NZ756401B2 - Liquid protein formulations containing ionic liquids - Google Patents

Abstract

Concentrated, low-viscosity, low-volume liquid pharmaceutical formulations of antibodies comprising 4-(3-butyl-l-imidazolio)-1-butane sulfonate (BIM) have been developed. Such formulations can be rapidly and conveniently administered by subcutaneous or intramuscular injection, rather than by lengthy intra-venous infusion. intra-venous infusion.

Description

/055245 LIQUID PROTEIN FORMULATIONS CONTAINING IONIC LIQUIDS CROSS-REFERENCE TO D APPLICATIONS This application claims priority to and the benefit ofUS Provisional Application No. 62/030,521, filed July 29, 2014, entitled “Low- Viscosity Protein Formulations Containing Hydrophobic Salts; ” U.S. Provisional Application No. 62/026,497, filed July 18, 2014, entitled “Low- Viscosity Protein Formulations Containing GRAS Viscosity-Reducing ; ” U.S. Provisional Application No. 62/008,050, filed June , 2014, entitled “Low- ity Protein Formulations Containing Ionic Liquids; ” U.S. Provisional ation No. ,005, filed May 2, 2014, entitled “Low— Viscosity Protein Formulations Containing Organophosphates,” U.S. Provisional Application No. 61/946,436, filed February 28, 2014, entitled “Concentrated, Low— Viscosity Infliximab Formulations; ” US Provisional Application No. 61/943,197, filed February 21, 2014, entitled ntrated, Low~ Viscosity, High-Molecular~ Weight-Protein Formulations; ” U.S. Provisional Application No. 61/940,227, filed February 14, 2014, entitled “Concentrated, Low- Viscosity High~Molecular~Weight Protein Formulations; " and U.S. Provisional ation No. 61,876,621, filed September 11, 2013, entitled “Concentrated, Low-Viscosity, High-Molecular- Weight Protein Formulations, ” the disclosures of which are expressly incorporated hereby by reference.

FIELD OF THE INVENTION The invention is generally in the field of injectable low-viscosity pharmaceutical formulations of highly concentrated proteins and methods of making and using f.

BACKGROUND OF THE INVENTION Monoclonal antibodies (mAbs) are important protein-based therapeutics for ng various human diseases such as cancer, ious diseases, inflammation, and autoimmune diseases. More than 20 InAb products have been approved by the U.S.

Food and Drug Administration (FDA), and approximately 20% of all biopharmaceuticals currently being evaluated in al trials are mAbs (Daugherty et al, Adv. Drug Deliv. Rev. 58:686-706, 2006; and Buss et al., Curr. Opinion in Pharmacol. 12:615-622, 2012). mAb-based therapies are usually administered repeatedly over an extended period of time and e several mg/kg dosing. Antibody solutions or suspensions can be administered via parenteral routes, such as by intravenous (IV) infusions, and subcutaneous (SC) or intramuscular (IM) injections. The SC or IM routes reduce the treatment cost, increase patient compliance, and improve convenience for patients and healthcare ers during stration compared to the IV route. To be effective and pharmaceutically acceptable, eral formulations should preferably be sterile, stable, inj ectable (e.g., via a syringe), and non-irritating at the site of injection, in compliance with FDA guidelines. Because ofthe small volumes required for subcutaneous (usually under about 2 mL) and intramuscular (usually under about 5 mL) injections, these routes of administration for high—dose protein therapies require concentrated protein solutions. These high concentrations often result in very s formulations that are difficult to administer by injection, cause pain at the site of injection, are often imprecise, and/or may have sed chemical and/or physical stability.

These characteristics result in manufacturing, storage, and usage requirements that can be challenging to achieve, in particular for formulations having high concentrations of high-molecular-weight ns, such as mAbs. All protein therapeutics to some extent are subject to physical and chemical instability, such as aggregation, denaturation, crosslinking, deamidation, isomerization, oxidation, and clipping (Wang 9161]., J. Pharm. Sci. 6, 2007). Thus, optimal formulation development is paramount in the development of commercially viable n pharmaceuticals.

High protein concentrations pose challenges relating to the physical and al stability of the protein, as well as lty with manufacture, storage, and delivery ofthe protein formulation. One problem is the cy of proteins to aggregate and form particulates during processing and/or storage, which makes manipulations during further processing and/or ry difficult. Concentrationdependent degradation and/or aggregation are major challenges in developing protein formulations at higher concentrations. In addition to the potential for non-native protein ation and particulate formation, reversible self-association in aqueous soiutions may occur, which contributes to, among other things, increased viscosity that complicates delivery by injection. (See, for example, Steven J. Shire et al, J.

Pharm. Sci. 93:1390—1402, 2004.) Increased viscosity is one of the key challenges encountered in concentrated protein compositions affecting both production processes and the ability to readily deliver such compositions by conventional means. (See, for example, J. Jezek at (.11., Advanced Drug Delivery Reviews 63:1107—1117, 2011.) Highly Viscous liquid formulations are difficult to manufacture, draw into a syringe, and inject aneously or intramuscularly. The use of force in manipulating the viscous formulations can lead to excessive ng, which may further denature and inactivate the eutically active protein. High viscosity solutions also require larger diameter s for injection and produce more pain at the inj ection site. tly available cial mAb products administered by SC or IM injection are usually formulated in aqueous buffers, such as a phosphate or L—histidine buffer, with ents or surfactants, such as ol, sucrose, lactose, ose, POLOXAMER® (nonionic triblock copolymers composed of a central hydrophobic chain ofpolyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene )) or POLYSORBATE® 80 - -- - 0)sorbitan monolaurate), to prevent aggregation and improve stability.

Reported antibody concentrations formulated as described above are typically up to about 100 mg/mL (Wang et al., J Pharm. Sci. 9621-26, 2007).

U.S. Patent No. 7,758,860 bes reducing the Viscosity in ations of low-molecular-weight proteins using a buffer'and a viscosity—reducing inorganic salt, such as calcium chloride or magnesium chloride. These same salts, however, showed little effect on the viscosity of a high-molecular-weight antibody (lMA-63 8) ' formulation. As described in U.S. Patent No. 7,666,413, the viscosity of aqueous formulations ofhigh-moleculafiweight proteins has been reduced by the addition of such salts as arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium de, calcium chloride, zinc chloride, or sodium acetate in a concentration of greater than about 100 mM or, as described in U.S.

Patent No. 7,740,842, by addition of organic or inorganic acids. However, these salts do not reduce the ity to a desired level and in some cases make the formulation so acidic that it is likely to cause pain at the site of inj ection.

U.S. Patent No. 7,666,413 describes reduced-viscosity formulations containing WO 38811 specific salts and a reconstituted anti-IgE mAb, but with a maximum antibody concentration of only up to about 140 mg/mL. U.S. Patent No. 7,740,842 describes E25 antinIgE mAb formulations containing acetate/acetic acid buffer with antibody concentrations up to 257 mg/mL. The on of salts such as NaCl, CaClg, or MgClz was demonstrated to decrease the dynamic viscosity under high—shear conditions; however, at low-shear the salts ed an undesirable and dramatic increase in the dynamic viscosity. Additionally, inorganic salts such as NaCl may lower solution viscosity and/or decrease aggregation (EP 1981824).

Non-aqueous antibody or protein formulations have also been described.

W02006/O71693 describes a non-aqueous sion of up to 100 mg/mL mAb in a formulation having a viscosity enhancer (polyvinylpyrrolidone, PVP) and a solvent (benzyl te or PEG 400). WO2004/089335 describes 100 mg/mL non~aqueous lysozyrne suspension formulations containing PVP, glycofurol, benzyl benzoate, benzyl alcohol, or PEG 400. U82008/0226689A1 describes 100 mg/mL human growth hormone (hGH) single phase, three vehicle component (polymer, surfactant, and a solvent), non-aqueous, viscous formulations. U.S. -No. 6,730,328 describes non-aqueous, hydrophobic, non—polar vehicles of low reactivity, such as perfluorodecalin, for protein ations. These formulations are non-optimal and have high viscosities that impair processing, manufacturing and injection; lead to the presence of multiple e components in the ations; and t ial regulatory challenges associated with using rs not yet approved by the FDA.

Alternative non-aqueous protein or antibody formulations have been described using organic solvents, such as benzyl benzoate (Miller et al., Langmuir 26: 1067- 1074, 2010), benzyl acetate, ethanol, or methyl ethyl ketone (Srinivasan et all, Pharm.

Res. 30:1749-1757, 2013). In both instances, viscosities of less than 50 centipoise (cP) were achieved upon formulation at protein rations of at least about 200 mg/mL. U.S. Patent No. 6,252,055 describes mAb formulations with concentrations ranging fi'om 100 mg/mL up to 257 mg/InL. Formulations with concentrations greater than about 189 mg/InL demonstrated dramatically increased viscosities, low ry rates, and difficulty in processing. U.S. Patent Application Publication No. 2012/0230982 describes antibody formulations with concentrations of 100 mg/mL to 200 mg/mL. None ofthese ations are low enough viscosity for ease of injection.

Du and Klibanov (Biotechnology and ineering 108:632-636, 2011) described reduced viscosity of concentrated aqueous solutions of bovine serum n with a maximum concentration up to 400 mg/mL and bovine gamma globulin with a maximum concentration up to 300 . Guo et al.

(Pharmaceutical Research 29:3102-3109, 2012) described low—viscosity s solutions of four model mAbs achieved using hydrophobic salts. The mAb formulation employed by Guo had an l viscosity, prior to adding salts, no greater than 73 cP. The viscosities of many phannaceutically important mAbs, on the other hand, can exceed 1,000 cP at therapeutically relevant concentrations.

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

The references cited above demonstrate that while many groups have attempted to prepare low-viscosity ations ofmAbs and other therapeutically important proteins, a truly useful formulation for many proteins has not yet been ed. Notably, many ofthe above reports employ agents for which safety and toxicity profiles have not been fully established. These formulations would therefore face a higher regulatory burden prior to approval than formulations containing compounds known to be safe. Indeed, even if a compound were to be shown to ntially reduce viscosity, the compound may ultimately be unsuitable for use in a formulation ed for injection into a human.

Many pharmaceutically important high-molecular-weight proteins, such as mAbs, are currently administered via IV infusions in order to deliver therapeutically effective amounts ofprotein 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 required.

It is, therefore, an object of the present invention to provide trated, low— viscosity liquid formulations of pharmaceutically important proteins, especially high- molecular-weight proteins, such as mAbs.

It is a further object of the present invention to provide concentrated low- viscosity liquid formulations of proteins, especially high-molecular—weight proteins, such as mAbs, capable of delivering therapeutically effective amounts ofthese proteins in volumes useful for SC and 1M injections.

It is a further object of the present invention to provide the concentrated liquid formulations of proteins, ally olecular-weight proteins, such as mAbs, with low viscosities that can improve inj ectability and/or patient compliance, convenience, and comfort.

It is also an object of the present invention to e methods for making and storing concentrated, low-viscosity formulations of ns, especially high- molecular—weight proteins, such as mAbs.

It is an additional obj ect of the present invention to provide methods of stering low-viscosity, concentrated liquid formulations of proteins, especially high-molecular-weight proteins, such as mAbs.

It is an additional object of the present invention to provide methods for processing reduced-viscosity, high-concentration biologics with concentration and ion techniques known to those d in the art.

SUMMARY OF THE INVENTION Concentrated, scosity, low—volume liquid pharmaceutical formulations of proteins have been developed. Such formulations can be rapidly and conveniently administered by aneous or intramuscular injection, rather than by lengthy intravenous infusion. These formulations include low-molecular-weight and/or high- molecular—weight proteins, such as mAbs, and viscosityureducing ionic liquids.

The concentration ofproteins is between about 10 mg/mL and about 5,000 mg/mL, more preferably from about 100 mg/mL to about 2,000 . In some ments, the concentration of proteins is between about 100 mg/mL to about 500 mg/mL, more preferably from about 300 mg/mL to about 500 mg/mL. Formulations containing proteins and viscosity-reducing 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 most ably at least three months. The viscosity of the formulation is less than about 75 CF, preferably below 50 CF, and most preferably below 20 cP at about 25° C.

In some embodiments, the viscosity is less than about 15 cP or even less than or about 10 cP at about 25° C. In certain embodiments, the viscosity of the formulation is about 10 cP.

Formulations ning proteins and ionic liquids typically are measured at shear rates from about 0.6 s-1 to about 450 s-1, and preferably from about 2 s-1 to about 400 s-1, when measured using a cone and plate viscometer. ations containing proteins and viscosity-reducing ionic liquids typically are measured at shear rates from about 3 s-1 to about 55,000 s-1, and preferably from about 20 s-1 to about 2,000s-1,when measured using a microfluidic viscometer.

The viscosity of the protein formulation is reduced by the presence of one or more viscosity-reducing ionic liquid(s). Unless specifically stated otherwise, the term “ionic liquid” includes both single nds and mixtures of more than one ionic liquid. It is preferred that the viscosity-reducing ionic liquid(s) is present in the formulation at a concentration less than about 1.0 M, preferably less than about 0.50 M, more ably less than about 0.30 M, and most preferably less than about 0.15 M. The formulations can have a viscosity that is at least about 30% less, preferably at least about 50% less, most preferably at least about 75% less, than the viscosity of the ponding formulation under the same conditions except for replacement of the ity-reducing ionic liquid with an appropriate buffer or salt of about the same concentration. In some embodiments, a low-viscosity formulation is provided where 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 above about 1,000 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 microfluidic viscometer.

The pharmaceutically acceptable liquid formulations contain one or more ionic s in an ive amount to icantly reduce the viscosity of the protein, e.g., mAb formulation. Representative ionic liquids include 4-(3-butylimidazolio)butane sulfonate (BIM), 1-butylmethylimidazolium methanesulfonate (BMI Mes), 4-ethyl methylmorpholinium methylcarbonate, (EMMC) and lmethylpyrrolidinium chloride (BMP Chloride), at concentrations preferably between about 0.10 and about 0.50 M, equivalent to about 20-150 mg/mL. The resultant formulations can exhibit Newtonian flow characteristics.

For embodiments in which the protein is a “high-molecular~w_eight protein”, the “high-molecular-weight n,” may have a molecular weight between about 100 kDa and about 1,000 kDa, preferably between about 120 kDa and about 500 kDa, and most preferably between about 120 kDa and about 250 kDa. The high-molecular- weight protein can be an antibody, such as a mAb, or a PEGylated or otherwise a tized form thereof. Preferred mAbs e natalizumab (TYSABRI®), cetuxi— mab (ERBITUX®), bevacizumab (AVASTIN®), trastuzumab (IIERCEPTIN®), inflix— imab (REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatu- mumab (ARZERRA®), and biosimiiars thereof. The high-molecular weight protein, optionally PEGylated, can be an enzyme. Other proteins and mixtures of proteins may also be formulated to reduce their viscosity.

In some embodiments, the n and viscosityureducing ionic liquid(s) are provided in a lized dosage unit, sized for reconstitution with a sterile aqueous ' pharmaceutically acceptable vehicle, to yield the concentrated low—viscosity liquid ations. The presence ofthe viscosity—reducing ionic liquid(s) facilitates and/or accelerates the reconstitution of the lyophilized dosage unit compared to a lyophilized dosage unit not containing a Viscosity-reducing ionic liquid.

Methods are provided herein for preparing concentrated, low-Viscosity liquid formulations of high-molecular-weight proteins such as mAbs, as well as s for storing the low-Viscosity, high-concentration protein formulations, and for stration thereof 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 the protein solutions.

DETAILED DESCRIPTION OF THE INVENTION I. - DEFINITIONS The term "protein," as generally used herein, refers to a r of amino acids linked to each other by e bonds to form a polypeptide for which the chain length is sufficient to produce at least a detectable tertiary ure. Proteins having a molecular weight (expressed in kDa n “Da” stands for “Daltons” and 1 kDa = 1,000 Da) greater than about 100 kDa may be designated molecular—weight proteins,” whereas proteins having a molecular weight less than about 100 kDa. may be designated “low-molecular-weight proteins.” The term “low-molecular—weight protein” excludes small peptides g the requisite of at least tertiary structure necessary to be ered a protein. Protein molecular weight may be determined using standard methods known to one skilled in the art, including, but not limited to, mass ometry (e.g., ESI, MALDI) or calculation from known amino acid sequences and glycosylation. Proteins can be naturally occurring or non-naturally occurring, synthetic, or semi-synthetic.

“Essentially pure protein(s)” and “substantially pure protein(s)” are used interchangeably herein and refer to a composition comprising at least about 90% by weight pure protein, preferably at least about 95% pure protein by weight. “Essentially homogeneous” and “substantially homogeneous” are used interchangeably herein and refer to a composition wherein at least about 90% by weight of the protein present is a combination of the monomer and reversible di- and oligo-meric associates (not irreversible aggregates), preferably at least about 95%.

The term “monoclonal antibody” or “mAb,” as generally used herein, refers to an antibody obtained from a population of ntially homogeneous antibodies, i.e., the dual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single e. These are typically synthesized by culturing oma cells, as described by Kohler et al. (Nature 256: 495, 1975), or may be made by inant DNA methods (see, e.g., U.S. Patent No. 4,816,567), or isolated from phage antibody libraries using the techniques described in Clackson et al. (Nature 352: 8, 1991) and Marks et al. (J. Mol. Biol. 222: 581-597, 1991), for example. As used herein, “mAbs” specifically e derivatized antibodies, antibody-drug conjugates, and ric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies d from a ular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is (are) identical with or gous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US. Patent No. 4,816,567; Morrison 61‘ al, Proc. Natl. Acad. Sci. USA 81 ;6851- 6855, 1984).

An “antibody fragmen ” comprises a portion of an intact dy, 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. 870; Zapata et ai, Protein Eng. 8: 1057-1062, 1995); singlenchain antibody les; multivalent single domain dies; and multispecific antibodies formed from antibody fragments.

“Humanized” forms ofnon—human (e. g., murine) antibodies are chimeric immunoglobulins, immunoglobuiin—chains, or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequences of dies) of mostly human sequences, which contain minimal sequences derived from non-human globulin. (See, e.g., Jones et al, Nature 321:522-525, 1986; Reichmann et (11., Nature 332:323-329, 1988; and , Curr. 0p. Struct. Biol. 2:593-596, 1992.) “Rheology" refers to the study of the deformation and flow of .

“Viscosity” refers to the resistance of a substance (typically a liquid) to flow.

Viscosity is related to the concept of shear force; it can be understood as the effect of different layers of the fluid exerting shearing force on each other, or on other surfaces, as they move against each other. There are several measures of viscosity. The units of viscosity are Ns/mz, known as Pascal-seconds (Pa-s). Viscosity can be "kinematic" or "absolute". tic viscosity is a measure of the rate at which momentum is transferred through a fluid. It is measured in Stokes (St). The tic viscosity is a measure of the resistive flow of a fluid under the ce 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 fluid takes longer than the less s fluid to flow through the capillary. If, for example, one fluid takes 200 seconds (5) to complete its flow and another fluid takes 400 s, the second fluid is called twice as viscous as the first on a kinematic Viscosity scale. The dimension of tic viscosity is lengch/time. Commonly, tic Viscosity is expressed in centiStokes (cSt). The SI unit of kinematic viscosity is mm2/s, which is equal to l cSt.

The ute viscosity," sometimes called ic Viscosity" or "simple viscosity,” is the product of kinematic viscosity and fluid density. Absolute viscosity is expressed in units of centipoise (CF). The SI unit of absolute viscosity is the milliPascal-second (mPa—s), where 1 OP = 1 InPa-s. Viscosity may be measured by using, for e, a viscometer at a given shear rate or multiple shear rates. An “extrapolated zero-shear" viscosity can be determined by ng a best fit line of the four highest- - shear points on a plot of absolute viscosity versus shear rate, and linearly extrapolating Viscosity back to zero-«shear. Alternatively, for a Newtonian fluid, Viscosity can be determined by averaging viscosity values at multiple shear rates.

Viscosity can also be measured using a microfluidic viscometer at single or multiple shear rates (also called flow rates), wherein te viscosity is derived from a change in pressure as a liquid flows through a channel. Viscosity equals shear stress over shear rate. Viscosities measured with microfluidic viscometers can, in some embodiments, be directly compared to extrapolated zero-shear ities, for example those extrapolated from viscosities measured at multiple shear rates using a cone and plate viscometer.

“Shear rate" refers to the rate of change of ty at which one layer of fluid passes over an adjacent layer. The velocity gradient is the rate of change of velocity with distance from the plates. This simple case shows the uniform velocity gradient with shear rate (v1 - vz)/h in units of (cm/sec)/(cm) = 1/sec. Hence, shear rate units are reciprocal s or, in general, reciprocal time. For a microfluidic viscometer, change in pressure and flow rate are d to shear rate. "Shear rate” refers to the speed with which a material is deformed. Formulations containing proteins and viscosity-lowering 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 a spindle appropriately chosen by one skilled in the art to accurately measure viscosities in the viscosity range of the sample of interest (i.e., a sample of 20 cP is most accurately measured on a CPE40 spindle affixed to a DV2T viscometer (Brookfield)); greater than about 20 s"1 to about 3,000 3'1 when measured using a microfluidic viscometer.

