BR122021023271B1 - LIQUID PHARMACEUTICAL FORMULATION COMPRISING THIAMINE OR ITS PHARMACEUTICALLY ACCEPTABLE SALT AND USE THEREOF - Google Patents

Abstract

Low-volume, low-viscosity, protein-concentrated liquid pharmaceutical formulations have been developed. Such formulations can be quickly and conveniently administered by subcutaneous or intramuscular injection rather than by prolonged intravenous infusion. These formulations include low molecular weight and/or high molecular weight proteins, such as mAbs, and viscosity reducing agents that are typically volumetric polar organic compounds, such as many of the GRAS (US Food and Drug Administration List of Compounds Generally Considered as safe) and FDA-approved therapeutic and inactive injectable ingredients.

Description

Split from BR112016005309-5, deposited on 9/11/2014 CROSS REFERENCE FOR RELATED ORDERS

[001] This application claims priority to and the benefit of U.S. Provisional Application No. 62/030,521, filed on 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. 61/946,436, filed February 28, 2014, entitled "Concentrated, Low-Viscosity Infliximab Formulations;" U.S. Provisional Application No. 61/943,197, filed February 21, 2014, entitled "Concentrated, Low-Viscosity, High-Molecular-Weight-Protein Formulations;" U.S. Provisional Application No. 61/940,227, filed 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 descriptions of which are hereby expressly incorporated by reference.

FIELD OF INVENTION

[002] The present invention generally relates to the field of injectable low viscosity pharmaceutical formulations of highly concentrated proteins and methods of preparing and using the same.

BACKGROUND OF THE INVENTION

[003] Monoclonal antibodies (mAbs) are important protein-based therapeutics for treating various human diseases such as cancer, infectious diseases, inflammation and autoimmune diseases. More than 20 mAb 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 mAbs (Daugherty et al., Adv. Drogue Deliv. Rev. 58: 686-706, 2006; and Buss et al., Curr. Opinion in Pharmacol. 12:615-622, 2012).

[004] mAb-based therapies are typically administered repeatedly over a prolonged period of time and require varying mg/kg dosages. Antibody solutions or suspensions can be administered via parenteral routines, such as intravenous (IV) infusions, and subcutaneous (SC) or intramuscular (IM) injections. SC or IM routines reduce the cost of treatment, increase patient compliance, and improve convenience for patients and healthcare providers during administration compared to routine IV. To be effective and pharmaceutically acceptable, parenteral formulations should preferably be sterile, stable, injectable (e.g., via a syringe), and non-irritating at the injection site, in accordance with FDA regulations. Because of the small volumes required for subcutaneous (typically about 2 mL) and intramuscular (typically about 5 mL) injections, these administration routines for high-dose protein therapies require concentrated protein solutions. These high concentrations often result in very viscous formulations that are difficult to administer by injection, cause pain at the injection site, are often imprecise, and/or may have decreased chemical and/or physical stability.

[005] These characteristics result in manufacturing, storage and use requirements that can be challenging to obtain, in particular for formulations that have high concentrations of high molecular weight proteins, such as mAbs. All protein therapeutics are to some extent subject to physical and chemical instability, such as aggregation, denaturation, cross-linking, deamidation, isomerization, oxidation, and shearing (Wang et al., J. Pharm. Sci. 96:1-26, 2007). Thus, optimal formulation development is paramount in the development of commercially viable protein pharmaceuticals.

[006] High concentrations of protein present challenges relating to the physical and chemical stability of the protein, as well as difficulty with the manufacture, storage and release of the protein formulation. One problem is the tendency of proteins to aggregate and form particulates during processing and/or storage that makes manipulations during other processing and/or release difficult. Concentration-dependent degradation and/or aggregation is the main challenge(s) in developing protein formulations at higher concentrations. In addition to the potential for non-native protein aggregation and particulate formation, reversible self-association can occur in aqueous solutions, which contribute among other things, to increased viscosity that complicates release by injection. (See, for example, Steven J. Shire et al., J. Pharm. Sci. 93:1390-1402, 2004.). Increased viscosity is one of the fundamental challenges encountered in concentrated protein compositions that equally affect production processes and the ability to easily release such compositions by conventional means. (See, for example, J. Jezek et al., Advanced Drug Delivery Reviews 63:1107-1117, 2011.)

[007] Highly viscous liquid formulations are difficult to manufacture, draw into a syringe and inject subcutaneously or intramuscularly. The use of force when handling viscous formulations can lead to excessive foaming, which can also denature and inactivate the therapeutically active protein. High viscosity solutions also require larger diameter needles for injection and produce more pain at the injection site.

[008] Currently, commercially available mAb products administered by SC or IM injection are typically formulated in aqueous buffers, such as a phosphate buffer or L-histidine, with excipients or surfactants, such as mannitol, sucrose, lactose, trehalose, POLOXAMER ® (non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene) oxide) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene) oxide)) or POLYSORBATE® 80 (PEG(80) monolaurate sorbitan), to prevent aggregation and improve stability. Reported antibody concentrations formulated as described above are typically up to about 100 mg/mL (Wang et al., J. Pharm. Sci. 96:1-26, 2007).

[009] U.S. Patent No. 7,758,860 describes the reduction of viscosity in low molecular weight protein formulations that use a buffer and a viscosity-reducing inorganic salt, such as calcium chloride or magnesium chloride. However, these same salts showed little effect on the viscosity of a high molecular weight antibody formulation (IMA-638). As described in U.S. Patent No. 7,666,413, the viscosity of aqueous formulations of high molecular weight proteins has been reduced by the addition of such salts as arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride or sodium acetate in a concentration of greater than about 100 mM or, as described in U.S. Patent No. 7,740,842, by addition of organic or inorganic acids. However, these salts do not reduce viscosity to a desired level and in some cases make the formulation so acidic that it is likely to cause pain at the injection site.

[0010] U.S. Patent No. 7,666,413 describes reduced viscosity formulations that contain specific salts and a reconstituted anti-IgE mAb, but with a maximum antibody concentration of only up to about 140 mg/mL. U.S. Patent No. 7,740,842 describes E25 anti-IgE mAb formulations that contain acetate/acetic acid buffer with antibody concentrations up to 257 mg/mL. The addition of salts such as NaCl, CaCl2 or MgCl2 has been shown to decrease dynamic viscosity under high shear conditions; however, at low shear the salts produced an undesirable and dramatic increase in dynamic viscosity. Additionally, inorganic salts such as NaCl can lower solution viscosity and/or decrease aggregation (EP 1981824).

[0011] Non-aqueous protein or antibody formulations were similarly described. WO2006/071693 describes a non-aqueous suspension of up to 100 mg/mL of mAb in a formulation that has a viscosity enhancer (polyvinylpyrrolidone, PVP) and a solvent (benzyl benzoate or PEG 400). WO2004/089335 describes 100 mg/mL nonaqueous lysozyme suspension formulations containing PVP, glycofurol, benzyl benzoate, benzyl alcohol or PEG 400. US2008/0226689A1 describes 100 mg/mL nonaqueous, viscous growth hormone formulations human (hGH) single phase, three component vehicle (polymer, surfactant and a solvent). U.S. Patent No. 6,730,328 describes low-reactivity, non-aqueous, hydrophobic, non-polar carriers, such as perfluorodecalin, for protein formulations. These formulations are non-ideal and have high viscosities that harm the process, manufacturing and injection; lead to the presence of multiple vehicle components in the formulations; and present potential regulatory challenges associated with using polymers not yet approved by the FDA.

[0012] Alternative non-aqueous antibody or protein formulations have been described using organic solvents, 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 examples, viscosities of less than 50 centipoise (cP) were obtained in formulation at protein concentrations of at least about 200 mg/mL. U.S. Patent No. 6,252,055 describes mAb formulations with concentrations ranging from 100 mg/mL to 257 mg/mL. Formulations with concentrations greater than about 189 mg/mL demonstrated dramatically increased viscosities, low recovery rates, and process difficulty. U.S. Patent Application Publication No. 2012/0230982 describes antibody formulations with concentrations of 100 mg/mL to 200 mg/mL. None of these formulations are of sufficiently low viscosity for ease of injection.

[0013] Du and Klibanov (Biotechnology and Bioengineering 108:632 636, 2011) described the reduced viscosity of concentrated aqueous solutions of bovine serum albumin with a maximum concentration of up to 400 mg/mL and bovine gamma globulin with a maximum concentration of up to 300 mg /mL. Guo et al. (Pharmaceutical Research 29:3102-3109, 2012) described low viscosity aqueous solutions of four model mAbs obtained using hydrophobic salts. The mAb formulation employed by Guo had an initial viscosity, before adding salts, of no greater than 73 cP. The viscosities of many pharmaceutically important mAbs, on the other hand, can exceed 1,000 cP at therapeutically relevant concentrations.

[0014] It is not a trivial matter to control aggregation and viscosity in high concentration mAb solutions (EP 2538973). This is proven by the few mAb products currently on the market as high concentration formulations (>100 mg/mL) (EP 2538973).

[0015] The references cited above demonstrate that while many groups have attempted to prepare low viscosity formulations of mAbs and other therapeutically important proteins, a truly useful formulation for many proteins has not yet been obtained. Notably, many of the previous reports employ agents for which the safety and toxicity profiles have not been completely established. These formulations would therefore face a higher regulatory burden prior to approval than formulations containing compounds known to be safe. In fact, even if a compound has been shown to substantially reduce viscosity, the compound may be definitely unsuitable for use in a formulation intended for injection into a human.

[0016] Many pharmaceutically important high molecular weight proteins, such as mAbs, are currently administered via IV infusions to deliver therapeutically effective amounts of protein due to problems with high viscosity and other properties of concentrated solutions of large proteins. For example, to deliver a therapeutically effective amount of many high molecular weight proteins, such as mAbs, in volumes less than about 2 mL, protein concentrations greater than 150 mg/mL are often required.

[0017] Therefore, it is an objective of the present invention to provide concentrated low-viscosity liquid formulations of pharmaceutically important proteins, especially high molecular weight proteins, such as mAbs.

[0018] It is another objective of the present invention to provide concentrated low viscosity liquid formulations of proteins, especially high molecular weight proteins, such as mAbs, capable of releasing therapeutically effective amounts of these proteins in volumes useful for SC and IM injections.

[0019] It is another objective of the present invention to provide concentrated liquid formulations of proteins, especially high molecular weight proteins, such as mAbs, with low viscosities, which can improve injectability and/or patient compliance, convenience and comfort.

[0020] It is also an object of the present invention to provide methods for preparing and storing low-viscosity, concentrated formulations of proteins, especially high molecular weight proteins, such as mAbs.

[0021] It is a further objective of the present invention to provide methods of administering low viscosity concentrated liquid formulations of proteins, especially high molecular weight proteins, such as mAbs. It is a further object of the present invention to provide methods for processing high concentration reduced viscosity biologics with concentration and filtration techniques known to those skilled in the art.

SUMMARY OF THE INVENTION

[0022] Low volume, low viscosity, protein concentrated liquid pharmaceutical formulations 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 include low molecular weight and/or high molecular weight proteins, such as mAbs, and viscosity reducing agents that are typically volumetric polar organic compounds, such as many of GRAS (US Food and Drug Administration list of compounds generally considered safe), inactive injectable ingredients, and FDA-approved therapeutics.

[0023] The protein concentration is between about 10 mg/mL and 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 between about 100 mg/mL to about 500 mg/mL, more preferably from about 300 mg/mL to about 500 mg/mL. Formulations containing proteins and viscosity reducing agents are stable when stored at a temperature of 4° C. for a period of at least one month, preferably at least two months, and preferably at least three months. The viscosity of the formulation is less than about 75 cP, preferably below 50 cP, and most preferably below 20 cP at about 25° C. In some embodiments, the viscosity is less than about 15 cP or even less than or about 10 cP at about 25° C. In certain embodiments, the viscosity of the formulation is about 10 cP. Formulations containing proteins and viscosity reducing agents typically are measured at shear rates from about 0.6 s-1 to about 450 s-1, and preferably from about 2 s-1 to about of 400 s-1, when measuring using a cone and plate viscometer. Formulations containing proteins and viscosity reducing agents typically are measured at shear rates from about 3 s-1 to about 55,000 s-1, and preferably from about 20 s-1 to about 2,000 s-1, when measuring using a microfluidic viscometer.

[0024] The viscosity of the protein formulation is reduced by the presence of one or more viscosity reducing agents. Unless specifically stated otherwise, the term "viscosity reducing agent" includes simple compounds and mixtures of two or more compounds. It is preferred that the viscosity reducing agent is present in the formulation at a concentration of less than about 1.0 M, preferably less than about 0.50 M, more preferably less than about 0.30 M, and most preferably less than about 0.15 M. In some embodiments, the viscosity reducing agent is present in the formulation in concentrations as low as 0.01 M. The formulations can have a viscosity that is at least about 30% lower, preferably by at least about 50% lower, more preferably at least about 75% lower, than the viscosity of the corresponding formulation under the same conditions with the exception of replacing the viscosity reducing agent with an appropriate buffer or salts at around the same concentration. In some embodiments, a low viscosity formulation is provided, where the viscosity of the corresponding formulation without the viscosity reducing agent is greater than about 200 cP, greater than about 500 cP, or even greater than about 1,000 cP. In a preferred embodiment, the shear rate of the formulation is at least about 0.5 s-1 when measuring using a cone and plate viscometer or at least about 1.0 s-1 when measuring using a microfluidic viscometer. .

[0025] For embodiments in which the protein is a "high molecular weight protein", the high molecular weight protein may have a molecular weight between about 100 kDa and about 1,000 kDa, preferably between about 120 kDa and about of 500 kDa, and more preferably between about 120 kDa and about 250 kDa. The high molecular weight protein may be an antibody, such as a mAb, or a PEGylated, or otherwise a derivative form thereof. Preferred MAbs include natalizumab (TYSABRI®), cetuximab (ERBITUX®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), infliximab (REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatumumab (ARZERRA®) and biosimilars thereof. The high molecular weight protein, optionally PEGylated, can be an enzyme. Other proteins and protein mixtures can also be formulated to reduce their viscosity.

[0026] In some embodiments, the protein and viscosity reducing agent are provided in a lyophilized dosage unit, sized for reconstitution with a sterile aqueous, pharmaceutically acceptable vehicle, to produce concentrated low viscosity liquid formulations. The presence of the viscosity reducing agent(s) facilitates and/or accelerates reconstitution of the lyophilized dosage unit compared to a lyophilized dosage unit that does not contain a viscosity reducing agent.

[0027] Methods are provided here for preparing low viscosity, concentrated liquid formulations of high molecular weight proteins such as mAbs, as well as methods for storing the low viscosity high concentration protein formulations, and for administering the same to patients. . In another embodiment, the viscosity reducing agent is added to facilitate the process (e.g., pumping, concentrating, and/or filtration) by reducing the viscosity of the protein solutions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 depicts viscosity in cP as a function of protein concentration (in mg/mL) for solutions of biosimilar cetuximab (ERBITUX®) in 0.25 M phosphate buffer (PB; diamonds) and a solution which contains 0.25 M camphorsulfonic acid L-lysine (CSAL; squares) at 25°C and final pH of 7.0. Data points incorporate standard deviations which, however, are often smaller than symbols.

[0029] Figure 2 depicts viscosity in cP as a function of protein concentration (in mg/mL) for solutions of biosimilar bevacizumab (AVASTIN®) in 0.25 M phosphate buffer (PB; diamonds) and CSAL at 0.25 M (square) at 25°C and final pH of 7.0. Data points incorporate standard deviations which, however, are often smaller than symbols.

[0030] Figure 3 is a graph of the viscosity (cP) of 200 ± 9 mg/mL aqueous solutions of biosimilar bevacizumab (AVASTIN®) as a function of pH along the x-axis containing phosphate-citrate buffer or arginine of camphorsulfonic acid (CSAA) at a concentration of 0.25 M.

[0031] Figure 4 is a bar graph comparing the doubling reduction in viscosity as a function of pH for aqueous solutions containing biosimilar bevacizumab (AVASTIN®; at about 200 mg/mL or 226 mg/mL) and arginine of 0.25 M camphorsulfonic acid (CSAA). Doubling reduction is computed as the ratio of the viscosity (cP) in phosphate-citrate buffer to the viscosity (cP) in the 0.25 M CSAA solution.

[0032] Figure 5 is a graph of the viscosity (cP) of aqueous solutions of biosimilar cetuximab (ERBITUX®; at 202 ± 5 mg/mL, 229 ± 5 mg/mL, or 253 ± 4 mg/mL) containing CSAA at 0.25 M as a function of pH along the x-axis at 25°C.

[0033] Figure 6 is a size exclusion chromatography trace depicting absorbance intensity (at 280 nm) as a function of elution time (in minutes) for a 220 mg/mL aqueous solution of stored REMICADE® at 4°C for up to 100 days, compared to a newly reconstituted commercial drug product.

[0034] Figure 7 depicts viscosity (cP) as a function of protein concentration (mg/mL) of aqueous solutions of biosimilar bevacizumab (AVASTIN®) in 0.25 M phosphate buffer, 0.25 M APMI*2HCl. 10 M or 0.25 M ((1-(3-aminopropyl)-2-methyl-1H-imidazole bis-HCl).

[0035] Figure 8 depicts viscosity (cP) as a function of protein concentration (mg/mL) of aqueous solutions of biosimilar bevacizumab (AVASTIN®) in 0.25 M phosphate buffer, thiamine pyrophosphate (TPP) at 0.10 M, or TPP1-(3-aminopropyl)-2-methyl-1H-imidazole (APMI) at 0.10 M.

[0036] Figure 9 depicts the viscosity (cP) of aqueous solutions of golimumab (SIMPONI ARIA®) as a function of protein concentration (mg/mL) with 0.15 M phosphate buffer or 0.15 M thiamine HCl. 15 M.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

[0037] The term "protein", when generally used herein, refers to a polymer of amino acids mutually linked by peptide bonds to form a polypeptide for which the chain length is sufficient to produce at least one detectable tertiary structure. Proteins that have a molecular weight (expressed in kDa where "Da" represents "Daltons" and 1 kDa = 1,000 Da) greater than about 100 kDa may be designated "high molecular weight proteins", whereas proteins that have a molecular weight molecular weight smaller than about 100 kDa may be termed "low molecular weight proteins". The term "low molecular weight protein" excludes small peptides without the requirement of at least tertiary structure necessary to be considered a protein. Protein molecular weight can be determined using standard methods known to one skilled in the art, including, but may not be limited to, mass spectrometry (e.g., ESI, MALDI) or calculation from known amino acid sequences and glycosylation. Proteins can be naturally occurring or non-naturally occurring, synthetic or semi-synthetic.

[0038] "Essentially pure protein(s)" and "substantially pure protein(s)" are used interchangeably herein and refer to a composition comprising at least about 90 % by weight pure protein, preferably at least about 95% by weight pure protein. "Essentially homogeneous" and "substantially homogeneous" are used interchangeably herein and refer to a composition, wherein at least about 90% by weight of the protein present is a combination of the monomer and reversible di- and oligomeric associates (non-binding aggregates). irreversible), preferably at least about 95%.

[0039] The term "antibody", when generally used herein, broadly encompasses mAbs (including full-sized antibodies that have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies, and chain antibody molecules. simple, as well as antibody fragments (e.g., Fab, Fab', F(ab')2 and Fv), single domain antibodies, multivalent single domain antibodies, Fab fusion proteins and fusions thereof.

[0040] The term "monoclonal antibody" or "mAb", when generally used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical, with with the exception of possible naturally occurring mutations that may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single epitope. These are typically synthesized by culturing hybridoma cells, as described by Kohler et al. (Nature 256:495, 1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), or isolated from phage antibody libraries using the techniques described in Clackson et al. al. (Nature 352: 624-628, 1991) and Marks et al. (J. Mol. Biol. 222: 581-597, 1991), for example. When used herein, "mAbs" specifically include derived antibodies, antibody-drug conjugates, and "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or that belongs to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from other species or that belong to another class or subclass of antibody, as well as fragments of such antibodies, 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 ).

[0041] An "antibody fragment" comprises a portion of an intact antibody, including the antigen binding and/or the variable region 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.

[0042] "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 subsequences of antibody antigen binding) of generally human sequences, which contain minimal sequences derived from non-human immunoglobulin. (See, for example, Jones et al., Nature 321:522-525, 1986; Reichmann et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593596, 1992 .)

[0043] "Rheology" refers to the study of the deformation and flow of matter.

[0044] "Viscosity" refers to the resistance of a substance (typically a liquid) to flow. Viscosity is related to the concept of shear force; can be understood as the effect of different layers of fluid exerting shear force on each other, or on other surfaces, as they move mutually. There are several viscosity measurements. The viscosity units are Ns/m2, known as Pascal-seconds (Pa-s). Viscosity can be "kinematic" or "absolute". Kinematic viscosity is a measure of the rate at which momentum is transferred through a fluid. It is measured in Stokes (St). Kinematic viscosity is a measure of the resistant flow of a fluid under the influence of gravity. When two fluids of equal volume and discrepant viscosity are placed in identical capillary viscometers and allowed to flow by gravity, the more viscous fluids take longer than the less viscous fluid to flow through the capillary vessel. For example, if one fluid takes 200 seconds to complete its flow and another fluid takes 400 s, the second fluid is called twice as viscous as the first on a kinematic viscosity scale. The kinematic viscosity dimension is length2/time. Generally, kinematic viscosity is expressed in centiStokes (cSt). The SI unit of kinematic viscosity is mm2/s, which is equal to 1 cSt. "Absolute viscosity", sometimes called "dynamic viscosity" or "simple viscosity", is the product of kinematic viscosity and fluid density. Absolute viscosity is expressed in centipoise units (cP). The SI unit of absolute viscosity is the milliPascal-second (mPas), where 1 cP = 1 mPa-s.

[0045] Viscosity can be measured using, for example, a viscometer at a given shear rate or multiple shear rates. An "extrapolated zero shear" viscosity can be determined by creating a best fit line of the four highest shear points on a plot of absolute viscosity against shear rate, and linearly extrapolating the viscosity again to zero shear. Alternatively, for a Newtonian fluid, viscosity can be determined by averaging viscosity values at multiple shear rates. Viscosity can similarly be measured using a microfluidic viscometer at single or multiple shear rates (also called flow rates), where absolute viscosity is derived from a change in pressure as a liquid flows through a channel. Viscosity equals shear stress over shear rate. Viscosities measured with microfluidic viscometers can, in some embodiments, be directly compared to extrapolated zero shear viscosities, for example, those extrapolated from viscosities measured at multiple shear rates using a cone and plate viscometer.

[0046] "Shear rate" refers to the rate of change in velocity at which a layer of fluid passes over an adjacent layer. The velocity gradient is the rate of change of velocity with distance from the plates. This simple case shows the uniform velocity gradient with shear rate (v1 - v2)/h in units of (cm/sec)/(cm) = 1/sec. Consequently, the units of shear rate are reciprocal seconds or, in general, reciprocal time. For a microfluidic viscometer, the change in flow rate and pressure is related to the shear rate. "Shear rate" is the speed at which a material is deformed. Formulations containing proteins and viscosity reducing agents are typically measured at shear rates ranging from about 0.5 s-1 to about 200 s-1, when measuring using a cone and plate viscometer and an appropriately selected spindle. by one skilled in the art of accurately measuring viscosities at the viscosity rate of the sample of interest (i.e., a 20 cP sample is accurately measured on a CPE40 spindle attached to a DV2T viscometer (Brookfield)); greater than about 20 s-1 to about 3,000 s-1 when measuring using a microfluidic viscometer.

[0047] For classical "Newtonian" fluids, when generally used here, viscosity is essentially independent of shear rate. For "non-Newtonian fluids", however, viscosity decreases or increases with increasing shear rate, e.g., the fluids are "shear-thinning" or "shear-thickening", respectively. In the case of concentrated (i.e., high concentration) protein solutions, this may manifest as pseudoplastic shear thinning behavior, i.e., a decrease in viscosity with shear rate.

[0048] The term "chemical stability", when generally used herein, refers to the ability of protein components in a formulation to resist degradation through series of chemical reactions, such as oxidation, deamidation or hydrolysis. A protein formulation is typically considered chemically stable if less than about 5% of the components are degraded after 24 months at 4°C.

