Liquid protein formulations containing viscosity-lowering agents

Abstract

To provide concentrated, low-viscosity liquid formulations of proteins, especially high-molecular-weight proteins, such as mAbs, capable of delivering therapeutically effective amounts of these proteins in volumes useful for SC and IM injections.SOLUTION: A liquid pharmaceutical formulation for injection comprises (i) antibodies, (ii) one or more viscosity-lowering agents, and (iii) a pharmaceutically acceptable solvent. When in a volume suitable for injection, the liquid pharmaceutical formulation has an absolute viscosity from about 1 cP to about 100 cP at 25°C as measured using a cone and plate viscometer or microfluidic viscometer. The absolute viscosity of the liquid pharmaceutical formulation is less than the absolute viscosity of a control composition comprising the antibodies and the pharmaceutically acceptable solvent and not comprising the one or more viscosity-lowering agents, where each absolute viscosity is an extrapolated zero-shear viscosity.SELECTED DRAWING: None

Description

[Technical field]

[0001] CITED RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 030,521, filed July 29, 2014, entitled "Low-Viscosity Protein Formulations Containing Hydrophobic Salts"; U.S. Provisional Application No. 62 / 026,497, filed July 18, 2014, entitled "Low-Viscosity Protein Formulations Containing GRAS Viscosity-Reducing Agents"; U.S. Provisional Application No. 62 / 008,050, filed June 5, 2014, entitled "Low-Viscosity Protein Formulations Containing Ionic Liquids"; U.S. Provisional Application No. 62 / 008,050, filed May 2, 2014, entitled "Low-Viscosity Protein Formulations Containing Ionic Liquids"; and U.S. Provisional Application No. 6, entitled "Protein Formulations Containing Organophosphates." No. 1 / 988,005, filed February 28, 2014, entitled "Concentrated, Low-Viscosity Infliximab Formulations"; U.S. Provisional Application No. 61 / 946,436, filed February 21, 2014, entitled "Concentrated, Low-Viscosity, High-Molecular-Weight-Protein Formulations"; No. 61 / 943,197, entitled "Concentrated, Low-Viscosity High-Molecular-Weight Protein Formulations," filed February 14, 2014; and U.S. Provisional Application No. 61 / 940,227, entitled "Concentrated, Low-Viscosity High-Molecular-Weight Protein Formulations," filed September 11, 2013, which claims priority to and the benefit of U.S. Provisional Application No. 61,876,621, entitled "Concentrated, Low-Viscosity High-Molecular-Weight Protein Formulations," filed September 11, 2013. The disclosures of these applications are expressly incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention is generally in the field of injectable, low viscosity pharmaceutical formulations of highly concentrated proteins and methods of making and using same. [Background technology]

[0003] 2. Background of the Invention Monoclonal antibodies (mAbs) are important protein-based therapeutics for treating various human diseases, such as cancer, infectious diseases, inflammation, and autoimmune diseases. More than 20 mAb products have been approved by the U.S. Food and Drug Administration (FDA), and approximately 20% of the biopharmaceuticals currently being evaluated in clinical trials are mAbs (Daugherty et al., Adv. Drug Deliv. Rev. 58:686-706, 2006, and Buss et al., Curr. Opinion in Pharmacol. 12:615-622, 2012).

[0004] mAb-based therapies are usually administered repeatedly over long periods of time and require the administration of several mg / kg. Antibody solutions or suspensions can be administered by parenteral routes, such as intravenous (IV) infusion, and subcutaneous (SC) or intramuscular (IM) injection. Compared with the IV route, the SC or IM route reduces treatment costs, increases patient compliance, and improves convenience for patients and healthcare providers during administration. According to FDA guidelines, to be effective and pharmacologic acceptable, parenteral formulations should preferably be sterile, stable, injectable (e.g., by syringe), and non-irritating at the injection site. Due to the small volumes required for subcutaneous (usually less than about 2 mL) and intramuscular (usually less than about 5 mL) injections, these administration routes for high-dose protein therapeutics require concentrated protein solutions. Such high concentrations often result in highly viscous formulations that are difficult to administer by injection, cause pain at the injection site, are often imprecise, and / or may have reduced chemical and / or physical stability.

[0005] These characteristics create manufacturing, storage and usage requirements that can be challenging to achieve, especially for formulations with high concentrations of high molecular weight proteins such as mAbs. All protein therapeutics are subject to some degree of physical and chemical instability, such as aggregation, denaturation, cross-linking, deamidation, isomerization, oxidation and clipping (Wang et al., J. Pharm. Sci. 96:1-26, 2007). Therefore, optimal formulation development is a priority in the development of commercially viable protein pharmaceuticals.

[0006] High protein concentrations pose challenges related to the physical and chemical stability of proteins, as well as difficulties with the manufacture, storage and delivery of protein formulations. One problem is the tendency of proteins to aggregate and form particles during processing and / or storage, which makes them difficult to handle during further processing and / or delivery. Concentration-dependent degradation and / or aggregation are major challenges in developing higher concentration protein formulations. In addition to the potential for aggregation and particle formation of non-native proteins, reversible self-association in aqueous solutions can occur, which contributes to increased viscosity, among other things, complicating delivery by injection (see, e.g., Steven J. Shire et al., J. Pharm. Sci. Vol. 93:1). (See, e.g., J. Jezek et al., Advanced Drug Delivery Reviews vol. 63:1107-1117, 2011). Viscosity increase is one of the key challenges faced in thick protein compositions, affecting both the production methods and the feasibility of easily delivering such compositions by conventional means (See, e.g., J. Jezek et al., Advanced Drug Delivery Reviews vol. 63:1107-1117, 2011).

[0007] Highly viscous liquid formulations are difficult to manufacture, draw into a syringe, and inject subcutaneously or intramuscularly. The use of force in manipulating a highly viscous formulation can result in excessive foaming, which can further denature and inactivate therapeutically active proteins. Highly viscous solutions also require larger diameter needles for injection, which creates more pain at the injection site.

[0008] Currently available commercial mAb products administered by SC or IM injection are typically formulated in aqueous buffers such as phosphate or L-histidine buffers with excipients or surfactants such as mannitol, sucrose, lactose, trehalose, POLOXAMER® (a non-ionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide))), or POLYSORBATE® 80 (PEG(80) sorbitan monolaurate) to prevent aggregation and improve stability. Reported antibody concentrations formulated as above are typically up to about 100 mg / mL (Wang et al., J. Pharm. Sci. 96:1-26, 2007).

[0009] US Patent No. 7,758,860 describes the viscosity reduction in low molecular weight protein formulations using buffers and viscosity-reducing inorganic salts such as calcium chloride or magnesium chloride.However, these same salts have been shown to have little effect on the viscosity of high molecular weight antibody (IMA-638) formulations.As described in US Patent No. 7,666,413, the viscosity of aqueous formulations of high molecular weight proteins is reduced by adding such salts as arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride or sodium acetate at a concentration of more than about 100 mM, or by adding organic or inorganic acids as described in US Patent No. 7,740,842.However, these salts do not reduce viscosity to the desired level, and in some cases make the formulation acidic enough to cause pain at the injection site.

[0010] US Patent No. 7,666,413 describes a reduced viscosity formulation containing certain salts and reconstituted anti-IgE mAb, but with a maximum antibody concentration of only up to about 140 mg / mL. US Patent No. 7,740,842 describes an E25 anti-IgE mAb formulation containing acetate / acetic acid buffer and with an antibody concentration of up to 257 mg / mL. It has been demonstrated that the addition of salts such as NaCl, CaCl2 or MgCl2 reduces the dynamic viscosity under high shear conditions. However, at low shear, these salts caused an undesirable dramatic increase in dynamic viscosity. Furthermore, inorganic salts such as NaCl can reduce solution viscosity and / or reduce aggregation (EP1981824).

[0011] Non-aqueous antibody or protein formulations have also been described. WO2006 / 071693 describes non-aqueous suspensions of up to 100 mg / mL mAbs in formulations with viscosity enhancers (polyvinylpyrrolidone, PVP) and solvents (benzyl benzoate or PEG400). WO2004 / 089335 describes non-aqueous lysozyme suspension formulations at 100 mg / mL containing PVP, glycofurol, benzyl benzoate, benzyl alcohol or PEG400. US2008 / 0226689A1 describes a single-phase, three-vehicle component (polymer, surfactant and solvent), non-aqueous viscous formulation of human growth hormone (hGH) at 100 mg / mL. US Patent No. 6,730,328 describes a non-aqueous, hydrophobic, non-polar vehicle with low reactivity, such as perfluorodecalin, for protein formulations. These formulations are not optimized and have high viscosities that make processing, manufacturing and injection difficult; they result in the presence of multiple vehicle components in the formulation; and there are potential regulatory challenges associated with the use of polymers that have not yet been approved by the FDA.

[0012] Benzyl benzoate (Miller et al., Langmuir 26:1067-1074, 2010), benzyl acetate, ethanol or methyl ethyl ketone (Srinivasan et al., Pharm. Res. Alternative non-aqueous protein or antibody formulations have been described using organic solvents such as glycerol, glycerol, sorbitol, sorbitol esters, etc. In both cases, viscosities of less than 50 centipoise (cP) were achieved when formulated at protein concentrations of at least about 200 mg / mL. U.S. Patent No. 6,252,055 describes mAb formulations with concentrations ranging from 100 mg / mL to 257 mg / mL. Formulations with concentrations above about 189 mg / mL demonstrated dramatic viscosity increases, low recovery rates, and processing difficulties. U.S. Patent Application Publication No. 2012 / 0230982 describes antibody formulations with concentrations of 100 mg / mL to 200 mg / mL. None of these formulations have a sufficiently low viscosity to facilitate injection.

[0013] Du and Klibanov (Biotechnology and Bioengineering 108:632-636, 2011) describe the reduction in viscosity of concentrated aqueous solutions of bovine serum albumin up to a maximum concentration of 400 mg / mL and bovine gamma globulin up to a maximum concentration of 300 mg / mL. Guo et al. (Pharmaceutical Research 29:3102-3109, 2012) (2003) described low-viscosity aqueous solutions of four model mAbs, achieved by using hydrophobic salts. The mAb formulations used by Guo had initial viscosities below 73 cP before the addition of salt. On the other hand, the viscosities of many pharma- ceutical important mAbs can exceed 1,000 cP at therapeutically relevant concentrations.

[0014] Control of aggregation and viscosity is not a trivial issue in high concentration mAb solutions (EP2538973), as evidenced by the fact that very few mAb products are currently marketed as high concentration formulations (>100 mg / mL) (EP2538973).

[0015] The references cited above clearly show that although many groups have attempted to prepare low viscosity formulations of mAbs and other therapeutically important proteins, truly useful formulations for many proteins remain elusive. Notably, many of the above reports have used agents whose safety and toxicity profiles have not been fully established. These formulations therefore likely face a higher regulatory burden before approval than formulations containing compounds known to be safe. Indeed, even if a compound is shown to substantially reduce viscosity, the compound may ultimately be unsuitable for use in formulations intended for injection into humans.

[0016] Many pharma- ceutical important high molecular weight proteins, such as mAbs, are currently administered by IV infusion to deliver therapeutically effective amounts of the proteins due to problems associated with the high viscosity and other properties of concentrated solutions of large proteins. For example, protein concentrations of greater than 150 mg / mL are often required to provide a therapeutically effective amount of many high molecular weight proteins, such as mAbs, in a volume of less than about 2 mL. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] U.S. Patent No. 7,758,860 [Patent Document 2] U.S. Pat. No. 7,666,413 [Patent Document 3] U.S. Patent No. 7,740,842 [Patent Document 4] International Publication No. 2006 / 071693 [Patent Document 5] International Publication No. 2004 / 089335 [Patent Document 6] US Patent Application Publication No. 2008 / 0226689 [Patent Document 7] U.S. Patent No. 6,730,328 [Patent Document 8] U.S. Patent No. 6,252,055 [Patent Document 9] US Patent Application Publication No. 2012 / 0230982 [Non-patent literature]

[0018] [Non-Patent Document 1] Daugherty et al., Adv. Drug Deliv. Rev. 58:686-706, 2006 [Non-Patent Document 2] Buss et al., Curr. Opinion in Pharmacol. 12:615-622, 2012 [Non-Patent Document 3] Wang et al., J. Pharm. Sci. 96:1-26, 2007 [Non-Patent Document 4] Steven J. Shire et al., J. Pharm. Sci. 93:1390-1402, 2004 [Non-Patent Document 5] J. Jezek et al., Advanced Drug Delivery Reviews, vol. 63:1107-1117, 2011 [Non-Patent Document 6] Miller et al., Langmuir 26:1067-1074, 2010 [Non-Patent Document 7] Srinivasan et al., Pharm. Res. 30:1749-1757, 2013 [Non-Patent Document 8] Du and Klibanov, Biotechnology and Bioengineering 108:632-636, 2011. [Non-Patent Document 9] Guo et al., Pharmaceutical Research 29:3102-3109, 2012 Summary of the Invention [Problem to be solved by the invention]

[0019] It is therefore an object of the present invention to provide low-viscosity concentrated liquid formulations of pharma- ceutical important proteins, especially high molecular weight proteins such as mAbs.

[0020] It is a further object of the present invention to provide low-viscosity, concentrated liquid formulations of proteins, particularly high molecular weight proteins such as mAbs, that are capable of delivering therapeutically effective amounts of these proteins in volumes useful for SC and IM injections.

[0021] It is a further object of the present invention to provide concentrated liquid formulations of proteins, especially high molecular weight proteins such as mAbs, that have low viscosity, which can improve injectability and / or patient compliance, convenience and comfort.

[0022] It is also an object of the present invention to provide methods for making and storing concentrated, low-viscosity formulations of proteins, especially high molecular weight proteins such as mAbs.

[0023] It is a further object of the present invention to provide a method for administering low viscosity concentrated liquid formulations of proteins, particularly high molecular weight proteins such as mAbs. It is a further object of the present invention to provide a method for administering low viscosity, highly concentrated biologics using concentration and filtration techniques known to those skilled in the art. The object of the present invention is to provide a method for processing the above-mentioned In certain embodiments, for example, the following are provided: (Item 1) (i) one or more proteins; (ii) one or more viscosity-lowering agents that are bulky polar organic compounds selected from the group consisting of hydrophobic compounds and active and inactive pharmaceutical ingredients approved for injection; (iii) a pharma- ceutically acceptable solvent; 1. An injectable pharmaceutical formulation comprising: when the protein is combined with a solvent and a viscosity-lowering agent in a volume suitable for injection, the formulation has an absolute viscosity of about 1 cP to about 50 cP at 25° C., as measured using a cone and plate viscometer, which absolute viscosity of the formulation is less than the absolute viscosity of an otherwise identical formulation that contains an equivalent amount of sodium phosphate instead of the viscosity-lowering agent; In each case, the absolute viscosity is the extrapolated zero shear viscosity. Injectable pharmaceutical preparations. (Item 2) 2. The formulation of item 1, wherein the protein(s) have a molecular weight between about 70 kDa and 100 kDa, between about 100 kDa and about 250 kDa, or between about 250 kDa and about 500 kDa. (Item 3) 3. The formulation according to any one of items 1 and 2, wherein the protein has a molecular weight of about 120 kDa to about 250 kDa. (Item 4) 4. The formulation of any one of items 1 to 3, wherein at least one of the proteins is an enzyme, an antibody or antibody fragment, a fusion protein, or a PEGylated protein. (Item 5) 5. The formulation of any one of items 1 to 4, wherein the protein(s) are present in a combined amount of about 100 mg to about 2,000 mg per mL (mg / mL), optionally greater than about 150 mg / mL. (Item 6) 6. The formulation according to any one of items 1 to 5, comprising at least two different proteins, preferably both of said proteins having a molecular weight of at least about 50 kDa. (Item 7) 7. The formulation of any one of items 1 to 6, wherein the initial absolute viscosity, prior to addition of a viscosity-lowering agent, at the same protein concentration is greater than about 50 cP, greater than about 80 cP, or greater than about 100 cP. (Item 8) 8. The formulation according to any one of items 1 to 7, wherein the liquid formulation is aqueous having a pH between about 5.0 and about 8.0. (Item 9) 9. The formulation of any one of items 1 to 8, comprising the viscosity-lowering agent present in a concentration of about 0.01 M to about 1.0 M. (Item 10) 10. The formulation of any one of the preceding items, comprising the viscosity-lowering agent present in an amount of less than 0.3 M, or less than 0.15 M. (Item 11) 11. The formulation according to any one of items 1 to 10, comprising one or more pharma- ceutically acceptable excipients for subcutaneous or intramuscular injection selected from the group consisting of sugars or sugar alcohols, buffers, preservatives, carriers, antioxidants, chelating agents, natural or synthetic polymers, cryoprotectants, lyoprotectants, surfactants, bulking agents and stabilizers. (Item 12) 12. The formulation of item 11, wherein one or more of the excipients are selected from the group consisting of polysorbates, poloxamer 188, sodium lauryl sulfate, polyols selected from the group consisting of sugar alcohols such as mannitol and sorbitol, poly(ethylene glycol), glycerol, propylene glycol and poly(vinyl alcohol). (Item 13) 12. The formulation of claim 11, wherein the surfactant is present in an amount of less than about 10 mg / mL. (Item 14) 13. The formulation of item 12, comprising a polyol present in an amount of about 2 mg / mL to about 900 mg / mL. (Item 15) 15. The formulation according to any one of items 1 to 14, wherein the absolute viscosity is from about 5 cP to about 50 cP at 25° C. (Item 16) 16. The formulation of any one of the preceding claims, wherein the absolute viscosity is at least about 30% lower than the absolute viscosity of a formulation without the viscosity-lowering agent, when measured under identical conditions except for replacing the viscosity-lowering agent with an appropriate buffer of about the same concentration. (Item 17) 17. The formulation of any one of items 1 to 16, wherein the absolute viscosity is at least about 2-fold or 4-fold lower than the absolute viscosity of a formulation without the viscosity-lowering agent, when measured under identical conditions except for replacing the viscosity-lowering agent with an appropriate buffer of about the same concentration. (Item 18) 18. The formulation according to any one of items 1 to 17 in a unit dose vial, container or prefilled syringe. (Item 19) 19. The formulation according to item 18, wherein the protein, viscosity-lowering agent and / or excipients are in dry form, preferably lyophilized. (Item 20) 20. The formulation of any of the preceding items, wherein the volume of the formulation when the viscosity-lowering agent, protein and solvent are combined is less than about 1.5 mL for SC injection and less than about 3 mL for IM injection. (Item 21) 21. The formulation according to any one of items 1 to 20, which is isotonic to human serum. (Item 22) 22. The formulation according to any one of items 1 to 21, which behaves rheologically essentially as a Newtonian liquid under the conditions under which it is administered to a human being in need thereof. (Item 23) 23. The formulation of any one of items 1 to 22, which achieves a therapeutically effective dosage compared to the same dose of said protein administered by intravenous infusion. (Item 24) 24. The formulation of any one of items 1 to 23, wherein the viscosity-lowering agent is present in a concentration that does not cause clinically significant signs of toxicity or injection site irritation when administered by subcutaneous or intramuscular injection. (Item 25) The absolute viscosity of the formulation is at least about 0.5 s when measured using a cone and plate viscometer. -1 25. The formulation according to any of the preceding items, measured at a shear rate of (Item 26) The absolute viscosity of the formulation is at least about 1.0 s when measured using a microfluidic viscometer. -125. The formulation according to any one of the preceding claims, wherein the shear rate is measured at a shear rate of (Item 27) 27. A method of administering a therapeutically effective amount of a protein comprising subcutaneous or intramuscular injection of the formulation of any one of items 1 to 26. (Item 28) 28. The method of claim 27, wherein the subcutaneous or intramuscular injection is performed using a syringe selected from the group consisting of a heated syringe, a self-mixing syringe, an autoinjector, a pre-filled syringe, and combinations thereof. (Item 29) 29. The method of claim 28, wherein the syringe is a heated syringe and the formulation is administered at a temperature between 25° C. and 40° C. (Item 30) 28. The method of claim 27, wherein the formulation produces a primary irritation index of less than 3 when evaluated using the Draize rating system. (Item 31) 31. The method of any one of items 27 to 30, wherein the injection force is at least 10% or 20% less than the injection force of an otherwise identical formulation administered in the same manner but without the viscosity-lowering agent. (Item 32) 32. The method according to any one of items 27 to 31, wherein the injection is performed using a needle having a diameter between gauge 27 and 31, and the injection force is less than 30 N when using a gauge 27 needle. (Item 33) 27. A method of preparing a pharmaceutical formulation comprising combining the protein, solvent and viscosity-lowering agent according to any of items 1 to 26 to form a liquid formulation. (Item 34) 34. The method of claim 33, wherein the dosage unit formulation is in a pre-filled syringe or cartridge. (Item 35) 11. A method for facilitating purification of a protein, comprising adding an effective amount of the viscosity-lowering agent according to any of items 1, or 7 to 10, to a protein solution to reduce the absolute viscosity of the protein solution. (Item 36) 36. The method of claim 35, wherein the protein-viscosity-lowering agent solution is purified or concentrated using a method selected from the group consisting of ultrafiltration / diafiltration, tangential flow filtration, centrifugal concentration, and dialysis. [Means for solving the problem]

[0024] Summary of the Invention We have developed low-viscosity, low-volume, concentrated liquid pharmaceutical formulations of proteins that can be administered quickly and conveniently by subcutaneous (SC) or intramuscular (IM) injection rather than by prolonged intravenous infusion. These formulations include low and / or high molecular weight proteins such as mAbs, as well as a number of GRAS (generally regarded as safe, a list of compounds by the U.S. Food and Drug Administration), injectable These include viscosity reducing agents, which are typically bulky polar organic compounds, such as inactive ingredients that may be useful in reducing the viscosity of a liquid, and FDA approved therapeutic agents.

[0025] The concentration of the protein is between about 10 mg / mL and about 5,000 mg / mL, more preferably between about 100 mg / mL and about 2,000 mg / mL. In some embodiments, the concentration of the protein is between about 100 mg / mL and about 500 mg / mL, more preferably between about 300 mg / mL and about 500 mg / mL. The formulation containing the protein and the viscosity-lowering agent is stable when stored at a temperature of 4° C. for a period of at least 1 month, preferably at least 2 months, and most preferably at least 3 months. The viscosity of the formulation is less than about 75 cP, preferably less than 50 cP, and most preferably less than 20 cP at about 25° C. In some embodiments, the viscosity is less than about 15 cP, or less than or about 10 cP at about 25° C. In certain embodiments, the viscosity of the formulation is about 10 cP. The formulation containing the protein and the viscosity-lowering agent typically has a viscosity of less than about 0.6 s when measured using a cone and plate viscometer. -1 ~About 450s -1 , preferably about 2 s -1 ~About 400s -1Formulations containing proteins and viscosity-lowering agents typically exhibit shear rates of approximately 3 s when measured using a microfluidic viscometer. -1 ~about 55,000s -1 , preferably about 20 s -1 ~about 2,000s -1 It is measured in.

[0026] The viscosity of the protein formulation is reduced by the presence of one or more viscosity-lowering agents. Unless otherwise specifically stated, the term "viscosity-lowering agent" includes both a single compound and a mixture of two or more compounds. The viscosity-lowering agent is preferably present in the formulation at a concentration of less than about 1.0M, preferably less than about 0.50M, more preferably less than about 0.30M, and most preferably less than about 0.15M. In some embodiments, the viscosity-lowering agent is present in the formulation at a concentration as low as 0.01M. The formulation can have a viscosity that is at least about 30% lower, preferably at least about 50% lower, and most preferably at least about 75% lower than the viscosity of the corresponding formulation under the same conditions except when the viscosity-lowering agent is replaced by a suitable buffer or salt of about the same concentration. In some embodiments, low-viscosity formulations are provided in which the viscosity of the corresponding formulation without the viscosity-lowering agent is greater than about 200 cP, greater than about 500 cP, or even greater than about 1,000 cP. In a preferred embodiment, the shear rate of the formulation is at least about 0.5 s when measured using a cone and plate viscometer. -1 or at least about 1.0 s when measured using a microfluidic viscometer -1 It is.

[0027] For embodiments in which the protein is a "high molecular weight protein," the high molecular weight protein can 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 derivatized form thereof. Preferred mAbs include natalizumab (TYSABRI®), cetuximab (ERBITUX®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), infliximab (REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatumumab (ARZERRA®), and biosimilars thereof. The high molecular weight protein, optionally PEGylated, can be an enzyme. Other proteins and mixtures of proteins can also be formulated to reduce their viscosity.

[0028] In some embodiments, the protein and viscosity-lowering agent are provided in a lyophilized dosage unit sized for reconstitution with a sterile, pharma- ceutically acceptable vehicle to yield a low-viscosity concentrated liquid formulation. The presence of the viscosity-lowering agent facilitates and / or expedites reconstitution of the lyophilized dosage unit compared to a lyophilized dosage unit that does not contain the viscosity-lowering agent.

[0029] Provided herein is a method for preparing a low-viscosity concentrated liquid formulation of a high molecular weight protein such as mAb, as well as a method for storing the low-viscosity high-concentration protein formulation and administering it to a patient.In another embodiment, a viscosity-reducing agent is added to reduce the viscosity of the protein solution, thereby facilitating processing (e.g., pumping, concentration and / or filtration). [Brief description of the drawings]

[0030] [Figure 1] Figure 1 shows the viscosity (cP) as a function of protein concentration (mg / mL) for solutions of biosimilar cetuximab (ERBITUX®) in 0.25 M phosphate buffer (PB; diamonds) and in a solution containing 0.25 M camphorsulfonic acid L-lysine (CSAL; squares) at 25°C and a final pH of 7.0. The data points incorporate standard deviations that are often smaller than the symbols.

[0031] [Diagram 2] Figure 2 shows the viscosity (cP) as a function of protein concentration (mg / mL) for solutions of biosimilar bevacizumab (AVASTIN®) in 0.25 M phosphate buffer (PB; diamonds) and 0.25 M CSAL (squares) at 25°C and a final pH of 7.0. The data points incorporate standard deviations that are often smaller than the symbols.

[0032] [Diagram 3] FIG. 3 is a graph of the viscosity (cP) of aqueous solutions of 200±9 mg / mL biosimilar bevacizumab (AVASTIN®) containing either 0.25 M phosphate-citrate buffer or camphorsulfonic acid arginine (CSAA) as a function of pH on the x-axis.

