The present invention relates to a method of manufacturing a lyophilized plasma-derived Factor VIII (FVIII) product and a corresponding FVIII product.

Factor VIII (FVIII) is an important co-factor in the coagulation cascade. Plasmatic human FVIII is synthesized as a single chain consisting of 2351 amino acids and comprises three A domains (A1-A3), one B domain and two C domains (C1 and C2), interrupted by short acidic sequences (a1-a3). The first 19 amino acids are the signal sequence, which is cleaved by intracellular proteases during secretion, leading to a FVIII molecule of 2332 amino acids.

Upon activation, FVIII is cleaved by thrombin at three positions, leading to a heterotrimer and loss of the B domain (heterotrimeric FVIIIa). The heterotrimer forms a complex with the activated coagulation Factor IXa and coagulation Factor X, and the light chain binds to a phospholipid bilayer, e. g., the cell membrane of (activated) platelets.

Hemophilia A mainly is a genetic bleeding disorder caused by a genetic deficiency in Factor VIII. The disorder is linked to the X-chromosome, occurring in 1 of 5000 newborn males. However, hemophilia A can also occur spontaneously due to an autoimmune response against FVIII. Patients with hemophilia A suffer from longer bleeding durations, spontaneous and internal bleedings, affecting their everyday life.

Hemophilia A patients are generally treated by administration of FVIII. Depending on the severity of the disease (mild, moderate or severe), treatment is on demand or prophylactic. Although there are already several recombinant FVIII product available, there is a strong need for plasma derived FVIII product for therapeutic purposes, with high margins of safety with respect to pathogenic viruses.

Factor VIII preparations from human plasma are known for many years. For manufacturing of plasma-derived Factor VIII preparations human plasma is used as a source material. As an important viral inactivation step the manufacturing process usually comprises a solvent/detergent (S/D) treatment using tri-n-butyl phosphate (TN BP) and polysorbate 80. This polysorbate 80 can then be removed in the next step using anionic exchange chromatography or affinity chromatography. From the chromatography eluate the corresponding drug substance is prepared by adjusting the concentration and buffer composition. As final steps the drug substance is typically lyophilized and may then be subjected to a dry heat treatment for virus inactivation. The obtained drug product usually comprises only minor amounts of polysorbate 80, usually 2 to 12 pg/mL. A typical production process is e. g. disclosed in EP 0 812 858 B1. The process comprises the steps of thawing and solubilizing a cryoprecipitate by adding a water/ethanol mixture and heparine, adsorption of impurities onto AI(OH)3, S/D (solvent/detergent) treatment with tri-n-butylphosphate and polysorbate 80 (as an inactivation step for lipid-enveloped viruses), anion exchange column chromatography and ultrafiltration. Then the concentration of Factor VIII is adjusted to a specific value and transferred by sterile filtration into different vials. The vials are lyophilized and subjected to dry heat treatment as final virus inactivating step.

It is common practice to add certain additives to the drug substance in order to stabilize the drug product during lyophilization and dry heat treatment. But additives, especially in higher amounts, are undesired, as they have the potential to cause other problems during these manufacturing steps. Certain additives also have the potential to cause undesired sideeffects in the patient.

The virus removal and/or inactivation steps are important for product safety. Usual process steps for virus removal are nanofiltration or S/D treatments. Other process steps like dry heat treatment can be used to further inactivate virus in a product.

A process step for virus removal or inactivation is considered successful when a virus can be robustly removed or inactivated by a factor of at least 2, preferably at least 3, especially at least 4 on a logarithmic (base 10) scale, or more. In general, the efficacy of the process step is measured by spiking a probe with model viruses and/or target viruses, performing the virus inactivation/removal step at laboratory scale, and measuring total infectivity in the virus- spiked test material and in the final fraction obtained after the step. For viruses difficult to remove an efficacy of at least 3 log™ is considered a good result.

EP 0 844 005 A1 describes that a high residual moisture (e. g. >0.7 %) increases the efficacy of virus inactivation, but the long-term stability of the drug product is reduced compared to drug product with lower residual moisture.

WO 93/05067 A1 describes the use of viral inactivating agent including Polysorbate 80, which is combined with organic solvents in an S/D treatment. The viral inactivating agent is removed after virus inactivation.

WO 2006/015704 A1 describes a dry heat treatment of solutions comprising fibrinogen with a stabilisation agent comprising ammonium groups or amino groups. WO 99/23111 A1 describes the addition of Tween 80 and other stabilizer agents like albumin before lyophilization and terminal dry heat treatment for a protein concentrate comprising fibrinogen, Factor XI 11 and fibronectin.

WO 94/13329 A1 describes the addition of a surfactant to a biological preparation. The surfactant is removed by chromatography after the heat treatment.

ON 104231073 B and ON 104225601 B describe the use of a non-ionic surfactant as protective agent for dry heat treatment in a composition also including salts and amino acids for preserving the activity of Factor VIII during the dry heat treatment.

It is object of the invention to provide an improved method of manufacturing a plasma- derived Factor VIII product and to provide a Factor VIII product obtained by this method. In particular, it is an object of the present invention to provide a FVIII product with improved virus safety with respect to more robust lipid-enveloped viruses such as represented by PRV (Porcine Pseudorabies Virus). It is also an object of the present invention to combine several advantageous properties in a single product, such as improved virus safety, stability and good solubility. It is a further object to provide a method which is easy to implement and results in a safe product with a good activity and a good stability and with only few side proteins or by-products.