[0049] The term "physical stability", when generally used herein, refers to the ability of a protein formulation to resist physical deterioration, such as aggregation. A formulation that is physically stable forms only an acceptable percentage of irreversible aggregates (e.g., dimers, trimers, or other aggregates) of the bioactive protein agent. The presence of aggregates can be assessed in several ways, including measuring the average particle size of the proteins in the formulation through dynamic light scattering. A formulation is considered physically stable if less than about 5% irreversible aggregates are formed after 24 months at 4°C. Acceptable levels of aggregate contaminants would ideally be less than about 2%. Levels as low as about 0.2% are obtainable, although about 1% is more typical.

[0050] The term "stable formulation", when generally used herein, means that a formulation is equally chemically stable and physically stable. A stable formulation may be one in which more than about 95% of the bioactive protein molecules retain bioactivity in a formulation after 24 months of storage at 4°C, or solution conditions equivalent to an elevated temperature, such as a month of storage at 40° C. Various analytical techniques for measuring protein stability are available in the art and are reviewed, 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 over a certain period of time. For rapid evaluation, for example, the formulation can be maintained at 40°C for 2 weeks to a month, during which time the residual biological activity is measured and compared to the initial condition to assess stability. When the formulation is to be stored at 2°C -8°C, generally the formulation should be stable at 30°C or 40°C for at least one month and/or stable at 2°C -8°C for at least 2 years. When the formulation must be stored at room temperature, about 25°C, generally the formulation should be stable for at least 2 years at about 25°C and/or stable at 40°C for at least about 6 months. The extent of aggregation after lyophilization and storage can be used as an indicator of protein stability. In some embodiments, stability is assessed by measuring the particle size of the proteins in the formulation. In some embodiments, stability can be assessed by measuring the activity of a formulation using standard biological activity or binding assays well within the capabilities of one ordinarily skilled in the art.

[0051] The term protein "particle size", when generally used herein, means the average diameter of the predominant population of bioactive molecule particulates, or particle size distributions thereof, in a formulation as determined using protein sizing instruments. well-known particle particles, e.g., dynamic light scattering, SEC (size exclusion chromatography), or other methods known to one ordinarily skilled in the art.

[0052] The term "concentrated" or "high concentration", when generally used herein, describes liquid formulations that have a final protein concentration 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 more preferably greater than about 250 mg/mL.

[0053] A "reconstituted formulation", when generally used herein, refers to a formulation that has been prepared by dissolving a dry powder, lyophilized, spray-dried or solvent-precipitated protein in a diluent, such that the protein is dissolved or dispersed in aqueous solution for administration.

[0054] A "lyoprotectant" is a substance that, when combined with a protein, significantly reduces the chemical and/or physical instability of the protein upon freeze-drying and/or subsequent storage. Exemplary lyoprotectants 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; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrins, modified gum and carboxymethyl cellulose. Preferred sugar alcohols are those compounds obtained by reduction of mono- and disaccharides, such as lactose, trehalose, maltose, lactulose and maltulose. Additional examples of sugar alcohols are glucitol, maltitol, lactitol, and isomaltulose. The lyoprotectant is usually added to the pre-lyophilized formulation in a "lyoprotectant amount." This means that, after lyophilization of the protein in the presence of the lyoprotectant amount of lyoprotectant, the protein essentially retains its physical and chemical stability and integrity.

[0055] A "diluent" or "vehicle", when generally used herein, is an ingredient that is pharmaceutically acceptable (i.e., safe and non-toxic for administration to a human or other mammal) and useful for the preparation of a liquid formulation, such as an aqueous formulation reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH-buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution, and combinations thereof.

[0056] A "preservative" is a compound that can be added here to formulations to reduce contamination by and/or the action of bacteria, fungi, or other infectious agent. The addition of a preservative can, for example, facilitate the production of a multi-use formulation (multiple doses). Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chloride, in which the alkyl groups are long chain), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenolic, butyl and benzyl alcohols, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

[0057] "Bulking agent", when generally used herein, is a compound that adds mass to a freeze-dried mixture and contributes to the physical structure of the freeze-dried dough (e.g., it facilitates the production of a physical structure of the freeze-dried dough that maintains a open pore structure). Exemplary bulking agents include mannitol, glycine, lactose, modified starch, poly(ethylene glycol) and sorbitol.

[0058] A "therapeutically effective amount" is the minimum concentration required to effect a measurable improvement or prevention of any symptom or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or generally improve the patient's quality of life . The therapeutically effective amount is dependent on the specific biologically active molecule and the specific condition or disorder being treated. Therapeutically effective amounts of many proteins, such as the mAbs described here, are well known in the art. The therapeutically effective amounts of proteins not yet established or to treat specific disorders with known proteins, such as mAbs, to be clinically applied to treat additional disorders, can be determined by standard techniques that are well within the skill of a skilled artisan, such as a doctor.

[0059] The term "injectability" or "syringability", when generally used herein, refers to the injection performance of a pharmaceutical formulation by a syringe equipped with an 18-32 gauge needle, optionally with thin walls. Injectability depends on factors such as pressure or force required for injection, equality of flow, suction qualities and freedom from clogging. The injectability of liquid pharmaceutical formulations can be assessed by comparing the injection force of a formulation with reduced viscosity to a standard formulation without added viscosity reducing agents. The reduction in injection force of the formulation containing a viscosity reducing agent reflects the improved injectability of that formulation. Reduced viscosity formulations have improved injectability when the injection force is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50% and most preferably by at least 75% when compared to a standard formulation that has the same protein concentration otherwise under the same conditions, with the exception of replacing the viscosity reducing agent with an appropriate buffer of approximately the same concentration. Alternatively, the injectability of liquid pharmaceutical formulations 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 the syringe is depressed with the same strength.

[0060] The term "injection force", when generally used herein, refers to the force required to push a given liquid formulation through a given syringe equipped with a given needle size at a given injection speed. Injection force is typically reported in Newtons. For example, injection force can be measured as the force required to push a liquid formulation through a 1 mL plastic syringe that has an internal diameter of 6.35 mm (0.25 inch), equipped with a 27 gauge needle. gauge of 12.70 mm (0.50 inch) at an injection speed of 250 mm/min. Test equipment can be used to measure injection force. When measured under the same conditions, a lower viscosity formulation will generally require a lower overall injection force.

[0061] The "viscosity gradient", when used herein, refers to the rate of change in the viscosity of a protein solution when the protein concentration increases. The viscosity gradient can be approximated from a plot of viscosity as a function of protein concentration for a series of formulations that are otherwise the same but have different protein concentrations. Viscosity increases approximately exponentially with increasing protein concentration. The viscosity gradient at a specific protein concentration can be approximated by the slope of a tangent line on the viscosity plot as a function of protein concentration. The viscosity gradient can be approximated from a linear approximation to plotting viscosity as a function of any protein concentration or within a narrow window of protein concentrations. In some embodiments, a formulation is said to have a decreased viscosity gradient if, when viscosity as a function of protein concentration is approximated as an exponential function, the exponent of the exponential function is smaller than the exponent otherwise obtained for the same formulation without viscosity reducing agent. In a similar manner, a formulation may be said to have a lower/higher viscosity gradient when compared to a second formulation, if the exponent for the formulation is lower/higher than the exponent for the second formulation. The viscosity gradient can be numerically approximated from a plot of viscosity as a function of protein concentration by other methods known to skilled formulation researchers.

[0062] The term "reduced viscosity formulation", when generally used herein, refers to a liquid formulation that has a high concentration of a high molecular weight protein, such as a mAb, or a low molecular weight protein, which is modified by the presence of one or more viscosity-lowering additives, when compared to a corresponding formulation that does not contain the viscosity-reducing additive(s).

[0063] The term "osmolarity", when generally used here, refers to the total number of dissolved components per liter. Osmolarity is similar to molarity, but includes the total number of moles of species dissolved in the solution. An osmolarity of 1 Osm/L means that there are 1 mol of dissolved components per L 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 thereby provides 2 moles of dissolved components per 1 mole of NaCl dissolved in solution. Physiological osmolarity is typically in the range of about 280 mOsm/L to about 310 mOsm/L.

[0064] The term "tonicity", when generally used here, refers to the osmotic pressure gradient that is the result of the separation of two solutions by a semipermeable membrane. In particular, tonicity is used to describe the osmotic pressure created by a cell membrane when a cell is exposed to an external solution. Solutes that can cross the cell membrane do not contribute to the final osmotic pressure gradient. Only those dissolved species that do not cross the cell membrane will contribute to osmotic pressure differences and thus tonicity.

[0065] The term "hypertonic", when generally used here, refers to a solution with a higher concentration of solutes than is present inside the cell. When a cell is immersed in a hypertonic solution, the tendency is for water to flow from the cell to balance the concentration of solutes.

[0066] The term "hypotonic", when generally used here, refers to a solution with a lower concentration of solutes, which is present within the cell. When a cell is immersed in a hypotonic solution, water flows into the cell to balance the concentration of solutes.

[0067] The term "isotonic", when generally used here, refers to a solution in which the osmotic pressure gradient across the cell membrane is essentially balanced. An isotonic formulation is one that has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 mOsm/kg to 350 mOsm/kg.

[0068] The term "liquid formulation", when used herein, is a protein that is provided in a pharmaceutically acceptable diluent or one that is reconstituted in a pharmaceutically acceptable diluent prior to administration to the patient.

[0069] The terms "trademark" and "reference", when used to refer to a protein or biologic, are used interchangeably herein to mean the only biologic product authorized under section 351(a) of the U.S. Public Health Service Act ( 42 U.S.C. § 262).

[0070] The term "biosimilar", when used herein, is generally used interchangeably with "a generic equivalent" or "follow-on". For example, a "biosimilar mAb" refers to a follow-on version of an innovator mAb typically made by a different company. "Biosimilar" when used in reference to a trademarked protein or trademarked biologic may refer to a biological product evaluated against the trademarked protein or trademarked biologic and licensed under section 351(k) of the U.S. Public Health Service Act (42 U.S.C. § 262). A biosimilar mAb may be one that satisfies one or more guidelines adopted on May 30, 2012 by the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency and published by the European Union as "Guideline on similar biological medicinal products containing monoclonal antibodies - non-clinical and clinical issues" (Document Reference EMA/CHMP/BMWP/403543/2010).

[0071] Biosimilars can be produced by microbial cells (prokaryotic, eukaryotic), cell lines of human or animal origin (e.g., mammals, birds, insects), or tissues derived from animals or plants. The term construct for a 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 that will not have an effect on safety, purity or potency, may be present.

[0072] A biosimilar mAb is similar to the reference mAb physiochemically or biologically equally in terms of safety and efficacy. The biosimilar mAb can be evaluated against a reference mAb using one or more in vitro studies, including assays that detail binding to the target antigen(s); binding to isoforms of Fc gamma receptors (FcyRI, FcyRII and FcyRIII), FcRn, and complement (C1q); Fab-associated functions (e.g., neutralization of a soluble ligand, blockade or activation of a receptor); or Fc-associated 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 of pharmacokinetics, pharmacodynamics and/or safety. Clinical evaluations of a biosimilar mAb against a reference mAb may include comparisons of pharmacokinetic properties (e.g., AUC0-inf, AUC0-t, Cmax, tmax, Ctrough); pharmacodynamic endpoints; or similarity of clinical efficacy (e.g., using randomized, parallel-group comparative clinical trials). Quality comparison between a biosimilar mAb and a reference mAb using established procedures can be assessed, including those described in the "Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: Quality issues" (EMEA/CHMP/BWP/ 49348/2005), and in the "Guideline on development, production, characterization and specifications for monoclonal antibodies and related substances" (EMEA/CHMP/BWP/157653/2007).

[0073] Differences between a biosimilar mAb and a reference mAb may include post-translational modification, for example, binding to the mAb other biochemical groups such as a phosphate, various lipids and carbohydrates; by proteolytic cleavage after translation; changing the chemical nature of an amino acid (e.g., formylation); or by many other mechanisms. Other post-translational modifications may be a consequence of manufacturing process operations - for example, glycation may occur upon exposure of the product to reducing sugars. In other cases, storage conditions may be permissive for certain series of degradation reactions such as oxidation, deamidation or aggregation. Since all these product-related variants can be included in a biosimilar mAb.

[0074] The term "viscosity reducing agent", when used herein, refers to a compound that acts to reduce the viscosity of a solution relative to the viscosity of the solution in the absence of the viscosity reducing agent. The viscosity reducing agent may be a single compound, or it may be a mixture of one or more compounds. When the viscosity reducing agent is a mixture of two or more compounds, the concentration listed refers to each individual agent unless otherwise specified. By way of example, a formulation that contains camphorsulfonic acid arginine at about 0.25 M as the viscosity reducing agent is a solution that has camphorsulfonic acid at a concentration of 0.25 M, and arginine at a concentration of 0. .25M.

[0075] Certain viscosity reducing agents contain acidic or basic functional groups. Whether or not these functional groups are completely or partially ionized depends on the pH of the formulation in which they are. Unless otherwise specified, reference to a formulation containing a viscosity reducing agent that has an ionizable functional group includes the parent compound and any possible ionized state.

[0076] When used herein, the term "hydrogen bond donor" refers to a hydrogen atom connected to a relatively electronegative atom, which creates a partial positive charge on the hydrogen atom.

[0077] When used herein, the term "hydrogen bond acceptor" refers to a relatively electronegative atom or functional group capable of interacting with a hydrogen atom that bears a partial positive charge.

[0078] When used herein, the term "free rotation bond" refers to any singly pair of non-hydrogen atoms.

[0079] When used herein, the term "molecular polar surface area" refers to the total exposed polar area on the surface of the molecule of interest.

[0080] When used herein, the term "molar volume" refers to the total volume, which that mole of the molecule of interest occupies in its native state (i.e. solid, liquid).

[0081] When used herein, the term "polarizability" refers to the dipole moment induced when the molecule of interest is placed in an electric field of unity resistance.

[0082] When used herein, the term "pharmaceutically acceptable salts" refers to salts prepared from non-toxic pharmaceutically acceptable bases and acids, including inorganic bases and acids and organic bases and acids. Suitable non-toxic acids include inorganic and organic acids such as acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic , pantothenic, phosphoric, succinic, sulfuric, tartaric acid, p-toluenesulfonic acid and the like. Suitable positively charged counterions include sodium, potassium, lithium, calcium, and magnesium.

[0083] When used herein, the term "ionic liquid" refers to a crystalline or amorphous salt, hybrid, or mixture thereof, which is a liquid at or near the temperatures where most conventional salts are solid: 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, for example, between 10°C and 40°C, or between 15°C and 35°C. The term "hybrid" is used here to describe an overall neutrally charged molecule that carries formal positive and negative charges on different chemical groups in the molecule. Examples of ionic liquids are described 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.

[0084] When used herein, the term "organophosphate" refers to a compound containing one or more phosphoryl groups, at least one of which is covalently connected to an organic group by a phosphoester bond.

[0085] When used herein, a "water-soluble organic dye" is an organic molecule that has a molar solubility of at least 0.001 M at 25°C and pH 7, and that absorbs certain wavelengths of light, preferably in the portion visible to infrared of the electromagnetic spectrum, while also possibly transmitting or reflecting other wavelengths of light.

[0086] When 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.

[0087] When used herein, the term "alkyl group" refers to straight-chain, branched-chain and cyclic hydrocarbon groups. Unless otherwise specified, the term alkyl group encompasses 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".

[0088] The term when used herein, "Aryl" refers to aromatic carbon ring systems, including fused ring systems. In an "aryl" group, each of the atoms forming the ring are carbon atoms.

[0089] The term when used herein "Heteroaryl" refers to aromatic ring systems, including fused ring systems, in which at least one of the atoms forming the ring is a heteroatom.

[0090] The term when used herein "Heterocycle" refers to ring systems that, including fused ring systems, are non-aromatic, in which at least one of the atoms forming the ring is a heteroatom.

[0091] The term when used herein, "heteroatom" is any non-carbon or non-hydrogen atom. Preferred heteroatoms include oxygen, sulfur, and nitrogen. Exemplary heteroaryl and heterocyclyl rings include: benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, cromanyl, cromenyl, cinolinyl, decahydroquinolinyl, 2H, 6H- 1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl , isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3 ,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxatinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidin yl, pyrazolinyl , pyrazolyl, pyridazinyl, pyridoxazole, pyridoimidazole, pyridinyl, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydro quinolinyl, tetrazolyl, 6H-1 ,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thiantrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl , thiophenyl, and xanthenyl.

II. FORMULATIONS

[0092] Low-viscosity, biocompatible protein solutions, such as those of mAbs, can be used to deliver therapeutically effective amounts of proteins in volumes useful for subcutaneous (SC) and intramuscular (IM) injections, 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 be of any molecular weight, although, in some embodiments, high molecular weight proteins are preferred. In other embodiments, the proteins are low molecular weight proteins.

[0093] Formulations can have protein concentrations between about 10 mg/mL and about 5,000 mg/mL. The formulations, including mAb 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, even more preferably greater than about 200 mg/mL. mL, even more preferably greater than about 225 mg/mL, even more preferably greater than about 250 mg/mL, and even more preferably greater than or about 300 mg/mL. In the absence of a viscosity reducing agent, the viscosity of a protein formulation increases exponentially when the concentration is increased. Such protein formulations, in the absence of a viscosity reducing agent, may have viscosities 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 often unsuitable for SC or IM injection. The use of one or more viscosity reducing agents allows the preparation of formulations that have a viscosity of less than or about 100 cP, preferably less than or about 75 cP, more preferably less than or about 50 cP, even more preferably less than or about 30 cP, even more preferably less than or about 20 cP, or even more preferably less than or about 10 cP, when measured at 25° C.

[0094] Although viscosity reducing agents can be used to reduce the viscosity of concentrated protein formulations, they can be used in less concentrated formulations as well. In some embodiments, the formulations may have protein concentrations between about 10 mg/mL and about 100 mg/mL. The formulations may have a protein concentration greater than about 20 mg/mL, greater than about 40 mg/mL, or greater than about 80 mg/mL.

[0095] For certain proteins, formulations that do not have a viscosity reducing agent may have viscosities greater than about 20 cP, greater than about 50 cP, or greater than about 80 cP. The use of one or more viscosity reducing agents allows the preparation of formulations that have a viscosity of less than or about 80 cP, preferably less than or about 50 cP, even more preferably less than about 20 cP, or preferably less that or about 10 cP when measured at 25° C.

[0096] In some embodiments, the aqueous protein formulations have a viscosity that is at least about 30% lower than the analogous formulation without the viscosity reducing agent(s), when measured under the same conditions. In other embodiments, the formulations have a viscosity that is 40% lower, 50% lower, 60% lower, 70% lower, 80% lower, 90% lower, or even greater than 90% lower than the analogous formulation without the ) viscosity reducing agent(s). In a preferred embodiment, the formulation contains a therapeutically effective amount of one or more high molecular weight proteins, such as mAbs, in a volume of less than about 2 mL, preferably less than about 1 mL, or more preferably less than about of 0.75 mL.

[0097] The reduced viscosity formulations improved injectability and required less injection force compared to the analogous formulation without the viscosity reducing agent (e.g., in phosphate buffer) under otherwise the same conditions. In some embodiments, the injection force is decreased by more than about 20%, more than about 30%, more than about 40%, more than about 50%, or more than about 2 times, compared to standard formulations without the viscosity reducing agent(s) under otherwise the same injection conditions. In some embodiments, the formulations have "Newtonian flow characteristics", defined as having viscosity that is substantially independent of shear rate. Protein formulations can be injected easily through needles of about 18-32 gauge. Preferred needle gauges for releasing low viscosity formulations include 27, 29, and 31 gauge, optionally thin walled.

[0098] Formulations may contain one or more additional excipients, such as buffers, surfactants, sugars and sugar alcohols, other polyols, preservatives, antioxidants, and chelating agents. The formulations have a suitable pH and osmolarity for administration without causing significant adverse side effects. In some embodiments, the low viscosity, concentrated formulations have a pH between 5 and 8, between 5.5 and 7.6, between 6.0 and 7.6, between 6.8 and 7.6, or between 5. 5 and 6.5.

[0099] Low viscosity protein formulations can allow greater flexibility in formulation development. Low viscosity formulations may exhibit changes in viscosity that are less dependent on protein concentration compared otherwise to the same formulation without the viscosity reducing agent. Low viscosity protein formulations may allow for increased concentrations and decreased dosing frequencies of the protein. In some embodiments, the low viscosity protein formulations contain 2 or more, 3 or more, or 4 or more different proteins. For example, combinations of 2 or more mAbs can be provided in a single low viscosity protein formulation.

[00100] Because protein formulations (such as mAb) can be administered to patients at higher protein concentrations than otherwise similar protein formulations that do not contain a viscosity reducing agent, the protein dosing frequency can be reduced. For example, proteins previously requiring once-daily administration may be administered once every two days, every three days, or even less frequently when the proteins are formulated with viscosity-reducing agents. Proteins that currently require multiple administrations on the same day (at the same time or at different times of the day) can be administered in fewer injections per day. In some examples, the frequency may be reduced to a single injection once per day. By increasing the dosage administered by injection multiple times, the dosage frequency can be decreased, for example, from once every 2 weeks to once every 6 weeks.

[00101] In some embodiments, liquid formulations have a physiological osmolarity, for example, between about 280 mOsm/L to about 310 mOsm/L. In some embodiments, the liquid formulations have an osmolarity greater than about 250 mOsm/L, greater than about 300 mOsm/L, greater than about 350 mOsm/L, greater than about 400 mOsm/L, or greater than about of 500 mOsm/L. In some embodiments, the formulations have an osmolarity of about 200 mOsm/L to about 2,000 mOsm/L or about 300 mOsm/L to about 1,000 mOsm/L. In some embodiments, the liquid formulations are essentially isotonic to human blood. Liquid formulations can in some cases be hypertonic.

[00102] Additives, including viscosity reducing agents, may be included in any amount to achieve the desired viscosity levels of the liquid formulation, provided that the amounts are not toxic or otherwise harmful, and do not substantially interfere with the chemical and/or physical stability of the formulation. The viscosity reducing agent(s) in some embodiments may be independently present at a concentration less than about 1.0 M, preferably less than about 0.50 M, less than or equal to about 0.30 M or less than or equal to 0.15 M. Especially preferred concentrations include about 0.15 M and about 0.30 M. For some embodiments having two or more viscosity reducing agents, the agents they are preferably, however, not necessarily present in the same concentration.

[00103] Viscosity reducing agents allow for faster reconstitution of a lyophilized dosage unit. The dosage unit is a lyophilized mass of protein, viscosity reducing agent and other excipients, to which water, saline or other pharmaceutically acceptable fluid is added. In the absence of viscosity reducing agents, periods of 10 minutes or more are often required to completely dissolve the freeze-dried mass at high protein concentration. When the freeze-dried dough contains one or more viscosity reducing agents, the period required to completely dissolve the dough is often reduced by a factor of two, five or even ten. In certain embodiments, less than one minute is required to completely dissolve a lyophilized mass that contains more than or about 150, 200 or even 300 mg/mL of protein.

[00104] Low viscosity protein formulations allow greater flexibility in formulation development. Low viscosity formulations exhibit viscosity that changes less with increasing protein concentrations compared otherwise to the same formulation without the viscosity reducing agent(s). Low viscosity protein formulations exhibit a decreased viscosity gradient compared otherwise to the same formulation without the viscosity reducing agent.

[00105] The viscosity gradient of the protein formulation may be 2 times less, 3 times less or even more than 3 times less than the viscosity gradient otherwise of the same protein formulation without the dehydrating agent(s). reduction of viscosity. The viscosity gradient of the protein formulation may 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 for a protein formulation that has a protein concentration between 10 mg/mL and 2,000 mg/mL. By reducing the viscosity gradient of the formulation, the protein concentration can be increased to a greater degree before an exponential increase in viscosity is observed.

A. Proteins

[00106] Any protein can be formulated, including recombinant, isolated, or synthetic proteins, glycoproteins or lipoproteins. These may be antibodies (including antibody fragments and recombinant antibodies), enzymes, growth factors or hormones, immunomodifiers, anti-infectives, antiproliferatives, vaccines, or other therapeutic, prophylactic, or diagnostic proteins. In certain 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 even greater than 200 kDa.

