 75 ° C, or about 35 ° C to about 60 ° C. In another embodiment, the high temperature annealing step is carried out at a temperature of about 50 ° C. In other embodiments, the high temperature annealing step is carried out at a temperature of about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60 ° C. In another embodiment, the temperature of the annealing step is a temperature or above the temperature observed by differential scanning calorimetry corresponding to the start of the crystallization event. A person skilled in the art may contemplate using a temperature slightly lower than the crystallization onset temperature recognizing that the kinetics of crystallization may be less and the duration of the step may be longer. Temperatures greater than the crystallization onset temperatures are preferred because the crystallization kinetics is faster and the duration of the step may be shorter. The regulation of the temperature increase from the primary drying of the annealing at high temperature is called "gradual change of annealing at high temperature" (the stages of gradual change are implicit in the present invention, since the changes in temperature maintenance at other temperature maintenance inherently include some kind of gradual change) and can be carried out at a rate of about 0.1 to about 20 ° C per minute. The duration of the high temperature annealing stage depends on many factors, including the filling volume. The high temperature annealing step can be carried out, for example for 1 hour to about 24 hours. In one embodiment, the high temperature annealing step is carried out for about 1 hour to about 15 hours. In another embodiment, the high temperature stage is carried out for about 10 hours. Surprisingly, the high temperature annealing step of the invention does not impair protein stability or activity (see example 2). This is unexpected because it is known that proteins are thermally unstable. In addition, Example 2 shows that the high temperature annealing step causes an increase in the crystallization of excipient (in this example, glycine). Secondary drying: Even if all of the free ice is separated by the sublimation procedure mentioned above, the sample may still contain enough water bound to limit its structural integrity and shelf life. During secondary drying, the water that is bound to the solids in the product is converted into steam. This can be a slow procedure since the remaining bound water has a lower pressure than the free liquid at the same temperature. Although some of the bound water is separated during pre-drying and annealing methods, secondary drying is required after separation of the free ice to obtain sufficiently low residual moisture concentrations that provide the desired biological and structural characteristics of the final product. Depending on the lyophilization process, mannitol may crystallize as mannitol hydrate. When stored, mannitol hydrate can be converted to crystalline mannitol, releasing water. The water that is released can then: (1) participate in chemical reactions and (2) decrease the Tg of the amorphous phase, allowing more molecular mobility and degradation reactions. The high temperature annealing step can be used to convert crystalline mannitol hydrate to crystalline mannitol so that the remaining water can be removed during secondary drying. In the present invention, the secondary drying step can be carried out at a temperature that is the same or lower than the temperature used in the high temperature stage. In one embodiment, the secondary drying step is carried out at a temperature from about 0 ° C to less than 35 ° C or about 15 ° C to about 35 ° C. In another embodiment, the secondary drying step is carried out at about 25 ° C. The step of regulating the temperature decrease from the high temperature annealing stage with respect to the secondary drying stage is called "gradual secondary drying change" which is an optional step in the present invention. The step of gradual change of secondary drying can be carried out at a rate of temperature decrease from about 0.1 ° C to about 10 ° C per minute. The secondary drying step can be carried out for a sufficient time to reduce the level of residual moisture in the lyophilized product to a desired level. In the invention, a desired residual moisture level is less than 2%. In one embodiment, the residual moisture level of the lyophilized product produced by the methods is less than 1%, 0.75%, 0.5%, 0.25% or 10%. To determine the level of residual moisture in the sample, the Karl Fischer method can be used. In addition, the pressure increase test or the measurement of the heat transfer rate can also be used to determine the end of the secondary drying stage. Alternatively, an electronic hygrometer or a residual gas analyzer can be used (Nail, S.L., Johnson, W., (1992), Dev. Biol. Stand 74, 137-150).

In addition, the minimum duration of the secondary drying can be determined systematically by using different combinations of shelf temperature (where the shelf temperature of the secondary drying stage is the same or lower than the temperature used in the high stage). temperature) and the durations. The residual moisture content of the lyophilized formulations can be determined by various methods including drying loss, Karl Fischer titration, thermal gravimetric analysis (TGA), gas chromatography (GC), or infrared spectroscopy. Lyophilization Formulations A formulation to be lyophilized consists of three basic components: (1) one or more active ingredients, (2) one or more excipients, and (3) one or more solvents. The excipients include pharmaceutically acceptable reagents to provide good freeze-dried cake properties (bulking agents) as well as to provide protection against lyophilization and / or cryoprotection of proteins ("stabilizer"), maintaining the pH (buffering agents) and proper conformation of the protein during storage so that a substantial retention of biological activity (which includes stability of the active ingredient, such as protein stability) is maintained. Thus, in relation to the excipients, an example of a formulation for including one or more of the buffering agents, one or more volume-generating agents, protein stabilizers and antimicrobials. The active ingredient, for example, refers to a reagent or a therapeutic medicament. When the active ingredient refers to a drug, the activity of the drug is related to its potency. When the active ingredient refers to a reagent, the activity of the reagent refers to its reactivity. Sugars / polyols: Many sugars and polyols are used as non-specific protein stabilizers, in solution and during freezing-heating and freezing-drying. The level of stabilization provided by sugars or polyols generally depends on their concentrations. In one embodiment, the present invention contemplates the use of disaccharides in formulations that are to be lyophilized by the methods described. The disaccharides may include, but are not limited to, trehalose, sucrose, maltose and lactose. Other sugars or polyols that can be used include, but are not limited to glycerol, xylitol, sorbitol, mannitol, glucose, inositol, raffinose and maltotriose. Mannitol is a crystallizing polyol that can also be used as a bulking agent.

