BR112016005287B1 - LIQUID PHARMACEUTICAL FORMULATION FOR INJECTION AND USE OF ONE OR MORE VISCOSITY REDUCING IONIC LIQUIDS - Google Patents

Abstract

LIQUID PROTEIN FORMULATIONS CONTAINING IONIC LIQUIDS. The present invention relates to concentrated, low viscosity, low protein volume pharmaceutical liquid formulations. Such formulations can be quickly and conveniently administered by subcutaneous or intramuscular injection, rather than by the longer intravenous infusion. These formulations include low molecular weight and/or high molecular weight proteins, such as mAbs, and viscosity-reducing ionic liquids.

Description

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

[001] This Patent Application claims the priority and benefit of US Provisional Patent Application No. 62/030,521, filed on July 29, 2014, entitled "Low viscosity protein formulations containing hydrophobic salts;" US Provisional Patent Application No. 62/026,497, filed July 18, 2014, entitled "Low Viscosity Protein Formulations Containing GRAS Viscosity Reducing Agents;" US Provisional Patent Application No. 62/008,050, filed on June 5, 2014, entitled "Low viscosity protein formulations containing ionic liquids;" US Provisional Patent Application No. 61/988,005, filed on May 2, 2014, entitled "Low viscosity protein formulations containing organophosphates;" US Provisional Patent Application No. 61/946,436, filed on February 28, 2014, entitled "Concentrated, low-viscosity Infliximab Formulations;" US Provisional Patent Application No. 61/943,197, filed on February 21, 2014, entitled "Concentrated protein formulations, low viscosity, high molecular weight;" US Provisional Patent Application No. 61/940,227, filed on February 14, 2014, entitled "Concentrated, low-viscosity, high molecular weight protein formulations", and US Provisional Patent Application No. 61,876,621, filed on September 11, 2013, entitled "Concentrated, Low Viscosity, High Molecular Weight Protein Formulations", the descriptions of which are expressly incorporated by reference herein.

FIELD OF THE PRESENT INVENTION

[002] The present invention relates, in general, to low viscosity injectable pharmaceutical formulations of highly concentrated proteins and the methods of production and use thereof.

BACKGROUND OF THE PRESENT INVENTION

[003] Monoclonal antibodies (mAbs) are important protein-based therapeutic agents for the treatment of various human diseases, such as cancer, infectious diseases, inflammation and autoimmune diseases. More than 20 mAb products have been approved by the US Food and Drug Administration (FDA), and approximately 20% of all biopharmaceutical products being evaluated in clinical trials are mAbs (Daugherty et al, Adv Drug Deliv Rev. 58 : 686 to 706, 2006; and Buss et al, Curr. Opinions in Pharmacol 12: 615 to 622, 2012).

[004] mAb-based therapies are generally administered repeatedly over a prolonged period of time and require several mg/kg dosage. Antibody solutions or suspensions can be administered parenterally, such as intravenous (iv) infusions, and subcutaneous (SC) or intramuscular (IM) injections. The SC or IM routes reduce the cost of treatment, increase patient compliance, and improve convenience for patients and healthcare providers during administration compared to the IV route. To be effective and pharmaceutically acceptable, parenteral formulations must preferably be sterile, stable, injectable (e.g., via a syringe), and non-irritating at the injection site, in accordance with FDA guidelines. Because of the small amounts required for administration of subcutaneous (generally less than about 2 mL) and intramuscular (generally less than about 5 mL) injections, these routes of therapeutic protein administration require high doses of concentrated protein solutions. These high concentrations typically result in very viscous formulations that are difficult to administer by injection, cause pain at the injection site, are often inaccurate, 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 achieve, in particular formulations with high concentrations of high molecular weight proteins, such as mAbs. All therapeutic proteins, to some extent, are subject to chemical and physical instability, such as aggregation, denaturation, cross-linking, deamidation, isomerization, oxidation, and shearing (Wang et al, J. Pharm Sci 96:1 to 26 , 2007) . Therefore, optimal formulation development is of paramount importance for the development of commercially viable protein drugs.

[006] High protein concentrations represent challenges relating to the physical and chemical stability of the protein, as well as difficulty in manufacturing, storing and delivering the protein formulation. One problem is the tendency of proteins to aggregate and form particles during processing and/or storage, which makes manipulations during further processing and/or delivery difficult. Concentration-dependent degradation and/or aggregation are the main challenges in developing protein formulations at higher concentrations. In addition to the possibility of non-native protein aggregation and particle formation, reversible self-association in aqueous solution can occur, which contributes to, among other things, increased viscosity that complicates delivery through injection. (See, for example, Steven J. Shire et al, J. Pharm Sci. 93:1390 to 1402, 2004.) Increased viscosity is one of the main challenges encountered in concentrated protein compositions that affect both production and production processes. the ability to readily deliver such compositions by conventional means. (See, for example, J. Jezek et al, Advanced Drug Delivery Reviews 63:1107 to 1117, 2011.)

[007] Highly viscous liquid formulations are difficult to manufacture, to be extracted with a syringe, and to be injected subcutaneously or intramuscularly. The use of force in manipulating viscous formulations can lead to excessive foam formation, which can further 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 available commercial mAb products administered via SC or IM injection are usually 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 ) sorbitan monolaurate), to prevent aggregation and improve stability. Formulated antibody concentrations reported as described above are typically up to about 100 mg/mL (Wang et al, J. Pharm Sci 96:1 to 26, 2007).

[009] US Patent No. 7,758,860 describes reducing viscosity in low molecular weight protein formulations using a buffer and an inorganic viscosity reducing salt, such as calcium chloride or magnesium chloride. These same salts, however, showed little effect on the viscosity of an antibody from a high molecular weight formulation (IMA-638). As described in US Patent No. 7,666,413, the viscosity of aqueous formulations of high molecular weight proteins has been reduced by adding salts such as arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride. , calcium chloride, zinc chloride, or sodium acetate in a concentration greater than about 100 mM or, as described in US Patent No. 7,740,842, through the 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] US Patent No. 7666413 describes reduced viscosity formulations that contain specific salts and a reconstituted anti-IgE mAb, but with a maximum antibody concentration only up to about 140 mg/mL. US Patent No. 7740842 describes E25 anti-IgE mAb formulations containing ethyl acid/acetic acid buffer with antibody concentrations up to 257 mg/mL. The addition of salts such as NaCl, CaCl2, or MgCl2 has been demonstrated to decrease dynamic viscosity under high shear conditions; However, the low shear of the salts produced an undesirable and dramatic increase in dynamic viscosity. Additionally, inorganic salts such as NaCl can decrease solution viscosity and/or decrease aggregation (EP 1981824).

[0011] Non-aqueous antibody or protein formulations have also been described. Document WO2006/071693 describes a non-aqueous suspension of up to 100 mg/ml mAb in a formulation having a viscosity promoter (polyvinylpyrrolidone, PVP) and a solvent (benzyl benzoate or PEG 400). WO2004/089335 describes formulations of 100 mg/mL non-aqueous lysozyme suspension containing PVP, glycofurol, benzyl benzoate, benzyl alcohol, or PEG 400. US2008/0226689A1 describes 100 mg/mL human growth hormone ( hGH) single-phase, three-component carrier (polymer, surface-active agent, and a solvent), non-aqueous, viscous formulations. US Patent No. 6,730,328 describes low-reactivity, non-aqueous, hydrophobic, non-polar carriers, such as perfluorodecalin, for protein formulations. These formulations are not ideal and have high viscosities that impair processing, production and injection; lead to the presence of multiple vehicle components in the formulations; and present potential regulatory challenges associated with the use of polymers which have not yet been approved by the FDA.

[0012] Alternative non-aqueous protein or antibody formulations have been described making use of organic solvents, such as benzyl benzoate (Miller et al, Langmuir 26: 1067 to 1074, 2010), benzyl acetate, ethanol, or methyl ethyl ketone (Srinivasan et al, Pharm Res 30:1749 to 1757, 2013). In both cases, viscosities of less than 50 centipoise (CP) were obtained by formulating at protein concentrations of at least about 200 mg/mL. US Patent No. 6252055 describes mAb formulations with concentrations ranging from 100 mg/ml to 257 mg/mL. Formulations with concentrations greater than about 189 mg/mL have demonstrated dramatically increased viscosity, low recovery rates, and difficulty in processing. US Patent Application Publication No. 2012/0230982 describes antibody formulations with concentrations of 100 mg/mL to 200 mg/mL. None of these formulations are sufficiently low viscosity for ease of injection.

[0013] Du and Klibanov (Biotechnology and Bioengineering 108:632 to 636, 2011) described 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 to 3109, 2012) described the low viscosity aqueous solutions of the four models of mAbs achieved using hydrophobic salts. The mAB formulation employed by Guo had an initial viscosity, before the addition of salts, of no greater than 73 cP. The viscosities of several pharmaceutically important mAbs, on the other hand, can be greater than 1,000 cP at therapeutically relevant concentrations.

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

[0015] The references cited above show that although 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 achieved. Notably, many of the above reports employ agents for which the safety and toxicity profiles have not been fully established. These formulations therefore face a higher regulatory burden prior to approval than formulations containing compounds known to be safe. Indeed, even if a compound is shown to substantially reduce viscosity, ultimately the compound may be 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 in order to provide therapeutically effective amounts of protein due to problems with high viscosity and other properties of concentrated solutions of large proteins. For example, to provide 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 necessary.

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

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

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

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

[0021] It is an additional 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.

[0022] It is a further object of the present invention to provide methods for processing viscosity reduction, high concentration biological products with concentration and filtration techniques known to those skilled in the art.

SUMMARY OF THE PRESENT INVENTION

[0023] Concentrated, low viscosity, low protein volume 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 ionic liquids.

[0024] The protein concentration is between about 10 mg/mL and about 5,000 mg/mL, more preferably from about 100 mg/mL to about 2000 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. The formulations containing the viscosity-reducing proteins and ionic liquids are stable when stored at a temperature of 4°C for a period of at least one month, preferably at least two months, and even more preferably at least least three months. The viscosity of the formulation is less than about 75 cP, preferably less than 50 cP, and more preferably less than 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 ionic liquids are typically measured at shear rates of about 0.6 s-1 to about 450 s-1, and preferably from about 2 s-1 to about 400 s-1, when measured using a cone and plate viscometer. Formulations containing viscosity-reducing proteins and ionic liquids are typically measured at shear rates of about 3 s-1 to about 55,000 s-1, and preferably from about 20 s-1 to about 2,000 s- 1, when measured using a microfluidic viscometer.

[0025] The viscosity of the protein formulation is reduced by the presence of one or more viscosity-reducing ionic liquid(s). Unless otherwise specifically indicated, the term "ionic liquid" includes both individual compounds and mixtures of more than one ionic liquid. It is preferred that the viscosity-reducing ionic liquid(s) be present in the formulation at a concentration of less than about 1.0 M, preferably less than about 0.50 M, more than preferably less than about 0.30 M, and more preferably less than about 0.15 M. The formulations may have a viscosity that is at least about 30% less, preferably at least about 50% less, more preferably at least about 75% less, than the viscosity of the corresponding formulation. , under the same conditions, except for replacing the viscosity-reducing ionic liquid with an appropriate buffer or salt of approximately the same concentration. In some embodiments, a low viscosity formulation is provided when the viscosity of the corresponding formulation without the viscosity reducing ionic liquid is greater than about 200 cP, greater than about 500 cP, or even greater than about 1000 cP. . In a preferred embodiment, the shear rate of the formulation is at least about 0.5 s-1 when measured using a cone and plate viscometer or at least about 1.0 s-1 when measured using a cone and plate viscometer. a microfluidic viscometer.

[0026] Pharmaceutically acceptable liquid formulations contain one or more IC-ion liquids in an amount effective to significantly reduce the viscosity of the protein, for example, mAb formulation. Representative ionic liquids include 4-(3-butyl-1-imidazolium)-1-butane sulfonate (BIM), 1-butyl-3-methylimidazolium methanesulfonate (BMI MES), 4-ethyl-4-methylmorpholinium methylcarbonate, (EMMC) and 1-butyl-1-methylpyrrolidinium chloride (BMP chloride), in concentrations preferably between about 0.10 and about 0.50 M, which is equivalent to about 20150 mg/mL. The resulting formulations can exhibit Newtonian flow characteristics.

[0027] 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 1000 kDa, preferably between about 120 kDa and about 500 kDa, and even more preferably between about 120 kDa and about 250 kDa. The high molecular weight protein may be an antibody, such as a mAb, or PEGylated or otherwise derivatized in the same way. Preferred mAbs include natalizumab (Tysabri®), cetuximAb (Erbitux®), bevacizumab (Avastin), trastuzumab (HERCEPTIN®), inflix-IMAB (REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatu-mumab (ARZERRA®) and similar biological products thereof. The high molecular weight protein, optionally PEGylated, may be an enzyme. Other proteins and protein mixtures can also be formulated to reduce their viscosity.

[0028] In some embodiments, the protein and viscosity-reducing ionic liquid(s) are provided in a lyophilized dosage unit, sized for reconstitution with a sterile aqueous pharmaceutically acceptable vehicle, to obtain the concentrated low viscosity liquid formulations. The presence of the viscosity-reducing ionic liquid(s) facilitates and/or accelerates the reconstitution of the lyophilized dosage unit compared to a lyophilized dosage unit containing an ionic viscosity-reducing liquid.

[0029] Methods are provided in the present invention for preparing concentrates, low viscosity liquid formulations of high molecular weight proteins, such as mAbs, as well as methods for storing low viscosity, high concentration protein formulations. , and for its administration to patients. In another embodiment, the viscosity-reducing ionic liquid is added to facilitate processing (e.g., pumping, concentration, and/or filtration) by reducing the viscosity of protein solutions.

DETAILED DESCRIPTION OF THE PRESENT INVENTION I. DEFINITIONS

[0030] The term "protein", as used in the present invention, generally refers to a polymer of amino acids linked to each other through peptide bonds to form a polypeptide for which the chain length is sufficient to produce at least one detectable tertiary structure. Proteins with a molecular weight (expressed in kDa, where "Da" stands for "Daltons" and 1 kDa = 1,000 Da) greater than about 100 kDa may be designated "high molecular weight proteins", while proteins with a molecular weight of less than about 100 kDa may be referred to as "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 weights can be determined using standard methods known to those skilled in the art, including, but not limited to, mass spectrometry (e.g., ESI, MALDI) or calculation from sequences of known amino acids and glycosylation. Proteins can be naturally occurring or non-naturally occurring, synthetic or semi-synthetic.

[0031] The "essentially pure protein(s)" and the "substantially pure protein(s)" are used in the present invention interchangeably and refer to a composition comprising at least about 90% by weight pure protein, preferably at least about 95% pure protein by weight. "Essentially homogeneous" and "substantially homogeneous" are used interchangeably herein and refer to a composition in which at least about 90% by weight of the protein present is a combination of the monomer and di- and oligomer reversible associates (not irreversible aggregates), preferably at least about 95%.

[0032] The term "antibody", as used in the present invention, generally encompasses mAbs (including full-length antibodies that have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies and single-chain antibody molecules, 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 their fusion.

[0033] The term "monoclonal antibody" or "the mAb," as used in the present invention, generally refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that constitute the population are identical, except for 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 can be produced by recombinant DNA methods (see, e.g., US Patent No. 4,816,567), or isolated from phage antibody libraries using the techniques described in Clackson et al. al. (Nature 352: 624 to 628, 1991) and Marks et al. (J. Mol Biol 222: 581 to 597, 1991), for example. As used herein, "mAb" specifically include derivatized antibodies, antibody-drug conjugates, and "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is (are) identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (US Patent No. 4816567; Morrison et al, Proc Natl Acad Sci US. 81: 6851 to 6855, 1984).

[0034] 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, US Patent No. 5,641,870; Zapata et al, Protein Eng. 8: 1057 to 1062, 1995); single-chain antibody molecules; Multivalent single domain antibodies; and multispecific antibodies formed from antibody fragments.

[0035] "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 antigen-binding antibodies) sequences primarily from humans, which contain minimal sequences derived from non-human immunoglobulin. (See, for example, Jones et al, Nature 321:522 to 525, 1986; Reichmann et al, Nature 332:323 to 329, 1988; and Presta, Curr Op Struct Biol 2:593 to 596, 1992.)

[0036] "Rheology" refers to the study of deformation and flow of matter.

[0037] "Viscosity" refers to the resistance of a substance (usually a liquid) to flow.

[0038] Viscosity is related to the concept of shear force; can be understood as the effect of different layers of fluids exerting shear force on each other, or on other surfaces, which move against each other. 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 speed at which motion 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 differing viscosity are placed in identical capillary viscometers and allowed to flow by gravity, the more viscous liquid takes longer than the less viscous fluid to flow through the capillary. If, for example, one fluid takes 200 seconds to complete its flow and another fluid takes 400 seconds, the second fluid is called twice as viscous as the first on a kinematic viscosity scale. The dimension of kinematic viscosity is length2/hour. 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 the kinematic viscosity and density of the fluid. Absolute viscosity expressed in centipoise units (cP). The SI unit of absolute viscosity is the milliPascal-second (mPa-s), where 1 cP = 1 mPa-s. Viscosity can be measured using, for example, a viscometer at a given shear rate or various shear rates. A "zero shear extrapolated" viscosity can be determined by creating a line of best fit of the four highest shear points in a terrain of absolute viscosity as a function of shear rate and extrapolating linear viscosity back to zero shear . Alternatively, for a Newtonian fluid, the viscosity can be determined by averaging the viscosity values at various shear rates. Viscosity can also 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 is equal to shear stress over shear rate. Viscosities measured with microfluidic viscometers can, in some embodiments, be directly extrapolated compared to zero shear viscosities, for example, those extrapolated from viscosities measured at various shear rates using a cone and plate viscometer.

