HK1210794B - Antibody and protein formulations - Google Patents

Description

Antibody and protein preparations

This application was filed on August 28, 2013 as a PCT International Patent Application and claims priority to U.S. Patent Application No. 13/601,598, filed on August 31, 2012, and U.S. Patent Application No. 13/843,780, filed on March 15, 2013, the disclosures of which are hereby incorporated by reference in their entireties.

background

The present disclosure generally relates to isotonic and low-viscosity formulations of antibodies and other proteins, including formulations for injection and general administration.

Hemophiliacs suffer from a bleeding disorder that causes delayed blood clotting after injury or surgery. Prolonged bleeding is caused by genetic defects in one or more blood clotting factors. Two common types of hemophilia are known: hemophilia A and hemophilia B. Hemophilia A is caused by a defect in factor VIII, while hemophilia B is caused by a defect in factor IX. Approximately 75-80% of all hemophiliacs have hemophilia A.

Tissue factor pathway inhibitor (TFPI) is a human inhibitor of the extrinsic pathway of blood coagulation and plays a role in anticoagulation. Antibodies against TFPI, including anti-TFPI monoclonal antibodies (aTFPI mAbs), have been developed in an effort to block TFPI function. One such aTFPI mAb is a human IgG2 anti-TFPI mAb, which is being developed for the treatment of patients with hemophilia A and B.

Antibodies and other proteins can be administered to patients via intravenous, intramuscular, and/or subcutaneous injection. To ensure patient compliance, it is desirable that subcutaneous dosage forms be isotonic and include a small injection volume (<2.0 ml/injection site). To reduce the injection volume, proteins are typically administered in the range of 1 mg/ml to 150 mg/ml.

While both liquid and lyophilized dosage forms are used for currently marketed antibody and other protein-based drug products, lyophilized forms are more frequently used for protein and antibody drug products with high protein concentrations.

Protein and antibody dosage forms can present many challenges in formulation development, especially for liquid formulations. For formulations where the protein concentration is close to its apparent solubility limit, phase separation can occur by precipitation, gelation and/or crystallization. At high protein concentrations, due to the formation of soluble and insoluble protein-protein aggregates, the stability of antibodies or other proteins can become problematic. Highly concentrated protein formulations are typically highly viscous, which presents difficulties in processing such as ultrafiltration and sterile filtration and injection of dosage solutions. And at the protein concentrations expected for formulations intended for intramuscular or subcutaneous administration, high concentrations of stabilizers such as sucrose and sodium chloride are required to achieve long-term protein stability. The resulting hypertonic solution typically causes injection pain due to tissue damage. Therefore, it is critical to balance the amount of stabilizers for the stability and osmotic pressure molarity of high protein concentration formulations.

For these reasons, there is a need in the art for therapeutic formulations based on antibodies and other proteins in liquid form that exhibit high protein concentrations without significantly increased protein aggregation, osmolality or viscosity and/or reduced protein stability. Therefore, it is desirable that formulations based on antibodies and other proteins contain limited amounts of excipients and small volumes for ease of therapeutic administration or delivery. It is further desirable that therapeutic formulations based on antibodies and other proteins be amenable to lyophilization to enhance protein stability under extended storage conditions.

Overview

The present disclosure provides liquid and lyophilized antibody and protein-based formulations that are substantially isotonic and low-viscosity and substantially free of inorganic salts. The antibody and other protein formulations presented herein contain about 0 mM to about 30 mM histidine at a pH of about pH 4.0 to about pH 6.0; about 50 ppm to about 200 ppm of a nonionic surfactant such as polysorbate (Tween®) 80 or polysorbate (Tween®) 20; about 88 mM to about 292 mM sugars or sugar alcohols such as mannitol, dextrose, glucose, trehalose, and/or sucrose; about 0 mM to about 50 mM arginine; about 0 mM to about 50 mM lysine; about 0 mM to about 133 mM glycine or alanine; about 0 mM to about 10 mM methionine; and about 1 mg/ml to about 150 mg/ml of protein. The formulations disclosed herein exhibit a viscosity ranging from about 1 mPa-S to about 20 mPa-S at 22°C, or from about 1 mPa-S to about 15 mPa-S at 22°C, or from about 1 mPa-S to about 10 mPa-S at 22°C, or from about 1 mPa-S to about 8 mPa-S at 22°C, or from about 1 mPa-S to about 6 mPa-S at 22°C and an osmolarity ranging from about 240 to about 380 mmol/kg.