[0033] [Figure 4] 4 is a bar graph comparing the fold reduction in viscosity as a function of pH for aqueous solutions containing biosimilar bevacizumab (AVASTIN®; approximately 200 mg / mL or 226 mg / mL) and 0.25 M camphorsulfonic acid arginine (CSAA). The fold reduction is calculated as the ratio of viscosity (cP) in phosphate-citrate buffer to viscosity (cP) in 0.25 M CSAA solution.

[0034] [Diagram 5]FIG. 5 is a graph of the viscosity (cP) of aqueous solutions of biosimilar cetuximab (ERBITUX®; at 202±5 mg / mL, 229±5 mg / mL, or 253±4 mg / mL) containing 0.25 M CSAA as a function of pH on the x-axis at 25° C.

[0035] [Figure 6] FIG. 6 is a size-exclusion chromatography trace showing absorbance intensity (at 280 nm) as a function of elution time (min) for a 220 mg / mL aqueous solution of REMICADE® stored at 4° C. for up to 100 days compared to freshly reconstituted commercial drug product.

[0036] [Figure 7] FIG. 7 illustrates the viscosity (cP) as a function of protein concentration (mg / mL) of aqueous solutions of biosimilar bevacizumab (AVASTIN®) in 0.25 M phosphate buffer, 0.10 M or 0.25 M APMI*2HCl ((1-(3-aminopropyl)-2-methyl-1H-imidazole bis-HCl).

[0037] [Figure 8] FIG. 8 illustrates the viscosity (cP) as a function of protein concentration (mg / mL) of aqueous solutions of biosimilar bevacizumab (AVASTIN®) in 0.25 M phosphate buffer, 0.10 M thiamine pyrophosphate (TPP), or 0.10 M TPP 1-(3-aminopropyl)-2-methyl-1H-imidazole (APMI).

[0038] [Figure 9] FIG. 9 illustrates the viscosity (cP) of aqueous solutions of golimumab (SIMPONI ARIA®) as a function of protein concentration (mg / mL) containing 0.15 M phosphate buffer or 0.15 M thiamine HCl. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed Description of the Invention

[0040] I. Definition The term "protein" as generally used herein refers to a polymer of amino acids linked together by peptide bonds to form a polypeptide whose chain length is at least sufficient to produce a detectable three-dimensional structure. Proteins with a molecular weight greater than about 100 kDa (expressed in kDa, where "Da" stands for "dalton" and 1 kDa=1,000 Da) can be referred to as "high molecular weight proteins", while proteins with a molecular weight less than about 100 kDa can be referred to as "low molecular weight proteins". The term "low molecular weight proteins" excludes small peptides that lack the requirement of at least three-dimensional structure necessary to be considered a protein. The molecular weight of a protein can be determined using standard methods known to those skilled in the art, including but not limited to mass spectrometry (e.g., ESI, MALDI) or calculation from known amino acid sequence and glycosylation. Proteins can be naturally occurring or non-naturally occurring, synthetic or semi-synthetic.

[0041] "Essentially pure protein" and "substantially pure protein" are used interchangeably herein and refer to a composition comprising at least about 90% by weight pure protein, preferably at least about 95% by weight pure protein. "Essentially homogeneous" and "substantially homogeneous" are used interchangeably herein and refer to a composition in which at least about 90% by weight, preferably at least about 95% by weight, of the protein present is a combination of monomers and reversible dimeric and oligomeric associations (as opposed to irreversible aggregates).

[0042] As generally used herein, the term "antibody" refers to any antibody that is capable of binding to any of a wide variety of antigens, including mAbs (including full-length antibodies having an immunoglobulin Fc region), antibodies with polyepitopic specificity, and antibodies with no known specificity. Antibody compositions, bispecific antibodies, diabodies and single chain antibody molecules comprising , as well as antibody fragments (eg, Fab, Fab', F(ab')2 and Fv), single domain antibodies, multivalent single domain antibodies, Fab fusion proteins, and fusions thereof.

[0043] The term "monoclonal antibody" or "mAb" as generally used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single epitope. They are usually synthesized by culturing hybridoma cells, as described, for example, by Kohler et al. (Nature 256:495, 1975), or can be produced by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), or can be produced by the methods described, for example, by Clackson et al. (Nature 352:624-628, 1991) and Marks et al. (Nature 352:624-628, 1991). (J. Mol. Biol. 222:581-597, 1991) The antibody may be isolated from a phage antibody library using techniques such as those described herein. As used herein, "mAb" specifically includes derivatized antibodies, antibody-drug conjugates, and "chimeric" antibodies (wherein a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass), as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984).

[0044] "Antibody fragments" include portions of an intact antibody, including the antigen binding and / or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8:1057-1062, 1995); Included are single chain antibody molecules; multivalent single domain antibodies; and multispecific antibodies formed from antibody fragments.

[0045] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) of mostly human sequence that contain minimal sequence derived from non-human immunoglobulin (see, e.g., Jones et al., Nature 321:522-533). 525, 1986; Reichmann et al., Nature 332:323-329, 1988 and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992).

[0046] "Rheology" refers to the study of the deformation and flow of materials.

[0047] "Viscosity" refers to the resistance of a substance (usually a liquid) to flow. Viscosity is related to the concept of shear force. Viscosity can be understood as the action of different layers of a fluid exerting shear forces on each other or on other surfaces as they move relative to each other. There are several viscosity scales. The unit of viscosity is Ns / m, known as the Pascal-second (Pa-s). 2 Viscosity can be "kinematic" or "absolute". Kinematic viscosity is a measure of the rate at which momentum is transferred through a fluid. Kinematic viscosity is measured in Stokes (St). Kinematic viscosity is a measure of the resistive flow of a fluid under the influence of gravity. If two fluids of equal volume and different viscosity are placed in the same capillary viscometer and allowed to flow by gravity, the more viscous fluid will take longer to flow through the capillary than the less viscous fluid. For example, if one fluid takes 200 seconds (s) to complete its flow and another takes 400s, the second fluid is said to be twice as viscous as the first on the kinematic viscosity scale. The dimensions of kinematic viscosity are length, 2 / hour. Kinematic viscosity is generally expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, which is equal to 1 cSt. "Absolute viscosity", sometimes called "dynamic viscosity" or "simple viscosity", is the product of kinematic viscosity and fluid density. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is millipascal-second (mPa-s), where 1 cP = 1 mPa-s.

[0048] Viscosity can be measured, for example, by using a viscometer at a given shear rate or multiple shear rates. The "extrapolated zero shear" viscosity can be determined by creating a best fit line of the four highest shear points based on a plot of absolute viscosity versus shear rate, and linearly extrapolating the viscosity back to zero shear. Alternatively, for Newtonian fluids, the viscosity can be determined by averaging the viscosity values ​​at multiple shear rates. Viscosity can also be measured using a microfluidic viscometer at single or multiple shear rates (also called flow rate), where the absolute viscosity is derived from the change in pressure as the liquid flows along a path. Viscosity is equal to the shear stress throughout the shear rate. In some embodiments, the viscosity measured using a microfluidic viscometer can be directly compared to the extrapolated zero shear viscosity, for example, that extrapolated from the viscosity measured at multiple shear rates using a cone and plate viscometer.

[0049] "Shear rate" refers to the rate of change of velocity at which one layer of fluid passes over an adjacent layer. The velocity gradient is the rate of change of velocity with distance from the plate. This simple case shows a constant velocity gradient with a shear rate (v1-v2) / h in units of (cm / sec) / (cm)=1 / sec. Thus, the units of shear rate are reciprocal seconds, or more commonly, reciprocal time. In a microfluidic viscometer, the change in pressure and flow rate are related to the shear rate. "Shear rate" refers to the speed at which a material deforms. Formulations containing proteins and viscosity-lowering agents typically exhibit a viscosity of approximately 0.5 s when measured using a cone and plate viscometer and a spindle appropriately selected by the skilled artisan to accurately measure viscosity in the viscosity range of the sample of interest (i.e., a 20 cP sample is most accurately measured with a CPE40 spindle mounted on a DV2T viscometer (Brookfield)). -1 ~about 200s -1 The shear rate was measured at a range of 20 s when measured using a microfluidic viscometer. -1 Super ~ about 3,000s -1 The shear rate is measured in the range of

[0050] For classical "Newtonian" fluids, as generally used herein, viscosity is essentially independent of shear rate. However, for "non-Newtonian fluids," as the shear rate increases, the viscosity either decreases or increases, e.g., the fluid becomes "shear-thinning" or "shear-thickening," respectively. For concentrated (i.e., highly concentrated) protein solutions, this can be expressed as pseudoplastic shear-thinning behavior, i.e., viscosity decreases with shear rate.

[0051] The term "chemical stability" as generally used herein refers to the ability of a protein component in a formulation to resist degradation by chemical pathways such as oxidation, deamidation or hydrolysis. A protein formulation is generally considered to be chemically stable if less than about 5% of the components are degraded after 24 months at 4°C.

[0052] The term "physical stability" as generally used herein refers to the ability of a protein formulation to resist physical deterioration, such as aggregation. A physically stable formulation forms only an acceptable percentage of irreversible aggregates (e.g., dimers, trimers or other aggregates) of a bioactive protein agent. The presence of aggregates can be assessed in several ways, including by measuring the average particle size of the protein in the formulation by dynamic light scattering. A formulation is considered physically stable if less than about 5% irreversible aggregates are formed after 24 months at 4°C. An acceptable level of aggregated contaminants is less than about 2%. A level as low as about 0.2% is achievable, but about 1% is more common.

[0053] The term "stable formulation" as generally used herein means that the formulation is both chemically and physically stable. A stable formulation can be one in which more than about 95% of the biologically active protein molecules retain their biological activity in the formulation after storage at 4°C for 24 months, or retain an equivalent solution state at an elevated temperature, such as storage at 40°C for 1 month. Various analytical techniques for measuring protein stability are available in the art, for example, see Peptide and Protein Drug Delivery, 24 Stability is reviewed in: pp. 7-301, Vincent Lee, (ed.), Marcel Dekker, Inc., New York, NY (1991) and Jones, A., Adv. Drug Delivery Revs. vol. 10:29-90, 1993. Stability can be measured for a period of time at a selected temperature. For rapid screening, for example, the formulation can be stored at 40°C for 2 weeks to 1 month, at which point the remaining biological activity is measured and compared to the initial state to evaluate stability. If 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 1 month, and / or at 2°C-8°C for at least 2 years. If the formulation is to be stored at room temperature, i.e., about 25°C, generally the formulation should be stable at about 25°C for at least 2 years, and / or at 40°C for at least about 6 months. The degree of aggregation after lyophilization and storage can be used as an indicator of protein stability. In some embodiments, stability is evaluated by measuring the particle size of the protein in the formulation. In some embodiments, stability can be evaluated by measuring the activity of the formulation using standard biological activity or binding assays, well within the capabilities of those skilled in the art.

[0054] As generally used herein, the term "particle size" of a protein refers to the average diameter of the major population of particles of a biologically active molecule in a formulation, or its particle size distribution, as determined by using well-known particle size measurement instruments, such as dynamic light scattering, SEC (size exclusion chromatography), or other methods known to those of skill in the art.

[0055] As generally used herein, the term "concentrated" or "highly concentrated" describes a liquid formulation having a final protein concentration of greater than about 10 mg / mL, preferably greater than about 50 mg / mL, more preferably greater than about 100 mg / mL, even more preferably greater than about 200 mg / mL, or most preferably greater than about 250 mg / mL.

[0056] As generally used herein, a "reconstituted formulation" refers to a formulation prepared by dissolving a dry powder, lyophilized protein, spray-dried protein or solvent-precipitated protein in a diluent such that the protein is dissolved or dispersed in an aqueous solution for administration.

[0057] A "lyoprotectant" is a compound that, when combined with a protein, prevents the protein from drying by lyophilization.

[0004] Lyophilization and / or subsequent storage of proteins with significantly reduced chemical and / or physical instability. Exemplary lyoprotectants include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; ); polyols such as propylene glycol, glycerol, poly(ethylene glycol) or poly(propylene glycol); and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrin, modified starch, and carboxymethylcellulose. Preferred sugar alcohols are those compounds obtained by reduction of mono- and disaccharides, such as lactose, trehalose, maltose, lactulose, and maltulose. Additional examples of sugar alcohols are glucitol, maltitol, lactitol, and isomaltulose. Lyoprotectants are generally added to the formulation before lyophilization in a "lyoprotective amount". This means that after lyophilization of the protein in the presence of a lyoprotective amount of lyoprotectant, the protein essentially retains its physical and chemical stability and integrity.

[0058] As generally used herein, a "diluent" or "carrier" is a useful ingredient for preparing a liquid formulation, such as an aqueous formulation that is pharma- ceutically acceptable (i.e., safe and non-toxic when administered to humans or another mammal) and reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution, and combinations thereof.

[0059] A "preservative" is a compound that can be added to the formulation herein to reduce contamination by bacteria, fungi, or other infectious agents or the action of bacteria, fungi, or other infectious agents. The addition of a preservative can, for example, facilitate the production of a multi-use (multiple dose) formulation. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides, where the alkyl group is long chain), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0060] As generally used herein, a "bulking agent" is a compound that provides bulk to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, lactose, modified starch, poly(ethylene glycol) and sorbitol.

[0061] "Therapeutically effective amount" is the minimum concentration required to measurably improve or prevent any symptoms or a particular condition or disorder, to measurably extend life expectancy, or to generally improve the quality of life of a patient. The therapeutically effective amount depends on the specific biologically active molecule and the specific condition or disorder to be treated. The therapeutically effective amount of many proteins, such as mAbs, described herein, is well known in the art. The therapeutically effective amount of a protein for treating a particular disorder with a known protein, such as mAb, that has not yet been established or is clinically applied to treat additional disorders, can be determined by suitable standard techniques that are well within the skill of a person skilled in the art, such as a physician.

[0062] The term "injectability" or "needleability" as generally used herein refers to the injectability of a pharmaceutical formulation, optionally with a thin-walled 18-32 gauge needle, in a syringe. Injectability depends on factors such as the pressure or force required for injection, uniform flow, aspiration quality, and lack of clogging. The injectability of the liquid pharmaceutical formulation can be evaluated by comparing the ejection force of a reduced-viscosity formulation to a standard formulation without added viscosity-reducing agent. A reduction in the ejection force of a formulation containing a viscosity-reducing agent reflects improved injectability of the formulation. A reduced-viscosity formulation has improved injectability if the ejection force is reduced by at least 10%, preferably at least 30%, more preferably at least 50%, and most preferably at least 75%, when compared to a standard formulation having the same concentration of protein under otherwise identical conditions, except that the viscosity-reducing agent is replaced by an appropriate buffer of approximately the same concentration. Alternatively, the injectability of the liquid pharmaceutical formulations can be assessed by comparing the time required to inject the same volume, such as 0.5 mL, or more preferably about 1 mL, of different liquid protein formulations using the same syringe pressure.

[0063] The term "ejection force" as generally used herein refers to the force required to extrude a given liquid formulation through a given syringe equipped with a given needle gauge at a given injection rate. This injection force is usually reported in Newton. For example, injection force can be measured as the force required to extrude a liquid formulation through a 1 mL plastic syringe with an inner diameter of 0.25 inches equipped with a 0.50 inch 27 gauge needle at an injection rate of 250 mm / min. A test device can be used to measure the injection force. When measured under the same conditions, a formulation with a lower viscosity generally requires a smaller overall injection force.

[0064] As used herein, "viscosity gradient" refers to the rate of change of viscosity of a protein solution as the protein concentration increases. Viscosity gradient can be estimated from a plot of viscosity as a function of protein concentration for a series of formulations with otherwise the same but different protein concentrations. The viscosity increases approximately exponentially with increasing protein concentration. The viscosity gradient at a particular protein concentration can be estimated from the slope of the tangent to the plot of viscosity as a function of protein concentration. Viscosity gradient can be estimated from a linear approximation to the viscosity plotted as a function of any protein concentration or over a narrow window of protein concentration. In some embodiments, a formulation is said to have a reduced viscosity gradient if the exponent of the exponential function is smaller than that obtained for the otherwise same formulation without the viscosity-lowering agent when the viscosity as a function of protein concentration is estimated as an exponential function. In a similar manner, a formulation can be said to have a smaller / larger viscosity gradient when compared to a second formulation if the exponent of the formulation is smaller / larger than that of the second formulation. The viscosity gradient can be numerically estimated from a plot of viscosity as a function of protein concentration by other methods known to the skilled formulation scientist.

[0065] As generally used herein, the term "reduced-viscosity formulation" refers to a liquid formulation having an elevated 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 that reduce the viscosity, compared to a corresponding formulation that does not contain the viscosity-reducing additive(s).

[0066] The term "osmolarity" as generally used herein refers to the total number of dissolved components per liter. Osmolarity is similar to molar concentration, but includes the total number of moles of dissolved species in a solution. An osmolarity of 1 Osm / L means that there is 1 mole of dissolved component per L of solution. Some solutes, such as ionic solutes that dissociate in solution, provide more than 1 mole of dissolved component per mole of solute in solution. For example, NaCl dissociates more than 1 mole of dissolved component per mole of solute in solution. + and Cl - i.e., each mole of NaCl dissolved in solution results in 2 moles of dissolved component. Physiological osmolarity is usually in the range of about 280 mOsm / L to about 310 mOsm / L.

[0067] The term "tonicity" generally used herein refers to the osmotic pressure gradient caused by the separation of two solutions by a semipermeable membrane.In particular, tonicity is used to describe the osmotic pressure that occurs across cell membrane when a cell is exposed to an external solution.Solutes that can pass through cell membrane do not contribute to the final osmotic pressure gradient.Only dissolved species that do not pass through cell membrane contribute to the osmotic pressure difference, i.e., tonicity.

[0068] As generally used herein, the term "hypertonic" refers to a solution that has a higher concentration of solutes than is present inside the cell. When a cell is immersed in a hypertonic solution, water tends to flow out of the cell to balance the concentration of the solutes.

[0069] As generally used herein, the term "hypotonic" refers to a solution that has a lower concentration of solutes than is present inside the cell. When a cell is immersed in a hypotonic solution, water flows into the cell to balance the concentration of the solutes.

[0070] The term "isotonic" as generally used herein refers to a solution in which the osmotic pressure gradient across the cell membrane is essentially balanced. An isotonic formulation is one that essentially has the same osmotic pressure as human blood. An isotonic formulation generally has an osmotic pressure of about 250 mOsm / kg to 350 mOsm / kg.

[0071] The term "liquid formulation," as used herein, is a protein that is either supplied in an acceptable pharmaceutical diluent or that is reconstituted with an acceptable pharmaceutical diluent prior to administration to a patient.

[0072] The terms "branded" and "reference" refer to a protein or biologic When used to refer to a biological product, they are used interchangeably herein and mean a single biological product that is licensed under section 351(a) of the US Public Health Service Act (42 U.S.C. § 262).

[0073] The term "biosimilar," as used herein, is generally used interchangeably with "generic equivalent" or "follow-on." For example, a "biosimilar mAb" refers to a follow-on version of an innovator's mAb, typically made by a different company. When used in reference to a branded protein or branded biologic, "biosimilar" can refer to a biological product that has been evaluated against that branded protein or branded biologic and that has been licensed under section 351(k) of the US Public Health Service Act (42 U.S.C. § 262). Biosimilar mAbs are defined as mAbs that are intended to be used in clinical trials or clinical trials, as defined in the Guidelines on Similar Biological Medicinal Products Containing Monoclonal Antibodies - Non-Clinical and Clinical Issues (reference document EMA / CHMP / BMAB), adopted on May 30, 2012 by the European Medicines Agency's Committee for Medicinal Products for Human Use. The present invention may comply with one or more of the guidelines issued by the European Union (UN 2010 / WP / 403543 / 2010).

[0074] Biosimilars can be produced by microbial cells (prokaryotic, eukaryotic), cell lines of human or animal origin (e.g., mammalian, avian, insect), tissues derived from animals or plants. The expression construct for a proposed biosimilar product generally encodes the same primary amino acid sequence as its reference product. Minor modifications, such as N- or C-terminal truncations, that do not affect safety, purity or potency, may be present.

[0075] Biosimilar mAbs are physiochemically or biologically similar to the reference mAb, both in terms of safety and efficacy. Biosimilar mAbs can be evaluated against the reference mAb using one or more in vitro studies, including assays that probe binding to target antigen(s); binding to isoforms of Fc gamma receptors (FcγRI, FcγRII and FcγRIII), FcRn, and complement (C1q); Fab-related functions (e.g., neutralization of soluble ligands, receptor activation or blockade); or Fc-related functions (e.g., antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, complement activation). In vitro comparisons can be combined with in vivo data, which demonstrate similarity in pharmacokinetics, pharmacodynamics and / or safety. Clinical evaluation of the biosimilar mAb against the reference mAb can be performed using pharmacokinetic properties (e.g., AUC 0-inf , AUC 0-t , C max , t max , C trough Quality comparisons between the biosimilar and reference mAb can include comparison of similarity of efficacy, pharmacodynamic endpoints, or clinical efficacy (e.g., using randomized, parallel-group clinical trials). Quality comparisons between the biosimilar and reference mAb can be assessed using established procedures, including those described in the "Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: Quality issues" (EMEA / CHMP / BWP / 49348 / 2005) and the "Guideline on development, production, characterization and specifications for monoclonal antibodies and related substances" (EMEA / CHMP / BWP / 157653 / 2007).

[0076] Differences between biosimilar and reference mAbs may include post-translational modifications, for example, by attaching other biochemical groups to the mAb, such as phosphates, various lipids and carbohydrates; by post-translational proteolytic cleavage; by changing the chemical nature of amino acids (e.g., formylation); or by many other mechanisms. Other post-translational modifications may be the result of manufacturing process manipulations. For example, glycation reactions may occur with exposure of the product to reducing sugars. In other cases, storage conditions may permit certain degradation pathways, such as oxidation, deamidation or aggregation. All of these product-related variants may be included in the biosimilar mAb.

[0077] The term "viscosity-reducing agent" as used herein refers to a compound that acts to reduce the viscosity of a solution, compared to the viscosity of the solution without the viscosity-reducing agent.A viscosity-reducing agent may be a single compound or a mixture of one or more compounds.When the viscosity-reducing agent is a mixture of two or more compounds, the listed concentrations refer to each individual agent unless otherwise specified.As an example, a formulation containing about 0.25M camphorsulfonic acid arginine as a viscosity-reducing agent is a solution having camphorsulfonic acid at a concentration of 0.25M and arginine at a concentration of 0.25M.

[0078] Certain viscosity-lowering agents contain acidic or basic functional groups. Whether these functional groups are fully or partially ionized depends on the pH of the formulation in which they are present. Unless otherwise specified, reference to a formulation containing a viscosity-lowering agent having an ionizable functional group includes both the parent compound and any possible ionized states.

[0079] As used herein, the term "hydrogen bond donor" refers to a hydrogen atom that is bonded to a relatively electronegative atom, which creates a partial positive charge on the hydrogen atom.

[0080] As used herein, the term "hydrogen bond acceptor" refers to a relatively electronegative atom or functional group capable of interacting with a hydrogen atom bearing a partial positive charge.

[0081] As used herein, the term "freely rotatable bond" refers to any single bond between a pair of non-hydrogen atoms.

[0082] As used herein, the term "polar surface area of ​​a molecule" refers to the total exposed polar area of ​​the surface of a molecule of interest.

[0083] As used herein, the term "molar volume" refers to the total volume occupied by one mole of a molecule of interest in its native state (i.e., solid, liquid).

[0084] As used herein, the term "polarizability" refers to the dipole moment induced when a molecule of interest is placed in an electric field of unit strength.

[0085] As used herein, the term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids and inorganic bases, and organic acids and organic bases.Suitable non-toxic acids include inorganic and organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc.Suitable positively charged counterions include sodium, potassium, lithium, calcium and magnesium.

[0086] As used herein, the term "ionic liquid" refers to a crystalline or amorphous salt, zwitterion, or mixture thereof, that is liquid at or near temperatures where most conventional salts are solid: below 200°C, preferably below 100°C, or more preferably below 80°C. Some ionic liquids have melting temperatures near room temperature, e.g., between 10°C and 40°C, or between 15°C and 35°C. The term "zwitterion" is used herein to describe an overall neutrally charged molecule that has formal positive and negative charges on different chemical groups in the molecule. Examples of ionic liquids can be found in Riduan et al., Chem. Soc. Rev., 42:9055-9070, 2013;Rantwijk et al., Chem. Rev., 107:2 757-2785, 2007; Earle et al., Pure Appl. Chem., 72(7):1 391-1398, 2000; and Sheldon et al., Green Chem., 4:147-1 51, 2002.

[0087] As used herein, the term "organophosphate" refers to a compound containing one or more phosphoryl groups, at least one of the phosphoryl groups being covalently attached to an organic group by a phosphate ester bond.

[0088] As used herein, a "water-soluble organic dye" is an organic molecule that has a molar solubility of at least 0.001 M at 25° C. and pH 7 and that absorbs certain wavelengths of light, preferably in the visible to infrared portions of the electromagnetic spectrum, while transmitting or reflecting other wavelengths of light.

[0089] As used herein, the term "chalcogen" refers to Group 16 elements, including oxygen, sulfur, and selenium, in any oxidation state. For example, unless otherwise specified, the term "chalcogen" also includes SO2.

[0090] As used herein, the term "alkyl group" refers to straight-chain, branched-chain and cyclic hydrocarbon groups. Unless otherwise specified, the term alkyl group includes hydrocarbon groups containing one or more double or triple bonds. An alkyl group containing at least one ring system is a "cycloalkyl" group. An alkyl group containing at least one double bond is an "alkenyl group" and an alkyl group containing at least one triple bond is an "alkynyl group."

[0091] As used herein, the term "aryl" refers to an aromatic carbocyclic ring system, including fused ring systems. In an "aryl" group, each of the atoms that form the ring are carbon atoms.

[0092] The term "heteroaryl" as used herein refers to an aromatic ring system, including fused ring systems, in which at least one of the atoms forming the ring is a heteroatom.

[0093] As used herein, the term "heterocycle" refers to a ring system, including fused ring systems, that is not aromatic and in which at least one atom that forms the ring is a heteroatom.

[0094] As used herein, the term "heteroatom" is any non-carbon or non-hydrogen atom. Preferred heteroatoms include oxygen, sulfur and nitrogen.Exemplary heteroaryl and heterocyclyl rings include benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, Furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrroly These include diphenyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

[0095] II. Preparations

[0096] Biocompatible low-viscosity protein solutions, such as those of mAbs, can be used to deliver therapeutically effective amounts of proteins in volumes useful for subcutaneous (SC) and intramuscular (IM) injection, typically less than or about 2 mL for SC and less than or about 5 mL for IM, more preferably less than or about 1 mL for SC and less than or about 3 mL for IM. Proteins can generally have any molecular weight, although in some embodiments, high molecular weight proteins are preferred. In other embodiments, the protein is a low molecular weight protein.