[0039] "Shear rate" refers to the rate of change in the speed at which a layer of fluid passes through 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. Thus, the units of shear rate are reciprocal seconds or, in general, reciprocal time. For a microfluidic viscometer, change in pressure and flow is related to the shear rate. "Shear rate" refers to 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 measured using a cone and plate viscometer and an appropriately chosen spindle through from a person who is skilled in the art of accurately measuring viscosities in the viscosity range of the sample of interest (i.e., a 20 cP sample is most accurately measured on a CPE40 axis attached to a DV2T viscometer (Brookfield)); greater than about 20 s-1 to about 3,000 s-1 when measured using a microfluidic viscometer.

[0040] For classical "Newtonian fluids", as used in the present invention, in general, viscosity is essentially independent of shear rate. By "non-Newtonian fluids", however, whether the viscosity decreases or increases with increasing shear rate, for example, the fluids are "shear thinning" or "shear thickening", respectively. In the case of concentrated (i.e., high concentration) protein solutions, this may manifest pseudoplasticity-like behavior, i.e., a decrease in viscosity with shear rate.

[0041] The term "chemical stability", as used in the present invention, generally refers to the ability of the protein components of a formulation to resist degradation by chemical pathways, 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.

[0042] The term "physical stability", as used in the present invention, generally 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 a number of ways, including by 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 achievable, but about 1% is more typical.

[0043] The term "stable formulation", as used in the present invention, generally means that a formulation is stable both chemically and physically. A stable formulation may be one in which more than about 95% of the bioactive protein molecules retain the bioactivity of a formulation after 24 months of storage at 4°C, or solution conditions equivalent to an elevated temperature, such as one month 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 to 301, Vincent Lee, Ed., Marcel Dekker, Inc., New York, New York (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 screening, for example, the formulation can be maintained at 40°C for 2 weeks to a month, at which time residual biological activity is measured and compared to the initial condition to assess stability. When the formulation is to be stored at 2°C to -8°C, in general, the formulation should be stable at 30°C or 40°C for at least one month and/or stable at 2°C to -8° C for at least 2 years. When the formulation is to be stored at room temperature, about 25°C, in general, 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 freeze-drying and storage can be used as an indicator of protein stability. In some embodiments, stability is determined 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 normal biological activity or binding assays well within the capabilities of a person skilled in the art.

[0044] The term "particle size" of the protein, as used in the present invention, generally means the average diameter of the predominant population of bioactive molecule particles, or particle size distributions thereof, in a formulation as determined through the use of well-known particle sizing instruments, for example, dynamic light scattering, SEC (size exclusion chromatography), or other methods known to a person skilled in the art.

[0045] The term "concentrate" or "high concentration", as used in the present invention, generally describes liquid formulations having a final protein concentration greater than about 10 mg/mL, preferably greater than about of 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.

[0046] A "reconstituted formulation", as used in the present invention, generally refers to a formulation that has been prepared by dissolving a dry, lyophilized, spray-dried powder or solvent-precipitated protein, in a diluent, such that the protein is dissolved or dispersed in the aqueous solution for administration.

[0047] A "lyoprotectant" is a substance that, when combined with a protein, significantly reduces the chemical and/or physical instability of the protein after 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, dextrin, modified starch, and carboxymethyl cellulose. Preferred sugar alcohols are compounds obtained through the reduction of mono- and disaccharides, such as lactose, trehalose, maltose, latulose and maltulose. Other examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. The lyoprotectant is generally added to the pre-lyophilized formulation in a "lyoprotectant amount". This means that, after lyophilization of proteins, in the presence of the lyoprotectant amount of lyoprotectant, the protein essentially maintains its physical and chemical stability and integrity.

[0048] A "diluent" or "vehicle", as used in the present invention, generally speaking, 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 preparing a liquid formulation, such as an aqueous solution of the formulation reconstituted after lyophilization. Examples of diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), sterile saline, dextrose solution, or Ringer's solution, and combinations thereof.

[0049] A "preservative" is a compound that can be added to the formulations in the present invention to reduce contamination and/or action by bacteria, fungi, or other infectious agent. The addition of a preservative can, for example, facilitate the production of a multi-use (multiple-dose) formulation. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long chain), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohols, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0050] A "thickening agent", as used in the present invention, generally speaking, is a compound that increases the mass of a freeze-dried mixture and contributes to the physical structure of the freeze-dried cake (e.g., facilitates the production of a cake essentially uniform freeze-dried product, which maintains an open pore structure). Exemplary thickening agents include mannitol, glycine, lactose, modified starch, poly(ethylene glycol), and sorbitol.

[0051] A "therapeutically effective amount" is the lowest concentration required to effect a measurable improvement or prevention of any symptom or a particular condition or disorder, to effect a measurable increase in life expectancy, or to improve the overall quality of life. of patients' lives. The therapeutically effective amount is dependent on the specific biologically active molecule and the specific condition or disease being treated. Therapeutically effective amounts of many proteins, such as the mAbs described in the present invention, are well known in the art. The therapeutically effective amounts of proteins not yet established or for the treatment of specific disorders with known proteins, such as mAbs, to be applied clinically to treat additional disorders can be determined using standard techniques that are well within a person's knowledge. who is skilled in the art, such as a doctor.

[0052] The term "injectability" or "syringability", as used in the present invention, generally refers to the injection performance of a pharmaceutical formulation through a syringe equipped with an 18 to 32 gauge needle, optionally, of thin walls. Injectability depends on factors such as the pressure or force required for injection, flow uniformity, aspiration qualities, and the absence of obstruction. The injectability of liquid pharmaceutical formulations can be assessed by comparing the injection force of a reduced viscosity formulation 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 with the same protein concentration otherwise under the same conditions, except for replacing the viscosity reducing agent with an appropriate buffer of about the same concentration. Alternatively, the injectability of liquid pharmaceutical formulations can be assessed 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 pressed with the same force.

[0053] The term "injection force", as used in the present invention, generally refers to the force necessary to push a given liquid formulation through a given syringe equipped with a given needle gauge at a given injection speed. injection. Injection force is generally expressed in Newtons. For example, injection force can be measured as the force required to push a liquid formulation through a 1 ml plastic syringe with a 0.63 cm (0.25 inch) internal diameter equipped with a 27 gauge needle. 1.27 cm (0.50 inch) at an injection speed of 250 millimeters/minute. Test equipment can be used to measure injection force. When measured under the same conditions, a formulation with lower viscosity will generally require a lower overall injection force.

[0054] The "viscosity gradient", as used in the present invention, refers to the rate of change in the viscosity of a protein solution with increasing protein concentration. The viscosity gradient can be approximated from a plot of viscosity versus 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 from the slope of a tangent line to the viscosity plot as a function of protein concentration. The viscosity gradient can be approximated from a linear approximation to the viscosity plot as a function of any protein concentration or over a narrow window of protein concentrations. In some embodiments, a formulation is said to have a reduced viscosity gradient if, when the viscosity as a function of protein concentration is approximated as an exponential function, the exponent of the exponential function is smaller than the exponent obtained for the other form. Same formulation without the viscosity decreasing agent. Similarly, a formulation can 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 versus protein concentration by other methods known to skilled formulation researchers.

[0055] The term "reduced viscosity formulation," as used in the present invention, generally refers to a liquid formulation having a high concentration of a high molecular weight protein, such as a mAb, or a protein low molecular weight, which is modified by the presence of one or more viscosity-lowering additives, as compared to a corresponding formulation that does not contain the viscosity-lowering additive(s).

[0056] The term "osmolarity," as used in the present invention, generally refers to the total number of components dissolved per liter. Osmolarity is similar to molarity but includes the total number of moles of species dissolved in solution. An osmolarity of 1 Osm/L means that there are one mole of dissolved components per L of solution. Some solutes, such as ionic solutes that dissociate in solution, will contribute more than one mole of dissolved components per mole of solute in solution. For example, NaCl dissociates into Na + and Cl @ in solution and thus provides 2 moles of dissolved components per 1 mole of NaCl dissolved in solution. Physiological osmolarity is typically in the range of about 280 mOsm/L to about 310 mOsm/L.

[0057] The term "tonicity", as used in the present invention, generally refers to the osmotic pressure gradient that results from the separation of two solutions by means of a semipermeable membrane. In particular, tonicity is used to describe the osmotic pressure created across a cell membrane when a cell is exposed to an external solution. Solutes that can cross the cell membrane do not contribute to the ultimate osmotic pressure gradient. Only dissolved species that do not cross the cell membrane will contribute to differences in osmotic pressure and thus tonicity.

[0058] The term "hypertonic," as used in the present invention, generally 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 out of the cell in order to balance the concentration of solutes.

[0059] The term "hypotonic," as used in the present invention, generally refers to a solution with a lower concentration of solutes than is present inside the cell. When a cell is immersed in a hypotonic solution, water flows into the cell in order to balance the concentration of solutes.

[0060] The term "isotonic", as used in the present invention, generally 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 of about 250 mOsm/kg to 350 mOsm/kg.

[0061] The term "liquid formulation", as used in the present invention, is a protein that is either provided in a pharmaceutically acceptable diluent or one that is reconstituted in a pharmaceutically acceptable diluent prior to administration to the patient.

[0062] The terms "brand" and "reference", when used to refer to a protein or biologic, are used herein interchangeably to mean the single biologic product under section 351(a) of the US Public Health Act Service (42 USC § 262).

[0063] The term "similar biological product", as used in the present invention, is generally used interchangeably with "a generic equivalent" or "follow-on". For example, a "mAb similar biologic" refers to a later version of an innovator's mAb typically made through a different company. "Similar biologics" when used in reference to a protein brand or biologic brand may refer to a biological product evaluated against the protein brand or biologic brand and under section 351(k) of the Health Service Act US Public (42 USC § 262). A similar biological product mAb may be one that satisfies one or more lineages of the guidelines adopted on 30 May 2012 by the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency and published by the European Union as "Guidance on medicinal products biological products similar products containing monoclonal antibodies - non-clinical and clinical issues" (Reference Document EMA/CHMP/BMWP/403543/2010).

[0064] Similar biological products can be produced using microbial cells (prokaryotes, eukaryotes), cell lines of human or animal origin (e.g., mammals, birds, insects), or tissues derived from animals or plants. The expression construct of a proposed similar biological product will generally encode the same primary amino acid sequence as its reference product. Minor modifications, such as truncations at the N or C terminals that will not have an effect on safety, purity, or potency, may be present.

[0065] A similar biological product mAb is similar to the reference mAb physiochemically or biologically both in terms of safety and in terms of efficacy. The similar biological product mAb can be evaluated against a reference mAb, using one or more, in in vitro studies, including assays that detail binding to target antigen(s) sequences; binding to Fc gamma receptor isoforms (Fc RI, FciRII and FciRIII), FcRn, and complementary (C1q); Fab-associated functions (e.g., neutralization of a soluble ligand, receptor activation or blockade); or Fc-associated functions (antibody-dependent cell-mediated cytotoxicity, e.g., complement-dependent cytotoxicity, complement activation). In vitro comparisons can be combined with in vivo data demonstrating similarity of pharmacokinetics, pharmacodynamics and/or safety. Clinical evaluations of a similar biologic mAb versus 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 parallel group randomized comparative clinical trials). The quality comparison between a similar biological product mAb and a reference mAb can be assessed using established procedures, including those described in the "Directive on medicinal products similar biological products containing biotechnology-derived proteins as active substance: quality issues" (EMEA/ CHMP/BWP/49348/2005), and the "Guidance on the development, production, characterization and specifications for monoclonal antibodies and related substances" (EMEA/CHMP/BWP/157653/2007).

[0066] Differences between a similar biological product mAb and a reference mAb may include post-translational modification, for example linking the mAb to other biochemical groups, such as a phosphate, various lipids and carbohydrates; through proteolytic cleavage following translation; by altering the chemical nature of an amino acid (e.g., formylation); or through various other mechanisms. Other post-translational modifications may be a consequence of manufacturing process operations - for example, glycosylation may occur with exposure of the product to reducing sugars. In other cases, storage conditions may be permissive for certain degradation pathways, such as oxidation, deamidation, or aggregation. As all these product-related variants can be included in a similar mAb biologic.

[0067] As used in the present invention, the term "pharmaceutically acceptable salts" refers to salts prepared from non-toxic pharmaceutically acceptable acids and bases, including inorganic acids and bases and organic acids and bases. 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, p-toluenesulfonic and the like. Suitable positively charged counterions include sodium, potassium, lithium, calcium and magnesium.

[0068] As used in the present invention, the term "ionic liquid" refers to a crystalline or amorphous salt, zwitterion, or a mixture thereof that is a liquid at or temperatures close to where most conventional salts are solids: at least 200°C, preferably less than 100°C or more preferably less than 80°C. Some ionic liquids have melting temperatures close to room temperature, for example between 10°C and 40°C, or between 15°C and 35°C. The term "zwitterionic" is used in the present invention to describe an overall neutral charge molecule that carries positive and negative charges on different chemical groups in the molecule. Examples of ionic liquids are described in Riduan et al, Chem Soe Rev., 42: 9055 to 9070, 2013; et al Rantwijk., Chem Rev., 107:2757 to 2785, 2007; Earle et al, Pure Appl Chem, 72 (7): 1391 to 1398, 2000; and Sheldon et al, Green Chem., 4:147 to 151, 2002.

[0069] As used in the present invention, the term "organophosphates" refers to a compound containing one or more phosphorylated groups, at least one of which is covalently linked to an organic group through a phosphoester bond.

[0070] As used in the present invention, 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 visible-to-infrared portion of the electromagnetic spectrum, while possibly transmitting or reflecting other wavelengths of light.

[0071] As used in the present invention, the term "Chalcogen" refers to the group of 16 elements, including oxygen, sulfur and selenium, in any oxidation state. For example, unless otherwise specified, the term "Chalcogen" also includes SO2.

[0072] As used in the present invention, the term "alkyl group" refers to straight-chain and branched-chain 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".

[0073] As used in the present invention, the term "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.

[0074] As used in the present invention, the term "heteroaryl" refers to aromatic ring systems, including fused ring systems, in which at least one of the atoms forming the ring is a heteroatom.

[0075] As used in the present invention, the term "heterocycle" refers to ring systems that, including fused ring systems, that are non-aromatic, in which at least one of the atoms forming the ring is a heteroatom .

[0076] As used in the present invention, a "heteroatom" is any non-carbon or non-hydrogen atom. Preferred heteroatoms include oxygen, sulfur, and nitrogen. Examples of heteroaryl and heterocyclyl rings include: benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, cromanyl, cromenyl, cynolin yl, 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, pyran ila , pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl , pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H- quinolizinyl, quinoxalinyl, qui nuclidinyl, tetrahydrofuranyl, tetra - hydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5- thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4- thiadiazolyl , thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thieno-oxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

II. FORMULATIONS

[0077] Biocompatible, low-viscosity protein solutions, such as mAb, can be used to deliver therapeutically effective amounts of proteins in volumes useful for subcutaneous (SC) administration and intramuscular (IM) injections, typically less than or about 2 ml SC and less than or about 5 ml IM, more preferably less than or about 1 ml SC and less than or about 3 ml IM. Proteins can generally be of any molecular weight, although in some preferred embodiments they are high molecular weight proteins. In other embodiments, the proteins are low molecular weight proteins.

[0078] 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, even more preferably greater than about 225 mg/ml, even more preferably greater than about 250 mg/ml, and most preferably greater than about 300 mg/ml. mL. In the absence of a viscosity-reducing ionic liquid, the viscosity of a protein formulation increases exponentially as the concentration is increased. Such protein formulations, in the absence of a viscosity reducing ionic liquid, 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 1000 cP when measured at 25°C. Such formulations are often unsuitable for SC or IM injection. The use of one or more ionic viscosity reducing liquids allows the preparation of formulations with 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 more preferably less than or about 10 cP, when measured at 25°C.

[0079] Although viscosity-reducing ionic liquids can be used to decrease 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.

[0080] For certain proteins, formulations that do not have an ionic liquid 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 ionic liquids 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 more preferably less than or about 10 cP when measured at 25°C.

[0081] In some embodiments, the aqueous protein formulations have a viscosity that is at least about 30% less than the analogous formulation without the ionic liquid(s), when measured under the same conditions. In other embodiments, the formulations have a viscosity that is 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, or even more than 90% less than the analogous formulation without the Viscosity reducing ionic liquid(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 0.75 mL.

[0082] The reduced viscosity formulations have improved injectability and require less injection force compared to the analogous formulation without the viscosity reducing ionic liquid (for example, in phosphate buffer), under the same conditions otherwise. In some embodiments, the injection force is reduced by more than about 20%, more than about 30%, more than about 40%, more than about 50%, or more than about two times compared to standard formulations without the viscosity-reducing ionic liquid(s) under otherwise the same injection conditions. In some embodiments, the formulations have "Newtonian flow characteristics", defined as having a viscosity that is substantially independent of shear rate. Protein formulations can be easily injected through needles of about 18 to 32 gauge. Preferred needle gauges for administering low viscosity formulations include 27, 29, and 31 gauge, optionally thin-walled.

[0083] The 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 pH and osmolarity suitable 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.

[0084] Low viscosity protein formulations can allow for greater flexibility in formulation development. Low viscosity formulations may exhibit changes in viscosity that are less dependent on protein concentration compared to the same formulation otherwise without the viscosity reducing ionic liquid. Low viscosity protein formulations may allow for increased concentrations and decreased protein dosing frequency. In some embodiments, the low viscosity protein formulations contain 2 or more, 3 or more, or four or more different proteins. For example, combinations of 2 or more mAbs can be provided in a single protein low viscosity formulation.

[0085] Since 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 ionic liquid, the frequency of Protein dosage 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-lowering agents. Proteins that currently require multiple administrations on the same day (either at the same time or at different times of the day) can be administered in fewer injections per day. In some cases, the frequency can be reduced to a single injection, once a day. By increasing the dosage administered by injection multiple times, the dosage frequency can be reduced, for example, from once every 2 weeks to once every 6 weeks.