For classical “Newtonian” fluids, as generally used herein, viscosity is essentially independent of shear rate. For “non—Newtonian fluids,” however, viscosity either decreases or increases with increasing shear rate, e. g., the fluids are "shear thinning" or "shear thickening", respectively. In the case of concentrated (i.e., high- coneentration) protein solutions, this may manifest as pseudoplastic shear~thinning or, i.e., a decrease in viscosity with shear rate.

The term "chemical stability," as generally used herein, refers to the y of the protein components in a formulation to resist degradation via chemical pathways, such as oxidation, deamidation, or hydrolysis. A protein formulation is typically ered ally stable if less than about 5% of the components are degraded after 24 months at 4°C.

The term "physical stability," as generally used herein, refers to the ability of a protein formulation to resist al deterioration, such as aggregation. A * , formulation that is physically stable forms only an acceptable percentage of irreversible aggregates (e.g., dimers, trimers, or other ates) of the bioactive protein agent. The ce of aggregates may be assessed in a number of ways, including by measuring the average particle size of the ns in the formulation by means of 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 ated contaminants ideally would be less than about 2%.

Levels as low as about 0.2% are achievable, although approximately 1% is more The term " stable formulation,” as generally used herein, means that a ation is both chemically stable and physically stable. A stable formulation may be one in which more than about 95% ofthe bioactive protein molecules retain ivity in a ation after 24 months of storage at 4° C, or equivalent solution conditions at 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, 1, Vincent Lee, 2014/055245 Ed, Marcel Dekker, Inc., New York, NY. (1991) and Jones, A., Adv. Drug Delivery Revs. 10:29-90, 1993. Stability can be measured at a selected temperature for a certain time period. For rapid screening, for example, the formulation may be kept at 40°C, for 2 weeks to one 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 -8°C, generally the formulation should be stable at 30°C or 40°C for at least one month and/or stable at 2°C -8°C for at least 2 years. When the formulation is to be stored at room temperature, about 25°C, generally the formulation should be stable for at least 2 years at about 25°C and/0r stable at 40°C for at least about 6 months. The extent of aggregation following lyophilization and storage can be used as an indicator of protein stability. In some embodiments, the stability is ed by ing the particle size of the proteins in the formulation. In some embodiments, stability may be assessed by measuring the activity of a formulation using standard biological activity or g assays well within the abilities of one ordinarily skilled in the art.

The term protein "particle size," as generally used herein, means the average diameter ofthe predominant tion ofbioactive molecule particulates, or particle size distributions thereof, in a formulation as determined by using well known particle sizing instruments, for example, dynamic light scattering, SEC (size exclusion chromatography), or other methods known to one ordinarily d in the art.

The term “concentrated” or "high-concentration", as lly used herein, describes liquid formulations having a final concentration of protein greater than about 10 mg/mL, preferably greater than about 50 mg/mL, more ably greater than about 100 mg/mL, still more preferably greater than about 200 mg/mL, or most preferably r than about 250 mg/mL.

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

A “lyoprotectant” is a substance which, when combined with a protein, significantly reduces chemical and/or physical instability of the protein upon lyophilization and/or subsequent storage. Exemplary tectants 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 line; lyotropic salts, such as magnesium e; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrins, modified starch, and carboxymethyl cellulose. Preferred sugar ls are those compounds obtained by reduction of mono- and di~saccharides, such as lactose, trehalose, maltose, lactulose, and maltulose. Additional examples of sugar ls are glucitol, ol, lactitol and isomaltulose. The lyoprotectant is generally added to the pre- lyophilized formulation in a “IyOprotecting amount.” This means that, following lization of the protein in the presence of the tecting amount of the lyoprotectant, the protein essentially retains its al and chemical stability and integrity.

A “diluent” 0r “carrier,” as generally used herein, is a pharmaceutically acceptable (i.e., safe and xic for administration to a human or another mammal) and useful ingredient for the preparation of a liquid formulation, such as an aqueous formulation reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered on (e.g., phosphate-buffered ), e saline on, Ringer's solution or se solution, and combinations thereof.

A “preservative” is a compoundwhich can be added to the formulations herein to reduce contamination by and/or action of bacteria, fungi, or another infectious agent. The addition of a preservative may, for example, facilitate the production of a multi—use (multiple-dose) formulation. Examples of potential preservatives include 0ctadecyldimethylbenzylammonium chloride, hexamethonium de, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long~chained), and benzethonium chloride. Other types of preservatives e aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m—cresol.

A “bulking agent,” as generally used herein, is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g. facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, lactose, modified starch, poly(ethylene glycol), and sorbitol.

A “therapeutically effective amount” is the lowest tration required to effect a measurable improvement or prevention of any m or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or to generally e patient quality of life. The therapeutically effective amount is dependent upon the specific biologically active molecule and the specific condition or disorder to be treated. eutically effective amounts of many proteins, such as the mAbs described herein, are well known in the art. The therapeutically effective amounts of proteins not yet established or for treating specific disorders with known proteins, such as mAbs, to be clinically applied to treat additional disorders may be determined by standard techniques which are well Within the craft of a skilled artisan, such as a physician.

The term "inj ectability" or “syringeability,” as generally used herein, refers to the inj ection performance of a ceutical formulation through a syringe equipped with an 18-32 gauge needle, optionally thin walled. Inj ectability depends upon factors such as pressure or force required for injection, evenness of flow, tion qualities, and freedom from ng. inj ectability of the liquid pharmaceutical formulations may be ed by comparing the injection force of a reduced-Viscosity formulation to a rd formulation t added Viscosity-lowering agents. The reduction in the injection force of the formulation containing a viscosity-lowering agent reflects improved inj ectability of that formulation. The reduced Viscosity formulations have ed inj ectability 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 rd formulation having the same concentration ofprotein under otherwise the same ions, except for ement of the viscosity-lowering agent with an appropriate buffer of about the same concentration. Alternatively, inj ectability of the liquid pharmaceutical formulations may be assessed by comparing the time ed to inject the same volume, such as 0.5 mL, or more preferably about 1 mL, of different liquid protein formulations when the syringe is depressed with the same force.

The term “injection force}: as generally used herein, refers to the force required to push a given liquid formulation h a given syringe equipped with a given needle gauge at a given ion speed. The injection force is typically reported in Newtons. For example, the injection force may be measured as the force required to push a liquid formulation through a 1 mL plastic syringe having a 0.25 inch inside diameter, equipped with a 0.50 inch 27 gauge needle at a 250 n injection speed. Testing equipment can be used to measure the injection force. When measured under the same conditions, a formulation with lower viscosity will generally require an overall lower injection force.

The “Viscosity nt,” as used herein, refers to the rate of change of the ity of a protein solution as protein concentration increases. The Viscosity gradient can be approximated from a plot ofthe Viscosity as a function of the protein concentration for a series of formulations that are otherwise the same but have different protein concentrations. The viscosity increases approximately exponentially with increasing protein concentration. The viscosity gradient at a specific n concentration can be imated from the slope of a line tangent to the plot of, A Viscosity as a function of protein concentration. The viscosity gradient can be approximated fi'om a linear approximation to the plot of viscosity as a on of any protein concentration or over a narrow Window of protein trations. In some embodiments a formulation is said to have a decreased viscosity gradient if, when the viscosity as a function of n concentration is approximated as an exponential function, the exponent of the exponential n is smaller than the exponent obtained for the otherwise same ation t the Viscosity-lowering agent In a similar manner, a formulation can be said to have a lower/higher ity gradient . when compared to a second formulation if the exponent for the formulation is higher than the exponent for the second formulation. The viscosity gradient can he numerically approximated from a plot ofthe Viscosity as a function of protein concentration by other methods known to the skilled formulation researchers.

The term “reduced-viscosity formulation,” as generally used herein, refers to a liquid formulation having a high concentration of a high—molecular-weight protein, such as- a mAb, or a low-molecular-weight protein that is modified by the presence of one or more additives to lower the viscosity, as compared to a corresponding formulation that does not contain the viscosity-lowering additive(s).

The term “osmolarity,” as generally used herein, refers to the total number of ved components per liter. Osmolarity is similar to molarity but includes the total number of moles of ved species in on. An osmolarity of 1 Osm/L means there is 1 mole of dissolved components per L of solution. Some solutes, such as ionic solutes that dissociate in solution, will contribute more than 1 mole of dissolved components per mole of solute in the solution. For example, NaCl dissociates into Na+ and CI' in solution and thus provides 2 moles of ved components per 1 mole of dissolved NaCl in solution. logical osmolarity is lly in the range of about 280 mOsm/L to about 310 m0sm/L.

The term “tonicity,” as generally used herein, refers to the osmotic pressure gradient resulting from the separation oftwo ons by a semi-permeable 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 cellular membrane do not bute to the final osmotic pressure gradient.

'Only those dissolved species that do not cross the cell membrane‘will contribute to c pressure differences and thus tonicity.

The term “hypertonic,” as lly used herein, refers to a solution with a higher concentration of solutes than is present on the inside of the cell. When a cell is immersed into a hypertonic solution, the tendency is for water to flow out of the cell in order to balance the concentration of the solutes.

The term “hypotonic,” as generally used herein, refers to a solution with a lower concentration of solutes than is present on the inside of the cell. When a cell is immersed into a hypotonic solution, water flows into the cell in order to balance the concentration of the solutes.

The term “isotonic,” as generally used , refers to a solution wherein the osmotic pressure gradient across the cell membrane is essentially balanced. An isotonic formulation is one which has essentially the same osmotic re as human blood. Isotonic formulations will generally have an osmotic re from about 250 mOsm/kg to 350 mOsm/kg.

The term “liquid formulation,” as used herein, is a protein that is either supplied in an acceptable pharmaceutical diluent or one that is reconstituted in an acceptable pharmaceutical diluent prior to administration to the patient.

The terms “branded” and “reference,” when used to refer to a protein or biologic, are used interchangeably herein to mean the single biological product licenSed under section 351(a) of the U.S. Public Health Service Act (42 U.S.C. § 262).

The term “biosirnilar,” as used herein, is generally used interchangeably with “a generic equivalen ” or “follow-on.” For example, a “biosimilar mAb” refers to a subsequent version of an innovator’s mAb lly made by a different y.

“Biosimilar” when used in reference to a branded protein or branded biologic can refer to a biological product evaluated against the branded protein or branded biologic and licensed under section 35 Mk) of the U.S. Public Health Service Act (42 U.S.C. § 262). A biosirnilar mAb can be one that satisfies one or more guidelines adopted May , 2012 by the Committee for Medicinal ts for Human Use (CHMP) ofthe European Medicines Agency and published by the European Union as “Guideline on similar biological medicinal products containing monoclonal antibodies — non-clinical and clinical issues” (Document Reference MP/BMWP/403543/2010).

Biosimilars can be produced by microbial cells ryotic, eukaryotic), cell lines of human or animal origin (e.g., mammalian, avian, ), or tissues derived from animals or plants. The expression construct for a proposed biosimilar t will generally encode the same primary amino acid sequence as its nce product.

Minor modifications, such as N— or C- terminal tions that will not have an effect on safety, purity, or potency, may be present.

A biosimilar mAb is r to the reference mAb physiochemically cr ically both in terms of safety and efficacy. The biosimilar mAb can be evaluated against a reference mAb using one or more in vitro studies including assays detailing binding to target antigen(s); binding to isoforms of the Fc gamma receptors (FcyRI, FcyRII, and Fc'yRIII), FcRn, and complement (Clq); Fab-associated functions (6.g. neutralization of a soluble ligand, receptor tion or de); or Fe— associated functions (e.g. dy-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, complement activation). In vitro comparisons may be combined with in vivo data demonstrating similarity of pharmacokinetics, pharmacodynamics, and/or safety. Clinical evaluations of a biosimilar mAb against a reference mAb can include isons ofpharmacokinetic properties (e.g. AUCO-jnf, AUCM, Cum, tmax, (Enough); pharmacodynamic endpoints; or similarity of clinical efficacy (e.g. using randomized, parallel group comparative clinical trials). The quality ison between a biosimilar mAb and a reference mAb Can be ted using established procedures, including those bed in the line on similar biological medicinal products containing biotechnology—derived proteins as active substance: Quality issues” (EMEA/CHMP/BWP/49348/2005), and the “Guideline on development, production, characterization and specifications for monoclonal antibodies and related substances” (EMEA/CHMP/BWP/l 57653/2007). ences between a biosimilar mAb and a reference mAb can include post- translational modification, e.g. by attaching to the mAb other biochemical groups such as a ate, various lipids and carbohydrates; by proteolytic cleavage following translation; by changing the al nature of an amino acid (e.g., formylation); or by many other mechanisms. Other post-translational modifications can be a consequence of manufacturing process operations —— for example, glycation In other cases, storage may occur with exposure of the product to reducing . conditions may be permissive for certain degradation pathways such as oxidation, ation, or aggregation. As all of these product-related variants may be included in a biosimilar mAb.

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

As used herein, the term "ionic liquid” refers to a crystalline or amorphous salt, rion, or mixture thereof that is a liquid at or near temperatures where most conventional salts are solids: at less than 200°C, preferably less than 100°C or more preferably less than 80°C. Some ionic liquids have g temperatures around room temperature, e.g. between 10°C and 40°C, or between 15°C and 35°C. The term "zwitterion" is used herein to describe an overall neutrally charged molecule which carries formal positive and negative s on different chemical groups in the ' molecule. Examples of ionic liquids are described in Riduan et 0]., Chem. Soc. Rev., 42:9055-9070, 2013; Rantwijk et at, Chem. Rev., 107:2757-2785, 2007; Earle et 511., Pure Appl. Chem, 72(7):1391-1398, 2000; and Sheldon er 611., Green Chem, 4:147- ] 51, 2002.

As used herein, the term -“organophosphate” refers to a compound ning one or more phosphoryl groups at least one of which is covalently connected to an organic group through a phosphoester bond.

As used herein, a “water e organic dye” is an organic molecule having a molar solubility of at least 0.001 M at 25°C and pH 7, and that absorbs certain wavelengths of light, ably in the visible-to-infrared n of the electromagnetic spectrum, while possibly transmitting or reflecting other wavelengths of light.

As used herein, the term “chalcogen” refers to Group 16 elements, including oxygen, sulfur and selenium, in any oxidation state. For instance, unless specified ' otherwise, the term “chalcogen” also include 802.

As used herein, the term “alkyl group” refers to straight-chain, branched-chain and cyclic hydrocarbon groups. Unless specified otherwise, the term alkyl group embraces 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 yl ” and an alkyl group containing at least one triple bond is an “alkynyl group.” As used herein, the term “aryl” refers to aromatic carbon ring systems, including fused ring systems. In an “aryl” group, each of the atoms that form the ring are carbon atoms.

As used herein, the term “heteroaryl” refers to aromatic ring s, including fused ring systems, n at least one of the atoms that form the ring is a heteroatom.

As used herein, the term “heterocycle” refers to ring s that, including fused ring systems, that are not aromatic, wherein at least one of the atoms that forms the ring is a heteroatom.

As used herein, a oatom” is any non-carbon or non—hydrogen atom.

Preferred heteroatoms include oxygen, sulfur, and nitrogen. Exemplary heteroaryl and heterocyclyl rings e: benzimidazolyl, benzofuranyl, hiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzirnidazolinyl, carbazolyl, 4aH carbazolyl, earbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H—1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, olinyl, imidazolyl, lH-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, l,2,5~oxadiazolyl, 1,3,4- oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyi, phenanthridinyl, phenanthrolinyl, phenazinyl, hiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, ridonyl, piperonyl, ‘ - pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoirnidazole, thiazole, nyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, ZH-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, ydroquinolinyl, tetrazolyl, 6H~1,2,5-thiadiaziny1, 1,2,3- thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5—thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoirnidazolyl, thiophenyl, and xanthenyl.

II. FORMULATIONS Biocompatible, low-Viscosity protein solutions, such as those of InAbs, can be used to r therapeutically effective amounts of ns in volumes useful for subcutaneous (SC) and intramuscular (1M) ions, typically less than or about 2 mL for SC and less than or about 5 mL for 1M, more preferably less than or about 1 mL for SC and less than or about 3 mL for IM. The proteins can generally have any molecular weight, although in some embodiments high-molecular-weight proteins are preferred. In other embodiments the ns are low-molecular-weight proteins. ations may have protein trations 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/rnL, more preferably greater than about 175 nag/ml, even more preferably greater than about 200 mg/mL, even more preferably greater than about 225 mg/mL, even more preferably r than about 250 mg/mL, and most preferably greater than or about 300 mg/mL. In the absence of a viscosity-reducing ionic liquid, the viscosity of a protein formulation increases exponentially as the concentration is increased. Such n formulations, in the absence of a viscosity-reducing ionic liquids, may have viscosities greater than 100 cP, greater than 150 01’, greater than 200 GP, greater than 300 cP, greater than 500 CR or even greater than 1,000 cP, when measured at 25° C.

Such formulations are often unsuitable for SC or IM injection; The use of one or more viscosity-reducing ionic liquids permits the preparation of ations having a viscosity less than or about 100 OF, preferably less than or about 75 GP, more preferably less than or about 50 GP, even more preferably less than or about 30 GP, even more preferably less than or about 20 CF, or most preferably less than or about 0?, when measured at 25° C. ' , Although the viscosity-reducing ionic liquids may be used to lower the viscosity of concentrated n formulations, they may be used in less-concentrated formulations as well. In some embodiments, 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.

For certain proteins, formulations not having an ionic liquid may have ities greater than about 20 01’, greater than about 50 CF, or r than about 80 CF. The use of one or more ionic liquids permits the ation of ations having a Viscosity less than or about 80 CF, preferably less than or about 50 GP, even more preferably less than about 20 CF, or most preferably less than or about 10 cP, when measured at 25° C.

In some embodiments, the aqueous protein formulations have a viscosity that is at least about 30% less than the analogous ation 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 ous formulation without the viscosity— reducing ionic liquid(s). In a preferred embodiment, the formulation contains a eutically effective amount ofthe 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 ably less than about 0.75 mL.

The reduced-viscosity formulations have improved inj ectability and require less injection force compared to the analogous formulation without the viscosity- reducing ionic liquid (e.g., in ate buffer) under otherwise the same ions.

In some ments, the force of injection is decreased by more than about 20%, more than about 30%, more than about 40%, more than about 50%, or more than about 2 fold, as compared to rd formulations without the viscosity—reducing ionic liquid(s) under otherwise the same injection conditions. In some embodiments, the formulations possess “Newtonian flow characteristics,” defined as having viscosity which is substantially independent of shear rate. The protein formulations can be readily injected through needles of about 18-32 gauge. Preferred needle gauges for the delivery of the low-viscosity fom‘lulations include 27, 29, and 31 gauge, ally thin walled.

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 concentrated, low-viscosity formulations have a pH between 5 and 8, between 5.5 and 7.6, n 6.0 and 7.6, between 6.8 and 7.6, or between 5.5 and 6.5.

The low-viscosity protein formulations can allow for greater flexibility in formulation pment. The low-viscosity formulations can t changes in viscosity that are less dependent upon the protein concentration as compared to the otherwise same formulation without the viscoshy-reducing ionic liquid. The low- viscosity protein formulations can allow for increased trations and sed dosage frequencies of the protein. In some embodiments the low—viscosity protein formulations contain 2 or more, 3 or more, or 4 or more different proteins. For example, combinations of 2 or more mAbs can be provided in a single low-viscosity protein formulation. e protein (such as mAb) formulations may be administered to patients at higher n concentrations than otherwise r protein formulations not containing a viscosity~reducing ionic liquid, the dosing frequency of the protein can be reduced. For instance, 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 which currently require multiple administrations on the same day (either at the same time or at different times of the day) may be administered in fewer ions per day. In some instances, the frequency may be reduced to a single injection once a day. By increasing the dosage administered per ion le-fold the dosing ncy can be decreased, for example from once every 2 weeks to once every 6 weeks.

In some embodiments, the liquid formulations have a logical osmolarity, for example, between about 280 mOsm/L to about 310 m0srn/L. In some embodiments, the liquid formulations have an osmolarity greater than about 250 mOsm/L, greater than about 300 , 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 about 200 mOsm/L to about 2,000 mOSm/L or about 300 mOsm/L to about 1,000 mOSIn/L. In some embodiments, the liquid formulations are essentially ic to human blood. The liquid formulations can in some cases be hypertonic.

The additives, including the viscosity-reducing ionic 1iquid(s), can be included in any amount to achieve the desired Viscosity levels of the liquid formulation, as long as the amounts are not toxic or otherwise harmful, and do not substantially interfere with the chemical and]or al stability of the formulation. The viscosity-reducing ionic liquid(s) in some embodiments can be independently 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 having two or more viscosity-reducing ionic liquids, the agents are ably, but not necessarily, present at the same concentration.