[0097] The formulations can have a protein concentration between about 10 mg / mL and about 5,000 mg / mL. The formulations, including mAb formulations, can have a protein concentration of more than 100 mg / mL, preferably more than 150 mg / mL, more preferably more than about 175 mg / ml, even more preferably more than about 200 mg / mL, even more preferably more than about 225 mg / mL, even more preferably more than about 250 mg / mL, and most preferably more than or about 300 mg / mL. In the absence of a viscosity-lowering agent, the viscosity of a protein formulation increases exponentially as its concentration increases. In the absence of a viscosity-lowering agent, such protein formulations can have a viscosity of more than 100 cP, more than 150 cP, more than 200 cP, more than 300 cP, more than 500 cP, or even more than 1,000 cP, measured at 25°C. Such formulations are often unsuitable for SC or IM injection. The use of one or more viscosity-lowering agents allows for the preparation of formulations having a viscosity, measured at 25° C., of less than or about 100 cP, preferably less than or about 75 cP, more preferably less than or about 50 cP, even more preferably less than or about 30 cP, even more preferably less than or about 20 cP, or most preferably less than or about 10 cP.

[0098] Viscosity-lowering agents can be used to reduce the viscosity of concentrated protein formulations, but they can also be used in less concentrated formulations as well. In some embodiments, the formulations can have a protein concentration between about 10 mg / mL and about 100 mg / mL. The formulations can have a protein concentration of more than about 20 mg / mL, more than about 40 mg / mL, or more than about 80 mg / mL.

[0099] For certain proteins, formulations without a viscosity-lowering agent can have a viscosity of greater than about 20 cP, greater than about 50 cP, or greater than about 80 cP. The use of one or more viscosity-lowering agents allows for the preparation of formulations with a viscosity of less than or about 80 cP, preferably less than or about 50 cP, even more preferably less than about 20 cP, or most preferably less than or about 10 cP, when measured at 25° C.

[0100] In some embodiments, the aqueous protein formulations have a viscosity at least about 30% lower than a similar formulation without the viscosity-lowering agent(s) when measured under the same conditions. In other embodiments, the formulations have a viscosity that is less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or even more than 90% lower than a similar formulation without the viscosity-lowering agent(s). In a preferred embodiment, the formulations contain a therapeutically effective amount of one or more high molecular weight proteins, such as mAbs, in a volume of less than about 2 mL, preferably less than about 1 mL, or more preferably less than about 0.75 mL.

[0101] The reduced viscosity formulations improve injectability and require less injection force than a similar formulation (e.g., in phosphate buffer) without the viscosity-lowering agent under otherwise identical conditions. In some embodiments, the injection force is reduced by more than about 20%, more than about 30%, more than about 40%, more than about 50%, or more than about 2-fold compared to a standard formulation without the viscosity-lowering agent(s) under otherwise identical injection conditions. In some embodiments, the formulations have "Newtonian flow characteristics," defined as having a viscosity that is substantially independent of shear rate. The protein formulations can be easily injected through needles of about 18-32 gauge. Preferred needle gauges for delivering low viscosity formulations include 27, 29, and 31 gauge, optionally with thin walls.

[0102] The formulation may contain one or more additional excipients, such as buffers, surfactants, sugars and sugar alcohols, other polyols, preservatives, antioxidants, and chelating agents. The formulation has a pH and osmolality suitable for administration without causing significant adverse side effects. In some embodiments, the low-viscosity concentrated formulation has a pH between 5-8, between 5.5-7.6, between 6.0-7.6, between 6.8-7.6, or between 5.5-6.5.

[0103] The low-viscosity protein formulation can allow greater flexibility in formulation development.The low-viscosity formulation can show less dependent viscosity change on protein concentration compared to the same formulation except that it does not contain viscosity-lowering agent.The low-viscosity protein formulation can allow for increased protein concentration and reduced frequency of administration.In some embodiments, the low-viscosity protein formulation contains two or more, three or more, or four or more different proteins.For example, the combination of two or more mAbs can be provided in a single low-viscosity protein formulation.

[0104] Because protein (such as mAb) formulations can be administered to patients at higher protein concentrations than otherwise similar protein formulations without the viscosity-lowering agent, the frequency of administration of the protein can be reduced. For example, a protein that previously required daily administration can be administered once every two days, once every three days, or even less frequently when the protein is formulated with a viscosity-lowering agent. Proteins that currently require multiple administrations on the same day (either at the same time of day or at different times) can be administered with fewer injections per day. In some cases, this frequency can be reduced to a single injection once a day. By increasing the dose administered per injection multiple-fold, the frequency of administration can be reduced, for example, from once every two weeks to once every six weeks.

[0105] In some embodiments, the liquid formulation has a physiological osmolality, for example, between about 280 mOsm / L and about 310 mOsm / L. In some embodiments, the liquid formulation has an osmolality of greater than about 250 mOsm / L, greater than about 300 mOsm / L, greater than about 350 mOsm / L, greater than about 400 mOsm / L, or greater than about 500 mOsm / L. In some embodiments, the formulation has an osmolality of about 200 mOsm / L to about 2,000 mOsm / L or about 300 mOsm / L to about 1,000 mOsm / L. In some embodiments, the liquid formulation is essentially isotonic to human blood. The liquid formulation can be hypertonic in some cases.

[0106] Additives, including viscosity-lowering agents, may be included in any amount that achieves the desired viscosity level of the liquid formulation, so long as the amount is not toxic or otherwise harmful and does not substantially interfere with the chemical and / or physical stability of the formulation. In some embodiments, the viscosity-lowering agent(s) may be present independently at a concentration of less than about 1.0M, preferably less than about 0.50M, less than or equal to about 0.30M, or less than or equal to 0.15M. Particularly preferred concentrations include about 0.15M and about 0.30M. For some embodiments having two or more viscosity-lowering agents, the viscosity-lowering agents are preferably, but not necessarily, present at the same concentration.

[0107] The viscosity-reducing agent allows the lyophilized dosage unit to be reconstituted more quickly. The dosage unit is a lyophilized cake of protein, viscosity-reducing agent and other excipients to which water, saline or another pharma- ceutically acceptable fluid is added. In the absence of a viscosity-reducing agent, 10 minutes or more are often required to completely dissolve a lyophilized cake with a high protein concentration. When the lyophilized cake contains one or more viscosity-reducing agents, the time required to completely dissolve the cake is often reduced by 2, 5, or even 10 times. In certain embodiments, less than 1 minute is required to completely dissolve a lyophilized cake containing more than 150 mg / mL, more than 200 mg / mL or even more than 300 mg / mL or about 150, about 200, or even about 300 mg / mL of protein.

[0108] The low-viscosity protein formulations can allow for greater flexibility in formulation development. The low-viscosity formulations exhibit less change in viscosity as protein concentration increases compared to the otherwise identical formulations without the viscosity-lowering agent(s). The low-viscosity protein formulations exhibit a reduced viscosity gradient compared to the otherwise identical formulations without the viscosity-lowering agent.

[0109] The viscosity gradient of the protein formulation may be 2-fold less, 3-fold less, or even more than 3-fold less than the viscosity gradient of an otherwise identical protein formulation without the viscosity-lowering agent(s). The viscosity gradient of the protein formulation may be less than 2.0 cPmL / mg, less than 1.5 cPmL / mg, less than 1.0 cPmL / mg, less than 0.8 cPmL / mg, less than 0.6 cPmL / mg, or less than 0.2 cPmL / mg for protein formulations having protein concentrations between 10 mg / mL and 2,000 mg / mL. By reducing the viscosity gradient of the formulation, the protein concentration can be increased to a significant extent before an exponential increase in viscosity is observed.

[0110] A. Protein

[0111] Any protein can be formulated, including recombinant, isolated or synthetic proteins, glycoproteins, or lipoproteins. These may be antibodies (including antibody fragments and recombinant antibodies), enzymes, growth factors or hormones, immunomodulators, anti-infectives, anti-proliferatives, vaccines, or other therapeutic, preventive or diagnostic proteins. In certain embodiments, the protein has a molecular weight of more than about 150 kDa, more than 160 kDa, more than 170 kDa, more than 180 kDa, more than 190 kDa, or even more than 200 kDa.

[0112] In certain embodiments, 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 via a chemical linker that may be different from the one or more polymer groups. PEGylated proteins are typically characterized by their reduced renal filtration, reduced uptake by the reticuloendothelial system, and reduced enzymatic degradation, resulting in, for example, an increased half-life and increased bioavailability. Stealth polymers include poly(ethylene glycol); poly(propylene glycol); poly(glutamic acid), poly(hydroxyethyl-L-asparagine), and poly(hydroxyethyl-L-asparagine). Poly(amino acid) polymers such as poly(amino acid) polymers, such as poly(2-oxazoline) and poly(2-ethyl-2-oxazoline); poly(acrylamide); poly(vinylpyrrolidone); poly(N-(2-hydroxypropyl)methacrylamide); and copolymers, and mixtures thereof. In a preferred embodiment, the stealth polymer in the PEGylated protein is poly(ethylene glycol) or a copolymer thereof. The PEGylated protein can be randomly PEGylated, i.e., having one or more stealth polymers covalently attached to a non-specific site(s) on the protein, or can be site-specifically PEGylated by covalently attaching a stealth polymer to a specific site(s) on the protein. Site-specific PEGylation can be performed, for example, using an activated stealth polymer, having one or more reactive functional groups. Examples include, for example, Hoffman et al., Progress in Polymer Science, 32:922-932, 2011. It was listed in 2007.

[0113] In a preferred embodiment, the protein is a high molecular weight antibody, most preferably a mAb, that has a high viscosity in aqueous buffer when concentrated sufficiently to inject a therapeutically effective amount in a volume not exceeding 1.0-2.0 mL for SC administration and not exceeding 3.0-5.0 mL for IM administration. High molecular weight proteins include those described in Scolnik, mAbs 1 179-184, 2009; Beck, mAbs 3:107-110, 2011; Baumann, Curr. Drug Meth. 7:15-21, 2006; or Federici, Biologicals 41:131-147, 2013. Proteins for use in the formulations described herein are preferably essentially pure and essentially homogeneous (i.e., substantially free of contaminating proteins and / or irreversible aggregates thereof).

[0114] Preferred mAbs herein include natalizumab (TYSABRI®), cetuximab (ERBITUX®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), infliximab (REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatumumab (ARZERRA®), and biosimilars thereof. Exemplary high molecular weight proteins may include tocilizumab (ACTEMRA®), alemtuzumab (marketed under several trademarks), brodalumab (developed by Amgen, Inc. ("Amgen")), denosumab (PROLIA® and XGEVA®), and biosimilars thereof.

[0115] Exemplary molecular targets for the antibodies described herein include CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD34; members of the HER receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mo1, p150,95, VLA-4, ICAM-1, VCAM and αv / β3 integrin (including either its α or β subunits) (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF; IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; protein C; PCSK9, and the like.

[0116] Antibody therapeutic drugs currently on the market

[0117] Many protein therapeutics currently on the market, especially antibodies as defined herein, require high dosage and are administered by IV infusion. The formulation can include one of the antibody therapeutics currently on the market or its biosimilars. Some protein therapeutics currently on the market are not high molecular weight, but still administered by IV infusion because they require high doses for therapeutic effectiveness. In some embodiments, liquid formulations of these low molecular weight proteins as defined herein are provided with a concentration for delivering therapeutically effective amounts for SC or IM injection.

[0118] Currently, antibody therapeutics on the market include belimumab (BENLYSTA®), golimumab (SIMPONI ARIA®), abciximab (REOPRO®), a 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®), and rifampinib (RIRI). KIT®), daclizumab (caclizumab) (ZENAPAX®), ibritumomab tiuxetan (ZEVALIN®), eculizumab (SOLIRIS®), ipilimumab (YERVOY®), muromonab-CD3 (ORTHOCLONE OKT3®), raxibacumab, nimotuzumab (THERACIM®), brentuximab vedotin (ADCETRIS®), adalimumab (HUMIRA®), golimumab (SIMPONI®), palivizumab (SYNAGIS®), omalizumab (XOLAIR®), and ustekinumab (STELARA®).

[0119] Natalizumab, a humanized mAb against the cell adhesion molecule α4-integrin, is used in the treatment of multiple sclerosis and Crohn's disease. Previously marketed under the trade name ANTEGREN®, natalizumab is now jointly marketed as TYSABRI® by Biogen Idec ("Biogen") and Elan Corp. ("Elan"). TYSABRI® is produced in mouse myeloma cells. Each 15 mL dose contains 300 mg natalizumab, 123 mg sodium chloride (USP), 17.0 mg monobasic sodium phosphate monohydrate (USP), 7.24 mg dibasic sodium phosphate heptahydrate (USP), 3.0 mg polysorbate 80 (USP / NF) in water for IV injection (USP) at pH 6.1. Natalizumab is usually administered by monthly intravenous (IV) infusion and has proven effective in treating the symptoms of both multiple sclerosis and Crohn's disease, as well as preventing relapses, vision loss, cognitive decline, and significantly improving patients' quality of life.

[0120] As used herein, the term "natalizumab" includes the mAb against the cell adhesion molecule α4-integrin, known under the international nonproprietary name "NATALIZUMAB", or an antigen-binding portion thereof. Natalizumab includes the antibodies described in U.S. Patent No. 5,840,299, U.S. Patent No. 6,033,665, U.S. Patent No. 6,602,503, U.S. Patent No. 5,168,062, U.S. Patent No. 5,385,839, and U.S. Patent No. 5,730,978. Natalizumab includes the active agent in products marketed under the trademark TYSABRI® by Biogen Idec and Elan Corporation, or biosimilar products thereof.

[0121] Cetuximab is an epidermal growth factor receptor (EGFR) inhibitor used to treat metastatic colorectal and head and neck cancer. Cetuximab is a chimeric (mouse / human) mAb that is usually administered by IV infusion. Cetuximab is marketed for IV use only under the trade name ERBITUX® by Bristol-Myers Squibb Company (North America; "Bristol-Myers Squibb"), Eli Lilly and Company (North America; "Eli Lilly"), and Merck KGaA. ERBITUX® is produced in mammalian (mouse myeloma) cell culture. Each single-use 50 mL vial of ERBITUX® contains 100 mg of cetuximab at a concentration of 2 mg / mL formulated in a preservative-free solution containing 8.48 mg / mL sodium chloride, 1.88 mg / mL dibasic sodium phosphate heptahydrate, 0.42 mg / mL monobasic sodium phosphate monohydrate, and Water for IV Injection, USP.

[0122] Cetuximab is indicated for the treatment of patients with epidermal growth factor receptor (EGFR)-expressing, KRAS wild-type metastatic colorectal cancer (mCRC), in combination with chemotherapy and as a single agent in patients who have failed oxaliplatin and irinotecan-based therapy or who are intolerant to irinotecan. Cetuximab is indicated for the treatment of patients with squamous cell carcinoma of the head and neck in combination with platinum-based chemotherapy for first-line treatment of recurrent and / or metastatic disease, and in combination with radiation therapy for locally advanced disease. Approximately 75% of patients with metastatic colorectal cancer have EGFR-expressing tumors and are therefore considered eligible for treatment with cetuximab or panitumumab according to FDA guidelines.

[0123] As used herein, the term "cetuximab" includes the mAb known by the International Nonproprietary Name "CETUXIMAB," or an antigen-binding portion thereof. Cetuximab includes the antibodies described in U.S. Pat. No. 6,217,866. Cetuximab includes the active agent in products marketed under the trademark ERBITUX® and in biosimilar products thereof. Biosimilars of ERBITUX® are available from Amgen, AlphaMab Co., Ltd. ("AlphaMab"), and Actavis. plc ("Actavis"), which is a wholly owned subsidiary of Actavis.

[0124] Bevacizumab, a humanized mAb that inhibits vascular endothelial growth factor A (VEGF-A), acts as an antiangiogenic agent. Bevacizumab is marketed under the trademark AVASTIN® by Genentech, Inc. ("Genentech") and F. Hoffmann-La Roche, LTD ("Roche"). Bevacizumab is approved for treating a variety of cancers, including colorectal cancer, lung cancer, breast cancer (outside the United States), glioblastoma (United States only), renal cancer, and ovarian cancer. AVASTIN® was approved by the FDA in 2004 for use in metastatic colorectal cancer when used in conjunction with standard chemotherapy treatment (as first-line treatment) and 5-fluorouracil-based therapy for second-line metastatic colorectal cancer. In 2006, the FDA approved AVASTIN® for use in first-line advanced non-squamous non-small cell lung cancer in combination with carboplatin / paclitaxel chemotherapy. AVASTIN® is administered as an IV infusion every three weeks at a dose of either 15 mg / kg or 7.5 mg / kg. With carboplatin-based chemotherapy, higher doses are usually administered, while with cisplatin-based chemotherapy, lower doses are administered. In 2009, the FDA approved AVASTIN® for use in metastatic renal cell carcinoma (a form of kidney cancer). The FDA also granted accelerated approval of AVASTIN® in 2009 for the treatment of recurrent glioblastoma multiforme. Treatment for early growth is still in Phase III clinical trials.

[0125] The National Comprehensive Cancer Network (NCCN) recommends that patients with BRAF should receive chemotherapy in combination with any of the platinum-based chemotherapy regimens, followed by chemotherapy for ≥ 1 year after disease progression. In 2010, NCCN updated its Clinical Practice Guidelines for Oncology (NCCN Guidelines) for breast cancer, recommending bevacizumab as standard first-line treatment, with bevacizumab maintained until progression. and reviewed recommendations regarding the use of bevacizumab (AVASTIN®, Genentech / Roche) in the treatment of metastatic breast cancer.

[0126] As used herein, the term "bevacizumab" includes the mAb that inhibits vascular endothelial growth factor A (VEGF-A), known under the international nonproprietary / common name "BEVACIZUMAB," or an antigen-binding portion thereof. Bevacizumab is described in U.S. Patent No. 6,054,297. Bevacizumab includes the active agent in products marketed under the trademark AVASTIN® and in biosimilar products thereof. Biosimilars of AVASTIN® can include those currently in development by Amgen, Actavis, AlphaMab, and Pfizer, Inc. ("Pfizer"). Biosimilars of AVASTIN® can include the biosimilar known as BCD-021, which is manufactured by Biocad and is currently in clinical trials in the United States.

[0127] Trastuzumab is a mAb that blocks the HER2 / neu receptor. Trastuzumab is marketed under the trademark HERCEPTIN® by Genentech, Inc. HERCEPTIN® is produced by a mammalian cell (Chinese Hamster Ovary (CHO)) system. HERCEPTIN® is a sterile, white to pale yellow, preservative-free, lyophilized powder for IV administration. Each HERCEPTIN® vial contains 440 mg trastuzumab, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, 400 mg a,a-trehalose dihydrate, and 1.8 mg polysorbate 20 (USP). Reconstitution with 20 mL water results in a multi-dose solution containing 21 mg / mL trastuzumab. HERCEPTIN® is currently administered by IV infusion at dosages ranging from about 2 mg / kg to about 8 mg / kg as frequently as weekly.

[0128] Trastuzumab is primarily used to treat certain types of breast cancer. The HER2 gene is amplified in 20-30% of early stage breast cancers, which results in overexpression of epidermal growth factor (EGF) receptors in the cell membrane. Trastuzumab is commonly administered as maintenance therapy to patients with HER2-positive breast cancer, usually for one year after chemotherapy. Trastuzumab is currently administered by IV infusion at doses ranging from about 2 mg / kg to about 8 mg / kg, as frequently as weekly.

[0129] As used herein, the term "trastuzumab" includes the mAb that blocks the HER2 / neu receptor, known under the International Nonproprietary / Common Name "TRASTUZUMAB", or an antigen-binding portion thereof. Trastuzumab is described in U.S. Patent No. 5,821,337. Trastuzumab includes the active agent in products marketed under the trade name HERCEPTIN®, and biosimilars thereof. The term "trastuzumab" includes the active agent in biosimilar HERCEPTIN® products marketed under the trade name HERTRAZ® by Mylan, Inc. ("Mylan"), and CANMAB® by Biocon, Ltd. ("Biocon"). Trastuzumab may include the active agent in biosimilar HERCEPTIN® products under development by Amgen and PlantForm Corporation, Canada.

[0130] Infliximab is a mAb against tumor necrosis factor alpha (TNF-α) that is used to treat autoimmune diseases. Infliximab is marketed under the trademark REMICADE® by Janssen Global Services, LLC ("Janssen") in the United States, Mitsubishi Tanabe Pharma Corporation 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 about 144 kDa. In some embodiments, the formulation contains a biosimilar of REMICADE®, such as REMSIMA™ or INFLECTRA™. Both REMSIMA™, developed by Celltrion, Inc. ("Celltrion"), and INFLECTRA™, developed by Hospira Inc, UK, have been recommended for regulatory approval in Europe. Celltrion has filed an application for REMSIMA™ with the FDA. Infliximab is currently administered by IV infusion at doses ranging from about 3 mg / kg to about 10 mg / kg.

[0131] Infliximab contains approximately 30% of the amino acid sequence of the mouse variable region, which confers antigen-binding specificity to human TNFα. The remaining 70% corresponds to the constant region of the human IgG1 heavy chain and the constant region of the human kappa light chain. Infliximab has high affinity for human TNFα, a cytokine with multiple biological actions, including mediating inflammatory responses and modulating the immune system.

[0132] Infliximab is a recombinant antibody that is generally produced and secreted from mouse myeloma cells (SP2 / 0 cells). The antibody is currently manufactured by continuous perfusion cell culture. Infliximab monoclonal antibody is expressed using a chimeric antibody gene consisting of variable region sequences cloned from mouse anti-TNFα hybridoma A2 and human antibody constant region sequences provided by a plasmid expression vector. Generation of mouse anti-TNFα hybridomas is performed by immunizing BALB / c mice with purified recombinant human TNFα. Heavy and light chain vector constructs are linearized and transfected into Sp2 / 0 cells by electroporation. Standard purification steps include chromatographic purification, viral inactivation, nanofiltration, and ultrafiltration / diafiltration.

[0133] As used herein, the term "infliximab" includes the chimeric murine / human monoclonal antibody known under the International Nonproprietary Name "INFLIXIMAB", or an antigen-binding portion thereof. Infliximab neutralizes the biological activity of TNFα by binding with high affinity to soluble and transmembrane forms of TNFα and inhibits the binding of TNFα to its receptor. Infliximab is described in U.S. Pat. No. 5,698,195. The term "infliximab" includes the active agent in products marketed or proposed to be marketed under the trademarks REMICADE® by several entities, REMSIMA™ by Celltrion, and INFLECTRA™ by Hospira, Inc. ("Hospira"). Infliximab is supplied as a sterile lyophilized cake for reconstitution and dilution. Each vial of infliximab contains 100 mg of infliximab and excipients such as monobasic sodium phosphate monohydrate, dibasic sodium phosphate dihydrate, sucrose and polysorbate 80.

[0134] Denosumab (PROLIA® and XGEVA®) is a human mAb and the first RANKL inhibitor approved for use in postmenopausal women at risk for osteoporosis and in patients with bone metastases from solid tumors. Denosumab is in Phase II trials for the treatment of rheumatoid arthritis.

[0135] Panitumumab is a fully human mAb approved by the FDA for the treatment of EGFR-expressing metastatic cancer with disease progression. Panitumumab is marketed by Amgen under the trademark VECTIBIX®. VECTIBIX® is packaged as a 20 mg / ml panitumumab concentrate in 5 ml, 10 ml and 15 ml vials for IV infusion. When prepared according to the package insert, the final panitumumab concentration does not exceed 10 mg / ml. VECTIBIX® is administered as an intravenous infusion at a dose of 6 mg / kg every 14 days. As used herein, the term "panitumumab" includes the anti-human epidermal growth factor receptor, known by the international nonproprietary name "PANITUMUMAB". The term "panitumumab" includes the active agent in products marketed by Amgen under the trademark VECTIBIX® and its biosimilars. The term "panitumumab" includes the monoclonal antibody described in U.S. Patent No. 6,235,883. The term "panitumumab" includes the active agent in biosimilar VECTIBIX® products, including the biosimilar VECTIBIX® being developed by BioXpress, SA ("BioXpress").

[0136] Belimumab (BENLYSTA®) is a human mAb with a molecular weight of about 151.8 kDa that inhibits B-cell activating factor (BAFF). Belimumab is approved in the United States, Canada, and Europe for the treatment of systemic lupus erythematosus. Belimumab is currently administered to lupus patients by IV infusion at a dose of 10 mg / kg. High molecular weight, low viscosity protein formulations can contain belimumab, preferably at a concentration of about 400 mg / mL to about 1,000 mg / mL. The preferred range is calculated based on a body weight of 40 to 100 kg (about 80 to 220 lbs) in a volume of 1 mL.

[0137] Abciximab (REOPRO®) is manufactured by Janssen Biologics BV and distributed by Eli Lilly & Company ("Eli Lilly"). Abciximab is the Fab fragment of the chimeric human-mouse monoclonal antibody 7E3. Abciximab inhibits platelet aggregation by binding to the glycoprotein (GP) IIb / IIIa receptor on human platelets and preventing the binding of fibrinogen, von Willebrand factor, and other adhesion molecules. Abciximab also binds to the vitronectin (αvβ3) receptor, which is found on platelets and endothelial and smooth muscle cells of the blood vessel wall. Abciximab is a platelet aggregation inhibitor primarily used during and after coronary artery surgery. Abciximab is administered initially by IV infusion at a bolus of 0.25 mg / kg, followed by a continuous IV infusion of 0.125 mcg / kg / min for 12 hours.

[0138] Tositumomab (BEXXAR®) is a drug for the treatment of follicular lymphoma. Tositumomab is an IgG2a anti-CD20 mAb derived from immortalized mouse cells. Tositumomab is administered in sequential infusions of a non-radioactive mAb followed by iodine (131I) tositumomab, the same antibody covalently linked to the radionuclide iodine-131. Clinical trials have established the efficacy of the tositumomab / iodine tositumomab regimen in patients with relapsed refractory follicular lymphoma. BEXXAR® is currently administered at a dose of 450 mg by IV infusion.

