EA045351B1 - LYOPHILIZED COMPOSITION OF FACTOR IX FOR PREVENTING OR REDUCING BLEEDING ATTACKS IN HEMOPHILIA B - Google Patents

Description

State of the art

The present invention generally relates to the field of therapeutic agents for the treatment of hemostatic disorders.

Hemophilia B (also known as Christmas disease) is one of the most common inherited bleeding disorders in the world. It entails a decrease in coagulating activity in vivo and in vitro and requires constant medical monitoring throughout the patient's life. Without intervention, the patient will suffer from spontaneous bleeding in the joints, which causes severe pain and limited mobility; bleeding into the muscles, which leads to the accumulation of blood in these tissues; spontaneous bleeding in the throat and neck, which can cause asphyxia in the absence of immediate treatment; renal bleeding; Heavy bleeding after surgery, minor accidental injuries, or tooth extraction is also common.

For normal blood coagulation in vivo, at a minimum, serine protease factors II (prothrombin), VII, IX, X and XI (soluble plasma proteins) are required; cofactors, including the transmembrane protein tissue factor and plasma proteins factors V and VIII; fibrinogen, transglutaminase factor XIII, phospholipid (including activated platelets) and calcium. Additional proteins, including kallikrein, high molecular weight kininogen, and factor XII, are sometimes required to determine coagulation in vitro and may play some role in vivo under pathological conditions. In hemophilia, blood clotting is impaired by a lack of certain clotting factors in the plasma. Hemophilia B is caused by factor IX deficiency, which occurs due to either decreased synthesis of the factor IX protein or a defective molecule with reduced activity. Hemophilia is treated by replacing the missing coagulation factor with an exogenous factor concentrate significantly enriched with factor IX. However, obtaining such concentrates from blood is fraught with technical difficulties, as described below.

Isolation of factor IX from plasma (plasma-derived factor IX; pdFIX) almost always produces active factor IX. However, such isolation of factor IX from plasma is very difficult, since factor IX is present in plasma at low concentrations (5 μg/ml). Andersson, Thrombosis Research 7: 451 459 (1975). Additionally, isolation from the blood requires elimination or inactivation of infectious agents such as HIV or HCV. In addition, pdFIX has a short half-life and therefore requires frequent administration. Recombinant factor IX (rFIX) is also available, but has the same disadvantages of short half-life and the need for frequent administration (eg, 2-3 times per week for prophylaxis) as pdFIX. rFIX also has a lower rate of gradual recovery of activity (K value) compared to pdFIX, which entails the use of higher doses of rFIX than pdFIX.

Reducing mortality, preventing joint damage, and improving quality of life are important advances resulting from the development of plasma-derived and recombinant factor IX. Long-term protection against bleeding would represent another important advance in the treatment of subjects with hemophilia B. However, to date, no drugs have been developed that provide long-term protection. Therefore, there remains a need for improved treatments for factor IX deficiency hemophilia that are better tolerated and more effective than existing treatments.

In particular, there remains a need for improved lyophilized formulations containing FIX with higher drug potency, increased shelf life, reduced lyophilization process time, and reduced reconstitution time.

The essence of the invention

In one aspect of the invention there is provided a pre-lyophilization composition comprising:

(a) a factor IX (FIX) polypeptide at a concentration of from about 80 IU/ml to about 2750 IU/ml, wherein the FIX polypeptide comprises FIX fused to an Fc region;

(b) L-histidine at a concentration of about 3 mg/ml to about 15 mg/ml;

(c) sucrose at a concentration of from about 10 mg/ml to about 50 mg/ml;

(d) mannitol at a concentration of about 20 mg/ml to about 100 mg/ml; and (e) polysorbate 20 at a concentration of about 0.01 mg/ml to about 5 mg/ml, the nominal volume of the formulation being less than 4 ml; and each of (a)-(e) is present in an amount per vial (mg/vial) sufficient to provide (1) increased stability of the FIX polypeptide after lyophilization of the composition;

(2) reducing splashing on the vial stopper after lyophilization of the composition;

(3) reducing the lyophilization cycle time;

(4) increasing the shelf life of the lyophilisate obtained from the pre-lyophilization composition at room temperature; or (5) any combination thereof, compared to a control prelyophilization composition, wherein the control composition contains (a)-(e) in an amount per vial identical to the given prelyo-

- 1 045351 to the filler composition, but has a nominal volume of 5 ml.

In some embodiments, L-histidine is present at a concentration of about 7.76 mg/ml; sucrose is present at a concentration of about 23.8 mg/ml; mannitol is present at a concentration of about 47.6 mg/ml; or polysorbate 20 is present at a concentration of about 0.2 mg/ml.

In some embodiments, the nominal volume of the composition per vial is about 2.65 ml.

In some embodiments, the FIX polypeptide contains an amino acid sequence that is at least 95% identical to amino acids 1-642 of SEQ ID NO: 2.

In some embodiments, the FIX polypeptide is a heterodimeric protein with one FIXFc chain (FIXFc-sc) and one Fc chain (Fc-sc) that are linked together through two disulfide bonds in the Fc hinge region, wherein FIXFc-sc has the amino acid sequence of SEQ ID NO: 2 without the C-terminal lysine, and Fc-sc has the amino acid sequence SEQ ID NO: 4 without the C-terminal lysine.

In another aspect of the invention, a lyophilized powder is provided which is obtained by lyophilizing the pre-lyophilization composition of the present invention.

In yet another aspect of the invention, there is provided a reconstituted formulation comprising the lyophilized powder of the present invention reconstituted with a reconstitution buffer solution.

In yet another aspect of the invention, there is provided a bottle containing a lyophilized powder of the present invention.

In yet another aspect of the invention, there is provided a kit comprising a first container containing a lyophilized powder of the present invention and a second container containing a reconstitution buffer solution in a volume sufficient to produce a reconstituted composition when combined with the lyophilization composition from the first container.

Another aspect of the invention provides the use of a reconstituted composition of the present invention to prevent or reduce the frequency or severity of bleeding episodes in a patient with hemophilia B.

In another aspect of the invention, there is provided a method for lyophilizing a pre-lyophilization composition of the present invention, comprising:

(a) a freezing step comprising freezing the pre-lyophilization composition having a nominal volume of less than 3 ml and containing the FIX polypeptide and an aqueous solvent to a freezing temperature of about -65°C to about -40°C;

(b) a vacuum step comprising reducing the pressure of the frozen pre-lyophilization composition by an amount sufficient to remove the aqueous solvent from the frozen pre-lyophilization composition; and (c) a single drying step involving raising the temperature of the frozen pre-lyophilization composition above the collapse temperature of about -1.5° C., resulting in a lyophilized powder. In some embodiments, (i) during the freezing step, the temperature of the pre-freezing composition is lowered from about 5°C to about -55°C, and during the freezing step, the freezing temperature is maintained for about 30 minutes to about 5 hours;

(ii) the frozen pre-lyophilization composition obtained in step (a) is further processed in an annealing step (a') before starting the vacuum step (b), and during the annealing step, the temperature of the frozen pre-lyophilization composition obtained in step (a) is increased to an annealing temperature of about 15°C to about -2°C, and wherein during the annealing step, the annealing temperature is maintained for about 30 minutes to about 5 hours;

(iii) during the annealing step, the temperature of the frozen pre-lyophilization composition is lowered from the annealing temperature to a temperature that is from about -65°C to about -40°C;

(iv) in the vacuum step, the frozen pre-lyophilization composition is exposed to a vacuum of about 0.05 to about 1 mbar for about 2 hours; or (v) the drying step comprises raising the temperature of the frozen pre-lyophilization composition from about -55°C to a drying temperature of about 40°C, wherein the drying temperature is maintained for about 10 hours to about 40 hours and wherein the drying step is carried out at a pressure of from about 0.05 mbar to about 1 mbar.

In some embodiments, the pre-lyophilized composition is filtered under sterile conditions and filled into a vial under sterile conditions prior to step (a), wherein the lyophilized powder is obtained from the pre-lyophilized composition in about 45 hours or less, or the residual moisture content of the lyophilized powder is less than 0.7 %.

Brief description of drawings

Fig. 1 illustrates a bottle loading diagram for each lyophilization cycle. Numbers 1, 2 and 3 indicate the location of the thermocouples.

Fig. Figure 2 illustrates the curve of the dependence of the residual moisture content on the lyophilization parameters - temperature, vacuum and time - predicted by ME.

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Fig. Figure 3 illustrates the curve of the dependence of the product temperature during sublimation on the lyophilization parameters - temperature and vacuum - predicted by ME.

Fig. Figure 4 illustrates the curve predicted by ME for the dependence of the mass flow in the vial during sublimation on the lyophilization parameters - temperature and vacuum.

Fig. 5 illustrates the lyophilization data obtained from the 8 ME test in Example 2, the conditions of which are similar to those proposed for the rFIXFc-2G lyophilization cycle (40°C shelf temperature, chamber vacuum corresponding to 250 mTorr (0.33 mbar) pressure, and drying time 25 h).

Detailed Description of the Invention

The present invention relates to a pre-lyophilization composition containing: (a) a factor IX (FIX) polypeptide having the coagulating activity of FIX; (b) a buffering agent; (c) a stabilizing agent; (d) a bulking agent; and (e) a surfactant, wherein the nominal volume for the formulation is less than about 5 ml, less than about 4 ml, or less than about 3 ml, and each of (a)-(e) is present in an amount per vial (mg/vial) , sufficient to (1) increase the stability of the FIX polypeptide. after lyophilization; (2) reduce the recovery time after lyophilization; (3) reduce splashing onto the stopper of the bottle containing the composition; (4) reduce the lyophilization cycle time; (5) increase the shelf life of the lyophilisate obtained from the pre-lyophilization composition at room temperature; or (6) any combination thereof, as compared to a control pre-lyophilization composition, wherein the control composition contains (a)-(e) in the same quantities per vial as the pre-lyophilization composition, but the nominal volume is at least 5 ml. In a particular embodiment of the invention, the nominal volume for the composition is about 2.65 ml.

In some embodiments, the pre-lyophilization composition contains at least 100 IU/vial of FIX polypeptide. In some embodiments, the pre-lyophilization composition contains from about 200 IU/vial to about 10,000 IU/vial of FIX polypeptide.

In some embodiments, the FIX polypeptide comprises wild-type FIX. In some embodiments, the FIX polypeptide further comprises a heterologous fragment fused to wild-type FIX. In one embodiment of the invention, the heterologous fragment is a fragment that increases the half-life of FIX. In other embodiments, the heterologous fragment includes a polypeptide or non-peptide fragment. In one embodiment, the FIX half-life increasing fragment includes an FcRn binding partner or an Fc fragment. In one embodiment, the FIX polypeptide is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% identical to SEQ ID NO: 2.

In some embodiments, the nominal volume is about 4 ml, about 3.5 ml, about 3.0 ml, about 2.9 ml, about 2.8 ml, about 2.7 ml, about 2.65 ml, about 2 .6 ml, about 2.5 ml, about 2.4 ml, about 2.3 ml, about 2.2 ml, about 2.1 ml or about 2.0 ml.

In some embodiments, the reduced recovery time is less than 1.5 minutes, less than 1 minute, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 20 seconds, or less than 10 seconds.

In some embodiments, the buffering agent is L-histidine. In one embodiment, the buffering agent is present at a concentration (mg/ml) of from about 3 mg/ml to about 15 mg/ml. In another embodiment, the buffering agent is present at a concentration of from about 8 mg to about 39 mg per vial.

In some embodiments, the stabilizing agent is sucrose. In one embodiment of the invention, the stabilizing agent is present at a concentration (mg/ml) of 10 mg/ml to about 50 mg/ml. In another embodiment, the stabilizing agent is present at a concentration of from about 27 mg to about 132 mg per vial.

In some embodiments, the bulking agent is mannitol. In one embodiment of the invention, the bulking agent is present at a concentration (mg/ml) of 20 mg/ml to about 100 mg/ml. In another embodiment, the bulking agent is present at a concentration of from about 53 mg per vial to about 265 mg per vial.

In some embodiments, the surfactant is polysorbate 20. In one embodiment, the surfactant is present at a concentration (mg/ml) of 0.01 mg/ml to about 5 mg/ml. In another embodiment, the surfactant is present at a concentration of from about 0.03 mg to about 13 mg per vial.

In one aspect, the present invention provides a pre-lyophilization composition containing: (a) from about 80 to about 2750 IU/ml rFIXFc; (b) about 7.76 mg/ml L-histidine; (c) about 47.6 mg/ml mannitol; (d) about 23.8 mg/ml sucrose; and (f) about 0.2 mg/ml polysorbate 20.

The present invention further relates to a lyophilized powder containing a FIX polypeptide, a buffering agent, a stabilizing agent, a bulking agent, a surfactant, or any combination thereof.

In some embodiments, the residual moisture content of the lyophilized powder is less than 1%.

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In one embodiment, the lyophilized powder contains: (a) a FIX polypeptide in an amount of from about 2 mg per vial to about 150 mg per vial; (b) a buffering agent in an amount of from 10 mg per vial to about 30 mg per vial; (c) a bulking agent in an amount of from about 70 mg per vial to about 200 mg per vial; (d) a stabilizing agent in an amount of from 30 mg per vial to 100 mg per vial; and (e) a surfactant in an amount of from 0.05 mg per vial to about 5 mg per vial.

In another embodiment, the lyophilized powder contains: (a) lyophilized FIX polypeptide in an amount of from about 2.2 mg per vial to about 125 mg per vial; (b) a buffering agent in an amount of from 12.5 mg per vial to about 25 mg per vial; (c) a stabilizing agent in an amount of from about 32.5 mg per vial to about 80 mg per vial; (d) a bulking agent in an amount of from about 75 mg per vial to 150 mg per vial; and (e) a surfactant in an amount of from about 0.1 mg/ml to about 2 mg/ml.

In another embodiment, the lyophilized powder contains: (a) from about 2.2 to about 125 mg/vial of FIX polypeptide; (b) about 20.6 mg/vial L-histidine; (c) about 126.1 mg/vial mannitol; (d) about 63.1 mg/vial sucrose; and (f) about 0.53 mg/vial of polysorbate 20.

The present invention also relates to a reconstituted formulation comprising the lyophilized powder described herein reconstituted with a reconstitution buffer solution.

In one embodiment, the reconstituted formulation contains: (a) a FIX polypeptide at a concentration of about 0.9 mg/ml to about 50 mg/ml; (b) a buffering agent at a concentration of from 1.5 mg/ml to about 7.5 mg/ml; (c) a bulking agent at a concentration of from 10 mg/ml to about 50 mg/ml; (d) a stabilizing agent at a concentration of about 5 mg/ml to 25 mg/ml per vial; and (f) a surfactant at a concentration of from 0.005 mg/ml to about 2.5 mg/ml.