The viscosity-reducing ionic liquid(s) permit faster reconstitution of a lyophilized dosage unit. The dosage unit is a lyophiiized cake of protein, viscosity- reducing ionic liquid(s) and other excipients, to Which water, saline or another ceutically acceptable fluid is added. In the absence of Viscosity-reducing ionic liquids, periods of 10 minutes or more are often required in order to completely dissolve the lyophilized cake at high protein concentration. When the lyophilized cake contains one or more viscosity-reducing ionic , the period 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 containing greater than or about 150, 200 or even 300 mg/mL of The low-viscosity protein formulations allow for greater flexibility in formulation development. The low«viscosity formulations exhibit a Viscosity that changes less with increasing protein concentrations as compared to the otherwise same formulation without the ionic iiquid(s). The low-viscosity protein formulations ' exhibit a decreased viscosity gradient as compared to the otherwise same formulation without the ionic liquid The viscosity nt ofthe protein ation may be 1ess,3-fold less, or even more than 3-fold less than the Viscosity gradient of the ise same protein formulation without the viscosity-reducing ionic liquid(s). The ity gra~ dient 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 having a protein concentration be- tween 10 mg/mL and 2,000 mg/mL. By reducing the viscosity gradient of the fonnu— , the protein concentration can be increased to a greater degree before an expo- nential se in viscosity is observed.

A. Proteins Any protein can be formulated, including recombinant, ed, or synthetic proteins, glycoproteins, or lipoproteins. These may be antibodies (including antibody fragments and recombinant antibodies), enzymes, growth s or es, im- munomodiflers, antiinfectives, antiproliferatives, vaccines, or other therapeutic, prophylactic, or diagnostic proteins. In certain ments, the protein has a molec- ular weight greater than about 150 kDa, greater than 160 kDa, greater than 170 kDa, greater than 180 kDa, greater than 190 kDa or even r than 200 kDa.

In certain embodiments, the protein can be a PEGylated protein. The term “PEGylated protein,” as used , refers to a protein having one or more poly(ethylene glycol) or other h polymer groups covalently attached thereto, optionally through a chemical linker that may be different from the one or more polymer groups. PEGylated proteins are characterized by their typically reduced renal filtra— tion, decreased uptake by the reticuloendothelial system, and diminished enzymatic degradation leading to, for example, prolonged half—lives and enhanced bioavailabil- ity. Stealth polymers include poly(ethylene ); poly(propylene ); poly(amino acid) rs such as poly(glutamic acid), poly(hydroxyethyl-L- asparagine), and poly(hydroxethyl-L—glutamine); poly(glycerol); poly(2~oxazoline) polymers such as -methyloxazoline) and poly(2—ethyloxazoline); poly(acrylarnide); poly(vinyl idone); polyCN—(2-hydr0xypropyl)methacrylamide); and mers and es thereof. In preferred embodiments the stealth polymer in a PEGylated protein is poly(ethylene glycol) or a copolymer thereof. ted proteins can be randomly PEGylated, z'. e. having one or more stealth polymers covalently attached at cific site(s) on the protein, or can be PEGylated in a pecific manner by cova- lently attaching the stealth polymer to specific site(s) on the protein. Site-specific PEGylation can be lished, for example, using activated stealth rs hav- ing one or more reactive functional groups. Examples are described, for instance, in Hoffman et 611., Progress in Polymer Science, 32:922—932, 2007.

In the preferred embodiment, the protein is high-molecular—weight and an an- tibody, most preferably a mAb, and has a high viscosity in aqueous buffered solution when concentrated sufficiently to inject a therapeutically effective amount in a vol- ume not exceeding 1.0 to 2.0 mL for SC and 3.0 to 5.0 mL for IM administration.

High-molecular-weight proteins can include those described in Scolnik, mAbs 1:179- 184, 2009; Beck, mAbs 3:107-110, 2011; Baumann, Curr. Drug Math. 7:15-21, 2006; or Federici, Biologicals -147, 2013. The proteins for use in the formulations described herein are preferably essentially pure and essentially homogeneous (i.e., substantially free from contaminating proteins and/or irreversible aggregates thereof).

Preferred mAbs herein include zumab (TYSABRI®), cetuximab (ERBL TUX®), zumab (AVASTIN®), trastuzumab (HERCEPTIN®), infliximab (REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX‘E), ofatumumab (ARZERRA® and biosimilars thereof. ary high-molecular—weight proteins can include tocilizumab RA®), alemtuzumab (marketed under several trade , brodalumab (developed by Amgen, Inc (“Amgen”)), denosumab (PROLIA® and XGEVA®), and biosirnilars thereof.

Exemplary molecular targets for antibodies described herein include CD pro— teins, such as CD3, CD4, CD8, CD19, CD20 and CD34; members of the HER recep- tor family such as the EGF receptor, HERZ, HER3 or HER4 receptor; cell adhesion molecules, such as LFA—l, M01, p150,95, VLA—4, ICAM-l, VCAM, and (xv/[33 integrin , including either a or B subunits thereof (e.g., D1 la, anti-CD18, or anti— CD111) antibodies); growth factors, such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; n C; PCSK9; etc.

Antibody Therapeutics Currentlyon the Market ' Many protein therapeutics currently on the , especially antibodies as defined herein, are administered Via IV infusions due to high dosing requirements.

Formulations can include one ofthe antibody therapeutics currently on the market or a biosimilar thereof. Some protein therapeutics currently on the market are not high- molecular—weight, but are still administered Via IV infusion e high doses are needed for therapeutic efficacy. In some embodiments, liquid formulations are pro- vided ofthese low-molecular—weight proteins as defined herein with concentrations to deliver therapeutically effective s for SC or IM ions.

Antibody therapeutics currently on the market include belimumab (BENLYSTA®), golimumab (SIMPONI ARIA®), abciximab (REOPRO®), the combination of tositumomab and iodine-131 tositumomab, marketed as BEXXAR®, alemtuzumab (CAMPATH®), zumab IS®), basiliximab (SIMULECT®), ado-trastuzumab emtansine (KADCYLA®), pertuzumab (PERJETA®), capromab pendetide (PROSTASCINT KIT®), caclizumab AX®), ibritumomabtiuxetan (ZEVALIN®), eculizumab (SOLIRIS®), ipilimumab (YERVOY®), muromonab-CD3 CLONE OKT3®), WO 38811 raxibacumab, nimotuzumab (THERACIM®), brentuximab vedotin RIS®), adalimumab,,(HUMlRA®), golimumab (SIMPONI®), palivizumab (SYNAGIS®), omalizumab R®), and ustekinumab (STELARA®).

Natalizumab, a humanized mAb against the cell adhesion molecule 0L4- integrin, is used in the treatment of multiple sclerosis and Crohn‘s disease. Previously marketed under the trade name ANTEGREN®, zumab is currently co-marketed as TYSABRI® by Biogen Idec (“Biogen”) and Elan Corp. (“Elan”) TYSABRI® is produced in murine myeloma cells. Each 15 mL dose contains 300 mg zumab; 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 1V ion, USP at pH 6.1. Natalizumab is typically administered by monthly intravenous (IV) infusions and has been proven effective in treating the symptoms of both multiple sclerosis and Crohn's disease, as well as for preventing relapse, vision loss, cognitive e, and significantly improving patient’s quality of life.

As used herein, the term “natalizuma ” es the mAb against the cell adhesion molecule 0L4-integrin known under the International Nonproprietary Name “NATALIZUMAB” or an antigen binding n thereof. zumab includes 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 US.

Patent No. 5,73 0,978. Natalizumab includes the active agent in products marketed under the trade name TYSABRI® by Biogen Idec and Elan Corporation or a biosimilar product thereof.

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 given by IV infusion. Cetuximab is marketed for IV use only under the trade name ERBITUX® by Bristol-Myers Squibb Company (North America; ol—Myers Squibb”), Eli Lilly and Company (North America; “Eli Lilly”), and Merck KGaA. ERBITUX® is produced in mammalian (murine myeloma) cell culture. Each single-use, 5O-mL Vial of ERBITUX® ns 100 mg of cetuximab at a concentration of 2 mg/mL and is formulated in a preservative—free solution ning 8.48 mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic heptahydrate, 0.42 mg/mL sodium phosphate monobasic drate, and water for IV Injection, USP.

Cetuximab is indicated for the ent of patients with epidermal growth factor receptor (EGFR)-expressing, KRAS wild-type atic ctal cancer (mCRC), in combination with chemotherapy, and as a single agent in patients who have failed oxaliplatin- and irinotecan—based therapy or who are rant to irinotecan. Cetuximab is indicated for the treatment of patients with squamous cell carcinoma ofthe head and neck in combination with platinum-based chemotherapy for the first-line ent of recurrent and/or metastatic disease and in combination with radiation therapy for locally advanced disease. Approximately 75% of patients with metastatic colorectal cancer have an EGFR—expressing tumor and are, therefore, considered eligible for ent with cetuximab or panitumumab, according to FDA guidelines.

As used herein, the term “cetuxima ” includes the mAb known under the International Nonproprietary Name “CETUXIMAB” or an antigen binding portion thereof.-Cetuximab includes antibodies described in US. Patent No. 6,217,866. ' * Cetuximab includes the active agent in products marketed under the trade name ERBITUX® and biosimilar products thereof. Biosimilars of ERBITUX® can include those tly being developed by Amgen, AlphaMab Co., Ltd. (“AlphaMab”), and Actavis plc (“Actavis”).

Bevacizumab, a humanized InAb that inhibits vascular endothelial growth factor A (VEGF-A), acts as an anti-angiogenic agent. It is ed under the trade name N® by Genentech, Inc. (“Genentech”) and F. HoffmaIm-La Roche, LTD (“Roche”). It is licensed to treat various cancers, ing colorectal, lung, breast (outside the U.S.A.), glioblastoma (U.S.A. only), kidney and ovarian.

AVASTIN® was approved by the FDA in 2004 for use in metastatic ctal cancer when used with rd chemotherapy treatment (as first-line treatment) and with 54 fluorouracil-based therapy for second-line metastatic colorectal cancer. In 2006, the FDA approved AVASTIN® for use in first-line advanced non-squamous non-small cell lung cancer in ation with carboplatin/paclitaxel chemotherapy.

AVASTIN® is given as an IV infusion every three weeks at the dose of either 15 mg/kg or 7.5 mg/kg. The higher dose is usually given with carboplatin—based herapy, whereas the lower dose is given with cisplatin—based chemotherapy. In 2009, the FDA approved AVASTIN® for use in metastatic renal cell carcinoma (a form of kidney cancer). The FDA also granted rated approval ofAVASTIN® for the ent of recurrent glioblastoma multiforme in 2009. Treatment for initial growth is still in phase III clinical trial.

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

As used herein, the term “bevacizumab” includes the mAb that inhibits vascular endothelial growth factor A (VEGF-A) known under the International Nonproprietary Name/Common Name “BEVACIZUMAB” or an antigen binding portion thereof. zumab is described in US. Patent No. 6,054,297.

Bevacizumab includes the active agent in products marketed under the trade name AVASTIN® and biosimilar products thereof. Biosimilars ofAVASTIN® can include those currently being developed by Amgen, Actavis, AlphaMab, and Pfizer, Inc (“Pfizer”). Biosimilars of AVASTIN® can include the biosimilar known as BCD-021 produced by Biocad and currently in clinical trials in the US. zumab is a mAb that interferes with the HERZ/neu receptor. zumab is marketed under the trade name HERCEPTIN® by Genentech, Inc.

TIN® is produced by a mammalian cell (Chinese Hamster Ovary (CHO)) line. TIN® is a sterile, white to pale-yellow, preservative-free lyophilized powder for IV stration. Each HERCEPTIN® vial contains 440 mg trastuzumab, 9.9 mg L-histidine HCl, 6.4 mg L—histidine, 400 mg a,a—trehalose dihydrate, and 1.8 mg polysorbate 20, USP. titution with 20 mL water yields a multi-dose solution containing 21 mg/mL zumab. HERCEPTIN® is currently administered via IV infusion as often as weekly and at a dosage ranging from about 2 mg/kg to about 8 mg/kg. zumab is mainly used to treat certain breast cancers. The HER2 gene is WO 38811 amplified in 20-30% of early-stage breast cancers, which makes it overexpress epi— epidermal growth factor (EGF) receptors in the cell membrane. Trastuznmab is generally administered as a nance therapy for patients with HERZ-positive breast cancer, typically for one year post-chemotherapy. Trastuzumab is currently administered via IV infiision as often as weekly and at a dosage ranging from about 2 mg/kg to about 8 mg/kg.

As used herein, the term “trastuzumab” es the mAb that interferes with the HER2/neu receptor known under the ational Nonproprietary Name/Common Name “TRASTUZUMAB” or an antigen binding portion thereof. zumab is described in US. Patent No. 337. Trastuzumab includes the active agent in products marketed under the trade name HERCEPTIN® and biosimilars thereof. The term “trastuzumab” includes the active agent in biosimilar HERCIE'ZPTIN® products marketed under the trade names Z® by Mylan, Inc. (“Mylan”) and CANMAB® by Biocon, Ltd. (“Biocon”). zurnab can include the active agent in biosimilar HERCEPTIN® products being developed by Amgen and by PlantForm ' Corporation, Canada.

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

Infliximab contains approximately 30% murine variable region amino acid ce, which confers antigen-binding specificity to human TNFcL. The remaining 70% correspond to a human IgG1 heavy chain constant region and a human kappa light chain constant region. Infliximab has high affinity for human TNFCL, which is a cytokine with multiple biologic actions including mediation of inflammatory respons- responses and modulation of the immune system.

Infliximab is a recombinant antibody generally produced and ed 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 dy genes consisting ofthe variable region sequences cloned from the murine anti-TNFd hybridoma A2, and human antibody constant region ces ed by the plasmid expression s. Generation of the murine anti- TNF u hybridoma is performed by immunization of BALB/c mice With purified inant human TNFOL. The heavy and light chain vector constructs are linearized and transfected into the Sp2/O cells by electroporation. Standard purification steps can e chromatographic purification, viral inactivation, nanofiltration, and ultrafiltration/diafiltration.

As used herein, the term “inflixirna ” includes the chimeric mouse/human monoclonal antibody known under the International Nonproprietary Name “INFLIXIMAB” or an antigen-binding portion thereof. Infliximab neutralizes the biological activity ofTNFd by binding with high affinity to the soluble and transmembrane forms of TNFd and ts binding of TNFd with its receptors. mab 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 REMICADE® by multiple entities; REMSIMATM by Celltrion and INFLECTRATM by Hospira, Inc (“Hospira”). Infliximab is supplied as a sterile lized cake for reconstitution and dilution. Each vial of infliximab contains 100 mg infliximab and excipients such as monobasic sodium phosphate monohydrate, dibasic sodium ate ate, sucrose, and polysorbate 80.

Denosumab (PROLIA® and XGEVA®) is a human mAb — and the first RANKL inhibitor - approved for use in postmenopausal women with risk of osteoporosis and patients with bone metastases from solid tumors. Denosumab is in Phase II trials for the treatment of rheumatoid arthritis. mumab is a fully hUman mAb approved by the FDA for treatment of EGFR—expressing atic cancer with disease progression. Panitumumab is marketed under the trade name VECTIBIX® by Amgen. VECTIBIX® is packaged as a 20 mg/ml paniturnumab concentrate in 5 ml, 10 ml, and 15 ml vials for IV infusion.

When ed according to the packaging instructions, the final panitumumab concentration does not exceed 10 mg/ml. VECTIBIX® is administered at a dosage of 6 mgfkg 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 Nonproprietary Name “PANITUMUMAB.” The term “panitumumab” includes the active agent in products marketed under the trade name VECTIBIX® by Amgen and biosimilars thereof. The term “panitumumab” includes monoclonal antibodies described in US. Patent No. 883. The term “panitumumab” includes the active agent in biosimilar VECTIBIX® products, including ilar VECTIBIX® being developed by BioXpress, SA (“BioXpress”).

Belimumab (BENLYSTA®) is a human mAb with a molecular weight of about 151.8 kDa that inhibits B-cell activating factor (BAPF). Belimumab is approved in the United States, Canada, and Europe for treatment of systemic lupus erythematosus.

Belimumab is currently stered to lupus patients by IV infusion at a 10 mg/kg dosage. A high-molecular—weight, low~viscosity protein formulation can e ' M -- Belimumab, preferably in a concentration of about 400 mg/mL to about 1,000 mg/mL. The preferred ranges are calculated based upon body weight of 40-100 kg (approximately 80-220 lbs) in a 1 mL volume. mab (REOPRO®) is manufactured by Janssen Biologics BV and distributed by Eli Lilly & Company (“Eli Lilly”). Abciximab is a Fab fragment ofthe chimeric human-murine monoclonal antibody 7E3. Abciximab binds to the glycoprotein (GP) IIb/IIIa receptor ofhuman platelets and inhibits platelet aggregation by preventing the binding of fibrinogen, von rand factor, and other adhesive molecules. It also binds to ectin (uvl33) or found on platelets and vessel wall endothelial and smooth muscle cells. mab is a platelet aggregation tor mainly used during and after coronary artery procedures. Abcixirnab is administered Via IV infusion, first in a bolus of 0.25 mg/kg and followed by continuous IV infusion of 0.125 mcg/kg/minute for 12 hours.

Tositumoma‘o (BEXXAR®) is a drug for the treatment of follicular lymphoma.

It is an IgG2a D20 mAb derived from immortalized mouse cells. Tositumomab is administered in tial infusions: cold mAb followed by iodine (1311) momab, the same antibody covalently bound to the radionuclide iodine-131.

Clinical trials have established the efficacy of the tositurnomab/iodine tositumomab regimen in patients with relapsed refractory follicular lymphoma. BEXXAR® is tly administered at a dose of 450 mg Via IV infusion.

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

Palivizumab (SYNAGIS®) is a humanized mAb directed against an epitope in the A antigenic site of the F protein of respiratory syncytial Virus. In two Phase III clinical trials in the pediatric population, palivizumab reduced the risk of hospitalization due to respiratory syncytial virus infection by 55% and 45%.

‘ - * ' Palivizumab is dosed once a month Via IM injection of 15 mg/kg.

Ofatumumab is a human D20 mAb which s to inhibit stage B lymphocyte activation. Ofaturnumab is ed under the trade name ARZERRA® by GlaxoSmithKline, plc (“GlaxoSmithKline”). ARZERRA® is distributed in singie- use Vials containing 100 mg/5 mL and 1,000 mg/SO mL ofatumumab for IV infusion.

Ofatumumab is FDA-approved for treating chronic lymphocytic leukemia and has also shown potential in treating Follicular dgkin’s lymphoma, e large B cell lymphoma, rheumatoid arthritis, and relapsing remitting multiple sclerosis.

Ofatumumab has a molecular weight of about 149 kDa. It is currently administered by IV infusion at an initial dose of 300 mg, foilowed by weekly infusions of 2,000 mg.

As usedherein, the term “ofatmnumab” includes the anti~CD20 mAb known by the International Nonproprietary Name “OFATUMUMAB.” The term muma ” includes the active agent in ts marketed under the trade name A® and biosimilars thereof. The term “ofatumumab” includes the active agent in biosimilar ARZERRA® products being developed by BioExpress. High-molecular~weight, low- viscosity liquid protein formulations can e ofatumumab, preferably in a concentration of about 300 mg/mL to about 2,000 mg/mL.

Trastuzumab emtansine (in the U.S., ado-trastuzurnab emtansine, marketed as KADCYLA®) is an antibody-drug ate consisting ofthe mAb trastuzumab linked to the cytotoxic agent mertansine (DIN/[163). Trastuzumab, described above, stops growth of cancer cells by binding to the HERZ/neu receptor, whereas mertansine enters cells and ys them by binding to tubulin. In the United States, trastuzumab emtansine was approved specifically for treatment ofrecurring HERZ- positive metastatic breast cancer. Multiple Phase III trials oftrastuzumab emtansine are planned or ongoing in 2014. Trastuzumab emtansine is currently administered by IV infusion of 3.6 rug/kg. High-molecular-weight, low~viscosity liquid formulations can include trastnzumab emtansine, preferably in a tration of about 144 mg/mL to about 360 mg/mL.

Pertuzumab (PERJETA®) is a mAb that inhibits HERZ dimerization. umab received FDA approval for the treatment of HERZ-positive metastatic breast cancer in 2012. The currently recommended dosage of Pertuzumab is 420 mg to 840 mg by IV infusion. High»molecular—weight, low-viscosity liquid formulations can include pertuzumab, preferably in a concentration of about 420 mg/mL to about 840 mg/mL.

Daclizumab is a humanized anti-CD25 mAb and is used to prevent rejection in organ transplantation, ally in kidney transplants. The drug is also under investigation for the ent of multiple sis. Daclizumab has a molecular weight of about 143 kDa. Daciizurnab was marketed in the US. by Hoffmann—La Roche, Ltd. e”) as ZENAPAX® and stered by IV infusion of 1 nag/kg.