The problem is solved by a method of manufacturing a lyophilized plasma-derived Factor VIII product comprising the following steps: a) providing a liquid plasma-derived Factor VIII preparation; b) adjusting the polysorbate 80 content of the preparation to obtain a resulting concentration of 20 to 120 pg polysorbate 80 per mg total protein, preferably 30 to 110 pg per mg total protein, more preferably 40 to 100 pg per mg protein, even more preferably 40 to 80 pg per mg total protein; b1) optionally transferring the preparation to one or more suitable container(s) each comprising a dosage unit of Factor VIII; c) lyophilization of the preparation obtained in step b) or optionally in step b1); d) dry heat treatment of the preparation obtained in step c) to obtain said lyophilized plasma-derived Factor VIII product.

The method is especially useful for virus inactivation, preferably to inactivate lipid-enveloped viruses. It has been shown by the inventors that the addition of a relatively small amount of polysorbate 80 in the above defined range improves the efficacy of the following dry heat treatment with respect to the virus inactivation. Thus, the inventive method includes the above-mentioned step b) of adjusting the polysorbate 80 content to a certain range. In general, the adjustment means an addition of polysorbate 80.

Virus inactivation by dry heat treatment, whereby the polysorbate 80 content is adjusted in the manner before lyophilization as described above, makes it possible to carry out the dry heat treatment under conditions which, on the one hand, are gentle on the product and, on the other hand, bring about very effective virus inactivation. Surprisingly, the inventors were able to show that this particular effect of the process according to the invention is associated with the adjustment of the polysorbate 80 content in the range as described. Without the adjusted polysorbate 80 range, more intense dry heat treatment conditions would be required to achieve comparable viral inactivation, but these would then cause much greater destruction of the active ingredient and possibly generation of neoantigenic structures. The method according to the invention thus allows substantially greater flexibility in the choice of conditions for the dry heat treatment, since even relatively mild conditions are sufficient to achieve the required viral inactivation. In general, the goal of virus inactivation by dry heat treatment is to achieve an inactivation of suitable model viruses by at least 4 log units.

The process of the invention leads to stable and safe Factor VIII product without the need of addition of further stabilization agents or other additives, especially proteins like albumin or oligopeptides. This reduces the risk of possible side effects in the patients.

The process allows to obtain a high yield of active Factor VIII product with a high margin of safety with respect to infectious viruses, and good stability by adding only a very low amount of polysorbate 80. In particular, the lyophilization as disclosed herein will result in a high degree of viral safety while retaining the integrity of the FVIII molecule. In a preferred embodiment, only polysorbate 80 and substantially no other excipients are added immediately before the step of lyophilization, wherein further excipients like glycine and/or CaCh may be comprised in the preparation from preceding steps for providing the liquid plasma-derived Factor VIII preparation. Thus, the process remains simple and is rather easy to implement.

The Factor VIII preparation in step a) is preferably derived from animal or human plasma, preferably human plasma. Methods for providing such Factor VIII preparations are known in the art.

The adjustment of the polysorbate 80 content according to step b) is preferably done by addition of a small amount of commercially available polysorbate 80 (polyoxyethylen(20)- sorbitan-monooleat) to the Factor VIII preparation. Depending from the total protein content of the preparation as outlined in more detail below e. g. about 5 to 100 pg polysorbate 80 per mL of the preparation may be used to adjust the polysorbate 80 content. Preferably 10 to 30 pg, more preferably 15 to 25 pg polysorbate 80, more preferably 20 pg polysorbate 80 may be added, especially for the preparation of a Factor VIII product with a dosage unit of 250 to 1000 Units Factor VIII, preferably 250 or 500 or 1000 Units Factor VIII. In another embodiment preferably 40 to 80 pg polysorbate 80 per mL of the preparation may be used, more preferably 50 to 70 pg, even more preferably 65 pg polysorbate 80 per mL may be added, especially for the preparation of a Factor VIII product with a dosage unit of more than 1000 Units Factor VIII, preferably 1000 to 2000 Units Factor VIII, preferably 2000 Units Factor VIII.

In especially preferred embodiments the liquid preparation of step b) is transferred into suitable containers, e. g. glass vials or other vials or other containers, according to the optional step b1) prior to lyophilization. Preferably, the steps a) and b) are still performed at the bulk stage. In preferred embodiments the containers contain the lyophilized drug product after the process.

The lyophilization according to step c) is preferably done under vacuum respectively under partial vacuum as it is known to a skilled person. After completion of lyophilization the containers are tightly closed still under vacuum. Preferably, commonly known lyophilization stoppers are used that are loosely placed on the containers before starting the lyophilization, so that vapor can escape. After completion of lyophilization, the stoppers are pressed firmly into the containers while still under the vacuum.

Each container preferably comprises a dosage unit of Factor VIII. In this context, a dosage unit is understood to be the unit to be administered to the patient. If the patient has a higher need, several dosage units can be administered under certain circumstances. Conversely, it may be envisaged that the patient receives only part of a dosage unit, depending on his or her needs.

Preferably, the single container represents the entity provided to the medical personnel for the administration of the preparation. The respective dosage unit may be adapted to the respective needs. E.g., each dosage unit may comprise between 250 units and 2000 units Factor VIII. In preferred embodiments the dosage unit may comprises about 250 Units Factor VIII or about 500 Units Factor VIII or about 1000 Units Factor VIII, or about 2000 Units Factor VIII. The information on Factor VIII activity is preferably based on a chromogenic activity assay as described in more detail below. In a preferred embodiment of the invention the product obtained by the process is a lyophilized drug product, which can be directly administered after reconstitution, preferably after reconstitution with water.

Preferably, the method of manufacturing contains further steps to inactivate or remove viruses, such as solvent/detergent treatment.

