HK1225980B - Liquid protein formulations containing water soluble organic dyes - Google Patents

Description

Liquid protein formulations containing water-soluble organic dyes

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Application No. 62/030,521, filed July 29, 2014, entitled “Low-Viscosity Protein Formulations Containing Hydrophobic Salts,” U.S. Provisional Application No. 62/026,497, filed July 18, 2014, entitled “Low-Viscosity Protein Formulations Containing GRAS Viscosity-Reducing Agents,” U.S. Provisional Application No. 62/008,050, filed June 5, 2014, entitled “Low-Viscosity Protein Formulations Containing Ionic Liquids,” U.S. Provisional Application No. 61/988,005, filed May 2, 2014, entitled “Low-Viscosity Protein Formulations Containing Organophosphates,” U.S. Provisional Application No. 61/988,005, filed February 28, 2014, entitled “Concentrated, Low-Viscosity Infusible Protein Formulations Containing Ionic Liquids,” Provisional Application No. 61/946,436, filed on February 21, 2014, entitled “Concentrated, Low-Viscosity, High-Molecular-Weight-Protein Formulations,” U.S. Provisional Application No. 61/943,197, filed on February 21, 2014, entitled “Concentrated, Low-Viscosity, High-Molecular-Weight-Protein Formulations,” U.S. Provisional Application No. 61/940,227, filed on February 14, 2014, entitled “Concentrated, Low-Viscosity High-Molecular-Weight Protein Formulations,” and U.S. Provisional Application No. 61,876,621, filed on September 11, 2013, entitled “Concentrated, Low-Viscosity, High-Molecular-Weight Protein Formulations,” the disclosures of which are expressly incorporated herein by reference.

Technical Field

The present invention is generally in the field of low-viscosity pharmaceutical formulations for injection containing highly concentrated proteins and methods for their preparation and use.

Background Art

Monoclonal antibodies (mAbs) are important protein-based therapeutic agents used to treat a variety of human diseases such as cancer, infectious diseases, inflammation, and autoimmune diseases. More than 20 monoclonal antibody products have been approved by the U.S. Food and Drug Administration (FDA), and approximately 20% of all biopharmaceuticals currently being evaluated in clinical trials are monoclonal antibodies (Daugherty et al., Adv. Drug Deliv. Rev. 58: 686-706, 2006; and Buss et al., Curr. Opinion in Pharmacol. 12: 615-622, 2012).

The therapeutic agent based on monoclonal antibody is usually repeatedly administered in the extended period and needs several mg/kg dosages.Antibody solution or suspension can be administered by parenteral route, such as by intravenous (IV) infusion and subcutaneous (SC) or intramuscular (IM) injection.Compared with intravenous route, subcutaneous or intramuscular route reduces treatment cost during administration, improves patient compliance and improves the convenience of patient and medical service provider.In order to become effective and pharmaceutically acceptable, parenteral preparation should preferably be sterile, stable, injectable (such as via syringe) and non-irritating at injection site, thereby meet FDA guidelines.Because subcutaneous (usually less than about 2mL) and intramuscular (usually less than about 5mL) injection need small volume, these routes of administration for high-dose protein therapeutics need concentrated protein solution.These high concentrations usually result in very sticky preparations, which are difficult to be administered by injection, cause pain at injection site, are usually imprecise and/or may have reduced chemical and/or physical stability.

These characteristics lead to preparation, storage and use requirements that may be difficult to meet, particularly for preparations with high concentrations of high molecular weight proteins such as monoclonal antibodies. All protein therapeutics are subject to physical and chemical instabilities to a certain extent, such as aggregation, denaturation, cross-linking, deamidation, isomerization, oxidation and tailoring (Wang et al., J. Pharm. Sci. 96: 1-26, 2007). Therefore, the development of optimized formulations is crucial in the development of commercial protein drugs.

High protein concentrations cause challenges related to the physical and chemical stability of proteins and the production, storage and delivery difficulties of protein formulations. One problem is the tendency of proteins to aggregate and form particles during processing and/or storage, which makes further processing and/or delivery difficult. Concentration-dependent degradation and/or aggregation are major challenges in developing higher concentration protein formulations. In addition to the possibility of non-natural protein aggregation and particle formation, reversible self-association in aqueous solution can occur, which also promotes the viscosity increase that makes injection delivery complicated (see, for example, Steven J. Shire et al., J. Pharm. Sci. 93: 1390-1402, 2004). Viscosity increase is one of the major challenges encountered in concentrated protein compositions, which affects both the production process and the ability to easily deliver such compositions by conventional methods (see, for example, J. Jezek et al., Advanced Drug Delivery Reviews 63: 1107-1117, 2011).

Highly viscous liquid formulations are difficult to prepare, draw up into syringes, and inject subcutaneously or intramuscularly. The use of force in manipulating viscous formulations can lead to excessive foaming, which can further denature and inactivate the therapeutically active protein. Highly viscous solutions also require larger diameter needles for injection and cause more pain at the injection site.

Currently marketed monoclonal antibody products for administration by subcutaneous or intramuscular injection are typically formulated in aqueous buffers, such as phosphate or L-histidine buffers, and excipients or surfactants to prevent aggregation and improve stability. The aqueous buffers are such as mannitol, sucrose, lactose, trehalose, (a nonionic triblock copolymer consisting of a central hydrophobic chain, polyoxypropylene (poly(propylene oxide)), and two hydrophilic chains, polyoxyethylene (poly(ethylene oxide)), located on either side) or 80 (PEG (80) sorbitan monolaurate). The reported concentration of the antibody formulated as described above is typically as high as about 100 mg/mL (Wang et al., J. Pharm. Sci. 96: 1-26, 2007).

United States Patent (USP) 7,758,860 has described using buffer and the inorganic salt such as calcium chloride or magnesium chloride that viscosity is reduced in the preparation of low molecular weight protein to reduce viscosity.However, these same salts demonstrate very little effect on the viscosity of high molecular weight antibody (IMA-638) preparation.As described in United States Patent (USP) 7,666,413, the viscosity of the aqueous preparation of high molecular weight protein has been reduced by adding salts such as arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride or sodium acetate greater than about 100mM concentration or reduced by adding organic acid or inorganic acid as described in United States Patent (USP) 7,740,842.However, these salts do not make viscosity be reduced to required level and make preparation be enough acidity so that probably cause pain at injection site in some cases.

U.S. Patent 7,666,413 describes formulations with reduced viscosity containing specific salts and reconstituted anti-IgE monoclonal antibodies, but the maximum antibody concentration is only about 140 mg/mL at most. U.S. Patent 7,740,842 describes formulations of E25 anti-IgE monoclonal antibodies containing acetate/acetic acid buffer with antibody concentrations as high as 257 mg/mL. The addition of salts such as NaCl, _CaCl2 , or _MgCl2 has been shown to reduce dynamic viscosity under high shear conditions; however, such salts produce an undesirable and significant increase in dynamic viscosity under low shear conditions. In addition, inorganic salts such as NaCl can reduce solution viscosity and/or reduce aggregation (EP1981824).

Non-aqueous antibody or protein formulations have also been described. WO2006/071693 describes non-aqueous suspensions of up to 100 mg/mL of monoclonal antibodies in formulations with a viscosity enhancer (polyvinyl pyrrolidone, i.e., PVP) and a solvent (benzyl benzoate or PEG 400). WO2004/089335 describes a 100 mg/mL non-aqueous lysozyme suspension formulation containing PVP, glycofurol, benzyl benzoate, benzyl alcohol, or PEG 400. US2008/0226689A1 describes a 100 mg/mL single-phase, three-vehicle component (polymer, surfactant, and solvent) non-aqueous viscous formulation of human growth hormone (hGH). U.S. Patent No. 6,730,328 describes non-aqueous, hydrophobic, non-polar, low-reactivity vehicles such as perfluorodecalin for protein formulations. These formulations are suboptimal and have high viscosities that hinder processing, preparation, and injection; result in the presence of multiple vehicle components in the formulation; and present potential regulatory challenges associated with the use of polymers that are not yet approved by the FDA.

Alternative non-aqueous protein or antibody formulations using organic solvents have been described, such as benzyl benzoate (Miller et al., Langmuir 26: 1067-1074, 2010), benzyl acetate, ethanol or methyl ethyl ketone (Srinivasan et al., Pharm. Res. 30: 1749-1757, 2013). In both cases, a viscosity of less than 50 centipoise (cP) is achieved when the configured protein concentration is at least about 200 mg/mL. U.S. Patent No. 6,252,055 describes monoclonal antibody formulations with a concentration range of 100 mg/mL to as high as 257 mg/mL. Formulations with a concentration greater than about 189 mg/mL show significantly increased viscosity, low recovery and processing difficulty. U.S. Patent Application Publication No. 2012/0230982 describes antibody formulations with a concentration of 100 mg/mL to 200 mg/mL. None of these formulations have a low viscosity sufficient to make injection easy.

Du and Klibanov (Biotechnology and Bioengineering 108:632-636, 2011) described the reduction in viscosity of concentrated aqueous solutions of bovine serum albumin at a maximum concentration of up to 400 mg/mL and bovine gamma globulin at a maximum concentration of up to 300 mg/mL. Guo et al. (Pharmaceutical Research 29:3102-3109, 2012) described the use of hydrophobic salts to achieve low-viscosity aqueous solutions of four model monoclonal antibodies. The monoclonal antibody formulation used by Guo had an initial viscosity of no more than 73 cP before the addition of salt. In addition, the viscosity of several pharmaceutically important monoclonal antibodies can exceed 1,000 cP at therapeutically relevant concentrations.

Controlling aggregation and viscosity in highly concentrated mAb solutions is not trivial (EP2538973). This is evidenced by the few mAb products currently on the market in the form of highly concentrated formulations (>100 mg/mL) (EP2538973).

The references cited above indicate that although multiple groups have attempted to prepare low-viscosity formulations of monoclonal antibodies and other therapeutically important proteins, truly useful formulations have not yet been achieved for many proteins. It is worth noting that many of the above reports use substances whose safety and toxicity profiles have not yet been fully established. Therefore, these formulations face a higher regulatory burden before approval than formulations containing compounds known to be safe. In fact, even if a compound is shown to significantly reduce viscosity, it may ultimately be unsuitable for formulations intended for injection into humans.

Due to the problems caused by the high viscosity and other properties of concentrated solutions of large proteins, many pharmaceutically important high molecular weight proteins, such as monoclonal antibodies, are currently administered via intravenous infusion to deliver therapeutically effective amounts of the protein. For example, to provide therapeutically effective amounts of many high molecular weight proteins, such as monoclonal antibodies, in a volume of less than about 2 mL, a protein concentration of greater than 150 mg/mL is generally required.

It is therefore an object of the present invention to provide concentrated, low viscosity liquid formulations of pharmaceutically important proteins, particularly high molecular weight proteins such as monoclonal antibodies.

Another object of the present invention is to provide concentrated, low-viscosity liquid formulations of proteins, particularly high molecular weight proteins such as monoclonal antibodies, which are capable of delivering therapeutically effective amounts of these proteins in volumes useful for subcutaneous and intramuscular injection.

Another object of the present invention is to provide concentrated low viscosity liquid formulations of proteins, especially high molecular weight proteins such as monoclonal antibodies, with low viscosity that can improve injectability and/or patient compliance, convenience and comfort.

It is also an object of the present invention to provide a method for preparing and storing concentrated, low-viscosity formulations of proteins, particularly high molecular weight proteins such as monoclonal antibodies.

Another object of the present invention is to provide a method for administering a concentrated, low-viscosity liquid formulation of a protein, particularly a high molecular weight protein such as a monoclonal antibody. Another object of the present invention is to provide a method for processing a high-concentration biological formulation having reduced viscosity using concentration and filtration techniques known to those skilled in the art.

Summary of the Invention

Concentrated, low-viscosity, low-volume liquid pharmaceutical formulations of proteins have been developed. Such formulations can be quickly and conveniently administered by subcutaneous (SC) or intramuscular (IM) injection rather than by prolonged intravenous infusion. These formulations contain low-molecular-weight and/or high-molecular-weight proteins such as monoclonal antibodies and water-soluble organic dyes that reduce viscosity.

The protein concentration is from about 10 mg/mL to about 5,000 mg/mL, more preferably from about 100 mg/mL to about 2,000 mg/mL. In some embodiments, the protein concentration is from about 100 mg/mL to about 500 mg/mL, more preferably from about 300 mg/mL to about 500 mg/mL. The formulation containing the protein and the viscosity-reducing water-soluble organic dye is stable for at least one month, preferably at least two months, and most preferably at least three months when stored at 4°C. The viscosity of the formulation is less than about 75 cP at about 25°C, preferably less than 50 cP, and most preferably less than 20 cP. In some embodiments, the viscosity is less than about 15 cP, or even less than or about 10 cP at about 25°C. In some embodiments, the viscosity of the formulation is about 10 cP. The formulation containing the protein and the viscosity-reducing water-soluble dye is typically measured at a shear rate of from about 0.6 s ⁻¹ to about 450 s ⁻¹ , and preferably from about 2 s ⁻¹ to about 400 s ⁻¹ , when measured using a cone-plate viscometer. Formulations containing a protein and a viscosity-lowering water-soluble dye are typically measured at shear rates of about 3 s ^"1 to about 55,000 s ^"1 and preferably about 20 s ^"1 to about 2,000 s ^"1 when measured using a microfluidic viscometer.

The viscosity of the protein formulation is reduced due to the presence of one or more water-soluble dyes that reduce the viscosity. Unless otherwise expressly stated, the term "water-soluble dye that reduces the viscosity" includes a mixture of a single compound and two or more compounds. Preferably, the concentration of the water-soluble dye that reduces the viscosity present in the formulation is less than about 1.0M, preferably less than about 0.50M, more preferably less than about 0.30M and most preferably less than about 0.15M. In some embodiments, the concentration of the water-soluble dye that reduces the viscosity present in the formulation is as low as 0.01M. The viscosity that the formulation has is lower than the viscosity of the corresponding formulation under the same conditions except that the appropriate buffer or salt of about the same concentration replaces the water-soluble dye that reduces the viscosity by at least about 30%, preferably lower by at least about 50%, most preferably lower by at least about 75%. Low-viscosity formulations are provided in some embodiments, wherein the viscosity of the corresponding formulation not containing the water-soluble dye that reduces the viscosity is greater than about 200cP, greater than about 500cP or even higher than about 1,000cP. In preferred embodiments, the formulation has a shear rate of at least about 0.5 s ^"1 when measured using a cone and plate viscometer or at least about 1.0 s ^"1 when measured using a microfluidic viscometer.

For embodiments in which the protein is a "high molecular weight protein," the high molecular weight protein may have a molecular weight of about 100 kDa to about 1,000 kDa, preferably about 120 kDa to about 500 kDa, and most preferably about 120 kDa to about 250 kDa. The high molecular weight protein may be an antibody such as a monoclonal antibody or a PEGylated or other derivative thereof. Preferred monoclonal antibodies include natalizumab, cetuximab, bevacizumab, trastuzumab, infliximab, rituximab, panitumumab, ofatumumab, and biosimilars thereof. The optionally PEGylated high molecular weight protein may be an enzyme. Other proteins and mixtures of proteins may also be formulated to reduce their viscosity.

In some embodiments, the protein and the viscosity-reducing water-soluble dye are provided in a lyophilized dosage unit that is sized to be reconstituted with a sterile aqueous pharmaceutical vehicle to produce a concentrated, low-viscosity liquid formulation. The presence of one or more viscosity-reducing water-soluble dyes facilitates and/or accelerates reconstitution of the lyophilized dosage unit compared to a lyophilized dosage unit that does not contain the viscosity-reducing water-soluble dye.

Provided herein are methods for preparing concentrated, low-viscosity liquid formulations of high molecular weight proteins, such as monoclonal antibodies, and methods for storing low-viscosity, high-concentration protein formulations and administering them to patients. In another embodiment, a viscosity-lowering water-soluble dye is added to aid processing (e.g., pumping, concentration, and/or filtration) by reducing the viscosity of the protein solution.

DETAILED DESCRIPTION

I. Definition

The term "protein" as generally used herein refers to a polymer of amino acids that are linked to each other by peptide bonds to form a polypeptide whose chain length is sufficient to produce a detectable at least tertiary structure. Proteins with a molecular weight (expressed in kDa, where "Da" stands for "Dalton" and 1kDa=1,000Da) greater than about 100kDa can be designated as "high molecular weight proteins", while proteins with a molecular weight of less than about 100kDa can be designated as "low molecular weight proteins". The term "low molecular weight protein" does not include small peptides that lack the necessary conditions required to be considered as proteins, i.e., at least tertiary structure. Protein molecular weight can be determined using standard methods known to those skilled in the art, including but not limited to mass spectrometry (e.g., ESI, MALDI) or calculated from known amino acid sequences and glycosylation. Proteins may be naturally occurring or non-naturally occurring, synthetic or semi-synthetic.

"Substantially pure protein" and "essentially pure protein" are used interchangeably herein and refer to a composition comprising at least about 90% pure protein by weight, preferably at least about 95% pure protein by weight. "Substantially homogeneous" and "essentially homogeneous" are used interchangeably herein and refer to a composition in which at least about 90% by weight, preferably at least about 95% by weight, of the protein present is a combination of monomers and reversible dimers and oligomers (not irreversible aggregates).

The term "antibody" as generally used herein broadly encompasses monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies and single-chain antibody molecules and antibody fragments (e.g., Fab, Fab', F(ab')2 and Fv), single domain antibodies, multivalent single domain antibodies, Fab fusion proteins and fusions thereof.

The terms "monoclonal antibody" or "mAb" as generally used herein refer to antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single epitope. For example, these antibodies are typically synthesized by culturing hybridoma cells as described by Kohler et al. (Nature 256:495, 1975) or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567) or isolated from phage antibody libraries using techniques described by Clackson et al. (Nature 352:624-628, 1991) and Marks et al. (J. Mol. Biol. 222:581-597, 1991). As used herein, "monoclonal antibody" specifically includes derivatized antibodies, antibody-drug conjugates, and "chimeric" antibodies (in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass), and fragments of these antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984).

"Antibody fragments" comprise a portion of an intact antibody, including the antigen-binding and/or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870; Zapata et al., Protein Eng. 8:1057-1062, 1995); single-chain antibody molecules; multivalent single-domain antibodies; and multispecific antibodies formed from antibody fragments.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequences of antibodies) that are largely human in sequence, with minimal sequence derived from non-human immunoglobulin (see, e.g., Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992).

"Rheology" refers to the study of the deformation and flow of matter.