Polymers Polymers can be used to stabilize proteins in solution and during freeze-reheat and freeze-dry. A popular polymer is serum albumin, which has been used as a cryoprotectant and as a protection against lyophilization. However, the concern about pathogens transported by the blood limits the application of serum albumin in therapeutic products and related to therapeutic treatment. Thus, in one embodiment, the invention provides formulations that are free of albumin, which are lyophilized by the methods described. Other polymers include, but are not limited to, dextran, polyvinyl alcohol (PVA), hydroxypropylmethylcellulose (HPMC), gelatin, polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP). Polymers are not crystallizing excipients since they form amorphous phases. Non-aqueous solvents: Non-aqueous solvents generally destabilize the proteins in solution. At low concentrations, some non-aqueous solvents may have a stabilizing effect. These non-aqueous stabilizing solvents include polyhydric alcohols such as PEG, ethylene glycol, glycerol and some polar and aprotic solvents such as dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). However, non-aqueous solvents are not preferred for use with the present invention. Surfactants: The formation of ice-water contact surface during freezing can cause protein surface denaturation. Surfactants can lower the surface tension of protein solutions and reduce the driving force of adsorption and / or protein aggregation within these limits. In addition, the surfactants may also compete with an active ingredient for the ice / water contact surface during lyophilization. Surfactants may include, for example, Tween 8O101 (Polysorbate 80; or other Polysorbate that is also contemplated), Brij15 35, Brij 30m, Lubrol-px1®, Triton X-IO101, Pluronic1 ^ F127 and sodium dodecyl sulfate (SOS). Salts as agents that provide volume: Various salts can be used as agents that provide volume. Exemplary salts as bulking agents include, for example, NaCl, MgCl 2 and CaCl 2. Amino Acids: Some amino acids can be used as cryoprotectants and / or protection elements against lyophilization and / or agents that provide volume. The amino acids that may be used include, but are not limited to glycine, proline, 4-hydroxyproline, L-serine, sodium glutamate, alanine, arginine and lysine hydrochloride. Short chains of amino acids such as di- or tri-amino acids including dilisin can also be used. Most amino acids are potential agents that provide volume since they are usually separated by crystallization with ease. However, the formation of acid salts reduces their tendency to crystallize. In addition, an amorphous excipient in a protein formulation can inhibit the crystallization of one or more bulking agents and in this way can alter the stability of the protein. Therefore, in the above methods, the combination of glycine and NaCl is not preferable, since NaCl has both low eutectic and low vitreous transition temperatures. In the present invention, the methods provide a high temperature annealing step and a secondary high temperature drying step without the prior annealing steps below zero which increases the degree of crystalline licina even when formulated in the presence of sodium. Buffering agents: Many buffering agents cover a wide pH range and are available for selection in formulations. Buffering agents include, for example, acetate, citrate, glycine, histidine, phosphate (sodium or potassium), diethanolamine and Tris. The buffering agents encompass those agents which maintain the pH of the solution in an acceptable range before lyophilization. Higher concentration limits are generally higher for "bulk" proteins than for "dosage" protein forms. For example, although the buffer concentrations may vary from several inimolars up to the upper limit of their solubility, for example histidine may be at a concentration as high as 200 M, a person skilled in the art will also take into account obtaining / maintaining of a physiologically adequate concentration. Active ingredient: The formulations lyophilized by the present methods can include essentially any active ingredient, such as proteins, nucleic acids, viruses and combinations thereof. The proteins may include, for example, coagulation factors, growth factors, cytokines, antibodies and chimeric constructions. In relation to proteins, the active ingredients present in a formulation may be recombinant proteins or proteins isolated from an organism. Glycine / NaCl Formulations: With the above lyophilization methods, the formulations comprising about 20 mM or more of sodium chloride in the presence of glycine (typically an amount of about 2% to about 250 mM), glycine