[00107] In certain embodiments, the protein may be a PEGylated protein. The term "PEGylated protein", when used herein, refers to a protein having one or more poly(ethylene glycol) or other stealth polymer groups covalently attached thereto, optionally by a chemical linker that may be other than a or more polymer groups. PEGylated proteins are characterized by their typically reduced renal filtration, decreased absorption by the reticuloendothelial system, and decreased enzymatic degradation leading, for example, to prolonged half-lives and enhanced 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(vinylpyrrolidone); poly(N-(2-hydroxypropyl)methacrylamide); and copolymers and mixtures thereof. In preferred embodiments, the stealth polymer in a PEGylated protein is poly(ethylene glycol) or a copolymer thereof. PEGylated proteins can be randomly PEGylated, that is, having one or more stealth polymers covalently linked at a non-specific site(s) on the protein, or can be PEGylated in a site-specific manner to covalently link the stealth polymer to the specific site(s) on the protein. Site-specific PEGylation can be carried out, for example, using activated stealth polymers having one or more reactive functional groups. Examples are described, for example, in Hoffman et al., Progress in Polymer Science, 32:922-932, 2007.

[00108] In the preferred embodiment, the protein is high molecular weight and an antibody, more preferably a mAb, and has a high viscosity in aqueous buffered solution when concentrated sufficiently to inject a therapeutically effective amount in a volume that does not exceed 1.0 to 2.0 mL for SC and 3.0 to 5.0 mL for IM administration. 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 for use in the formulations described herein are preferably essentially pure and essentially homogeneous (that is, substantially free of contaminating proteins and/or irreversible aggregates thereof).

[00109] Preferred mAbs include natalizumab here (TYSABRI®), cetuximab (ERBITUX®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), infliximab (REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatumumab (ARZERRA®), and biosimilars thereof. Exemplary high molecular weight proteins may include tocilizumab (ACTEMRA®), alemtuzumab (marketed under various trade names), brodalumab (developed by Amgen, Inc ("Amgen")), denosumab (PROLIA® and XGEVA®), and biosimilars thereof. .

[00110] Exemplary molecular targets for antibodies described here include CD proteins, such as CD3, CD4, CD8, CD19, CD20 and CD34; members of the HER receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules, such as LFA-1, Mol, p150.95, VLA-4, ICAM-1, VCAM, and αv/β3 integrin, including α or β subunits thereof (e.g., anti-CD11a antibodies, anti -CD18, or anti-CD11b); growth factors, such as VEGF; IgE; blood group antigens; flk2/flt3 receiver; obesity receptor (OB); protein C; PCSK9; etc. Antibody Therapeutics Currently on the Market

[00111] Many protein therapeutics currently on the market, especially antibodies as defined here, are administered via IV infusions due to high dosage requirements. Formulations may include one of the antibody therapeutics currently on the market or a biosimilar thereof. Some protein therapeutics are currently not high molecular weight, however, they are still administered via IV infusion because high doses are required for therapeutic efficacy. In some embodiments, liquid formulations are provided of these low molecular weight proteins as defined herein with concentrations to deliver therapeutically effective amounts for SC or IM injections.

[00112] Antibody therapeutics currently on the market include belimumab (BENLYSTA®), golimumab (SIMPONI ARIA®), abciximab (REOPRO®), the combination of tositumomab and iodine-131 tositumomab, marketed as BEXXAR®, alemtuzumab (CAMPATH®) , palivizumab (SYNAGIS®), basiliximab (SIMULECT®), ado-trastuzumab emtansine (KADCYLA®), pertuzumab (PERJETA®), capromab pendetide (PROSTASCINT KIT®), caclizumab (ZENAPAX®), ibritumomab tiuxetam (ZEVALIN®), eculizumab (SOLIRIS®) ®), omalizumab (XOLAIR®), and ustekinumab (STELARA®).

[00113] Natalizumab, a humanized mAb with the cell adhesion molecule 4-integrin, is used in the treatment of multiple sclerosis and Crohn's disease. Previously marketed under the trade name ANTEGREN®, natalizumab is currently co-marketed as TYSABRI® by Biogen Idec ("Biogen") and Elan Corp. ("Elan") TYSABRI® is produced in murine myeloma cells. Each 15 mL dose contains 300 mg of natalizumab; 123 mg sodium chloride, USP; 17.0 mg sodium phosphate monohydrate, monobasic, USP; 7.24 mg heptahydrate, sodium phosphate dibasic, USP; 3.0 mg polysorbate 80, USP/NF, in water for IV injection, USP at pH 6.1. Natalizumab is typically administered by monthly intravenous (IV) infusions and has been proven to be effective in treating the symptoms of multiple sclerosis and Crohn's disease, as well as preventing relapse, vision loss, cognitive decline, and significantly improving the patient's quality of life. .

[00114] When used herein, the term "natalizumab" includes the mAb to the α4-integrin cell adhesion molecule known under 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 under the trade name TYSABRI® by Biogen Idec and Elan Corporation or a biosimilar product thereof.

[00115] Cetuximab is an epidermal growth factor receptor (EGFR) inhibitor used for the treatment of metastatic colorectal cancer and head and neck cancer. Cetuximab is a chimeric (mouse/human) mAb typically administered by IV infusion. Cetuximab is marketed for IV use only under the trade name ERBITUX® by Bristol-Myers Squibb Company (North America; "Bristol-Myers Squibb"), Eli Lilly and Company (North America; "Eli Lilly"), and Merck KGaA. ERBITUX® is produced in mammalian cell culture (murine myeloma). Each 50 mL single-use vial of ERBITUX® contains 100 mg 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 of sodium phosphate dibasic heptahydrate, 0.42 mg/mL of sodium phosphate monobasic monohydrate, and water for IV Injection, USP.

[00116] Cetuximab is indicated for the treatment of patients with KRAS wild-type, epidermal growth factor receptor (EGFR)-expressing metastatic colorectal cancer (mCRC), in combination with chemotherapy, and as a single agent in patients who have not responded to therapy based on oxaliplatin and irinotecan or who are intolerant to irinotecan. Cetuximab is indicated for the treatment of patients with squamous cell carcinoma of the head and neck in combination with platinum-based chemotherapy for the first-line treatment of recurrent and/or metastatic disease and in combination with radiotherapy for locally advanced disease. Approximately 75% of patients with metastatic colorectal cancer have an EGFR-expressing tumor and are therefore considered eligible for treatment with cetuximab or panitumumab, according to FDA guidelines.

[00117] When used herein, the term "cetuximab" includes the mAb known under the International Nonproprietary Name "CETUXIMAB" or an antigen-binding portion thereof. Cetuximab includes antibodies described in U.S. Patent No. 6,217,866. Cetuximab includes the active agent in products marketed under the trade name ERBITUX® and biosimilar products thereof. ERBITUX® biosimilars may include those currently being developed by Amgen, AlphaMab Co., Ltd. ("AlfaMab"), and Actavis plc ("Actavis").

[00118] Bevacizumab, a humanized mAb that inhibits vascular endothelial growth factor A (VEGF-A), acts as an antiangiogenic agent. It is marketed under the trade name AVASTIN® by Genentech, Inc. ("Genentech") and F. Hoffmann-La Roche, LTD ("Roche"). It is authorized to treat several cancers, including colorectal, lung, breast (outside the U.S.A.), glioblastoma (U.S.A. only), kidney, and ovarian. AVASTIN® was approved by the FDA in 2004 for use in metastatic colorectal cancer when used with standard chemotherapy treatment (as first-line treatment) and with 5-fluorouracil-based therapy for second-line metastatic colorectal cancer. In 2006, the FDA approved AVASTIN® for use in first-line, advanced, non-small cell lung cancer in combination with carboplatin/paclitaxel chemotherapy. AVASTIN® is given as an IV infusion every three weeks at a dose of 15 mg/kg or 7.5 mg/kg. The highest dose is typically determined with carboplatin-based chemotherapy, whereas the lowest dose is determined with cisplatin-based chemotherapy. In 2009, the FDA approved AVASTIN® for use in metastatic renal cell carcinoma (a form of kidney cancer). The FDA also granted accelerated approval of AVASTIN® for the treatment of recurrent glioblastoma multiforme in 2009. The early growth treatment is still in phase III clinical trials.

[00119] The National Comprehensive Cancer Network ("NCCN") recommends bevacizumab as standard first-line treatment in combination with any platinum-based chemotherapy, followed by maintenance bevacizumab until disease progression. The NCCN updated its Oncology Clinical Practice Guidelines (NCCN Guidelines) for Breast Cancer in 2010 to affirm the recommendation regarding the use of bevacizumab (AVASTIN®, Genentech/Roche) in the treatment of metastatic breast cancer.

[00120] When used herein, the term "bevacizumab" includes the mAb that inhibits vascular endothelial growth factor A (VEGF-A) known under the Nonproprietary Name/International Common Name "BEVACIZUMAB" or an antigen-binding moiety the same. Bevacizumab is described in U.S. Patent No. 6,054,297. Bevacizumab includes the active agent in products marketed under the trade name AVASTIN® and biosimilar products thereof. AVASTIN® biosimilars may include those currently being developed by Amgen, Actavis, AlphaMab, and Pfizer, Inc ("Pfizer"). AVASTIN® biosimilar may include the biosimilar known as BCD-021 produced by Biocad and currently in clinical trials in the U.S.

[00121] Trastuzumab is a mAb that interferes with the HER2/neu receptor. Trastuzumab is marketed under the trade name HERCEPTIN® by Genentech, Inc. HERCEPTIN® is produced by a mammalian cell line (Chinese Hamster Ovary (CHO)). HERCEPTIN® is a sterile, white to light yellow, preservative-free lyophilized powder for IV administration. Each bottle of HERCEPTIN® contains 440 mg of trastuzumab, 9.9 mg of L-histidine HCl, 6.4 mg of L-histidine, 400 mg of a,a-trehalose dihydrate, and 1.8 mg of polysorbate 20 , USP. Reconstitution with 20 mL of water produces a multidose solution containing 21 mg/mL trastuzumab. HERCEPTIN® is currently administered as frequently as weekly via IV infusion and at a dosage ranging from about 2 mg/kg to about 8 mg/kg.

[00122] Trastuzumab is primarily used to treat certain breast cancers. The HER2 gene is amplified in 20-30% of early-stage breast cancers, which overexpress epidermal growth factor (EGF) receptors on the cell membrane. Trastuzumab is generally administered as maintenance therapy to HER2-positive breast cancer patients, typically for a year post-chemotherapy. Trastuzumab is currently administered via IV infusion as frequently as weekly at a dosage ranging from about 2 mg/kg to about 8 mg/kg.

[00123] When used herein, the term "trastuzumab" includes the mAb that interferes with the HER2/neu receptor known under the Nonproprietary Name/International Common Name "TRASTUZUMAB" or an antigen-binding portion thereof. Trastuzumab is described in U.S. Patent No. 5,821,337. Trastuzumab includes the active agent in products marketed under the trade name HERCEPTIN® and biosimilars thereof. The term "trastuzumab" includes the active agent in HERCEPTIN® biosimilar products marketed under the trade name HERTRAZ® by Mylan, Inc. ("Mylan") and CANMAB® by Biocon, Ltd. ("Biocon"). Trastuzumab may include the active agent in HERCEPTIN® biosimilar products that are developed by Amgen and PlantForm Corporation, Canada.

[00124] Infliximab is a mAb against tumor necrosis factor alpha (TNF-a) used to treat autoimmune diseases. It is marketed under the trade name REMICADE® by Janssen Global Services, LLC ("Janssen") in the U.S., Mitsubishi Tanabe Pharma in Japan, Xian Janssen in China, and Merck & Co ("Merck"); in other places. Infliximab is a chimeric mouse/human monoclonal antibody with a high molecular weight of about 144 kDa. In some embodiments, the formulations contain a biosimilar REMICADE®, such as REMSIMATM or INFLECTRATM. Both REMSIMATM, developed by Celltrion, Inc. ("Celltrion"), and INFLECTRATM, developed by Hospira Inc, UK, have been recommended for regulatory approval in Europe. Celltrion has submitted a filing for REMSIMATM to the FDA. Infliximab is currently administered via IV infusion at doses ranging from about 3 mg/kg to about 10 mg/kg.

[00125] Infliximab contains approximately 30% murine variable region amino acid sequence, which confers antigen binding specificity to human TNFa. The remaining 70% corresponds to a human IgG1 heavy chain constant region and a human kappa light chain constant region. Infliximab has high affinity for human TNFa, which is a cytokine with multiple biological actions including mediation of inflammatory responses and modulation of the immune system.

[00126] Infliximab is generally a recombinant antibody produced and secreted from mouse myeloma cells (SP2/0 cells). The antibody is currently manufactured by continuous perfusion cell culture. The infliximab monoclonal antibody is expressed using chimeric antibody genes consisting of variable region sequences cloned from murine anti-TNF hybridoma A2, and human antibody constant region sequences provided by plasmid expression vectors. Generation of the murine anti-TNF α hydrodome is accomplished by immunizing BALB/c mice with purified recombinant human TNFα. The light and heavy 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.

[00127] When used herein, the term "infliximab" includes the chimeric mouse/human monoclonal antibody known under the International Nonproprietary Name "INFLIXIMAB" or an antigen-binding portion thereof. Infliximab neutralizes the biological activity of TNFα by binding with high affinity to soluble and transmembrane forms of TNFα and inhibits the binding of TNFα to its receptors. Infliximab is described in U.S. Patent No. 5,698,195. The term "Infliximab" includes the active agent in products marketed or proposed to be marketed under the trade name REMICADE® by multiple entities; REMSIMATM by Celltrion and INFLECTRATM by Hospira, Inc ("Hospira"). Infliximab is supplied as a sterile lyophilized mass for reconstitution and dilution. Each vial of infliximab contains 100 mg of infliximab and excipients such as monobasic sodium phosphate monohydrate, dibasic sodium phosphate dihydrate, sucrose, and polysorbate 80.

[00128] Denosumab (PROLIA® and XGEVA®) is a human mAb - and the first RANKL inhibitor - approved for use in postmenopausal women at risk of osteoporosis and patients with bone metastasis from solid tumors. Denosumab is in Phase II trials for the treatment of rheumatoid arthritis.

[00129] Panitumumab is a fully human mAb approved by the FDA for the treatment of metastatic EGFR-expressing cancer with disease progression. Panitumumab is marketed under the trade name VECTIBIX® by Amgen. VECTIBIX® is packaged as a 20 mg/ml panitumumab concentrate in 5 ml, 10 ml, and 15 ml vials for IV infusion. When prepared according to package instructions, the final panitumumab concentration does not exceed 10 mg/ml. VECTIBIX® is administered at a dosage of 6 mg/kg every 14 days as an intravenous infusion. When 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 products marketed under the trade name VECTIBIX® by Amgen and biosimilars thereof. The term "panitumumab" includes monoclonal antibodies described in U.S. Patent No. 6,235,883. The term "panitumumab" includes the active agent in VECTIBIX® biosimilar products, including biosimilar VECTIBIX® being developed by BioXpress, SA ("BioXpress").

[00130] Belimumab (BENLYSTA®) is a human mAb with a molecular weight of about 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 by IV infusion at a dosage of 10 mg/kg. A high molecular weight, low viscosity protein formulation may include Belimumab, preferably at a concentration of about 400 mg/mL to about 1,000 mg/mL. Preferred ranges are calculated based on body weight of 40-100 kg (about 80-220 lbs) in a 1 mL volume.

[00131] Abciximab (REOPRO®) 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 on human platelets and inhibits platelet aggregation by preventing the binding of fibrinogen, von Willebrand factor, and other adhesive molecules. Likewise, it binds to the vitronectin receptor (αvβ3) found on platelets and smooth muscle and epithelial cells of the wall. Abciximab is a platelet aggregation inhibitor primarily used during and after coronary artery procedures. Abciximab is administered via IV infusion, first as a bolus of 0.25 mg/kg and followed by a continuous IV infusion of 0.125 mcg/kg/minute over 12 hours.

[00132] Tositumomab (BEXXAR®) is a drug for the treatment of follicular lymphoma. It is an IgG2a anti-CD20 mAb derived from immortalized mouse cells. Tositumomab is administered in sequential infusions: cold mAb followed by iodine tositumomab (131I), the same antibody covalently linked to the radionuclide iodine-131. Clinical experience has established the efficacy of the tositumomab/tositumomab iodine regimen in patients with relapsed refractory follicular lymphoma. BEXXAR® is currently administered at a dose of 450 mg via IV infusion.

[00133] Alemtuzumab (marketed as CAMPATH®, MABCAMPATH®, or CAMPATH-1H® and currently under further development as LEMTRADA®) is a mAb used in the treatment of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), and T-cell lymphoma. It is also used under clinical trial protocols for the treatment of some autoimmune diseases, such as multiple sclerosis. Alemtuzumab has a weight of about 145.5 kDa. It is administered as daily IV infusions of 30 mg to patients with B-cell chronic lymphocytic leukemia.

[00134] Palivizumab (SYNAGIS®) is a humanized mAb targeted at an epitope in the antigenic site A of the F protein of the respiratory syncytial virus. In two Phase III clinical trials in the pediatric population, palivizumab reduced the risk of hospitalization due to respiratory syncytial virus infection by 55% and 45%. Palivizumab is dosed once monthly via 15 mg/kg IM injection.

[00135] Ofatumumab is a human anti-CD20 mAb that appears to inhibit early stage B lymphocyte activation. Ofatumumab is marketed under the trade name ARZERRA® by GlaxoSmithKline, plc ("GlaxoSmithKline"). ARZERRA® is distributed in single-use vials containing 100 mg/5 mL and 1,000 mg/50 mL ofatumumab for IV infusion. Ofatumumab is FDA approved to treat 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 multiple sclerosis. Ofatumumab has a molecular weight of approximately 149 kDa. It is currently administered by IV infusion at an initial dose of 300 mg, followed by weekly infusions of 2,000 mg. When used herein, the term "ofatumumab" includes the anti-CD20 mAb known by the International Nonproprietary Name "OFATUMUMAB." The term "ofatumumab" includes the active agent in products marketed under the trade name ARZERRA® and biosimilars thereof. The term "ofatumumab" includes the active agent in ARZERRA® biosimilar products that are developed by BioExpress. Low viscosity, high molecular weight liquid protein formulations may include ofatumumab, preferably at a concentration of about 300 mg/mL to about 2,000 mg/mL.

[00136] Trastuzumab emtansine (in the U.S., ado-trastuzumab emtansine, marketed as KADCYLA®) is an antibody-drug conjugate consisting of the mAb trastuzumab linked to the cytotoxic agent mertansine (DM1®). Trastuzumab, described above, stops the growth of cancer cells by binding to the HER2/neu receptor, whereas mertansine enters cells and destroys them by binding to tubulin. In the United States, trastuzumab emtansine has been specifically approved for the treatment of recurrent HER2-positive metastatic breast cancer. Multiple Phase III trials of trastuzumab emtansine are planned or ongoing in 2014. Trastuzumab emtansine is currently administered by 3.6 mg/kg IV infusion. High molecular weight, low viscosity liquid formulations may include trastuzumab emtansine, preferably at a concentration of about 144 mg/mL to about 360 mg/mL.

[00137] Pertuzumab (PERJETA®) is a mAb that inhibits HER2 dimerization. Pertuzumab received FDA approval for the treatment of HER2-positive metastatic breast cancer in 2012. The currently indicated dosage of Pertuzumab is 420 mg to 840 mg by IV infusion. High molecular weight, low viscosity liquid formulations may include pertuzumab, preferably at a concentration of about 420 mg/mL to about 840 mg/mL.

[00138] Daclizumab is a humanized anti-CD25 mAb and is used to prevent rejection in organ transplants, especially in kidney transplants. The drug is also under investigation for the treatment of multiple sclerosis. Daclizumab has a molecular weight of approximately 143 kDa. Daclizumab was marketed in the U.S. by Hoffmann-La Roche, Ltd. ("Roche") as ZENAPAX® and administered by 1 mg/kg IV infusion. The Daclizumab High Yield Process (DAC HYP; BIIB019; Biogen Idec ("Biogen") and AbbVie, Inc. ("AbbVie")) is in Phase III clinical trials as a once-monthly, subcutaneous injection of 150 mg to treat relapsing, remitting multiple sclerosis. High molecular weight, low viscosity liquid formulations may include daclizumab, preferably at a concentration of about 40 mg/mL to about 300 mg/mL.

[00139] Eculizumab (SOLIRIS®) is a humanized mAb approved for the treatment of rare blood diseases, such as paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab, with a molecular weight of approximately 148 kDa, is being developed by Alexion Pharmaceutics, Inc ("Alexion"). It is administered by IV infusion in an amount of about 600 mg to about 1,200 mg. High molecular weight, low viscosity liquid formulations may include eculizumab, preferably at a concentration of about 500 mg/mL to about 1,200 mg/mL.

[00140] Tocilizumab (ACTEMRA®) is a humanized mAb against the interleukin-6 receptor. It is an immunosuppressive drug, mainly for the treatment of rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis, a severe form of RA in children. Tocilizumab is generally administered by IV infusion in doses 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.

[00141] Rituximab (RITUXAN®) is a chimeric anti-CD20 mAb 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 predominant subtype. It has been shown to be an effective rheumatoid arthritis treatment. Rituximab is widely used off-label to treat difficult cases of multiple sclerosis, systemic lupus erythematosus, and autoimmune anemias.

[00142] Rituximab is jointly marketed in the U.S. under the trade name RITUXAN® by Biogen and Genentech and outside the U.S. under the trade name MABTHERA® by Roche. RITUXAN® is distributed in single-use vials containing 100 mg/10 mL and 500 mg/50 mL. RITUXAN® is typically administered by IV infusion of approximately 375 mg/m2. The term "rituximab", when used herein, includes the anti-CD20 mAb known under the Non-Proprietary Name/International Common Name "RITUXIMAB". Rituximab includes mAbs described in U.S. Patent No. 5,736,137. Rituximab includes the active agent in products marketed under the trade names RITUXAN® and MABTHERA® and biosimilars thereof.

[00143] High molecular weight, low viscosity liquid formulations may include rituximab, preferably at a concentration of about 475 mg/mL to about 875 mg/mL (approximate using a body surface area range of 1.3 to 2.3 square meters, derived from Mosteller's formula for people ranging from 5 ft, 40 kg to 6 ft, 100 kg). Concentrations are calculated for a 1 mL formulation.

[00144] Ipilimumab is a human mAb developed by Bristol-Myers Squibb Company ("Bristol-Myers Squibb"). Marketed as YERVOY®, it is used for the treatment of melanoma and is also undergoing clinical trials for the treatment of non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), and refractory prostate cancer. to metastatic hormone. Ipilimumab is currently administered as a 3 mg/kg IV infusion. High molecular weight, low viscosity liquid formulations may include ipilimumab, preferably at a concentration of about 120 mg/mL to about 300 mg/mL.

[00145] Raxibacumab (ABtrax®) is a human mAb intended for the prophylaxis and treatment of inhaled anthrax. It is currently administered by IV infusion. The suggested dosage for adults and children over 50 kg is 40 mg/kg. High molecular weight, low viscosity liquid formulations may include raxibacumab, preferably at a concentration of about 1,000 mg/mL to about 4,000 mg/mL.

[00146] Nimotuzumab (THERACIM®, BIOMAB EGFR®, THERALOC®, CIMAher®) is a humanized mAb with a molecular weight of about 151 kDa used to treat squamous cell carcinomas of the head and neck, recurrent high-grade malignant glioma or refractory, anaplastic astrocytomas, glioblastomas, and diffuse intrinsic pontine glioma. Nimotuzumab is typically administered as an IV infusion of about 200 mg weekly. High molecular weight, low viscosity liquid formulations may include nimotuzumab, preferably at a concentration of about 200 mg/mL.

[00147] Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate targeting the CD30 protein, expressed in classical Hodgkin's lymphoma and systemic anaplastic large cell lymphoma. It is administered by IV infusion of about 1.8 mg/kg. High molecular weight, low viscosity liquid formulations may include brentuximab vedotin, preferably at a concentration of about 80 mg/mL to about 200 mg/mL.

[00148] Itolizumab (ALZUMAB®) is a humanized IgG1 mAb developed by Biocon. Itolizumab has successfully completed Phase III studies in patients with moderate to severe psoriasis. Itolizumab has received marketing approval in India; an application for FDA approval has not been submitted.

[00149] Obinutuzumab (GAZYVA®), originally developed by Roche and also being developed under a collaboration agreement with Biogen, is a humanized anti-CD20 mAb approved for the treatment of chronic lymphocytic leukemia. It is similarly being investigated in Phase III clinical trials for patients with various lymphomas. Dosages of about 1,000 are being administered via IV infusion.

