Liquid protein formulations containing organophosphates

Citing Related Applications This application is US Provisional Application No. 62 / 030,521 entitled "Low-Viscosity Protein Formulations Containing Hydrophobic Salts" filed July 29, 2014; "Low-Viscosity" filed July 18, 2014. US Provisional Application No. 62 / 26,497 entitled "Protein Formulations Containing GRAS Viscosity-Reducing Agents"; US Provisional Application No. 62 / entitled "Low-Viscosity Protein Formulations Containing Ionic Liquids" filed June 5, 2014 008,050; US Provisional Application No. 61 / 988,005 entitled "Low-Viscosity Protein Formulations Containing Organophosphates" filed May 2, 2014; "Concentrated," filed February 28, 2014. US Provisional Application No. 61 / 946,436 entitled "Low-Viscosity Infliximab Formulations"; US Provisional Application entitled "Concentrated, Low-Viscosity, High-Molecular-Weight-Protein Formulations" filed February 21, 2014 No. 61 / 943,197; U.S. Provisional Application Nos. 61 / 940,227 entitled "Concentrated, Low-Viscosity High-Molecular-Weight Protein Formulations" filed February 14, 2014, and filed September 11, 2013. Claims the priority and benefits of US Provisional Application No. 61,876,621 entitled "Concentrated, Low-Viscosity, High-Molecular-Weight Protein Formulations". The disclosures of these applications are expressly incorporated herein by reference.

Field of Invention The present invention is generally in the field of injectable pharmaceutical formulations of proteins such as monoclonal antibodies, as well as methods of making and using them.

Background of the Invention Monoclonal antibodies (mAbs) are important protein-based therapeutic agents for the treatment of various human diseases such as cancer, infectious diseases, inflammation and autoimmune diseases. Over 20 mAb products have been approved by the US Food and Drug Administration (FDA), and approximately 20% of biopharmacy 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).

mAb-based therapies usually require repeated doses and doses of several mg / kg over a long period of time. Antibodies solutions or suspensions can be administered by parenteral routes, such as by intravenous (IV) injection and subcutaneous (SC) or intramuscular (IM) injection. Compared to the IV route, the SC or IM route reduces treatment costs during administration, increases patient compliance, and improves patient and healthcare provider convenience. According to FDA guidelines, to be effective and pharmaceutically acceptable, the parenteral formulation is preferably sterile, stable, injectable (eg, by syringe) and non-irritating to the injection site. Should be. Due to the low volume required for subcutaneous (usually less than about 2 mL) and intramuscular (usually less than about 5 mL) injection, a concentrated protein solution is required for these routes of administration for high-dose protein therapies. .. These high concentrations are very difficult to administer by injection, cause pain at the injection site, are often inaccurate, and / or can reduce chemical and / or physical stability. It is often a highly viscous formulation.

These characteristics create requirements for manufacture, storage, and use 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 pages, 2007). Therefore, in the development of commercially feasible protein drugs, the development of optimal formulations is prioritized.

High protein concentrations pose challenges related to the physical and chemical stability of proteins, as well as the difficulties associated with the manufacture, storage and delivery of protein formulations. One of the problems is that during processing and / or storage, proteins tend to aggregate to form particles, which makes handling during further processing and / or delivery difficult. 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 unnatural proteins, reversible self-association in aqueous solution can occur, which contributes to increased viscosity, which complicates delivery by injection, among other things ( See, for example, Steven J. Shire et al., J. Pharm. Sci., Vol. 93, pp. 1390-1402, 2004). Increased viscosity is one of the key challenges faced in concentrated protein compositions and affects both the method of production by conventional means and the ease of delivery of such compositions (eg,). , J. See Jezek et al., Advanced Drug Delivery Reviews Vol. 63, pp. 1107-1117, 2011).

Very viscous liquid formulations are difficult to manufacture, withdraw into syringes, and inject subcutaneously or intramuscularly. The use of force when manipulating a viscous formulation can result in excessive foaming, which can further denature and inactivate therapeutically active proteins. High viscosity solutions also require larger diameter needles for injection, causing more pain at the injection site.

Currently available commercially available mAb products administered by SC or IM injections are mannitol, sucrose, lactose, trehalose, POLOGAMER® (two registered trademarks) to prevent aggregation and improve stability. A nonionic triblock copolymer consisting of a central polyoxypropylene (poly (propylene oxide)) hydrophobic chain sandwiched between polyoxyethylene (poly (ethylene oxide)) hydrophilic chains), or POLYSORBATE®. ) 80 (PEG (80) sorbitan monolaurate) and other excipients or surfactants are usually formulated in aqueous buffers such as phosphate buffers or L-histidine buffers. The reported antibody concentration formulated as described above is usually up to about 100 mg / mL (Wang et al., J. Pharm. Sci. 96: 1-26, 2007).

US Pat. No. 7,758,860 describes a viscosity reduction using a buffer and a viscosity reducing inorganic salt such as calcium chloride or magnesium chloride in the formulation of low molecular weight proteins. 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 Pat. No. 7,666,413, the viscosity of aqueous formulations of high molecular weight proteins is such as arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride or sodium acetate. It is reduced by adding salts in concentrations above about 100 mM, or by adding organic or inorganic acids, as described in US Pat. No. 7,740,842. However, these salts do not reduce the viscosity to the desired level and, in some cases, make the formulation acidic enough to cause pain at the injection site.

U.S. Pat. No. 7,666,413 describes a viscosity-reducing formulation that contains certain salts and a reconstituted anti-IgE mAb, but with a maximum antibody concentration of only about 140 mg / mL. US Pat. No. 7,740,842 describes an E25 anti-IgE mAb formulation containing acetate / acetate buffer with antibody concentrations up to 257 mg / mL. NaCl, CaCl₂Or MgCl₂It was demonstrated that the addition of such salts reduces the dynamic viscosity under high shear conditions. However, at low shear, these salts produced an undesired dramatic increase in dynamic viscosity. In addition, inorganic salts such as NaCl may reduce solution viscosity and / or agglomeration (EP1981824).

Non-aqueous antibody preparations or protein preparations are also described. WO2006 / 071693 describes a non-aqueous suspension of up to 100 mg / mL mAb in a formulation with a viscosity enhancer (polyvinylpyrrolidone, PVP) and a solvent (benzyl benzoate or PEG400). WO2004 / 089335 describes a 100 mg / mL non-aqueous lysozyme suspension formulation containing PVP, glycoflor, benzyl benzoate, benzyl alcohol or PEG400. US2008 / 0226689A1 describes a non-aqueous viscous formulation of three vehicle components (polymers, surfactants and solvents) in a single phase of 100 mg / mL human growth hormone (hGH). U.S. Pat. No. 6,730,328 describes a less reactive, non-aqueous, hydrophobic, non-polar vehicle, such as perfluorodecalin, for protein formulations. These formulations are not optimized and have high viscosities that are detrimental to processing, manufacturing and injection; resulting in the presence of multiple vehicle components in the formulations; polymers not yet approved by the FDA. There are potential regulatory challenges associated with the use of.

Organic solvents such as benzyl benzoate (Miller et al., Langmuir 26: 1067-1074, 2010), benzyl acetate, ethanol or methyl ethyl ketone (Srinivasan et al., Pharm. Res. 30: 1749-1757, 2013). Alternative non-aqueous protein or antibody preparations to be used are described. In both cases, viscosities of less than 50 centipores (cP) were achieved when formulated at a protein concentration of at least about 200 mg / mL. U.S. Pat. No. 6,252,055 describes an mAb formulation with a concentration in the range of 100 mg / mL to 257 mg / mL. Formulations with concentrations above about 189 mg / mL demonstrated dramatic viscosity increase, low recovery, and difficulty in processing. U.S. Patent Application Publication No. 2012/0230982 describes antibody formulations with concentrations ranging from 100 mg / mL to 200 mg / mL. None of these formulations are sufficiently low in viscosity to facilitate injection.

Du and Klibanov (Biotechnology and Bioengineering Vol. 108: 632-636, 2011) found the viscosity of bovine serum albumin up to a maximum concentration of 400 mg / mL and concentrated aqueous solutions of bovine gamma globulin up to a maximum concentration of 300 mg / mL. It describes a decline. Guo et al. (Pharmaceutical Research Vol. 29: 3102-3109, 2012) described four low-viscosity aqueous solutions of model mAbs realized using hydrophobic salts. The mAb formulation used by Guo had an initial viscosity of 73 cP or less before adding the salt. On the other hand, the viscosity of many pharmaceutically important mAbs can exceed 1,000 cP at treatment-related concentrations.

Control of aggregation and viscosity is not a trivial issue in high concentration mAb solutions (EP 2538973). This is evident in the fact that few mAb products are currently on the market as high-concentration formulations (> 100 mg / mL) (EP 2538973).

In the references cited above, many groups have attempted to prepare low-viscosity formulations of mAbs and other therapeutically important proteins, but formulations that are truly useful for many proteins are still realized. It clearly shows that it is not. In particular, many of the above reports use agents whose safety and toxicity profiles have not been fully established. Therefore, these formulations appear to face a higher regulatory burden prior to approval than formulations containing compounds known to be safe. In fact, even if a compound has been shown to substantially reduce its viscosity, it may ultimately be unsuitable for use in a formulation intended for human injection. is there.

Many of the pharmaceutically important high molecular weight proteins, such as mAbs, are currently IV because they deliver therapeutically effective amounts of protein due to problems and other properties associated with the high viscosity of concentrated solutions of large proteins. It is administered by infusion. For example, protein concentrations greater than 150 mg / mL are often required to provide therapeutically effective amounts of large numbers of high molecular weight proteins, such as mAbs, in volumes less than about 2 mL.

<p><patcit num="1"><text>U.S. Pat. No. 7,758,860</text></patcit><patcit num="2"><text>U.S. Pat. No. 7,666,413</text></patcit><patcit num="3"><text>U.S. Pat. No. 7,740,842</text></patcit><patcit num="4"><text>International Publication No. 2006/071693</text></patcit><patcit num="5"><text>International Publication No. 2004/089335</text></patcit><patcit num="6"><text>U.S. Patent Application Publication No. 2008/0226689</text></patcit><patcit num="7"><text>U.S. Pat. No. 6,730,328</text></patcit><patcit num="8"><text>U.S. Pat. No. 6,252,055</text></patcit><patcit num="9"><text>U.S. Patent Application Publication No. 2012/0230982</text></patcit></p>

<p><nplcit num="1"><text>Daugherty et al., Adv. Drug Deliv. Rev. Vol. 58: pp. 686-706, 2006</text></nplcit><nplcit num="2"><text>Buss et al., Curr. Opinion in Pharmacol. Volume 12, pp. 615-622, 2012</text></nplcit><nplcit num="3"><text>Wang et al., J. Pharm. Sci. Vol. 96: pp. 1-26, 2007</text></nplcit><nplcit num="4"><text>Steven J. Shire et al., J. Pharm. Sci. Volume 93: 1390-1402, 2004</text></nplcit><nplcit num="5"><text>J. Jezek et al., Advanced Drug Delivery Reviews Vol. 63, pp. 1107-1117, 2011</text></nplcit><nplcit num="6"><text>Miller et al., Langmuir Vol. 26: 1067-1074, 2010</text></nplcit><nplcit num="7"><text>Srinivasan et al., Pharm. Res. Vol. 30, pp. 1749-1757, 2013</text></nplcit><nplcit num="8"><text>Du and Klibanov, Biotechnology and Bioengineering Vol. 108: pp. 632-636, 2011</text></nplcit><nplcit num="9"><text>Guo et al., Pharmaceutical Research Vol. 29: 3102-3109, 2012</text></nplcit></p>

<p> Therefore, an object of the present invention is to provide a low-viscosity concentrated liquid preparation of a pharmaceutically important protein, particularly a high molecular weight protein such as mAb.</p><p> A further object of the present invention is a low viscosity concentrated liquid preparation 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. , To provide a low-viscosity concentrated liquid preparation.</p><p> A further object of the present invention is a concentrated liquid formulation of a protein, particularly a high molecular weight protein such as mAb, which has a low viscosity that can improve injectability and / or patient compliance, convenience and comfort. It is to provide a concentrated liquid preparation.</p><p> It is also an object of the present invention to provide a method for making and storing a low viscosity concentrated preparation of a protein, particularly a high molecular weight protein such as mAb.</p><p> An additional object of the present invention is to provide a method for administering a low viscosity concentrated liquid preparation of a protein, particularly a high molecular weight protein such as mAb. An additional object of the present invention is to provide a method of treating low-viscosity, high-concentration biologics using concentration and filtration techniques known to those of skill in the art.</p>

<p> In certain embodiments, for example, the following are provided: (Item 1) (i) One or more proteins; A liquid pharmaceutical formulation for injection comprising (ii) one or more reduced viscosity organic phosphates; and (iii) a pharmaceutically acceptable solvent; the protein being solvent and organic in a volume suitable for injection. When combined with phosphate, the formulation has an absolute viscosity of about 1 cP to about 50 cP at 25 ° C as measured using a conical plate viscous meter, and the absolute viscosity of the formulation is that of the organic phosphate. An injectable pharmaceutical formulation, which is lower than the absolute viscosity of the same formulation, except that it instead contains an equal amount of sodium phosphate, and in each case the absolute viscosity is an extrapolated zero shear viscosity. (Item 2) The preparation according to item 1, wherein the protein (s) is a high molecular weight protein having a molecular weight between about 70 kDa and 100 kDa, about 100 kDa and about 250 kDa, or about 250 kDa and about 500 kDa. .. (Item 3) The preparation 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) The preparation according to 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) Any one of items 1 to 4 in which the protein (s) are present in a combined amount of about 100 mg to about 2,000 mg (mg / mL) per mL, and optionally more than about 150 mg / mL. The formulation described in the section. (Item 6) The preparation according to any one of items 1 to 5, which comprises at least two different proteins, preferably both of which have a molecular weight of at least about 50 kDa. (Item 7) The item according to any one of items 1 to 6, wherein the initial absolute viscosity at the same protein concentration before the addition of the organic phosphate exceeds about 60 cP, more than about 80 cP, or more than about 100 cP. Formulation. (Item 8) The preparation according to any one of items 1 to 7, wherein the liquid preparation is an aqueous product having a pH between about 5.0 and about 8.0. (Item 9) The preparation according to any one of items 1 to 8, which contains the organic phosphate present at a concentration of about 0.01 M to about 1.0 M. (Item 10) The preparation according to any one of items 1 to 9, which comprises the organic phosphate present in an amount of less than 0.30 M or less than 0.15 M. (Item 11) One or more pharmaceutically acceptable excipients for subcutaneous or intramuscular injection, which are sugars or sugar alcohols, buffers, preservatives, carriers, antioxidants, chelating agents, natural or From item 1 comprising one or more pharmaceutically acceptable excipients selected from the group consisting of synthetic polymers, lyophilizers, lyophilizers, surfactants, bulking agents and stabilizers. The preparation according to any one of 10. (Item 12) One or more of the excipients consist of polyolbate, poloxamer 188, sodium lauryl sulfate, sugar alcohols such as mannitol and sorbitol, poly (ethylene glycol), glycerol, propylene glycol and poly (vinyl alcohol). The preparation according to item 11, which is selected from the group consisting of polyols selected from the group. (Item 13) The preparation according to item 11, wherein the surfactant is present in an amount of less than about 10 mg / mL. (Item 14) The preparation according to item 12, which comprises a polyol present in an amount of about 2 mg / mL to about 900 mg / mL. (Item 15) The preparation according to any one of items 1 to 14, wherein the absolute viscosity is about 5 cP to about 50 cP at 25 ° C. (Item 16) The absolute viscosity is at least about 30% higher than the absolute viscosity of the organic phosphate-free preparation when measured under the same conditions except that the organic phosphate is replaced with an appropriate buffer having almost the same concentration. Low, the formulation according to any one of items 1 to 15. (Item 17) When the absolute viscosity is measured under the same conditions except that the organic phosphate is replaced with an appropriate buffer having almost the same concentration, the absolute viscosity is at least about twice as high as the absolute viscosity of the preparation containing no organic phosphate. Or the formulation according to any one of items 1 to 16, which is 4 times lower. (Item 18) The preparation according to any one of items 1 to 17 in a unit dose vial, container or prefilled syringe. (Item 19) The preparation according to item 18, wherein the protein, organic phosphate and / or excipient is in a dry form, preferably lyophilized. (Item 20) The volume of the preparation when the organic phosphate, protein and solvent are combined is less than about 1.5 mL in the case of SC injection and less than about 3 mL in the case of IM injection, according to any one of items 1 to 19. Formulation. (Item 21) The preparation according to any one of items 1 to 20, which is isotonic with human serum. (Item 22) The preparation according to any one of items 1 to 21, which behaves rheologically and essentially as a Newtonian liquid under the condition of being administered to a human who needs it. (Item 23) The preparation according to any one of items 1 to 22, which realizes a therapeutically effective dose as compared with the same dose of the protein administered by intravenous injection. (Item 24) When the organic phosphate is administered by subcutaneous injection or intramuscular injection, it is present at a concentration that does not cause clinically significant signs of toxicity or irritation at the injection site, any one of items 1 to 23. The formulation described in the section. (Item 25) When the absolute viscosity of the formulation is measured using a conical plate viscometer, at least about 0.5 s.^-1The preparation according to any one of items 1 to 24, which is measured at the shear rate of. (Item 26) The absolute viscosity of the pharmaceutical product is at least about 1.0 s when measured using a microfluidic viscometer.^-1The formulation according to any one of items 1 to 24, which is measured at the shear rate of. (Item 27) A method for administering a therapeutically effective amount of protein, including subcutaneous injection or intramuscular injection, of the preparation according to any one of items 1 to 26. 28. Item 27, wherein the subcutaneous or intramuscular injection is performed using a syringe selected from the group consisting of heated syringes, self-mixing syringes, autoinjectors, prefilled syringes, and combinations thereof. Method. (Item 29) The method of item 28, wherein the syringe is a heated syringe and the formulation is administered at a temperature between 25 ° C and 40 ° C. (Item 30) The method according to any one of items 27 to 29, wherein the formulation induces a primary stimulus index of less than 3 when evaluated using the Draize scoring system. (Item 31) Any one of items 27 to 30, wherein the emission output is at least 10% or 20% smaller than the emission output when the same preparation is administered in the same manner but does not contain the organic phosphate. The method described in. (Item 32) Any one of items 27-31, wherein the injection is performed using a needle with a diameter between 27 and 31 gauges and the firing power is less than 30 N when using a needle with the gauge 27. The method described in. (Item 33) A method for preparing a pharmaceutical preparation, which comprises the step of combining the protein, solvent and organic phosphate according to any one of items 1 to 26. (Item 34) The method of item 33, wherein the formulation is in a prefilled syringe or cartridge. (Item 35) To facilitate protein purification, including adding an effective amount of the organic phosphate according to any one of items 1 or 7 to 10 to the protein solution to reduce the viscosity of the protein solution. Method. (Item 36) The protein-organic phosphate solution is purified or concentrated using a method selected from the group consisting of ultrafiltration / diafiltration, tangential flow filtration, centrifugal concentration, and dialysis. The method described in 35. Outline of the invention We have developed a low-viscosity, low-volume concentrated liquid pharmaceutical formulation of protein. These formulations can be administered rapidly and conveniently by subcutaneous (SC) or intramuscular (IM) injection rather than by prolonged intravenous infusion. These formulations contain low molecular weight and / or high molecular weight proteins such as mAbs, and organic phosphates. Typical organic phosphates include thiamine pyrophosphate (TPP), adenosine triphosphate (ATP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dTTP), Deoxycytidine triphosphate (dCTP), cyclic adenosine monophosphate (cAMP), nicotine amide adenine dinucleotide phosphate (NADP)⁺) And pyridoxal phosphate, and salts thereof, are preferably contained in concentrations between about 0.01M and about 0.50M, most preferably between about 0.05M and about 0.25M.</p><p> Protein concentrations range from about 10 mg / mL to about 5,000 mg / mL, more preferably between about 100 mg / mL and up to about 2,000 mg / mL. In some embodiments, the protein concentration is between about 100 mg / mL and about 500 mg / mL, more preferably between about 300 mg / mL and up to about 500 mg / mL. Formulations containing proteins and organic phosphates are 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. Viscosity formulations degree at about 25 ° C, less than about 75 cP, preferably less than 50 cP, and most preferably less than 20 cP. In some embodiments, the viscosity is less than about 15 cP, or less than about 10 cP or about 10 cP at about 25 ° C. In certain embodiments, the viscosity of the formulation is about 10 cP. Formulations containing proteins and organic phosphates typically have a shear rate of approximately 0.6 s when measured using a conical plate viscometer.^-1~ About 450s^-1, Preferably about 2s^-1~ About 400s^-1Measured at. Formulations containing proteins and organic phosphates typically have a shear rate of approximately 3 s when measured using a microfluidic viscometer.^-1~ About 55,000s^-1, Preferably about 20s^-1~ About 2,000s^-1Measured at.</p><p> The viscosity of this protein preparation is reduced by the presence of one or more reduced viscosity organic phosphates. Unless otherwise stated, the term "decayed organic phosphate" includes both single compounds and mixtures of two or more compounds. The reduced viscosity organic phosphate (s) are preferably present in the formulation at a concentration of less than about 1.0 M, preferably less than about 0.50 M, more preferably less than about 0.30 M, most preferably less than about 0.15 M. The present formulation is at least about 30% lower, preferably at least about 50%, lower than the viscosity of the corresponding formulation under the same conditions except when the reduced viscosity organic phosphate is replaced with a suitable buffer or salt of approximately the same concentration. It can have a low, most preferably at least about 75% lower viscosity. In some embodiments, a low viscosity formulation is provided in which the viscosity of the corresponding formulation without the reduced viscosity organic phosphate is greater than about 200 cP, greater than about 500 cP, or even greater than about 1,000 cP. Will be done. In a preferred embodiment, the shear rate of the present formulation is at least about 0.5 s when measured using a conical plate viscometer.^-1Or at least about 1.0 s when measured using a microfluidic viscometer^-1Is.</p><p> In the case of embodiments where the protein is a "high molecular weight protein", the high molecular weight protein is between about 100 kDa and about 500 kDa, preferably between about 120 kDa and about 1,000 kDa, most preferably between about 120 kDa and about 250 kDa. Can have a molecular weight of. This high molecular weight protein can be in antibody such as mAb, or in a PEGylated or otherwise derivatized form thereof. Preferred mAbs include naturizumab (TYSABRI®), cetuximab (ERBITUX®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), infliximab (REMICADE®), Includes rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatumumab (ARZERRA®), and their biosimulators. The high molecular weight protein, which is optionally PEGylated, can be an enzyme. Other proteins and mixtures of proteins can also be formulated to reduce their viscosity.</p><p> In some embodiments, the protein and the reduced viscosity organic phosphate are lyophilized and sized to be reconstituted with a sterile, pharmaceutically acceptable vehicle to produce a low viscosity concentrated liquid formulation. Provided in dosage units. The presence of the reduced viscosity organic phosphate facilitates and / or facilitates the reconstruction of the lyophilized dose unit as compared to the lyophilized dose unit that does not contain the reduced viscosity organic phosphate.</p><p> A method for preparing a low-viscosity concentrated liquid preparation of a high-molecular-weight protein such as mAb, and a method for storing a low-viscosity high-concentration protein preparation and administering it to a patient are provided herein. .. In another embodiment, a reduced viscosity organic phosphate is added to facilitate processing (eg, pumping, concentrating and / or filtering) by reducing the viscosity of the protein solution. The concentration of high molecular weight protein herein is from about 10 mg / mL to about 5,000 mg / mL, most preferably from about 100 mg / mL to about 2,000 mg / mL. The viscosity of the formulation is less than about 75 cP, preferably less than 50 cP, most preferably less than 20 cP. In some embodiments, the viscosity is less than about 15 cP, or even less than about 10 cP.</p>