crystallization is inhibited for sodium chloride. In formulations comprising NaCl in a concentration greater than 30 mM, the crystallization of glycine decreases significantly when the above lyophilization methods are used. In . In contrast, the present invention provides lyophilization methods wherein the salts do not substantially inhibit the crystallization of an amino acid excipient (eg, sodium chloride and glycine). Factor IX Formulations: In one embodiment, the present invention provides lyophilized factor IX products made by the methods described. Suitable formulations of factor IX that can be lyophilized by the present methods include formulations of factor IX described in the patent of E.U.A. No. 6,372,716; the patent of E.U.A. No. 5,770,700 and the publication of patent application of E.U.A. No. US2001 / 0031721. For example, a factor IX formulation that can be lyophilized comprises factor IX, a bulking agent and a cryoprotectant. The concentration of factor IX can be, for example, from about 0.1 mg / ml to about 20 mg / ml (equivalent to about 20 to at least 400 U / ml) or from about 0.4 mg / ml to about 20 mg / ml . The bulking agents for factor IX formulations may include, for example glycine and / or a magnesium salt, a calcium salt, a sodium salt or a chloride salt, wherein the concentration of one or more of the agents that provide volume is from about 0.5 mM to about 400 mM. In one embodiment, the bulking agent is glycine, wherein the glycine concentration is from about 0.1 M to about 0.3 M, from about 0.2 M to about 0.3 M, or from about 0.25 M to about 0.27 M. In another embodiment, the bulking agents are glycine in a concentration from about 0.25 M to about 0.27 M and sodium chloride at a concentration of about 50 mM. Suitable cryoprotectants for factor IX formulations include, for example, polyols such as mannitol and sucrose in a concentration of about 0.5% to about 2%. Factor IX formulations may further comprise a surfactant and / or detergent, such as Polysorbate (e.g., Tween-80) or polyethylene glycol (PEG), which may also serve as a cryoprotectant during one or more freezing steps. The surfactant can vary from about 0.005% to about 0.05%. the concentrations of the excipients may have, for example, a combined osmolarity of about 250 mOsM to about 350 mOsM, or about 300 mOsM + 50 mOsM and may also contain an appropriate buffering agent to maintain a physiologically adequate pH, for example in the range from about 6.0 to 8.0. Buffering agents may include, for example, histidine, sodium phosphate or potassium phosphate with a target pH of from about 6.5 to about 7.5, all at about 5 mM to about 50 mM In one embodiment, the factor IX formulation comprises factor IX, 10 mM histidine, 1% sucrose, 50 mM sodium chloride, 0.005% Polysorbate 80 and 250 to 270 mM glycine The final concentration of NaCl in a reconstituted lyophilized factor IX formulation should be> 40 mM with the In order to reduce agglutination / aggregation of erythrocytes when the factor IX formulation is administered, thus, in one embodiment, the factor IX formulations that are lyophilized by the present methods comprise sodium chloride in a concentration of at least 40 mM. It should be understood and expected that variations in the principles of the invention as described herein in an exemplary embodiment may be elaborated by a person skilled in the art. the art and it is intended that said modifications, changes and substitutions are included within the scope of the present invention.

The examples' which are set forth in the following illustrate various embodiments of the invention. These examples are for illustrative purposes only and in no way mean that they are limiting. EXAMPLES EXAMPLE 1: NON-HIGH TEMPERATURE RELATED LYOPHILIZATION METHODS Three different lyophilization cycles were carried out in order to identify a lyophilization method that improves glycine crystallization and maintains protein stability. An additional benefit that was examined to include it when the lyophilized product has a residual moisture content of less than 1%. The lyophilization cycles performed in this example are indicated as "Lyo G", Lyo H "and" Lyo I "as described in Tables 3, 4 and 5. These cycles do not include the high temperature annealing step and as a consequence the crystallization of glycine is not complete or is less complete compared to the methods that include the high temperature annealing step.In addition, the cycles without the high temperature annealing step resulted in lyophilized products with lower residual moisture contents of 2% Table 2 shows the three formulations that were used in this example Each formulation contains factor IX at 250 IU / ml at pH 6.8.