[0086] 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 500 mOsm/L. In some embodiments, the formulations have an osmolarity of from about 200 mOsm/L to about 2000 mOsm/L or from about 300 mOsm/L to about 1000 mOsm/L. In some embodiments, the liquid formulations are essentially isotonic with human blood. Liquid formulations may in some cases be hypertonic.

[0087] Additives, including viscosity-reducing ionic liquid(s), may be included in any amount to achieve desired viscosity levels of the liquid formulation, while the values are non-toxic or otherwise harmful, and does not substantially interfere with the chemistry and/or physical stability of the formulation. The ionic viscosity reducing liquid(s), in some embodiments, may independently be present in 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 with two or In most ionic viscosity reducing liquids, the agents are preferably, but not necessarily, present in the same concentration.

[0088] The viscosity-reducing ionic liquid(s) allow for rapid reconstitution of a lyophilized dosage unit. The dosage unit is a lyophilized cake of proteins, the viscosity-reducing ionic liquid(s) and other excipients, which is added to water, saline or other pharmaceutically acceptable fluid. In the absence of viscosity-reducing ionic liquids, periods of 10 minutes or more are often required in order to completely dissolve the freeze-dried cake with a high protein concentration. When the freeze-dried cake contains one or more viscosity-reducing ionic liquid, the period of time required to completely dissolve the cake 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 cake that contains greater than or about 150, 200 or even 300 mg/mL of protein.

[0089] Low viscosity protein formulations allow greater flexibility in formulation development. Low viscosity formulations have a viscosity that changes less with increasing protein concentrations, compared to the same formulation otherwise without the ionic liquid(s). Low viscosity protein formulations exhibit a decreased viscosity gradient compared to the same formulation otherwise without the ionic liquid.

[0090] The viscosity gradient of the protein formulation can be 2 times less, 3 times less, or even more than three times less than the viscosity gradient of the protein formulation otherwise even without the liquid(s). s) ionic viscosity reduction. The gradient viscosity 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 with a protein concentration being Tween 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.

A. Proteins

[0091] 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.

[0092] In certain embodiments, the protein may be a PEGylated protein. The term "PEGylated protein", as used in the present invention, refers to a protein having one or more poly(ethylene glycol) or other stealth polymer groups covalently linked, optionally via a chemical linker, which may be different from that of one or more polymer groups. PEGylated proteins are characterized by reduced typically renal filtration, decreasing absorption through the reticuloendothelial system, and decreasing enzymatic degradation leading to, for example, prolonged half-lives and enhanced bioavailability. Polymer descriptions 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(vinilidone); poly(N-(2-hydroxypropyl) methacrylamide); and copolymers and mixtures thereof. In preferred embodiments, the polymer disc in a PEGylated protein is poly(ethylene glycol) or a copolymer thereof. PEGylated proteins can be randomly PEGylated, i.e., by having one or more stealth polymers covalently attached to the non-specific site(s) of the protein, or can be PEGylated in a site-specific manner by covalently attaching the stealth polymer to the( s) specific site(s) on the protein. Site-specific PEGylation can be carried out, for example, using stealth activated polymers having one or more reactive functional groups. Examples are described, for example, in Hoffman et al, Progress in Polymer Science, 32: 922 to 932, 2007.

[0093] In the preferred embodiment, the protein is high molecular weight and an antibody, more preferably a mAb, and has a high viscosity in buffered aqueous solution when concentrated sufficiently to inject a therapeutically effective amount of a volume no greater than 1 .0 to 2.0 mL SC and 3.0 to 5.0 mL for IM administration. High molecular weight proteins may include those described in Scolnik, mAbs 1: 179 to 184, 2009; Beck, mAbs 3: 107 to 110, 2011; Baumann, Curr. Drug Meth. 7: 15 to 21, 2006; or Federici, Biologicals 41: 131 to 147, 2013. The proteins for use in the formulations in the present invention described are preferably essentially pure and essentially homogeneous (that is, substantially free of contaminating proteins and/or irreversible aggregates thereof).

[0094] Preferred mAbs in the present invention include natalizumab (Tysabri®), cetuximab (ERBI-TUX®), bevacizumab (Avastin), trastuzumab (HERCEPTIN®), infliximab (REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX® ), ofatumumab (ARZERRA®), and similar biological products 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 the products similar biological substances.

[0095] Examples of molecular targets for the antibodies described in the present invention 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, Mo1, p150.95, VLA-4, ICAM-1, VCAM and αv/β3 integrin, including either the α or β subunits thereof (e.g., anti-CD11a, anti - CD18 or anti-CD11b); growth factors such as VEGF; IgE; blood group antigens; flk2/FLT3 receiver; obesity receptor (OB); protein C; PCSK9; etc.

Therapeutic antibodies currently on the market

[0096] Many therapeutic proteins currently on the market, especially antibodies as defined in the present invention, are administered via IV infusions due to high dosage requirements. Formulations may include one of the therapeutic antibodies currently on the market or a similar biological product thereof. Some protein therapeutics currently on the market are not high molecular weight, but are still administered via intravenous infusion, as high doses are necessary for therapeutic efficacy. In some embodiments, liquid formulations are provided of these low molecular weight proteins, as defined in the present invention, in concentrations to provide therapeutically effective amounts for SC or IM injections.

[0097] Therapeutic antibodies currently on the market include belimumab (BEN-LYSTA®), golimumab (SIMPONI ARIA®), abciximab (ReoPro®), the combination of tositumomab and tositumomab iodine-131, marketed as BEXXAR®, alemtuzumab (CAMPATH® ), palivizumab (SYNAGIS®), basiliximab (SIMULECT®), delay-trastuzumab emtansine (KADCILA®), pertuzumab (PERJETA®), capromab Pende-tide (PROSTASCINT kit®), caclizumab (ZENAPAX®), ibritumomab tiuxetan (ZEVALIN® ), eculizumab (SOLIRIS®), ipilimumab (YERVOY®), muromonab-CD3 (Orthoclone OKT3®), raxibacumab, nimotuzumab (THERACIM®), brentuximab vedotin (ADCETRIS®), adalimumab (Humira®), golimumab (SIMPONI®), palivi-zumab (SYNAGIS®), omalizumab (XOLAIR®) and ustekinumab (STELARA®).

[0098] Natalizumab, a humanized mAb against the cell adhesion molecule α4-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 Tysabri® is produced in murine myeloma Corp. cells. ( "Not her"). Each 15 ml dose contains 300 mg of natalizumab; 123 mg sodium chloride, USP; 17.0 mg sodium phosphate monobasic, monohydrate, USP; 7.24 mg Sodium phosphate dibasic heptahydrate, 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 shown to be effective in treating the symptoms of both multiple sclerosis and Crohn's disease, as well as for preventing relapse, vision loss, cognitive decline, and improving, significantly, the patient's quality of life.

[0099] As used in the present invention, the term "natalizumab" includes the mAb against the α4-integrin cell adhesion molecule known under the International Name "natalizumab" or an antigen-binding portion thereof. Natalizumab includes the antibodies described in US Patent No. 5,840,299, US Patent No. 6,033,665, US Patent No. 6,602,503, US Patent No. 5,168,062, US Patent No. 5,385,839, and in US 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.

[00100] 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 via 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 single-use, 50 mL vial of Erbitux® contains 100 mg of cetuximab at a concentration of 2 mg/mL and is formulated in a preservative-free solution containing 8.48 mg/mL sodium chloride, 1.88 mg/mL sodium phosphate diheptabasic, 0.42 mg/mL sodium phosphate monobasic monohydrate, and water for IV injection, USP.

[00101] Cetuximab is indicated for the treatment of patients with epidermal growth factor receptor (EGFR)-expressing KRAS wild-type metastatic colorectal cancer (mCRC), in combination with chemotherapy, and as a single agent, in patients who have failed oxaliplatin and irinotecan-based therapy 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 radiation therapy for recurrent and/or metastatic disease. locally advanced. 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.

[00102] As used in the present invention, the term "cetuximab" includes the mAb known under the National Trade Name "cetuximab" or an antigen-binding portion thereof. Cetuximab includes the antibodies described in US Patent No. 6,217,866. Cetuximab includes the active agent in products marketed under the trade name RE-BITUX® and similar biologics thereof. Similar Erbitux® biologics may include those currently being developed by Amgen, AlphaMab Co., Ltd. ("AlphaMab") and Actavis plc ("Actavis").

[00103] Bevacizumab, a humanized mAb that inhibits vascular endothelial growth factor A (VEGF-A), acts as an anti-angiogenic agent. It is marketed under the trade name Avastinfi by Genentech, Inc. ("Genentech") and F. Hoffmann-La Roche, LTD ("Roche"). He is licensed to treat several types of cancer, including colorectal, lung, breast (outside the US), glioblastoma (USA 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 treatment of colorectal cancer. -metastatic rectal. In 2006, the FDA approved Avastin® for first-line use in advanced non-squamous non-small cell lung cancer in combination with carboplatin/paclitaxel chemotherapy. AVAS-TIN® is given as an IV infusion every three weeks at a dose of either 15 mg/kg or 7.5 mg/kg. The higher dose is usually given with carboplatin-based chemotherapy, while the lower dose is given with cisplatin chemotherapy. In 2009, the FDA approved Avastin® for use in metastatic renal cell carcinoma (a type of kidney cancer). The FDA also granted accelerated approval of Avastin® for the treatment of recurrent glioblastoma multiforme in 2009. The initial growth treatment is still in phase III clinical trials.

[00104] The National Comprehensive Cancer Network ("NCCN") recommends bevacizumab as standard first-line treatment in combination with any platinum-based chemotherapy, followed by bevacizumab maintenance until disease progression. The NCCN updated its Oncology Clinical Practice Guidelines (NCCN Guidelines) for breast cancer in 2010 to affirm the recommendation for the use of bevacizumab (Avastin®, Genentech/Roche) in the treatment of metastatic breast cancer.

[00105] As used in the present invention, the term "Bevacizumab" includes the mAb that inhibits vascular endothelial growth factor A (VEGF-A), known under the International Trade Name/Common Name "Bevacizumab" or a binding moiety to their antigen. Bevacizumab is described in US Patent No. 6,054,297. Bevacizumab includes the active agent in products marketed under the trade name Avastin® and similar biologics thereof. Similar biologic products to Avastin® may include those currently being developed by Amgen, Actavis, AlphaMab, and Pfizer, Inc ("Pfizer"). Similar biologics to Avastin® may include the similar biologic known as BCD-021 produced by Biocad and currently in clinical trials in the US.

[00106] 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 pale yellowish, lyophilized powder without preservatives for IV administration. Each vial contains 440 mg HERCEPTIN® trastuzumab, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, 400 mg a, a-di-trehalose, 1.8 mg polysorbate 20, USP. Reconstitution with 20 mL of water produces a multi-dose solution containing 21 mg/mL trastuzumab. HERCEPTIN® is currently administered via IV infusion as frequently as weekly and at a dosage ranging from about 2 mg/kg to about 8 mg/kg.

[00107] Trastuzumab is mainly used to treat certain types of breast cancer. The HER2 gene is amplified in 20 to 30% of early-stage breast cancers, which causes epidermal growth factor (EGF) receptors to be overexpressed in the cell membrane. Trastuzumab is generally administered as maintenance therapy to patients with HER2-positive breast cancer, typically one year post-chemotherapy. Trastuzumab is currently administered via IV infusion as frequently as weekly and at a dosage ranging from about 2 mg/kg to about 8 mg/kg.

[00108] As used in the present invention, the term "trastuzumab" includes the mAb that interferes with the HER2/neu receptor known under the International Common Name "trastuzumab" or an antigen-binding portion thereof. Trastuzumab is described in US Patent No. 5,821,337. Trastuzumab includes the active agent in products marketed under HERCEPTIN® and similar biologics under its trade name. The term "trastuzumab" includes the active agent in similar biologics Herceptin® marketed under the trade names HERTRAZ® by Milan, Inc. ("Milan") and CANMAB® by Biocon, Ltd. ("Biocon"). Trastuzumab may include the active agent in similar Herceptin® biologics to be developed by Amgen and PlantForm Corporation, Canada.

[00109] Infliximab is a mAb against tumor necrosis factor alpha (TNF-α) used to treat autoimmune diseases. It is marketed under the trade name REMICADE® by Janssen Global Services, LLC ("Janssen") in the US, Mitsubishi Tanabe Pharma in Japan, Xian Janssen in China, and Merck & Co ("Merck"); in another place. Infliximab is a chimeric mouse/human monoclonal antibody with a high molecular weight of approximately 144 kDa. In some embodiments, the formulations contain a biologic similar to REMI-CADE®, such as REMSIMA™ or INFLECTRA™. Both REMSIMATM, developed by Celltrion, Inc. ("Celltrion") and INFLECTRATM, developed by Hospira Inc, United Kingdom, have been recommended for regulatory approval in Europe. Celltrion has submitted a REMSIMATM declaration to the FDA. Infliximab is currently administered via IV infusion at doses ranging from about 3 mg/kg to about 10 mg/kg.

[00110] Infliximab contains approximately 30% of the murine variable region of the amino acid sequence, which confers antigen binding specificity to human TNFa. The remaining 70% corresponds to a human IgG1 constant region heavy chain and a human kappa light chain constant region. Infliximab has high affinity for human TNFa, which is a cytokine with multiple biological actions including mediating inflammatory disease responses and modulating the immune system.

[00111] Infliximab is a general 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 the variable region sequences cloned from the anti-TNFα murine A2 hybridoma, and human antibody constant region sequences provided through the plasmid expression vectors. Generation of HY-bridoma murine anti-TNF α is accomplished by immunizing BALB/c mice with purified recombinant human TNFα. Heavy and light chain vector constructs are linearized and transfected into Sp2/0 cells via electroporation. Conventional purification steps may include chromatographic purification, viral inactivation, nanofiltration, and ultrafiltration/diafiltration.

[00112] As used in the present invention, the term "infliximab" includes the chimeric mouse/human monoclonal antibody known under the International Trade Name "INFLIX-IMAB" or an antigen-binding portion thereof. Infliximab neutralizes the biological activity of TNFa by binding with high affinity to the soluble and transmembrane forms of TNFa and inhibits the binding of TNFa with its receptors. Infliximab is described in US Patent No. 5,698,195. The term "Infliximab" includes the active agent in products marketed or proposed to be marketed under the trade names REMI-CADE® through various entities; REMSIMA™ by Celltrion and INFLECTRA™ by Hospira, Inc ("Hospira"). Infliximab is supplied as a sterile lyophilized bolus 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.

[00113] 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 metastases from solid tumors. Denosumab is in Phase II trials for the treatment of rheumatoid arthritis.

[00114] Panitumumab is a human mAb fully approved by the FDA for the treatment of metastatic cancer with EGFR expression with disease progression. Panitumumab is marketed under the trade name VECTIBIX® by Amgen. VECTIBIX® is packaged as a 20 mg/ml solution of panitumumab concentrate in 5 ml, 10 ml and 15 ml IV infusion vials. When prepared according to packaging 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. As used herein, the term "panitumumab" includes the anti-human epidermal growth factor receptor known by the International Trade Name "panitumumab." The term "panitumumab" includes the active agent in products marketed under the trade name VECTIBIX® by Amgen and similar biologics thereof. The term "panitumumab" includes the monoclonal antibodies described in US Patent No. 6,235,883. The term "panitumumab" includes the active agent in similar biologics VECTIBIX®, including the similar biologic product VECTIBIX® being developed by BioXpress, SA ("BioXpress").

[00115] Belimumab (BENLYSTA®) is a human mAb with a molecular weight of about 151.8 kDa that inhibits B cell activating factor (FABF). Belimumab is approved in the United States, Canada and Europe for the treatment of systemic lupus erythematosus. Belimumab is currently administered to lupus patients via 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 1000 mg/mL. Preferred ranges are calculated based on body weight of 40 to 100 kg (about 80-220 pounds) in a volume of 1 ml.

[00116] 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 7E3 monoclonal antibody. Abciximab binds to the glycoprotein (GP) IIb/IIIa receptor on human platelets and inhibits platelet aggregation by pre-aerating the binding of fibrinogen, von Willebrand factor, and other adhesive molecules. It also binds to the vitronectin receptor (aVp3) found on platelets and in the walls of endothelial vessels and smooth muscle cells. Abciximab is a platelet aggregation inhibitor used primarily during and after coronary artery procedures. Abciximab is administered via intravenous infusion, first as a bolus of 0.25 mg/kg and followed by continuous intravenous infusion of 0.125 mcg/kg/minute for 12 hours.

[00117] Tositumomab (BEXXAR®) is a drug for the treatment of follicular lymphoma. It is an anti-CD20 mouse IgG2a mAb derived from immortalized cells. Tositumomab is administered in sequential infusions: cold mAb followed by iodine (131I) tositumomab, the same antibody covalently linked to the radionuclide iodine-131. Clinical trials have established the efficacy of tositumomab/iodine tositumomab regimens in patients with relapsed refractory follicular lymphoma. BEXXAR® is currently administered at a dose of 450 mg via IV infusion.

[00118] Alemtuzumab (marketed as CAMPATH®, MABCAMPATH®, or CAMPATH-1H® and currently in 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 to treat some autoimmune diseases, such as multiple sclerosis. Alemtuzumab has a weight of about 145.5 kDa. It is administered in daily IV infusions of 30 mg to patients with B-cell chronic lymphocytic leukemia.

[00119] Palivizumab (SYNAGIS®) is a humanized mAb directed against an epitope in the antigenic site of the respiratory syncytial virus F protein. 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 a month via IM injection at 15 mg/kg.

