NZ717918B2 - Liquid protein formulations containing ionic liquids - Google Patents

Abstract

Concentrated, low-viscosity, low-volume liquid pharmaceutical formulations of antibodies comprising 1-butyl-3-methylimidazolium methanesulfonate (BMI-Mes) have been developed. Such formulations can be rapidly and conveniently administered by subcutaneous or intramuscular injection, rather than by lengthy intra-venous infusion. ngthy intra-venous infusion.

Description

LIQUID PROTEIN FORMULATIONS CONTAINING IONIC LIQUIDS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit ofUS Provisional ation No. 62/030,521, filed July 29, 2014, entitled “Low- Viscosity Protein Formulations Containing hobic Salts; ” U.S. Provisional Application No. 62/026,497, filed July 18, 2014, entitled “Low- Viscosity Protein Formulations Containing GRAS Viscosity-Reducing Agents; ” U.S. ional ation No. 62/008,050, filed June , 2014, entitled “Low- Viscosity Protein Formulations ning Ionic Liquids; ” U.S. Provisional Application No. 61/988,005, filed May 2, 2014, entitled “Low— Viscosity Protein Formulations Containing Organophosphates,” U.S. Provisional ation No. 61/946,436, filed February 28, 2014, entitled “Concentrated, Low— Viscosity mab Formulations; ” US Provisional Application No. 61/943,197, filed February 21, 2014, entitled “Concentrated, Low~ Viscosity, olecular~ Weight-Protein Formulations; ” U.S. Provisional Application No. 61/940,227, filed February 14, 2014, entitled “Concentrated, Low- Viscosity High~Molecular~Weight Protein Formulations; " and U.S. Provisional Application No. 61,876,621, filed September 11, 2013, entitled “Concentrated, Low-Viscosity, High-Molecular- Weight n 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 thereof.

BACKGROUND OF THE ION Monoclonal antibodies (mAbs) are important n-based therapeutics for ng various human diseases such as cancer, infectious 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 clinical trials are mAbs erty 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 2014/055245 period of time and require 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 providers during administration ed to the IV route. To be effective and pharmaceutically acceptable, parenteral formulations should preferably be e, stable, inj ectable (e.g., via a syringe), and non-irritating at the site of injection, in ance 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 viscous formulations that are difficult to administer by ion, cause pain at the site of injection, are often imprecise, and/or may have decreased chemical and/or al 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 proteins, such as mAbs. All protein therapeutics to some extent are t to physical and al instability, such as aggregation, denaturation, crosslinking, deamidation, isomerization, oxidation, and clipping (Wang 9161]., J. Pharm. Sci. 96:1-26, 2007). Thus, optimal formulation development is paramount in the development of cially viable protein pharmaceuticals.

High protein concentrations pose challenges ng to the physical and chemical stability of the protein, as well as difficulty with manufacture, storage, and delivery ofthe protein formulation. One problem is the tendency of proteins to aggregate and form particulates during processing and/or storage, which makes manipulations during further processing and/or delivery difficult. Concentrationdependent degradation and/or aggregation are major challenges in developing protein formulations at higher concentrations. In addition to the ial for non-native n aggregation 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 tion processes and the ability to readily deliver such compositions by conventional means. (See, for example, J. Jezek at (.11., Advanced Drug Delivery Reviews 7—1117, 2011.) Highly Viscous liquid formulations are difficult to manufacture, draw into a syringe, and inject subcutaneously or intramuscularly. The use of force in manipulating the viscous formulations can lead to excessive frothing, which may further denature and inactivate the therapeutically active protein. High viscosity solutions also require larger diameter needles for injection and produce more pain at the inj ection site.

Currently available commercial mAb products administered by SC or IM injection are usually formulated in aqueous buffers, such as a phosphate or L—histidine buffer, with excipients or tants, such as mannitol, sucrose, lactose, trehalose, POLOXAMER® (nonionic ck copolymers composed of a central hobic chain oxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide))) or POLYSORBATE® 80 - -- - (PEG(80)sorbitan monolaurate), to prevent aggregation and improve stability. ed 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 describes reducing the Viscosity in formulations of low-molecular-weight proteins using a buffer'and a viscosity—reducing nic salt, such as calcium chloride or magnesium chloride. These same salts, however, showed little effect on the viscosity of a high-molecular-weight dy (lMA-63 8) ' formulation. As described in U.S. Patent No. 7,666,413, the viscosity of s formulations ofhigh-moleculafiweight proteins has been reduced by the addition of such salts as arginine hydrochloride, sodium thiocyanate, ammonium anate, ammonium e, um 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 Viscosity 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 specific salts and a tituted anti-IgE mAb, but with a m antibody concentration of only up to about 140 mg/mL. U.S. Patent No. 7,740,842 describes E25 antinIgE mAb formulations containing e/acetic acid buffer with antibody trations up to 257 mg/mL. The addition of salts such as NaCl, CaClg, or MgClz was demonstrated to decrease the dynamic viscosity under hear conditions; however, at low-shear the salts produced an undesirable and dramatic increase in the dynamic viscosity. onally, 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 suspension of up to 100 mg/mL mAb in a formulation having a viscosity enhancer inylpyrrolidone, PVP) and a solvent l benzoate or PEG 400). WO2004/089335 describes 100 mg/mL non~aqueous lysozyrne suspension formulations containing PVP, urol, 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. Patent-No. 6,730,328 describes ueous, hydrophobic, non—polar vehicles of low reactivity, such as perfluorodecalin, for protein formulations. These formulations are non-optimal and have high viscosities that impair processing, manufacturing and ion; lead to the presence of multiple vehicle components in the formulations; and present potential regulatory challenges associated with using polymers not yet approved by the FDA.

Alternative non-aqueous protein or antibody formulations have been described using organic solvents, such as benzyl benzoate (Miller et al., ir 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 g fi'om 100 mg/mL up to 257 mg/InL. Formulations with concentrations greater than about 189 mg/InL demonstrated dramatically increased viscosities, low recovery 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 formulations are low enough viscosity for ease of injection.

Du and Klibanov (Biotechnology and Bioengineering 108:632-636, 2011) described reduced viscosity of concentrated aqueous solutions of bovine serum albumin 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 2-3109, 2012) described low—viscosity aqueous solutions of four model mAbs achieved using hydrophobic salts. The mAb formulation employed by Guo had an initial viscosity, prior to adding salts, no greater than 73 cP. The viscosities of many ceutically important mAbs, on the other hand, can exceed 1,000 cP at therapeutically relevant trations.

It is not a trivial matter to control aggregation and ity in high- tration 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 formulations ofmAbs and other therapeutically important proteins, a truly useful formulation for many proteins has not yet been achieved. y, many ofthe above reports employ agents for which safety and toxicity profiles have not been fully established. These formulations would ore 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 tely be unsuitable for use in a formulation intended 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 e 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 concentrated, low— ity liquid formulations of pharmaceutically important proteins, especially high- molecular-weight ns, such as mAbs.

It is a further object of the present invention to provide concentrated low- viscosity liquid ations 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 ions.

It is a further object of the present invention to provide the concentrated liquid formulations of proteins, especially high-molecular-weight proteins, such as mAbs, with low viscosities that can improve inj lity and/or patient compliance, ience, and comfort.

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

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

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

SUMMARY OF THE ION Concentrated, low-Viscosity, lume liquid ceutical formulations of proteins have been developed. Such formulations can be rapidly and conveniently stered by subcutaneous 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 mg/rnL. In some embodiments, 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. ations 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 preferably 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 ments, the viscosity of the formulation is about 10 cP.

Formulations containing proteins and ionic liquids typically are ed 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 eter. 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 compounds and es of more than one ionic liquid. It is red 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 preferably 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, ably at least about 50% less, most preferably at least about 75% less, than the viscosity of the corresponding formulation under the same conditions except for replacement of the viscosity-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 t the viscosity-reducing ionic liquid is greater than about 200 cP, r 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 liquids in an effective amount to significantly reduce the viscosity of the n, e.g., mAb formulation. Representative ionic liquids include 4-(3-butylimidazolio)butane sulfonate (BIM), 1-butylmethylimidazolium methanesulfonate (BMI Mes), 4-ethyl morpholinium methylcarbonate, (EMMC) and 1-butylmethylpyrrolidinium 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. 2014/055245 For ments in which the protein is a “high-molecular~w_eight protein”, the “high-molecular-weight protein,” may have a molecular weight between about 100 kDa and about 1,000 kDa, preferably between about 120 kDa and about 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 derivatized form thereof. Preferred mAbs include natalizumab (TYSABRI®), cetuxi— mab (ERBITUX®), bevacizumab (AVASTIN®), trastuzumab EPTIN®), inflix— imab (REMICADE®), rituximab AN®), 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 protein and viscosityureducing ionic liquid(s) are ed in a lyophilized dosage unit, sized for reconstitution with a sterile aqueous ' pharmaceutically acceptable vehicle, to yield the concentrated low—viscosity liquid formulations. 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 .

Methods are provided herein for ing concentrated, low-Viscosity liquid formulations of high-molecular-weight proteins such as mAbs, as well as methods for storing the low-Viscosity, oncentration protein formulations, and for administration thereof to patients. In another ment, 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 PTION OF THE INVENTION I. - DEFINITIONS The term "protein," as generally used herein, refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce at least a detectable tertiary structure. Proteins having a molecular weight (expressed in kDa wherein “Da” stands for “Daltons” and 1 kDa = 1,000 Da) greater than about 100 kDa may be designated “high-molecular—weight proteins,” whereas proteins having a molecular weight less than about 100 kDa. may be designated “low-molecular-weight ns.” The term “low-molecular—weight protein” excludes small peptides lacking the requisite of at least tertiary structure necessary to be considered a protein. Protein lar weight may be determined using standard s known to one skilled in the art, including, but not limited to, mass spectrometry (e.g., ESI, MALDI) or calculation from known amino acid sequences and glycosylation. Proteins can be naturally occurring or turally 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 n 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 lly used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies sing the population are identical, except for le naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being ed against a single epitope. These are typically synthesized by culturing hybridoma cells, as described by Kohler et al. (Nature 256: 495, 1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), or isolated from phage antibody libraries using the techniques described in on et al. (Nature 352: 624-628, 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 “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or gous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the der of the chain(s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or ss, 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 antibody, including the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', 2, and Fv fragments; diabodies; linear antibodies (see US. Patent No. 5,641,870; Zapata et ai, n Eng. 8: 1057-1062, 1995); singlenchain antibody molecules; multivalent single domain antibodies; and pecific antibodies formed from antibody fragments.

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

“Viscosity” refers to the resistance of a substance (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 es 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 kinematic viscosity is a measure of the resistive flow of a fluid under the ce of gravity. When two fluids of equal volume and differing ity are placed in identical capillary eters and allowed to flow by gravity, the more viscous fluid takes longer than the less viscous fluid to flow through the capillary. If, for example, one fluid takes 200 seconds (5) to te its flow and another fluid takes 400 s, the second fluid is called twice as s as the first on a kinematic Viscosity scale. The dimension of kinematic viscosity is lengch/time. Commonly, kinematic Viscosity is expressed in centiStokes (cSt). The SI unit of kinematic viscosity is mm2/s, which is equal to l cSt.

The "absolute viscosity," sometimes called "dynamic 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 ed by using, for example, a viscometer at a given shear rate or multiple shear rates. An “extrapolated hear" viscosity can be determined by creating a best fit line of the four highest- - shear points on a plot of te viscosity versus shear rate, and linearly extrapolating Viscosity back to zero-«shear. Alternatively, for a Newtonian fluid, Viscosity can be determined by ing viscosity values at multiple shear rates.

Viscosity can also be measured using a microfluidic eter at single or multiple shear rates (also called flow rates), wherein absolute viscosity is derived from a change in pressure as a liquid flows through a channel. Viscosity equals shear stress over shear rate. Viscosities measured with microfluidic viscometers can, in some embodiments, be ly compared to extrapolated zero-shear viscosities, for e 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 ty 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 seconds or, in general, reciprocal time. For a microfluidic viscometer, change in pressure and flow rate are related 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 riately 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 eter (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 ng" or "shear thickening", respectively. In the case of concentrated (i.e., high- coneentration) protein ons, this may manifest as plastic shear~thinning behavior, i.e., a se in viscosity with shear rate.