Table 2: Formulations Tables 3, 4 and 5 show the lyophilization steps for Lyo G, H and I. All the bottles were covered under vacuum. Table 3: Lyophilization cycle wLyo G " Formulations 1, 2 and 3 were lyophilized each in duplicate, according to the protocols of Lyo G, Lyo H and Lyo I. The residual moisture content of each lyophilized product was then determined using the Karl Fisher method. As can be seen in table 6, none of Lyo G, Lyo H or Lyo I lyophilized the formulations in such a way that the moisture content was less than 1%. Table 6: Residual moisture content In addition, the lyophilized cakes of the formulations 1, 2 and 3 produced by the Lyo G, H and I protocols were determined for complete crystallization. The determination for complete crystallization was carried out by DSC. What is observed in the first exploration (Figures IA, 2A and 3A) is an irreversible exothermic event. The absence of the exothermic event on the second exploration confirms that it is irreversible. A person skilled in the art recognizes that this irreversible exothermic event represents a crystallization event. This indicates that the crystallization excipient does not completely crystallize during lyophilization. Fig. 1A shows a first scan and Fig. IB shows a second scan of a lyophilized formulation 1, where the scans are representative of Lyo G, H and I. Fig. 2A shows a first scan and Fig. 2B shows a second scan of a lyophilized formulation 2, wherein the scans are representative of Lyo G, H and I. Figure 3A shows a first exploration and Figure 3B shows a second exploration of the lyophilized formulation 3, where the scans are representative of Lyo G , H and I. As can be seen in figures 1, 2 and 3, all lyophilized samples prepared by the Lyo G, H and I protocols show a crystallization event during the first exploration (and without transition of the second exploration) indicating that glycine does not completely crystallize during lyophilization. EXAMPLE 2: METHODS OF LYOPHILIZATION WITH HIGH TEMPERATURE RECYCLE In order to provide complete crystallization or improve the crystallization of the excipients in the cake, lyophilization methods comprising the step of annealing at high temperature were tested. An additional objective is to improve the final percentage of residual moisture so that it is below 1%. Table 7 shows the formulations used in example 2. Each formulation contains factor IX at 250 IU / ml, pH 6.8. Table 7: Formulations used in example 2 Tables 8, 9 and 10 show the lyophilization steps for Lyo J, K and L. All the bottles were covered under vacuum. Table 8: Lyophilization cycle "Lyo J" Formulations 4, 5 and 6 were lyophilized each in duplicate, according to the protocols of Lyo J, Lyo K and Lyo L. The moisture content of each lyophilized product was then determined using the Karl Fisher method (see Table 11). The lyophilized cakes of formulations 4-6 produced by the Lyo J, and L cycles are then analyzed to determine complete crystallization. The determination of complete crystallization is carried out by DSC. Again, if a crystallization event is observed on the first scan, then the sample does not completely crystallize during lyophilization. Figure 4A shows the first scan and Figure 4B shows a second scan of the lyophilized formulation 4 ("fix9271yoJ.001"), the formulation 5 lyophilized ("fix502501yoja.001") and formulation 6 lyophilized ("fix502701yoja.001"), wherein the formulations were lyophilized by Lyo J. Figure 5A shows a first scan and Figure 5B shows a second scan of the lyophilized formulation 4 ("fix927yoK.002"), the formulation 5 lyophilized ("fix50250yok.001") and the freeze-dried formulation 6 ("fix50270yok.001") where the formulations were lyophilized according to Lyo K. Figure 5C represents data from another second formulation 4 exploration ("fix927 lyo K") lyophilized by the Lyo K cycle. Figure 6A shows a first exploration and Figure 6B shows a second exploration of the lyophilized formulation 4 ("fix9271yol.001"), formulation 5 lyophilized ("fix502501yoL.001") and formulation 6 lyophilized ("fix502701yoL.001"), where the formulations were lyophilized according to LyoL. Figure 6C shows another second exploration of formulation 4 ("fix 927 Lyo L") lyophilized according to LyoL. Table 11 presents a summary of the first and second) DSC scanning data of the formulations 4, 5 and 6 lyophilized according to Lyo J, K or L. Table 11: Summary of moisture percentage and first and second data of DSC scan for example 2 fifteen 0 In relation to the formulations 5 and 6, Lyo J does not completely crystallize the excipients as in a case of crystallization that is observed with the first exploration. 5 Unwittingly joining any theory, this is probably due to the fact that the secondary drying stage in Lyo J lasts for 0 hours and that the high temperature annealing step is only 3 hours. Lyo J is capable of complete crystallization of the excipients in formulation 4, but formulation 4 does not contain a combination of sodium chloride and glycine, while formulations 5 and 6 have a combination of sodium chloride and glycine. Lyo K shows complete crystallization and the residual moisture in the final cakes is 1.2%. Lyo L shows superior results, since full crystallization occurs "and the residual moisture in the final layers is less than 1%. Additionally, stability studies for 3 months in lyophilized cakes according to the Lyo L cycle show that the moisture content remains at less than 1%. In addition, X-ray diffraction analysis and lyophilized samples with the high-temperature annealing step show an increase in glycine crystallization when compared with freeze-dried samples without the high-temperature annealing step (greater than 35 ° C) (see Figure 10A and Figure 10B). The stability of lyophilized products was also determined when testing for: (1) the percentage of high molecular weight species (HMW) present in the final cakes, (2) the recovery percentage of the factor IX coagulation activity and (3) the recovery rate of specific activity of factor IX. The percentage of the high molecular weight is determined by