[00120] Ofatumumab is a human anti-CD20 mAb, which appears to inhibit the early phase of 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 1000 mg/50 ml ofatumumab for IV infusion. Ofatumumab is FDA approved for the treatment of chronic lymphocytic leukemia and has also demonstrated potential in the treatment of follicular non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and remitting multiple sclerosis. Ofa-tumumab has a molecular weight of approximately 149 kDa. It is currently administered via IV infusion at an initial dose of 300 mg, followed by weekly infusions of 2,000 mg. As used in the present invention, the term "ofatumumab" includes the anti-CD20 mAb known by the International Trade Name "Ofatumumab". The term "ofatumumab" includes the active agent in products marketed under ARZERRA® and similar biologics with the same trade names. The term "ofatumumab" includes the active agent in similar biologics ARZERRA® being developed by BioExpress. High molecular weight, low viscosity liquid protein formulations may include ofatumumab, preferably at a concentration of about 300 mg/mL to about 2000 mg/mL.

[00121] Trastuzumab emtansine (in the US, emtansine ADO-trastuzumab, marketed as KADCILA®) is an antibody-drug conjugate consisting of trastuzumab mAb linked to the cytotoxic agent mertansine (DM1®). Trastuzumab, described above, stops the growth of cancer cells by binding to the HER2/neu receptor, while mertansine enters the cells and destroys them by binding to tubulin. In the United States, trastuzumab emtansine has been approved specifically for the treatment of recurrence of HER2-positive metastatic breast cancer. Multiple Phase III trials of trastuzumab emtansine are planned or ongoing in 2014. Trastuzumab emtansine is currently administered via intravenous infusion at 3.6 mg/kg. Liquid, high molecular weight, low viscosity formulations may include trastuzumab emtansine, preferably at a concentration of about 144 mg/mL to about 360 mg/mL.

[00122] 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 recommended dosage of Pertuzumab is 420 mg to 840 mg via 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.

[00123] Daclizumab is an anti-humanized CD25 mAb and is used to prevent rejection in organ transplantation, especially in kidney transplantation. 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 US by Hoffmann-La Roche, Ltd. ("Roche") as ZENAPAX® and administered via 1 mg/kg intravenous infusion. Daclizumab High Yield Process (DAC HYP; BIIB019; Biogen Idec ("Biogen") and Abbvisto é, Inc. ("Abbvie")) is in phase III clinical trials as a 150 mg, once-monthly subcutaneous injection to treat sclerosis multiple repeat offender, sender. Liquid, high molecular weight, low viscosity formulations may include daclizumab, preferably at a concentration of about 40 mg/mL to about 300 mg/mL.

[00124] 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 about 148 kDa, is the syringe developed by Alexion Pharmaceuticals, Inc ("Alexion"). It is administered via IV infusion in an amount of about 600 mg to about 1200 mg. High molecular weight, low viscosity liquid formulations may include eculizumab, preferably at a concentration of about 500 mg/mL to about 1200 mg/mL.

[00125] 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 via 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.

[00126] Rituximab (RITUXAN®) is a chimeric anti-CD20 mAb used to treat a variety of diseases characterized by an excessive number of B cells, hyperactive B cells or dysfunctional B cells. Rituximab is used to treat cancers of the white blood system, such as leukemias and lymphomas, including Hodgkin's lymphoma and its predominant lymphocyte subtype. It has been shown to be an effective rheumatoid arthritis treatment. Rituximab is widely used without label to treat difficult cases of multiple sclerosis, systemic lupus erythematosus, and autoimmune anemias.

[00127] Rituximab is jointly marketed in the US under the trade name RITUXAN® by Biogen and Genentech and outside the US 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 via IV infusion of approximately 375 mg/m2. The term "rituximab" as used herein includes the anti-CD20 mAb known under the International Trade Name "rituximab." Rituximab includes mAbs described in US Patent 5,736,137. Rituximab includes the active agent in products marketed under the trade name RITUXAN® MABTHERA® and similar biological products thereof.

[00128] High molecular weight, low viscosity liquid formulations may include rituximab, preferably at a concentration of about 475 mg/mL to about 875 mg/mL (approximately using a range of 1.3 to 2.3 square meters of body surface area, derived from the Mosteller formula for people ranging from 5 feet, 40 kg to 6 feet, 100 kg). Concentrations are calculated for a 1 mL formulation.

[00129] Ipilimumab is a human mAb developed by the Bristol-Myers Squibb Company ("Bristol-Myers Squibb"). Marketed as YERVOY®, which 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 metastatic prostate cancer refractory to hormones. Ipilimumab is currently administered via 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.

[00130] Raxibacumab (ABthrax®) is a human mAb intended for the prophylaxis and treatment of inhaled anthrax. Currently, it is administered via IV infusion. The suggested dosage in 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 4000 mg/mL.

[00131] Nimotuzumab (THERACIM®, BIOMAB EGFR®, THERALOC®, CIMAher®) is a humanized mAb with a molecular weight of approximately 151 kDa used to treat recurrent or refractory high-quality malignant head and neck squamous cell carcinomas of the head and neck. quality glioma, anaplastic astrocytoma, glioblastomas, and diffuse intrinsic pontine glioma. Nimotuzumab is typically administered via IV infusion of about 200 mg per week. High molecular weight, low viscosity liquid formulations may include nimotuzumab, preferably at a concentration of about 200 mg/mL.

[00132] Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate directed to the CD30 protein, expressed in classical Hodgkin's lymphoma and anaplastic cell lymphoma. It is administered via 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.

[00133] 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 filed.

[00134] Obinutuzumab (GAZYVA®), originally developed by Roche and being developed under a collaboration agreement with Biogen, is a humanized anti-CD20 mAb approved for the treatment of chronic lymphocytic leukemia. It is also being investigated in Phase III clinical trials for patients with various lymphomas. Dosages of around 1,000 mg are being administered via intravenous infusion.

[00135] Certolizumab Certolizumab (CIMZIA®) is a recombinant form, a humanized Fab' antibody fragment, with specificity for the human tumor of tumor necrosis factor alpha (TNFa), conjugated to a polyethylene glycol of approximately 40 kDa (PEG2MAL40K). The molecular weight of Certolizumab pegol is approximately 91 kDa.

[00136] Other antibody therapeutics that can be formulated with low viscosity ionic liquids include CT-P6 from Celltrion, Inc. (Celltrion).

Antibody Therapeutics in Trials and Final Phase Development

[00137] The progression of antibody therapeutics to 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 therapeutic antibodies under ongoing evaluation in late-phase studies. The first marketing applications for two mAbs (Vedolizumab and ramucirumab) were recently submitted to the FDA. Amgen is currently sponsoring multiple-course Phase III trials on the use of brodalumab in patients with plaque psoriasis, with additional trials planned or recruiting patients. XBi- OTECH, Inc. has sponsored two Phase I clinical trials of MABp1 (Xilanix) for patients with advanced cancer or type-2 diabetes. Additional MABp1 trials are recruiting patients. Multiple trials are sponsored by MedImmune, LLC ("MedImmune") and refer or enroll patients for the treatment of leukemia with pasudotox moxetumomab. Long-term safety and efficacy studies are ongoing for the use of tildrakizumab for the treatment of chronic plaque psoriasis. Multiple phase II trials were recently completed using rilatumumab for the treatment of several cancers.

[00138] At least 28 mAbs are high molecular weight proteins, at this time, or have recently completed Phase III studies for the treatment of inflammatory or immunological disorders, cancers, high cholesterol, osteoporosis, Alzheimer's disease, and infectious diseases. mAbs in or having recently completed Phase III trials include AMG 145, elotuzumab, epratuzumab, farletuzumab (MORAb-003), gantenerumab (RG1450), gevokizumab, inotuzumab ozogamicin, itolizumab, ixekizumab, lebriki-zumab, mepolizumab, stafenatox naptumomab, necitumumab, nivolumab, ocreli-zumab, onartuzumab, racotumomab, ramucirumab, reslizumab, romosozumab, sarilumab, secukinumab, sirukumab, solanezumab, tabalumab and Vedolizumab. A mAb mixture (actoxumab and bezlotoxumab) is also being evaluated in Phase III trials. See, for example, Reichert, MAb 5: 1 to 4, 2013.

[00139] Vedolizumab is a mAb being developed by Millenium Pharmaceuticals, 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 showed it to meet the goals of inducing a clinical response and maintaining remission in Crohn's disease and ulcerative colitis patients. Studies evaluating long-term clinical results show around 60% of patients achieving clinical remission. A common dose of Vedolizumab is 6 mg/kg via IV infusion.

[00140] Ramucirumab is a human mAb being developed for the treatment of solid tumors. Phase III clinical trials are underway for the treatment of breast cancer, metastatic gastric adenocarcinoma, non-small cell lung cancer, and other types of cancer. Ramucirumab, in some Phase III trials, is administered at about 8 mg/kg by IV infusion.

[00141] Rilatumumab is a human mAb that inhibits the action of hepatocyte growth factor/dispersion factor. Developed by Amgen, it is in Phase III trials as a treatment for solid tumors. An open-label Phase III trial of rilatumumab treatment in patients with advanced or metastatic esophageal cancer will administer rilatumumab at approximately 15 mg/kg via IV infusion.

[00142] Evolocumab (AMG 145), also developed by Amgen, is a mAb that binds to PCSK9. Evolocumab is indicated for hypercholesterolemia and hyperlipidemia.

[00143] Alirocumab (REGN727) is a human mAb from Regeneron Pharmaceuticals, Inc. ("Regeneron") and Sanofi-Aventis USA LLC ("Sanofi"), indicated for hypercholesterolemia and acute coronary syndrome.

[00144] Naptumomab stafenatox, ABR-217620 from Active Biotech AB ("Active BIOTECH") is a mAb indicated for renal cell carcinoma.

[00145] Racotumomab from CIMAB, SA ("CIMAB"); Laboratorio Elea S.A.C.I.F.y A. is a mAb indicated for non-small cell lung cancer.

[00146] Other antibodies that can be formulated with viscosity-reducing ionic liquids include bococizumab (PF-04950615) and tanezumab; Amgen's ganitumab, blinatumomab, trebananib; Anthrax immunoglobulin from Cangene Corporation; teplizumab from MacroGenics, Inc.; MK-3222, MK-6072 from Merck & Co ("Merck"); girentuximab from WILEX AG; RIGScan from Navidea Biopharmaceuticals ("Navidea"); PF-05280014 from Pfizer; SA237 from Chugai Pharmaceutical Co. Ltd. ("Chugai"); guselkumab from Janssen/Johnson and Johnson Services, Inc. ("J&J"); Antithrombin Gamma (KW-3357) from Kyowa; and CT-P10 from Celltrion.

Antibodies in early phase clinical trials

[00147] Many mAbs have recently entered, or are entering, clinical trials. They may include proteins currently administered via intravenous infusion, preferably those having 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 albumin-conjugated drugs or peptides 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 jointly developed by Amgen and AstraZeneca and currently in Phase I clinical 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 a mAb to the epithelial cell adhesion molecule; AMG 157, jointly developed by Amgen and AstraZeneca, is a human mAb currently in Phase I for the treatment of asthma; AMG 167 is a humanized mAb that has been evaluated in several Phase I clinical trials for the treatment of osteopenia; AMG 334, having completed dosing Phase I studies and currently in Phase II studies for the treatment of migraines and hot flashes, is a human mAb that inhibits the gene-related peptide calcitonin; AMG 780 is a human anti-angiopoietin mAb that inhibits the interaction between the selective endothelial cell receptor Tie2 and the ANG1 and ANG2 ligands thereof, and recently completed Phase I trials as a cancer treatment; AMG 811 is a human monoclonal antibody that inhibits interferon gamma being 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.

[00148] 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 the human interleukin-17 family of cytokines. Phase I clinical trials evaluating RG7624 for autoimmune disease are ongoing. BIIB033 is an anti-LINGO-1 anti-mAb by Biogen currently in Phase II trials for the treatment of multiple sclerosis.

[00149] High molecular weight proteins may also include AGS-009, an IFN-alpha mAb targeting developed by Argos Therapeutics, Inc., which recently completed phase I studies 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 in patients with rheumatoid arthritis. Certolizumab pegol (CIMZIA®) is a mAb in Phase II trials for ankylasing spondylitis and juvenile rheumatoid arthritis. Clazakizumab, an anti-IL-6 mAb, is in Phase II trials by Bristol-Myers Squibb.

[00150] CNTO-136 (sirukumab) and CNTO-1959 are mABs with 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 Hu-mAb targeting interleukin-1 beta. It has been approved for the treatment of cyropyrin-associated periodic syndromes. Canakinumab is in Phase I trials as a possible treatment for chronic obstructive pulmonary disease, gout and coronary artery disease. Mavrilimumab is a human mAb designed for the treatment of rheumatoid arthritis. Discovered as CAM-3001 by Cambridge Antibody Technology, mavrilimumab is being developed by MedImmune.

[00151] 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 via regular IV infusions of 300-1,000 mg. MEDI-551, another mAb developed by AstraZeneca for numerous indications, is also currently administered via IV infusion. NN8209, a C5aR receptor blocking mAb to be developed by Novo Nordisk A/S ("Novo Nordisk"), has been completed as 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 NKG2A-targeted mAb being developed by Novo Nordisk to treat patients with inflammatory conditions and autoimmune diseases. NN8765 recently completed Phase I trials.

[00152] Olokizumab is a humanized mAb that potently targets the cytokine IL-6. IL-6 is involved in several autoimmune and inflammatory pathways. Olokizumab has completed Phase II trials for the treatment of rheumatoid arthritis. Otelixizumab, also known as TRX4, is a 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.

[00153] Pfizer currently has phase I trials for mAbs PD-360324 and PF-04236921 for the treatment of lupus. A similar biologic rituximab, PF-05280586, was developed by Pfizer and is in Phase I/Phase II trials for rheumatoid arthritis.

[00154] Rontalizumab is a humanized mAb to be developed by Genentech. It recently completed Phase II trials for the treatment of lupus. SAR113244 (anti-CXCR5) is a Sanofi mAb in Phase I trials. Sifalimumab (anti-IFN-alpha mAb) is a mAb in Phase II trials for the treatment of lupus.

[00155] A low viscosity, high molecular weight liquid formulation may include one of the mAbs in early stage clinical development for the treatment of various blood diseases. 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 mAb and hepcidin from Eli Lily, and SelG1 from Selexys Pharmaceuticals, Corp. ("Selexys").

[00156] One or more mAbs in early phase development for the treatment of various cancers and related conditions can be included in a low viscosity, high molecular weight liquid formulation. United Therapeutics, Corporation has two 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 Pharmaceuticals, Inc has been conducting early-stage trials 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 diseases, including anti CD22-ADC (RG7593; pinatuzumab vedotin), anti CD79b-ADC (RG7596), anti- STEAP1 ADC (RG7450), ASG-5ME and ASG-22ME from Agensys, Inc. ("Agensys") antibody-drug conjugate RG7458, and mafodotin vorsetuzumab. Genentech's early stage cancer therapeutics can be included in low viscosity formulations, including ALT-836, the antibody-drug conjugate RG7600 and DEDN6526A, anti-CD22 ADC (RG7593), anti-EGFL7 mAb (RG7414), anti -HER3/DAF EGFR mAb (RG7597), anti-PD-L1 mAb (RG7446), DFRF4539A, a MINT1526A. Bristol-Myers Squibb is developing mAbs in early-stage 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 Pharmaceuticals, Inc. ("AVEO"), AVX701 and AVX901 from AlphaVax, BAX -69 from Baxter International, Inc. ("Baxter"), BAY 79-4620 and BAY 20-10,112 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 SpA

[00157] Other antibody therapeutics that can be formulated with viscosity-reducing ionic liquids include alzumab, GA101, daratumumab, siltuximab, ALX-0061, ALX-0962, ALX-0761, bimagumab (BYM338), CT-011 (pidilizumab) , AC- toxumab/bezlotoxumab (MK-3515A), MK-3475 (pembrolizumab), dalotuzumab (MK-0646), icrucumab (IMC-18F1, LY3012212), AMG 139 (MEDI2070), SAR339658, dupilumab (REGN668), SAR156597, SAR256212, SAR279356, SAR3419, SAR153192 (REGN421, enoticumab), SAR307746 (nesvacumab), SAR650984, SAR566658, SAR391786, SAR228810, SAR252067, SGN-CD19A, SGN-CD33A, SGN -LIV1A, ASG 15ME, Anti-LINGO, BIIB037, ALXN1007 , Tepro-tumumab, concizumab, anrukinzumab (IMA-638), ponezumab (PF- 04360365), PF-03446962, PF-06252616, etrolizumab (RG7413), quilizumab, ranibizumab, lampali-zumab, onclacumab, gentenerumab, crenezumab (RG74 12) , IMC-RON8 (narnatu-mAb), tremelimumab, vantictumab, eemcizumab, ozanezumab, lojatumumab, tralokinumab, XmAb5871, DI573, MEDI0639, MEDI3617, MEDI4736 , MEDI6469, MEDI0680, MEDI5872, PF-05236812 (AAB-003), PF-05082566, BI 1,034,020, RG7116, RG7356, RG7155, RG7212, RG7599, RG7636, RG7221, RG7652 ( MPSK3169A), RG7686, HuMaxTFADC, MOR103, AGS -16M8F, AGS-16C3F, LY3016859, LY2495655 , LY2875358, and LY2812176.