The term "chemical stability," as generally used herein, refers to the ability of the protein components in a formulation to resist degradation via chemical pathways, such as ion, ation, or hydrolysis. A protein formulation is lly considered chemically stable if less than about 5% of the components are degraded after 24 months at 4°C.

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

Levels as low as about 0.2% are achievable, although approximately 1% is more typical.

The term " stable formulation,” as generally used herein, means that a formulation is both chemically stable and ally stable. A stable formulation may be one in which more than about 95% ofthe bioactive protein molecules retain bioactivity in a formulation 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 ed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, Ed, Marcel Dekker, Inc., New York, NY. (1991) and Jones, A., Adv. Drug Delivery Revs. 10:29-90, 1993. Stability can be ed 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 ed to the l 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 assessed by measuring 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 binding assays well within the abilities of one ordinarily skilled in the art.

The term protein "particle size," as generally used , means the average diameter ofthe inant population 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 generally used herein, describes liquid formulations having a final concentration of protein r 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 stituted formulation,” as lly used herein, refers to a formulation which has been prepared by dissolving a dry powder, lyophilized, spray-dried or solvent-precipitated protein in a t, such that the n 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 lyoprotectants include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, ol, xylitol, sorbitol, and mannitol; amino acids, such as ne or histicline; lyotropic salts, such as magnesium sulfate; polyols, such as propylene , glycerol, poly(ethylene glycol), or poly(propylene glycol); and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrins, modified starch, and carboxymethyl cellulose. Preferred sugar alcohols are those compounds obtained by reduction of mono- and di~saccharides, such as lactose, trehalose, maltose, lactulose, and maltulose. Additional examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. The lyoprotectant is generally added to the pre- lized formulation in a “IyOprotecting amount.” This means that, following lyophilization of the protein in the presence of the lyoprotecting amount of the lyoprotectant, the protein essentially s its physical and chemical stability and integrity.

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

A “preservative” is a compoundwhich can be added to the ations herein to reduce contamination by and/or action of bacteria, fungi, or r 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 chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long~chained), and honium de. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl ns such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and ol.

A ng 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 lized cake which maintains an open pore structure). ary bulking agents include mannitol, e, lactose, d starch, poly(ethylene glycol), and sorbitol.

A “therapeutically effective amount” is the lowest concentration required to effect a measurable improvement or prevention of any m or a particular condition or disorder, to effect a measurable enhancement of life ancy, or to generally improve patient quality of life. The eutically effective amount is ent upon the specific biologically active molecule and the specific condition or disorder to be treated. Therapeutically effective amounts of many proteins, such as the mAbs described herein, are well known in the art. The eutically 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 ined by standard techniques which are well Within the craft of a skilled artisan, such as a physician.

The term "inj lity" or “syringeability,” as generally used herein, refers to the inj ection performance of a pharmaceutical formulation through a syringe equipped with an 18-32 gauge , optionally thin walled. Inj ectability depends upon factors such as pressure or force required for injection, evenness of flow, aspiration qualities, and freedom from clogging. inj ectability of the liquid pharmaceutical ations may be assessed by comparing the injection force of a reduced-Viscosity formulation to a standard formulation without 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 ity formulations have improved 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 standard formulation having the same concentration ofprotein under otherwise the same conditions, except for replacement 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 required to inject the same volume, such as 0.5 mL, or more preferably about 1 mL, of ent 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 through a given syringe equipped with a given needle gauge at a given injection 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 e having a 0.25 inch inside diameter, equipped with a 0.50 inch 27 gauge needle at a 250 mrn/min injection speed. g equipment can be used to measure the ion force. When measured under the same conditions, a formulation with lower viscosity will generally require an overall lower injection force.

The sity gradient,” as used herein, refers to the rate of change of the viscosity 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 ses approximately exponentially with increasing protein concentration. The viscosity gradient at a c protein concentration can be approximated from the slope of a line tangent to the plot of, A Viscosity as a function of protein concentration. The viscosity gradient can be imated fi'om a linear approximation to the plot of ity as a function of any protein concentration or over a narrow Window of protein concentrations. In some embodiments a formulation is said to have a decreased viscosity nt if, when the viscosity as a function of protein concentration is approximated as an exponential function, the nt of the exponential fimction is smaller than the exponent obtained for the otherwise same formulation without the Viscosity-lowering agent In a similar manner, a formulation can be said to have a lower/higher viscosity gradient . when compared to a second ation if the exponent for the formulation is lower/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 ed-viscosity formulation,” as generally used , refers to a liquid ation 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 2014/055245 formulation that does not contain the ity-lowering additive(s).

The term “osmolarity,” as generally used , refers to the total number of dissolved components per liter. Osmolarity is similar to molarity but includes the total number of moles of dissolved species in solution. An osmolarity of 1 Osm/L means there is 1 mole of dissolved components per L of solution. Some solutes, such as ionic s 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 dissolved components per 1 mole of dissolved NaCl in solution. Physiological osmolarity is typically in the range of about 280 mOsm/L to about 310 m0sm/L.

The term ity,” as generally used herein, refers to the osmotic pressure gradient resulting from the separation oftwo solutions by a semi-permeable membrane. In particular, tonicity is used to describe the osmotic pressure created across a cell ne when a cell is exposed to an external solution. Solutes that can cross the cellular membrane do not contribute to the final osmotic pressure gradient.

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

The term “hypertonic,” as generally 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 ed into a hypertonic solution, the tendency is for water to flow out of the cell in order to balance the tration 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 nic,” as generally used herein, 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. ic formulations will generally have an osmotic pressure from about 250 mOsm/kg to 350 mOsm/kg.

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

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 lly used interchangeably with “a generic equivalen ” or “follow-on.” For example, a “biosimilar mAb” refers to a subsequent version of an innovator’s mAb typically made by a different company.

“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 es one or more guidelines adopted May , 2012 by the Committee for nal Products for Human Use (CHMP) ofthe European Medicines Agency and published by the European Union as “Guideline on similar biological medicinal products ning monoclonal antibodies — non-clinical and clinical issues” (Document Reference EMA/CHMP/BMWP/403543/2010).

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

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

A biosimilar mAb is similar to the nce mAb chemically cr biologically 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); g to isoforms of the Fc gamma receptors (FcyRI, , 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. antibody-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 comparisons ofpharmacokinetic ties (e.g. AUCO-jnf, AUCM, Cum, tmax, (Enough); pharmacodynamic nts; or similarity of clinical efficacy (e.g. using randomized, parallel group comparative clinical trials). The y comparison between a biosimilar mAb and a reference mAb Can be ted using established procedures, including those described in the line on similar biological medicinal products containing biotechnology—derived proteins as active nce: 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).

Differences n 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 phosphate, various lipids and carbohydrates; by proteolytic ge following translation; by changing the chemical 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 sive for certain degradation pathways such as oxidation, deamidation, or aggregation. As all of these product-related variants may be included in a biosimilar mAb.

As used herein, the term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids and bases, and c acids and bases. Suitable non-toxic acids include inorganic and organic acids such as , benzenesulfonic, benzoic, camphorsulfonic, 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 , potassium, lithium, calcium and ium.

As used herein, the term "ionic liquid” refers to a crystalline or amorphous salt, zwitterion, or mixture thereof that is a liquid at or near temperatures where most tional salts are solids: at less than 200°C, preferably less than 100°C or more preferably less than 80°C. Some ionic liquids have melting temperatures around room temperature, e.g. n 10°C and 40°C, or between 15°C and 35°C. The term 2014/055245 erion" is used herein to describe an overall neutrally charged molecule which carries formal positive and ve charges on ent 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 nd containing 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 soluble 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, preferably in the visible-to-infrared portion 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 ion 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 “alkenyl group,” 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 systems, 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, n at least one of the atoms that forms the ring is a heteroatom.

As used herein, a “heteroatom” is any non-carbon or drogen atom.

Preferred heteroatoms include oxygen, , and nitrogen. Exemplary heteroaryl and heterocyclyl rings include: idazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzirnidazolinyl, carbazolyl, 4aH carbazolyl, earbolinyl, chromanyl, nyl, cinnolinyl, decahydroquinolinyl, 2H,6H—1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H—indolyl, isatinoyl, zofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, azolyl, olyl, methylenedioxyphenyl, linyl, 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, phenothiazinyl, phenoxathinyl, azinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, ‘ - inyl, purinyl, pyranyl, nyl, pyrazolidinyl, pyrazolinyl, lyl, pyridazinyl, pyridooxazole, pyridoirnidazole, pyridothiazole, pyridinyl, 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, thiadiazolyl, hrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoirnidazolyl, thiophenyl, and xanthenyl.

II. FORMULATIONS Biocompatible, low-Viscosity protein solutions, such as those of InAbs, can be used to deliver therapeutically effective amounts of proteins in volumes useful for subcutaneous (SC) and intramuscular (1M) injections, 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 proteins are low-molecular-weight proteins.

Formulations may have protein concentrations between about 10 mg/mL and about 5,000 mg/mL. The formulations, including mAb formulations, may have a protein concentration greater than 100 mg/mL, ably greater than 150 mg/rnL, more preferably greater than about 175 nag/ml, even more ably greater than about 200 mg/mL, even more preferably greater than about 225 mg/mL, even more preferably greater than about 250 mg/mL, and most preferably greater than or about 300 mg/mL. In the e of a viscosity-reducing ionic liquid, the viscosity of a protein formulation increases exponentially as the tration is increased. Such n formulations, in the absence of a viscosity-reducing ionic liquids, may have viscosities greater than 100 cP, r 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 ations are often unsuitable for SC or IM injection; The use of one or more viscosity-reducing ionic liquids permits the preparation of formulations 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 ity of concentrated protein formulations, they may be used in less-concentrated formulations as well. In some embodiments, ations may have protein concentrations between about 10 mg/mL and about 100 mg/mL. The ations may have a protein concentration greater than about 20 mg/mL, greater than about 40 mg/mL, or r than about 80 mg/mL.

For certain proteins, formulations not having an ionic liquid may have viscosities greater than about 20 01’, greater than about 50 CF, or greater than about 80 CF. The use of one or more ionic liquids permits the preparation of formulations 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 formulation without the ionic liquid(s), when measured under the same conditions. In other embodiments, the formulations have a viscosity that is 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, or even more than 90% less than the analogous formulation without the viscosity— reducing ionic liquid(s). In a preferred embodiment, the formulation contains a eutically ive 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 preferably less than about 0.75 mL.

The reduced-viscosity formulations have improved inj ectability and require less injection force compared to the analogous formulation t the viscosity- reducing ionic liquid (e.g., in phosphate buffer) under otherwise the same ions.

In some embodiments, 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, optionally thin walled.

The ations may contain one or more onal excipients, such as buffers, surfactants, sugars and sugar alcohols, other polyols, preservatives, idants, and chelating agents. The formulations have a pH and osmolarity suitable for administration without causing significant e side effects. In some embodiments, the trated, 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 lity in formulation development. The low-viscosity formulations can exhibit 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 concentrations and decreased dosage frequencies of the protein. In some embodiments the low—viscosity n 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.

Because protein (such as mAb) formulations may be administered to patients at higher protein concentrations than otherwise r protein formulations not containing a viscosity~reducing ionic , the dosing frequency of the protein can be reduced. For instance, proteins previously ing once daily administration may be administered once every two days, every three days, or even less frequently when the ns 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 injection multiple-fold the dosing frequency can be decreased, for e from once every 2 weeks to once every 6 weeks.