<figref num="1">Figure 1 shows 0.25 M phosphate buffer, 0.10 M thiaminepyrophosphate (TPP), or 0.10 M TPP1- (3-aminopropyl) -2- as a function of protein concentration (mg / mL). The viscosity (cP) of an aqueous solution of biosimilar AVASTIN® containing methyl-1H-imidazole (APMI) is illustrated.</figref>

Detailed description of the invention

I. Definitions As commonly used herein, the term "protein" is linked to each other by peptide bonds to form a polypeptide whose chain length is sufficient to give rise to at least detectable three-dimensional structure. , Refers to a polymer of amino acids. A protein having a molecular weight greater than about 100 kDa (represented by kDa, where "Da" stands for "Dalton" and 1 kDa = 1,000 Da) can be referred to as a "high molecular weight protein", while about. A protein having a molecular weight of less than 100 kDa can be referred to as a "low molecular weight protein". The term "low molecular weight protein" excludes small peptides that lack the requirement of at least three-dimensional structure to be considered a protein. The molecular weight of a protein can be determined using standard methods known to those of skill in the art, including but not limited to mass spectrometry (eg, ESI, MALDI) or calculations from known amino acid sequences and glycosylation. .. Proteins can be naturally occurring or non-naturally occurring, synthetic or semi-synthetic.

"Intrinsically pure protein" and "substantially pure protein" are used interchangeably herein to include at least about 90% by weight pure protein, preferably at least about 95% by weight pure protein. Including, refers to a composition. "Essentially homogeneous" and "substantially homogeneous" are used interchangeably herein and at least about 90% by weight, preferably at least about 95%, of the proteins present are reversible with monomers. Refers to a composition that is a combination of dimer and oligomeric aggregates (not irreversible aggregates).

As commonly used herein, the term "antibody" refers to mAbs (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with polyepitopic specificity, bispecific antibodies, Diabody and single chain antibody molecules, as well as antibody fragments (eg, Fab, Fab', F (ab') 2 and Fv), single domain antibodies, polyvalent single domain antibodies, Fab fusion proteins, and them. Widely covers the fusion of.

As commonly used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up that population are in trace amounts. Except for possible naturally occurring variants that may be present, they are identical. Monoclonal antibodies are highly specific and target a single epitope. These are usually synthesized or recombinant DNA methods (eg, US Pat. No. 4,816,567), for example, by culturing hybridoma cells described by Kohler et al. (Nature 256: 495, 1975). Can be produced by Clackson et al. (Nature 352: 624-628, 1991) and Marks et al. (J. Mol. Biol.). It can be isolated from a phage antibody library using the techniques described in Vol. 222: pp. 581-597, 1991). As used herein, "mAb" is specifically a derivatized antibody, antibody-drug conjugate, and "chimeric" antibody, in which parts of the heavy and / or light chain are derived from a particular species. Is the same or homologous to the corresponding sequence in an antibody or antibody belonging to a particular antibody class or subclass, while the rest of the strand (s) is in an antibody derived from another species or another. Contains (US Pat. No. 4,816,567, Morrison et al., US Pat. No. 4,816,567, US Pat. No. 4,816,567, US Pat. No. 4,816,567, US Pat. No. 4,816,567, Morrison et al. Proc. Natl. Acad. Sci. USA Volume 81: pp. 6851-6855, 1984).

An "antibody fragment" comprises a portion of an intact antibody, including antigen binding and / or a variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F (ab') 2 and Fv fragments; Diabodies; Linear antibodies (US Pat. No. 5,641,870; Zapata et al., Protein Eng. 8: 1057-1062, 1995. Year); single chain antibody molecules; polyvalent single domain antibodies; and multispecific antibodies formed from antibody fragments.

The "humanized" form of a non-human (eg, murine) antibody is a chimeric immunoglobulin, an immunoglobulin chain, or a fragment of those fragments (Fv, Fab, Fab', F (ab') 2 or antibody that are mostly human sequences. Other antigen-binding subsequences, etc.), which contain the smallest sequences derived from non-human immunoglobulins (eg, Jones et al., Nature 321, pp. 522-525, 1986; Reichmann et al., Nature 332). Volumes: 323-329, 1988, and Presta, Curr. Op. Struct. Biol. 2: 593-596, 1992).

"Rheology" refers to the study of material deformation and flow.

"Viscosity" refers to the resistance to the flow of a substance (usually a liquid). Viscosity is related to the concept of shear force. Viscosity can be understood as the action of different layers of fluid, exerting shear forces on each other or other surfaces as different layers of fluid move relative to each other. There are several viscosity measures. The unit of viscosity is Ns / m, known as Pascal-second (Pa-s).²Is. The viscosity can be "kinematic" or "absolute". The kinematic viscosity is a measure of the velocity at which momentum is transferred through a fluid. The kinematic viscosity is measured by Stokes (St). The kinematic viscosity is a measure of the resistance flow of a fluid under the influence of gravity. If two fluids of equal volume and different viscosities were placed in the same capillary viscometer and flowed by weight, the more viscous fluid would take longer to flow through the capillary than the less viscous fluid. It takes. For example, if one fluid takes 200 seconds (s) to complete its flow and another fluid takes 400 s, the second fluid is twice as viscous as the first fluid on the kinematic viscosity scale. Is called high. The dimension of kinematic viscosity is length²/ Time. The kinematic viscosity is generally expressed in centi-stokes (cSt). The SI unit of kinematic viscosity is mm²/ s, which is equal to 1cSt. "Absolute viscosity", sometimes referred to as "dynamic viscosity" or "simple viscosity", is the product of kinematic viscosity and fluid density. Absolute viscosity is expressed in units of centipores (cP). The SI unit of absolute viscosity is millipascal-seconds (mPa-s), where 1cP = 1mPa-s. Viscosity can be measured, for example, by using a viscometer at a given shear rate or at multiple shear rates. The "externalized zero shear" viscosity is determined by creating an optimal straight line of the four highest shear points based on the absolute viscosity vs. shear rate plot and linearly extrapolating the viscosity back to zero shear. be able to. Alternatively, in the case of 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 velocity), where absolute viscosity is the absolute viscosity as the liquid flows along a passage. Derived from changes in pressure. Viscosity is equal to shear stress throughout the shear rate. In some embodiments, the viscosities measured using a microfluidic viscometer are extrapolated from the externalized zero shear viscosities, eg, viscosities measured at multiple shear velocities using a conical plate viscometer. It can be compared directly with the one.

"Shear velocity" refers to the rate of change of velocity at which one of the layers of fluid passes over an adjacent layer. The velocity gradient is the rate of change of velocity with distance from the plate. In this simple case, the shear rate (v) in units of (cm / sec) / (cm) = 1 / sec₁-v₂) / H, showing a constant velocity gradient. Therefore, the unit of shear rate is the reciprocal of seconds, or generally the reciprocal of time. For microfluidic viscometers, changes in pressure and flow velocity are related to shear rate. "Shear velocity" is used to refer to the speed at which a material deforms. Formulations containing proteins and reduced viscosity organic phosphates are typically spindles (ie, 20 cP samples) appropriately selected by those skilled in the art to accurately measure viscosity within the viscosity range of conical plate viscometers and samples of interest. Is most accurately measured with a CPE40 spindle mounted on a DV2T viscometer (Brookfield)), about 0.5s^-1~ About 200s^-1Measured at shear rates in the range of, about 20 s when measured using a microfluidic viscometer^-1Super ~ about 3,000s^-1Measured at shear rates in the range of.

For the classic "Newtonian" fluids commonly used herein, the viscosity is essentially independent of the shear rate. However, in the case of "non-Newtonian fluids", as the shear rate is increased, the viscosity either decreases or increases, for example, the fluids are either "slip fluidized" or "slip sticky", respectively. ". In the case of concentrated (ie, high concentration) protein solutions, this can be expressed as pseudoplastic shear fluidization behavior, i.e., where the viscosity decreases with shear rate.

As commonly used herein, the term "chemical stability" refers to the ability of a protein component in a pharmaceutical product to resist degradation by chemical pathways such as oxidation, deamidation or hydrolysis. A protein preparation is usually considered to be chemically stable if less than about 5% of its constituents are degraded after 24 months at 4 ° C.

As commonly used herein, the term "physical stability" refers to the ability of a protein preparation to resist physical deterioration such as aggregation. A physically stable formulation forms only acceptable proportions of irreversible aggregates of bioactive protein agents (eg, dimers, trimers or other aggregates). The presence of aggregates can be assessed by several methods, including by measuring the average particle size of the protein in the formulation by dynamic light scattering. The formulation is considered to be physically stable if less than about 5% of irreversible aggregates form after 24 months at 4 ° C. The acceptable level of agglomerated contaminants is less than about 2%. A low level of about 0.2% is feasible, but about 1% is more common.

As commonly used herein, the term "stable formulation" means that the formulation is both chemically stable and physically stable. A stable formulation is one in which more than about 95% of the bioactive protein molecules retain their bioactivity in the formulation after storage at 4 ° C for 24 months or at high temperatures such as storage at 40 ° C for 1 month. , Can be a formulation that maintains an equivalent solution state. Various analytical techniques for measuring protein stability are available in the art, such as Peptide and Protein Drug Delivery, pp. 247-301, Vincent Lee, (ed.), Marcel Dekker, Inc., New. York, NY (1991) and Jones, A., Adv. Drug Delivery Revs. Volume 10: pp. 29-90, outlined in 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 residual biological activity is measured and the stability is assessed compared to the initial state. .. Generally, if the formulation is to be stored at 2 ° C to 8 ° C, the formulation should be stable at 30 ° C or 40 ° C for at least 1 month, and / or 2 °. At C-8 ° C, it should be stable for at least 2 years. Generally, if the formulation is to be stored at room temperature, i.e. about 25 ° C, the formulation should be stable at about 25 ° C for at least 2 years, and / or at least at 40 ° C. It should be stable for about 6 months. The degree of lyophilization and aggregation after storage can be used as an indicator of protein stability. In some embodiments, stability is assessed by measuring the particle size of the protein in the formulation. In some embodiments, stability can be assessed by measuring the activity of the pharmaceutical product using standard biological activity or binding assays, well within the capacity of those skilled in the art.

The term protein "particle size", commonly used herein, refers to well-known particle size measuring instruments such as dynamic light scattering, SEC (size exclusion chromatography), or other known to those skilled in the art. It means the average diameter of a major population of particles of a bioactive molecule in a formulation, or its particle size distribution, as determined by using the method.

As commonly used herein, the term "concentrated" or "high concentration" means that the final concentration of protein is greater than about 10 mg / mL, preferably greater than about 50 mg / mL, more preferably about 100 mg / mL. Described are liquid formulations having ultra, even more preferably greater than about 200 mg / mL, or most preferably greater than about 250 mg / mL.

As commonly used herein, the "reconstituted formulation" is the solution of a dry powder, lyophilized protein, spray-dried protein or solvent-precipitated protein in a diluent as a result. , Refers to a pharmaceutical product prepared by dissolving or dispersing this protein in an aqueous solution for administration.

A "lyoprotectant" is a substance that, when combined with a protein, significantly reduces the chemical and / or physical instability of the protein during lyophilization and / or subsequent storage. Is. Exemplary cryoprotectants include sugars such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol and mannitol and their corresponding sugar alcohols; amino acids such as arginine or histidine; release solutions such as magnesium sulfate. Sex salt (lyotropic) Salt); polyols such as propylene glycol, glycerol, poly (ethylene glycol) or poly (propylene glycol); and combinations thereof. Additional exemplary lyophilizers include gelatin, dextrin, modified starch, and carboxymethyl cellulose. Preferred sugar alcohols are such compounds obtained by reduction of mono and disaccharides such as lactose, trehalose, maltose, lactulose and maltulose. Additional examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. The lyophilization protective agent is generally added to the pre-lyophilized formulation in a "lyophilized amount". This means that after lyophilizing the protein in the presence of a lyophilized protective amount, the protein essentially retains its physical and chemical stability, as well as its integrity. ..

Commonly used herein, a "diluent" or "carrier" is pharmaceutically acceptable (ie, safe and non-toxic when administered to a human or another mammal) and is re-dried after lyophilization. It is a useful ingredient for the preparation of liquid preparations such as the constituent aqueous preparations. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline solution, Ringer's solution or dextrose solution, and combinations thereof.

A "preservative" is a compound that can be added to the formulations 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 may facilitate the production of, for example, multiple use (multiple dose) formulations. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chloride with a long alkyl group), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohols, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

As commonly used herein, a "bulking agent" is a compound that imparts bulk to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (eg, maintaining an open pore structure, essentially. Facilitates the production of uniform freeze-dried cakes). Exemplary bulking agents include mannitol, glycine, lactose, modified starch, poly (ethylene glycol) and sorbitol.

A "therapeutically effective amount" is used to provide measurable improvement or prevention of any symptom, or specific condition or disorder, to prolong measurable life expectancy, or to generally improve a patient's quality of life. The minimum required concentration. The therapeutically effective amount depends on the specific biologically active molecule and the specific condition or disorder being treated. Therapeutically effective amounts of many proteins, such as mAbs, described herein are well known in the art. A therapeutically effective amount of protein for treating a particular disorder with a known protein, such as mAb, which has not yet been established or is clinically applied to treat further disorders, is known to physicians and others. It can be determined by appropriate standard techniques that are well within the skill of the person skilled in the art.

As commonly used herein, the term "injectability" or "needle passability" refers to the ability of a syringe equipped with an 18-32 gauge needle to inject a pharmaceutical product. Injectability depends on factors such as the pressure or force required for the injection, uniform flow, suction quality, and absence of clogging. The injectability of this liquid pharmaceutical product can be evaluated by comparing the shot output of the hypoviscosity preparation and the standard preparation to which the viscosity-reducing organic phosphate is not added. Decreased viscosity The reduced output of a pharmaceutical product containing an organic phosphate is reflected in the improved injectability of the pharmaceutical product. The reduced-viscosity formulation has an injection output of at least 10 when compared to a standard formulation with the same concentration of protein under the same conditions, except that the reduced-viscosity organic phosphate is replaced with an appropriate buffer of approximately the same concentration. If%, preferably at least 30%, more preferably at least 50%, and most preferably at least 75%, decrease, the injectability is improved. Alternatively, the injectability of this liquid pharmaceutical formulation compares the time required to inject the same volume, such as 0.5 mL, or more preferably about 1 mL, of different liquid protein formulations when the syringe is pressed with the same force. By doing so, it can be evaluated.

As commonly used herein, the term "firing output" is the force required to push out a given liquid formulation with a given syringe equipped with a given needle gauge at a given infusion rate. Point to. This morphism is usually reported in Newton. For example, the firing power can be measured as the force required to push out the liquid formulation with 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. Test equipment can be used to measure the emission power. Formulations with lower viscosities, when measured under the same conditions, generally require a smaller emission output as a whole.

In one embodiment, the injection is performed using a 27 gauge needle and the firing power is less than 30 N. In most cases, the present formulation can be administered using a very small gauge needle, eg, between 27-31 gauge, usually a thin-walled needle with an optional 27, 29 or 31 gauge.

As used herein, "viscosity gradient" refers to the rate of change in the viscosity of a protein solution as the protein concentration increases. The viscosity gradient can be estimated from plotting the viscosity as a function of protein concentration for a series of formulations that are otherwise the same but have different protein concentrations. This viscosity increases almost 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. The viscosity gradient can be estimated as a function of any protein concentration or from a linear approximation to the viscosity plotted over a narrow window of protein concentration. In some embodiments, the formulations were obtained for the same formulation where the exponential index does not contain the reduced viscosity organic phosphate when the viscosity as a function of protein concentration is estimated as an exponential function. If it is less than the exponential, the viscosity gradient is said to have decreased. In a similar manner, a formulation can be said to have a smaller / larger viscosity gradient if the index of the formulation is less / greater than the index of the second formulation when compared to the second formulation. The viscosity gradient can be estimated numerically from a plot of viscosity as a function of protein concentration by another method known to skilled pharmaceutical researchers.

As commonly used herein, the term "viscosity-reducing formulation" is one or more additives that reduce the viscosity as compared to the corresponding formulation that does not contain the reducing-viscosity additive (s). Refers to a liquid preparation having a high concentration of a high molecular weight protein such as mAb or a low molecular weight protein that is modified by the presence of.

As commonly used herein, the term "volume osmolality" refers to the total number of constituents dissolved per liter. Volume osmolality is similar to molarity but includes the total number of moles of dissolved species in solution. A volume osmolal concentration of 1 Osm / L means that 1 mol of the constituents are dissolved per L of the solution. Some solutes, such as ionic solutes that are dissociated in solution, give more than 1 mole of dissolved constituents per mole of solute in solution. For example, NaCl is in solution, Na⁺And Cl^-Dissociates into, i.e., results in 2 moles of dissolved constituents per mole of NaCl dissolved in solution. Physiological volume The osmolal concentration is usually in the range of about 280 mOsm / L to about 310 mOsm / L.