[0139] Alemtuzumab (marketed as CAMPATH®, MABCAMPATH® or CAMPATH-1H®, and currently under further development as LEMTRADA®) is a mAb used in the treatment of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), and T-cell lymphoma. Alemtuzumab is also used under clinical trial protocols for the treatment of several autoimmune diseases, such as multiple sclerosis. Alemtuzumab has a weight of approximately 145.5 kDa. Alemtuzumab is administered daily as an IV infusion of 30 mg to patients with B-cell chronic lymphocytic leukemia.

[0140] Palivizumab (SYNAGIS®) is a humanized mAb directed against an epitope in the A antigenic site of the F protein of respiratory syncytial virus. In two Phase III clinical trials in pediatric populations, palivizumab reduced the risk of hospitalization due to respiratory syncytial virus infection by 55% and 45%. Palivizumab is administered by IM injection at 15 mg / kg once a month.

[0141] Ofatumumab is a human anti-CD20 mAb that appears to inhibit early B-lymphocyte activation. Ofatumumab is marketed under the trademark ARZERRA® by GlaxoSmithKline, plc ("GlaxoSmithKline"). ARZERRA® is sold in single-use vials containing 100 mg / 5 mL and 1,000 mg / 50 mL ofatumumab for IV infusion. Ofatumumab is FDA approved to treat chronic lymphocytic leukemia and has also shown potential to treat follicular non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing-remitting multiple sclerosis. Ofatumumab has a molecular weight of approximately 149 kDa. Ofatumumab is currently administered at an initial dose of 300 mg by IV infusion, followed by weekly infusions of 2,000 mg. As used herein, the term "ofatumumab" includes the anti-CD20 mAb known by the International Nonproprietary Name "OFATUMUMAB". The term "ofatumumab" includes the active agent in products marketed under the trademark ARZERRA® and biosimilars thereof. The term "ofatumumab" includes the active agent in the biosimilar ARZERRA® product being developed by BioExpress. High molecular weight, low viscosity liquid protein formulations can include ofatumumab, preferably at a concentration of about 300 mg / mL to about 2,000 mg / mL.

[0142] Trastuzumab emtansine (in the United States, ado-trastuzumab emtansine, marketed as KADCYLA®) is an antibody-drug conjugate consisting of the mAb trastuzumab linked to the cytotoxic agent mertansine (DM1®). Trastuzumab, described above, stops the growth of cancer cells by binding to the HER2 / neu receptor, while mertansine enters cells and destroys them by binding to microtubules. In the United States, trastuzumab emtansine has been specifically approved for the treatment of recurrent, HER2-positive, metastatic breast cancer. Several phase III trials of trastuzumab emtansine are planned or ongoing in 2014. Trastuzumab emtansine is currently administered by IV infusion of 3.6 mg / kg. High molecular weight, low viscosity liquid formulations can contain trastuzumab emtansine, preferably at a concentration of about 144 mg / mL to about 360 mg / mL.

[0143] Pertuzumab (PERJETA®) is a mAb that inhibits HER2 dimerization. Pertuzumab received FDA approval for the treatment of HER2-positive metastatic breast cancer in 2012. The currently recommended dosage of pertuzumab is 420 mg to 840 mg by IV infusion. High molecular weight, low viscosity liquid formulations can contain pertuzumab at a concentration of preferably about 420 mg / mL to about 840 mg / mL.

[0144] Daclizumab is a humanized anti-CD25 mAb that has been used to prevent rejection in organ transplants, especially kidney transplants. The drug is also under investigation for the treatment of multiple sclerosis. Daclizumab has a molecular weight of approximately 143 kDa. Daclizumab is marketed in the United States by Hoffmann-La Roche, Ltd. ("Roche") as ZENAPAX® and is administered by IV infusion at 1 mg / kg. Daclizumab High-Yield Process (DAC HYP; BIIB019; Biogen) is being used to treat relapsing-remitting multiple sclerosis. It is in Phase III clinical trials by Idec ("Biogen") and AbbVie, Inc. ("AbbVie") as a subcutaneous injection of 150 mg once per month. High molecular weight, low viscosity liquid formulations can contain daclizumab, preferably at a concentration of about 40 mg / mL to about 300 mg / mL.

[0145] Eculizumab (SOLIRIS®) is a humanized mAb approved for the treatment of rare blood disorders such as paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab, which has a molecular weight of about 148 kDa, is in development by Alexion Pharmaceuticals, Inc. ("Alexion"). Eculizumab is administered by IV infusion in an amount of about 600 mg to about 1,200 mg. High molecular weight, low viscosity liquid formulations can contain eculizumab, preferably in a concentration of about 500 mg / mL to about 1,200 mg / mL.

[0146] Tocilizumab (ACTEMRA®) is a humanized mAb against the interleukin-6 receptor. Tocilizumab is an immunosuppressant drug primarily for treating rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis, a severe form of RA in children. Tocilizumab is commonly administered by IV infusion at a dose of about 6 mg / kg to about 8 mg / kg. High molecular weight, low viscosity liquid formulations can contain tocilizumab, preferably at a concentration of about 240 mg / mL to about 800 mg / mL.

[0147] 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 subtypes. Rituximab has been shown to be an effective rheumatoid arthritis treatment. Rituximab is widely used off-label to treat difficult cases of multiple sclerosis, systemic lupus erythematosus, and autoimmune anemia.

[0148] Rituximab is jointly marketed in the United States by Biogen and Genentech under the trade name RITUXAN®, and outside the United States by Roche under the trade name MABTHERA®. RITUXAN® is sold in single-use vials containing 100 mg / 10 mL and 500 mg / 50 mL. RITUXAN® is available in a concentration of approximately 375 mg / m 2 It is usually administered by IV infusion. As used herein, the term "rituximab" includes the anti-CD20 mAb known by the International Nonproprietary Name / Common Name "RITUXIMAB." Mabs include the mAbs described in U.S. Patent No. 5,736, 137. Rituximab includes the active agent in products marketed under the trade names RITUXAN® and MABTHERA®, and their biosimilars.

[0149] High molecular weight, low viscosity liquid formulations can contain rituximab, preferably in a concentration of about 475 mg / mL to about 875 mg / mL (approximated using a body surface area of ​​1.3 to 2.3 square meters derived from the Mosteller formula for a human ranging from 5 ft, 40 kg to 6 ft, 100 kg). Concentrations are calculated for a 1 mL formulation.

[0150] Ipilimumab is a human mAb developed by Bristol-Myers Squibb Company ("Bristol-Myers Squibb"). Marketed as YERVOY®, ipilimumab is used to treat melanoma and is in clinical trials for the treatment of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and metastatic hormone-refractory prostate cancer. Ipilimumab is currently administered by IV infusion of 3 mg / kg. High molecular weight, low viscosity liquid formulations can contain ipilimumab, preferably at a concentration of about 120 mg / mL to about 300 mg / mL.

[0151] Raxibacumab (ABthrax®) is a human mAb intended for the prevention and treatment of inhalation anthrax. Raxibacumab is currently administered by IV infusion. In adults and children over 50 kg, the suggested dosage is 40 mg / kg. High molecular weight, low viscosity liquid formulations can contain raxibacumab, preferably in a concentration of about 1,000 mg / mL to about 4,000 mg / mL.

[0152] Nimotuzumab (THERACIM®, BIOMAB EGFR®, THERALOC®, CIMAher®) is a humanized mAb with a molecular weight of about 151 kDa that has been used to treat squamous cell carcinoma of the head and neck, recurrent or refractory high-grade malignant glioma, anaplastic astrocytoma, glioblastoma, and diffuse intrinsic pontine glioma. Nimotuzumab is typically administered by IV infusion of about 200 mg weekly. High molecular weight, low viscosity liquid formulations can contain nimotuzumab, preferably at a concentration of about 200 mg / mL.

[0153] Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate directed against the protein CD30, which is expressed in classical Hodgkin's lymphoma and systemic anaplastic large cell lymphoma. Brentuximab vedotin has been administered by IV infusion at about 1.8 mg / kg. High molecular weight, low viscosity liquid formulations can contain brentuximab vedotin, preferably at a concentration of about 80 mg / mL to about 200 mg / mL.

[0154] Itolizumab (ALZUMAB®) is a humanized IgG1 mAb developed by Biocon. Itolizumab has successfully completed Phase III trials in patients with moderate to severe psoriasis. Itolizumab has received marketing approval in India. No application for FDA approval has been submitted.

[0155] Obinutuzumab (GAZYVA®), originally developed by Roche and further developed in a collaboration agreement with Biogen, is a humanized anti-CD20 mAb approved for the treatment of chronic lymphocytic leukemia. Obinutuzumab is also being investigated in Phase III clinical trials in patients with various lymphomas. Doses of approximately 1,000 mg are administered by IV infusion.

[0156] Certolizumab pegol (CIMZIA®) is a recombinant humanized antibody Fab′ fragment with specificity for human tumor necrosis factor alpha (TNFα) conjugated to a polyethylene glycol of approximately 40 kDa (PEG2MAL40K). The molecular weight of certolizumab pegol is approximately 91 kDa.

[0157] Other antibody therapeutics that can be formulated with viscosity-lowering agents include CT-P6 from Celltrion, Inc. (Celltrion).

[0158] Antibody therapeutics in late-stage trials and development

[0159] Progression of antibody therapeutics into late-stage clinical development; and regulatory review are proceeding at a rapid pace. In 2014, there are over 300 mAbs in clinical trials and 30 commercially sponsored antibody therapeutics being evaluated in late-stage studies. The first marketing applications for two mAbs (vedolizumab and ramucirumab) were recently submitted to the FDA. Amgen is currently sponsoring multiple ongoing Phase III trials of the use of brodalumab in patients with plaque psoriasis, including planning or recruiting additional trials. XBiotech, Inc. is sponsoring two Phase I clinical trials of MABp1 (Xilonix) in patients with advanced cancer or type 2 diabetes. Additional trials of MABp1 are recruiting patients. Several trials are underway or recruiting patients for the treatment of leukemia with moxetumomab pasudotox, sponsored by MedImmune, LLC ("MedImmune"). Long-term safety and efficacy studies are underway for the use of tildrakizumab for the treatment of chronic plaque psoriasis. Several Phase II trials of the use of rilotumumab for the treatment of various cancers have recently been completed.

[0160] At least 28 mAbs are high molecular weight proteins currently in or have recently completed Phase III studies for the treatment of inflammatory or immunological disorders, cancer, high cholesterol, osteoporosis, Alzheimer's disease, and infectious diseases. mAbs in or that have recently completed Phase III trials include AMG145, elotuzumab, epratuzumab, farletuzumab (MORAb-003), gantenerumab (RG1450), gevokizumab, inotuzumab ozogamicin, itolizumab, ixekizumab, lebrikizumab, mepolizumab, naptumomab estafenatox, necitumumab, nivolumab, ocrelizumab, onartuzumab, racotumomab, ramucirumab, reslizumab, romosozumab, sarilumab, secukinumab, sirukumab, solanezumab, tabalumab, and vedolizumab. A mAb mixture (actoxumab and bezlotoxumab) is also being evaluated in Phase III trials. See, e.g., Reichert, MAbs 5:1-4, 2013.

[0161] Vedolizumab is a mAb being developed by Millennium Pharmaceuticals, Inc. ("Millennium"; a subsidiary of Takeda Pharmaceutical Company Limited ("Takeda"). Vedolizumab has been found to be safe and highly effective in inducing and maintaining clinical remission in patients with moderate to severe ulcerative colitis. Phase III clinical trials have demonstrated that vedolizumab meets the objectives of inducing clinical response and maintaining remission in patients with Crohn's disease and ulcerative colitis. Studies evaluating long-term clinical outcomes have shown that nearly 60% of patients achieved clinical remission. The typical dose of vedolizumab is 6 mg / kg by IV infusion.

[0162] Ramucirumab is a human mAb in development 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 is administered at approximately 8 mg / kg by IV infusion in some Phase III trials.

[0163] Rilotumumab is a human mAb that inhibits the action of hepatocyte growth factor / scatter factor. Developed by Amgen, rilotumumab 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 administers approximately 15 mg / kg of rilotumumab by IV infusion.

[0164] Evolocumab (AMG145), also developed by Amgen, is a mAb that binds to PCSK9. Evolocumab is indicated for hypercholesterolemia and hyperlipidemia.

[0165] Alirocumab (REGN727) is a human mAb from Regeneron Pharmaceuticals, Inc. ("Regeneron") and Sanofi-Aventis US LLC ("Sanofi") indicated for hypercholesterolemia and acute coronary syndromes.

[0166] ABR-217620, naptumomab estafenatox, from Active Biotech AB ("Active Biotech"), is a mAb indicated for renal cell carcinoma.

[0167] CIMAB, SA ("CIMAB"), from Laboratorio Elea SACIFy A, is a mAb indicated for the treatment of non-small cell lung cancer.

[0168] Other antibodies that may be formulated with viscosity-lowering agents include bococizumab (PF-04950615) and tanezumab; ganitumab, blinatumomab, trebananib from Amgen; anthrax immune globulin from Cangene Corporation; teplizumab from MacroGenics, Inc.; MK-3222, MK-6072 from Merck & Co. ("Merck"); girentuximab from Wilex AG; RIGScan from Navidea Biopharmaceuticals ("Navidea"); PF-05280014 from Pfizer; SA237 from Chugai Pharmaceutical Co., Ltd. ("Chugai"); guselkumab from Janssen / Johnson and Johnson Services, Inc. ("J&J"); antithrombin gamma (KW-3357) from Kyowa; and CT-P10 from Celltrion.

[0169] Antibodies in early-stage clinical trials

[0170] Many mAbs have recently entered or are entering clinical trials. They can include proteins currently administered by IV infusion, preferably with a molecular weight of more than about 120 kDa, usually between about 140 kDa and about 180 kDa. They can also include such high molecular weight proteins, such as albumin-conjugated drugs or peptides, also in clinical trials or approved by the FDA. Many mAbs from Amgen are currently in clinical trials. These can be high molecular weight proteins, such as AMG557, a human monoclonal antibody jointly developed by Amgen and AstraZeneca and currently in Phase I trials for the treatment of lupus. Similarly, AMG729 is a humanized mAb developed by Amgen and currently in Phase I trials for the treatment of lupus and rheumatoid arthritis. Additionally, AMG110 is a mAb against epithelial cell adhesion molecule. AMG157, co-developed by Amgen and AstraZeneca, is a human mAb currently in Phase I for the treatment of asthma. AMG167 is a humanized mAb being evaluated in multiple Phase I trials for the treatment of osteopenia. AMG334 is a human mAb that inhibits calcitonin gene-related peptide, which has completed a Phase I dose study and is currently in Phase II studies for the treatment of migraine and flushing. AMG780 is a human anti-angiopoietin mAb that inhibits the interaction between the endothelial cell-selective Tie2 receptor and its ligands Ang1 and Ang2, and has recently completed Phase I trials as a cancer treatment. AMG811 is a human monoclonal antibody that inhibits interferon gamma, which is being investigated as a treatment for systemic lupus erythematosus. AMG820 is a human mAb that inhibits c-fms and reduces tumor-associated macrophage (TAM) function, and is being investigated as a cancer treatment. Co-developed by Amgen and AstraZeneca, AMG181 is a human mAb that blocks the action of alpha4 / beta7 and is in Phase II trials as a treatment for ulcerative colitis and Crohn's disease.

[0171] Many mAbs are currently in clinical trials for the treatment of autoimmune disorders. These mAbs can be included in high molecular weight, low viscosity liquid formulations. RG7624 is a fully human mAb designed to specifically and selectively bind to the human interleukin-17 family of cytokines. Phase I clinical trials evaluating RG7624 for autoimmune diseases are ongoing. BIIB033 is an anti-LINGO-1 mAb by Biogen currently in Phase II trials for the treatment of multiple sclerosis.

[0172] High molecular weight proteins can also include AGS-009, an IFN-alpha targeting mAb developed by Argos Therapeutics, Inc., which recently completed Phase I trials for the treatment of lupus. Patients receive up to 30 mg / kg of AGS-009 by IV infusion. BT-061, developed by AbbVie, 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-IL-6 mAb, is in Phase II trials by Bristol-Myers Squibb.

[0173] CNTO-136 (sirukumab) and CNTO-1959 are mABs that have recently completed Phase II and Phase III trials by Janssen. Daclizumab (previously marketed by Roche as ZENAPAX®) is currently in or has recently completed multiple Phase III trials by AbbVie for the treatment of multiple sclerosis. Epratuzumab is a humanized mAb in Phase III trials for the treatment of lupus. Canakinumab (ILARIS®) is a human mAb that targets interleukin-1 beta. Canakinumab has been approved for the treatment of cryopyrin-associated periodic syndromes. Canakinumab is a treatment for chronic obstructive pulmonary disease (COPD). It is in Phase I trials as a potential treatment for rheumatoid arthritis, gout, and coronary artery disease. Mavrilimumab is a human mAb designed for the treatment of rheumatoid arthritis. Discovered by Cambridge Antibody Technology as CAM-3001, mavrilimumab is being developed by MedImmune.

[0174] MEDI-546 and MEDI-570 are mAbs currently in Phase I and Phase II trials by AstraZeneca for the treatment of lupus. MEDI-546 is being administered in a Phase II study by regular IV infusion of 300-1,000 mg. MEDI-551, another mAb being developed by AstraZeneca for various indications, is also currently administered by IV infusion. NN8209, a mAb that blocks the C5aR receptor being developed by Novo Nordisk A / S ("Novo Nordisk"), has completed a Phase II dose trial for the treatment of rheumatoid arthritis. NN8210 is another anti-C5aR mAb being developed by Novo Nordisk and is currently in Phase I trials. IPH2201 (NN8765) is a humanized mAb targeting NKG2A being developed by Novo Nordisk to treat patients with inflammatory conditions and autoimmune diseases. NN8765 has recently completed Phase I trials.

[0175] Olokizumab is a humanized mAb that potently targets the cytokine IL-6. IL-6 is involved in several autoimmune and inflammatory pathways. Olokizumab has completed Phase II trials for the treatment of rheumatoid arthritis. Otelixizumab, also known as TRX4, is a mAb in development for the treatment of type I diabetes, rheumatoid arthritis, and other autoimmune diseases. Ozolarizumab is a humanized mAb that has completed Phase II trials.

[0176] Pfizer currently has mAbs PD-360324 and PF-04236921 in Phase I trials for the treatment of lupus. A rituximab biosimilar, PF-05280586, also developed by Pfizer, is in Phase I / II trials for rheumatoid arthritis.

[0177] Rontalizumab is a humanized mAb being developed by Genentech. Rontalizumab recently completed Phase II trials for the treatment of lupus. SAR113244 (anti-CXCR5) is a mAb by Sanofi in Phase I trials. Sifalimumab (anti-IFN alpha mAb) is a mAb in Phase II trials for the treatment of lupus.

[0178] The high molecular weight, low viscosity liquid formulations can include one of the mAbs in early stage clinical development for treating various hematological disorders. For example, belimumab (BENLYSTA®) recently completed Phase I trials in patients with vasculitis. Other mAbs in early stage trials for hematological disorders include BI-655075 from Boehringer Ingelheim GmbH "Boehringer Ingelheim", Eli Ferroportin mAb and hepcidin mAb from Lily, and Selexys and SelG1 from Selexys Pharmaceuticals, Corp. ("Selexys").

[0179] One or more mAbs in early stages of development to treat various cancers and related conditions may be included in the low viscosity, high molecular weight liquid formulation. United Therapeutics, Corporation has two mAbs in Phase I trials, 8H9 mAb and ch14.18 mAb. mAbs from AbbVie, ABT-806, enavatuzumab, and volociximab, are in early stages of development. Actinium Pharmaceuticals, Inc. has conducted early stage trials with mAbs actimab-A (M195 mAb), anti-CD45 mAb, and iomab-B. Seattle Genetics, Inc. ("Seattle Genetics") has several mAbs in early stage trials for cancer and related conditions, including an anti-CD22 ADC (RG7593; pinatuzumab vedotin), an anti-CD79b ADC (RG7596), an anti-STEAP1 ADC (RG7450), ASG-5ME and ASG-22ME from Agensys, Inc. ("Agensys"), the antibody-drug conjugate RG7458, and borsetuzumab mafodotin. Early stage cancer therapeutics from Genentech may be included in the low viscosity formulation, including ALT-836, antibody-drug conjugates RG7600 and DEDN6526A, anti-CD22 ADC (RG7593), anti-EGFL7 mAb (RG7414), anti-HER3 / EGFR DAF mAb (RG7597), anti-PD-L1 mAb (RG7446), DFRF4539A, MINT1526A. Bristol-Myers Squibb is developing early stage mAbs for cancer therapeutics, including those identified as anti-CXCR4, anti-PD-L1, IL-21 (BMS-982470), lirilumab and urelumab (anti-CD137).Other mAbs in early stage trials as treatments for cancer include APN301 (hu14.18-IL2) from Apeiron Biologics AG, AV-203 from AVEO Pharmaceuticals, Inc. ("AVEO"), AVX701 and AVX901 from AlphaVax, BAX-69 from Baxter International, Inc. ("Baxter"), BAY79-4620 and BAY20-10112 from Bayer HealthCare AG, BHQ880 from Novartis AG, 212-Pb-TCMC-trastuzumab from AREVA Med, AbGn-7 from AbGenomics International Inc, and ABIO-0501 (TALL-104) from Abiogen Pharma SpA.

[0180] Other antibody therapeutics that can be formulated with viscosity-lowering agents include alzumab, GA101, daratumumab, siltuximab, ALX-0061, ALX-0962, ALX-0761, bimagumab (BYM338), CT-011 (pidilizumab), actoxumab / bezlotoxumab (MK-3515A), MK-3475 (pembrolizumab), dalotuzumab (MK-0646), icrucumab (IMC-18F1, LY3012212), AMG139 (M EDI2070), SAR339658, dupilumab (REGN668), SAR156597, SAR256212, SAR279356, SAR3419, SAR153192 (REGN421, enoticumab), SAR307746 (nesbacumab), SAR650984, SAR566658, SAR391786, SAR228810, SAR252067, SGN-CD19A, SGN-CD33A, SGN-LIV1A, ASG15ME, anti-LIN GO, BIIB037, ALXN1007, teprotumumab, concizumab, anrukinzumab (IMA-638), ponezumab (PF-04360365), PF-03446962, PF-06252616, etrolizumab (RG7413), quilizumab, ranibizumab, lampalizumab, onclacumab, gentenerumab, crenezumab (RG7412), IMC-RON8 (narutomab), tremelimumab, vanticutumab, emucizumab (eem cizumab), ozanezumab, mapatumumab, tralokinumab, XmAb5871, XmAb7195, cixutumumab (LY3012217), LY2541546 (brosozumab), olaratumab (LY3012207), MEDI4893, MEDI573, MEDI0639, MEDI3617, MEDI4736, MEDI6469, MEDI0680, MEDI5872, PF-05236812 (AAB-003), PF-05082566, BI 1034020, RG7116, RG7356, RG7155, RG7212, RG7599, RG7636, RG7221, RG7652 (MPSK3169A), RG7686, HuMaxTFADC, MOR103, BT061, MOR208, OMP59R5 (anti-notch2 / 3), VAY736, MOR202, BAY94-9343, LJM716, OMP52M51, GSK933776, GSK249320, GSK1070806, NN8828, CEP-37250 / KHK2804 AGS-16M8F, AGS-16C3F, LY3016859, LY2495655, LY2875358, and LY2812176.