"Viscosity" refers to the resistance of a substance (usually a liquid) to flow. Viscosity is related to the concept of shear force; it can be understood as the effect of different layers of a fluid exerting shear forces on each other or other surfaces as they move relative to each other. There are several ways to measure viscosity. The unit of viscosity is Ns/ ^m2 , called Pascal-seconds (Pa-s). Viscosity can be "dynamic" or "absolute." Dynamic viscosity is a measure of the rate at which momentum is transferred through a fluid. It is measured in Störgs (St). Dynamic viscosity is a measure of the resistance of a fluid to flow under the influence of gravity. When two fluids of equal volume but different viscosities are placed in the same capillary viscometer and allowed to flow by gravity, the more viscous fluid takes longer to flow through the capillary than the less viscous fluid. For example, if one fluid takes 200 seconds (s) to complete its flow and the other takes 400s, the viscosity of the second fluid is said to be twice that of the first fluid on the dynamic viscosity scale. The dimension of dynamic viscosity is ^length² /time. Typically, dynamic viscosity is expressed in centistosters (cSt). The SI unit for dynamic viscosity is ^mm² /s, which is equal to 1 cSt. "Absolute viscosity" (sometimes called "dynamic viscosity" or "simple viscosity") is the product of dynamic viscosity and the density of the fluid. Absolute viscosity is expressed in centipoise (cP). The SI unit for absolute viscosity is milliPascal-second (mPa-s), where 1 cP = 1 mPa-s.

Viscosity can be measured at a given shear rate or a plurality of shear rates by using, for example, a viscometer." extrapolating zero shear " viscosity can be determined as follows: create the best fit line of four highest shear points on the curve of absolute viscosity to shear rate and linear extrapolation viscosity back to zero shear. Alternatively, for Newtonian fluids, viscosity can be determined by the average viscosity value at a plurality of shear rates. Viscosity also can be measured using a microfluidic viscometer at a single or multiple shear rates (also referred to as flow velocity), wherein absolute viscosity derives from the change in pressure when liquid flows through the channel. Viscosity equals shear stress to shear rate. In some embodiments, the viscosity measured with the microfluidic viscometer can directly be compared with those viscosities of extrapolating zero shear viscosity, for example, by using a cone-plate viscometer to extrapolate the viscosity of a plurality of shear rate measurements.

"Shear rate" refers to the rate of change in viscosity as one layer of fluid passes over an adjacent layer. The velocity gradient is the rate of change of velocity with distance from the plate. This simple case shows a uniform velocity gradient with units of (cm/second)/(cm) for shear rate (v ₁ -v ₂ )/h = 1/second. Therefore, the units of shear rate are reciprocal seconds or, more commonly, reciprocal time. For microfluidic viscometers, changes in pressure and flow rate are related to shear rate. "Shear rate" refers to the rate at which a material is deformed. When measured using a cone-plate viscometer and a shuttle rod appropriately selected by one skilled in the art, formulations containing proteins and water-soluble dyes that reduce viscosity are typically measured at shear rates of about 0.5 s ⁻¹ to about 200 s ⁻¹ to accurately measure viscosity within the viscosity range of the sample of interest (i.e., a 20 cP sample is most accurately measured using a CPE 40 shuttle rod mounted on a DV2T viscometer (Brookfield)); when measured using a microfluidic viscometer, shear rates are greater than about 20 s ⁻¹ to about 3,000 s ⁻¹ .

For the classical "Newtonian" fluids generally used herein, viscosity is essentially independent of shear rate. However, for "non-Newtonian fluids," viscosity decreases or increases with increasing shear rate, e.g., the fluid is "shear-thinning" or "shear-thickening," respectively. In the case of concentrated (i.e., high-concentration) protein solutions, this can manifest as pseudoplastic shear-thinning behavior, i.e., viscosity decreases with shear rate.

As used herein, the term "chemical stability" refers to the ability of a protein component in a formulation to resist degradation by chemical pathways such as oxidation, deamidation, or hydrolysis. A protein formulation is generally considered chemically stable if less than about 5% of the components degrade after storage at 4°C for 24 months.

The term "physical stability" as generally used herein refers to the ability of a protein formulation to resist physical deterioration such as polymerization. Physically stable formulations form only an acceptable percentage of irreversible aggregates (e.g., dimers, trimers, or other aggregates) of the biologically active protein. The presence of aggregates can be evaluated in a variety of ways, including by measuring the average particle size of the protein in the formulation by dynamic light scattering. A formulation is considered to be physically stable if the irreversible aggregates formed are less than about 5% after being stored at 4°C for 24 months. The acceptable level of aggregated impurities should ideally be less than about 2%. Although levels as low as about 0.2% are achievable, about 1% is more typical.

As generally used herein, the term "stable formulation" refers to a formulation that is both chemically and physically stable. A stable formulation can be one in which more than about 95% of the biologically active protein molecules in the formulation retain biological activity after storage for 24 months at 4°C or under high temperature equivalent processing conditions, such as after storage for 1 month at 40°C. Various analytical techniques for measuring protein stability are available in the art and are described, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, Ed., Marcel Dekker, Inc., New York, N.Y. (1991) and Jones, A., Adv. Drug Delivery Revs. 10:29-90, 1993. Stability can be measured at a selected temperature for a certain period of time. For example, for rapid screening, the formulation can be stored at 40°C for 2 weeks to 1 month, and then the remaining biological activity can be measured and compared to the initial conditions to evaluate stability. When the formulation is stored at 2°C-8°C, the formulation should generally be stable at 30°C or 40°C for at least 1 month and/or at 2°C-8°C for at least 2 years. When the formulation is stored at room temperature, i.e., about 25°C, the formulation should generally be stable at about 25°C for at least 2 years and/or at 40°C for at least about 6 months. The degree of aggregation after lyophilization and storage can be used as an indicator of protein stability. In some embodiments, stability is evaluated by measuring the particle size of the protein in the formulation. In some embodiments, stability can be evaluated as follows: the activity of the formulation is measured using standard biological activity or binding assays known to those skilled in the art.

The term "protein particle size" as generally used herein refers to the average diameter of the major population of particles of the biologically active molecule in a formulation, or its size distribution, when measured by using a known particle size analyzer such as dynamic light scattering, SEC (size exclusion chromatography), or other methods known to those skilled in the art.

As used generally herein, the terms "concentrated" or "high concentration" describe liquid formulations having a final concentration of protein greater than about 10 mg/mL, preferably greater than about 50 mg/mL, more preferably greater than about 100 mg/mL, even more preferably greater than about 200 mg/mL, or most preferably greater than about 250 mg/mL.

As used generally herein, a "reconstituted formulation" refers to a formulation prepared by dissolving a dry powder, ie, lyophilized, spray-dried, or solvent-precipitated protein, in a diluent to dissolve or disperse the protein in an aqueous solution for administration.

"Lyophilization protectants" are substances that, when combined with proteins, significantly reduce the chemical and/or physical instability of proteins during freeze-drying and/or subsequent storage. Exemplary lyophilization protectants include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; readily soluble salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol) or poly(propylene glycol); and combinations thereof. Other exemplary lyophilization protectants include gelatin, dextrin, modified starch, and carboxymethyl cellulose. Preferred sugar alcohols are those compounds obtained by reducing monosaccharides and disaccharides such as lactose, trehalose, maltose, lactulose, and maltulose. Other examples of sugar alcohols are glucitol, maltitol, lactitol, and isomaltulose. Lyophilization protectants are typically added to pre-lyophilized formulations in a "lyophilization protective amount." This means that after the protein is lyophilized in the presence of a lyoprotectant amount of a lyoprotectant, the protein substantially retains its physical and chemical stability and integrity.

As generally used herein, a "diluent" or "carrier" is a component that is pharmaceutically acceptable (i.e., safe and non-toxic for administration to humans or other mammals) and can be used to prepare a liquid formulation (such as an aqueous formulation that is reconstituted after lyophilization). Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution, and combinations thereof.

"Preservatives" are compounds that can be added to the formulations of the present invention to reduce contamination and/or effects caused by bacteria, fungi, or other infectious agents. For example, the addition of preservatives can aid in the production of multi-use (multi-dose) formulations. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long chain), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl alcohol, and benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol.

As generally used herein, "bulking agents" are compounds that increase the mass of the lyophilized mixture and contribute to the physical structure of the lyophilized cake (e.g., help produce a substantially uniform lyophilized cake that maintains an open-pore structure). Exemplary bulking agents include mannitol, glycine, lactose, modified starches, poly(ethylene glycol), and sorbitol.

A "therapeutically effective amount" is the minimum concentration required to achieve a measurable improvement or prevention of any symptom or specific condition or disorder, to achieve a measurable extension of life expectancy, or to generally improve the patient's quality of life. The therapeutically effective amount depends on the specific biologically active molecule and the specific condition or disorder to be treated. Therapeutically effective amounts of various proteins, such as the monoclonal antibodies described herein, are known in the art. Therapeutically effective amounts of undetermined proteins or known proteins, such as monoclonal antibodies, for treating specific disorders and clinically for treating other disorders can be determined by standard techniques known to those skilled in the art, such as physicians.

The terms "injectability" or "needleability" as commonly used herein refer to the ability of a pharmaceutical formulation to pass through a syringe equipped with an optional thin-walled 18-32 gauge needle. Injectability depends on a variety of factors, such as the pressure or force required for injection, the uniformity of flow, the amount of suction, and the avoidance of clogging. The injectability of a liquid pharmaceutical formulation can be evaluated by comparing the injection force of a viscosity-reducing formulation with the injection force of a standard formulation that does not add a water-soluble dye that reduces viscosity. The reduction in injection force of a formulation containing a water-soluble dye that reduces viscosity reflects the improved injectability of the formulation. When compared with a standard formulation with the same protein concentration under the same conditions, except that the water-soluble dye that reduces viscosity is replaced with an appropriate buffer of approximately the same concentration, the viscosity-reducing formulation has improved injectability, the injection force is reduced by at least 10%, preferably at least 30%, more preferably at least 50%, and most preferably at least 75%. Alternatively, the injectability of a liquid pharmaceutical formulation can be evaluated by comparing the time required to inject the same volume, such as 0.5 mL or more preferably about 1 mL, of different liquid protein formulations when pressing the syringe with the same force.

The term "injection force" generally used herein refers to the force required for a given syringe of a needle of a given size to push a given liquid preparation at a given injection speed. Injection force is reported in Newtons usually. For example, injection force can be measured as the force required for a 1mL plastic syringe equipped with a 0.50 inch 27 gauge needle of 0.25 inch in internal diameter to push a liquid preparation at an injection speed of 250mm/min. Test equipment can be used to measure injection force. When measured under the same conditions, preparations with lower viscosity will generally require an overall lower injection force.

" Viscosity gradient " used herein refers to the rate of change of the viscosity of a protein solution when protein concentration increases. The viscosity gradient can be approximated by the following graph, which is a function of viscosity to the protein concentration of a series of preparations that are otherwise identical but different in protein concentration. As protein concentration increases, viscosity increases in an approximate exponential manner. The viscosity gradient during a specific protein concentration can be approximated by the slope of the tangent of the following graph, which is a function of viscosity to protein concentration. The viscosity gradient can be approximated by the linear approximation of the following graph, which is a function of viscosity to any protein concentration or a graph at a narrow window of protein concentration. In some embodiments, when the function of viscosity to protein concentration is approximated as an exponential function, if the exponent of the exponential function is less than the index obtained with respect to the preparation that is otherwise identical but does not contain the water-soluble dye that reduces viscosity, then it is believed that the preparation has a reduced viscosity gradient. In a similar manner, when compared with the second preparation, if the index of the preparation is lower than/higher than the index of the second preparation, then it is believed that the preparation has a lower/higher viscosity gradient. The viscosity gradient can be numerically approximated by the graph of viscosity to the function of protein concentration using other methods known to skilled formulation researchers.

As generally used herein, the term "reduced viscosity formulation" refers to a liquid formulation having a high concentration of high molecular weight proteins, such as monoclonal antibodies, or low molecular weight proteins, which is modified by the presence of one or more viscosity-lowering additives compared to a corresponding formulation without the one or more viscosity-lowering additives.

The term "osmolarity" as generally used herein refers to the total number of dissolved components per liter. Osmolarity is similar to molar concentration, but includes the total number of moles of various substances dissolved in a solution. An osmolarity of 1 Osm/L means that there is 1 mole of dissolved components per liter of solution. Some solutes, such as ionic solutes that dissociate in solution, will contribute more than 1 mole of dissolved components per mole of solute in solution. For example, NaCl dissociates into Na ⁺ and ^Cl- in solution and thus provides 2 moles of dissolved components per 1 mole of dissolved NaCl in solution. Physiological osmolarity is typically about 280 mOsm/L to about 310 mOsm/L.

As used generally herein, the term "tonicity" refers to the osmotic pressure gradient caused by a semipermeable membrane separating two solutions. Specifically, tonicity describes the osmotic pressure generated across a cell membrane when a cell is exposed to an external solution. Solutes that can pass through the cell membrane do not contribute to the resulting osmotic pressure gradient. Only dissolved substances that cannot pass through the cell membrane contribute to the osmotic pressure difference and, therefore, tonicity.

As used herein, the term "hypertonic" refers to a solution having a higher concentration of solutes than that present inside cells. When cells are immersed in a hypertonic solution, the tendency is for water to flow out of the cell to balance the concentration of solutes.

As used herein, the term "hypotonic" refers to a solution having a solute concentration that is lower than that present inside a cell. When a cell is immersed in a hypotonic solution, water flows into the cell to balance the concentration of the solutes.

As used generally herein, the term "isotonic" refers to a solution in which the osmotic pressure gradient across a cell membrane is substantially balanced. An isotonic formulation is one that has an osmotic pressure substantially the same as human blood. An isotonic formulation will typically have an osmotic pressure of approximately 250 mOsm/kg to 350 mOsm/kg.

As used herein, the term "liquid formulation" is a protein that is provided in an acceptable pharmaceutical diluent or that is reconstituted in an acceptable pharmaceutical diluent prior to administration to a patient.

The terms "brand" and "reference" when used to refer to a protein or a biological product are used interchangeably herein and refer to a single biological product licensed under section 351(a) of the United States Public Health Service Act (42 U.S.C. §262).

The term "biosimilar" as used herein is usually used interchangeably with "general equivalent" or "second-generation product". For example, a "biosimilar monoclonal antibody" refers to a subsequent variant of an innovator's monoclonal antibody that is usually produced by another company. "Biosimilar" when used to refer to a brand protein or a brand biological product can refer to a biological product that is evaluated relative to a brand protein or a brand biological product and is licensed according to Article 351 (k) of the U.S. Public Health Service Act (42 U.S.C. § 262). Biosimilar monoclonal antibodies can be biosimilar monoclonal antibodies that meet one or more guidelines (reference EMA/CHMP/BMWP/403543/2010) formally adopted by the European Medicines Agency's Committee for Medicinal Products for Human Use (CHMP) on May 30, 2012 and disclosed by the European Union as "Guideline on similar biological medicinal products containing monoclonal antibodies-non-clinical and clinical issues".

Biosimilars can be produced by microbial cells (prokaryotic, eukaryotic), cell lines of human or animal origin (e.g., mammalian, avian, insect), or tissues derived from animals or plants. The expression construct of the proposed biosimilar product will generally encode the same primary amino acid sequence as its reference product. Minor modifications such as N- or C-terminal truncations may exist that do not affect safety, purity, or potency.

Biosimilar monoclonal antibodies are physicochemically or biologically similar to the reference monoclonal antibody in terms of safety and efficacy. Biosimilar monoclonal antibodies can be evaluated relative to the reference monoclonal antibody using one or more in vitro studies, including assays detailing: binding to one or more target antigens; binding to isoforms of Fcγ receptors (FcγRI, FcγRII, and FcγRIII), FcRn, and complement (C1q); Fab-related functions (e.g., neutralization of soluble ligands, receptor activation, or blocking); or Fc-related functions (e.g., antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, complement activation). In vitro comparisons can be combined with in vivo data that demonstrate similarity in pharmacokinetics, pharmacodynamics, and/or safety. Clinical evaluation of biosimilar monoclonal antibodies relative to reference monoclonal antibodies may include comparison of pharmacokinetic properties (e.g., AUC _0-inf , AUC _0-t , C _max , t _max , C _trough ); pharmacodynamic endpoints; or similarity of clinical effects (e.g., using randomized parallel group comparative clinical trials). The quality comparison between biosimilar monoclonal antibodies and reference monoclonal antibodies can be evaluated using established procedures, including those described in the "Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substances: Quality issues" (EMEA/CHMP/BWP/49348/2005) and the "Guideline on development, production, characterization and specifications for monoclonal antibodies and related substances" (EMEA/CHMP/BWP/157653/2007).

The differences between a biosimilar monoclonal antibody and a reference monoclonal antibody may include post-translational modifications, for example, by attaching other biochemical groups such as phosphates, various lipids, and carbohydrates to the monoclonal antibody; by proteolytic cleavage after translation; by changing the chemical properties of amino acids (e.g., formylation); or by a variety of other mechanisms. Other post-translational modifications may be the result of manufacturing process manipulations, for example, glycosylation may occur when the product is exposed to reducing sugars. In other cases, storage conditions may allow some degradation pathways such as oxidation, deamidation, or aggregation. All of these product-related variants may be included in a biosimilar monoclonal antibody.

As used herein, the term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases. Suitable non-toxic acids include inorganic and organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like. Suitable positively charged counterions include sodium, potassium, lithium, calcium, and magnesium.

As used herein, the term "ionic liquid" refers to a crystalline or amorphous salt, zwitterion, or mixture thereof that is liquid at or near the temperature at which most conventional salts are solid, which is less than 200° C., preferably less than 100° C., or more preferably less than 80° C. Some ionic liquids have melting temperatures around room temperature, e.g., 10° C. to 40° C. or 15° C. to 35° C. The term "zwitterion" is used herein to describe an overall neutrally charged molecule that carries both formal positive and formal negative charges on different chemical groups of the molecule. Examples of ionic liquids are found in Riduan et al., Chem. Soc. Rev., 42:9055-9070, 2013; Rantwijk et al., Chem. Rev., 107:2757-2785, 2007; Earle et al., Pure Appl. Chem., 72(7):1391-1398, 2000; and Sheldon et al., Green Chem., 4:147-151, 2002.

As used herein, the term "organophosphate" refers to a compound containing one or more phosphoryl groups, at least one of which is covalently attached to an organic group through a phosphate linkage.

As used herein, "water-soluble organic dye" is used interchangeably with "water-soluble dye" and is an organic molecule that has a molar solubility of at least 0.001 M at 25°C and pH 7 and absorbs some wavelengths of light, preferably in the visible to infrared portion of the electromagnetic spectrum, while transmitting or reflecting other wavelengths of light.

As used herein, the term "chalcogen" refers to Group 16 elements, including oxygen, sulfur, and selenium in any oxidation state. For example, unless otherwise specified, the term "chalcogen" also includes _SO2 .

As used herein, the term "alkyl" refers to straight-chain, branched-chain, and cyclic hydrocarbon groups. Unless otherwise indicated, the term "alkyl" includes hydrocarbon groups containing one or more double or triple bonds. An alkyl group containing at least one ring system is a "cycloalkyl group." An alkyl group containing at least one double bond is an "alkenyl group," and an alkyl group containing at least one triple bond is an "alkynyl group."

As used herein, the term "aryl" refers to an aromatic carbocyclic ring system, including fused ring systems. In an "aryl" group, each atom forming the ring is a carbon atom.

As used herein, the term "heteroaryl" refers to aromatic ring systems, including fused ring systems, in which at least one atom forming the ring is a heteroatom.

As used herein, the term "heterocycle" refers to non-aromatic ring systems, including fused ring systems, in which at least one atom forming the ring is a heteroatom.