Daclizumab High—Yield Process (DAC HYP; BIIB019; Biogen Idec (“Biogen”) and Abeie, Inc. (“Abeie”)) is in phase III clinical trials as a 150 mg, once-monthly subcutaneous injection to treat relapsing, remitting multiple-sclerosis. High- molecular—weight, low-viscosity liquid formulations can include daclizumab, ably in a tration of about 40 mg/mL to about 300 mg/rnL. umab (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 being ped by Alexion Pharmaceuticals, Inc (“Alexion”). It is administered by IV infilsion in the amount of about 600 mg to about 1,200 mg. High-molecular- weight, low-viscosity liquid formulations can include eculizumab, preferably in a concentration of about 500 mg/mL to about 1,200 rug/mL.

Tocilizumab (ACTEMRA®) is ahumanized mAb against the interleukin-6 or. it is an immunosuppressive drug, mainly for the treatment of rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis, a severe form ofRA in children. Tocilizumab is commonly administered by IV infusion in doses of about 6 mg/kg to about 8 mg/kg. High-molecular-weight, low-viscosity liquid formulations can include tocilizumab, preferably in a concentration of about 240 mg/mL to about 800 mg/mL.

Rituximab (RITUXAN®) is a ic anti-CD20 mAb used to treat a variety of diseases characterized by excessive numbers of B cells, overactive B cells, or ctional B cells. Rituximab is used to treat cancers of the white blood system, such as leukemias and mas, including Hodgkin‘s lymphoma and its lymphocyte—predominant subtype. It has been shown to be an effective rheumatoid arthritis treatment. Rituximab is widely used off-label to treat difficult cases of multiple sclerosis, systemic lupus eryfilematosus, and autoimmune anemias.

Rituximab is y marketed in the US. under the trade name N® by Biogen and Genentech and outside the U.S. under the trade name RA® by Roche. RITUXAN® is distributed in single-use vials containing 100 mg/10 mL and 500 mg/SO mL. RITUXAN® is typically administered by IV infusion of about 375 rug/m2. The term “rituximab,” as used herein, es the anti-CD20 mAb known under the International Nonproprietary Name/Common Name “RITUXIMAB.” Rituxirnab includes mAbs described in US. Patent No. 5,736,137. Rituximab includes the active agent in products marketed under the trade name RITUXAN® and MABTHERA® and biosimilars f.

High-molecular—weight, low-viscosity liquid ations can include mab, preferably in a concentration of about 475 mg/mL to about 875 mg/mL (approximated using a body e area range of 1.3 to 2.3 square meters, derived from the Mosteller formula for persons ranging from 5 ft, 40 kg to 6 ft, 100 kg).

Concentrations are calculated for a 1 mL formulation.

Ipilimumab is a human mAb developed by Bristol-Myers Squibb Company (“Bristol-Myers Squibb”). Marketed as YERVOY®, it is used‘for the treatment of WO 38811 melanoma and is also undergoing clinical trials for the treatment of non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), and atic hormone- refiactory prostate cancer. Ipilimumab is currently administered by IV infusion of 3 mg/kg. High—molecular—weight, scosity liquid formulations can include ipilimurnab, preferably in a concentration of about 120 mg/mL to about 300 mg/mL. cumab (ABthraX®) is a human mAb intended for the prophylaxis and treatment of inhaled anthrax. It is currently administered by IV infusion. The suggested dosage in adults and children over 50 kg is 40 mg/kg. High-molecular- weight, scosity liquid formulations can include raxibacumab, preferably in a concentration of about 1,000 mg/mL to about 4,000 mg/mL.

Nimotuzumab (THERACIM®, BIOMAB EGFR®, oc®, CIMAher®) is a humanized mAb with a molecular weight of about 151 kDa used to treat squamous cell carcinomas of the head and neck, recurrent or refractory high—grade malignant glioma, anaplastic astrocytomas, glioblastomas, and diffuse sic e glioma. Nimotuzumab is typically administered by IV infusion of about 200 mg weekly. High-'molecular-Weight, low—viscosity liquid formulations can include nimotuzumab, preferably in a concentration of about 200 mg/mL.

Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate directed to the protein CD30, expressed in classical Hodgkin’s lymphoma and systemic anaplastic large cell lymphoma. It is administered by IV infusion of about 1.8 mg/kg.

High»molecular-weight, low—viscosity liquid formulations can include brentuximab vedotin, preferably in a concentration of about 80 mg/mL to about 200 mg/mL.

Itolizumab (ALZUMAB®) is a humanized IgGl mAb ped by Biocon.

Itolizumab completed successful Phase III studies in patients With moderate to severe psoriasis. ltolizumab has received marketing approval in India; an ation for FDA approval has not been submitted.

Obinutuzumab (GAZYVA®), originally developed by Roche and being further developed under a collaboration agreement with Biogen is a humanized anti—CD20 mAb approved for ent of chronic lymphocytic leukemia. It is also being investigated in Phase III clinical trials for patients With various mas. Dosages of about 1,000 mg are being administered via IV on. izumab pegol (CIMZIA®) is a recombinant, humanized antibody Fab’ fiagment, with city for human tumor necrosis factor alpha , conjugated to an approximately 40kDa polyethylene glycol (PEGZMAL40K). The lar weight of certelizumab pegol is approximately 91 kDa.

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

Antibody Therapeutics in Late~Srage Trials and Development The progression of antibody therapeutics to late-stage clinical development and regulatory review are proceeding at a rapid pace. In 2014, there are more than 300 mAbs in clinical trials and 30 commercially-sponsored antibody therapeutics undergoing evaluation in late-stage studies. First marketing ations for two mAbs (vedolizumab and ramucirumab) were recently submitted to the FDA. Arngen is currently sponsoring multiple ongoing Phase III trials on the use of brodalumab in patients with plaque psoriasis, with additional trials d or recruiting ts.

XBiotech, Inc. has sponsored two Phase I clinical trials ofMABpl (Xilonix) for patients with advanced cancer or type—2 diabetes. onal trials of MABpl are recruiting patients. Multiple trials are sponsored by Medlmmune, LLC 7, - (“Medlmmune”) and underway or recruiting patients for the treatment of ia with moxetumomab pasudotox. Long—term safety and efficacy studies are underway for the use of tildrakizurnab for the treatment of chronic plaque psoriasis. Multiple phase II trials have ly completed for the use of mumab for the treatment of s cancers.

At least 28 mAbs are olecular—weight proteins currently in or having recently completed Phase III studies for the treatment of inflammatory or immunological disorders, cancers, high cholesterol, osteoporosis, Alzheimer’s disease, and infectious diseases. The mAbs in or having recently completed Phase III trials include AMG 145, elotuzumab, epratuzumab, farletuzumab (MORAb-003), gantenerurnab (RG1450), zumab, inotuzumab ozogamicin, itolizumab, ixekizumab, lebrikizumab, mepolizumab, naptumomab estafenatox, necitumumab, nivolumab, ocrelizumab, onartuzumab, racotumomab, ramucirnmab, reslizumab, romosozumab, sarilmnab, secukinumab, sirukumab, solanezurnab, tabalumab, and vedolizumab. A mAb mixture umab and bezlotoxurnab) is also being evaluated in Phase III trials. See, e.g., Reichert, Mb: 5:1-4, 2013. zumab is a mAb being developed by Millennium Pharmaceuticals, Inc ennium”; a subsidiary of Takeda ceuticals Company, Ltd. (“Takeda”)). zumab was found safe and highly effective for inducing and maintaining clinical remission in patients with moderate to severe tive colitis. Phase III clinical trials showed it to meet the objectives of inducing a clinical response and maintaining remission in Crohn's and ulcerative colitis patients. s evaluating long—term clinical outcomes show close to 60% of patients ing clinical remission. A common dose of vedolizumab are 6 mg/kg by IV infusion.

Ramucirumab is a human InAb being developed for the treatment of solid tumors. Phase III al trials are ongoing 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 via IV infusion. mumab is a human mAb that inhibits the action ofhepatocyte growth factor/scatter factor. Developed by Amgen, it is in Phase III trials as a treatment for solid tumors. An open Phase III study of mumab treatment in patients with advanced or metastatic esophageal cancer will administer rilotumurnab at about 15 mg/kg via IV infusion.

Evolocumab (AMG 145), also developed by Amgen, is a mAb that binds to PC8K9. Evolocumab is indicated for hypercholesterolemia and hyperlipidemia.

Alirocumab (REGN727) is a human mAb from Regeneron Pharmaceuticals, Inc. (“Regeneron”) and Sanofi-Aventis US. LLC (“Sanofi”), indicated for hypercholesterolemia and acute coronary syndrome.

Naptumomab estafenatox, AER-217620 from Active Biotech AB (“Active Biotech”) is a mAb ted for renal cell carcinoma.

Racotum'omab from CIMAB, SA (“CIMAB”); Laboratorio Elea S.A.C.I.F.y A. is a mAb indicated for non—small cell lung cancer.

Other antibodies which may be formulated with viscosity-lowering ionic liquids e bococizumab (PF-04950615) and tanezumab; ganitumab, blinatumomab, trebananib from Arngen; Anthrax immune globulin from Cangene Corporation; teplizumab from enics, Inc.; MK-3222, MRI-6072 from Merck & Co (“Merck"); girentuximah from Wilex AG; RIGScan fifom a Biopharma- Biopharmaceuticals C'Navidea”); PF~05280014 from Pfizer; SA237 from Chugai Pharmaceutical Co. Ltd. (“Chugai”); guselkumab from Janssen/ Johnson and Johnson Services, Inc. (“18d"); Antithrombin Gamma 57) from Kyowa; and CT~P10 from Celltrion.

Antibodies in Early—Stage Clinical Trials Many mAbs have ly entered, or are entering, clinical trials. They can include proteins currently administered via IV infusion, preferably those having a molecular weight greater than about 120 kDa, typicallyfrom about 140 kDa to about 180 kDa. They can also include such high-molecular-weight proteins such as Albumin-conjugated drugs or peptides that are also entering al trials or have been approved by the FDA. Many mAbs fiom Amgen are currently in clinical trials.

These can be high-molecular-weight proteins, for example, AMG 557, Which is a human monoclonal dy developed jointly by Amgen and AstraZeneca and currently in Phase I trials for treatment of lupus. Likewise, AMG 729 is a humanized InAb developed by Amgen and currently in Phase I trials for the treatment of lupus and rheumatoid arthritis. In addition, AMG 110 is a mAb for epithelial cell adhesion molecuie; 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 multiple Phase I trials for the treatment of osteopenia; AMG 334, having completed Phase I dosing studies and tly in in Phase II studies for the treatment of migraines and hot flashes, is a human mAb that inhibits Calcitonin Gene-Related Peptide; AMG 780 is a human antiuangiopoietin mAb that ts the interaction between the endothelial cell-selective Tie2 receptor and its s Angl and Ang2, and recently completed Phase I trials as a cancer ent; 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 ses tumor associated macrophage (TAM) function and is being investigated as a cancer ent; AMG 181, jointly developed by Amgen and AstraZeneca, is a human mAb that inhibits the action of alpha4/beta7 and is in Phase 2014/055245 II trials as a treatment for ulcerative colitis and Crohn's disease.

Many mAbs are currently in clinical trials for the treatment of autoimmune disorders. These mAbs can be included in low-viscosity, high-molecular-weight liquid formulations. RG7624 is a fully human mAb designed to specifically and selectively bind to the human interleukin-l7 family of cytokines. A Phase I al trial evaluating RG7624 for autoimmune disease is ongoing. 3 is an anti- LINGO-l mAb by Biogen currently in Phase II trials for treating multiple sclerosis.

High-molecular-weight proteins also can include AGS-009, a mAb targeting IFN—alpha developed by Argos Therapeutics, Inc. that ly completed phase I trials for the treatment of lupus. Patients are administered up to 30 mg/kg ofAGS—009 via IV infusion. BT-061, developed by Abeie, is in Phase II trials for patients with rheumatoid tis. Certolizumab pegol (CIMZIA®) is a mAb in Phase II trials for ankylosing spondylitis and juvenile rheumatoid arthritis. Clazakizumab, an anti-1L6 mAb, is in Phase II trials by Bristol-Myers Squibb.

ONTO-136 (sirukumab) and CNTO-1959 are mABs having recently completed Phase II and Phase III trials by Janssen. Daclizumab (previously marketed as ZENAPAX® by Roche) is currently in or has recently completed multiple Phase III trials by Abeie for the ent ofmultiple sclerosis. Epratuzumab is a humanized mAb in Phase III trials for the treatment of lupus. Canakinumab (ILARIS®) is a human mAb targeted at interleukin-1 beta. It was approved for the treatment of cryopyrin—associated periodic syndromes. Canakinumab is-in Phase I trials as a possible treatment for chronic obstructive ary disease, gout and coronary artery e. Mavrilimumab is a human mAb ed for the treatment of rheumatoid arthritis. Discovered as CAM—3001 by Cambridge Antibody Technology, mavrilimumab is being developed by MedImmune.

MEDI-546 are MEDI-57O are mAbs currently in Phase I and Phase II trials by AstraZeneca for the treatment of lupus. MEDI-546 is administered in the Phase II study by regular IV infusions of BOO-1,000 mg. MEDI—SSI, another mAb being developed by AstraZeneca for numerous indications, is also tly administered by IV infusion. NN8209, a mAb ng the C5aR receptor being developed by Novo Nordisk A/S( “Novo k”), has completed a Phase II dosing study for treatment ofrheumatoid tis. NN8210 is r antiCSaR mAb being developed by Novo Nordisk and currently is in Phase I . 1 CNN8765) is a humanized mAb targeting NKG2A being developed by Novo Nordisk to treat patients with inflammatory conditions and autoimmune diseases. NN8765 recently completed Phase I trials.

Olokizumab is a humanized mAb that potently targets the cytokine IL-6. IL-6 is involved in several autoimmune and inflammatory ys. Olokizumab has completed Phase II trials for the treatment ofrheumatoid arthritis. Otelixizumab, also known as TRX4, is a mAb, which is being developed for the ent of type 1 diabetes, rheumatoid tis, and other autoimmune diseases. Ozoralizumab is a humanized mAb that has completed Phase II .

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

Rontalizumab is a zed mAb being developed by Genentech. It recently completed Phase II trials for the treatment of lupus. SAR113244 (anti—CXCRS) is a mAb by Sanofi in Phase I trials. Sifaiimumab (anti-IFN—alpha mAb) is a mAb in . - Phase II trials for the treatment of lupus.

A high-molecular-weight low-viscosity liquid formulation can include one of the mAbs in early stage clinical development for treating various blood disorders. For example, Belimurnab (BENLYSTA®) has recently completed Phase I trials for ts with vasculitis. Other mAbs in stage triais for blood disorders include BI—655075 from Boehringer Ingelheim GmbH “Boehringer Ingelheim”, ferroportin mAb and hepcidin mAb from Eli Lily, and SelGl from Selexys Pharmaceuticals, Corp. (“Selexys”).

One or more mAbs in early-stage development for treating various cancers and related conditions can be included in a scosity, high-molecular-weight iiquid formulation. United Therapeutics, Corporation has two mAbs in Phase I trials, 8H9 mAb and ch14.18 mAb. The mAbs ABT-806, enavatuzumab, and volociximab from Abeie are in early—stage development. Actinium Pharmaceuticals, Inc has conducted early-stage trials for the mAbs Actimab-A (M195 mAb), anti-CD45 mAb, and Iornab- B. Seattle Genetics, Inc. (“Seattle Genetics”) has several mAbs in early-stage trials for cancer and related conditions, ing anti-CD22 ADC (RG7593; pinatuzumab vedotin), anti-CD79b ADC (RG7596), anti-STEAPI ADC (RG7450), ASG—SME and ASG-ZZME from Agensys, Inc. (“Agensys”) the antibody-drug conjugate RG7458, and vorsetuzumab mafodotin. The early-stage cancer therapeutics from Genentech can be included in low-Viscosity formulations, including ALT-836, the antibody-drug conjugates RG7600 and DEDN6526A, D22 ADC (RG7593), GFL7 mAb (RG7414), anti-HER3/EGFR DAF InAb (RG7597), anti—PD-Ll mAb (RG7446), 39A, an MINT1526A. Bristol-Myers Squibb is developing early—stage mAbs for cancer therapeutics, including those identified as anti—CXCR4, anti-PD-Ll, IL—21 (EMS-982470), lirilumab, and urelumab (anti-CD13 7). Other mAbs in early-stage trials as cancer therapeutics include APN301(hnl4.18-IL2) from Apeiron Biologics AG, AV-203 from AVEO ceuticals, Inc. (“AVEO”), AVX701 and AVX901 from AlphaVax, BAX-69 from Baxter International, Inc. (“Baxter”), BAY 0 and BAY 20-10112 from Bayer HealthCare AG, BHQ880 from Novartis AG, 212- Pb-TCMCtrastuzumab from AREVA Med, AbGn-7 from AbGenornics International Inc, and ABIO-0501 (TALL-104) from Abiogen Pharma S.p.A.

Other antibody therapeutics that can be formulated with ity—lowering ionic s include alzumab, GAIOI, daraturnurnab, siltuxirnab, ALX—006l, ALX- 0962, ALX~0761, bimagumab (BYM338), CT—Oll izumab), actoxumab/bezlotoxumab (MK-3515A), MIC-3475 (pembrolizumab), dalotuzumab (MK-0646), icrucurnab (IMO-18F 1, LY3012212), AMG 139 070), SAR339658, dupilumab (REGN668), SAR156597, SAR256212, SAR279356, SAR3419, SAR153192 (REGN421, enoticumab), SAR307746 (nesvacurnab), SAR650984, SAR566658, SAR391786, SAR228810, SAR252067, SGN-CDIQA, SGN-CD3 3A, SGN—LIVIA, ASG 15MB, Anti-LINGO, BIIB037, ALXN1007, teprotumurnab, concizumab, anrukinzumab (IMA-63 8), ponezumab (PF-04360365), PF-03446962, PF-062526l6, zumab (RG7413), quilizurnab, ranibizurnab, lampalizumab, onclacumab, gentenerumab, urnab (RG7412), IMC-RONS (namatumab), tremelimumab, turnab, eemcizumab, ozanezumab, mapatumurnab, tralokinumab, XmAbS871, XmAb7 195 , cixutumumab (LY30122 1 7), LY2541546 (blosozumab), olaratumab (LY3012207), MEDI4893, 3, 39, MEDI3 617, MEDI4736, MEDI6469, MEDIO680, MEDIS872, PF- 05236812 (AAB-003), PF-05082566, BI 0, RG7116, RG7356, RG7155, WO 38811 RG7212, RG7599, RG7636, RG7221, RG7652 (MPSK3169A), RG7686, HuMaX- HuMaXTFADC, MOR103, BT061, MORZOS, OMP59R5 (antimotch 2/3), VAY736, MOR202, BAY94-9343, LJM716, 51, 776, 320, GSK1070806, NN8828, CEP-37250/KHK2804 AGS—16M8F, AGS—16C3F, LY3016859, LY2495655, LY2875358, and LY2812176.