In some embodiments, the liquid formulations have a logical osmolarity, for e, between about 280 mOsm/L to about 310 m0srn/L. In some embodiments, the liquid ations have an osmolarity r than about 250 mOsm/L, greater than about 300 mOsm/L, greater than about 350 mOsm/L, greater than about 400 mOsm/L, or greater than about 500 mOsm/L. In some embodiments, the formulations have an osmolarity of 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 isotonic 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 physical ity 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 trations include about 0.15 M and about 0.30 M. For some embodiments having two or more viscosity-reducing ionic liquids, the agents are preferably, 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 pharmaceutically acceptable fluid is added. In the e of Viscosity-reducing ionic s, periods of 10 minutes or more are often required in order to completely dissolve the lized cake at high protein concentration. When the lyophilized cake contains one or more viscosity-reducing ionic liquid, 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 r than or about 150, 200 or even 300 mg/mL of protein.

The low-viscosity protein formulations allow for greater lity in formulation pment. The low«viscosity formulations exhibit a ity that changes less with increasing protein concentrations as compared to the ise 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 gradient ofthe protein formulation may be 2-fold1ess,3-fold less, or even more than 3-fold less than the Viscosity gradient of the otherwise same protein formulation without the viscosity-reducing ionic liquid(s). The viscosity 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— lation, the protein concentration can be sed to a greater degree before an expo- nential increase in viscosity is observed.

A. Proteins Any protein can be formulated, including inant, isolated, or synthetic proteins, glycoproteins, or lipoproteins. These may be antibodies (including antibody fragments and recombinant antibodies), enzymes, growth factors or hormones, im- munomodiflers, antiinfectives, oliferatives, es, or other therapeutic, prophylactic, or stic proteins. In certain embodiments, the n 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 greater than 200 kDa.

In certain ments, the protein can be a PEGylated protein. The term “PEGylated protein,” as used herein, refers to a protein having one or more poly(ethylene glycol) or other stealth polymer groups covalently attached thereto, optionally h a chemical linker that may be different from the one or more polymer groups. PEGylated proteins are characterized by their typically reduced renal — tion, sed 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 glycol); poly(amino acid) polymers such as poly(glutamic acid), poly(hydroxyethyl-L- asparagine), and poly(hydroxethyl-L—glutamine); poly(glycerol); poly(2~oxazoline) polymers such as poly(2-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. PEGylated proteins can be randomly PEGylated, z'. e. having one or more stealth polymers covalently attached at nonspecific site(s) on the protein, or can be PEGylated in a site-specific manner by cova- lently attaching the stealth polymer to specific site(s) on the n. Site-specific PEGylation can be accomplished, for example, using activated stealth polymers 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- , 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. olecular-weight proteins can include those bed in Scolnik, mAbs 1:179- 184, 2009; Beck, mAbs 3:107-110, 2011; n, Curr. Drug Math. 7:15-21, 2006; or Federici, icals 41:131-147, 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 f).

Preferred mAbs herein include zumab (TYSABRI®), mab (ERBL TUX®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), mab ADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX‘E), umab (ARZERRA® and biosimilars thereof. Exemplary high-molecular—weight proteins can include tocilizumab (ACTEMRA®), alemtuzumab (marketed under several trade names), brodalumab (developed by Amgen, Inc (“Amgen”)), denosumab (PROLIA® and XGEVA®), and biosirnilars thereof.

Exemplary molecular targets for antibodies described herein e 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 , ing either a or B subunits thereof (e.g., anti-CD1 la, anti-CD18, or anti— CD111) antibodies); growth s, such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; y (OB) receptor; protein C; PCSK9; etc.

Antibody Therapeutics Currentlyon the Market ' Many protein therapeutics currently on the market, especially antibodies as defined herein, are administered Via IV infusions due to high dosing requirements. ations can e one ofthe antibody therapeutics currently on the market or a ilar thereof. Some protein therapeutics currently on the market are not high- molecular—weight, but are still administered Via IV infusion because 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 injections.

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

Natalizumab, a zed 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®, natalizumab is currently co-marketed as TYSABRI® by Biogen Idec (“Biogen”) and Elan Corp. (“Elan”) TYSABRI® is produced in murine a cells. Each 15 mL dose contains 300 mg natalizumab; 123 mg sodium chloride, USP; 17.0 mg sodium phosphate, monobasic, monohydrate, USP; 7.24 mg sodium phosphate, dibasic, heptahydrate, USP; 3.0 mg polysorbate 80, USP/NF, in water for 1V injection, 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 decline, and significantly improving patient’s quality of life.

As used herein, the term “natalizuma ” includes the mAb against the cell adhesion molecule 0L4-integrin known under the International Nonproprietary Name “NATALIZUMAB” or an antigen binding portion thereof. Natalizumab includes dies bed in US. Patent No. 5,840,299, US. Patent No. 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 ed under the trade name TYSABRI® by Biogen Idec and Elan Corporation or a ilar product thereof.

Cetuximab is an epidermal growth factor or (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; “Bristol—Myers Squibb”), Eli Lilly and Company (North a; “Eli Lilly”), and Merck KGaA. X® is ed in mammalian (murine myeloma) cell e. Each -use, 5O-mL Vial of ERBITUX® contains 100 mg of cetuximab at a concentration of 2 mg/mL and is formulated in a preservative—free solution containing 8.48 mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic heptahydrate, 0.42 mg/mL sodium phosphate monobasic monohydrate, and water for IV Injection, USP.

Cetuximab is indicated for the treatment of patients with epidermal growth factor receptor (EGFR)-expressing, KRAS wild-type metastatic colorectal cancer (mCRC), in combination with chemotherapy, and as a single agent in ts who have failed oxaliplatin- and irinotecan—based therapy or who are intolerant to irinotecan. Cetuximab is indicated for the treatment of patients with squamous cell carcinoma ofthe head and neck in combination with um-based chemotherapy for the first-line treatment of recurrent and/or metastatic disease and in combination with radiation therapy for y advanced disease. Approximately 75% of patients with metastatic colorectal cancer have an EGFR—expressing tumor and are, ore, considered eligible for treatment 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 dies described in US. Patent No. 6,217,866. ' * mab includes the active agent in products marketed under the trade name ERBITUX® and biosimilar products thereof. Biosimilars of X® can include those currently being developed by Amgen, AlphaMab Co., Ltd. (“AlphaMab”), and Actavis plc (“Actavis”).

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

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

AVASTIN® is given as an IV on 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 chemotherapy, whereas the lower dose is given with cisplatin—based chemotherapy. In 2009, the FDA approved N® for use in metastatic renal cell carcinoma (a form of kidney cancer). The FDA also granted accelerated approval ofAVASTIN® for the treatment 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 herapy, followed by nance bevacizumab until disease progression. The NCCN updated its Clinical ce 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 elial growth factor A A) known under the International Nonproprietary Name/Common Name “BEVACIZUMAB” or an antigen binding portion thereof. Bevacizumab is described in US. Patent No. 6,054,297.

Bevacizumab includes the active agent in products ed under the trade name AVASTIN® and biosimilar ts thereof. Biosimilars ofAVASTIN® can e those currently being developed by Amgen, s, 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.

Trastuzumab is a mAb that interferes with the HERZ/neu receptor.

Trastuzumab is ed under the trade name HERCEPTIN® by Genentech, Inc.

HERCEPTIN® is produced by a mammalian cell (Chinese Hamster Ovary (CHO)) line. HERCEPTIN® is a sterile, white to pale-yellow, preservative-free lyophilized powder for IV administration. Each TIN® vial contains 440 mg trastuzumab, 9.9 mg L-histidine HCl, 6.4 mg L—histidine, 400 mg ehalose dihydrate, and 1.8 mg polysorbate 20, USP. Reconstitution with 20 mL water yields a multi-dose solution containing 21 mg/mL trastuzumab. 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.

Trastuzumab is mainly used to treat certain breast cancers. The HER2 gene is amplified in 20-30% of early-stage breast cancers, which makes it overexpress epi— epidermal growth factor (EGF) ors in the cell membrane. Trastuznmab is generally administered as a maintenance therapy for patients with HERZ-positive breast cancer, typically for one year post-chemotherapy. Trastuzumab is tly administered via IV on 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” includes the mAb that eres with the HER2/neu receptor known under the International Nonproprietary Name/Common Name “TRASTUZUMAB” or an antigen binding portion f. Trastuzumab is described in US. Patent No. 5,821,337. Trastuzumab includes the active agent in products marketed under the trade name HERCEPTIN® and biosimilars thereof. The term “trastuzumab” includes the active agent in biosimilar HERCIE'ZPTIN® products marketed under the trade names HERTRAZ® by Mylan, Inc. (“Mylan”) and CANMAB® by Biocon, Ltd. (“Biocon”). Trastuzurnab can include the active agent in biosimilar HERCEPTIN® products being developed by Amgen and by PlantForm ' Corporation, Canada.

Infliximab is a mAb t tumor necrosis factor alpha (TNF-cr) used to treat autoimmune diseases. It is marketed under the trade name REMICADE® by Janssen Global Services, LLC (“Janssen”) in the U.S., Mitsubishi Tanabe Pharma in Japan, Xian Janssen in China, and Merck & Co (“Merck”); 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 REMSIMATM, developed by Celltrion, Inc. (“Celltrion”), and INFLECTRATM, developed by Hospira Inc, UK, have been recommended for regulatory approval in Europe. Celltrion has ted a filing for REMSIMATM to the FDA. Infliximab is currently stered via IV infusion at doses ranging from about 3 mg/kg to about 10 mgfkg.

Infliximab ns approximately 30% murine le region amino acid sequence, which s antigen-binding specificity to human TNFcL. The ing 70% correspond to a human IgG1 heavy chain constant region and a human kappa light chain constant . 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 secreted from mouse myeloma cells (SP2/0 cells). The antibody is currently manufactured by continuous perfusion cell culture. The infliximab monoclonal antibody is sed using chimeric antibody genes consisting ofthe variable region sequences cloned from the murine anti-TNFd hybridoma A2, and human antibody constant region sequences supplied by the plasmid expression vectors. Generation of the murine anti- TNF u hybridoma is performed by zation of BALB/c mice With d recombinant human TNFOL. The heavy and light chain vector constructs are linearized and transfected into the Sp2/O cells by electroporation. Standard purification steps can include chromatographic purification, viral vation, nanofiltration, and ultrafiltration/diafiltration.

As used , 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 inhibits g of TNFd with its receptors.

Infliximab is described in US. Patent No. 5,698,195. The term “Infliximab” includes the active agent in products ed or ed to be marketed under the trade names REMICADE® by multiple es; REMSIMATM by Celltrion and INFLECTRATM by Hospira, Inc (“Hospira”). Infliximab is supplied as a sterile lyophilized cake for reconstitution and dilution. Each vial of infliximab ns 100 mg infliximab and excipients such as monobasic sodium ate monohydrate, dibasic sodium phosphate dihydrate, sucrose, and polysorbate 80.

Denosumab (PROLIA® and ) 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 . Denosumab is in Phase II trials for the treatment of rheumatoid arthritis.

Panitumumab is a fully hUman mAb approved by the FDA for ent of EGFR—expressing metastatic 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 prepared 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 uman mal 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 onal antibodies described in US. Patent No. 6,235,883. The term “panitumumab” es the active agent in biosimilar VECTIBIX® products, including biosimilar VECTIBIX® being developed by BioXpress, SA (“BioXpress”).

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

Belimumab is currently administered to lupus patients by IV infusion at a 10 mg/kg dosage. A high-molecular—weight, scosity protein formulation can include ' 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.

Abciximab (REOPRO®) is ctured 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 Willebrand factor, and other adhesive molecules. It also binds to vitronectin ) receptor found on platelets and vessel wall endothelial and smooth muscle cells. mab is a et aggregation inhibitor mainly used during and after coronary artery ures. 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 ent of follicular lymphoma.

It is an IgG2a anti—CD20 mAb derived from immortalized mouse cells. Tositumomab is administered in sequential infusions: cold mAb followed by iodine (1311) tositumomab, the same antibody covalently bound to the uclide -131.