The total protein content of the preparation in step a) and/or in step b) is preferably at or below 1.6 mg/mL, more preferably at or below 1 mg/mL, even more preferably at or below 0.7 mg/mL, even more preferably from 0.1 to 0.7 mg/mL, even more preferably from 0.2 to 0.7 mg/mL, even more preferably from 0.4 to 0.6 mg/mL. The total protein content is measured using OD280 as described in more detail below. The mentioned higher protein contents, e. g. in the range of 1.0 to 1.6 mg/mL, are especially preferred in cases where a higher dosage unit is prepared, e. g. 2000 Units Factor VIII per dosage.

If a step of solvent/detergent treatment is included in the inventive method, this may have the consequence that a certain amount of polysorbate 80 is already present in the FVIII preparation of step a) prior to the adjustment respectively addition of polysorbate 80 according to the inventive method. Thus, in certain embodiments the preparation of step a) already comprises up to 12 pg polysorbate 80 per mL prior to the addition of the additional polysorbate 80, e. g. 2 to 12 pg polysorbate 80 per mL. Relating to the preferred protein concentration in the preparation of step a) and/or step b) which is at or below 1 mg/mL, the preparation of step a) already comprises up to 12 pg polysorbate 80 per mg total protein prior to the addition of the additional polysorbate 80 according to step b). Thus, in case of a preceding solvent/detergent treatment, the preparation of step a) may comprise polysorbate 80 in a range of up to 2 to 12 pg polysorbate 80 per mg total protein prior to the adjustment of the polysorbate 80 content according to step b) of the inventive method.

Nevertheless, adjusting the polysorbate 80 concentration in the described range according to step b) of the inventive method has been shown to clearly increase the efficacy of virus inactivation during the terminal dry heat treatment after lyophilization. The amounts in step b) always refer to the total amount of polysorbate 80 in relation to the amount of total protein.

Given a preferred total protein concentration in the preparation of step a) of up to 1.6 mg/mL, in a preferred embodiment the amount of the polysorbate 80 added in step b) is an amount of 5 to 100 pg/mL, preferably 10 to 80 pg/mL. In one preferred embodiment, especially in such cases where a dosage unit of 250 to 1000 Units Factor VIII, preferably 250 or 500 or 1000 Units Factor VIII, per dosage unit are to be prepared, 10 to 30 pg/mL, preferably 15 pg/mL to 25 pg/mL, more preferably 20 pg/mL are added. In another preferred embodiment, especially in such cases where a dosage unit of more than 1000, preferably 1000 to 2000 Units Factor VIII, preferably 2000 Units Factor VIII, per dosage unit are to be prepared, 40 to 80 pg/mL, preferably 60 pg/mL to 70 pg/mL, more preferably 65 pg/mL are added. These higher amounts of added polysorbate 80 are especially useful when the total protein content of the preparation is in a range of 0.8 to 1.6 mg/mL. Thus, in case the total protein concentration of the preparation is relatively high, e. g. in a range of 0.8 to 1.6 mg/mL, it may be preferred to add a larger amount of polysorbate 80, e. g. up to 100 pg/mL, in order to adjust the polysorbate 80 content relating to mg total protein as defined above.

Given a preferred total protein concentration in the preparation of step a) of up to 1.6 mg/mL, especially up to 0.8 mg/mL, in a preferred embodiment the method includes a preceding solvent/detergent treatment and the resulting polysorbate 80 concentration after addition of polysorbate 80 in step b) is 10 to 100 pg/mL, preferably 10 to 80 pg/mL, preferably 10 pg/mL to 50 pg/mL, preferably 10 pg/mL to 40 pg/mL. More preferably, the concentration after addition of polysorbate 80 in step b) is 10 pg/mL to 39 pg/mL, preferably 12 pg/mL to 36 pg/mL, even more preferably 12 pg/mL to 35 pg/mL, especially 17 pg/mL to 27 pg/mL.

In a preferred embodiment in step b) polysorbate 80 is added to obtain a resulting concentration of 20 to 120 pg polysorbate 80 per mg total protein, preferably 30 to 110 pg per mg total protein, more preferably 40 to 100 pg per mg protein, even more preferably 40 to 80 pg per mg total protein, while the concentration of polysorbate 80 after addition of polysorbate 80 in step b) is 10 pg/mL to 100 pg/mL, even more preferably 10 pg/mL to 39 pg/mL, especially 12 pg/mL to 36 pg/mL.

In preferred embodiments the Factor VIII concentration respectively Factor VIII activity within the liquid plasma-derived Factor VIII preparation in step a) and/or step b) of the inventive method is within a range of 50 to 250 Units of Factor VIII per mg total protein, preferably 100 to 200 Units Factor VIII per mg total protein, more preferably 120 to 150 Units Factor VIII per mg total protein. The Factor VIII activity is preferably measured by a chromogenic assay, especially by a two-step chromogenic assay, as described below in more detail.

Preferably, in step b) polysorbate 80 is adjusted respectively added to obtain a resulting concentration of 0.1 to 0.9 pg polysorbate 80 per Unit Factor VIII, preferably 0.1 to 0.8 pg polysorbate 80 per Unit Factor VIII, preferably 0.1 to 0.7 pg polysorbate 80 per Unit Factor VIII, preferably 0.1 to 0.6 pg/Unit Factor VIII, even more preferably 0.1 to 0.5 pg/Units Factor VIII.

In a preferred embodiment in step b) polysorbate 80 is added to obtain a resulting concentration of 0.1 to 0.9 pg polysorbate 80 per Unit Factor VIII, preferably 0.1 to 0.8 pg polysorbate 80 per Unit Factor VIII, preferably 0.1 to 0.7 pg polysorbate 80 per Unit Factor VIII, preferably 0.1 to 0.6 pg/Unit Factor VIII, even more preferably 0.1 to 0.5 pg/Units Factor VIII, while the concentration of polysorbate 80 after addition of polysorbate 80 in step b) is 5 pg/mL to 100 pg/mL.