In another embodiment, the reconstituted formulation contains: (a) a FIX polypeptide at a concentration of about 0.9 mg/ml to about 50 mg/ml; (b) a buffering agent at a concentration of about 3.88 mg/ml; (c) a bulking agent at a concentration of about 23.8 mg/ml; (d) a stabilizing agent at a concentration of about 11.9 mg/ml; (e) a surfactant at a concentration of about 0.1 mg/ml; and (f) reduction buffer solution.

In another embodiment, the reconstituted formulation contains: (a) a FIX polypeptide at a concentration of about 80 IU/ml to about 2750 IU/ml; (b) a buffering agent at a concentration of about 25 mM; (c) a bulking agent at a concentration of about 131 mM; (d) a stabilizing agent at a concentration of about 35 mM; (e) surfactant at a concentration of 0.01% (w/v); and (f) reduction buffer solution.

The present invention further provides a method of administering a FIX polypeptide to a hemophilia B patient in need thereof, or a method of preventing, treating, ameliorating or managing hemophilia B in a patient in need thereof, which includes administering to the patient the reconstituted formulations described herein.

The present invention also relates to a method for producing a lyophilized powder containing a FIX polypeptide, which includes lyophilizing the pre-lyophilization compositions described herein. In one aspect, the present invention provides a method for lyophilizing a FIX polypeptide, comprising: (a) a freezing step comprising freezing a pre-lyophilization composition containing a FIX polypeptide and an aqueous solvent; (b) a vacuum step comprising reducing the pressure in the chamber containing the frozen pre-lyophilization composition by an amount sufficient to remove the aqueous solvent from the frozen pre-lyophilization composition; and (c) one drying step including raising the temperature of the frozen pre-lyophilization composition above the collapse temperature, resulting in the formation of a lyophilized powder. In some embodiments, the pre-lyophilization composition is filtered under sterile conditions and filled into a vial under sterile conditions prior to step (a).

In another aspect, the present invention provides methods for preparing a lyophilized powder containing a FIX polypeptide, comprising: (a) a freezing step comprising freezing the pre-lyophilization composition containing the FIX polypeptide by reducing the temperature over about 2 hours to a freezing temperature of about -55 °C, and kept at freezing temperature for about 2 hours; (a') an annealing step comprising raising the temperature of the frozen pre-lyophilization composition obtained in step (a) to an annealing temperature of about -6° C. for about 1.5 hours, maintaining at the annealing temperature for about 3 hours, and lowering the temperature over about 1.5 hours to about -55°C; (b) a vacuum step comprising maintaining the frozen pre-lyophilization composition obtained in step (a') at about -55° C. for two hours at atmospheric pressure and reducing the pressure over about 2 hours to about 0.33 mbar; and (c) one drying step comprising raising the temperature of the frozen pre-lyophilization composition obtained in step (b) for 3 hours to about 40° C. while maintaining the pressure at about 0.33 mbar, and maintaining the frozen pre-lyophilization composition at temperature of about 40°C for 25 hours, while maintaining the pressure at

- 4 045351 level of about 0.33 mbar, which results in a lyophilized powder. In a further aspect of the invention, the lyophilized powder has one or more characteristics selected from the group consisting of: (1) increased stability of the FIX polypeptide after lyophilization; (2) reduced recovery time after lyophilization; (3) reduced splashing onto the stopper of the bottle containing the composition; (4) reduced lyophilization cycle time; (5) increased shelf life of the lyophilisate obtained from the pre-lyophilization composition at room temperature; or (6) any combination thereof.

In one aspect, the invention provides a method for stabilizing a lyophilized powder containing a FIX polypeptide, comprising lyophilizing the pre-lyophilization composition according to the methods described herein, wherein the increase in stability of the lyophilized powder is determined by size exclusion chromatography (SEC) relative to the lyophilized powder prepared using the lyophilization method, including more than one drying stage.

In another aspect, the invention provides a method for increasing the shelf life of a lyophilized powder containing a FIX polypeptide, comprising lyophilizing the pre-lyophilized composition according to the methods described herein, wherein the increase in the shelf life of the lyophilized powder is determined by SEC and/or analysis of the coagulating activity of FIX relative to the shelf life of the lyophilized powder prepared using a lyophilization process involving more than one drying step.

The invention also provides a method for reducing the reconstitution time of a lyophilized powder containing a FIX polypeptide, comprising lyophilizing the pre-lyophilization composition according to the methods described herein, wherein the reconstitution time of the lyophilized powder is reduced relative to the reconstitution time of a lyophilized powder prepared using a lyophilization process comprising more than one drying step. .

The invention further provides a method for reducing the duration of the lyophilization process when preparing a lyophilized powder containing a FIX polypeptide, comprising lyophilizing a pre-lyophilization composition according to the methods described herein, wherein the duration of the lyophilization process of the pre-lyophilization composition is reduced relative to the duration of the lyophilization process when obtaining a lyophilized powder using a lyophilization method, including more than one drying stage. The invention provides, among other things, pre-lyophilization compositions, reconstituted compositions and lyophilized powder compositions containing factor IX (FIX) polypeptide. The invention also provides lyophilization methods for producing a lyophilized powder containing the FIX polypeptide. Also proposed are methods for stabilizing a lyophilized powder containing a FIX polypeptide, a method for increasing the shelf life of a lyophilized powder containing a FIX polypeptide, a method for reducing the recovery time for a lyophilized powder containing a FIX polypeptide, and a method for reducing the duration of the lyophilization process of a pre-lyophilization composition containing a FIX polypeptide. Additionally, the invention provides methods for preventing, treating, ameliorating or regulating hemophilia B in a patient in need thereof by administering a reconstituted composition containing a FIX polypeptide.

Definitions.

Throughout this specification, "object" in the singular refers to one or more objects; for example, a polynucleotide is understood to represent one or more polynucleotides. Thus, the terms singular, one or more, and at least one may be used interchangeably herein.

Moreover, the use of and/or herein is to be taken as a specific description of either of the two specified properties or components separately from or together with the second property or component. Therefore, the term and/or, when used herein in an expression such as A and/or B, is intended to include A and B, A or B, A only and B only. Likewise, the term and/or, used in an expression such as A, B and/or C, covers each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; only A; only in; and C only. It should be understood that throughout this document where the wording "comprises" is used to describe aspects, other similar aspects described by the terms "consists of" and/or "substantially consists of" are also implied.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, PeiShow, ^2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, ^3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one skilled in the art with a common vocabulary for many of the terms used herein.

Units, prefixes and symbols are specified in the form adopted in the International System of Units (Systeme International de Unites - SI). Numeric ranges include numbers that define a range. If

- 5 045351 not otherwise stated, amino acid sequences are written from left to right in the direction from the amino-carboxyl end. The headings set forth herein are not intended to limit the various aspects of the invention that may be apparent by reference to the description as a whole. Accordingly, terms that are defined immediately below are more fully defined by reference to the description in its entirety. The term "about" as used herein means about, in the ballpark, approximately, or on the order of magnitude, and the meaning will depend on the limitations of the measurement system. When the term about is used in conjunction with a range of numeric values, it modifies that range by extending the boundaries of the numeric values above and below. In general, the term about can tilt the numerical value above and below by some amount, such as 10 percent or 20 percent, upward or downward (above or below). Unless otherwise stated, a value of about is assumed to be within the acceptable error range for a particular value for the formulation or composition.

The terms polypeptide, peptide and protein are used interchangeably and refer to a polymeric compound consisting of covalently linked amino acid residues.

The terms polynucleotide and nucleic acid are used interchangeably and refer to a polymeric compound consisting of covalently linked nucleotide residues. The polynucleotides may be DNA, cDNA, RNA, single- or double-stranded, vectors, plasmids, phages or viruses. As used herein, the term administration means, for example, to prescribe or give a pharmaceutical composition containing a FIX polypeptide to a subject. Examples of routes of administration include, but are not limited to, intravenous, such as intravenous injection, and intravenous infusion, such as via central venous access. Additional routes of administration include subcutaneous, intramuscular, oral, nasal and inhalation. A pharmaceutical composition containing a FIX polypeptide may contain one or more excipients as described herein. The advantages of the methods, compositions and pharmaceutical kits proposed herein include: improved adherence to treatment; reduction of breakthrough bleeding; increasing joint protection from bleeding; prevention of joint damage; reduction of morbidity; reduction in mortality; long-term protection against bleeding; reduction of thrombosis; and improving quality of life. Administration includes parenteral administration. In some embodiments, parenteral administration is intravenous or subcutaneous administration. The term treatment, as used herein, means reducing the intensity or alleviation of one or more symptoms of a blood clotting disorder or disorder, including, but not limited to, hemophilia B. In one embodiment of the invention, treating a blood clotting disorder or disorder includes preventing one or more symptoms of a blood clotting disorder or disorder. For a blood clotting disorder or disorder caused by FIX deficiency (eg, low baseline FIX activity), the term treatment may refer to replacement therapy with FIX. Following administration of a FIXFc polypeptide to a subject, the subject may achieve and/or maintain a minimum level of plasma FIX activity of about 1 IU/dL or greater than 1 IU/dL. In other embodiments, treatment means reducing the incidence of one or more symptoms of bleeding disorders or disorders, such as spontaneous or uncontrolled bleeding episodes. However, treatment does not necessarily have to lead to recovery.

Patient, as used herein, includes an individual who has had at least one episode of uncontrolled bleeding, who has been diagnosed with a disease or disorder associated with uncontrolled bleeding episodes, such as a bleeding disorder or disorder, such as hemophilia B, who is susceptible to uncontrolled bleeding. bleeding episodes, such as hemophilia, or which has any combination of these features. Patients may also include an individual who is at risk of experiencing one or more uncontrolled bleeding episodes prior to a particular activity, such as surgery, sports, or any other strenuous activity. The patient may have a baseline FIX activity that is less than 1%, less than 0.5%, less than 2%, less than 2.5%, less than 3%, or less than 4%. Patients also include children. Pediatric patients are patients aged from birth to 20 years, preferably from birth to 18 years, from birth to 16 years, from birth to 15 years, from birth to 12 years, from birth to 11 years, from birth to 6 years , from birth to 5 years, from birth to 2 years and from 2 to 11 years.

Baseline, as used herein, is the lowest measured factor IX level in a subject before dosing. Plasma levels of factor IX can be measured two times before dosing: at assessment and immediately before dosing. Alternatively, (a) the baseline level in subjects for whom pre-treatment FIX activity is <1%, who do not have FIX antigen detected, and who have nonsense mutations in the genotype may be set to 0%, (b) baseline the level for subjects with pre-treatment FIX activity <1% who have FIX antigen detected can be set to 0.5%, (c) the baseline level for subjects for

- 6 045351 whose pre-treatment FIX activity is 1 - 2%, represents the Cmin (lowest activity during the PK study) and (d) the baseline for subjects for whom pre-treatment FIX activity is >2% may be set to 2%. Activity above baseline before dosing can be considered the presence of residual drug from previous treatment and can be reduced to baseline and subtracted from PK data after rFIXFBP administration. A trough level, as used herein, is the lowest level of factor IX plasma activity that is achieved after administration of a dose of a chimeric polypeptide of the present invention or another factor IX molecule and before administration of the next dose, if any. Minimum level is used interchangeably with threshold in this document. To calculate trough levels, baseline factor IX levels are subtracted from measured factor IX levels.

As used herein, the term half-life refers to the biological half-life of a particular polypeptide in vivo. The half-life can be thought of as the time required for half of the amount administered to a subject to be eliminated from the bloodstream and/or other tissues of the animal. The terms long-acting and extended-release are used interchangeably in this document. In one embodiment, the terms long-acting and extended-release indicate that the FIX activity resulting from administration of the rFIXFBP polypeptide lasts longer than the activity of wild-type FIX (eg, BENEFIX® or plasma-derived FIX (pdFIX)). The longer duration of action of FIX can be measured by any method known in the art, for example, by aPTT assay, chromogenic assay, ROTEM (rotational thromboelastometry), TGT, etc. In one embodiment, the longer duration of action of FIX can be demonstrated by T _1/2beta (activity). In another embodiment, a longer duration of action of FIX can be predicted based on the level of FIX antigen present in the plasma, for example, by T _1/2beta (antigen).

The terms lyophilisate, lyophilized powder, lyophilized product, or compacted tablet, as used herein, refer to a formulation that is prepared using freeze-drying methods. The solvent (eg water) is removed by freezing followed by sublimation under vacuum and desorption of the residual water at elevated temperature. In the pharmaceutical field, the lyophilisate is present in the form of a powder or a physically stable mass compacted into a tablet. The lyophilisate is characterized by rapid dissolution after the addition of a reducing medium. The terms pre-lyophilization composition or initial pre-lyophilization mixture, as used herein, refer to the liquid composition before removal of the solvent (eg, water) using freeze-drying. The fill volume of the pre-lyophilization formulation is the total volume of the liquid formulation before lyophilization.

_Tw , or T _{1/2 beta} , or beta PP, as used herein, is the half-life associated with the elimination phase, t _1/2 β=(ln2)/the elimination _rate constant associated with the terminal phase. T1/2 beta can be determined by FIX activity or plasma FIX antigen levels. T _{1/2 beta} based on activity is designated T _{1/2 beta} (activity), and T _{1/2 beta} based on FIX antigen level can be designated T _{1/2 beta} (antigen). Both T _{1/2 beta} (activity) and T _{1/2 beta} (antigen) can be expressed as intervals or a geometric mean. The term reconstituted or reconstituted composition, as used herein, means a composition that has been lyophilized and redissolved by the addition of a solvent. The solvent may contain, without limitation, water for injection (WFI), bacteriostatic water for injection (WFI), sodium chloride solutions (for example, 0.9% (w/v) NaCl solution), glucose solutions (for example, 5% glucose) , solutions containing surfactants (for example, 0.01% polysorbate 20 or polysorbate 80), pH buffer solution (for example, phosphate buffer solutions) and combinations thereof.

Lyophilization Process in General Lyophilization, or freeze drying, is a process widely used in the pharmaceutical industry to preserve biological and pharmaceutical substances. The lyophilization process, also known as the lyophilization cycle, is traditionally divided into three distinct stages: freezing, primary drying, and secondary drying. Lyophilization, as used herein, refers to the complete lyophilization process including both freezing and drying steps.