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

Alemtuzumab (marketed as CAMPATH®, PATH®, or CAMPATH- lH® and currently under further development as LEMTRADA®) is a mAb used in the treatment of chronic cytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), and T-cell lymphoma. It is also used under clinical trial ols for treatment of some autoimmune 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. zumab (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 anti-CD20 mAb which appears to inhibit early-stage B lymphocyte activation. Ofaturnumab is marketed 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 non-Hodgkin’s lymphoma, Diffuse 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 l dose of 300 mg, ed by weekly infusions of 2,000 mg.

As usedherein, the term “ofatmnumab” includes the anti~CD20 mAb known by the ational Nonproprietary Name “OFATUMUMAB.” The term “ofatumuma ” includes the active agent in ts marketed under the trade name ARZERRA® 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 include 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 conjugate consisting ofthe mAb zumab linked to the cytotoxic agent sine (DIN/[163). Trastuzumab, described above, stops growth of cancer cells by g to the HERZ/neu receptor, whereas mertansine enters cells and destroys them by binding to tubulin. In the United States, zumab 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 concentration of about 144 mg/mL to about 360 mg/mL.

Pertuzumab (PERJETA®) is a mAb that inhibits HERZ dimerization.

Pertuzumab received FDA approval for the treatment of HERZ-positive metastatic breast cancer in 2012. The currently recommended dosage of umab 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 t rejection in organ transplantation, ally in kidney transplants. The drug is also under investigation for the treatment of multiple sclerosis. Daclizumab has a molecular weight of about 143 kDa. Daciizurnab was marketed in the US. by Hoffmann—La Roche, Ltd. (“Roche”) as ZENAPAX® and stered by IV infusion of 1 nag/kg.

Daclizumab ield 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 umab, preferably in a tration of about 40 mg/mL to about 300 mg/rnL.

Eculizumab (SOLIRIS®) is a humanized mAb approved for the treatment of rare blood diseases, such as paroxysmal nal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab, with a molecular weight of about 148 kDa, is being developed 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 receptor. it is an immunosuppressive drug, mainly for the ent of toid 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 e tocilizumab, preferably in a concentration of about 240 mg/mL to about 800 mg/mL.

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

Rituximab is jointly marketed in the US. under the trade name R1TUXAN® by Biogen and Genentech and outside the U.S. under the trade name MABTHERA® by Roche. RITUXAN® is buted in single-use vials containing 100 mg/10 mL and 500 mg/SO mL. N® is lly administered by IV infusion of about 375 . The term imab,” as used herein, includes 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 es the active agent in products ed under the trade name RITUXAN® and MABTHERA® and biosimilars thereof.

High-molecular—weight, low-viscosity liquid formulations can include mab, preferably in a concentration of about 475 mg/mL to about 875 mg/mL (approximated using a body surface 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 tol-Myers Squibb”). Marketed as YERVOY®, it is used‘for the treatment of melanoma and is also undergoing clinical trials for the treatment of non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), and metastatic hormone- refiactory prostate cancer. Ipilimumab is currently administered by IV infusion of 3 mg/kg. High—molecular—weight, low-viscosity liquid formulations can include ipilimurnab, preferably in a concentration of about 120 mg/mL to about 300 mg/mL.

Raxibacumab (ABthraX®) is a human mAb intended for the laxis 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, low-viscosity 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®, THERALoc®, CIMAher®) is a humanized mAb with a molecular weight of about 151 kDa used to treat us cell carcinomas of the head and neck, recurrent or refractory high—grade malignant glioma, anaplastic astrocytomas, glioblastomas, and diffuse intrinsic pontine glioma. Nimotuzumab is typically stered by IV infusion of about 200 mg weekly. High-'molecular-Weight, low—viscosity liquid formulations can include nimotuzumab, preferably in a tration of about 200 mg/mL.

Brentuximab vedotin (ADCETRIS®) is an antibody-drug ate directed to the n 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 developed by Biocon.

Itolizumab completed sful Phase III studies in patients With te to severe psoriasis. ltolizumab has received marketing approval in India; an application for FDA approval has not been ted.

Obinutuzumab A®), originally developed by Roche and being r developed under a collaboration agreement with Biogen is a humanized anti—CD20 mAb approved for treatment of chronic lymphocytic leukemia. It is also being investigated in Phase III clinical trials for ts With various mas. Dosages of about 1,000 mg are being administered via IV infusion.

Certolizumab pegol (CIMZIA®) is a recombinant, humanized antibody Fab’ fiagment, with specificity for human tumor necrosis factor alpha (TNFu), conjugated to an approximately 40kDa polyethylene glycol (PEGZMAL40K). The molecular weight of certelizumab pegol is approximately 91 kDa.

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

Antibody Therapeutics in Late~Srage Trials and Development The ssion 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 s. First marketing applications 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 planned or ting patients.

XBiotech, Inc. has sponsored two Phase I clinical trials ofMABpl ix) for patients with advanced cancer or type—2 diabetes. Additional trials of MABpl are ting patients. Multiple trials are sponsored by Medlmmune, LLC 7, - (“Medlmmune”) and underway or recruiting ts for the treatment of leukemia 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 recently completed for the use of rilotumumab for the treatment of various cancers.

At least 28 mAbs are high-molecular—weight ns tly in or having recently completed Phase III studies for the treatment of inflammatory or immunological disorders, cancers, high terol, osteoporosis, Alzheimer’s disease, and ious diseases. The mAbs in or having recently completed Phase III trials include AMG 145, elotuzumab, epratuzumab, farletuzumab (MORAb-003), gantenerurnab (RG1450), gevokizumab, inotuzumab ozogamicin, itolizumab, ixekizumab, lebrikizumab, mepolizumab, naptumomab estafenatox, necitumumab, mab, ocrelizumab, onartuzumab, racotumomab, rnmab, reslizumab, romosozumab, sarilmnab, secukinumab, sirukumab, solanezurnab, tabalumab, and vedolizumab. A mAb mixture (actoxumab and bezlotoxurnab) is also being ted in Phase III trials. See, e.g., Reichert, Mb: 5:1-4, 2013.

WO 38811 Vedolizumab is a mAb being developed by Millennium Pharmaceuticals, Inc (“Millennium”; a subsidiary of Takeda ceuticals Company, Ltd. (“Takeda”)).

Vedolizumab was found safe and highly effective for inducing and ining al remission in patients with moderate to severe ulcerative 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. Studies evaluating erm clinical outcomes show close to 60% of patients achieving clinical ion. A common dose of vedolizumab are 6 mg/kg by IV infusion. rumab is a human InAb being developed for the treatment of solid tumors. Phase III clinical trials are ongoing for the treatment of breast cancer, metastatic gastric adenocarcinoma, non-small cell lung cancer, and other types of cancer. Ramucirumab, in some Phase III trials, is administered at about 8 mg/kg via IV on.

Rilotumumab 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 rilotumumab 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 holesterolemia and acute coronary syndrome.

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

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

Other antibodies which may be formulated with viscosity-lowering ionic liquids include bococizumab (PF-04950615) and tanezumab; mab, blinatumomab, trebananib from Arngen; Anthrax immune globulin from Cangene ation; teplizumab from MacroGenics, Inc.; MK-3222, MRI-6072 from Merck & Co (“Merck"); girentuximah from Wilex AG; n fifom Navidea 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 (KW-6357) 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 stered 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 n-conjugated drugs or peptides that are also entering clinical 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 antibody developed jointly by Amgen and AstraZeneca and tly in Phase I trials for treatment of lupus. Likewise, AMG 729 is a zed 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 currently in in Phase II studies for the treatment of migraines and hot flashes, is a human mAb that inhibits Calcitonin Gene-Related e; AMG 780 is a human antiuangiopoietin mAb that inhibits the interaction between the endothelial cell-selective Tie2 or and its ligands Angl and Ang2, and recently completed Phase I trials as a cancer treatment; AMG 811 is a human monoclonal dy that inhibits interferon gamma being investigated as a treatment for systemic lupus erythematosus; AMG 820 is a human mAb that inhibits c-fms and decreases tumor associated hage (TAM) function and is being investigated as a cancer treatment; AMG 181, jointly ped 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 tive colitis and Crohn's disease.

Many mAbs are currently in clinical trials for the ent of autoimmune disorders. These mAbs can be included in low-viscosity, high-molecular-weight liquid formulations. RG7624 is a fully human mAb designed to cally and selectively bind to the human eukin-l7 family of cytokines. A Phase I clinical trial evaluating RG7624 for autoimmune disease is ongoing. BIIB033 is an anti- LINGO-l mAb by Biogen currently in Phase II trials for treating multiple sclerosis.

High-molecular-weight ns also can include AGS-009, a mAb targeting IFN—alpha ped by Argos Therapeutics, Inc. that recently 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 arthritis. 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 ly ted 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 treatment 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 rin—associated periodic syndromes. Canakinumab is-in Phase I trials as a possible treatment for chronic obstructive pulmonary disease, gout and ry artery disease. imumab is a human mAb designed for the treatment of rheumatoid arthritis. Discovered as CAM—3001 by Cambridge dy 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 currently stered by IV infusion. NN8209, a mAb blocking the C5aR receptor being developed by Novo Nordisk A/S( “Novo Nordisk”), has completed a Phase II dosing study for treatment ofrheumatoid arthritis. NN8210 is another antiCSaR mAb being developed by Novo WO 38811 Nordisk and currently is in Phase I trials. IPH2201 CNN8765) is a humanized mAb targeting NKG2A being ped by Novo Nordisk to treat patients with inflammatory conditions and autoimmune diseases. NN8765 ly 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 pathways. Olokizumab has completed Phase II trials for the treatment ofrheumatoid arthritis. Otelixizumab, also known as TRX4, is a mAb, which is being ped for the treatment of type 1 diabetes, rheumatoid arthritis, and other autoimmune diseases. Ozoralizumab is a humanized mAb that has completed Phase II trials.

Pfizer currently 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 humanized mAb being developed by Genentech. It recently completed Phase II trials for the ent 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 ent of lupus.

A high-molecular-weight low-viscosity liquid formulation can e one of the mAbs in early stage clinical development for treating various blood disorders. For example, rnab (BENLYSTA®) has recently completed Phase I trials for patients with vasculitis. Other mAbs in early—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. xys”).

One or more mAbs in early-stage development for treating various cancers and related conditions can be included in a low-viscosity, 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, uzumab, and volociximab from Abeie are in stage development. Actinium Pharmaceuticals, Inc has conducted early-stage trials for the mAbs b-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, including anti-CD22 ADC (RG7593; pinatuzumab vedotin), anti-CD79b ADC (RG7596), TEAPI 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, anti-CD22 ADC (RG7593), anti—EGFL7 mAb (RG7414), anti-HER3/EGFR DAF InAb (RG7597), anti—PD-Ll mAb (RG7446), DFRF4539A, an MINT1526A. Bristol-Myers Squibb is developing early—stage mAbs for cancer therapeutics, ing 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 Pharmaceuticals, Inc. (“AVEO”), AVX701 and AVX901 from AlphaVax, BAX-69 from Baxter International, Inc. (“Baxter”), BAY 79-4620 and BAY 20-10112 from Bayer HealthCare AG, BHQ880 from Novartis AG, 212- Ctrastuzumab 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 liquids include alzumab, GAIOI, daraturnurnab, siltuxirnab, ALX—006l, ALX- 0962, ALX~0761, bimagumab 8), CT—Oll (pidilizumab), actoxumab/bezlotoxumab (MK-3515A), 75 (pembrolizumab), dalotuzumab (MK-0646), icrucurnab (IMO-18F 1, LY3012212), AMG 139 (MED12070), SAR339658, dupilumab (REGN668), SAR156597, SAR256212, 356, SAR3419, SAR153192 (REGN421, enoticumab), SAR307746 curnab), SAR650984, SAR566658, 786, SAR228810, SAR252067, SGN-CDIQA, SGN-CD3 3A, SGN—LIVIA, ASG 15MB, Anti-LINGO, BIIB037, ALXN1007, teprotumurnab, concizumab, anrukinzumab (IMA-63 8), mab (PF-04360365), PF-03446962, PF-062526l6, etrolizumab (RG7413), quilizurnab, ranibizurnab, lampalizumab, onclacumab, gentenerumab, crenezurnab (RG7412), IMC-RONS (namatumab), tremelimumab, turnab, eemcizumab, ozanezumab, mapatumurnab, tralokinumab, 71, XmAb7 195 , cixutumumab 22 1 7), LY2541546 (blosozumab), olaratumab 2207), MEDI4893, MEDIS73, MED10639, MEDI3 617, MEDI4736, MEDI6469, MEDIO680, MEDIS872, PF- 05236812 (AAB-003), PF-05082566, BI 1034020, RG7116, RG7356, RG7155, RG7212, RG7599, RG7636, RG7221, RG7652 (MPSK3169A), RG7686, HuMaX- HuMaXTFADC, MOR103, BT061, MORZOS, OMP59R5 (antimotch 2/3), VAY736, MOR202, BAY94-9343, LJM716, OMP52M51, GSK933776, GSK249320, GSK1070806, NN8828, CEP-37250/KHK2804 AGS—16M8F, AGS—16C3F, 859, LY2495655, LY2875358, and LY2812176.