In a preferred embodiment the preparation of step a) comprises an activity of Factor VIII of 50 to 120 Units/mL, preferably 50 to 100 Units/mL, more preferably 50 to 80 Units/mL.

In such cases where a dosage unit of 250 to 1000 Units Factor VIII, preferably 250 or 500 or 1000 Units Factor VIII, are to be prepared, preferably the preparation of step a) has a total protein content of 0.1 to 0.7 mg/mL and a Factor VIII activity of 50 to 120 Units/mL, preferably 0.2 to 0.7 mg/mL and a Factor VIII activity of 50 to 100 Units/mL.

In a preferred embodiment in the case of a Factor VIII activity of 50 to 120 Units/mL in step a) polysorbate 80 in step b) is added to obtain a resulting concentration of 20 to 120 pg polysorbate 80 per mg total protein.

The amounts or concentrations of polysorbate 80 can be measured by methods known to the skilled person. Preferably, in the context of the present invention, the content of polysorbate 80 is measured by a photometric assay, especially by an assay which is based on the formation of a blue-colored complex with ammonium cobalt thiocyanate and extraction with dichloromethane and measurement of absorption at 620 nm.

Preferably, the content of total protein is measured by a photometric measurement at 280 nm corrected for the extinction at 360 nm and by multiplication of the result with the productspecific factor of 1/1.49 (this factor is used to normalize the results in relation to the Bradford method). Alternatively, the protein concentration can be determined according to the Bradford method as known in the art (e. g. using a reference curve determined using human albumin).

Preferably, the Factor VIII activity is measured by a chromogenic assay, especially by a two- step assay. More preferably, in the two-step assay FX is activated by FIXa and FVIIIa in the first step. The activated FX hydrolyses a chromogenic substrate in the second step. The resulting color change of the chromogenic substrate is preferably measured at 405 nm.

In a preferred embodiment the dry heat treatment in step d) is performed at a temperature in a range of 90 to 110 °C, preferably 95 to 105 °C, more preferably at 100 °C +/- 1.5 °C, especially preferred at 100 °C +/- 1.0 °C. In routine production, the temperature is preferably measured outside the vials that contain the preparation.

The dry heat treatment is performed preferably for at least 25 minutes, more preferably for 25 minutes to 100 minutes, even more preferably for 30 to 90 minutes, especially 40 to 90 minutes, preferably under conditions using water-saturated steam (e. g. using an autoclave). As the product is comprised in closed containers, the heat treatment is considered a “dry” heat treatment. Most preferred is a treatment for 60 minutes or about 60 minutes.

In a preferred embodiment the dry heat treatment is performed preferably at a temperature of 100 °C +/- 1.5 °C for at least 25 minutes, more preferably for 25 minutes to 100 minutes, even more preferably for 30 to 90 minutes, especially 40 to 90 minutes. In a preferred embodiment, sufficient virus inactivation was reached by a heat treatment of up to 60 minutes, preferably 30 to 60 minutes, more preferably 60 minutes or about 60 minutes.

In a preferred embodiment of the invention the residual moisture after the lyophilization according to step c) of the inventive method is in the range of 0.2 to 1.3 % (w/w), preferably in the range of 0.3 to 1.0 % (w/w), more preferably in the range of 0.3 to 0.7 % (w/w), even more preferably in the range of 0.3 to 0.6 % (w/w). It was surprising that virus inactivation was very effective even at low residual moisture, wherein the low residual moisture has the advantage that it increases the long-term stability of the product.

The residual moisture is preferably tested using Near Infrared spectroscopy (NIR) calibrated using the Karl-Fischer method as described in more detail below. For the Karl-Fischer method, the water is extracted from the lyophilizate and chemically quantified with a following reaction.

The addition of polysorbate 80 allows a shorter heat treatment at a lower residual moisture, which improves the yield of the process and the long-term stability of the product. The product is also as natural as possible, since polysorbate 80 is only added in a minor amount.

In a preferred embodiment of the invention the preparation of step a) comprises arginine, lysine or histidine in a concentration of less than 4 mmol/L, preferably less than 2 mmol/L, more preferably less than 0.5 mmol/L. In a preferred embodiment the preparation does not comprise any arginine, lysine or histidine. The preparation is preferably derived from natural plasma source without addition of arginine, lysine or histidine.

In a preferred embodiment the preparation of step a) comprises a Factor VIII protein content in an amount of 5 % (w/w) or less in relation to the total protein content of the preparation. Preferably, the other protein compounds are vWF in an amount of approx. 60 % (w/w) ± 5 % (w/w) and/or fibrinogen in an amount of approx. 30 % (w/w) ± 5 % (w/w) and/or fibronectin in an amount of less than 10 % (w/w) and/or IgM in an amount of less than 10 % (w/w). Moreover, traces of other proteins may be present.

In a preferred embodiment of the invention the concentration of glycine in the preparation of step a) is from 40 mmol/L to 160 mmol/L. In a preferred embodiment of the invention the concentration of sodium in the preparation of step a) is from 40 mmol/L to 165 mmol/L.

In a preferred embodiment of the invention the concentration of calcium in the preparation of step a) is from 0.3 mmol/L to 1.7 mmol/L.

In a preferred embodiment of the invention the concentration of chloride in the preparation of step a) is from 30 mmol/L to 120 mmol/L.

In a preferred embodiment of the invention the concentration of citrate in the preparation of step a) is from 3.5 mmol/L to 17 mmol/L.

In a preferred embodiment of the invention the dry heat treatment is performed at a temperature of 100 °C +/- 1.0 °C and for at least 30 min, preferably at least 40 min, while the sample has a residual moisture of at least 0.2 %. Preferably, the dry heat treatment is performed at a temperature of 100 °C +/- 1.0 °C for 60 min at a residual moisture in the range of 0.3 - 0.5 %.