[0181] Other early stage mAbs that can be formulated with viscosity-lowering agents include benralizumab, MEDI-8968, anifrolumab, MEDI7183, sifalimumab, MEDI-575, tralokinumab from AstraZeneca and MedImmune; BAN2401 from Biogen Idec / Eisai Co., Ltd. ("Eisai") / BioArctic Neuroscience AB; CDP7657, a monovalent PEGylated Fab antibody fragment of anti-CD40L, and STX-100, BIIB059, anti-TWEAK (BIIB023), and BIIB022, anti-avB6 mAbs from Biogen; furanumab from Janssen and Amgen; BI-204 / RG7418 from BioInvent International / Genentech; BT-062 (indatuximab ravtansine) from Biotest Pharmaceuticals Corporation; Boehringer XmAb from Ingelheim / Xencor; anti-IP10 from Bristol-Myers Squibb; J591Lu-177 from BZL Biologics LLC; CDX-011 (glembatumumab vedotin), CDX-0401 from Celldex Therapeutics; foravirumab from Crucell; tigatuzumab from Daiichi Sankyo Co., Ltd; MORAb-004, MORAb-009 (amatuximab) from Eisai; LY2382770 from Eli Lilly; DI17E6 from EMD Serono Inc; zanolimumab from Emergent BioSolutions, Inc.; FG-3019 from FibroGen, Inc.; Fresenius Catumaxomab from SE&Co. KGaA; pateclizumab, lontalizumab from Genentech; fresolimumab from Genzyme&Sanofi; GS-6624 (simtuzumab) from Gilead; CNTO-328, bapineuzumab (AAB-001), carlumab, CNTO-136 from Janssen; KB003 from KaloBios Pharmaceuticals, Inc.; ASKP1240 from Kyowa; Labrys Biologics Inc.RN-307 from;ecloneximab from Life Science Pharmaceuticals;LY2495655, LY2928057, LY3015014, LY2951742 from Eli Lilly;MBL-HCV1 from MassBiologics;AME-133v from MENTRIK Biotech, LLC;avituzumab from Merck KGaA;MM-121 from Merrimack Pharmaceuticals;MCS110, QAX576, QBX258, QGE031 from Novartis AG;HCD122 from Novartis AG and XOMA Corporation ("XOMA");NN8555 from Novo Nordisk;bavituximab, Kotara from Peregrine Pharmaceuticals, Inc.;PSMA-ADC from Progenics Pharmaceuticals, Inc.;Quest oregovomab from Pharmatech, Inc.; fasinumab (REGN475), REGN1033, SAR231893, REGN846 from Regeneron; RG7160, CIM331, RG7745 from Roche; ibalizumab (TMB-355) from TaiMed Biologics Inc.; TCN-032 from Theraclone Sciences; TRC105 from TRACON Pharmaceuticals, Inc.; UB-421 from United Biomedical Inc.; VB4-845 from Viventia Bio, Inc.; ABT-110 from AbbVie; caplacizumab, ozoralizumab from Ablynx; PRO140 from CytoDyn, Inc.; GS-CDA1, MDX-1388 from Medarex, Inc.; AMG827, AMG888 from Amgen; TG ublituximab from Tolera Therapeutics, Inc.; TOL101 from Tolera Therapeutics, Inc.; huN901-DM1 (lorvotuzumab mertansine) from ImmunoGen Inc.; Immunomedics, Inc.epratuzumab Y-90 / veltuzumab combination (IMMU-102) from Biogen; anti-fibrin mAb / 3B6 / 22 Tc-99m from Agenix, Limited; and Alder. ALD403 from Biopharmaceuticals,Inc.;RN6G / PF-04382923 from Pfizer;CG201 from CG Therapeutics,Inc.;KB001-A from KaloBios Pharmaceuticals / Sanofi;KRN-23 from Kyowa;Y-90hPAM4 from Immunomedics,Inc.;talexitumab from Morphosys AG&OncoMed Pharmacetuicals,Inc.;LFG316 from Morphosys AG&Novartis AG;CNTO3157, CNTO6785 from Morphosys AG&Jannsen;RG6013 from Roche&Chugai;Merrimack Pharmaceuticals,Inc.MM-111 from Merrimack; GSK2862277 from GlaxoSmithKline; AMG282, AMG172, AMG595, AMG745, AMG761 from Amgen; BVX-20 from Biocon; CT-P19, CT-P24, CT-P25, CT-P26, CT-P27, CT-P4 from Celltrion; GSK284933, GSK2398852, GSK2618960, GSK1223249, GSK933776A from GlaxoSmithKline; anetumab ravtansine from Morphosys AG & Bayer AG; BI-836845 from Morphosys AG & Boehringer Ingelheim; Morphosys AG & Novartis NOV-7, NOV-8 from AG; MM-302, MM-310, MM-141, MM-131, MM-151 from Merrimack; RG7882 from Roche&Seattle Genetics; RG7841 from Roche / Genentech; PF-06410293, PF-06438179, PF-06439535, PF-04605412, PF-05280586 from Pfizer; RG7716, RG7936, gentenerumab, RG7444 from Roche; MEDI-547, MEDI-565, MEDI1814, MEDI4920, MEDI8897, MEDI-4212, MEDI-5117, MEDI-7814 from Astrazeneca; Bristol-Myers Urocupulumab, PCSK9 Adnectin from Squibb; FPA009, FPA145 from FivePrime Therapeutics,Inc.; GS-5745 from Gilead; BIW-8962, KHK4083, KHK6640 from Kyowa Hakko Kirin Co., Ltd.; MM-141 from Merck KGaA; REGN1154, REGN1193, REGN1400, REGN1500, REGN1908-1909, REGN2009, REGN2176-3, REGN728 from Regeneron; SAR307746 from Sanofi; SGN-CD70A from Seattle Genetics; ALX-0141, ALX-0171 from Ablynx; Immunomedics,Inc.milatuzumab-DOX, milatuzumab, TF2 from; MLN0264 from Millennium; ABT-981 from AbbVie; AbGn-168H from AbGenomics International Inc.; ficlatuzumab from AVEO; BI-505 from BioInvent International; CDX-1127, CDX-301 from Celldex Therapeutics; CLT-008 from Cellerant Therapeutics Inc.; VGX-100 from Circadian; U3-1565 from Daiichi Sankyo Co., Ltd.; DKN-01 from Dekkun Corp.; framvotumab (TYRP1 protein), IL-1β antibody, IMC-CS4 from Eli Lilly; VEGFR3 mAb, IMC-TR1 (LY3022859) from Eli Lilly and ImClone, LLC; anti-mem from Elusys Therapeutics Inc.; Galaxy HuL2G7 from Biotech LLC; IMGB853, IMGN529 from ImmunoGen Inc.; CNTO-5, CNTO-5825 from Janssen; KD-247 from Kaketsuken; KB004 from KaloBios Pharmaceuticals; MGA271, MGAH22 from MacroGenics, Inc.; XmAb5574 from MorphoSys AG / Xencor; ensituximab (NPC-1C) from Neogenix Oncology, Inc.; LFA102 from Novartis AG and XOMA; ATI355 from Novartis AG; SAN-300 from Santarus Inc.; SelG1 from Selexys; HuM195 / rGel from Targa Therapeutics, Corp.; Teva Pharmaceuticals, Industries Ltd. ("Teva") and Vaccinex Inc.These include VX15 from Theraclone Sciences; TCN-202 from Theraclone Sciences; XmAb2513, XmAb5872 from Xencor; XOMA3AB from XOMA and the National Institute of Allergy and Infectious Diseases; neuroblastoma antibody vaccine from MabVax Therapeutics; Cytolin from CytoDyn, Inc.; Thravixa from Emergent BioSolutions Inc.; and FB301 from Cytovance Biologics; rabies mAbs from Janssen and Sanofi; influenza mAbs from Janssen and funded in part by the National Institutes of Health; MB-003 and ZMapp from Mapp Biopharmaceutical, Inc.; and ZMab from Defyrus Inc..

[0182] Other protein therapeutics

[0183] The protein can be an enzyme, a fusion protein, a stealth or PEGylated protein, a vaccine, or an otherwise biologically active protein (or mixture of proteins). The term "enzyme," as used herein, refers to a protein or functional fragment thereof that catalyzes the biochemical conversion of a target molecule to a desired product.

[0184] Enzymes as drugs have at least two important characteristics: i) they often bind and act on their targets with high affinity and specificity, and ii) they are catalytic, converting multiple target molecules into desired products. In certain embodiments, proteins can be PEGylated, as defined herein.

[0185] The term "fusion protein" as used herein refers to a protein made from two different genes that code for two separate proteins. Fusion proteins are generally produced by recombinant DNA techniques known to those skilled in the art. The two proteins (or protein fragments) are covalently fused together to exhibit properties from both parent proteins.

[0186] Several fusion proteins are available on the market.

[0187] ENBREL® (etanercept) is a fusion protein marketed by Amgen that competitively inhibits TNF.

[0188] ELOCTATE® (Recombinant), Antihemophilic Factor Fc Fusion Protein, is a recombinant DNA-derived antihemophilic factor indicated for the control and prevention of bleeding episodes, perioperative management, and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with hemophilia A (congenital factor VIII deficiency).

[0189] EYLEA® (aflibercept) is a recombinant fusion protein consisting of a portion of the extracellular domain of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1, formulated as an isotonic solution for intravitreal administration. EYLEA (aflibercept) is a recombinant fusion protein consisting of a portion of the extracellular domain of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1, formulated as an isotonic solution for intravitreal administration. Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation that constitutes an additional 15% of the total molecular mass, resulting in a total molecular weight of 115 kDa. Aflibercept is produced in recombinant Chinese hamster ovary (CHO) cells and is marketed by Regeneron.

[0190] ALPROLIX™ (Recombinant), a coagulation factor IX Fc fusion protein, is a recombinant DNA-derived coagulation factor IX concentrate indicated for the control and prevention of bleeding episodes, perioperative management, and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with hemophilia B.

[0191] Pegloticase (KRYSTEXXA®) is a drug developed by Savient Pharmaceuticals, Inc. for the treatment of severe, treatment-refractory chronic gout and is the first drug approved for this indication. Pegloticase is a PEGylated recombinant porcine-like uricase with a molecular weight of about 497 kDa. Pegloticase is currently administered by IV infusion at about 8 mg / kg. High molecular weight, low viscosity liquid formulations can contain pegloticase, preferably at a concentration of about 300 mg / mL to about 800 mg / mL.

[0192] Alteplase (ACTIVASE®) is a tissue plasminogen activator produced by recombinant DNA technology. Alteplase is a purified glycoprotein containing 527 amino acids and is synthesized by using complementary DNA (cDNA) to native human tissue-type plasminogen activator obtained from a human melanoma cell line. Alteplase is administered by IV infusion of about 100 mg immediately after stroke symptoms. In some embodiments, a low viscosity formulation containing alteplase is provided, preferably at a concentration of about 100 mg / mL.

[0193] Glucarpidase (VORAXAZE®) is an FDA approved drug for the treatment of high levels of methotrexate (defined as at least 1 micromol / L) during the treatment of cancer patients with impaired renal function. Glucarpidase is administered by IV in a single dose of about 50 IU / kg. In some embodiments, a low viscosity formulation containing glucarpidase is provided.

[0194] Alglucosidase alfa (LUMIZYME®) is an orphan drug in enzyme replacement therapy for the treatment of Pompe disease (Glycogen storage disease type II), a rare lysosomal storage disorder. Alglucosidase alfa has a molecular weight of about 106 kDa and is currently administered by IV infusion of about 20 mg / kg. In some embodiments, low viscosity pharmaceutical formulations of alglucosidase alfa are provided, preferably having a concentration of about 100 mg / mL to about 2,000 mg / mL.

[0195] Pegdamase bovine (ADAGEN®) is an adenosine Bovine pegademase is a modified enzyme used in enzyme replacement therapy for the treatment of severe combined immunodeficiency (SCID), which is associated with a deficiency of adenosine deaminase. Bovine pegademase is a conjugate of various chains of monomethoxypolyethylene glycol (PEG) (molecular weight 5,000 Da) covalently linked to the adenosine deaminase enzyme derived from bovine intestine.

[0196] Alpha-galactosidase is a lysosomal enzyme that catalyzes the hydrolysis of the glycolipid globotriaosylceramide (GL-3) to galactose and ceramide dihexosides. Fabry disease is a rare inherited lysosomal storage disease characterized by subnormal enzyme activity of alpha-galactosidase and the resulting accumulation of GL-3. Agalsidase alpha (REPLAGAL®) is a human alpha-galactosidase A enzyme produced by a human cell line. Agalsidase beta (FABRAZYME®) is a recombinant human alpha-galactosidase expressed in a CHO cell line. Replagal is administered by intravenous infusion at a dose of 0.2 mg / kg every other week for the treatment of Fabry disease and (off-label) for the treatment of Gaucher disease. FABRAZYME® is administered by IV infusion at a dose of 1.0 mg / kg body weight every other week. Other lysosomal enzymes can also be used. For example, the protein can be a lysosomal enzyme as described in US2012 / 0148556.

[0197] Rasburicase (ELITEK®) is a recombinant urate oxidase indicated for the initial management of plasma uric acid levels in pediatric and adult patients with leukemia, lymphoma and solid malignancies undergoing anticancer therapy expected to result in tumor lysis and subsequent elevation of plasma uric acid. ELITEK® is administered daily by IV infusion at a dose of 0.2 mg / kg.

[0198] Imiglucerase (CEREZYME®) is a recombinant analog of human β-glucocerebrosidase. Initial dosages range from 2.5 U / kg body weight three times per week to 60 U / kg once every two weeks. CEREZYME® is administered by IV infusion.

[0199] Abraxane, a paclitaxel-conjugated albumin, is approved for metastatic breast cancer, non-small cell lung cancer, and late-stage pancreatic cancer.

[0200] Taliglucerase alfa (ELEYSO®) is a hydrolytic lysosomal glucocerebroside-specific enzyme indicated for long-term enzyme replacement therapy of Gaucher disease type I. The recommended dose is 60 U / kg body weight administered by intravenous infusion once every 2 weeks.

[0201] Laronidase (ALDURAZYME®) is a polymorphic variant of the human enzyme α-L-iduronidase produced by a CHO cell line. The recommended ALDURAZYME® dosage regimen is 0.58 mg / kg administered once weekly as an intravenous infusion.

[0202] Erosphase alfa (VIMIZIM®) is a human N-acetylgalactosamine-6-sulfatase produced by a CHO cell line by BioMarin Pharmaceuticals Inc. ("BioMarin"). Erosphase alfa was approved by the FDA on February 14, 2014 for the treatment of mucopolysaccharidosis type IVA. Erosphase alfa is administered weekly by intravenous infusion at a dosage of 2 mg / kg.

[0203] Other biologics that can be formulated with viscosity-reducing agents include asparaginase erwinia chrysanthemi (ERWINAZE® ), incobotulinumtoxin A (XEOMIN®), EPOGEN® (epoetin alfa), PROCRIT® (epoetin alfa), ARANESP® (darbepoetin alfa), ORENCIA® (abatacept), BATASERON® (interferon beta-1b), NAGLAZYME® (galsulfase); ELAPRASE® (idursulfase); MYOZYME® (LUMIZYME®, algucosidase alfa); VPRIV® (velaglucerase), abobotulinumtoxin A (DYSPORT®); BAX-326, octocog alfa from Baxter; synclear from GlaxoSmithKline; reprotamase from Eli Lilly; Auxilium and BioSpecifics Technologies Xiaflex Corp. collagenase clostridium histolyticum); anakinra from Swedish Orphan Biovitrum AB; metreleptin from Bristol-Myers Squibb; Avonex, Pregridi (BIIB017) from Biogen; NN1841, NN7008 from Novo Nordisk; KRN321 (darbepoetin alfa), AMG531 (romiplostim), KRN125 (pegfilgrastim), KW-0761 (mogamulizumab) from Kyowa; IB1001 from Inspiration Biopharmaceuticals; and iprivasc from Canyon Pharmaceuticals Group.

[0204] Protein therapeutics under development

[0205] Versartis, Inc.'s VRS-317 is a recombinant human growth hormone (hGH) fusion protein that utilizes XTEN half-life extension technology. VRS-317 is intended to reduce the frequency of hGH injections required for hGH-deficient patients. VRS-317 has completed a Phase II study comparing its efficacy to daily injections of underivatized hGH with positive results. Phase III studies are planned.

[0206] Vibriolysin is a proteolytic enzyme secreted by the gram-negative marine microorganism Vibrio proteolyticus. This endoprotease has a specific affinity for hydrophobic regions of proteins and can cleave proteins near hydrophobic amino acids. Vibriolysin is currently being investigated by Biomarin for the cleansing and / or treatment of burns. Vibriolysin formulations are described in patent WO02 / 092014.

[0207] PEG-PAL (PEGylated recombinant phenylalanine ammonia lyase or "PAL") is an investigational enzyme replacement therapy for the treatment of phenylketonuria (PKU), an inherited metabolic disease caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). PEG-PAL is being developed as a potential treatment for patients whose blood phenylalanine (Phe) levels are not adequately controlled by KUVAN®. PEG-PAL is currently in Phase 2 clinical development to treat patients who do not adequately respond to KUVAN®.

[0208] Other protein therapeutics that can be formulated with viscosity-lowering agents include alprolix / rFIXFc, eloctate / rFVIIIFc, BMN-190; BMN-250; lamazyme; galazyme; ZA-011; sebelipase alfa; SBC-103; and HGT-1110. Additionally, fusion proteins containing XTEN half-life extension technology, including but not limited to the following fusion proteins: VRS-317 GH-XTEN; Factor VIIa, Factor VIII, Factor IX; PF05280602, VRS-859; Exenatide-XTEN; AMX-256; GLP2-2G / XTEN; and AMX-179 folate-XTEN-DM1 can be formulated with a viscosity-lowering agent.

[0209] Other late-stage protein therapeutics that can be formulated with viscosity-reducing agents include CM-AT from CureMark LLC; NN7999, NN7088, liraglutide (NN8022), NN9211, semaglutide (NN9535) from Novo Nordisk; AMG386, filgrastim from Amgen; CSL-654, factor VIII from CSL Behring; LA-EP2006 (pegfilgrastim biosimilar) from Novartis AG; murchikin (leukocyte interleukin) from CEL-SCI Corporation; LY2605541, teriparatide (recombinant PTH1-34) from Eli Lilly; NU-100 from Nuron Biotech, Inc.; calaspargase pegol from Sigma-Tau Pharmaceuticals, Inc.; and Polaris ADI-PEG-20 from BioMarin; BMN-110, BMN-702 from Molmed SpA; NGR-TNF from Pharming Group / Santarus Inc.; Recombinant Human C1 Esterase Inhibitor 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 alpha from Agennix AG; Desmoteplase from Lundbeck; Cinryze from Shire; R RG7421 and Roche and Exelixis, Inc.; midostaurin (PKC412) from Novartis AG; damoctocog alfa pegol from Bayer AG , BAY86-6150, BAY94-9027; pegylated interferon lambda-1a, Nulojix (belatacept) from Bristol-Myers Squibb; k Pergoveris, corifollitropin alfa (MK-8962) from KGaA; recombinant coagulation factor IX Fc fusion protein (rFIXFc; BIIB029) and recombinant coagulation factor VIII Fc fusion protein (rFVIIIFc; BIIB031) from Biogen; and Myalept from AstraZeneca.

[0210] Other early stage protein biologics that can be formulated with viscosity-lowering agents include Alferon LDO from Hemispherx BioPharma, Inc.; SL-401 from Stemline Therapeutics, Inc.; PRX-102 from Protalix Biotherapeutics, Inc.; KTP-001 from Kaketsuken / Teijin Pharma Ltd.; Vericiguat from Bayer AG; BMN-111 from BioMarin; ACC-001 (PF-05236806) from Janssen; LY2510924, LY2944876 from Eli Lilly; NN9924 from Novo Nordisk; INGAP peptides from Exsulin; ABT-122 from Abbvie; AZD9412 from AstraZeneca; NEUBLASTIN (BG00010) from Biogen; Celgene Luspatercept (ACE-536), Sotatercept (ACE-011) from GlaxoSmithKline Corporation; PRAME immunotherapy from ithKline; Provamel from Merck KGaA plovamer acetate (PI-2301); Premiplex from Shire ( 607);BMN-701 from BioMarin;Ontak from Eisai;Haloz rHuPH20 / insulin from PhaseBio Pharmaceuticals, Inc.; PB-1023 from PhaseBio Pharmaceuticals, Inc.; ALV-003 from Alvine Pharmaceuticals Inc. and Abbvie; NN8717 from Novo Nordisk; PRT-201 from Proteon Therapeutics Inc.; PEGPH20 from Halozyme, Inc.; Amevive® Alefacept from Astellas Pharma; F-627 from Regeneron; AGN-214868 (senrebotase) from Allergan, Inc.; BAX-817 from Baxter; Portola Pharmaceuticals PRT4445 from Nuticals, Inc.; VEN100 from Ventria Bioscience; Onconase / Ranpirnase from Tamir Biotechnology Inc.; Interferon alfa-2b Infusion from Medtronic, Inc; Sebelipase alfa from Synageva BioPharma; IRX-2 from IRX Therapeutics, Inc; GSK2586881 from GlaxoSmithKline; SI-6603 from Seikagaku Corporation; ALXN1101, asfotase alfa from Alexion; SHP611, SHP609 (elaprase, idursulfase) from Shire; PF-04856884, PF-05280602 from Pfizer; ACE-031, Dalantercept from Acceleron Pharma; ALT-801 from Altor BioScience Corp.; BioAxone BA-210 from Biosciences, Inc.; WT1 immunotherapy from GlaxoSmithKline; GZ402666 from Sanofi; MSB0010445, atacicept from Merck KGaA; Leukine (monkey) from Bayer AG grammostim); KUR-211 from Baxter; fibroblast growth factor-1 from CardioVascular BioTherapeutics Inc.; Hanmi SPI-2012 from Spectrum Pharmaceuticals; FGF-18 (Sprifermin) from Merck KGaA; MK-1293 from Merck; Interferon-alpha-2b from HanAll Biopharma; CYT107 from Cytheris SA; RT001 from Revance Therapeutics, Inc.; MEDI6012 from AztraZeneca; E2609 from Biogen; BMN-190, BMN-270 from BioMarin; ACE-661 from Acceleron Pharma; AMG876 from Amgen; GSK3052230 from GlaxoSmithKline; RG7813 from Roche; SAR342434, Lantus from Sanofi; AZ01 from Allozyne Inc.; ARX424 from Ambrx, Inc.; FivePrime FP-1040, FP-1039 from Therapeutics, Inc.; ATX-MS-1467 from Merck KGaA; XTEN fusion proteins from Amunix Operating Inc.; entolimod from Cleveland BioLabs, Inc. (CBLB502);HGT2310 from Shire;HM10760A from Hanmi Pharmaceuticals Co., LTD;ALXN1102 / ALXN1103 from Alexion;CSL-689, CSL-627 from CSL Behring;Glial Growth Factor 2 from Acorda Therapeutics,Inc.;NX001 from Nephrx Corporation;NN8640, NN1436, NN1953, NN9926, NN9927, NN9928 from Novo Nordisk;NHS-IL12 from EMD Serono;3K3A-APC from ZZ Biotech LLC;PB-1046 from PhaseBio Pharmaceuticals,Inc.;RU-101 from R-Tech Ueno Co., Ltd.;Insulin lispro / BC106 from Adocia;hl-con1 from Iconic Therapeutics,Inc.;Protalix PRT-105 from BioTherapeutics, Inc; PF-04856883, CVX-096 from Pfizer; ACP-501 from AlphaCore Pharma LLC; BAX-855 from Baxter; CDX-1135 from Celldex Therapeutics; PRM-151 from Promedior, Inc.; TS01 from Thrombolytic Science International; TT-173 from Thrombotargets Corp.; QBI-139 from Quintessence Biosciences, Inc.; Vatelizumab, GBR500, GBR600, GB from Glenmark Pharmaceuticals R830 and GBR900; and CYT-6091 from Cytimmune Sciences, Inc.

[0211] Other biologic agents

[0212] Other biological drugs that may be formulated with viscosity-lowering agents include PF-05285401, PF-05231023, RN317 (PF-05335810), PF-06263507, PF-05230907, Dekavil, PF-06342674, PF06252616, RG7598, RG7842, RG7624d, OMP54F28, GSK1995057, BAY1179470, IMC-3G3, IMC-18F1, IMC-35C, IMC-20D7S, PF-06480605, PF-06647263, PF-06650808, PF-05335810 (RN317) from Pfizer. PD-0360324, PF-00547659; MK-8237 from Merck; BI033 from Biogen; GZ402665, SAR438584 / REGN2222 from Sanofi; IMC-18F1, and Icrucumab, IMC-3G3 from ImClone LLC; Ryzodeg, Tresiba, Xultophy from Novo Nordisk; Toujeo (U300), LixiLan, Lixumia (lixenatide) from Sanofi; GlaxoSmithKlin These include MAGE-A3 immunotherapy drugs from Eklund; tesemotide from Merck KGaA; serelaxin (RLX030) from Novartis AG; erythropoietin; pegfilgrastim; LY2963016, dulaglutide (LY2182965) from Eli Lilly; and insulin glargine from Boehringer Ingelheim.

[0213] B. Viscosity reducer

[0214] The viscosity of liquid protein formulations containing low and / or high molecular weight proteins is reduced by the addition of one or more viscosity-lowering agents. The pharmaceutical formulations can be converted from non-Newtonian to Newtonian fluids by adding an effective amount of one or more viscosity-lowering agents.

[0215] When used in a formulation intended for administration to humans or other mammals, the viscosity-lowering agent, like the formulation itself, must be pharma- ceutically acceptable. Viscosity-lowering agents are usually organic compounds that contain at least one non-carbon, non-hydrogen atom. Preferably, the viscosity-lowering agent contains hydrogen, carbon, oxygen, and at least one other type of atom. In certain embodiments, the viscosity-lowering agent is characterized by at least one of the following: 1) an organic compound having at least four carbon atoms and four hydrogen atoms, and at least one sulfur, oxygen, nitrogen, or phosphorus atom; 2) Molecular weight between about 85 and 1,000 Da; 3) the presence of at least one charged or other hydrophilic moiety; 4) the presence of at least one, preferably two, and more preferably three freely rotatable bonds; 5) the presence of at least one substituted ring; 6) At least 24 Å 2 , preferably at least 50 Å 2 , more preferably at least 80 Å 2 The polar surface area of ​​the molecule, 7) At least 75cm 3 , preferably at least 85 cm 3 , more preferably at least 100 cm 3 , and most preferably at least 120 cm 3 Molar volume of, 8) At least 10cm 3 , preferably at least 15 cm 3 , more preferably at least 20 cm 3 , and most preferably at least 25 cm 3 The polarizability of 9) The presence of at least one, preferably two, more preferably three hydrogen bond donors and / or acceptors.

[0216] In certain embodiments, the viscosity-lowering agents are characterized by at least two, three, four, five, six, seven, eight, or all nine of the above-listed attributes. In certain embodiments, the viscosity-lowering agents are further characterized in that they do not contain aldehyde or carbon-carbon triple bond functional groups.

[0217] In other embodiments, the viscosity-lowering agent is a combination of two or more compounds, each of which is characterized by at least two, three, four, five, six, seven, eight, or all nine of the attributes listed above.

[0218] In some embodiments, the viscosity-lowering agent is listed as GRAS by the U.S. Food and Drug Administration ("FDA") as of September 11, 2014. "GRAS" is an acronym for Generally Recognized As Safe. Under sections 201(s) and 409 of the Federal Food, Drug, and Cosmetic Act (Act), any substance intentionally added to food is a food additive and is subject to premarket review and approval by the FDA unless the substance is generally recognized among competent experts as adequately shown to be safe under the conditions of its intended use or the use of the substance is otherwise excluded from the definition of a food additive. Another source of compounds is the equivalent of those listed in the FDA's Inactive Ingredient Guide (IIG) as of September 11, 2014, as well as by the International Pharmaceutical Excipients Council (IPEC) and the European Medicines Agency (EMA). Substances used in the formulation must be safe for injection. Preferably, a GRAS-listed viscosity-lowering agent is characterized by at least two, three, four, five, six, seven, eight, or all nine of the above-listed attributes.

[0219] In other embodiments, the viscosity-lowering agent is an FDA or EMA approved formulation as of September 11, 2014. Like compounds from the GRAS and IIG lists, the toxicity and safety profiles of FDA and EMA approved formulations are well established. In addition to reducing the viscosity of protein solutions, the use of FDA or EMA approved formulations provides opportunities for combination therapy. Preferably, the viscosity-lowering agent of the FDA or EMA approved formulation is characterized by at least two, three, four, five, six, seven, eight, or all nine of the attributes listed above.