[00158] Other early stage mAbs that can be formulated with viscosity-reducing ionic liquids 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 pegylated monovalent anti-CD40L 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 (ravtansine indatuximab) from Biotest Pharmaceuticals Corporation; XmAb from Boehringer Ingelheim/Xencor; anti-IP10 from Bristol-Myers Squibb; J 591 Lu-177 from BZL Biologics LLC; CDX-011 (glembatumumab vedotin), CDX0401 from Celldex Therapeutics; Foravirumab from Crucell; tigatuzumab from Daiichi Sankyo Company Limited; MORAb-004, MORAb-009 (amatuximab) from Eisai; LY2382770 from Eli Lilly; DI17E6 from Merck Serono Inc; zanolimumab from Emergent BioSolutions, Inc.; FG-3019 from FibroGen, Inc.; catumaxomab from Fresenius SE & Co. KGaA; pateclizumab, rontalizumab from Genentech; fresolimumab from Genzyme &Sanofi; GS-6624 (simtuzumab) from Gilead; CNTO-328, bapineuzumab (AAB-001), carlumab, CNTO-136 from Janssen; KB003 from Kalo-BIOS Pharmaceuticals, Inc.; ASKP1240 from Kyowa; RN-307 from Labrys Biologics Inc.; ecromeximab from Vida Pharmaceuticals Science; LY2495655, LY2928057, LY3015014, LY2951742 from Eli Lilly; MBL-VHC1 from MassBiologics; AME-133V from MENTRIK Biotech, LLC; abituzumab from Merck KGaA; MM-121 from Merrimack Pharmaceuticals, Inc.; MCS110, QAX576, QBX258, QGE031 from Novartis AG; HCD122 from Novartis AG and XOMA Corporation ("XOMA"); NN8555 from Novo Nordisk; bavituximab, Cotara from Peregrine Pharmaceuticals, Inc.; PSMA-ADC from Progenics Pharmaceuticals, Inc.; Oregovomab from Quest Phar-Matech, 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 Pharmaceuticals, Inc.; UB-421 from United Biomedical Inc.; VB4-845 from Viventia Bio, Inc.; ABT-110 from AbbVie; Caplacizumab, Ozoralizumab from Ablynx; 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 (mertansine lorvotuzumab) from ImmunoGen Inc.; Epratuzumab Y-90/veltuzumab combination (IMMU-102) from Immunomedics, Inc.; anti-fibrin/3B6/22 Tc-99m mAb from Agenix, limited; ALD403 from Alder Biopharmaceuticals, Inc; RN6G/PF-04382923, from Pfizer; CG201 from CG Therapeutics, Inc; KB001-A from KaloBios Pharmaceuticals/Sanofi; KRN-23 from Kyowa; Y- 90 hPAM 4 from Immunomedics, Inc.; Tarextumab from Morphosys AG & OncoMed Pharmacetuicals, Inc.; LFG316 from Morphosys AG & Novartis AG; CNTO3157, CNTO6785 from Morphosys AG &Jannsen; RG6013 from Roche &Chugai; MM-111 from Merrimack Pharmaceuticals, Inc. ("Merrimack"); GSK2862277 from GlaxoSmithKline; AMG 282, AMG 172, AMG 595, AMG 745, AMG 761 from Amgen; BVX-20 from Biocon; CT-P19, P24-CT, CT-P25, P26-CT, CT-P27, CT-P4 from Celltrion; GSK284933, GSK2398852, GSK2618960, GSK1223249, GSK933776A from GlaxoSmithKline; ravtansine anetumab from Morphosys AG & Bayer AG; BI-836845 from Morphosys AG & Boehringer Ingelheim; NOV-7, NOV-8 Morphosys AG & Novartis AG; MM-302, MM-310, MM-141, MM-131, MM-151 from Merrimack, RG7882 from Roche & Seattle Genet-ics; 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 DNAectin from Bristol-Myers Squibb; FPA009, FPA145 from FivePrime Therapeutics, Inc.; Gilead GS-5745; BIW-8962, KHK4083, KHK6640 from Kyowa Hakko Kirin; MM141 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 Millennium; ABT-981from Abbvie; Abgn-168h from ABGE-nomics International Inc.; ficlatuzumab from AVEO; BI-505 from BioInvent International; CDX1127, 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 beta antibody, IMC-CS4 from Eli Lilly; VEGFR3 mAb, IMC-TR1 (LY3022859) from Eli Lilly and ImClone, LLC; Antim from Elusys Therapeutics Inc.; HuL2G7 from Galaxy Biotech LLC; IMGB853, IMGN529 from ImmunoGen Inc.; CNTO-5, CNTO-5825 from Janssen; KD-247 from Kaketsuken; KaloBios medicines u-cals KB004; 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 National Institute of Allergy and Infectious Diseases; neuroblastoma antibody vaccine from MabVax Therapeutics; Cytolin from CitoDyn, Inc.; Thravixa from Emergent BioSolutions Inc.; and FB 301 from Cytovance Biologics; anti-rabies mAb from Janssen and Sanofi; influenza mAb from Janssen and partially funded by the National Institutes of Health; MB-003 and zmapp from Mapp Biofarmacêutica, Inc.; and ZMAb from Defyrus Inc.

Other therapeutic proteins

[00159] The protein may be an enzyme, a fusion protein, a pegylated or stealth protein, the vaccine or otherwise a biologically active protein (mixture or protein). The term "enzyme", as used in the present invention, refers to the protein or functional fragment thereof that catalyzes a biochemical transformation of a target molecule to a desired product.

[00160] Enzymes like drugs have at least two important characteristics, namely i) they often bind and act on their targets with high affinity and specificity, and ii) they are catalytic and convert various target molecules to the desired products. In certain embodiments, the protein may be PEGylated, as defined in the present invention.

[00161] The term "fusion protein", as used in the present invention, refers to a protein that is created from two different genes that code for two separate proteins. Fusion proteins are generally 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.

[00162] There are a number of fusion proteins that are on the market.

[00163] Enbrel® (etanercept), is a fusion protein that is marketed by Amgen that competitively inhibits TNF.

[00164] ELOCTATE®, Antihemophilic Factor (recombinant), Fc fusion protein, is a recombinant DNA derivative, antihemophilic factor indicated in adults and children with hemophilia A (congenital factor VIII deficiency) for case control and prevention of bleeding, perioperative management, routine prophylaxis to prevent or reduce the frequency of bleeding cases.

[00165] EILEA® (aflibercept) is a recombinant fusion protein consisting of human receptor portions of the VEGF extracellular domains 1 and 2 fused with the Fc portion of human IgG1 formulated as an iso-osmotic solution for intravitreal administration. EILEA (aflibercept) is a recombinant fusion protein consisting of human receptor portions of the VEGF 1 and 2 extracellular domains 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, which constitutes 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.

[00166] ALPROLIX™, coagulation factor IX (recombinant), Fc fusion protein, is a recombinant DNA derivative, coagulation factor IX concentrate is indicated for adults and children with hemophilia B for the control and prevention of cases bleeding, perioperative management, routine prophylaxis to prevent or reduce the frequency of bleeding cases.

[00167] Pegloticase (KRYSTEXXA®) is a drug for the treatment of severe chronic gout, refractory treatment, developed by Savient Pharmaceuticals, Inc. and is the first drug approved for this indication. Pegloticase is a similar pegylated recombinant porcine uricase with a molecular weight of about 497 kDa. Pegloticase is currently being 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.

[00168] Alteplase (Activase®) is a tissue plasminogen activator produced through recombinant DNA technology. It is a purified glycoprotein comprising 527 amino acids and synthesized using natural-type human plasminogen activator complementary DNA (cDNA), tissue obtained from a human melanoma cell line. Alteplase is administered via intravenous infusion of approximately 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.

[00169] 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 compromised kidney function. Glucarpidase is administered IV, in a single dose of approximately 50 IU/kg. In some embodiments, low viscosity formulations are provided containing glucarpidase.

[00170] Alglucosidase alfa (LUMIZYME®) is an orphan enzyme replacement therapeutic drug for the treatment of Pompe disease (glycogen storage disease type II), a rare lysosomal storage disease. 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 pharmaceutical formulation of alglucosidase alfa is preferably provided at a concentration of about 100 mg/mL to about 2000 mg/mL.

[00171] 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), with a molecular weight of 5000 Da, covalently linked to the enzyme adenosine deaminase, which was derived from bovine intestine.

[00172] α-galactosidase is a lysosomal enzyme that catalyzes the hydrolysis of the glycolipid, globotriaosylceramide (GL-3), galactose and dihexosylceramide. Fabry disease is a rare hereditary lysosomal storage disease characterized by subnormal enzymatic activity of α-galactosidase and consequent accumulation of GL-3. Agalsidase alfa (REPLAGAL®) is a human α-galactosidase A enzyme produced through a human cell line. Agalsidase beta (FABRAZYME®) is a recombinant human α-galactosidase A expressed in a CHO cell line. Replagal is administered at a dose of 0.2 mg/kg weekly OTH-er via 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 via IV infusion. Other lysosomal enzymes can also be used. For example, the protein may be a lysosomal enzyme, as described in US 2012/0148556.

[00173] Rasburicase (ELITEK®) is a recombinant urate oxidase indicated for the initial management of plasma uric acid levels in pediatric and adult patients with leukemia, lymphoma and malignant solid tumors who are receiving anti-cancer therapy expected to result in tumor lysis and subsequent elevation of uric acid in plasma. ELITEK® is administered via IV infusion daily, at a dose of 0.2 mg/kg.

[00174] Imiglucerase (CEREZYME®) is a recombinant analogue of human β-glucocerebrosidase. Initial doses ranging from 2.5 L/kg of body weight 3 times a week to 60 U/kg once every 2 weeks CEREZYME® is administered via IV infusion.

[00175] Abraxane, albumin conjugated to paclitaxel, is approved for metastatic breast cancer, non-small cell lung cancer and late stage pancreatic cancer.

[00176] Taliglucerase alfa (ELEYSO®) is a hydrolytic lysosomal glucocerebroside-specific enzyme indicated for long-term enzyme replacement therapy for type 1 Gaucher disease. The recommended dose is 60 U/kg of body weight, administered once once every 2 weeks via intravenous infusion.

[00177] Laronidase (ALDURAZYME®) is a polymorphic variant of the human α-L-iduronidase enzyme, which is produced through the CHO cell line. The recommended dose of ALDURAZYME® is 0.58 mg/kg administered once weekly as an intravenous infusion.

[00178] Elosufase alfa (VIMIZIM®) is a human N-acetylgalactosamine-6-sulfatase produced through the CHO cell line by BioMarin Pharmaceuticals Inc ("BioMarin"). It was approved by the FDA on February 14, 2014 for the treatment of Type IVA Mucopolysaccharidosis. It is administered weekly via intravenous infusion, at a dosage of 2 mg/kg.

[00179] Other biologics that can be formulated with viscosity-reducing ionic liquids include Erwinia chrysanthemi asparaginase (ERWINAZE®), incobotulinumtoxin A (XEOMIN®), EPOGEN® (epoetin alfa), Procrit® (epoetin alfa), Aranesp® ( darbepoetin alfa), Orencia® (abatacept), BATASERON® (interferon beta-1b), NAGLAZYME® (galsulfase); ELAPRASE® (Idursulfase); MYOZYME® (LU-MIZYME®, algucosidase alfa); VPRIV® (velaglucerase), abobotulinumtoxin A (DYSPORT®); BAX-326, Octocog alfa from Baxter; Syncria from GlaxoSmithKline; liprotamase from Eli Lilly; Xiaflex (collagenase clostridium histolyticum) from Aid 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; Canyon Pharma-Ceuticals Group Iprivask.

Therapeutic proteins in development

[00180] Versartis, Inc. VRS-317 is a recombinant human growth hormone (hGH) fusion protein using XTEN half-life extension technology. It is intended to reduce the frequency of hGH injections required for patients with hGH deficiency. VRS-317 has completed a Phase II study, comparing its effectiveness to daily injections of non-derivative hGH, with positive results. Phase III studies are planned.

[00181] Vibriolysin is a proteolytic enzyme secreted by Gram-negative marine microorganisms, 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 being investigated by BioMarin for cleaning and/or treating burns. Vibriolysin formulations are described in patent WO 02/092014.

[00182] 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 through a deficiency of phenyl alanine hydroxylase enzyme (HAP). 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 for the treatment of patients who do not respond adequately to KUVAN®.

[00183] Other therapeutic proteins that can be formulated with viscosity-reducing ionic liquids include Alprolix/rFIXFc, Eloctate/rFVIIIFc, BMN-190; BMN-250; Lamazyme; Galazyme; ZA-011; Sebelipase alfa; SBC-103; and HGT-1110. Additionally, fusion proteins containing half-life extension technology include XTEN, but not limited to: VRS-317 GH-XTEN; Vila Factor, 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 ionic liquids.

[00184] Other late-phase therapeutic proteins that can be formulated with viscosity-reducing ionic liquids include CM-AT from CureMark LLC; NN7999, NN7088, Li-raglutide (NN8022), NN9211, Semaglutide (NN9535) from Novo Nordisk; AMG 386, Amgen Filgrastim; CSL-654, CSL Behring Factor VIII; LA-EP2006 (pegfilgrastim similar biological product) 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.; similar somatropin biologic from LG Life Sciences LTD; Natpara from NPS Pharmaceuticals, Inc.; ART123 from Asahi Kasei Corporation; BAX-111 from Baxter; OBI-1 from Inspiration Biopharmaceuti-cals; Wilate from Octapharma AG; Talactoferrin alfa from Agennix AG; Desmotep-lase from Lundbeck; Cinryze from 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 protein (rFIXFc; BIIB029) and recombinant coagulation factor VIII Fc fusion protein (rFVIIIFc; BIIB031) from Biogen; and AstraZeneca's Myalept.

[00185] Other early-stage protein biologics that can be formulated with viscosity-reducing water ionic liquids include Alferon LDO from Hemispherx BioPharma, Inc.; SL-401 from Stemline Therapeutics, Inc.; PRX-102 from Protalix Biotherapeutics, Inc.; KTP-001 from Kaketsuken/Teijin Pharma Limited; Vericiguat from Bayer AG; BMN-111 from BioMarin; ACC-001 (PF- 05236806) from Janssen; LY2510924, LY2944876 from Eli Lilly; NN9924 from Novo Nordisk; INGAP peptide from Exsu-lin; ABT-122 from Abbvie; AZD9412 from AstraZeneca; Neublastin (BG00010) from Biogen; Luspatercept (ACE-536), Sotatercept (ACE-011) from Celgene Corporation; PRAME immunotherapeutics from GlaxoSmithKline; Ethyl Plovamer (PI-2301) from Merck KGaA; PREMIPLEX (607) from Shire; BMN-701 from BioMarin; Ontak from Eisai; rHuPH20/insulin from Halozyme, Inc.; PB-1023 from PhaseBio Pharmaceuticals, Inc.; ALV-003 from Alvine Pharmaceuticals Inc. and Abbvie; NN8717 from Novo Nordisk; PRT-201 from Proteon Therapeutics Inc.; PEGPH20 from Halozyme, Inc.; Amevive® alefacept from Astellas Pharma Inc.; F-627 from Regeneron; AGN-214868 (senrebotase) from Allergan, Inc.; BAX-817 from Baxter; PRT4445 from Portola Pharmaceuticals, Inc.; VEN100 from Ventria Bioscience; On-conase/Ranpirnase from Tamir Biotechnology Inc.; interferon alfa-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, idursul-phase) from Shire; PF-04856884, PF-05280602 from Pfizer; ACE-031, Dalantercept from Acceleron Pharma; ALT-801 from Altor BioScience Corp.; BA-210 from Bio-Axone Biosciences, Inc.; WT1 immunotherapeutic from GlaxoSmithKline; GZ402666 from Sanofi; MSB0010445, Atacicept from Merck KGaA; Leukine (sar-gramostim) from Bayer AG; Kur-211 from Baxter; 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 alfa-2b from HanAll Biopharma; CIT107 from Citheris SA; RT001 from Revance Therapeutics, Inc.; MEDI6012 from AztraZeneca; E2609 in 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; Amunix Operational Inc. XTen fusion proteins; entolimod (CBLB502) from Cleveland BioLabs, Inc.; HGT2310 from Shire; HM10760A from Hanmi Pharmaceuticals Co., LTD; ALXN1102/ALXN1103 from 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 Merck 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, CVX-096 from Pfizer; ACP-501 from AlphaCore Pharma LLC; BAX-855 from Baxter; CDX-1135 from Celldex Therapeutics; PRM-151 from Promedior, Inc.; TS01 of Thrombolytic Science International; TT-173 from Thrombotargets Corp.; QBI-139 from Quintessença Biosciences, Inc.; Vatelizumab, GBR500, GBR600, GBR830 and GBR900 from Glenmark Pharmaceuticals; and CIT-6091 from Citimmune Sciences, Inc.

Other biological agents

[00186] Other biological medicines that can be formulated with the viscosity-reducing ionic liquids include PF-05285401, PF-05231023, RN317 (PF-05335810), PF-06263507, PF-05230907, Dekavil, PF-06342674, PF0625261 6, RG7598, RG7842, RG7624d, OMP54F28, GSK1995057, BAY1179470, IMC-3G3, IMC- 18F1, IMC-35C, IMC-20D7S, PF-06480605, PF-06647263, PF- 06650808, PF-05335810 (RN317) PD-0360324, PF-00547659 from Pfizer; MK-8237 from Merck; BI033 in Biogen; GZ402665, SAR438584/ REGN2222 from Sanofi; BMI-18F1; and Icrucumab, IMC-3G3 from ImClone LLC; Ryzod, for example, Tresiba, Xultophy from Novo Nordisk; Toujeo (U300), LixiLan, Lyxumia (lixisenatide) from Sanofi; MAGE-A3 immunotherapeutics from GlaxoSmithKline; Tecemotide from Merck KGaA; Sereleaxin (RLX030) from Novartis AG; erythropoietin; pegfilgrastim; LY2963016, Dulaglutide (LY2182965) from Eli Lilly; and insulin glargine from Boehringer Ingelheim.

B. Ionic liquids

[00187] The viscosity of liquid protein formulations, including low molecular weight and/or high molecular weight proteins, is reduced through the addition of one or more viscosity reducing ionic liquids. Pharmaceutical formulations can be converted from non-Newtonian to Newtonian fluids by adding an effective amount of one or more viscosity-reducing ionic liquids.