Other early stage mAbs that can be formulated with Viscosity-lowering ionic liquids include benralizumab, MEDI-8968, anifrolumab, MEDI7183, sifalimumab, MEDI—575, tralokinumab from AstraZeneca and Medlmmune; BAN2401 from Biogen Idec/Eisai Co. LTD ("Eisai”)/ BioArctic Neuroscience AB; CDP7657 an anti- CD4OL monovalent ted Fab antibody fragment, STX~100 an anti-aVB6 mAb, BIIB059, Anti-TWEAK (BHB023), and BIIBO22 from Biogen; fulranumab from Janssen and Amgen; BI—204/RG741 8 from BioInvent ational/Genentech; BT- 062 (indatuximab ravtansine) from Biotest Pharmaceuticals Corporation; XmAb from Boehringer Ingelheimeencor; anti-IP10 from Bristol-Myers Squibb; J 591 Lu-l77 from BZL Biologics LLC; CDX—Oll (glembatumumab vedotin), CDX—O401 from Celldex Therapeutics; foravirumab from CrucellI;-t-igatuzumab from Daiichi Sankyo Company Limited; MORAb-004, MORAb-009 (amatuximab) from Eisai; LY2382770 from Eli Lilly; DIl7E6 fiom EMD Serono Inc; zanolimumab from Emergent BioSolutions, Inc.; FG—301 9 from FibroGen,Inc.; catumaxomab from Fresenius SE & Co. KGaA; pateclizumab, rontalizumab from ech; fresolimumab from Genzyme & Sanofi; (38-6624 (simtuzurnab) from Gilead; CNTO— 328, bapineuzurnab (AAB—OOl), carlumab, CNTO—136 from Janssen; KB003 from KaloBios ceuticals, Inc.; ASKP1240 from Kyowa; RN—307 from Labrys Biologics Inc.; ecromeximab from Life Science Pharmaceuticals; LY2495655, LY2928057, LY3015014, LY2951742 from Eli Lilly; MBL-HCV] from ologics; AME—l3 3v from K h, LLC; abituzurnab from Merck KGaA; MM-121 from Merrimack Pharmaceuticals, Inc.; MCSl 10, , , QGE031 from Novartis AG; HCD122 from Novartis AG and XOMA ation ("XOMA”); NNSSSS from Novo Nordisk; bavituximab, cotara from Peregrine Pharmaceuticals, Inc.; PSMA—ADC from Progenics Pharmaceuticals, Inc.; oregovomab from Quest Pharmatech, Inc.; fasinumab (REGN475), REGN1033, SAR231893, REGN846 from Regeneron; RG7160, CIM331, RG7745 from Roche; ibalizumab (TMB—355) from TaiMed Biologics Inc.; TON-032 from Theraclone Sciences; TRC105 from TRACON ceuticals, Inc; UB—421 from United Biomedical Inc; VB4-845 from Viventia Bio, Inc.; ABT—l 10 from Abeie; izumab, Ozoraiizurnab from Ablynx; PRO 140 from CytoDyn, Inc; GS- CDAI, MDX-1388 from Medarex, Inc; AMG 827, AMG 888 from Amgen; ublituximab from TG Therapeutics Inc.; TOL 1 01 from Tolera Therapeutics, 1110.; huN901—DM1 (lorvotuzurnab mertansine) from ImmunoGen Inc. ; epratuzumab Y- 90/Veltuzumab combination (WU—102)from medics, Inc.; brin mAb/ 3B6/22 Tc—99m from Agenix, Limited; ALD403 from Alder Biopharmaceuticais, Inc.; RN6G/ PF-04382923 from Pfizer; CG201 from CG Therapeutics, 1110.; KBOOI— A from os Pharmaceuticals/Sanofi; KRN—23 from Kyowa.; Y—90 hPAM 4 from Immunomedics, Inc.; Tarextumab from Morphosys AG & OncoMed Pharmacetuicals, Inc; LFG316 fiom Morphosys AG & Novartis AG; CNTO3157, CNT06785 from Morphosys AG & Jarmsen; RG6013 fiom 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, CT—P24, CT-P25, CT-P26, CT~P27, CT—P4 from Celltrion; GSK284933, GSK23 98852, 8960, GSK1223249, 'GSK933776A from GlaxoSmithKline; anetumab ravtansine fi'orn Morphosys AG & Bayer AG; BI—836845 from Morphosys AG & Boehringer eim; NOV-7, NOV- 8 from Morphosys AG & Novartis AG; , MM-310, MM-Ml, MM-131, MM—151 from Merrimack, RG7882 from Roche & Seattle cs; RG7841 from Roche/ Genentech; PF-06410293, PF-06438179, PF-0643 9535, PRO-4605412, PF- 05280586 from Pfizer; RG7716, RG7936, gentenerurnab, RG7444 from Roche; MEDI-547, MEDI-565, MEDI1814, MEDI4920, MED18897, MEDI—4212, MEDI- 51 17, MEDI—78 14 from Astrazeneca; ulocuplumab, PCSK9 adnectin from Bristol- Myers Squibb; FPA009, FPA145 from irne eutics, Inc; GS—5745 from Gilead; BIW-8962, KHK4083, KHK6640 from Kyowa Hakko Kirin; MM—141 from Merck KGaA; REGN1154, REGN1193, REGN1400, REGNISOO, REGN1908—1909, REGN2009, REGN2176~3, REGN728 from Regeneron; SAR307746 from Sanofi; SGN—CD70A fiom Seattle Genetics; 41, ALX-0171 from AblynX; milatuzurnab-DOX, milatuzumab, TF2, from Immunomedics, Inc.; MLN0264 from Millennium; ABT-981from Abeie; AbGn—168H from AbGenornics International Inc.; ficlatuzumab from AVEO; BI-SOS from BioInvent International; CDX-1127, CDX-301 from Celldex Therapeutics; CLT-008 from Cellerant Therapeutics Inc.; VGX-IOO from Circadian; U3-1565 from Daiichi Sankyo Company d; DKN~ 01 from Dekkun Corp; flanvotumab (TYRPI protein), IL-1 [3 antibody, IMC-CS4 from Eli Lilly; VEGFR3 mAb, IMC—TRI (LY30228b59) from Eli Lilly and IrnClone, LLC; Anthirn from Elusys Therapeutics Inc.; HuL2G7 from Galaxy Biotech LLC; IMGB853, IMGN529 from ImmuncGen Inc. ; CNTO-S, CNTO—5825 from Janssen; KD-247 from Kaketsuken; KB004 from KaloBios Pharmaceuticals; MGA271, MGAH22 from MacroGenics, Inc.; XmAb5574 from Sys AG/Xencor; ensituximab (NFC-1C) from Neogenix Oncology, Inc.; LFA102 from Novartis AG and XOMA; ATI355 from Novartis AG; SAN-300 from us Inc.; SelGl from Selexys; HuM195/rGel from Targa Therapeutics, Corp; VX15 from Teva ceuticals, Industries Ltd. (“Teva”) and Vaccinex Inc.; TCN—202 from Theraclone Sciences; XmAb2513, XmAb5872 from Xencor; XOMA 3AB from XOMA and National Institute for Allergy and ious Diseases; lastorna antibody vaccine from MabVax Therapeutics; Cytolin from CytoDyn, Inc.; Thravixa fiom Emergent BioSolutions Inc.; and FE 301 from Cytovance Biologics; rabies mAb from n and Sanofi; flu mAb from Janssen and partly funded by National Institutes of ; MB—003 and ZMapp from Mapp Biophannaceutical, Inc.; and ZMAb from Defyrus Inc.

Other Protein Therapeutics The protein can be an enzyme, a fusion protein, a stealth or ated protein, vaccine or otherwise a biologically active protein (or protein mixture). The term “enzyme,” as used herein, refers to the protein or functional fragment thereof that catalyzes a biochemical transformation of a target molecule to a d product. s as drugs have at least two important features, namer i) often bind and act on their targets with high affinity and specificity, and ii) are catalytic and convert multiple target molecules to the desired products. In certain embodiments, WO 38811 the protein can be PEGylated, as defined herein.

The term “fusion protein,” as used herein, refers to a protein that is created from two different genes encoding for two separate proteins. Fusion proteins are generally produced through recombinant DNA techniques known to those skilled in the art. Two ns (or n fragments) are filsed together covalently and exhibit properties from both parent proteins.

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

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

ELOCTATE®, Antihemophilic Factor (Recombinant), Fc Fusion n, is a recombinant DNA derived, antihemophilic factor indicated in adults and children with Hemophilia A (congenital Factor VIII deficiency) for control and prevention of bleeding episodes, perioperative ment, e prophylaxis to prevent or reduce the frequency of bleeding episodes.

EYLEA® (aflibercept) is a recombinant fusion protein consisting of portions *- ofhuman VEGF receptors 1 and 2 extracellular domains fused to the Fc portion of human lgGl formulated as an iso-osmotic solution for intravitreal administration.

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

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

Pegloticase (KRYSTEXXAQQ) is a drug for the treatment of severe, treatment- refractory, chronic gout, developed by t ceuticals, Inc. and is the first drug approved for this indication. Pegloticase is a pegylated recombinant porcine—like uricase with a molecular weight of about 497 kDa. icase is currently adminis— administered by IV infusions of about 8 mg/kg. High-molecular—weight, low-viscosity liquid formulations can include pegloticase, ably in a concentration of about 300 mg/mL to about 800 mg/mL.

Alteplase (ACTIVASE®) is a tissue plasminogen activator produced by recombinant DNA technology. It is a purified glycoprotein comprising 527 amino acids and synthesized using the complementary DNA (cDNA) for natural human tissue-type plasminogen tor obtained from a human melanoma cell line.

Alteplase is stered via IV infusion of about 100 mg immediately ing symptoms of a stroke. In some embodiments, low-viscosity formulations are provided containing alteplase, preferably in a concentration of about 100 mg/mL.

Glucarpidase (VORAXAZE®) is a FDA-approved drug for the treatment of elevated levels of methotrexate (defined as at least 1 micromol/L) during treatment of cancer patients who have impaired kidney function. Glucarpidase is administered via IV in a single dose of about 50 lU/kg. In some embodiments, low-viscosity formulations are provided containing glucarpidase. - - Algiucosidase alfa (LUMIZYME®) is an enzyme replacement therapy orphan drug for treatment e disease (glycogen storage disease type II), a rare lysosomal e disorder. It has a molecular weight of about 106 kDa and is currently administered by IV infusions of about 20 mg/kg. In some embodiments, a low-viscosity pharmaceutical formulation of osidase alfa is provided, preferably with a tration of about 100 mg/mL to about 2,000 mg/mL.

Pegdamase bovine (ADAGEN®) is a modified enzyme used for enzyme replacement therapy for the ent of severe combined immunodeficiency disease (SCID) associated with a deficiency of adenosine deaminase. Pegdamase bovine is a conjugate of numerous strands methoxypolyethylene glycol (PEG), molecular weight 5,000 Da, covalently attached to adenosine deaminase enzyme that has been derived from bovine intestine. o-Galactosidase is a lysosomal enzyme that catalyses the hydrolysis of the giycolipid, globotriaosylceramide (GL-S), to galactose and ceramide dihexoside.

Fabry disease is a rare table lysosomal e disease characterized by subnormal enzymatic activity of u-Galactosidase and resultant accumulation of GL-3. dase alfa (REPLAGAL®) is a human d-galactosidase A enzyme produced by a human cell line. Agalsidase beta ZYME®) is a recombinant human 01-— galactosidase expressed in a CHO cell line. Replagal is administered at a dose of 0.2 mg/kg every other week by intravenous infusion for the treatment of Fabry disease and, off label, for the treatment of Gaucher disease. FABRAZYME® is administered at a dose of 1.0 mg/kg body weight every other week by IV infusion. Other lysosomal enzymes can also be used. For example, the protein can be a lysosomai enzyme as described in US 148556.

Rasburicase (ELITEK®) is a recombinant urate-oxidase indicated for initial management ofplasma uric acid levels in pediatric and adult patients with leukemia, lymphoma, and solid tumor malignancies who are ing ancer therapy expected to result in tumor lysis and subsequent elevation of plasma uric acid.

ELITEK® is stered by daily IV on at a dosage of 0.2 mg/kg. cerase (CEREZYME®) is a recombinant analogue of human B- glucocerebrosidase. Initial dosages range from 2.5 U/kg bodylweight 3 times a week to 60 U/kg once every 2 weeks. ME® isadministered by IV infusion.

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

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

Laronidase (ALDURAZYME®) is a polymorphic variant of the human enzyme d—L—iduronidase that is produced via CHO cell line. The recommended dosage regimen ofALDURAZYME® is 0.58 mg/kg administered once weekly as an intravenous infusion.

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

Other biologics which may be formulated With viscosity-lowering ionic liq- uids include asparaginase erwinia chrysanthemi (ERWINAZE®), incobotulinumtoxin A (XEOMIN®), EPOGEN® (epoetin Alfa), PROCRIT® (epoetin Alta), ARANESP®' (darbepoetin alfa), ORENCIA® (abatacept), BATASERON® (interferon beta-1b), NAGLAZYME® (galsulfase); ELAPRASE® (Idursulfase); E® (LUMIZYME®, algucosidase alfa); VPRJV‘:D (velagiucerase), abobotulinumtoxin A RT®); BAX—326, Octocog alfa from Baxter; Syncria from GlaxoSmithKline; liprotamase from Eli Lilly; Xiaflex (collagenase clostridium histolyticum) from Aux- ilium and BioSpecifics Technologies Corp; anakinra from Swedish Orphan Biovitrum AB; metreleptin from Bristol-Myers Squibb; Avonex, Plegridy l7) from Biogen; NN1841, NN7008 from Novo Nordisk; KRN321 (darbepoetin alfa), AMG531 lostim), KRN125 (pegfilgrastim), KW—076l ulizumab) from Kyowa; IB 1001 from Inspiration Biophannaceuticals; lprivask from Canyon Phannan ceuticals Group.

Protein Therapeutics in Development Versartis, Inc—37's VRS—3 17 is a recombinant human growth hormone (hGH) fusion n utilizing the XTEN half—life extension technology. It aims to reduce the frequency of hGH inj ections necessary for patients With hGH deficiency. VRS—3 l 7 has completed a Phase II study, comparing its efficacy to daily injections of non- derivatized hGH, with positive results. Phase III studies are d.

Vibriolysin is a proteolytic enzyme secreted by the Gram-negative marine microorganism, Vibrio proreolyticus. This endoprotease has specific affinity for the hydrophobic regions ofproteins and is capable of ng ns nt to hydrophobic amino acids. Vibriolysin is currently being investigated by BioMarin for the cleaning and/or treatment of burns. Vibriolysin formulations are described in patent W0 02/092014.

PEG-PAL (PEGylated recombinant phenylalanine ammonia lyase or “PAL”) is an investigational enzyme substitution y for the treatment of phenylketonuria (PKU), an ted metabolic disease caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). PEG-PAL is being developed as a potential treatment for ts Whose blood alanine (Phe) levels are not adequately controlled by . PEG-PAL is now in Phase 2 clinical development to treat patients who do not adequately respond to KUVAN®.

Other protein therapeutics which may be formulated with Viscosity-lowering ionic liquids include Alprolixf rFlXFc, Eloctate/ rFVIIIFc, BMN—190; BMN—250; Lamazyme; Galazyme; ZA-Ol I; pase alfa; SEC-103; and HGT-11 10.

Additionally, fusion-proteins containing the XTEN half-life extension technology including, but not limited to: VRS~317 GH-XTEN; Factor VIIa, Factor VIII, Factor IX; PF05280602, 9; Exenatide-XTEN; AMX—256; GLPZ-ZG/XTEN; and AMX—179 Folate—XTEN—DMI can be formulated with viscosity-lowering ionic liquids.

Other late-stage protein therapeutics which can be ated with Viscosity- lowering ionic liquids include CM—AT from CureMark LLC; NN7999, , Liraglutide (NNS022), NN9211, Semaglutide (NN953 5) from Novo Nordisk; AMG 386, Filgrastim from Amgen; CSL-654, Factor VIII from CSL Behring; LA—EP2006 (pegfilgrastim biosimilar) from Novartis AG; Multikine (leukocyte interleukin) from GEL-SCI Corporation; 541, Teriparatide (recombinant PTH 1-34) fromEli Lilly; NU—lOO from Nuron Biotech, Inc; Calaspargase PegoI from Sigma-Tau Pharmaceuticals, Inc.; ADI-PEG—20 from Polaris Pharmaceuticals, Inc. ; O, BMN—702 from BioMarin; NGR—TNF from Molmed S.p.A.; recombinant human Cl esterase tor from Pharming Group/Santarus Inc.; Somatropin biosimilar from LG Life Sciences LTD; Natpara from NPS Pharmaceuticals, Inc; ART123 from Asahi Kasei Corporation; BAX-111 from Baxter; OBI—1 from Inspiration Biopharmaceuticals; Wilate from Octapharma AG; oferrin alfa from Agennix AG; Desmoteplase from Lundbeck; e from Shire; RG7421 and Roche and Exelixis, Inc; aurin (PKC412) fiom Novartis AG; Darnoctocog alfa pegol, BAY 0, BAY 94-9027 from Bayer AG; Peginterferon lambdad a, Nulojix (Belatacept) from Bristol-Myers Squibb; Pergoveris, Corifollitropin alfa (MK-8962) from Merck KGaA; recombinant ation Factor IX Fc fusion protein (rFIXFc; BIIB029) and recombinant coagulation Factor VIII Fc fusion protein (rFVIIIFc; BHB031) from ; and Myalept from AstraZeneca.

Other early stage protein biologics which can be formulated with viscosity- lowering water ionic liquids include Alferon LDO from Hemispherx BioPharma, Inc.; SL-401 from Stemline Therapeutics, Inc; PRX-102 fiom Protalix Biotherapeutics, Inc; KTP—OOl from Kaketsuken/Teijin Pharma Limited; Vericiguat from Bayer AG; BMN—l i 1 from BioMarin; ACC-OO] (PF—05236806) from Janssen; LY2510924, LY2944876 from Eli Lilly; NN9924 from Novo Nordisk; INGAP peptide from Exsulin; 2 from Abbvie; AZD9412 from AstraZeneca; NEUBLASTIN (BGOOOlO) from Biogen; ercept (ACE—536), Sotatercept (ACE-011) from Celgene Corporation; PRAME immunotherapeutic from GlaxoSmithKline; Plovamer acetate (PI-2301) from Merck KGaA; PREMIPLEX (607) from Shire; BMN-701 from BioMarin; Ontak from Eisai; rHuPH20/insu1in from Halozyme, Inc; PB-1023 from PhaseBio Pharmaceuticals, Inc; ALV-003 from Alvine Pharmaceuticals Inc. and Abbvie; NN8717 from Novo Nordisk; PRT-201 from n Therapeutics Inc; PEGPH20 from Halozyme, Inc; Amevive® alefacept from Astellas Pharma Inc; F- 627 from Regeneron; AGN—214868 botase) from Allergan, Inc; BAX-817 from Baxter; PRT4445 from Portola ceuticals, Inc; VENl 00 from a Bioscience; Onconase/ ranpirnase from Tamir Biotechnology Inc; interferon alpha-2b infilsion from Medtronic, Inc; sebelipase alfa fiom Synageva BioPharma; IRX-2 from IRX Therapeutics, Inc; GSK2586881 from GlaxoSmithKline; 3 from Seikagaku Corporation; ALXNI 101, asfotase alfa from Alexion; SHP611, SHP609 (Elaprase, idursulfase) from Shire; 56884, PF-05280602 from Pfizer; ACE- 031, Dalantercept from Acceleron Pharma; ALT-801 from Altor ence Corp; BA-210 from BioAxone Biosciences, Inc; WTl immunotherapeutic from GlaxoSmithKline; 666 from Sanofi; MSBOOIO445, Atacicept from Merck KGaA; Leukine (sargramostim) from Bayer AG; KUR-211 from Baxter; fibroblast growth factor-1 from CardioVascular BioTherapeutics Inc; SPI—2012 from Hanmi Pharmaceuticals Cc, LTD fSpectrum Pharmaceuticals; FGF-18 (sprifermin) from Merck KGaA; MK—1293 from Merck; interferon-alpha-Qb from HanAll Biopharma; CYT107 from Cytheris SA; RTOOl from Revance Therapeutics, Inc; MEDI6012 from AztraZeneca; E2609 from ; BMN—190, BMN-270 from BioMarin; ACE- 661 from Aceeleron ; AMG 876 from Amgen; GSK3052230 from GlaxoSInithKline; RG7813 from Roche; SAR342434, Lantus fiom Sanofi; A201 from Allozyne Inc; ARX424 from Ambrx, Inc; FP-1040, FP-1039 from FivePrime eutics, Inc.; ATX-MS—1467 from Merck KGaA; XTEN fusion proteins from Amunix Operating 1110.; entolimod (CBLBSOZ) fiom Cleveland BioLabs, Inc.; 0 from Shire; HM10760A from Hanmi Pharmaceuticals Co., LTD; ALXNl 102/ ALXN1103 from Alexion; CSL~689, CSL-627 fiom CSL g; glial growth factor 2 from Acorda Therapeutics, Inc.;NX001 from Nephrx Corporation; , NN1436, NN1953, NN9926, NN9927, NN9928 from Novo Nordisk; NHS— IL 12 from EMD Serono; 3K3A—APC from 22 Biotech LLC; PB-1046 from PhaseBio ceuticals, Inc.; RU—1 01 from R—Tech Ueno, Ltd. ; insulin lispro/BC106 from Adocia; hl—conl from Iconic Therapeutics, Inc.; PRT-105 from Protalix BioTherapeutics, Inc.; 56883, CVX—096 from Pfizer; ACP-501 from AlphaCore Pharma LLC; BAX-855 from Baxter; CDX-1135 from Celldex Therapeutics; PRM-lSl from Prornedior, Inc.; T801 from Thrombolytic Science ational; TT»173 from Thrombotargets Corp; QBI-l39 from Quintessence ences, Inc.; Vatelizumab, GBRSOO, GBR600, GBR830, and GBR900 from Glenmark Pharmaceuticals; and CYT—6091 from une Sciences, Inc..

Other Biologic Agents Other biologic drugs that can be formulated with viscosity-lowering ionic liquids include PF-05285401, PF-05231023, RN317 (PF-05335810), PF-06263507, PF-05230907, Dekavil, PF-06342674, PF06252616, RG7598, RG7842, RG7624d, OMP54F28, GSK1995057, BAY1179470, IMO-3G3, IMC-l 8F1, C, IMC- 20D7S, PF~06480605, PF-06647263, PF—06650808, PF—05335810 (RN317) PD— 03 60324, PF-00547659 from Pfizer; MK-8237 fiom Merck; 81033 from Biogen; GZ402665, SAR43 8584/ REGN2222 from Sanofi; lMC-18F1; and Icrucumab, IMC- 3G3 from ImClone LLC; Ryzodeg, Tresiba, Xultophy from Novo Nordisk; Touje0 (U300), LixiLan, Lyxurnia (lixisenatide) from Sanofi; MAGE~A3 immunotherapeutic from GlaxoSmithKline; Tecemotide from Merck KGaA; Sereleaxin (RLX030) from Novartis AG; Erythropoietin; Pegfilgrastim; LY2963016, Dulaglutide (LY2182965) from Eli Lilly; and Insulin Glargine from nger Ingelheim.