[0220] In some embodiments, the viscosity-lowering agent comprises at least one compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, [ka] represents either a single or double bond, and A is O, S, SO2, or NR 3 , C(R 3 )2, or [ka] is selected from In the formula, R 3 is hydrogen, R 2 , -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R 4a , -C(=NR 4a )R 4 , -C(=O)OH, -C(=O)OR 4 , -OC(=O)R 4 , -OC(=O)OR 4 , -SO3H, -SO2N(R 4a )2, -SO2R 4 , -SO2NR 4a C(=O)R 4 , -PO3H2, -R 4a C(=NR4a )N(R 4a )2, -NHC(=NR 4a )NH-CN, -NR 4a C(=O)R 4 , -NR 4a SO2R 4 , -NR 4a C(=NR 4a )NR 4a C(=NR 4a )N(R 4a )2, -NR 4a C(=O)N(R 4a )2, -C(=O)NH2, -C(=O)N(R 4a )2, -OR 4 , -SR 4a and -N(R 4a )2 are independently selected from R 2 is C 1~12 Alkyl, C 3~12 Cycloalkyl, C 6~12 Aryl, C 1~12 Heteroaryl and C 2~12 heterocyclyl; C 1~12 The alkyl groups are C 3~12 Cycloalkyl, C 6~12 Aryl, C 1~12 Heteroaryl, C 2~12 Heterocyclyl, -OH, NH2, (=O), (=NR 4a ), -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R 4a , -C(=NR 4a )R 4 , -C(=O)OH, -C(=O)OR 4 , -OC(=O)R 4 , -OC(=O)OR 4 , -SO3H, -SO2N(R 4a )2, -SO2R 4 , -SO2NR 4a C(=O)R 4 , -PO3H2, -R 4a C(=NR 4a )N(R 4a )2, -NHC(=NR 4a )NH-CN, -NR 4aC(=O)R 4 , -NR 4a SO2R 4 , -NR 4a C(=NR 4a )NR 4a C(=NR 4a )N(R 4a )2, -NR 4a C(=O)N(R 4a )2, -C(=O)NH2, -C(=O)N(R 4a )2, -OR 4 , -SR 4a or -N(R 4a ) may be substituted one or more times by C 3~12 Each cycloalkyl is C 1~12 Alkyl, C 6~12 Aryl, C 1~12 Heteroaryl, C 2~12 Heterocyclyl, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R 4a , -C(=NR 4a )R 4 , -C(=O)OH, -C(=O)OR 4 , -OC(=O)R 4 , -OC(=O)OR 4 , -SO3H, -SO2N(R 4a )2, -SO2R 4 , -SO2NR 4a C(=O)R 4 , -PO3H2, -R 4a C(=NR 4a )N(R 4a )2, -NHC(=NR 4a )NH-CN, -NR 4a C(=O)R 4 , -NR 4a SO2R 4 , -NR 4a C(=NR 4a )NR 4a C(=NR 4a )N(R 4a )2, -NR 4a C(=O)N(R 4a )2, -C(=O)NH2, -C(=O)N(R 4a )2, -OR4 , -SR 4a or -N(R 4a ) may be substituted one or more times by C 6~12 Each aryl is C 1~12 Alkyl, C 3~12 Cycloalkyl, C 1~12 Heteroaryl, C 2~12 Heterocyclyl, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R 4a , -C(=NR 4a )R 4 , -C(=O)OH, -C(=O)OR 4 , -OC(=O)R 4 , -OC(=O)OR 4 , -SO3H, -SO2N(R 4a )2, -SO2R 4 , -SO2NR 4a C(=O)R 4 , -PO3H2, -R 4a C(=NR 4a )N(R 4a )2, -NHC(=NR 4a )NH-CN, -NR 4a C(=O)R 4 , -NR 4a SO2R 4 , -NR 4a C(=NR 4a )NR 4a C(=NR 4a )N(R 4a )2, -NR 4a C(=O)N(R 4a )2, -C(=O)NH2, -C(=O)N(R 4a )2, -OR 4 , -SR 4a , or -N(R 4a ) may be substituted one or more times by C 1~12 Each heteroaryl is C 1~12 Alkyl, C 3~12 Cycloalkyl, C 6~12 Aryl, C 2~12Heterocyclyl, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R 4a , -C(=NR 4a )R 4 , -C(=O)OH, -C(=O)OR 4 , -OC(=O)R 4 , -OC(=O)OR 4 , -SO3H, -SO2N(R 4a )2, -SO2R 4 , -SO2NR 4a C(=O)R 4 , -PO3H2, -R 4a C(=NR 4a )N(R 4a )2, -NHC(=NR 4a )NH-CN, -NR 4a C(=O)R 4 , -NR 4a SO2R 4 , -NR 4a C(=NR 4a )NR 4a C(=NR 4a )N(R 4a )2, -NR 4a C(=O)N(R 4a )2, -C(=O)NH2, -C(=O)N(R 4a )2, -OR 4 , -SR 4a , or -N(R 4a ) may be substituted one or more times by C 2~12 Heterocyclyl is C 1~12 Alkyl, C 3~12 Cycloalkyl, C 6~12 Aryl, C 1~12 Heteroaryl, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R 4a , -C(=NR 4a )R 4 , -C(=O)OH, -C(=O)OR 4 , -OC(=O)R 4 , -OC(=O)OR 4 , -SO3H, -SO2N(R 4a )2, -SO2R 4 , -SO2NR4a C(=O)R 4 , -PO3H2, -R 4a C(=NR 4a )N(R 4a )2, -NHC(=NR 4a )NH-CN, -NR 4a C(=O)R 4 , -NR 4a SO2R 4 , -NR 4a C(=NR 4a )NR 4a C(=NR 4a )N(R 4a )2, -NR 4a C(=O)N(R 4a )2, -C(=O)NH2, -C(=O)N(R 4a )2, -OR 4 , -SR 4a , or -N(R 4a ) may be substituted one or more times by R 4 is C 1~12 Alkyl, C 3~12 Cycloalkyl, C 6~12 Aryl, C 1~12 Heteroaryl and C 2~12 heterocyclyl, each of which is optionally substituted one or more times with -OH, -NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)OH, -SO3H, -PO3H2, or -C(=O)NH2; R 4a is R 4 or hydrogen, R 2 , R 3 , R 4 and R 4a any two or more of the groups may be taken together to form a ring; The Two R's 3 If the R groups are attached to the same carbon atom, the two R 3 The groups taken together are (=O), (=NR 4a ) or (=C(R 4a ) 2) may be formed, z, at each occurrence, is independently selected from 1 or 2, provided that (R 3 ) z Substituents are sp 2 When attached to a hybridized carbon, z is 1, and (R 3 ) z Substituents are sp 3 If attached to a hybridized carbon, z is 2.

[0221] Substituent -NR 4a C(=NR 4a )NR 4a C(=NR 4a )N(R 4a )2, R 4a is preferably selected to give -NHC(=NH)NHC(=NH)NH2.

[0222] In certain embodiments, the compound of formula (1) is -C(=O)OH, -SO3H, -SONHC(=O)R 4 and -PO3H2. In some embodiments, the compound of formula (1) contains at least one -SO3H group.

[0223] In certain embodiments, R 3 One or more of the substituents is [ka] may be In the formula, R 3a and R 3b is hydrogen, C 1~12 Alkyl, C 3~12 Cycloalkyl, C 6~12 Aryl, C 1~12 Heteroaryl and C 2~12 Heterocyclyl, C(=O)R 4a , -C(=O)OH, -C(=O)OR 4 , -SO3H, -SO2N(R 4a )2, -SO2R 4 , -SO2NHC(=O)R 4 , C(=O)NH2, -C(=O)N(R4a )2, -OR 4 , -SR 4 , and -N(R 4a )2 are independently selected from R 3b If any two of R are bonded to the same carbon atom, 3b The groups together are (=O), (=NR 4a ) or (=C(R 4a ) 2) may be formed, C 1~12 Alkyl, C 3~12 Cycloalkyl, C 6~12 Aryl, C 1~12 Heteroaryl and C 2~12 Heterocyclyl is -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R 4a , -C(=NR 4a )R 4 , -C(=O)OH, -C(=O)OR 4 , -OC(=O)R 4 , -OC(=O)OR 4 , -SO3H, -SO2N(R 4a )2, -SO2R 4 , -SO2NR 4a C(=O)R 4 , -PO3H2, -R 4a C(=NR 4a )N(R 4a )2, -NHC(=NR 4a )NH-CN, -NR 4a C(=O)R 4 , -NR 4a SO2R 4 , -NR 4a C(=NR 4a )NR 4a C(=NR 4a )N(R 4a )2, -NR 4a C(=O)N(R 4a )2, -C(=O)NH2, -C(=O)N(R 4a )2, -OR 4 , -SR 4a , or -N(R 4a ) may be substituted one or more times by R4 and R 4a is as defined above, x is selected from 1, 2, 3, 4, 5, 7, 8, 9 or 10; R 3 , R 3a , R 4 and R 4a Any two or more of the groups may be joined to form a ring.

[0224] In certain embodiments, the compound of formula (1) is either a compound of formula (1a) or (1b) [ka] It can be expressed by In the formula, R 3 has the meaning given above.

[0225] In certain embodiments, the compound of formula (1a) is a compound of formula (1a-i-iv) [ka] It can be expressed by In the formula, R 3 is hydrogen, NH2, CH3, Cl, OR 4 and N.H.R. 4 are independently selected from x is 1 or 2; R 3a and R 3b is hydrogen and C 1~12 independently selected from alkyl, Said C 1~12 Alkyl is C 3~12 Cycloalkyl, C 6~12 Aryl, C 1~12 Heteroaryl, C 2~12 Heterocyclyl, -OH, NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R 4a , -C(=NR 4a )R 4 , -C(=O)OH, -C(=O)OR 4, -OC(=O)R 4 , -OC(=O)OR 4 , -SO3H, -SO2N(R 4a )2, -SO2R 4 , -SO2NR 4a C(=O)R 4 , -PO3H2, -R 4a C(=NR 4a )N(R 4a )2, -NHC(=NR 4a )NH-CN, -NR 4a C(=O)R 4 , -NR 4a SO2R 4 , -NR 4a C(=NR 4a )NR 4a C(=NR 4a )N(R 4a )2, -NR 4a C(=O)N(R 4a )2, -C(=O)NH2, -C(=O)N(R 4a )2, -OR 4 , -SR 4a , or -N(R 4a ) may be substituted one or more times by R 4 and R 4a is as defined above, R 3a , R 3b , R 4 , R 4a Any two or more of may be joined together to form a ring.

[0226] The compound of formula (1) is a compound of formula (1a-v, vi or vii) [ka] It can be expressed by In the formula, R 3f is -C(=O)OH, -SO3H, -SO2NHC(=O)R 4 and -PO3H2, R 3 is as defined above. In certain preferred embodiments, R 3are hydrogen, OH, NH2, C 1~6 is independently selected from alkyl and COOH.

[0227] In other embodiments, the compound of formula (1) is any of the compounds of formula (1c), (1d), (1e) or (1f) [ka] It can be expressed by In the formula, R 3 has the meaning given above.

[0228] In another embodiment, the compound of formula (1) is a compound of formula (1g) [ka] It can be expressed by In the formula, R 3c is hydrogen and R 2 are independently selected from R 2 has the meaning given above, R 3d are hydrogen, OH, NH2, NH(C 1~6 alkyl), N(C 1~6 alkyl)2, NHC(=O)(C 1~6 alkyl), COOH and CHOH; Or, R attached to the same carbon 3c and R 3d Any two of the groups taken together can be oxo (=O), imino (=NR 4a ) or olefin (=C(R 4a )2), R 4a has the meaning given above, R 3e is hydrogen, -OH or OR 4 is selected from R 4 has the meaning given above.

[0229] In certain embodiments, the viscosity-lowering agent is a compound of formula (1g-i) [ka] Including, In the formula, R 3e -OH and -OC 1~12 alkyl, which are further substituted with at least one OH and at least one COOH; R 3d is selected from COOH and CHOH.

[0230] In some embodiments, the viscosity-lowering agent is a compound of formula (2) [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, [ka] represents a single or double bond, X is chalcogen, N(R 3 ) z and C(R 3 ) z are independently selected from X 1 is absent or chalcogen, N(R 3 ) z , C(R 3 ) z or [ka] and In the formula, R 3 has the meaning given for compounds of formula (1), but However, (R 3 ) z Substituents are sp 2 When attached to a hybridized nitrogen, z is 0 or 1, and (R 3 ) z Substituents are sp 2 Hybridized carbon or sp 3 When attached to a nitrogen hybrid, z is 1, and (R3 ) z The substituent is sp 3 If attached to a hybridized carbon, z is 2, X or X 1 At least one of the groups is chalcogen or N(R 3 ) z It is.

[0231] In certain embodiments, the compound may be an aromatic ring. Exemplary aromatic rings include compounds of formula (2a-e): [ka] Includes: In the formula, R 3 and X has the above meaning, 2 is N(R 3 ) z and C(R 3 ) z is selected from.

[0232] In certain embodiments, the viscosity-lowering agent is a compound of formula (2a-i) [ka] and In the formula, R 4 is as defined above, preferably hydrogen or CH3, R 6 is C 1~12 Heteroaryl, which is C 1~6 may be substituted one or more times with alkyl; Said C 1~6 Alkyl is OH, -NH2, -F, -Cl, -Br, -I, -NO2, -CN, -C(=O)R 4 , -C(=NR 4a )R 4 , -C(=O)OH, -C(=O)OR 4 , -SO3H, -SO2NR 4 -, -SO2R4, -PO3H2, -NHC(=O)R 4 , -NHC(=O)N(R 4)2, -C(=O)NH2, -C(=O)N(R 4 )2, -OR 4b , -SR 4b , -N(R 4b )2 may be substituted one or more times by R 4 has the meaning given above, or [ka] It has In the formula, R 4 is as defined above, and R 7 SR 4 and -C(=O)R 4 The double bond in the above groups may be in either the E or Z configuration.

[0233] In a preferred embodiment, R 6 The structure [ka] is a heterocycle having the formula In the formula, X 4 is chalcogen and R 6a is hydrogen or C 1~6 This C is an alkyl group. 1~6 The alkyl may be substituted one or more times with -OH, -NH, -F, -Cl, -Br, -I, -NO, -CN, -C(=O)OH. In an even more preferred embodiment, R 6 The structure [ka] is a heterocycle having the formula In the formula, R 6a is unsubstituted C 1~6 C substituted one or more times with alkyl and -OH 1~6 is selected from alkyl.

[0234] The viscosity reducing agent is an imidazole of formula (2b-i) [ka] and In the formula, R 3 is as defined above. In certain embodiments, R 3 is hydrogen, NO2 and R 4 In certain preferred embodiments, the compound of formula (2b-i) is independently selected from the structure [ka] It has In the formula, R 3 is C 1~6 alkyl, which may be unsubstituted or selected from OH, NH, SR 4 , optionally substituted one or more times with a group selected from F, Cl, Br and I; R 3g is either hydrogen or NO2.

[0235] In other embodiments, the viscosity-lowering agent has the structure of Formula (2a-ii) or Formula (2c-i): [ka] It has In the formula, R 3 are OH, Cl, Br, F, I, N(R 4a )2, C(=O)OH, C(=O)NH2.

[0236] In a further embodiment, at least one R 3 The substituent is NHR. 4 and R 4 are Cl, Br, F, I, OH, C(=O)OH, NH2, NH(C 1~6 alkyl) and N(C 1~6 C optionally substituted with one or more groups selected from alkyl 1~6 It is an alkyl.

[0237] In other embodiments, the viscosity-lowering agent is a pyridinium salt of formula (2a-iii): [ka] and In the formula, R 3 and R 4 is as defined above.

[0238] In other embodiments, the heterocyclic ring is not a heteroaryl ring. Exemplary non-aromatic rings include compounds of formula (2f-k): [ka] Includes: In the formula, R 5 and X has the above meaning, 3 is chalcogen or N(R 3 ) z It is.

[0239] In certain embodiments, the compound of formula (2f) is a β-lactam of formula (2f-i): [ka] It is.

[0240] β-lactams of formula (2f-i) include penicillin-type compounds, as well as cephalosporin-type and cephamycin-type compounds of formulae (2f-ii) and (2f-iii). [ka] Includes: wherein X and R3 are as defined above. In a preferred embodiment, X is sulfur.

[0241] In certain embodiments, the compound of formula (2i) is a compound of formula (2i-i) [ka] and In the formula, X and R 3is as defined above. In certain embodiments, X is, in both instances, NR 4 and R 4 has the meaning given above, and R 3 is hydrogen in both cases.

[0242] In another embodiment, the compound of formula (2) is a compound of formula (2i-ii) [ka] is represented by In the formula, X, 1 and R 3 is as defined above.

[0243] The compound of formula (2j) is a compound of formula (2j-i) [ka] It can be expressed by In the formula, X 3 and R 3 is as defined above, and R 8 is NHC(=O)R 2 and OC(=O)R 2 In a preferred embodiment, X 3 is N + (CH3)2, R 3 are both hydrogen or R 3 together form an epoxide or a double bond.

[0244] The compound of formula (2k) is a compound of formula (2k-i) [ka] It can be expressed by In the formula, X 3 and R 8 is as defined above.

[0245] In other embodiments, the viscosity-lowering agent is a compound having the structure of formula (3): [ka] or a pharma- ceutically acceptable salt thereof, In the formula, R 5 is, in each case, hydrogen and R 2 are independently selected from R 5’ is R 5 or absent, but However, at least one R 5 R, provided that the substituent is not hydrogen. 2 has the same meaning as given in the case of the compound or formula (1).

[0246] In certain embodiments, the viscosity-lowering agent is a mixture of two or more compounds selected from compounds of Formula (1), Formula (2), and Formula (3).

[0247] In a preferred embodiment, the viscosity-lowering agent is camphorsulfonic acid (CSA) or a pharma- ceutically acceptable salt thereof, such as an alkali metal salt or an alkaline earth metal salt. Camphorsulfonic acid or a salt thereof is combined with one or more compounds of formula (1), (2) or (3) to provide mixtures such as CSA-piperazine, CSA-TRIS, CSA-4-aminopyridine, CSA-1-(o-tolyl)biguanide, CSA-procaine, CSA-Na-aminocyclohexanecarboxylic acid, CSA-Na-creatinine and CSA-Na-ornidazole. Further preferred viscosity-lowering agents include thiamine, procaine, biotin, creatinine, metoclopramide, scopolamine, cimetidine, chloroquine phosphate, mepivacaine, granisetron, sucralose, HEPES-tris, nicotinamide, lactobionate-TRIS, glucuronate-TRIS, sulfacetamide, CSA-4-aminopyridine, CSA-piperazine and cefazolin. Any two or more of the above listed viscosity-lowering agents may further be combined in the same formulation.

[0248] In other embodiments, the viscosity reducing agent is an organic sulfonic acid. Exemplary organic sulfonic acids include camphorsulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, toluenesulfonic acid, cyclohexylsulfonic acid, xylenesulfonic acid (including p-xylene-2-sulfonic acid, m-xylene-2-sulfonic acid, m-xylene-4-sulfonic acid, and o-xylene-3-sulfonic acid), methanesulfonic acid, 1,2 ethane disulfonic acid, 4-(2-hydroxyethyl)-1-piperazine ethoxylate sulfonic acid, 1,2 ethane disulf ... The organic sulfonic acids include, but are not limited to, benzenesulfonic acid, 2-hydroxyethane-1-sulfonic acid, 3-hydroxypropane-1-sulfonic acid, cymenesulfonic acid, 4-hydroxybutane-1-sulfonic acid and pharma- ceutically acceptable salts thereof. The organic sulfonic acids may be in the form of an alkali metal salt or an alkaline earth metal salt, such as lithium, sodium, potassium, magnesium and calcium salts. The organic sulfonic acid (or a salt thereof) may be combined with one or more compounds of formula (2) or formula (3).

[0249] In certain embodiments, the viscosity-lowering agent contains at least one carboxylic acid. The carboxylic acid may be in the form of an alkali metal salt or an alkaline earth metal salt, such as lithium, sodium, potassium, magnesium, and calcium salts. Exemplary carboxylic acid compounds include lactobionic acid, glucuronic acid, 1-aminocyclohexane carboxylic acid, biotin, brocrinat, cyclopentanepropionic acid, hydrazine, glycerol ... Examples of suitable carboxylic acids include aryloxynaphthoic acid, phenylpropionic acid, gentisic acid, salicylic acid, camphoric acid, mandelic acid, sulfosalicylic acid, hydroxybenzoylbenzoic acid, phenylacetic acid, acetylsalicylic acid, cinnamic acid, t-butylacetic acid, phthalic acid, trimethylacetic acid, anthrallic acid, and pharma- ceutically acceptable salts thereof. The carboxylic acid (or salt thereof) may be combined with one or more compounds of formula (2) or formula (3).

[0250] The following compounds may also be used as viscosity-lowering agents: colistin, articaine, tetracaine, proxymethacaine, metoclopramide, procaine, lidocaine, cyclomethylcaine, piperocaine, chloroprocaine, etidocaine, benzocaine, phenylephrine, bupivacaine, mepivacaine, cinchocaine, mixtures thereof, and pharma- ceutically acceptable salts thereof.

[0251] Other agents that can be used as viscosity reducers include 1-aminocyclohexanecarboxylic acid, 1-(o-tolyl)biguanide, benzethonium chloride, benzoic acid, brocrinat, calcium carrageenan, calcium cyclamate, and calcobutrol. , caroxetic acid, camphorsulfonic acid, creatinine, dalfampridine, dehydroacetic acid, diazolidinyl urea, dichlorobenzyl alcohol, dimethyl isosorbide, epitetracycline, ethyl maltol, ethyl vanillin, ornidazole, gentisic acid ethanolamide, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), gentisic acid, glucuronic acid, iodoxamic acid, menthol, galactose, medronic acid, m-cresol, glutathione, lactobionic acid, maltitol, octisalate, oxyquinoline, pentetic acid, piperazine, propenyl guaethol, propyl gallate, propylene carbonate, propylparaben, protamine sulfate, quaternium-15, quaternium-52, satialgine H, 1,2-ethoxycaprylylsilane, ethylparaben ... sodium disulfonate, sodium cocoyl sarcosinate, sodium lauroyl sarcosinate, sodium polymetaphosphate, sodium pyrophosphate, pyroglutamic acid, sodium trimetaphosphate, sodium tripolyphosphate, sorbitan, tartaric acid, lactic acid, iofetamine, sucralose, 1-(4-pyridyl)pyridinium chloride, aminobenzoic acid, sodium sulfacetamide, naphthalene-2-sulfonic acid, tert-butylhydroquinone, thimerosal, trolamine, tromantadine, vanillin, versetamide, nioxime, niacinamide, methylisothiazolinone, mannose D, maltose, lidofenine, lactose, lactitol, isomalt, imidurea, gluconolactone, methanesulfonic acid, xylenesulfonic acid, sulfobutyl ether beta-cyclodextrin, and pharma- ceutically acceptable salts thereof.

[0252] In certain embodiments, the viscosity-reducing agent comprises an organic base. Exemplary organic bases include N-methylglucamine, morpholine, piperidine, and primary, secondary, tertiary and quaternary amines, substituted amines, and cyclic amines. For example, they can be isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, lidocaine, hydrabamine, cholines, betaines, choline, betaine, ethylenediamine, theobromine, purine, piperazine, N-ethylpiperidine, N-methylpiperidine polyamine. Particularly preferred organic bases are arginine, histidine, lysine, ethanolamine, thiamine, 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), 4-aminopyridine, aminocyclohexanecarboxylic acid, 1-o-tolybiguanide, ornidazole, urea, nictoinamide, benzethonium chloride, 5-amino-1-pentanol, 2-(2-amino ethoxy)ethanol, trans-cyclohexane-1,4-diamine, trans-cyclohexane-1R,2R-diamine, ethylenediamine, propane-1,3-diamine, butane-1,4-diamine, pentane-1,5-diamine, hexane-1,6-diamine, octane-1,8-diamine, 5-amino-1-pentanol, 2-(2-aminoethoxy)ethanamine, 2-(2-(2-aminoethoxy)-ethoxy)ethanamine, 3-(4-(3-aminopropoxy)-butoxy)propan-1-amine, 3-(2-(2-(3-aminopropoxy)-ethoxy)-ethoxy)propan-1-amine, N-(2-(2-aminoethylamino)ethyl)ethane-1,2-diamine, N-(2-a and 2-(2-aminoethyl)ethane-1,2-diamine, N-(2-(2-(2-aminoethylamino)ethylamino)-ethyl)ethane-1,2-diamine, N,N-dimethylhexane-1,6-diamine, N,N,N,N-tetramethylbutane-1,4-diamine, phenyltrimethylammonium salts, isopropylamine, diethylamine, ethanolamine, trimethamine, choline, 1-(3-aminopropyl)-2-methyl-1H-imidazole, piperazine, 1-(2-aminoethyl)piperazine, 1-[3-(dimethylamino)propyl]piperazine, 1-(2-aminoethyl)piperidine, 2-(2-aminoethyl-1-methylpyrrolidine, mixtures thereof, and pharma- ceutically acceptable salts thereof.

[0253] Exemplary β-lactams include benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), cloxacillin, dicloxacillin, flucloxacillin, methicillin, nafcillin, oxacillin, temocillin, amoxicillin, ampicillin, mecillinam, carbenicillin, ticarcillin, azlocillin, mezlocillin, piperacillin, cefoxitin, cefazolin, cephalexin, cephalosporin C, cephalothin, cefaclor, cefamandole, cefuroxime, cefotetan, cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, cefepime, cefpirome, ceftobiprole, biapenem, doripenem, ertapenem. These include faropenem, imipenem, meropenem, panipenem, raspenem, tebipenem, thienamycin, aztreonam, tigemonam, nocardicin a, tabtoxinine, clavulanic acid, clavulanic acid, tazobactam, sulbactam, and pharmaceutically acceptable salts thereof.

[0254] Other viscosity-lowering agents include tropane N-heterocycles and their salts, such as atropine, hyoscyamine, scopolamine, as well as tiotropium and ipratropium salts, thiamine, allithiamine, prosultiamine, fursultiamine, benfotiamine, sulbutiamine, quaternium 15; 1-(3-aminopropyl)-2-methyl-1H-imidazole dihydrochloride, creatinine; biotin, cimetidine, piperocaine, cyclomethylcaine, granisetron, moxifloxacin, chloroquine, mepivacaine, levetiracetam, bupivacaine, cinchocaine, clindamycin, and pharma- ceutically acceptable salts thereof. Thiamine is a particularly preferred viscosity-lowering agent.

[0255] In certain formulations, the following compounds are not preferred and are excluded from the scope of the above formula and definition of useful viscosity-lowering agents: creatinine, cadaverine, lidocaine, arginine and lysine.

[0256] C. Excipients

[0257] A wide range of pharmaceutical excipients useful for liquid protein formulations are known to those skilled in the art. They include one or more additives such as liquid solvents or cosolvents; sugars or sugar alcohols such as mannitol, trehalose, sucrose, sorbitol, fructose, maltose, lactose or dextran; surfactants such as TWEEN® 20, 60 or 80 (polysorbate 20, 60 or 80); buffers; preservatives such as benzalkonium chloride, benzethonium chloride, tertiary ammonium salts and chlorhexidine diacetate; carriers such as poly(ethylene glycol) (PEG); antioxidants such as ascorbic acid, sodium metabisulfite and methionine; chelating agents such as EDTA or citric acid; or biodegradable polymers such as water-soluble polyesters; cryoprotectants; lyoprotectants; bulking agents; and stabilizers.