In lyophilization, the water present in a substance is converted to ice during the freezing step and then removed from the material by direct low-pressure sublimation during the primary drying step. However, during freezing, not all water turns into ice. Some of the water is retained in a matrix of solids containing, for example, the components of the formulation and/or the active ingredient. Excess bound water in the matrix can be reduced to the desired residual moisture level during the secondary drying step. All stages of lyophilization: freezing, primary drying and secondary drying determine the properties of the finished product. Primary drying is typically the longest step in the freeze-drying process, therefore optimizing this part of the process has significant economic benefits.

In some aspects of the invention, the lyophilization process includes only a primary drying step.

In some aspects of the invention, the lyophilization process also includes a separate vacuum step between the freezing step and the primary drying step.

In other aspects of the invention, the lyophilization process further includes an annealing step between the freezing step and the primary drying step. The term annealing step, as used herein, refers to a step in the lyophilization process of the polypeptide drug that is being lyophilized, prior to the drug drying step, in which the temperature of the drug is raised from a lower temperature to a higher temperature and, after a period of time, lowered again.

Traditionally, cycle and composition optimization are performed so that the product temperature during primary drying never exceeds the collapse temperature. The term collapse temperature, as used herein, refers to the temperature of the product during freeze-drying, above which the mass compacted into a tablet begins to lose its original structure. Above the collapse temperature, the product may experience slow irregular bubbling, swelling, foaming, cavitation, pinhole formation, macroscopic breakdown, shrinkage and granulation, which may affect the appearance of the product. Consequently, degradation can lead to poor product stability, long drying times, uneven drying and loss of structure; see, for example, US 2010/0041870.

The lyophilized product of the present invention can be evaluated based on analysis of product quality, reconstitution time, reconstitution quality, high molecular weight, moisture content, glass transition temperature (Tg), and biological or biochemical activity. Typically, product quality analysis involves determining the rate of product degradation using techniques including, but not limited to, size exclusion chromatography (SEC), cation exchange HPLC (CEX-HPLC), X-ray diffraction (XRD), modulated differential scanning calorimetry (MDSC) , reverse phase HPLC (RP HPLC), multi-angle light scattering (MALS), fluorescence, ultraviolet absorption, nephelometry, capillary electrophoresis (CE), SDS-PAGE electrophoresis and combinations thereof. In some embodiments, evaluation of a lyophilized product in accordance with the present invention includes the step of evaluating the appearance of the mass compacted into a tablet. Additionally, the lyophilized product can be evaluated based on the biological or biochemical activity of the product, typically after reconstitution.

Lyophilized formulations containing factor IX.

The present invention provides pre-lyophilized compositions, lyophilized and reconstituted compositions or pharmaceutical compositions containing a FIX polypeptide.

In some aspects of the invention, the compositions described herein contain a FIX polypeptide, a buffering agent, a stabilizing agent, a bulking agent and a surfactant, or any combination thereof. The composition may also contain any other agents that are suitable for a pharmaceutical formulation.

Polypeptide factor IX (FIX).

The FIX polypeptide or FIX protein used in the formulation is a functional FIX factor protein in its normal role in the coagulation process unless otherwise stated. Thus, a FIX polypeptide includes variant polypeptides that are functional and polynucleotides that encode such functional variant polypeptides. In one embodiment, the FIX polypeptides are human, bovine, porcine, canine, feline and mouse FIX polypeptides. The full length polypeptide and polynucleotide sequences of FIX are known, as are many other functional variants, such as fragments, mutants and modifications. FIX polypeptides include full-length FIX, full-length FIX without methionine at the N-terminus, full-length FIX without a signal sequence, mature FIX (without signal sequence and propeptide), mature FIX with an additional methionine at the N-terminus. FIX can be produced recombinantly (recombinant factor IX or rFIX), i.e. it does not occur naturally or is derived from plasma.

A large number of functional variants of FIX are known. International Publication No. WO 02/040544 A3, which is incorporated herein by reference in its entirety, describes mutants that exhibit increased resistance to heparin inhibition on page 4, lines 9-30 and page 15, lines 6-31. In international publication No. WO 03/020764 A2, which is incorporated into this document in its entirety by reference, in table. 2 and 3 (on pages 14-24) and on page 12, lines 1-27, FIX mutants with reduced T-cell immunogenicity are described. In international publication No. WO 2007/149406 A2, which is included in this document in its entirety

- 8 045351 by reference, page 4, line 1 to page 19, line 11 describes a functional FIX mutant that exhibits increased protein stability, increased half-life in vivo and in vitro, and increased resistance to proteases. WO 2007/149406 A2 page 19, line 12 to page 20, line 9 also describes chimeric FIX molecules and other variants. International Publication No. WO 08/118507 A2, which is incorporated herein by reference in its entirety, from page 5, line 14 to page 6, line 5, describes FIX mutants that exhibit increased coagulation activity. International Publication No. WO 09/051717 A2, which is incorporated herein by reference in its entirety, from page 9, line 11 to page 20, line 2, describes FIX mutants with an increased number of N- and/or O-glycosylation sites, which leads to an increase in half-life and/or level of recovery of activity. In International Publication No. WO 09/137254 A2, which is incorporated herein in its entirety by reference, from page 2, paragraph [006] to page 5, paragraph [011] and from page 16, paragraph [044] to page 24, paragraph [057] also describes factor IX mutants with an increased number of glycosylation sites. International Publication No. WO 09/130198 A2, which is incorporated herein by reference in its entirety, from page 4, line 26 to page 12, line 6, describes a functional FIX mutant with an increased number of glycosylation sites, resulting in an increased half-life. International publication No. WO 09/140015 A2, which is incorporated herein in its entirety by reference, from page 11, paragraph [0043] to page 13, paragraph [0053] describes functional FIX mutants with an increased number of cysteine residues, which can be used for binding to a polymer (eg PEG). FIX polypeptides are also described in International Application No. PCT/US2011/043569, filed July 11, 2011 and published as WO 2012/006624 on January 12, 2012, which is incorporated herein by reference in its entirety.

In some embodiments, the FIX polypeptide comprises wild-type FIX. In some embodiments, the FIX polypeptide further comprises a heterologous fragment fused to wild-type FIX. In some embodiments, the heterologous fragment is a fragment that increases the half-life of FIX. In some embodiments, the heterologous fragment includes a polypeptide or non-peptide fragment.

In other embodiments, the FIX polypeptide is a long-acting FIX polypeptide. A long-acting FIX polypeptide may comprise a FIX region and another region, such as a heterologous fragment, that is capable of increasing the in vivo or in vitro half-life of the FIX polypeptide. Examples of fragments that are not FIX include, for example, Fc, albumin, PAS sequence, transferrin, CTP (28-amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) with 4 O-glycans), polyethylene glycol (PEG) , hydroxyethyl starch (HES), albumin-binding polypeptide, albumin-binding small molecules, or any combination thereof. Examples of long-acting FIX polypeptides of the present invention include, for example, Factor IX-Fc polypeptides, Factor IX-Albumin polypeptides, Factor IX-PAS polypeptides, Factor IX-Transferrin polypeptides, Factor IX-CTP polypeptides, Factor IX-PEG polypeptides, Factor IX polypeptides. IX - HES, factor IX polypeptides - albumin-binding polypeptide, or factor IX polypeptides - small molecule albumin binding.

In one embodiment, the FIX polypeptide is rFIXFc, a recombinant fusion protein consisting of human coagulation factor IX (FIX) and a human antibody Fc domain (IgG1 isotype); see, for example, PCT Application No. PCT/US2011/043569, filed July 11, 2011 and published as WO 2012/006624, which is incorporated herein by reference in its entirety. The rFIXFc polypeptide is a heterodimeric protein with one FIXFc chain (FIXFc-sc) and one Fc chain (Fc-sc) that are linked together through two disulfide bonds at the Fc hinge region. rFIXFc requires two protein subunits, FIXFc-sc (642 amino acids, SEQ ID NO: 2) and Fc-sc (227 amino acids, SEQ ID NO: 4), to accumulate in the transfected cell line to form the target rFIXFc protein produced. The polynucleotide sequences encoding FIXFc-sc and Fc-sc are shown as SEQ ID NO: 1 and SEQ ID NO: 3, respectively.

In some embodiments, the factor IX region of rFIXFc has a primary amino acid sequence identical to the allelic form of plasma-derived factor IX with a threonine at position 148, and structural and functional characteristics similar to endogenous factor IX. The Fc domain of rFIXFc includes the hinge, CH2, and CH3 regions of IgG1. The mature form of the combined rFIXFc heterodimer contains 869 amino acids and has a molecular weight of approximately 98 kilodaltons. In some embodiments, the rFIXFc polypeptide contains an amino acid sequence that is at least 90%, 95%, or 100% identical to amino acids 1-642 of SEQ ID NO: 2. In one embodiment, the second fragment fused to FIX is a partner FcRn binding. In another embodiment, the FcRn binding partner fused to FIX is an Fc fragment. Partner

- 9 045351 FcRn binding is any molecule that can specifically bind to an FcRn receptor followed by active transport through the FcRn receptor of an FcRn binding partner. Thus, the term Fc includes any IgG Fc variants that are functional. The region of the Fc region of IgG that binds to the FcRn receptor has been described on the basis of x-ray crystallography (Burmeister et al., Nature 372:379 (1994), incorporated herein by reference in its entirety). The main area of contact between Fc and FcRn is located near the junction of the CH2 and CH3 domains. All Fc-FcRn contacts are within one Ig heavy chain. FcRn binding partners include, for example, whole IgG, an IgG Fc fragment, and other IgG fragments that have a complete FcRn binding region. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of the CH2 domain and amino acid residues 385-387, 428 and 433-436 of the CH3 domain. Said numbering of amino acids of immunoglobulins or fragments of immunoglobulins or regions of immunoglobulins is based on the work of Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda; MD, which is incorporated herein by reference in its entirety. The FcRn receptor has been derived from several mammalian species, including humans. The sequences of human FcRn, rat FcRn and mouse FcRn are known (Story et al., J. Exp. Med. 180: 2377 (1994), incorporated herein by reference in its entirety.) The Fc may contain the CH2 and CH3 domains of an immunoglobulin hinge immunoglobulin region or without it. Exemplary embodiments of Fc are presented in WO 2004/101740 and WO 2006/074199, which are incorporated herein by reference in their entirety.

The Fc (or Fc region of a chimeric polypeptide) may contain one or more mutations and combinations of mutations.

The Fc (or Fc region of a chimeric polypeptide) may contain mutations that provide an increase in half-life, such as M252Y, S254T, T256E and combinations thereof, as described in Oganesyan et al., Mol. Immunol. 46:1750 (2009), which is incorporated herein by reference in its entirety; H433K, N434F and combinations thereof, as described in Vaccaro et al., Nat. Biotechnol. 23:1283 (2005), which is incorporated herein by reference in its entirety; mutants are described on pages 1-2, paragraph [0012] and in examples 9 and 10 of US 2009/0264627 A1, which is incorporated herein by reference in its entirety; Mutants are also described on page 2, paragraphs [0014]-[0021] of US 20090163699 A1, which is incorporated herein by reference in its entirety.

The Fc (or Fc region of the chimeric polypeptide) may also contain, for example, the following mutations. The Fc fragment of an IgG may be modified according to well-known procedures such as site-directed mutagenesis and the like to produce modified IgG or Fc fragments, or portions thereof, that will bind to FcRn. Such modifications include, for example, modifications at sites distant from contact with FcRn, as well as modifications within contact sites that maintain or even enhance binding to FcRn. For example, the following single amino acid residues in the human IgG1 Fc (Fcy1) can be replaced without significant loss of Fc binding affinity to FcRn:

P238A, S239A, K246A, K248A, D249A, M252A,

T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A,

Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A,

Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A,

D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A33OQ,

A330S, P331A, P331S, EZZZA, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y37 3A, S375A

D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F,

K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A,

S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A and K447A, where, for example, P238A represents a substitution of wild-type proline for alanine at position number 238. Wild-type amino acids can be replaced with other amino acids in addition to alanine, in the positions stated above. Mutations can be introduced into Fc one at a time, resulting in over a hundred FcRn binding partners different from the native Fc. Additionally, combinations of two, three, or more of these individual mutations can be introduced together, resulting in hundreds more FcRn binding partners. Some of these mutations may confer new functionality on the FcRn binding partner. For example, one embodiment of the invention includes the N297A mutation, which removes a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby increasing the half-life from the blood

- 10 045351 FcRn binding partner, and obtaining an FcRn binding partner that is unable to bind FcyRI, FcyRIIA, FcyRIIB and FcyRIIIA without compromising affinity for FcRn (Routledge et al., 1995, Transplantation 60:847, which is included in this document is incorporated by reference in its entirety; Friend et al., 1999, Transplantation 68:1632, which is incorporated herein in its entirety by reference; Shields et al., 1995, J. Biol. Chem. 276:6591, which is incorporated herein by reference this document in its entirety by reference). Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG in the lower hinge region, which typically corresponds to amino acids 234-237. Thus, another example of new functionality and potential reduction in immunogenicity may arise from mutations in this region, such as by replacing amino acids 233-236 of human IgG1 ELLG with the corresponding sequence from IgG2 PVA (removing one amino acid). It has been shown that FcyRI, FcyRII and FcyRIII, which mediate various effector functions, will not bind to IgG1 when such mutations are introduced (Ward and Ghetie, Therapeutic Immunology 2:11 (1995), which is incorporated herein by reference in its entirety ; and Armor et al., Eur J Immunol 29:2613 (1999), which is incorporated herein by reference in its entirety). An additional example of new functionality arising from the mutations described above is that in some cases the affinity for FcRn can be increased beyond wild-type levels. This increased affinity may reflect an increase in the rate of association, a decrease in the rate of dissociation, or both an increase in the rate of association and a decrease in the rate of dissociation. Mutations believed to confer increased affinity for FcRn include, for example, T256A, T307A, E380A, and N434A (Shields et al., J. Biol. Chem. 276:6591 (2001), which is incorporated herein in full volume by reference).

The Fc (or Fc region of the chimeric polypeptide) may be at least about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence of Fc shown in table. 14 (eg, amino acids 21-247 SEQ ID NO: 4). The Fc (or Fc region of the chimeric polypeptide) may be identical to the amino acid sequence of the Fc shown in table. 14 (eg, amino acids 21-247 SEQ ID NO: 4). As discussed above, exemplary long-acting polypeptides also include FIX fused to one or more albumin polypeptides, albumin-binding polypeptides, or albumin-binding small molecules. In one embodiment of the invention, the albumin is human albumin. Albumin or albumin binding protein can be attached to the N-terminus of FIX or the C-terminus of FIX or inserted between two amino acids in FIX. Examples of albumins, for example, fragments thereof, which can be used in the present invention are known and are shown, for example, in US patent No. 7592010; US Patent No. 6686179; and Schulte, Thrombosis Res. 124 Suppl. 2:S6-S8 (2009), each of which is incorporated herein by reference in its entirety.