Other early stage mAbs that can be formulated with Viscosity-lowering ionic liquids include benralizumab, MEDI-8968, anifrolumab, MEDI7183, mumab, MEDI—575, tralokinumab from AstraZeneca and Medlmmune; BAN2401 from Biogen Idec/Eisai Co. LTD ("Eisai”)/ BioArctic cience AB; CDP7657 an anti- CD4OL monovalent pegylated 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 International/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 lI;-t-igatuzumab from Daiichi Sankyo y 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 Genentech; fresolimumab from e & Sanofi; (38-6624 (simtuzurnab) from ; CNTO— 328, bapineuzurnab (AAB—OOl), carlumab, CNTO—136 from Janssen; KB003 from KaloBios Pharmaceuticals, Inc.; 40 from Kyowa; RN—307 from Labrys Biologics Inc.; ecromeximab from Life Science Pharmaceuticals; LY2495655, LY2928057, 014, LY2951742 from Eli Lilly; MBL-HCV] from MassBiologics; AME—l3 3v from MENTRIK Biotech, LLC; abituzurnab from Merck KGaA; MM-121 from ack Pharmaceuticals, Inc.; MCSl 10, QAX576, QBX258, QGE031 from is AG; HCD122 from Novartis AG and XOMA Corporation ("XOMA”); NNSSSS from Novo Nordisk; ximab, cotara from Peregrine Pharmaceuticals, Inc.; PSMA—ADC from Progenics Pharmaceuticals, Inc.; oregovomab from Quest Pharmatech, Inc.; mab (REGN475), REGN1033, SAR231893, REGN846 from Regeneron; RG7160, CIM331, RG7745 from Roche; ibalizumab 55) from TaiMed Biologics Inc.; TON-032 from lone Sciences; TRC105 from TRACON Pharmaceuticals, Inc; UB—421 from United Biomedical Inc; VB4-845 from Viventia Bio, Inc.; ABT—l 10 from Abeie; Caplacizumab, 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.; anti-fibrin 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 KaloBios Pharmaceuticals/Sanofi; KRN—23 from Kyowa.; Y—90 hPAM 4 from Immunomedics, Inc.; Tarextumab from sys 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, GSK2618960, GSK1223249, 'GSK933776A from GlaxoSmithKline; anetumab ravtansine fi'orn Morphosys AG & Bayer AG; BI—836845 from Morphosys AG & nger Ingelheim; NOV-7, NOV- 8 from Morphosys AG & Novartis AG; MM-302, MM-310, MM-Ml, , MM—151 from Merrimack, RG7882 from Roche & Seattle Genetics; RG7841 from Roche/ Genentech; PF-06410293, PF-06438179, PF-0643 9535, PRO-4605412, PF- 05280586 from Pfizer; RG7716, RG7936, gentenerurnab, RG7444 from Roche; 47, MEDI-565, MEDI1814, MEDI4920, MED18897, MEDI—4212, MEDI- 51 17, MEDI—78 14 from Astrazeneca; ulocuplumab, PCSK9 adnectin from l- Myers ; , FPA145 from irne Therapeutics, 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; ALX-0141, ALX-0171 from AblynX; milatuzurnab-DOX, milatuzumab, TF2, from Immunomedics, Inc.; MLN0264 from Millennium; ABT-981from Abeie; AbGn—168H from AbGenornics ational Inc.; ficlatuzumab from AVEO; BI-SOS from BioInvent International; 27, 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; 3, IMGN529 from ImmuncGen Inc. ; CNTO-S, CNTO—5825 from Janssen; KD-247 from Kaketsuken; KB004 from KaloBios Pharmaceuticals; , MGAH22 from MacroGenics, Inc.; XmAb5574 from MorphoSys AG/Xencor; ensituximab (NFC-1C) from ix Oncology, Inc.; LFA102 from Novartis AG and XOMA; ATI355 from Novartis AG; SAN-300 from Santarus Inc.; SelGl from Selexys; HuM195/rGel from Targa Therapeutics, Corp; VX15 from Teva Pharmaceuticals, Industries Ltd. (“Teva”) and Vaccinex Inc.; TCN—202 from Theraclone Sciences; XmAb2513, 72 from Xencor; XOMA 3AB from XOMA and National Institute for Allergy and ious Diseases; neuroblastorna antibody vaccine from MabVax Therapeutics; Cytolin from CytoDyn, Inc.; Thravixa fiom nt BioSolutions Inc.; and FE 301 from Cytovance Biologics; rabies mAb from Janssen and Sanofi; flu mAb from n and partly funded by National Institutes of Health; 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 plegylated protein, vaccine or otherwise a biologically active protein (or n 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 desired 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 le target molecules to the desired products. In n embodiments, the protein can be PEGylated, as defined herein.

The term n protein,” as used herein, refers to a protein that is created from two different genes encoding for two separate ns. Fusion proteins are generally produced h recombinant DNA techniques known to those skilled in the art. Two proteins (or protein 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 itively inhibits TNF.

ELOCTATE®, Antihemophilic Factor (Recombinant), Fc Fusion Protein, 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 management, routine prophylaxis to t or reduce the frequency of bleeding es.

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 (aflibercept) is a recombinant fusion protein consisting of ns ofhuman VEGF receptors 1 and 2 extracellular domains fused to the Fe n of human IgG1 formulated as an iso-osmotic solution for intravitreal administration. Afiibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation, constituting an additional 15% of the total molecular mass, resulting in a total molecular weight of 115 kDa. Aflibercept is produced in inant 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 ng episodes, perioperative management, routine prophylaxis to prevent or reduce the ncy of bleeding episodes.

Pegloticase (KRYSTEXXAQQ) is a drug for the treatment of severe, treatment- refractory, chronic gout, ped by Savient Pharmaceuticals, Inc. and is the first drug approved for this indication. Pegloticase is a pegylated inant 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, scosity liquid ations can include pegloticase, preferably in a concentration of about 300 mg/mL to about 800 mg/mL.

Alteplase (ACTIVASE®) is a tissue plasminogen activator ed by recombinant DNA technology. It is a purified glycoprotein comprising 527 amino acids and synthesized using the mentary DNA (cDNA) for natural human -type plasminogen activator obtained from a human melanoma cell line. ase is administered via IV on of about 100 mg immediately following 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 ofPompe disease (glycogen storage disease type II), a rare lysosomal storage 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 scosity pharmaceutical formulation of alglucosidase alfa is provided, preferably with a concentration 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 treatment of severe combined immunodeficiency disease (SCID) associated with a deficiency of adenosine deaminase. Pegdamase bovine is a conjugate of numerous strands ofmonomethoxypolyethylene glycol (PEG), molecular weight 5,000 Da, covalently attached to adenosine ase enzyme that has been derived from bovine intestine. o-Galactosidase is a lysosomal enzyme that catalyses the hydrolysis of the ipid, riaosylceramide , to galactose and ceramide dihexoside.

Fabry disease is a rare inheritable lysosomal storage e characterized by subnormal enzymatic activity of u-Galactosidase and resultant accumulation of GL-3.

Agalsidase alfa (REPLAGAL®) is a human d-galactosidase A enzyme produced by a human cell line. Agalsidase beta (FABRAZYME®) is a inant 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 mal enzymes can also be used. For e, the protein can be a lysosomai enzyme as described in US 2012/0148556.

Rasburicase (ELITEK®) is a inant urate-oxidase indicated for initial management ofplasma uric acid levels in pediatric and adult patients with ia, lymphoma, and solid tumor malignancies who are receiving anti-cancer therapy expected to result in tumor lysis and uent elevation of plasma uric acid.

ELITEK® is administered by daily IV infusion at a dosage of 0.2 mg/kg.

Imiglucerase (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. CEREZYME® isadministered by IV infusion.

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

Taliglucerase alfa (ELEYSO®) is a hydrolytic lysosomal glucocerebroside— specific enzyme indicated for long-term enzyme ement 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 t of the human enzyme d—L—iduronidase that is ed 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 in 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 ated With viscosity-lowering ionic liq- uids include asparaginase erwinia chrysanthemi AZE®), incobotulinumtoxin A (XEOMIN®), EPOGEN® (epoetin Alfa), T® (epoetin Alta), P®' (darbepoetin alfa), ORENCIA® (abatacept), BATASERON® (interferon beta-1b), NAGLAZYME® (galsulfase); ELAPRASE® (Idursulfase); E® (LUMIZYME®, algucosidase alfa); VPRJV‘:D (velagiucerase), abobotulinumtoxin A (DYSPORT®); 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; eptin from Bristol-Myers Squibb; Avonex, Plegridy (BIIBOl7) from Biogen; NN1841, NN7008 from Novo Nordisk; KRN321 (darbepoetin alfa), AMG531 (romiplostim), KRN125 (pegfilgrastim), KW—076l (mogamulizumab) from Kyowa; IB 1001 from Inspiration Biophannaceuticals; lprivask from Canyon Phannan ceuticals Group.

Protein eutics in Development Versartis, 's VRS—3 17 is a recombinant human growth hormone (hGH) fusion protein 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 planned.

Vibriolysin is a proteolytic enzyme secreted by the Gram-negative marine rganism, Vibrio proreolyticus. This endoprotease has specific affinity for the hydrophobic regions ofproteins and is capable of cleaving proteins adjacent to hobic amino acids. Vibriolysin is currently being investigated by BioMarin for the ng and/or treatment of burns. Vibriolysin formulations are described in patent W0 014.

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

Other protein eutics which may be formulated with Viscosity-lowering ionic liquids include ixf rFlXFc, Eloctate/ Fc, 0; BMN—250; Lamazyme; Galazyme; ZA-Ol I; Sebelipase alfa; SEC-103; and HGT-11 10.

Additionally, fusion-proteins ning the XTEN half-life ion technology including, but not limited to: VRS~317 GH-XTEN; Factor VIIa, Factor VIII, Factor IX; PF05280602, VRS-859; Exenatide-XTEN; 6; 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 formulated with Viscosity- lowering ionic liquids include CM—AT from CureMark LLC; NN7999, NN7088, Liraglutide (NNS022), NN9211, Semaglutide (NN953 5) from Novo Nordisk; AMG 386, Filgrastim from Amgen; CSL-654, Factor VIII from CSL Behring; 006 (pegfilgrastim biosimilar) from Novartis AG; Multikine (leukocyte interleukin) from GEL-SCI Corporation; LY2605541, Teriparatide binant 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. ; BMN-llO, BMN—702 from BioMarin; NGR—TNF from Molmed S.p.A.; recombinant human Cl esterase inhibitor from Pharming Group/Santarus Inc.; opin biosimilar from LG Life Sciences LTD; Natpara from NPS Pharmaceuticals, Inc; ART123 from Asahi Kasei Corporation; BAX-111 from Baxter; OBI—1 from Inspiration Biopharmaceuticals; Wilate from Octapharma AG; Talactoferrin alfa from Agennix AG; Desmoteplase from Lundbeck; Cinryze from Shire; RG7421 and Roche and Exelixis, Inc; Midostaurin (PKC412) fiom Novartis AG; tocog alfa pegol, BAY 86—6150, BAY 94-9027 from Bayer AG; Peginterferon lambdad a, Nulojix (Belatacept) from l-Myers Squibb; Pergoveris, Corifollitropin alfa (MK-8962) from Merck KGaA; recombinant coagulation Factor IX Fc fusion protein (rFIXFc; BIIB029) and recombinant coagulation Factor VIII Fc fusion protein (rFVIIIFc; BHB031) from Biogen; and Myalept from AstraZeneca.