[0258] Remington: “The Science and Practice of Pharmacy” 20th edition, Alfonso R. Gennaro, ed., Lippincott Williams & Wilkins (2000) Other pharma- ceutically acceptable carriers, excipients, or stabilizers, such as those described herein, can also be included in the protein formulations described herein, provided they do not adversely affect the desired characteristics of the formulation.

[0259] The viscosity-lowering agents described herein may be used in combination with one or more other types of viscosity-lowering agents, such as, for example, the organophosphates described in co-filed PCT application by Arsia Therapeutics entitled "LIQUID PROTEIN FORMULATIONS CONTAINING ORGANOPHOSPHATES"; the organophosphates described in co-filed PCT application by Arsia Therapeutics entitled "LIQUID PROTEIN FORMULATIONS CONTAINING WATER SOLUBLE ORGANIC DYES"; water soluble dyes, as described in U.S. Pat. No. 6,399,423; and ionic liquids, as described in co-filed PCT application by Arsia Therapeutics entitled "LIQUID PROTEIN FORMULATIONS CONTAINING IONIC LIQUIDS."

[0260] III. Preparation method

[0261] Proteins, such as mAbs, to be formulated can be produced by any known technique, such as by culturing cells transformed or transfected with a vector containing one or more nucleic acid sequences encoding the protein, as known in the art, or by synthetic techniques (such as recombinant techniques and peptide synthesis, or a combination of these techniques), or can be isolated from endogenous sources of the protein.

[0262] Purification of formulated proteins can be carried out by any suitable technique known in the art, such as, for example, precipitation with ethanol or ammonium sulfate, reverse-phase HPLC, chromatography on silica or cation exchange resins (e.g., DEAE-cellulose), dialysis, chromatofocusing, gel filtration using a Protein A SEPHAROSE® column (e.g., SEPHADEX® G-75) to remove contaminants, metal chelating columns for binding to epitope-tagged forms, and ultrafiltration / diafiltration (non-limiting examples include centrifugal filtration and tangential flow filtration (TFF)).

[0263] Inclusion of a viscosity-reducing agent at a viscosity-reducing concentration, such as 0.010 M to 1.0 M, preferably 0.050 M to 0.50 M, and most preferably 0.10 M to 0.30 M, allows the solution of the pharma- ceutical active mAb to be purified and / or concentrated to higher concentrations of the mAb using common methods known to those of skill in the art, including, but not limited to, tangential flow filtration, centrifugal concentration, and dialysis.

[0264] In some embodiments, a lyophilized formulation of a protein is provided and / or used in the preparation and manufacture of a concentrated low-viscosity protein formulation. In some embodiments, the pre-lyophilized protein in powder form is reconstituted by dissolving in an aqueous solution. In this embodiment, the liquid formulation is delivered in a vial or a pre-filled mixing syringe. The drug is packaged in a specific dosage unit container, such as a syringe, and optionally contains a lyoprotectant, It is lyophilized together with preservatives, antioxidants and other conventional pharma- ceutically acceptable excipients and then stored under sterile storage conditions until immediately prior to use, at which point it is reconstituted with a defined volume of diluent to give the liquid the desired concentration and viscosity.

[0265] The formulations described herein may be stored by any suitable method known to those skilled in the art.Non-limiting examples of methods for preparing protein formulations for storage include freezing, lyophilization, and spray drying of liquid protein formulations.In some cases, the lyophilized formulations are frozen for storage at subzero temperatures, such as at about -80°C or in liquid nitrogen.In some cases, the lyophilized formulations or aqueous formulations are stored at 2-8°C.

[0266] Non-limiting examples of diluents useful for reconstituting the lyophilized formulation prior to injection include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, dextrose solution, or aqueous solutions of salts and / or buffers. In some cases, the formulations are spray dried and then stored.

[0267] IV. Administration to Individuals in Need Thereof

[0268] Protein formulations, including but not limited to reconstituted formulations, are administered to a human in need thereof by intramuscular, intraperitoneal (i.e., into a body cavity), intracerebrospinal or subcutaneous injection using an 18-32 gauge needle (optionally, a thin-walled needle) in a volume of less than about 5 mL, less than about 3 mL, preferably less than about 2 mL, and more preferably less than about 1 mL.

[0269] The appropriate dosage of a protein, such as a mAb (a "therapeutically effective amount") will depend on the condition being treated, the severity and course of the disease or condition (whether the protein is administered for prophylactic or therapeutic purposes), previous therapy, the patient's medical history and response to the protein, the type of protein used, and the judgment of the attending physician. The protein is suitably administered as the sole treatment or in combination with other drugs or therapies, either once in a single injection or in multiple injections, or over a series of treatments.

[0270] The dosage formulation is designed such that the injection does not cause significant signs of irritation at the injection site, e.g., where the primary irritation index is less than 3 as assessed using the Draize scoring system. In an alternative embodiment, the injection causes a similar level of macroscopic irritation when compared to an injection of an equal volume of saline. In another embodiment, the bioavailability of the protein is higher when compared to an otherwise identical formulation administered in the same manner but without the viscosity-lowering agent(s). In another embodiment, the formulation is at least about as pharmacologic effective as the protein administered at about the same dose by intravenous infusion.

[0271] In a preferred embodiment, the formulation is injected to produce high levels of therapeutic protein, for example, AUC values ​​can be at least 10%, preferably at least 20%, higher than the AUC values ​​calculated for the otherwise same formulation, but without the viscosity-lowering agent(s), administered in the same manner.

[0272] Viscosity-lowering agents may also affect bioavailability: for example, the bioavailability of a protein may be at least 1.1-fold, and preferably at least 1.2-fold, greater than the bioavailability of an otherwise identical formulation without the viscosity-lowering agent(s) administered in the same manner.

[0273] Viscosity-reducing agents may also affect pharmacokinetics. For example, the C MAX is the C of a pharmacologically effective dose administered intravenously in approximately equal amounts. MAX is at least 10%, preferably at least 20% smaller than

[0274] In some embodiments, the protein is administered at higher dosages and less frequently than the otherwise same formulation without the viscosity-lowering agent(s).

[0275] A lower viscosity formulation requires less injection force. For example, the injection force is at least 10%, preferably at least 20%, lower than the injection force of the otherwise same formulation without the viscosity-lowering agent(s) administered in the same manner. In one embodiment, the injection is administered using a 27 gauge needle, which has an injection force of less than 30 N. The formulation can be administered in most cases using a very small gauge needle, for example, between gauge 27 and 31, usually gauge 27, 29 or 31.

[0276] The viscosity-lowering agent can be used to prepare a dosage unit formulation suitable for reconstitution to produce a liquid pharmaceutical formulation for subcutaneous or intramuscular injection. The dosage unit can contain dry powders of one or more proteins, one or more viscosity-lowering agents, and other excipients. The protein is present in the dosage unit such that, after reconstitution in a pharma-ceutically acceptable solvent, the resulting formulation has a protein concentration of about 100 mg to about 2,000 mg per mL (mg / mL). Such reconstituted formulations can have an absolute viscosity of about 1 cP to about 50 cP at 25°C.

[0277] The low-viscosity formulation can be provided as a solution or in a dosage unit form, where the protein is lyophilized with or without the viscosity-lowering agent and other excipients in one vial and the solvent, with or without the viscosity-lowering agent and other excipients, is provided in a second vial. In this embodiment, the solvent is added to the protein just before or at the time of injection to ensure uniform mixing and dissolution.

[0278] The viscosity-lowering agent(s) are present in the formulation at a concentration that does not cause significant and / or irreversible signs of toxicity when administered by subcutaneous, intramuscular or other type of injection. As used herein, "significant signs of toxicity" includes intoxication, lethargy, behavioral modification, such as those that occur with damage to the central nervous system, infertility, arrhythmia, cardiomyopathy, myocardial infarction and serious cardiac toxicity signs such as heart failure or congestive heart failure, renal failure, hepatic failure, respiratory distress and death.

[0279] In a preferred embodiment, the formulation does not cause significant irritation when administered twice daily, once daily, twice weekly, once weekly, or once monthly or less. The protein formulation can be administered without causing significant signs of irritation at the injection site as measured by a primary irritation index of less than 3, less than 2, or less than 1 when assessed using the Draize scoring system. As used herein, "significant signs of irritation" include erythema, redness and / or swelling at the site of injection with a diameter of greater than 10 cm, greater than 5 cm, or greater than 2.5 cm, necrosis at the injection site, exfoliative dermatitis at the injection site, and severe pain that interferes with daily activities and / or requires medical attention or hospitalization. In some embodiments, injection of the protein formulation causes a similar level of macroscopic irritation when compared to injection of an equal volume of saline.

[0280] The protein formulations may exhibit increased bioavailability when administered by subcutaneous or intramuscular injection, compared to the otherwise identical protein formulations without the viscosity-lowering agent(s). "Bioavailability" refers to the extent and rate at which a bioactive species, such as a mAb, reaches the circulation or site of action. In the case of SC or IM injection, the overall bioavailability may be increased compared to the otherwise identical formulations without the viscosity-lowering agent(s). "Bioavailability" refers to the percentage of an administered dose of a bioactive species that enters the circulation, determined with respect to the dose administered intravenously. One method of measuring bioavailability is by comparing the "area under the curve" (AUC) in plots of plasma concentration as a function of time. AUC can be calculated, for example, using the linear trapezoidal rule. "AUC ∞ " As used herein, "AUC" refers to the area under the plasma concentration curve from time 0 to the time when the plasma concentration returns to the baseline level. 0-t " as used herein refers to the area under the plasma concentration curve from time 0 to time t after, e.g., the time to reach baseline. Time is usually measured in days, but time can also be used as clear from the context. For example, the AUC can be 10%, 20%, 30%, 40% or 50% greater than the otherwise same formulation without the viscosity-lowering agent(s) administered in the same manner.

[0281] As used herein, "t max " refers to the time after administration at which the plasma concentration reaches its maximum value.

[0282] As used herein, "C max " refers to the maximum plasma concentration after administration of a dose and before administration of any subsequent doses.

[0283] As used herein, "C min " or "C trough " refers to the minimum plasma concentration after administration of a dose and before administration of a subsequent dose.

[0284] C after SC or IM injectionmax is the C of the intravenously administered dose max It can be, for example, at least 10%, more preferably at least 20% smaller than this C max A reduction in may result in reduced toxicity.

[0285] Pharmacokinetic and pharmacodynamic parameters can be estimated across species using techniques known to those skilled in the art. The pharmacokinetics and pharmacodynamics of antibody therapeutics can vary considerably based on the specific antibody. Approved murine mAbs have been shown to have a half-life of about 1 day in humans, while human mAbs usually have a half-life of about 25 days (Waldmann et al., Int. Immunol., 2001, vol. 13:1551-1559). The pharmacokinetics and pharmacodynamics of antibody therapeutics can vary considerably based on the route of administration. After IM or SC injection of IgG, the time to reach maximum plasma concentrations usually ranges from 2 to 8 days, but can be shorter or longer (Wang et al., Clin. Pharm. Ther., 2008, vol. 84(5):548-558). The pharmacokinetics and pharmacodynamics of antibody therapeutics can vary considerably based on the formulation.

[0286] The low-viscosity protein formulations allow for increased flexibility of administration and reduced frequency of administration compared to those protein formulations that do not include viscosity-lowering agent(s).In some embodiments, the frequency of administration can be reduced, for example, from once every 2 weeks to once every 6 weeks, by increasing the dosage administered per injection multiple times.The protein formulations, including but not limited to reconstituted formulations, can be administered using heated and / or self-mixing syringes or autoinjectors.The protein formulations can also be preheated in a separate warming unit before being loaded into a syringe.

[0287] i. Heated Syringe

[0288] Heated syringes are preheated using a syringe warmer. The syringe warmer may be a standard syringe that is used in a syringe warmer. The syringe warmer typically has one or more openings, each of which can receive a syringe containing a protein formulation, and a means for warming and maintaining the syringe at a particular (usually above ambient) temperature prior to use. This is referred to herein as a pre-heated syringe. Suitable heated syringe warmers include those available from Vista Dental Products and Inter-Med. The warmers are capable of accommodating syringes of various sizes and heating to any temperature, typically within 1° C., up to about 130° C. In some embodiments, the syringe is preheated in a heating bath, such as a water bather, that is maintained at the desired temperature.

[0289] The heated syringe can be a self-heating syringe, i.e., it can heat and maintain the liquid formulation inside the syringe at a certain temperature.The self-heating syringe can also be a standard medical syringe with a heating device attached to it.Suitable heating devices that can be attached to the syringe include syringe heaters or syringe heater tapes available from Watlow Electric Manufacturing Co., St. Louis, MO, and syringe heater blocks, stage heaters and in-line perfusion heaters, such as the SW-61 model syringe warmer available from Warner Instruments, Hamden, CT.The heater can be controlled by a central controller, for example, the TC-324B or TC-344B model heater controller available from Warner Instruments.

[0290] The heated syringe maintains the liquid protein formulation at a designated temperature or within 1° C., 2° C., or 5° C. of a designated 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., so long as the protein formulation is sufficiently stable at that temperature. The heated syringe can maintain the protein formulation at a temperature between 20° C. and 60° C., between 21° C. and 45° C., between 22° C. and 40° C., between 25° C. and 40° C., or between 25° C. and 37° C. Maintaining the protein formulation at an elevated temperature during injection reduces the viscosity of the liquid formulation, increases the solubility of the protein in the formulation, or both.

[0291] ii. Self-mixing syringes

[0292] The syringe can be self-mixing or can be equipped with a mixer. The mixer can be a static mixer or a dynamic mixer. Examples of static mixers include those disclosed in U.S. Pat. Nos. 5,819,988, 6,065,645, 6,394,314, 6,564,972 and 6,698,622. Examples of some dynamic mixers can include those disclosed in U.S. Pat. Nos. 6,443,612 and 6,457,609, and U.S. Patent Application Publication No. US2002 / 0190082. The syringe can include multiple barrels for mixing components of a liquid protein formulation. U.S. Pat. No. 5,819,998 describes a syringe with two barrels and a mixing tip for mixing two component viscous substances.

[0293] iii. Autoinjectors and prefilled syringes for protein preparations

[0294] The liquid protein formulations can be administered using a prefilled syringe autoinjector or needleless injection device. An autoinjector includes a portable, often pen-like, cartridge holder for holding a replaceable prefilled cartridge, and a spring-based or similar mechanism for subcutaneously or intramuscularly injecting a dose of liquid drug from the prefilled cartridge. Autoinjectors are usually designed for self-administration or administration by untrained personnel. Autoinjectors are available for dispensing either single or multiple doses from a prefilled cartridge. Autoinjectors can be configured for a variety of user settings, including, among others, injection depth, injection speed, etc. Other injection systems can include those described in U.S. Pat. No. 8,500,681.

[0295] Lyophilized protein formulations can be provided in pre-filled or unit dose syringes.US Patent Nos. 3,682,174, 4,171,698 and 5,569,193 describe a sterile syringe that contains two chambers, which can be pre-filled with dry formulation and liquid, which can be mixed immediately before injection.US Patent No. 5,779,668 describes a syringe system for lyophilization, reconstitution and administration of pharmaceutical compositions.In some embodiments, the protein formulations are provided in lyophilized form in pre-filled or unit dose syringes, which are reconstituted in syringes before administration and administered as a single subcutaneous or intramuscular injection.An autoinjector for delivering unit doses of lyophilized drugs is described in WO2012 / 010,832. Autoinjectors such as Safe Click Lyo™ (marketed by Future Injection Technologies, Ltd., Oxford, UK) can be used to administer unit doses of protein formulations, where the formulations are stored in lyophilized form and reconstituted immediately prior to administration. In some embodiments, the protein formulations are provided in unit dose cartridges for lyophilized drugs (sometimes called Vetter cartridges). Examples of suitable cartridges can include those described in U.S. Pat. Nos. 5,334,162 and 5,454,786.

[0296] V. Purification and Concentration Methods

[0297] The viscosity-reducing agents can also be used to aid in the purification and concentration of proteins. An effective amount of the viscosity-reducing agent(s) and excipients that reduces the viscosity of the protein solution is added to the protein. For example, the viscosity-reducing agents are added to a concentration of between about 0.01M and about 1.0M, preferably between about 0.01M and about 0.50M, and most preferably between about 0.01M and about 0.25M.

[0298] The protein-containing viscosity-lowering agent solution is then purified or concentrated using a method selected from the group consisting of ultrafiltration / diafiltration, tangential flow filtration, centrifugal concentration, and dialysis. EXAMPLES

[0299] The above will be further understood by the following non-limiting examples.

[0300] The viscosities of all well-mixed aqueous mAb solutions were measured after 5 min equilibration at 25° C. (unless otherwise indicated) using either an mVROC microfluidic viscometer (RheoSense) or a DV2T cone and plate viscometer (Brookfield; “C&P”). The mVROC viscometer is equipped with chips “A” or “B”, each of which is manufactured with a 50 micron channel. Typically, 0.10 mL of protein solution was back-loaded into a gas-tight microlab instrument syringe (Hamilton; 100 μL) attached to the chip, and multiple flow rates, i.e., 100 μL for each chip, were measured. For example, samples of about 50 cP were run at about 10, 20 and 30 μL / min (about 180, 350 and 530 s for chip "A", respectively) until the viscosity stabilized (usually after at least 30 s). -1 The mean absolute viscosity and standard deviation were then calculated from at least these three measurements. A C&P viscometer was fitted with a CPE40 or CPE52 spindle (0.8° and 3.0° cone angles, respectively) and 0.50 mL of sample was added at 2–400 s. -1 Specifically, the samples were run at shear rates starting at a rate that provided at least 10% torque and continuing until the instrument torque reached 100%, at shear rates of 22.58, 24.38, 26.25, 28.13, 30, 31.88, 45, 67.5, 90, 112.5, 135, 157.5, 180, 202.5, 247, 270, 292.5, 315, 337.5, 360, 382, ​​and 400 s, respectively. -1The viscosity was measured at 100° C. for 30 seconds. The extrapolated zero shear viscosity was then determined from a plot of dynamic viscosity versus shear rate for the samples measured on a DV2T cone and plate viscometer. The extrapolated zero shear viscosity reported is the average and standard deviation of at least three measurements.

[0301] Example 1 Effect of the Viscosity-Lowering Agent Camphorsulfonic Acid Lysine (CSAL) on the Viscosity of Solutions of Biosimilar ERBITUX®

[0302] material and method

[0303] Commercially obtained biosimilar ERBITUX® (100-400 mg) containing pharmaceutical excipients (polysorbate 80, phosphate buffer, and NaCl) was purified. First, polysorbate 80 was removed using a DETERGENT-OUT® TWEEN® medicolumn (G-Biosciences). The resulting solution was then extensively buffer exchanged into 20 mM sodium phosphate buffer (PB; pH 7.0) or 20 mM CSAL (pH 7.0) and purified using a Jumbo The mixture was concentrated in a sep centrifugal concentrator (Pall Corp.) to a final volume of less than 10 mL. The collected protein solution was freeze-dried. Protein and buffer salts or agents were removed. The dried protein cakes containing mAb were reconstituted to a final volume of 0.15-1.3 mL. These samples were reconstituted with enough additional PB (pH 7.0) or CSAL (pH 7.0) to bring the final concentration of PB or CSAL to 0.25 M. The final concentration of mAb in solution was determined by light absorbance at 280 nm. The protein concentrations reported represent the range of all protein samples included in each table or figure. Specifically, the values ​​reported are the median plus or minus half of the range. Zero shear extrapolated using an experimentally determined extinction coefficient of 1.4 L / g cm, and the reported viscosities were measured with a DV2T cone and plate viscometer.

[0304] result

[0305] The data in FIG. 1 demonstrates the viscosity-reducing effect of CSAL on aqueous solutions of biosimilar ERBITUX®. The viscosity of solutions of biosimilar ERBITUX® in phosphate buffer (PB) increased exponentially as the mAb concentration increased. The viscosity of solutions of biosimilar ERBITUX® in the presence of CSAL was observed to increase exponentially as the mAb concentration increased, but not as much as the formulation in PB. That is, the viscosity gradient is reduced. The data in FIG. 1 show that the higher the concentration of mAb, the greater the viscosity-reducing effect. The magnitude of the viscosity-reducing effect brought about by the replacement of PB with CSAL varied from 1.1-fold at 100±5 mg / mL mAb to 10.3-fold at 227±5 mg / mL mAb.

[0306] Example 2 Viscosity-reducing effect of the viscosity-reducing agent camphorsulfonic acid lysine (CSAL) as a function of biosimilar AVASTIN® concentration

[0307] material and method

[0308] Commercially obtained biosimilar AVASTIN® containing pharmaceutical excipients (polysorbate 20, phosphate buffer, citrate buffer, mannitol, and NaCl) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed (using an extinction coefficient of 1.7 L / g cm at 280 nm) as described above in Example 1. The protein was formulated to contain either 0.25 M phosphate buffer or 0.25 M CSAL.

[0309] result

[0310] FIG. 2 illustrates the viscosity of aqueous mAb solutions in aqueous buffered solutions and with CSAL as a function of mAb concentration. The viscosity of biosimilar AVASTIN® in aqueous phosphate buffer and in the presence of CSAL increases exponentially with increasing concentration. However, as with biosimilar ERBITUX®, this increase is significantly lower for CSAL-containing formulations. That is, the viscosity gradient is reduced. In general, a greater viscosity-reducing effect was observed with higher mAb concentrations. The magnitude of the viscosity-reducing effect brought about by the replacement of PB with CSAL varied from 2.1-fold at 80 mg / mL mAb to 3.7-fold at 230±5 mg / mL mAb.

[0311] Example 3 Viscosity-reducing effect as a function of CSAL concentration for aqueous solutions of biosimilar ERBITUX®

[0312] material and method

[0313] Samples were purified, buffer exchanged, concentrated, dried, reconstituted and analyzed as above in Example 1. Final concentrations of CSAL upon reconstitution in aqueous CSAL ranged from 0.25M to 0.50M.

[0314] result

[0315] Table 1 shows the viscosity of solutions of biosimilar ERBITUX® formulated in 0.25 M phosphate buffer (no CSAL as a control) and with varying concentrations of CSAL. The viscosity-lowering effect of CSAL increases by 8.4-12.1 fold with increasing concentration of the viscosity-lowering agent. The data in Table 1 show that, at least within the range of viscosity-lowering agent concentrations tested, the higher the concentration of CSAL, the greater the viscosity-lowering effect.

[0316] [Table 1]

[0317] Example 4 Viscosity of solutions of biosimilar ERBITUX® as a function of temperature in the presence of various viscosity-reducing agents

[0318] material and method

[0319] Aqueous solutions of biosimilar ERBITUX® containing various viscosity-lowering agents were prepared as described in Example 1. Specifically, a 20 mM solution of the viscosity-lowering agent of interest was used for buffer exchange, and the lyophilized cake was reconstituted to 0.25 M of the respective viscosity-lowering agent. For samples containing CSA-APMI, the biosimilar ERBITUX® was fully buffer exchanged into 2 mM PB (pH 7.0) and concentrated in a Jumbosep centrifugal concentrator (Pall Corp.) to a final volume of less than 10 mL. The samples were first divided into aliquots. An appropriate amount of CSAAPMI solution (pH 7.0) was then added to each aliquot, resulting in a final excipient concentration of 0.25 M upon reconstitution with water. The protein solution was then freeze-dried. The dried protein cake containing the protein and viscosity-lowering agent (and negligible amounts of buffer salts) was reconstituted to a final volume of approximately 0.10 mL and the viscosity-lowering agent concentration described above.

[0320] result

[0321] Table 2 shows the viscosity data of biosimilar ERBITUX® in the presence of six viscosity-lowering agents: camphorsulfonic acid lysine (CSAL), camphorsulfonic acid arginine (CSAA), benzenesulfonic acid lysine (BSAL), benzenesulfonic acid arginine (BSAA), naphthalenesulfonic acid arginine (NSAA), and camphorsulfonic acid 1-(3-aminopropyl)-2-methyl-1H-imidazole (CSAAPMI). The data in Table 2 shows at least about a 9-fold viscosity reduction for all six viscosity-lowering agents compared to a solution of biosimilar ERBITUX® in phosphate buffer under otherwise identical conditions. The most effective viscosity-lowering agent, CSAAPMI, reduced the viscosity by >40-fold.

[0322] Furthermore, the data in Table 3 show that at temperatures ranging from 20° C. to 30° C., the 225 mg / mL solution of biosimilar ERBITUX® prepared with 0.25 M CSAA had the lowest viscosity among the five viscosity-lowering agents. Thus, the viscosity trends observed at 25° C. appear to be predictive of viscosities at temperatures of at least 20° C. and 30° C.

[0323] [Table 2]

[0324] [Table 3]

[0325] Example 5 Effect of temperature on the viscosity of aqueous solutions of biosimilar AVASTIN® formulated with various viscosity-reducing agents

[0326] material and method

[0327] Solutions of biosimilar AVASTIN® containing various viscosity-lowering agents were prepared as described above in Example 1. In particular, a 20 mM solution of the viscosity-lowering agent of interest was used for buffer exchange and the lyophilized cake was reconstituted to 0.15 M or 0.25 M of the viscosity-lowering agent.

[0328] result

[0329] As seen in Table 4, 0.25 M CSAL reduced the viscosity of a 230±5 mg / mL solution of biosimilar AVASTIN® at all three temperatures between 20 and 30° C. Furthermore, at 20° C. and 25° C., 0.15 M CSAL reduced the viscosity to nearly the same absolute value as 0.25 M CSAL, and was equally effective at 30° C.

[0330] The data in Table 5 compares the effectiveness of CSAL and BSAL at a concentration of 0.15 M. CSAL is the superior viscosity reducer compared to BSAL at all three temperatures.

[0331] [Table 4]

[0332] [Table 5]

[0333] Example 6 In the mAb solution, removal of CSAL reverses the viscosity-lowering effect.