Albumin-binding polypeptides may include, without limitation, bacterial albumin-binding domains, albumin-binding peptides, or albumin-binding antibody fragments that can bind albumin. Domain 3 from streptococcal protein G, as described in Kraulis et al., FEBSLett. 378:190-194 (1996) and Linhult et al., Protein Sci. 11:206-213 (2002), are an example of a bacterial albumin-binding domain. Examples of albumin-binding peptides include a number of peptides having the core sequence DICLPRWGCLW (SEQ ID NO: 5); see, for example, Dennis et al., J. Biol. Chem. 2002, 277: 35035-35043 (2002). Examples of albumin-binding antibody fragments are described in Muller and Kontermann, Curr. Opin. Mol. Ther. 9:319-326 (2007); Rooverset et al., Cancer Immunol. Immunother. 56:303-317 (2007) and Holt et al., Prot. Eng. Design Sci., 21:283–288 (2008), which are incorporated herein by reference in their entirety.

In some aspects of the invention, the recombinant FIX polypeptide of the present invention contains at least one attachment site for a small molecule, non-polypeptide, variant or derivative that can bind to albumin. An example of such albumin-binding moieties is 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate (designation Albu), as described in Trusselet et al, Bioconjugate Chem. 20:2286-2292 (2009). As discussed above, exemplary long-acting polypeptides also include FIX fused to at least one C-terminal peptide (CTP) of the β-subunit of human chorionic gonadotropin or a fragment, variant or derivative thereof. CTP can be attached to FIX either through the N-terminus of FIX, or through the C-terminus of FIX. It is known that one or more CTP peptides attached to or inserted into a recombinant protein increase the in vivo half-life of that protein; see, for example, US Pat. No. 5,712,122, which is incorporated herein by reference in its entirety. Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 6) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ (seq id NO: 7); see, for example, US Patent Application Publication No. US2009/0087411 A1, incorporated by reference. As discussed above, exemplary long-acting polypeptides also include FIX fused to at least one sequential

- 11 045351 stu PAS or its fragment, variant or derivative. The PAS sequence can be attached to either the N-terminus of FIX or the C-terminus of FIX. A PAS peptide or PAS sequence, as used herein, refers to an amino acid sequence consisting primarily of alanine and serine residues, or containing principally alanine, serine and proline residues, wherein under physiological conditions the amino acid sequence is in a random helix conformation. Therefore, a PAS sequence is a building block, an amino acid polymer, or a sequence cassette containing, essentially consisting of, or consisting of alanine, serine, and proline, which can be used as part of a heterologous fragment in a chimeric protein. An amino acid polymer can also be in a random helix conformation when residues other than alanine, serine, and proline are added to the PAS sequence as a minor component. By minor component it is meant that amino acids other than alanine, serine and proline can be added to the PAS sequence to a certain level, for example up to about 12%, i.e. about 12 of the 100 amino acids of the PAS sequence, up to about 10%, up to about 9%, up to about 8%, about 6%, about 5%, about 4%, about 3%, i.e. about 2% or about 1% amino acids. Amino acids other than alanine, serine and proline may be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr and Val. Under physiological conditions, the PAS peptide is in a random helix conformation and thus can mediate an increase in in vivo and/or in vitro stability of the recombinant protein of this invention and has procoagulant activity.

Non-limiting examples of PAS peptides include

ASPAAPAPASPAAPAPSAPA (SEQ Ш No.: 8), AAP ASP AP AAP S AR AP AAP S (SEQ ID No.: 9),

APSSPSPSAPSSPSPASPSS (SEQ Ш No.: 10), APSSPSPSAPSSPSPASPS (SEQ Ш No.: И), SSPSAPSPSSPASPSPSSPA (SEQ Ш No.: 12), AASPAAPSAPPAAASPAAPSAPPA (SEQ Ш No.: 13), ASAAAPAAASAAASAPSAAA (SEQ Ш No.: 14) or any derivatives thereof , fragments or combinations thereof. Additional examples of PAS sequences are provided, for example, in US Patent Publication No. 2010/0292130 A1, PCT Application Publication No. WO 2008/155134 A1, and European Granted Patent No. EP 2173890.

As discussed above, exemplary long-acting polypeptides also include FIX fused to at least one transferrin peptide or a fragment, variant or derivative thereof. At least one transferrin peptide may be attached to either the N-terminus of FIX or the C-terminus of FIX or inserted between two amino acids in FIX. Any transferrin can be attached to or inserted into the recombinant FIX protein of the present invention. For example, wild-type human Tf (Tf) is a 679 amino acid protein of approximately 75 kDa (ignoring glycosylation) with two major domains: N (about 330 amino acids) and C (about 340 amino acids), which appear to arise from - for gene duplication; see GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM039847, and S95936 (www.ncbi.nlm.nih.gov), each of which is incorporated herein by reference in its entirety.

Transferrin transports iron via transferrin receptor (TfR)-mediated endocytosis. After iron is released into the endosomal compartment and the Tf-TfR complex returns to the cell surface, Tf is released back into the extracellular space for the next cycle of iron transport. Tf has a long half-life of more than 14-17 days (Li et al., Trends Pharmacol. Sci. 23:206-209 (2002)). Transferrin-containing fusion proteins have been explored for increased half-life, targeted delivery for cancer treatment, oral delivery, and long-term activation of proinsulin (Brandsma et al., Biotechnol. Adv., 29: 230-238 (2011); Bai et al., Proc. Natl. Acad. Sci. USA 102:7292-7296 (2005); Kim et al., J. Pharmacol. Exp. Ther., 334:682-692 (2010); Wang et al., J. Controlled Release 155:386-392 (2011)).

As discussed above, exemplary long-acting polypeptides also include FIX fused to at least one polyethylene glycol (PEG) moiety. PEGylated FIX can refer to a conjugate that is formed between FIX and at least one polyethylene glycol (PEG) molecule. PEG is commercially available in a variety of molecular weights and average molecular weight ranges. Typical examples of average PEG molecular weight ranges include, but are not limited to, about 200, about 300, about 400, about 600, about 1000, about 1300-1600, about 1450, about 2000, about 3000, about 3000-3750, about 3350 , about 3000-7000, about 3500-4500, about 50007000, about 7000-9000, about 8000, about 10000, about 8500-11500, about 16000-24000, about 35000, about 40000, about 60000 and about 80000 dalton. These average molecular weights are provided as examples only and are not intended to be limiting.

The long-acting recombinant FIX protein of the present invention may be PEGylated to include one or more (eg, 2-4) PEG moieties. PEGylation can be accomplished using any of the PEGylation reactions known in the art. Methods for preparing PEGylated protein derivatives generally involve: (a) contacting the polypeptide with a polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions in which the peptide of the invention is attached to one or more PEG groups ; and (ii) obtaining the reaction product(s). Typically, the optimal reaction conditions are determined for each case separately based on known parameters and the desired result.

There are a number of methods for attaching PEG available to those skilled in the art, for example Malik F et al., Exp. Hematol. 20:1028-35 (1992); Francis, Focus on Growth Factors 3(2):4-10 (1992); publications of European patents No. EP 0401384, EP 0154316 and EP 0401384; and publication of international patent applications No. WO92/16221 and WO95/34326. As a non-limiting example, FIX variants may contain cysteine substitutions at one or more insertion sites in FIX, and these cysteine residues may be further attached to the PEG polymer; see Mei et al., Blood 116:270-279 (2010) and US Patent No. 7,632,921, which are incorporated herein by reference in their entirety.

As discussed above, exemplary long-acting polypeptides also include FIX fused to at least one hydroxyethyl starch (HES) polymer. HES is a derivative of natural amylopectin and is broken down in the body by alpha-amylase. HES exhibits beneficial biological properties and is used in clinical settings as a blood volume restoring agent and for hemodilution; see, for example, Sommermeyer et al., Krankenhauspharmazie 8:271-278 (1987); and Weidler et al., Arzneim.-Forschung/Drug Res. 41:494.498 (1991).

HES is characterized mainly by its molecular weight distribution and degree of substitution. HES has an average molecular weight (weighted average) of 1 to 300 kDa, 2 to 200 kDa, 3 to 100 kDa, or 4 to 70 kDa. Hydroxyethyl starch may be further characterized by a molar degree of substitution that is 0.1 to 3, 0.1 to 2, 0.1 to 0.9, or 0.1 to 0.8, and the ratio between C2:C6 substitution is in the range from 2 to 20 in relation to hydroxyethyl groups. HES with an average molecular weight of about 130 kDa is VOLUVEN® from Fresenius. VOLUVEN® is an artificial colloid used, for example, for fluid replacement when indicated in the treatment and prevention of hypovolemia. There are a number of HES coupling methods available to those skilled in the art, for example similar to the PEG coupling methods described above.

The coagulating activity of factor IX is expressed in international units (IU). One ME of factor IX activity approximately corresponds to the amount of factor IX in one milliliter of normal human plasma. Several methods are available to measure factor IX activity, including the one-step coagulation assay (activated partial thromboplastin time; aPTT), the thrombin generation test (TGT), and rotational thromboelastometry (ROTEM®).

Buffer agents.

Buffering agents useful in the present invention may be a weak acid or base used to maintain the acidity (pH) of a solution close to a selected value after the addition of another acid or base. Suitable buffering agents can increase the stability of pharmaceutical formulations by maintaining a constant pH of the formulation. Suitable buffering agents can also provide physiological compatibility or optimize solubility. Rheological, viscosity and other properties may also depend on the pH of the formulation. Commonly known buffering agents include, but are not limited to, histidine, citrate, succinate, acetate and phosphate. In some embodiments, the buffering agent includes L-histidine or mixtures of L-histidine and L-histidine hydrochloride with agents to ensure that the solution is isotonic, the pH being adjustable with an acid or base known in the art. In some embodiments, the buffering agent is L-histidine. In some embodiments, the pH of the composition is maintained between about 6 and about 8, or between about 6.5 and about 7.5.

Stabilizing agents.

Stabilizing agents are added to a pharmaceutical preparation in order to stabilize the drug. Such agents can stabilize proteins in several different ways. Commonly known stabilizing agents include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine or threonine, carbohydrates such as glucose, sucrose, trehalose, raffinose or maltose, polyhydric alcohols such as glycerol, mannitol, sorbitol, cyclodextrins or dextrans of any type and with any molecular weight, or PEG. In one aspect of the invention, the stabilizing agent is selected to maximize the stability of the FIX polypeptide in lyophilized preparations. In some embodiments, the stabilizing agent is sucrose.

Volume-forming agent.

Bulking Agents Bulking agents can be added to a pharmaceutical preparation to increase the volume and weight of the drug, facilitating its accurate dosing and handling. Commonly known bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate or magnesium stearate. In some embodiments, the bulking agent is mannitol.

Surfactants.

Surfactants are amphiphilic substances with lyophilic and lyophobic groups. The surfactant may be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

Prelyophilization composition.

In one aspect, the present invention provides a pre-lyophilization composition comprising:

(a) a factor IX (FIX) polypeptide with the coagulating activity of FIX;

(b) a buffering agent;

(c) a stabilizing agent;

(d) a bulking agent; and (e) a surfactant, wherein the nominal volume of the formulation is less than about 5 ml, less than about 4 ml, or less than about 3 ml, and each of (a)-(e) is present in an amount per vial ( mg/vial), sufficient to provide:

(1) increasing the stability of the FIX polypeptide after lyophilization;

(2) reducing recovery time after lyophilization;

(3) reducing splashing onto the stopper of the bottle containing the composition;

(4) reducing the lyophilization cycle time;

(5) increasing the shelf life of the lyophilisate obtained from the pre-lyophilization composition at room temperature; or (6) any combination thereof, compared to a control pre-lyophilization composition, wherein the control composition contains (a)-(e) in amounts per vial identical to the pre-lyophilization composition, but the nominal volume is at least 5 ml. In some embodiments, the nominal volume for the control formulation is 5.3 ml or 5 ml.

In other embodiments, the pre-lyophilization composition provides at least two, at least three, at least four, or at least five characteristics selected from (1) increased stability of the FIX polypeptide after lyophilization; (2) reduced recovery time after lyophilization; (3) reduced splashing onto the stopper of the bottle containing the composition; (4) reduced lyophilization cycle time; and (5) increased shelf life of the lyophilisate obtained from the pre-lyophilization composition at room temperature. In some embodiments, the pre-lyophilization composition provides (1) increased stability of the FIX polypeptide after lyophilization. In some embodiments, the pre-lyophilization composition provides (2) reduced recovery time after lyophilization. In some embodiments, the pre-lyophilization composition provides (3) reduced splashing onto the stopper of the vial containing the composition. In some embodiments, the pre-lyophilization composition provides (4) a reduction in lyophilization cycle time. In some embodiments, the pre-lyophilization composition provides (5) increased shelf life of the lyophilisate obtained from the pre-lyophilization composition at room temperature. In some embodiments, the pre-lyophilization composition provides (6) any combination of the characteristics described herein.

In some embodiments, the pre-lyophilization composition contains at least about 100 IU/vial of FIX polypeptide. In some embodiments, the pre-lyophilization composition contains at least about 200 IU/vial to about 10,000

IU/vial of FIX polypeptide, from about 200 IU/vial to about 6000 IU/vial or from about 500

IU/vial up to about 5000 IU/vial. In some embodiments, the pre-lyophilization composition contains at least about 220 IU/vial, about 250 IU/vial, about 300

IU/vial, about 400 IU/vial, about 500 IU/vial, about 600 IU/vial, about 700 IU/vial, about 800 IU/vial, about 900 IU/vial, about 1000 IU/vial, about 1100 IU/vial vial, about 1200 IU/vial, about 1300 IU/vial, about 1400 IU/vial, about 1500 IU/vial, about 2000

IU/vial, about 2500 IU/vial, about 3000 IU/vial, about 4000 IU/vial, about 5000

IU/vial, about 5500 IU/vial, about 6000 IU/vial, about 6500 IU/vial, about 7000

IU/vial, approximately 7500 IU/vial, approximately 8000 IU/vial, approximately 8500 IU/vial, approximately 9000

IU/vial, approximately 9,500 IU/vial or approximately 10,000 IU/vial of FIX polypeptide.

In some embodiments, a higher concentration in the pre-lyophilization composition is achieved by reducing the fill volume. In some embodiments, the nominal volume of the pre-lyophilization composition is about 4.0 ml, about 3.5 ml, about 3.0 ml, about 2.9 ml, about 2.8 ml, about 2.7 ml, about 2.65 ml, about 2.6 ml, about 2.5 ml, about 2.4 ml, about 2.3 ml, about 2.2 ml, about 2.1 ml or about 2.0 ml. In some embodiments, the nominal volume of the pre-lyophilization composition is about 2.65 ml. In some embodiments, the nominal volume of the pre-lyophilization composition is less than about 5 ml.