size exclusion chromatography (SEC-CLAP). The coagulation activity is determined using the part time assay of. Activated thromboplastin of a stage. The specific activity is calculated by dividing the coagulation activity between the protein concentration and determining the protein concentration using SEC-CLAP. Figure 7 shows the% of HMW for formulations 4-6 lyophilized by Lyo J, K and L. Figures 8A and 8B show the. Coagulation activity data of factor IX before and after lyophilization (due to a filling with 4 ml and a reconstitution dilution of 5 ml, a recovery of 80% is the highest recovery value expected in figure 8B ). Figure 9 shows the specific activity of recovery percentage. These results indicate that factor IX lyophilized by the methods having high temperature annealing and drying steps are not adversely affected, as shown by% HMW and by potency tests. EXAMPLE 3: HIGH TEMPERATURE RECYCLED LYOPHILIZATION METHODS PROVIDE LONG-TERM STABILITY The results in Example 2 indicate that the formulations are not adversely affected by the heat treatment step at 50 ° C (or "annealing maintenance").; see tables 9 and 10). In fact, this heat treatment step resulted in a lower percentage of residual moisture values for the formulations comprising sodium chloride and glycine. This reduction in the percentage of moisture is related to an increase in the crystallization of glycine. To provide additional evidence that lyophilization cycles carried out with a high temperature heat treatment step do not impair the stability of the active ingredient, the following experiments were carried out. Table 12 shows the formulation filled and lyophilized in this example. The formulation contains recombinant factor IX either at 69 IU / ml, pH 6.8 or 550 IU / ml, pH 6.8. Table 12: Formulation used in example 3 Two freeze-drying cycles were carried out (Lyo P and Lyo Q) with the only difference between the cycles of which one set was covered under full vacuum and the other was covered with a nitrogen-free space in the bottles. Tables 13 and 14 present the list of lyophilization cycles for Lyo P and Lyp Q. For Lyo P, all the bottles were covered under vacuum. For Lyo Q, all the bottles were covered with a nitrogen-free space.

Table 13: Lyophilization cycle "Lyo P" Multiple cakes were produced (the appearance of all the cakes is good) by Lyo P and Lyo Q in order to test the residual moisture levels of these cakes in storage at various times and at different temperatures. In all times and temperatures tested, the cakes showed residual moisture concentrations lower than 1.5% (residual moisture concentrations lower than 2% are generally acceptable). Even after 9 months and 12 months at 50 ° C, the cakes have residual moisture levels less than 1%. The results are presented below in table 15: Table 15: Residual moisture levels The stability of the cakes produced by Lyo P and Lyo Q are tested when performing tests to determine the percentage of HMW species present in the cakes. These stability tests were carried out when the cakes were stored at 2-8 ° C and higher storage temperatures. Less than 3% of HMW is acceptable. Experiments were carried out using a single vial per time point, each time point is made in duplicate using SEC-CLAP. The results are presented below in tables 16A-D: Table 16A: HMW percentage results for Lyo P (69 IU / ml or 250 IU / vial) Table 16B: HMW percentage results for Lyo P (550 IU / ml or 2000 IU / vial) Table 16C: HMW percentage results for Lyo Q (69 IU / ml or 250 IU / vial) Table 16D: HMW percentage results for Lyo Q (550 IU / ml or 2000 IU / vial) The stability of lyophilized drug products by Lyo P and Lyo Q is also tested when testing factor IX concentrations. These stability tests were carried out when the cakes were stored at 2-8 ° C and at accelerated temperatures. The experiments were carried out using one flask per time point, and each time point was tested in triplicate. The results (in units of μg / ml) are presented below in tables 17A-D. Note *: the time points at -80 ° C in tables 17A-D are controls, where the control of pre-lyophilized bulk drug product (BDP) is the one being tested. 4 ml of BDP are filled into each bottle and lyophilized. The resulting flasks are reconstituted with 5 ml of water for injection (WFl). This results in a medication product that is 20% less concentrated than the control BDP. The concentration of factor IX in the reconstituted bottles is determined by SEC-CLAP. Table 17A: Results of the protein concentration Factor IX for Lyo P (69 IU / ml or 250 IU / vial) Table 17B: Results of the protein concentration Factor IX for Lyo P (550 IU / ml or 2000 IU / vial) Table 17C: Results of the protein concentration Factor IX for Lyo Q (69 IU / ml or 250 IU / vial) Table 17D: Results of the protein concentration Factor IX for Lyo Q (550 IU / ml or 2000 IU / vial) The stability of the cakes produced by Lyo P and Lyo Q is also tested by tests of the coagulation activity potency of factor IX. These stability tests are carried out when the cakes are stored at 2-8 ° C and at accelerated temperatures. The experiments are carried out with a single flask per time point and the results, presented below in tables 18A-D, are in units of Ul / ml. In addition, in tables 18A-D, data points -80 ° C are controls with a pre-lyophilized bulk drug product (BDP) control. 4 ml of BDP are filled into each bottle and lyophilized. The resulting flasks are reconstituted with 5 ml of WFl. This results in a medication product that has 20% less potency than the BDP control. Therefore, for Table 18A, 44 IU / mL is equivalent to 100% activity for reconstituted BDP (20% less power than the BDP control -80 ° C at 12 months.) Similarly, for Table 18B, 593 IU / ml is equivalent to 100% activity for reconstituted BDP; for table 18C, 49 IU / ml is equivalent to 100% activity for reconstituted BDP; and for Table 18B, 697 IU / ml is equivalent to 100% activity for reconstituted BDP. The potency of factor IX is determined by assaying the coagulation activity using the one-step activated partial thromboplastin time assay.