As commonly used herein, the term "tonicity" refers to the gradient of osmotic pressure due to the separation of the two solutions by a semipermeable membrane. In particular, tonicity is used to describe the osmotic pressure that occurs throughout the cell membrane when cells are exposed to an external solution. Solutes that can cross the cell membrane do not contribute to the final osmotic gradient. Only lysed species that do not cross the cell membrane contribute to the osmotic pressure difference, or tonicity.

As commonly used herein, the term "hypertonic" refers to a solution that has a higher concentration of solute than is present inside the cell. When cells are immersed in a hypertonic solution, water tends to flow out of the cells in order to balance the concentration of solute.

As commonly used herein, the term "hypotonic" refers to a solution that has a lower concentration of solute than is present inside the cell. When the cells are immersed in a hypotonic solution, water flows into the cells to balance the concentration of solute.

As commonly used herein, the term "isotonic" refers to a solution in which the osmotic gradient across the cell membrane is essentially balanced. The isotonic preparation essentially has the same osmotic pressure as human blood. The isotonic preparation generally has an osmotic pressure of about 250 mOsm / kg to 350 mOsm / kg.

The term "liquid formulation" as used herein is either supplied with an acceptable pharmaceutical diluent or reconstituted with an acceptable pharmaceutical diluent prior to administration to a patient. That protein.

The terms "branded" and "reference" are used interchangeably herein when used to refer to a protein or biologic, according to the US Public Health Service Act (42 U.SC § 262). Means a single approved biological product under Section 351 (a).

The term "biosimilar" is commonly used as used herein, compatible with "generic equivalent" or "successor". For example, "biosimilar mAb" refers to a subsequent version of a developer's mAb, usually made by a different company. A "biosimilar", when used in reference to a branded protein or branded biologic, is a bioproduct evaluated for that branded protein or branded biologic and is US. Under Section 351 (k) of the Public Health Service Act (42 U.SC § 262), it can refer to licensed biological products. The biosimilar mAb was adopted by the European Medicines Agency's Human Medicines Commission (CHMP) on May 30, 2012, and was adopted by the European Medicines Agency on May 30, 2012. Products containing monoclonal antibodies --non-clinical and clinical issues) "(reference document EMA / CHMP / BMWP / 403543/2010) may satisfy one or more guidelines issued by the European Union.

Biosimilars can be produced by microbial cells (prokaryotes, eukaryote), cell lines of human or animal origin (eg, mammals, birds, insects), tissues of animal or plant origin. The expression construct for the proposed biosimilar product generally encodes the same primary amino acid sequence as its reference product. Small modifications, such as N-terminal or C-terminal shortening, that do not affect safety, purity or efficacy may be present.

Biosimilar mAbs are biochemically or biologically similar to reference mAbs in terms of both safety and efficacy. Biosimilar mAb binds to target antigen (s); binds to isoforms of Fc gamma receptors (FcγRI, FcγRII and FcγRIII), FcRn, and complement (C1q); Fab-related functions (eg, soluble) Ligand neutralization, receptor activation or blockade); or Fc-related functions (eg, antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, complement activation) were included in the detailed assay. , Can be evaluated against reference mAbs using one or more in vitro studies. In vitro comparisons can be combined with in vivo data, which demonstrates similarities in pharmacokinetics, pharmacodynamics and / or safety. Clinical evaluation of biosimilar mAbs for reference mAbs is based on pharmacokinetic properties (eg, AUC)._0-inf, AUC_0-t, C_max, T_max, C_trough), Pharmacodynamic endpoints, or comparisons of clinical efficacy similarity (eg, using randomized, parallel-group comparative clinical trials) can be included. A quality comparison between biosimilar mAbs and reference mAbs is described in "Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance. :: Quality issues "(EMEA / CHMP / BWP / 49348/2005) and" Guideline on development, production, characterization and specifications for monoclonal antibodies and related Substances) "(EMEA / CHMP / BWP / 157653/2007) can be evaluated using established procedures, including those described.

Differences between biosimilar mAbs and reference mAbs are due, for example, by binding other biochemical groups to the mAbs, such as phosphates, various lipids and carbohydrates; by cleavage by post-translational proteolysis; amino acids Post-translational modifications may be included by altering the chemistry of the (eg, formylation); or by many other mechanisms. Other post-translational modifications can be the result of manipulation in the manufacturing process. For example, a saccharification reaction can occur with exposure of the product to reducing sugars. In other cases, storage conditions may allow certain degradation pathways such as oxidation, deamidation or aggregation. All of these product-related variants can be included in biosimilar mAbs.

As used herein, the term "pharmaceutically acceptable salt" refers to inorganic acids and bases, as well as from pharmaceutically acceptable non-toxic acids and bases, including organic acids and bases. Refers to the salt to be prepared. Suitable non-toxic acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isetionic acid, lactic acid, maleine. Includes inorganic and organic acids such as acids, malic acid, mandelic acid, methanesulfonic acid, mucilage, nitrate, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Suitable positively charged counterions include sodium, potassium, lithium, calcium and magnesium.

As used herein, the term "ionic liquid" means that the most conventional salt is a solid, temperature at or near temperature: less than 200 ° C, preferably less than 100 ° C, or more preferably. Refers to crystalline or amorphous salts, zwitterions, or mixtures thereof that are liquid below 80 ° C. Some ionic liquids have melting temperatures near room temperature, such as between 10 ° C and 40 ° C or between 15 ° C and 35 ° C. The term "zwitterion" is used herein to describe a molecule that has formal positive and negative charges on different chemical groups in the molecule and is totally neutral. Examples of ionic liquids are Riduan et al., Chem. Soc. Rev., Vol. 42: 9055-9070, 2013; Rantwijk et al., Chem. Rev., 107: 2757-2785, 2007; Earle et al., Pure Appl. Chem., Vol. 72 (No. 7): pp. 1391-1398, 2000; and Sheldon et al., Green Chem., Vol. 4: pp. 147-151, 2002.

As used herein, a "water-soluble organic dye" is an organic molecule having a molar solubility of at least 0.001 M at 25 ° C and pH 7, and is visible at certain wavelengths of light, preferably electromagnetic spectra. It is an organic molecule that can absorb light of wavelengths in the infrared part from the part while transmitting or reflecting light of other wavelengths.

As used herein, the term "chalcogen" refers to a Group 16 element, including oxygen, sulfur and selenium, in any oxidation state. For example, unless otherwise specified, the term "chalcogen" includes SO.₂Is also included.

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 a hydrocarbon group containing one or more double or triple bonds. Alkyl groups containing at least one ring system are "cycloalkyl" groups. An alkyl group containing at least one double bond is an "alkenyl group" and an alkyl group containing at least one triple bond is an "alkynyl group".

As used herein, "aryl" refers to an aromatic carbocyclic ring system, including fused ring systems. In the "aryl" group, each of the atoms forming the ring is a carbon atom.

As used herein, "heteroaryl" refers to an aromatic ring system, including a fused ring system, wherein at least one atom forming the ring is a heteroatom. Point to.

As used herein, a "heterocycle" is a non-aromatic ring system that includes a fused ring system in which at least one atom forming the ring is a heteroatom. Point.

As used herein, the term "heteroatom" is either a non-carbon atom or a non-hydrogen atom. Preferred heteroatoms include oxygen, sulfur and nitrogen. Exemplary heteroaryl and heterocyclyl rings include benzoimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzoisooxazoli. Lu, benzoisothiazolyl, benzoimidazolinyl, carbazolyl, 4aH carbazolyl, carborinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H, 6H-1,5,2-dithiadinyl, dihydroflo [2,3b] tetrahydrofuran, Furanyl, Frazanyl, Imidazolidinyl, Imidazolinyl, Imidazolyl, 1H-Indazolyl, Indolenyl, Indolinyl, Indridinyl, Indolyl, 3H-Indrill, Isatinoyl, Isobenzofuranyl, Isochromanyl, Isoindazolyl, Isoindolinyl, Isoindrill, Isoquinolinyl, Isoquinolinyl Phenyl, morpholinyl, naphthyldinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,

As used herein, the term "organic phosphate" is a compound containing one or more phosphoryl groups in which at least one of the phosphoryl groups is covalently linked to the organic group by a phosphate ester bond. Refers to a compound. Organic phosphates can be synthetic or semi-synthetic, either naturally occurring or non-naturally occurring.

The term "nucleobase", as used herein, preferably has both a hydrogen bond donor and a hydrogen bond acceptor and is Watson-click with a complementary nucleobase. Broadly refers to substituted and unsubstituted nitrogen-containing heteroaromatic rings capable of forming type hydrogen bonds. Nucleic acid bases include naturally occurring nucleobases and non-naturally occurring nucleobases. Non-naturally occurring nucleobases include nucleobases that are rarely or only transiently found in naturally occurring nucleic acids, such as hypoxanthin, 6-methyladenine, 5-methylpyrimidine (such as 5-methylcytosine), 5-. Hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, and synthetic nucleobases such as 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7- Derazaguanine, N⁶-Contains (6-aminohexyl) adenine and 2,6-diaminopurine. Nucleic acid bases include purines and purine bases such as adenine, guanine, hypoxanthine, xanthine and 7-methylguanine. Nucleic acid bases include pyrimidines and pyrimidine bases such as thymine, cytosine, uracil, 5,6-dihydrouracil, 5-methylcytosine and 5-hydroxymethylcytosine.

Certain organic phosphates contain acidic or basic functional groups. Whether these functional groups are fully or partially ionized depends on the pH of the pharmaceutical product in which they are present. Unless otherwise specified, the reference to a preparation containing an organic phosphate having an ionizable functional group includes both the parent compound and any possible ionized state.

The term "nucleoside" is used herein to refer to any compound in which the nucleobase is covalently coupled to a pentacarbon sugar, preferably in its cyclic form. Five-carbon sugars include ribose, deoxyribose, arabinose, xylose, lyxose and their derivatives.

The term "nucleotide" means that the nucleoside is covalently coupled to a phosphate or polyphosphate group, such as a diphosphate or triphosphate group, at one or more positions on the sugar. Used to refer to a compound.

II. Formulation

A biocompatible low-viscosity protein solution, such as that of mAb, provides a therapeutically effective amount of protein in a volume useful for subcutaneous (SC) and intramuscular (IM) injection, usually less than 2 mL for SC or It can be used to deliver at about 2 mL, and less than 5 mL or about 5 mL for IM, more preferably less than 1 mL or about 1 mL for SC, and less than 3 mL or about 3 mL for IM. The protein can generally have any molecular weight, but in some embodiments, high molecular weight proteins are preferred. In other embodiments, the protein is a low molecular weight protein.

The formulation can have a protein concentration between about 10 mg / mL and about 5,000 mg / mL. This product, including the mAb product, is more than 100 mg / mL, preferably more than 150 mg / mL, more preferably more than about 175 mg / ml, preferably even larger than about 200 mg / mL, even more preferably about 225 mg / mL. It can have a protein concentration of greater than, even more preferably greater than about 250 mg / mL, most preferably greater than 300 mg / mL or about 300 mg / mL. In the absence of organic phosphate, the viscosity of the protein preparation increases exponentially as its concentration increases. In the absence of organic phosphate, these protein formulations can have viscosities greater than 100cP, greater than 150cP, greater than 200cP, greater than 300cP, greater than 500cP, or even greater than 1,000cP when measured at 25 ° C. These formulations are often unsuitable for SC or IM injections. By using one or more reduced viscosity organic phosphates, when measured at 25 ° C, less than about 100 cP or about 100 cP, preferably less than about 75 cP or about 75 cP, more preferably less than about 50 cP or about 50 cP, less than about 30 cP. Alternatively, it is possible to prepare a preparation having a viscosity of about 30 cP, less than about 20 cP or about 20 cP, or even more preferably less than about 10 cP or about 10 cP.

Viscosity-reducing organic phosphates can be used to reduce the viscosity of concentrated protein formulations, but they can also be used in less concentrated formulations. In some embodiments, the formulation can have a protein concentration between about 10 mg / mL and about 100 mg / mL. The formulation can have a protein concentration of greater than about 20 mg / mL, greater than about 40 mg / mL, or greater than about 80 mg / mL.

For certain proteins, a formulation that does not have a reduced viscosity organic phosphate can have a viscosity greater than about 20 cP, greater than about 50 cP, or greater than about 80 cP. By using one or more reduced viscosity organic phosphates, when measured at 25 ° C, less than about 80 cP or about 80 cP, preferably less than about 50 cP or about 50 cP, even more preferably less than about 20 cP, or most preferably about. It is possible to prepare a preparation having a viscosity of less than 10 cP or about 10 cP.

In some embodiments, the aqueous protein preparation has a viscosity that is at least about 30% lower than similar preparations that do not contain the reduced viscosity organic phosphate (s) when measured under the same conditions. In other embodiments, the present formulation is less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, than a similar formulation that does not contain the reduced viscosity organic phosphate (s). Or even more viscous, less than 90%. In a preferred embodiment, the pharmaceutical product contains a therapeutically effective amount of one or more high molecular weight proteins, such as mAb, in a volume of less than about 2 mL, preferably less than about 1 mL, or more preferably less than about 0.75 mL. ..

The reduced-viscosity formulation improves injectability and, under the same conditions, requires a smaller firing output than similar formulations that do not contain the reduced-viscosity organic phosphate (eg, in sodium phosphate buffer). .. In some embodiments, the morphism is about 20%, more than 30%, about 40%, under the same injection conditions, as compared to a standard formulation that does not contain the reduced viscosity organic phosphate (s). It drops by more than 50%, or more than 2 times. In some embodiments, the present formulation has "Newtonian flow characteristics" defined as having a viscosity that is substantially independent of shear rate. This protein preparation can be easily injected with a needle of about 18 to 32 gauge. Preferred needle gauges for delivering low viscosity formulations include optionally thin-walled 27, 29 and 31 gauges.

The present formulation may contain one or more additional excipients such as buffers, surfactants, sugars and sugar alcohols, other polyols, preservatives, antioxidants, and chelators. This product has a pH and volume of osmolality suitable for administration without causing serious adverse side effects. In some embodiments, the low viscosity concentrated formulation has a pH between 5-8, 5.5-7.6, 6.0-7.6, or 5.5-6.5.

This low-viscosity protein formulation can enable greater adaptability in formulation development. This low-viscosity preparation may exhibit a change in viscosity that is less dependent on protein concentration than the same preparation except that it does not contain a reduced-viscosity organic phosphate. This low-viscosity protein preparation may enable an increase in protein concentration and a decrease in the frequency of administration thereof. In some embodiments, the low-viscosity protein preparation contains two or more different proteins, three or more, or four or more different proteins. For example, a combination of two or more mAbs can be provided in a single low viscosity protein formulation.

Since protein (such as mAb) preparations can be administered to patients at higher protein concentrations than other similar protein preparations, which are free of viscosity-reducing organic phosphates, the frequency of protein administration can be reduced. For example, a protein that previously required a daily dose can be given once every two days, once every three days, or even less frequently if the protein is formulated with a reduced viscosity organic phosphate. it can. Currently, proteins that require multiple doses on the same day (either at the same time or at different times of the day) can be given with fewer injections per day. In some cases, this frequency can be reduced to a single injection once daily. By doubling the dose administered per injection, the frequency of administration can be reduced, for example, from once every two weeks to once every six weeks.

In some embodiments, the liquid formulation herein has, for example, a physiological volume osmolal concentration between about 280 mOsm / L and about 310 mOsm / L. In some embodiments, the liquid formulation has a volume 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 pharmaceutical product has a volumetric osmolal concentration 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 present liquid preparation can be hypertonic in some cases.

Additives, including reduced viscosity organic phosphates, are desired in liquid formulations unless their amounts are toxic or otherwise harmful and substantially interfere with the chemical and / or physical stability of the formulation. Can be included in any amount to achieve the viscosity level of. In some embodiments, the reduced viscosity organic phosphate (s) is less than about 1.0 M, preferably less than about 0.50 M, less than about 0.30 M or equal to about 0.30 M, or less than 0.15 M or 0.15 M. Can exist independently at a concentration equal to. Particularly preferred concentrations include about 0.10 M and about 0.30 M. For some embodiments having two or more deviscosified organic phosphates, the compounds preferably do not necessarily have to be present at the same concentration.

The reduced viscosity organic phosphate allows the lyophilized dosage unit to be reconstituted more quickly. The dosage unit is a lyophilized cake of protein, deviscosified organic phosphate and other excipients, to which water, saline solution or another pharmaceutically acceptable fluid is added. In the absence of reduced viscosity organic phosphate, it often takes 10 minutes or more to completely dissolve a lyophilized cake with a high protein concentration. When lyophilized cakes contain one or more reduced viscosity organic phosphates, the time required to completely dissolve the cake is often reduced by a factor of 2, 5, or even 10 times. In certain embodiments, 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. Requires less than 1 minute.

This low-viscosity protein preparation can enable greater adaptability in preparation development. This low-viscosity preparation does not contain the reduced-viscosity organic phosphate (s), but exhibits a viscosity that does not change so much as the protein concentration increases as compared with the same preparations other than the same. This low-viscosity protein preparation does not contain the reduced-viscosity organic phosphate, but otherwise exhibits a lower viscosity gradient than the same preparation.

The viscosity gradient of a protein preparation may be two times less, three times less, or even more than three times less than the viscosity gradient of the same protein preparation, otherwise it does not contain the reduced organic phosphate (s). The viscosity gradient of this protein preparation is less than 2.0 cPmL / mg, less than 1.5 cPmL / mg, less than 1.0 cPmL / mg, 0.8 cPmL / mg for protein preparations having a protein concentration between 10 mg / mL and 2,000 mg / mL. It can be less than, less than 0.6 cPmL / mg, or less than 0.2 cPmL / mg. By lowering the viscosity gradient of the present formulation, the protein concentration can be increased to a considerable extent before an exponential increase in viscosity is observed.

A. Protein

Any protein can be formulated, including recombinant, isolated or synthetic proteins, glycoproteins, or lipoproteins. These are antibodies (including antibody fragments and recombinant antibodies), enzymes, growth factors or hormones, immunomodulators, anti-infective agents, anti-proliferative agents, vaccines, or other therapeutic, prophylactic or diagnostic proteins. You may. In certain embodiments, the protein has a molecular weight of greater than about 150 kDa, greater than 160 kDa, greater than 170 kDa, greater than 180 kDa, greater than 190 kDa, or even greater than 200 kDa.

In certain embodiments, the protein can be a PEGylated protein. The term "PEGylated protein", as used herein, is optionally covalently attached to it via a chemical linker, which may be different from one or more polymeric groups. , A protein having one or more poly (ethylene glycol) or other stealth polymer groups. PEGylated proteins are usually characterized by reduced filtration of their kidneys, reduced uptake by the reticuloendotheli system, and reduced enzymatic degradation, resulting in, for example, increased half-life and increased bioavailability. Stealth polymers include poly (ethylene glycol); poly (propylene glycol); poly (glutamic acid), poly (hydroxyethyl-L-asparagin), and poly (hydroxyethyl-L-glutamine) ((poly (hydroxethyl-L-). Poly (amino acid) polymers such as glutamine))); poly (glycerol); poly (2-methyl-2-oxazoline) and poly (2-oxazoline) polymers such as poly (2-ethyl-2-oxazoline); poly (2-oxazoline) Acrylamide); Poly (vinylpyrrolidone); Poly (N- (2-hydroxypropyl) methacrylamide); And copolymers, as well as 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 specific on the protein. By covalently bonding the stealth polymer to the site (s), it can be site-specifically PEGylated. Site-specific PEGylation can be performed using, for example, activated stealth polymers with one or more reactive functional groups. Examples are described, for example, in Hoffman et al., Progress in Polymer Science, Vol. 32: pp. 922-932, 2007.

In a preferred embodiment, the protein is high molecular weight and antibody, most preferably mAb, not exceeding 1.0-2.0 mL for SC administration and greater than 3.0-5.0 mL for IM administration. It has a high viscosity in buffered aqueous solution when concentrated enough to inject a therapeutically effective amount in no volume. For high molecular weight proteins, Scolnik, mAbs Volume 1: 179-184, 2009; Beck, mAbs Volume 3: 107-110, 2011; Baumann, Curr. Drug Meth. Volume 7: 15-21, 2006 Year; or Federici, Biologicals Vol. 41: pp. 131-147, 2013. The proteins for use in the formulations described herein are preferably essentially pure and essentially homogeneous (ie, substantially contaminating proteins and / or irreversible thereof). No agglomerates).

