STABLE, LOW-VISCOSITY ANTIBODY FORMULATIONS AND USES THEREOF

Provided herein are aqueous antibody formulations that exhibit improved stability and low viscosity. The formulations include an antibody or an antigen-binding fragment, a buffer, and a salt selected from the group of magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, or lithium sulfate, where the formulations have a pH of about 4 to about 8 and, optionally an osmolality of about 250 mOsm/kg to about 1500 mOsm/kg

TECHNICAL FIELD

The present invention relates generally to antibody formulations. Specifically, the present invention relates to monoclonal antibody formulations with improved stability and low viscosity.

BACKGROUND

Antibody formulations can lose their efficacy over time due, for example, to the effects of denaturation, oxidation, aggregation, or other degradation reactions. Degradation and aggregation of antibodies in an antibody formulation can also pose risks such as toxicity or immunogenicity. High solution viscosity can negatively impact the manufacturability and performance of protein therapeutic agents, especially those formulated at high protein concentrations.

The low level of stability exhibited by currently available pharmaceutical antibody formulations is disadvantageous due to, for example, loss of efficacy of the antibody formulation before administration and possible toxicity and immunogenicity due to the degradation/aggregation. Traditional excipients used to stabilize proteins in solution can often increase the viscosity of the solution. Therefore, there is a need in the art for an antibody formulation that will allow for improved stability of antibodies. Additionally, there is a need in the art of antibody formulation that will allow for the development of stable and low-viscosity antibody solutions.

SUMMARY

The present invention is based on the discovery that antibodies can be stabilized in solution by including a salt selected from the group of magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate. The resulting stabilized antibody solutions are also less viscous compared to formulations which do not include one of these salts or that contain traditional excipients, for example sugars like sucrose, used to stabilize proteins. The osmolality of stabilized antibody solutions with one of these salts is also less than that with sugars and polyols such as sucrose, trehalose, sorbitol, mannitol etc. when formulated at equipotent concentrations.

In view of this discovery, provided herein are aqueous antibody formulations that include about 0.1 mg/mL to about 500 mg/mL of an antibody or an antigen-binding fragment thereof (e.g., any of the exemplary antibodies or antigen-binding fragments described herein or known in the art); about 1 mM to about 100 mM of a buffer (e.g., any of the exemplary buffers described herein or known in the art); and about 1 mM to 750 mM of a salt selected from the group consisting of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate, where the formulations have a pH of about 4 to about 8.

Also provided are an aqueous antibody formulations that include about 0.1 mg/mL to about 500 mg/mL of an antibody or an antigen-binding fragment thereof (e.g., any of the exemplary antibodies or antigen-binding fragments described herein or known in the art); and about 1 mM to 750 mM of a salt selected from the group of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate, where the formulations have a pH of about 4 to about 8. In some embodiments of the aqueous antibody formulations, the aqueous antibody formulation is a buffer-free aqueous antibody formulation.

Some embodiments of any of the aqueous antibody formulations described herein can further include a stabilizer (e.g., any of the exemplary stabilizers described herein or known in the art) and/or a surfactant (e.g., any of the exemplary surfactants described herein or known in the art). Also provided are injection devices that include any of these formulations, and kits including one or more vials containing any of these formulations.

Also provided are methods of making an aqueous antibody formulation that include mixing or combining: (i) an antibody or an antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein or known in the art); (ii) a buffer (e.g., any of the exemplary buffers described herein or known in the art); (iii) a salt selected from the group consisting of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate; (iv) a stabilizer (e.g., any of the exemplary stabilizers described herein or known in the art); (v) a surfactant (e.g., any of the exemplary surfactants described herein or known in the art); and (vi) sterile water, where (i) to (vi) are mixed or combined in amounts sufficient to generate any of the formulations described herein.

Also provided herein are methods of making an aqueous antibody formulation that include mixing or combining: (i) an antibody or an antigen-binding fragment thereof (e.g., any of the exemplary antibodies or antigen-binding fragments described herein or known in the art); and (ii) a salt selected from the group of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate, (iii) a stabilizer (e.g., any of the exemplary stabilizers described herein or known in the art); (iv) a surfactant (e.g., any of the exemplary surfactants described herein or known in the art); and (v) sterile water; wherein (i) to (v) are mixed or combined in amounts sufficient to generate any of the formulations described herein. In some embodiments of these methods, the method does not include mixing or combining a buffer with (i) and (ii), and the method results in a buffer-free aqueous antibody formulation.

Also provided are methods of treating a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the formulations described herein.

Provided herein are aqueous antibody formulations that include: about 0.1 mg/mL to about 500 mg/mL of an antibody or an antigen-binding fragment thereof; about 1 mM to about 100 mM of a buffer; and about 1 mM to about 750 mM of a salt selected from the group consisting of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate, wherein the formulation has a pH of about 4 to about 8 and optionally, an osmolality of about 250 mOsm/kg to about 1500 mOsm/kg.

Also provided herein are aqueous antibody formulations that include: about 0.1 mg/mL to about 500 mg/mL of an antibody or an antigen-binding fragment thereof; and about 1 mM to about 750 mM of a salt selected from the group of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate, where the formulation has a pH of about 4 to about 8. In some embodiments of any of the aqueous antibody formulations described herein, the formulation is a buffer-free aqueous antibody formulation.

In some embodiments of any of the aqueous antibody formulations described herein, the salt is magnesium glutamate, magnesium acetate, magnesium aspartate, or magnesium sulfate, or a combination thereof. In some embodiments of any of the aqueous antibody formulations described herein, the salt is magnesium glutamate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is magnesium acetate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is magnesium aspartate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is magnesium sulfate. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 10 mM to about 750 mM of the salt. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 20 mM to about 750 mM of the salt.

In some embodiments of any of the aqueous antibody formulations described herein, the salt is sodium acetate, sodium aspartate, sodium glutamate or sodium sulfate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is sodium acetate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is sodium aspartate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is sodium glutamate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is sodium sulfate. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 10 mM to about 750 mM of the salt. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 20 mM to about 750 mM of the salt.

In some embodiments of any of the aqueous antibody formulations described herein, the salt is lithium acetate, lithium aspartate, lithium glutamate, or lithium sulfate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is lithium acetate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is lithium aspartate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is lithium glutamate. In some embodiments of any of the aqueous antibody formulations described herein, the salt is lithium sulfate. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 10 mM to about 750 mM of the salt. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 20 mM to about 750 mM of the salt.