Other early stage protein biologics which can be formulated with viscosity- lowering water ionic s include Alferon LDO from Hemispherx BioPharma, Inc.; SL-401 from Stemline Therapeutics, Inc; PRX-102 fiom Protalix rapeutics, Inc; KTP—OOl from Kaketsuken/Teijin Pharma Limited; Vericiguat from Bayer AG; BMN—l i 1 from BioMarin; ACC-OO] 236806) from Janssen; LY2510924, LY2944876 from Eli Lilly; NN9924 from Novo Nordisk; INGAP peptide from Exsulin; ART-122 from Abbvie; AZD9412 from AstraZeneca; NEUBLASTIN (BGOOOlO) from Biogen; Luspatercept (ACE—536), Sotatercept 11) from Celgene ation; PRAME immunotherapeutic from GlaxoSmithKline; er acetate (PI-2301) from Merck KGaA; PREMIPLEX (607) from Shire; BMN-701 from BioMarin; Ontak from Eisai; rHuPH20/insu1in from me, Inc; 3 from PhaseBio Pharmaceuticals, Inc; ALV-003 from Alvine Pharmaceuticals Inc. and Abbvie; NN8717 from Novo Nordisk; PRT-201 from Proteon Therapeutics Inc; PEGPH20 from Halozyme, Inc; Amevive® ept from Astellas Pharma Inc; F- 627 from Regeneron; AGN—214868 (senrebotase) from Allergan, Inc; 7 from Baxter; PRT4445 from Portola Pharmaceuticals, Inc; VENl 00 from Ventria Bioscience; Onconase/ ranpirnase from Tamir Biotechnology Inc; interferon 2b infilsion from Medtronic, Inc; sebelipase alfa fiom Synageva BioPharma; IRX-2 from IRX Therapeutics, Inc; 6881 from mithKline; 81-6603 from Seikagaku Corporation; ALXNI 101, asfotase alfa from Alexion; SHP611, SHP609 ase, idursulfase) from Shire; PF-04856884, PF-05280602 from Pfizer; ACE- 031, Dalantercept from Acceleron Pharma; ALT-801 from Altor BioScience Corp; BA-210 from BioAxone Biosciences, Inc; WTl immunotherapeutic from GlaxoSmithKline; (32402666 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 Biogen; BMN—190, BMN-270 from BioMarin; ACE- 661 from Aceeleron Pharma; 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 Therapeutics, Inc.; ATX-MS—1467 from Merck KGaA; XTEN fusion proteins from Amunix ing 1110.; entolimod (CBLBSOZ) fiom Cleveland BioLabs, Inc.; HGT2310 from Shire; HM10760A from Hanmi Pharmaceuticals Co., LTD; ALXNl 102/ ALXN1103 from Alexion; CSL~689, CSL-627 fiom CSL Behring; glial growth factor 2 from Acorda Therapeutics, Inc.;NX001 from Nephrx Corporation; NN8640, , NN1953, NN9926, NN9927, NN9928 from Novo Nordisk; NHS— IL 12 from EMD ; 3K3A—APC from 22 Biotech LLC; PB-1046 from PhaseBio Pharmaceuticals, 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.; PF-04856883, 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 International; TT»173 from Thrombotargets Corp; QBI-l39 from Quintessence Biosciences, Inc.; Vatelizumab, GBRSOO, GBR600, , and GBR900 from Glenmark Pharmaceuticals; and CYT—6091 from Cytimmune 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), 63507, PF-05230907, Dekavil, PF-06342674, PF06252616, RG7598, , RG7624d, OMP54F28, GSK1995057, BAY1179470, IMO-3G3, IMC-l 8F1, IMO-35C, IMC- 20D7S, PF~06480605, PF-06647263, PF—06650808, 35810 (RN317) PD— 03 60324, PF-00547659 from Pfizer; MK-8237 fiom Merck; 81033 from Biogen; 65, SAR43 8584/ REGN2222 from Sanofi; lMC-18F1; and Icrucumab, IMC- 3G3 from ImClone LLC; Ryzodeg, Tresiba, Xultophy from Novo Nordisk; Touje0 (U300), LixiLan, ia (lixisenatide) from Sanofi; MAGE~A3 immunotherapeutic from GlaxoSmithKline; tide from Merck KGaA; Sereleaxin (RLX030) from Novartis AG; opoietin; Pegfilgrastim; LY2963016, Dulaglutide 2965) from Eli Lilly; and Insulin Glargine from Boehringer eim.

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 Newtonian fluids by the addition of an effective amount of one or more ity~reducing ionic liquids.

Ionic Liquid Salts The ionic liquid can be a salt. Representative ionic liquid salts include salts with imidazolium cations, including N,N-dialkyl-irnidazoliums. Ionic liquids e salts with N—alkyiated unsaturated or saturated nitrogen-containing heterocyclic cations, including N-alkylpyridinium salts, lpyrrolidinium 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, heteroalkyi, 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 c tuents include halide ions, sulfate, sulfonate, sulfite, sulfinate, phosphate, phosphonate, ite, phosphonite, carbonate, and carboxylate anions ally 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 heterocyclic group can be saturated or unsaturated. Saturated cationic heterocyclic groups include pyrrolidinium, oxazolidinium, piperidinium, piperazinium, liniurn, thiomorpholinium, and azepanium groups, and the like.

Unsaturated cationic heterocyclic groups include pyrrolinium, imidazolinium, 1,2,3- triazolium, triazolium, thiazolium, 1,2,4-dithiazolium, 1,4,2—dithiazolium, tetrazolium, linium, oxazolinium, pyridinium, and azepinium groups, and the like. The ic 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, including hydroxyl and substituted and tituted 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 l~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, ing one or more carbons with a atom, replacing the N-butyl or N—methyl group with one or more 'higherworder N—alkyl groups, attaching additional tuents to one or more carbon atoms, or a combination thereof. Exemplary anionic constituents are described above.

Exemplary atoms include N, O, P, and S. Exemplary higher-order l 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 er—order l groups include N—ethyl, anropyl, N-butyl, N-Sec- butyl, and N~tert-butyl. Additional substituents can include hydroxyl and substituted and unsubstituted alkoxy, heteroalkoxy, alkyl, aryl, aralkyl, 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, replacing the N,N-butyl-Inethyl group with one or more higher- 2014/055245 order N,N—dialkyl groups, attaching one or more additional substituents to a carbon atom, or a combination thereof. Exemplary anionic constituents include those described above. Exemplary heteroatoms include N, O, P, and S. Exemplary higher- order alkyl groups include linear, branched, and cyclic l and N- heteroalkyl groups ning 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— ; ropyl-N-methyl; N—butyl-N—methyl; N,N—diethyl; N—ethyl-N-isopropyl; N,N—diisopropyl groups, and the like. Additional substituents can include hydroxyl, and substituted and unsubstituted alkoxy, heteroaikoxy, alkyl, heteroalkyl, aryl, aryloxy, aralkyl, loxy, 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, halide, hydroxyl, and substituted and unsubstituted alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryi, aryloxy, aralkyl, araikyloxy, alkenyl, and alkynyl 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. ia I R2 may also be ndently selected from hydrogen, R1, -OH, NHz, ~F, -Cl, - Br, -I, -N02, -CN, -C(=O)R4a, -C(=NR4a)R4, 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, - WO 38811 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, C3_1g_cycloa1ky1, C6- Igaryl, C1_Igheteroary1 and C2_12heterocyclyl, wherein each C1_Izalky1 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, R4a 2, , -SO;NR4aC(=O)R4, -PO3H2, -R4aC(=NR4a)N(R4a 2, -NHC(=NR4a)NH-CN, —NR4aC(=O)R4, ~NR4aso2R4, - NR4HC(=NR4“)NR4EC(=NR43)N(R43 2, -NR4"C(=O)N(R43 2, -C(=0)NH2, — (R4a 2, OR“, -SR“3, or -N(R43 2; wherein each C3_ucycloalkyl may be substituted one or more times with C1- lgaikyl, C5_12ary1, C1-1gheteroaryl, C2_12heterocyclyl, -OH, NH2, -F, -Cl, -Br, -I, ~N02, - CN, -C(:O)R4a, -C(:NR4‘1)R4, —C(:0)0H, —C(=0)0R4, -OC(=O)R4, -0C(=0)0R4, - s02H, -so2N(R4a)2, -so2R4, -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, _—.

C(:O)N(R4a 2, -OR4, -SR““, or -N(R4a 2; wherein each C5_12aryl may be tuted one or more times with C1_12alky1, Cgiucycloalkyl, C1.1zheteroaryl, eterocyclyl, -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, 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, R4a 2, , 4aC(=O)R“, , 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; 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, )OR4, - so2H, -so2N(R4a 2, —so2R4, -so2NR4aC(=0)R4, -Po2H2, _We(=NR4a)1~~I(1r*a 2, _ NHC(=NR4a)NH-CN, -NR4aC(=O)R4, -NR4aso2R4, — NR4aC(=NR4a)NR4aC(=NR43)N(R4a 2, (=0)N(R4a 2, —C(:O)NH2, — C(=0)N(R4a 2, -OR4, sat: or -N(R4a)2; R4 is ndently 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 — 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 contains a cationic tuent 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 ).

The ionic liquid may also contain a cationic constituent having the structure according 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 2014/055245 structure according to Formula IV: RZQMR‘ a? a? a (IV) wherein R1 and R2 are as defined above.

In some embodiments, the ionic liquid ns a cationic constituent having a ure according to any one of Formulas 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 ed to the same atom and taken together, each R10 and R10” is =0 or together with the atom to which they are attached form a carbocycle or cycle having from 2 to 30, preferably from 3 to 12 carbon atoms; so long as at least one occurrence 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 methoxy, ethoxy, and butoxy groups.

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

T\Rl° Rm”? ° 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 Formula VIII a 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, , and substituted and unsubstituted alkoxy, heteroalkoxy, alkyl, heteroalkyl, aryl, aryloxy, l, 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 ed form a carbocycle or heterocycle having from 1 to 30, preferably from 3 to 12 carbon atoms. In preferred embodiments at least one occurrence of R10 or R10” has at least two, at least three, at least four, or at least five carbon atoms. Exemplary alkyl groups include ethyl, propyl, butyl, hexyl, octyl, and decyl groups, as well as isomers thereof. Exemplary heteroalkyl groups include cyanobutyl and cyanopropyl groups. Exemplary alkoxy groups include methoxy, ethoxy, and butoxy groups.

The ionic liquid can be an ammonium salt: ”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 hydrogen 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 ments, the ionic liquid contains a cationic constituent having a ure according to Formula XI where Ar is a substituted or unsubstituted benzyl group; where R12 is a tuted or unsubstituted C1-C12 alkyl group, or both.

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

Air—sit’L-rst—Fz‘3 Formula XI The ionic liquid can be a phosphonium salt. In some embodiments, the ionic liquid contains a cationic constituent having a structure according to Formula XII Where each occurrence of R14 is independently 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 ence 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 ce of at least one group selected from - COOH, -SOgH and -PO3H2.

Formuia XII In some embodiments, the ionic liquid contains a cationic tuent 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 hydrogen and substituted and unsubstituted aikoxy, aikoxy, alkyi, heteroalkyl, aryl, y, aralkyl, aralkyloxy, aikenyi, 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 ments, the ionic liquid contains a cationic constituent having a structure accordingito Formula XIII Where Ar is a substituted or unsubstituted benzyl group; where R15 is a tuted 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 ing to Formula XV: R? R2 Formula XV wherein R1 and R2 are as defined above, and X may be 0, S, 802, NR1 or .