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

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

Antibodies currently on the market

Many protein therapeutics currently on the market, especially the antibodies defined herein, are administered by IV infusion due to their high dosage requirements. The pharmaceutical product can include one of the antibody therapeutic agents currently on the market or one of its biosimilars. Some protein therapeutics currently on the market are not high molecular weight, but are still administered by IV infusion due to the high dose required for therapeutic efficacy. In some embodiments, liquid formulations of such low molecular weight proteins as defined herein are provided that, in the case of SC or IM injection, have concentrations to deliver a therapeutically effective amount.

Currently marketed antibody therapeutics include belimumab (BENLYSTA®), golimumab (SIMPONI ARIA®), absiximab (REOPRO®), and BEXXAR®. , Combination of tocitumomab and iodine-131 tocitumomab, alemtuzumab (CAMPATH®), paribismab (SYNAGIS®), basiliximab (SIMULECT®), ad-tras tubemtancin (KADCYLA®) ), PERJETA (registered trademark), PROSTASCINT KIT (registered trademark), daclizumab (ZENAPAX (registered trademark)), Ibritsumomabuchiukisetan (ZEVALIN (registered trademark)) , Eculizumab (SOLIRIS®), Ipirimumab (YERVOY®), Muromonab-CD3 (ORTHOCLONE) OKT3 (registered trademark)), luximab, nimotuzumab (THERACIM (registered trademark)), brentuximab vedotin (ADCETRIS (registered trademark)), adalimbab (HUMIRA (registered trademark)), golimumab (SIMPONI (registered trademark)), palivizumab (SYNAGIS®), omalizumab (XOLAIR®), and ustekinumab (STELARA®) are included.

Natalizumab, a humanized mAb against the cell adhesion molecule α4-integrin, is used in the treatment of multiple sclerosis and Crohn's disease. Natalizumab, previously marketed under the trademark ANTEGREN®, is now jointly marketed as TYSABRI® by Biogen Idec ("Biogen") and Elan Corp. ("Elan"). There is. TYSABRI® is produced in mouse myeloma cells. The 15 mL doses are 300 mg sodium lysumab, 123 mg sodium chloride (USP), 17.0 mg sodium monophosphate monohydrate (USP), 7.24 mg, respectively, at pH 6.1 in IV water for injection (USP). Contains 3.0 mg of sodium dibasic sodium phosphate (USP) and 3.0 mg of polysorbate 80 (USP / NF). Natalizumab is usually administered monthly by intravenous (IV) infusion in the treatment of both multiple sclerosis and Crohn's disease symptoms, as well as in the prevention of recurrence, vision loss, cognitive decline, and the patient's quality of life. Has proven to be effective for significant improvement in.

As used herein, the term "natalizumab" includes mAbs to the cell adhesion molecule α4-integrin, or antigen-binding moieties thereof, known by the international nonproprietary name "NATALIZUMAB". Natalizumab contains the antibodies described in US Pat. No. 5,840,299, US Pat. No. 6,033,665, US Pat. No. 6,602,503, US Pat. No. 5,168,062, US Pat. No. 5,385,839, and US Pat. No. 5,730,978. Natalizumab contains an activator in products marketed by Biogen Idec and Elan Corporation under the trademark TYSABRI®, or in its biosimilar products.

Cetuximab is an epidermal growth factor receptor (EGFR) inhibitor used in the treatment of metastatic colorectal cancer and head and neck cancer. Cetuximab is a chimeric (mouse / human) mAb, usually administered by IV infusion. Cetuximab is a trademark of Bristol-Myers Squibb Company (North America; "Bristol-Myers Squibb"), Eli Lilly and Company (North America; "Eli Lilly"), and Merck KGaA under the trademark ERBITUX® for IV use. Only on the market. ERBITUX® is produced in cell cultures of mammals (mouse myeloma). The single-use 50 mL vials of ERBITUX® each contain 100 mg of cetuximab at a concentration of 2 mg / mL, sodium chloride 8.48 mg / mL and sodium diphosphate heptahydrate 1.88. Formulated with a preservative-free solution containing mg / mL, sodium chloride monohydrate 0.42 mg / mL, and IV water for injection (USP).

Cetuximab, in combination with chemotherapy and as a single agent, expresses epithelial growth factor receptor (EGFR), in patients who have been unsuccessful in treatment with oxaliplatin and irinotecan, or who are intolerant to irinotecan. It is indicated for the treatment of patients with KRAS wild-type metastatic colorectal cancer (mCRC). Cetuximab in patients with head and neck squamous cell carcinoma in combination with platinum-based chemotherapy for first-line treatment of recurrent and / or metastatic disease and in combination with radiation therapy for locally progressive disease There is an indication for the treatment of. 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.

As used herein, the term "cetuximab" includes the mAb known by the international nonproprietary name "CETUXIMAB", or an antigen-binding portion thereof. Cetuximab contains the antibody described in US Pat. No. 6,217,866. Cetuximab contains an activator in products marketed under the trademark ERBITUX® and its biosimilar products. ERBITUX® biosimilars can include those currently under development by Amgen, AlphaMab Co., Ltd. ("AlphaMab"), and Actavis plc ("Actavis").

Bevacizumab, a humanized mAb that inhibits vascular endothelial growth factor A (VEGF-A), acts as an anti-angiogenic agent. Bevacizumab is Genentech, Inc. ("Genentech") and F. Hoffmann-La. It is marketed by Roche, LTD ("Roche") under the trademark AVASTIN®. Bevacizumab is approved to treat a variety of cancers, including colorectal cancer, lung cancer, breast cancer (non-US), glioblastoma (US only), kidney and ovarian cancer. AVASTIN® is metastatic colorectal cancer when used with standard chemotherapy treatment (as first-line treatment) and 5-fluorouracil-based treatment for second-line metastatic colorectal cancer. Approved by the FDA in 2004 for use in. 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 3 weeks at a dose of either 15 mg / kg or 7.5 mg / kg. Higher doses are usually given for carboplatin-based chemotherapy, while lower doses are given for cisplatin-based chemotherapy. In 2009, the FDA approved AVASTIN® for use in metastatic renal cell carcinoma (a form of renal cancer). The FDA also granted accelerated approval of AVASTIN® in 2009 for the treatment of recurrent polymorphic glioblastoma. Treatment for early growth is still in Phase III clinical trials.

The National Comprehensive Cancer Network ("NCCN") combines bevacizumab as a standard first-line treatment that is combined with any platinum-based chemotherapy and then maintains bevacizumab until the disease progresses. Is recommended. In 2010, NCCN updated its Clinical Practice Guidelines for Oncology (NCCN Guidelines) for bevacizumab (AVASTIN®, Genentech / Roche) in the treatment of metastatic breast cancer. Confirmed recommendations for use.

As used herein, the term "bevacizumab" is an mAb that inhibits vascular endothelial growth factor A (VEGF-A), or antigen binding thereof, known by the international generic name / generic name "BEVACIZUMAB". Including the part. Bevacizumab is described in US Pat. No. 6,054,297. Bevacizumab contains an activator in products marketed under the trademark AVASTIN® and its biosimilar products. AVASTIN® biosimilars can include those currently under development by Amgen, Actavis, AlphaMab and Pfizer, Inc ("Pfizer"). AVASTIN® biosimilars are manufactured by Biocad and can include biosimilars known as BCD-021 that are currently in clinical trials in the United States.

Trastuzumab is an mAb that interferes with the HER2 / neu receptor. Trastuzumab is marketed by Genentech, Inc. under the trademark HERCEPTIN®. HERCEPTIN® is produced by the 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 trastuzumab 440 mg, L-histidine HCl 9.9 mg, L-histidine 6.4 mg, a, a-trehalose dihydrate 400 mg, and polysorbate 20 (USP) 1.8 mg, respectively. Reconstitution with 20 mL of water yields a multi-dose solution containing 21 mg / mL trastuzumab. HERCEPTIN® is currently administered by IV infusion at doses ranging from about 2 mg / kg to about 8 mg / kg, as often as weekly.

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 causes epidermal growth factor (EGF) receptor overexpression in the cell membrane. Trastuzumab is commonly given as maintenance therapy for patients with HER2-positive breast cancer for 1 year after chemotherapy. Trastuzumab is currently administered by IV infusion at doses ranging from about 2 mg / kg to about 8 mg / kg, as often as weekly.

As used herein, the term "trastuzumab" includes the mAb that interferes with the HER2 / neu receptor, or antigen-binding portion thereof, known by the international nonproprietary / generic name "TRASTUZUMAB". Trastuzumab is described in US Pat. No. 5,821,337. Trastuzumab contains activators in products marketed under the trademark HERCEPTIN® and its biosimilars. The term "trastuzumab" is marketed by Mylan, Inc. ("Mylan") under the trademark HERTRAZ® and by Biocon, Ltd. ("Biocon") under the trademark CANMAB®, biosimilar HERCEPTIN. (Registered Trademark) product contains an activator. Trastuzumab can contain an activator in a biosimilar HERCEPTIN® product under development by Amgen, PlantForm Corporation, Canada.

Infliximab is a mAb against tumor necrosis factor alpha (TNF-α) used to treat autoimmune diseases. Infliximab was 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 &amp; Co ("Merck") elsewhere. ing. Infliximab is a chimeric mouse / human monoclonal antibody with a high molecular weight of about 144 kDa. In some embodiments, the formulation contains a REMICADE® biosimilar, such as REMSIMA or INFLECTRA . REMSIMA and Hospira developed by Celltrion, Inc. ("Celltrion") Both Inc and INFLECTRA , developed by the United Kingdom, are recommended for regulatory approval in Europe. Celltrion has filed an application with the FDA for approval of REMSIMA . Infliximab is currently administered by IV infusion at doses ranging from about 3 mg / kg to about 10 mg / kg.

Approximately 30% of infliximab contains the amino acid sequence of the murine variable region, which confer antigen binding specificity for 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 a high affinity for human TNFα, a cytokine with multiple biological effects, including mediation of the inflammatory response and modulation of the immune system.

Infliximab is a recombinant antibody that is commonly produced and secreted by mouse myeloma cells (SP2 / 0 cells). This antibody is currently produced by continuous perfusion cell culture. Infliximab monoclonal antibodies are expressed using a chimeric antibody gene consisting of a variable region sequence cloned from mouse anti-TNFα hybridoma A2 and a human antibody constant region sequence supplied by a plasmid expression vector. Mice anti-TNFα hybridoma production 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 can include chromatographic purification, virus inactivation, nanofiltration, and ultrafiltration / diafiltration.

As used herein, the term "infliximab" includes a chimeric mouse / human monoclonal antibody known by the international nonproprietary name "INFLIXIMAB", or an antigen-binding portion thereof. Infliximab neutralizes the biological activity of TNFα and inhibits the binding of TNFα to its receptor by binding to the soluble and transmembrane morphology of TNFα with high affinity. Infliximab is described in US Pat. No. 5,698,195. The term "infliximab" is marketed or planned to be marketed under the trademarks REMICADE® by multiple entities, REMSIMA by Celltrion, and INFLECTRA by Hospira, Inc ("Hospira"). Contains an activator in the product being used. Infliximab is supplied as a sterile lyophilized cake for reconstruction and dilution. Each vial of infliximab contains 100 mg of infliximab and excipients such as sodium monophosphate monohydrate, sodium dibasic dihydrate, sucrose and polysorbate 80, respectively.

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

Panitumumab is an FDA-approved, fully human mAb 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 injection. When prepared according to the package insert, the final panitumumab concentration does not exceed 10 mg / ml. VECTIBIX® is administered as an intravenous infusion every 14 days at a dose of 6 mg / kg. 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 activators in products marketed by Amgen under the trademark VECTIBIX® and their biosimilars. The term "panitumumab" includes monoclonal antibodies described in US Pat. No. 6,235,883. The term "panitumumab" includes activators in biosimilar VECTIBIX® products, including Biosimilar VECTIBIX® under development by BioXpress, SA ("BioXpress").

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

Abciximab (REOPRO®) is manufactured by Janssen Biologics BV and marketed by Eli Lilly &amp; Company ("Eli Lilly"). Abciximab is a 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 blocking the binding of fibrinogen, von Willebrand factor and other adhesion molecules. Abciximab also binds to the vitronectin (αvβ3) receptor found on platelets and vascular wall endothelial cells and smooth muscle cells. Abciximab is a platelet aggregation inhibitor that is primarily used during and after coronary artery surgery. Abciximab is first administered by IV infusion with a 0.25 mg / kg bolus, followed by a 12-hour continuous IV infusion of 0.125 mcg / kg / min.

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 by sequential infusion of non-radioactive mAbs, followed by iodine (131I) tositumomab, the same antibody that is covalently bound to the radionuclide iodine-131. Clinical trials have established the efficacy of tositumomab / iodine tositumomab regimen in patients with recurrent refractory follicular lymphoma. BEXXAR® is currently administered at a dose of 450 mg by IV infusion.

Alemtuzumab (marketed as CAMPATH®, MABCAMPATH® or CAMPATH-1H® and currently under development as LEMTRADA®) is a chronic lymphocytic leukemia (CLL). ), Cutaneous T cell lymphoma (CTCL), and mAbs used in the treatment of T cell lymphoma. Alemtuzumab has also been used under clinical trial protocols for the treatment of several autoimmune diseases such as multiple sclerosis. Alemtuzumab weighs about 145.5 kDa. Alemtuzumab is given daily to patients with B-cell chronic lymphocytic leukemia with a 30 mg IV infusion.

Palivizumab (SYNAGIS®) is a humanized mAb that targets an epitope at the A antigen site of the F protein of respiratory syncytial virus. In two phase III clinical trials in the pediatric population, palivizumab reduced the risk of hospitalization for respiratory syncytological virus infection by 55% and 45%. Palivizumab is administered once a month by 15 mg / kg IM injection.

Ofatumumab is a human anti-CD20 mAb that appears to inhibit early B lymphocyte activation. Ofatumumab is marketed by GlaxoSmithKline, plc ("GlaxoSmithKline") under the trademark ARZERRA®. ARZERRA® is sold in single-use vials containing 100 mg / 5 mL and 1,000 mg / 50 mL ofatumumab for IV injection. Ofatumumab has been approved by the FDA to treat chronic lymphocytic leukemia and may be able to treat follicular non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing-remitting multiple sclerosis. Shown. Ofatumumab has a molecular weight of about 149 kDa. Ofatumumab is currently administered by IV injection at an initial dose of 300 mg, followed by weekly injections of 2,000 mg. As used herein, the term "ofatumumab" is an anti-CD20 known by the international nonproprietary name "OFATUMUMAB". Contains mAb. The term "ofatumumab" includes an activator in a product marketed under the trademark ARZERRA® and its biosimilars. The term "ofatumumab" contains an activator in a biosimilar ARZERRA® product under development by BioExpress. The high molecular weight low viscosity liquid protein preparation can contain ofatumumab at a concentration of preferably from about 300 mg / mL to about 2,000 mg / mL.

Trastuzumab emtansine (ad-trastuzumab emtansine, marketed as KADCYLA® in the United States) is a mAb trastuzumab linked to the cytotoxic agent mertansine (DM1®). It is an antibody-drug conjugate consisting of. Trastuzumab, described above, arrests the growth of cancer cells by binding to the HER2 / neu receptor, while mertansine enters the 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. Multiple phase III trials of trastuzumab emtansine are planned or underway in 2014. Trastuzumab emtansine is currently being administered by IV infusion of 3.6 mg / kg. The high molecular weight low viscosity liquid preparation can contain trastuzumab emtansine at a concentration of preferably from about 144 mg / mL to about 360 mg / mL.

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

Daclizumab is a humanized anti-CD25 mAb and is 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 about 143 kDa. Daclizumab was marketed in the United States by Hoffmann-La Roche, Ltd. ("Roche") as ZENAPAX® and was administered by IV infusion of 1 mg / kg. To treat relapsing-remitting multiple sclerosis, the Daclizumab High-Yield Process (DAC HYP; BIIB019; Biogen Idec ("Biogen") and AbbVie, Inc. ("AbbVie")) It is in a phase III clinical trial as a subcutaneous injection of 150 mg once a month. The high molecular weight low viscosity liquid formulation can contain daclizumab at a concentration of preferably from about 40 mg / mL to about 300 mg / mL.

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 with a molecular weight of approximately 148 kDa is under development by Alexion Pharmaceuticals, Inc ("Alexion"). Eculizumab is administered by IV infusion in an amount of about 600 mg to about 1,200 mg. The high molecular weight low viscosity liquid preparation can contain eculizumab at a concentration of preferably from about 500 mg / mL to about 1,200 mg / mL.

Tocilizumab (ACTEMRA®) is a humanized mAb for the interleukin-6 receptor. Tocilizumab is an immunosuppressive drug primarily for the treatment of rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis, a serious form of RA in children. Tocilizumab is generally administered by IV infusion at doses of about 6 mg / kg to about 8 mg / kg. The high molecular weight low viscosity liquid formulation can contain tocilizumab at a concentration of preferably from about 240 mg / mL to about 800 mg / mL.

Rituximab (RITUXAN®) is a chimeric anti-CD20 mAb used to treat a variety of diseases characterized by an excess of B cells, hyperactive B cells, or dysfunctional B cells. .. Rituximab is used to treat leukocyte-based cancers such as leukemia and lymphoma, including Hodgkin lymphoma and its lymphocyte-dominant subtypes. Rituximab has been shown to be an effective treatment for rheumatoid arthritis. Rituximab is widely used off-label to treat difficult cases of multiple sclerosis, systemic lupus erythematosus, and autoimmune anemia.

Rituximab is jointly marketed in the United States by Biogen and Genentech under the trademark RITUXAN® and outside the United States by Roche under the trademark MABTHERA®. RITUXAN® is sold in single-use vials containing 100 mg / 10 mL and 500 mg / 50 mL. RITUXAN® is approximately 375 mg / m²Is usually administered by IV infusion. As used herein, the term "rituximab" includes the anti-CD20 mAb known under the international nonproprietary / generic name "RITUXIMAB". Rituximab contains the mAbs described in US Pat. No. 5,736,137. Rituximab contains activators in products marketed under the trademarks RITUXAN® and MABTHERA®, and their biosimilars.

The high-molecular-weight, low-viscosity liquid formulation can contain rituximab at a concentration of preferably from about 475 mg / mL to about 875 mg / mL (for humans in the range of 5 ft, 40 kg to 6 ft, 100 kg, derived from Mosteller's equation. Estimated using a body surface area of 1.3-2.3 square meters). Concentration is calculated for 1 mL formulation.

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

Raxibacumab (ABthrax®) is a human mAb intended for the prevention and treatment of inhaled anthrax. Raxibacumab is currently being administered by IV infusion. In adults and children over 50 kg, the suggested dose is 40 mg / kg. The high molecular weight low viscosity liquid preparation can contain raxibacumab at a concentration of preferably from about 1,000 mg / mL to about 4,000 mg / mL.

Nimotuzumab (THE RACIM®, BIOMAB EGFR®, THERALOC®, CIMAher®) is a head and neck squamous cell carcinoma, recurrent or refractory high-grade malignant glioma, A humanized mAb with a molecular weight of approximately 151 kDa used to treat undifferentiated astrocytomas, gliomas, and diffuse endogenous pontine gliomas. Nimotuzumab is usually administered by an IV infusion of about 200 mg weekly. The high molecular weight low viscosity liquid formulation can contain nimotuzumab at a concentration of preferably about 200 mg / mL.

Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate for the protein CD30 expressed in classical Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Brentuximab vedotin is administered by IV infusion of approximately 1.8 mg / kg. The high molecular weight low viscosity liquid preparation can contain brentuximab vedotin at a concentration of preferably from about 80 mg / mL to about 200 mg / mL.

Itrizumab (ALZUMAB®) is a humanized IgG1 mAb developed by Biocon. Itrizumab successfully completed a phase III trial in patients with moderate to severe psoriasis. Itrizumab has received marketing approval in India. No application has been submitted for FDA approval.