[00150] Certolizumab pegol (CIMZIA®) is a humanized, recombinant antibody Fab' fragment with specificity for human tumor necrosis factor alpha (TNFa), conjugated to a polyethylene glycol of approximately 40kDa (PEG2MAL40K). The molecular weight of Certolizumab pegol is about 91 kDa.

[00151] Other antibody therapeutics that can be formulated with viscosity reducing agents include CT-P6 from Celltrion, Inc. (Celltrion).

Antibody Therapeutics in Late-Stage and Developmental Experiments

[00152] The progress of antibody therapeutics toward late-stage clinical development and regulatory review is proceeding at a rapid pace. As of 2014, there are more than 300 mAbs in clinical trials and 30 commercially sponsored antibody therapeutics undergoing evaluation in late-stage studies. The first marketing applications for two mAbs (vedolizumab and ramucirumab) were recently submitted to the FDA. Amgen is currently sponsoring multiple ongoing Phase III trials in the use of brodalumab in patients with plaque psoriasis, with additional trials planned or patients being enrolled. XBiotech, Inc. has sponsored two Phase I clinical trials of MABp1 (Xilonix) for patients with advanced cancer or type 2 diabetes. Additional MABp1 trials are recruiting patients. Multiple trials are sponsored by MedImmune, LLC ("MedImmune") and patients enrolled or enrolled in the treatment of leukemia with moxetumomab pasudotox. Long-term efficacy and safety studies are underway for the use of tildracizumab for the treatment of chronic plaque psoriasis. Multiple Phase II trials have recently concluded the use of rilotumumab for the treatment of several cancers.

[00153] At least 28 mAbs are high molecular weight proteins currently in or having recently completed Phase III studies for the treatment of inflammatory or immunological disorders, cancers, high cholesterol, osteoporosis, Alzheimer's disease, and infectious diseases. MABS EM or having recently completed phase III experiences include AMG 145, ELOTUZUMAB, EPRATUZUMABE, FARLETUZUMABE (MoraB-003), GANTENRUMABE (RG1450), GEVOCIZUMABE, INOTUZUMABE OZOGAMICIN, ITALIUM, IXECIZUMAB, LEBRICIZUMABE, MEPOLYMABE, NAPTUMMEBE X, necitumumab , ocrelizumab, onartuzumab, racotumomab, ramucirumab, reslizumab, romosozumab, sarilumab, secukinumab, sirucumab, solanezumab, tabalumab, and vedolizumab. A mixture of mAb (actoxumab and bezlotoxumab) is also being evaluated in Phase III trials. See, for example, Reichert, MAbs 5:1-4, 2013.

[00154] Vedolizumab is a mAb that is developed by Millennium Pharmaceutics, Inc ("Millennium"; a subsidiary of Takeda Pharmaceuticals Company, Ltd. ("Takeda")). Vedolizumab has been found safe and highly effective for inducing and maintaining clinical remission in patients with moderate to severe ulcerative colitis. Phase III clinical trials have demonstrated the goals of inducing a clinical response and maintaining remission in patients with Crohn's and ulcerative colitis. Studies evaluating long-term clinical results show close to 60% of patients achieving clinical remission. A common dose of vedolizumab is 6 mg/kg by IV infusion.

[00155] Ramucirumab is a human mAb that is developed for the treatment of solid tumors. Phase III clinical trials are ongoing for the treatment of breast cancer, metastatic gastric adenocarcinoma, non-small cell lung cancer, and other cancers. Ramucirumab, in some Phase III trials, is administered at about 8 mg/kg via IV infusion.

[00156] Rilotumumab is a human mAb that inhibits the action of hepatocyte growth factor/scatter factor. Developed by Amgen, it is in Phase III trials as a treatment for solid tumors. An open-label Phase III study of rilotumumab treatment in patients with advanced or metastatic esophageal cancer will administer rilotumumab at approximately 15 mg/kg via IV infusion.

[00157] Evolocumab (AMG 145), also developed by Amgen, is a mAb that binds to PCSK9. Evolocumab is indicated for hypercholesterolemia and hyperlipidemia.

[00158] Alirocumab (REGN727) is a human mAb from Regeneron Pharmaceutics, Inc. ("Regeneron") and Sanofi-Aventis U.S. of LLC ("Sanofi"), indicated for hypercholesterolemia and acute coronary syndrome.

[00159] Naptumomab stafenatox, ABR-217620 from Active Biotech AB ("Active Biotech") is a mAb indicated for renal cell carcinoma.

[00160] Racotumomab from CIMAB, SA ("CIMAB"); Laboratorio Elea S.A.C.I.F.y A. is a mAb indicated for non-small cell lung cancer.

[00161] Other antibodies that can be formulated with viscosity-lowering agents include bococizumab (PF-04950615) and tanezumab; Amgen's ganitumab, blinatumomab, trebananib; 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 Biopharmaceutics ("Navidea"); PF-05280014 from Pfizer; SA237 from Chugai Pharmaceutical Co. Ltd. ("Chugai"); guselkumab from Janssen/Johnson and Johnson Services, Inc. ("J&J"); Antithrombin Gamma (KW-3357) from Kiowa; and CT-P10 from Celltrion.

Antibodies in Early Stage Clinical Trials

[00162] Many mAbs have recently entered, or are entering clinical trials. They may include proteins currently administered via IV infusion, preferably those that have a molecular weight greater than about 120 kDa, typically from about 140 kDa to about 180 kDa. They may also include such high molecular weight proteins as peptides or albumin-conjugated drugs that are also entering clinical trials or have been approved by the FDA. Many Amgen mAbs are currently in clinical trials. These can be high molecular weight proteins, for example, AMG 557, which is a human monoclonal antibody developed jointly by Amgen and AstraZeneca and currently in Phase I trials for the treatment of lupus. Similarly, AMG 729 is a humanized mAb developed by Amgen and currently in Phase I trials for the treatment of lupus and rheumatoid arthritis. Furthermore, AMG 110 is an epithelial cell adhesion molecule mAb; AMG 157, jointly developed by Amgen and AstraZeneca, is currently a Phase I human mAb for the treatment of asthma; AMG 167 is a humanized mAb that has been evaluated in multiple Phase I trials for the treatment of osteopenia; AMG 334, having completed Phase I dosing studies and currently in Phase II studies for the treatment of migraines and hot flashes, is a human mAb that inhibits Calcitonin Gene-Related Peptide; AMG 780 is a human anti-angiopoietin mAb that inhibits the interaction between the endothelial cell-selective Tie2 receptor and its ligands Ang1 and Ang2, and recently completed Phase I trials as a cancer treatment; AMG 811 is a human monoclonal antibody that inhibits interferon gamma that is investigated as a treatment for systemic lupus erythematosus; AMG 820 is a human mAb that inhibits c-fms and decreases tumor-associated macrophage (TAM) function and is being investigated as a cancer treatment; AMG 181, jointly developed by Amgen and AstraZeneca, is a human mAb that inhibits the action of alpha4/beta7 and is in Phase II trials as a treatment for ulcerative colitis and Crohn's disease.

[00163] Many mAbs are currently in clinical trials for the treatment of autoimmune disorders. These mAbs can be included in low viscosity, high molecular weight liquid formulations. RG7624 is a fully human mAb designed to specifically and selectively bind to the human interleukin-17 family of cytokines. A Phase II clinical trial evaluating RG7624 for autoimmune disease is ongoing. BIIB033 is an anti-LINGO-1 mAb by Biogen currently in Phase II trials to treat multiple sclerosis.

[00164] High molecular weight proteins may also include AGS-009, a mAb that targets IFN-alpha developed by Argos Therapeutics, Inc. which recently completed phase I trials for the treatment of lupus. Patients are administered up to 30 mg/kg of AGS-009 via IV infusion. BT-061, developed by AbbVie, is in Phase II trials for patients with rheumatoid arthritis. Certolizumab pegal (CIMZIA®) is a mAb in Phase II trials for ankylosing spondylitis and juvenile rheumatoid arthritis. Clazakizumab, an anti-IL6 mAb, is in Phase II trials by Bristol-Myers Squibb.

[00165] CNTO-136 (sirucumab) and CNTO-1959 are mABs that have recently completed Phase II and Phase III trials by Janssen. Daclizumab (previously marketed as ZENAPAX® by Roche) is currently in or recently completed multiple Phase III trials by AbbVie for the treatment of multiple sclerosis. Epratuzumab is a humanized mAb in Phase III trials for the treatment of lupus. Canakinumab (ILARIS®) is a human mAb targeting interleukin-1 beta. It has been approved for the treatment of cryopyrin-associated periodic syndromes. Canakinumab is in Phase I trials as a potential treatment for chronic obstructive pulmonary disease, gout and coronary artery disease. Mavrilimumab is a human mAb designed for the treatment of rheumatoid arthritis. Discovered as CAME-3001 by Cambridge Antibody Technology, mavrilimumab is being developed by MedImmune.

[00166] MEDI-546 are MEDI-570 are mAbs currently in Phase I and Phase II trials by AstraZeneca for the treatment of lupus. MEDI-546 is administered in the Phase II study by regular IV infusions of 300-1,000 mg. MEDI-551, another mAb being developed by AstraZeneca for numerous indications, is likewise currently administered by IV infusion. NN8209, a mAb that blocks the C5aR receptor being developed by Novo Nordisk A/S ("Novo Nordisk"), has completed a Phase II dosing study for the treatment of rheumatoid arthritis. NN8210 is another antiC5aR mAb being developed by Novo Nordisk and is currently in Phase I trials. IPH2201 (NN8765) is a humanized mAb that targets NKG2A being developed by Novo Nordisk to treat patients with inflammatory conditions and autoimmune diseases. NN8765 recently completed Phase I experiments.

[00167] Olocizumab is a humanized mAb that potentially targets the cytokine IL-6. IL-6 is involved in several series of autoimmune and inflammatory reactions. Olocizumab has completed Phase II trials for the treatment of rheumatoid arthritis. Otelixizumab, also known as TRX4, is a mAb being developed for the treatment of type 1 diabetes, rheumatoid arthritis, and other autoimmune diseases. Ozoralizumab is a humanized mAb that has completed Phase II trials.

[00168] Pfizer currently has Phase I trials for mAbs PD-360324 and PF-04236921 for the treatment of lupus. A biosimilar rituximab, PF-05280586, was developed by Pfizer and is in Phase I/Phase II trials for rheumatoid arthritis.

[00169] Rontalizumab is a humanized mAb being developed by Genentech. It recently completed Phase II trials for the treatment of lupus. SAR113244 (anti-CXCR5) is a mAb by Sanofi in Phase I trials. Sifalimumab (anti-IFN-alpha mAb) is a mAb in Phase II trials for the treatment of lupus.

[00170] A low viscosity liquid formulation with high molecular weight may include one of the mAbs in early stage clinical development to treat various blood disorders. For example, Belimumab (BENLYSTA®) recently completed Phase I trials for patients with vasculitis. Other mAbs in early-stage trials for blood disorders include BI-655075 from Boehringer Ingelheim GmbH "Boehringer Ingelheim", ferroportin mAb and hepcidin mAb from Eli Lily, and SelG1 from Selexys Pharmaceuticals, Corp. ("Selexys").

[00171] One or more mAbs in early stage development to treat various cancers and related conditions can be included in a low viscosity, high molecular weight liquid formulation. United Therapeutics, Corporation has two mAbs in Phase I trials, 8H9 mAb and ch14.18 mAb. AbbVie's mAbs ABT-806, enavatuzumab, and volociximab are in early-stage development. Actinium Pharmaceutics, Inc conducted early stage experiments for the mAbs Actimab-A (M195 mAb), anti-CD45 mAb, and Iomab-B. Seattle Genetics, Inc. ("Seattle Genetics") has several mAbs in early-stage trials for cancer and related conditions, including anti-CD22 ADC (RG7593; pinatuzumab vedotin), anti-CD79b ADC (RG7596), anti-STEAP1 ADC ( RG7450), ASG-5ME and ASG-22ME from Agesis, Inc. ("Agensis") the antibody-drug conjugate RG7458, and vorsetuzumab mafodotin. Genentech's early stage cancer therapeutics can be included in low viscosity formulations, including ALT-836, antibody-drug conjugates RG7600 and DEDN6526A, anti-CD22 ADC (RG7593), anti-EGFL7 mAb (RG7414), anti-CD22 mAb -HER3/EGFR DAF (RG7597), anti-PD-L1 mAb (RG7446), DFRF4539A, a MINT1526A. Bristol-Myers Squibb is developing early-stage mAbs for cancer therapeutics, including those identified as anti-CXCR4, anti-PD-L1, IL-21 (BMS-982470), lirilumab, and urelumab (anti-CD137). Other mAbs in early-stage trials as cancer therapeutics include APN301(hu14.18-IL2) from Apeiron Biologics AG, AV-203 from AVEO Pharmaceutics, Inc. ("AVEO"), AVX701 and AVX901 from AlfaVax, 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.

[00172] Other antibody therapeutics that can be formulated with viscosity-lowering agents 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, SAR2562 12, SAR279356 , SAR3419, SAR153192 (REGN421,enoticumab), SAR307746 (nesvacumab), SAR650984, SAR566658, SAR391786, SAR228810, SAR252067, SGN-CD19A, SGN-CD33A,SGN-LIV1A, ASG 15ME, Anti-LIN GO, BIIB037, ALXN1007, teprotumumab, concizumab, anrucinzumab (IMA-638), ponezumab (PF-04360365), PF-03446962, PF-06252616, etrolizumab (RG7413), quilizumab, ranibizumab, lampalizumab, onclacumab, gentenerumab, crenezumab (RG7412), IMC-RON8 (narnatumab ) , tremelimumab, vantictumab, eencizumab, ozanezumab, lojatumumab, tralokinumab, XmAb5871, 3617, MEDI4736, MEDI6469, MEDI0680, MEDI5872, PF- 05236812 (AAB-003) , BT061, MOR208, OMP59R5 (anti-notching 2/ 3) LY2495655, LY2875358, and LY2812176.

[00173] Other early stage mAbs that can be formulated with viscosity-lowering agents 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 is a monovalent anti-CD40L pegylated Fab antibody fragment, STX-100 is an anti-avB6 mAb, BIIB059, Anti-TWEAK (BIIB023), and BIIB022 from Biogen; fulranumab from Janssen and Amgen; BI-204/RG7418 from BioInvent International/Genentech; BT-062 (indatuximab ravtansine) from Biotest Pharmaceutics Corporation; XmAb from Boehringer Ingelheim/Xencor; Bristol-Myers Squibb anti-IP10; J 591 Lu-177 from BZL Biologics LLC; CDX-011 (glembatumumab vedotin), CDX-0401 from Celldex Therapeutics; Crucell's foravirumab; tigatuzumab from Daiichi Sankio Company Limited; MORAb-004, MORAb-009 (amatuximab) from Eisai; LY2382770 from Eli Lilly; DI17E6 from EMD Serono Inc; zanolimumab from Emergente BioSolutions, Inc.; FG-3019 from FibroGen, Inc.; catumaxomab from Fresenius SE & Co. KGaA; pateclizumab, Genentech's rontalizumab; fresolimumab from Genzyme &Sanofi; GS-6624 (simtuzumab) from Gilead; CNTO-328, bapineuzumab (AAB-001), carlumab, Janssen's CNTO-136; KB003 from KaloBios Pharmaceutics, Inc.; ASKP1240 from Kyowa; RN-307 from Labrys Biologics Inc.; ecromeximab from Life Science Pharmaceutics; LY2495655, LY2928057, LY3015014, LY2951742 from Eli Lilly; MBL-HCV1 from MassBiologics; AME-133v from MENTRIK Biotech, LLC; abituzumab from Merck KGaA; MM-121 from Merrimack Pharmaceutics, Inc.; MCS110, QAX576, QBX258, QGE031 from Novartis AG; HCD122 from Novartis AG and XOMA Corporation ("XOMA"); NN8555 from Novo Nordisk; bavituximab, cotara from Peregrine Pharmaceutics, Inc.; PSMA-ADC from Progenics Pharmaceutics, 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.; TCN-032 from Theraclone Sciences; TRC105 from TRACON Pharmaceutics, Inc.; UB-421 from United Biomedical Inc.; VB4-845 from Viventia Bio, Inc.; ABT-110 from AbbVie; Caplacizumab, Ozoralizumab from Ablinx; PRO 140 from CytoDyn, Inc.; GS-CDA1, MDX-1388 from Medarex, Inc.; AMG 827, AMG 888 from Amgen; ublituximab from TG Therapeutics Inc.; TOL101 from Tolera Therapeutics, Inc.; huN901-DM1 (lorvotuzumab mertansine) from ImmunoGen Inc.; epratuzumab Y-90/veltuzumab combination (IMMU-102) from Immunomedics, Inc.; anti-fibrin mAb/3B6/22 Tc-99m from Agenix, Limited; ALD403 from Alder Biopharmaceutics, Inc.; RN6G/PF-04382923 from Pfizer; CG201 from CG Therapeutics, Inc.; KB001-A from KaloBios Pharmaceutics/Sanofi; KRN- 23 from Kyowa; Y-90 hPAM 4 from Immunomedics, Inc.; Tarextumab from Morphosys AG & OncoMed Pharmaceuticals, Inc.; LFG316 from Morphosys AG & Novartis AG; CNTO3157, CNTO6785 from Morphosys AG &Jannsen; RG6013 from Roche &Chugai; MM-111 from Merrimack Pharmaceutics, Inc. ("Merrimack"); GSK2862277 from GlaxoSmithKline; AMG 282, AMG 172, AMG 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; anetumab ravtansine from Morphosys AG & Bayer AG; BI-836845 from Morphosys AG & Boehringer Ingelheim; NOV-7, NOV - 8 from Morphosys AG & Novartis AG; MM-302, MM-310, MM-141, MM-131, MM-151 from Merrimack, RG7882 from Roche & Seattle Genetics; RG7841 from Roche/Genentech; PF-06410293, PF-06438179, PF-06439535, PF-04605412, PF-05280586 from Pfizer; RG7716, RG7936, gentenerumab, RG7444 from Roche; MEDI-547, MEDI-565, MEDI1814, MEDI4920, MEDI8897, MEDI-4212, MEDI-5117, MEDI-7814 from Astrazeneca; ulocuplumab, PCSK9 adnectin from Bristol-Myers Squibb; FPA009, FPA145 from FivePrime Therapeutics, Inc.; GS-5745 from Gilead; BIW-8962, KHK4083, KHK6640 from Kyowa Hakko 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, ALX-0171 from Ablynx; milatuzumab-DOX, milatuzumab, TF2, from Immunomedics, Inc.; MLN0264 from Millenium; ABT-981 from AbbVie; AbGn-168H from AbGenomics International Inc.; ficlatuzumab from AVEO; BI-505 from BioInvent International; CDX-1127, 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-1 β antibody, IMC-CS4 from Eli Lilly; VEGFR3 mAb, IMC-TR1 (LY3022859) from Eli Lilly and ImClone, LLC; Anthim from Elusys Therapeutics Inc.; HuL2G7 from Galaxy Biotech LLC; IMGB853, IMGN529 from ImmunoGen Inc.; CNTO-5, CNTO-5825 from Janssen; KD-247 from Kaketsuken; KB004 from KaloBios Pharmaceutics; MGA271, MGAH22 from MacroGenics, Inc.; XmAb5574 from MorphoSys AG/Xencor; ensituximab (NPC-1C) from Neogenix Oncology, Inc.; LFA102 from Novartis AG and XOMA; ATI355 from Novartis AG; SAN-300 from Santarus Inc.; SelG1 from Selexys; HuM195/rGel from Targa Therapeutics, Corp.; VX15 from 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 for Allergy and Infectious Diseases; neuroblastoma antibody vaccine from MabVax Therapeutics; Cytolin from CytoDyn, Inc.; Thravixa from Emergent BioSolutions Inc.; and FB 301 from Cytovance Biologics; Janssen and Sanofi rabies mAb; influenza mAb from Janssen and 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

[00174] The protein may be an enzyme, a fusion protein, a stealth or pegylated protein, vaccine or otherwise a biologically active protein (or protein mixture). The term "enzyme", when used herein, refers to the protein or functional fragment thereof that catalyzes a biochemical transformation of a target molecule to a desired product.

[00175] Enzymes as drugs have at least two important aspects, that is, i) they often bind and act on their targets with affinity and specificity, and ii) they are catalytic and convert multiple target molecules to the desired products. In certain embodiments, the protein may be PEGylated, as defined herein.

[00176] The term "fusion protein", when used herein, refers to a protein that is created from two different genes that code for two separate proteins. Fusion proteins generally are produced by recombinant DNA techniques known to those skilled in the art. Two proteins (or protein fragments) are fused together covalently and exhibit properties of both parent proteins.

[00177] There are several fusion proteins that are on the market.

[00178] ENBREL® (Etanercept), is a fusion protein marketed by Amgen that competitively inhibits TNF.

[00179] ELOCTATE®, Antiemophilic Factor (Recombinant), Fc Fusion Protein, is an antiemophilic factor, derived from recombinant DNA indicated in adults and children with Hemophilia A (congenital Factor VIII deficiency) for control and prevention of bleeding episodes, perioperative control, routine prophylaxis to prevent or reduce the frequency of bleeding episodes.

[00180] EYLEA® (aflibercept) is a recombinant fusion protein consisting of portions of extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1 formulated as an iso-osmotic solution for intravitreal administration. EYLEA (aflibercept) is a recombinant fusion protein consisting of portions of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1 formulated as an iso-osmotic solution for intravitreal administration. Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation constituting an additional 15% of the total molecular mass, resulting in a total molecular weight of 115 kDa. Aflibercept is produced in recombinant Chinese hamster ovary (CHO) cells, marketed by Regeneron.

[00181] ALPROLIXTM, Coagulation Factor IX (Recombinant), Fc Fusion Protein, is a concentrate of coagulation Factor IX, derived from recombinant DNA, is indicated in adults and children with hemophilia B for control and prevention of bleeding episodes, perioperative control, routine prophylaxis to prevent or reduce the frequency of bleeding episodes.

[00182] Pegloticase (KRYSTEXXA®) is a drug for the treatment of severe, treatment-refractory, chronic gout, developed by Savient Pharmaceutics, Inc. and is the first drug approved for this indication. Pegloticase is a pegylated recombinant porcine type uricase with a molecular weight of about 497 kDa. Pegloticase is currently administered via IV infusions of about 8 mg/kg. High molecular weight, low viscosity liquid formulations may include pegloticase, preferably at a concentration of about 300 mg/mL to about 800 mg/mL.

[00183] Alteplase (ACTIVASE®) is a tissue plasminogen activator produced by recombinant DNA technology. It is a purified glycoprotein comprising 527 amino acids and synthesized using complementary DNA (cDNA) for natural human tissue-type plasminogen activator obtained from a human melanoma cell line. Alteplase is administered immediately via IV infusion of about 100 mg immediately following symptoms of a stroke. In some embodiments, low viscosity formulations are provided containing alteplase, preferably at a concentration of about 100 mg/mL.

[00184] Glucarpidase (VORAXAZE®) is a drug approved by the FDA for the treatment of elevated levels of methotrexate (defined as at least 1 micromol/L) during the treatment of cancer patients who have impaired renal function. Glucarpidase is administered IV in a single dose of about 50 IU/kg. In some embodiments, low viscosity formulations are provided containing glucarpidase.

[00185] Alglucosidase Alfa (LUMIZYME®) is an orphan enzyme replacement therapy drug for the treatment of Pompe disease (glycogen storage disease type II), a rare lysosomal storage disorder. It has a molecular weight of about 106 kDa and is currently administered via IV infusions of about 20 mg/kg. In some embodiments, a low viscosity alglucosidase alfa pharmaceutical formulation is provided, preferably at a concentration of about 100 mg/mL to about 2,000 mg/mL.

[00186] Bovine pegdamase (ADAGEN®) is a modified enzyme used for enzyme replacement therapy for the treatment of severe combined immunodeficiency disease (SCID) associated with an adenosine deaminase deficiency. Bovine pegdamase is a conjugate of numerous strands of monomethoxypolyethylene glycol (PEG), molecular weight 5,000 Da, covalently linked to the enzyme adenosine deaminase that was derived from the bovine intestine.