In a preferred embodiment a sterile filtration of the preparation is performed between step b) and c), preferably during the optional step b1) of transferring the preparation into one or more suitable containers respectively vials. This step may inadvertently educe the content of polysorbate 80 in the preparation in the final product, but only by an amount of less than 3 pg/mL.

In a preferred embodiment the Factor FVIII product manufactured with this method comprises Factor VIII in an amount of up to 2500 Units per container or vial or the like, preferably in an amount of up to 2000 Units per container, even more preferably of at least 200 Units per container. Preferred dosage units comprise about 250 Units per container or about 500 Units per container or about 1000 Units per container or about 2000 Units per container. The preferred volume of the reconstitution medium, e. g. water for injection, may be in the range of 1 mL to 10 mL, preferably 5 mL.

In preferred embodiments of the method according to the invention the method comprises further preparation steps. These further steps may include: cryoprecipitation of plasma, especially for providing cryoprecipitate, adsorption methods, especially AI(OH)3 adsorption, chromatographic enrichment or purification methods, especially ion exchange chromatography, preferably anion exchange chromatography, filtration steps, especially ultrafiltration and/or the above already mentioned sterile filtration.

In especially preferred embodiments the above-mentioned further steps are combined with each other, especially preferred is a combination of one or more, and preferably all of the above-mentioned steps with a solvent/detergent treatment as mentioned above.

Cryoprecipitation

Cryoprecipitation is a widely used method step to enrich cold-insoluble plasma proteins (such as FVIII and Fibrinogen) and is preferably also applied in the inventive method. Generally, the plasma donations are provided in frozen form. For cryoprecipitation the frozen plasma donations are thawn at 0°C to +4 °C and pooled. The cryoprecipitate is obtained by centrifugation as known to a skilled person.

For thawing and suspension of the cryoprecipitate a solution with ethanol and heparin in water may be used, especially 1 % ethanol and 900 IE heparin per liter.

Adsorption

In certain preferred embodiments an adsorption of the suspended cryoprecipitate with AI(OH)3 adsorption is performed as a purification step. For example, after adjustment of the pH value to pH 6.9 to 7.1 , more particularly 7.0 to 7.1 , e. g. by addition of 0.1 M acetic acid, the suspension is brought to room temperature and stirred, e. g. for about 30 min. AI(OH)a is added and stirred, e. g. for further 15 min. After a minor lowering of the pH value, e. g. pH 6.55 and cooling, e. g. to about 15 °C, the supernatant is obtained for further processing after centrifugation.

Solvent/detergent treatment

In especially preferred embodiments a solvent/detergent (S/D) treatment is performed, wherein S/D treatment provides one important step for virus inactivation. Preferably, the S/D treatment is performed by addition of tri-n-butyl phosphate (TNPB) and polysorbate 80 as it is known to a skilled person.

Chromatographic methods

For further enrichment and/or purification of the Factor VIII preparation chromatographic methods are used in preferred embodiments, especially for removal of other coagulation factors and, if applicable, TN BP and polysorbate 80 from a preceding S/D treatment. Especially useful for the inventive method are anion exchange materials, especially weak anion exchange materials like resins with diethylaminoethyl (DEAE) as ligand, wherein elution of the target protein fractions may be obtained by increased ionic strength. In especially preferred embodiments column chromatographic methods may be used. Filtration

Several filtration steps may be included in the inventive process. Especially preferred is a sterile filtration step, that may be performed during transferring the preparation of step b) into single containers after adjustment of the polysorbate 80 concentration and prior to lyophilization. For performing the sterile filtration suitable membranes with a pore diameter of 0.2 pm may be used, as it is known to a skilled person.

Moreover, in especially preferred embodiments at least one ultrafiltration step may be applied and may be used to adjust the buffer conditions and the desired protein and/or Factor VIII activity within the solution.

In an especially preferred embodiment of the inventive manufacturing method the above- mentioned preparation steps are carried out in the following order to obtain and provide the liquid plasma-derived Factor VIII preparation of step a) a1) Providing cryoprecipitate from human plasma, a2) Suspension of the cryoprecipitate, a3) AI(OH)3 adsorption, a4) Solvent/detergent treatment, a5) Anion exchange chromatography (e. g. binding at a NaCI concentration of 110 to 130 mM and elution of impurities at 150 to 170 mM NaCI and elution of FVIII at 230 to 250 mM NaCI), a6) Ultrafiltration, a7) Adjustment of the protein concentration and/or Factor VIII concentration to the desired total protein concentration and/or desired Factor VIII activity.

In a preferred embodiment the Factor VIII concentration is adjusted to about 50 to 120, preferably 50 to 100 Units FVIII/mL in step a6) and/or a7).

In a preferred embodiment the plasma-derived FVIII preparation as provided in step a) is obtained by a method comprising each of the steps a1) to a7).

In an especially preferred embodiment of the inventive method the method comprises the steps a1) to a7) for providing the plasma-derived FVIII preparation of step a), followed by step b), step b1) including a sterile filtration, step c) and step d).

In a preferred embodiment the method of the invention comprises no further precipitation steps or chromatographic steps after providing the Factor VIII preparation according to step a). In especially preferred and advantageous embodiments of the present invention the dry heat treatment according to step d) is capable of inactivating Porcine Pseudorabies Virus (PRV, also named Suid Herpesvirus 1) by at least 2 log units, preferably at least 3 log units. In general, the goal of virus inactivation at this level is to achieve an inactivation by at least 4 log units.

As a common practice the inactivation of viruses in plasma preparations is measured on a logarithmic scale (base 10), wherein generally the measurement is based on spiking experiments using model viruses.