The ionic liquid can be an imidazolium salt such as l-ally1 methylimidazolium bis(trifluor0methylsulfonyl); l-allyl—3 -methylirnidazolium bromide; 1-ally1n1ethylirnidazolimn de; l-allyl«methylimidazolium dicyanamide; 1-allylmethylimidazolium iodide; l-benzylmethylimidazolium chloride; l-benzyl-3 -rnethylirnidazoliurn hexafluorophosphate; l—benzyl—3- methylimidazolium tetrafluoroborate; 1,3-bis(cyanomethyl)imidazolium bis(trifluoromethylsulfonylfimide; 1,3-bis(cyanomethyl)irnidazolium chloride; 1- butyl-2;3 -dimethyli1nidazoliun1 chloride; 1-butyl-2,3-dimethyli1nidazolium hexafluorophosphate; 1-butyl-2,3-dimethylimidazolium tetrafluoroborate; l-buty1 imidazolium acetate; 1-butyl—3—rnethyli1nidazolium bis(trifluoromethylsulfony1)imide; 1-butylmethyli1nidazolium bromide; 1-butyl—3- methylimidazoliurn chloride; 1~butyl—3—methylimidazolium dibutyl ate; 1- butyl-3~methylimidazolium dicyanamide; l-butylmethylirnidazolium hexafluoroantimonate; l-butyl1nethylimidazolium hexafluorophosphate; 1—butyl~3~ methylimidazolium en sulfate; l—3-methylirnidazolium iodide; 1—butyl methylimidazolimn methanesulfonate; l-‘outyl-«3 ~methyl-imidazolium methyl carbonate; l—butylmethylimidazolium methyl sulfate; l methylimidazolium nitrate; l-butylmethylimjdazoliu1n octyl e; 1~butyl~3~ methylimidazolium tetrachloroaluminate; 1—butylmethylimidazolium tetrafluoroborate; l-butylmethyli1nidazoliurn thiocyanate; 1-butyl-«3- methylimidazolium tosylate; 1-butylmethylimidazolium trifluoromethanesulfonate; 1—(3 -cyan0pr0pyl)—3-methylimidazolium rifluoromethylsulfonyl)amide; 1~(3 ~ cyanopropyl)—3-methylimidazolium chloride; 1-(3-cyanopropyl) methylimidazolium dicyanamide; l-decyl—3-1nethyli1nidazolium; l-decyl methylimidazoliurn tetrafluoroborate; 1,3-diethoxyirnidazolium bis(trifluoromethylsulfonyl)imide; 1,3-diethoxyimidazolium hexafluorophosphate; 1,3-dihydroxyimidazolium bi3(uifiuoromethylsulfonyl)imide; 1,3-dihydroxy methylimidazolium ifluoromethylsulfonylfimide; 1,3-dimethoxyimidazolium bis(trifluoromethylsulfonyl)imide; methoxyimidazolium 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 bis(trifluoromethylsulfonyl)imide; 1-dodecyl-3~methylimidazolium iodide; l-ethyl- 2,3-dimethylimidazoliurn tetrafluoroborate; 1-ethyl-2,3-dimethylirnidazolium chloride; l-ethyl-2,3-dimethylimidazolium ethyl e; 1-ethyl-2,3- ylimidazoliom hexafluorophosphate; l~ethyl—3-methylimidazolium acetate; 1rnethylimidazolium aminoacetate; 1-ethyl-3—methylirrfidazolium (S)—2- aminopropionate; l-ethyl—3-rnethylimidazolium bis(pentafluoroethylsulfonyl)imide; 1-ethy1rnethylimidazolium bromide; l-ethyl—3—methylimidazolium chloride; 1- ethyl—3 -n1ethylirnidazolium 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 ; l-ethyl~3-methylimidazolium L-(+)-lactate; l-ethyl—S- methylimidazolium methanesulfonate; 1-ethyl-3—methylimidazolium methyl sulfate; l-ethylmethylimidazolium nitrate; 1-ethy1methylimidazolium tetrachloroaluminate; lwethyl—3—methylimidazoliurn tetrachloroaluminate; l-ethyl-3— methylimidazolium tetrafluoroborate; 1—ethy1—3-methylimidazolium 1,1,2,2- tetrafluoroethanesulfonate ; 1-ethylmethylirnidazolium anate; 1—3- imidazolium tosylate; l—ethyl—3-methylimidazolium romethanesulfonate; 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; 1-methylimidazolium chloride; 1— methylirnidazolium en sulfate; 1-methyloctylimidazoliurn chloride; 1- methyloctylirnidazolium hexafluorophosphate; 1-methyloctylimidazolium tetrafluoroborate; yloctylimidazolium trifluoromethanesulfonate; 1-methyl- ylimidazolium iodide; l-methyl-3—propylimidazolium methyl carbonate; 1,2,3- trimethylimidazolium methyl sulfate; derivatives thereof and combinations thereof.

Derivatives can include substituting the anionic constituent for other anionic constituents, replacing one or more carbons with a heteroatom, replacing an N—alkyl group with one or more higher-order N—alkyl groups, or a combination f.

Exemplary anionic constituents and atoms are described above. Exemplary higher—order N—alkyl groups can include linear and branched N—alkyl and N- heteroalkyl groups containing from 1 to 30 carbon atoms, preferably from 2 to 12 carbon atoms. Examples of higher-order N—alkyl groups include N—ethyl, N—propyl, N—iospropyl, N—butyl, N—Sec-butyl, and -butyl.

The ionic liquid can be a pyrrolidinium salt suoh as l-butyl- l - pyrrolidiniurn bis(trifluoromethylsulfonyl)imide; i-butyl-l- methylpyrrolidinium bromide; l-butyl—1-methylpyrrolidinium chloride; l-butyl-l- methylpyrrolidinium dicyanamide; l —butylInethylpyrrolidiniurn orophosphate; 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; lmethylpyrrolidinium tetrafluoroborate; derivatives thereof and ations thereof. Derivatives can include substituting the anionic constituent for other anionic constituents, replacing one or more carbons With a heteroatom, replacing an N—alkyl or N~methyl group with one or more higher-order N-alkyl groups, or a combination thereof. ary anionic constituents, heteroatoms, and higher-order N—alkyl groups are described above. rionic 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 tive 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 , attaching additional substituents to one or more carbon atoms, or a combination thereof.

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

Exemplary heteroatoms and higher—order N—alkyl groups are described above.

Additional substituents can include yl, 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 embodiments, the ionic liquid is a rion containing a cationic heterocyciic substituent and an anionic substituent connected by a substituted or unsubstituted alkyl, heteroaikyl, aryl, aralkyl, alkenyl, or alkynyl group having from 2 to 50 carbon atoms, from 3 to 30 carbon atoms, or from 4 to 12 carbon atoms. The cationic heterocyclic substituent can be saturated or unsaturated. Examples include pyrrolidinium, oiinium, oxazolidiniurn, piperidinium, piperaziniurn, morpholinium, thiornorpholinium, azepanium, pyrrolinium, 1,2,3-triazolium, 1,2,4- triazolium, lium, 1,2, 4-dithiazolium, 1,4,2-dithiazolium, tetrazolium, linium, oxazolinium, pyridinium, and azepinjum groups. The cationic heterocyclic substituent can be a fused ring structure having two or more fused rings.

WO 38811 The ic heterocyclie substituent can be a bicyclic cationic heterocycle, such as benzoxazolium, benzothiazoliurn, riazoliurn, idazoliurn, and indolium.

The cationic heterocyclic substituent can additionally be substituted with one or more additional tuents. Exemplary anionic substituents include sulfate [-0803'], ate [-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'], alkylphosphite {-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 zwitterion 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 Formula 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 n R1, R2 and R3 are as defined above, provided that the compounds of a XVI, XVII, XVIII, XVIV, XX and XXI each contain at least one ~COOH, - SO3H, or -P03H2 substituent.

C. Excipients A Wide variety ofpharmaceutical excipients useful for liquid protein ations are known to those skilled in the art. They include one or more additives, such as liquid solvents or co-solvents; sugars or sugar alcohols such as mannitol, trehalose, sucrose, sorbitol, se, maltose, lactose, or dextrans; surfactants such as TWEEN® 20, 60, or 80 (polysorbate 20, 60, or 80); buffering agents; preservatives such as benzalkonium chloride, benzethoniurn chloride, tertiary ammonium salts, and 2014/055245 chlorhexidinediacetate; carriers such as poly(ethylene glycol) (PEG); antioxidants such as ascorbic acid, sodium sulfite, and methionine; chelating agents such as EDTA or citric acid; or biodegradable polymers such as water soluble polyesters; cryoprotectants; lyoprotectants; bulking agents; and izing agents.

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

The viscosity—lowering agents described herein can be combined with one or more other types of ity—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 nds, such as hydrophobic compounds, many ofthe GRAS (US Food and Drug Administration List of compounds Generally Regarded As Safe) and inactive able ingredients and FDA approved therapeutics, described in d 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 formulated may be produced by any known technique, such as by culturing cells transformed or transfected with a vector containing one or more nucleic acid ces encoding the protein, as is well known in the art, or through synthetic techniques (such as recombinant techniques and e synthesis or a combination ofthese techniques), or may be isolated from an endogenous source ofthe n.

Purification of the protein to be formulated may be conducted by any suitable que known in the art, such as, for example, ethanol or ammonium sulfate precipitation, reverse phase HPLC, chromatography on silica or cation-exchange resin (e.g., DEAE-cellulose), dialysis, chromatofocusing, gel filtration using protein A SE- SEPHAROSE® s (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, preferably 0.050 M to 0.50 M, most ably 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 limited to tangential flow filtration, centrifugal concentration, and dialysis.

In some embodiments, lized formulations ofthe proteins are provided and/or are used in the preparation and manufacture of the low-viscosity, concentrated protein ations. 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, preservatives, - antioxidants, and other l phannaceutically acceptable excipients, then stored under sterile storage ions 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 ations for storage include freezing, lyophilizing, and spray drying the liquid protein formulation. 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. miting examples of ts useful for reconstituting a lyophilized formulation prior to injection include sterile water, bacteriostatic water for ion (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), e 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 ations, including, but not limited to, reconstituted formulations, are administered to a person in need thereof by intramuscular, intraperitoneal (i.e., into a body cavity), erobrospinal, 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 protein, such as a mAb, will depend on the condition to be treated, the ty and course of the disease or condition, Whether the protein is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the protein, the type of protein used, and the discretion of the attending physician. The protein is suitably administered at one time in single or multiple injections, or over a series of ents, as the sole treatment, or in conjunction with other drugs or therapies.

Dosage formulations are designed so that the injections cause no cant signs of irritation at the site of injection, for example; wherein the primary irritation index is less than 3 when evaluated using a Draize scoring . In an alternative embodiment, the injections cause macroscopically similar levels of irritation when compared to ions 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 imately as effective pharmaceutically as about the same dose of the protein administered by intravenous infusion.

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) stered in the same way.

The viscosity—lowering agent may also affect bioavailability. For example, the percent ilability of the protein may be at least 1.1 times, preferably at least 1.2 times the percent bioavailability of the otherwise same ation without the WO 38811 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%, preferably at least %, less than the CMAX of an approximately equivalent pharmaceutically effective intravenously 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 require less injection force. For e, ‘ the injection force may be at least 10%, preferably at least 20%, less than the ion force for the otherwise same formulation without the viscosity-reducing ionic liquid administered in the same way. In one embodiment, the injection is administered with a 27 gauge needle and the injection force is less than 30 N. The formulations can be administered in most cases using a very small gauge needle, 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 ns are present in the dosage unit such that after titution in a pharmaceutically able solvent, 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 ation 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- lowering 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 t in the formulations at concentrations that cause no significant signs of toxicity and/or no irreversible signs of toxicity When administered via aneous, intramuscular, or other types of in- injection. As used herein, “significant signs of toxicity” include cation, lethargy, behavioral ations such as those that occur with damage to the central nervous system, infertility, signs of serious cardiotoxicity such as cardiac arrhythmia, cardiomyopathy, myocardial infarctions, and cardiac 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 weekly, once weekly or once monthly. The protein formulations can be administered causing no cant signs of tion at the site of injection, as ed 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, redness, 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 copically similar levels of irritation when compared to injections of equivalent s of saline solution.