In a further aspect, the present disclosure provides methods for treating a condition in a patient comprising administering to the patient a therapeutically effective amount of one or more formulations described herein. For example, provided are methods for treating a condition in a patient comprising administering to the patient a therapeutically effective amount of an antibody or other protein formulation as described in more detail herein.

These and other features of the teachings herein are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled person should understand that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

FIG1 is a graph showing the effect of sodium chloride (NaCl) concentration on the turbidity of a 20 mg/ml anti-TFPI mAb formulation at pH 5.5.

FIG2 is a graphical representation showing the effect of pH on the turbidity of an anti-TFPI mAb drug product.

Description of various embodiments

As described above, the present disclosure provides antibody and other protein formulations that stabilize the antibody or other protein in liquid or lyophilized form under the expected storage conditions. The formulations described herein include one or more pharmaceutically acceptable excipients or stabilizers and are contained in a buffered medium at an appropriate pH and are substantially isotonic with physiological solutions. For systemic administration, injection is one route of administration, including intramuscular, intravenous, intraperitoneal, and subcutaneous injections.

Due to their low viscosity, the protein formulations disclosed herein can be conveniently processed via, for example, ultrafiltration and sterile filtration, and can be administered to patients via injection, including subcutaneous injection. In addition, because they are substantially isotonic, the antibodies and protein formulations disclosed herein reduce tissue damage or other adverse physiological effects, and thereby achieve favorable patient tolerance and increased patient compliance.

The formulations described herein are characterized by the substantial absence of added salts, which provides flexibility in increasing the concentration of other stabilizers, such as sucrose, while maintaining the osmolality of the formulation for improved in vivo tolerability and thereby increased patient compliance. In addition, the low viscosity of the formulations described herein allows for convenient processing, including but not limited to ultrafiltration and sterile filtration, and injection of the drug product solution through a needle.

As used herein, the term "viscosity" refers to, for example, the resistance of a liquid formulation to flow when injected by a syringe during application to a patient. Viscosity measurement can be accomplished by a cone-plate technique using a Peltier element (Peltier element) set at a defined temperature, for example, 22°C as described herein. Typically, a well-defined shear stress gradient is applied to the liquid formulation, and the resulting shear rate is measured. Viscosity is the ratio of shear stress to shear rate. As used herein, viscosity is represented by mPa-S units at 22°C, where 1 mPa-S = 1 cP. Low viscosity, substantially isotonic formulations disclosed herein are generally characterized in that they have a range of about 1 mPa-S - about 20 mPa-S at 22°C, or about 1 mPa-S - about 15 mPa-S at 22°C, or about 1 mPa-S - about 10 mPa-S at 22°C, or about 1 mPa-S - about 8 mPa-S at 22°C, or about 1 mPa-S - about 6 mPa-S at 22°C.

As used herein, the term "osmolarity" refers to a measure of solute concentration defined as the number of solute millimeters per kg of solution. Required osmolarity levels can be achieved by adding one or more stabilizers, such as sugars or sugar alcohols, including but not limited to mannitol, dextrose, glucose, trehalose, and/or sucrose. Other stabilizers suitable for providing osmolarity are described in the handbook of references such as Pharmaceutical Excipients (4th edition, Royal Pharmaceutical Society of Great Britain, Science & Practice Publishers) or Remingtons: The Science and Practice of Pharmacy (19th edition, Mack Publishing Company).

As used herein, the term "about" refers to +/- 10% of the unit value provided. As used herein, the term "substantially" refers to a qualitative condition that shows a characteristic or property of interest to a total or approximate degree. Those of ordinary skill in the biological field will appreciate that biological and chemical phenomena rarely, if ever, achieve or avoid absolute results. The term substantially is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. As used herein, the terms "isotonic" and "isotonic" can be used interchangeably with the terms "substantially isotonic" and "substantially isotonic," and refer to a formulation characterized by having an osmotic pressure that is the same as, or at least substantially equal to, the osmotic pressure of another solution, achieved by a formulation wherein the total concentration of solutes, including both permeable and impermeable solutes, in the formulation is the same as, or at least substantially equal to, the total number of solutes in the other solution. Thus, while those skilled in the art will understand that "isotonic" and "isotonic" formulations for in vivo administration generally have an osmolality in the range of about 270 mmol/kg to about 310 mmol/kg, in the context of the low viscosity formulations of the present disclosure, the terms "isotonic," "isotonic," "substantially isotonic," and "substantially isotonic" are used interchangeably to refer to formulations having an osmolality in the range of about 240 mmol/kg to about 380 mmol/kg, or about 270 mmol/kg to about 370 mmol/kg, or about 300 mmol/kg to about 330 mmol/kg.