[00187] α-Galactosidase is a lysosomal enzyme that catalyzes the hydrolysis of the glycolipid, globotriaosylceramide (GL-3), into galactose and ceramide diexoside. Fabry disease is a rare heritable lysosomal storage disease characterized by subnormal a-Galactosidase enzyme activity and resultant accumulation of GL-3. Agalsidase Alfa (REPLAGAL®) is a human a-galactosidase A enzyme produced by a human cell line . Agalsidase beta (FABRAZYME®) is a recombinant human α-galactosidase expressed in a CHO cell line. Replagal is administered every two weeks at a dose of 0.2 mg/kg by intravenous infusion for the treatment of Fabry disease and, off-label, for the treatment of Gaucher disease. FABRAZYME® is administered at a dose of 1.0 mg/kg body weight every two weeks by IV infusion. Other lysosomal enzymes can also be used. For example, the protein may be a lysosomal enzyme as described in US 2012/0148556.

[00188] Rasburicase (ELITEK®) is a recombinant urate oxidase indicated for initial administration of plasma uric acid levels in pediatric and adult patients with leukemia, lymphoma, and solid tumor malignancies who are receiving anticancer therapy expected to result in tumor lysis and subsequent elevation of plasma uric acid. ELITEK® is administered by daily IV infusion at a dosage of 0.2 mg/kg.

[00189] Imiglucerase (CEREZYME®) is a recombinant analogue of human β-glucocerebrosidase. Initial dosages range from 2.5 U/kg of body weight 3 times per week to 60 U/kg once every 2 weeks. CEREZYME® is administered by IV infusion.

[00190] Abraxane, paclitaxel-conjugated albumin, is approved for metastatic breast cancer, non-small cell lung cancer, and late-stage pancreatic cancer.

[00191] Taliglucerase alfa (ELEYSO®) is a hydrolytic lysosomal glucocerebroside-specific enzyme indicated for long-term enzyme replacement therapy for Type 1 Gaucher disease. The indicated dose is 60 U/kg of body weight administered once every 2 weeks via intravenous infusion.

[00192] Laronidase (ALDURAZYME®) is a polymorphic variant of the human enzyme α-L-iduronidase that is produced through the CHO cell line. The indicated dosage regimen of ALDURAZYME® is 0.58 mg/kg administered once weekly as an intravenous infusion.

[00193] Elosufase alfa (VIMIZIM®) is a human N-acetylgalactosamine-6-sulfatase produced by CHO cell line by BioMarin Pharmaceutics 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 dosage of 2 mg/kg.

[00194] Other biologics that can be formulated with viscosity reducing agents include asparaginase erwinia chrysantemi (ERWINAZE®), incobotulinumtoxin A (XEOMIN®), EPOGEN® (epoetin Alfa), PROCRIT® (epoetin Alfa), ARANESP® (darbepoetin alfa ), ORENCIA® (abatacept), BATASERON® (interferon beta-1b), NAGLAZYME® (galsulfase); ELAPRASE® (Idursulfase); MYOZYME® (LUMIZYME®, algucosidase alfa); VPRIV® (velaglucerase), abobotulinumtoxin A (DYSPORT®); BAX-326, Baxter's Octocog alfa; Syncria by GlaxoSmitKline; liprotamase from Eli Lilly; Xiaflex (collagenase clostridium histolyticum) from Auxilium and BioSpecifics Technologies Corp.; anakinra from Swedish Orphan Biovitrum AB; metreleptin from Bristol-Myers Squibb; Avonex, Plegridy (BIIB017) from Biogen; NN1841, NN7008 from Novo Nordisk; KRN321 (darbepoetin alfa), AMG531 (romiplostim), KRN125 (pegfilgrastim), KW-0761 (mogamulizumab) from Kyowa; IB1001 from Inspiration Biopharmaceuticals; Iprivask from Canyon Pharmaceuticals Group.

Protein Therapeutics in Development

[00195] Versartis, Inc.'s VRS-317 is a recombinant human growth hormone (hGH) fusion protein utilizing XTEN half-life extension technology. It aims to reduce the frequency of hGH injections needed for patients with hGH deficiency. VRS-317 has completed a Phase II study comparing its effectiveness to daily injections of non-derivative hGH, with positive results. Phase III studies are planned.

[00196] Vibriolysin is a proteolytic enzyme secreted by the Gram-negative marine microorganism, Vibrio proteolyticus. This endoprotease has specific affinity for the hydrophobic regions of proteins and is capable of cleaving proteins adjacent to hydrophobic amino acids. Vibriolysin is generally being investigated by Biomarin for cleaning and/or treating burns. Vibriolysin formulations are described in WO 02/092014.

[00197] PEG-PAL (PEGylated recombinant phenylalanine ammonia lyase or "PAL") is an investigational enzyme replacement therapy for the treatment of phenylketonuria (PKU), an inherited metabolic disease 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 by KUVAN®. PEG-PAL is now in Phase 2 clinical development to treat patients who do not respond adequately to KUVAN®.

[00198] Other protein therapeutics that can be formulated with viscosity reducing agents include Alprolix/ rFIXFc, Eloctate/ rFVIIIFc, BMN-190; BMN-250; Lamazyme; Galazyme; ZA-011; Sebelipase alfa; SBC-103; and HGT-1110. Additionally, fusion proteins containing XTEN half-life extension technology including, but not limited to: VRS-317 GH-XTEN; Factor VIIa, Factor VIII, Factor IX; PF05280602, VRS-859; Exenatide-XTEN; AMX-256; GLP2-2G/XTEN; and AMX-179 Folate-XTEN-DM1 can be formulated with viscosity reducing agents.

[00199] Other late-stage protein therapeutics that can be formulated with viscosity reducing agents include CM-AT from CureMark LLC; NN7999, NN7088, Liraglutide(NN8022), NN9211, Semaglutide (NN9535) from Novo Nordisk; AMG 386, Amgen Filgrastim; CSL-654, CSL Behring Factor VIII; LA-EP2006 (pegfilgrastim biosimilar) from Novartis AG; Multikine (leukocyte interleukin) from CEL-SCI Corporation; LY2605541, Teriparatide (recombinant PTH 1-34) from Eli Lilly; NU-100 from Nuron Biotech, Inc.; Calaspargase Pegol from Sigma-Tau 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.; Somatropin biosimilar from LG Life Sciences LTD; Natpara from NPS Pharmaceuticals, Inc.; ART123 from Asahi Kasei Corporation; Baxter's BAX-111; OBI-1 from Inspiration Biopharmaceuticals; Wilate from Octapharma AG; Alpha talactoferrin from Agennix AG; Lundbeck desmoteplase; Cinryze of Shire; RG7421 and Roche and Exelixis, Inc.; Midostaurin (PKC412) from Novartis AG; Damoctocog alfa pegal, BAY 86-6150, BAY 94-9027 from Bayer AG; Peginterferon lambda-1a, Nulojix (Belatacept) from Bristol-Myers Squibb; Pergoveris, Corifollitropin alfa (MK-8962) from Merck KGaA; recombinant coagulation Factor IX Fc fusion protein (rFIXFc; BIIB029) and recombinant coagulation Factor VIII Fc fusion protein (rFVIIIFc; BIIB031) from Biogen; and Myalept from AstraZeneca.

[00200] Other early stage protein biologics that can be formulated with viscosity reducing agents 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; Exsulin INGAP peptide; Abbvie's ABT-122; AZD9412 from AstraZeneca; NEUBLASTIN (BG00010) from Biogen; Luspatercept (ACE-536), Sotatercept (ACE-011) from Celgene Corporation; PRAME immunotherapeutic from GlaxoSmithKline; Plovamer Acetate (PI-2301) from Merck KGaA; PREMIPLEX (607) from Shire; BMN-701 from BioMarin; Eisai's Ontak; 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.; Amevive® alefacept from Astellas Pharma Inc.; F-627 from Regeneron; AGN-214868 (senrebotase) from Allergan, Inc.; Baxter's BAX-817; PRT4445 from Portola Pharmaceuticals, Inc.; VEN100 from Ventria Bioscience; Onconase/ranpirnase from Tamir Biotechnology Inc.; interferon alpha-2b infusion from Medtronic, Inc; sebelipase alfa from Synageva BioPharma; IRX-2 from IRX Therapeutics, Inc; GSK2586881 from GlaxoSmithKline; SI-6603 from Seikagaku Corporation; ALXN1101, Alexion asfotase alpha; 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 immunotherapeutic from GlaxoSmithKline; GZ402666 from Sanofi; MSB0010445, Atacicept from Merck KGaA; Leukine (sargramostim) from Bayer AG; Baxter's KUR-211; fibroblast growth factor-1 from CardioVascular BioTherapeutics Inc.; SPI-2012 from Hanmi Pharmaceuticals Co., LTD /Spectrum Pharmaceuticals; FGF-18 (sprifermin) from Merck KGaA; MK-1293 from Merck; interferon-alpha-2b from HanAll Biopharma; CYT107 from Cytheris SA; RT001 from Revance Therapeutics, Inc.; MEDI6012 from AztraZeneca; E2609 from Biogen; BMN-190, BMN-270 from BioMarin; ACE-661 from Acceleron Pharma; AMG 876 from Amgen; GSK3052230 from GlaxoSmithKline; RG7813 from Roche; SAR342434, Lantus from Sanofi; AZ01 from Allozyne Inc.; ARX424 from Ambrx, Inc.; FP-1040, FP-1039 from FivePrime Therapeutics, Inc.; ATX-MS-1467 from Merck KGaA; XTEN fusion proteins from Amunix Operating Inc.; entolimod (CBLB502) from Cleveland BioLabs, Inc.; HGT2310 from Shire; HM10760A from Hanmi Pharmaceuticals Co., LTD; ALXN1102/ ALXN1103 by Alexion; CSL-689, CSL-627 from CSL Behring; glial growth factor 2 from Acorda Therapeutics, Inc.; NX001 from Nephrx Corporation; NN8640, NN1436, NN1953, NN9926, NN9927, NN9928 from Novo Nordisk;NHS-IL 12 from EMD Serono; 3K3A-APC from ZZ Biotech LLC; PB-1046 from PhaseBio Pharmaceuticals, 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, CVX096 from Pfizer; ACP-501 from AlphaCore Pharma LLC; Baxter's BAX-855; CDX-1135 from Celldex 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 Agents

[00201] Other biological drugs that can be formulated with viscosity reducing agents include PF-05285401, PF-05231023, RN317 (PF-05335810), PF-06263507, PF-05230907, Dekavila, PF-06342674, PF06252616, RG7598, RG7842, RG7624d, OMP54F28, GSK1995057, BAY1179470, IMC-3G3, IMC-18F1, IMC35C, IMC-20D7S, PF-06480605, PF-06647263, PF-06650808, PF-05335810 (RN317) PD-0360324, PF-00547659 from Pfizer; MK-8237 from Merck; BI033 from Biogen; GZ402665, SAR438584/ REGN2222 from Sanofi; BMI-18F1; and Icrucumab, IMC-3G3 from ImClone LLC; Ryzodeg, Tresiba, Xultophy from Novo Nordisk; Toujeo (U300), LixiLan, Lyxumia (lixisenatide) from Sanofi; MAGE-A3 immunotherapeutic from GlaxoSmithKline; Merck Wecemotide KGaA; Sereleaxin (RLX030) from Novartis AG; Erythropoietin; Pegfilgrastim; LY2963016, Dulaglutide (LY2182965) from Eli Lilly; and Insulin Glargine from Boehringer Ingelheim.

B. Viscosity Reducing Agents

[00202] The viscosity of liquid protein formulations, including low molecular weight and/or high molecular weight proteins, is reduced by the addition of one or more viscosity reducing agents. Pharmaceutical formulations can be converted from non-Newtonian to Newtonian fluids by adding an effective amount of one or more viscosity reducing agents.

[00203] When employed in a formulation intended for administration to a human or other mammal, viscosity reducing agents, like the formulation itself, must be pharmaceutically acceptable. Viscosity reducing agents are typically organic compounds containing at least one non-carbon, non-hydrogen atom. Preferably, the viscosity reducing agents contain hydrogen, carbon, oxygen and at least one other type of atom. In certain embodiments, the viscosity reducing agents are characterized by at least one of the following:

[00204] 1) organic compounds having at least four carbon and four hydrogen atoms, and at least one sulfur, oxygen, nitrogen or phosphorus atom;

[00205] 2) a molecular weight between about 85 and 1,000 Da;

[00206] 3) the presence of at least one charged, or other hydrophilic, portion;

[00207] 4) the presence of at least one, preferably two, and more preferably three, freely rotating bonds;

[00208] 5) the presence of at least one substituted ring;

[00209] 6) a molecular polar surface area of at least 24 A2, preferably at least 50 A2, and more preferably at least 80 A2;

[00210] 7) a molar volume of at least 75 cm3, preferably at least 85 cm3, more preferably at least 100 cm3, and even more preferably at least 120 cm3;

[00211] 8) a polarizability of at least 10 cm3, preferably at least 15 cm3, more preferably at least 20 cm3, and even more preferably at least 25 cm3; It is

[00212] 9) the presence of at least one, preferably two, and more preferably three hydrogen bond donors and/or acceptors.

[00213] In certain embodiments, the viscosity reducing agent is characterized by at least two, three, four, five, six, seven, eight or all nine of the previous listed attributes. In certain embodiments, the viscosity reducing agent is also characterized by the fact that it does not contain an aldehyde or carbon-carbon triple bond functional group.

[00214] In other embodiments, the viscosity reducing agent is a combination of two or more compounds, each of which is characterized by at least two, three, four, five, six, seven, eight or all nine of the attributes listed above.

[00215] In some embodiments, viscosity reducing agents are listed as GRAS by the U.S. Food and Drug Administration ("the FDA"), as of September 11, 2014. "GRAS" is an acronym for the phrase Generally Recognized As Safe. Under sections 201(s) and 409 of the Federal Food, Drug, and Cosmetic Act (the Act), any substance that is intentionally added to food is a food additive and is subject to premarket review and approval by the FDA at unless the substance is generally recognized, among qualified experts, as having been adequately shown to be safe under the conditions of its intended use, or unless the use of the substance is otherwise excluded from the definition of a food additive. Another source of compounds is the FDA Inactive Ingredient Guide (IIG), and equivalents listed by the International Pharmaceutical Excipients Council (IPEC) and the European Medicines Agency (EMA), as of September 11, 2014. Substances used in formulations must be safe for injection. Preferably, the GRAS-listed viscosity reducing agent is characterized by at least two, three, four, five, six, seven, eight or all nine of the attributes listed above.

[00216] In other embodiments, the viscosity reducing agent is a drug product approved by the FDA or EMA as of September 11, 2014. As compounds removed from the GRAS and IIG lists, the toxicity and safety profiles of the Drug products approved by the FDA and EMA are well established. In addition to reducing the viscosity of the protein solution, the use of an FDA or EMA approved drug product provides the opportunity for combination therapies. Preferably, an FDA or EMA approved drug product viscosity reducing agent is characterized by at least two, three, four, five, six, seven, eight or all nine of the attributes listed above.

[00217] In some embodiments, the viscosity reducing agent includes at least one compound of Formula (I):

[00218] or a pharmaceutically acceptable salt thereof;

[00219] where represents a single or double bond, A is selected from O, S, SO2, NR3, C(R3)2 or:

[00220] wherein R3 is independently selected from hydrogen, R2, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R4a, - C( =NR4a)R4, -C(=O)OH, -C(=O)OR4, -OC(=O)R4, -OC(=O)OR4, -SO3H, -SO2N(R4a)2, -SO2R4, -SO2NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a)2, -NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4aSO2R4, -NR4aC(=NR4a) NR4aC(=NR4a)N(R4a)2, -NR4aC(=O)N(R4a)2, -C(=O)NH2, - C(=O)N(R4a)2, -OR4, -SR4a, and -N(R4a)2;

[00221] wherein R2 is independently selected from C1-12alkyl, C3-12cycloalkyl, C6-12aryl, C1-12heteroaryl and C2-12heterocyclyl;

[00222] wherein each C1-12alkyl can be substituted one or more times with C3-12cycloalkyl, C6-12aryl, C1-12heteroaryl, C2-12heterocyclyl, -OH, NH2, (=O), (=NR4a), -F , -Cl, -Br, -I, -NO2, -CN, - C(=O)R4a, -C(=NR4a)R4, -C(=O)OH, -C(=O)OR4, -OC (=O)R4, - OC(=O)OR4, -SO3H, -SO2N(R4a)2, -SO2R4, -SO2NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a)2, -NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4aSO2R4, -NR4aC(=NR4a)NR4aC(=NR4a)N(R4a)2, -NR4aC(=O)N(R4a)2, -C(=O)NH2, -C(=O)N(R4a)2, -OR4, -SR4a, or -N(R4a)2;

[00223] wherein each C3-12cycloalkyl can be substituted one or more times with C1-12alkyl, C6-12aryl, C1-12heteroaryl, C2-12heterocyclyl, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R4a, - C(=NR4a)R4, -C(=O)OH, -C(=O)OR4, -OC(=O)R4, -OC(= O)OR4, -SO3H, -SO2N(R4a)2, -SO2R4, -SO2NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a)2, -NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4aSO2R4, -NR4aC(=NR4a)NR4aC(=NR4a)N(R4a)2, -NR4aC(=O)N(R4a)2, -C(=O)NH2, - C (=O)N(R4a)2, -OR4, -SR4a, or -N(R4a)2;

[00224] wherein each C6-12aryl can be substituted one or more times with C1-12alkyl, C3-12cycloalkyl, C1-12heteroaryl, C2-12heterocyclyl, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R4a, -C(=NR4a)R4, -C(=O)OH, -C(=O)OR4, -OC(=O)R4, -OC(= O)OR4, -SO3H, -SO2N(R4a)2, -SO2R4, -SO2NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a)2, -NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4aSO2R4, -NR4aC(=NR4a)NR4aC(=NR4a)N(R4a)2, -NR4aC(=O)N(R4a)2, -C(=O)NH2, - C (=O)N(R4a)2, -OR4, -SR4a, or -N(R4a)2;

[00225] wherein each C1-12heteroaryl can be substituted one or more times with C1-12alkyl, C3-12cycloalkyl, C6-12aryl, C2-12heterocyclyl, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R4a, -C(=NR4a)R4, -C(=O)OH, -C(=O)OR4, -OC(=O)R4, -OC(= O)OR4, -SO3H, -SO2N(R4a)2, -SO2R4, -SO2NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a)2, -NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4aSO2R4, -NR4aC(=NR4a)NR4aC(=NR4a)N(R4a)2, -NR4aC(=O)N(R4a)2, -C(=O)NH2, - C (=O)N(R4a)2, -OR4, -SR4a, or -N(R4a)2;

[00226] wherein each C2-12heterocyclyl can be substituted one or more times with C1-12alkyl, C3-12cycloalkyl, C6-12aryl, C1-12heteroaryl, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R4a, -C(=NR4a)R4, -C(=O)OH, -C(=O)OR4, -OC(=O)R4, -OC(= O)OR4, -SO3H, -SO2N(R4a)2, -SO2R4, -SO2NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a)2, -NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4aSO2R4, -NR4aC(=NR4a)NR4aC(=NR4a)N(R4a)2, -NR4aC(=O)N(R4a)2, -C(=O)NH2, - C (=O)N(R4a)2, -OR4, -SR4a, or -N(R4a)2;

[00227] wherein R4 is independently selected from C1-12alkyl, C3-12cycloalkyl, C6-12aryl, C1-12heteroaryl and C2-12heterocyclyl, each of which can be substituted one or more times by -OH, -NH2, - F, -Cl, -Br, -I, -NO2, -CN, -C(=O)OH, -SO3H, -PO3H2, or - C(=O)NH2;

[00228] where R4a can be R4 or hydrogen;

[00229] in which any two or more of groups R2, R3, R4 and R4a can together form a ring;

[00230] wherein when two R3 groups are attached to the same carbon atom, the two R3 groups can together form a (=O), (=NR4a), or (=C(R4a)2);

[00231] wherein z is in each case independently selected from 1 or 2, provided that when the substituent of (R3)z is attached to an sp2 hybridized carbon, z is 1, and when the substituent of (R3) z is connected to an sp3 hybridized carbon, z is 2.

[00232] When the substituent -NR4aC(=NR4a)NR4aC(=NR4a)N(R4a)2 is present, it is preferred that R4a is selected to produce - NHC(=NH)NHC(=NH)NH2.

[00233] In certain embodiments, the compound of Formula (1) contains at least one substituent selected from -C(=O)OH, -SO3H, -SO2NHC(=O)R4, and -PO3H2. In some embodiments, the compound of Formula (1) contains at least one -SO3H group.

[00234] In certain embodiments, one or more of the substituents of R3 may be: wherein R3a and R3b are independently selected from hydrogen, C1-12alkyl, C3-12cycloalkyl, C6-12aryl, C1-12heteroaryl and C2-12heterocyclyl, C(=O)R4a, -C(=O)OH, -C (=O)OR4, -SO3H, - SO2N(R4a)2, -SO2R4, -SO2NHC(=O)R4, C(=O)NH2, -C(=O)N(R4a)2, - OR4, - SR4, and -N(R4a)2, and when any two R3b are bonded to the same carbon atom, the two R3b groups can together form (=O), (=NR4a), or (=C(R4a)2) ;

[00235] wherein each C1-12alkyl, C3-12cycloalkyl, C6-12aryl, C1-12heteroaryl and C2-12heterocyclyl can be substituted one or more times with -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R4a, - C(=NR4a)R4, -C(=O)OH, -C(=O)OR4, -OC(=O)R4, -OC(= O)OR4, -SO3H, -SO2N(R4a)2, -SO2R4, -SO2NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a)2, -NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4aSO2R4, - NR4aC(=NR4a)NR4aC(=NR4a)N(R4a)2, -NR4aC(=O)N(R4a)2, -C(=O)NH2, - C (=O)N(R4a)2, -OR4, -SR4a, or -N(R4a)2;

[00236] wherein R4 and R4a are as defined above;

[00237] where x is selected from 1, 2, 3, 4, 5, 7, 8, 9 or 10; It is

[00238] in which any two or more of groups R3, R3a, R4 and R4a can together form a ring.

[00239] In certain embodiments, the compound of Formula (1) can be represented by the compound of Formula (1a) or (1b):

[00240] where R3 has the meanings given above.

[00241] In certain embodiments, the compound of Formula (1a) can be represented by the compounds of Formulas (1a-i-iv):

[00242] wherein R3 is independently selected from hydrogen, NH2, CH3, Cl, OR4 and NHR4;

[00243] where x is 1 or 2;

[00244] wherein R3a and R3b are independently selected from hydrogen and C1-12 alkyl;

[00245] wherein said C1-12alkyl may be substituted one or more times by C3-12cycloalkyl, C6-12aryl, C1-12heteroaryl, C2-12heterocyclyl, -OH, NH2, -F, -Cl, -Br, -I , -NO2, -CN, -C(=O)R4a, - C(=NR4a)R4, -C(=O)OH, -C(=O)OR4, -OC(=O)R4, -OC( =O)OR4, -SO3H, -SO2N(R4a)2, -SO2R4, -SO2NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a)2, -NHC(=NR4a)NH-CN , -NR4aC(=O)R4, -NR4aSO2R4, - NR4aC(=NR4a)NR4aC(=NR4a)N(R4a)2, -NR4aC(=O)N(R4a)2, -C(=O)NH2, - C(=O)N(R4a)2, -OR4, -SR4a, or -N(R4a)2;

[00246] R4 and R4a are as defined above; It is

[00247] wherein any two or more R3a, R3b, R4, R4a can together form a ring.

[00248] The compound of Formula (1) can be represented by the compound of Formula (1a-v, vi or vii):

[00249] wherein R3f is selected from -C(=O)OH, -SO3H, -SO2NHC(=O)R4, and -PO3H2, and R3 is as defined above. In certain preferred embodiments, R3 is independently selected from hydrogen, OH, NH2, C1-6alkyl and COOH.

[00250] In other embodiments, the compound of Formula (1) can be represented by any of the compounds of Formulas (1c), (1d), (1e) or (1f):

[00251] where R3 has the meanings given above.

[00252] In other embodiments, the compound of Formula (1) can be represented by a compound of Formula (1g):

[00253] wherein R3c is independently selected from hydrogen and R2, wherein R2 has the meanings given above;

[00254] wherein R3d is independently selected from hydrogen, OH, NH2, NH(C1-6alkyl), N(C1-6alkyl)2; NHC(=O)(C1-6alkyl), COOH and CH2OH;

[00255] or any two groups R3c and R3d connected to the same carbon can together form an oxo (=O), imino (=NR4a), or an olefin (=C(R4a)2), where R4a has the meanings given above ;

[00256] wherein R3e is selected from hydrogen, -OH or OR4; It is

[00257] where R4 has the meanings given above.