As used herein, the term "heteroatom" is any non-carbon or non-hydrogen atom. Preferred heteroatoms include oxygen, sulfur, and nitrogen. Exemplary heteroaryl and heterocyclic rings include benzimidazolyl, benzofuranyl, benzothiophenyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuranyl, furanyl, Furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, 3H-indolyl, dihydroindolinyl, indolizinyl, indolyl, 3H-indolyl, isatoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthazinyl, decahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenothioxazinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyrazoloxazolyl, pyrazoloimidazolyl, pyrazolothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolidinyl, pyrazolothiazolyl, pyridinyl, pyrimidin ...olidinyl, pyrazolidinyl, pyrazolidinyl, pyrazolidinyl, pyrazolidinyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrazolidinyl, pyridinyl, pyrimidinyl, pyridinyl, pyridinyl, pyrimidinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, py pyrrolyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thienyl, and xanthenyl.

II. Preparation

Biocompatible low-viscosity protein solutions, such as those of monoclonal antibodies, can be used to deliver therapeutically effective amounts of proteins in volumes suitable for subcutaneous (SC) and intramuscular (IM) injection, typically less than or about 2 mL for SC and less than or about 5 mL for IM, more preferably less than or about 1 mL for SC and less than or about 3 mL for IM. Proteins can generally have any molecular weight, although in some embodiments, high molecular weight proteins are preferred. In other embodiments, the protein is a low molecular weight protein.

The formulations may have a protein concentration of about 10 mg/mL to about 5,000 mg/mL. Formulations, including monoclonal antibody formulations, may have a protein concentration greater than 100 mg/mL, preferably greater than 150 mg/mL, more preferably greater than about 175 mg/mL, more preferably greater than about 200 mg/mL, more preferably greater than about 225 mg/mL, more preferably greater than about 250 mg/mL, and most preferably greater than or about 300 mg/mL. In the absence of a water-soluble dye that reduces viscosity, the viscosity of the protein formulation increases exponentially with increasing concentration. Such protein formulations, in the absence of a water-soluble dye that reduces viscosity, may have a viscosity greater than 100 cP, greater than 150 cP, greater than 200 cP, greater than 300 cP, greater than 500 cP, or even greater than 1,000 cP when measured at 25°C. Such formulations are generally not suitable for subcutaneous or intramuscular injection. The use of one or more viscosity-lowering water-soluble dyes allows for the preparation of formulations having a viscosity of less than or about 100 cP, preferably less than or about 75 cP, more preferably less than or about 50 cP, more preferably less than or about 30 cP, more preferably less than or about 20 cP, or most preferably less than or about 10 cP when measured at 25°C.

Although water-soluble dyes that reduce viscosity can be used to reduce the viscosity of concentrated protein formulations, they can also be used for formulations with less concentration. In some embodiments, the protein concentration of the formulation can be from about 10 mg/mL to about 100 mg/mL. The protein concentration of the formulation can be greater than about 20 mg/mL, greater than about 40 mg/mL, or greater than about 80 mg/mL.

For some proteins, formulations without a viscosity-lowering water-soluble dye may have a viscosity greater than about 20 cP, greater than about 50 cP, or greater than about 80 cP. The use of one or more viscosity-lowering water-soluble dyes allows for the preparation of formulations having a viscosity less than or about 80 cP, preferably less than or about 50 cP, more preferably less than about 20 cP, or most preferably less than or about 10 cP when measured at 25°C.

In some embodiments, when measuring under the same conditions, the viscosity ratio of the aqueous protein formulation does not contain similar formulations of one or more water-soluble dyes that reduce viscosity and is at least about 30% low. In other embodiments, the viscosity ratio of the preparation does not contain similar formulations of one or more water-soluble dyes that reduce viscosity and is 40% low, 50% low, 60% low, 70% low, 80% low, 90% or even more than 90% low. In preferred embodiments, the preparation contains one or more high molecular weight proteins such as monoclonal antibodies less than about 2mL, preferably less than about 1mL or more preferably less than about 0.75mL of a volume for the treatment of significant dose.

Compared with similar formulations that are otherwise identical but do not contain water-soluble dyes that reduce viscosity (e.g., in phosphate buffered saline), the viscosity-reducing formulation has improved injectability and requires less injection force. In some embodiments, compared with standard formulations that are otherwise identical but do not contain one or more water-soluble dyes that reduce viscosity, the injection force reduces by more than about 20%, more than about 30%, more than about 40%, more than about 50% or more than about 2 times. In some embodiments, the formulation has "Newtonian flow characteristics," which is defined as having a viscosity that is substantially unrelated to shear rate. The protein formulation can be easily injected through a needle of about 18-32 in size. Preferred needle sizes for delivering the low-viscosity formulation include 27, 29, and 31, optionally thin-walled.

The formulation may contain one or more other excipients such as buffers, surfactants, sugars and sugar alcohols, other polyols, preservatives, antioxidants, and chelating agents. The formulation has a pH and osmolarity that are suitable for administration but does not cause significant adverse side effects. In some embodiments, the concentrated low-viscosity formulation has a pH of 5 to 8, 5.5 to 7.6, 6.0 to 7.6, 6.8 to 7.6, or 5.5 to 6.5.

Low viscosity protein formulations can allow greater flexibility in formulation development. Compared with formulations that are otherwise identical but do not contain water-soluble dyes that reduce viscosity, low viscosity formulations can exhibit less viscosity variations that depend on protein concentration. Low viscosity protein formulations can allow increasing the concentration of protein and reducing the dosing frequency of protein. In some embodiments, low viscosity protein formulations contain 2 or more, 3 or more, or 4 or more different proteins. For example, a combination of 2 or more monoclonal antibodies can be provided in a single low viscosity protein formulation.

Because can be by the protein concentration that is similar but does not contain the protein preparation that makes viscosity reduce high protein concentration to patient administration protein (such as monoclonal antibody) preparation than otherwise, so can reduce the dosing frequency of protein.For example, when protein is prepared together with the water-soluble dye that makes viscosity reduce, previously need the protein that is administered once a day can be administered once every two days, once every three days or even less frequently.Currently need the protein that multiple administrations on the same day (at the same time of day or different time) can be administered by injection less times every day.In some cases, frequency can be reduced to single injection once a day.By making the dosage of each injection administration increase many times, can reduce the dosing frequency, for example, by being reduced to once every 6 weeks once every 2 weeks.

In some embodiments, the physiological molar osmotic pressure concentration of the liquid preparation is, for example, about 280mOsm/L to about 310mOsm/L. In some embodiments, the molar osmotic pressure concentration of the liquid preparation is greater than about 250mOsm/L, greater than about 300mOsm/L, greater than about 350mOsm/L, greater than about 400mOsm/L or greater than about 500mOsm/L. In some embodiments, the molar osmotic pressure concentration of the preparation is about 200mOsm/L to about 2,000mOsm/L or about 300mOsm/L to about 1,000mOsm/L. In some embodiments, the liquid preparation is substantially isotonic with human blood. In some cases, the liquid preparation may be hypertonic.

Can introduce the additive that comprises the water-soluble dye that makes viscosity reduction to realize the required viscosity level of liquid preparation by any amount, as long as described amount is not poisonous or harmful and does not interfere with the chemical and/or physical stability of preparation substantially.In some embodiments, one or more water-soluble dyes that make viscosity reduction can independently exist by following concentration: be less than about 1.0M, preferably be less than about 0.50M, be less than or equal to about 0.30M or be less than or equal to 0.15M.Especially preferred concentration comprises about 0.01M and about 0.10M.For some embodiments with two or more water-soluble dyes that make viscosity reduction, described material preferably but not necessarily exists with same concentrations.

The water-soluble dye that viscosity is reduced allows the faster redissolution of lyophilized dosage unit.Dosage unit is the lyophilized cake of protein, the water-soluble dye that viscosity is reduced and other excipient, in described lyophilized cake, add water, saline or other medicinal fluid.Under the situation that does not have the water-soluble dye that viscosity is reduced, usually need 10 minutes or longer time so that the lyophilized cake of high protein concentration is dissolved fully.When lyophilized cake contained one or more water-soluble dyes that viscosity is reduced, lyophilized cake is dissolved needed time and is reduced to original 1/2, 1/5 or 1/10th usually.In some embodiments, make protein concentration greater than or be about 150,200 or even 300mg/mL lyophilized cake and dissolve need less than 1 minute fully.

Low viscosity protein formulations allow for greater flexibility in formulation development. Compared to otherwise identical formulations lacking one or more viscosity-lowering water-soluble dyes, low viscosity formulations exhibit less viscosity change as protein concentration increases. Compared to otherwise identical formulations lacking one or more viscosity-lowering water-soluble dyes, low viscosity formulations exhibit a reduced viscosity gradient.

The viscosity gradient of the protein formulation can be one-half, one-third, or even less than one-third the viscosity gradient of an otherwise identical protein formulation that does not contain one or more water-soluble dyes that reduce viscosity. For protein formulations with a protein concentration of 10 mL/mg to 2,000 mL/mg, the viscosity gradient of the protein formulation can be less than 2.0 cP mL/mg, less than 1.5 cP mL/mg, less than 1.0 cP mL/mg, less than 0.8 cP mL/mg, less than 0.6 cP mL/mg, or less than 0.2 cP mL/mg. By reducing the viscosity gradient of the formulation, the protein concentration can be increased to a higher level before an exponential increase in viscosity is observed.

Some water-soluble organic dyes contain acidic or basic functional groups. Whether these functional groups are fully or partially ionized depends on the pH of the formulation in which they are present. Unless otherwise specified, the parent compound of a water-soluble organic dye with ionizable functional groups and all possible ionization states may be present in the formulation.

A. Protein

Any protein can be formulated, including recombinant, isolated or synthetic proteins, glycoproteins or lipoproteins. These proteins can be antibodies (including antibody fragments and recombinant antibodies), enzymes, growth factors or hormones, immunomodulatory factors, anti-infective factors, anti-proliferative factors, vaccines or other therapeutic, prophylactic or diagnostic proteins. In some embodiments, the protein has a molecular weight greater than about 150 kDa, greater than 160 kDa, greater than 170 kDa, greater than 180 kDa, greater than 190 kDa or greater than 200 kDa.

In some embodiments, the protein may be a PEGylated protein. As used herein, the term "PEGylated protein" refers to a protein having one or more poly(ethylene glycol) or other stealth polymer groups covalently attached to the protein, optionally via a chemical linker that may be different from the one or more polymer groups. PEGylated proteins are characterized by generally reduced renal filtration, decreased reticuloendothelial uptake, and attenuated enzymatic degradation, resulting in, for example, an extended half-life and improved bioavailability. Stealth polymers include poly(ethylene glycol); poly(propylene glycol); poly(amino acid) polymers such as poly(glutamic acid), poly(hydroxyethyl-L-asparagine), and poly(hydroxyethyl-L-glutamine); poly(glycerol); poly(2-oxazoline) polymers such as poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline); poly(acrylamide); poly(vinyl pyrrolidone); poly(N-(2-hydroxypropyl)methacrylamide); and copolymers and mixtures thereof. In a preferred embodiment, the stealth polymer in the PEGylated protein is poly(ethylene glycol) or a copolymer thereof. The PEGylated protein can be randomly PEGylated, i.e., with one or more stealth polymers covalently attached to one or more non-specific sites on the protein, or can be PEGylated in a site-specific manner by covalently attaching the stealth polymer to one or more specific sites on the protein. Site-specific PEGylation can be achieved, for example, using activated stealth polymers having one or more reactive functional groups. For examples, see, eg, Hoffman et al., Progress in Polymer Science, 32:922-932, 2007.

In a preferred embodiment, the protein is of high molecular weight and is an antibody (most preferably a monoclonal antibody) and has a high viscosity in a buffered aqueous solution when concentrated to a concentration sufficient to inject a therapeutically effective amount in a volume of no more than 1.0 to 2.0 mL for subcutaneous injection and no more than 3.0 to 5.0 mL for intramuscular injection. High molecular weight proteins may include those described in Scolnik, mAbs 1: 179-184, 2009; Beck, mAbs 3: 107-110, 2011; Baumann, Curr. Drug Meth. 7: 15-21, 2006; or Federici, Biologicals 41: 131-147, 2013. Proteins used in the formulations described herein are preferably substantially pure and substantially homogeneous (i.e., substantially free of contaminating proteins and/or irreversible aggregates thereof).

Preferred monoclonal antibodies herein include natalizumab, cetuximab, bevacizumab, trastuzumab, infliximab, rituximab, panitumumab, ofatumumab, and biosimilars thereof. Exemplary high molecular weight proteins may include tocilizumab, alemtuzumab (sold under several trade names), brodalumab (developed by Amgen, Inc. ("Amgen")), denosumab (and ) and biosimilars thereof.

Exemplary molecular targets of the antibodies described herein include CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD34; members of the HER receptor family, such as EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules, such as LFA-1, Mo1, p150,95, VLA-4, ICAM-1, VCAM and αv/β3 integrin, including its α or β subunit (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; protein C; PCSK9, etc.

Antibody therapeutics currently on the market

Many protein therapeutics currently on the market, particularly antibodies as defined herein, are administered via intravenous infusion due to the need for high doses. The formulation may comprise one of the antibody therapeutics currently on the market or a biosimilar thereof. Some protein therapeutics currently on the market are not high molecular weight, but are still administered via intravenous infusion due to the need for high doses for efficacy. In some embodiments, liquid formulations of these low molecular weight proteins as defined herein are provided at concentrations suitable for delivering a therapeutically effective amount for subcutaneous or intramuscular injection.

Antibody therapeutics currently on the market include belimumab, golimumab (Simponi), abciximab (marketed as a combination of tositumomab and iodine-131 tositumomab), alemtuzumab, palivizumab, basiliximab, ado-trastuzumab, bemtansine, pertuzumab, capromab pendetide (ProstaScint), caclizumab, ibritumomab tiuxetan, eculizumab, ipilimumab, and muromonab-CD3 (Orthoclone). ), raxibacumab, nimotuzumab, brentuximab vedotin, adalimumab, golimumab, palivizumab, omalizumab, and ustekinumab

Natalizumab is a humanized monoclonal antibody directed against the cell adhesion molecule α4-integrin used to treat multiple sclerosis and Crohn's disease. Natalizumab was previously marketed under the trade name ANTIBASE and is currently co-marketed by Biogen Idec ("Biogen") and Elan Corp. ("Elan"). It is produced in murine myeloma cells. Each 15 mL dose contains 300 mg of natalizumab; 123 mg of sodium chloride USP; 17.0 mg of sodium phosphate monobasic monohydrate USP; 7.24 mg of sodium phosphate dibasic heptahydrate USP; and 3.0 mg of polysorbate 80 USP/NF in water for intravenous injection USP pH 6.1. Natalizumab is typically administered by monthly intravenous (IV) infusion and has been shown to be effective in treating the symptoms of multiple sclerosis and Crohn's disease and for preventing relapses, vision loss, cognitive decline, and significantly improving patients' quality of life.

As used herein, the term "natalizumab" includes monoclonal antibodies directed against the cell adhesion molecule α4-integrin known by the international nonproprietary name "NATALIZUMAB," or an antigen-binding portion thereof. Natalizumab includes antibodies described in U.S. Patent No. 5,840,299, U.S. Patent No. 6,033,665, U.S. Patent No. 6,602,503, U.S. Patent No. 5,168,062, U.S. Patent No. 5,385,839, and U.S. Patent No. 5,730,978. Natalizumab includes the active agent in products marketed by Biogen Idec and Elan Corporation under the trade name NATALIZUMAB, or biosimilar products thereof.

Cetuximab is an epidermal growth factor receptor (EGFR) inhibitor used to treat metastatic colorectal cancer and head and neck cancer. Cetuximab is a chimeric (murine/human) monoclonal antibody that is usually administered by intravenous infusion. Cetuximab is marketed under the trade name Cetuximab by Bristol-Myers Squibb Company (North America; "Bristol-Myers Squibb"), Eli Lilly and Company (North America; "Eli Lilly"), and Merck KGaA for intravenous use only. It is produced in mammalian (murine myeloma) cell culture. Each single-use 50 mL vial contains 100 mg of cetuximab at a concentration of 2 mg/mL and is formulated in a preservative-free solution containing 8.48 mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic heptahydrate, 0.42 mg/mL sodium phosphate monobasic monohydrate, and water for intravenous injection, USP.

Cetuximab is indicated for use in combination with chemotherapy for patients with KRAS wild-type metastatic colorectal cancer (mCRC) expressing epidermal growth factor receptor (EGFR) and as a single agent in patients who cannot be treated with oxaliplatin- and irinotecan-based therapies or who are intolerant to irinotecan. Cetuximab is indicated for use in combination with platinum-based chemotherapy for first-line treatment of recurrent and/or metastatic disease and in combination with radiation therapy for locally advanced disease in patients with head and neck squamous cell carcinoma. Approximately 75% of patients with metastatic colorectal cancer have tumors that express EGFR and are therefore believed to be suitable for treatment with cetuximab or panitumumab according to FDA guidelines.

As used herein, the term "cetuximab" includes the monoclonal antibody known by the international nonproprietary name "CETUXIMAB," or an antigen-binding portion thereof. Cetuximab includes the antibody described in U.S. Patent No. 6,217,866. Cetuximab includes the active agent in products marketed under the trade name Cetuximab and biosimilar products thereof. Biosimilars of Cetuximab may include those currently being developed by Amgen, AlphaMab Co., Ltd. ("AlphaMab"), and Actavis plc ("Actavis").

Bevacizumab is a humanized monoclonal antibody that inhibits vascular endothelial growth factor A (VEGF-A) and acts as an anti-angiogenic agent. It is marketed under the trade name by Genentech, Inc. ("Genentech") and F. Hoffmann-La Roche, LTD ("Roche"). It is licensed for the treatment of a variety of cancers, including colorectal cancer, lung cancer, breast cancer (excluding the United States), glioblastoma (only in the United States), renal cancer, and ovarian cancer. In 2004, it was approved by the FDA for use in metastatic colorectal cancer when used with standard chemotherapy (as a first-line treatment) and for second-line metastatic colorectal cancer when used with 5-fluorouracil-based therapy. The FDA approved it in 2006 for use in combination with carboplatin/paclitaxel chemotherapy for first-line advanced non-squamous non-small cell lung cancer. It is administered once every three weeks at a dose of 15 mg/kg or 7.5 mg/kg by intravenous infusion. Higher doses are usually given with carboplatin-based chemotherapy, while lower doses are given with cisplatin-based chemotherapy. The FDA approved it in 2009 for the treatment of metastatic renal cell carcinoma (a form of kidney cancer). The FDA also granted accelerated approval in 2009 for the treatment of recurrent glioblastoma multiforme. The treatment of initial growths is still in Phase III clinical trials.

The National Comprehensive Cancer Network ("NCCN") recommends bevacizumab in combination with any platinum-based chemotherapy as standard first-line therapy, followed by maintenance bevacizumab until disease progression. In 2010, the NCCN updated its Clinical Practice Guidelines in Oncology for Breast Cancer (NCCN Guidelines) to affirm the recommendation for the use of bevacizumab (Genentech/Roche) in the treatment of metastatic breast cancer.