Table 18A: Factor IX potency results for Lyo P (69 IU / ml or 250 IU / vial) Table 18B: Power Factor IX results for Lyo P (550 IU / ml or 2000 IU / vial) Table 18C: Results of the power Factor IX for Lyo Q (69 IU / ml or 250 IU / bottle) Table 18D: Results of the power Factor IX for Lyo Q (550 IU / ml or 2000 IU / bottle) The stability of the cakes produced by Lyo P and Lyo Q is also determined by determining the specific activity of factor IX. The specific activity of factor IX is calculated by dividing the clotting activity by the protein concentration. The data points below are in units of Ul / mg. Table 19A: Specific activity results of factor IX for Lyo P (69 IU / ml or 250 IU / vial) Table 19B: Factor IX specific activity results for Lyo P (550 IU / ml or 2000 IU / vial) Table 19C: Factor IX specific activity results for Lyo Q (69 IU / ml or 250 IU / vial) Table 19D: Factor IX specific activity results for Lyo Q (550 IU / ml or 2000 IU / vial) X-ray diffraction (XRD) was also performed on the lyophilized cakes to see if the high temperature heat treatment step improves glycine crystallization (see Figure 11). XRD is used to identify the crystal structures present in freeze-dried cakes. Lyo G is used for comparison since it includes the same lyophilization cycle parameters as Lyo Q, except that Lyo G does not have the heat treatment step at 50 ° C before secondary drying. Figure 11 shows that Lyo Q provides an increase in the crystallization of glycine. In summary, the results of Example 3 show: (1) that the high temperature heat treatment step does not affect the stability or activity of the active ingredient, the factor IX measured by% HMW, the potency and specific activity; (2) that the stability data show that factor IX is stable for the heat treatment process and is stable by long-term storage at accelerated temperatures; and (3) that the high temperature thermal treatment step increases the amount of crystalline glycine. As an additional benefit, the high temperature thermal treatment stage decreases the final residual moisture value of the samples. In this way, the results of Example 3 provide further evidence that high temperature annealing or heat treatment step before secondary drying does not destabilize the active ingredients but rather serves to improve the crystallization of excipient and lyophilization efficiency general . EXAMPLE 4: THE LOW TEMPERATURE RECOMMENDED STAGE IS OPTIONAL A freeze-drying cycle is performed with and without an annealing step at -15 ° C and 50 ° C to determine if the low temperature annealing step is required to improve the crystallization of glycine. The buffer of the formulation consists of 10 mM histidine, 1% sucrose, 260 M glycine, 50 mM NaCl, 0. Polysorbate 0.005%, pH 6.8 is used for all cycles. The analysis of the lyophilized cakes consists of DSC, XRD and residual moisture percentage. Table 20 shows the lyophilization cycles that were carried out. Table 20: Lyophilization cycles Table 21 below shows the analytical results of the experiments.

Table 21: Results * Average of two jars Based on the 'DSC data, there is evidence that the evidence that the inclusion of the low temperature annealing step, (here, -15 ° C) does not affect if a crystallization event is detected in the first scan. The high temperature annealing step (here, 50 ° C) is sufficient to eliminate the recrystallization event as observed in a first DSC scan. Although a lower annealing step is optional, its inclusion is beneficial, because a peak at 17.5 ° from the XRD data suggests that the annealing step at -15 ° C further improves glycine crystallization. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims

CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for lyophilizing an aqueous pharmaceutical formulation, characterized in that it comprises: (a) freezing the aqueous pharmaceutical formulation; (b) drying the pharmaceutical formulation of step (b); (c) annealing the pharmaceutical formulation of step (c) at a temperature greater than about 252C; and (d) drying the pharmaceutical formulation of step (c) at a temperature lower than the temperature used in step (c).
2. The method according to claim 1, characterized in that the freezing in step (a) is carried out at a temperature of less than -10SC.