In some embodiments of any of the aqueous antibody formulations described herein, the buffer is selected from the group consisting of: acetate, succinate, gluconate, histidine, citrate, and phosphate. In some embodiments of any of the aqueous antibody formulations described herein, the buffer is a histidine buffer. In some embodiments of any of the aqueous antibody formulations described herein, the buffer is an acetate buffer. In some embodiments of any of the aqueous antibody formulations described herein, the buffer is a citrate buffer. In some embodiments of any of the aqueous antibody formulations described herein, the buffer is a phosphate buffer. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 1 mM to about 100 mM of the buffer. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 1 mM to about 75 mM of the buffer. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 1 mM to about 50 mM of the buffer. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 1 mM to about 20 mM of the buffer.

In some embodiments of any of the aqueous antibody formulations described herein, the formulation has a pH of about 5 to about 6. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has a pH of about 5.5.

In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes an antibody. In some embodiments of any of the aqueous antibody formulations described herein, the antibody is a monoclonal antibody (mAb). In some embodiments of any of the aqueous antibody formulations described herein, the mAb is a human antibody or a humanized antibody. In some embodiments of any of the aqueous antibody formulations described herein, the mAb has an Fc amino acid substitution that decreases its conformational stability as compared to a similar antibody not including the Fc amino acid substitution. In some embodiments of any of the aqueous antibody formulations described herein, the mAb is an IgG1 or an IgG4 antibody.

In some embodiments of any of the aqueous antibody formulations described herein, the mAb is an anti-C—X—C motif chemokine receptor 3 (CXCR3) mAb. In some embodiments of any of the aqueous antibody formulations described herein, the anti-CXCR3 mAb includes a heavy chain including SEQ ID NO: 1 and a light chain including SEQ ID NO: 2.

In some embodiments of any of the aqueous antibody formulations described herein, the mAb is an anti-cluster of differentiation 38 (CD38)-Fc engineered mAb. In some embodiments of any of the aqueous antibody formulations described herein, the anti-CD38-Fc engineered mAb includes a heavy chain including SEQ ID NO: 3 and a light chain including SEQ ID NO: 4.

In some embodiments of any of the aqueous antibody formulations described herein, the monoclonal antibody is an anti-carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) monoclonal antibody. In some embodiments of any of the aqueous antibody formulations described herein, the anti-CEACAM5 monoclonal antibody comprises a heavy chain comprising SEQ ID NO: 7 and a light chain comprising SEQ ID NO: 8.

In some embodiments of any of the aqueous antibody formulations described herein, the monoclonal antibody is an anti-carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)-Fc engineered monoclonal antibody. In some embodiments of any of the aqueous antibody formulations described herein, the anti-CEACAM5-Fc engineered monoclonal antibody comprises a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10. In some embodiments of any of the aqueous antibody formulations described herein, the anti-CEACAM5-Fc engineered monoclonal antibody comprises a heavy chain comprising SEQ ID NO: 11 and a light chain comprising SEQ ID NO: 12. In some embodiments of any of the aqueous antibody formulations described herein, the anti-CEACAM5-Fc engineered monoclonal antibody comprises a heavy chain comprising SEQ ID NO: 13 and a light chain comprising SEQ ID NO: 14.

In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 0.1 mg/mL to 400 mg/mL of the antibody or the antigen-binding antibody fragment. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 0.1 mg/mL to 250 mg/mL of the antibody or the antigen-binding antibody fragment. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 0.1 mg/mL to about 200 mg/mL of the antibody or the antigen-binding antibody fragment. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 0.1 mg/mL to about 150 mg/mL of the antibody or the antigen-binding antibody fragment. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 0.1 mg/mL to about 100 mg/mL of the antibody or the antigen-binding antibody fragment. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 0.1 mg/mL to about 50 mg/mL of the antibody or the antigen-binding antibody fragment. In some embodiments of any of the aqueous antibody formulations described herein, the formulation includes about 0.1 mg/mL to about 25 mg/mL of the antibody or the antigen-binding antibody fragment.

In some embodiments of any of the aqueous antibody formulations described herein, the formulation has a viscosity of about 1 cP to about 50 cP. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has a viscosity of about 1 cP to about 40 cP. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has a viscosity of about 1 cP to about 30 cP. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has a viscosity of about 1 cP to about 20 cP.

In some embodiments of any of the aqueous antibody formulations described herein, the formulation has an osmolality of about 250 mOsm/kg to about 1500 mOsm/kg. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has an osmolality of about 250 mOsm/kg to about 1500 mOsm/kg. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has an osmolality of about 250 mOsm/kg to about 750 mOsm/kg. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has an osmolality of about 250 mOsm/kg to about 500 mOsm/kg. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has an osmolality of about 250 mOsm/kg to about 400 mOsm/kg. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has an osmolality of about 500 mOsm/kg to about 1500 mOsm/kg. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has an osmolality of about 500 mOsm/kg to about 1000 mOsm/kg. In some embodiments of any of the aqueous antibody formulations described herein, the formulation has an osmolality of about 1000 mOsm/kg to about 1500 mOsm/kg.

In some embodiments of any of the aqueous antibody formulations described herein, the formulation is stable (e.g., % of high molecular weight (HMW) by SEC ≤5%) at 25° C. for about 1 week to about 2 years. In some embodiments of any of the aqueous antibody formulations described herein, the formulation is stable (e.g., % HMW by SEC ≤5%) at 40° C. for about 1 hour to about 8 weeks.

In some embodiments of any of the aqueous antibody formulations described herein, the formulation is suitable for intravenous, intramuscular, or subcutaneous administration. In some embodiments of any of the aqueous antibody formulations described herein, the formulation is suitable for intravenous administration. In some embodiments of any of the aqueous antibody formulations described herein, the formulation is suitable for subcutaneous administration.

Some embodiments of any of the aqueous antibody formulations described herein further includes one or more of a stabilizer, an anti-oxidant, a metal chelator, a viscosity modifier, an amino acid, and a surfactant. In some embodiments of any of the aqueous antibody formulations described herein, the stabilizer is fructose, maltose, galactose, glucose, O-mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, a maltodextrin, a dextran, starch, mannitol, xylitol, maltitol, lactitol, glucitol, sucrose, trehalose, raffinose, maltose, sorbitol, mannitol, an amino sugar, sodium chloride, and glycerol.

In some embodiments of any of the aqueous antibody formulations described herein, the antioxidant is methionine, ascorbic acid, or N-acetyl cysteine. In some embodiments of any of the aqueous antibody formulations described herein, the metal chelator is sodium ethylenediaminetetraacetic acid (EDTA), calcium EDTA, or diethylenetriamine pentaacetate (DTPA). In some embodiments of any of the aqueous antibody formulations described herein, the viscosity modifier is arginine, histidine, lysine, proline, glycine, or sodium chloride.