The low-viscosity, substantially isotonic antibody and other protein formulations disclosed herein contain about 0 mM to about 30 mM histidine at a pH of about pH 4 to about pH 6; about 50 ppm to about 200 ppm of a nonionic surfactant, such as polysorbate (Tween®) 80 or polysorbate (Tween®) 20; about 88 mM to about 292 mM sugar or sugar alcohol, such as mannitol, dextrose, glucose, trehalose and/or sucrose; about 0 mM to about 50 mM arginine; about 0 mM to about 50 mM lysine; about 0 mM to about 133 mM glycine or alanine; about 0 mM to about 10 mM methionine; and about 1 mg/ml to about 150 mg/ml of protein. The formulations disclosed herein exhibit a viscosity ranging from about 1 mPa-S to about 20 mPa-S at 22°C, or from about 1 mPa-S to about 15 mPa-S at 22°C, or from about 1 mPa-S to about 10 mPa-S at 22°C, or from about 1 mPa-S to about 8 mPa-S at 22°C, or from about 1 mPa-S to about 6 mPa-S at 22°C and an osmolarity ranging from about 240 to about 380 mmol/kg.

In these formulations, histidine is a buffering agent that can be used to maintain the pH of the formulation at about pH 4 to about pH 6.0, or about pH 5 to about pH 6, for example, about pH 5, about pH 5.5, or about pH 6. Sugars or sugar alcohols such as mannitol, dextrose, glucose, trehalose, and/or sucrose are used separately or in combination as cryoprotectants and stabilizers for the antibody in the liquid formulation and during and after lyophilization. Nonionic surfactants such as polysorbates, including polysorbate 20 and polysorbate 80; polyoxamers, including poloxamer 184 and 188; pluronic® polyols; and other ethylene/polypropylene block polymers stabilize the antibody during processing and storage by reducing interfacial interactions and preventing protein adsorption. Arginine is a protein solubilizer and stabilizer that reduces aggregation of antibodies and other proteins, such as aTFPI mAb aggregation, and glycation. Methionine is an antioxidant that prevents oxidation of the antibody during processing and storage.

Sugars and inorganic salts are commonly used as protein stabilizers; however, both sugars and inorganic salts are also effective tonicity agents. If a formulation requires high concentrations of one or more sugars to stabilize the protein, the inorganic salt concentration should be zero or kept very low to maintain the osmolality of the formulation so that injection pain is reduced upon administration. Surprisingly, sodium chloride was found to increase the turbidity of antibody formulations. Thus, inorganic salts are essentially excluded from the formulations described herein. These salt-free formulations maintain the osmolality of antibodies and other protein formulations with increased stability and reduced phase changes such as precipitation or aggregation.

As used herein, the term "salt" _{refers to inorganic salts, including but not limited to sodium chloride (NaCl), sodium sulfate (Na2SO4 )} , sodium thiocyanate (NaSCN), magnesium chloride (MgCl), magnesium sulfate ( _MgSO4 ), ammonium thiocyanate ( _NH4SCN ), ammonium sulfate (( _NH4 ) _2SO4 ), ammonium chloride ( _NH4Cl ), calcium chloride ( _CaCl2 ), calcium sulfate ( _CaSO4 ), zinc chloride ( _ZnCl2 ), and the like, or combinations _thereof . The antibody and other protein formulations disclosed herein are characterized by the substantial absence of added salts and are therefore referred to herein as salt-free antibody and/or protein formulations. One skilled in the art will understand that the presence of inorganic salts introduced by pH adjustment in the formulations disclosed herein is not considered added salt, and that if present in formulations according to the present disclosure, such inorganic salts should not exceed a concentration of about 2 mM.