As a main advantage of the inventive method, the method is capable of inactivating PRV (Porcine Pseudorabies Virus) in a FVIII protein product comprising the method according to steps a) to d) as described above. PRV is a widely used model virus for enveloped viruses like hepatitis B virus (HBV). Thus, if the process is capable of inactivating PRV, it should also be capable of inactivating HBV.

In an especially preferred embodiment, the inventive method is capable of inactivating PRV with a reduction capacity of at least 3 log units for PRV, preferably at least 4 log units. The temperature of the dry heat treatment, duration of the dry heat treatment and/or the amount of polysorbate 80 may be further adjusted to optimize the virus inactivation. The higher the temperature or duration of dry heat treatment, or the higher the amount of polysorbate 80, the more effective is the virus inactivation. However, the conditions as mentioned within the present specification are especially preferred in order to avoid other undesired effects.

Other viruses that are difficult to remove or to inactivate, especially other enveloped viruses, can also be effectively inactivated using the method of the invention, like Bovine Viral Diarrhea Virus (BVDV) and Hepatitis B virus (HBV), and certain non-enveloped viruses such as Hepatitis A virus (HAV).

Further, the invention comprises a Factor VIII product obtained by the inventive method as described above. The inventive Factor VIII product is characterized by an especially safe virus inactivation and moreover by a very good stability and activity.

Preferably, the Factor VIII product comprises at least one pharmaceutically acceptable carrier and/or excipient. Suitable pharmaceutically acceptable carrier and/or excipients are known to a skilled person. According to the inventive method the pharmaceutical product is a lyophilized product which should be reconstituted prior to use, preferably with water for injection or other suitable solutions, e. g. a buffered solution. Depending on the volume of the reconstitution medium it is possible to provide a more or less concentrated product. In preferred embodiments the reconstituted Factor VIII product has a concentration of about 50 U/rnL or about 100 U/rnL or about 200 U/rnL, wherein the term “about” in this context means that the mentioned concentrations are nominal concentrations and in fact, the product may contain a higher content of enzymatic activity (e. g. + 10 % or + 20 %) prior to lyophilization in order to compensate for possible loss of activity by heat treatment and/or during storage.

Preferably, the Factor VIII product is provided for intravenous administration, preferably to a human patient. More preferably, said human patient is suffering from hemophilia A.

Additionally, the invention comprises a pharmaceutical product or kit, comprising a container comprising the lyophilized Factor VIII product as obtainable by the inventive method as described above and, preferably, a container comprising a suitable solvent respectively reconstitution medium, e. g. water for injection or a suitable solution, e. g. a buffered solution.

The containers may be separate or connected, e. g. separate vials, particularly glass vials, or may be part of an administration device, e. g. a two-chamber syringe.

In especially preferred embodiments the pharmaceutical product comprises a leaflet with instructions for use.

Finally, the invention comprises a use of the described FVIII product and/or the described pharmaceutical product or kit for treating a patient suffering from hemophilia A. Thereby, the Factor VIII product of the invention is especially useful for the treatment of mild, moderate or severe Hemophilia A. The treatment may be on demand and/or the treatment may be a prophylactic treatment depending on the demands of the patient.

In the figures it is shown:

Fig. 1 PRV reduction capacity for different samples with 500 Units and 10 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 30 min.;

Fig. 2 PRV reduction capacity for different samples with 500 Units and 10 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 45 min.;

Fig. 3 PRV reduction capacity for different samples with 500 Units and 10 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 60 min.; Fig. 4 PRV reduction capacity for different samples with 500 Units and 30 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 30 min.;

Fig. 5 PRV reduction capacity for different samples with 500 Units and 30 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 60 min.;

Fig. 6 PRV reduction capacity for different samples with 500 Units and 30 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 90 min.;

Fig. 7 PRV reduction capacity for different samples with 500 Units and 30 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 30 min.;

Fig. 8 PRV reduction capacity for different samples with 500 Units and 30 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 60 min.;

Fig. 9 PRV reduction capacity for different samples with 500 Units and 30 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 90 min.;

Fig. 10 PRV reduction capacity for different samples with 1000 Units and 10 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 30 min.;

Fig. 11 PRV reduction capacity for different samples with 1000 Units and 10 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 60 min.;

Fig. 12 PRV reduction capacity for different samples with 1000 Units and 10 pg/mL polysorbate 80 added using a dry heat treatment at 99 °C for 90 min.;

Examples

Manufacturing process of the Factor VIII product

The manufacturing process was principally based on the process as disclosed in EP 0 812 858 B1. In brief, the manufacturing process of the FVIII product used human plasma as the source material. Preferably, the process started with cryoprecipitate, which was obtained from a plasma pool using known methods. Adsorption on AI(OH)3 may also be used as a further purification step As an important virus inactivating step an S/D treatment with TNBP/polysorbate 80 was performed. TN BP, polysorbate 80 and further coagulation factors were then largely removed by ion exchange column chromatography. The next step was a diafiltration and/or ultrafiltration. In this step and/or after this step the concentration of Factor VIII and the electrolytes were adjusted. To this preparation polysorbate 80 was added according to the invention.

Subsequently, after sterile filtration, the preparation was filled in suitable containers, especially in glass vials, shock frozen in liquid nitrogen and lyophilized under vacuum (preferably 40 pbar) to obtain a residual moisture of 0.2 to 1.3 % (w/w) and closed under vacuum. After the sterile filtration the amount of polysorbate 80 in the final product was slightly decreased, but not more than 3 pg/mL, due to absorption during filtration. The lyophilized preparation was subjected to dry heat treatment in an autoclave at 100°C for 30 to 60 minutes, preferably for 60 minutes.

The Factor VIII content of the resulting product was about 5 % (w/w) or less of the total protein content. Further proteins in the product were about 60 % (w/w) vWF and about 30 % (w/w) fibrinogen. Fibronectin and IgM were each less than 10 % (w/w). Thus, as it is typical for plasma-derived FVIII products, the majority of the protein content was not FVIII.