As used herein, the term "bevacizumab" includes a monoclonal antibody or antigen-binding portion thereof that inhibits vascular endothelial growth factor A (VEGF-A) known by the international nonproprietary name/common name "BEVACIZUMAB." Bevacizumab is described in U.S. Patent No. 6,054,297. Bevacizumab includes the active agent in products marketed under the trade name BEVACIZUMAB and biosimilar products thereof. Biosimilars of BEVACIZUMAB may include those currently being developed by Amgen, Actavis, AlphaMab, and Pfizer, Inc. ("Pfizer"). Biosimilars of BEVACIZUMAB may include a biosimilar known as BCD-021 produced by Biocad and currently undergoing clinical trials in the United States.

Trastuzumab is a monoclonal antibody that interferes with the HER2/neu receptor. Trastuzumab is marketed by Genentech, Inc. under the trade name Trastuzumab. It is produced by a mammalian cell line (Chinese hamster ovary (CHO)). It is a sterile, white to pale yellow, preservative-free, lyophilized powder for intravenous administration. Each vial contains 440 mg of trastuzumab, 9.9 mg of L-histidine HCl, 6.4 mg of L-histidine, 400 mg of α,α-trehalose dihydrate, and 1.8 mg of polysorbate 20 USP. Reconstitution with 20 mL of water yields a multiple-dose solution containing 21 mg/mL trastuzumab. Currently, it is administered via intravenous infusion once a week at a dose of about 2 mg/kg to about 8 mg/kg.

Trastuzumab is mainly used to treat some breast cancers. The HER2 gene is amplified in 20-30% of early breast cancers, which causes it to overexpress epidermal growth factor (EGF) receptors in the cell membrane. Trastuzumab is usually administered as a maintenance therapy for patients with HER2-positive breast cancer, usually for one year after chemotherapy. Trastuzumab is currently administered via intravenous infusion at a frequency of once a week and at a dose of about 2 mg/kg to about 8 mg/kg.

The term "trastuzumab" as used herein includes monoclonal antibodies or antigen-binding portions thereof that interfere with the HER2/neu receptor known by the international nonproprietary name/common name "TRASTUZUMAB". Trastuzumab is described in U.S. Patent No. 5,821,337. Trastuzumab includes active agents in products listed under the trade name HER2/neu and biosimilars thereof. The term "trastuzumab" includes active agents in biosimilar products listed under the trade name HER2/neu and biosimilars listed under the trade name HER2/neu. Trastuzumab may include active agents in biosimilar products being developed by Amgen and PlantForm Corporation, Canada.

Infliximab is a monoclonal antibody against tumor necrosis factor alpha (TNF-α) used to treat autoimmune diseases. It is marketed under the trade name by Janssen Global Services, LLC ("Janssen") (USA), Mitsubishi Tanabe Pharma (Japan), Xian Janssen (China) and Merck & Co ("Merck") (other countries and regions). Infliximab is a chimeric mouse/human monoclonal antibody of high molecular weight, i.e., about 144 kDa. In some embodiments, the formulation contains biosimilars such as REMSIMA ^™ or INFLECTRA ^™ . REMSIMA ^™ developed by Celltrion, Inc. ("Celltrion") and INFLECTRA ^™ developed by Hospira Inc., UK have both been recommended for administrative approval in Europe. Celltrion has submitted REMSIMA ^™ to the FDA. Infliximab is currently administered via intravenous infusion at a dose of about 3 mg/kg to about 10 mg/kg.

Infliximab contains approximately 30% murine variable region amino acid sequence, which confers antigen-binding specificity for human TNFα. The remaining 70% corresponds to the human IgG1 heavy chain constant region and the human kappa light chain constant region. Infliximab has high affinity for human TNFα, a cytokine with diverse biological effects, including mediating inflammatory responses and regulating the immune system.

Infliximab is a recombinant antibody typically produced and secreted by mouse myeloma cells (SP2/0 cells). The antibody is currently produced by continuous pre-perfusion cell culture. Infliximab is expressed using a chimeric antibody gene consisting of a variable region sequence cloned from mouse anti-TNFα hybridoma cell A2 and a human antibody constant region sequence provided by a plasmid expression vector. The production of mouse anti-TNFα hybridomas is performed by immunizing BALB/c mice with purified recombinant human TNFα. The heavy and light chain vector constructs are linearized and transfected into Sp2/0 cells by electroporation. Standard purification steps may include chromatographic purification, viral inactivation, nanofiltration, and ultrafiltration/diafiltration.

The term "infliximab" as used herein includes the chimeric mouse/human monoclonal antibody known by the international nonproprietary name "INFLIXIMAB" or its antigen-binding portion. Infliximab neutralizes the biological activity of TNFα and inhibits the binding of TNFα to its receptor by binding to the soluble transmembrane form of TNFα with high affinity. Infliximab is described in U.S. Patent No. 5,698,195. The term "infliximab" includes the active agent in products marketed or proposed for marketing under the trade names of various companies, REMSIMA ^™ by Celltrion, and INFLECTRA ^™ by Hospira, Inc. ("Hospira"). Infliximab is provided in the form of a sterile lyophilized cake for reconstitution and dilution. Each vial of infliximab contains 100 mg of infliximab and excipients such as sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, sucrose, and polysorbate 80.

Denosumab (and ) is a human monoclonal antibody and the first RANKL inhibitor approved for use in postmenopausal women at risk for osteoporosis and patients with bone metastases from solid tumors. Denosumab is in Phase II clinical trials for the treatment of rheumatoid arthritis.

Panitumumab is a fully human monoclonal antibody approved by the FDA for the treatment of metastatic EGFR-expressing cancers associated with disease progression. Panitumumab is marketed by Amgen under the trade name Panitumumab. It is packaged as a 20 mg/ml panitumumab concentrate in 5 ml, 10 ml, and 15 ml vials for intravenous infusion. When formulated according to the package insert, the final concentration of panitumumab does not exceed 10 mg/ml. It is administered by intravenous infusion every 14 days at a dose of 6 mg/kg. As used herein, the term "panitumumab" includes the anti-human epidermal growth factor receptor known by the international nonproprietary name "PANITUMUMAB." The term "panitumumab" includes the active agent in the product marketed by Amgen under the trade name Panitumumab and its biosimilars. The term "panitumumab" includes the monoclonal antibody described in U.S. Patent 6,235,883. The term "panitumumab" includes biosimilar products, including biosimilars being developed by BioXpress, SA ("BioXpress").

Belimumab is a human monoclonal antibody with a molecular weight of approximately 151.8 kDa that inhibits B-cell activating factor (BAFF). Belimumab is approved in the United States, Canada, and Europe for the treatment of systemic lupus erythematosus. Belimumab is currently administered to lupus patients at a dose of 10 mg/kg by intravenous infusion. High molecular weight, low viscosity protein formulations may contain belimumab, preferably at a concentration of about 400 mg/mL to about 1,000 mg/mL. The preferred range is calculated based on a body weight of 40-100 kg (about 80-220 pounds) in a volume of 1 mL.

Abciximab is manufactured by Janssen Biologics BV and distributed by Eli Lilly & Company ("Eli Lilly"). Abciximab is a Fab fragment of the chimeric human/murine monoclonal antibody 7E3. Abciximab binds to the glycoprotein (GP) IIb/IIIa receptor of human platelets and inhibits platelet aggregation by preventing the binding of fibrinogen, von Willebrand factor, and other adhesion molecules. It also binds to the vitronectin (αvβ3) receptor found on platelets, endothelial cells of blood vessel walls, and smooth muscle cells. Abciximab is a platelet aggregation inhibitor used primarily during and after coronary procedures. Abciximab is administered via intravenous infusion, initially as a 0.25 mg/kg bolus, followed by a continuous intravenous infusion of 0.125 mcg/kg/minute for 12 hours.

Tositumomab is a drug used to treat follicular lymphoma. It is an IgG2a anti-CD20 monoclonal antibody derived from immortalized mouse cells. Tositumomab is administered by sequential infusion: cold monoclonal antibody, followed by iodine ( ^131I ) tositumomab, the same antibody covalently bound to the radionuclide iodine-131. Clinical trials have determined the efficacy of the tositumomab/iodine tositumomab dosing regimen in patients with relapsed refractory follicular lymphoma. Currently, it is administered via intravenous infusion at a dose of 450mg.

Alemtuzumab (marketed as FDA-approved and currently under development) is a monoclonal antibody used to treat chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), and T-cell lymphomas. It is also being used to treat some autoimmune diseases such as multiple sclerosis under clinical trial protocols. Alemtuzumab has a molecular weight of approximately 145.5 kDa. Patients with B-cell chronic lymphocytic leukemia are administered 30 mg daily by intravenous infusion.

Palivizumab is a humanized monoclonal antibody directed against an epitope in the A antigenic site of the F protein of respiratory syncytial virus. In two Phase III clinical trials in pediatric patients, palivizumab reduced the risk of hospitalization due to respiratory syncytial virus infection by 55% and 45%. Palivizumab is administered once monthly via intramuscular injection at a dose of 15 mg/kg.

Ofatumumab is a human anti-CD20 monoclonal antibody that appears to inhibit early B lymphocyte activation. Ofatumumab is marketed by GlaxoSmithKline, plc ("GlaxoSmithKline") under the trade name of Ofatumumab. It is distributed in single-use vials containing 100 mg/5 mL and 1,000 mg/50 mL ofatumumab for intravenous infusion. Ofatumumab is FDA-approved for the treatment of chronic lymphocytic leukemia and has also shown potential in the treatment of follicular non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing-remitting multiple sclerosis. Ofatumumab has a molecular weight of approximately 149 kDa. It is currently administered by intravenous infusion at an initial dose of 300 mg, followed by weekly intravenous infusions of 2,000 mg. As used herein, the term "ofatumumab" includes the anti-CD20 monoclonal antibody known by the international nonproprietary name "OFATUMUMAB." The term "ofatumumab" includes the active agent in products marketed under the trade name ANTIFATUMAB® and biosimilars thereof. The term "ofatumumab" also includes the active agent in biosimilar products currently under development by BioExpress. A high molecular weight, low viscosity liquid protein formulation may include ofatumumab, preferably at a concentration of about 300 mg/mL to about 2,000 mg/mL.

Trastuzumab emtansine (marketed in the United States as ado-trastuzumab emtansine) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent mertansine. Trastuzumab halts the growth of cancer cells by binding to the HER2/neu receptor, while mertansine enters cells and destroys them by binding to tubulin. Trastuzumab emtansine is approved in the United States specifically for the treatment of recurrent HER2-positive metastatic breast cancer. Several Phase III clinical trials of trastuzumab emtansine are planned or underway in 2014. Trastuzumab emtansine is currently administered via intravenous infusion at a dose of 3.6 mg/kg. High-molecular-weight, low-viscosity liquid formulations can contain trastuzumab emtansine, preferably at a concentration of about 144 mg/mL to about 360 mg/mL.

Pertuzumab is a monoclonal antibody that inhibits HER2 dimerization. It was approved by the FDA in 2012 for the treatment of HER2-positive metastatic breast cancer. The current recommended dose of pertuzumab is 420 mg to 840 mg via intravenous infusion. High-molecular-weight, low-viscosity liquid formulations can contain pertuzumab, preferably at a concentration of about 420 mg/mL to about 840 mg/mL.

Daclizumab is a humanized anti-CD25 monoclonal antibody and is used to prevent rejection in organ transplants, particularly kidney transplants. The drug is also being studied for the treatment of multiple sclerosis. The molecular weight of daclizumab is approximately 143 kDa. Daclizumab is marketed in the United States by Hoffmann-La Roche, Ltd. ("Roche") and is administered by intravenous infusion of 1 mg/kg. The daclizumab high-yield method (DAC HYP; BIIB019; Biogen Idec ("Biogen") and AbbVie, Inc. ("AbbVie")) is in Phase III clinical trials for a monthly subcutaneous injection of 150 mg for the treatment of relapsing-remitting multiple sclerosis. A high molecular weight, low viscosity liquid formulation may contain daclizumab, preferably at a concentration of about 40 mg/mL to about 300 mg/mL.

Eculizumab is a humanized monoclonal antibody approved for the treatment of rare blood disorders such as paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab, with a molecular weight of approximately 148 kDa, was developed by Alexion Pharmaceuticals, Inc. ("Alexion"). It is administered by intravenous infusion in an amount of about 600 mg to about 1,200 mg. High-molecular-weight, low-viscosity liquid formulations can contain eculizumab, preferably at a concentration of about 500 mg/mL to about 1,200 mg/mL.

Tocilizumab is a humanized monoclonal antibody directed against the interleukin-6 receptor. It is an immunosuppressive drug primarily used to treat rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (a severe form of rheumatoid arthritis in children). Tocilizumab is typically administered by intravenous infusion at a dosage of about 6 mg/kg to about 8 mg/kg. High molecular weight, low viscosity liquid formulations may include Tocilizumab, preferably at a concentration of about 240 mg/mL to about 800 mg/mL.

Rituximab is a chimeric anti-CD20 monoclonal antibody used to treat a variety of diseases characterized by excessive numbers of B cells, overactive B cells, or dysfunctional B cells. Rituximab is used to treat cancers of the white blood cell system, such as leukemias and lymphomas, including Hodgkin lymphoma and its lymphocyte-dominant subtype. It has also been shown to be an effective treatment for rheumatoid arthritis. Rituximab is widely used off-label to treat difficult cases of multiple sclerosis, systemic lupus erythematosus, and autoimmune anemia.

Rituximab is marketed in the United States by Biogen and Genentech under the trade name Rituxan and outside the United States by Roche under the trade name Rituximab. It is distributed in single-use vials containing 100 mg/10 mL and 500 mg/50 mL. It is typically administered by intravenous infusion at approximately 375 mg/ ^m² . As used herein, the term "rituximab" includes the anti-CD20 monoclonal antibody known by the international nonproprietary name/common name "RITUXIMAB." Rituximab includes the monoclonal antibody described in U.S. Patent No. 5,736,137. Rituximab includes the active agent in products marketed under the trade names Rituxan and Rituxan, as well as biosimilars thereof.

A high molecular weight, low viscosity liquid formulation can contain rituximab, preferably at a concentration of about 475 mg/mL to about 875 mg/mL (approximated using the body surface area range of 1.3 to 2.3 square meters obtained from Mosteller's formula for a 5 foot 40 kg to 6 foot 100 kg person). The concentration is calculated based on 1 mL of the formulation.

Ipilimumab is a human monoclonal antibody developed by Bristol-Myers Squibb Company ("Bristol-Myers Squibb"). It is currently marketed for the treatment of melanoma and is in clinical trials for the treatment of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and metastatic hormone-refractory prostate cancer. Ipilimumab is currently administered by intravenous infusion at 3 mg/kg. High molecular weight, low viscosity liquid formulations can contain ipilimumab, preferably at a concentration of about 120 mg/mL to about 300 mg/mL.

Rexibacumab is a human monoclonal antibody designed to prevent and treat inhalation anthrax. It is currently administered by intravenous infusion. The recommended dose for adults and children over 50 kg is 40 mg/kg. High-molecular-weight, low-viscosity liquid formulations can contain rexibacumab, preferably at a concentration of about 1,000 mg/mL to about 4,000 mg/mL.

Nimotuzumab (BIOMAB®) is a humanized monoclonal antibody with a molecular weight of approximately 151 kDa used to treat head and neck squamous cell carcinoma, recurrent or refractory high-grade malignant gliomas, anaplastic astrocytomas, glioblastomas, and diffuse intrinsic pontine gliomas. Nimotuzumab is typically administered by weekly intravenous infusion of approximately 200 mg. High-molecular-weight, low-viscosity liquid formulations can contain nimotuzumab, preferably at a concentration of approximately 200 mg/mL.

Brentuximab vedotin is an antibody-drug conjugate directed against CD30, a protein expressed in classical Hodgkin lymphoma and systemic anaplastic large cell lymphoma. It is administered by intravenous infusion at approximately 1.8 mg/kg. High-molecular-weight, low-viscosity liquid formulations can contain brentuximab vedotin, preferably at a concentration of about 80 mg/mL to about 200 mg/mL.

Itolizumab is a humanized IgG1 monoclonal antibody developed by Biocon. Itolizumab successfully completed a Phase III clinical trial in patients with moderate to severe psoriasis. Itolizumab has received marketing authorization in India; an application for FDA approval has not yet been submitted.

Originally developed by Roche and further developed under a collaboration agreement with Biogen, obinutuzumab is a humanized anti-CD20 monoclonal antibody approved for the treatment of chronic lymphocytic leukemia. It is also being studied in a Phase III clinical trial in patients with various lymphomas. A dose of approximately 1,000 mg is administered via intravenous infusion.

Certolizumab pegol is a recombinant humanized antibody Fab' fragment specific for human tumor necrosis factor alpha (TNFα) conjugated to approximately 40 kDa polyethylene glycol (PEG2MAL40K). The molecular weight of certolizumab pegol is approximately 91 kDa.

Other antibody therapeutics that can be formulated with water-soluble dyes to reduce viscosity include CT-P6 from Celltrion, Inc. (Celltrion).

Antibody therapeutics in late-stage trials and development

The pipeline of antibody therapeutics progressing to late-stage clinical development and regulatory approval is advancing at a rapid pace. In 2014, over 300 monoclonal antibodies were in clinical trials, and 30 commercially launched antibody therapeutics were undergoing late-stage research evaluation. Two monoclonal antibodies (vedolizumab and ramucirumab) recently received their first marketing authorization applications (MAA) with the FDA. Amgen is currently sponsoring multiple Phase III trials for brodalumab in patients with plaque psoriasis, with additional trials planned or patient recruitment. XBiotech, Inc. has initiated two Phase I clinical trials of MABp1 (Xilonix) in patients with advanced cancer or type 2 diabetes. Other MABp1 trials are currently recruiting patients. MedImmune, LLC ("MedImmune") is sponsoring multiple trials for moxetumomab pasudotox in leukemias, and these trials are ongoing or recruiting patients. The long-term safety and efficacy of tildrakizumab in the treatment of chronic plaque psoriasis are being investigated, and multiple Phase II trials of rilotumumab in the treatment of various cancers have recently been completed.

At least 28 monoclonal antibodies currently in or recently completed Phase III studies for the treatment of inflammatory or immune disorders, cancer, high cholesterol, osteoporosis, Alzheimer's disease, and infectious diseases are high molecular weight proteins. Monoclonal antibodies currently undergoing or recently completed phase III trials include AMG145, elotuzumab, epratuzumab, farletuzumab (MORAb-003), gantenerumab (RG1450), gevokizumab, inotuzumab ozogamicin, itolizumab, ixekizumab, lebrikizumab, mepolizumab, naptumomab estafenatox, necitumumab, nivolumab, ocrelizumab, onartuzumab, racotumomab, ramucirumab, reslizumab, romosozumab, sarilumab, secukinumab, sirukumab, solanezumab, tabalumab, and vedolizumab. A monoclonal antibody cocktail (actoxumab and bezlotoxumab) is also being evaluated in phase III trials. See, e.g., Reichert, MAbs 5:1-4, 2013.