Originally developed by Roche and further developed in partnership with Biogen, obinutuzumab (GAZYVA®) is a humanized anti-CD20 mAb approved for the treatment of chronic lymphocytic leukemia. Obinutuzumab is also under investigation in a phase III clinical trial in patients with various lymphomas. A dose of about 1,000 mg has been administered by IV infusion.

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

Other antibody therapeutics that can be formulated with the reduced viscosity organic phosphate include CT-P6 from Celltrion, Inc. (Celltrion).

Antibody therapeutics in late-stage trials and development

Progress in late-stage clinical development of antibody therapeutics and regulatory review are proceeding at a rapid pace. In 2014, there were over 300 mAbs in clinical trials and 30 commercially sponsored antibody therapies evaluated in late-stage studies. The first launch application for two mAbs (vedolizumab and ramucirumab) was recently submitted to the FDA. Amgen is currently sponsoring multiple ongoing Phase III trials of brodalumab use in patients with plaque psoriasis, including planning additional trials or recruiting patients. XBiotech, Inc is sponsoring two Phase I clinical trials of MABp1 (Xilonix) in patients with advanced cancer or type 2 diabetes. An additional study of MABp1 is in the process of recruiting patients. Multiple trials sponsored by MedImmune, LLC ("MedImmune"), moxetumomab Treatment of leukemia with pasudotox) is in progress or is being recruited for it. Long-term safety and efficacy studies are ongoing using chilled akizumab for the treatment of chronic plaque psoriasis. Several phase II trials of the use of lyrotumumab for the treatment of various cancers have recently been completed.

At least 28 mAbs are high, currently in or recently completed, in Phase III studies for the treatment of inflammation or immunological disorders, cancer, high cholesterol, osteoporosis, Alzheimer's disease, and infections. It is a molecular weight protein. MAbs that are in or have recently completed Phase III trials include AMG145, Elotuzumab, Eplatuzumab, Farretsuzumab (MORAb-003), Gantenerumab (RG1450), Gebokizumab, Inotsumab Ozogamycin, Itrizumab, Ixekizumab, Lebrikizumab, mepolizumab, naptumomab estaphenatox, necitumumab, nivolumab, ocrelizumab, onaltuzumab, lacotumomab, ramucirumab, reslizumab, romosozumab, sarilumab, sekkinumab, silkmab, solanezumab. mAb mixtures (actoxumab and bezlotoxumab) are also being evaluated in Phase III trials. See, for example, Reichert, MAbs Vol. 5, pp. 1-4, 2013.

Vedolizumab is an mAb under development 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 shown that vedolizumab meets the goals of inducing a clinical response and maintaining remission in patients with Crohn's disease and ulcerative colitis. Studies assessing long-term clinical outcomes have shown that nearly 60% of patients have achieved clinical remission. A typical dose of vedolizumab is 6 mg / kg by IV infusion.

Ramucirumab is a human mAb under development for the treatment of solid tumors. Phase III clinical trials to treat breast cancer, metastatic gastric adenocarcinoma, non-small cell lung cancer, and other types of cancer are underway. Ramucirumab is infused at approximately 8 mg / kg by IV infusion in some Phase III trials.

Lilotumumab is a human mAb that inhibits the action of hepatocyte growth factor / dispersant. Lirotumumab, developed by Amgen, is in Phase III trials for the treatment of solid tumors. An open phase III study of lyrotumumab treatment in patients with advanced or metastatic esophageal cancer administers approximately 15 mg / kg of lyrotumumab by IV infusion.

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

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

ABR-217620 from Active Biotech AB ("Active Biotech"), or naptumomabu estaphenatox, is a mAb indicated for renal cell carcinoma.

CIMAB, SA ("CIMAB"); Lacotumomab from Laboratorio Elea SACIFy A is a mAb indicated for non-small cell lung cancer.

Other antibodies that can be formulated with reduced viscosity organic phosphate include bocosizumab (PF-04950615) and tanezumab; ganitzumab from Amgen, blinatumomab, trevananib; anthrax immunoglobulin from Cangene Corporation; from MacroGenics, Inc. Teprizumab; MK-3222 from Merck &amp; Co ("Merck"), MK-6072; Gilentuximab from Wilex AG; RIGScan from Navidea Biopharmaceuticals ("Navidea"); Includes SA237 from "Chinagai"); Guselkumab from Janssen / Johnson and Johnson Services, Inc. ("J &amp; J"); anti-thrombin gamma from Kyowa (KW-3357); and CT-P10 from Celltrion.

Antibodies in the early stages of clinical trials

Many mAbs have recently entered or are in clinical trials. They can preferably contain proteins currently being administered by IV infusion of those having a molecular weight of greater than about 120 kDa, typically about 140 kDa to about 180 kDa. They can also include these high molecular weight proteins, such as albumin-conjugated drugs or peptides, which are also in clinical trials or approved by the FDA. Numerous mAbs from Amgen are currently in clinical trials. These can be high molecular weight proteins such as AMG557, a human monoclonal antibody that was jointly developed by Amgen and AstraZeneca and is 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. In addition, AMG110 is a mAb for epithelial cell adhesion molecules. Co-developed by Amgen and AstraZeneca, AMG157 is a human mAb currently in Phase I for the treatment of asthma. AMG167 is a humanized mAb evaluated in multiple Phase I trials for the treatment of osteopenia. AMG334, which has completed a phase I dose study and is currently in a phase II study for the treatment of migraine and flushing, is a human mAb that inhibits a calcitonin gene-related peptide. 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 recently completed a phase I trial as a cancer treatment. .. AMG811 is a human monoclonal antibody that inhibits interferon gamma, which is being investigated for the treatment of systemic lupus erythematosus. AMG820 is a human mAb that inhibits c-fms and reduces tumor-related macrophage (TAM) function and is under investigation as a cancer treatment. Amg

Many mAbs are currently in clinical trials for the treatment of autoimmune disorders. These mAbs can be contained in a high molecular weight low viscosity liquid formulation. 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 underway. BIIB033 is an anti-LINGO-1 mAb by Biogen, which is currently in Phase II trials for the treatment of multiple sclerosis.

High molecular weight proteins can also include AGS-009, an IFN alpha-targeted mAb developed by Argos Therapeutics, Inc., which recently completed a phase I study 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 a phase II trial in patients with rheumatoid arthritis. Certolizumab pegol (CIMZIA®) is a mAb in a phase II trial for ankylosing spondylitis and juvenile rheumatoid arthritis. Kurazakizumab, an anti-IL-6 mAb, is in Phase II trials by Bristol-Myers Squibb.

CNTO-136 (Silkumab) and CNTO-1959 are mABs that have recently completed Phase II and Phase III trials by Janssen. Daclizumab (formerly launched by Roche as ZENAPAX®) is currently in or recently completed in multiple Phase III trials by AbbVie for the treatment of multiple sclerosis. Eplatzumab is a humanized mAb in a phase III trial for the treatment of lupus. Canakinumab (ILARIS®) is a human mAb that targets interleukin-1 beta. Canakinumab was approved for the treatment of cryopyrin-associated periodic syndromes. Canakinumab is in Phase I trials as it may be able to treat chronic obstructive pulmonary disease, gout and coronary artery disease. Mabrilimumab is a human mAb designed for the treatment of rheumatoid arthritis. Discovered as CAM-3001 by Cambridge Antibody Technology, Mabrilimumab is under development by Med Immunone.

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

Orokizumab is a humanized mAb that strongly targets the cytokine IL-6. IL-6 is involved in several autoimmune and inflammatory pathways. Orokizumab has completed a phase II trial for the treatment of rheumatoid arthritis. Otelixizumab, also known as TRX4, is a mAb under development to treat type I diabetes, rheumatoid arthritis, and other autoimmune diseases. Ozolarisumab is a humanized mAb that has completed a phase II trial.

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

Rontalismab is a humanized mAb under development by Genentech. Rontalizumab recently completed a phase II trial for the treatment of lupus. SAR113244 (anti-CXCR5) is a Sanofi mAb during Phase I trials. Ciphalimumab (anti-IFN alpha mAb) is a mAb under phase II trials for the treatment of lupus.

High molecular weight low viscosity liquid formulations can contain one of the mAbs that are in the early stages of clinical development for the treatment of various blood disorders. For example, belimumab (BENLYSTA®) has recently completed a phase I trial in patients with vasculitis. Other mAbs in the early stages of trials for hematological disorders include BI-655075 from Boehringer Ingelheim GmbH "Boehringer Ingelheim", ferroportin mAbs and hepcidin mAbs from Eli Lilly, and Selexys Pharmaceuticals, Corp. ("Selexys"). SelG1 is included.

One or more mAbs, which are in the early stages of development for treating various cancers and related conditions, may be included in a low viscosity, high molecular weight liquid formulation. United Therapeutics, Corporation has two mAbs under Phase I study, 8H9 mAb and ch14.18 mAb. The mAbs from AbbVie, ABT-806, enabatsuzumab, and borosiximab, are in the early stages of development. Actinium Pharmaceuticals, Inc conducted early-stage trials on the mAbs Actimab-A (M195 mAb), anti-CD45 mAb, and Iomab-B. Seattle Genetics, Inc. ("Seattle Genetics") is in early-stage trials for cancer and related conditions, anti-CD22 ADC (RG7593; pinatsuzumabbedotin), anti-CD79b ADC (RG7596), anti-STEAP1 Several mAbs, including ADC (RG7450), ASG-5ME and ASG-22ME from Agensys, Inc. ("Agensys"), the antibody-drug conjugate RG7458, and bolsetzumab mafodotin. Have. 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, A therapeutic agent for early stage cancer from Genentech, including MINT1526A, may be included in the low viscosity formulation. Bristol-Myers Squibb is an early-stage mAb for cancer treatments, including those identified as anti-CXCR4, anti-PD-L1, IL-21 (BMS-982470), lililumab and urerumab (anti-CD137). Is under development. Other mAbs that are in the early stages of trial as a cancer treatment include APN301 (hu14.18-IL2) from Apeiron Biologics AG, AVEO. AV-203 from 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.

Other antibody therapeutics that can be formulated with the reduced viscosity organic phosphate include alzumab, GA101, daratumumab, siltuzumab, ALX-0061, ALX-0962, ALX-0761, bimagumab (BYM338), CT-011 ( Ranibizumab), Actoxumab / Vezrotoxumab (MK-3515A), MK-3475 (Pembrolizumab), Darotuzumab (MK-0646), Ikulkumab (IMC-18F1, LY3012212), AMG139 (MEDI2070), SAR339658, Dupilumab (REGN668) , SAR279356, SAR3419, SAR153192 (REGN421, Enochikumab), SAR307746 (Nesvacumab), SAR650984, SAR566658, SAR391786, SAR228810, SAR252067, SGN-CD19A, SGN-CD33A, SGN-LIV1A, ASG15ME, Anti-LINGO, BIIB037 Concizumab, Anlucinzumab (IMA-638), Ponezumab (PF-04360365), PF-03446962, PF-06252616, Etrolizumab (RG7413), Quirizumab, Ranibizumab, Ramparizumab, Onkrakumab, Gentenerumab, Crenezumab (RG7412) , Tremelimumab, Vantictumab, emcizumab, obinutuzumab, mapatumumab, tralokinumab, XmAb5871, XmAb7195, sixtumumab (LY3012217), LY2541546 (brosozumab), LY2541546 (brosozumab), LY2541546 (brosozumab), LY2541546 (brosozumab), LY2541546 (brosozumab), LY2541546 (brosozumab), LY2541546 , PF-05236812 (AAB-003), PF-05082566, BI1034020, RG7116, RG7356, RG7155, RG7212, RG7599, RG7636, RG7221, RG7652 (MPSK3169A), RG7686, HuMaxTFADC, MOR103, BT061, MOR208, OMP59R5 (anti-notch 2/3), VAY736, MOR202, BAY94-9343, LJM716 Includes OMP52M51, GSK933776, GSK249320, GSK1070806, NN8828, CEP-37250 / KHK2804 AGS-16M8F, AGS-16C3F, LY3016859, LY2495655, LY2875358, and LY2812176.

Other early-stage mAbs that can be formulated with reduced-viscosity organic phosphates include Benlarizumab, MEDI-8968, Aniflorumab, MEDI7183, Ciphalimumab, MEDI-575, Traloquinumab; Biogen Idec / Eisai from AstraZeneca and MedImmune. BAN2401 from BioArctic Neuroscience AB; CDP7657, an anti-CD40L monovalent PEGylated Fab antibody fragment from Biogen, STX-100, BIIB059, anti-TWEAK (BIIB023) and BIIB022, which are anti-avB6 mAbs. Furlanumab from Janssen and Amgen; BI-204 / RG7418 from BioInvent International / Genentech; BT-062 from Biotest Pharmaceuticals Corporation; XmAb from Boehringer Ingelheim / Xencor; Bristol-Myers Anti-IP10 from Squibb; J591Lu-177 from BZL Biologics LLC; CDX-011 (Grembatumumab Bedochin) from Celldex Therapeutics, CDX-0401; Forabirumab from Crucell; Chigatsuzumab from Daiichi Sankyo Corporation; MORAb-004 from Eisai, MORAb-009 (Amatsuximab); LY2382770 from Eli Lilly; DI17E6 from EMD Serono Inc.; Zanolimmab from Emergent BioSolutions, Inc.; FG-3019 from FibroGen, Inc.; Fresenius SE &amp; Co. Katsumakisomab from KGaA; Patecrizumab from Genentech, Rontalizumab; Fresolimmab from Genzyme &amp; Sanofi; GS-6624 (Simtuzumab) from Giled; CNTO-328 from Janssen, Bappineuzumab (AAB-001), Karlumab, CNTO-136 KB003 from Pharmaceuticals, Inc.; ASKP1240 from Kyowa; RN-307 from Labrys Biologics Inc.; eclomeximab from Life Science Pharmaceuticals; LY2495655, LY2928057, LY3015014, LY2951742; MBL-HCV1 from Mass Biologics; MENTRIK AME-133v from Biotech, LLC; Abitsuzumab from Merck KGaA; MM-121 from Merrimack Pharmaceuticals; MCS110, QAX576, QBX258, QBX258, QGE031 from Novartis AG; HCD122; Novo from Novartis AG and XOMA Corporation ("XOMA") NN8555 from Nordisk; Babyximab from Peregrine Pharmaceuticals, Inc., Kotara; PSMA-ADC from Progenics Pharmaceuticals, Inc.; Quest Oregobomab from Pharmatech, Inc.; Facinumab from Regeneron (REGN475), REGN1033, SAR231893, REGN846; RG7160 from Roche, CIM331, RG7745; Ibarizumab from TaiMed Biologics Inc. (TMB-355); TCN from Theraclone Sciences- 032; TRC 105 from TRACON Pharmaceuticals, Inc.; UB-421 from United Biomedical Inc.; VB4-845 from Viventia Bio, Inc.; ABT-110 from AbbVie; Kaprasizumab, Ozora Rituximab from Ablynx; CytoDyn, Inc. PRO140 from PRO140; GS-CDA1 from Medarex, Inc., MDX-1388; AMG827 from Amgen, AMG888; Ubrituximab from TG Therapeutics Inc.; TOL101 from Torera Therapeutics, Inc.; ImmunoGen HuN901-DM1 from Inc. (Rolbotzumab mertancin); Eplatzzumab Y-90 / Bertzzumab combination from Immunomedics, Inc. (IMMU-102); Anti-fibrin mAb / 3B6 / 22 Tc from Agenix, Limited -99m; ALD403 from Alder Biopharmaceuticals, Inc.; RN6G / PF-04382923 from Pfizer; CG201 from CG Therapeutics, Inc.; KB001-A from Kalo Bios Pharmaceuticals / Sanofi; KRN-23 from Kyowa; Immunomedics, Inc. Y-90h PAM4 from.; Tarexitumab from Morphosys AG &amp; OncoMed Pharmaceuticals, Inc.; LFG316 from Morphosys AG &amp; Novartis AG; CNTO3157, CNTO6785; Roche &amp; RG6013 from China and outside; Merrimack MM-111 from Pharmaceuticals, Inc. ("Merrimack"); GSK2862277 from GlaxoSmithKline; AMG282, AMG172, AMG595, AMG745, AMG761 from Amgen; BVX-20 from Biocon; CT-P19, CT-P24 from Celltrion , CT-P25, CT-P26, CT-P27, CT-P4; GSK284933 from GlaxoSmithKline, GSK2398852, GSK2618960, GSK1223249, GSK933776A; Anetsuma Brabtansin from Morphosys AG &amp; Bayer AG; NOV-7, NOV-8 from AG &amp; Novartis AG; MM-302, MM-310, MM-141, MM-131, MM-151; Roche &amp; Seattle from Merrimack RG7882 from Genetics; RG7841 from Roche / Genentech; PF-06410293, PF-06438179, PF-06439535, PF-04605412, PF-05280586 from Roche; RG7716, RG7936, Gentenermab, RG7444; MEDI from Astrazeneca -547, MEDI-565, MEDI1814, MEDI4920, MEDI8897, MEDI-4212, MEDI-5117, MEDI-7814; Urokpurumab from Bristol-Myers Squibb, PCSK9 Adnectin; FPA009 from Five Prime Therapeutics, Inc., FPA145; from Gilead GS-5745; BIW-8962, KHK4083, KHK6640; Merck from Kyowa Hakko Kirin Co., Ltd. MM-141 from KGaA; REGN1154, REGN1193, REGN1400, REGN1500, REGN1908-1909, REGN2009, REGN2176-3, REGN728; SAR307746 from Sanofi; SGN-CD70A from Seattle Genetics; ALX-0141 from Ablynx, ALX-0171; Miratuzumab from Immunomedics, Inc.-DOX, Miratuzumab, TF2; MLN0264 from Millennium; ABT-981 from AbbVie; AbGn-168H from AbGenomics International Inc.; Fikratuzumab from AVEO; BI from BioInvent International -505; CDX-1127 from Celldex Therapeutics, CDX-301; CLT-008 from Cellerant Therapeutics Inc.; VGX-100 from Circadian; U3-1565 from Daiichi Sankyo Co., Ltd .; Dekkun DKN-01 from Corp.; Franbotumab (TYRP1 protein) from Eli Lilly, IL-1β antibody, IMC-CS4; Eli Lilly and ImClone, VEGFR3 mAb from LLC, IMC-TR1 (LY3022859); from Elusys Therapeutics Inc. Antibodies; HuL2G7 from Galaxy Biotech LLC; IMGB853 from ImmunoGen Inc., IMGN529; CNTO-5, CNTO-5825 from Janssen; KD-247 from Kaseiken; KB004 from KaloBios Pharmaceuticals; from MacroGenics, Inc. MGA271, MGAH22; XmAb5574 from MorphoSys AG / Xencor; Encituximab (NPC-1C) from Neogenix Oncology, Inc.; LFA102 from Novartis AG and XOMA; ATI355 from Novartis AG; SAN-300 from Santarus Inc.; SelG1 from Selexys; Targa HuM195 / rGel from Therapeutics, Corp.; Teva Pharmaceuticals, Industries Ltd. ("Teva") and VX15 from Vaccinex Inc.; TCN-202 from Theraclone Sciences; XmAb2513 from Xencor, XmAb5872; XOMA3AB from the Institute of Diseases; Neuroblastoma antibody vaccine from MabVax Therapeutics; Cytolin from CytoDyn, Inc.; Travixa from Emergent BioSolutions Inc.; FB301 from Cytovance Biologics; FB301 from Janssen and Sanofi Includes MB-003 and ZMapp; from Mapp Biopharmaceutical, Inc. and ZMab from Defyrus Inc., which are from mad dog disease mAb; Janssen and are partly funded by the National Institute of Health. ..

Other protein treatments

The protein can be an enzyme, fusion protein, stealth or PEGylated protein, vaccine, or other bioactive protein (or protein mixture). As used herein, the term "enzyme" refers to a protein or functional fragment thereof that catalyzes the biochemical conversion of a target molecule into a desired product.

Enzymes as drugs have at least two important characteristics. That is, i) they often bind to and act on their targets with high affinity and specificity, and ii) they are catalytic and convert multiple target molecules into the desired product. In certain embodiments, the protein can be PEGylated, as defined herein.

As used herein, the term "fusion protein" refers to a protein made up of two different genes that encode two separate proteins. Fusion proteins are commonly produced by recombinant DNA techniques known to those of skill in the art. The two proteins (or protein fragments) are covalently fused together to exhibit properties from both parent proteins.

Several fusion proteins are on the market.