In some embodiments of any of the aqueous antibody formulations described herein, the amino acid is selected from the group of: arginine, lysine, histidine, proline, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid, and aspartic acid.

Also provided herein are injection devices including any of the aqueous antibody formulations described herein.

Also provided herein are kits including one or more vials containing any of the aqueous antibody formulations described herein. In some embodiments of any of the kits described herein, the kit further includes an injection device for administration of the aqueous antibody formulation to a subject in need thereof.

Provided herein are methods of making an aqueous antibody formulation that include mixing or combining: (i) an antibody or an antigen-binding fragment thereof; (ii) a buffer; (iii) a salt selected from the group consisting of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate, (iv) a stabilizer; (v) a surfactant; and (vi) sterile water, wherein (i) to (vi) are mixed or combined in amounts sufficient to generate any of the aqueous antibody formulations described herein.

Also provided herein are methods of making an aqueous antibody formulation that include mixing or combining: (i) an antibody or an antigen-binding fragment thereof; and (ii) a salt selected from the group consisting of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate; (iii) a stabilizer); (iv) a surfactant); and (v) sterile water, wherein (i) to (v) are mixed or combined in amounts sufficient to generate any of the aqueous antibody formulations described herein. In some embodiments of any of the aqueous antibody formulations described herein, the method does not include mixing a buffer with (i) to (v) and the method results in the generation of a buffer-free aqueous antibody formulation.

Some embodiments of any of the methods described herein further include mixing or combining one or more (e.g., one, two or three) of an antioxidant, a metal chelator, and a viscosity modifier to (i) and (vi).

Also provided herein are methods of treating a subject in need thereof, the method includes administering to the subject a therapeutically effective amount of any of the aqueous antibody formulations described herein.

The term “stabilizer” refers to an additional agent (e.g., not including any of the salts of magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate) that improves or otherwise enhances stability of a protein (e.g., an antibody or an antigen-binding antibody fragment) in a formulation. Non-limiting examples of stabilizers are described herein. Additional examples of stabilizers are known in the art. The term “surfactant” generally includes an agent that protects a protein (e.g., an antibody or an antigen-binding antibody fragment) from air/solution interface-induced stress and/or solution/surface induced-stress. In some embodiments, a surfactant may protect a protein (e.g., an antibody or an antigen-binding antibody fragment) from aggregation. Non-limiting examples of surfactants are described herein. Additional examples of surfactants are known in the art.

The term “subject” refers to any mammal. In some embodiments, the subject or “subject in need of treatment” can be a canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), ovine, bovine, porcine, caprine, primate, e.g., a simian (e.g., a monkey (e.g., a marmoset, baboon), or an ape (e.g., a gorilla, chimpanzee, orangutan, or gibbon), a human; or a rodent (e.g., a mouse, a guinea pig, a hamster, or a rat). In some embodiments, the subject or “subject suitable for treatment” may be a non-human mammal, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, lapine, porcine, canine or primate animals) may be employed.

As used herein, “treating” means a reduction in the number, severity, or frequency of one or more symptoms of a medical disease or condition in a subject (e.g., any of the exemplary subjects described herein).

As used herein, “buffer-free” means no or trace amount of a buffer (e.g., any of the buffers described herein).

DETAILED DESCRIPTION

Over the past few decades, therapies involving the use of monoclonal antibodies and other Fc-derived antigen-binding proteins have become a mainstay of modern medicine. There is an ever-increasing reliance on these complex molecules in various therapeutic areas including but not limited to oncology, immunology, immuno-oncology, cardiology with nearly 100 molecules approved for therapeutic use to date and more than 500 at various stages of development or clinical trials.

A fundamental aspect for ensuring the transition of these therapeutic entities from the lab into manufacturable and marketable products of high and consistent quality is their stability in the dosage form. Owing to their complex chemistry and structure, proteins are susceptible to various forms of physical and chemical degradation that can compromise the biological efficacy and safety of the final drug product. Protein aggregation for example is a key quality attribute that is routinely monitored for protein-based products and is critical to the determination of product shelf life. At a fundamental level, protein aggregation is linked to the stability of the native form of the protein, with a growth in non-native cell (e.g., a non-native mammalian cell) generally linked to an increased rate and extent of aggregation. Thus, it is no surprise that attempts to control and minimize aggregation during product shelf life (kinetic stability) are often mediated through the use of excipients or formulation conditions intended to increase conformational stability of the protein. Essentially, the intent is to stabilize the protein in its native conformation in order to minimize the population of aggregation-competent “non-native” species. Sugars and polyols, such as sucrose, trehalose, mannitol, sorbitol etc. are often used to stabilize proteins in their native state and reduce rates of aggregation. However an unwanted effect of using these stabilizers is the concentration-dependent increase in solution viscosity.

Solution viscosity is a key attribute of protein products especially those that are formulated at high protein concentrations (for example ≥100 mg/mL for an antibody or Fc-derived proteins of similar molecular weight) and it can critically impact the utility and success of the product. The manufacturability of a product and the end use by the patient or healthcare practitioner is intimately linked to the ability of a solution to flow seamlessly. High viscosity, for example, can necessitate the use of specialized administration devices or protocols which may not always be suitable for the desired population thereby limiting the use of the product. In other instances, high solution viscosity may require the application of manufacturing technologies which may negatively impact the stability of the protein (for example high-temperature processing). It is thus not unusual to employ viscosity-reducing excipients, such as salts and amino acids, in high protein concentration solutions. However, these excipients can negatively impact the stability of the protein thereby resulting in solutions with an increased aggregation rate compared to high-viscosity control solutions lacking the viscosity-reducing agent. In essence, commonly employed stabilizers and the viscosity-reducing excipients can have an opposite effect on product performance thereby complicating its development.

Another critical attribute for injectable products (most protein-based products) that needs to be considered is its osmolality. While intravenous solutions generally need to be isotonic, it is not unusual for subcutaneous solutions to be hypertonic. In fact, there is evidence in literature of hypertonic formulations resulting in enhanced protein bioavailability following subcutaneous administration (Fathallah, A. M. et al, Biopharm Drug Dispos. 2015 March; 36(2):115-25). Thus, the impact of solution osmolality (and thus tonicity) on injection site discomfort and/or reaction as well as bioavailability in the patient population needs to be carefully monitored and characterized during clinical development phases.

Thus, there is a need in the art of formulating antibody and other Fc-derived products for developing stable, low-viscosity solution formulations with well characterized osmotic properties.