Vedolizumab is a monoclonal antibody developed by Millennium Pharmaceuticals, Inc. ("Millennium"; a subsidiary of Takeda Pharmaceuticals Company, Ltd. ("Takeda"). Vedolizumab was found to be safe and highly effective in inducing and maintaining clinical remission in patients with moderate to severe ulcerative colitis. Phase III clinical trials showed that it achieved its goals of inducing clinical response and maintaining remission in patients with Crohn's disease and ulcerative colitis. Studies evaluating long-term clinical outcomes showed that nearly 60% of patients achieved clinical remission. The usual dose of vedolizumab is 6 mg/kg intravenously.

Ramucirumab is a human monoclonal antibody being developed for the treatment of solid tumors. Phase III clinical trials are underway for the treatment of breast cancer, metastatic gastric adenocarcinoma, non-small cell lung cancer, and other types of cancer. In some Phase III trials, ramucirumab is administered at approximately 8 mg/kg via intravenous infusion.

Rilotumumab is a human monoclonal antibody that inhibits the effects of hepatocyte growth factor/scatter factor. It is being developed by Amgen and is in Phase III trials for the treatment of solid tumors. An open-label Phase III study of rilotumumab in patients with advanced or metastatic esophageal cancer will administer rilotumumab at approximately 15 mg/kg via intravenous infusion.

Evolocumab (AMG 145), also developed by Amgen, is a monoclonal antibody that binds to PCSK9. Evolocumab is indicated for the treatment of hypercholesterolemia and hyperlipidemia.

Alirocumab (REGN727) is a human monoclonal antibody from Regeneron Pharmaceuticals, Inc. ("Regeneron") and Sanofi-Aventis U.S. LLC ("Sanofi"), indicated for the treatment of hypercholesterolemia and acute coronary syndrome.

Naptumomab estafenatox, ABR-217620, from Active Biotech AB ("Active Biotech"), is a monoclonal antibody indicated for the treatment of renal cell carcinoma.

Racotumomab from CIMAB, SA ("CIMAB"); Laboratorio Elea S.A.C.I.F.y A. is a monoclonal antibody indicated for the treatment of non-small cell lung cancer.

Other antibodies that can be formulated with water-soluble dyes that reduce viscosity include bococizumab (PF-04950615) and tanezumab; ganitumab, blinatumomab, trebananib from Amgen; anthrax immune globulin from Cangene Corporation; teplizumab from MacroGenics, Inc.; MK-3222, MK-6072 from Merck & Co ("Merck"); girentuximab from Wilex AG; RIGScan from Navidea Biopharmaceuticals ("Navidea"); PF-05280014 from Pfizer; SA237 from Chugai Pharmaceutical Co. Ltd. ("Chugai"); and dapoxetine from Janssen/Johnson and guselkumab from Johnson Services, Inc. ("J&J"); antithrombin gamma (KW-3357) from Kyowa; and CT-P10 from Celltrion.

Antibodies in early clinical trials

A variety of monoclonal antibodies have recently entered or are entering clinical trials. They may include proteins currently administered via intravenous infusion, preferably those with a molecular weight greater than about 120 kDa, typically about 140 kDa to about 180 kDa. They may also include high molecular weight proteins such as drugs or peptides conjugated to albumin that are also entering clinical trials or have been approved by the FDA. A variety of monoclonal antibodies from Amgen are currently in clinical trials. These monoclonal antibodies may be high molecular weight proteins such as AMG 557, a human monoclonal antibody jointly developed by Amgen and AstraZeneca and currently in Phase I trials for the treatment of lupus. Similarly, AMG 729 is a humanized monoclonal antibody developed by Amgen and currently in Phase I trials for the treatment of lupus and rheumatoid arthritis. In addition, AMG 110 is a monoclonal antibody against epithelial cell adhesion molecules; AMG 157, jointly developed by Amgen and AstraZeneca, is a human monoclonal antibody currently in Phase I trials for the treatment of asthma; AMG 167 is a humanized monoclonal antibody that has been evaluated in multiple Phase I trials for the treatment of osteoporosis; AMG334, which has completed Phase I dosing studies and is currently in Phase II studies for the treatment of migraine and hot flashes, is a human monoclonal antibody that inhibits calcitonin gene-related peptide; AMG 780 is a human anti-angiopoietin monoclonal antibody that inhibits the interaction of the endothelial cell-selective Tie2 receptor with its ligands Ang1 and Ang2 and recently completed Phase I trials as a cancer therapy; AMG 811 is a human monoclonal antibody that inhibits interferon gamma and is being studied as a therapy for systemic lupus erythematosus; AMG 820 is a human monoclonal antibody that inhibits c-fms and reduces tumor-associated macrophage (TAM) function and is being studied as a cancer therapy; AMG 181, jointly developed by Amgen and AstraZeneca, is a human monoclonal antibody that inhibits the action of α4/β7 and is in Phase II trials as a therapy for ulcerative colitis and Crohn's disease.

Several monoclonal antibodies are currently in clinical trials for the treatment of autoimmune disorders. These monoclonal antibodies can be formulated as low-viscosity, high-molecular-weight liquid formulations. RG7624 is a fully human monoclonal antibody designed to specifically and selectively bind to cytokines of the human interleukin-17 family. A Phase I clinical trial evaluating RG7624 for autoimmune diseases is underway. BIIB033 is an anti-LINGO-1 monoclonal antibody developed by Biogen and currently in Phase II trials for the treatment of multiple sclerosis.

High molecular weight proteins may also include AGS-009, a monoclonal antibody targeting IFN-α developed by Argos Therapeutics, Inc., which recently completed a Phase I trial for the treatment of lupus. Patients are administered up to 30 mg/kg of AGS-009 via intravenous infusion. BT-061, developed by AbbVie, is in a Phase II trial for patients with rheumatoid arthritis. Certolizumab pegol is a monoclonal antibody in a Phase II trial for ankylosing spondylitis and juvenile rheumatoid arthritis. Clazakizumab is an anti-IL6 monoclonal antibody in a Phase II trial conducted by Bristol-Myers Squibb.

CNTO-136 (sirukumab) and CNTO-1959 are monoclonal antibodies that have recently completed Phase II and Phase III trials by Janssen. Daclizumab (previously marketed by Roche) is currently in or has recently completed multiple Phase III trials by AbbVie for the treatment of multiple sclerosis. Epratuzumab is a humanized monoclonal antibody in Phase III trials for the treatment of lupus. Canakinumab is a human monoclonal antibody that targets interleukin-1β. It is approved for the treatment of cryopyrin-associated periodic syndromes. Canakinumab is in Phase I trials as a possible treatment for chronic obstructive pulmonary disease, gout, and coronary artery disease. Mavrilimumab is a human monoclonal antibody designed to treat rheumatoid arthritis. Mavrilimumab, discovered by Cambridge Antibody Technology as CAM-3001, is being developed by MedImmune.

MEDI-546 and MEDI-570 are monoclonal antibodies currently in Phase I and Phase II trials being conducted by AstraZeneca for the treatment of lupus. MEDI-546 is administered by conventional intravenous infusion of 300-1,000 mg in Phase II studies. MEDI-551 is another monoclonal antibody developed by AstraZeneca for multiple indications and is also currently administered by intravenous infusion. NN8209 is a monoclonal antibody developed by Novo Nordisk A/S ("Novo Nordisk") for blocking the C5aR receptor and has completed Phase II dosing studies for the treatment of rheumatoid arthritis. NN8210 is another anti-C5aR monoclonal antibody being developed by Novo Nordisk and is currently in Phase I trials. IPH2201 (NN8765) is a humanized monoclonal antibody targeting NKG2A being developed by Novo Nordisk for the treatment of patients with inflammatory conditions and autoimmune diseases. NN8765 recently completed a Phase I trial.

Olokizumab is a potent humanized monoclonal antibody that targets the cytokine IL-6. IL-6 is involved in several autoimmune and inflammatory pathways. Olokizumab has completed Phase II trials for the treatment of rheumatoid arthritis. otelixizumab (also known as TRX4) is a monoclonal antibody being developed for the treatment of type 1 diabetes, rheumatoid arthritis, and other autoimmune diseases. Ozoralizumab is a humanized monoclonal antibody that has completed Phase II trials.

Pfizer is currently conducting Phase I trials for the monoclonal antibodies PD-360324 and PF-04236921 for the treatment of lupus. PF-05280586, a rituximab biosimilar, has been developed by Pfizer and is in Phase I/II trials for rheumatoid arthritis.

Rontalizumab is a humanized monoclonal antibody being developed by Genentech. It recently completed a Phase II trial for the treatment of lupus. SAR113244 (anti-CXCR5) is a monoclonal antibody being developed by Sanofi and is in Phase I trials. Sifalimumab (anti-IFN-α monoclonal antibody) is a monoclonal antibody in Phase II trials for the treatment of lupus.

High molecular weight, low viscosity liquid formulations may include one of the monoclonal antibodies in early clinical development for the treatment of various blood disorders. For example, belimumab recently completed a Phase I trial for patients with vasculitis. Other monoclonal antibodies in early trials for blood disorders include BI-655075 from Boehringer Ingelheim GmbH ("Boehringer Ingelheim"), monoclonal antibodies to ferroportin and hepcidin from Eli Lily, and SelG1 from Selexys Pharmaceuticals, Corp. ("Selexys").

One or more monoclonal antibodies in early development for the treatment of various cancers and related conditions can be included in a low-viscosity, high-molecular-weight liquid formulation. United Therapeutics, Corporation has two monoclonal antibodies in Phase I trials: 8H9 and ch14.18. AbbVie's monoclonal antibodies ABT-806, enavatuzumab, and volociximab are in early development. Actinium Pharmaceuticals, Inc. has conducted early trials for the monoclonal antibodies Actimab-A (M195mAb), anti-CD45mAb, and lomab-B. Seattle Genetics, Inc. (“Seattle Genetics”) has several monoclonal antibodies in early-stage trials for cancer and related disorders, including an anti-CD22 ADC (RG7593; pinatuzumab vedotin), an anti-CD79b ADC (RG7596), an anti-STEAP1 ADC (RG7450), ASG-5ME and ASG-22ME from Agensys, Inc. (“Agensys”), the antibody-drug conjugate RG7458, and vorsetuzumab mafodotin. Early-stage cancer therapeutics from Genentech that can be included in low-viscosity formulations include ALT-836, antibody-drug conjugates RG7600 and DEDN6526A, anti-CD22 ADC (RG7593), anti-EGFL7 mAb (RG7414), anti-HER3/EGFR DAF mAb (RG7597), anti-PD-L1 mAb (RG7446), DFRF4539A, and MINT1526A. Bristol-Myers Squibb is developing early-stage monoclonal antibodies for cancer therapeutics, including those identified as anti-CXCR4, anti-PD-L1, IL-21 (BMS-982470), lirilumab, and urelumab (anti-CD137). Other monoclonal antibodies in early-stage trials as cancer therapeutics include APN301 (hu14.18-IL2) from Apeiron Biologics AG, AV-203 from AVEO Pharmaceuticals, Inc. (“AVEO”), AVX701 and AVX901 from AlphaVax, BAX-69 from Baxter International, Inc. (“Baxter”), BAY 79-4620 and BAY 20-10112 from Bayer HealthCare AG, BHQ880 from Novartis AG, 2¹²-Pb-TCMCtrastuzumab from AREVA Med, AbGn-7 from AbGenomics International Inc., and ABIO-0501 (TALL-104) from Abiogen Pharma S.p.A.

Other antibody therapeutics that can be formulated with water-soluble dyes that reduce viscosity include alzumab, GA101, daratumumab, siltuximab, ALX-0061, ALX-0962, ALX-0761, bimagumab (BYM338), CT-011 (pidilizumab), actoxumab/bezlotoxumab (MK-3515A), MK-3475 (pembrolizumab), dalotuzumab (MK-0646), icrucumab (IMC-18F1, LY3012212), AMG 139(MEDI2070), SAR339658, dupilumab(REGN668), SAR156597, SAR256212, SAR279356, SAR3419, SAR153192(REGN421,enoticuma b), SAR307746 (nesvacumab), SAR650984, SAR566658, SAR391786, SAR228810, SAR252067, SGN-CD19A, SGN-CD33A, SGN-LIV1A, ASG 15ME, anti-LINGO, BIIB037, ALXN1007, teprotumumab, concizumab, anrukinzumab(IMA-638), ponezumab(PF-04360365), PF-03446962, PF-06252616, etrol izumab(RG7413), quilizumab, ranibizumab, lampalizumab, onclacumab, gentenerumab, crenezumab(RG7412), IMC-RON8(narnatumab), tremeli mumab, vantictumab, eemcizumab, ozanezumab, mapatumumab, tralokinumab, XmAb5871, XmAb7195, cixutumumab (LY3012217), LY2541546 (blosozumab), olaratumab(LY3012207), MEDI4893, MEDI573, MEDI0639, MEDI3617, MEDI4736, MEDI6469, MEDI0680, MEDI5872, PF-05236812(AAB-003), PF-05082566, BI 1034020, RG7116, RG7356, RG7155, RG7212, RG7599, RG7636, RG7221, RG7652(MPSK3169A), RG7686, HuMaxTFADC, MOR103, BT061, MOR208, OMP59R5 (anti-notch 2/3), VAY736, MOR202, BAY94-9343, LJM716, OMP52M51, GSK933776, GSK249320, GSK1070806, NN8 828, CEP-37250/KHK2804AGS-16M8F, AGS-16C3F, LY3016859, LY2495655, LY2875358 and LY2812176.

Other early-stage monoclonal antibodies that can be formulated with water-soluble dyes that reduce viscosity include benralizumab, MEDI-8968, anifrolumab, MEDI7183, sifalimumab, MEDI-575, tralokinumab from AstraZeneca and MedImmune; BAN2401 from Biogen Idec/Eisai Co. LTD ("Eisai")/BioArctic Neuroscience AB; CDP7657 (an anti-CD40L monovalent PEGylated antibody Fab fragment), STX-100 (an anti-avB6 monoclonal antibody), BIIB059, anti-TWEAK (BIIB023), and BIIB022 from Biogen; fullanumab from Janssen and Amgen; BI-204/RG7418 from BioInvent International/Genentech; and BIIB023 from Biotest Pharmaceuticals. Corporation's BT-062 (indatuximabravtansine); XmAb from Boehringer Ingelheim/Xencor; anti-IP10 from Bristol-Myers Squibb; J 591Lu-177 from BZL Biologics LLC; CDX-011 (glembatumumab vedotin), CDX-0401 from Celldex Therapeutics; foravirumab from Crucell; tigatuzumab from Daiichi Sankyo Company Limited; MORAb-004, MORAb-009 (amatuximab) from Eisai; LY2382770 from Eli Lilly and Company; DI17E6 from EMD Serono Inc.; zanolimumab from Emergent BioSolutions, Inc.; FG-3019 from FibroGen, Inc.; and FG-3019 from Fresenius. SE & Co. KGaA's catumaxomab; Genentech's pateclizumab and rontalizumab; Genzyme and Sanofi's fresolimumab; Gilead's GS-6624 (simtuzumab); Janssen's CNTO-328, bapineuzumab (AAB-001), carlumab, and CNTO-136; KaloBios Pharmaceuticals, Inc.'s KB003; Kyowa's ASKP1240; Labrys Biologics Inc.'s RN-307; Life Science Pharmaceuticals' ecoromeximab; Eli Lilly's LY2495655, LY2928057, LY3015014, and LY2951742; MassBiologics' MBL-HCV1; MENTRIK Biotech, LLC's AME-133v; and Merck's abituzumab from KGaA; MM-121 from Merrimack Pharmaceuticals, Inc.; MCS110, QAX576, QBX258, QGE031 from Novartis AG; HCD122 from Novartis AG and XOMA Corporation ("XOMA"); NN8555 from Novo Nordisk; bavituximab, cotara from Peregrine Pharmaceuticals, Inc.; PSMA-ADC from Progenics Pharmaceuticals, Inc.; oregovomab from Quest Pharmatech, Inc.; fasinumab (REGN475), REGN1033, SAR231893, REGN846 from Regeneron; RG7160, CIM331, RG7745 from Roche; ibalizumab (TMB-355) from TaiMed Biologics Inc.; and dapoxetine from Theraclone. Sciences' TCN-032; TRC105 from TRACON Pharmaceuticals, Inc.; UB-421 from United Biomedical Inc.; VB4-845 from Viventia Bio, Inc.; ABT-110 from AbbVie; caplacizumab and ozoralizumab from Ablynx; PRO 140 from CytoDyn, Inc.; GS-CDA1 and MDX-1388 from Medarex, Inc.; AMG827 and AMG 888 from Amgen; ublituximab from TG Therapeutics Inc.; TOL101 from Tolera Therapeutics, Inc.; huN901-DM1 (lorvotuzumab mertansine) from ImmunoGen Inc.; and epratuzumab from Immunomedics, Inc. Y-90/veltuzumab combination (IMMU-102); anti-fibrin monoclonal antibody/3B6/22Tc-99m from Agenix, Limited; ALD403 from Alder Biopharmaceuticals, Inc.; RN6G/PF-04382923 from Pfizer; CG201 from CG Therapeutics, Inc.; KB001-A from KaloBios Pharmaceuticals/Sanofi; KRN-23 from Kyowa; Y-90hPAM 4 from Immunomedics, Inc.; Tarextumab from Morphosys AG and OncoMed Pharmaceuticals, Inc.; LFG316 from Morphosys AG and Novartis AG; CNTO3157, CNTO6785 from AG and Jannsen; RG6013 from Roche and Chugai; MM-111 from Merrimack Pharmaceuticals, Inc. ("Merrimack"); GSK2862277 from GlaxoSmithKline; AMG 282, AMG172, G 595, AMG 745, AMG 761 from Amgen; BVX-20 from Biocon; CT-P19, CT-P24, CT-P25, CT-P26, CT-P27, CT-P4 from Celltrion; GSK284933, GSK2398852, GSK2618960, GSK1223249, GSK933776A from GlaxoSmithKline; and GSK-1777 from Morphosys AG and Bayer. AG's anetumab ravtansine; BI-836845 from Morphosys AG and Boehringer Ingelheim; NOV-7 and NOV-8 from Morphosys AG and Novartis AG; MM-302, MM-310, MM-141, MM-131, and MM-151 from Merrimack; and MM-7 and MM-8 from Roche and Seattle. RG7882 from Genetics; RG7841 from Roche/Genentech; PF-06410293, PF-06438179, PF-06439535, PF-04605412, and PF-05280586 from Pfizer; RG7716, RG7936, gentenerumab, and RG7444 from Roche; MEDI-547, MEDI-565, MEDI1814, MEDI4920, MEDI8897, MEDI-4212, MEDI-5117, and MEDI-7814 from Astrazeneca; ulocuplumab and a PCSK9 adnectin from Bristol-Myers Squibb; FPA009 and FPA145 from FivePrime Therapeutics, Inc.; GS-5745 from Gilead; and GS-5745 from Kyowa Hakko Pharmaceuticals. BIW-8962, KHK4083, KHK6640 from Kirin; MM-141 from Merck KGaA; REGN1154, REGN1193, REGN1400, REGN1500, REGN1908-1909, REGN2009, REGN2176-3, REGN728 from Regeneron; SAR307746 from Sanofi; SGN-CD70A from Seattle Genetics; ALX-0141 and ALX-0171 from Ablynx; milatuzumab-DOX, milatuzumab, and TF2 from Immunomedics, Inc.; MLN0264 from Millennium; ABT-981 from AbbVie; AbGn-168H from AbGenomics International Inc.; ficlatuzumab from AVEO; and International's BI-505; CDX-1127 and CDX-301 from Celldex Therapeutics; CLT-008 from Cellerant Therapeutics Inc.; VGX-100 from Circadian; U3-1565 from Daiichi Sankyo Company Limited; DKN-01 from Dekkun Corp.; flanvotumab (TYRP1 protein), IL-1β antibody, IMC-CS4 from Eli Lilly and Company; VEGFR3 monoclonal antibody, IMC-TR1 (LY3022859) from Eli Lilly and ImClone, LLC; Anthim from Elusys Therapeutics Inc.; and from Galaxy Biotech. LLC's HuL2G7; ImmunoGen Inc.'s IMGB853 and IMGN529; Janssen's CNTO-5 and CNTO-5825; Kaketsuken's KD-247; KaloBios Pharmaceuticals' KB004; MacroGenics, Inc.'s MGA271 and MGAH22; MorphoSys AG/Xencor's XmAb5574; Neogenix Oncology, Inc.'s ensituximab (NPC-1C); Novartis AG and XOMA's LFA102; Novartis AG's ATI355; Santarus Inc.'s SAN-300; SelG1; Targa Therapeutics, Corp.'s HuM195/rGel; and Teva Pharmaceuticals, Industries Ltd. (“Teva”) and Vaccinex Inc.; TCN-202 from Theraclone Sciences; XmAb2513, XmAb5872 from Xencor; XOMA 3AB from XOMA and the National Institute of Allergy and Infectious Diseases; a neuroblastoma antibody vaccine from MabVax Therapeutics; cytolin from CytoDyn, Inc.; thravixa from Emergent BioSolutions Inc.; FB 301 from Cytovance Biologics; rabies monoclonal antibodies from Janssen and Sanofi; an influenza monoclonal antibody from Janssen funded in part by the National Institutes of Health; MB-003 and ZMapp from Mapp Biopharmaceutical, Inc.; and ZMAb from Defyrus Inc.