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

ELOCTATE® (recombinant), an Fc fusion protein of the anti-hemophilia factor, controls and prevents bleeding episodes in adults and children with hemophilia A (congenital factor VIII deficiency). An anti-hemophilia factor derived from recombinant DNA that is indicated for staging, routine prophylaxis to prevent or reduce the frequency of bleeding episodes.

EYLEA® (Aflibercept) is one of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc moiety of human IgG1, which is formulated as an isotonic solution for intravitreal administration. It is a recombinant fusion protein consisting of parts. EYLEA is a set of parts of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1, which is formulated as an isotonic solution for intravitreal administration. It is a transfusion protein. Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation that makes up an additional 15% of the total molecular weight, resulting in a total molecular weight. It will be 115 kDa. Aflibercept is produced in the ovarian (CHO) cells of recombinant Chinese hamsters and is marketed by Regeneron.

Does ALPROLIX (Recombinant), an Fc fusion protein of coagulation factor IX, control and prevent bleeding episodes, perioperative management, and prevent bleeding episodes in adults and children with hemophilia B? Or a coagulation factor IX concentrated preparation derived from recombinant DNA that is indicated for routine prevention to reduce its frequency.

Pegloticase (KRYSTEX XA®) is a drug developed by Savient Pharmaceuticals, Inc. for the treatment of severely refractory chronic gout and is the first approved drug for this indication. Pegloticase is a PEGylated recombinant porcine-like uricase with a molecular weight of approximately 497 kDa. Pegloticase is currently being administered by IV infusion of approximately 8 mg / kg. The high molecular weight, low-viscosity liquid preparation can contain pegloticase at a concentration of preferably from about 300 mg / mL to about 800 mg / mL.

Alteplase (ACTIVASE®) is a tissue plasminogen activator produced by recombinant DNA technology. Alteplase is a purified glycoprotein containing 527 amino acids by using complementary DNA (cDNA) to the naturally occurring human tissue plasminogen activator from the human melanoma cell line. It is synthesized. Alteplase is administered by IV infusion of approximately 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.

Glucalpitase (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. Glucalpitase is administered by IV in a single dose of about 50 IU / kg. In some embodiments, a low viscosity formulation containing glucarpitase is provided.

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

Bovine pegdamase bovine (ADAGEN®) is a modifying enzyme used in enzyme replacement therapy for the treatment of severe combined immunodeficiency disease (SCID) associated with adenosine deaminase deficiency. Bovine pegademase is a conjugate of various chains of monomethoxypolyethylene glycol (PEG) (molecular weight 5,000 Da) that is covalently attached to the adenosine deaminase enzyme derived from bovine intestines.

α-galactosidase is a lysosomal enzyme that catalyzes the hydrolysis of the glycolipid globotriaosylceramide (GL-3) to galactose and ceramide dihexoside. Fabry disease is a rare hereditary lysosomal storage disease characterized by subnormal enzymatic activity of α-galactosidase and the resulting accumulation of GL-3. Agarsidase alpha (REPLAGAL®) is a human α-galactosidase A enzyme produced by human cell lines. Agarsider beta (FABRAZYME®) is a recombinant human α-galactosidase expressed in the CHO cell line. Lepragal is administered by intravenous infusion at a dose of 0.2 mg / kg every other week for the treatment of Fabry's disease and Gaucher's disease (off label). 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 the lysosomal enzyme described in US2012 / 0148556.

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

Imiglucerase (CEREZYME®) is a recombinant analog of human β-glucocerebrosidase. The initial dose ranges from 3 times a week at 2.5 U / kg body weight to once every 2 weeks at 60 U / kg. CEREZYME® is administered by IV infusion.

Abraxane, a paclitaxel-conjugated albumin, has been approved for metastatic breast cancer, non-small cell lung cancer, and end-stage pancreatic cancer.

Taliglucerase alpha (ELEYSO®) is a hydrolyzable lysosomal glucocerebroside-specific enzyme indicated for long-term enzyme replacement therapy for type I Gaucher's disease. The recommended dose is 60 U / kg body weight, administered by intravenous infusion once every two weeks.

ALDURAZYME® is a polymorphic variant of the human enzyme α-L-iduronidase produced by the CHO cell line. The recommended dose regimen for ALDURAZYME® is 0.58 mg / kg, given once weekly as an intravenous infusion.

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

Other biologics that can be formulated with reduced viscosity organic phosphate include asparaginase erwinia chrysanthemi (ERWINAZE®), Incobotulinum toxin A (XEOMIN®), EPOGEN® (Epoetin Alpha), PROCRIT® (Epoetin Alpha), ARANESP® (Darbepoetin Alpha), ORENCIA® (Abatacept), BATASERON® (Interferon Beta-1b) , NAGLAZYME®; ELAPRASE®; MYOZYME® (LUMIZYME®, Algucosidase Alpha); VPRIV® (Bellaglucerase), Abobotulinumtoxin A (DYSPORT®); BAX-326 from Baxter, Octocog Alpha; Thin Clear from GlaxoSmith Kline; Eli Liprotamase from Lilly; Xiaflex from Auxilium and BioSpecifics Technologies Corp. (collagenase clostridium histolyticum); Anakinra from Swedish Orphan Biovitrum AB; Metrereptin from Bristol-Myers Squibb; Avonex from Biogen (BIIB017); NN1841 from Novo Nordisk, NN7008; KRN321 (Dalbepoetin alpha) from Kyowa, AMG531 (Romiprostim), KRN125 (Pegfilgrastim), KW-0761 (Mogamurizumab); IB1001 from Inspiration Biopharmaceuticals; Canyon Pharmaceuticals Group Includes Iprivasc from.

Protein therapy under development

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

Vibriolysin is a proteolytic enzyme secreted by the Gram-negative marine microorganism, Vibrio proteolyticus. This endoprotease has a specific affinity for the hydrophobic region of the protein and can cleave the protein in the vicinity of the hydrophobic amino acid. Vibriolysin is currently being investigated by Biomarin for the cleansing and / or treatment of burns. The vibriolysin preparation is described in Japanese Patent WO 02/092014.

PEG-PAL (PEGylated recombinant phenylalanine ammonia-lyase or "PAL") is a treatment for phenylketonuria (PKU), a hereditary metabolic disease caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). Is an enzyme replacement therapy that is being tested for. PEG-PAL is under development as it may treat patients whose blood phenylalanine (Phe) levels are not adequately controlled by KUVAN®. PEG-PAL is currently in Phase II clinical development to treat patients who do not respond appropriately to KUVAN®.

Other protein therapeutics that can be formulated with reduced viscosity organic phosphates include Alprolix / rFIXFc, Eroctate / rFVIIIFc, BMN-190; BMN-250; Lamazyme; Galazyme; ZA-011; Severipase Alpha. Includes SBC-103; and HGT-1110. In addition, fusion proteins that include, but are not limited to, XTEN half-life extension techniques: VRS-317 GH-XTEN; Factor VIIa, Factor VIII, Factor IX; PF05280602, VRS- 859; exenatide-XTEN; AMX-256; GLP2-2G / XTEN; and AMX-179 forate-XTEN-DM1 can be formulated with a half-life organic phosphate.

Other late-stage protein therapeutics that can be formulated with reduced viscosity organic phosphates include CM-AT from CureMark LLC; NN7999, NN7088, Rilaglutide (NN8022), NN9211, Semaglutide (NN8022) from Novo Nordisk. NN9535); AMG386 from Amgen, Filgrastim; CSL-654 from CSL Behring, Factor VIII; LA-EP2006 (Pegfilgrastim biosimilar) from Novartis AG; Interroykin); LY2605541 from Eli Lilly, teriparatide (recombinant PTH1-34); NU-100 from Nuron Biotech, Inc.; Karaspargaze Pegol from Sigma-Tau Pharmaceuticals, Inc.; Polaris Pharmaceuticals, Inc. ADI-PEG-20 from BioMarin, BMN-110, BMN-702; Molmed from BioMarin NGR-TNF from SpA; Recombinant Human C1 Esterase Inhibitor from Pharmacing Group / Santarus Inc.; Somatropin Biosimilar from LG Life Sciences LTD; Natpara from NPS Pharmaceuticals, Inc.; ART123 from Asahi Kasei Co., Ltd. BAX-111 from Baxter; OBI-1 from Inspiration Biopharmaceuticals; Wilate from Octapharma AG; Talactoferrin alpha from Agennix AG; Desmoteprase from Lundbeck; Cinryze from Shire; Roche and Exelixis, RG7421 from Inc. (RG7421 and Roche and Exelixis, Inc.); Midostaurin from Novartis AG (PKC412); Damoctocog alfa pegol from Bayer AG, BAY86-6150, BAY94-9027; Peginterferon Lambda-1a from Bristol-Myers Squibb, Nulojix (Belatacept); Pergoveris from Merck KGaA, corifoliatropin alpha (MK-8962); recombinant coagulation factor IX Fc fusion protein (rFIXFc; BIIB029) and recombinant coagulation factor VIII Fc fusion protein (rFVIIIFc; BIIB031); and Myalept from AstraZeneca are included.

Another early stage protein biologics that can be formulated with reduced viscosity organic phosphate is Alferon LDO from Hemispherx BioPharma, Inc.; SL-401 from Stemline Therapeutics, Inc.; Protalix Biotherapeutics, PRX-102 from Inc.; KTP-001 from Kaseiken / Teijin Pharma Co., Ltd .; Vericiguat from Bayer AG; BMN-111 from BioMarin; ACC-001 from Janssen (PF-05236806); from Eli Lilly LY2510924, LY2944876; NN9924 from Novo Nordisk; INGAP peptide from Exsulin; ABT-122 from Abbvie; AZD9412 from AstraZeneca; NEUBLASTIN (BG00010) from Biogen; Celgene Luspatercept (ACE-536) from Corporation, Sotatercept (ACE-011); PRAME immunotherapeutic agent from GlaxoSmithKline; Plovamer acetate from Merck KGaA (PI-2301); from Shire Premiplex (607); BMN-701 from BioMarin; Ontak from Eisai; rHuPH20 / insulin from Halozyme, Inc.; PB-1023 from Phase Bio Pharmaceuticals, Inc.; ALV from Alvine Pharmaceuticals Inc. and Abbvie- 003; NN8717 from Novo Nordisk; Protein Therapeutics PRT-201 from Inc.; PEGPH20 from Halozyme, Inc.; Amevive® Alefacept from Astellas Pharma; F-627 from Regeneron; AGN-214868 (senrebotase) from Allergan, Inc.; BAX-817 from Baxter; PRT4445 from Portola Pharmaceuticals, Inc.; VEN100 from Ventria Bioscience; Onconase / Lampyrnase from Tamir Biotechnology Inc.; Interferon Alpha-2b Infusion from Medtronic, Inc; Severipase from Synageva BioPharma Alpha; IRX IRX-2 from Therapeutics, Inc; GSK2586881 from GlaxoSmithKline; SI-6603 from Seikagaku Corporation; ALXN1101 from Alexion, Asphotase Alpha; SHP611, SHP609 from Shire (Eraplase, Idursulfase) PF-04856884 from Pfizer, PF-05280602; ACE-031 from Acceleron Pharma, Dalantercept; ALT-801 from Altor BioScience Corp.; BA-210 from BioAxone Biosciences, Inc.; from GlaxoSmithKline WT1 immunotherapeutic agent; GZ402666 from Sanofi; MSB0010445 from Merck KGaA, Atacicept; Leukine (Sargramostim) from Bayer AG; KUR-211 from Baxter; Fibroblast growth factor from Cardio Vascular BioTherapeutics Inc.- 1; Hanmi SPI-2012 from Pharmaceuticals Co., LTD / Spectrum Pharmaceuticals; FGF-18 (Sprifermin) from Merck KGaA; MK-1293 from Merck; Interferon from Han All Biopharma-Alfa-2b; CYT107 from Cytheris SA; Revance RT001 from Therapeutics, Inc.; MEDI6012 from AztraZeneca; E2609 from Biogen; BMN-190 from BioMarin, BMN-270; ACE-661 from Acceleron Pharma; AMG876 from Amgen; GSK3052230 from GlaxoSmithKline; RG7813; SAR342434 from Sanofi, Lantus; AZ01 from Allozyne Inc.; ARX424 from Ambrx, Inc.; FP-1040, FP-1039; Merck from Five Prime Therapeutics, Inc. ATX-MS-1467 from KGaA; XTEN fusion protein from Amunix Operating Inc.; entry mod (entolimod) (CBLB502) from Cleveland BioLabs, Inc.; HGT2310 from Shire; Hanmi Pharmaceuticals Co., HM10760A from LTD; Alexion ALXN1102 / ALXN1103 from CSL Behring; CSL-689 from CSL Behring, CSL-627; Greer Growth Factor from Acorda Therapeutics, Inc. 2; NX001 from Nephrx Corporation; NN8640, NN1436, NN1953, NN9926, NN9927, from Novo Nordisk NN9928; NHS-IL12 from EMD Serono; 3K3A-APC from ZZ Biotech LLC; PB-1046 from Phase Bio Pharmaceuticals, Inc.; RU-101 from R-Tech Ueno Co., Ltd .; Insulin Lispro / BC106 from Adocia; Iconic Hl-con1 from Therapeutics, Inc.; PRT-105 from Protalix BioTherapeutics, Inc; PF-04856883 from Pfizer, CVX-096; ACP-501 from AlphaCore Pharma LLC; BAX-855 from Baxter; from Celldex Therapeutics CDX-1135; PRM-151 from Promedior, Inc.; TS01 from Thrombolytic Science International; TT-173 from Thrombolytic Science Corp.; QBI-139 from Quintessence Biosciences, Inc.; Baxterizumab from Glenmark Pharmaceuticals. , GBR500, GBR600, GBR830 and GBR900; and CYT-6091 from Cytimmune Sciences, Inc.

Other biological agents

Other biodrugs that can be formulated with reduced viscosity organic phosphates include PF-05285401, PF-05231023, RN317 (PF-05335810), PF-06263507, PF-05230907, Dekavil, PF- from Pfizer. 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) PD-0360324, PF-00547659; MK-8237 from Merck; BI033 from Biogen; GZ402665 from Sanofi, SAR438584 / REGN2222; IMC-18F1 from ImClone LLC, and Icrucumab, IMC-3G3; Novo Ryzodeg from Nordisk, Tresor, Xultophy; Toujeo (U300) from Sanofi, LixiLan, Lixisenatide; MAGE-A3 immunotherapeutic agent from GlaxoSmithKline; Tesemo from Merck KGaA Includes Celeraxin from AG (RLX030); Erythropoetin; Pegfilgrastim; LY2963016 from Eli Lilly, Duraglutide (LY2182965); and Insulin glargine from Boehringer Ingelheim.

B. Organic phosphate

The viscosity of liquid protein preparations containing low molecular weight and / or high molecular weight proteins is reduced by the addition of one or more organic phosphates. In some cases, this pharmaceutical product is converted from a non-Newtonian fluid to a Newtonian fluid by adding an effective amount of one or more organic phosphates. As used herein, an "organic phosphate" is a compound containing one or more phosphoryl groups, in which at least one of the phosphoryl groups is covalently linked to the organic group by a phosphate ester bond. , A compound. The organic phosphate can be a monoester of phosphoric acid or polyphosphoric acid. The organic phosphate can be a diester of phosphoric acid or polyphosphoric acid. The organic phosphate can be a salt or zwitterion. The term "zwitterion" is used herein to describe a chemical molecule that has positive and negative formal charges on different chemical groups in the molecule and is totally neutral.

The organic phosphate can be a salt or zwitterion. The term "zwitterion" is used herein to describe a chemical molecule that has positive and negative formal charges on different chemical groups in the molecule and is totally neutral.

When the organic phosphate is in the form of a salt, its counterion can be an alkali metal such as sodium, calcium, lithium, potassium or an alkaline earth metal. In other embodiments, the counterion is a continuous methylene group and / or methine group, benzene, naphthalene, Drosophila, adamantan, toluene, quinone, anthracene, phenanthrene, pyridine, pyrazine, piperazine, pyrrolidine, piperidine, imidazole, pyrazole, oxazole. , Thiophen, benzimidazole, or their substitution analogs, including nitrogen-containing compounds. Exemplary nitrogen-containing compounds include, but are not limited to, L-lysine, L-arginine, L-histidine, pentane-1,5-diamine and hexane-1,6-diamine, adamantylamine, 1- (3). Includes -aminopropyl) -2-methyl-1H-imidazole, aminomethylethylpyrrolidin, dimethylaminopropylpiperazine, aminoethylpiperazine, aminoethylpiperazine, and ethanolamine. For example, this organic phosphate can be a salt of thiamine pyrophosphate called TPP-APMI and 1- (3-aminopropyl) -2-methyl-1H-imidazole.

In general, any organic phosphate may reduce the viscosity of the protein formulation, but in some embodiments the viscosity-reducing organic phosphate is a nucleotide or nucleotide derivative or contains a nucleotide or nucleotide derivative. The viscosity-reducing organic phosphate can be nucleotide monophosphate, nucleotide diphosphate, nucleotide triphosphate, or derivatives thereof. The viscosity-reducing organic phosphate can be nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, or derivatives thereof. The viscosity-reducing organic phosphate can contain a nucleobase or a derivative thereof. In some embodiments, the reducing organic phosphate is a conjugate of a nucleobase and a phosphoryl group; a conjugate of a sugar and a phosphoryl group; or a conjugate of a nucleobase, a sugar and a phosphoryl group. The sugar can optionally be a 5-carbon sugar, a 6-carbon sugar or a 7-carbon sugar having one or more substituents. Nucleobases are purine, adenine, guanine, hypoxanthine, xanthine, 7-methylguanine, pyrimidine, thymine, cytosine, uracil, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, or derivatives thereof. Can be. Nucleosides can be adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, or derivatives thereof. The nucleotide can be a monophosphate, diphosphate, or triphosphate of any of the above nucleosides.

The viscosity-reducing organic phosphate can have a structure according to formula I, where X is phosphate, preferably diphosphate or triphosphate, and Y is absent or sugar, preferably ribose, deoxyribose, Or derivatives thereof, where Z is a nucleobase, preferably one of the above or a derivative thereof.<chemistry num="1"><img file="JP6564512B2_D0001.tif" /></chemistry>

This viscosity-reducing organic phosphate can have a structure according to Formula II, where n is an integer of 1-20, 1-10, 2-10 or 2-6, R.¹Is an organic group having 3 to 50 carbon atoms, 5 to 30 carbon atoms or 7 to 20 carbon atoms, preferably R.¹Is a nucleobase, a nucleoside, or a derivative thereof, R²Each appearance of is absent, hydrogen, monovalent cationic group, and 1 to 50 carbon atoms, 1 to 30 carbon atoms, 3 to 30 carbon atoms or 7 to 20 carbon atoms. It is independently selected from the group consisting of organic groups having. Monovalent cationic groups include potassium, sodium, lithium, ammonium and alkylammonium groups. R¹And R²Is R²Whenever is an organic group, it should be a substituted or unsubstituted carbon ring or heterocycle having 3 to 50 carbon atoms, 5 to 30 carbon atoms or 7 to 20 carbon atoms. Can be done. R¹Can be a nucleoside, such as one of those listed above, or a derivative thereof.<chemistry num="2"><img file="JP6564512B2_D0002.tif" /></chemistry>

This viscosity-reducing organic phosphate can have a structure according to formula III, in which n is an integer of 1 to 20, 1 to 10, 2 to 10 or 2 to 6, and R.³Is a monosaccharide or disaccharide that is absent or has a sugar, preferably 1 to 30 carbon atoms, 1 to 20 carbon atoms or 4 to 20 carbon atoms, R.⁴Is substituted, having a bulky cyclic group, preferably 3-50 carbon atoms, 5-30 carbon atoms or 7-20 carbon atoms, which can be substituted or unsubstituted. Or unsubstituted carbocycle or heterocycle, R⁵Each appearance of is absent, hydrogen, monovalent cationic group, and 1 to 50 carbon atoms, 1 to 30 carbon atoms, 3 to 30 carbon atoms or 7 to 20 carbon atoms. It is independently selected from the group consisting of organic groups having. R³Contains one or more substituents, optionally deoxyribose, fructose, galactose, gentiobiose, gentiobiose, glucose, kestose, isomaltose, isomalttriose, kojibiose, laminaribiose, maltose, With maltose, maltotriose, maltotriose, mannobiose, mannose, melibiose, melibiose, nigerose, nigerotriose, raffinose, ribose, rutinose, rutinulose, sofolus, trehalose, β, β-trehalose, α, β-trehalose or turanose can do. In certain embodiments, the substituent R³And R⁵May form a ring together, for example, as found in cyclic adenosine monophosphate.