Vials with different amounts of Factor VIII (250, 500 or 1000 Units per dosing unit) were used in the experiments as described below.

The concentration of Factor VIII before lyophilization was varied depending on the amount of the desired Factor VIII in the final pharmaceutical preparation (sample). For samples with 250 and 500 Units the concentration was adjusted to 0.2 to 0.5 mg total protein/mL, and corresponding to around 60 Units/mL. For samples with 1000 Units the concentration was adjusted to 0.47 to 0.63 mg total protein/mL, corresponding to around 90 Units/mL. To these solutions polysorbate 80 was added. In order to reach the desired nominal amounts of Units per vial after reconstitution the samples may contain up to 130 % of the nominal amount of FVIII expressed in International Units (IU), e. g. 1200 IU or 1270 IU filled in the vial of the 1000 Unit sample. This did not affect the relation between the polysorbate 80 and the total protein or the Factor VIII.

After lyophilization, for further testing e. g. of the virus inactivation, the samples in the vials were reconstituted to a nominal concentration of 40 lU/mL to 480 lU/mL, depending on the experiment performed. In a preferred embodiment the sample with 250 Units was reconstituted to 40 to 60 lU/mL or 80 to 120 lU/mL, the sample with 500 Units was reconstituted to 80 to 120 lU/mL, the sample with 1000 Units to 160 to 240 lU/mL.

The effect on inactivating PRV by adjustment of polysorbate 80 to the described range prior to lyophilization and dry heat treatment was further investigated using several different conditions and combinations of conditions, especially several durations of dry heat treatment (30 min, 45 min, 60 min, 90 min), different amounts of residual moisture, and different amounts of polysorbate 80. Subsequently, the samples were tested to assess the efficacy of virus inactivation under the different conditions.

Virus inactivation

In order to be able to verify the virus inactivation capacity of the heat treatment step, the samples were spiked with viruses in a predefined manner. The membrane-enveloped pseudorabies virus PRV (Porcine Pseudorabies virus - Suide Herpesvirus 1), which is generally accepted as a model virus for hepatitis B viruses, was used for this purpose. Generally, the inactivation of this virus by heat treatment is especially difficult among the typically used group of lipid-enveloped viruses used for virus reduction studies for plasma products.

Due to the low protein content of the Factor FVIII preparation analyzed, (< 1 mg/mL) the virus solutions were purified by ultracentrifugation. This approach excludes the possibility that proteins introduced by the spiking might have a stabilizing effect on the virus.

To the virus-spiked samples an aqueous polysorbate 80 solution was added to increase the concentration of polysorbate 80. 10 pg or 30 pg polysorbate 80/mL were added. For an increase by 10 pg polysorbate 80/mL, 1 pL of a 1 % (w/v) solution of polysorbate 80 per mL was added. For 30 pg/mL 1 pL of a 3 % (w/v) solution of polysorbate 80 per mL was added.

The amount of polysorbate 80 resulting from the preceding S/D treatment was considered before the addition of the polysorbate 80 according to the inventive approach to reach the corresponding final respectively resulting concentration. Before addition of polysorbate 80 the content of polysorbate 80 was measured to be in a range of 2 to 12 pg/mL. Thus, the addition of 10 pg polysorbate 80/mL resulted in a final concentration of 12 to 22 pg/mL and the addition of 30 pg/mL resulted in a final concentration of 32 to 42 pg/mL.

The temperature during the dry heat treatment was measured in the vial. The dry heat treatment was conducted at 99.0 °C.

Purification of the virus solution

The virus solution used for the spiking experiments was purified by ultracentrifugation. For this purpose, the respective virus stock solution (PRV) was centrifuged at 2 - 16 °C (setpoint 5 °C) with 25,000 rpm and 90 min (Beckman 70 Ti rotor). The pellets were resuspended in the same volume of phosphate buffered saline (PBS) as before ultracentrifugation and then filtered using syringe filters with 0.45 pm pore size. Analytical methods

FVIII activity chromogenic assay

The activity of FVIII was determined by a chromogenic assay. In this two-step assay, FIXa and FVIIIa activated FX in the first step. In the second step, the activated FX hydrolysed a chromogenic substrate, resulting in a color change, which could be measured at 405 nm. Due to the fact that calcium and phospholipids were present in optimal amounts and an excess of FIXa and FX was available, the activation rate of FX was only dependent on the amount of active FVIII in the sample.

The reagents for this chromogenic FVIII activity assay were taken from the Coatest® SP FVIII Kit. The kit contained phospholipids, calcium chloride (CaCh), trace amounts of thrombin, the substrate S-2765, a mixture of FIXa and FX and the thrombin inhibitor 1-2581. The inhibitor was added, in order to prevent hydrolysis of the substrate by thrombin, which was built during the reaction. All dilutions were performed in distilled water or Tris-BSA (TBSA) buffer, containing 25 mM Tris, 150 mM sodium chloride (NaCI) and 1 % bovine serum albumin (BSA), set to pH 7.4. Each sample was diluted at least 1 :2 with FVI Il-depleted plasma. Further dilutions were performed using the TBSA Buffer.

The assay was performed using the BCS XP (Siemens Healthcare, Erlangen, Germany), a fully automated hemostasis analyzer. All reagents including water, TBSA Buffer and the samples were inserted into the analyzer. For each sample the analyzer mixed 34 pL calcium chloride, 20 pL TBSA Buffer, 10 pL sample, 40 pL water, 11 pL phospholipids and 56 pL FIXa-FX-mixture. This mixture was incubated for 300 seconds. Afterwards, 50 pL of S-2765 + 1-2581 were added to the reaction. Upon addition of the substrate, the absorption at 405 nm was measured for 200 seconds.