In some embodiments, the FIX polypeptide may be further concentrated by a further purification step, such as a second ultrafiltration step.

In some embodiments, the reduced recovery time is less than 1.5 minutes, less than 1 minute, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 20 seconds, or less than 10 seconds. In specific embodiments of the invention, the reduced recovery time is less than 30 seconds. In some embodiments, the reduced lyophilization cycle time of the pre-lyophilization composition is about 4 days or less, about 3 days or less, about 2 days or less, or about 1 day or less.

In some embodiments, the concentration of the buffering agent in the pre-lyophilization composition is from about 3 mg/ml to about 15 mg/ml, from 4 mg/ml to about 12 mg/ml, from about 5 mg/ml to about 10 mg/ml, or from about 5.82 mg/ml to about 9.7 mg/ml. In one embodiment, the buffering agent is present at a concentration of from about 3.88 mg/ml to about 9.7 mg/ml. In one embodiment of the invention, the buffering agent is present at a concentration of about 7.76 mg/ml. In some embodiments, the pre-lyophilization composition contains L-histidine at a concentration of about 7.76 mg/ml.

In some embodiments, the concentration of the stabilizing agent in the pre-lyophilization composition is from about 10 mg/ml to about 50 mg/ml, from about 13 mg/ml to about 40 mg/ml, from about 15 mg/ml to about 35 mg/ml or from about 17.85 mg/ml to about 29.95 mg/ml. In one embodiment of the invention, the buffering agent is present at a concentration of about 23.8 mg/ml. In some embodiments, the pre-lyophilization composition contains sucrose at a concentration of 23.8 mg/ml.

In some embodiments, the concentration of the bulking agent in the pre-lyophilization composition is from about 20 mg/ml to about 100 mg/ml, from about 30 mg/ml to about 70 mg/ml, from about 30 mg/ml to about 60 mg/ml or from about 35.7 mg/ml to about 59.5 mg/ml. In one embodiment of the invention, the bulking agent is present at a concentration of about 47.6 mg/ml. In some embodiments, the pre-lyophilization composition contains mannitol at a concentration of about 47.6 mg/ml.

In some embodiments, the concentration of surfactant in the pre-lyophilization composition is from about 0.01 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4 mg/ml, from about 0.1 mg /ml to about 3 mg/ml, from about 0.01 mg/ml to about 2 mg/ml, or from about 0.05 mg/ml to about 1 mg/ml. In one embodiment of the invention, the surfactant is present at a concentration of about 0.2 mg/ml. In some embodiments, the pre-lyophilization composition contains polysorbate 20 or polysorbate 80 at a concentration of about 0.2 mg/ml.

In some embodiments, the concentration of the FIX polypeptide in the pre-lyophilization composition is from about 80 IU/ml to about 2750 IU/ml. In some embodiments, the concentration of the FIX polypeptide in the pre-lyophilization composition is at least about 100 IU/ml, at least about 200 IU/ml, at least about 300 IU/ml, at least about 400 IU/ml, at least at least about 500 IU/ml, at least about 600 IU/ml, at least about 700 IU/ml, at least about 800 IU/ml, at least about 900 IU/ml, at least about 1000 IU/ ml, at least about 1500 IU/ml, at least about 2000 IU/ml, or at least about 2500 IU/ml.

In one aspect, the present invention provides a pre-lyophilization composition comprising:

(a) from about 80 to about 2750 IU/ml rFIXFc;

(b) about 7.76 mg/ml L-histidine;

(c) about 47.6 mg/ml mannitol;

(d) about 23.8 mg/ml sucrose and (e) about 0.2 mg/ml polysorbate 20.

In some embodiments, the nominal volume of such pre-lyophilization composition is about 3 ml, about 2.9 ml, about 2.8 ml, about 2.7 ml, about 2.65 ml, about 2.6 ml, about 2.5 ml , about 2.4 ml, about 2.3 ml, about 2.2 ml, about 2.1 ml or about 2.0 ml. In one embodiment of the invention, the nominal volume of such a pre-lyophilization composition is about 2.65 ml.

Additionally, this invention provides a lyophilized powder that is obtained by lyophilizing any of the above pre-lyophilization compositions. In some embodiments, the pre-lyophilization composition is any composition described herein.

- 15 045351

Lyophilized powder.

The present invention also provides a lyophilized powder containing a FIX polypeptide, a buffering agent, a stabilizing agent, a bulking agent, a surfactant, or any combination thereof. In some embodiments, the lyophilized powder contains from about 8 mg to about 39 mg per vial, from about 9 mg to about 35 mg per vial, from about 10 mg to about 30 mg per vial, from about 12 mg to about 25 mg per vial. vial, from about 15 mg to about 23 mg per vial of buffering agent (eg, L-histidine). In one embodiment, the lyophilized powder contains about 25 mg per vial, about 24 mg per vial, about 23 mg per vial, about 22 mg per vial, about 21 mg per vial, about 20 mg per vial, about 19 mg per vial, about 18 mg per vial, about 17 mg per vial, about 16 mg per vial, about 15 mg per vial buffering agent. In another embodiment, the lyophilized powder contains about 20.6 mg per vial of buffering agent. In some embodiments, the buffering agent is L-histidine.

In some embodiments, the lyophilized powder contains from about 27 mg to about 132 mg per vial, from about 30 mg to about 120 mg per vial, from about 40 mg to about 110 mg per vial, from about 50 mg to about 100 mg per vial. vial, from about 60 mg to about 90 mg per vial of stabilizing agent. In one embodiment, the lyophilized powder contains about 68 mg per vial, about 67 mg per vial, about 66 mg per vial, about 65 mg per vial, about 64 mg per vial, about 63 mg per vial, about 62 mg per vial, about 61 mg per vial, about 60 mg per vial, about 59 mg per vial stabilizing agent. In another embodiment, the lyophilized powder contains about 63.1 mg per vial of stabilizing agent. In some embodiments, the stabilizing agent is sucrose.

In some embodiments, the lyophilized powder contains from about 50 mg to about 265 mg per vial, from about 53 mg to about 265 mg per vial, from about 50 mg to about 250 mg per vial, from about 53 mg to about 265 mg per vial. vial, about 80 mg to about 200 mg per vial, about 100 mg to about 150 mg per vial, or about 110 mg to about 140 mg per vial of bulking agent. In one embodiment, the lyophilized powder contains about 131 mg per vial, about 130 mg per vial, about 129 mg per vial, about 128 mg per vial, about 127 mg per vial, about 126 mg per vial, about 125 mg per vial, about 124 mg per vial, about 123 mg per vial, or about 122 mg per vial of bulking agent. In another embodiment, the lyophilized powder contains about 126.1 mg per vial of bulking agent. In some embodiments, the bulking agent is mannitol.

In some embodiments, the lyophilized powder contains from about 0.03 mg to about 13 mg per vial, from about 0.05 mg to about 10 mg per vial, from about 0.07 mg to about 8 mg per vial, from about 0 .1 mg to about 2 mg per vial of surfactant. In one embodiment, the lyophilized powder contains about 1 mg per vial, about 0.9 mg per vial, about 0.8 mg per vial, about 0.7 mg per vial, about 0.6 mg per vial, about 0.5 mg per vial, about 0.4 mg per vial, about 0.3 mg per vial, about 0.2 mg per vial, or about 0.1 mg per vial surfactant. In another embodiment, the lyophilized powder contains about 0.5 mg per vial of surfactant. In one embodiment, the lyophilized powder contains about 0.53 mg per vial of surfactant. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

In some embodiments, the lyophilized powder contains:

(a) a FIX polypeptide in an amount of from about 2 mg per vial to about 150 mg per vial; (b) a buffering agent in an amount of from about 10 mg per vial to about 30 mg per vial.

(c) a bulking agent in an amount of from about 70 mg per vial to about 200 mg per vial;

(d) a stabilizing agent in an amount from about 30 mg per vial to about 100 mg per vial; and (e) a surfactant in an amount from about 0.05 mg per vial to about 5 mg per vial.

In some embodiments, the lyophilized powder contains:

(a) a FIX polypeptide in an amount of from about 2.2 mg per vial to about 125 mg per vial;

(b) a buffering agent in an amount of from about 8 mg per vial to about 39 mg per vial;

(c) a bulking agent in an amount of from about 53 mg per vial to about 265 mg per vial;

(d) a stabilizing agent in an amount from about 27 mg per vial to about 132 mg per vial; and (e) a surfactant in an amount from about 0.03 mg per vial to about 13 mg per vial.

In some embodiments, the lyophilized powder contains:

(a) lyophilized FIX polypeptide in an amount of from about 2.2 mg per vial to about 125 mg per vial;

(b) a buffering agent in an amount of from about 12.5 mg per vial to about 25 mg per vial;

(c) a stabilizing agent in an amount of from about 32.5 mg per vial to about 80 mg per vial;

- 16 045351 (d) a bulking agent in an amount of about 75 mg per vial to 150 mg per vial and (e) a surfactant in an amount of about 0.1 mg/ml to about 2 mg/ml.

In one embodiment of the invention, the lyophilized powder contains:

(a) from about 2.2 to about 125 mg/vial of FIX polypeptide;

(b) about 20.6 mg/vial L-histidine;

(c) about 126.1 mg/vial mannitol;

(d) about 63.1 mg/vial sucrose and (e) about 0.53 mg/vial polysorbate 20.

Restored composition

The invention further provides a reconstituted formulation comprising any of the above-described lyophilized powders reconstituted with a reconstitution buffer solution.

In some embodiments, the recovery buffer solution is a NaCl solution. In some embodiments, the volume of the recovery buffer solution is 5 ml.

In some embodiments of the invention, the reconstituted composition contains:

(a) a FIX polypeptide at a concentration of about 0.9 mg/ml to about 50 mg/ml;

(b) a buffering agent at a concentration of from 2 mg/ml to about 5 mg/ml;

(c) a bulking agent at a concentration of from 20 mg/ml to about 30 mg/ml;

(d) a stabilizing agent at a concentration of 8 mg/ml to 15 mg/ml per vial; and (e) a surfactant at a concentration of 0.05 mg/ml to about 0.4 mg/ml.

In some embodiments of the invention, the reconstituted composition contains:

(a) a FIX polypeptide at a concentration of about 0.9 mg/ml to about 50 mg/ml;

(b) a buffering agent at a concentration of about 3.88 mg/ml;

(c) a bulking agent at a concentration of about 23.8 mg/ml;

(d) a stabilizing agent at a concentration of about 11.9 mg/ml;

(e) a surfactant at a concentration of about 0.1 mg/ml; and (f) a reducing buffer solution containing about 3.25 mg/ml NaCl.

In some embodiments of the invention, the reconstituted composition contains:

(a) a FIX polypeptide at a concentration of about 80 IU/ml to about 2750 IU/ml;

(b) a buffering agent at a concentration of about 25 mM;

(c) a bulking agent at a concentration of about 131 mM;

(d) a stabilizing agent at a concentration of about 35 mM;

(e) surfactant at a concentration of 0.01% (w/v) and (f) reducing buffer solution.

Examples of formulation compositions are additionally presented in table. 2-4.

In one aspect, the invention further provides a vial containing pre-lyophilization compositions, lyophilized powder or reconstituted compositions described herein.

In another aspect, the invention provides a kit comprising a first container containing the lyophilized powder described herein and a second container containing a reconstitution buffer solution in a volume sufficient to produce a reconstituted formulation when combined with the lyophilized powder of the first container. In some embodiments, the volume of the recovery buffer solution in the kit is 5 ml. In some embodiments, the volume is about 5.3 ml. In some embodiments, the kit's recovery buffer solution contains NaCl. In some embodiments, the kit is used to treat hemophilia B.

In yet another aspect, the invention provides a method of administering a FIX polypeptide to a hemophilia B patient in need thereof, comprising administering to the patient a reconstituted formulation described herein, which administration prevents bleeding episodes in the patient or reduces their frequency or severity.

The invention further provides a method for preventing, treating, ameliorating or managing hemophilia B in a patient in need thereof, comprising administering a reconstituted formulation described herein.

Methods for producing lyophilized powder containing FIX polypeptide.

The invention provides methods for producing a lyophilized powder containing a FIX polypeptide. In one aspect, the invention provides lyophilization methods including lyophilization of the pre-lyophilization compositions described herein. In another aspect, the invention provides a lyophilization process comprising a single drying step.

In one aspect, the invention provides a method for lyophilizing a FIX polypeptide, comprising:

(a) a freezing step comprising freezing the pre-lyophilization composition containing the FIX polypeptide and an aqueous solvent;

- 17 045351 (b) a vacuum step, including reducing the pressure in the chamber containing the frozen pre-lyophilization composition sufficient to remove aqueous solvent from the frozen pre-lyophilization composition; and (c) one drying step, including raising the temperature of the frozen pre-lyophilization composition above the collapse temperature, resulting in obtaining lyophilized powder. In another aspect of the invention, the duration of the lyophilization process is reduced compared to a control method, for example, a lyophilization process with two or more drying steps. In other aspects of the invention, the lyophilized powder obtained using the present method has one or more of the following characteristics: (1) increased stability of the FIX polypeptide; (2) reduced recovery time; (3) reduced splashing onto the stopper of the bottle containing the composition; or (4) extended shelf life of the lyophilized powder at room temperature. In some embodiments of the invention, the collapse temperature is -1.5°C.