Ionic liquid salts

[00188] The ionic liquid can be a salt. Representative ionic liquid salts include salts with imidazolium cations, including N,N-dialkyl imidazolium. Ionic liquids include salts with N-alkylated saturated or unsaturated nitrogen-containing heterocyclic cations, including N-alkylpyridinium salts, N-alkylpyrrolidinium salts, and N-alkylpiperidinium salts. In preferred embodiments, the ionic liquid is pharmaceutically acceptable and miscible with water.

[00189] In some embodiments, the ionic liquid contains a cationic constituent having a cationic heterocyclic group with one or more alkyl, heteroalkyl, alkenyl or alkynyl substituents having from 2 to 50 carbon atoms, from 3 to 30 carbon atoms , or from 4 to 12 carbon atoms. Suitable anionic components include halide, sulfate, sulfonate, sulfite, sulfinate, phosphate, phosphonate, phosphite, phosphonite, carbonate, and carboxylate ions optionally substituted with one or more alkyl, heteroalkyl, alkenyl, alkynyl, carbocyclic groups, or heterocyclic, preferably having from 1 to 20 or from 1 to 12 carbon atoms. Exemplary anionic components include chloride, bromide, methylphosphate, methyl ethyl phosphate, methylsulfate, methylsulfonate, formate, acetate, butyrate, citrate, carbonate, methyl carbonate, and lactate. The cationic heterocyclic group can be saturated or unsaturated. Saturated cationic heterocyclic groups include pyrrolidinium, oxazolidinium, piperidinium, piperazinium, morpholinium, thiomorpholinium, and azepanium groups, and the like. Unsaturated cationic heterocyclic groups include pyrrolinium, imidazolinium, 1,2,3-triazolium, 1,2,4-triazolium, thiazolium, 1,2,4-dithiazolium, 1,4,2-dithiazolium, tetrazolium, pyrazolinium, oxazolinium groups , pyridinium, and azepinium, and the like. The cationic heterocyclic group may be a fused ring structure having two, three, four, or more fused rings. The cationic heterocyclic group may be a cationic bicyclic heterocycle, such as benzoxazolium, benzothiazolium, benzotriazolium, benzimidazolium, and indole groups, and the like. The cationic heterocyclic group may be substituted with one or more additional substituents, including substituted and unsubstituted hydroxyl and alkoxy, heteroalkoxy, alkyl, heteroalkyl, alkenyl, and alkynyl having from 1 to 30, preferably from 3 to 20 carbon atoms.

[00190] The ionic liquid can be 1-butyl-3-methylimidazolium methanesulfonate (BMI MES) which has the structure shown below or a derivative thereof.

[00191] BMI Mes derivatives can be obtained, for example, by replacing the methanesulfonate constituent with other anionic components, replacing one or more carbons with a heteroatom, which replaces the n-butyl or N-methyl group with one or more plus higher order N-alkyl groups, attaching additional substituents to one or more carbon atoms, or a combination thereof. Exemplary anionic components are described above. Exemplary include N, O, P and S heteroatoms. Examples of higher order N-alkyl groups include substituted and unsubstituted N-alkyl and N-heteroalkyl groups contain from 1 to 30 carbon atoms, preferably from 1 to 12 atoms. of carbon. Examples of higher order N-alkyl groups include N-ethyl, N-propyl, N-butyl, N-sec-butyl, and N-tert-butyl. Additional substituents may include hydroxyl and alkoxy groups, substituted and unsubstituted heteroalkoxy, alkyl, aryl, aralkyl, aryloxy, aralkyloxy, heteroalkyl, alkenyl, and alkynyl groups of from 1 to 30, preferably 3 to 20, carbon atoms.

[00192] The ionic liquid can be 1-butyl-1-methylpyrrolidinium chloride (BMP chloride) which has the structure shown below or a derivative thereof.

[00193] BMP chloride derivatives can be obtained, for example, by replacing the chloride component with other anionic components, replacing one or more ring carbons with a heteroatom, replacing the N,N-butyl group - methyl with one or more higher order N, N-dialkyl groups, attaching one or more additional substituents to a carbon atom, or a combination thereof. Exemplary anionic components include those described above. Exemplary include N, O, P and S heteroatoms. Examples of higher order N, N-dialkyl groups include linear and branched N-heteroalkyl groups, containing from 2 to 30 carbon atoms, preferably from 3 to 12 carbon atoms. cyclic N-alkyl carbon. Examples of higher order N,N-dialkyl groups include N-ethyl-N-methyl; N-isopropyl-N-methyl; N-butyl-N-methyl; N, N-diethyl; N-ethyl-N-isopropyl; N,N-diisopropyl groups, and the like. Additional substituents may include hydroxyl, and substituted and unsubstituted alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyloxy, alkenyl, and alkynyl having from 1 to 30, preferably from 3 to 20 carbon atoms.

[00194] In some embodiments, the ionic liquid contains a cationic constituent having a structure according to Formula I in which each occurrence of R1 is independently selected from substituted hydrogen and alkyl, heteroalkyl, aryl, aralkyl, alkenyl, and alkynyl unsubstituted having 1 to 30 carbon atoms, 3 to 20 carbon atoms, or 4 to 12 carbon atoms; wherein each occurrence of R2 is independently selected from hydrogen, halide, substituted hydroxyl, and unsubstituted alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyloxy, alkenyl, and alkynyl groups having from 1 to 30 carbon atoms , from 3 to 20 carbon atoms, or from 4 to 12 carbon atoms. In some embodiments, at least one, at least two, or at least three occurrences of R1 or R2 are not hydrogen.

[00195] R2 can be independently selected from hydrogen, R1, -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;

[00196] wherein R1 is independently selected from C1-12alkyl, C3-12cycloalkyl, C6-12aryl, C1-12heteroaryl and C2-12heterocyclyl,

[00197] wherein each C1-12alkyl can be selected 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;

[00198] wherein each C3-12cycloalkyl can be selected 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;

[00199] wherein each C6-12aryl can be selected 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;

[00200] wherein each C1-12heteroaryl can be selected 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;

[00201] wherein each C2-12heterocyclyl can be selected 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;

[00202] R4 is independently selected from C1-12alkyl, C3-12cycloalkyl, C6-12aryl, C1-12heteroaryl and C2-12heterocyclyl, each of which can be selected one or more times by means of - OH, -NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)OH, -SO3H, -PO3H2, or -C(=O)NH2;

[00203] R4a can be R4 or hydrogen;

[00204] wherein any two or more of the groups R2, R3, R4 and R4 can together form a ring.

[00205] In some embodiments, the ionic liquid contains a cationic constituent having a structure according to Formula II:

[00206] wherein R1 as defined above and R3 can be either R2 as defined above, or two R3 substituents on the same carbon atom can together form a (= O), (= NR4a) or (= CR22). The ionic liquid may also contain a cationic constituent having the structure according to Formula III:

[00207] wherein R1 and R2 are as defined above.

[00208] In some embodiments, the ionic liquid contains a cationic constituent having a structure according to general formula IV:

[00209] wherein R1 and R2 are as defined above.

[00210] In some embodiments, the ionic liquid contains a cationic constituent having a structure according to any one of Formulas V to IX, where each occurrence of one is independently selected from C, N, O, S and P; where each dashed line (-------) can be a single, double or triple bond; and wherein each R10 and R10', when taken separately, is independently selected from none, H, hydroxyl, halide, and alkoxy, heteroalkoxy, alkyl, aryl, heteroalkyl, alkenyl, and alkynyl having from 1 to 30 carbon atoms, from 2 to 20 carbon atoms, or from 3 to 12 carbon atoms or, when bonded to the same atom and taken together, each R10 and R10' is = O or together with the atom to which they are bonded form a carbocycle or heterocycle having from 2 to 30, preferably from 3 to 12 carbon atoms; provided that at least one occurrence of A has a formal positive charge. In preferred embodiments, at least one occurrence of R10 or R10' has at least two, at least three, at least four, or at least five carbon atoms. Examples of alkyl groups include ethyl, propyl, isopropyl, butyl, tert-butyl, hexyl, octyl, and decyl groups. Exemplary heteroalkyl groups include cyanoethyl, cyanobutyl, and cyanopropyl groups. Exemplary alkoxy groups include methoxy, ethoxy and butoxy groups.

[00211] In some embodiments, the ionic liquid contains a cationic constituent having a structure according to any one of Formulas V to IX wherein at least one occurrence of A is a nitrogen atom has a formal positive charge with the remainder each independently selected from C, N, O, S and P; each dashed line (-----------) is a single or double bond; and wherein each R10 and R10', when taken separately, is independently selected from none, H, hydroxyl, halide, and alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyloxy, alkenyl, and substituted or unsubstituted alkynyl groups substituted having from 1 to 30 carbon atoms, from 2 to 20 carbon atoms, or from 3 to 12 carbon atoms or, when bonded to the same atom and taken together, each R10 and R10' is = O or together with the atom to which they are linked, they form a carbocycle or heterocycle that has between 1 and 30, preferably 3 to 12 carbon atoms. In preferred embodiments, at least one occurrence of R10 or R10' has at least two, at least three, at least four, or at least five carbon atoms. Examples of alkyl groups include ethyl, propyl, butyl, hexyl, octyl, and decyl groups, as well as their isomers. Exemplary heteroalkyl groups include cyanopropyl and cyanobutyl groups. Exemplary alkoxy groups include methoxy, ethoxy, and butoxy.

[00212] The ionic liquid can be an ammonium salt:

[00213] where R1 is as defined above.

[00214] In some embodiments, the ionic liquid contains a cationic constituent having a structure according to Formula XI, in which Ar is a substituted or unsubstituted aryl group; R12 is either none or an alkyl, heteroalkyl, aryl, aralkyl, alkenyl or alkynyl group having from 1 to 30 carbon atoms, from 3 to 20 carbon atoms, or from 4 to 12 carbon atoms; and each occurrence of R13 is independently selected from hydrogen and substituted and unsubstituted alkyl, heteroalkyl, aryl, aralkyl, alkenyl and alkynyl groups, having from 1 to 30 carbon atoms, from 3 to 20 carbon atoms, or from 4 to 12 carbon atoms. In some embodiments, the ionic liquid contains a cationic constituent having a structure according to Formula XI, wherein Ar is a substituted or unsubstituted benzyl group; wherein R12 is a substituted or unsubstituted C1-C12 alkyl group, or both. In some embodiments, the compound of Formula XI is characterized by the presence of at least one group selected from -COOH, -SO3H and -PO3H2.

[00215] The ionic liquid can be a phosphonium salt. In some embodiments, the ionic liquid contains a cationic constituent having a structure according to Formula XII, wherein each occurrence of R14 is independently selected from hydrogen groups alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyloxy, substituted or unsubstituted alkenyl or alkynyl having from 1 to 30 carbon atoms, from 3 to 20 carbon atoms, or from 4 to 12 carbon atoms; wherein at least one, at least two, or at least three occurrences of R14 are not hydrogen. In some embodiments, at least one occurrence of R14 is an aryl group, aralkyl group, or aralkoxy group having from 2 to 30 carbon atoms or from 4 to 12 carbon atoms. In some embodiments, the compound of formula XII is characterized by the presence of at least one group selected from -COOH, -SO3H and -PO3H2.

[00216] In some embodiments, the ionic liquid contains a cationic constituent having a structure according to formula XIII in which Ar is a substituted or unsubstituted aryl group; R15 is either none or an alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyloxy, alkenyl, or alkynyl group having from 2 to 30 carbon atoms, from 3 to 20 carbon atoms, or from 4 to 12 carbon atoms of carbon; and each occurrence of R16 is independently selected from hydrogen and alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyloxy, alkenyl, and substituted and unsubstituted alkynyl having from 1 to 30 carbon atoms, from 3 to 20 carbon atoms, or from 4 to 12 carbon atoms. In some embodiments, the ionic liquid contains a cationic constituent having a structure according to formula XIII in which Ar is an unsubstituted or substituted benzyl group; wherein R15 is a substituted or unsubstituted C1-C12 alkyl group, or both. In some embodiments, the compound of Formula XIII is characterized by the presence of at least one group selected from -COOH, -SO3H and -PO3H2.

[00217] The ionic liquid may be a guanidinium salt having a structure according to Formula XIV:

[00218] wherein R1 and R2 are as defined above.

[00219] The ionic liquid can be a salt that has a structure according to Formula XV

[00220] wherein R1 and R2 are as defined above, and X can be O, S, SO2, NR1 or C(R2)2.

[00221] The ionic liquid can be an imidazolium salt such as 1-allyl-3-methylimidazolium bis(trifluoromethylsulfonyl); 1-allyl-3-methylimidazolium bromide; 1-allyl-3-methylimidazolium chloride; 1-allyl-3-methylimidazolium dicyanamide; 1-allyl-3-methylimidazolium iodide; 1-benzyl-3-methylimidazolium chloride; 1-benzyl-3-methylimidazolium hexafluorophosphate; 1-benzyl-3-methylimidazolium tetrafluoroborate; 1,3-bis(cyanomethyl)imidazolium bis(trifluoromethylsulfonyl)imide; 1,3-bis(cyanomethyl)imidazolium chloride; 1-butyl-2,3-dimethylimidazolium chloride; 1-butyl-2,3-dimethylimidazolium hexafluorophosphate; 1-butyl-2,3-dimethylimidazolium tetrafluoroborate; 1-butyl-3-methylimidazolium acetate; 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide; 1-butyl-3-methylimidazolium bromide; 1-butyl-3-methylimidazolium chloride; 1-butyl-3-methylimidazolium dibutyl phosphate; 1-butyl-3-methylimidazolium dicyanamide; 1-butyl-3-methylimidazolium hexafluoroantimonate; 1-butyl-3-methylimidazolium hexafluorophosphate; 1-butyl-3-methylimidazolium hydrogen sulfate; 1-butyl-3-methylimidazolium iodide; 1-butyl-3-methylimidazolium methanesulfonate; 1-butyl-3-methyl-imidazolium methyl carbonate; 1-butyl-3-methylimidazolium methyl sulfate; 1-butyl-3-methylimidazolium nitrate; 1-butyl-3-methylimidazolium octyl sulfate; 1-butyl-3-methylimidazolium tetrachloroaluminate; 1-butyl-3-methylimidazolium tetrafluoroborate; 1-butyl-3-methylimidazolium thiocyanate; 1-butyl-3-methylimidazolium tosylate; 1-butyl-3-methylimidazolium trifluoromethanesulfonate; 1-(3-cyanopropyl)-3-methylimidazolium bis(trifluoromethylsulfonyl)amide; 1-(3-cyanopropyl)-3-methylimidazolium chloride; 1-(3-cyanopropyl)-3-methylimidazolium dicyanamide; 1-decyl-3-methylimidazolium; 1-decyl-3-methylimidazolium tetrafluoroborate; 1,3-diethoxymidazolium bis(trifluoromethylsulfonyl)imide; 1,3-diethoxymidazolium hexafluorophosphate; 1,3-dihydroxymidazolium bis(trifluoromethylsulfonyl)imide; 1,3-dihydroxy-2-methylimidazolium bis(trifluoromethylsulfonyl)imide; 1,3-dimethoxymidazolium bis(trifluoromethylsulfonyl)imide; 1,3-dimethoxymidazolium hexafluorophosphate; 1,3-dimethoxy-2-methylimidazolium bis(trifluoromethylsulfonyl)imide; 1,3-dimethoxy-2-methylimidazolium hexafluorophosphate; 1,3-dimethylimidazolium dimethyl phosphate; 1,3-dimethylimidazolium methanesulfonate; 1,3-dimethylimidazolium methyl sulfate; 1,2-dimethyl-3-propylimidazolium bis(trifluoromethylsulfonyl)imide; 1-dodecyl-3-methylimidazolium iodide; 1-ethyl-2,3-dimethylimidazolium tetrafluoroborate; 1-ethyl-2,3-dimethylimidazolium chloride; 1-ethyl-2,3-dimethylimidazolium ethyl sulfate; 1-ethyl-2,3-dimethylimidazolium hexafluorophosphate; 1-ethyl-3-methylimidazolium acetate; 1-ethyl-3-methylimidazolium aminoacetate; 1-ethyl-3-methylimidazolium(s)-2-aminopropionate; 1-ethyl-3-methylimidazolium bis(pentafluoroethylsulfonyl)imide; 1-ethyl-3-methylimidazolium bromide; 1-ethyl-3-methylimidazolium chloride; 1-ethyl-3-methylimidazolium dibutyl phosphate; 1-ethyl-3-methylimidazolium dicyanamide; 1-ethyl-3-methylimidazolium diethyl phosphate; 1-ethyl-3-methylimidazolium ethyl sulfate; 1-ethyl-3-methylimidazolium hexafluorophosphate; 1-ethyl-3-methylimidazolium hydrogen carbonate; 1-ethyl-3-methylimidazolium hydrogen sulfate; 1-ethyl-3-methylimidazolium hydroxide; 1-ethyl-3-methylimidazolium iodide; 1-ethyl-3-methylimidazolium L-(+)-lactate; 1-ethyl-3-methylimidazolium methanesulfonate; 1-ethyl-3-methylimidazolium methyl sulfate; 1-ethyl-3-methylimidazolium nitrate; 1-ethyl-3-methylimidazolium tetrachloroaluminate; 1-ethyl-3-methylimidazolium tetrachloroaluminate; 1-ethyl-3-methylimidazolium tetrafluoroborate; 1-ethyl-3-methylimidazolium 1,1,2,2-tetrafluoroethanesulfonate; 1-ethyl-3-methylimidazolium thiocyanate; 1-ethyl-3-methylimidazolium tosylate; 1-ethyl-3-methylimidazolium trifluoromethanesulfonate; 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide; 1-hexyl-3-methylimidazolium chloride; 1-hexyl-3-methylimidazolium hexafluorophosphate; 1-hexyl-3-methylimidazolium iodide; 1-hexyl-3-methylimidazolium tetrafluoroborate; 1-hexyl-3-methylimidazolium trifluoromethansulfonate; 1-(2-hydroxyethyl)-3-methylimidazolium dicyanamide; 1-methylimidazolium chloride; 1-methylimidazolium hydrogen sulfate; 1-methyl-3-octylimidazolium chloride; 1-methyl-3-octylimidazolium hexafluorophosphate; 1-methyl-3-octylimidazolium tetrafluoroborate; 1-methyl-3-octylimidazolium trifluoromethanesulfonate; 1-methyl-3-propylimidazolium iodide; 1-methyl-3-propylimidazolium methyl carbonate; 1,2,3-trimethylimidazolium methyl sulfate; derivatives thereof and combinations thereof. Derivatives may include substituting the anionic constituent for other anionic constituents, replacing one or more carbons with a heteroatom, replacing an N-alkyl group with one or more higher order N-alkyl groups, or a combination thereof. Exemplary anionic and heteroatom constituents are described above. Exemplary higher order N-alkyl groups may include linear and branched N-alkyl and N-heteroalkyl groups containing from 1 to 30 carbon atoms, preferably from 2 to 12 carbon atoms. Examples of higher order N-alkyl groups include N-ethyl, N-propyl, N-iospropyl, N-butyl, N-sec-butyl, and N-tert-butyl.