In order to calculate the amount of active FVIII, the software of the analyzer evaluated the slope of the measured kinetic between 30 seconds and 190 seconds after starting the reaction. This result was correlated to a calibration curve, generated with a biological reference preparation (BRP) of FVIII. The activity of the BRP is indicated in lU/mL. However, lU/mL can be assumed equivalent to U/mL.

Protein concentration

The protein concentration was determined by photometric measurement at 280 nm (OD 280, A280) corrected for the extinction at 360 nm (A360) and by multiplication of the result with the product-specific factor of 1/1.49 (protein in g/l = (A280 - A360) 1 1.49). Usually, the extinction at 360 nm is zero. The factor of 1/1.49 was used to normalize the results of the A280 method to the Bradford method as known in the art using a reference curve determined using human albumin.

Detection of polysorbate 80

Polysorbate 80 was detected using a photometric measurement with a detection range of 10 to 100 pg/mL. The measurement relied on the formation of a blue-colored complex with ammonium cobalt thiocyanate, which was extracted with dichloromethane. The absorption at 620 nm was measured.

Residual moisture

The residual moisture was determined by the Karl Fischer method and the non-destructive near-infrared (NIR) spectroscopy.

For the Karl Fischer method, the water was extracted from the lyophilizate and then quantified by means of a chemical (Karl-Fischer) detection reaction.

Virus-spiked samples for inactivation experiments were measured by the non-destructive near-infrared (NIR) spectroscopy only. Each experiment included control samples which were first measured by the non-destructive NIR spectroscopy and then by the Karl Fischer method in order to have calibration of NIR spectroscopy with the Karl Fischer method in each experiment. The Karl Fischer assay is considered the gold standard for measuring residual moisture in lyophilized samples.

Experimental results

The following table summarizes the Factor VIII activity loss by dry heat treatment under various conditions and the corresponding results of virus inactivation of PRV.

As can be seen for Factor VIII (500 Units) and for Factor VIII (1000 Units) the prolongation of the heat treatment without polysorbate 80 (PS80) from 60 min to 90 min increased the loss of Factor VIII activity (11 % compared to 16.4 % respectively 6.5 % compared to 9.4 %), wherein at the same time the virus inactivation is improved as expected (3.58 log™ compared to 4.38 log for Factor VIII (500 Units) respectively 2.91 log™ compared to 3.47 logwfor Factor VIII (1000 Units)). Surprisingly, about the same or even a better improvement in virus inactivation (4.43 log™ for Factor VIII (500 Units) respectively 4.11 log for Factor VIII (1000 Units) was achieved by addition of polysorbate 80 according to the inventive method without prolongation of the time of dry heat treatment (60 min heat treatment, with PS80). The FVIII activity loss for both Factor VIII (500 Units) and Factor VIII (1000 Units) was within an acceptable range over the 60 min heat treatment with polysorbate 80 (about 11 %).

Figures 1, 2 and 3 show the results for samples with 500 Units, addition of 10 pg/mL polysorbate 80 with purified PRV for different times of the dry heat treatment. The content of polysorbate 80 before addition of additional polysorbate 80 was 2 pg/mL. All samples in this study had a residual moisture from 0.2 to 0.4 %. The reduction factor for the virus was determined for different times of dry-heat treatment.

Before the lyophilization the content of Factor VIII was 60 Units/mL. A heat treatment of 30 minutes, 45 minutes and 60 minutes was performed. For experimental purposes the time of dry heat treatment was each reduced by 5 min (e. g. 25 min instead of 30 min) when in the laboratory environment the temperature was measured directly in the vial in contrast to the production environment where the temperature was measured is the ambient temperature outside the vial. The 30, 45 and 60 minutes correspond to a temperature measurement outside the vial.

A positive effect on virus inactivation of polysorbate 80 was clearly visible at 45 and 60 minutes. At low residual moisture of 0.2 % (w/w) the positive effect of polysorbate 80 in a treatment of 30 minutes was less visible. Figures 4, 5 and 6 show the results for samples with 500 Units, addition of 30 pg/mL polysorbate 80 with purified PRV for different times of the dry heat treatment. The content of polysorbate 80 before addition of additional polysorbate 80 was 3 pg/mL. The residual moisture and the reduction factor for the virus was determined. The samples had a residual moisture from 0.3 to 0.7 % (w/w).

Figures 7, 8 and 9 show the results for samples with 500 Units, addition of 30 pg/mL polysorbate 80 with purified PRV for different times of the dry heat treatment including the samples from figures 4, 5 and 6. The residual moisture and the reduction factor for the virus was determined. The samples had a residual moisture from 0.3 to 0.7 % (w/w).

With the addition of polysorbate 80 a high virus inactivation was achieved for the whole range of residual moisture even at low dry heat treatment times. A lower residual moisture increased the long-term stability of the lyophilized product.

Figures 10, 11 and 12 show the results for samples with 1000 Units, 10 pg/mL polysorbate 80 with purified PRV for different times of the dry heat treatment. The content of polysorbate 80 before addition of additional polysorbate 80 was 3.1 pg/mL. The residual moisture and the reduction factor for the virus was determined. The samples had a residual moisture from 0.2 to 0.5 % (w/w).

Before the lyophilization the content of Factor VIII was 90 Units/mL. An improvement of virus inactivation was visible for all samples. At 30 minutes the virus inactivation in samples with a residual moisture of 0.3 % (w/w) was better than in samples with residual moisture < 0.3 % (w/w).

The loss of activity before or after dry heat treatment with or without the addition of additional polysorbate 80 was similar for all samples. No protective effect on enzymatic activity of the additional polysorbate 80 was observed.