In some embodiments, during the freezing step, the pre-lyophilization composition is frozen to a freezing temperature that is from about -65 to about -40°C, from about -65 to about -45°C, from about -65 to about -55°C , from about -60 to about -40°C, from about -60 to about -50°C, or from about -60 to about -55°C. In some embodiments, during the freezing step, the pre-lyophilization composition is frozen to a freezing temperature of about -55°C. In some embodiments, during the freezing step, the freezing temperature is lowered from about 5°C to about -55°C. In some embodiments, during the freezing step, the freezing temperature is maintained for about 30 minutes to about 5 hours, about 1 hour to about 5 hours, about 1.5 hours to about 5 hours, about 1.5 hours to about 4 hours, about 1.5 hours to about 3 hours, or about 1.5 hours to 2.5 hours. In some embodiments, the freezing temperature is maintained for about 2 hours during the freezing step. In some embodiments, the frozen pre-lyophilization composition obtained in step (a) is further processed in the annealing step (a') before the vacuum step (b). In some embodiments, during the annealing step, the temperature of the frozen pre-lyophilization composition obtained in step (a) is raised to an annealing temperature that is from about -15°C to about -2°C. In some embodiments, during the annealing step, the temperature of the frozen pre-lyophilization composition obtained in step (a) is raised to the annealing temperature, which is about -6°C. In some embodiments, during the annealing step, the annealing temperature is maintained for about 2 hours to about 4 hours. In some embodiments, during the annealing step, the annealing temperature is maintained for about 30 minutes to about 5 hours, from about 1 hour to about 5 hours, from about 2 hours to about 5 hours, from about 2 hours to about 4 hours, or from about 2.5 hours to 3.5 hours. In some embodiments, during the annealing step, the annealing temperature is maintained for about 3 hours. In some embodiments, during the annealing step, the temperature of the frozen pre-lyophilization composition is lowered from the annealing temperature to a temperature that is from about -65°C to about -40°C. In some embodiments, during the annealing step, the temperature of the frozen pre-lyophilization composition is reduced from the annealing temperature to a temperature of -55°C. In some embodiments, the vacuum step includes subjecting the frozen pre-lyophilization composition to a vacuum that corresponds to a pressure of from about 0.05 to about 1 mbar, from about 0.05 to about 0.50 mbar, from about 0.10 to about 0.50 mbar, about 0.15 to about 0.50 mbar, about 0.20 to about 0.50 mbar, or about 0.25 to about 0.50 mbar. In some embodiments, the vacuum during the evacuation step corresponds to a pressure of about 0.33 mbar.

In some embodiments, the vacuum stage is maintained for about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour. In some embodiments, the vacuum stage is maintained for about 2 hours.

In some embodiments, the drying step includes raising the temperature of the frozen pre-lyophilization composition from about -55°C to a drying temperature of about 40°C. In some embodiments, the drying temperature is at least about 30°C, at least about 32°C, at least about 34°C, at least about 35°C, at least about 36°C, at least at least about 38°C, at least about 39°C, at least about 40°C. In other embodiments, the drying temperature is about 35°C, about 40°C, about 32°C, or about 45°C.

In some embodiments, the drying step further includes maintaining the drying temperature for from about 10 hours to about 40 hours, from about 10 hours to about 30 hours, or from about 20 hours to about 30 hours. In some embodiments, the drying temperature is maintained at for about 25 hours.

In some embodiments, the drying step is carried out at a pressure that is from about 0.05 to about 1 mbar, from about 0.05 to about 0.50 mbar, from about 0.10 to about 0.50 mbar, from about 0 .15 to about 0.50 mbar, about 0.20 to about 0.50 mbar or about 0.20 to about 0.45

- 18 045351 mbar. In some embodiments, pressure is maintained during the drying step

0.33 mbar. The unit of measurement mbar can be converted to torr or any other units. For example, 1 mbar can be converted to 0.75006375541921 torr.

In one aspect, the invention provides a method for producing a lyophilized powder containing a FIX polypeptide comprising:

(a) a freezing step comprising freezing the pre-lyophilization composition containing the FIX polypeptide by lowering the temperature over about 2 hours to a freezing temperature of about -55° C. and maintaining the freezing temperature for 2 hours;

(a') an annealing step comprising raising the temperature of the frozen pre-lyophilization composition obtained in step (a) for about 1.5 hours to an annealing temperature of about -6°C, maintaining the annealing temperature for 3 hours, and lowering the temperature for about 1.5 hours to about -55°C;

(b) a vacuum step comprising maintaining the frozen pre-lyophilization composition obtained in step (a') at about -55° C. for two hours at atmospheric pressure and reducing the pressure over about 2 hours to about 0.33 mbar; and (c) one drying step comprising raising the temperature of the frozen pre-lyophilization composition obtained in step (b) to about 40°C for 3 hours while maintaining the pressure at about 0.33 mbar, and maintaining the temperature of the frozen pre-lyophilization composition at about 40°C for 25 hours while maintaining the pressure at about 0.33 mbar, resulting in a lyophilized powder.

In some embodiments, the lyophilization process has a shorter cycle time. In some embodiments, the lyophilized powder produced using the methods described herein has the following characteristics:

(1) increased stability of the FIX polypeptide;

(2) reduced recovery time;

(3) reducing splashing onto the stopper of the bottle containing the composition;

(4) increased shelf life of lyophilized powder at room temperature; or (5) any combination thereof.

In some embodiments, the lyophilization cycle length may be less than about 4.5 days, about 4 days, about 3.5 days, about 3 days, about 2.5 days, or about 2 days. In other embodiments, the lyophilization cycle time is about 3 days or less. In some embodiments, the nominal volume of the pre-lyophilization composition used in the lyophilization process is less than about 5 ml. In some embodiments, the nominal volume is about 4 ml, about 3.5 ml, about 3.0 ml, about 2.9 ml, about 2.8 ml, about 2.7 ml, about 2.65 ml, about 2 .6 ml, about 2.5 ml, about 2.4 ml, about 2.3 ml, about 2.2 ml, about 2.1 ml or about 2.0 ml. In one embodiment of the invention, the nominal volume is about 2.65 ml. In some embodiments, the reduced recovery time for the lyophilized powder produced by the lyophilization process is less than about 1.5 minutes, less than about 1 minute, less than about 50 seconds, less than about 40 seconds, less than about 30 seconds, less than about 20 seconds, or less than about 10 s. In some embodiments, the reduced recovery time for the lyophilized powder produced by the lyophilization process is less than about 30 seconds.

In some embodiments, the reduced lyophilization cycle time of the prelyophilization composition used in the lyophilization process is about 4 days or less, about 3 days or less, about 2 days or less, or about 1 day or less.

In some embodiments, the lyophilized powder is obtained from the pre-lyophilization composition in about 90 hours or less, about 80 hours or less, about 70 hours or less, about 60 hours or less, about 50 hours or less, about 45 hours or less, about 40 h or less or about 30 h or less.

In some embodiments, the lyophilized powder is obtained from the pre-lyophilized composition in about 45 hours or less.

In some embodiments, the residual moisture content of the lyophilized powder is less than about 1.0%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, or about 0.3%. In some embodiments, the residual moisture content of the lyophilized powder is less than about 0.5%.

In one aspect, the invention provides a method for stabilizing a lyophilized powder containing a FIX polypeptide, comprising lyophilizing the pre-lyophilization composition according to the methods described herein, wherein the increase in stability of the lyophilized powder is determined by size exclusion chromatography (SEC) relative to the lyophilized powder prepared using the lyophilization method, including more than one drying stage.

In another aspect, the invention provides a method for increasing the shelf life of a lyophilized powder containing a FIX polypeptide, comprising lyophilizing the pre-lyophilization composition according to the methods described herein, wherein the increased shelf life of the lyophilized powder is determined by SEC and/or FIX coagulating activity assay regarding the shelf life of lyophilized powder prepared using a lyophilization process involving more than one drying step.

The invention also provides a method for reducing the reconstitution time of a lyophilized powder containing a FIX polypeptide, comprising lyophilizing a pre-lyophilization composition according to the methods described herein, wherein the reconstitution time of the lyophilized powder is reduced relative to the reconstitution time of a lyophilized powder prepared using a lyophilization process comprising more than one drying step. .

The invention further provides a method of reducing the duration of the lyophilization process when preparing a lyophilized powder containing a FIX polypeptide, comprising lyophilizing a pre-lyophilization composition according to the methods described herein, wherein the duration of the lyophilization process of the pre-lyophilization composition is reduced relative to the duration of the lyophilization process when obtaining a lyophilized powder using a lyophilization method, including more than one drying stage. Having described the present invention in detail, the same will be more clearly understood on the basis of the following examples, which are included for purposes of illustration only and are not intended to limit the present invention. All patents and publications mentioned herein are expressly incorporated herein by reference.

Examples

Example 1. Drug and drug compositions containing factor IX-Fc.

Description of rFIXFc.

rFIXFc is a fully recombinant, long-acting fusion protein consisting of coagulation factor IX (FIX) covalently linked to the Fc domain of human immunoglobulin Gl (IgG1). The factor IX region of rFIXFc has a primary amino acid sequence identical to the allelic form of plasma-derived factor IX with a threonine at position 148, and has structural and functional characteristics similar to endogenous factor IX. The Fc domain of rFIXFc includes the hinge, CH2, and CH3 regions of IgG1. rFIXFc contains 869 amino acids and has a molecular weight of approximately 98 kilodaltons. rFIXFc is produced using recombinant DNA technology in a cell line derived from human embryonic kidney (HEK) and then purified. The formulation of the rFIXFc drug formulation of the present invention containing rFIXFc may enable the development of highly concentrated drug formulations, for example, with a drug formulation dosage of 4000+ IU/vial. This requires a higher concentration of rFIXFc protein in the drug, as shown in Table. 1 below. The composition of the rFIXFc medicinal product according to this invention makes it possible to increase the shelf life of the medicinal product with dosages of 250 and 500 IU/vial and increase stability at room temperature. Development data demonstrates that the 250 and 500 IU/vial formulations are significantly more stable under accelerated testing conditions than the control formulation.

The composition of the rFIXFc medicinal product according to this invention also makes it possible to reduce the recovery time after lyophilization. The recovery time of the control drug varies in the range of 1-2 minutes. Development data demonstrates that the LCM drug reduces recovery time to less than 30 seconds.

The composition of the rFIXFc medicinal product according to this invention also makes it possible to reduce the duration of the lyophilization process. Currently the freeze drying cycle is ~4.5 days. With a smaller nominal vial volume, the duration can be reduced to ~3 days or less for more cost-effective production.

Medicine (medicine).

The drug formulation for the rFIXFc drug of this invention will use the same excipients as the control drug. Higher concentrations will be achieved using a second ultrafiltration step during drug production; see table 1.

- 20 045351

Table 1

Drug compositions

Component Control _ „ „ Medicinal drug drug Recombinant factor IX-Fc L-histidine 10-13 mg/ml 65-75 mg/ml 3.88 mg/ml (25 mmol) 3.88 mg/ml (25 mmol) Polysorbate 20 0.1 mg/ml (0.01%) 0.1 mg/ml (0.1%)

Medicinal product (LP).

In order to achieve the above objectives, the drug product was formulated to contain double the concentrations of all components of the control drug product (protein and excipients), which was achieved by reducing the nominal volume of the vial before lyophilization. This ensures that the doses of all components are maintained for the patient, and at the same time improves the performance parameters of the drug mentioned above.

In table 2-4 below describe in detail the composition of the initial pre-lyophilization mixture, the composition of the solid mass in the vial after lyophilization and the composition after reconstitution. It is important to note the wide range of rFIXFc protein concentrations. Because each batch of Factor IX varies slightly in IU/mg activity, the stock mixture is formulated based on the measured activity. This results in a range of protein concentrations, and this variation, added to the various dosages in the drug product, gives the ranges shown below.

table 2

Medicinal product - compositions of pre-lyophilization formulations.

Initial compositions for lyophilization to obtain a medicinal product

Control drug Control Drug Component drug 250-2000 medicinal product drug 250-5500 IU/bottle 3000 IU/bottle IU/bottle Recombinant 0.45-9.5 mg/ml 5.5-13.7 mg/ml 0.9-50 mg/ml factor IX-Fc 40-500 IU/ml 480-750 IU/ml 80-2750 IU/ml L-histidine 3.88 mg/ml (25 mmol) 3.88 mg/ml (25 mmol) 7.76 mg/ml (50 mM) Mannitol 23.8 mg/ml (131 mM) 23.8 mg/ml (131 mM) 47.6 mg/ml (261 mM) Sucrose 11.9 mg/ml (35 mM) 11.9 mg/ml (35 mM) 23.8 mg/ml (70 mM) Polysorbate 20 0.1 mg/ml (0.01%) 0.1 mg/ml (0.01%) 0.2 mg/ml (0.2%) Nominal volume for original 5.3 ml 7.4 ml 2.65 ml mixtures

Table 3 Drug product - lyophilized compositions.

Compositions of lyophilized solid mass in vials for obtaining a medicinal product

Control Control Medicinal PPDpotut drug drug Component a drug a drug irsidrd 1 250-5500 250-2000 IU/bottle 3000 IU/bottle IU/vial* Recombinant 2.2-46 mg/vial 28-69 mg/vial 2.2-125 mg/vial factor IX-Fc L-histidine 19.4 mg/vial 27.2 mg/vial 19.4 mg/vial

- 21 045351

Mannitol 119 mg/vial 167 mg/vial 119 mg/vial Sucrose 59.5 mg/vial 83.3 mg/vial 59.5 mg/vial Polysorbate 20 0.5 mg/vial 0.7 mg/vial 0.5 mg/vial

*Values are nominal values and do not include overflow. The values including overfill are: 20.6 mg/vial (L-histidine), 126.01 mg/vial (mannitol), 63.1 mg/vial (sucrose), and 0.53 mg/vial (polysorbate).

Table 4

Medicinal product - compositions of reconstituted compounds.

Compositions of the medicinal product in vials after reconstitution

Control drug Control drug Drug ΠΤΙΛΠΟ ΙΊΟΤ Component a drug a drug prspdrd1 250-5500 250-2000 IU/bottle 3000 IU/bottle IU/bottle

Solvent 5.2 ml NaCl in water for injection Recombinant factor IX-Fc L-histidine 0.45-9.5 mg/ml 5.5-13.7 mg/ml 0.9-50.0 mg/ml 40-500 IU/ml 480-750 IU/ml 80-2750 IU/ml 3.88 mg/ml (25 mmol) 5.43 mg/ml (35 mmol) 3.88 mg/ml (25 mmol) Mannitol 23.8 mg/ml (131 mmol) 33.3 mg/ml (183 mmol) 23.8 mg/ml (131 mmol) Sucrose 11.9 mg/ml (35 mmol) 16.7 mg/ml (48 mmol) 11.9 mg/ml (35 mmol) Polysorbate 20 0.1 mg/ml (0.01%) 0.14 mg/ml (0.014%) 0.1 mg/ml (0.01%)

Restorative_, _ , „ 3.25 mg/ml 3.25 mg/ml buffer solution Volume after 5.3 ml 5.3 ml recovery 3.25 mg/ml 5.3 ml

Example 2. Development of lyophilization cycle parameters for the second generation drug rFIXFc.

Summary.

The purpose of this study was to evaluate the parameter ranges of the drying phase of the lyophilization cycle for the rFIXFc drug formulation of this invention. This report summarizes a statistical simulation experiment (SE) to evaluate lyophilization process parameters (drying shelf temperature, chamber vacuum level, and drying time) and their effect on product drying temperature, residual moisture content, or drug product drying rate.