[00222] The ionic liquid can be a pyrrolidinium salt such as 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide; 1-butyl-1-methylpyrrolidinium bromide; 1-butyl-1-methylpyrrolidinium chloride; 1-butyl-1-methylpyrrolidinium dicyanamide; 1-butyl-1-methylpyrrolidinium hexafluorophosphate; 1-butyl-1-methylpyrrolidinium iodide; 1-butyl-1-methylpyrrolidinium methyl carbonate; 1-butyl-1-methylpyrrolidinium tetrafluoroborate; 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate; 1-ethyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide; 1-ethyl-1-methylpyrrolidinium bromide; 1-ethyl-1-methylpyrrolidinium hexafluorophosphate; 1-ethyl-1-methylpyrrolidinium tetrafluoroborate; derivatives thereof and combinations thereof. Derivatives may include substituting the anionic constituent for the anionic constituents, replacing one or more carbons with a heteroatom, replacing an N-alkyl or N-methyl group with one or more higher order N-alkyl groups, or a combination thereof. The constituents, exemplary heteroatoms, and higher order N-alkyl groups are described above.

Zwittenonic Liquids

[00223] The ionic liquid can be a zwitterionic (that is, an internal salt), for example, 4-(3-butyl-1-imidazolium)-1-butane sulfonate; 3-(1-methyl-3-imidazolium)propanesulfonate; 4-(3-methyl-1-imidazolium)-1-butanesulfonate; or 3-(triphenylphosphonium)propane-1-sulfonate.

[00224] The zwitterionic liquid can be 4-(3-butyl-1-imidazolium)-1-butane sulfonate (BIM) having the structure shown below or a derivative thereof.

[00225] BIM derivatives may include replacing the sulfonate group with a different anionic substituent, replacing one or more carbons with a heteroatom, which replaces the N-butyl group with one or more N-alkyl groups of higher order. low or higher order by attaching additional substituents on one or more carbon atoms, or a combination thereof. Exemplary anionic substituents include sulfate [-OSO3], sulfonate [-SO3], sulfite [-OSO2-], sulfinate [-SO2-], phosphate [-OP(OH)O2], alkylphosphate [-OP(OR2)O2] , phosphonate [P (OH) O2], alkylphosphonate [-P (OR2) O2], phosphite [-OP (OH) O-], alkylphosphite [OP (OR 2) O-alkyl]. Phosphonite [-P(OH)O-], alkylphosphonite [-P(OR2)O-], carbonate [-OCO2-], and ethyl carboxylate [-CO2], wherein R2 is as defined above. Examples of heteroatoms and higher order N-alkyl groups are described above. Additional substituents may include hydroxyl, and alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyloxy, alkenyl, and substituted and unsubstituted alkynyl having from 1 to 30, preferably 3 to 12 carbon atoms.

[00226] In some embodiments, the ionic liquid is an amphoteric ion containing a cationic heterocyclic substituent and an anionic substituent linked through a substituted or unsubstituted alkyl, heteroalkyl, aryl, aralkyl, alkenyl, or alkynyl group having from 2 to 50 carbon atoms, from 3 to 30 carbon atoms, or from 4 to 12 carbon atoms. The cationic heterocyclic substituent can be saturated or unsaturated. Examples include the groups pyrrolidinium, imidazolinium, oxazolidinium, piperidilinium, piperazinium, morpholinium, thiomorpholinium, azepanium, pyrrolinium, 1,2,3-triazolium, 1,2,4-triazolium, thiazolium, 1,2, 4-dithiazolium, 1, 4,2-dithiazolium, tetrazolium, pyrazolinium, oxazolinium, pyridinium and azepine. The cationic heterocyclic substituent may be a fused ring structure having two or more fused rings. The cationic heterocyclic substituent can be a cationic bicyclic heterocycle, such as benzoxazolium, benzothiazolium, benzotriazolium, benzimidazolium, and indole. The cationic heterocyclic substituent may be additionally substituted with one or more substituents. Exemplary anionic substituents include sulfate [-OSO3], sulfonate [-SO3], sulfide [-OSO2-], sulfinate [-SO2-], phosphate [-OP(OH)O2], alkylphosphate [-OP(OR2)O2] , phosphonate [P (OH) O2], alkylphosphonate [-P (OR2) O2], phosphite [-OP (OH) O-], alkylphosphite [OP (OR 2) O-alkyl]. phosphonite [-P(OH)O-], alkylphosphonite [-P(OR2)O-], carbonate [-OCO2-], and ethyl carboxylate [-CO2], wherein R2 is as described above.

[00227] In some embodiments, the ionic liquid is a zwitterionic liquid that has a structure according to Formula XVI, XVII, XVIII or XIV:

[00228] Wherein R1, R2 and R3 are as defined above, provided that the compounds of formula XVI, XVII, XVIII, XVIV, XX and XXI each contain at least one substituent -COOH, -SO3H, or - PO3H2.

C. Excipients

[00229] 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 chlorhexidinediacetate; 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; thickening agents; and stabilizing agents.

[00230] Other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington: "The Science and Practice of Pharmacy", 20th edition, Alfonso R. Gennaro, Ed, Lippincott Williams & Wilkins (2000) may also be included in a protein formulation in the present invention described, provided that they do not adversely affect the desired characteristics of the formulation.

[00231] The viscosity reducing agents described in the present invention can be combined with one or more other types of viscosity reducing agents, for example, organophosphates described in the co-filed PCT application entitled "Liquid protein formulations containing organophosphates " by ARSIA Therapeutics; water-soluble dyes described in the co-filed PCT application entitled "Liquid Protein Formulations Containing Organic Water-Soluble Dyes" by ARSIA Therapeutics; the typically bulky polar organic compounds, such as hydrophobic compounds, many of the GRAS (United States Food and Drug Administration Listing Generally Regarded as Safe) and FDA-approved therapeutic and inactive injectable ingredients described in the co-filed PCT application titled: "Liquid protein formulations containing viscosity reducing agents" by Arsia Therapeutics.

III. Production methods

[00232] The protein, such as a mAb, to be formulated can be produced by any known technique, such as by means of cells transformed or transfected with a culture vector containing one or more nucleic acid sequences encoding the protein, as is well known in the art, or through 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.

[00233] Purification of the protein to be formulated can be carried out by means of any suitable technique known in the literature, such as, for example, ethanol or ammonium sulfate precipitation, reversed-phase HPLC, silica chromatography or cation exchange resin (e.g. DEAE- cellulose), dialysis columns, chromatofocusing, gel filtration, using protein A-SE PHAROSE® (e.g. SEPHADEX® G-75) to remove contaminants, metal chelating columns to bind forms labeled with epitope, and ultrafiltration/diafiltration (non-limiting examples include centrifugal filtration and tangential flow filtration (TFF)).

[00234] The inclusion of the ionic liquid in viscosity reducing concentrations, such as 0.010 M to 1.0 M, preferably 0.050 M to 0.50 M, more preferably 0.10 M to 0.30 M, allows that a solution of the pharmaceutically active mAb is purified and/or concentrated to higher concentrations of mAb using common methods known to those skilled in the art, including but not limited to tangential flow filtration, centrifugal concentration, and dialysis.

[00235] In some embodiments, lyophilized formulations of proteins are provided and/or are used in the preparation and manufacture of low viscosity protein formulations. In some embodiments, the pre-lyophilized protein in powder form is reconstituted by dissolving it in an aqueous solution. In this embodiment, the liquid formulation is poured into a specific dosage unit container such as a vial or filled pre-mixing syringe, lyophilized, optionally with lyoprotectants, preservatives, antioxidants and other typical pharmaceutically acceptable excipients, then stored under storage. under sterile conditions shortly before use, at which time it is reconstituted with a defined volume of diluent, to bring the liquid to the desired concentration and viscosity.

[00236] The formulations described in the present invention can be stored by any suitable method known to a person skilled in the art. Examples of non-limiting methods for preparing protein formulations for storage include freezing, lyophilization and spray drying of 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 formulation or aqueous solution is stored at 2-8°C.

[00237] Non-limiting examples of diluents useful for reconstituting a lyophilisate formulation prior to injection include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), Ringer's solution, sterile saline 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

[00238] Protein formulations, including but not limited to reconstituted formulations, are administered to a person in need thereof by intramuscular, intraperitoneal (i.e., within a body cavity), intracerebrospinal, or subcutaneous injection using a 18-32 gauge needle (optionally a thin-walled needle), in a volume of less than about 5 mL, less than about 3 mL, preferably less than about 2 mL, more preferably less than which is about 1 mL.

[00239] The appropriate dosage ("therapeutically effective amount") of the protein, such as a mAb, will depend on the condition being treated, the severity and evolution of the disease or condition, whether the protein is administered for preventive purposes or therapy, prior therapy, the patient's clinical history and response to the protein, the type of protein used, and the discretion of the attending physician. The protein is suitably administered at once, in single or multiple injections, or through a series of treatments, as the sole treatment or in conjunction with other drugs or therapies.

[00240] Dosage formulations are designed such that injections cause no significant signs of irritation at the injection site, for example, where the primary irritation index is less than 3 when evaluated using a Draize scoring system . In an alternative embodiment, the injections cause macroscopically similar levels of irritation when compared to injections of equivalent volumes of saline. In another embodiment, the bioavailability of the protein is higher when compared to the same formulation otherwise without the viscosity-reducing ionic liquid(s) administered in the same manner. In another embodiment, the formulation is at least approximately as pharmaceutically effective as the same dose of protein administered via intravenous infusion.

[00241] In a preferred embodiment, the formulation is injected to obtain 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 calculated otherwise, even the formulation without the ionic reducing liquid(s) of viscosity(s) administered in the same way.

[00242] The viscosity lowering agent can also affect bioavailability. For example, the percent bioavailability of the protein may be at least 1.1 times, preferably at least 1.2 times the percent bioavailability of the formulation otherwise even without the viscosity-reducing ionic liquid administered in the same form.

[00243] The viscosity reducing agent can also affect pharmacokinetics. For example, the Cmax following SC or IM injection may be at least 10%, preferably at least 20%, less than the Cmax of an approximately equivalent pharmaceutically effective intravenously administered dose.

[00244] In some embodiments, the proteins are administered at a higher dosage and a lower frequency than the same formulations otherwise without the viscosity-reducing ionic liquid(s).

[00245] Lower viscosity formulations require less injection force. For example, the injection force may be at least 10%, preferably at least 20%, lower than the injection force for the same formulation otherwise without the viscosity-reducing ionic liquid administered in the same form. 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.

[00246] The viscosity reducing ionic liquid can be used to prepare a dosage unit form suitable for reconstitution to make 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 ionic viscosity reducing liquids; 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 2000 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.

[00247] 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 lowering agent and the other excipients, and the solvent, with or without the viscosity reducing agent and other excipients, is supplied in a second bottle. In this embodiment, the solvent is added to the protein immediately before or at the time of injection to ensure uniform mixing and dissolution.

[00248] The viscosity-reducing ionic liquid(s) are present in the formulations at concentrations that cause no significant signs of toxicity and/or no signs of irreversible toxicity when administered subcutaneously, intramuscularly, or other types of injection. As used in the present invention, "significant signs of toxicity" include intoxication, lethargy, behavioral changes such as those occurring with damage to the central nervous system, infertility, signs of severe cardiotoxicity such as cardiac arrhythmia, cardiomyopathy, myocardial infarction , cardiac and/or congestive failure, kidney failure, liver failure, respiratory distress and death.

[00249] In preferred embodiments, the formulations do not cause significant irritation when administered no more than twice a day, once a day, twice a week, once a week or once a month. Protein formulations can be administered without causing significant signs of irritation at the injection site, as measured by a primary irritation index of less than 3, less than 2, or less than 1 when assessed using a Draize scoring system. . As used in the present invention, "significant signs of irritation" include erythema, redness and/or swelling at the injection site that has 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 macroscopically protein formulations cause similar levels of irritation when compared to injections of equivalent volumes of saline.

[00250] Protein formulations may exhibit greater bioavailability compared to the same formulation otherwise than protein without the viscosity-reducing ionic liquid(s) when administered subcutaneously or intramuscularly. "Bioavailability" refers to the extent and rate at which bioactive species, such as a mAb, reach the circulation or site of action. Total bioavailability can be increased by SC or IM injections, compared to the same formulations otherwise without the viscosity-reducing ionic liquid(s). "Percent bioavailability" refers to the fraction of the administered dose of the bioactive species that enters the 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 graph of plasma concentration as a function of time. The AUC can be calculated, for example, using the linear trapezoidal rule. "AUC~", as used in the present invention, refers to the area under a plasma concentration curve from time zero to a time at which the plasma concentration returns to baseline levels. "AUC 0-t", as used in the present invention, refers to the area under the plasma concentration curve from time zero to a later time, t, for example, to the time of reaching the threshold. base. Time will typically be measured in days, although hours may also be used, as will be evident from the context. For example, the AUC may be increased by more than 10%, 20%, 30%, 40%, or 50% compared to the otherwise same formulation without the viscosity-reducing ionic liquid(s) and administered in the same way.

[00251] As used in the present invention, "tmax" refers to the time after administration at which the plasma concentration reaches a maximum.

[00252] As used in the present invention, "Cmax" means the maximum concentration in plasma after administration of the dose, and before administration of a subsequent dose.

[00253] As used in the present invention, "Cmin" or "Catravés" refers to the minimum concentration in plasma after administration of the dose, and before administration of a subsequent dose.

[00254] The Cmax after SC or IM injection may be lower, for example, at least 10%, more preferably at least 20%, less than the Cmax of a dose administered intravenously. This reduction in Cmax may also result in decreased toxicity.

[00255] Pharmacokinetic and pharmacodynamic parameters can be approximated across species using approaches that are known to those skilled in the art. The pharmacokinetic and pharmacodynamic parameters of therapeutic antibodies 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, whereas a human mAb will typically have a half-life of ~25 days (Waldmann et al, Int Immunol, 2001, 13:1551 to 1559) . The pharmacokinetics and pharmacodynamics of therapeutic antibodies may differ markedly based on the route of administration. The time to reach maximum plasma concentration after IM or SC injection of IgG generally ranges from 2 to 8 days, although shorter or longer times may be encountered (Wang et al, Clin Pharm Ther, 2008, 84(5): 548 to 558). The pharmacokinetics and pharmacodynamics of labeled therapeutic antibodies may differ based on formulation.

[00256] Low viscosity protein formulations can allow for greater flexibility in dosing and decreased dosing frequency compared to protein formulations without the viscosity reducing ionic liquid(s). For example, by increasing the dosage administered by injecting 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 syringe or autoinjector and/or self-mixing. Protein formulations can also be preheated in a separate heating unit prior to filling the syringe.

I. Heated syringes

[00257] The syringe can be heated using a standard syringe that is preheated using a heated syringe. The warmer syringe will generally have one or more openings, each capable of receiving a syringe containing the protein formulation and a means for heating and maintaining the syringe at a specific temperature (typically above room temperature) prior to use. This will be referred to in the present invention as a preheated syringe. Suitable heated syringe warmers include those available from Vista Dental Products and Inter-Med. The heaters are capable of accommodating various syringe 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 water bath maintained at the desired temperature.

[00258] The syringe can be heated using a self-heating syringe, this is capable of heating and maintaining the liquid formulation inside the syringe at a specific temperature. The self-heating syringe may also be a standard medical syringe having a heating device attached to it. Suitable heating devices capable of being attached to a syringe include syringe heaters or a syringe heating strip available from Watlow Electric Manufacturing Co. of St. Louis, MO, and syringe heating blocks, phase heaters, and in the infusion line of warmers available from Warner Instruments of Hamden, CT, such as the SW-61 model syringe warmer. The heater can be controlled via a central controller, for example the TC-324B or TC-344B heater controller models available from Warner Instruments.

[00259] 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 this 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 reduced, the solubility of the protein in the formulation is increased, or both.

II. Self-mixing syringes

[00260] 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 US 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 US Patent Nos. 6,443,612 and 6,457,609, as well as US Patent Application Publication No. US 2002/0190082. The syringe may include multiple barrels for mixing the components of the liquid protein formulation. US Patent No. 5,819,998 describes syringes with two barrels and a mixing tip for mixing two-component viscous substances.

iii.Autoinjectors and pre-filled syringes of protein formulations

[00261] The liquid protein formulation can be administered using a prefilled syringe autoinjector or a needleless injection device. Autoinjectors include a handheld, often pen-type, cartridge holder for holding replaceable prefilled cartridges, and a base spring or similar mechanism for subcutaneous or intramuscular injections of liquid drug dosages from a cartridge. pre-filled. Autoinjectors are typically designed for self-administration or administration by untrained personnel. Autoinjectors are available to dispense single dosages or multiple doses from a prefilled cartridge. Autoinjectors allow for different user configurations, including, inter alia, injection depth, injection speed, and the like. Other injection systems may include those described in US Patent No. 8,500,681.