The protein formulations can exhibit increased bioavailability compared to the ise same protein formulation without the viscosity—reducing ionic liquid(s) when administered via subcutaneous or intramuscular injection. “Bioavailability” refers to the extent and rate at which the bioactive species such as a mAb, s circulation or the site of . The overall bioavailability can be increased for SC or IM injections as compared to the otherwise same formulations without the viscosity- reducing ionic 1iquid(s). “Percent ilability” refers to the fraction of the administered dose of the bioactive species 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 concentration as a function of time. The AUC can be calculated, for example, using the linear trapezoidal rule. “AUCQO”, as used , refers to the area under the plasma concentration curve from time zero to a time where the plasma concentration s to baseline levels. “AUCM”, as used herein, refers to the area under the plasma WO 38811 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 ed 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 stration at which the plasma concentration reaches a m.

Asused herein, “ max” refers to the m plasma concentration after dose administration, and before administration 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 ably at least 20%, less than the Cmax of an enously administered dose. This reduction in Cm, may also result in decreased toxicity.

' " - The pharmacokinetic and pharmacodynamic parameters may be approximated - across species using ches that are known to the skilled artisan. The pharmacokinetics and pharmacodynamics of antibody therapeutics can differ markedly based upon the specific 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 therapeutics 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 increasing the dosage stered 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, tituted formulations, 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 syringe can be a standard syringe that is pre-heated using a syringe warmer. The syringe warmer will lly have one or more openings each e of ing a syringe containing the protein formulation and a means for heating and maintaining the syringe at a specific ally above the ambient) ature prior to use. This will be referred to herein as a premheated syringe. Suitable heated syringe s include those available from Vista Dental Products and Inter-Med. The warmers are capable of accommodating s 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 desired temperature.

The heated syringe can be a self-heating syringe, i.e capable of g and maintaining the liquid formulation inside the syringe at a specific temperature. The self-heating syringe can also be a standard medical syringe having attached thereto a heating device. Suitable heating s capable of being ed to a syringe include syringe heaters or e heater tape available from Watlow Electric Manufacturing Co. of St. Louis, MO, and syringe heater blocks, stage heaters, and in—line perfusion s 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 B or TC-344B model heater controllers available from Warner Instruments.

The heated syringe 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 syringe can maintain the protein formulation at a temperature between °C and 60°C, between 21°C and 45°C, between 22°C and 40°C, between 25° C and 40° C, or between 25°C and 37°C. By maintaining the protein formulations at an elevated temperature during ion, the ity of the liquid formulation is de- decreased, the solubility of the protein in the formulation is increased, or both. ii. Self-Mixing Syringes The syringe can be self—mixing or can have a mixer attached. The mixer can be a static mixer or a dynamic mixer. es of static mixers include those disclosed in US. Patent Nos. 5,819,988, 6,065,645, 314, 972, and 6,698,622.

Examples 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 Application Publication No. US 2002/0190082.The syringe can e multiple barrels for mixing the components of the liquid protein formulation. US. Patent No. 5,819,998 describes syringes with two barrels and a mixing tip for mixing two-component Viscous substances. oinieciors and Free—tilled Syringes of Protein ations The liquid protein formulation can be administered using a prevfilled syringe autoinj ector or a needleless injection device. Autoinj ectors include a handheld, often pen-like, cartridge holder for holding replaceable lled cartridges and a spring based or ous mechanism for subcutaneous or intramuscular injections of liquid drug dosages from a pre-filled cartridge. Autoinj ectors are typically designed for self- administration or administration by untrained personnel. Autoinjectors are available to dispense either single dosages or multiple dosages from a pre-filled cartridge.

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

The lyophiiized protein formulation can be provided in pre-filied 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 ion. 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 lized form in a pre-filled or unit-dose syringe, tituted 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., Oxford, U.K.) can be used to ad- administer a unit—dose protein formulation where the ation 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 cartridges). Examples of suitable dges can include those described in U.S. Patent Nos. 5,334,162 and 5,454,786.

V. Methods of ation 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 example, 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 protein is then purified or concentrated using a method selected from the group consisting of ultrafiltration/diafiltration, tangential flow ion, centrifugal concentration, and dialysis.

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

All viscosities of well-mixed aqueous mAb solutions were ed using either a mVROC uidic viscometer (RheoSense) or a DVZT cone and plate viscometer (Brookfield; “C & P”) after a 5 minute equilibration at 25°C (unless otherwise indicated). The mVROC eter was ed with an “A” or “B” chip, each manufactured with a 50 micron l. Typically, 0.10 mL of protein solution was back-loaded into a gastight microlab instrument e (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 absolute viscosity and WO 38811 standard deviation was then calculated from at least these three ements. The C & P viscometer 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 olated 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 eter. The extrapolated zero~shear viscosities reported are the average and standard deviation 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® containing pharmaceutical excipients (Polysorbate 20, ate and citrate buffers, mannitol, and NaCl) was purified. First, Polysorbate 20 was removed using DETERGENT-OUTID TWEEN Medi Columns (G-Biosciences). Next, the resulting solutions were extensively buffer- exchanged into 20 mM sodium phosphate buffer (PB) or 20 mM viscosity-reducing ionic liquid solutions and concentrated to a final volume of less than 10 mL 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 ged into 20 mM PB (PB control samples) or 20 mM viscosity-reducing ionic liquid, the collected protein on was freeze-dried. The dried protein cakes, containing protein and buffer salts or viscosity-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 ity-reducing ionic liquid (pH 7.0), as appropriate, sufficient to bring the final concentration of PB to 0.25 M and the final concentration of viscosity- ng ionic liquid as indicated in the tables below. s buffer exchanged into 2 mM PB were first aliquoted. 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 protein solutions were then freeze-dried. The dried protein cakes, containing protein and viscosity- 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 ance at 280 nm using an experimentally determined extinction coefficient of 1.7 L/g‘crn and viscosities reported were measured on a RheoSense mVROC microfluidic viscometer.

Results The data in Table 1 demonstrate that the Viscosity of aqueous solutions of biosimilar AVASTIN® can be reduced by up to 6.5~fold (compared to phosphate buffered samples) in the ce of 0.20—0.50 M ity-reducing ionic liquids.

Viscosities over 200 CP in the absence of viscosity—reducing ionic liquids were reduced to less than 50 cP by the on 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 viscosity-reducing ionic .

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

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

Ionic * [IL] (M) [Protein] (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 = utylimidazolio)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 ilar RITUXAN® Materials and s Commercially-obtained biosimilar RITUXAN® containing pharmaceutical excipients (citrate buffer, NaCl, and Tween 80) was purified, buffer ged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction ient of 1.7 L/g-cm). Viscosities were measured 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 RITUXAN® 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. ities (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 = 4—(3-butylimidazolio)—1~butane sulfonate; n.d. = not determined.

Example 3: Ionic s reduce the viscosity of concentrated aqueous solutions of I® Materials and Methods Commercially-obtained I® containing pharmaceutical excipients (sodium phosphate buffer, NaCl, Polysorbate 80) was buffer exchanged, concentrated, dried, reconstituted, 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 viscometer equipped with an “A” or “B” chip.

Results The data in Table 3 demonstrate that the viscosity of aqueous solutions 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 de 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 Imethylimidazolium methanesulfonate; BMP Chloride = 1-buty1—1-methylpyrrolidinium C1; EMMC = 4-ethyl methylmorpholinium methyl carbonate.

Example 4: utyl~1~imidazolio)hl-butane sulfonate reduces the viscosity of trated REMICADE® and VECTIBIX® solutions Materials and Methods Commercially-obtained DE® ning pharmaceutical excipients- (sucrose, Polysorbate 80, sodium phosphate buffer) was prepared 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 aqueous protein drug products were purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1 above (using the tion 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 measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip.

Rinks The s in Table 4 demonstrate that BIM is ive 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. .

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 s solutions of HERCEPTIN® Materials and Methods Commercially—obtained HERCEPTIN® containing pharmaceutical excipients (histidine , trehalose, Polysorbate 20) was prepared as per ctions in the . prescribing information sheet. The aqueous protein drug product was buffer exchanged, trated, dried, reconstituted, and analyzed as described in Example 1 above (using the extinction coefficient of: 1.5 L/g-cm). The protein was formulated either with phosphate buffer or with various ity-reducing ionic liquids at concentrations in the table listed below. Viscosities were measured using a RheoSense mVROC microfluidic viscometer equipped with an “A” or “B” chip.

The results in Table 5 demonstrate that Viscosity-reducing ionic liquids are effective at reducing the viscosity of concentrated, 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.

Viscosity-reducing [IL] (M) [Protein] (mg/mL) Viscosity (cP) ionic * 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 biosimilar ERBITUX®.

Commercially-obtained biosimilar ERBITUX® containing pharmaceutical ents (Phosphate buffer, sodium chloride, Polysorbate 80) was buffer exchanged, trated, dried, reconstituted, and analyzed as described in Example 1 above (using the tion coefficient of: 1.4 L/g'cm). The protein was formulated either with ate buffer or with various trations 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 dependent 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 tration 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 ed subcutaneously Thirty 11-week old Sprague-Dawley rats were separated into 6 groups of 5 rats each (3 saline control groups and 3 BIM groups). The rats were injected aneously with 0.5 mL of endotoxin—free either phosphate-buffered saline or 0.25 M BIM according to the ing le: 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 evaluation scores pre—dose, ately post-dose, at 1 hour (i15 minutes) post close, and prior to sacrifice.

Overall, the observed consequences ofthe injections of saline and BIM were macroscopically similar throughout the course of the study. Both d 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 injection sites suggests a very minor, clinically ificant, irritative 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 ly 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 injection comprising: (i) an antibody; (ii) l-butylmethylimidazolium esulfonate es) or a pharmaceutically able 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 solvent but without the BMI-Mes or pharmaceutically acceptable salt thereof; and wherein the absolute viscosity is an extrapolated zero-shear ity.

2. The liquid pharmaceutical formulation 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, comprising from about 100 mg/ml to about 300 mg/ml of the antibody.

5. The liquid pharmaceutical formulation of any one of the previous claims, comprising from about 214 mg/ml to about 232 mg/ml of the antibody.

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

7. The liquid pharmaceutical formulation of any one of the us claims, wherein the BMI-Mes or pharmaceutically acceptable salt thereof is present at a tration of from about 0.01 M to about 1.0 M.

8. The liquid pharmaceutical formulation of any one of the previous , wherein the BMI-Mes or pharmaceutically acceptable salt thereof is present at a concentration of from about 0.40 M to about 0.50 M.

9. The liquid pharmaceutical ation of any one of the previous claims, r comprising one or more pharmaceutically acceptable excipients, the one or more pharmaceutically acceptable ents comprising a sugar, sugar alcohol, buffering agent, preservative, carrier, antioxidant, chelating agent, natural polymer, synthetic polymer, cryoprotectant, lyoprotectant, surfactant, g agent, stabilizing agent, or any combination thereof.

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

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

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

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

15. The liquid ceutical formulation of any one of the previous claims, 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 viscometer, 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 us claims in the manufacture of a medicament for administering to a subject a eutically 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 led syringe, or combinations thereof.

19. The use of claim 18, wherein the syringe is a heated syringe and the medicament is formulated 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 .

21. 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 10% less than an injection force for a control composition comprising the antibody and the ceutically acceptable solvent but without the BMI-Mes or 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 sing the antibody and the pharmaceutically acceptable solvent but without the s or 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 injection 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 acceptable solvent, and the BMI-Mes or pharmaceutically acceptable salt thereof.

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

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