As used herein, the term "surfactant" includes nonionic surfactants, including but not limited to polysorbates such as polysorbate 20 or 80, and poloxamers such as poloxamer 184 or 188, Pluronic® polyols and other ethylene/polypropylene block polymers. The amount of surfactant effective to provide stable antibody and other protein formulations is generally in the range of 50 ppm to 200 ppm. The use of nonionic surfactants allows the formulation to be exposed to shear and surface stress without causing denaturation of the antibody or other protein, and also reduces adsorption on the surface during processing and storage. The formulations disclosed herein include but are not limited to formulations with one or more nonionic surfactants, including, for example, one or more polysorbates, such as polysorbate 20 or 80; one or more poloxamers, such as poloxamer 184 or 188; one or more Pluronic® polyols; and/or one or more ethylene/polypropylene block polymers. Exemplified herein are formulations with polysorbates, such as polysorbate 20 (Tween® 20) or polysorbate 80 (Tween® 80).

As used herein, the term "protein" refers to an amino acid polymer containing at least 5 constituent amino acids covalently linked by peptide bonds. The constituent amino acids can be from the group of amino acids encoded by the genetic code, including: alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, arginine, histidine, lysine, aspartic acid and glutamic acid. As used herein, the term "protein" is synonymous with the related terms "peptide" and "polypeptide".

As used herein, the term "antibody" refers to a class of proteins generally known as immunoglobulins. Antibodies include full-length monoclonal antibodies (mAbs), such as _IgG2 monoclonal antibodies, which include an immunoglobulin Fc region. The term antibody also includes bispecific antibodies, diabodies, single-chain molecules, and antibody fragments such as Fab, F(ab') ₂ , and Fv.

As used herein, the term "anti-TFPI antibody" refers to an antibody having binding specificity for human TFPI protein, as well as fragments and variants of the human TFPI protein. The anti-TFPI antibodies presented herein can be IgG2 antibodies, and include anti-TFPI IgG2 monoclonal antibodies, such as chimeric, humanized, and fully human anti-TFPI IgG2 monoclonal antibodies. Anti-TFPI antibodies are exemplified in the present disclosure by human anti-TFPI IgG2 monoclonal antibodies having a light chain comprising the sequence presented herein as SEQ ID NO: 1 and/or a heavy chain presented herein as SEQ ID NO: 2. Other anti-TFPI monoclonal antibodies, including full-length antibodies and antigen-binding fragments and variants thereof (which are also suitable for use in the formulations disclosed herein), are presented in PCT Patent Publication Nos. WO 2011/109452 and WO 2010/017196, both of which are incorporated herein by reference in their entirety.

"Monoclonal antibodies" are characterized by having specificity for a single antigenic determinant. Monoclonal antibodies can be prepared, for example, by the hybridoma method described by Kohler and Milstein, Nature 256 :495 (1975) or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage display libraries using techniques such as those described in Clackson et al., Nature 352 :624-628 (1991) and Marks et al., J. Mol. Biol. 222 : 581-597 (1991).

Monoclonal antibodies include "chimeric monoclonal antibodies," in which a portion of the heavy and/or light chain comprises sequences from an antibody derived from one species, while the remainder of the antibody (including the Fc region) comprises sequences from an antibody derived from a second species, typically human. See, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81 : 6851-6855 (1984).

Monoclonal antibodies also include "humanized monoclonal antibodies," in which one or more complementarity determining regions (CDRs) from the heavy and/or light chain sequences of an antibody derived from one species replace one or more CDRs from the heavy and/or light chain sequences of an antibody derived from a second species, typically human. The "humanization" process is often applied to the development of monoclonal antibodies for administration to humans. See, for example, Riechmann et al., Nature 332(6162) :323-27 (1988) and Queen et al., Proc. Natl. Acad. Sci. USA 86(24):10029-33 (1989).

Monoclonal antibodies also include "fully human monoclonal antibodies" in which the entire heavy and light chain sequences are derived from human antibody sequences. Fully human monoclonal antibodies can be generated by phage display technology and can be isolated from mice that have been genetically modified to express the human antibody repertoire. See, for example, McCafferty et al., Nature 348 (6301) : 552-554 (1990), Marks et al., J. Mol. Biol. 222 (3) : 581-597 (1991), and Carmen and Jermutus, Brief Funct. Genomic Proteomic 1 (2) : 189-203 (2002).