R⁴Can be a nitrogen-containing heterocycle. Nitrogen-containing heterocycles can be saturated or unsaturated. Nitrogen-containing heterocycles include substituted and unsubstituted pyrrolidine, pyrrol, imidazolidine, pyrrolidine, imidazole, pyrazole, oxazolidine, isooxazolidine, oxazolidine, isoxazolidine, piperidine, tetrahydropyridine, dihydropyridine, pyridine, pyrimidine, piperazine, They can include polycyclic and fused ring structures, and derivatives thereof. R⁴Can be a bulky cyclic group. Suitable bulky cyclic groups are cyclopentane, cyclopentene, cyclopentadiene, pyrrolidine, pyrrol, imidazolidine, pyrazolidine, imidazole, pyrazole, oxazolidine, isooxazolidine, oxazole, isooxazole, tetrahydrofuran, furan, dioxolan, thiolane, thiophene, dithiolane. , Thiazol, isothiazole, tetrahydrofuran, silol, triazole, oxazolidine and the like, and can include 5-membered carbocyclce and heterocycles, as well as derivatives thereof. Suitable bulky cyclic groups are cyclohexane, cyclohexene, cyclohexa-1,3-diene, cyclohexa-1,4-diene, benzene, piperidine, tetrahydropyridine, dihydropyridine, pyridine, oxane, pyran, piperazine, pyrazine, pyrimidine, pyridazine. , Morpholine, 1,3,5-triazine and other 6-membered carbocycles and heterocycles, as well as derivatives thereof. Suitable bulky cyclic groups can include 7-membered carbocycles and heterocycles such as cycloheptane, cycloheptene, azepane, azepine, thiepine, diazepine, thiazepine, and derivatives thereof. Suitable bulky cyclic groups include polycyclic and fused ring structures of any of the above carbocycles and heterocycles such as naphthalene, anthracene, tetracene, acrydin, dibenzothiophene, carbazole, dibenzofuran, decalin and the like. Cyclic compounds, bridging carbocycles and heterocycles such as norbornan, adamantan, and spirocyclic compounds such as spiro [2.2] pentane can be included.<chemistry num="3"><img file="JP6564512B2_D0003.tif" /></chemistry>

The viscosity-reducing organic phosphate can be dinucleotide phosphoric acid. This viscosity-reducing organic phosphate can have a structure according to Formula IV, where n is an integer of 1-20, 1-10, 2-10 or 2-6, R.⁶Each appearance of is selected independently of monosaccharides or disaccharides that are absent or have sugars, preferably 1-30 carbon atoms, 1-20 carbon atoms or 4-20 carbon atoms. , R⁷Each appearance of a bulky cyclic group, which can be independently substituted or unsubstituted, preferably 3 to 50 carbon atoms, 5 to 30 carbon atoms or 7 to 20 carbon atoms. A substituted or unsubstituted carbocycle or heterocycle having R⁸Each appearance of is absent, hydrogen, monovalent cationic group, and 1 to 50 carbon atoms, 1 to 30 carbon atoms, 3 to 30 carbon atoms or 7 to 20 carbon atoms. It is independently selected from the group consisting of organic groups having. R⁶Each independently contains one or more substituents, deoxyribose, fructose, galactose, gentiobiose, gentiobiose, glucose, kestose, isomaltose, isomalttriose, kojibiose, Laminaribiose, maltos, maltorose, maltotriose, maltotriose, mannobiose, mannose, melivioce, meribiurose, nigerose, nigerotriose, raffinose, ribose, lucinose, rutinulose, sofolus, trehalose, β, β-trehalose, α, It can be β-trehalose or turanose. R⁷Can independently be nitrogen-containing heterocycles. Nitrogen-containing heterocycles can be saturated or unsaturated. Nitrogen-containing heterocycles include substituted and unsubstituted pyrrolidine, pyrrol, imidazolidine, pyrrolidine, imidazole, pyrazole, oxazolidine, isooxazolidine, oxazolidine, isoxazolidine, piperidine, tetrahydropyridine, dihydropyridine, pyridine, pyrimidine, piperazine, They can include polycyclic and fused ring structures, and derivatives thereof. R⁷Can each independently be a bulky cyclic group. Suitable bulky cyclic groups are cyclopentane, cyclopentene, cyclopentadiene, pyrrolidine, pyrrol, imidazolidine, pyrazolidine, imidazole, pyrazole, oxazolidine, isooxazolidine, oxazole, isooxazole, tetrahydrofuran, furan, dioxolan, thiolane, thiophene, dithiolane. , Thiazol, isothiazole, tetrahydrofuran, silol, triazole, oxazolidine and the like, and can include 5-membered carbocyclce and heterocycles, as well as derivatives thereof. Suitable bulky cyclic groups are cyclohexane, cyclohexene, cyclohexa-1,3-diene, cyclohexa-1,4-diene, benzene, piperidine, tetrahydropyridine, dihydropyridine, pyridine, oxane, pyran, piperazine, pyrazine, pyrimidine, pyridazine. , Morpholine, 1,3,5-triazine and other 6-membered carbocycles and heterocycles, as well as derivatives thereof. Suitable bulky cyclic groups can include 7-membered carbocycles and heterocycles such as cycloheptane, cycloheptene, azepane, azepine, thiepine, diazepine, thiazepine, and derivatives thereof. Suitable bulky cyclic groups include polycyclic and fused ring structures of any of the above carbocycles and heterocycles such as naphthalene, anthracene, tetracene, acrydin, dibenzothiophene, carbazole, dibenzofuran, decalin and the like. Cyclic compounds, bridging carbocycles and heterocycles such as norbornan, adamantan, and spirocyclic compounds such as spiro [2.2] pentane can be included.<chemistry num="4"><img file="JP6564512B2_D0004.tif" /></chemistry>

The viscosity-reducing organic phosphate can be thiamine pyrophosphate (TPP), or a derivative thereof, whose structure is shown below as a chloride salt. Derivatives of TPPs replace diphosphates with different phosphates, such as triphosphates, from monophosphates, replace chlorine anions with other anionic constructs, and substitute one or more methyl substituents (substitutents) at higher levels. Substituents with alkyl or higher N-alkyl substituents, substituted or unsubstituted alkyl, aminoalkyl, heterocyclyl, aryl having 1 to 30 carbon atoms in one or more amino substituents Alternatively, it can be replaced with a heteroaryl group, one or more hydroxyl groups can be replaced with an O-acyl or O-alkyl group, or a combination thereof can be included. Suitable anionic constructs are preferably by one or more alkyl, heteroalkyl, alkenyl, alkynyl, carbocyclic or heterocyclic groups having 1 to 20 or 1 to 12 carbon atoms. Multiple halide ions, sulfate anions, sulfonic acid anions, sulfite anions, sulfinate anions, phosphate anions, phosphonic acid anions, phosphite anions, phosphite, optionally substituted Includes anions, carbonate anions and carboxylate anions. An exemplary anionic construct (anionic Constituent) includes chlorine ion, bromine anion, methylphosphate anion, methyl-ethyl-phosphate anion, methylsulfate anion, methylsulfonic acid anion, formate anion, acetate anion, butyric acid anion, and quen. Acid anions and lactate anions, and camphor sulfonic acid (CSA), benzene sulfonic acid (BSA), toluene sulfonic acid (TSA), 1- (3-aminopropyl) -2-methyl-1H-imidazole (APMI), Or it contains bulky hydrophobic anions such as methanesulfonic acid (MSA). Derivatives can include common inorganic bases such as NaOH or base addition salts of TPP using the above exemplary hydrophobic bases.<chemistry num="5"><img file="JP6564512B2_D0005.tif" /></chemistry>

In other embodiments, the present viscosity reducing organic phosphate may be benfotiamine or its corresponding diphosphate or triphosphate analog. The viscosity-reducing organic phosphate may be fursultiamine monophosphate, prosultiamine monophosphate or allithiamine monophosphate, and the corresponding diphosphate or triphosphate of any of the above.

The viscosity-reducing organic phosphate can be adenosine triphosphate (ATP), or a derivative thereof, whose structure is shown below as a sodium salt. Derivatives of ATP replace triphosphates with different phosphates such as monophosphates or diphosphates, and the amino substituents are substituted or unsubstituted alkyls, aminoalkyls, aryls, having 1-30 carbon atoms. It can include replacement with a heterocyclyl or heteroaryl group, replacement of one or more hydroxyl groups with an O-acyl or O-alkyl group, or a combination thereof.<chemistry num="6"><img file="JP6564512B2_D0006.tif" /></chemistry>

The viscosity-reducing organic phosphate can be deoxyadenosine triphosphate (dATP), whose structure is shown below, or a derivative thereof. Derivatives of dATP have triphosphates replaced with different phosphates such as monophosphates or diphosphates, amino substituents having 1 to 30 carbon atoms, substituted or unsubstituted alkyls, aminoalkyls, aryls, It can include replacement with a heterocyclyl or heteroaryl group, replacement of one or more hydroxyl groups with an O-acyl or O-alkyl group, or a combination thereof.<chemistry num="7"><img file="JP6564512B2_D0007.tif" /></chemistry>

The viscosity-reducing organic phosphate can be deoxyguanosine triphosphate (dGTP), whose structure is shown below, or a derivative thereof. Derivatives of dGTP replace triphosphates with different phosphates such as monophosphates or diphosphates, substituted or unsubstituted alkyls with 1-30 carbon atoms as amino substituents, aminoalkyls, heterocyclyls, It can be replaced with an aryl or heteroaryl group, one or more hydroxyl groups can be replaced with an O-acyl or O-alkyl group, or a combination thereof.<chemistry num="8"><img file="JP6564512B2_D0008.tif" /></chemistry>

The viscosity-reducing organic phosphate can be deoxythymidine triphosphate (dTTP), whose structure is shown below, or a derivative thereof. Derivatives of dTTP include substituting triphosphates with different phosphates such as monophosphates or diphosphates, substituting methyl substituents with higher-order alkyl or higher N-alkyl substituents, one or more amino substituents. Is replaced with a substituted or unsubstituted alkyl, aminoalkyl, heterocyclyl, aryl or heteroaryl group having 1 to 30 carbon atoms, and one or more hydroxyl groups are replaced with O-acyl or O-alkyl. It can be replaced with a group, or a combination thereof can be included.<chemistry num="9"><img file="JP6564512B2_D0009.tif" /></chemistry>

The viscosity-reducing organic phosphate can be deoxycytidine triphosphate (dCTP), whose structure is shown below, or a derivative thereof. Derivatives of dCTP have triphosphates replaced with different phosphates such as monophosphates or diphosphates, amino substituents having 1 to 30 carbon atoms, substituted or unsubstituted alkyls, aminoalkyls, aryls, It can include replacement with a heterocyclyl or heteroaryl group, replacement of one or more hydroxyl groups with an O-acyl or O-alkyl group, or a combination thereof.<chemistry num="10"><img file="JP6564512B2_D0010.tif" /></chemistry>

The viscosity-reducing organic phosphate can be cyclic adenosine monophosphate (cAMP), the structure of which is shown below, or a derivative thereof. Derivatives of cAMP are monophosphates replaced with different phosphates such as diphosphates or triphosphates, amino substituents having 1 to 30 carbon atoms, substituted or unsubstituted alkyls, aminoalkyls, aryls, It can include replacement with a heterocyclyl or heteroaryl group, replacement of a hydroxyl group with an O-acyl or O-alkyl group, or a combination thereof.<chemistry num="11"><img file="JP6564512B2_D0011.tif" /></chemistry>

The viscosity-reducing organic phosphate can be cyclic guanosine monophosphate (cGMP), the structure of which is shown below, or a derivative thereof. Derivatives of cGMP include substituting monophosphonates with different phosphates such as diphosphates or triphosphates, substituted or unsubstituted alkyls with 1-30 carbon atoms as amino substituents, aminoalkyls, aryls, It can include replacement with a heterocyclyl or heteroaryl group, replacement of one or more hydroxyl groups with an O-acyl or O-alkyl group, or a combination thereof.<chemistry num="12"><img file="JP6564512B2_D0012.tif" /></chemistry>

The viscosity-reducing organic phosphate can be cyclic thymidin monophosphate (cTMP), the structure of which is shown below, or a derivative thereof. Derivatives of cTMP are monophosphonates replaced with different phosphates such as diphosphates or triphosphates, methyl substituents replaced with higher alkyl or higher N-alkyl substituents, one or more amino substituents. Is replaced with a substituted or unsubstituted alkyl, aminoalkyl, aryl, heterocyclyl or heteroaryl group having 1 to 30 carbon atoms, and one or more hydroxyl groups are replaced with O-acyl or O-alkyl. It can be replaced by a group, or a combination thereof can be included.<chemistry num="13"><img file="JP6564512B2_D0013.tif" /></chemistry>

The viscosity-reducing organic phosphate can be cyclic cytidine monophosphate (cCMP), the structure of which is shown below, or a derivative thereof. Derivatives of cCMP include substituting monophosphonates with different phosphates such as diphosphates or triphosphates, substituted or unsubstituted alkyls with 1-30 carbon atoms as amino substituents, aminoalkyls, aryls, It can include replacement with a heterocyclyl or heteroaryl group, replacement of one or more hydroxyl groups with an O-acyl or O-alkyl group, or a combination thereof.<chemistry num="14"><img file="JP6564512B2_D0014.tif" /></chemistry>

The viscosity-reducing organic phosphate can be nicotinamide adenine dinucleotide phosphate (NADP), whose structure is shown below as a sodium salt, or a derivative thereof. Derivatives of NADP replace diphosphate with a different phosphate such as monophosphate or triphosphate, replace diphosphate with a different phosphate, or replace one or more amino substituents with 1-30 carbon atoms. Substituting with or not substituted alkyl, aminoalkyl, heterocyclyl, aryl or heteroaryl groups, substituting one or more hydroxyl groups with O-acyls or O-alkyl groups, or including combinations thereof. Can be done.<chemistry num="15"><img file="JP6564512B2_D0015.tif" /></chemistry>

The viscosity-reducing organic phosphate can be pyridoxal phosphate, or a derivative thereof, whose structure is shown below. Derivatives of pyridoxal phosphate replace monophosphates with different phosphates such as diphosphates or triphosphates, replace methyl substituents with higher-order alkyl or higher N-alkyl substituents, one or more hydroxyl groups. The group can be replaced with an O-acyl or O-alkyl group, or a combination thereof can be included.<chemistry num="16"><img file="JP6564512B2_D0016.tif" /></chemistry>

The viscosity-reducing organic phosphate can be riboflavin-5'-phosphoric acid, the structure of which is shown below, or a derivative thereof. Derivatives of riboflavin-5'-phosphate replace the phosphate with a different phosphate such as diphosphate or triphosphate, replace the sodium pair ion with another cationic construct, replace one or more methyl substituents. Substituents with higher-order alkyl or higher N-alkyl substituents, one or more hydroxyl groups with O-acyls or O-alkyl groups, or combinations thereof can be included.<chemistry num="17"><img file="JP6564512B2_D0017.tif" /></chemistry>

C. Excipients

A wide range of pharmaceutical excipients useful for liquid protein formulations are known to those of skill in the art. They are one or more additives such as liquid solvents or co-solvents; sugars or sugar alcohols such as mannitol, trehalose, sucrose, sorbitol, fructose, maltose, lactose or dextran; TWEEN® 20, 60 or 80. Surfactants such as (polysolvate 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 citrate; or biodegradable polymers such as water-soluble polyester; cryoprotectant; cryoprotectant; bulking agent ; And contains stabilizers.

Remington: Other pharmaceutically acceptable carriers, forms, such as those described in The Science and Practice of Pharmacy, 20th Edition, Alfonso R. Gennaro, ed., Lippincott Williams &amp; Wilkins (2000). Agents or stabilizers may also be included in the protein preparations described herein, provided that they do not adversely affect the desired characteristics of the preparation.

The present formulation may contain one or more additional viscosity-reducing excipients in addition to the viscosity-reducing organic phosphate described above. The organic phosphate reducing agents described herein are jointly filed by one or more other types of viscosity reducing agents, such as Asia Therapeutics, under the title LIQUID PROTEIN FORMULATIONS CONTAINING WATER SOLUBLE ORGANIC DYES. Water-soluble organic dyes described in PCT applications; hydrophobic compounds of LIQUID PROTEIN FORMULATIONS CONTAINING VISCOSITY-LOWERING AGENTS by Arsia Therapeutics, numerous GRAS (generally considered safe, compounds of the US Food and Drug Administration) LIQUID PROTEIN FORMULATIONS by Asia Therapeutics, as well as normally bulky polar organic compounds, such as (list), and injectable inactive ingredients, and FDA-approved therapeutic agents. It can be combined with the ionic liquids described in the PCT application jointly filed under the title CONTAINING IONIC LIQUIDS.

III. Manufacturing method

A. Protein preparation

The protein to be formulated, such as mAb, is obtained by culturing transformed or transfected cells using a vector containing one or more nucleic acid sequences encoding the protein, which is well known in the art. Alternatively, it can be produced by any known technique, such as by synthetic techniques (such as recombinant and peptide synthesis, or a combination of these techniques), or can be isolated from an endogenous source of protein.

Purification of the formulated proteins is for, for example, precipitation with ethanol or ammonium sulfate, reverse phase HPLC, chromatography of silica or cation exchange resins (eg DEAE-cellulose), dialysis, chromatofocusing, and removal of impurities. Gel filtration using a Protein A SEPHAROSE® column (eg, SEPHADEX® G-75), a metal chelate forming column for binding to an epitope-tagged morphology, and ultrafiltration / diafiltration ( Non-limiting examples include centrifugal filtration and tangential flow filtration (TFF)), which can be done by any suitable technique known in the art.

A solution of a pharmaceutically active mAb that contains a viscosity-reducing organic phosphate at a viscosity-reducing concentration, such as 0.010M to 1.0M, preferably 0.050M to 0.50M, most preferably 0.10M to 0.30M. It will be possible to purify and / or concentrate to higher concentrations of mAbs using common methods known to those of skill in the art, including but not limited to tangential flow filtration, centrifugal concentration and dialysis.

In some embodiments, lyophilized proteins are provided and / or used in the preparation and production of low viscosity concentrated protein formulations. In some embodiments, the pre-lyophilized protein in powder form is reconstituted by dissolving it in aqueous solution. In this embodiment, the liquid formulation is filled in a container of a specific dosage unit, such as a vial or a pre-filled mixing syringe, and optionally a lyophilization protectant, preservative, antioxidant. And lyophilized with other conventional pharmaceutically acceptable excipients, then stored under sterile storage conditions until just before use, at the time of use, with a defined volume of diluent. Reconstituted, the liquid is made to the desired concentration and viscosity.

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

Non-limiting examples of diluents useful for reconstitution of lyophilized formulations prior to injection include sterile water, bacteriostatic water for injection (BWFI), pH buffer solution (eg, phosphate buffer saline), sterilization. Includes saline solution, Ringer's solution, dextrose solution, or aqueous solution of salt and / or buffer. In some cases, the product is spray dried and then stored.

IV. Administration to individuals who need it

Protein preparations, including but not limited to, use 18-32 gauge needles (optionally thin-walled needles) for humans in need of them, less than about 5 mL, about 3 mL. It is administered by intramuscular injection, intraperitoneal (ie, intracoelomic) injection, intracranial or subcutaneous injection in a volume of less than, preferably less than about 2 mL, more preferably less than about 1 mL.

The appropriate dose of protein, such as mAb (the "therapeutic effective dose"), is independent of the condition being treated, the severity and course of the condition (whether the protein is administered for prophylactic or therapeutic purposes). ), Previous treatment, patient history and response to protein, type of protein used, and judgment of the attending physician. The protein is preferably administered as a single treatment or in combination with another drug or treatment, in a single injection, in a single or multiple injections, or over a series of treatments.

The dosing formulation is designed so that the injection does not cause significant signs of irritation at the injection site, eg, in this case, the primary irritation index is less than 3 when evaluated using the Draize scoring system. In an alternative embodiment, the injection causes macroscopically similar levels of irritation when compared to an injection of equal volume saline. In another embodiment, the bioavailability of the protein is higher when compared to an organic phosphate-free but otherwise identical formulation administered in the same manner.