Provided herein are aqueous antibody formulations that include about 0.1 mg/mL to about 500 mg/mL of an antibody or an antigen-binding fragment thereof (e.g., any of the exemplary antibodies or antigen-binding fragments described herein or known in the art); about 1 mM to about 100 mM of a buffer (e.g., any of the exemplary buffers described herein or known in the art); and about 1 mM to 750 mM of a salt selected from the group consisting of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate, where the formulations have a pH of about 4 to about 8.

Also provided herein are aqueous antibody formulations include about 0.1 mg/mL to about 500 mg/mL (e.g., any of the subranges of this range described herein) of an antibody or an antigen-binding fragment thereof (e.g., any of the exemplary antibodies or antigen-binding fragments described herein or known in the art); and about 1 mM to 750 mM (e.g., any of the subranges of this range described herein) of a salt selected from the group of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate, where the formulations have a pH of about 4 to about 8. In some embodiments, the aqueous antibody formulation is a buffer-free aqueous antibody formulation.

Also provided are injection devices that include any of these formulations, and kits including one or more vials containing any of these formulations.

Also provided are methods of making an aqueous antibody formulation that include mixing or combining: (i) an antibody or an antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein or known in the art); (ii) a buffer (e.g., any of the exemplary buffers described herein or known in the art); (iii) a salt selected from the group consisting of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate, (iv) a stabilizer; (v) a surfactant; and (vi) sterile water, where (i) to (vi) are mixed or combined in amounts sufficient to generate any of the formulations described herein.

Also provided herein are methods of making an aqueous antibody formulations that include mixing or combining: (i) an antibody or an antigen-binding fragment thereof; and (ii) a salt selected from the group of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate; (iii) a stabilizer); (iv) a surfactant); and (v) sterile water, wherein (i) to (v) are mixed or combined in amounts sufficient to generate any of the aqueous antibody formulations described herein. In some embodiments of the methods described herein, the method does not include mixing or combining a buffer with (i) to (v) and the method results in a buffer-free aqueous antibody formulation.

Also provided are methods of treating a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the formulations described herein.

Non-limiting aspects of these formulations, injection devices, kits, and methods are described below. As can be appreciated by those in the field, the exemplary aspects listed below can be used in any combination, and can be combined with other aspects known in the field.

Antibodies and Antigen-Binding Antibody Fragments

The term “antibody” as used herein is used broadly to mean any polypeptide that includes an antigen-binding domain. Non-limiting examples of types of antibodies are described herein. Additional examples of antibodies are known in the art.

The term “antigen-binding antibody fragment” refers to a fragment of a mammalian (e.g., human) IgG1, IgG2, IgG3, IgG4, IgM, IgE, or IgA that retains its ability to bind specifically to an antigen. Non-limiting examples of antigen-binding antibody fragments are described herein. Additional examples of antigen-binding antibody fragments are known in the art.

In some embodiments, an antibody can be a VHH domain, a VNAR domain, a scFv, a BiTe, a (scFv)2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv.

In some embodiments, an antibody can be a VHH-scAb, a VHH-Fab, a Dual scFab, a diabody, a crossMab, a DAF (two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a κλ-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv, a F(ab′)2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a lmmTAC, an IgG-IgG conjugate, a Cov-X-Body, and a scFv1-PEG-scFv2.

Non-limiting examples of an antigen-binding antibody fragments include an Fv fragment, a Fab fragment, a F(ab′)2 fragment, and a Fab′ fragment. Additional examples of antigen-binding antibody fragments include any antigen-binding fragment of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM).

A “Fv” fragment includes a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

A “Fab” fragment includes, the constant domain of the light chain and the first constant domain (CH1) of the heavy chain, in addition to the heavy and light chain variable domains of the Fv fragment.

A “F(ab′)2” fragment includes two Fab fragments joined, near the hinge region, by disulfide bonds.

A “dual variable domain immunoglobulin” or “DVD-Ig” refers to multivalent and multispecific binding proteins as described, e.g., in DiGiammarino et al.,Methods Mol. Biol.899:145-156, 2012; Jakob et al.,MABs5:358-363, 2013; and U.S. Pat. Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each of which is incorporated by reference in its entirety.

An antibody or an antigen-binding antibody fragment can bind to its epitope or antigen with a dissociation equilibrium constant (KD) of less than 1×10−7M, less than 1×10−8M, less than 1×10−9M, less than 1×10−1° M, less than 1×10−11M, less than 1×10−12M, or less than 1×10−13M. In some embodiments, the antibody or the antigen-binding antibody fragment can bind to its antigen or epitope with a KDof about 1×10−3M to about 1×10−5M, about 1×10−4M to about 1×10−6M, about 1×10−5M to about 1×10−7M, about 1×10−6M to about 1×10−8M, about 1×10−7M to about 1×10−9M, about 1×10−8M to about 1×10−10M, or about 1×10−9M to about 1×10−11M (inclusive).

In some examples, the antibody can be a mAb (e.g., a monoclonal human or humanized antibody).

In some embodiments, the mAb can have an Fc region comprising one or more amino acid substitutions that result in low CH2 domain unfolding temperature compared to an antibody having a wildtype Fc region. In some embodiments, the mAb can have an Fc region comprising one or more amino acid substitutions that decrease the stability of the antibody, e.g., as compared to the stability of a similar antibody lacking the one or more amino acid substitutions.

In some embodiments, the mAb can be an IgG1, IgG2, IgG3, or IgG4 antibody (e.g., a human or humanized antibody). In some preferred embodiments, the mAb is an IgG1 or IgG4 antibody.

In some embodiments, the mAb is an anti-C—X—C motif chemokine receptor 3 (CXCR3) mAb (e.g., a human or humanized antibody). In some embodiments, the anti-CXCR3 mAb comprises a heavy chain comprising or consisting of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO: 1 and a light chain comprising or consisting of a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical SEQ ID NO: 2. In some embodiments, the anti-CXCR3 antibody includes the three CDRs present in SEQ ID NO: 1 and the three CDRs present in SEQ ID NO: 2.

In some embodiments, the mAb is an anti-cluster of differentiation 38 (CD38) mAb (e.g., a human or humanized anti-CD38 antibody). In some embodiments, the anti-CD38 mAb comprises a heavy chain comprising or consisting of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO: 3 and a light chain comprising or consisting of a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical SEQ ID NO: 4. In some embodiments, the anti-CD38 antibody includes the three CDRs present in SEQ ID NO: 3 and the three CDRs present in SEQ ID NO: 4.

In some embodiments, the mAb is an anti-cluster of differentiation 38 (CD38)-Fc engineered mAb (e.g., a human or humanized antibody). In some embodiments, the anti-CD38-Fc engineered mAb comprises a heavy chain comprising or consisting of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO: 5 and a light chain comprising or consisting of a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical SEQ ID NO: 6. In some embodiments, the anti-CD38-Fc engineered mAb includes the three CDRs present in SEQ ID NO: 5 and the three CDRs present in SEQ ID NO: 6.