[00258] In certain embodiments, the viscosity reducing agent includes a compound of Formula (1g-i):

[00259] wherein R3e is selected from OH and -OC1-12alkyl, which is also substituted with at least one OH and at least one COOH; It is

[00260] where R3d is selected from COOH and CH2OH.

[00261] In some embodiments, the viscosity reducing agent includes a compound of Formula (2):

[00262] or a pharmaceutically acceptable salt thereof;

[00263] wherein represents a single or double bond;

[00264] X is independently selected from chalcogen, N(R3)z and C(R3)z;

[00265] X1 is absent, or is chalcogen, N(R3)z, C(R3)z or:

[00266] wherein R3 has the meanings given for the compound of Formula (1);

[00267] provided that when the substituent of (R3)z is connected to an sp2 hybridized nitrogen, z is 0 or 1, when the substituent of (R3)z is connected to an sp2 hybridized carbon or an sp3 hybridized nitrogen , z be 1, and when the substituent of (R3)z is connected to an sp3 hybridized carbon, z be 2;

[00268] wherein at least one of X or X1 is chalcogen or N(R3)z.

[00269] In certain embodiments, the compound may be an aromatic ring. Exemplary aromatic rings include the compounds of Formulas (2a-e): where R3 and X have the above meanings, and X2 is selected from N(R3)z and C(R3)z.

[00270] In certain embodiments, the viscosity reducing agent is a compound of Formula (2a-i):

[00271] wherein R4 is as defined above and is preferably hydrogen or CH3;

[00272] where R6 is C1-12heteroaryl, which can be substituted one or more times by C1-6alkyl;

[00273] wherein said C1-6alkyl may be substituted one or more times by OH, -NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R4, - C(=NR4a)R4, -C(=O)OH, -C(=O)OR4, -SO3H, -SO2NR4-, -SO2R4, - PO3H2, -NHC(=O)R4, -NHC(=O) N(R4)2, -C(=O)NH2, -C(=O)N(R4)2, - OR4b, -SR4b, -N(R4b)2, where R4 has the meanings given above; or

[00274] where R4 is as defined above, and R7 is selected from SR4 and -C(=O)R4. The double bond in the above group can be in E or Z geometry.

[00275] In preferred embodiments, R6 is a heterocycle having the structure:

[00276] wherein -NO2, -CN, -C(=O)OH. In an even more preferred embodiment, R6 is a heterocycle having the structure:

[00277] wherein R6a is selected from unsubstituted C1-6alkyl and C1-6alkyl substituted one or more times with -OH.

[00278] The viscosity reducing agent can be an imidazole of Formula (2b-i)

[00279] where R3 is as defined above. In certain embodiments, R3 is independently selected from hydrogen, NO2, and R4. In certain preferred embodiments, the compound of Formula (2b-i) has the structure:

[00280] wherein R3 is independently selected from C16 alkyl, which may be unsubstituted or substituted one or more times with a group selected from OH, NH2, SR4, F,Cl, Br and

[00281] R3g is hydrogen or NO2.

[00282] In other embodiments, the viscosity reducing agent has the structure of Formula (2a-ii) or Formula (2c-i):

[00283] wherein R3 is independently selected from OH, Cl, Br, F, I, N(R4a)2, C(=O)OH, C(=O)NH2.

[00284] In other embodiments, at least one substituent of R3 is NHR4, wherein R4 is a C1-6alkyl, optionally substituted by one or more groups selected from Cl, Br, F, I, OH, C(=O )OH, NH2, NH(C1-6alkyl) and N(C1-6alkyl)2.

[00285] In other embodiments, the viscosity reducing agent is a pyridinium salt of Formula (2a-iii):

[00286] wherein R3 and R4 are as defined above.

[00287] In other embodiments, the heterocyclic ring is not a heteroaryl ring. Exemplary non-aromatic rings include compounds of Formulas (2f-k): where R5 and X have the above meanings, and X3 is chalcogen or N(R3)z

[00288] In certain embodiments, the compound of Formula (2f) is a beta-lactam of Formula (2f-i),

[00289] The beta lactam of Formula (2f-i) includes penicillin-type compounds, as well as cephalosporin-type and cephamycin-type compounds of Formulas (2f-ii) and (2f-iii): where X and R3 are as defined above. In preferred embodiments, X is sulfur.

[00290] In certain embodiments, the compound of Formula (2i) is a compound of Formula (2i-i): where X and R3 are as defined above. In certain embodiments, X is in both cases NR4, wherein R4 has the meanings given above, and R3 is in both cases hydrogen.

[00291] In other embodiments, the compound of Formula (2) is represented by a compound of Formula (2i-ii): wherein X, X1 and R3 are as defined above.

[00292] The compound of Formula (2j) can be represented by the compound of Formula (2j-i): where X3 and R3 are as defined above, and R8 is selected from NHC(=O)R2 and OC(=O)R2. In preferred embodiments, X3 is N+(CH3)2, R3 is also hydrogen, or R3 together forms an epoxide or double bond.

[00293] The compound of Formula (2k) can be represented by the compound of Formula (2k-i): where X3 and R8 are as defined above.

[00294] In other embodiments, the viscosity reducing agent includes a compound of the structure of Formula (3):

[00295] or a pharmaceutically acceptable salt thereof;

[00296] wherein R5 is in each case independently selected from hydrogen, and R2,

[00297] R5' is R5 or absent;

[00298] provided that at least one substituent of R5 is not hydrogen, where R2 has the same meanings given for the compound or Formula (1).

[00299] In certain embodiments, the viscosity reducing agent is a mixture of two or more compounds selected from the compounds of Formula (1), Formula (2) and Formula (3).

[00300] In preferred embodiments, the viscosity reducing agent is camphorsulfonic acid (CSA), or a pharmaceutically acceptable salt thereof, such as an alkali or alkaline earth metal salt. Camphorsulfonic acid or salt thereof is combined with one or more compounds of Formula (1), (2) or (3) to produce mixtures such as CSA-piperazine, CSA-TRIS, CSA-4-amino pyridine, CSA-1 -(o-tolyl)biguanide, CSA-procaine, CSA-Na-aminocyclohexane carboxylic acid, CSA-Na-cretinin and CSA-Na-ornidazole. Other preferred viscosity reducing agents include thiamine, procaine, biotin, cretinin, metoclopramide, scopolamine, cimetidine, chloroquine phosphate, mepivacaine, granisetron, sucralose, HEPES-tris, nicotinamide, lactobionic acid-TRIS, glucuronic acid-TRIS, sulfacetamide, CSA-4-aminopyridine, CSA-piperazine and cefazolin. Any two or more of the viscosity reducing agents listed above can also be combined in the same formulation.

[00301] In other embodiments, the viscosity reducing agent is an organosulfonic acid. Exemplary organosulfonic acids include, but are not limited to, camphorsulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, toluenesulfonic acid, cyclohexylsulfonic acid, xylenesulfonic acid (including, p-xylene-2-sulfonic acid, m-xylene-2-sulfonic acid, -sulfonic acid, m-xylene-4-sulfonic acid and o-xylene-3-sulfonic acid), methanesulfonic acid, 1,2-ethanedisulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-hydroxyethane- 1-sulfonic acid, 3-hydroxypropane-1-sulfonic acid, cymenesulfonic acid, 4-hydroxybutane-1-sulfonic acid and pharmaceutically acceptable salts thereof. Organosulfonic acid can be in the form of an alkali or alkaline earth metal salt, such as lithium, sodium, potassium, magnesium, and calcium salt. Organosulfonic acid (or salts thereof) can be combined with one or more compounds of Formula (2) or Formula (3).

[00302] In certain embodiments, the viscosity reducing agent contains at least one carboxylic acid. The carboxylic acid may be in the form of an alkali or alkaline earth metal salt, such as lithium, sodium, potassium, magnesium and calcium salt. Exemplary carboxylic acid compounds include lactobionic acid, glucuronic acid, 1-aminocyclohexane carboxylic acid, biotin, brocrinate, cyclopentane propionic acid, hydroxynaphthoic acid, phenylpropionic acid, gentisic acid, salicylic acid, camphoric acid, mandelic acid, sulfosalicyclic acid, hydroxybenzoyl benzoic acid , phenyl acetic acid, acetyl salicylic acid, cinnamic acid, t-butyl acetic acid, phthalic acid, trimethylacetic acid, anthralic acid and pharmaceutically acceptable salts thereof. The carboxylic acid (or salts thereof) can be combined with one or more compounds of Formula (2) or Formula (3).

[00303] The following compounds can also be used as viscosity reducing agents: colistin, articaine, tetracaine, proximethacaine, metoclopramide, procaine, lidocaine, cyclomethylcaine, piperocaine, chloroprocaine, etidocaine, benzocaine, phenylephrine, bupivacaine, mepivacaine, cinchocaine , mixtures thereof and pharmaceutically acceptable salts thereof.

[00304] Other agents that can be employed as viscosity reducing agents include 1-aminocyclohexanecarboxylic acid, 1-(o-tolyl)biguanide, benzethonium chloride, benzoic acid, brocrinate, calcium carrageenan, calcium cyclamate, calcobutrol, acid caloxetic, camphorsulfonic acid, cretinine, dalfampridine, dehydroacetic acid, diazolidinyl urea, dichlorobenzyl alcohol, dimethyl isosorbide, epitetracycline, ethyl maltol, ethyl vanillin, ornidazole, gentilic acid ethanolamide, HEPES (4-(2-hydroxyethyl)-1- acid piperazineethanesulfonic acid), gentisic acid, glucuronic acid, iodoxamic acid, menthol, galactose, medronic acid, m-cresol, glutathione, lactobionic acid, maltitol, octissalate, oxyquinoline, pentetic acid, piperazine, propenyl guaethol, propyl gallate, propylene carbonate, propylparaben, protamine sulfate, QUATERNIUM-15, QUATERNIUM-52, satialgin H, sodium 1,2-ethanedisulfonate, sodium cocoyl sarcosinate, sodium lauroyl sarcosinate, sodium polymetaphosphate, sodium pyrophosphate, pyroglutamic acid, sodium trimetaphosphate, sodium tripolyphosphate, sorbitan, tartaric acid, lactic acid, iofetamine, sucralose, 1-(4-pyridyl)pyridinium chloride, aminobenzoic acid, sodium sulfacetamide, naphthalene-2-sulfonic acid, tert-butylhydroquinone, thimerosal, trolamine, tromantadine , vanillin, verstamide, nioxime, niacinamide, methylisothiazolinone, mannose D, maltose, lidofenine, lactose, lactitol, isomalt, imidurea, gluconolactone, methanesulfonic acid, xylenesulfonic acid, sulfobutylether β-cyclodextrin and pharmaceutically acceptable salts thereof.

[00305] In certain embodiments, the viscosity reducing agent includes an organic base. Exemplary organic bases include N-methylglyamine, morpholine, piperidine, and primary, secondary, tertiary, and quaternary amines, substituted amines, and cyclic amines. For example, they may be isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, lidocaine, hydrabamine, cholines, betaines, choline, betaine, ethylenediamine, theobromine, purines, piperazine, N-ethylpiperidine, N-methylpiperidinepolyamine. Particularly preferred organic bases are arginine, histidine, lysine, ethanolamine, thiamine, 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), 4-aminopyridine, aminocyclohexane carboxylic acid, 1-o-toliguanide, ornidazole, urea, nicotinamide, benzethonium chloride, 5-amino-1-pentanol, 2-(2-aminoethoxy)ethanol, trans-cyclohexane-1,4-diamine, trans-cyclohexane-1R, 2R-diamine, ethylenediamine, propane-1 ,3-diamine, butane-1,4-diamine, pentane-1,5-diamine, hexane-1,6-diamine, octane-1,8-diamine, 5-amino-1-pentanol, 2-(2- aminoethoxy)ethanamine, 2-(2-(2-aminoethoxy)-ethoxy)ethanamine, 3-(4-(3-aminopropoxy)-butoxy)propan-1-amine, 3-(2-(2-(3-aminopropoxy) )-ethoxy)-ethoxy)propan-1-amine, N-(2-(2-aminoethylamino)ethyl)ethane-1,2-diamine, N-(2-aminoethyl)ethane-1,2-diamine, N- 1-(2-(2-(2-aminoethylamino)ethylamino)-ethyl)ethane-1,2-diamine, N,N-dimethylhexane-1,6-diamine, N,N,N,N-tetramethylbutane-1, 4-diamine, phenyltrimethylammonium salts, isopropylamine, diethylamine, ethanolamine, trimethamine, choline, 1-(3-aminopropyl)-2-methyl-1H-imidazole, piperazine, 1-(2-aminoethyl)piperazine, 1-[3- (dimethylamino)propyl]piperazine, 1-(2-aminoethyl)piperidine, 2-(2-aminoethyl)-1-methylpyrrolidine, mixtures thereof, and pharmaceutically acceptable salts thereof.

[00306] Exemplary beta-lactans include benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), cloxacillin, dicloxacillin, flucloxacillin, methicillin, nafcillin, oxacillin, temocillin, amoxicillin, ampicillin, mecillinam, carbenicillin, ticarcillin, azlocillin, mezlocillin, piperacillin, cefoxitin, cefazolin, cephalexin, cephalosporin C, cephalothin, cefaclor, cefamandole, cefuroxime, cefotetam, cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, cefepime, cefpirome, ceftobiprol, biapenem, doripenem, ertapenem faropenem, imipenem, meropenem, panipenem , razupenem, tebipenem, thienamycin, aztreonam, tigemonam, nocardicin a, tabtoxinin, clavulanic acid, clavulanic acid, tazobactam, sulbactam and pharmaceutically acceptable salts thereof.

[00307] Other viscosity reducing agents include N-tropane heterocycles, such as atropine, hyoscyamine, scopolamine, and salts thereof, as well as salts of tiotropium and ipratropium, thiamine, allithiamine, prosultiamine, fursultiamine, benfotiamine, sulbutiamine, quaternion 15; 1-(3-aminopropyl)-2-methyl-1H-imidazole dihydrochloride; creatinine; biotin, cimetidine, piperocaine, cyclomethylcaine, granisetron, moxifloxacin, chloroquine, mepivacaine, levetiracetam, bupivacaine, cinchocaine, clindamycin and pharmaceutically acceptable salts thereof. Thiamine is an especially preferred viscosity reducing agent.

[00308] In certain formulations, the following compounds are not preferred: cretinine, cadaverine, lidocaine, arginine and lysine, and are excluded from the scope of the preceding formulas and definitions of useful viscosity reducing agents.

C. Excipients

[00309] A wide variety of pharmaceutical excipients useful for liquid protein formulations are known to those skilled in the art. They include one or more additives, such as liquid solvents or co-solvents; sugars or sugar alcohols such as mannitol, trehalose, sucrose, sorbitol, fructose, maltose, lactose, or dextrans; surfactants such as TWEEN® 20, 60, or 80 (polysorbate 20, 60, or 80); buffering agents; 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 stabilizing agents.

[00310] 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 likewise can be included in a protein formulation described herein, as long as they do not adversely affect the desired characteristics of the formulation.

[00311] The viscosity reducing agents described here can be combined with one or more other types of viscosity reducing agents, for example, organophosphates described in the co-deposited PCT application entitled "LIQUID PROTEIN FORMULATIONS CONTAINING ORGANOPHOSPHATES" by Arsia Therapeutics; water soluble dyes described in co-filed PCT application entitled "LIQUID PROTEIN FORMULATIONS CONTAINING WATER SOLUBLE ORGANIC DYES" by Arsia Therapeutics; ionic liquids described in co-filed PCT application entitled "LIQUID PROTEIN FORMULATIONS CONTAINING IONIC LIQUIDS" by Arsia Therapeutics.

III. Preparation Methods

[00312] The protein, such as a mAb, to be formulated 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 well known in the art, or by synthetic techniques (such as recombinant techniques and peptide synthesis or a combination of these techniques), or may be isolated from an endogenous source of the protein.

[00313] Purification of the protein to be formulated can be conducted by any suitable technique known in the art, such as, for example, ethanol or ammonium sulfate precipitation, reversed-phase HPLC, silica chromatography or cation exchange resin (e.g. e.g., DEAE-cellulose), dialysis, chromatofocusing, gel filtration using SEPHAROSE® protein A columns (e.g., SEPHADEX® G-75) to remove contaminants, metal chelation columns to bind epitope-tagged forms, and ultrafiltration/ diafiltration (non-limiting examples include centrifugal filtration and tangential flow filtration (TFF)).

[00314] The inclusion of viscosity reducing agents at viscosity reducing concentrations such as 0.010 M to 1.0 M, preferably 0.050 M to 0.50 M, preferably 0.10 M to 0.30 M, allows for a solution of the pharmaceutically active mAb to be purified and/or concentrated to higher mAb concentrations using common methods known to those skilled in the art, including, but not limited to, tangential flow filtration, centrifugal concentration, and dialysis.

[00315] In some embodiments, lyophilized formulations of proteins are provided and/or are used in the preparation and manufacture of low viscosity concentrated protein formulations. In some embodiments, the pre-lyophilized protein in a powder form is reconstituted by dissolving it in an aqueous solution. In this embodiment, the liquid formulation is loaded into a specific dosage unit container such as a pre-filled mixing vial or syringe, lyophilized, optionally with lyoprotectants, preservatives, antioxidants, and other typical pharmaceutically acceptable excipients, then stored under conditions sterile storage until immediately before use, at which time it is reconstituted with a defined volume of diluent, to bring the liquid to the desired concentration and viscosity.

[00316] The formulations described here can be stored by any suitable method known to one skilled in the art. Non-limiting examples of methods for preparing protein formulations for storage include freezing, lyophilization, and spray drying the liquid protein formulation. In some cases, the lyophilized formulation is frozen for storage at subzero temperatures, such as about -80°C or in liquid nitrogen. In some cases, a lyophilized or aqueous formulation is stored at 2-8oC.

[00317] Non-limiting examples of diluents useful for reconstituting a lyophilized formulation prior to injection include sterile water, bacteriostatic water for injection (BWFI), a pH-buffered solution (e.g., phosphate-buffered saline), sterile saline, 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 an Individual in Need of the Same

[00318] Protein formulations, including, but not limited to, reconstituted formulations, are administered to a person in need thereof by intramuscular, intraperitoneal (i.e., into a body cavity), intracerobrospinal, or subcutaneous injection using a needle. 8-32 gauge (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, more preferably less than about 1 mL.

[00319] The appropriate dosage ("therapeutically effective amount") of the protein, such as a mAb, will depend on the condition being treated, the severity and course of the disease or condition, whether the protein is administered for preventive or therapeutic purposes, prior therapy , the patient's clinical history and response to the protein, the type of protein used, and the discretion of the treating physician. The protein is appropriately administered once in single or multiple injections, or in a series of treatments, as sole treatment, or together with other drugs or therapies.

[00320] Dosage formulations are designed so that injections do not cause any significant signs of irritation at the injection site, for example, where the primary irritation index is less than 3 when evaluated using a Draize classification system. In an alternative embodiment, the injections cause macroscopically similar levels of irritation compared to injections of equivalent volumes of saline. In another embodiment, the bioavailability of the protein is higher compared otherwise to the same formulation without the viscosity reducing agent(s) administered in the same manner. In another embodiment, the formulation is at least approximately as pharmaceutically effective as about the same dose of the protein administered by intravenous infusion.

[00321] In a preferred embodiment, the formulation is injected to produce increased levels of the therapeutic protein. For example, the AUC value may be at least 10%, preferably at least 20%, greater than the same value computed for otherwise the same formulation without the viscosity reducing agent(s) administered. Likewise.

[00322] The viscosity reducing agent can also affect bioavailability. For example, the percentage bioavailability of the protein may be at least 1.1 times, preferably at least 1.2 times the percentage bioavailability otherwise of the same formulation without the administered viscosity reducing agent(s). s) in the same way.

[00323] The viscosity reducing agent can also affect pharmacokinetics. For example, the CMAX after SC or IM injection may be at least 10%, preferably at least 20%, lower than the CMAX of an approximately equivalent pharmaceutically effective intravenously administered dose.

[00324] In some embodiments, the proteins are administered at a higher dosage and a lower frequency than otherwise the same formulations without the viscosity reducing agent(s).

[00325] Lower viscosity formulations require less injection force. For example, the injection force may be at least 10%, preferably at least 20%, less than the injection force for otherwise the same formulation without the viscosity reducing agent(s) administered. Likewise. In one embodiment, the injection is administered with a 27 gauge needle and the injection force is less than 30 N. The formulations can be administered in most cases using a very small gauge needle, e.g., between 27 and 31 gauge. , typically 27, 29 or 31 gauge.

[00326] The viscosity reducing agent can be used to prepare a dosage unit formulation suitable for reconstitution to prepare a liquid pharmaceutical formulation for subcutaneous or intramuscular injection. The dosage unit may contain a dry powder of one or more proteins; one or more viscosity reducing agents; and other excipients. The proteins are present in the dosage unit such that after reconstitution in a pharmaceutically acceptable solvent, the resulting formulation has a protein concentration of about 100 mg to about 2,000 mg per 1 mL (mg/mL). Such reconstituted formulations may have an absolute viscosity of about 1 cP to about 50 cP at 25°C.

[00327] The low viscosity formulation may be provided as a solution or in a unit dosage form where the protein is lyophilized in a vial, with or without the viscosity reducing agent and the other excipients, and the solvent with or without viscosity reducing agent and other excipients, it is supplied in a second vial. In this embodiment, the solvent is added to the protein just before or at the time of injection to ensure uniform mixing and dissolution.

[00328] The viscosity reducing agent(s) are present in the formulations at concentrations that do not cause any significant signs of toxicity and/or any irreversible signs of toxicity when administered via subcutaneous, intramuscular, or other types of injection. When used herein, "significant signs of toxicity" include intoxication, lethargy, behavioral changes such as those that occur with damage to the central nervous system, infertility, signs of serious cardiotoxicity such as cardiac arrhythmia, cardiomyopathy, myocardial infarctions, and cardiac arrest or congestive disease, kidney failure, liver failure, difficulty breathing, and death.

[00329] In preferred embodiments, the formulations do not cause any significant irritation when administered no more than twice a day, once a day, twice a week, once a week or once a month. Protein formulations that do not cause significant signs of irritation at the injection site may be administered as measured by a primary irritation index of less than 3, less than 2, or less than 1 when evaluated using a Draize scoring system. When used herein, "significant signs of irritation" include erythema, redness, and/or swelling at the injection site having a diameter greater than 10 cm, greater than 5 cm, or greater than 2.5 cm, necrosis at the injection site, exfoliative dermatitis at the injection site, and severe pain that prevents daily activity and/or requires medical attention or hospitalization. In some embodiments, injections of the protein formulations cause macroscopically similar levels of irritation compared to injections of equivalent volumes of saline.

[00330] Protein formulations may exhibit increased bioavailability compared otherwise to the same protein formulation without the viscosity reducing agent(s) when administered via subcutaneous or intramuscular injection. "Bioavailability" refers to the extent and rate at which bioactive species, such as a mAb, reach the circulation or site of action. Overall bioavailability may be increased for SC or IM injections compared otherwise to the same formulations without the viscosity reducing agent(s). "Percent bioavailability" refers to the fraction of the administered dose of bioactive species that enters circulation, as determined with respect to an intravenously administered dose. One way to measure bioavailability is by comparing the "area under the curve" (AUC) on a plot of plasma concentration as a function of time. The AUC can be calculated, for example, using the linear trapezoidal rule. "AUC«", when used herein, refers to the area under the plasma concentration curve from time zero to a time where the plasma concentration returns to reference levels. "AUC0-t", when used herein, refers to the area under the plasma concentration curve from time zero to a time, t, later, for example, the time of reaching the reference line. Time will typically be measured in days, although hours may also be used as this will be evident from the context. For example, the AUC may be increased by more than 10%, 20%, 30%, 40%, or 50% compared otherwise to the same formulation without the viscosity reducing agent(s) and administered as Similarly.

[00331] When used herein, "tmax" refers to the time after administration at which the plasma concentration reaches a maximum.

[00332] When used herein, "Cmax" refers to the maximum plasma concentration after administration of the dose, and before administration of a subsequent dose.

[00333] When used herein, "Cmin" or "Ctrough" refers to the minimum plasma concentration after administration of the dose, and before administration of a subsequent dose.

[00334] The Cmax after SC or IM injection may be lower, for example at least 10%, more preferably at least 20%, lower than the Cmax of an intravenously administered dose. This reduction in Cmax may similarly result in decreased toxicity.