In a preferred embodiment, the product is injected to produce high levels of therapeutic protein. For example, the AUC value is at least 10%, preferably at least 20% higher than the AUC value calculated for the same formulation, which does not contain the reduced viscosity organic phosphate (s) but otherwise calculated in the same manner. obtain.

Viscosity-reducing organic phosphates can also affect bioavailability. For example, the bioavailability of a protein is at least 1.1 times, preferably at least 1.2 times, the bioavailability of the same formulation, which does not contain the reduced viscosity organic phosphate (s) administered in the same manner, but otherwise the same. Can be.

Reduced viscosity organic phosphates can also affect pharmacokinetics. For example, C after SC or IM injection_MAXIs a pharmaceutically effective dose of C, administered intravenously in approximately equal doses._MAXAt least 10% less, preferably at least 20% less than.

In some embodiments, the protein does not contain the reduced viscosity organic phosphate (s), but otherwise it is administered at a higher dose and less frequently than the same formulation.

Lower viscosity formulations require less firing power. For example, the shot output is at least 10%, preferably at least 20% lower than the shot power for the same formulation, which does not contain the reduced viscosity organic phosphate (s) administered in the same manner but otherwise the same. In one embodiment, the injection is administered using a 27 gauge needle and the firing power is less than 30 N. The formulation can be administered using a very small gauge needle, for example, between gauges 27-31, usually gauges 27, 29 or 31 in most cases.

This viscosity-reducing organic phosphate can be used to prepare a reconstitution-friendly, dose-unit-unit formulation for making liquid pharmaceutical formulations for subcutaneous or intramuscular injection. This dosage unit can contain a dry powder of one or more proteins, one or more deviscosified organic phosphates, and other excipients. This protein is reconstituted in a pharmaceutically acceptable solvent so that the resulting formulation has a protein concentration of about 100 mg to about 2,000 mg (mg / mL) per mL in the dosage unit. Exists in. Such reconstituted formulations can have an absolute viscosity of about 1 cP to about 50 cP at 25 ° C.

The low-viscosity formulation can be provided as a solution or in the form of a dose unit, in which case the protein is in one vial with a reduced viscosity organic phosphate and other excipients or The solvent, which is lyophilized without being combined and contains or does not contain the reduced viscosity organic phosphate and other excipients, is provided in the second vial. In this embodiment, the solvent is added to the protein immediately before or at the time of injection to ensure uniform mixing and dissolution.

The reduced viscosity organic phosphate (s) is present in the formulation at a concentration that does not cause significant and / or irreversible signs of toxicity when administered subcutaneously, intramuscularly or by other types of injection. To do. As used herein, "significant signs of toxicity" include damage to the central nervous system, infertility, arrhythmias, cardiomyopathy, myocardial infarction and heart failure or congestive heart failure, renal failure, hepatic failure, dyspnea and death. Includes addiction, dyspnea, and behavioral modifications, such as those that occur with signs of severe cardiotoxicity.

In a preferred embodiment, the product does not cause significant irritation when administered twice daily, once daily, twice weekly, once weekly, or less than once monthly. The protein preparation can be administered without causing significant signs of irritation at the injection site, eg, less than 3, less than 2, or less than 1 primary irritation index when evaluated using the Draize scoring system. As used herein, "significant signs of irritation" include erythema, redness and / or swelling, at the site of injection, at the site of injection with a diameter greater than 10 cm, greater than 5 cm, or greater than 2.5 cm. Includes necrosis, 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 preparation causes macroscopically similar levels of irritation when compared to injection of equal volume saline.

When this protein preparation is administered by subcutaneous injection or intramuscular injection, it may show an increase in bioavailability as compared with the same protein preparation, although it does not contain the viscosity-reducing organic phosphate (s). "Bioavailability" refers to the extent and rate at which a bioactive species (eg, mAb) reaches the circulation or site of action. For SC or IM injections, the overall bioavailability does not contain the reduced viscosity organic phosphate (s), but others can be increased compared to the same formulation. "Bioavailability" refers to the percentage of administered dose of bioactive species that enters the circulation as determined with respect to the intravenously administered dose. One way to measure bioavailability is to compare the "area under the curve" (AUC) in a plot of plasma concentration as a function of time. AUC can be calculated using, for example, the linear trapezoidal rule. "AUC_0-tAs used herein, refers to the area under the plasma concentration curve from time 0 to after time t. Time is usually measured in days, but as the context makes clear, time can also be used. "AUC_∞As used herein, refers to the area under the plasma concentration curve from time 0 to the time the plasma concentration returns to baseline levels. One way to measure bioavailability is by comparing the "area under the curve" (AUC) in a plot of plasma concentration as a function of time. AUC can be calculated using, for example, the linear trapezoidal rule. "AUC_∞As used herein, refers to the area under the plasma concentration curve from time 0 to the time the plasma concentration returns to baseline levels. "AUC_0-tAs used herein, refers to the area under the plasma concentration curve from time 0 to time t, for example, the time to reach baseline. Time is usually measured in days, but as the context makes clear, time can also be used. For example, AUC can be more than 10%, 20%, 30%, 40% or 50% larger than the same formulation, which does not contain the reduced viscosity organic phosphate (s) administered in the same manner but otherwise the same. ..

As used herein, "t"_max"" Refers to the time when the plasma concentration reaches the maximum value after administration.

As used herein, "C_max"" Refers to the maximum plasma concentration after administration of the dose and before administration of subsequent doses.

As used herein, "C_minOr "C_trough"" Refers to the minimum plasma concentration after administration of a dose and before administration of a subsequent dose.

C after SC or IM injection_maxIs the dose of intravenously administered C_maxFor example, it can be at least 10%, more preferably at least 20% smaller than. This C_maxA reduction in toxicity can result in a reduction in toxicity.

Pharmacodynamic and pharmacodynamic parameters can be estimated species-wide using techniques known to those of skill in the art.

The pharmacokinetics and pharmacodynamics of antibody therapeutics can vary considerably based on the specific antibody. Approved mouse mAbs have been shown to have a half-life of about 1 day in humans, while human mAbs typically have a half-life of about 25 days (Waldmann et al., Int. Immunol., 2001). , Vol. 13, pp. 1551 ~ 1559). The pharmacokinetics and pharmacodynamics of antibody therapeutics can vary considerably based on the route of administration. The time to reach maximum plasma levels after IM or SC injection of IgG is usually in the range of 2-8 days, but can be shorter or longer (Wang et al., Clin. Pharm). . Ther., 2008, Vol. 84 (No. 5): pp. 548-558). The pharmacokinetics and pharmacodynamics of antibody therapeutics can vary considerably depending on the formulation. A protein preparation containing an organic phosphate can show an improvement in pharmacokinetics in the case of SC or IM injection as compared with the same preparation, which does not contain the organic phosphate (s). C after SC or IM administration_maxIs intravenously administered and does not contain the viscosity-reducing organic phosphate (s), but can be reduced when compared to the same other. For example, C_MAXC, which is intravenously administered and does not contain the viscosity-reducing organic phosphate, but is otherwise the same._MAXIt can be reduced to more than 1 day, 2 days, 3 days or 4 days, or to more than 10%, 20%, 30%, 40% or 50%. Overall bioavailability can be increased with SC or IM injections when compared to the same formulation, which does not contain the reducing organic phosphate (s). Overall bioavailability can be assessed, for example, by comparing one or more AUC values for intravenously administered doses, or by comparing calculated bioavailability. .. For example, AUC may increase by more than 10%, 20%, 30%, 40% or 50% when compared to the same formulation, which does not contain the intravenously administered hypoviscosity organic phosphate (s). ..

This low-viscosity protein preparation makes it possible to increase the adaptability of administration and reduce the frequency of administration as compared with such a protein preparation that does not contain the reduced viscosity organic phosphate (s). In some embodiments, the frequency of administration may be reduced, for example, from once every two weeks to once every six weeks by multiplying the dose administered per injection. it can.

Protein formulations, including, but not limited to, reconstituted formulations can be administered using heated and / or self-mixing syringes or autoinjectors. The protein preparation can also be preheated in a separate heating unit prior to filling the syringe.

i. Heating syringe

The heated syringe can be a standard syringe that has been preheated using a syringe warmer. This syringe warmer has one or more openings, each of which can accept a syringe containing a protein formulation, and to warm and maintain the syringe at a specific (usually above ambient temperature) temperature prior to use. Generally has the means of. 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 warmer can accommodate syringes of various sizes and can be heated 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, which is maintained at the desired temperature.

The heating syringe can be a self-heating syringe, i.e., the liquid formulation inside the syringe can be heated and maintained at a particular temperature. The self-heating syringe can also be a standard medical syringe to which a heating device is attached. Suitable heating devices that can be attached to a syringe include a syringe-heater or syringe heater tape available from St. Louis, MO Watlow Electric Manufacturing Co., and Warner Instruments from Hamden, CT. Includes syringe heater blocks, stage heaters and in-line perfusion heaters, such as the SW-61 model syringe warmer. The heater can be controlled by a central controller, eg, a TC-324B or TC-344B model heater controller available from Warner Instruments.

The heated syringe maintains the liquid protein formulation at the specified temperature, or within 1 ° C, 2 ° C, or 5 ° C of the specified temperature. A heated syringe can be used to move the protein product from room temperature up to about 80 ° C, up to about 60 ° C, up to about 50 ° C, or up to about 45 °, as long as the protein preparation is sufficiently stable at that temperature. It can be maintained at any temperature up to C. The heated syringe allows the protein preparation to be heated between 20 ° C and 60 ° C, between 21 ° C and 45 ° C, between 22 ° C and 40 ° C, or between 25 ° C and 37 ° C. Can be maintained at. Maintaining the protein preparation at a high temperature during injection reduces the viscosity of the liquid preparation and increases the solubility of the protein in the preparation, or both.

ii. Self-mixing syringe

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

iii. Auto-injector and prefilled syringe of protein preparation

The liquid protein preparation can be administered using an autoinjector of a prefilled syringe or a needleless injection device. The autoinjector is a portable, often pen-like cartridge holder for holding a replaceable prefilled cartridge, and a spring for injecting a dose of liquid drug subcutaneously or intramuscularly from the prefilled cartridge. Includes or similar mechanisms based on. Autoinjectors are usually designed to be self-administered or administered by untrained personnel. Auto-injectors are available for administering either single or multiple doses from prefilled cartridges. The auto-injector can be set to various user settings, including, among other things, injection depth, injection rate, and the like. Other injection systems may include those described in US Pat. No. 8,500,681.

The lyophilized protein preparation can be provided in a prefilled syringe or a unit dose syringe. U.S. Pat. Nos. 3,682,174, 4,171,698 and 5,569,193 describe sterile syringes containing two chambers that can be prefilled with dry formulations and liquids that can be mixed immediately prior to injection. doing. U.S. Pat. No. 5,779,668 describes a syringe system for lyophilization, reconstruction and administration of pharmaceutical compositions. In some embodiments, the protein preparation is reconstituted in a syringe prior to administration and is administered in lyophilized form in a prefilled syringe or unit dose syringe, which is administered as a single subcutaneous or intramuscular injection. Supplied. Autoinjectors for delivering unit doses of lyophilized drugs are described in WO2012 / 010,832. Safe Click Lyo (Future Injection) Auto-injectors such as (marketed by Technologies, Ltd., Oxford, UK) can be used to administer a unit dose of a protein formulation, in which case the formulation is stored in lyophilized form. , Reconstituted just before administration. In some embodiments, the protein preparation is provided in a unit dose cartridge for lyophilized drugs (sometimes referred to as a Vetter cartridge). Examples of suitable cartridges may include those described in US Pat. Nos. 5,334,162 and 5,454,786.

V. Purification and concentration method

The reduced viscosity organic phosphate can also be used to assist in the purification and concentration of proteins. The present viscosity-reducing organic phosphate (s) and excipients are added to the protein in an effective amount of the viscosity-reducing organic phosphate that reduces the viscosity of the protein solution. For example, the reduced viscosity organic phosphate is added to a concentration between about 0.01M and about 1M, preferably between about 0.01M and about 0.5M, and most preferably between about 0.01M and about 0.25M.

The organic phosphate solution is then purified or concentrated using a method selected from the group consisting of ultrafiltration / diafiltration, tangential flow filtration, centrifugal concentration, and dialysis.

<p> The above is further understood by the following non-limiting examples.</p><p> All viscosities of well-mixed mAb aqueous solutions are after equilibration at 25 ° C for 5 minutes (unless otherwise indicated) with the mVROC Microfluidic Viscometer (RheoSense) or DV2T Conical Plate Viscometer (Brookfield; "C &amp; P"). Measured using any of. 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 is back-loaded into an airtight microlab instrument syringe (Hamilton; 100 μL) attached to the tip above, with multiple flow rates, or about 20% of the maximum pressure on each tip. , 40% and 60%. For example, a sample of about 50 cP will have about 10, 20 and 30 μL / min (on chip "A", about 180, 350 and 530 s, respectively) until the viscosity stabilizes (usually after at least 30 seconds).^-1) Will be measured. The average absolute viscosity and standard deviation were then calculated from at least these three measurements. A CPE40 or CPE52 spindle (0.8 ° and 3.0 ° cone angles, respectively) is attached to the C &amp; P viscometer, and 0.50 mL of sample is used for 2 to 400 s.^-1Measured at multiple shear rates during. Specifically, the sample starts at a shear rate that gives at least 10% torque and continues until the equipment torque reaches 100%, 22.58, 24.38, 26.25, 28.13, 30, 31.88, 45, respectively. , 67.5, 90, 112.5, 135, 157.5, 180, 202.5, 247, 270, 292.5, 315, 337.5, 360, 382, 400s^-1Then, it was measured for 30 seconds. The extrapolated zero shear viscosity was then determined from a dynamic viscosity vs. shear rate plot for the sample measured with a DV2T conical plate viscometer. The reported extrapolated zero shear viscosities are the mean and standard deviation of at least three measurements.</p><p> (Example 1) Organic phosphate reduces the viscosity of a concentrated aqueous solution of biosimilar AVASTIN (registered trademark).</p><p> A commercially obtained biosimilar AVASTIN® (100-400 mg) containing pharmaceutical excipients (polysorbate 20, phosphate and citrate buffers, mannitol and NaCl) was purified. First, polysorbate 20 was removed using a DETERGENT-OUT® TWEEN Medicolumn (G-Biosciences). The resulting solution is then adequately buffered to 20 mM sodium phosphate buffer (PB; pH 7.0) for PB samples and 2 mM PB (pH 7.0) for samples of reduced viscosity organic phosphate. Liquid exchange, Jumbosep centrifugal concentrator (Pall) Concentrated to a final volume of less than 10 mL with Corp.). The sample, which had been buffer-exchanged with 2 mM PB, was first divided into aliquots. Next, an appropriate amount of a solution of reduced viscosity organic phosphate (pH 7.0) was added to each aliquot. As a result, the final concentration of the excipient during reconstitution with water was 0.10 to 0.25 M. The protein solution was then lyophilized. The dried protein cake containing the protein and the reduced viscosity organic phosphate (and a negligible amount of buffer salt) was reconstituted to a final volume of about 0.1 mL and the concentration of the reduced viscosity organic phosphate described above. In the case of a sample (PB control sample) in which the buffer solution was exchanged with 20 mM PB, the collected protein solution was freeze-dried. Dried protein cakes containing protein and buffer salts were reconstituted to a final volume of approximately 0.10 to 0.50 mL. These samples were reconstituted with additional PB (pH 7.0), sufficient to bring the final concentration of PB to 0.25M. The final concentration of mAbs in solution was determined by a protein quantification assay by Kumasy by comparing samples of unknown concentration with the standard curve of biosimilar AVASTIN®. Reported viscosities were measured with a RheoSense mVROC microfluidic viscometer. Using the same protocol, formulations containing biosimilar ERBITUX®, TYSABRI®, HERCEPTIN® and REMICADE® were also prepared.</p><p> The data in Table 1 demonstrate that the viscosity of an aqueous solution of biosimilar AVASTIN® can be reduced by at least 2-fold in the presence of a devisible organic phosphate of 0.10 to 0.25 M. Viscosity above 200 cP in phosphate buffer was reduced to less than 50 cP in some cases by the addition of 0.10 to 0.25 M degrading organic phosphate. The TPP demonstrates that it has the highest viscosity-reducing capacity (lowest viscosity) of the viscosity-reducing organic phosphates tested here.</p><p><tables num="1-1"><img file="JP6564512B2_D0018.tif" /></tables><tables num="1-2"><img file="JP6564512B2_D0019.tif" /></tables></p><p> (Example 2) The decrease in viscosity of the aqueous solution of biosimilar AVASTIN (registered trademark) depends on the concentration of organic phosphate.</p><p> A commercially obtained aqueous solution of biosimilar AVASTIN® was prepared as described in Example 1. The dried protein cake was reconstituted in phosphate buffer or water to a final volume of about 0.1 mL and a final concentration of reducing viscosity organic phosphate from 0.02 M to 0.5 M. The final concentration of mAb in solution was determined by a protein quantification assay by Kumashi by comparing samples of unknown concentration with the standard curve of biosimilar AVASTIN®. Reported viscosities were measured with a RheoSense mVROC microfluidic viscometer.</p><p> The data in Table 2 demonstrate that the viscosity of an aqueous solution of biosimilar AVASTIN® is initially reduced by the addition of a viscosity-reducing organic phosphate. However, as the concentration of de-viscosity organic phosphate increases above a certain value, the addition of more de-viscosity organic phosphate can be counterproductive (causing an increase in viscosity). In the case of the aqueous solution of biosimilar AVASTIN (registered trademark) tested here, the viscosity begins to increase when the concentration of the viscosity-reducing organic phosphate exceeds about 0.20 M.</p><p><tables num="2"><img file="JP6564512B2_D0020.tif" /></tables></p><p> (Example 3) Organic phosphate reduces the viscosity of many therapeutically relevant monoclonal antibodies.</p><p> A commercially obtained aqueous solution of biosimilar AVASTIN® was prepared as described in Example 1. The dried protein cake was reconstituted in phosphate buffer or water to a final volume of about 0.10 mL and a final viscosity reduced organic phosphate concentration of 0.02 M to 0.50 M. The final concentration of mAb in solution was determined by a protein quantification assay by Kumashi by comparing samples of unknown concentration with the standard curve of biosimilar AVASTIN®.</p><p> Commercially obtained TYSABRI® containing pharmaceutical excipients (sodium phosphate buffer, NaCl, polysorbate 80) purified, buffer exchanged, concentrated, dried, reconstituted and analyzed in the same manner. did. Commercially obtained biosimilar ERBITUX® containing pharmaceutical excipients (sodium phosphate buffer, NaCl, polysorbate 80) is purified, buffer exchanged, concentrated, dried and reconstituted in the same manner. Constructed and analyzed. A commercially obtained REMICADE® containing pharmaceutical excipients (sucrose, polysorbate 80, sodium phosphate buffer) was prepared according to the instructions in the prescription information sheet and purified, buffered in the same manner. Exchanged, concentrated, dried, reconstituted, and analyzed. A commercially obtained HERCEPTIN® containing pharmaceutical excipients (histidin buffer, trehalose, polysorbate 20) was prepared according to the instructions in the prescription information sheet and purified, buffer exchange, in the same manner. Concentrated, dried, reconstituted, and analyzed. Commercially obtained biosimilar RITUXAN® containing pharmaceutical excipients (citrate buffer, sodium chloride and TWEEN® 80) is purified, buffer exchanged and concentrated in the same manner. , Dryed, reconstituted, and analyzed. Reported viscosities were measured with a RheoSense mVROC microfluidic viscometer.</p><p> The data in Table 3 demonstrate that reduced viscosity organic phosphates can reduce the viscosity of many concentrated aqueous solutions of treatment-related mAbs. In some cases, cAMP-Tris reduces viscosity by up to about 9 times.</p><p><tables num="3-1"><img file="JP6564512B2_D0021.tif" /></tables><tables num="3-2"><img file="JP6564512B2_D0022.tif" /></tables></p><p> Unless specifically defined above, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. One of ordinary skill in the art will recognize or confirm many equivalents to the specific embodiments of the invention described herein using only routine experiments. Such equivalents are intended to be covered by the claims below.</p>