As used herein, the term "pharmaceutically effective amount" of an antibody or other protein formulation refers to that amount of the formulation that provides a therapeutic effect within the administration regimen. The antibody and protein formulations disclosed herein typically include an antibody or other protein at a concentration range of about 1 mg/ml to about 150 mg/ml, or about 1 mg/ml to about 100 mg/ml, or about 1 mg/ml to about 50 mg/ml, or about 1 mg/ml to about 20 mg/ml, or about 1 mg/ml to about 10 mg/ml, or about 10 mg/ml to about 20 mg/ml, or about 20 mg/ml to about 150 mg/ml, or about 50 mg/ml to about 150 mg/ml, or about 60 mg/ml to about 150 mg/ml, or about 70 mg/ml to about 150 mg/ml, or about 80 mg/ml to about 150 mg/ml, or about 90 mg/ml to about 150 mg/ml, or about 100 mg/ml to about 150 mg/ml, or about 120 mg/ml to about 150 mg/ml. In some aspects, the protein or antibody concentration in these formulations is about 150 mg/ml. When administered subcutaneously, such formulations are typically administered in a volume of less than about 2.0 ml, or about 1.5 ml, or about 1 ml, or about 0.5 ml per injection site.

In certain aspects, the antibody or other protein formulation contains about 30 mM histidine, about 100 ppm Tween 80, about 292 mM sucrose, and about 20 mg/ml of the antibody or other protein at a pH in the range of about pH 5.0 to about pH 6.0. In related aspects, the antibody or other protein formulation further contains about 30 mM to about 50 mM arginine.

In other aspects, the antibody or other protein formulation contains about 10 mM histidine, about 75 ppm Tween 80, about 234 mM sucrose, and about 50 mg/ml of the antibody or other protein at a pH in the range of about pH 5.0 to about pH 6.0. In related aspects, the antibody or other protein formulation further contains about 30 mM to about 50 mM arginine.

In other aspects, the antibody or other protein formulation contains about 10 mM histidine, about 75 ppm Tween 80, about 234 mM sucrose, and about 100 mg/ml of the antibody or other protein at a pH in the range of about pH 5.0 to about pH 6.0. In related aspects, the antibody or other protein formulation further contains about 30 mM to about 50 mM arginine.

In other aspects, the antibody or other protein formulation contains about 10 mM histidine, about 75 ppm Tween 80, about 88 mM sucrose, about 133 mM glycine, and about 100 mg/ml of the antibody or other protein at a pH in the range of about pH 5.0 to about pH 6.0. In related aspects, the antibody or other protein formulation further contains about 30 mM to about 50 mM arginine.

In other aspects, the antibody or other protein formulation contains about 10 mM histidine, about 75 ppm Tween 20, about 88 mM sucrose, about 133 mM glycine, and about 100 mg/ml of the antibody or other protein at a pH in the range of about pH 5.0 to about pH 6.0. In related aspects, the antibody or other protein formulation further contains about 30 mM to about 50 mM arginine.

In other aspects, the antibody or other protein formulation contains about 10 mM histidine, about 200 ppm Tween 20, about 88 mM sucrose, about 133 mM glycine, and about 100 mg/ml of the antibody or other protein at a pH in the range of about pH 5.0 to about pH 6.0. In related aspects, the antibody or other protein formulation further contains about 30 mM to about 50 mM arginine.

In other aspects, the antibody or other protein formulation contains about 10 mM histidine, about 75 ppm Tween 80, about 88 mM sucrose, and about 100 mg/ml of the antibody or other protein at a pH in the range of about pH 5.0 to about pH 6.0. In related aspects, the antibody or other protein formulation further contains about 10 mM to about 50 mM arginine.

In other aspects, the antibody or other protein formulations contain about 10 mM histidine, about 75 ppm Tween 80, about 88 mM sucrose, about 133 mM glycine, about 10 mM arginine, and about 100 mg/ml of the antibody or other protein at a pH in the range of about pH 5.0 to about pH 6.0. In related aspects, the antibody and other protein formulations also contain about 0 mM to about 10 mM methionine.

In other aspects, the antibody or other protein formulation contains about 10 mM histidine, about 75 ppm Tween 80, about 88 mM sucrose, about 133 mM glycine, about 30 mM lysine, about 100 mg/ml of the antibody or other protein at a pH ranging from about pH 5.0 to about pH 6.0.

In other aspects, the antibody or other protein formulations contain about 10 mM histidine, about 75 ppm of Tween 80, about 234 mM sucrose, about 30 mM arginine, about 100 mg/ml of the antibody or other protein at a pH in the range of about pH 5.0 to about pH 6.0. In related aspects, the antibody or other protein formulations also contain about 0 mM to 10 mM methionine.