[00335] Pharmacokinetic and pharmacodynamic parameters can be approximated by species using methods that are known to the skilled artisan. The pharmacokinetics and pharmacodynamics of antibody therapeutics can differ markedly based on the specific antibody. An approved murine mAb has been shown to have a half-life in humans of ~1 day, while a human mAb will typically have a half-life of ~25 days (Waldmann et al., Int. Immunol., 2001, 13:1551-1559 ). The pharmacokinetics and pharmacodynamics of antibody therapeutics may differ markedly based on routine administration. The time to reach maximum plasma concentration after IM or SC injection of IgG typically ranges from 2 to 8 days, although shorter or longer times may be encountered (Wang et al., Clin. Pharm. Ther., 2008 , 84(5):548-558). The pharmacokinetics and pharmacodynamics of antibody therapeutics can differ markedly based on formulation.

[00336] Low viscosity protein formulations can allow for greater flexibility in dosing and decreased dosing frequencies compared to those protein formulations without the viscosity reducing agent(s). For example, by increasing the dosage administered by injection multiple times, the dosage frequency can in some embodiments be decreased from once every 2 weeks to once every 6 weeks. Protein formulations, including but not limited to reconstituted formulations, can be administered using a heated and/or self-mixing syringe or autoinjector. Protein formulations can also be preheated in a separate heating unit before filling the syringe.

i. Heated Syringes

[00337] The heated syringe may be a standard syringe that is preheated using a syringe heater. The syringe heater will generally have one or more openings each capable of receiving a syringe containing the protein formulation and a means of heating and maintaining the syringe at a specific temperature (typically above ambient) prior to use. This will be referred to here as a preheated syringe. Suitable heated syringe heaters include those available from Vista Dental Products and Inter-Med. The heaters are capable of accommodating syringes of various sizes and heating, typically within 1°C, to any temperature up to about 130°C. In some embodiments, the syringe is preheated in a heating bath such as a bathtub of water maintained at the desired temperature.

[00338] The heated syringe may be a self-heating syringe, that is, capable of heating and maintaining the liquid formulation within the syringe at a specific temperature. The self-heating syringe may also be a standard medical syringe having a heating device attached thereto. Suitable heating devices capable of being attached to a syringe include syringe heaters or syringe heater strips available from Watlow Electric Manufacturing Co. of St. Louis, MO, and syringe heater blocks, stage heaters, and in-line perfusion heaters. available from Warner Instruments of Hamden, CT, such as the model SW-61 syringe heater. The heater can be controlled by a central controller, for example, the model TC-324B or TC-344B heater controls available from Warner Instruments.

[00339] The heated syringe maintains the liquid protein formulation at a specified temperature or within 1°C, within 2°C, or within 5°C of a specified temperature. The heated syringe can maintain the protein formulation at any temperature from room temperature to about 80°C, to about 60°C, to about 50°C, or to about 45°C as long as the protein formulation protein is sufficiently stable at that temperature. The heated syringe can maintain the protein formulation at a temperature between 20°C and 60°C, between 21°C and 45°C, between 22°C and 40°C, between 25°C and 40°C, or between 25°C and 37°C. By maintaining protein formulations at an elevated temperature during injection, the viscosity of the liquid formulation is decreased, the solubility of the protein in the formulation is increased, or both.

ii. Automix Syringes

[00340] The syringe may be self-mixing or may have a mixer attached. The mixer can be a static mixer or a dynamic mixer. Examples of static mixers include those described in U.S. Patent Nos. 5,819,988, 6,065,645, 6,394,314, 6,564,972, and 6,698,622. Examples of some dynamic mixers may include those described in U.S. Patent Nos. 6,443,612 and 6,457,609, as well as U.S. Patent Application Publication No. US 2002/0190082. The syringe may include multiple buffers for mixing the components of the liquid protein formulation. U.S. Patent No. 5,819,998 describes syringes with two barrels and a mixing end for mixing two-component viscous substances.

iii. Autoinjectors and Prefilled Syringes for Protein Formulations

[00341] The liquid protein formulation can be administered using a pre-filled syringe auto-injector or a needle-free injection device. Autoinjectors include a portable cartridge holder, often pen-like, to hold replaceable prefilled cartridges and a spring-based or analog mechanism for subcutaneous or intramuscular injections of liquid drug dosages from a preloaded cartridge. . Autoinjectors are typically designed for self-administration or administration by untrained personnel. Autoinjectors are available to deliver single dosages or multiple dosages from a prefilled cartridge. Autoinjectors allow different user applications including interalia injection depth, injection speed, and the like. Other injection systems may include those described in U.S. Patent No. 8,500,681.

[00342] The lyophilized protein formulation can be supplied in pre-filled or single-dose syringes. U.S. Patent Nos. 3,682,174; 4,171,698; and 5,569,193 describes sterile syringes containing two chambers that can be pre-filled with a dry formulation and a liquid that can be mixed immediately before injection. U.S. Patent No. 5,779,668 describes a syringe system for lyophilizing, reconstituting, and administering a pharmaceutical composition. In some embodiments, the protein formulation is provided in lyophilized form in a prefilled or single-dose syringe, reconstituted in the syringe prior to administration, and administered as a single subcutaneous or intramuscular injection. Autoinjectors for single-dose lyophilized drug delivery are described in WO 2012/010.832. Autoinjectors such as the Safe Click LyoTM (marketed by Future Injection Technologies, Ltd., Oxford, U.K.) can be used to administer a single-dose protein formulation where the formulation is stored in lyophilized form and only reconstituted prior to administration. In some embodiments, the protein formulation is provided in single-dose lyophilized drug cartridges (sometimes referred to as Vetter cartridges). Examples of suitable cartridges may include those described in U.S. Patent Nos. 5,334,162 and 5,454,786.

V. Purification and Concentration Methods

[00343] Viscosity reducing agents can also be used to help with protein purification and concentration. The viscosity reducing agent(s) and excipients are added to the protein in an amount effective to reduce the viscosity of the protein solution. For example, the viscosity reducing agent is added at a concentration of between about 0.01 M and about 1.0 M, preferably between about 0.01 M and about 0.50 M, and preferably between about 0.01 M and about 0.25 M.

[00344] The protein-containing viscosity reducing agent solution is then purified or concentrated using a method selected from the group consisting of ultrafiltration/diafiltration, tangential flow filtration, centrifugal concentration, and dialysis.

Examples

[00345] The foregoing will also be understood by the following non-limiting examples.

[00346] All viscosities of the well-mixed aqueous mAb solutions were measured using an mVROC microfluidic viscometer (RheoSense) or a DV2T plate and cone viscometer (Brookfield; "C&P") after a 5 minute equilibration at 25oC (unless otherwise noted). 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 loaded movably into a gas-tight Microlab instrument syringe (Hamilton; 100 μL), attached to the chip, and measured at multiple flow rates, about 20%, 40%, and 60% of maximum pressure for each chip. For example, a sample of approximately 50 cP would be measured at around 10, 20, and 30 μL/min (approximately 180, 350, and 530 s-1, respectively, on an "A" chip) until the viscosity stabilizes, typically after at least 30 seconds. A mean absolute viscosity and standard deviation were then calculated from at least these three measurements. The C&P viscometer was equipped with a CPE40 or CPE52 spindle (cone angle of 0.8° and 3.0°, respectively) and 0.50 mL of samples were measured at multiple shear rates between 2 and 400 s. -1. Specifically, the samples were measured for 30 seconds each 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, starting at a shear rate that produced at least 10% torque, and continuing until the instrument torque reaches 100%. An extrapolated zero shear viscosity was then determined from a plot of dynamic viscosity versus shear rate for the samples measured on a DV2T plate and cone viscometer. The reported extrapolated zero-shear viscosities are the mean and standard deviation of at least three measurements.

Example 1: Effect of a viscosity reducing agent, lysine camphorsulfonic acid (CSAL), on the viscosity of biosimilar ERBITUX® solutions Materials and Methods

[00347] A commercially obtained biosimilar ERBITUX® (100-400 mg) containing pharmaceutical excipients (Polysorbate 80, phosphate buffer, and NaCl) was purified. First, Polysorbate 80 was removed using DETERGENT-OUT® TWEEN® Medi Columns (G-Biosciences). Then, the resulting solutions were extensively buffer exchanged into 20 mM sodium phosphate buffer (PB; pH 7.0) or 20 mM CSAL (pH 7.0) and concentrated to a final volume of less than 10 mL in Jumbosep centrifugal concentrators (Pall Corp.). The collected protein solution was freeze-dried. The dried protein masses, containing protein and salts or buffering agent or agent, were reconstituted to a final volume of 0.15 - 1.3 ml. These samples were reconstituted using enough additional PB (pH 7.0) or CSAL (pH 7.0) to bring the final concentration of PB or CSAL to 0.25 M. The final concentration of mAb in the solution was determined by light absorbance in 280nm. Reported protein concentrations represent the range of all protein samples included in each Table or Figure. Specifically, the reported values are the average plus or minus half of the range. Zero shear extrapolated using an experimentally determined extinction coefficient of 1.4 L/gxm and reported viscosities were measured on a DV2T plate and cone viscometer. Results

[00348] The data in Figure 1 demonstrates the viscosity reducing effect of CSAL on aqueous solutions of biosimilar ERBITUX®. The viscosity of a solution of biosimilar ERBITUX® in phosphate buffer (PB) increases exponentially with increasing mAb concentration. The viscosity of a biosimilar ERBITUX® solution in the presence of CSAL is seen to increase exponentially with increasing mAb concentration, however, to a lesser extent than the PB formulation, i.e., the viscosity gradient is reduced. The data in Figure 1 shows that the higher the mAb concentration, the greater the viscosity reduction effect. The magnitude of the viscosity reduction effects provided by replacing PB with CSAL ranged from 1.1 times at 100 + 5 mg/mL to 10.3 times at 227 + 5 mg/mL mAb.

Example 2: Viscosity-lowering effect of a viscosity-lowering agent, camphorsulfonic acid lysine (CSAL), as a function of concentration of AVASTIN® biosimilar Materials and Methods

[00349] A commercially obtained biosimilar AVASTIN® containing pharmaceutical excipients (Polysorbate 20, phosphate buffer, citrate buffer, mannitol, and NaCl) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficient of 1.7 L/gxm at 280 nm). The protein was formulated to contain 0.25 M phosphate buffer or 0.25 M CSAL.Results

[00350] Figure 2 depicts the viscosity of aqueous mAb solutions as a function of mAb concentration in aqueous buffered solution and with CSAL. The viscosity of AVASTIN® biosimilar in aqueous phosphate buffer and in the presence of CSAL increases exponentially with increasing concentration; however, as in the case of biosimilar ERBITUX®, this increase is much less commercialized for the formulation containing CSAL, that is, the viscosity gradient is reduced. In general, the higher the mAb concentration, the greater the viscosity reduction effect observed. The magnitude of the viscosity reduction effects provided by replacing PB with CSAL ranged from 2.1-fold at 80 mg/mL to 3.7-fold at 230 + 5 mg/mL mAb.

Example 3: Viscosity decreasing effect as a function of CSAL concentration for aqueous solutions of biosimilar ERBITUX® Materials and Methods

[00351] Samples were purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed similarly to Example 1 above. The final concentration of CSAL upon reconstitution in an aqueous CSAL solution ranged from 0.25 M to 0.50 M. Results

[00352] Table 1 shows the viscosity of biosimilar ERBITUX® solutions formulated in 0.25 M phosphate buffer (in CSAL as a control) and with varying concentrations of CSAL. The viscosity reducing effect of CSAL is observed to increase by 8.4- to 12.1-fold with increasing viscosity reducing agent concentration. The data in Table 1 shows that the higher the concentration of CSAL, the greater the viscosity reducing effect, at least within the concentration range of the agent tested. Table 1. The viscosities of aqueous solutions of biosimilar ERBITUX® (155 + 5 mg/mL, pH 7.0) in the presence of different concentrations of CSAL at 25oC.

Example 4: Viscosities of biosimilar ERBITUX® solutions as a function of temperature in the presence of various viscosity reducing agents Materials and Methods

[00353] Aqueous solutions of biosimilar ERBITUX® containing various viscosity reducing agents were prepared as described in Example 1. Specifically, 20 mM solutions of the viscosity reducing agents of interest were used for buffer exchange, and the lyophilized masses were reconstituted in 0.25 M of each viscosity reducing agent. For the sample containing CSA-APMI, ERBITUX® biosimilar was extensively buffer exchanged into 2 mM PB (pH 7.0), and concentrated to a final volume of less than 10 mL in Jumbosep centrifugal concentrators (Pall Corp.). The sample was first aliquoted. Next, an appropriate amount of CSAAPMI solution (pH 7.0) was added to each aliquot such that upon reconstitution with water, the final excipient concentration is 0.25 M. The protein solutions were then freeze-dried. The dried protein masses, containing protein and viscosity reducing agent (and a negligible amount of buffer salts) were reconstituted to a final volume of approximately 0.10 mL and viscosity reducing agent concentration as previously described. Results

[00354] Table 2 shows viscosity data for biosimilar ERBITUX® in the presence of six viscosity reducing agents - lysine camphorsulfonic acid (CSAL), arginine camphorsulfonic acid (CSAA), benzenesulfonic acid lysine (BSAL), arginine acid arginine benzenesulfonic acid (BSAA), naphthalenesulfonic acid arginine (NSAA), and camphorsulfonic acid 1-(3-aminopropyl)-2-methyl-1H-imidazole (CSAAPMI). The data in Table 2 show a reduction in viscosity of at least about 9 times for all six viscosity reducing agents compared to a solution of biosimilar ERBITUX® in phosphate buffer otherwise under the same conditions. The most effective viscosity reducing agent - CSAAPMI - decreased viscosity by >40 times.

[00355] Additionally, the data in Table 3 shows that at multiple temperatures ranging from 20oC to 30oC, a 225 mg/mL solution of biosimilar ERBITUX® prepared with 0.25 M CSAA had the lowest viscosity of the five decontamination agents. viscosity reduction. Thus, the attempts observed at viscosities at 25 oC proved to be predictable from those at temperatures of at least 20 oC and 30 oC. Table 2. Reduction in viscosity of aqueous solutions of biosimilar ERBITUX® (226 + 6 mg/mL, pH 7, 0) formulated with various viscosity reducing agents at 0.25 M, compared to that in 0.25 M sodium phosphate buffer (PB) at 25oC. Table 3. The viscosities of aqueous solutions of biosimilar ERBITUX® (225 + 5 mg/mL, pH 7.0) formulated with various viscosity reducing agents at 0.25 M.

Example 5: The effect of temperature on the viscosity of aqueous solutions of biosimilar AVASTIN® formulated with various viscosity reducing agents Materials and Methods

[00356] Biosimilar AVASTIN® solutions containing different viscosity reducing agents were prepared as described in Example 1 above. In particular, 20 mM solutions of the viscosity reducing agents of interest were used for buffer exchange, and the lyophilized masses were reconstituted in 0.15 or 0.25 M viscosity reducing agent.Results

[00357] As seen in Table 4, CSAL at 0.25 M decreased the viscosity of a 230 + 5 mg/mL solution of biosimilar AVASTIN® at all three temperatures between 20 and 30oC. Furthermore, CSAL at 0.15 M reduces viscosity by approximately the same absolute value as CSAL at 0.25 M at 20 and 25oC and is equally effective at 30oC.

[00358] The data in Table 5 compares the effects of CSAL and BSAL at a concentration of 0.15 M. CSAL is a superior viscosity reducing agent compared to BSAL at all three temperatures. Table 4. The viscosities of aqueous solutions of biosimilar AVASTIN® (230 + 5 mg/mL, pH 7.0) formulated with 0.25 and 0.15 M CSAL at different temperatures. Table 5. The viscosities of aqueous solutions of biosimilar AVASTIN® (230 + 5 mg/mL, pH 7.0) formulated with 0.15 M CSAL and BSAL at different temperatures.

Example 6: CSAL Removal Reverses the Viscosity Reduction Effect in mAb Solutions Materials and Methods

[00359] Three samples each of ERBITUX® biosimilar and AVASTIN® biosimilar were prepared. First, Polysorbate was removed from commercially obtained mAb solutions. The resulting solution with remaining pharmaceutical excipients was (i) concentrated in a centrifugal device with a molecular weight cutoff of 100-kDa (MWCO) (Pall Corp.) as a control sample (original excipients), (ii) buffer exchanged in 0.25 M CSAL as described in Example 1, or (iii) buffer exchanged in 0.25 M CSAL as described in Example 1, reconstituted, and then also exchanged in 0.25 M PB. In this third For example, exchange in 0.25 M phosphate buffer first proceeded by overnight dialysis against 20 mM PB (50-kDa MWCO, Spectrum Labs). Partially dialyzed samples were then diluted to 60 mL in 0.25 M PB and subjected to centrifugal concentration (30-kDa MWCO Jumbosep (Pall Corp.), followed by a 100-kDa MWCO Macrosep device (Pall Corp.) ). The viscosities of these three aqueous solutions were determined as described in Example 1 above. Results

[00360] The viscosities of aqueous solutions of both ERBITUX® biosimilar and AVASTIN® biosimilar decreased in the presence of CSAL - 2.7- and 1.5 times, respectively - but then increased when CSAL was removed (see Tables 6 and 7) . Furthermore, upon removal of CSAL, the viscosities of the mAb solution returned to approximately the same level as the original solutions, suggesting that CSAL does not damage the protein and showing that it is necessary for the observed viscosity reduction. Table 6. The viscosities of aqueous solutions of biosimilar ERBITUX® (80 + 5 mg/mL, pH 7.0) at 25oC. Table 7. Viscosities of aqueous solutions of biosimilar AVASTIN® (101 + 5 mg/mL, pH 7.0) at 25oC.

Example 7: Viscosity reducing agents containing camphorsulfonic acid provide greater viscosity reductions in aqueous solutions of AVASTIN® and biosimilar AVASTIN® Materials and Methods

[00361] AVASTIN® and a commercially obtained biosimilar AVASTIN® containing pharmaceutical excipients (AVASTIN®: trehalose, sodium phosphate buffer, and Polysorbate 20; AVASTIN® biosimilar: Polysorbate 20, phosphate buffer, citrate buffer, mannitol, and NaCl ) were purified, buffer exchanged, concentrated, freeze-dried, and reconstituted as described above. The samples in Table 8 were prepared as described in Example 1 above (using the protein extinction coefficient of 1.7 L/gxm at 280 nm) and measured on a C&P viscometer. The viscosity reduced samples in Table 9 were prepared as described in Example 4 above, but mAb was extensively buffer exchanged into 2 mM PB. Subsequently, the appropriate amount of viscosity reducing agent was added to result in a final viscosity reducing agent concentration of 0.15-0.35 M upon reconstitution. Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an "A" or "B" chip. Results

[00362] The data in Tables 8 and 9 demonstrate the viscosity reducing effect of different viscosity reducing agents on aqueous solutions of biosimilar AVASTIN®. Viscosity reductions of up to 2.5-fold (compared to mAb solutions in PB) are observed for aqueous solutions of AVASTIN® biosimilar in the presence of CSA-containing viscosity reducing agents. Table 8. The viscosities of aqueous solutions of AVASTIN® biosimilar (200 ± 5 mg/mL, pH 7.0) at 25°C with various viscosity reducing agents. Contains equimolar NaCl; CSA = Camphorsulfonic acid, BSA = Benzenesulfonic acid, MSA = Methanesulfonic acid, PB = Phosphate buffer Table 9. The viscosities of aqueous solutions of AVASTIN® biosimilar (pH 7.0) at 25°C with viscosity reducing agents at 0 .15M (unless otherwise noted).

[00363] The viscosity of a 200 ± 9 mg/mL aqueous solution of AVASTIN® biosimilar with CSAA was measured as a function of pH as described in Figure 3. As the pH increases, the magnitude of the viscosity reduction effect resulting from The presence of CSAA in aqueous solutions of biosimilar AVASTIN® likewise increases, reaching a minimum viscosity and maximum viscosity reduction effect around pH 7. Viscosity reduction by CSAA was compared as a function of pH for two different concentrations of AVASTIN® biosimilar. Figure 4 demonstrates that CSAA at 0.25 M results in a greater reduction in viscosity with (i) increasing concentration of AVASTIN® biosimilar and (ii) pH.

[00364] Table 10 compares the viscosity reduction of biosimilar AVASTIN® to that of registered AVASTIN® with and without CSAL. The registered AVASTIN® solution has a much higher viscosity than a solution of the biosimilar mAb in the absence of the agent. However, the presence of CSAL at 0.25 M results in a reduction in viscosity of 1.8 and 3.3 times of biosimilar and registered AVASTIN® respectively; the viscosities of biosimilar and registered AVASTIN® were observed to be similar in the presence of CSAL at 0.25 M. Table 10. The viscosities of aqueous solutions containing 205 ± 5 mg/mL of biosimilar AVASTIN® or registered AVASTIN® with or without CSAL at 0.25 M measured at 25°C and pH 7.0.

[00365] As demonstrated in Table 11, CSA 1-(3-aminopropyl)-2-methyl-1H-imidazole (CSAAPMI) with HCl provides superior viscosity reduction than CSAL, reducing viscosity more than 5 times compared to PB control for a 210 mg/mL solution of AVASTIN® biosimilar. Table 11. The viscosities of aqueous solutions of AVASTIN® biosimilar with various viscosity reducing agents at 25°C and pH 70.

[00366] For a solution containing ~ 230 mg/mL of biosimilar AVASTIN®, Table 12 demonstrates approximately 5-fold viscosity reduction with viscosity reducing agents containing sulfosalicylic acid as well as for CSAAPMI and CSA thiamine. Table 12. The viscosities of aqueous solutions containing 228 ± 5 mg/mL of AVASTIN® biosimilar with viscosity reducing agents at 25°C and pH 7.0.

Example 8. The effect of viscosity reducing agents on aqueous solutions of ERBITUX® and ERBITUX® biosimilar Materials and Methods

[00367] Aqueous solutions of biosimilar and registered ERBITUX® containing various viscosity reducing agents were prepared as described in Example 1. Specifically, 20 mM solutions of the salts of interest were used for buffer exchange, and the lyophilized masses were reconstituted in contain 0.25 M of each agent. Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an "A" or "B" chip or a DV2T cone and plate viscometer. Results

[00368] Table 13 shows data for biosimilar ERBITUX® (222 ± 5 mg/mL) in the presence of five viscosity reducing agents: CSAA, CSAL, BSAA, BSAL, and NSAA. Table 14 compares the viscosity reduction of biosimilar ERBITUX® solutions using CSAA and CSAL to arginine or lysine alone. Table 13. The viscosities of biosimilar ERBITUX® aqueous solutions (222 ± 5 mg/mL, pH 7.0) with viscosity reducing agents at 0.25 M at 25°C. Table 14. The viscosities of aqueous solutions of biosimilar ERBITUX® (222 ± 5 mg/mL, pH 7.0) with viscosity reducing agents at 0.25 M at 25°C.

[00369] The data in Table 13 shows a reduction in viscosity of at least 9.0 times for all five viscosity reducing agents compared to an aqueous solution of biosimilar ERBITUX® in otherwise phosphate buffer under the same conditions . The most effective viscosity reducing agents, CSAA and BSAA, decreased the viscosity of the solution by 21 times. The viscosities of aqueous solutions of biosimilar ERBITUX® containing 0.25 M CSAA were compared as a function of pH at varying protein concentrations. Figure 5 demonstrates that a minimum viscosity is observed around pH 7.0 for all protein concentrations. The effect of pH on viscosity is more pronounced for higher protein concentrations (253 mg/mL in the example).

[00370] As seen in Table 15, aqueous solutions of biosimilar and registered ERBITUX® have similar viscosities in the presence of the BSAA arginine salt at 0.25 M. Table 15. The viscosities of 224 ± 4 mg/mL of aqueous solutions of biosimilar ERBITUX® or registered ERBITUX® with or without BSAA at 0.25 M at 25°C and pH 7.0.

[00371] The impact of viscosity reducing agents on the formation of irreversible protein aggregates was examined for biosimilar ERBITUX®. Aqueous liquid formulations were prepared from (i) ERBITUX® biosimilar and (ii) ERBITUX® biosimilar containing CSAL at 0.25 M. These solutions were stored for 90 days at 4oC and pH 5.4 and 7.0, respectively. Stored samples were examined using size exclusion chromatography (column: Tosoh TSKgel UltraSW Aggregate; mobile phase: 0.1 M potassium phosphate/0.1 M sodium sulfate, pH 6.8 in 0.8 mL/ min; injection: 20 μL of a 5 mg/mL mAb solution). The data in Table 16 do not reveal significant aggregate formation in the commercial drug product or formulation decreased by high concentration viscosity. Table 16. Percentage of protein aggregate formation after 90 days of storage at 4°C when measured by chromatography size exclusion for aqueous solutions containing biosimilar ERBITUX® with or without CSAL at 0.25 M.