Other protein therapeutics

The protein may be an enzyme, a fusion protein, a stealth or PEGylated protein, a vaccine or other biologically active protein (or protein mixture). As used herein, the term "enzyme" refers to a protein or a functional fragment thereof that catalyzes the biochemical conversion of a target molecule into a desired product.

Enzymes as pharmaceuticals have at least two important characteristics, namely i) they bind and act on their target, usually with high affinity and specificity, and ii) they catalyze and convert a variety of target molecules into desired products. In some embodiments, the protein may be PEGylated as defined herein.

As used herein, the term "fusion protein" refers to a protein produced by two different genes that encode two separate proteins. Fusion proteins are typically produced by recombinant DNA techniques known to those skilled in the art. Two proteins (or protein fragments) are covalently fused together and exhibit the properties of both parent proteins.

There are a variety of fusion proteins available on the market.

Etanercept is a fusion protein marketed by Amgen that competitively inhibits TNF.

Antihemophilic Factor (Recombinant) Fc Fusion Protein is an antihemophilic factor produced by recombinant DNA and is indicated for the control and prevention of bleeding episodes, perioperative management, and routine prophylaxis aimed at preventing or reducing the frequency of bleeding episodes in adults and children with hemophilia A (congenital deficiency of coagulation factor VIII).

Aflibercept is a recombinant fusion protein composed of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1, formulated in an isotonic solution for intravitreal administration. Aflibercept is a recombinant fusion protein composed of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1, formulated in an isotonic solution for intravitreal administration. Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation that contributes an additional 15% of the total molecular weight, resulting in a total molecular weight of 115 kDa. Aflibercept is produced in recombinant Chinese hamster ovary (CHO) cells and is marketed by Regeneron.

ALPROLIX ^™ , or Coagulation Factor IX (Recombinant) Fc Fusion Protein, is a coagulation Factor IX concentrate produced by recombinant DNA and is indicated for the control and prevention of bleeding episodes, perioperative management, and routine prophylaxis aimed at preventing or reducing the frequency of bleeding episodes in adults and children with hemophilia B.

Pegloticase is a drug being developed by Savient Pharmaceuticals, Inc. for the treatment of severe, refractory chronic gout and is the first drug approved for this indication. Pegloticase is a pegylated recombinant porcine uricase with a molecular weight of approximately 497 kDa. Pegloticase is currently administered via intravenous infusion at a dose of 8 mg/kg. High-molecular-weight, low-viscosity liquid formulations can contain pegloticase, preferably at a concentration of about 300 mg/mL to about 800 mg/mL.

Alteplase is a tissue plasminogen activator produced by recombinant DNA technology. It is a purified glycoprotein comprising 527 amino acids and synthesized using the complementary DNA (cDNA) of natural human tissue plasminogen activator derived from a human melanoma cell line. Alteplase is administered via an intravenous infusion of approximately 100 mg immediately after the onset of stroke symptoms. In some embodiments, a low-viscosity formulation containing alteplase is provided, preferably at a concentration of about 100 mg/mL.

Glucarpidase is an FDA-approved drug for the treatment of elevated methotrexate levels (defined as at least 1 μmol/L) during treatment of cancer patients with impaired renal function. Glucarpidase is administered intravenously in a single dose of about 50 IU/kg. In some embodiments, a low-viscosity formulation containing glucarpidase is provided.

Alglucosidase α is an orphan drug for enzyme replacement therapy for the treatment of Pompe disease (glycogen storage disease type II), a rare lysosomal storage disease. It has a molecular weight of approximately 106 kDa and is currently administered by intravenous infusion at approximately 20 mg/kg. In some embodiments, a low-viscosity pharmaceutical formulation of alglucosidase α is provided, preferably at a concentration of about 100 mg/mL to about 2,000 mg/mL.

Pegdamase bovine is a modified enzyme used in enzyme replacement therapy for the treatment of severe combined immunodeficiency (SCID) associated with adenosine deaminase deficiency. Pegdamase bovine is a conjugate of 5,000 Da multi-stranded monomethoxypolyethylene glycol (PEG) covalently attached to adenosine deaminase obtained from bovine intestine.

α-galactosidase is a lysosomal enzyme that catalyzes the hydrolysis of the glycolipid ceramide trihexoside (GL-3) into galactose and ceramide dihexoside. Fabry disease is a rare, inherited lysosomal storage disease characterized by subnormal α-galactosidase activity and the resulting accumulation of GL-3. α-Galactosidase is a human α-galactosidase A produced by a human cell line. β-Galactosidase is a recombinant human α-galactosidase expressed in a CHO cell line. It is administered by intravenous infusion every other week at a dose of 0.2 mg/kg to treat Fabry disease and is used off-label for the treatment of Gaucher disease. It is administered by intravenous infusion every other week at a dose of 1.0 mg/kg body weight. Other lysosomal enzymes may also be used. For example, the protein may be a lysosomal enzyme described in US 2012/0148556.

Rasburicase is a recombinant uricase indicated for the initial control of plasma uric acid levels in children and adults with malignant leukemias, lymphomas, and solid tumors who are receiving anticancer therapy that is expected to result in tumor lysis and subsequent elevation of plasma uric acid. It is administered at a dose of 0.2 mg/kg by daily intravenous infusion.

Imiglucerase is a recombinant analog of human β-glucocerebrosidase. The initial dose is 2.5 U/kg body weight three times per week, increasing to 60 U/kg every two weeks. It is administered by intravenous infusion.

Abraxane, albumin conjugated to paclitaxel, is approved for metastatic breast cancer, non-small cell lung cancer, and advanced pancreatic cancer.

Taliglucerase alfa is an enzyme specific for hydrolyzing lysosomal glucocerebrosides and is indicated for long-term enzyme replacement therapy in Gaucher disease type 1. The recommended dose is 60 units/kg body weight administered via intravenous infusion every 2 weeks.

Laronidase is a polymorphic variant of human α-L-iduronidase produced in a CHO cell line. The recommended dosage regimen of laronidase is 0.58 mg/kg administered once weekly by intravenous infusion.

Elosulfase α is a human N-acetylgalactosamine-6-sulfatase produced in a CHO cell line by BioMarin Pharmaceuticals Inc ("BioMarin"). It was approved by the FDA on February 14, 2014, for the treatment of mucopolysaccharidosis type IVA. It is administered weekly via intravenous infusion at a dose of 2 mg/kg.

Other biologics that can be formulated with viscosity-lowering water-soluble dyes include asparaginase, erwinia chrysanthemi, cobotulinumtoxin A (epoetin alfa), (epoetin alfa), (darbepoetin alfa), (abatacept), (interferon beta-1b), (galactosidase); (idursulfatase); (alpha-glucosidase); (velaglucerase), abobotulinumtoxin A from Baxter's BAX-326, octocogol alfa; syncria from GlaxoSmithKline; liprotamase from Eli Lilly; xiaflex (collagenase from Clostridium histolyticum) from Auxilium and BioSpecifics Technologies Corp.; anakinra from SwedishOrphan Biovitrum AB; metreleptin from Bristol-Myers Squibb; avonex, plegridy (BIIB017) from Biogen; and dapoxetine (Dapoxetine) from Novo. NN1841 and NN7008 from Nordisk; KRN321 (darbepoetin alfa), AMG531 (romiplostim), KRN125 (pegfilgrastim), and KW-0761 (mogamulizumab) from Kyowa; IB1001 from Inspiration Biopharmaceuticals; and iprivask from Canyon Pharmaceuticals Group.

Protein therapeutics in development

Versartis, Inc.'s VRS-317 is a recombinant human growth hormone (hGH) fusion protein using XTEN half-life extension technology. It is intended to reduce the frequency of hGH injections required for patients with hGH deficiency. VRS-317 has completed a Phase II study comparing its efficacy to daily injections of underivatized hGH, with positive results. A Phase III study is planned.

Vibriolysin is a proteolytic enzyme secreted by the Gram-negative marine microorganism Vibrio proteolyticus. This endoprotease has a specific affinity for hydrophobic regions of proteins and is capable of cleaving proteins adjacent to hydrophobic amino acids. Vibriolysin is currently being studied by Biomarin for the debridement and/or treatment of burns. Vibriolysin preparations are described in patent WO 02/092014.

PEG-PAL (PEGylated recombinant phenylalanine ammonia lyase or "PAL") is an investigational enzyme replacement therapy for the treatment of phenylketonuria (PKU), an inherited metabolic disorder caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). PEG-PAL is being developed as a potential treatment for patients whose blood phenylalanine (Phe) levels are not adequately controlled. PEG-PAL is currently in Phase 2 clinical development for patients who have not adequately responded to treatment.

Other protein therapeutics that can be formulated with water-soluble dyes that reduce viscosity include Alprolix/rFIXFc, Eloctate/rFVIIIFc, BMN-190; BMN-250; Lamazyme; Galazyme; ZA-011; Sebelipase α; SBC-103; and HGT-1110. In addition, fusion proteins containing XTEN half-life extension technology can be formulated with water-soluble dyes that reduce viscosity, including but not limited to VRS-317GH-XTEN; coagulation factor VIIa, coagulation factor VIII, coagulation factor IX; PF05280602, VRS-859; Exenatide-XTEN; AMX-256; GLP2-2G/XTEN; and AMX-179 folate-XTEN-DM1.

Other late-stage protein therapeutics that can be formulated with viscosity-lowering water-soluble dyes include CM-AT from CureMark LLC; NN7999, NN7088, liraglutide (NN8022), NN9211, semaglutide (NN9535) from Novo Nordisk; AMG 386, filgrastim from Amgen; CSL-654, coagulation factor VIII from CSL Behring; LA-EP2006 (pegylated filgrastim biosimilar) from Novartis AG; Multikine (interleukin) from CEL-SCI Corporation; LY2605541, teriparatide (recombinant PTH 1-34) from Eli Lilly; NU-100 from Nuron Biotech, Inc.; and Sigma-Tau. Calaspargase Pegol from Polaris Pharmaceuticals, Inc.; ADI-PEG-20 from Polaris Pharmaceuticals, Inc.; BMN-110, BMN-702 from BioMarin; NGR-TNF from Molmed S.p.A.; recombinant human C1 esterase inhibitor from Pharming Group/Santarus Inc.; growth hormone biosimilar from LG Life Sciences LTD; Natpara from NPS Pharmaceuticals, Inc.; ART123 from Asahi Kasei Corporation; BAX-111 from Baxter; OBI-1 from Inspiration Biopharmaceuticals; Wilate from Octapharma AG; Talactoferrin α from Agennix AG; Desmoteplase from Lundbeck; Cinryze from Shire; RG7421 from Roche and Exelixis, Inc.; and RG7421 from Novartis. AG's midostaurin (PKC412); Damoctocog alpha pegol, BAY 86-6150, BAY 94-9027; Bristol-Myers Squibb's pegylated interferon lambda-1a, Nulojix (Belatacept); Merck KGaA's Pergoveris, corifollitropin alpha (MK-8962); Biogen's recombinant coagulation factor IX Fc fusion protein (rFIXFc; BIIB029) and recombinant coagulation factor VIII Fc fusion protein (rFVIIIFc; BIIB031); and AstraZeneca's Myalept.

Other early-stage protein biologics that can be formulated with viscosity-lowering water-soluble dyes include Alferon LDO from Hemispherx BioPharma, Inc.; SL-401 from Stemline Therapeutics, Inc.; PRX-102 from Protalix Biotherapeutics, Inc.; KTP-001 from Kaketsuken/Teijin Pharma Limited; Vericiguat from Bayer AG; BMN-111 from BioMarin; ACC-001 (PF-05236806) from Janssen; LY2510924, LY2944876 from Eli Lilly; NN9924 from Novo Nordisk; INGAP peptide from Exsulin; ABT-122 from Abbvie; AZD9412 from AstraZeneca; NEUBLASTIN (BG00010) from Biogen; and VEGF (BioNTech) from Celgene. Corporation's Luspatercept (ACE-536), Sotatercept (ACE-011); PRAME immunotherapeutic from GlaxoSmithKline; Plovamer acetate (PI-2301) from Merck KGaA; PREMIPLEX (607) from Shire; BMN-701 from BioMarin; Ontak from Eisai; rHuPH20/insulin from Halozyme, Inc.; PB-1023 from PhaseBio Pharmaceuticals, Inc.; ALV-003 from Alvine Pharmaceuticals Inc. and Abbvie; NN8717 from Novo Nordisk; PRT-201 from Proteon Therapeutics Inc.; PEGPH20 from Halozyme, Inc.; and alefacept; F-627 from Regeneron; AGN-214868 (senrebotase) from Allergan, Inc.; BAX-817 from Baxter; PRT4445 from Portola Pharmaceuticals, Inc.; VEN100 from Ventria Bioscience; Onconase/ranpirnase from Tamir Biotechnology Inc.; Interferon alpha-2b fusion protein from Medtronic, Inc.; sebelipase alpha from Synageva BioPharma; IRX-2 from IRX Therapeutics, Inc.; GSK2586881 from GlaxoSmithKline; and phenobarbital from Seikagaku Corporation's SI-6603; ALXN1101, asfotase α from Alexion; SHP611, SHP609 (elaprase, idursulfase) from Shire; PF-04856884, PF-05280602 from Pfizer; ACE-031, Dalantercept from Acceleron Pharma; ALT-801 from Altor BioScience Corp.; BA-210 from BioAxone Biosciences, Inc.; WT1 immunotherapeutics from GlaxoSmithKline; GZ402666 from Sanofi; MSB0010445, Atacicept from Merck KGaA; Leukine (sargramostim) from Bayer AG; KUR-211 from Baxter; and from CardioVascular BioTherapeutics. Inc.'s fibroblast growth factor-1; SPI-2012 from Hanmi Pharmaceuticals Co., LTD/Spectrum Pharmaceuticals; FGF-18 (sprifermin) from Merck KGaA; MK-1293 from Merck; interferon-α-2b from HanAll Biopharma; CYT107 from Cytheris SA; RT001 from Revance Therapeutics, Inc.; MEDI6012 from AztraZeneca; E2609 from Biogen; BMN-190 and BMN-270 from BioMarin; ACE-661 from Acceleron Pharma; AMG 876 from Amgen; GSK3052230 from GlaxoSmithKline; RG7813 from Roche; SAR342434 and Lantus from Sanofi; and HER2 from Allozyne. Inc.'s AZ01; Ambrx, Inc.'s ARX424; FivePrime Therapeutics, Inc.'s FP-1040 and FP-1039; Merck KGaA's ATX-MS-1467; Amunix Operating Inc.'s XTEN fusion protein; Cleveland BioLabs, Inc.'s entolimod (CBLB502); Shire's HGT2310; Hanmi Pharmaceuticals Co., LTD.'s HM10760A; Alexion's ALXN1102/ALXN1103; CSL-689 and CSL-627; Acorda Therapeutics, Inc.'s glial growth factor 2; Nephrx Corporation's NX001; and Novo Nordisk's NX001. NN8640, NN1436, NN1953, NN9926, NN9927, NN9928 from Nordisk; NHS-IL 12 from EMD Serono; 3K3A-APC from ZZ Biotech LLC; PB-1046 from PhaseBioPharmaceuticals, Inc.; RU-101 from R-Tech Ueno, Ltd.; insulin lispro/BC106 from Adocia; hl-con1 from Iconic Therapeutics, Inc.; PRT-105 from Protalix BioTherapeutics, Inc.; PF-04856883, CVX-096 from Pfizer; ACP-501 from AlphaCorePharma LLC; BAX-855 from Baxter; and from Celldex CDX-1135 from the Therapeutics; PRM-151 from Promedior, Inc.; TS01 from Thrombolytic Science International; TT-173 from Thrombotargets Corp.; QBI-139 from Quintessence Biosciences, Inc.; Vatelizumab, GBR500, GBR600, GBR830, and GBR900 from Glenmark Pharmaceuticals; and CYT-6091 from Cytimmune Sciences, Inc.

Other biological drugs

Other biopharmaceuticals that can be formulated with viscosity-lowering water-soluble dyes include PF-05285401, PF-05231023, RN317 (PF-05335810), PF-06263507, PF-05230907, Dekavil, PF-06342674, PF06252616, RG7598, RG7842, RG7624d, OMP54F28, GSK1995057, BAY1179470, IMC-3G from Pfizer. 3. IMC-18F1, IMC-35C, IMC-20D7S, PF-06480605, PF-06647263, PF-06650808, PF-05335810 (RN317), PD-0360324, PF-00547659; MK-8237 from Merck; BI033 from Biogen; GZ402665, SAR438584/REGN2222 from Sanofi; IMC-18F1 from ImClone LLC's Icrucumab, IMC-3G3; Ryzodeg, Tresiba, Xultophy from Novo Nordisk; Toujeo (U300), LixiLan, Lyxumia (lixisenatide) from Sanofi; MAGE-A3 immunotherapeutic from GlaxoSmithKline; Tecemotide from Merck KGaA; Sereleaxin (RLX030) from Novartis AG; erythropoietin; pegfilgrastim; LY2963016, dulaglutide (LY2182965) from Eli Lilly; and insulin glargine from Boehringer Ingelheim.