[00262] The lyophilized protein formulation can be supplied in pre-filled syringes or unit doses. US Patent No. 3,682,174; 4,171,698; and 5,569,193 describe sterile syringes containing two chambers, which can be pre-loaded with a dry formulation and a liquid that can be mixed immediately before injection. US Patent No. 5,779,668 describes a syringe system for lyophilization, reconstitution and administration of a pharmaceutical composition. In some embodiments, the protein formulation is provided in lyophilized form in a pre-filled or single-dose syringe, reconstituted in the syringe prior to administration, and administered as a single subcutaneous or intramuscular injection. Autoinjectors for delivering lyophilized unit dose drugs are described in WO 2012/010.832. Auto injectors such as the Safety Click Lyo™ (marketed by Future Injection Technologies, Ltd., Oxford, UK) can be used to administer a unit dose formulation of the protein in which the formulation is stored in lyophilized form and reconstituted. immediately before administration. In some embodiments the protein formulation is provided in unit dose cartridges for lyophilized medicines (sometimes referred to as Vetter CARTRIDGES). Examples of suitable cartridges may include those described in US Patent Nos. 5,334,162 and 5,454,786.

V. Purification and concentration methods

[00263] Viscosity-reducing ionic liquids can also be used to help with protein purification and concentration. The ionic viscosity reducing liquid(s) and excipients are added to the protein in an amount effective to reduce the viscosity of the protein solution. For example, the viscosity lowering agent is added at a Tween concentration of about 0.01 M and about 1.0 M, preferably between about 0.01 M and about 0.50 M, and more preferably between about 0.01 M and about 0.25 M.

[00264] The liquid solution containing the viscosity-reducing ionic protein is then purified or concentrated using a method selected from the group consisting of ultrafiltration/diafiltration, tangential flow filtration, centrifugal concentration, and dialysis.

Examples

[00265] The foregoing will be better understood through the following non-limiting examples.

[00266] All viscosities of the well-mixed aqueous mAb solutions were measured using both an mVROC microfluidic viscometer (RheoSense) and a DV2T cone and plate viscometer (Brookfield; "C&P") after a 5 minute equilibration at 25°C (except where indicated). 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 again loaded into a Microlab gas-tight instrument syringe (Hamilton; 100 mL), affixed to the chip, and measured at multiple flow rates of approximately 20%, 40%, and 60%. % of maximum pressure for each chip. For example, a sample of approximately 50 cP would be measured at about 10, 20, and 30 uL/min (about 180, 350, and 530 s-1, respectively, by an "A" chip) until the viscosity stabilizes, typically after less than 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 samples were measured at various shear speeds 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, from a shear rate that gave torque at least 10%, and continued until the binary instrument reached 100%. An extrapolated zero shear viscosity was then determined from a plot of viscosity versus dynamic shear rate for the measurement samples on a DV2T cone and plate viscometer. The reported extrapolated zero shear viscosities are the mean and standard deviation of at least three measurements.

Example 1: Ionic liquids reduce the viscosity of concentrated aqueous solutions of similar biological product AVASTIN® Materials and methods

[00267] A similar commercially obtained AVASTIN® biological product containing pharmaceutical excipients (Polysorbate 20, phosphate and citrate buffers, mannitol, and NaCl) was purified. First, Polysorbate 20 was removed using DETERGENTOUT® TWEEN Medi columns (G-Biosciences). Then, the resulting solutions were extensively buffer exchanged into 20 mM sodium phosphate buffer (PB) or 20 mM viscosity-reducing ionic liquid solutions and concentrated to a final volume of less than 10 ml in concentrators. Jum-bosep centrifuges (Pall Corp.). For samples containing 4-ethyl-4-methyl methylmorpholinium carbonate (EMMC), the protein was carefully exchanged into outlet buffer in 2 mM PB (pH 7.0). For buffer-exchanged samples to 20 mM PB (PB control samples) or 20 mM viscosity-reducing ionic liquid, the collected protein solution was freeze-dried. The dried protein cakes, containing the protein salts and ionic or viscosity-reducing liquid buffer, were reconstituted to a final volume of about 0.10 to 1.30 mL. These samples were reconstituted using additional PB (pH 7.0) or the ionic viscosity reducing liquid (pH 7.0), as appropriate, sufficient to bring the final concentration of PB to 0.25 M and the final concentration of viscosity reducing ionic liquid as indicated in the tables below. Samples exchanged in 2 mM PB buffer were aliquoted first. Next, an appropriate amount of viscosity reducing ionic liquid solution (pH 7.0) was added to each aliquot such that after reconstitution with water, the final excipient concentration was 0.1 to 0. 5 M. The protein solutions were then lyophilized. The dried protein cakes, containing protein and the ionic viscosity-reducing liquid (and a small amount of buffer salts) were reconstituted to a final volume of approximately 0.1 ml and the ionic viscosity-reducing liquid as indicated. in the tables below. The final mAb concentration in solution was determined via light absorbance at 280 nm using an experimentally determined extinction coefficient of 1.7 L/g • cm and reported viscosities were measured on a RheoSense mVROC microfluidic viscometer.

Results

[00268] The data in Table 1 demonstrate that the viscosity of aqueous solutions of the similar biological product AVASTIN® can be reduced by up to 6.5 times (compared to phosphate buffered samples) in the presence of ionic liquids of 0.20-0.50 M of reduction of viscosity. Viscosities greater than 200 cP in the absence of viscosity-reducing ionic liquids were reduced to less than 50 cP by adding 0.20-0.50 M viscosity-reducing ionic liquids. It can be seen that in this example the magnitude of viscosity reduction is in some cases dependent on the concentration of the viscosity reducing ionic liquid. The viscosity reduction increases (i.e., viscosity decrease) with increasing viscosity-reducing ionic liquid concentration. Table 1. The viscosities of aqueous solutions of similar biological product Avastin®, in the presence of various concentrations of ionic liquids ("ILs") at 25°C and pH 7. * PB = phosphate buffer; BIM = 4-(3-butyl-1-imidazolium)sulfonate-1-butane; BMI Mes = 1-butyl-3-methylimidazolium methanesulfonate; BMP = 1-Butyl-1-methylpyrrolidinium chloride; EMMC = 4-ethyl-4-methylmorpholinium methyl carbonate.

Example 2: Ionic liquids reduce the viscosity of concentrated aqueous solutions of the similar biological product RITUXAN® Materials and methods

[00269] The commercially obtained RITUXAN® similar biological product containing the pharmaceutical excipients (citrate buffer, NaCl, and Tween 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described above in Example 1 (using the extinction coefficient of 1.7 L/g-cm). Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip.

Results

[00270] The data in Table 2 demonstrate that the viscosity of aqueous solutions of the similar biological product of RITUXAN® can be reduced by up to 8.5 times in the presence of ionic liquids of 0.400.50 M of the viscosity reducing ionic liquids, in comparison with PB samples. Table 2. Viscosities (in cP) of aqueous solutions of the similar biological product RITUXAN®, in the presence of various concentrations of ionic liquid BIM, at 25°C and pH 7. PB = phosphate buffer; BIM = 4-(3-butyl-1-imidazolium)sulfonate-1-butane; nd = Not determined.

Example 3: Ionic liquids reduce the viscosity of concentrated aqueous solutions of Tysabri® Materials and methods

[00271] TYSABRI® containing commercially obtained pharmaceutical excipients (phosphate buffer, NaCl, Polysorbate 80) was buffer exchanged, concentrated, dried, reconstituted, and analyzed as described above in Example 1 (using the extinction coefficient of 1.5 L/g •cm). Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip.

Results

[00272] The data in Table 3 demonstrate that the viscosity of TYSABRI® aqueous solutions can be reduced by up to 7 times in the presence of 0.10 M EMMC. Table 3. The viscosities (in cP) of Tysabri® aqueous solutions in the presence of various excipients at 25°C and pH 7. PB = phosphate buffer; Arg-HCl = Arginine-HCl; BIM = 4-(3-butyl-1-imidazolium)sulfonate-1-butane; BMI Mes = 1-butil-3-methylimidazolium methanesulfonate; BMP chloride = 1-Butyl-1-methyl-pyrrolidinium chloride Cl; EMMC = 4-ethyl-4-methylmorpholinium methyl carbonate.

Example 4: 4-(3-butyl-1-imidazolium)-1-butane sulfonate reduces the viscosity of VECTIBIX® REMICADE® concentrate solutions Materials and methods

[00273] REMICADE® containing commercially obtained pharmaceutical excipients (sucrose, polysorbate 80, sodium phosphate buffer) was prepared according to the instructions in the prescribing information sheet. Commercially obtained VECTIBIX® containing pharmaceutical excipients was prepared according to the instructions in the prescribing information sheet. Subsequently, the aqueous protein drug products were purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described above in Example 1 (using the extinction coefficients: 1.4 L/g cm for REMICADE and 1.25 L/g • cm for VECTIBIX®). Proteins were formulated with either phosphate buffer or 0.50 M 4-(3-butyl-1-imidazolium)-1-butane sulfonate (BIM). Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip.

Results

[00274] The results in Table 4 demonstrate that BIM is effective in reducing the viscosity of concentrated aqueous solutions of both mAbs tested. Viscosity reductions with 0.50 M BIM are up to 22 times in the proteins examined in the present invention. Table 4. Viscosities (in cP) of aqueous solutions of REMICADE® and Vecti-BIX® at 25°C and pH 7 with and without BIM.

Example 5: Ionic liquids reduce the viscosity of concentrated aqueous solutions of HERCEPTIN® Materials and methods

[00275] Commercially obtained HERCEPTIN® containing pharmaceutical excipients (histidine buffer, trehalose, Polysorbate 20) was prepared according to the instructions in the prescribing information sheet. The aqueous product was buffer exchange protein drug, concentrated, dried, reconstituted, and analyzed as described above in Example 1 (using the extinction coefficient of: 1.5 L/g-cm). The protein was formulated with either phosphate buffer or various viscosity-reducing ionic liquids relative to the concentration in the table listed below. Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip.

[00276] The results in Table 5 demonstrate that viscosity reducing ionic liquids are effective in reducing the viscosity of concentrated aqueous solutions of HERCEPTIN®. EMMC can reduce viscosity by almost three times when present at 0.10 M. Table 5. The viscosities of aqueous solutions of HERCEPTIN® in the presence of various concentrations of ionic liquids (IL) at 25°C and pH 7. PB = phosphate buffer; BIM = 4-(3-butyl-1-imidazolium)sulfonate-1-butane; BMI Mes = 1-butyl-3-methylimidazolium methanesulfonate; BMP chloride = 1-butyl-1-methylpyrrolidinium chloride; EMMC = 4-ethyl-4-methylmorpholinium methyl carbonate.

Example 6: Dependence of the viscosity reduction effect on the concentration of ionic liquid for aqueous solutions of the similar biological product of ERBITUX®.

[00277] The similar commercially obtained ERBITUX® biological product containing pharmaceutical excipients (phosphate buffer, sodium chloride, Polysorbate 80) was buffer exchanged, concentrated, dried, reconstituted, and analyzed as described above in Example 1 (using the coefficient extinguishing capacity: 1.4 L/g •cm). The protein was formulated with both phosphate buffer and various concentrations of BIM. Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip.

[00278] The results in Table 6 demonstrate that the BIM viscosity reducing ionic liquid is effective in reducing the viscosity of concentrated aqueous solutions of the similar biological product Erbitux® in a dose-dependent manner up to about 0.50 M, where at a certain point, the effect of BIM becomes less and less effective. This demonstrates that in some embodiments there is an optimal concentration of viscosity-reducing ionic liquid. Table 6. The viscosities of aqueous solutions of the similar biological product Erbitux®, in the presence of various concentrations of BIM at 25°C and pH 7.

Example 7. Reducing viscosity shows no signs of toxicity when injected subcutaneously

[00279] Thirty 11-week-old Sprague-Dawley rats were divided into 6 groups of 5 rats each (3 saline control groups and 3 BIM groups). Rats were injected subcutaneously with 0.5 mL of free endotoxin, either 0.25 M phosphate-buffered saline or BIM according to the following scheme: one group of each condition was injected once on day 1 and then then sacrificed an 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.

[00280] Clinical observations were recorded for any toxic-pharmacological signs at pre-dosing, immediately after dosing, at 1 and 4 hours (± 15 minutes) after dosing, and daily thereafter. Irritation, if applicable, at injection sites was scored using the Draize assessment score from pre-dose, immediately after administration, to 1 hour (±15 minutes) post-dose, and before sacrifice.

[00281] In general, the observed consequences of saline and BIM injections were macroscopically similar throughout the course of the study. Both induced from no irritation to mild irritation with edema scores from 0 to 2 at various time points. The appearance of a slight irritation appeared to occur after the second subcutaneous injection of control and BIM. Microscopic examination of injection sites suggests a very minimal, clinically insignificant, irritative effect with BIM that was no longer evident by day 4.

[00282] Unless expressly defined otherwise above, all technical and scientific terms used in the present invention have the same meanings as commonly understood by a person skilled in the art. The person skilled in the art will recognize, or be able to determine using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (17)

1. Liquid pharmaceutical formulation for injection, characterized by the fact that it comprises: (i) an antibody; (ii) one or more ionic viscosity reducing liquids, comprising: 0.2 M to 0.5 M 4-(3-butyl-1-imidazolium)-1-butane sulfonate (BIM) or a pharmaceutically acceptable salt thereof, 0.4 M to 0.5 M 1-butyl-3-methylimidazolium methanesulfonate (BMI MES) or a pharmaceutically acceptable salt thereof, 0.4 M to 0.5 M 1-butyl-chloride 1-methylpyrrolidinium (BMP chloride) or a pharmaceutically acceptable salt thereof, 0.1 M to 0.4 M 1-ethyl-1-methylmorpholinium methylcarbonate (EMMC) 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, measured using a cone and plate 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 pharmaceutically acceptable solvent, but without the one or more viscosity-reducing ionic liquids; where absolute viscosity is a shear viscosity extrapolated from zero. 2. Formulation according to claim 1, characterized in that the antibody comprises a monoclonal antibody. 3. Formulation according to claim 1 or 2, characterized by the fact that the one or more viscosity reducing ionic liquids comprise 0.2 M to 0.5 M 4-(3-butyl-1- imidazolium)-1-butane (BIM) or a pharmaceutically acceptable salt thereof. 4. Liquid pharmaceutical formulation according to claim 1 or 2, characterized in that the one or more viscosity-reducing ionic liquids comprise 0.4 M to 0.5 M 1-butyl-3-methylimidazolium methanesulfonate (BIM Mes) or a pharmaceutically acceptable salt thereof. 5. Liquid pharmaceutical formulation according to claim 1 or 2, characterized in that the one or more ionic viscosity reducing liquids comprise 0.4 M to 0.5 M 1-butyl-1-methylpyrrolidinium chloride (BMP Chloride) or a pharmaceutically acceptable salt thereof. 6. Liquid pharmaceutical formulation according to claim 1 or 2, characterized in that the one or more ionic viscosity reducing liquids comprise 0.1 M to 0.4 M 1-ethyl-1- methyl carbonate methylmorpholinium (EMMC) or a pharmaceutically acceptable salt thereof. 7. Liquid pharmaceutical formulation according to any one of claims 1 to 6, characterized by the fact that the antibody has a molecular weight of 120 kDa to 250 kDa. 8. Liquid pharmaceutical formulation according to any one of claims 1 to 7, characterized in that it comprises from 100 mg/mL to 300 mg/mL of antibody. 9. Liquid pharmaceutical formulation according to any one of claims 1 to 8, characterized in that it comprises from 180 mg/mL to 232 mg/ml of antibody. 10. Liquid pharmaceutical formulation according to any one of claims 1 to 9, characterized in that the liquid pharmaceutical formulation is aqueous. 11. Liquid pharmaceutical formulation according to any one of claims 1 to 10, characterized in that it further comprises one or more pharmaceutically acceptable excipients comprising a sugar, sugar alcohol, buffering agent, preservative, carrier, antioxidant, chelating agent, natural polymer, synthetic polymer, cryoprotectant, lyoprotectant, surfactant, thickening agent, stabilization agent, or any combination thereof. 12. Liquid pharmaceutical formulation according to claim 11, characterized by the fact that the one or more of the excipients is a polysorbate, poloxamer 188, sodium lauryl sulfate, a polyol, a poly (ethylene glycol), glycerol, a propylene glycol , or a poly (vinyl alcohol). 13. Liquid pharmaceutical formulation according to claim 11, characterized in that the sugar alcohol is sorbitol or mannitol. 14. Liquid pharmaceutical formulation according to any one of claims 1 to 13, characterized by the fact that it is a unit dose vial, cartridge, container, or pre-filled syringe. 15. Liquid pharmaceutical formulation according to any one of claims 1 to 14, characterized in that the liquid pharmaceutical formulation is isotonic with human blood serum. 16. Formulation according to any one of claims 1 to 15, 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 cone and plate, or a shear velocity of at least 1.0 s-1 when measured using a microfluidic viscometer. 17. Use of 4-(3-butyl-1-imidazolium)-1-butane sulfonate (BIM), 1-butyl-3-methylimidazolium methanesulfonate (BIM Mes), 1-butyl-1-methylpyrrolidinium chloride (Chloride BMP), 1-ethyl-1-methylmorpholinium methyl carbonate (EMMC), or a pharmaceutically acceptable salt thereof, characterized in that it is for manufacturing a subcutaneous or intramuscular liquid pharmaceutical formulation, as defined in any one of claims 1 to 16.