[0334] material and method

[0335] Three samples of biosimilar ERBITUX® and biosimilar AVASTIN® were prepared, respectively. First, polysorbate was removed from a commercially obtained mAb solution. The resulting solution containing the remaining pharmaceutical excipients was either (i) concentrated in a centrifugation device with a 100-kDa molecular weight cut-off (MWCO) (Pall Corp.) as a control sample (original excipients), (ii) buffer exchanged into 0.25 M CSAL as described in Example 1, or (iii) buffer exchanged into 0.25 M CSAL, reconstituted, and then further exchanged into 0.25 M PB as described in Example 1. In this third case, exchange into 0.25 M phosphate buffer was proceeded by first dialysis overnight into 20 mM PB (50-kDa MWCO, Spectrum Labs). The partially dialyzed sample was then diluted to 60 mL of 0.25 M PB and subjected to centrifugal concentration (30-kDa MWCO Jumbosep (Pall Corp.) followed by a 100-kDa MWCO Macrosep device (Pall Corp.)). The viscosities of these three aqueous solutions were determined as described in Example 1 above.

[0336] result

[0337] In the presence of CSAL, the viscosity of aqueous solutions of both biosimilar ERBITUX® and biosimilar AVASTIN® was reduced by 2.7-fold and 1.5-fold, respectively. However, subsequent removal of CSAL increased (see Tables 6 and 7). Furthermore, removal of CSAL returned the viscosity of the mAb solution to nearly the same level as the original solution, suggesting that CSAL is not damaging to the protein and indicating that CSAL is necessary for the observed viscosity reduction.

[0338] [Table 6]

[0339] [Table 7]

[0340] Example 7 Camphorsulfonic acid-containing viscosity-lowering agents provide significant viscosity reduction in aqueous solutions of AVASTIN® and biosimilar AVASTIN®.

[0341] material and method Commercially obtained AVASTIN® and biosimilar AVASTIN® containing pharmaceutical excipients (AVASTIN®: trehalose, sodium phosphate buffer, and polysorbate 20; biosimilar AVASTIN®: polysorbate 20, phosphate buffer, citrate buffer, mannitol, and NaCl) were purified, buffer exchanged, concentrated, lyophilized, and reconstituted as described above. Samples in Table 8 were prepared as described in Example 1 above (using an extinction coefficient of protein of 1.7 L / g·cm at 280 nm) and measured on a C&P viscometer. Viscosity-reduced samples in Table 9 were prepared as described in Example 4 above, except that the mAb was fully buffer exchanged into 2 mM PB. An appropriate amount of viscosity-reducing agent was then added to achieve a final viscosity-reducing agent concentration of 0.15-0.35 M upon reconstitution. Viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with chip "A" or "B". Results

[0342] The data in Tables 8 and 9 demonstrate the viscosity-reducing effect of various viscosity-reducing agents on aqueous solutions of biosimilar AVASTIN®. In the presence of viscosity-reducing agents, including CSA, up to a 2.5-fold viscosity reduction (compared to mAb solutions in PB) is observed for aqueous solutions of biosimilar AVASTIN®.

[0343] [Table 8-1] [Table 8-2]

[0344] [Table 9]

[0345] As illustrated in Figure 3, the viscosity of aqueous solutions of 200 ± 9 mg / mL biosimilar AVASTIN® containing CSAA was measured as a function of pH. As the pH increased, the magnitude of the viscosity-reducing effect due to the presence of CSAA in aqueous solutions of biosimilar AVASTIN® also increased, reaching a minimum and maximum viscosity-reducing effect at about pH 7. Viscosity reduction by CSAA was compared as a function of pH for two different concentrations of biosimilar AVASTIN®. Figure 4 demonstrates that 0.25 M CSAA results in greater viscosity reduction as (i) the concentration of biosimilar AVASTIN® and (ii) the pH increase.

[0346] Table 10 compares the viscosity reduction of biosimilar AVASTIN® with and without CSAL to that of branded AVASTIN®. The branded AVASTIN® solution has a significantly higher viscosity than the solution of the biosimilar mAb in the absence of a viscosity-lowering agent. However, the presence of 0.25M CSAL reduces the viscosity of biosimilar AVASTIN® and branded AVASTIN® by 1.8-fold and 3.3-fold, respectively. The viscosities of biosimilar and branded AVASTIN® are observed to be similar in the presence of 0.25M CSAL.

[0347] [Table 10]

[0348] As demonstrated in Table 11, CSA 1-(3-aminopropyl)-2-methyl-1H-imidazole (CSAAPMI) with HCl provided better viscosity reduction than CSAL, with a greater than 5-fold reduction in viscosity compared to the PB control for the 210 mg / mL biosimilar AVASTIN® solution.

[0349] [Table 11]

[0350] For a solution containing approximately 230 mg / mL of biosimilar AVASTIN®, Table 12 demonstrates that there is approximately a 5-fold viscosity reduction with a sulfosalicylic acid-containing viscosity-lowering agent, as well as CSAAPMI and CSA thiamine.

[0351] [Table 12]

[0352] Example 8 Effect of Viscosity-Lowering Agents on Aqueous Solutions of ERBITUX® and Biosimilar ERBITUX®

[0353] material and method

[0354] Aqueous solutions of biosimilar ERBITUX® and branded ERBITUX® containing various viscosity-lowering agents were prepared as described in Example 1. Specifically, 20 mM solutions of the salts of interest were used for buffer exchange and the lyophilized cakes were reconstituted to contain 0.25 M of the respective viscosity-lowering agent. Viscosity was measured using either a RheoSense mVROC microfluidic viscometer equipped with chip "A" or "B" or a DV2T cone and plate viscometer.

[0355] result

[0356] Table 13 shows data for biosimilar ERBITUX® (222±5 mg / mL) in the presence of five viscosity-lowering agents: CSAA, CSAL, BSAA, BSAL and NSAA. Table 14 compares the viscosity reduction of biosimilar ERBITUX® solutions using CSAA and CSAL with arginine or lysine alone.

[0357] [Table 13]

[0358] [Table 14]

[0359] The data in Table 13 show at least a 9.0-fold viscosity reduction for all five viscosity-reducing agents compared to aqueous solutions of biosimilar ERBITUX® in phosphate buffer under otherwise identical conditions. The most effective viscosity-reducing agents, CSAA and BSAA, reduced solution viscosity by nearly 21-fold. The viscosity of aqueous solutions of biosimilar ERBITUX® containing 0.25 M CSAA was compared as a function of pH at various protein concentrations. Figure 5 demonstrates that a viscosity minimum is observed at about pH 7.0 for all protein concentrations. The effect of pH on viscosity is most pronounced at higher protein concentrations (253 mg / mL in this example).

[0360] As can be seen in Table 15, aqueous solutions of biosimilar ERBITUX® and branded ERBITUX® have similar viscosities in the presence of 0.25 M arginine salt BSAA.

[0361] [Table 15]

[0362] The effect of viscosity-reducing agents on the formation of irreversible protein aggregates was tested for biosimilar ERBITUX®. Aqueous liquid formulations of (i) biosimilar ERBITUX® and (ii) biosimilar ERBITUX® containing 0.25 M CSAL were prepared. These solutions were stored at 4° C. for 90 days at pH 5.4 and 7.0, respectively. The stored samples were tested using size exclusion chromatography (column: Tosoh TSKgel UltraSW Aggregate; mobile phase: 0.1 M potassium phosphate / 0.1 M sodium sulfate, pH 6.8, 0.8 mL / min; injection: 20 μL of 5 mg / mL mAb solution). The data in Table 16 reveal the absence of significant aggregate formation in either the commercial formulation or the high concentration viscosity-reducing formulation.

[0363] [Table 16]

[0364] Example 9 Effect of Viscosity-Lowering Agents on Aqueous Solutions of REMICADE®

[0365] material and method

[0366] Commercially obtained REMICADE® containing pharmaceutical excipients (sucrose, polysorbate 80, sodium phosphate buffer) was prepared according to the instructions in the prescribing information sheet. It was then prepared as described in Example 1 above (1.4 L / g at 280 nm). * Using an extinction coefficient of 1000 nm (cm), this aqueous formulation was purified, buffer exchanged, concentrated, dried, reconstituted and analyzed. Viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with either chip "A" or "B".

[0367] result

[0368] The data for aqueous solutions of REMICADE® in Table 17 demonstrates that (i) viscosity-lowering agents containing bulky cyclic groups provide greater than 15-fold viscosity reduction, and (ii) CSAA, CSAAPMI, and sulfosalicylic acid diarginine (SSA DiArg) provide the greatest viscosity reduction of approximately 29-fold. Solution viscosities in the presence of ArgHCl alone are significantly higher than those with bulky cyclic groups.

[0369] [Table 17]

[0370] The dependence of viscosity reduction on viscosity-reducing agent concentration was tested for aqueous solutions of REMICADE® in the presence of CSAA. The results, shown in Table 18, demonstrate that the viscosity reduction increases with increasing viscosity-reducing agent concentration. For example, the viscosity reduction is more than two times greater with 0.35M viscosity-reducing agent compared to 0.20M viscosity-reducing agent (which is less than half the viscosity).

[0371] [Table 18]

[0372] The biophysical properties of solutions of REMICADE® formulated with 0.25M CSAA were evaluated over a 90-day period. Samples of REMICADE® formulated with 0.25M CSAA were prepared as described in Example 1 above. As can be seen in Table 19 and FIG. 6, the monomer content of concentrated solutions of REMICADE® in 0.25M CSAA, as determined by size exclusion chromatography (Tosoh TSKgel UltraSW Aggregate column; 0.1M potassium phosphate / 0.1M sodium sulfate buffer, pH 6.8, 0.8 mL / min; 20 μL injection of approximately 4.5 mg / mL solution), is similar to the formulation at all time points, with no detectable aggregation observed after 100 days of storage at 4° C. Viscosity measured using a microfluidic viscometer demonstrated to remain stable even after 30 days of storage at 4° C. (Table 20). Additionally, antigen binding of the REMICADE® proteins so treated was measured using a REMICADE®-specific ELISA assay, and no loss in binding was observed between days 0 and 100 (Table 20). Similarly, after one week of storage at room temperature, the monomer content (Table 21) and antigen binding (normalized to that of the formulation, Table 22) of a concentrated solution of REMICADE® in 0.25 M CSAA is comparable to that of the formulation. Finally, Table 23 demonstrates that storage of lyophilized cakes containing CSAA at 4° C. for 75 days does not negatively affect the viscosity of the solution or the degree of protein aggregation when the sample is reconstituted. The results in Tables 19-23 and FIG. 6 demonstrate the biophysical stability of REMICADE® formulated with CSAA before storage and after storage at 4° C. for at least 100 days.

[0373] [Table 19]

[0374] [Table 20]

[0375] [Table 21]

[0376] [Table 22]

[0377] [Table 23]

[0378] Example 10 Effect of Viscosity-Lowering Agents on Aqueous Solutions of HERCEPTIN(R)

[0379] material and method

[0380] Commercially obtained HERCEPTIN® containing pharmaceutical excipients (histidine buffer, trehalose, polysorbate 20) was prepared according to the instructions in the prescribing information sheet. This aqueous formulation was then purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed (1.5 L / g at 280 nm) as described in Example 1 above. * The viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with chip "A" or "B".

[0381] result

[0382] The data presented in Table 24 indicates that the viscosity of aqueous solutions of HERCEPTIN® containing a viscosity-lowering agent is minimal in the presence of CSAA when compared to aqueous solutions of HERCEPTIN® containing PB. At higher protein concentrations (i.e., >250 mg / mL), arginine HCl alone significantly reduces viscosity, and CSA further enhances the effect.

[0383] [Table 24]

[0384] Example 11 Effect of Viscosity Reducers on Aqueous Solutions of TYSABRI®

[0385] material and method

[0386] Commercially obtained TYSABRI® containing pharmaceutical excipients (sodium phosphate buffer, sodium chloride, polysorbate 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed (1.5 L / g at 280 nm) as described in Example 1 above. * The viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with chip "A" or "B".

[0387] result

[0388] The data presented in Table 25 indicates that the viscosity reduction of aqueous solutions of TYSABRI® containing a viscosity-lowering agent (compared to solutions containing PB) is approximately 2.5-fold at around 276 mg / mL protein.

[0389] [Table 25]

[0390] Example 12 Effect of Viscosity-Lowering Agents on Aqueous Solutions of Biosimilar RITUXAN®

[0391] material and method

[0392] Commercially obtained biosimilar RITUXAN® containing pharmaceutical excipients (citrate buffer, sodium chloride, and TWEEN® 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed (1.7 L / g at 280 nm) as described in Example 1 above. * The viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with chip "A" or "B".

[0393] result

[0394] The data presented in Table 26 demonstrate that the viscosity reduction of aqueous solutions of biosimilar RITUXAN® containing a viscosity-lowering agent is greater than 13-fold at about 213 mg / mL protein and greater than 5-fold at about 202 mg / mL compared to mAb formulated in PB.

[0395] [Table 26]

[0396] Example 13 Effect of Viscosity-Lowering Agents on Aqueous Solutions of VECTIBIX®

[0397] material and method

[0398] Commercially obtained VECTIBIX® containing pharmaceutical excipients was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed (1.25 L / g at 280 nm) as described in Example 1 above. * The viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with chip "A" or "B".

[0399] result

[0400] The data presented in Table 27 show that the viscosity reduction of aqueous solutions of VECTIBIX® containing a viscosity-lowering agent is approximately 2-fold at 291 mg / mL and 3-fold at 252 mg / mL when compared to solutions containing PB but no viscosity-lowering agent.

[0401] [Table 27]

[0402] Example 14 Effect of Viscosity-Reducing Agents on Aqueous Solutions of ARZERRA®

[0403] material and method

[0404] Commercially obtained ARZERRA® containing pharmaceutical excipients was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed (1.5 L / g at 280 nm) as described in Example 1 above. * The viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with chip "A" or "B".

[0405] result

[0406] The data presented in Table 28 show that the viscosity reduction of aqueous solutions of ARZERRA® containing a viscosity-lowering agent is approximately three-fold at 274 mg / mL and two-fold at 245 mg / mL when compared to solutions containing PB but no viscosity-lowering agent.

[0407] [Table 28]

[0408] Example 15 Comparison of different methods for measuring viscosity

[0409] material and method

[0410] An aqueous solution containing 220 mg / mL REMICADE® and 0.25 M CSAA was prepared as described in Example 1 above. Table 29 reports the viscosities at 25° C. and pH 7.0 as extrapolated zero shear viscosities from cone and plate viscometer measurements and as absolute viscosities measured by microfluidic viscometer. Cone and plate measurements were performed using a DV2T cone and plate viscometer (Brookfield) equipped with a CPE40 or CPE52 spindle at multiple shear rates from 2 to 400 s -1 The zero shear viscosity was measured between 0.01 and 0.05. The extrapolated zero shear viscosity was determined from a plot of absolute viscosity versus shear rate. Microfluidic viscometer measurements were performed using a RheoSense mVROC microfluidic viscometer equipped with chip "A" or "B" at multiple flow rates (approximately 20%, 40% and 60% of maximum pressure for each chip).

[0411] result

[0412] The data in Table 29 demonstrate that the absolute viscosity from the microfluidic viscometer can be directly compared to the extrapolated zero shear viscosity determined from the cone and plate viscometer.

[0413] [Table 29]

[0414] To compare a wider range of viscosities and protein concentrations, aqueous solutions of a model antibody, bovine gamma globulin, were prepared with and without 0.25 M CSAL. These viscosities were measured as described above at protein concentrations ranging from 110 mg / mL to 310 mg / mL. The data shown in Table 30 demonstrate that the absolute viscosities from the microfluidic viscometer can be directly compared to the extrapolated zero shear viscosities for both low and high viscosity protein solutions.

[0415] [Table 30]

[0416] Example 16 The viscosity-lowering agent shows no signs of toxicity when injected subcutaneously.

[0417] material and method

[0418] Thirty 11-week-old Sprague-Dawley rats were divided into six groups of five rats each (three saline control groups and three CSAA groups). The rats were injected subcutaneously with 0.5 mL of either endotoxin-free phosphate buffered saline or endotoxin-free 0.25 M CSAA according to the following schedule: one group from each condition was injected once on day 1 and then sacrificed 1 hour later; one group from each condition was injected once on day 1 and once on day 2 and then sacrificed 24 hours after the second injection; and one group from each condition was injected once on day 1, once on day 2, and once on day 3 and then sacrificed 24 hours after the third injection.

[0419] Clinical observations for any pharmacological or toxicological signs were recorded pre-dose, immediately after dosing, 1 hour and 4 hours (± 15 minutes) after dosing, and daily thereafter. Irritation at the injection site, if present, was scored using the Draize rating scale pre-dose, immediately after dosing, 1 hour (± 15 minutes) after dosing, and before sacrifice.

[0420] result

[0421] Overall, the observed outcomes of saline and CSAA injections were macroscopically similar throughout the course of the study, with both ranging from no irritation to slight irritation accompanied by edema, scored from 0 to 2, at various time points. Microscopic examination of the injection sites suggested a very slight, clinically insignificant, irritant effect of CSAA that was no longer evident by day 4.

[0422] (Example 17) Concentrated aqueous solutions of REMICADE® formulated with viscosity-reducing agents exhibit low syringe ejection forces and high monomer content when dispensed through various gauge needles.

[0423] material and method

[0424] Commercially obtained REMICADE® containing pharmaceutical excipients (sucrose, polysorbate 80, sodium phosphate buffer) was prepared according to the instructions in the prescribing information sheet. This aqueous formulation was then purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed (1.4 L / g at 280 nm) as described in Example 1 above. * The viscosity of the concentrated REMICADE® solution was measured using an extinction coefficient of 1000 nm (using an extinction coefficient of 1000 nm in cm). A 20 mM solution of either phosphate buffer, CSAAPMI or CSAA was used for buffer exchange, and the lyophilized cake was reconstituted to 0.25 M of each viscosity-lowering agent. After reconstitution, the viscosity of each solution was measured using a microfluidic viscometer as described in the previous example. These solutions were then back-filled into 1 mL BD insulin syringes with 27, 29 or 31 gauge fixed needles. The force required to extrude the concentrated REMICADE® solution was then measured using an Instron at an ejection rate equivalent to a fluid flow rate of 3 mL / min. The solutions ejected from the syringes were collected and analyzed by size exclusion chromatography.

[0425] result

[0426] All of the REMICADE® solutions containing the viscosity-lowering agent were able to be expelled from the syringe with relatively low extrusion forces (Table 31). The solution containing the phosphate buffer could not be expelled due to high viscosity. Both solutions containing the viscosity-lowering agent retained high monomer content after extrusion, regardless of needle gauge, as shown in Table 31.

[0427] [Table 31]

[0428] (Example 18) The viscosity-reducing agent reduces the viscosity of concentrated aqueous solutions of biosimilar AVASTIN®.

[0429] material and method

[0430] Commercially obtained biosimilar AVASTIN® containing pharmaceutical excipients (polysorbate 20, phosphate and citrate buffers, mannitol, and NaCl) was purified. First, polysorbate 20 was removed using DETERGENT-OUT® TWEEN medi-columns (G-Biosciences). The resulting solutions were then thoroughly buffer exchanged into 20 mM sodium phosphate buffer (PB) for the PB samples and 2 mM PB for the viscosity-reducing agent samples, and concentrated in Jumbosep centrifugal concentrators (Pall Corp.) to a final volume of less than 10 mL. Viscosity-reducing agents were then added to the 2 mM PB samples as described in Example 4 above. The viscosity-reducing agent(s) were added in sufficient amounts to provide the concentrations specified below upon reconstitution. When the agents were combined, the concentration of each component was 0.15 M. These protein solutions were then freeze-dried. The dried protein cake was reconstituted in phosphate buffer (for PB samples) or water (for samples containing a viscosity-lowering agent) to a final volume of approximately 0.10 mL. The final concentration of mAb in solution was determined by Coomassie protein quantification assay, by comparison of samples of unknown concentration to a standard curve of biosimilar AVASTIN®, or by 1.7 L / g mAb when possible. * The viscosity was determined either by A280 using an extinction coefficient of 1000 nm in cm. The reported viscosities were measured with a RheoSense mVROC microfluidic viscometer. The results are reported in Table 32.

[0431] result

[0432] Many of the GRAS, IIG, and API compounds can reduce the viscosity of concentrated biosimilar AVASTIN® solutions when compared to phosphate-buffered samples. Of those compounds included in Table 32, local anesthetics such as procaine and lidocaine, and GRAS agents such as biotin, are the most effective viscosity-reducing excipients.

[0433] [Table 32-1] [Table 32-2] [Table 32-3]

[0434] (Example 19) The viscosity reduction is an agent concentration dependent effect.

[0435] material and method

[0436] Aqueous solutions of commercially obtained biosimilar AVASTIN® were prepared as described in Example 4. The dried protein cake was reconstituted in phosphate buffer or water to a final volume of approximately 0.10 mL and final concentrations of either 0.10 or 0.25 M 1-(3-aminopropyl)-2-methyl-1H-imidazole dihydrochloride (APMI). * 2HCl). The final concentration of mAb in solution was determined by comparing samples of unknown concentration to a standard curve of biosimilar AVASTIN® using a Coomassie protein quantification assay. Viscosity reported was measured with a RheoSense mVROC microfluidic viscometer.

[0437] result

[0438] As shown in Figure 7, APMI * As the concentration of 2HCl increased, the viscosity reducing effect increased.

[0439] (Example 20) A single viscosity-lowering agent reduces the viscosity of many therapeutically relevant monoclonal antibodies.

[0440] material and method

[0441] Aqueous solutions of commercially obtained biosimilar AVASTIN® were prepared as described in Example 4. The dried protein cake was reconstituted in phosphate buffer or water to a final volume of approximately 0.10 mL and a final thiamine HCl concentration of 0.10 or 0.25 M. The final concentration of mAb in solution was determined by comparing samples of unknown concentration to a standard curve of biosimilar AVASTIN® by Coomassie protein quantification assay.

[0442] Commercially obtained TYSABRI® containing pharmaceutical excipients (sodium phosphate buffer, NaCl, polysorbate 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed in the same manner. Commercially obtained HERCEPTIN® containing pharmaceutical excipients (sodium phosphate buffer, NaCl, polysorbate 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed in the same manner. Commercially obtained biosimilar ERBITUX® containing pharmaceutical excipients (polysorbate 80, phosphate buffer, and NaCl) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed in the same manner. Commercially obtained REMICADE® containing pharmaceutical excipients (sucrose, polysorbate 80, sodium phosphate buffer salt) was prepared according to the instructions in the prescribing information sheet. This aqueous formulation was then purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed in the same manner as described. The reported viscosities were measured with a RheoSense mVROC microfluidic viscometer.

[0443] result

[0444] The data in Table 33 demonstrate that thiamine HCl can reduce the viscosity of concentrated aqueous solutions of many therapeutically relevant mAbs. Thiamine HCl can produce a greater than four-fold reduction in viscosity for each mAb.

[0445] [Table 33]

[0446] (Examples 21 to 24) Viscosity-lowering agents reduce the viscosity of aqueous solutions of many therapeutically relevant monoclonal antibodies.

[0447] material and method

[0448] Aqueous solutions of commercially obtained biosimilar RITUXAN®, TYSABRI®, HERCEPTIN®, biosimilar ERBITUX®, and REMICADE® were prepared as described in Examples 18 and 19. Tables 34-38 demonstrate that the viscosity-lowering agents can be used advantageously with many different monoclonal antibodies.

[0449] result

[0450] [Table 34-1] [Table 34-2] [Table 34-3]

[0451] [Table 35]

[0452] [Table 36]

[0453] [Table 37-1] [Table 37-2]

[0454] [Table 38]

[0455] (Example 25) Viscosity-reducing effect of TPP and TPPAPMI as a function of biosimilar AVASTIN® concentration

[0456] Aqueous solutions of commercially obtained biosimilar AVASTIN® were prepared as described above in Example 1. The protein was formulated to contain either 0.25 M phosphate buffer, 0.10 M thiamine pyrophosphate (TPP), or 0.10 M TPP-1-(3-aminopropyl)-2-methyl-1H-imidazole (TPPAPMI).

[0457] Figure 8 illustrates the viscosity of aqueous solutions of biosimilar AVASTIN® as a function of mAb concentration, containing either phosphate buffer, TPP or TPPAPMI. The viscosity of biosimilar AVASTIN® in phosphate buffer increases exponentially within the range of protein concentrations tested. In the presence of TPP-containing excipients, the increase in viscosity is attenuated; i.e., the viscosity gradient is reduced.

[0458] (Example 26) Viscosity-reducing effect of the viscosity-reducing agent thiamine HCl as a function of the concentration of biosimilar SIMPONI ARIA®

[0459] material and method

[0460] Commercially obtained SIMPONI ARIA® containing pharmaceutical excipients (histidine, sorbitol, polysorbate 80) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed (using an extinction coefficient of 1.4 L / g cm at 280 nm) as described above in Example 1. The protein was formulated to contain either 0.15 M phosphate buffer or 0.15 M thiamine HCl.

[0461] result

[0462] Figure 9 illustrates the viscosity of aqueous solutions of SIMPONI ARIA® as a function of mAb concentration, containing either phosphate buffer or thiamine HCl. The viscosity of SIMPONI ARIA® in phosphate buffer increases exponentially within the range of protein concentrations tested. In the presence of thiamine HCl, the increase in viscosity is attenuated; i.e., the viscosity gradient is reduced.

[0463] Example 27 Viscosity-Lowering Effect of Thiamine HCl as a Function of ENBREL® Concentration

[0464] material and method

[0465] Commercially obtained ENBREL® containing pharmaceutical excipients (mannitol, sucrose, tromethamine) was purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed (using an extinction coefficient of 0.96 L / g cm at 280 nm) as described above in Example 1. The protein was formulated to contain either 0.15 M phosphate buffer or 0.15 M thiamine HCl.

[0466] result

[0467] Figure 39 illustrates the viscosity of aqueous solutions of ENBREL® containing either phosphate buffer or thiamine HCl. The addition of thiamine HCl reduces the viscosity of ENBREL® by up to about two-fold.

[0468] [Table 39]

[0469] (Example 28) Isotonic solutions of viscosity-reducing excipients reduce the viscosity of concentrated solutions of REMICADE®.

[0470] material and method

[0471] Commercially obtained REMICADE® containing pharmaceutical excipients (sucrose, polysorbate 80, sodium phosphate buffer) was prepared according to the instructions in the prescribing information sheet. This aqueous formulation was then purified, buffer exchanged, concentrated, dried, reconstituted, and analyzed as described in Example 1, except that an isotonic amount of a charged hydrophobic compound was added.

[0472] result

[0473] As demonstrated in Table 40, isotonic amounts of both CSAA and CSAAPMI can substantially reduce the viscosity of concentrated solutions of REMICADE®, in some cases by up to about 10-fold.

[0474] [Table 40]

[0475] Unless otherwise expressly defined above, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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

[Claim 1] The invention as depicted in the drawings.

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