B. Medicinal water-soluble organic dyes

The viscosity of the liquid protein formulation that comprises low molecular weight and/or high molecular weight protein is reduced by adding one or more water-soluble organic dyes.Can be by adding one or more water-soluble organic dyes of effective dose, pharmaceutical preparation is transformed into Newtonian fluid by non-Newtonian fluid." water-soluble organic dye " is following organic molecule, and it has the molar solubility of at least 0.001M and absorbs the light of some wavelengths of light preferably electromagnetic spectrum to infrared part simultaneously but can transmit or reflect the light of other wavelengths.Water-soluble organic dye can be acridine dye, anthraquinone dye, diarylmethane dye, triarylmethane dye, azo dye, diazo dye, nitrophenyl dye, nitrosophenyl dye, phthalocyanine dye, quinone dye, thiazole dye, xanthene dye or its combination.Organic dye can be salt or zwitterion.

While generally any water-soluble organic dye can reduce the viscosity of a protein formulation, in some embodiments, the organic dye has a molar absorptivity in the visible to infrared portion of the electromagnetic spectrum greater than 500 ^M-1 cm ^-1 , greater than 1,000 M ^{-1 cm-1, greater than 10,000 M - 1} cm- ¹ , greater than 20,000 ^M-1 cm ^-1 , or greater than 50,000 ^M-1 cm ^-1 .

The organic dye may have a fused ring structure of Formula I, wherein X is a carbon atom or a heteroatom, optionally with one or more substituents; and each A is independently a substituted or unsubstituted aryl group having 3 to 50 carbon atoms, 3 to 30 carbon atoms, or 6 to 25 carbon atoms. It should be understood that X may have one or more hydrogen atoms or other substituents to satisfy the valence. For example, when X is carbon, X may be a CH group, a _CH2 group, a CHR group, a CR group, or a _CR2 group, wherein each R is independently any organic group having any number of carbon atoms.

In some embodiments, the organic dye has a structure of Formula I, wherein X is C, N, O, or S; or wherein A is a substituted or unsubstituted phenyl or naphthyl, or both. An example of such a compound is acridine, wherein X is a CH group and each A is an unsubstituted phenyl group.

The organic dye may be an acridine dye. The organic dye may have a structure of Formula II:

Each R ¹ is independently selected from hydrogen, R ² , -OH, NH ₂ , -F, -Cl, -Br, -I, -NO ₂ , -CN, -C(=O)R ^4a , -C(=NR ^4a )R ⁴ , -C(=O)OH, -C(=O)OR ⁴ , -OC(=O)R ⁴ , -OC(=O)OR ⁴ , -SO ₃ H, -SO ₂ N(R ^4a ) ₂ , -SO ₂ R ⁴ , -SO ₂ NR ^4a C(=O)R ⁴ , -PO ₃ H ₂ , -R ^4a C(=NR ^4a )N(R ^4a ) ₂ , -NHC(=NR ^4a )NH-CN, -NR ^4a C(=O)R ⁴ , -NR ^4a SO ₂ R ⁴ ,-NR ^4a C(=NR ^4a )NR ^4a C(=NR ^4a )N(R ^4a ) ₂ , -NR ^4a C(=O)N(R ^4a ) ₂ , -C(=O)NH ₂ , -C(=O)N(R ^4a ) ₂ , -OR ⁴ , -SR ^4a and -N(R ^4a ) ₂ ;

wherein R ² is independently selected from C _1-12 alkyl, C _3-12 cycloalkyl, C _6-12 aryl, C _1-12 heteroaryl and C _2-12 heterocyclyl,

wherein each C _1-12 alkyl group may be replaced by a C _3-12 cycloalkyl group, a C _6-12 aryl group, a C _1-12 heteroaryl group, a C _2-12 heterocyclyl group, -OH, NH ₂ , (═O), (═NR ^4a ), -F, -Cl, -Br, -I, -NO ₂ , -CN, -C(═O)R ^4a , -C(═NR ^4a )R ⁴ , -C(═O)OH, -C(═O)OR ⁴ , -OC(═O)R ⁴ , -OC(═O)OR ⁴ , -SO ₃ H , -SO ₂ N(R ^4a ) ₂ , -SO ₂ R ⁴ , -SO ₂ NR ^4a C(═O)R ⁴ , -PO ₃ H ₂ , -R ^4a C(═NR ^4a )N(R ^4a ) ₂ , -NHC(＝NR ^4a )NH-CN, -NR ^4a C(＝O)R ⁴ , -NR ^4a SO ₂ R ⁴ , -NR ^4a C(＝NR ^4a )NR ^4a C(＝NR ^4a )N(R ^4a ) ₂ , -NR ^4a C(＝O)N(R ^4a ) ₂ , -C(＝O)NH ₂ , -C(＝O)N(R ^4a ) ₂ , -OR ⁴ , -SR ^4a or -N(R ^4a ) ₂ ;

wherein each C _3-12 cycloalkyl group may be replaced by a C _1-12 alkyl group, a C _6-12 aryl group, a C _1-12 heteroaryl group, a C _2-12 heterocyclyl group, -OH, NH ₂ , -F, -Cl, -Br, -I, -NO ₂ , -CN, -C(═O)R ^4a , -C(═NR ^4a )R ⁴ , -C(═O)OH, -C(═O)OR ⁴ , -OC(═O)R ⁴ , -OC(═O)OR ⁴ , -SO ₃ H , -SO ₂ N(R ^4a ) ₂ , -SO ₂ R ⁴ , -SO ₂ NR ^4a C(═O)R ⁴ , -PO ₃ H ₂ , -R ^4a C(═NR ^4a )N(R ^4a ) ₂ , -NHC(═NR ^4a )NH-CN, -NR ^4a C(＝O)R ⁴ , -NR ^4a SO ₂ R ⁴ , -NR ^4a C(＝NR ^4a )NR ^4a C(＝NR ^4a )N(R ^4a ) ₂ , -NR ^4a C(＝O)N(R ^4a ) ₂ , -C(＝O)NH ₂ , -C(＝O)N(R ^4a ) ₂ , -OR ⁴ , -SR ^4a or -N(R ^4a ) ₂ ;

wherein each C _6-12 aryl group may be replaced by a C _1-12 alkyl group, a C _3-12 cycloalkyl group, a C _1-12 heteroaryl group, a C _2-12 heterocyclyl group, -OH, NH ₂ , -F, -Cl, -Br, -I, -NO ₂ , -CN, -C(═O)R ^4a , -C(═NR ^4a )R ⁴ , -C(═O)OH, -C(═O)OR ⁴ , -OC(═O)R ⁴ , -OC(═O)OR ⁴ , -SO ₃ H , -SO ₂ N(R ^4a ) ₂ , -SO ₂ R ⁴ , -SO ₂ NR ^4a C(═O)R ⁴ , -PO ₃ H ₂ , -R ^4a C(═NR ^4a )N(R ^4a ) ₂ , -NHC(═NR ^4a )NH-CN, -NR ^4a C(＝O)R ⁴ , -NR ^4a SO ₂ R ⁴ , -NR ^4a C(＝NR ^4a )NR ^4a C(＝NR ^4a )N(R ^4a ) ₂ , -NR ^4a C(＝O)N(R ^4a ) ₂ , -C(＝O)NH ₂ , -C(＝O)N(R ^4a ) ₂ , -OR ⁴ , -SR ^4a or -N(R ^4a ) ₂ ;

wherein each C _1-12 heteroaryl group may be replaced by a C _1-12 alkyl group, a C _3-12 cycloalkyl group, a C _6-12 aryl group, a C _2-12 heterocyclyl group, -OH, NH ₂ , -F, -Cl, -Br, -I, -NO ₂ , -CN, -C(═O)R ^4a , -C(═NR ^4a )R ⁴ , -C(═O)OH, -C(═O)OR ⁴ , -OC(═O)R ⁴ , -OC(═O)OR ⁴ , -SO ₃ H , -SO ₂ N(R ^4a ) ₂ , -SO ₂ R ⁴ , -SO ₂ NR ^4a C(═O)R ⁴ , -PO ₃ H ₂ , -R ^4a C(═NR ^4a )N(R ^4a ) ₂ , -NHC(═NR ^4a )NH-CN, -NR ^4a C(＝O)R ⁴ , -NR ^4a SO ₂ R ⁴ , -NR ^4a C(＝NR ^4a )NR ^4a C(＝NR ^4a )N(R ^4a ) ₂ , -NR ^4a C(＝O)N(R ^4a ) ₂ , -C(＝O)NH ₂ , -C(＝O)N(R ^4a ) ₂ , -OR ⁴ , -SR ^4a or -N(R ^4a ) ₂ ;

wherein each C _2-12 heterocyclic group may be replaced by a C _1-12 alkyl group, a C _3-12 cycloalkyl group, a C _6-12 aryl group, a C _1-12 heteroaryl group, -OH, NH ₂ , -F, -Cl, -Br, -I, -NO ₂ , -CN, -C(═O)R ^4a , -C(═NR ^4a )R ⁴ , -C(═O)OH, -C(═O)OR ⁴ , -OC(═O)R ⁴ , -OC(═O)OR ⁴ , -SO ₃ H , -SO ₂ N(R ^4a ) ₂ , -SO ₂ R ⁴ , -SO ₂ NR ^4a C(═O)R ⁴ , -PO ₃ H ₂ , -R ^4a C(═NR ^4a )N(R ^4a ) ₂ , -NHC(═NR ^4a )NH-CN, -NR ^4a C(＝O)R ⁴ , -NR ^4a SO ₂ R ⁴ , -NR ^4a C(＝NR ^4a )NR ^4a C(＝NR ^4a )N(R ^4a ) ₂ , -NR ^4a C(＝O)N(R ^4a ) ₂ , -C(＝O)NH ₂ , -C(＝O)N(R ^4a ) ₂ , -OR ⁴ , -SR ^4a or -N(R ^4a ) ₂ ;

R ⁴ is independently selected from C _1-12 alkyl, C _3-12 cycloalkyl, C _6-12 aryl, C _1-12 heteroaryl, and C _2-12 heterocyclyl, each of which may be substituted one or more times with -OH, -NH ₂ , -F, -Cl, -Br, -I, -NO ₂ , -CN, -C(=O)OH, -SO ₃ H, -PO ₃ H ₂ , or -C(=O)NH ₂ ;

R ^4a may be R ⁴ or hydrogen;

Any two or more of the R ² , R ³ , R ⁴ and R ^4a groups may together form a ring.

In some embodiments, at least one, at least two, or at least three occurrences of R ¹ are not hydrogen. In some embodiments, at least one, at least two, at least three, or at least four occurrences of R ¹ are amine groups such as dimethylamine or other dialkylamines. Other preferred R ¹ groups include NO ₂ , SO ₃ H, and CO ₂ H.

The water-soluble organic dye can also be anthraquinone dye:

Diarylmethane dyes:

or triarylmethane dyes:

wherein R ¹ has the above meaning. In other embodiments, the water-soluble organic dye may be an azo dye represented by the following formula:

wherein R ¹ is as defined above, and R ^a is an aryl or heteroaryl ring. Exemplary ring systems include:

wherein X is O, S, SO ₂ or NR ² , X ¹ may be a nitrogen atom or CR ¹ , and R ² is as defined above.

The water-soluble organic dye may also be a nitrophenyl dye, a nitrosophenyl dye, a phthalocyanine dye, a quinone dye, a triazole dye or a xanthene dye.

Exemplary dyes include, but are not limited to, Yellow 5, Orange G, Quinolone Yellow, Betanin, Red 40, carmosin, Azure C, Congo Red, amaranth, Ponceau S, erythrosine, patent blue, brilliant black BN, acid fuchsine, napthol yellow S, quinoline yellow, indigo carmine, fast green FCF, Orange 2, Natural Red, Xylene Cyanol FF, Cresyl violet acetate, Light Green SF Yellowish, Thiazolyl Yellow G, and the like. G), crystal violet, Nile blue A, and indocyanine green (Cardiogreen).

C. Excipients

A wide variety of pharmaceutical excipients that can be used in liquid protein formulations are known to those skilled in the art. These include one or more additives such as liquid solvents or cosolvents; sugars or sugar alcohols such as mannitol, trehalose, sucrose, sorbitol, fructose, maltose, lactose, or dextran; surfactants such as 20, 60, or 80 (polysorbate 20, 60, or 80); buffers; preservatives such as benzalkonium chloride, benzethonium chloride, tertiary ammonium salts, and chlorhexidine diacetate; carriers such as poly(ethylene glycol) (PEG); antioxidants such as ascorbic acid, sodium metabisulfite, and methionine; chelating agents such as EDTA or citric acid; or biodegradable polymers such as water-soluble polyesters; cryoprotectants; lyoprotectants; bulking agents; and stabilizers.

Other pharmaceutically acceptable carriers, excipients, or stabilizers such as those described in Remington: "The Science and Practice of Pharmacy", 20th edition, Alfonso R. Gennaro, Ed., Lippincott Williams & Wilkins (2000) may also be included in the protein formulations described herein, provided that they do not adversely affect the desired properties of the formulation.

The viscosity-lowering water-soluble dyes described herein can be combined with one or more other types of viscosity-lowering substances, for example, typical generally positive organic compounds, such as hydrophobic compounds, various substances on the GRAS (U.S. Food and Drug Administration's list of compounds generally regarded as safe) list and inactive injectable ingredients and FDA-approved therapeutic agents; viscosity-lowering agents described in a PCT application co-filed by Arsia Therapeutics entitled "LIQUID PROTEIN FORMULATIONS CONTAINING VISCOSITY-LOWERING AGENTS"; ionic liquid viscosity-lowering agents described in a PCT application co-filed by Arsia Therapeutics entitled "LIQUID PROTEIN FORMULATIONS CONTAINING IONIC LIQUIDS"; and organophosphate viscosity-lowering agents described in a PCT application co-filed by Arsia Therapeutics entitled "LIQUID PROTEIN FORMULATIONS CONTAINING ORGANOPHOSPHATES."

III. Preparation Method

The protein to be formulated, such as a monoclonal antibody, can be produced by any known technique, such as by culturing cells transformed or transfected with a vector containing one or more nucleic acid sequences encoding the protein, as is known in the art, or by synthetic techniques (such as recombinant techniques and peptide synthesis or a combination of these techniques) or can be isolated from an endogenous source of the protein.

Purification of the protein to be formulated can be carried out by any suitable technique known in the art, such as ethanol or ammonium sulfate precipitation, reverse phase HPLC, chromatography on silica gel or cation exchange resins (e.g., DEAE-cellulose), dialysis, chromatofocusing, gel filtration using a Protein A column (e.g., G-75) to remove impurities, metal chelating columns that bind to epitope-tagged forms, and ultrafiltration/diafiltration (non-limiting examples include centrifugal filtration and tangential flow filtration (TFF)).

The viscosity-lowering water-soluble organic dye introduced at a viscosity-lowering concentration, such as 0.010 M to 1.0 M, preferably 0.050 M to 0.50 M, most preferably 0.01 M to 0.10 M, allows for the purification and/or concentration of solutions of pharmaceutically active monoclonal antibodies to higher monoclonal antibody concentrations using conventional methods known to those skilled in the art, including but not limited to tangential flow filtration, centrifugal concentration, and dialysis.

In some embodiments, the lyophilized formulations of protein are provided and/or used in the preparation and manufacture of concentrated low-viscosity protein formulations. In some embodiments, the pre-lyophilized protein in powder form is redissolved by being dissolved in the aqueous solution. In this embodiment, the liquid formulation is filled into a specific dosage unit container such as a bottle or a pre-filled hybrid syringe, freeze-dried, optionally together with a lyoprotectant, preservative, antioxidant and other typical pharmaceutical excipients, then stored under sterile storage conditions until before use, now redissolved with a defined volume of diluent so that the liquid reaches desired concentration and viscosity.

The formulations described herein can be stored by any suitable method known to those skilled in the art. Non-limiting examples of methods for preparing protein formulations for storage include freezing, lyophilizing, and spray drying the liquid protein formulation. In some cases, lyophilized formulations are stored frozen at subzero temperatures, such as at about -80°C or in liquid nitrogen. In some cases, lyophilized or aqueous formulations are stored at 2-8°C.

Non-limiting examples of diluents that can be used to reconstitute the lyophilized formulation prior to injection include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, dextrose solution, or aqueous solutions of salts and/or buffers. In some cases, the formulation is spray dried and then stored.

IV. Administration to a Subject in Need thereof

The protein formulations, including but not limited to reconstituted formulations, are administered to a human in need thereof by intramuscular, intraperitoneal (i.e., injection into a body cavity), intracerebrospinal, or subcutaneous injection using an 18-32 gauge needle (optionally a thin-walled needle) in a volume of less than about 5 mL, less than about 3 mL, preferably less than about 2 mL, and more preferably less than about 1 mL.

The appropriate dosage ("therapeutically effective amount") of a protein, such as a monoclonal antibody, will depend on the severity and course of the condition, disease or disorder to be treated, whether the protein is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the protein, the type of protein used, and the judgment of the attending physician. The protein is suitably administered all at once in single or multiple injections or over a series of treatments as a monotherapy or in combination with other drugs or therapies.

The dosage formulation is designed so that the injection does not cause significant signs of irritation at the injection site, for example, wherein the major irritation index is less than 3 when evaluated using the Draize scoring system. In a selectable embodiment, the injection causes a visually similar level of irritation when compared to an injection of an equal volume of saline solution. In another embodiment, the bioavailability of the protein is higher when compared to an otherwise identical formulation administered in the same manner but not containing one or more water-soluble organic dyes that reduce viscosity. In another embodiment, the formulation is pharmaceutically effective at least roughly as effective as the protein administered by intravenous infusion at about the same dose.

In a preferred embodiment, the formulation is injected to obtain an increased level of the therapeutic protein. For example, the AUC value can be at least 10%, preferably at least 20%, higher than the AUC value calculated for an otherwise identical formulation administered in the same manner but without the one or more viscosity-lowering water-soluble organic dyes.

One or more water-soluble dyes that reduce viscosity can also affect bioavailability. For example, the percent bioavailability of a protein can be at least 1.1 times, preferably at least 1.2 times, the percent bioavailability of an otherwise identical formulation administered in the same manner but without the one or more water-soluble dyes that reduce viscosity.

One or more viscosity-lowering water-soluble dyes may also affect pharmacokinetics. For example, the _CMAX following subcutaneous or intramuscular injection may be at least 10%, preferably at least 20%, lower than the _CMAX of a substantially equivalent pharmaceutically effective intravenous dose.

In some embodiments, the protein is administered at a higher dose and less frequently than an otherwise identical formulation that does not contain the one or more viscosity-lowering water-soluble organic dyes.

Preparations with lower viscosity require less injection force. For example, the injection force can be at least 10% less, preferably at least 20% less, than the injection force required for the preparation administered in the same manner that is otherwise identical but does not contain one or more water-soluble organic dyes that reduce viscosity. In one embodiment, injection is performed with a 27-gauge needle, and the injection force is less than 30N. In most cases, the preparation can be administered using a very small needle, for example, 27 to 31, typically 27, 29, or 31.