Example 9. The effect of viscosity reducing agents on aqueous solutions of REMICADE® Materials and Methods

[00372] Commercially obtained REMICADE® containing pharmaceutical excipients (sucrose, Polysorbate 80, sodium phosphate buffer) was prepared according to the instructions in the prescribing information leaflet. Subsequently, the aqueous drug product was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficient of 1.4 L/g*cm at 280 nm). Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip. Results

[00373] The data for aqueous REMICADE® solutions in Table 17 demonstrate that (i) viscosity reducing agents containing a bulky cyclic group provide viscosity reductions greater than 15-fold, and (ii) CSAA, CSAAPMI, and sulfosalicylic acid diarginine (SSA DiArg) provides the greatest viscosity reduction of about 29 times. Solution viscosities in the presence of ArgHCl alone are significantly higher than those with bulky cyclic groups. Table 17. Viscosities of aqueous solutions of REMICADE® containing viscosity reducing agents at 0.25M at 25°C and pH 7 ,0.

[00374] The dependence of viscosity reduction on agent concentration was examined for aqueous solutions of REMICADE® in the presence of CSAA. The results presented in Table 18 demonstrate that viscosity reduction increases with increasing agent concentration. The viscosity reduction, for example, is more than twice as great (viscosity is less than half) with 0.35 M agent compared to 0.20 M agent. Table 18. Viscosity of an aqueous solution of REMICADE®(215 ± 5 mg/mL) in the presence of various concentrations of CSAA measured at 25°C and pH 7.0.

[00375] Biophysical properties of REMICADE® solutions formulated with 0.25 M CSAA were evaluated for 90 days. Samples of REMICADE® formulated with 0.25 M CSAA were prepared as described in Example 1 above. As seen in Table 19 and Figure 6, the monomer content of concentrated solutions of REMICADE® in 0.25 M CSAA as determined by size exclusion chromatography (Tosoh TSKgel UltraSW Aggregate column; 0.1 M potassium phosphate buffer M/0.1 M sodium sulfate pH 6.8 at 0.8 mL/min; 20 μL injection of ~4.5 mg/mL solutions), is similar to the drug product at all time points and no detectable aggregation is observed after storage for 100 days at 4°C. Viscosity, when measured using a microfluidic viscometer, has been demonstrated to remain stable after storage for 30 days at 4°C (Table 20). Additionally, antigen binding of this processed REMICADE® protein was measured with a REMICADE® specific ELISA assay and no decrease in binding was seen between day 0 and 100 (Table 20). Similarly, the monomer content (Table 21) and antigen binding (normalized to that of the drug product, Table 22) of the concentrated solutions of REMICADE® in 0.25 M CSAA are comparable to those of the drug product after 1 week of storage. at room temperature. Finally, Table 23 demonstrates that storing a freeze-dried mass containing CSAA at 4°C for 75 days had no negative effects on the viscosity of the solution or extent of protein aggregation when the sample is reconstituted. The results in Tables 19-23 and Figure 6 demonstrate the biophysical etability of REMICADE® formulated with CSAA before and after storage for at least 100 days at 4°C. Table 19. No increased aggregation (compared to drug product) is observed in an aqueous solution of REMICADE® (227 mg/mL, pH 7) after formulation with CSAA at 0.25 Mea and storage at 4°C. Table 20. Reduced viscosity and antigen binding are maintained over time in an aqueous solution of REMICADE® (227 mg/mL, pH 7) after formulation with 0.25 M CSAA and storage at 4°C. Table 21. No increased aggregation (compared to drug product) is observed in an aqueous solution of REMICADE® (219 mg/mL, pH 7) after formulation with CSAA at 0.25 Mea and storage at room temperature. Table 22. Antigen binding persists in an aqueous solution of REMICADE® (219 mg/mL, pH 7) after formulation with 0.25 M CSAA and storage at room temperature. Table 23. REMICADE® stored as a lyophilized powder maintains low viscosity and monomer content upon reconstitution after storage at 4°C for 75 days

Example 10. The effect of viscosity reducing agents on aqueous solutions of HERCEPTIN® Materials and Methods

[00376] Commercially obtained HERCEPTIN® containing pharmaceutical excipients (histidine buffer, trehalose, Polysorbate 20) was prepared according to the instructions in the prescribing information leaflet. Subsequently, the aqueous drug product was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficient of 1.5 L/g*cm at 280 nm). Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an "A" or "B" chip. Results

[00377] The data presented in Table 24 shows that the viscosity of an aqueous solution of HERCEPTIN® containing viscosity reducing agents - compared to those containing PB - is lower in the presence of CSAA. At higher protein concentrations (i.e., >250 mg/mL) Arginine HCl alone reduces viscosity significantly and CSA also enhances the effect. Table 24. The viscosities of aqueous solutions of

Example 11. The effect of viscosity reducing agents on aqueous solutions of TYSABRI® Materials and Methods

[00378] Commercially obtained TYSABRI® containing commercially obtained pharmaceutical excipients (sodium phosphate buffer, sodium chloride, Polysorbate 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the coefficient of extinction of 1.5 L/g*cm at 280 nm). Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip. Results

[00379] The data presented in Table 25 shows that the viscosity reduction of an aqueous solution of TYSABRI® containing viscosity reducing agents is approximately 2.5 times (compared to the solution containing PB) close to 276 mg/mL of protein .Table 25. The viscosities of aqueous solutions of TYSABRI® containing viscosity reducing agents at 0.25 M at 25°C and pH 7.0.

Example 12. The effect of viscosity reducing agents on aqueous solutions of RITUXAN® biosimilar Materials and Methods

[00380] Commercially obtained biosimilar RITUXAN® containing pharmaceutical excipients (citrate buffer, sodium chloride, and TWEEN® 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the coefficient extinction rate of 1.7 L/g*cm at 280 nm). Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip. Results

[00381] The data presented in Table 26 shows that the viscosity reduction for an aqueous solution of biosimilar RITUXAN® containing viscosity reducing agents is more than 13 times at approximately 213 mg/mL of protein and more than 5 times at approximately 202 mg/mL, compared to the mAb formulated in PB.Table 26. The viscosities of aqueous solutions of RITUXAN® biosimilar with viscosity reducing agents at 25°C and pH 7.0.

Example 13. The effect of viscosity reducing agents on aqueous solutions of VECTIBIX® Materials and Methods

[00382] Commercially obtained VECTIBIX® containing pharmaceutical excipients was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficient of 1.25 L/g*cm at 280 nm) . Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip. Results

[00383] The data presented in Table 27 shows that the reduction in viscosity of an aqueous solution of VECTIBIX® containing viscosity reducing agents is approximately 2 times at 291 mg/mL and 3 times at 252 mg/mL, compared to solutions with PB, however, no viscosity reducing agent. Table 27. The viscosities of aqueous solutions of VECTIBIX® with viscosity reducing agents at 0.25 M at 25°C and pH 7.0.

Example 14. The effect of viscosity reducing agents on aqueous solutions of ARZERRA® Materials and Methods

[00384] Commercially obtained ARZERRA® containing pharmaceutical excipients was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficient of 1.5 L/g*cm at 280 nm) . Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an "A" or "B" chip. Results

[00385] The data presented in Table 28 shows that the reduction in viscosity of an aqueous solution of ARZERRA® containing viscosity reducing agents is approximately 3 times at 274 mg/mL and 2 times at 245 mg/mL, compared to solutions with PB, however, no viscosity reducing agent. Table 28. The viscosities of aqueous solutions of ARZERRA® with viscosity reducing agents at 0.25 M at 25°C and pH 7.0.

Example 15. Comparison of Different Methods for Measuring Viscosity Materials and Methods

[00386] Aqueous solutions containing 220 mg/mL of REMICADE® and 0.25 M CSAA were prepared as described in Example 1 above. Viscosities at 25°C and pH 7.0 are reported in Table 29 as zero shear viscosities extrapolated from plate and cone viscometer measurements and as absolute viscosities measured with a microfluidic viscometer. Plate and cone measurements used a DV2T plate and cone viscometer (Brookfield) equipped with a CPE40 or CPE52 spindle measured at multiple shear rates between 2 and 400 s-1. An extrapolated zero shear viscosity was determined from a plot of absolute viscosity versus shear rate. Microfluidic viscometer measurements were performed using a RheoSense mVROC microfluidic viscometer equipped with an "A" or "B" chip at multiple flow rates (approximately 20%, 40%, and 60% of maximum pressure for each chip).Results

[00387] The data in Table 29 demonstrates that the absolute viscosities of the microfluidic viscometer can be directly compared to the extrapolated zero shear viscosities determined from the plate and cone viscometer. Table 29. The viscosities of aqueous solutions of REMICADE® (220 mg /mL) with CSAA at 0.25 M at 25 °C and pH 7.0 measured in two different viscometers.

[00388] To compare a wider range of viscosities and protein concentrations, aqueous solutions of a model antibody, bovine gamma globulin, were prepared with and without CSAL at 0.25 M. Viscosities were measured as described above at protein concentrations. ranging from 110 mg/mL to 310 mg/mL. The data presented in Table 30 demonstrates that absolute viscosities from the microfluidic viscometer can be directly compared to zero shear viscosities extrapolated equally for low and high viscosity protein solutions. Table 30. The viscosities of aqueous gamma globulin solutions with and without CSAL at 0.25 M at 25°C and pH 7.0 measured in two different viscometers.

Example 16. Viscosity reducing agents show no signs of toxicity when injected subcutaneously Materials and Methods

[00389] Thirty 11-week-old Sprague-Dawley rats were separated into 6 groups of 5 rats each (3 saline control groups and 3 CSAA groups). Rats were injected subcutaneously with 0.5 mL of endotoxin-free phosphate-buffered saline or 0.25 M endotoxin-free CSAA according to the following schedule: One group from each condition was injected once on day 1 and on then sacrificed 1 hour later; one group from each condition was injected once on day 1 and once on day 2 and then sacrificed 24 hours after the second injection; and one group from each condition was injected once on day 1, once on day 2, and once on day 3, and then sacrificed 24 hours after the third injection.

[00390] Clinical observations were recorded for any pharmaco-toxicological signs pre-dose, immediately post-dose, at 1 and 4 hours (± 15 minutes) post-dose, and daily thereafter. Irritation, if any, at injection sites was scored using Draize assessment scores pre-dose, immediately post-dose, at 1 hour (±15 minutes) post-dose, and before sacrifice. Results

[00391] Generally, the observed consequences of saline and CSAA injections were macroscopically similar throughout the course of the study. Both induced from no irritation to mild irritation with edema scores of 0-2 at various time points. Microscopic examination of the injection sites suggests a much smaller, clinically insignificant, irritant effect with CSAA that was no longer evident by day 4.

Example 17. Concentrated aqueous solutions of REMICADE® formulated with viscosity reducing agents exhibit low syringe extrusion forces and high monomer content when expelled through needles of various gauges. Materials and methods

[00392] Commercially obtained REMICADE® containing pharmaceutical excipients (sucrose, Polysorbate 80, sodium phosphate buffer) was prepared per instructions in the prescribing information leaflet. Subsequently, the aqueous drug product was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficient of 1.4 L/g*cm at 280 nm). 20 mM solutions of phosphate buffer, CSAAPMI, or CSAA were used for buffer exchange, and the lyophilized masses were reconstituted in 0.25 M of each viscosity reducing agent. After reconstitution, the viscosity of each solution was measured using the microfluidic viscometer as described in the previous examples. The solutions were then reloaded into 1 mL BD insulin syringes with needles set at 27, 29, or 31 gauge. The force required to extrude the concentrated REMICADE® solutions was then measured using an Instron at a displacement rate equivalent to a fluid flow rate of 3 mL/min. The expelled solution was collected from the syringe and analyzed by size exclusion chromatography. Results

[00393] All REMICADE® solutions containing viscosity reducing agents were able to be expelled through the syringes at relatively low extrusion forces (Table 31). The solution containing phosphate buffer cannot be expelled due to high viscosity. Both solutions containing viscosity reducing agents maintained high post-extrusion monomer content regardless of needle gauge, as indicated in Table 31.Table 31. Syringability of concentrated aqueous REMICADE® solutions extruded through various gauge needles.

Example 18: Viscosity reducing agents reduce the viscosity of concentrated aqueous solutions of AVASTIN® biosimilar Materials and Methods

[00394] A commercially obtained biosimilar AVASTIN® containing pharmaceutical excipients (Polysorbate 20, phosphate and citrate buffer, mannitol, and NaCl) was purified. First, Polysorbate 20 was removed using DETERGENT-OUT® TWEEN Medi Columns (G- Biosciences). Then, the resulting solutions were extensively buffer exchanged into 20 mM sodium phosphate buffer (PB) for PB samples and 2 mM PB for viscosity reducing agent samples, and concentrated to a final volume of less than 10 mL in Jumbosep centrifugal concentrators (Pall Corp.). The viscosity reducing agent was then added to the PB samples at 2 mM as described in Example 4 above. The viscosity reducing agent(s) was added in an amount sufficient to produce the reconstitution concentration as specificity. In the case of combinations of agents, the concentration of each component is 0.15 M. The protein solutions were then freeze-dried. The dried protein masses were reconstituted in phosphate buffer (for PB samples) or water (for samples containing viscosity reducing agents) to a final volume of approximately 0.10 mL. The final concentration of mAb in the solution was determined by a Coomassie protein quantification assay comparing unknown sample concentrations to a biosimilar AVASTIN® standard curve or by A280 using the extinction coefficient of 1.7 L/g*cm, when possible. Reported viscosities were measured on a RheoSense mVROC microfluidic viscometer. The results are reported in Table 32. Results

[00395] Many GRAS, IIG, and API compounds are capable of reducing the viscosity of concentrated AVASTIN® biosimilar solutions relative to phosphate-buffered samples. Of these compounds included in Table 32, local anesthetics such as procaine and lidocaine, as well as GRAS agents such as biotin are among the most effective viscosity reducing agents. Table 32: Effect of Viscosity Reducing Agents on AVASTIN® Biosimilar Solutions .

Example 19. Viscosity Reduction is an Effect Dependent on Agent Concentration Materials and Methods

[00396] Aqueous solutions of a commercially obtained biosimilar AVASTIN® were prepared as described in Example 4. The dried protein masses were reconstituted in phosphate buffer or water to a final volume of about 0.10 mL and a concentration of dihydrochloride of final 1-(3-aminopropyl)-2-methyl-1H-imidazole (APMI*2HCl) of 0.10 or 0.25 M. The final concentration of mAb in the solution was determined by a Coomassie protein quantification assay comparing Unknown sample concentrations are applied to an AVASTIN® biosimilar standard curve. Reported viscosities were measured on a RheoSense mVROC microfluidic viscometer.Results

[00397] As described in Figure 7, the viscosity reduction effect was increased when the concentration of APMI*2HCl was increased.

Example 20. A simple viscosity-lowering agent decreases the viscosity of many therapeutically relevant monoclonal antibodies Materials and Methods

[00398] Aqueous solutions of a commercially obtained biosimilar AVASTIN® were prepared as described in Example 4. The dried protein masses were reconstituted in phosphate buffer or water to a final volume of about 0.10 mL and an HCl concentration of final thiamine of 0.10 or 0.25 M. The final concentration of mAb in the solution was determined by a Coomassie protein quantification assay by comparing the unknown concentrations of the samples to a biosimilar AVASTIN® standard curve.

[00399] Commercially obtained TYSABRI® containing pharmaceutical excipients (sodium phosphate buffer, NaCl, Polysorbate 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed in the same way. Commercially obtained HERCEPTIN® containing pharmaceutical excipients (sodium phosphate buffer, NaCl, Polysorbate 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed in the same manner. Commercially obtained biosimilar ERBITUX® containing pharmaceutical excipients (Polysorbate 80, phosphate buffer, and NaCl) was purified, buffer exchanged, concentrated, dried, reconstituted and analyzed in the same manner. Commercially obtained REMICADE® containing pharmaceutical excipients (sucrose, Polysorbate 80, sodium phosphate buffer) was prepared according to the instructions in the prescribing information leaflet. Subsequently, the aqueous drug product was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in the same manner. Reported viscosities were measured on a RheoSense mVROC microfluidic viscometer. Results

[00400] The data in Table 33 demonstrate that thiamine HCl can decrease the viscosity of concentrated aqueous solutions of many therapeutically relevant mAbs. Thiamine HCl can produce a viscosity reduction greater than 4 times for each mAb. Table 33: Effect of Thiamine HCl on Solution Viscosity.

Examples 21-24. Viscosity-lowering agents reduce the viscosity of aqueous solutions of many therapeutically relevant monoclonal antibodies Materials and Methods

[00401] Aqueous solutions of commercially obtained RITUXAN® biosimilar, TYSABRI®, HERCEPTIN®, ERBITUX® biosimilar, and REMICADE® were prepared as described in Examples 18 and 19. Tables 34-38 demonstrate that viscosity reducing agents can be advantageously employed for many different monoclonal antibodies. Results Table 34: Viscosities of Aqueous Solutions of RITUXAN® Biosimilar in the Presence of Viscosity Reducing Agents at 0.15 M Table 35: Viscosities of TYSABRI® Aqueous Solutions in the Presence of Viscosity Reducing Agents at 0.15 M (Unless Otherwise Indicated). Table 36: Viscosities of HERCEPTIN® Aqueous Solutions in the Presence of Viscosity Reducing Agents at 0.15 M (Unless Otherwise Indicated). Table 37: Viscosities of ERBITUX® Aqueous Solutions in the Presence of Viscosity Reducing Agents at 0.15 M (Unless Otherwise Indicated). Table 38: Viscosities of REMICADE® Aqueous Solutions in the Presence of Viscosity Reducing Agents at 0.15 M (Unless Otherwise Indicated).

Example 25. Effect of viscosity decrease of TPP and TPPAPMI, as a function of concentration of AVASTIN® biosimilar

[00402] Aqueous solutions of a commercially obtained biosimilar AVASTIN® were prepared as described in Example 1 above. The protein was formulated to contain 0.25 M phosphate buffer, 0.10 M thiamine pyrophosphate (TPP), or 0.10 M TPP-1-(3-aminopropyl)-2-methyl-1H-imidazole (TPPAPMI).

[00403] Figure 8 depicts the viscosity of the aqueous biosimilar AVASTIN® solution as a function of the concentration of phosphate-buffered mAb, TPP, or TPPAPMI. The viscosity of AVASTIN® biosimilar in phosphate buffer increases exponentially within the tested protein concentration range. In the presence of excipients containing TPP, the increase in viscosity is attenuated, i.e., the viscosity gradient is reduced.

Example 26: Viscosity reducing effect of a viscosity reducing agent, thiamine HCl, as a function of concentration of SIMPONI ARIA® biosimilar Materials and Methods

[00404] SIMPONI ARIA® commercially obtained and containing pharmaceutical excipients (Histidine, Sorbitol, Polysorbate 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficient of 1. 4 L/gxm at 280 nm). The protein was formulated to contain 0.15 M phosphate buffer or 0.15 M thiamine HCl. Results

[00405] Figure 9 depicts the viscosity of aqueous SIMPONI ARIA® solutions as a function of mAb concentration with phosphate buffer or thiamine HCl. The viscosity of SIMPONI ARIA® in phosphate buffer increases exponentially within the tested protein concentration range. In the presence of thiamine HCl, the increase in viscosity is attenuated, i.e., the viscosity gradient is reduced.

Example 27. Effect of viscosity decrease of Thiamine HCl as a function of ENBREL® concentration Materials and Methods

[00406] ENBREL® obtained commercially and containing pharmaceutical excipients (Mannitol, Sucrose, Tromethamine) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficient of 0.96 L /gxm at 280 nm). The protein was formulated to contain 0.15 M phosphate buffer or 0.15 M Thiamine HCl. Results

[00407] Table 39 describes the viscosity of aqueous ENBREL® solutions with phosphate buffer or thiamine HCl. The addition of Thiamine HCl reduces the viscosity of ENBREL® up to approximately 2 times. Table 39: Viscosities of Aqueous ENBREL® Solutions in the Presence of 0.15 M PB or Thiamine HCl

Example 28. Isotonic solutions of viscosity-reducing excipients reduce the viscosity of concentrated solutions of REMICADE® Materials and Methods

[00408] Commercially obtained REMICADE® containing pharmaceutical excipients (sucrose, Polysorbate 80, sodium phosphate buffer) was prepared according to the instructions in the prescribing information leaflet. Subsequently, the aqueous drug product was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1, except that isotonic amounts of charged hydrophobic compounds were added. Results

[00409] As demonstrated in Table 40, isotonic amounts of both CSAA and CSAAPMI are capable of substantially reducing the viscosity of concentrated REMICADE® solutions, in some cases by up to about 10 times. Table 40. The viscosities of REMICADE® solutions in the presence of isotonic viscosity reducing excipients (0.3 molal)

[00410] Unless expressly defined otherwise, all technical and scientific terms used herein have the same meanings as generally understood by one of skill in the art. Those skilled in the art will recognize, or be able to verify using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims (15)

1. Liquid pharmaceutical formulation for injection, characterized by the fact that it comprises: (i) at least 100 mg/mL of an antibody; (ii) thiamine or a pharmaceutically acceptable salt thereof; and (iii) a pharmaceutically acceptable solvent; wherein the liquid pharmaceutical formulation, when in a volume suitable for injection, has an absolute viscosity of 1 cP to 100 cP at 25°C when measured using a plate and cone viscometer or a microfluidic viscometer; and the absolute viscosity of the liquid pharmaceutical formulation is less than the absolute viscosity of a liquid pharmaceutical formulation comprising the antibody and the thiamine-free pharmaceutically acceptable solvent or a pharmaceutically acceptable salt thereof; where the absolute viscosity is an extrapolated zero shear viscosity. 2. Formulation according to claim 1, characterized in that the antibody is a monoclonal antibody. 3. Formulation according to claim 1 or 2, characterized by the fact that the antibody has a molecular weight of 120 kDa to 250 kDa. 4. Formulation according to any one of claims 1 to 3, characterized in that it comprises from 100 mg/mL to 300 mg/mL of the antibody. 5. Formulation according to any one of claims 1 to 4, characterized in that it comprises 178 mg/mL to 245 mg/mL of the antibody. 6. Formulation according to any one of claims 1 to 5, characterized in that the liquid pharmaceutical formulation is aqueous. 7. Formulation according to any one of claims 1 to 6, characterized in that thiamine or a pharmaceutically acceptable salt thereof is present at a concentration of 0.01 M to 1.0 M. 8. Formulation according to any one of claims 1 to 7, characterized in that thiamine or a pharmaceutically acceptable salt thereof is present at a concentration of 0.10 M to 0.25 M. 9. Formulation according to any one of claims 1 to 8, characterized in that it further comprises one or more pharmaceutically acceptable excipients comprising sugar, sugar alcohol, buffering agent, preservative, carrier, antioxidant, chelating agent, polymer natural, synthetic polymer, cryoprotectant, lyoprotectant, surfactant, bulking agent, stabilization agent, or any combination thereof. 10. Formulation according to claim 9, characterized in that the one or more of the excipients is a polysorbate, poloxamer 188, sodium lauryl sulfate, a polyol, a polyethylene glycol, glycerol, a propylene glycol, or an alcohol polyvinyl. 11. Formulation according to claim 9, characterized by the fact that the sugar alcohol is sorbitol or mannitol. 12. Formulation according to any one of claims 1 to 11, characterized by the fact that it is in a single dose vial, cartridge, container, or pre-filled syringe. 13. Formulation according to any one of claims 1 to 12, characterized by the fact that it is isotonic to human blood serum. 14. Formulation according to any one of claims 1 to 13, characterized by the fact that the absolute viscosity of the liquid pharmaceutical formulation is measured at a shear rate of at least 0.5 s-1, when measured using a viscometer of plate and cone, or a shear rate of at least 1.0 s-1 when measured using a microfluidic viscometer. 15. Use of thiamine or a pharmaceutically acceptable salt thereof, characterized by the fact that it is for the manufacture of a subcutaneous or intramuscular liquid pharmaceutical formulation, as defined in any one of claims 1 to 14.