Exemplified herein are antibody preparations wherein the antibodies include IgG2 antibodies, such as anti-tissue factor pathway inhibitor antibodies (aTFPI Abs), comprising a human IgG2 monoclonal antibody having a light chain comprising the sequence of SEQ ID NO: 1 and a heavy chain comprising the sequence of SEQ ID NO: 2.

Thus, the present disclosure provides anti-TFPI mAb formulations, including anti-TFPI IgG ₂ mAb formulations, in which the anti-TFPI mAbs are soluble at high protein concentrations. Generally, the anti-TFPI mAbs in the formulations disclosed herein remain soluble at concentrations of about 1 mg/ml to about 150 mg/ml, remain stable under isotonic storage conditions, and exhibit reduced viscosity compared to currently available antibody formulations.

Anti-TFPI antibodies having a light chain comprising the sequence of SEQ ID NO: 1 and a heavy chain comprising the sequence of SEQ ID NO: 2 are _IgG2 antibodies that block tissue factor pathway inhibitor (TFPI). Because TFPI downregulates extrinsic coagulation, anti-TFPI antibodies can promote extrinsic pathway-driven coagulation by blocking TFPI, thereby bypassing FVIII or FIX deficiencies in the intrinsic pathway for the treatment of hemophilia. The salt-free anti-TFPI antibody formulations presented herein can be administered to patients via subcutaneous injection or other injection routes.

As part of the present disclosure, it was discovered that the solubility and stability of anti-TFPI antibodies are affected by excipients. The solubility of anti-TFPI antibodies increases as the concentration of NaCl decreases. In the absence of NaCl, the solubility of anti-TFPI antibodies is higher than in formulations that include NaCl. In addition, it was discovered that positively charged amino acids, such as arginine and lysine, can improve the stability of anti-TFPI antibodies, and that pH greatly affects anti-TFPI antibody solubility. The turbidity of the antibody solution increases with increasing pH; however, precipitation is reversible when the pH is decreased. The optimal pH range for stabilizing the anti-TFPI antibodies presented herein is about pH 4 to about pH 6 or about pH 5 to about pH 6, for example, about pH 5, about pH 5.5, or about pH 6.

Exemplified herein are formulations as described above, wherein the antibody is an anti-TFPI antibody (aTFPI Ab). In at least one aspect, the anti-TFPI antibody is a human IgG2 monoclonal antibody. For example, human anti-TFPI IgG2 monoclonal antibodies include antibodies comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2.

The present disclosure also provides methods for treating a disorder in a patient, comprising administering to the patient a therapeutically effective amount of one or more formulations described herein. For example, provided are methods for treating a disorder in a patient, comprising administering to the patient a therapeutically effective amount of an antibody or other protein formulation containing about 0 mM to about 30 mM histidine, and about 50 ppm to about 200 ppm polysorbate (Tween®) 80 or polysorbate (Tween®) 20, about 88 mM to about 292 mM sucrose, about 0 mM to about 50 mM arginine, about 0 mM to about 50 mM lysine, about 0 mM to about 133 mM glycine or alanine, about 0 mM to about 10 mM methionine, and about 1 mg/ml to about 150 mg/ml of protein at a pH ranging from about pH 4.0 to about pH 6.0. In at least one aspect of these methods, the antibody or other protein formulation can be administered intravenously. In other aspects of these methods, the antibody or other protein formulation can be administered subcutaneously. In other aspects of these methods, the antibody or other protein formulation can be administered intramuscularly.

In related aspects, the present disclosure provides methods for treating hemophilia A or hemophilia B in a patient, comprising administering to the patient a therapeutically effective amount of an anti-TFPI antibody formulation comprising about 0 mM to about 30 mM histidine, and about 50 ppm to about 200 ppm polysorbate (Tween®) 80 or polysorbate (Tween®) 20, about 88 mM to about 292 mM sucrose, about 0 mM to about 50 mM arginine, about 0 mM to about 50 mM lysine, about 0% (0 mM) to about 1% (133 mM) glycine, about 0 mM to about 10 mM methionine, and about 1 mg/ml to about 150 mg/ml of protein at a pH in the range of about pH 4.0 to about pH 6.0. In at least one aspect of these methods, the anti-TFPI antibody formulation can be administered intravenously. Within other aspects of these methods, the anti-TFPI antibody formulation can be administered subcutaneously. Within other aspects of these methods, the antibody or other protein formulation can be administered intramuscularly.