One or more viscosity-lowering water-soluble organic dyes can be used to prepare dosage unit formulations suitable for reconstitution to form liquid pharmaceutical formulations for subcutaneous or intramuscular injection. The dosage unit may contain a dry powder of one or more proteins; one or more viscosity-lowering water-soluble organic dyes; and other excipients. The protein is present in the dosage unit such that, upon reconstitution in a pharmaceutically acceptable solvent, the resulting formulation has a protein concentration (mg/mL) of about 100 mg to about 2,000 mg/mL. Such reconstituted formulations may have an absolute viscosity of about 1 cP to about 50 cP at 25°C.

Low viscosity formulations can be provided as solutions or dosage units in which the protein is lyophilized in one vial with or without one or more water-soluble organic dyes to reduce viscosity and other excipients, and the solvent, with or without one or more water-soluble organic dyes to reduce viscosity and other excipients, is provided in a second vial. In this embodiment, the solvent is added to the protein immediately before or at the time of injection to ensure uniform mixing and dissolution.

One or more water-soluble organic dyes that reduce viscosity are present in the formulation at concentrations that do not cause significant toxicity signs and/or irreversible toxicity signs when administered via subcutaneous, intramuscular, or other injection types." significant toxicity signs " as used herein include poisoning, lethargy, behavioral changes such as those that occur when the central nervous system is damaged, infertility, severe cardiotoxicity signs such as arrhythmias, cardiomyopathy, myocardial infarction, and cardiogenic or congestive heart failure, renal failure, liver failure, dyspnea, and death.

In preferred embodiments, the formulation does not cause significant irritation when administered no more than twice a day, once a day, twice a week, once a week, or once a month. Administration of the protein formulation may result in no significant signs of irritation at the injection site as measured by a major irritation index of less than 3, less than 2, or less than 1 when evaluated using the Draize scoring system. As used herein, "significant signs of irritation" include erythema, redness, and/or swelling greater than 10 cm, greater than 5 cm, or greater than 2.5 cm in diameter at the injection site, necrosis at the injection site, exfoliative dermatitis at the injection site, and severe pain that interferes with daily activities and/or requires medical attention or hospitalization. In some embodiments, injection of the protein formulation results in visually similar levels of irritation when compared to injection of an equal volume of saline solution.

When administered via subcutaneous or intramuscular injection, the protein formulation can exhibit improved bioavailability compared to formulations that are otherwise identical but do not contain one or more water-soluble organic dyes that reduce viscosity. "Bioavailability" refers to the degree and rate at which a bioactive substance, such as a monoclonal antibody, enters the circulation or reaches the site of action. The overall bioavailability of a subcutaneous or intramuscular injection is improved compared to formulations that are otherwise identical but do not contain one or more water-soluble organic dyes that reduce viscosity. "Percent bioavailability" refers to the fraction of a dose of a bioactive substance that enters the circulation, which is determined relative to an intravenous dose. One way to measure bioavailability is by comparing the "area under the curve" (AUC) of a function curve of plasma concentration versus time. AUC can be calculated, for example, using the linear trapezoidal rule. "AUC _∞ " used herein refers to the area under the plasma concentration curve from time zero to the time when plasma concentration returns to baseline. "AUC _0-t " used herein refers to the area under the plasma concentration curve from time zero to a later time t, for example, to the time when baseline is reached. Time is typically measured in days, although hours may also be used when the context is clear. For example, the AUC may be increased by more than 10%, 20%, 30%, 40% or 50% compared to an otherwise identical formulation administered in the same manner but lacking one or more viscosity-lowering water-soluble organic dyes.

As used herein, "t _max " refers to the time after administration at which the plasma concentration reaches a maximum.

As used herein, " _Cmax " refers to the maximum plasma concentration following administration of a dose and prior to administration of a subsequent dose.

As used herein, "C _min " or "C _trough " refers to the minimum plasma concentration following administration of a dose and prior to administration of the subsequent dose.

The _Cmax following subcutaneous or intramuscular injection may be, for example, at least 10%, more preferably at least 20%, less than the _Cmax of the intravenously administered dose. This reduction in _Cmax may also result in reduced toxicity.

Pharmacokinetic and pharmacodynamic parameters can be approximated across species using measures known to those skilled in the art. The pharmacokinetics and pharmacodynamics of antibody therapeutics can be significantly different based on the specific antibody. The half-life of approved mouse monoclonal antibodies in humans is about 1 day, while the half-life of human monoclonal antibodies will generally be about 25 days (Waldmann et al., Int. Immunol., 2001, 13: 1551-1559). The pharmacokinetics and pharmacodynamics of antibody therapeutics can be significantly different based on the route of administration. The time to maximum plasma concentration after intramuscular or subcutaneous injection of IgG is generally 2 to 8 days, although shorter or longer times may occur (Wang et al., Clin. Pharm. Ther., 2008, 84 (5): 548-558). The pharmacokinetics and pharmacodynamics of antibody therapeutics can be significantly different based on the formulation.

Low viscosity protein formulations can allow greater flexibility in administration and reduce the frequency of administration compared to those protein formulations that do not contain one or more water-soluble organic dyes that reduce viscosity. For example, by increasing the dose per injection multiple-fold, the frequency of administration can be reduced from once every 2 weeks to once every 6 weeks in some embodiments.

Protein formulations, including but not limited to reconstituted formulations, can be administered using heated and/or self-mixing syringes or autoinjectors. Protein formulations can also be preheated in a separate warming unit before filling the syringe.

i. Heated syringe

The heated syringe can be a standard syringe that is preheated using a syringe warming device. The syringe warming device will typically have one or more openings that are each capable of receiving a syringe containing a protein formulation and a device for heating and maintaining the syringe at a specific temperature (usually above ambient temperature) before use. It will be referred to herein as a preheated syringe. Warming devices suitable for heated syringes include those available from Vista Dental Products and Inter-Med. The warming device can accommodate syringes of various sizes and heat to any temperature up to about 130°C (usually within 1°C). In some embodiments, the syringe is preheated in a heating bath, such as a water bath maintained at the desired temperature.

Heated syringe can be self-heating syringe promptly can heat the liquid preparation in syringe and be maintained at specific temperature.Self-heating syringe can also be its standard medical syringe attached with heating device.The suitable heating device that can be attached with syringe comprises the syringe heater or syringe heating belt available from Watlow Electric Manufacturing Co., St.Louis, MO and the syringe heating module, stage type heater and online pre-infusion heater such as SW-61 type syringe warmer available from Warner Instruments, Hamden, CT.Heater can be controlled by central controller, for example, can be available from TC-324B or TC-344B type heater controller of Warner Instruments.

The heated syringe maintains the liquid protein formulation at a specific temperature or a specific temperature that varies within 1°C, 2°C, or 5°C. The heated syringe can maintain the protein formulation at any temperature from room temperature up to about 80°C, up to about 60°C, up to about 50°C, or up to about 45°C, as long as the protein formulation is sufficiently stable at that temperature. The heated syringe can maintain the protein formulation at a temperature of 20°C to 60°C, 21°C to 45°C, 22°C to 40°C, 25°C to 40°C, or 25°C to 37°C. By maintaining the protein formulation at an elevated temperature during injection, the viscosity of the liquid formulation is reduced, the solubility of the protein in the formulation is increased, or both.

ii. Self-mixing syringe

The syringe can be self-mixing or can have an attached mixer. The mixer can be a static mixer or a dynamic mixer. Examples of static mixers include those disclosed in U.S. Patents 5,819,988, 6,065,645, 6,394,314, 6,564,972 and 6,698,622. Examples of some dynamic mixers may include those disclosed in U.S. Patents 6,443,612 and 6,457,609 and U.S. Patent Application Publication No. US2002/0190082. The syringe can include a plurality of barrels for mixing the components of the liquid protein formulation. U.S. Patent No. 5,819,998 describes a syringe with two barrels and a mixing tip for mixing a two-component viscous substance.

iii. Autoinjectors and prefilled syringes for protein formulations

Liquid protein formulations can be administered using pre-filled syringe auto-injectors or needle-free injection devices. Auto-injectors include handheld, typically pen-shaped barrel grips for gripping a replaceable pre-filled barrel and spring-based or similar mechanisms for subcutaneous or intramuscular injection of liquid drug doses from pre-filled barrels. Auto-injectors are typically designed for self-administration or administration by untrained personnel. Auto-injectors can be used to dispense a single dose or multiple doses from a pre-filled barrel. Auto-injectors can implement different user settings, particularly including injection depth, injection speed, etc. Other injection systems may include those described in U.S. Patent No. 8,500,681.

Lyophilized protein formulations can be provided in prefilled or unit dose syringes. U.S. Patents 3,682,174, 4,171,698 and 5,569,193 describe a sterile syringe containing two chambers, which can be prefilled with a dry formulation and a liquid that can be mixed immediately before injection. U.S. Patent 5,779,668 describes a syringe system for freeze-drying, reconstitution and administration of a pharmaceutical composition. In some embodiments, the protein formulation is provided in a prefilled or unit dose syringe in a lyophilized form, reconstituted in the syringe before administration and administered by a single subcutaneous or intramuscular injection. An automatic syringe for delivering a unit dose of lyophilized drug is described in WO 2012/010,832. Automatic syringes such as Safe Click Lyo ^™ (listed by Future Injection Technologies, Ltd., Oxford, UK) can be used for administering a unit dose of protein formulation, wherein the formulation is stored in a lyophilized form and reconstituted immediately before administration. In some embodiments, the protein formulation is provided in unit dose cartridges for lyophilized pharmaceuticals (sometimes referred to as Vetter cartridges). Examples of suitable cartridges may include those described in US Patents 5,334,162 and 5,454,786.

V. Purification and Concentration Methods

Water-soluble organic dyes can also be used to assist in protein purification and concentration. One or more water-soluble organic dyes and excipients are added to the protein in effective amounts to reduce the viscosity of the protein solution. For example, the water-soluble organic dye is added to a concentration of about 0.01 M to about 1.0 M, preferably about 0.01 M to about 0.50 M, more preferably about 0.01 M to 0.25 M, and most preferably about 0.01 M to about 0.10 M.

The protein-containing aqueous organic dye solution is then purified or concentrated using a method selected from ultrafiltration/diafiltration, tangential flow filtration, centrifugal concentration, and dialysis.

Example

The foregoing will be further understood by way of the following non-limiting examples.

After equilibration at 25°C for 5 minutes, the viscosity of all well-mixed aqueous monoclonal antibody solutions was measured using an mVROC microfluidic viscometer (RheoSense) or a DV2T cone-plate viscometer (Brookfield; "C&P") (unless otherwise stated). The mVROC viscometer was equipped with an "A" or "B" chip, each manufactured with a 50-micron channel. Typically, 0.10 mL of protein solution was rear-loaded into a gas-tight microlaboratory instrument syringe (Hamilton; 100 μL), fixed to the chip and measured at multiple flow rates (approximately 20%, 40%, and 60% of the maximum pressure of each chip). For example, a sample of approximately 50 cP was measured at approximately 10, 20, and 30 μL/min (approximately 180, 350, and 530 s ^-1 on the "A" chip, respectively) until the viscosity stabilized, typically after at least 30 seconds. The average absolute viscosity and standard deviation were then calculated from at least three such measurements. The C&P viscometer was equipped with a CPE40 or CPE52 shuttle bar (cone angles of 0.8° and 3.0°, respectively), and 0.50 mL of sample was measured at multiple shear rates from 2 to 400 s ⁻¹ . Specifically, samples were measured at 22.58, 24.38, 26.25, 28.13, 30, 31.88, 45, 67.5, 90, 112.5, 135, 157.5, 180, 202.5, 247, 270, 292.5, 315, 337.5, 360, 382, 400 s ⁻¹ for 30 seconds each, starting at a shear rate at which at least 10% torque was achieved and continuing until the instrument torque reached 100%. The extrapolated zero shear viscosity was then determined from a plot of dynamic viscosity versus shear rate for samples measured on the DV2T cone and plate viscometer. The extrapolated zero-shear viscosity reported is the average and standard deviation of at least three measurements.

Example 1: Water-soluble organic dyes reduce the viscosity of concentrated aqueous solutions of high molecular weight proteins

Commercially available biosimilars (100-400 mg) containing pharmaceutical excipients (polysorbate 20, phosphate and citrate buffer, mannitol, and NaCl) were purified. Polysorbate 20 was first removed using TWEEN Medi Columns (G-Biosciences). The resulting solution was subsequently buffer-exchanged into 20 mM sodium phosphate buffer (PB; pH 7.0) for PB samples and 2 mM PB (pH 7.0) for water-soluble dye samples and concentrated to a final volume of less than 10 mL on a Jumbosep centrifugal concentrator (Pall Corp.). The sample buffered into 2 mM PB was first divided into equal portions. An appropriate solution of a water-soluble organic dye (pH 7.0) was then added to each aliquot so that, after redissolving with water, the final excipient concentration was 0.03-0.1 M. The protein solution was then freeze-dried. The dried protein cake containing protein and water-soluble dye (and negligible amount of buffer salt) was reconstituted to a final volume of about 0.1 mL and the water-soluble dye concentration as described above. For samples in which the buffer was exchanged into 20 mM PB (PB control sample), the collected protein solution was freeze-dried. The dried protein cake containing protein and buffer salt was reconstituted to a final volume of about 0.10-0.50 mL. These samples were reconstituted using additional PB (pH 7.0) sufficient to give a final concentration of 0.25 M PB. The final concentration of the monoclonal antibody in the solution was determined by comparing samples of unknown concentration with a standard curve of the biosimilar using the Coomassie protein quantification method. The reported viscosity was measured on a RheoSense mVROC microfluidic viscometer. Formulations containing biosimilars and were also prepared using the same protocol.

The table below demonstrates that water-soluble organic dyes significantly reduce the viscosity of monoclonal antibody formulations compared to phosphate buffer controls.

Unless otherwise expressly defined above, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art. Those skilled in the art will recognize or be able to ascertain, using only routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. The appended claims are intended to encompass such equivalents.

Claims (27)

1. A liquid pharmaceutical preparation for injection, comprising... (i) Antibody at least 100 mg/ml; (ii) Yellow 5 or Orange-yellow G; and (iii) Pharmaceutical solvents; The liquid pharmaceutical preparation has an absolute viscosity of 1 cP to 271 cP at 25°C when in an injectable volume, which is measured using a cone-plate viscometer or a microfluidic viscometer; and the absolute viscosity of the liquid pharmaceutical preparation is less than that of the control composition containing the antibody and the pharmaceutical solvent but excluding the Yellow 5 or Orange G. The absolute viscosity mentioned therein is the extrapolated zero-shear viscosity. 2. The liquid pharmaceutical formulation of claim 1, wherein the molecular weight of the antibody is from 120 kDa to 250 kDa. 3. The liquid pharmaceutical preparation of claim 1, wherein the antibody is a monoclonal antibody. 4. The liquid pharmaceutical preparation of claim 1, comprising the antibody at a concentration of 150 mg/ml to 300 mg/ml. 5. The liquid pharmaceutical preparation of claim 1, wherein the pharmaceutical solvent is aqueous. 6. The liquid pharmaceutical preparation of claim 1, further comprising one or more pharmaceutical excipients. 7. The liquid pharmaceutical formulation of claim 6, wherein the one or more pharmaceutical excipients comprise a diluent or a carrier. 8. The liquid pharmaceutical formulation of claim 6, wherein the one or more pharmaceutical excipients comprise sugars, sugar alcohols, buffers, natural polymers, synthetic polymers, surfactants, fillers, or any combination thereof. 9. The liquid pharmaceutical preparation of claim 8, wherein the sugar alcohol is sorbitol or mannitol. 10. The liquid pharmaceutical formulation of claim 6, wherein the one or more pharmaceutical excipients include stabilizers. 11. The liquid pharmaceutical formulation of claim 10, wherein the stabilizer comprises a preservative, an antioxidant, a chelating agent, a cryoprotectant, a lyophilization protectant, or any combination thereof. 12. The liquid pharmaceutical formulation of claim 6, wherein one or more pharmaceutical excipients are polysorbate, poloxamer 188, sodium lauryl sulfate, or polyols. 13. The liquid pharmaceutical preparation of claim 12, wherein the polyol is poly(ethylene glycol), glycerol, propylene glycol or poly(vinyl alcohol). 14. The liquid pharmaceutical preparation of claim 1, wherein it is in the form of a single-dose vial, a multi-dose vial, a tube, or a pre-filled syringe. 15. The liquid pharmaceutical preparation of claim 1, wherein the liquid pharmaceutical preparation is isotonic with respect to human serum. 16. The liquid pharmaceutical formulation of claim 1, wherein the absolute viscosity is measured at a shear rate of at least 0.5 ^s⁻¹ when measured using a cone-plate viscometer. 17. The liquid pharmaceutical formulation of claim 1, wherein the absolute viscosity is measured at a shear rate of at least 1.0 ^s⁻¹ when measured using a microfluidic viscometer. 18. A liquid pharmaceutical preparation of any one of claims 1-17, wherein the liquid pharmaceutical preparation is reconstituted from a lyophilized composition. 19. Use of Yellow 5 or Orange G in the preparation of a liquid pharmaceutical formulation of any one of claims 1-17, wherein the liquid pharmaceutical formulation is prepared for subcutaneous or intramuscular injection. 20. The use of claim 19, wherein the liquid pharmaceutical preparation is prepared for subcutaneous or intramuscular injection using a syringe, wherein the syringe is a heated syringe, a self-mixing syringe, an auto-injector, a pre-filled syringe, or a combination thereof. 21. The use of claim 20, wherein the syringe is a heated syringe, and the liquid pharmaceutical preparation is prepared to have a temperature of 25°C to 40°C. 22. The use of claim 19, wherein when evaluated using the Draize scoring system, the liquid pharmaceutical preparation is prepared to cause a major irritation index of less than 3. 23. The use of claim 19, wherein the liquid pharmaceutical formulation is prepared to have an injection force that is at least 10% less than that of a control composition containing the antibody and the pharmaceutical solvent but excluding the Yellow 5 or Orange G. 24. The use of claim 19, wherein the liquid pharmaceutical formulation is prepared to have an injection force that is at least 20% less than that of a control composition containing the antibody and the pharmaceutical solvent but excluding the Yellow 5 or Orange G. 25. The use of claim 19, wherein the liquid pharmaceutical preparation is prepared for injection with a needle of diameter 27 to 31, and the liquid pharmaceutical preparation is prepared to have an injection force of less than 30 N with a 27-gauge needle. 26. A method for preparing a liquid pharmaceutical formulation according to any one of claims 1-17, the method comprising the step of combining the antibody, the pharmaceutical solvent, and the yellow 5 or orange-yellow G. 27. A freeze-dried composition comprising: (i) Antibody; (ii) Yellow 5 or Orange-yellow G; and (iii) Pharmaceutical excipients; The lyophilized composition can be reconstituted with a pharmaceutical solvent to have at least 100 mg/ml of the antibody and an absolute viscosity of 1 cP to 271 cP at 25°C, which is measured using a cone-plate viscometer or a microfluidic viscometer, and the absolute viscosity after reconstitution is less than the absolute viscosity of the control composition containing the antibody and the pharmaceutical solvent but excluding the Yellow 5 or Orange G, wherein the absolute viscosity is an extrapolated zero-shear viscosity.