In some embodiments, the mAb is an anti-carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) mAb (e.g., a human or humanized antibody). In some embodiments, the anti-CEACAM5 mAb comprises a heavy chain comprising or consisting of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO: 9 and a light chain comprising or consisting of a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical SEQ ID NO: 10. In some embodiments, the anti-CEACAM5 antibody includes the three CDRs present in SEQ ID NO: 9 and the three CDRs present in SEQ ID NO: 10.

In some embodiments, the mAb is an anti-carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)-Fc engineered mAb (e.g., a human or humanized antibody). In some embodiments, the anti-CEACAM5-Fc engineered mAb comprises a heavy chain comprising or consisting of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO: 9 and a light chain comprising or consisting of a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical SEQ ID NO: 10. In some embodiments, the anti-CEACAM5-Fc engineered mAb includes the three CDRs present in SEQ ID NO: 9 and the three CDRs present in SEQ ID NO: 10.

In some embodiments, the mAb is an anti-carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)-Fc engineered mAb (e.g., a human or humanized antibody). In some embodiments, the anti-CEACAM5-Fc engineered mAb comprises a heavy chain comprising or consisting of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO: 11 and a light chain comprising or consisting of a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical SEQ ID NO: 12. In some embodiments, the anti-CEACAM5-Fc engineered mAb includes the three CDRs present in SEQ ID NO: 11 and the three CDRs present in SEQ ID NO: 12.

In some embodiments, the mAb is an anti-carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)-Fc engineered mAb (e.g., a human or humanized antibody). In some embodiments, the anti-CEACAM5-Fc engineered mAb comprises a heavy chain comprising or consisting of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO: 13 and a light chain comprising or consisting of a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical SEQ ID NO: 14. In some embodiments, the anti-CEACAM5-Fc engineered mAb includes the three CDRs present in SEQ ID NO: 13 and the three CDRs present in SEQ ID NO: 14.

An antibody can be produced by introducing into a cell a nucleic acid sequence encoding the antibody to produce a recombinant cell; and culturing the recombinant cell under conditions sufficient for the expression of the antibody. In some embodiments, the introducing step includes introducing into a cell an expression vector including a sequence encoding the antibody to produce a recombinant cell.

An antigen described herein can be produced by any cell, e.g., a eukaryotic cell. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such asSaccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells.

Methods of culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor proliferation, differentiation and growth. Briefly, cells can be cultured by contacting a cell (e.g., any cell) with a cell culture medium that includes the necessary growth factors and supplements to support cell viability and growth.

Methods of introducing nucleic acids and expression vectors into a cell (e.g., a eukaryotic cell) are known in the art. Non-limiting examples of methods that can be used to introduce a nucleic acid into a cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection.

Provided herein are methods that further include isolation of the antibody from a cell (e.g., a eukaryotic cell) using techniques well-known in the art (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation, ion-exchange chromatography (anion or cation), chromatography based on hydrophobic interaction, metal-affinity chromatography, ligand-affinity chromatography, size exclusion chromatography).

Buffers

The formulations described herein can include a buffer (e.g., one or more buffers) (e.g., any of the non-limiting buffers described herein or known in the art). In some embodiments, the antibody or antigen-binding antibody fragment present in the formulation does not significantly buffer the pH of the formulation.

Non-limiting examples of a buffer (e.g., one or more buffers) that can be present in any of the formulations described herein include: acetate, succinate, gluconate, histidine, citrate, phosphate, and Tris. In some embodiments of any of the formulations described herein, the formulation can include acetate, histidine, or phosphate. Additional examples of buffers that can be present in any of the formulations described herein are known in the art.

Salts

The aqueous antibody formulations described herein include a salt (e.g., one or more salts) selected from the group of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate. In some examples, the aqueous antibody formulations described herein include a salt (e.g., one or more salts) selected from the group of: magnesium glutamate, magnesium acetate, magnesium aspartate, and magnesium sulfate.

Formulation Stability

In some embodiments of any of the aqueous antibody formulations described herein, the formulation is a stable formulation. A “stable” formulation is one in which a protein of interest (e.g., an antibody or an antigen-binding antibody fragment) therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage at about 4° C. to about 25° C. Various analytical techniques for measuring protein stability are known in the art. See, e.g., Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Additional methods for determining the stability of a protein (e.g., an antibody or antigen-binding antibody fragment) in a formulation are described in the Examples section. In some examples, the stability of a protein (e.g., an antibody or an antigen-binding antibody fragment) is determined according to the percentage of monomer protein in the solution, with a low percentage of degraded (e.g., fragmented) and/or aggregated protein. For example, an aqueous formulation comprising a stable protein may include at least 95% monomer protein. Alternatively, an aqueous formulation of the invention may include no more than 5% (e.g., no more than 4.5%, no more than 4.0%, no more than 3.5%, no more than 3.0%, no more than 2.5%, no more than 2.0%, no more than 1.5%, no more than 1.0%, or no more than 0.5%) aggregates and/or degraded protein.

In some embodiments of any of the aqueous antibody formulations described herein, the formulation has improved stability as compared to a control antibody or antigen-binding antibody fragment (e.g., as compared a control antibody formulation that includes all of the same components, except it does not include any of the following salts: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate).

In some embodiments, the formulation, e.g., before and/or after the addition of a salt (e.g., any of the salts described herein), has a viscosity of about 1 cP to about 50 cP, about 1 cP to about 45 cP, about 1 cP to about 40 cP, about 1 cP to about 35 cP, about 1 cP to about 30 cP, about 1 cP to about 25 cP, about 1 cP to about 20 cP, about 1 cP to about 15 cP, about 1 cP to about 10 cP, about 1 cP to about 5 cP, about 5 cP to about 50 cP, about 5 cP to about 45 cP, about 5 cP to about 40 cP, about 5 cP to about 35 cP, about 5 cP to about 30 cP, about 5 cP to about 25 cP, about 5 cP to about 20 cP, about 5 cP to about 15 cP, about 5 cP to about 10 cP, about 10 cP to about 50 cP, about 10 cP to about 45 cP, about 10 cP to about 40 cP, about 10 cP to about 35 cP, about 10 cP to about 30 cP, about 10 cP to about 25 cP, about 10 cP to about 20 cP, about 10 cP to about 15 cP, about 15 cP to about 50 cP, about 15 cP to about 45 cP, about 15 cP to about 40 cP, about 15 cP to about 35 cP, about 15 cP to about 30 cP, about 15 cP to about 25 cP, about 15 cP to about 20 cP, about 20 cP to about 50 cP, about 20 cP to about 45 cP, about 20 cP to about 40 cP, about 20 cP to about 35 cP, about 20 cP to about 30 cP, about 20 cP to about 25 cP, about 25 cP to about 50 cP, about 25 cP to about 45 cP, about 25 cP to about 40 cP, about 25 cP to about 35 cP, about 25 cP to about 30 cP, about 30 cP to about 50 cP, about 30 cP to about 45 cP, about 30 cP to about 40 cP, about 30 cP to about 35 cP, about 35 cP to about 50 cP, about 35 cP to about 45 cP, about 35 cP to about 40 cP, about 40 cP to about 50 cP, about 40 cP to about 45 cP, or about 45 cP to about 50 cP (e.g., as measured using a viscometer).