According to certain aspects of these methods for treating hemophilia A or hemophilia B in a patient, the anti-TFPI antibody is a human anti-TFPI IgG2 monoclonal antibody, e.g., a human anti-TFPI IgG2 monoclonal antibody containing a light chain having the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2.

For the purposes of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular also include the plural, and vice versa. To the extent any definition set forth below conflicts with the use of that term in any other document (including any document incorporated herein by reference), for the purposes of interpreting this specification and its associated claims, the definition set forth below shall always control unless a contrary meaning is expressly indicated (e.g., in the document in which the term is first used). The term "or" means "and/or" unless otherwise indicated. The term "a/kind" herein means "one or more" unless otherwise indicated or where the use of "one or more" is clearly inappropriate. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are used interchangeably and are not intended to be limiting. Furthermore, when the description of one or more embodiments uses the term "comprising," those skilled in the art will understand that in some specific cases, one or more embodiments may alternatively be described using the language "consisting essentially of" and/or "consisting of."

Aspects of the present disclosure can be further understood in light of the following examples, which should not be construed as limiting the scope of the teachings herein in any way.

Example

Example 1

Effects of NaCl concentration and pH on the turbidity of antibody solutions

This example discloses the effect of salt (NaCl) concentration and pH on the turbidity of a solution containing an anti-TFPI human monoclonal antibody having a light chain comprising the amino acid sequence presented in SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence presented in SEQ ID NO: 2.

The turbidity of the solution was measured by nephelometry to quickly assess the effects of salt concentration and pH on the aTFPI Ab solution. The formulation of the anti-TFPI antibody used in this example contained 10 mM acetate buffer, 88 mM sucrose, and 200 ppm Tween 80. The NaCl concentration was varied from 0 mM to 30 mM. The results of NaCl-dependent turbidity measurements of the anti-TFPI formulation at pH 5.5 are presented in Figure 1. These data demonstrate that the turbidity of the anti-TFPI mAb formulation increases significantly with increasing NaCl concentration. Increasing the salt concentration from 0 to 300 mM resulted in an increase of 72 FNU in the turbidity value by nephelometry, which can be attributed to precipitation, aggregation, or insolubility of the aTFPI Ab in the solution. Due to this finding, solutions without sodium chloride are recommended for use in the anti-TFPI antibody formulations disclosed herein.

Without being bound by theory, it is believed that the increased turbidity of the anti-TFPI mAb formulation with increasing NaCl concentration arises from the neutralization of the positive charge on the arginine side chain of the anti-TFPI mAb. The phase behavior of the aTFPI mAb at different pH levels, along with the influence of monovalent salt (NaCl), explains why aTFPI mAb formulations that are stable, soluble, salt-free, and essentially isosmolal can be achieved.

The aTFPI mAb molecule disclosed herein has 116 amino acids (42 arginines and 74 lysines) with side chains that carry a positive charge at pH values below 0.01. This anti-TFPI antibody has a pH value of ~7.9. At pH values below 0.01, such as pH 4-6, the anti-TFPI antibody has a net positive charge. The repulsion of positive charges on the anti-TFPI antibody surface may prevent protein-protein binding between individual molecules and thereby significantly increase solubility. It is hypothesized that salt anions (Cl ⁻ ) bind to guanidine groups on arginine side chains on the anti-TFPI antibody surface to neutralize the positive charge, enhancing protein-protein interactions and, therefore, contributing to lower solubility and solution turbidity. By shifting the pH to 4-6, the salt-free formulation described herein was developed to achieve increased antibody solubility and stability (see Figure 1). In the absence of salt, the concentration of other stabilizers, such as sucrose, can be increased to >150 mM and <300 mM without compromising osmolarity.