B. Ionic Liquids The Viscosity of liquid protein formulations, including low—molecular-weight and/or high-molecular-weight proteins, is reduced by the addition of one or more viscositynreducing ionic liquids. The pharmaceutical formulations may be converted fi‘om non-Newtonian to ian fluids by the on of an effective amount of one or more viscosity~reducing ionic liquids.

Ionic Liquid Salts The ionic liquid can be a salt. Representative ionic liquid salts e salts with imidazolium cations, including N,N-dialkyl-irnidazoliums. Ionic liquids include salts with N—alkyiated unsaturated or saturated nitrogen-containing heterocyclic cations, including N-alkylpyridinium salts, N—alkylpyrrolidinium salts, and N- alkylpiperidinium salts. In preferred embodiments, the ionic liquid is phannaceutically acceptable and miscible with water.

In some embodiments, the ionic liquid contains a cationic constituent having a ic heterocyclic group with one or more alkyl, alkyi, 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 constituents include halide ions, sulfate, sulfonate, sulfite, sulfinate, ate, phosphonate, phosphite, phosphonite, carbonate, and carboxylate anions optionally substituted with one or more alkyl, heteroalkyl, alkenyl, alkynyl, carbocyclic, or heterocyclic groups, preferably having from 1 to 20 or from 1 to 12 carbon atoms. Exemplary anionic constituents include chloride, bromide, methylphosphate, methyl-ethyl-phosphate, methylsulfate, methylsulfonate, formate, acetate, butyrate, citrate, carbonate, methyl carbonate, and lactate. The cationic cyclic group can be saturated or rated. Saturated cationic heterocyclic groups include pyrrolidinium, oxazolidinium, piperidinium, piperazinium, morpholiniurn, thiomorpholinium, and azepanium groups, and the like.

Unsaturated ic heterocyclic groups e inium, olinium, 1,2,3- triazolium, 1,2,4-triazolium, thiazolium, 1,2,4-dithiazolium, 1,4,2—dithiazolium, tetrazolium, pyrazolinium, oxazolinium, pyridinium, and azepinium groups, and the like. The cationic heterocyclic group can be a fiised ring structure having two, three, four, or more fused rings. The cationic heterocyclic group can be a bicyclic cationic heterocycle, such as benzoxazolium, benzothiazolium, benzotriazolium, benzirnidazoliurn, and indolium groups, and the like. The cationic heterocyclic group can be substituted with one or more additional substituents, ing hydroxyl and tuted and unsubstituted alkoxy, heteroalkoxy, alkyi, heteroalkyl, alkenyl, and alkynyl groups having from 1 to 30, preferably from 3 to 20 carbon atoms.

The ionic liquid can be 1-butyl~3-methylimidazolium methanesulfonate (BMI Mes) having the structure shown below or a derivative thereof. / 3 i 0WSm N I K/\ O Derivatives ofBMI Mes can be obtained, for example, by substituting the methanesulfonate constituent for other anionic constituents, replacing one or more carbons with a heteroatom, replacing the N-butyl or N—methyl group with one or more 'higherworder N—alkyl groups, attaching onal substituents to one or more carbon atoms, or a combination f. ary anionic constituents are described above.

Exemplary heteroatoms include N, O, P, and S. Exemplary higher-order N—alkyl groups include substituted and unsubstituted N—alkyl and N~heteroalkyl groups containing from 1 to 30 carbon atoms, preferably from 1 to 12 carbon atoms.

Examples ofhigher—order N—alkyl groups include N—ethyl, anropyl, N-butyl, N-Sec- butyl, and N~tert-butyl. Additional substituents can e hydroxyl and substituted and unsubstituted alkoxy, heteroalkoxy, alkyl, aryl, l, aryloxy, aralkyloxy, heteroalkyl, alkenyl, and l groups having from 1 to 30, preferably from 3 to 20 carbon atoms.

The ionic liquid can be l-butylmethylpyrrolidinium chloride (BMP chloride) having the structure shown below or a derivative thereof.

Derivatives of BMP chloride can be obtained, for example, by substituting the chloride constituent for other anionic constituents, replacing one or more ring carbons with a heteroatom, ing the tyl-Inethyl group with one or more higher- order N,N—dialkyl groups, ing one or more additional substituents to a carbon atom, or a combination thereof. Exemplary anionic constituents e those described above. Exemplary atoms include N, O, P, and S. Exemplary higher- order N,N—dialkyl groups include linear, branched, and cyclic N—alkyl and N- heteroalkyl groups containing from 2 to 30 carbon atoms, preferably from 3 to 12 carbon atoms. 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 tuents can include hydroxyl, and substituted and unsubstituted alkoxy, heteroaikoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyloxy, alkenyl, and alkynyl groups having from 1 to 30, preferably from 3 to 20 carbon atoms.

In some embodiments, the ionic liquid contains a cationic constituent having a structure according to Formula I where each occurrence of R1 is independently selected from hydrogen and substituted and unsubstituted alkyl, heteroalkyl, aryl, aralkyl, alkenyl, and alkynyi groups having from 1 to 30 carbon atoms, from 3 to 20 carbon atoms, or from 4 to 12 carbon atoms; Where each occurrence of R2 is independently selected from hydrogen, , yl, and substituted and unsubstituted alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryi, aryloxy, aralkyl, araikyloxy, alkenyl, and l groups having from 1 to 30 carbon atoms, from 3 to carbon atoms, or from 4 to 12 carbon atoms. In some embodiments at least one, at least two, or at least three occurrences of Rlor R2 are not hydrogen.

Fantasia I R2 may also be independently selected from hydrogen, R1, -OH, NHz, ~F, -Cl, - Br, -I, -N02, -CN, -C(=O)R4a, -C(=NR4a)R4, -C(=O)OH, —C(=O)OR4, —OC(=O)R4, - OC(=0)0R4, -SO3H, -SOzN(R4a 2, «south -S02NR4“C(=O)R4, -P03H2, - R4aC(=NR4a)N(R4a 2, -NHC(=NR4a)NH—CN, —NR4aC(:O)R4, —NR4as02R4, - NR4aC(=NR43)NR4EC(=NR4a)N(R4a)2, -NR4aC(=O)N(R4a 2, -C(=0)NH2, - C(:0)N(R4a 2, -OR4, 61143, and -N(R4a)2; wherein R1 is independently selected from C1-Igalkyl, cycloa1ky1, C6- Igaryl, C1_Igheteroary1 and eterocyclyl, wherein each lky1 may be substituted one or more times with C3- lgcycloalkyl, C5_12ary1, C1_12heteroaryl, C2_12heterocyc1yl, -OH, NHg, (=0), (=NR4a), - F, -Cl, “Br, J, ~N02, —CN, —C(:0)R4a, »C(=NR4H)R4, -C(=O)OH, -C(=O)OR4, - OC(=0)R4, —OC(:O)0R4, _so2H, —so2N(R4a 2, -so2R4, -SO;NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a 2, -NHC(=NR4a)NH-CN, (=O)R4, ~NR4aso2R4, - =NR4“)NR4EC(=NR43)N(R43 2, -NR4"C(=O)N(R43 2, -C(=0)NH2, — C(=O)N(R4a 2, OR“, -SR“3, or -N(R43 2; wherein each C3_ucycloalkyl may be tuted one or more times with C1- lgaikyl, C5_12ary1, C1-1gheteroaryl, eterocyclyl, -OH, NH2, -F, -Cl, -Br, -I, ~N02, - CN, R4a, -C(:NR4‘1)R4, —C(:0)0H, —C(=0)0R4, -OC(=O)R4, -0C(=0)0R4, - s02H, -so2N(R4a)2, , -so2NR4aC(:0)R4, -PO3H2, —R4aC(:NR4a)N(R4a 2, .

NHC(=NR43)NH—CN, —NR4“C(:O)R4, —NR4flso2R4,— — NR4aC(=NR4a)NR4aC(=NR4a)N(R4a 2, -NR4aC(=0)N(R4a)2, —C(:0)NH2, _—.

(R4a 2, -OR4, -SR““, or -N(R4a 2; wherein each ryl may be substituted one or more times with C1_12alky1, Cgiucycloalkyl, C1.1zheteroaryl, €2.12heterocyclyl, -OH, NH;, -F, -Cl, -Br, -I, -N02, - CN, -C(=O)R43, -C(=NR4a)R4, -C(=O)OH, -C(:O)OR4, ~OC(=O)R4, —OC(:0)0R4, — so2H, —SOgN(R4a)2, -SO2R4, -SO2NR4aC(=0)R4, ~PO3H2, -R4aC(=NR4a)N(R4a 2, - NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4“802R4, - NR4ac(:NR4“)NR4aC(=NR4a)N(R4a)2, ~NR4aC(=O)N(R4a 2, —C(=0)NH2, — C(=O)N(R4a 2, -0R4, -SR4a, or -N(R4a 2; wherein each C1-12heteroaryl may be substituted one or more times with C1- lzalkyl, C3.lgcycloalkyl, C6-12a1'y1, C2.1zheterocyclyl, —OH, NH;, ~F, Cl, «Br, J, ~N02, -CN, _C(=0)R4a, —C(=NR4“)R4, —C(=0)OH, -C(:O)OR4, —OC(~—~0)R4, -OC(=O)OR4, - so2H, -so2N(R4a 2, -so2R4, -so2NR4aC(=O)R“, —P02H2, 414%:(=1\1R43‘)N(R4a 2, _ NHC(=NR43)NH—CN, —NR4aC(=O)R4, —NR4flso2R4, — NR4aC(=NR4a)NR4aC(=NR43)N(R43)2, -NR4aC(:0)N(R4a 2, -C(=O)NH2, - C(20)N(R4a)2, -0R4, -SR4“, or -N(R4a 2; 2014/055245 wherein each C2_12heterocyclyl may be substituted one or more times with C1- lgalkyl, C3.lgcycloalkyl, C6.1zaryl, C1.1gheteroaryl, -OH, NHz, -F, -Cl, -Br, -I, -N02, - CN, —C(=O)R4a, -C(=NR4a)R4, -C(=O)OH, —C(:O)OR4, -OC(:O)R4, —OC(=O)OR4, - so2H, -so2N(R4a 2, —so2R4, -so2NR4aC(=0)R4, , _We(=NR4a)1~~I(1r*a 2, _ NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4aso2R4, — =NR4a)NR4aC(=NR43)N(R4a 2, -NR4aC(=0)N(R4a 2, —C(:O)NH2, — (R4a 2, -OR4, sat: or -N(R4a)2; R4 is independently selected fi‘om CHZalkyl, C3.1zcycloalkyl, C6.12aryl, C1. gheteroaryl and C2_12heterocyclyl, each ofwhich may be substituted one or more times by -OH, -NH2, -F, -Cl, -Br, -I, -N02, -CN, -C(=O)OH, -SO3H, -P03H2, or — C(ZO)NH2; R43 may be R4 or hydrogen; wherein any two or more of R2, R3, R4 and R4"1 groups may together form a ring.

In some embodiments, the ionic liquid ns a cationic constituent having a structure according to Formula Hr R3 R3 Formula (11), wherein R1 as defined above and R3 may either be R2 as defined above, or two R3 substituents on the same carbon atom may together form 3 (=0), (=NR4a) or (=CR22).

The ionic liquid may also contain a cationic constituent having the structure ing to Formula III: R2 1% ,2 R2 R3 R2 Formula (111) wherein R1 and R2 are as defined above.

In some embodiments, the ionic liquid contains a cationic constituent having a WO 38811 structure according to Formula IV: RZQMR‘ a? a? Formula (IV) n R1 and R2 are as defined above.

In some embodiments, the ionic liquid contains a cationic constituent having a structure according to any one of as V—IX, where each occurrence ofA is independently selected from C, N, O, S, and P; where each dashed line (-~~~~~ ) can be a single, double, or triple bond; and where each R10 and R10”, when taken separately, is independently selected from none, H, hydroxyl, halide, and substituted and unsubstituted alkoxy, heteroalkoxy, alkyl, aryl, heteroalkyl, alkenyl, and alkynyl groups having from 1 to 30 carbon atoms, from 2 to 20 carbon atoms, or from 3 to 12 carbon atoms or, when attached to the same atom and taken together, each R10 and R10” is =0 or er with the atom to which they are attached form a carbocycle or heterocycle having from 2 to 30, preferably from 3 to 12 carbon atoms; so long as at least one ence ofA 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. Exemplary alkyl groups include ethyl, propyl, isopropyl, butyl, tart-butyl, hexyl, octyl, and decyl groups. Exemplary heteroalkyl groups include cyanoethyl, cyanobutyl, and cyanopropyl groups. Exemplary alkoxy groups include y, ethoxy, and butoxy groups.

R10 30! R10! R10 R I Rm‘ R10 R1;) \ I / /Rw- \A/ R” R \A/"l10. N..."l .mh"‘l‘ ’;‘ ‘5 R‘°’"A. «‘AC: " ~\ : i i 1:) ‘i fl R R10 V.

T\Rl° Rm”? A\“‘R‘° i 'l ~ 10/? "*A\ to R10 Ra; R10: R10 9230' R Formula V Formula VI Formula VII 10.

R10 R10 R R 19 R R10- \ / R39 Rw' \ / \ i / 0\ ’lwA‘h~~~ LR“). " ale—A “eA—R’io R1 m. : : 1 a R16 l t R,0,"m. ‘ A me/‘lu ,‘AMRw / “a x’ \ 10. a ’4 l R R10 A"- R10 / Rifl- /l |\R16 R10 R10: R10 R10 g1!)I a VIII Formula IX In some embodiments, the ionic liquid contains a cationic constituent having a structure according to any one of Formulas V-IX where at least one occurrence ofA ‘is a nitrogen atom having a formal positive charge with the remaining A each - independently selected from C, N, O, S, and P; each dashed line - ) is a single or double bond; and where each R10 and RIO’, when taken separately, is independently selected from none, H, hydroxyl, halide, and substituted and tituted alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyloxy, alkenyl, and alkynyl groups having from 1 to 30 carbon atoms, from 2 to 20 carbon atoms, or from 3 to 12 carbon atoms or, when attached to the same atom and taken together, each R10 and Rm” is :0 or together with the atom to which they are attached form a ycle or heterocycle having from 1 to 30, preferably from 3 to 12 carbon atoms. In red 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. Exemplary alkyl groups include ethyl, propyl, butyl, hexyl, octyl, and decyl groups, as well as isomers thereof. Exemplary heteroalkyl groups include cyanobutyl and cyanopropyl . Exemplary alkoxy groups include methoxy, ethoxy, and butoxy groups.

The ionic liquid can be an ammonium salt: WO 38811 ”it“ wherein R1 is as defined above.

In some embodiments, the ionic liquid contains a cationic constituent having a structure according to Formula X1 where Ar is a substituted or unsubstituted aryl group; R12 is either none or an alkyi, alkyl, aryl, aralkyl, alkenyl, or alkynyl group having from 1 to 30 carbon atoms, from 3 to 20 carbon atoms, or fiom 4 to 12 carbon atoms; and each occurrence of R13 is independently selected from en and substituted and unsubstituted alkyl, heteroalkyl, aryl, aralkyl, antenyl, 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 where Ar is a substituted or unsubstituted benzyl group; where R12 is a substituted or unsubstituted C1-C12 alkyl group, or both.

In some embodiments, the nd of Formula X1 is characterized by the presence of at least one group selected from -COOH, -SO3H and 4303112.

Air—sit’L-rst—Fz‘3 a XI The ionic liquid can be a phosphonium salt. In some embodiments, the ionic liquid contains a cationic tuent having a structure according to Formula XII Where each occurrence of R14 is ndently selected from hydrogen and substituted 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; wherein at least one, at least two, or at least three occurrences of R9 are not hydrogen. In some embodiments, at least one occurrence of R14 is an aryl, aralkyl, or aralkoxy group having from 2 to 30 carbon atoms or fi‘orn 4 to 12 carbon atoms. In some embodiments, the compound of a XII is characterized by the presence of at least one group selected from - COOH, -SOgH and .

Formuia XII In some ments, the ionic liquid contains a cationic constituent having a structure according to Formula XIII Where Ar is a substituted or unsubstituted aryl group; R15 is either none or an alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, aralkyioxy, alkenyl, or alkynyi group having from 2 to 30 carbon atoms, from 3 to 20 carbon atoms, or from 4 to 12 carbon atoms; and each occurrence of R16 is independently selected from en and substituted and unsubstituted aikoxy, heteroaikoxy, alkyi, alkyl, aryl, aryloxy, aralkyl, aralkyloxy, i, and alkynyl groups having irom 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 accordingito a XIII Where Ar is a substituted or unsubstituted benzyl group; where R15 is a substituted or unsubstituted C1-C1; alky 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 -P03H2.

NHR‘E5““PI'WR18 Formula XIII The ionic liquid can be a guanidinium salt having a structure according to Formula XIV: 521%le 32 R2 Formula XIV, wherein R1 and R2 are as defined above.

The ionic liquid can be a salt having a structure according to Formula XV: R? R2 Formula XV wherein R1 and R2 are as defined above, and X may be 0, S, 802, NR1 or C(R2)2.

The ionic liquid can be an imidazolium salt such as l-ally1 methylimidazolium bis(trifluor0methylsulfonyl); l-allyl—3 -methylirnidazolium bromide; 1-ally1n1ethylirnidazolimn chloride; l-allyl«methylimidazolium dicyanamide; 1-allylmethylimidazolium iodide; l-benzylmethylimidazolium chloride; yl-3 -rnethylirnidazoliurn orophosphate; l—benzyl—3- methylimidazolium tetrafluoroborate; 1,3-bis(cyanomethyl)imidazolium bis(trifluoromethylsulfonylfimide; 1,3-bis(cyanomethyl)irnidazolium chloride; 1- butyl-2;3 hyli1nidazoliun1 chloride; 1-butyl-2,3-dimethyli1nidazolium hexafluorophosphate; 1-butyl-2,3-dimethylimidazolium tetrafluoroborate; l-buty1 methylimidazolium acetate; 1-butyl—3—rnethyli1nidazolium bis(trifluoromethylsulfony1)imide; 1-butylmethyli1nidazolium bromide; 1-butyl—3- methylimidazoliurn chloride; 1~butyl—3—methylimidazolium dibutyl phosphate; 1- butyl-3~methylimidazolium dicyanamide; lmethylirnidazolium hexafluoroantimonate; l-butyl1nethylimidazolium hexafluorophosphate; 1—butyl~3~ imidazolium en sulfate; 1-butyl—3-methylirnidazolium iodide; 1—butyl methylimidazolimn methanesulfonate; l-‘outyl-«3 ~methyl-imidazolium methyl carbonate; l—butylmethylimidazolium methyl sulfate; 1-butyl methylimidazolium nitrate; l-butylmethylimjdazoliu1n octyl sulfate; 1~butyl~3~ methylimidazolium tetrachloroaluminate; 1—butylmethylimidazolium tetrafluoroborate; l-butylmethyli1nidazoliurn thiocyanate; 1-butyl-«3- methylimidazolium tosylate; 1-butylmethylimidazolium trifluoromethanesulfonate; 1—(3 pr0pyl)—3-methylimidazolium ‘ois(trifluoromethylsulfonyl)amide; 1~(3 ~ cyanopropyl)—3-methylimidazolium chloride; 1-(3-cyanopropyl) methylimidazolium dicyanamide; l-decyl—3-1nethyli1nidazolium; l methylimidazoliurn tetrafluoroborate; 1,3-diethoxyirnidazolium bis(trifluoromethylsulfonyl)imide; 1,3-diethoxyimidazolium orophosphate; 1,3-dihydroxyimidazolium bi3(uifiuoromethylsulfonyl)imide; 1,3-dihydroxy methylimidazolium bis(trifluoromethylsulfonylfimide; 1,3-dimethoxyimidazolium bis(trifluoromethylsulfonyl)imide; 1,3-dimethoxyimidazolium hexafluorophosphate; 1,3-dimethoxy—2~methylimidazolium bis(trifluoromethylsulfonyl)imide; 1,3- dimethoxymethylimidazolium hexafluorophosphate; 1,3-dimethylimidazolium dimethyl phosphate; 1,3-dimethy1imidazolium methanesulfonate; 1,3- dimethylimidazolium methyl sulfate; l,2~dimethyl—3-propylimidazolium ifluoromethylsulfonyl)imide; cyl-3~methylimidazolium iodide; l-ethyl- 2,3-dimethylimidazoliurn tetrafluoroborate; 1-ethyl-2,3-dimethylirnidazolium chloride; l-ethyl-2,3-dimethylimidazolium ethyl sulfate; 1-ethyl-2,3- dimethylimidazoliom hexafluorophosphate; l~ethyl—3-methylimidazolium acetate; 1- ethyl—3 -rnethylimidazolium aminoacetate; 1-ethyl-3—methylirrfidazolium (S)—2- aminopropionate; l—3-rnethylimidazolium bis(pentafluoroethylsulfonyl)imide; 1-ethy1rnethylimidazolium bromide; l-ethyl—3—methylimidazolium chloride; 1n1ethylirnidazolium dibutyl phosphate; l-ethylInethylimidazolium dicyanarnide; 1-ethylmethylimidazolium diethyl phosphate; l-ethyl-B— methylimidazolium ethyl sulfate; 1-ethylrnethylimidazolium hexafluorophosphate; l-ethyl-3 -rnethylimidazolium hydrogen carbonate; 1-ethyl—3-methylimidazolium hydrogen sulfate; l-ethyl~3-methylimidazolium hydroxide; l-ethyl-3— methylimidazolium iodide; l-ethyl~3-methylimidazolium L-(+)-lactate; l-ethyl—S- methylimidazolium esulfonate; l-3—methylimidazolium methyl e; l-ethylmethylimidazolium nitrate; 1methylimidazolium tetrachloroaluminate; lwethyl—3—methylimidazoliurn tetrachloroaluminate; l-ethyl-3— methylimidazolium uoroborate; 1—ethy1—3-methylimidazolium 1,1,2,2- tetrafluoroethanesulfonate ; 1-ethylmethylirnidazolium thiocyanate; 1-ethy1—3- methylimidazolium tosylate; l—ethyl—3-methylimidazolium trifluoromethanesulfonate; l-3—methylimidazolium bis(trifluormethylsulfonyl)irnide; l-hexyl-3— methylirnidazolium chloride; l—hexylmethylimidazolium hexafluorophosphate; l- hexylmethylimidazolium iodide; l-hexyl—3~methylimidazolium tetrafluoroborate; 1-hexylmethylirnidazolium trifluoromethansulfonate; 1-(2-hydroxyethyl)—3- methylimidazoliurn dicyanamide; ylimidazolium chloride; 1— irnidazolium hydrogen sulfate; 1-methyloctylimidazoliurn chloride; 1- methyloctylirnidazolium hexafluorophosphate; 1-methyloctylimidazolium tetrafluoroborate; l—methyloctylimidazolium trifluoromethanesulfonate; 1-methyl- 3-propylimidazolium iodide; l-methyl-3—propylimidazolium methyl carbonate; 1,2,3- hylimidazolium methyl sulfate; derivatives thereof and combinations thereof. tives can include substituting the anionic tuent for other anionic constituents, replacing one or more carbons with a heteroatom, replacing an N—alkyl group with one or more -order N—alkyl groups, or a combination thereof.