Some embodiments of any of the formulations described herein can further include a stabilizer (e.g., one or more stabilizers). Non-limiting examples of stabilizers include fructose, maltose, galactose, glucose, 0-mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, a maltodextrin, a dextran, starch, mannitol, xylitol, maltitol, lactitol, glucitol, sucrose, trehalose, raffinose, maltose, sorbitol, mannitol, an amino sugar, sodium chloride, and glycerol, and combinations thereof. Additional examples of stabilizers are known in the art. The final concentration of a stabilizer (or the final total concentration of one or more stabilizers) in any of the formulations described herein can be about 0.01 mM to about 1500 mM (or any of the subranges of this range described herein).

Amino Acids

Some embodiments of any of the formulations described herein can further include an amino acid (e.g., one or more amino acids). Non-limiting examples of amino acids include arginine, lysine, histidine, proline, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid, and aspartic acid, and combinations thereof. Additional examples of amino acids are known in the art. The final concentration of an amino acid (or a final total concentration of one or more amino acids) in any of the formulations described herein can be about 0.01 mM to about 750 mM (or any of the subranges of this range described herein).

Injection Devices

Also provided herein are injection devices that include any of the aqueous antibody formulations described herein (e.g., one or more doses of any of the aqueous antibody formations described herein). For example, the injection device can be a pre-loaded syringe. Non-limiting examples of such pre-loaded syringes are known in the art.

Also provided herein are kits that include any of the injection devices provided herein. Also provided herein are kits that include one or more vials containing any of the aqueous antibody formulations described herein. In some embodiments, any of the kits provided herein can further include instructions for administration of any of the aqueous antibody formulations to a subject in need thereof.

Methods of Making a Formulation

Also provided herein are methods of making any of the aqueous antibody formulations described herein that include mixing or combining: (i) an antibody or an antigen-binding fragment thereof (e.g., any of the exemplary antibodies or antigen-binding antibody fragments described herein); (ii) a buffer (e.g., any of the buffers or one or more of any of the buffers described herein); (iii) a salt (or one or more salts) selected from the group of magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate; (iv) a stabilizer; (v) a surfactant; and (vi) water (e.g., sterile water), where (i) to (vi) are mixed or combined in amounts sufficient to generate any of the aqueous antibody formulations described herein.

Some embodiments of these methods can further include filtering mixed or combined (i) to (vi). Some embodiments of these methods can further include disposing or placing the formulation into a sterile vile (e.g., a vacuum-sealed, sterile vial) or a syringe (e.g., a sterile syringe).

Some embodiments of any of these methods can further include adding or mixing with (i) to (vi), one or more (e.g., two or three) of a stabilizer (e.g., one or more of any of the exemplary stabilizers described herein or known in the art), an amino acid (e.g., one or more of any of the exemplary amino acids described herein or known in the art), and a surfactant (e.g., one or more of any of the exemplary surfactants described herein or known in the art), e.g., in amounts sufficient to result in any of the aqueous antibody formulations described herein.

Some embodiments of any of these methods can further include adding or mixing together with the other components one or more additional therapeutic agent(s).

As can be appreciated by those in the art, the order that each component of the formulation is added can be varied. For example, the antibody or antigen-binding antibody fragment can be a lyophilized solid that is dissolved in a buffered aqueous solution including the salt and the buffer. In another example, a solid comprising the antibody or antigen-binding antibody fragment (in the form of a lyophilized powder) and the salt, can be dissolved in a buffered aqueous solution (comprising the buffer). In some embodiments, a solid comprising the antibody or antigen-binding fragment (in the form of a lyphophilized powder), the buffer, and the salt, is dissolved in water (e.g., sterile water).

Also provided herein are methods of making an aqueous antibody formulations that include mixing or combining: (i) an antibody or an antigen-binding fragment thereof; and (ii) a salt selected from the group of: magnesium glutamate, magnesium acetate, magnesium aspartate, magnesium sulfate, arginine acetate, arginine aspartate, arginine glutamate, arginine sulfate, lysine acetate, lysine aspartate, lysine glutamate, lysine sulfate, sodium acetate, sodium aspartate, sodium glutamate, sodium sulfate, lithium acetate, lithium aspartate, lithium glutamate, and lithium sulfate; (iii) a stabilizer); (iv) a surfactant); and (v) sterile water, wherein (i) to (v) are mixed or combined in amounts sufficient to generate any of the aqueous antibody formulations described herein. In some embodiments of the methods described herein, the method does not include mixing or combining a buffer with (i) to (v) and the methods results in a buffer-free aqueous antibody formulation.

Some embodiments of these methods can further include filtering mixed or combined (i) to (v). Some embodiments of these methods can further include disposing or placing the formulation into a sterile vile (e.g., a vacuum-sealed, sterile vial) or a syringe (e.g., a sterile syringe).

Some embodiments of any of these methods can further include adding or mixing with (i) to (v), one or more (e.g., two or three) of a stabilizer (e.g., one or more of any of the exemplary stabilizers described herein or known in the art), an amino acid (e.g., any of the exemplary amino acids described herein except for histidine and arginine), and a surfactant (e.g., one or more of any of the exemplary surfactants described herein or known in the art), e.g., in amounts sufficient to result in any of the aqueous antibody formulations described herein.

Some embodiments of any of these methods can further include adding or mixing together with the other components one or more additional therapeutic agent(s). The mixing or combining can be formed by pipetting, vortexing, rocking agitation, or hand agitation. Other means for performing the mixing or combining are known in the art.

Methods of Treating a Subject

Also provided herein are methods of treating a subject in need thereof that include administering to the subject (e.g., any of the subjects described herein) a therapeutically effective amount of any of the aqueous antibody formulations provided herein. In some embodiments of any of these methods, the aqueous antibody formulation can be administered by intravenous, intramuscular, intraperitoneal, subcutaneous, intraarterial, intraocular, intraocular, intraarticular, or interlaminar administration. In some embodiments of any of these methods, the subject can be administered one or more doses of the aqueous antibody formulation.