3. The method according to claim 1, characterized in that the freezing in step (a) is carried out at a temperature of less than -352C.
4. The method according to claim 1, characterized in that the drying in step (b) is carried out at a temperature between about -352C and about 202C.
5. The method according to claim 1, characterized in that the drying in step (b) is carried out at a temperature between about -252C and about 102C.
The method according to claim 1, characterized in that the drying in step (b) is carried out at a temperature between about -20 ° C and about 0 ° C.
7. The method according to claim 1, characterized in that the drying in the stage (b) is carried out at a temperature of about 02C.
8. The method according to claim 1, characterized in that the annealing in the stage (c) it is carried out at a temperature between about 25 aC and about 752C.
9. The method according to claim 1, characterized in that the annealing in the stage (c) is carried out at a temperature between about 352C and about 60SC.
The method according to claim 1, characterized in that the annealing in step (c) is carried out at a temperature of about 50aC.
The method according to claim 1, characterized in that the drying in step (d) is carried out at a temperature of about 25 aC.
12. A method for lyophilizing an aqueous pharmaceutical formulation, characterized in that it comprises: (a) freezing the aqueous pharmaceutical formulation; (b) annealing the pharmaceutical formulation of step (a) at a temperature between about -35aC and about 02C. (c) drying the pharmaceutical formulation of the stage (b) at a temperature between about -352C and about 102C; (d) annealing the pharmaceutical formulation of the stage (c) at a temperature between about 252C and about 752C; and (e) drying the pharmaceutical formulation of step (c) at a temperature lower than the temperature used in step (d).
13. The method according to claim 12, characterized in that the freezing in step (a) is carried out at a lower temperature of -102C.
14. The method according to claim 12, characterized in that the freezing in step (a) is carried out at a temperature of less than -352C.
15. The method according to claim 12, characterized in that the annealing in step (b) is carried out at a temperature between about -252C and about 102C.
16. The method according to claim 12, characterized in that the annealing in step (b) is carried out at a temperature between about -20aC and about -102C.
17. The method according to claim 12, characterized in that the annealing in step (b) is carried out at a temperature of about -152C.
18. The method according to claim 12, characterized in that the drying in the stage (c) is carried out at a temperature between about -252C and about -10aC. 1 .
The method according to claim 12, characterized in that the drying in step (c) is carried out at a temperature between about -202C and about -10aC.
The method according to claim 12, characterized in that the drying in step (c) is carried out at a temperature of about 02C.
21. The method according to claim 12, characterized in that the annealing in step (d) is carried out at a temperature between about 352C and about 602C.
22. The method according to claim 12, characterized in that the annealing in step (d) is carried out at a temperature of about 502C.
23. The method according to claim 12, characterized in that the drying in step (e) is carried out at a temperature of about 252C.
The method according to claim 12, characterized in that it further comprises a refreezing step which is carried out after step (b) and before step (c), wherein the refreezing step is carried out at a temperature of less than -352C.
25. The method according to claim 24, characterized in that the refreezing step is carried out at a temperature between about -402C and about -502C.
26. The method according to claim 1 or 12, characterized in that the aqueous pharmaceutical formulation comprises at least one crystallization excipient.
27. The method according to claim 26, characterized in that one or more crystallization excipients are selected from the group consisting of an amino acid, a salt and a polyol.
28. The method according to claim 27, characterized in that the amino acid is glycine or histidine.
29. The method according to claim 27, characterized in that the salt is sodium chloride.
30. The method according to claim 27, characterized in that the polyol is mannitol.
31. The method according to claim 1 or 12, characterized in that the aqueous pharmaceutical formulation comprises a combination of crystallization excipients, wherein the combination is a salt and an amino acid.
32. The method according to claim 31, characterized in that the salt is sodium chloride.
33. The method according to claim 32, characterized in that the sodium chloride is present in the formulation in a concentration greater than about 25 mM.
34. The method according to claim 32, characterized in that the sodium chloride is present in the formulation in a concentration between approximately 25 mM and 200 M.
35. The method according to claim 32, characterized in that the sodium chloride it is present in the formulation in a concentration between about 30 mM and 100 mM.
36. The method according to claim 32, characterized in that the sodium chloride is present in the formulation in a concentration between approximately 40 mM and 60 mM.
37. The method according to claim 32, characterized in that the sodium chloride is present in the formulation at a concentration of approximately 50 mM.
38. The method according to claim 31, characterized in that the amino acid is present in the formulation in a concentration between about 1% and about 10%.
39. The method according to claim 31, characterized in that the amino acid is present in the formulation in a concentration between about 1.5% and about 5%.
40. The method according to claim 31, characterized in that the amino acid is present in the formulation in a concentration between about 1.5% and about 3%.
41. The method according to claim 31, characterized in that the amino acid is present in the formulation in a concentration of approximately 2%.