Preliminary modeling of the lyophilization cycle for the second generation rFIXFc drug product demonstrated that separate primary and secondary drying steps are not necessary due to the high formulation collapse temperature of approximately -1.5°C To evaluate the influence of shelf temperature, vacuum degree and drying time on residual content moisture and product temperature during the placebo lyophilization process, 12 model experiments (ME) were developed. Analysis of the data showed that in order for the residual moisture content to be less than 1%, a minimum shelf temperature of 30°C is required during the drying phase. The analysis also demonstrated that drying times greater than 25 h did not further reduce the residual moisture content of the vials, and the vacuum level in the chamber only had a minor effect on the residual moisture content. Product temperature during drying is significantly affected by shelf temperature and chamber vacuum level, but the most severe drying conditions in the study (40°C shelf temperature and 1000 mTorr vacuum level) produced product temperatures that were greater than 10°C. lower than the collapse temperature. Vial mass flow is primarily a function of shelf temperature, and to maintain vacuum levels, the commercial lyophilizer must be capable of handling moisture flow rates of 0.7 g/h/vial. A lyophilization cycle has been proposed that results in a product with a residual moisture content of <0.5%, in which the shelf temperature is 40°C, the vacuum degree in the chamber is 250 mTorr (0.33 mbar), and the drying temperature is 25 h.

Introduction.

The second generation factor IX-Fc drug formulation (rFIXFc-2G) was designed to increase protein stability under accelerated testing conditions, decrease recovery time, and reduce nominal volume to reduce plug spatter.

This was accomplished by reducing the nominal volume from 5.3 ml to 2.65 ml and doubling the concentrations of protein and excipients in the formulation so that the reconstituted formulation is the same as the first generation formulation. Another advantage of reducing the nominal volume was the reduction in the amount of water that must be removed during the lyophilization process.

Since the development of a new drug product requires re-validation of the drug production process, the lyophilization cycle was re-developed. The collapse temperature of the placebo formulation was measured to be approximately -1.5°C, which would allow the use of a shorter lyophilization cycle than that designed for the lyophilization process of the rFIXFc control formulation. In initial experiments it was determined that there was no need for a separate primary drying step, as the product did not deteriorate even at shelf temperatures above 40°C. The cycle was developed using freezing parameters for the control drug in combination with going directly to the primary drying temperature after applying vacuum. Placebo is a good substitute for vials containing rFIXFc-2G, as mannitol provides a crystalline structure to the mass and its appearance is the same as in vials containing the active ingredient. Amorphous sugar is also more difficult to dry than protein, so the resulting residual moisture content is slightly higher, representing the worst value for the process. The lack of protein in vials also reduces water vapor resistance, giving the worst rating for vial mass flow rate.

A statistical simulation experiment was conducted to evaluate the parameters of the lyophilization process (drying shelf temperature, chamber vacuum level, and drying time) and their effect on product drying temperature, residual moisture content, or drug product drying rate.

Materials and methods.

The purpose of this study was to evaluate the parameter ranges of the drying phase of the lyophilization cycle for the second generation rFIXFc drug product. The drying phase parameters and intervals used for ME planning using JMP 9 software are presented in Table. 5. The resulting ME plan, consisting of 12 experiments, which demonstrates the specified parameter values for an individual test, is shown in Table. 6.

Table 5

Drying parameter intervals used when planning ME

Simulated drying parameter Minimum value Maximum value Shelf temperature (°C) 0 40 Chamber vacuum (mtorr) 100 1000 Drying time (h) 15 35

Table 6

Research plan based on the results of template calculations in JMP9

Trial Temperature (°C) Vacuum (mtorr) Time (h) 1 40 100 35 2 0 100 15 3 40 1000 25 4 20 100 28.2 5 20 500 25 6 0 100 25 7 20 550 25 8 40 250 15 9 20 1000 15 10 0 1000 35 AND 10 500 25 12 40 500 25

For each lyophilization cycle in the study, eighty Schott 10 mL vials (cat. no. 68000320) were filled with 2.75 mL of second-generation rFIXFc placebo, as shown in Table 1. 7, which is the worst nominal volume for estimating moisture content. For each experiment, filled vials were placed on the same shelf with three thermocouples, as shown in FIG. 1.

- 23 045351

Table 7

Placebo formulation for second generation rFIXFc

Component FW g/l mm % (mass/volume) Histidine 155.15 7.76 50.00 0.776 PS-20 1227.54 0.2 0.1629 0.020 Mannitol 182.172 47.6 261.29 4,760 Sucrose 342.30 23.8 69.53 2,380 P= 1.029 g/ml

The lyophilization cycles used were modifications of the cycle shown in table. 8. The temperature of the freeze dryer shelves during drying, the duration of the drying stage and the vacuum level during drying were varied based on the experimental design shown in Table. 6. For each lyophilization cycle, Lyostar II (SP Industries) was used and the vials were placed on the middle shelf.

Table 8

Lyophilization cycle parameters showing ME input temperature, time and vacuum

Phase Stage Pace. (°C) Speed (°C/min) Wetting (min.) Vacuum (mtorr) Loading Loading 25 - of necessity - Freezing Balancing 0 0.5 60 - Freezing -55 0.5 120 - Annealing -6 0.5 180 - Freezing -55 0.5 120 - Decreased pressure Initial application of vacuum -55 - - simulated pressure Drying G/2° drying simulated temperature does not change simulated time simulated pressure Final Final 5 5.0 of necessity simulated pressure

For each lyophilization cycle, five vials were selected from the corners and middle, and their residual moisture content was determined using the TDMP-74 procedure and averaged for the entire shelf. The product temperature during the drying step and the mass flow rate in the vial were measured using thermocouples and by manometric temperature measurement using Lyostar II software. These results were analyzed using JMP 9 software to evaluate the effect of drying parameters on the second generation rFIXFc lyophilization process. JMP stage analysis was performed to determine significant variables, and these variables were then analyzed using a standard least squares influence algorithm that shows how process outputs (residual moisture content, product temperature, and mass flow during drying) respond to inputs. variables.

- 24 045351

Results and its discussion.

The results of twelve lyophilization experiments are shown in Table. 9.

Table 9

ME results for residual moisture content, vial mass flow and product temperature

Not tested Pace. (°C) Vacuum m (mtorr) Time I(h) Link NB Humidity (%) dm/dt (g/h/bottle) Tproduct (MTM, °C) 1 40 100 35 16322-117 0.33 0.53 -26.5 2 0 100 15 16322-110 2.82 0.28 -32.2 3 40 1000 25 16322-090 0.57 0.45 -15.2 4 20 100 28.2 16322-139 1.28 0.41 -29.5 5 20 500 25 16322-098 1.58 0.57 -20.3 6 0 100 25 16322-151 2.18 0.27 -32.5 7 20 550 25 16322-146 1.49 0.56 -19.7 8 40 250 15 18266-004 0.59 0.70 -21.9 9 20 1000 15 16322-131 1.95 0.38 -15.6 10 0 1000 35 16322-124 2.56 0.17 -15.8 AND 10 500 25 16322-104 2.01 0.35 -21.8 12 40 500 25 16322-083 0.49 0.56 -19.8

1. Effect of lyophilization cycle parameters on residual moisture content.

The placebo for rFIXFc-2G was used as a substitute for the worst performing drug since it is generally more difficult to remove residual moisture from sugars during the secondary drying step than from protein. The final predicted curve showing the results of the ME analysis is shown in FIG. 2. Several observational findings are clear. Shelf temperature has the most significant effect on the residual moisture content of the drug product. This is expected based on the fact that secondary drying, which removes tightly bound water, is a process controlled by diffusion and desorption. The model predicts with high confidence that to achieve a residual moisture level of less than 1%, a shelf temperature above 30°C is required. The degree of vacuum appears to have a small but measurable effect on the final residual moisture content. The drying time appears to have a point of diminishing influence observed after 25 hours, where further increases in drying time do not result in a further reduction in the residual moisture content. This type of behavior is consistent with the kinetic approach to equilibrium boundaries determined by the shelf temperature, and the moisture content asymptotically approaches a certain value when additional drying is not possible. Based on this ME residual moisture content analysis, the drying shelf temperature should be 30°C or higher and the drying time should be 25 hours or less.

2. The influence of lyophilization cycle parameters on the temperature of the product during sublimation.

The freeze-dried placebo breakdown temperature for rFIXFc-2G was measured to be approximately -1.5°C. In practical terms, this means that the bulk structure of the drug product will be maintained provided that the product temperature is maintained below this collapse temperature as bulk water is removed from the vial during lyophilization. Using ME, it was determined that both shelf temperature and chamber vacuum degree have a significant effect on product temperature, as shown in FIG. 3. The degree of vacuum in the chamber has the greatest influence, with higher pressures resulting in higher product temperatures during sublimation. Even at 1000 mTorr (1.33 mbar), the highest measured product temperature was -15.2°C, approximately 13°C lower than the product collapse temperature. Shelf temperature also has some effect on product temperature, but this effect is less pronounced than the effect of vacuum. The analysis demonstrates that the risk of collapse is small even at a shelf temperature of 40°C and a vacuum degree in the chamber of 1000 mTorr, which virtually eliminates the possibility of collapse using any practicable freeze-drying cycle parameters.

3. Influence of lyophilization cycle parameters on the mass flow rate in the vial during sublimation.

Vial mass flow rate (dm/dt) is a measure of the rate at which water is removed from vials during the sublimation process. When faster drying is desired to reduce the time spent on a lyophilization cycle, too much moisture can enter the condensers of industrial lyophilizers and cause a loss of vacuum in the product chamber. The placebo represents the worst case of mass flow in the vial. Since the composition does not contain protein, the proportions of solid

- 25 045351 substances in the mass after lyophilization are minimized, and this leads to a lower stability of the mass flow from the lyophilized mass. Shelf temperature has a significant effect on the mass flow in the vial, as shown in FIG. 4. Moreover, an increase in temperature leads to faster sublimation. The degree of vacuum in the chamber is included in the model for ME, but the p value is 0.136, which is not significant at 95%. The highest measured dm/dt in the study was 0.7 g/h/vial.

4. Second-generation rFIXFc lyophilization cycle proposed based on ME placebo study.

Data from the ME placebo study suggest that it is possible to design a lyophilization cycle to achieve a target residual moisture content of <0.5% while maintaining the product temperature below the collapse temperature using a single drying step. The proposed lyophilization cycle is shown in Table. 10, and data from ME trial 8, under conditions similar to the proposed rFIXFc-2G lyophilization cycle, are shown in FIG. 5. The target moisture content was chosen to be 0.5% as this is the average value for the first generation rFIXFc drug series. This moisture content is selected with a margin, therefore, although the product will absorb moisture under accelerated stability testing conditions, the quality characteristics of the product will not be affected.

The freeze-anneal steps of the lyophilization cycle designed for the lyophilization cycle of the control drug rFIXFc were used, and the separate primary and secondary drying steps were replaced by a single drying step at a shelf temperature of 40°C and a vacuum corresponding to a pressure of 250 mTorr for 25 hours.

Table 10

Proposed lyophilization cycle for rFIXFc-2G

Phase Stage Pace. (°C) Speed (°C/min) Wetting (min.) Vacuum (mtorr) Loading Loading 25 - of necessity - Freezing Balancing 0 0.5 60 - Freezing -55 0.5 120 - Annealing -6 0.5 180 - Freezing -55 0.5 120 - Decreased pressure Initial application of vacuum -55 - - 250 Drying 1°/2° drying 40 0.5 1500 250 Final Final 5 5.0 of necessity 250

Conclusion.

A 12-run simulation was performed to evaluate the effects of second-generation rFIXFc lyophilization process parameters on residual moisture content, product temperature, and vial mass flow rate when using a placebo. Analysis of the data showed that in order for the residual moisture content to be less than 1%, a minimum shelf temperature of 30°C is required during the drying phase. The analysis also demonstrated that drying times greater than 25 h did not significantly reduce the residual moisture content of the vials, and the degree of vacuum only had a minor effect on the residual moisture content. The temperature of the product during drying is significantly influenced by the temperature of the shelves and the degree of vacuum in the chamber. The most severe conditions in the study (40°C shelf temperature and 1000 mTorr vacuum) produced product temperatures that were more than 10°C lower than the collapse temperature.

A lyophilization cycle has been proposed that results in a product with a residual moisture content of <0.5%, in which the shelf temperature is 40°C, the degree of vacuum in the chamber is 250 mTorr (0.33 mbar), and the drying temperature is 25 hours. Previous the description of specific embodiments of the invention so fully discloses the general nature of the invention that others can, using knowledge in the art, easily modify and/or adapt for various applications such specific embodiments of the invention, without undue experimentation, without departing from the general concept of the present invention. It is therefore intended that such adaptations and modifications are within the scope and range of equivalents of the disclosed embodiments based on the ideas and teachings presented herein. It should be understood that the language or terminology used herein is for purposes of description and not limitation; therefore, terminology or language herein should be interpreted by one skilled in the art in light of the ideas and guidelines presented.

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The scope and scope of the present invention should not be limited by any of the above illustrative embodiments of the invention, but should be determined only in accordance with the following claims and their equivalents. Other embodiments of the invention will be apparent to those skilled in the art from consideration of this specification and practice of the invention described herein.

All documents, articles, publications, patents and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual publication or patent application had been specifically and separately identified as being incorporated by reference.

This application claims priority to U.S. Provisional Patent Application No. 61/969801, filed March 24, 2014, which is incorporated herein by reference in its entirety.

List of sequences

Table 11 FIX polynucleotide sequences

FIX-Fc DNA chain sequence (FIX signal peptide is underlined, FIX sequence is double underlined, Fc fragment is in bold) (SEQ ID NO: 1, which encodes SEQ ID NO: 2).