In some embodiments, the subject has been identified or diagnosed (e.g., previously identified or diagnosed as having a disease or condition that will benefit from treatment with the antibody or the antigen-binding antibody fragment that is present in the aqueous antibody formulation).

In some embodiments of any of the methods described herein, the subject can also be administered one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents can be administered to the subject at the substantially the same time as the aqueous antibody formulation. In some embodiments, the one or more additional therapeutic agents can be administered to the subject to the subject before or after the administration of the aqueous antibody formulation to the subject.

EXAMPLES

Exemplary Components of Antibody Formulations

Exemplary Sample Preparation

All sugar, polyol, amino acid, amino acid salt and metal salt solutions were prepared in 10 mM histidine buffer between pH 5.5 and pH 6.2. For thermal (conformational) stability measurements, 1 mg/mL mAb formulations were prepared by spiking excipient solutions with stock mAb solution at a ratio of ˜ 1/50 volume to volume. For kinetic stability studies and viscosity measurements, stock mAb solutions were concentrated and buffer exchanged into the various excipient solutions using Vivaspin-20 30 kilodalton molecular weight cut off (MWCO) membranes (General Electric). MAb formulations were concentrated up to >180 mg/mL and subsequently diluted down to 150 mg/mL with the corresponding excipient solution.

Exemplary Aqueous Antibody Formulations

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 400 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 0 to about 100 mM of a buffer (e.g., any of the buffers described herein), about 1 to about 750 mM of a stabilizing salt or a viscosity salt (e.g., any of the stabilizing salts or viscosity salts described herein), about 0.001 to about 0.2% of a surfactant (e.g., any of the surfactants described herein), about 0% to about 10% of a sugar or a polyol, wherein the aqueous antibody formulation has a pH of about 4 to about 8. See, e.g., Embodiment A in Table 1.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 300 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 0 to about 50 mM of a buffer (e.g., any of the buffers described herein), about 5 to about 500 mM of a stabilizing salt or a viscosity salt (e.g., any of the stabilizing salts or viscosity salts described herein), about 0.01 to about 0.1% of a surfactant (e.g., any of the surfactants described herein), about 1% to about 8% of a sugar or a polyol, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. See, e.g., Embodiment B in Table 1.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 0 to about 25 mM of a buffer (e.g., any of the buffers described herein), about 25 to about 300 mM of a stabilizing salt or a viscosity salt (e.g., any of the stabilizing salts or viscosity salts described herein), about 0.01 to about 0.06% of a surfactant (e.g., any of the surfactants described herein), about 1.5% to about 8% of a sugar or a polyol, wherein the aqueous antibody formulation has a pH of about 5 to about 7. See, e.g., Embodiment C in Table 1.

In some embodiments, the aqueous antibody formulation comprises about 0 to about 10 mM of a buffer (e.g., any of the buffers described herein), about 300 to about 750 mM of a stabilizing salt or a viscosity salt (e.g., any of the stabilizing salts or viscosity salts described herein), wherein the aqueous antibody formulation has a pH of about 5 to about 6.5. See, e.g., Embodiment D in Table 1.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium acetate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium acetate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 1 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium aspartate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 2 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium glutamate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 3 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium sulfate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium sulfate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 4 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 5 mM of a histidine buffer, about 200 mM of magnesium aspartate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 5 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 5 mM of a histidine buffer, about 200 mM of magnesium glutamate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 6 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of an acetate buffer, about 100 to about 300 mM of magnesium aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of an acetate buffer, about 200 mM of magnesium aspartate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.0. See, e.g., Embodiment 7 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of an acetate buffer, about 100 to about 300 mM of magnesium glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of an acetate buffer, about 200 mM of magnesium glutamate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.0. See, e.g., Embodiment 8 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium aspartate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 6.0. See, e.g., Embodiment 9 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium glutamate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 6.0. See, e.g., Embodiment 10 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of lithium aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of lithium aspartate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 11 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of lithium glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of lithium glutamate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 12 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of sodium aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of sodium aspartate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 13 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of sodium glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of sodium glutamate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 14 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 200 to about 750 mM of arginine aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 300 mM of arginine aspartate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 15 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 200 to about 750 mM of arginine glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 300 mM of arginine glutamate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 16 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 200 to about 750 mM of lysine aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 300 mM of lysine aspartate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 17 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 200 to about 750 mM of lysine glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 300 mM of lysine glutamate, about 2% of sucrose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 18 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium aspartate, about 1% to about 8% of trehalose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium aspartate, about 2% of trehalose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 19 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium glutamate, about 1% to about 8% of trehalose, about 0.01% to about 0.1% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium glutamate, about 2% of trehalose, about 0.05% of polysorbate 80, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 20 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 20, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium aspartate, about 2% of sucrose, about 0.05% of polysorbate 20, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 21 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of polysorbate 20, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium glutamate, about 2% of sucrose, about 0.05% of polysorbate 20, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 22 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium aspartate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of poloxamer 188, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium aspartate, about 2% of sucrose, about 0.05% of poloxamer 188, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 23 in Table 2.

In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 1 to about 100 mM of a histidine buffer, about 100 to about 300 mM of magnesium glutamate, about 1% to about 8% of sucrose, about 0.01% to about 0.1% of poloxamer 188, wherein the aqueous antibody formulation has a pH of about 4.5 to about 7.5. In some embodiments, the aqueous antibody formulation comprises about 0.1 to about 200 mg/mL of an antibody or antigen-binding fragment thereof (e.g., any of the antibodies or antigen-binding fragments described herein), about 10 mM of a histidine buffer, about 200 mM of magnesium glutamate, about 2% of sucrose, about 0.05% of poloxamer 188, wherein the aqueous antibody formulation has a pH of about 5.5. See, e.g., Embodiment 24 in Table 2.

Thermal (conformational) stability was assessed by measuring the lowest unfolding temperature, Tm1, of mAbs on a capillary DSC system (Malvern). The impact of both the identity and concentration of different excipients on increasing the Tm1value was used to rank order their effectiveness at improving conformational stability. Measurements were conducted by ramping the temperature of the solution from 15° C. to 110° C. at a rate of 1° C./min. The temperature value at the maximum of the first peak was designated as Tm1.