The effect of pH on the turbidity of anti-TFPI antibodies was also studied. As shown in Figure 2, pH can also greatly affect the turbidity of aTFPI Ab solutions. When the pH increased from 4 to 6.5, the turbidity of the aTFPI Ab solution increased by 81 FNU. Further increasing the pH to 7, the turbidity of the solution exceeded the range for accurate measurement. However, when the pH was lowered, the precipitate formed in the solution was reversible. When the pH was increased to a value close to the PI of the aTFPI Ab, this result can be attributed to surface charge neutralization, as the PI value of the aTFPI Ab is approximately 7.9. According to this study, a lower pH is preferred for aTFPI Ab formulations. However, a low pH can cause tissue irritation during injection. Therefore, from the perspective of patient compliance, a neutral pH is preferred. Balancing these two factors, the optimal pH for aTFPI Ab formulations is pH 5-pH 6.

Example 2

Anti-TFPI antibody preparations

Substantially isotonic anti-TFPI Ab formulations were prepared that did not contain NaCl based on the unexpected findings presented in Example 1. These formulations typically utilize high sucrose concentrations to help stabilize the anti-TFPI Ab.

Frozen anti-TFPI antibodies were thawed and reconstituted by dialysis according to the formulations presented in Table 1. The formulations were prepared and filtered with a 0.22 μm filter and filled into glass tubing vials and stoppered with rubber stoppers.

It was also found that positively charged amino acids such as arginine (10–50 mM) could effectively inhibit aTFPI Ab glycation in the absence of NaCl and in the presence of sucrose (88 mM–292 mM) and polysorbate 80 or polysorbate 20 (50–200 ppm).

Each of these anti-TFPI mAb formulations was analyzed by HPLC-SEC for protein aggregation and degradation, LC-MS for structural changes in aTFPI (glycation and oxidation), nephelometry for turbidity assessment, viscometer for viscosity measurement, and osmolarity instrumentation for osmolarity measurement. The results of these analyses are presented in Table 2.

Example 3

Arginine inhibits TFPI Ab glycation

The Examples demonstrate that anti-TFPI Ab formulations containing arginine exhibit reduced antibody glycation compared to anti-TFPI Ab formulations without added arginine.

Anti-TFPI Ab formulations with and without arginine were stored at 40°C for 14 days at pH 6 and tested by LC-MS. The results presented in Table 3 demonstrate that the positively charged amino acid arginine reduces glycation of the anti-TFPI Ab to levels found in the reference standard, likely due to the unique structure of the aTFPI Ab.

Based on the results of this study, it can be concluded that the stability of aTFPI Ab is highly affected by the pH of the formulation, and the optimal pH for formulation stability is pH 5-pH 6 when considering IV, IM, and subcutaneous injections. Arginine appears to be able to prevent the glycation of aTFPI Ab. The formulation development and stability studies presented in Examples 4 and 5 were designed based on these findings.

Example 4

Stability of anti-TFPI Ab formulations

The HPLC-SEC results and LC-MS results of the anti-TFPI Ab preparations are summarized in Tables 4-7.

¹ Calculations based on 2-month values.

² If the rate is negative, enter zero.

Y: Comparable to the reference standard

Y*: Comparable to the reference standard with minor modifications

¹ Calculations based on 2-month values.

² If the rate is negative, enter zero.

Y: Comparable to the reference standard

Y*: Comparable to the reference standard with minor modifications

¹ Calculations based on 2-month values.

² If the rate is negative, enter zero.

Y: Comparable to the reference standard

Y*: Comparable to the reference standard with minor modifications

¹ Calculations based on 2-month values.

Y: Comparable to the reference standard

Y*: Comparable to the reference standard with minor modifications.

Claims (5)

1. A salt-free anti-TFPI antibody preparation, comprising: a.10 mM histidine, b.75ppm Tween 80, c. 234 mM sucrose, d.30 mM arginine, e. 10 mM methionine, and f. 100 mg/ml anti-TFPI antibody; wherein the anti-TFPI antibody formulation has a pH of 5.5, wherein the anti-TFPI antibody is a human _IgG2 monoclonal antibody having a light chain with the amino acid sequence of SEQ ID NO: ₁ and a heavy chain with the amino acid sequence of SEQ ID NO: 2. 2. Use of the salt-free anti-TFPI antibody formulation of claim 1 in the preparation of a medicament for treating a condition in a patient. 3. The use according to claim 2, wherein the antibody preparation is administered intravenously, subcutaneously or intramuscularly. 4. Use of the salt-free anti-TFPI antibody formulation of claim 1 in the preparation of a medicament for treating hemophilia A or hemophilia B in a patient. 5. The use of claim 4, wherein the anti-TFPI antibody preparation is administered intravenously, intramuscularly or subcutaneously.