42. The method according to claims 38, 39, 40 or 41, characterized in that the amino acid is glycine.
43. A method for lyophilizing an aqueous pharmaceutical formulation comprising sodium chloride and glycine, characterized in that it comprises: (a) freezing the aqueous pharmaceutical formulation at a temperature of less than -352C; (b) optionally annealing the pharmaceutical formulation of step (a) at a temperature between about -20 ° C and about -10 ° C; (c) drying the pharmaceutical formulation of the stage (b) at a temperature between about -10 ° C and about 10 ° C; (d) annealing the pharmaceutical formulation of the stage (c) at a temperature between about 352C and about 502C; and (e) drying the pharmaceutical formulation of the stage (d) at a temperature lower than the temperature used in step (d).
44. The method according to claim 43, characterized in that the temperature in step (e) is approximately 252C.
45. A method for lyophilizing an aqueous pharmaceutical formulation comprising sodium chloride in a concentration greater than 35 mM and glycine between approximately 250 mM and approximately 300 mM, characterized in that it comprises: (a) freezing the aqueous pharmaceutical formulation at a temperature of less than from -35 aC; (b) annealing the pharmaceutical formulation of the stage (a) at about -15 BC; (c) drying the pharmaceutical formulation of the stage (b) at approximately DaC; (d) annealing the pharmaceutical formulation of the stage (c) at about 502C; and (e) drying the pharmaceutical formulation of the stage (d) at about 25aC; 46. The method according to claim 45, characterized in that it further comprises a refreezing step after step (b) and before step (c), wherein the refreezing step comprises refreezing the pharmaceutical formulation of the stage (b) at about -502C. 47. The method according to claim 46, characterized in that step (a) is carried out for approximately 5 hours; step (b) is carried out for about 5 hours; stage (c) is carried out for approximately 38 hours; step (d) is carried out for about 5 hours; and step (e) is carried out for approximately .5 hours. 48. A method for increasing the crystallization of excipient during lyophilization, characterized in that it comprises: (a) providing an aqueous pharmaceutical formulation comprising glycine and sodium chloride; (b) freezing the aqueous pharmaceutical formulation; (c) optionally annealing the pharmaceutical formulation of step (b) at a temperature between about -35aC and about 02C; (d) drying the pharmaceutical formulation of step (b) or step (c) at a temperature between about -352C and about 102C; (e) annealing the pharmaceutical formulation of step (d) at a temperature between about 25aC and about 75aC such that the glycine is more crystallized after step (c) than before step (e); and (f) drying the pharmaceutical formulation of step (e) at a temperature that is the same as or lower than the temperature used in step (e) and thereby increasing the crystallization of excipient. 49. A lyophilized product produced by a process characterized in that it comprises: (a) providing a formulation comprising glycine and sodium chloride; (b) freeze the formulation; (c) optionally annealing the pharmaceutical formulation of step (b) at a temperature between about -35aC and about 02C; (d) "drying the pharmaceutical formulation of step (b) or step (c) at a temperature between about -35 ° C and about 10 ° C; (e) annealing the pharmaceutical formulation of step (d) at a temperature between about 25 ° C. and about 752C, and (f) drying the pharmaceutical formulation of step (e) at a temperature that is the same as or lower than the temperature used in step (e) and thereby providing the lyophilized product. lyophilized according to claim 49, characterized in that the formulation further comprises an active ingredient. 51. The lyophilized product according to claim 50, characterized in that the active ingredient is a protein, a nucleic acid or a virus. 52. The lyophilized product according to claim 50, characterized in that the active ingredient is factor IX. 53. The lyophilized product according to claim 49, characterized in that the glycine in the lyophilized product after step (f) is more crystallized than a lyophilized product made by a process without an annealing step at high temperature, before drying secondary. 54. A lyophilized product produced by a process characterized in that it comprises: (a) providing a formulation comprising glycine and sodium chloride; (b) freeze the formulation; (c) annealing the formulation of step (b) at a temperature between about -202C and about -102C; (d) drying the formulation of step (c) at a temperature between about 0aC and about 52C; (e) annealing the formulation of step (d) at a temperature between about 352C and about 502C; Y (f) drying the formulation of step (e) at a temperature that is the same or lower than the temperature used in step (e) and thus providing the lyophilized product. 55. The lyophilized product according to claim 54, characterized in that the glycine in the lyophilized product after step (f) is more crystallized than a lyophilized product made by a process without an annealing step at high temperature before secondary drying . 56. The lyophilized product according to claim 54, characterized in that it is substantially stable for long term at high storage temperature. 57. The lyophilized product according to claim 56, characterized in that the long term comprises between approximately 3 months and approximately 1 year, and wherein the accelerated temperature comprises between approximately 252C and approximately 502C.