Kinetic Stability by Size Exclusion Chromatography (SEC)

Kinetic stability was assessed by periodically measuring the amount of aggregates generated in mAb formulations stored at 40° C. for 4 weeks. SEC was used to quantify the levels of both aggregated and non-aggregated mAb molecules after 0, 1, 2, and 4 weeks of storage. Samples were measured by eluting 50 μg of total mAb off of a TSKgel UP-SW3000 column (Tosoh Co.) at a rate 0.43 mL/min with 40 mM Phosphate and 150 mM Sodium Chloride at pH 7.2 (detection wavelength: 280 nm). Aggregates were designated as high molecular weight species (HMWS) that eluted faster than non-aggregated mAb monomers; HMWS % was calculated by dividing the amount of aggregate generated by the sum of total aggregated+non-aggregated mAb molecules. The initial aggregation rate, kagg, was taken as the slope of a plot of HMWS % vs. storage time.

Viscosity Measurements of mAb Formulations

The viscosities of 150 mg/mL mAb formulations were measured on an initium rheometer (Rheosense). MAb formulations were forced through a microchannel at a single shear rate and pressure was measured at 20° C.

Example 1: Stabilization of an Fc-Mutant mAb

An Fc engineered mAb (antibody A) with a relatively low CH2 domain unfolding temperature (Tm1), compared to traditional mAbs, was selected. Solutions of the mAb were prepared at 1 mg/mL in 10 or 20 mM histidine buffer at pH 5.5. The excipient solutions were prepared at 20 mM and 200 mM. Thermodynamic/conformational stability was evaluated by differential scanning calorimetry (DSC). Further, solutions of the mAb were prepared at 25 or 150 mg/mL concentration in the same buffer-excipient-pH solutions and kinetic or storage stability, as measured by rates of aggregation (kagg), was evaluated at 5° C., 25° C. and 40° C. Excipients were then assessed based on their effectiveness in increasing Tm1(ΔTm1) and/or decreasing kaggfor antibody A in solution. Based on the identity of the salt used, Tm1could be increased by as much as 7° C. with magnesium salts generally resulting in the most effect (FIG. 1). Arginine salts generally resulted in the least but still significant increase for antibody A. Of the anions, aspartates and glutamates were generally most effective in increasing the Tm1. For kagg(40° C., 150 mg/mL antibody A), a general decrease was observed with increasing ΔTm1(FIG. 2). Based on the identity of the salt, a roughly 35× decrease in the aggregation rate was noted compared to the control formulation. Magnesium salts generally resulted in the most decrease in kaggfollowed by arginine, lysine, lithium and sodium.

Example 2: Stabilization of Other Fc-Mutant mAb

Based on the results of Example 1, a subset of excipients was selected and their effectiveness as stabilizers was evaluated using another Fc-engineered mAb, with a relatively low Tm1(antibody B).

Solutions of antibody B (1 mg/mL) in 10 mM histidine buffer at pH 5.5 and 6.2 were prepared for conformational stability measurements. Solutions of antibody B (50 mg/mL) were prepared in the same buffer-excipient at pH 6.2 for kinetic stability (40° C.) analysis. Similar to the effect observed for antibody A, an increase in Tm1was observed for antibody B in a salt-dependent manner (FIG. 3). Magnesium salts generally exhibited the most increase. For anions, glutamates were observed to be most effective in increasing Tm1followed by aspartates and sulfates. Consistent with the effect on Tm1, a decrease in kaggwas also noted in the presence of salts (FIG. 4). Magnesium salts generally were most effective in increasing kagg.

Example 3: Stabilization of an Fc-Mutant mAb (Antibody A) as a Function of Excipient Concentration

Excipients were evaluated at higher concentrations, 500 mM and 750 mM, for their stabilizing effect on antibody A in solution. All excipients were shown to improve the conformational stability of antibody A by increasing Tm1to in a concentration dependent manner (FIG. 5). Magnesium salts generally exhibited the most increase in Tm1; ˜15° C. at 750 mM for magnesium glutamate. Furthermore, results from kinetic stability study (FIG. 6) showed that these same excipients at 500 mM further decreased aggregation and kaggcompared to 200 mM concentration used in Example 1.

Example 4: Stabilization of a Wild-Type mAb

The same excipients as those used in Example 2 were also evaluated for their stabilizing effect on a traditional mAb (antibody C), which has a much higher Tm1than the Fc-mutants used in our studies. Most excipients moderately improved the conformational stability with increasing concentration (200 mM, 500 mM, and 750 mM) with the exception of sulfates (FIG. 7). Aspartate and glutamate salts were generally better in stabilizing wild-type antibodies.

Example 5: Stabilization of Another Wild-Type mAb and its Single and Double Amino Acid Mutants

Aspartic and glutamic acid salts of magnesium, lithium and sodium at 200 mM concentration were evaluated for their stabilizing effect on wild-type anti-CEACAM5 antibody and its antibody D, antibody E and antibody DE mutants. All salts improved the conformational stability of the four antibodies studied with the effect being most prominent for the antibody DE mutant (FIG. 8). Of the salts studied, magnesium salts were most effective at increasing Tm1.

Example 6: Effects of Excipients on Viscosity and Osmolality of mAb Solutions

Effect of excipients at 200 mM concentration on viscosity of antibody solutions was also evaluated. Viscosity of antibody A and antibody C solutions (˜150 mg/mL) in presence and absence of aforementioned excipients was measured and compared with that in presence of traditional stabilizing excipients such as sucrose. Results indicated that the selected excipients either (i) significantly reduced solution viscosity compared to the control solutions (FIG. 9) and/or (ii) resulted in solutions of lower viscosity (FIG. 10) and lower osmolality (FIG. 11) in comparison to sucrose at equipotent concentrations. For antibody C, solution viscosity could be reduced by 10×, compared to control, with arginine, lysine and magnesium salts being most effective followed by sodium and lithium (FIG. 9). Antibody A solutions on the contrary were not too viscous to begin with (Control formulation inFIG. 10). Addition of sucrose to these solutions, for improving conformational stability resulted in a significant increase in solution viscosity especially at concentrations higher than 10%. Such an increase in solution viscosity was not observed with any of the salts studied. The effect of the two excipient classes on solution viscosity was most apparent for solutions exhibiting a ΔTm1of >3° C. Results indicate that the viscosity liability associated with the use of sucrose at concentrations that are not too far from those generally employed for stabilizing protein formulations (˜8%) is essentially absent for excipients listed in this invention. A near-identical effect was observed in the context of solution osmolality as well (FIG. 11).

Solutions containing 15% and 30% sucrose exhibited higher osmolality compared to 200 mM salt solutions without necessarily having a significantly higher stabilizing effect as measured by ΔTm1.

OTHER EMBODIMENTS

SEQUENCE INFORMATION

Amino acid sequences are provided, corresponding to heavy and light chains of the exemplary antibodies disclosed herein.