Nucleotide sequence of pSYN-FIX-030 (nucleotides 1-7583):

exon 1 FIX (signal peptide, propeptide from the first amino acid): nucleotides 690-777, mini-intron FIX: nucleotides 778-1076, FIX sequence: nucleotides 1077-2371,

Fc: nucleotides 2372-3052.

gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac

101 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt

151 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga

201 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca

251 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat

301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact

351 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt

401 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc

451 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat

501 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat

551 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc

601 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac

651 tatagggaga cccaagcttc gcgacgtacg gccgccacca tgcagcgcgt

701 gaacatgatc atggcagaat caccaggcct catcaccatc tgccttttag

751 gatatctact cagtgctgaa tgtacaggtt tgtttccttt tttaaaatac

801 attgagtatg cttgcctttt agatatagaa atatctgatg ctgtcttctt

- 27 045351

851 cactaaattt tgattacatg atttgacagc aatattgaag agtctaacag 901 ccagcacgca ggttggtaag tactgtggga acatcacaga ttttggctcc 951 atgccctaaa gagaaattgg ctttcagatt atttggatta aaaacaaaga 1001 ctttcttaag agat gtaaaa ttttcatgat gttttctttt ttgctaaaac 1051 taaagaatta ttcttttaca tttcaqtttt tcttqatcat qaaaaccfcca 1101 acaaaattct gaatcggcca aagaggtata attcaggtaa attggaagag 1151 tttqttcaacf qcfaat ctacfa qacfacfaatcft atqcfaaqaaa aqtcftacfttt 1201 tgaagaagca cgagaagttt ttgaaaacac tgaaagaaca actgaatttt 1251 ggaagcagta tgttgatgga gatcagtgtg agtccaatcc atgtttaaat 13 01 cfcfccfcfcacftt cfcaaqqatcfa cattaattcc tatqaatqtt cfcftcftccctt 1351 tggatttgaa ggaaagaact gtgaattaga tgtaacatgt aacattaaga 14 01 atcfcfca cratcf cqacfcacfttt tqtaaaaata cftgctgataa caacfcftcfcftt 1451 tgctcctgta ctgagggata tcgacttgca gaaaaccaga agtcctgtga 1501 accagcagtg ccatttccat gtggaagagt ttctgtttca caaacttcta 1551 acfct caccccf tgctcfaqact qtttttcctcf atcftcfcracta tqtaaattct 1601 actgaagctg aaaccatttt ggataacatc actcaaagca cccaatcatt 1651 taatqacttc actccfcfcfttcf ttcfcftgcfaqa aqatqccaaa ccacfcftcaat 1701 tcccttggca ggttgttttg aatggtaaag ttgatgcatt ctgtggaggc 1751 tctatcgtta atgaaaaatg gattgtaact gctgcccact gtgttgaaac 18 01 tcfcftcfttaaa attacaqttq tccfcagcftga acataatatt qacfcfaqacacf 1851 aacatacaga gcaaaagcga aatgtgattc gaattattcc tcaccacaac 1901 tacaatcfcacf ctattaataa qtacaaccat qacattgccc ttctqcfaact 1951 ggacgaacc c ttagtgctaa acagctacgt tacacctatt tgcattgctg 2001 acaaggaata cacgaacatc ttcctcaaat ttggatctgg ctatgtaagt 2 051 cfcfctcfcfcfcraa gagtcttcca caaagcfcfaqa tcaqctttacf ttcttcaqta 2101 ccttacfacftt ccacttcfttcf acccfacfccac atcftcttccfa tctacaaacft 2151 tcaccatcta taacaacatq ttctcftcfctcf gcttccatqa aqqacfcftacfa 2201 gattcatgtc aaggagatag tgggggaccc catgttactg aagtggaagg 2251 gaccagtttc ttaactggaa ttattagctg gggtgaagag tgtgcaatga 2301 aacfcfcaaata tqqaatatat accaacfcftcft ccccfcftatcft caactcfcratt 2351 aaggaaaaaa caaagctcac tqacaaaact cacacatgcc caccgtgccc 2401 agctccggaa ctcctgggcg gaccgtcagt cttcctcttc cccccaaaac

- 28 045351

2451 ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg

2501 gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga

2551 cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca

2601 acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg

2651 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc

2701 ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac

2751 aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc

2801 agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga

2851 gtggggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg

2901 tgttggactc cgacggctcc ttcttcctct acagcaagct caccgtggac

2951 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga

3001 ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta

3051 aatgagaatt cagacatgat aagatacatt gatgagtttg gacaaaccac

3101 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta

3151 ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc

3201 gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt

3251 cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa

3301 tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca cgaagtgcac

3351 gcagttgccg gccgggtcgc gcagggcgaa ctcccgcccc cacggctgct

3401 cgccgatctc ggtcatggcc ggcccggagg cgtcccggaa gttcgtggac

3451 acgacctccg accactcggc gtacagctcg tccaggccgc gcaccacac

3501 ccaggccagg gtgttgtccg gcaccacctg gtcctggacc gcgctgatga

3551 acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc ctccacgaag

3601 tcccgggaga acccgagccg gtcggtccag aactcgaccg ctccggcgac

3651 gtcgcgcgcg gtgagcaccg gaacggcact ggtcaacttg gccatggttt

3701 agttcctcac cttgtcgtat tatactatgc cgatatacta tgccgatgat

3751 taattgtcaa cacgtgctga tcagatccga aaatggatat acaagctccc

3801 gggagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt

3851 ctggaatagc tcagaggcag aggcggcctc ggcctctgca taaataaaaa

3901 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg

3951 ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag

4001 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac

- 29 045351

4051 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct

4101 ggggagcctg gggactttcc acaccctcgt cgagctagct tcgtgaggct

4151 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag

4201 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg

4251 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg

4301 gtggggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt

4351 cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg

4401 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt

4451 ccacctggct ccagtacgtg attcttgatc ccgagctgga gccaggggcg

4501 ggccttgcgc tttaggagcc ccttcgcctc gtgcttgagt tgaggcctgg

4551 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg

4601 tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg

4651 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaggat

4701 ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg

4751 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac

4801 cgagaatcgg acggggggtag tctcaagctg gccggcctgc tctggtgcct

4851 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg

4901 gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctc

4951 cagggggctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag

5001 tcacccacac aaaggaaagg ggcctttccg tcctcagccg tcgcttcatg

5051 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctgga

5101 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg

5151 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca

5201 cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt

5251 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag

5301 gtgtcgtgaa cacgtggtcg cggccgcgcc gccaccatgg agacagacac

5351 actcctgcta tgggtactgc tgctctgggt tccaggttcc actggtgaca

5401 aaactcacac atgcccaccg tgcccagcac ctgaactcct gggaggaccg

5451 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg

5501 gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg

5551 aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag

5601 acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt

- 30 045351

5651 cctcaccgtc ctgcaccagg actggctgaa tggcaaggag tacaagtgca

5701 aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa

5751 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg

5801 cgatgagctg accaagaacc aggtcagcct gacctgcctg gtcaaaggct

5851 tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag

5901 aacaactaca agaccacgcc tcccgtgttg gactccgacg gctccttctt

5951 cctctacagc aagctcaccg tggacaagag caggtggcag caggggaacg

6001 tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag

6051 aagagcctct ccctgtctcc gggtaaatga ctcgagagat ctggccggct

6101 gggcccgttt cgaaggtaag cctatcccta accctctcct cggtctcgat

6151 tctacgcgta ccggtcatca tcaccatcac cattgagttt aaacccgctg

6201 atcagcctcg actngtgcctt ctagttgcca gccatctgtt gtttgcccct

6251 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc

6301 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat

6351 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca

6401 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa

6451 agaaccagtg gcggtaatac ggttatccac agaatcaggg gataacgcag

6501 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag

6551 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca

6601 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa

6651 gataccaggc gtttccccct agaagctccc tcgtgcgctc tcctgttccg

6701 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt

6751 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg

6801 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc

6851 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga

6901 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt

6951 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac

7001 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt

7051 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta

7101 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga

7151 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa

7201 cgaaaactca cgttaaggga ttttggtcat gacattaacc tataaaaata

- 31 045351

7251 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa

7301 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc

7351 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg

7401 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga

7451 gtgcaccata tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa

7501 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg

7551 gcgatcggtg cggggcctctt cgctattacg cca

Table 12

FIX polypeptide sequence

Hybrid monomeric FIX-Fc: produced by co-expression of FIX-Fc and Fc chains.

A. FIX-Fc chain (SEQ ID NO: 2).

The C-terminal lysine is not present in any of the subunits; this processing is often observed in recombinant proteins obtained in mammalian cell culture, as well as in proteins obtained from plasma.

FIX-Fc-SC subunit (the Fc region in FIX-Fc is in bold).

YNSGKLEEFV QGNLERECME EKCSFEEARE VFENTERTTE FWKQYVDGDQ

CESNPCLNGG SCKDDINSYE CWCPFGFEGK NCELDVTCNI KNGRCEQFCK

101 NSADNKWCS CTEGYRLAEN QKSCEPAVPF PCGRVSVSQT SKLTRAETVF

151 PDVDYVNSTE AETILDNITQ STQSFNDFTR WGGEDAKPG QFPWQWLNG

201 KVDAFCGGSI VNEKWIVTAA HCVETGVKIT WAGEHNIEE TEHTEQKRNV

251 IRIIPHHNYN AAINKYNHDI ALLELDEPLV LNSYVTPICI ADKEYTNIFL

301 KFGSGYVSGW GRVFHKGRSA LVLQYLRVPL VDRATCLRST KFTIYNNMFC

351 AGFHEGGRDS CQGDSGGPHV TEVEGTSFLT GIISWGEECA MKGKYGIYTK

401 VSRYVNWIKE KTKLTDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR

451 TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV

501 LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR

551 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF

601 LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

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Claims (13)

Table 13 Fc Polynucleotide Sequences Fc DNA sequence (mouse IgK signal peptide is underlined) (SEQ ID NO: 3, which encodes SEQ ID NO: 4). (1) increasing the stability of the FIX polypeptide after lyophilization of the composition; 1. Pre-lyophilization composition containing: (a) a factor IX (FIX) polypeptide at a concentration of about 80 to about 2750 IU/ml, wherein the FIX polypeptide comprises FIX fused to an Fc region; (b) L-histidine at a concentration of about 3 to about 15 mg/ml; (c) sucrose at a concentration of about 10 to about 50 mg/ml; (d) mannitol at a concentration of about 20 to about 100 mg/ml; and (e) polysorbate 20 at a concentration of about 0.01 to about 5 mg/ml, the nominal volume of the formulation being less than 4 ml; and each of (a) to (e) is present in an amount per vial (mg/vial) sufficient to provide: 1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED 51 PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK 101 CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK 151 GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG 201 NVFSCSVMHE ALHNHYTQKS LSLSPGK CLAIM 1 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg 51 ttccactggt gacaaaactc acacatgccc accgtgccca gcacctgaac 101 tcctgggagg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 151 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag 201 ccacgaagac cctgaggtca agttcaactg gtacgtggac ggcgtggagg 251 tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 301 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa 351 ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga 401 aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 451 ctgcccccat cccgcgatga gctgaccaag aaccaggtca gcctgacctg 501 cctggtcaaa ggcttctatc ccagcgacat cgccgtggag tggggagagca 551 atgggcagcc ggagaacaac tacaagacca cgcctcccgt gttggactcc 601 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg 651 gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca 701 accactacac gcagaagagc ctctccctgt ctccgggtaa a Table 14 Fc chain polypeptide sequence (SEQ ID NO: 4) 2. Prelyophilization composition according to claim 1, characterized in that L-histidine is present at a concentration of about 7.76 mg/ml; sucrose is present at a concentration of about 23.8 mg/ml; mannitol is present at a concentration of about 47.6 mg/ml or polysorbate 20 is present at a concentration of about 0.2 mg/ml. (2) reducing splashing on the vial stopper after lyophilization of the composition; 3. Prelyophilization composition according to claim 1 or 2, characterized in that the nominal volume of the composition per bottle is about 2.65 ml. (3) reducing the lyophilization cycle time; - 33 045351 (4) increasing the shelf life of the lyophilisate obtained from the pre-lyophilization composition at room temperature, or (5) any combinations thereof, compared to the control pre-lyophilization composition, wherein the control composition contains (a)-(e) in quantity per bottle, identical to this pre-lyophilization composition, but has a nominal volume of 5 ml. 4. Pre-lyophilization composition according to any one of claims 1-3, characterized in that the FIX polypeptide contains an amino acid sequence that is at least 95% identical to amino acids 1-642 of the sequence SEQ ID NO: 2. 5. Pre-filization composition according to any one of claims 1 to 4, characterized in that the FIX polypeptide is a heterodimeric protein with one FIXFc chain (FIXFc-sc) and one Fc chain (Fc-sc), which are linked together through two disulfide bonds in hinge region of the Fc, wherein FIXFc-sc has the amino acid sequence SEQ ID NO: 2 without the C-terminal lysine, and Fc-sc has the amino acid sequence SEQ ID NO: 4 without the C-terminal lysine. 6. Lyophilized powder, which is obtained by lyophilizing the pre-lyophilization composition according to any one of claims 1-5. 7. A reconstituted formulation comprising the lyophilized powder of claim 6 reconstituted with a reconstitution buffer solution. 8. A bottle containing lyophilized powder according to claim 6. 9. A kit comprising a first container containing the lyophilized powder according to claim 6, and a second container containing a reconstitution buffer solution in a volume sufficient to obtain the reconstituted composition when combined with the lyophilization composition from the first container. 10. Use of the reconstituted composition according to claim 7 to prevent or reduce the frequency or severity of bleeding attacks in a patient with hemophilia B. 11. The method of lyophilization of a pre-lyophilization composition according to claim 1, including: (a) a freezing step comprising freezing the pre-lyophilization composition having a nominal volume of less than 3 ml and containing the FIX polypeptide and an aqueous solvent to a freezing temperature of about -65 to about -40° C.; (b) a vacuum step comprising reducing the pressure of the frozen pre-lyophilization composition by an amount sufficient to remove the aqueous solvent from the frozen pre-lyophilization composition; and (c) a single drying step involving raising the temperature of the frozen pre-lyophilization composition above the collapse temperature of about -1.5° C., resulting in a lyophilized powder. 12. Method according to claim 11, characterized in that: (i) during the freezing step, the temperature of the pre-freezing composition is lowered from about 5 to about -55° C., and during the freezing step, the freezing temperature is maintained for about 30 minutes to about 5 hours; (ii) the frozen pre-lyophilization composition obtained in step (a) is further processed in an annealing step (a') before starting the vacuum step (b), and during the annealing step, the temperature of the frozen pre-lyophilization composition obtained in step (a) is increased to an annealing temperature of about -15 to about -2°C, and wherein during the annealing step, the annealing temperature is maintained for about 30 minutes to about 5 hours; (iii) during the annealing step, the temperature of the frozen pre-lyophilization composition is lowered from the annealing temperature to a temperature that is from about -65 to about -40°C; (iv) the vacuum step involves subjecting the frozen pre-lyophilization composition to a vacuum of about 0.05 to about 1 mbar for about 2 hours; or (v) the drying step involves raising the temperature of the frozen pre-lyophilization composition from about -55° C. to a drying temperature of about 40° C., wherein the drying temperature is maintained for a period of from about 10 to about 40 hours and wherein the drying step is carried out at a pressure of from about 0.05 to about 1 mbar. 13. The method according to claim 11 or 12, characterized in that the pre-lyophilization composition is filtered under sterile conditions and filled into a vial under sterile conditions prior to step (a), wherein the lyophilized powder is obtained from the pre-lyophilization composition in about 45 hours or less or the residual content The moisture content of the lyophilized powder is less than 0.7%. -