Exemplary anionic constituents and heteroatoms are described above. Exemplary higher—order N—alkyl groups can e 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.

The ionic liquid can be a pyrrolidinium salt suoh as l-butyl- l - methylpyrrolidiniurn bis(trifluoromethylsulfonyl)imide; i-butyl-l- methylpyrrolidinium e; l-butyl—1-methylpyrrolidinium chloride; l-butyl-l- methylpyrrolidinium dicyanamide; l —butylInethylpyrrolidiniurn hexafluorophosphate; 1-butyl-l-methylpyrrolidiniurn iodide; '1-butyl—l- methylpyrrolidinium methyl carbonate; l-buty1methylpyrrolidiniurn luoroborate; 1-butyl—1-methylpyrrolidinium trifluoromethanesulfonate; 1-ethyl- l-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide; 1-ethy1-l — methylpyrrolidinium bromide; l-ethylmethylpyrrolidinium hexafluorophosphate; 1—ethylmethylpyrrolidinium tetrafluoroborate; derivatives thereof and combinations thereof. Derivatives can include substituting the anionic tuent for other anionic tuents, 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 f. ary c tuents, heteroatoms, and higher-order N—alkyl groups are described above.

Zwitferionic Liquids The ionic liquid can be a zwitterion (i.e., an internal salt), for example, 4-(3- butyl~1-imidazolio)-1—butane sulfonate; 3 -(l -methyl-3 -imidazolio)propanesulfonate; 4-(3 -methyl—l -imidazolio)— 1 -butanesulfonate; or 3 -(triphenylphosphonio)propane—l ~ sulfonate. ' The zwitterionic liquid can be 4-(3 ubutyl-l-imidazolio)~1-butane sulfonate (BIM) having the structure shown below or a derivative thereof. ‘303 @/\/\/ ' N Derivatives of BlM can include substituting the sulfonate group for a different anionic substituent, replacing one or more carbons with a heteroatom, replacing the N-butyl group with one or more lower-order or higher-order N—alkyl groups, attaching additional substituents to one or more carbon atoms, or a combination thereof.

Exemplary anionic substituents e sulfate [-OSO3'], sulfonate [-SOg'], sulfite [— 0802‘], sulfinate {-SOZ'], phosphate [—OP(OH)OZ'], alkylphosphate [-OP(OR2)02'], onate [-P(OH)02'], alkylphosphonate.[——P(OR2)02'], phosphite [-OP(OH)O'], alkylphosphite [-OP(OR2)O']. phosphonite [-P(OH)O'], alkylphosphonite [-P(0R2)O' ], carbonate [-OCOfl, and carboxylate L Where R2 is as defined above.

Exemplary heteroatoms and higher—order l groups are described above.

Additional substituents can include hydroxyl, and substituted and unsubstituted alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl; aralkyloxy, alkenyl, and alkynyl groups having from 1 to 30, preferably from 3 to 12 carbon atoms.

In some ments, the ionic liquid is a zwitterion ning a cationic cyciic substituent and an anionic substituent connected by a substituted or unsubstituted alkyl, heteroaikyl, aryl, aralkyl, alkenyl, or l group having from 2 to 50 carbon atoms, from 3 to 30 carbon atoms, or from 4 to 12 carbon atoms. The cationic heterocyclic tuent can be saturated or unsaturated. Examples e pyrrolidinium, imidazoiinium, oxazolidiniurn, piperidinium, piperaziniurn, morpholinium, thiornorpholinium, ium, inium, 1,2,3-triazolium, 1,2,4- triazolium, thiazolium, 1,2, 4-dithiazolium, 1,4,2-dithiazolium, tetrazolium, pyrazolinium, oxazolinium, pyridinium, and azepinjum groups. The cationic heterocyclic substituent can be a fused ring structure having two or more fused rings.

The cationic heterocyclie substituent can be a bicyclic cationic heterocycle, such as benzoxazolium, benzothiazoliurn, benzotriazoliurn, benzimidazoliurn, and indolium.

The cationic heterocyclic substituent can additionally be substituted with one or more additional substituents. Exemplary anionic substituents include sulfate [-0803'], sulfonate [-SOg'], sulfite [-OSOZ'], sulfinate [—SOZ'], phosphate [-OP(OH)02‘], alkylphosphate [-OP(OR2)02'], phosphonate {-P(OH)OZ"], alkylphosphonate [- P(OR2)02'], phosphite [—OP(OH)O'], hosphite {-OP(OR2)O‘]. phosphonite [- P(OH)O'], alkylphosphonite [-P(OR2)O"], carbonate [-OCOg'], and carboxylate [-COZ' ], Where R2 is as described above.

In some embodiments, the ionic liquid is a rion having a structure according to Formula XVI, XVII, XVIII or XIV: 1 R‘ R1 R1 F? R3 \N/ RE R3 Ker“ M2 _ R2 rlq“ R1 N‘“ R R3 in” R2 R”t 3 3 Formula XVI, R 3 a XVII, R2 R2 . $31 _ at N- R2 l 6:, R133".

R2 R2 k Formula XVIII, R2 Formula XVIV, R2 32 Formula XX, Rip-fil—Rj “ Formula XXI Wherein R1, R2 and R3 are as defined above, ed that the compounds of Formula XVI, XVII, XVIII, XVIV, XX and XXI each contain at least one ~COOH, - SO3H, or -P03H2 tuent.

C. ents A Wide variety ofpharmaceutical excipients useful for liquid protein formulations are known to those skilled in the art. They include one or more ves, such as liquid solvents or co-solvents; sugars or sugar alcohols such as mannitol, trehalose, sucrose, sorbitol, fructose, maltose, lactose, or dextrans; tants such as TWEEN® 20, 60, or 80 (polysorbate 20, 60, or 80); buffering ; preservatives such as benzalkonium chloride, benzethoniurn chloride, tertiary ammonium salts, and 2014/055245 chlorhexidinediacetate; carriers such as thylene 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 e polyesters; cryoprotectants; lyoprotectants; bulking agents; and stabilizing agents.

Other pharmaceutically acceptable rs, excipients, or stabilizers, such as those described in Remington: “The e and Practice of Pharmacy”, 20th edition, Alfonso R. Gennaro, Ed, Lippincott Williams & Wilkins (2000) may also be ed in a protein formulation described herein, provided that they do not adversely affect the desired characteristics of the formulation.

The ity—lowering agents described herein can be combined with one or more other types of viscosity—lowering agents, for example, organophosphates described in co-filed PCT application entitled “LIQUID PROTEIN FORMULATIONS CONTAINING ORGANOPHOSPHATES” by Arsia Therapeutics; water soluble organic dyes described in co~filed PCT application entitled “LIQUID PROTEIN FORMULATIONS CONTAINING WATER SOLUBLE’ORGANIC DYES” by Arsia Therapeutics; the typically bulky polar - - * organic compounds, such as hobic nds, many ofthe GRAS (US Food and Drug Administration List of compounds Generally ed As Safe) and inactive injectable ients and FDA approved eutics, described in co-filed PCT application entitled: “LIQUID PROTEIN FORMULATIONS CONTAINING VISCOSITY-LOWERING AGENTS” by Arsia Therapeutics.

III. Methods of making The protein, such as a mAb, to be ated may be produced by any known technique, such as by culturing cells transformed or transfected with a vector containing one or more nucleic acid sequences encoding the protein, as is well known in the art, or through synthetic techniques (such as recombinant techniques and peptide synthesis or a combination ofthese techniques), or may be isolated from an endogenous source ofthe protein.

Purification of the protein to be formulated may be conducted by any suitable technique known in the art, such as, for example, ethanol or ammonium sulfate precipitation, reverse phase HPLC, chromatography on silica or cation-exchange resin WO 38811 (e.g., DEAE-cellulose), dialysis, tofocusing, gel filtration using protein A SE- SEPHAROSE® columns (e.g., SEPHADEX® G—75) to remove contaminants, metal chelating columns to bind epitope-tagged forms, and Ifltrafiltration/diafiltration (nonlimiting examples include centrifugal filtration and tangential flow filtration (TFFD.

Inclusion of the ionic liquid at viscosity-reducing concentrations such as 0.010 M to 1.0 M, ably 0.050 M to 0.50 M, most preferably 0.10 M to 0.30 M, allows a solution of the pharmaceutically active mAb to be purified and!or concentrated at higher mAb concentrations using common methods known to those skilled in the art, including but not d to tangential flow filtration, centrifugal concentration, and In some embodiments, lyophilized formulations ofthe proteins are provided and/or are used in the preparation and manufacture of the low-viscosity, concentrated protein formulations. In some embodiments, the pre-lyophilized protein in a powder form is reconstituted by dissolution in an aqueous solution. In this embodiment, the liquid formulation is filled into a specific dosage unit container such as a vial or pre- filled mixing syringe, lyophilized, optionally with lyoprotectants, vatives, - idants, and other typical ceutically acceptable excipients, then stored under sterile storage conditions until 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.

The formulations described herein may be stored by any suitable method known to one skilled in the art. Non—limiting examples ofmethods for ing the protein formulations for storage include freezing, lizing, and spray drying the liquid protein ation. In some cases, the lyophilized formulation is frozen for storage at subzero temperatures, such as at about -80°C or in liquid nitrogen. In some cases, a lyophilized or aqueous formulation is stored at 2—8°C.

Non-limiting examples of diluents useful for tituting a lyophilized formulation prior to injection include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's on, 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 Thereof The protein formulations, including, but not limited to, reconstituted ations, are administered to a person in need thereof by intramuscular, intraperitoneal (i.e., into a body cavity), intracerobrospinal, or subcutaneous injection using an 18-32 gauge needle (optionally a thin-walled needle), in a volume of less than about 5 mL, less that about 3 mL, preferably less than about 2 mL, more preferably less than about 1 mL.

The appropriate dosage (“therapeutically effective amount”) of the n, such as a mAb, will depend on the condition to be treated, the severity and course of the disease or condition, Whether the protein is administered for preventive or eutic purposes, us therapy, the patient's al history and response to the protein, the type of protein used, and the discretion of the attending physician. The protein is suitably administered at one time in single or le injections, or over a series of treatments, as the sole treatment, or in conjunction with other drugs or ies.

Dosage ations are designed so that the injections cause no significant signs of irritation at the site of injection, for example; n 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 s of saline solution. In another embodiment, the bioavailability of the protein is higher when compared to the otherwise same formulation Without the viscosity-reducing ionic liquid(s) administered in the same way. in another embodiment, the formulation is at least approximately as effective ceutically as about the same dose of the protein administered by intravenous on.

In a preferred embodiment, the formulation is injected to yield increased levels of the therapeutic protein. For example, the AUC value may be at least 10%, preferably at least 20%, larger than the same value computed for the otherwise same formulation without the viscosity-reducing ionic liquid(s) administered in the same way.

The ity—lowering agent may 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 otherwise same formulation without the viscosity-lowering ionic liquid administered in the same way.

The viscosity-lowering agent may also affect the pharmacokinetics. For example, the CM after SC or IM injection may be at least 10%, ably at least %, less than the CMAX of an approximately equivalent pharmaceutically effective enously administered dose.

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

The lower viscosity formulations e less ion force. For example, ‘ the injection force may be at least 10%, ably at least 20%, less than the injection force for the otherwise same formulation without the viscosity-reducing ionic liquid administered in the same way. In one embodiment, the injection is stered with a 27 gauge needle and the injection force is less than 30 N. The formulations can be stered in most cases using a very small gauge needle, for example, between 27 and 31 gauge, typically 27, 29 or 31 gauge.

The viscosity~reducing ionic liquid may be used to prepare a dosage unit formulation 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 viscosity-reducing ionic liquids; and other excipients. The proteins are t in the dosage unit such that after reconstitution in a pharmaceutically acceptable t, the resulting formulation has a protein concentration from about 100 mg to about 2,000 mg per 1 mL (mg/mL).

Such reconstituted formulations may have an absolute viscosity offiom about 1 CF to about 50 CP at 25°C.

The low viscosity formulation can be provided as a solution or in a dosage unit form Where the protein is lyophilized in one vial, with or without the viscosity- ng agent and the other excipients, and the soivent, with or without the Viscositylowering agent and other excipients, is provided in a second vial. In this embodiment, the solvent is added to the protein shortly before or at the time of injection to ensure uniform mixing and dissolution.

The viscosity-reducing ionic liquid(s) are present in the formulations at concentrations that cause no significant signs of toxicity and/or no irreversible signs of toxicity When administered via subcutaneous, intramuscular, or other types of in- ion. As used herein, ficant signs of toxicity” include intoxication, lethargy, behavioral modifications such as those that occur with damage to the central s system, infertility, signs of serious toxicity such as cardiac hmia, cardiomyopathy, myocardial infarctions, and cardiac or congestive heart failure, kidney failure, liver failure, difficulty breathing, and death.

In preferred embodiments the formulations cause no significant irritation when administered not more than twice daily, once daily, twice , once weekly or once y. The n ations can be administered causing no significant signs of irritation at the site of injection, as measured by a primary irritation index of less than 3, less than 2, or less than 1 when evaluated using a Draize scoring system.

As used herein, “significant signs of irritation” include erythema, s, and/or swelling at the site of injection having a diameter of greater than 10 cm, greater than 5 cm, or greater than 2.5 cm, necrosis at the site of injection, exfoliative dermatitis at the site of injection, and severe pain that prevents daily activity and/or requires medical attention or hospitalization. In some embodiments, injections of the protein formulations cause macroscopically similar levels of irritation when compared to injections of equivalent volumes of saline solution.

The protein formulations can exhibit increased bioavailability compared to the otherwise same protein formulation without the viscosity—reducing ionic liquid(s) when administered via aneous or intramuscular injection. “Bioavailability” refers to the extent and rate at which the bioactive species such as a mAb, reaches circulation or the site of action. The overall bioavailability can be increased for SC or IM injections as compared to the otherwise same formulations t the viscosity- reducing ionic 1iquid(s). “Percent bioavailability” refers to the fraction of the administered dose of the bioactive s which enters circulation, as determined with respect to an intravenously administered dose. One way of measuring the bioavailability is by comparing the “area under the curve” (AUC) in a plot of the plasma tration as a function of time. The AUC can be calculated, for example, using the linear trapezoidal rule. “AUCQO”, as used herein, refers to the area under the plasma concentration curve from time zero to a time where the plasma concentration returns to ne levels. “AUCM”, as used herein, refers to the area under the plasma concentration curve from time zero to a time, t, later, for example to the time of reach— reaching baseline. The time will typically be measured in days, although hours can also be used as will be apparent by context. For example, the AUC can be increased by more than 10%, 20%, 30%, 40%, or 50% as compared to the otherwise same formulation without the Viscosity—reducing ionic liquid(s) and administered in the same way.

As used herein, “tmax” refers to the time after administration at which the plasma concentration s a maximum.

Asused , “ max” refers to the maximum plasma tration after dose administration, and before stration Of a subsequent dose.

As used herein, "Cm," or "CmmghH refers to the minimum plasma concentration after dose administration, and before administration of a subsequent dose.

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

' " - The pharmacokinetic and pharmacodynamic parameters may be imated - across species using ches that are known to the skilled artisan. The pharmacokinetics and pharmacodynamics of antibody eutics can differ markedly based upon the ic antibody. An approved murine mAb was shown to have a half-life in humans of ~ 1 day, while a human mAb will typically have a half— life of~ 25 days (Waldmann et at, Int. Immunol., 2001, 13:1551-1559). The pharmacokinetics and pharmacodynamics of antibody therapeutics can differ markedly based upon the route of administration. The time to reach maximal plasma concentration after IM or SC injection of IgG typically ranges from 2 to 8 days, although shorter or longer times may be encountered (Wang et 6111., Clin. Pharm.

Ther., 2008, 84(5):548-558). The pharmacokinetics and pharmacodynamics of antibody eutics can differ markedly based upon the formulation.

The low-Viscosity protein formulations can allow for greater flexibility in dosing and decreased dosing frequencies compared to those protein formulations without the Viscosity-reducing ionic liquid(s). For example, by sing the dosage administered per injection multiple—fold, the dosing frequency can in some embodiments be decreased fiom once every 2 weeks to once every 6 weeks. The protein formulations, including, but not limited to, reconstituted ations, can be administered using a heated and!or self-mixing syringe or autoinj ector. The protein formulations can also be pre-heated in a separate warming unit prior to filling the syringe. z'. Heated Syringes The heated e can be a standard syringe that is pre-heated using a e warmer. The syringe warmer will generally have one or more openings each e of receiving a syringe containing the protein formulation and a means for g and maintaining the syringe at a specific (typically above the ambient) temperature prior to use. This will be referred to herein as a premheated syringe. Suitable heated syringe warmers include those ble from Vista Dental Products and Inter-Med. The warmers are capable of accommodating various sized syringes and heating, typically to within 1°C, to any temperature up to about 130°C. In some embodiments the syringe is pre-heated in a heating bath such as a water bather maintained at the d temperature.

The heated syringe can be a eating syringe, i.e capable of heating and maintaining the liquid formulation inside the syringe at a specific temperature. The self-heating syringe can also be a standard l syringe having attached thereto a heating device. Suitable heating devices capable of being attached to a syringe include syringe heaters or syringe heater tape available from Watlow Electric Manufacturing Co. of St. Louis, MO, and syringe heater blocks, stage heaters, and in—line perfusion heaters available from Warner Instruments of Hamden, CT, such as the SW-61 model syringe warmer. The heater may be controlled through a central controller, e.g. the TC-324B or B model heater controllers available from Warner Instruments.

The heated e maintains the liquid protein formulation at a specified temperature or to 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 up to about 80°C, up to about 60°C, up to about 50°C, or up to about 45°C as long as the protein formulation is sufficiently stable at that temperature.

The heated e can maintain the protein formulation at a temperature between °C and 60°C, between 21°C and 45°C, n 22°C and 40°C, between 25° C and 40° C, or between 25°C and 37°C. By maintaining the protein formulations at an 2014/055245 elevated temperature during injection, the viscosity of the liquid formulation is de- decreased, the lity of the protein in the formulation is increased, or both. ii. Self-Mixing es The syringe can be self—mixing or can have a mixer attached. The mixer can be a static mixer or a dynamic mixer. Examples of static mixers include those disclosed in US. Patent Nos. 988, 6,065,645, 6,394,314, 6,564,972, and 6,698,622. es of some dynamic mixers can include those disolosed in US. Patent Nos. 6,443,612 and 6,457,609, as well as U.S. Patent ation Publication No. US 2002/0190082.The syringe can 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 mponent Viscous substances. iiiAutoinieciors and Free—tilled es of Protein Formulations The liquid protein formulation can be administered using a prevfilled e autoinj ector or a needleless injection device. Autoinj ectors include a handheld, often pen-like, cartridge holder for holding replaceable lled dges and a spring based or analogous mechanism for subcutaneous or intramuscular injections of liquid drug dosages from a pre-filled cartridge. Autoinj ectors are typically designed for self- stration or administration by untrained personnel. Autoinjectors are available to dispense either single dosages or multiple dosages from a pre-filled cartridge.

Autoinj ectors enable ent user settings ing inter alia injection depth, injection speed, and the like. Other injection systems can include those described in U.S. Patent No. 8,500,681.

The lyophiiized protein formulation can be provided in ied or unit-dose syringes. U.S. Patent Nos. 3,682,174; 4,171,698; and 5,569,193 describe sterile syringes containing two-chambers that can be pie-filled with a dry formulation and a liquid that can be mixed immediately prior to injection. U.S. 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 unit-dose syringe, reconstituted in the syringe prior to administration, and administered as a single subcutaneous or intramuscular injection. Autoinj ectors for delivery of unit-dose lyophilized drugs are described in W0 2012/010,832. Auto injectors such as the Safe Click LyoTM 2014/055245 (marketed by Future Injection Technologies, Ltd., , U.K.) can be used to ad- administer a unit—dose protein formulation where the formulation is stored in lyophilized form and reconstituted just prior to administration. In some embodiments the protein formulation is provided in ose cartridges for lyophilized drugs (sometimes referred to as Vetter dges). Examples of suitable cartridges can include those described in U.S. Patent Nos. 5,334,162 and 5,454,786.

V. Methods of Purification and Concentration The viscosity-reducing ionic liquids can also be used to assist in protein purification and concentration. The viscosity-reducing ionic liquid(s) and excipients are added to the protein in an effective amount reduce the viscosity of the protein solution. For e, the viscosity.lowering agent is added to a concentration of between about 0.01 M and about 1.0 M, preferably between about 0.01 M and about 0.50 M, and most preferably between about 0.01 M and about 0.25 M.

The Viscosity—reducing ionic liquid solution containing n is then purified or concentrated using a method selected from the group consisting of ltration/diafiltration, tial flow filtration, fugal concentration, and dialysis.

Examples The foregoing will be filrther understood by the following nonwlimiting examples.

All viscosities of well-mixed aqueous mAb solutions were measured using either a mVROC microfluidic viscometer (RheoSense) or a DVZT cone and plate viscometer field; “C & P”) after a 5 minute equilibration at 25°C (unless otherwise 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 on was back-loaded into a gastight microlab instrument syringe (Hamilton; 100 uL), affixed to the chip, and measured at multiple flow rates, approximately 20%, 40%, and 60% ofthe maximum pressure for each chip. For example a sample of approximately 50 cP would be measured at around 10, 20, and 30 uL/min (approximately 180, 350, and 530 5'1, respectively, on an “A” chip) until viscosity stabilized, typically after at least 30 seconds. An average te viscosity and standard deviation was then calculated from at least these three measurements. The C & P eter was equipped with a CPE40 or CPE52 spindle (cone angle of 08° and 3.0°, respectively) and 0.50 mL samples were measured at multiple shear rates between 2 and 400 5'1. Specifically, samples were measured for 30 seconds each at 22.58, 24.38, 26.25, 28.13, 30, 31.88, 45, 67.5, 90,1125, 135,157.5, 180, 202.5, 247, 270, 292.5, 315, 337.5, 360, 382, 400 5", starting at a shear rate that gave at least 10% torque, and continuing until instrument torque reached 100%. An extrapolated zero- shear Viscosity was then determined from a plot of dynamic viscosity versus shear rate for the samples measured on a DV2T cone and plate viscometer. The extrapolated hear viscosities reported are the average and rd ion of at least three measurements.

Example 1: Ionic liquids reduce the viscosity of concentrated aqueous solutions of biosimilar AVASTIN® Materials and Methods * *A corrunercially-obtained biosimilar AVASTIN® ning pharmaceutical excipients (Polysorbate 20, phosphate and citrate buffers, ol, and NaCl) was purified. First, Polysorbate 20 was removed using DETERGENT-OUTID TWEEN Medi Columns sciences). Next, the resulting ons 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 on Jumbosep centrifugal concentrators (Pall Corp). For samples containing 4—ethyl methylmorpholinium methyl carbonate (EMMC), protein was thoroughly buffer exchanged into 2 mM PB (pH 7.0). For samples buffer exchanged into 20 mM PB (PB control samples) or 20 mM viscosity-reducing ionic , the collected protein solution was freeze-dried. The dried protein cakes, containing protein and buffer salts or ity-reducing ionic liquid, were reconstituted to a final volume of approximately 0.10-1.30 mL. These samples were reconstituted using additional PB (pH 7.0) or viscosity-reducing ionic liquid (pH 7.0), as appropriate, sufficient to bring the final concentration of PB to 0.25 M and the final tration of viscosity- reducing ionic liquid as indicated in the tables below. Samples buffer exchanged into 2 mM PB were first ted. Then, an appropriate amount of viscosity-reducing ionic liquid solution (pH 7.0) was added to each aliquot such that upon reconstitution with water, the final excipient concentration was 0.1 - 0.5 M. The n solutions were then freeze-dried. The dried protein cakes, containing protein and ity- reducing ionic liquid (and a negligible amount of buffer salts) were reconstituted to a final volume of approximately 0.1 mL and Viscosity-deducing ionic liquid concentration as indicated in the tables below. The final concentration of mAb in solution was determined by light absorbance at 280 nm using an experimentally determined extinction coefficient of 1.7 L/g‘crn and viscosities reported were measured on a nse mVROC microfluidic viscometer.

Results The data in Table 1 demonstrate that the Viscosity of aqueous solutions of ilar AVASTIN® can be reduced by up to 6.5~fold (compared to phosphate buffered samples) in the presence of .50 M viscosity-reducing ionic liquids.

Viscosities over 200 CP in the absence of viscosity—reducing ionic liquids were reduced to less than 50 cP by the addition of 0.20-0.50 M viscosity-reducing ionic liquids. One can see that in this example the magnitude of viscosity reduction is, in some cases, dependent upon the tration of the ity-reducing ionic liquid.

The viscosity reduction rises (i.e., viscosity decreases) with increasing Viscosity~ reducing ionic liquid concentration.

Table 1. Viscosities of aqueous solutions of biosimilar AVASTIN® in the presence of various trations of ionic liquids (“ILs”) at 25°C and pH 7.

Ionic Liquid* [IL] (M) in] (mg/mL) Viscosity (0P) PB 0.25 215 213 i: 10 PB 0.25 235 398 i 4 BIM 0.2 215 61.8i0.3 BIM 0.4 215 47.3 :1: 2.3 BIM 0.5 215 41.9i 0.8 BIM 0.5 226 64.3 -_I- 3.7 BMI Mes 0.4 214 36.3 d: 0.2 BMI Mes 0.5 221 46.5 i 1.7 BMI Mes 0.4 229 69.2 i 5.2 7 7 ___ - BMI Mes 0.5 230 82.0 d: 3.0 BMP Chloride 0.5 213 42.0 i 1.2 BMP Chloride 0.4 227 63.0 :I: 8.4 BMP Chloride 0.5 230 60.8 i 0.1 EMMC 0.4 217 38.7 d: 0.3 * PB = phosphate buffer; BIM = 4-(3-butylimidazolio)butane sulfonate; BMI Mes = l-butyimethylimidazolium methanesulfonate; BMP Chloride "—“ 1-butyl—1- methylpyrrolidinium; EMMC = 4-ethyl~4~methylmorpholinium methyl carbonate.

Example 2: Ionic liquids reduce the viscosity of concentrated aqueous Solutions of biosimilar RITUXAN® Materials and Methods Commercially-obtained biosimilar RITUXAN® containing pharmaceutical excipients (citrate buffer, NaCl, and Tween 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in e 1 above (using the tion coefficient of 1.7 L/g-cm). Viscosities were ed using a RheoSense mVROC microfluidic viscometer ed with an “A” or “B” chip. hulls The data in Table 2 demonstrate the viscosity of aqueous solutions of biosimilar N® can be reduced by up to 85-fold in the presence of 0.40050 M Viscosity-«reducing ionic liquids, compared to PB samples.

Table 2. Viscosities (in cP) of aqueous solutions of biosimilar RITUXAN® in the presence of various concentration of the ionic liquid BIM at 25°C and pH 7.

[Protein] PB 0.40M 0.50M (mg/mL) 0.25M BIM BIM 75.4 :h 83.9 i 213 :3: 4 636 i 32 1.0 0.8 ' " ' 65.43: 203 d: 4 251 :1: 1 n.d. 43.9i 191 :t 2 n.d. n.d.

PB = phosphate buffer; BIM = utylimidazolio)—1~butane sulfonate; n.d. = not determined.

Example 3: Ionic liquids reduce the viscosity of concentrated aqueous solutions of TYSABRI® Materials and Methods Commercially-obtained TYSABRI® containing pharmaceutical excipients (sodium phosphate buffer, NaCl, Polysorbate 80) was buffer ged, concentrated, dried, tituted, and analyzed as described in Example 1 above (using the extinction coefficient of 1.5 L/g-cm). Viscosities were measured using a RheoSense mVROC microfluidic eter equipped with an “A” or “B” chip.

Results The data in Table 3 trate that the viscosity of aqueous ons of TYSABRI® can be reduced by up to 7—fold in the presence of 0.10 M EMMC.

Table 3. Viseosities (in cP) of aqueous solutions of TYSABRI® in the presence of various excipients at 25°C and pH 7.

Ionic Liquid [IL] (M) [Protein] (mg/mL) Viscosity (cP) PB 0.25 237 182 :l: 6 Arg HCl 0.25 228 37 :t 0.1 BIM 0.4 234 43.6 :J: 1.1 BMI Mes 0.4 232 35.2 i 5.0 BMP Chloride 0.4 249 42.7 i 1.9 EMMC 0.1 232 24.7 i 0.3 PB = phosphate buffer; Arg—HCI =1Arginine-HC1 ; BIM = 4-(3 —buty1—1-imidazolio)—l- butane sulfonate; BMI Mes 2 i—butyImethylimidazolium methanesulfonate; BMP Chloride = 1-buty1—1-methylpyrrolidinium C1; EMMC = 4-ethyl methylmorpholinium methyl carbonate.

Example 4: 4—(3~butyl~1~imidazolio)hl-butane sulfonate reduces the viscosity of concentrated REMICADE® and VECTIBIX® solutions als and Methods cially-obtained REMICADE® containing pharmaceutical ents- (sucrose, Polysorbate 80, sodium phosphate buffer) was ed as per instructions in the presoribing information sheet. Commercially—obtained VECTlBIX® containing pharmaceutical excipients was prepared as per instructions in the prescribing information sheet. Subsequently, the s protein drug products were purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficients of: 1.4 L/g-em for REMICADE® and 1.25 L/g-cm for VECTIBIX®). The proteins were formulated either with phosphate buffer or with 0.50 M of 4-(3-butyl-l-imidazolio)—i-butane sulfonate (BIM). Viscosities were ed using a RheoSense mVROC uidic viscometer equipped with an “A” or “B” chip.

Rinks The results in Table 4 demonstrate that BIM is effective at reducing the viscosity of trated, aqueous solutions of both mAbs tested. Viscosity reductions with 0.50 M BIM are up to 22-fold in the proteins examined here.

Table 4. Viseosities (in CF) of aqueous solutions of REMICADE® and VECTIBIX® at 25°C and pH 7 with and without BIM.

[Protein] Protein. Exc1p1ent. . (mg/HID REMICADE® 222 a 6 1557 a 22 71.2 a 2.9 291 :|:3 328i 12 l70:|:2 VECTIBIX® 233 i4 38.7i1.8 51.l:|:3.7 Example 5: Ionic liquids reduce the viscosity of concentrated aqueous solutions of HERCEPTIN® Materials and Methods Commercially—obtained HERCEPTIN® containing pharmaceutical excipients (histidine buffer, ose, Polysorbate 20) was prepared as per instructions in the . prescribing information sheet. The aqueous protein drug product was buffer exchanged, conCentrated, dried, reconstituted, and analyzed as bed in Example 1 above (using the extinction coefficient of: 1.5 L/g-cm). The n was formulated either with phosphate buffer or with various viscosity-reducing ionic liquids at concentrations in the table listed below. Viscosities were measured using a RheoSense mVROC microfluidic viscometer ed with an “A” or “B” chip.

The results in Table 5 demonstrate that Viscosity-reducing ionic liquids are effective at reducing the ity of trated, aqueous solutions of HERCEPTIN®i EMMC can reduce the viscosity by almost 3-fold when present at 0.10 M.

Table 5. Viscosities of aqueous solutions of HERCEPTIN® in the presence of var- ious concentrations of ionic liquids (IL) at 25°C and pH 7. ity-reducing [IL] (M) in] (mg/mL) ity (cP) ionic liquid* PB 0.25 253 172 :l: 4 PB 0.25 218 71.6i3.9 BIM 0.40 255 97.9 :I: 3.5 BIM 0.40 223 43.8 i 0.4 BMI Mes 0.40 227 47.8 i 1.0 BMP Chloride 0.40 244 99.2 i 2.2 BMP Chloride. 0.40 210 55.6 :t 2.0 EMMC 0.10 253 60.2 2|: 4.3 PB = phosphate buffer; BIM m 4—(3-buty1imidazolio)butane sulfonate; BMI Mes = 1-butylmethylimidazolium methanesulfonate; BMP Chloride = l—butyl-l- methylpyrrolidiniurn chloride; EMMC = 4-ethylmethy1morpholinium methyl carbonate.

Example 6: Dependence of viscosity-lowering effect on ionic liquid concentration for aqueous solutions of ilar ERBITUX®.

Commercially-obtained biosimilar X® containing pharmaceutical excipients (Phosphate buffer, sodium chloride, Polysorbate 80) was buffer exchanged, concentrated, dried, reconstituted, and analyzed as bed in Example 1 above (using the extinction coefficient of: 1.4 L/g'cm). The protein was formulated either with phosphate buffer or with various concentrations of BIM. Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip.

The results in Table 6 demonstrate that the Viscosity—reducing ionic liquid BIM is effective at reducing the Viscosity of concentrated, aqueous solutions of biosimilar ERBITUX® in a dose ent manner up to about 0.50 M, at which point, the effect of BIM becomes decreasingly effective. This demonstrates that in some embodiments there is an optimal concentration of Viscosity-reducing ionic liqi1id.

Table 6. Viscosities of s solutions of biosimilar ERBITUX® in the presence of various concentrations of BIM at 25°C and pH 7. [biosimilar {BIM}, M ERBITUX®], ' Viscosity, cP mg/mL 0 280 3630 0.3 263 96.8 :I: 2.2 0.4 270 86.7 :I: 2.1 0.5 257 75.0 :I: 0.3 0.75 263 148 :b 2 1.0 279 145 :l: 2 1.5 267 347 :l: 5 Example 7. Viscosity-reducing show no signs of toxicity when injected aneously Thirty 11-week old e-Dawley rats were separated into 6 groups of 5 rats each (3 saline control groups and 3 BIM groups). The rats were injected subcutaneously with 0.5 mL of endotoxin—free either phosphate-buffered saline or 0.25 M BIM according to the following schedule: One group from each condition was ed once on day 1 and then sacrificed 1 hour later; one group from each condition was injected once on day l and once on day 2 andthen 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.

Clinical observations were recorded for any pharmaco-toxicological signs at pro-dose, immediately post-dose, at 1 and 4 hours (i 15 minutes) post-dose, and daily thereafter. Irritation, if any, at injection sites was scored using the Draize tion scores se, immediately ose, at 1 hour (i15 s) post close, and prior to sacrifice.

Overall, the ed consequences ofthe injections of saline and BIM were macroscopically similar throughout the course of the study. Both induced from no irritation to slight irritation with edema scores of 0-2 at various time points. The onset of slight irritation seemed to occur after the second subcutaneous injections of the saline control and BIM. Microscopic examination of ion sites suggests a very minor, clinically insignificant, tive effect with BIM that was no longer evident by day 4.

Unless expressly defined otherwise above, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art. Those skilled in the art will recognize, or will be able to ascertain 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 (26)

We claim :

1. A liquid pharmaceutical formulation for ion comprising: (i) an antibody; (ii) 4-(3-butylimidazolio)butane sulfonate (BIM) 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 from about 1 cP to about 100 cP at 25°C as measured using a cone and plate viscometer or a microfluidic viscometer; and the absolute viscosity of the liquid pharmaceutical formulation is less than an absolute viscosity of a control composition comprising the antibody and the pharmaceutically acceptable t but without the BIM or a pharmaceutically acceptable salt thereof; wherein the absolute viscosity is an extrapolated zero-shear viscosity.

2. The liquid pharmaceutical ation of claim 1, wherein the antibody has a molecular weight of from about 120 kDa to about 250 kDa.

3. The liquid pharmaceutical formulation of claim 1 or 2, wherein the antibody is a monoclonal antibody.

4. The liquid pharmaceutical formulation of any one of the previous claims, sing from about 100 mg/ml to about 300 mg/ml of the antibody.

5. The liquid pharmaceutical formulation of any one of the previous , comprising from about 191 mg/ml to about 270 mg/ml of the antibody.

6. The liquid pharmaceutical formulation of any one of the previous claims, wherein the pharmaceutically acceptable solvent is aqueous.

7. The liquid pharmaceutical formulation of any one of the previous claims, n the BIM or a pharmaceutically able salt thereof is present at a concentration of from about 0.01 M to about 1.0 M.

8. The liquid pharmaceutical formulation of any one of the us claims, wherein the BIM or a pharmaceutically able salt thereof is present at a concentration of from about 0.20 M to about 0.50 M.

9. The liquid pharmaceutical formulation of any one of the previous claims, further comprising one or more pharmaceutically acceptable excipients, the one or more ceutically acceptable excipients comprising a sugar, sugar alcohol, ing agent, preservative, carrier, antioxidant, chelating agent, natural polymer, synthetic polymer, cryoprotectant, lyoprotectant, surfactant, bulking agent, stabilizing agent, or any combination thereof.

10. The liquid ceutical formulation of claim 9, wherein the one or more pharmaceutically acceptable ents comprise a polysorbate, poloxamer 188, sodium lauryl sulfate, a polyol, a thylene glycol), glycerol, a propylene glycol, or a poly(vinyl alcohol).

11. The liquid pharmaceutical formulation of claim 9, wherein the sugar alcohol is sorbitol or ol.

12. The liquid pharmaceutical formulation of any one of the previous claims, in a unitdose vial, multi-dose vial, cartridge, or pre-filled syringe.

13. The liquid pharmaceutical formulation of any one of the previous claims, wherein the liquid pharmaceutical formulation is reconstituted from a lyophilized composition.

14. The liquid pharmaceutical formulation of any one of the previous claims, n the liquid pharmaceutical ation is isotonic to human blood serum.

15. The liquid pharmaceutical formulation of any one of the previous , wherein the absolute viscosity is measured at a shear rate of at least about 0.5 s-1 when measured using a cone and plate eter, or a shear rate of at least about 1.0 s-1 when measured using a microfluidic viscometer.

16. Use of the liquid pharmaceutical formulation of any one of the previous claims in the manufacture of a medicament for administering to a subject a therapeutically effective amount of an antibody, wherein the medicament is formulated for subcutaneous or intramuscular injection.

17. The use of claim 16, wherein the medicament is formulated for injection with a syringe.

18. The use of claim 17, wherein the e is a heated syringe, a self-mixing syringe, an auto-injector, a prefilled syringe, or combinations thereof.

19. The use of claim 18, wherein the syringe is a heated syringe and the medicament is ated to have an administration temperature between 25°C and 40°C.

20. The use of any one of claims 16-19, wherein the medicament is formulated to produce a primary irritation index of less than 3 when evaluated using a Draize scoring system.

21. The use of any one of claims 16-20, wherein the medicament is formulated to be administered with an ion force that is at least 10% less than an injection force for a control composition comprising the dy and the pharmaceutically acceptable solvent but without the BIM or a pharmaceutically acceptable salt thereof, when administered in the same way.

22. The use of any one of claims 16-20, wherein the medicament is formulated to be administered with an injection force that is at least 20% less than an injection force for a control composition comprising the antibody and the pharmaceutically acceptable t but without the BIM or a pharmaceutically acceptable salt thereof, when administered in the same way.

23. The use of any one of claims 16-22, wherein the medicament is formulated for administration with a needle between 27 and 31 gauge in diameter and with an ion force less than 30 N with the 27 gauge needle.

24. A method of preparing the liquid pharmaceutical formulation of any one of claims 1- 15, comprising the step of combining the antibody, the pharmaceutically able solvent, and the BIM or a ceutically acceptable salt thereof.

25. A lyophilized composition comprising: (i) an antibody; (ii) BIM or a pharmaceutically able salt thereof; and (iii) a pharmaceutically acceptable excipient.

26. The lyophilized composition of claim 25, wherein, once reconstituted, the antibody has a concentration of at least 100 mg/ml.