EA043928B1 - HIGHLY CONCENTRATED LOW-VISCOSITY MASP-2 INHIBITORY ANTIBODIES, KITS AND METHODS - Google Patents

Description

Cross reference to related applications

This application claims priority based on a provisional patent application

U.S. No. 62/382156, filed Aug. 31, 2016, the entire contents of which are incorporated herein by reference.

Field of technology to which the invention relates

The present invention relates to stable, highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations, kits comprising the preparations, and therapeutic methods of using the preparations and kits for inhibiting the adverse effects of MASP-2-dependent complement activation.

Sequence List Statement

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence list is named MP_1_0261_PCT_SequenceListing_20170809_ST25. The text file is 17 KB in size; it was created on August 9, 2017 and submitted via EFS-Web when filing this application.

Prerequisites for creating the invention

Antibody therapeutics are typically administered on a regular basis and often require injection doses of several mg/kg. The preferred form of delivery for the treatment of chronic conditions is the outpatient administration of a high dose of monoclonal antibodies (several mg per kg body weight) by subcutaneous (SC) injection (Stockwin & Holmes, Expert Opin. Biol. Ther., 3:1133-1152 (2003) ; Shire et al., J. Pharm. Sci., 93:1390-1402 (2004)). It is desirable to use highly concentrated pharmaceutical formulations of the therapeutic antibody because it allows for a smaller volume and/or fewer administrations, which therefore means less discomfort for the patient. Additionally, smaller volumes allow therapeutic doses of the monoclonal antibody to be packaged into individual single-dose prefilled syringes for self-administration. The use of a pre-filled syringe or auto-injector for subcutaneous delivery allows injection to be performed at home, which promotes patient compliance with the drug administration regimen.

However, when developing a drug with a high protein concentration, problems arise related to the physical and chemical stability of the protein, as well as the complexity of production, storage and delivery of the protein drug (see, for example, Wang et al., J. of Pharm. Sci., vol. 96(1):l-26 (2007)). One of the challenges in developing drugs with high protein concentrations is the concentration-dependent viscosity of the solution. At a certain protein concentration, the viscosity changes dramatically depending on the preparation. In particular, monoclonal antibodies are known to exhibit distinctive and different viscosity-concentration profiles, which are characterized by a sharp exponential increase in solution viscosity with increasing monoclonal antibody concentration (see, for example, Connolly B.D. et al., Biophysical Journal, vol. 103:69 -78 (2012)). Another problem with liquid formulations at high monoclonal antibody concentrations is the physical stability of the protein (Alford et al., J. Pharm. Sci., 97:3005-3021 (2008); Salinas et al., J. Pharm. Sci., 99:82 -93 (2010); Sukumar et al., Pharm. Res., 21:1087-1093 (2004)). As a result, the high viscosity of monoclonal antibody pharmaceuticals at high concentrations, along with a potential decrease in stability, may hinder their development as drugs suitable for subcutaneous and/or intravenous delivery.

The complement system plays a role in the inflammatory response and is activated as a result of tissue damage or microbial infection. Complement activation must be highly regulated to ensure selective targeting of invading microorganisms and avoid causing harm to the body (Ricklin et al., Nat. Immunol., 11:785-797, 2010). It is now known that the complement system can be activated through three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is typically initiated by a complex consisting of host antibodies bound to a foreign particle (i.e., antigen), and usually requires prior exposure to the antigen to produce specific antibodies. Because activation of the classical pathway depends on the host's prior adaptive immune response, the classical pathway is part of acquired immunity. In contrast, both the lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

Mannan-binding lectin-associated serine protease-2 (MASP-2) has been shown to be essential for the functioning of the lectin pathway, one of the major pathways of complement activation (Vorup-Jensen et al., J. Immunol., 165:2093-2100, 2000; Ambrus et al., J. Immunol., 170:1374-1382, 2003; Schwaeble et al., PNAS, 108:7523-7528, 2011). It is important to note that MASP-2 inhibition does not appear to interfere with the functioning of the antibody-dependent classical complement pathway, which is an extremely important component of the acquired immune response to infection. As described in US Pat. No. 9,011,860 (assigned to Omeros Corporation), the contents of which are incorporated herein by reference, a fully human monoclonal antibody, OMS646, targeting human MASP-2, which binds

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Human MASP-2 has high affinity and blocks the lectin pathway of complement activation and is therefore useful for the treatment of various diseases and disorders associated with the lectin pathway of complement activation.

As also described in US Patent No. 7919094, US Patent No. 8840893, US Patent No. 8652477, US Patent No. 8951522, US Patent No. 9011860, US Patent No. 9644035, US Patent Application Publications US2013/0344073, US2013/0266560, US 201 5/ 0166675, US2017/0189525 and concurrently pending US Patent Applications Serial Numbers 15/476154, 15/347434, 15/470647, 62/315857, 62/275025 and 62/527926 (assigned in each case to Omeros Corporation, assignee nickname for of this application, all referenced documents are incorporated herein by reference), MASP-2-dependent complement activation contributes to the pathogenesis of numerous acute and chronic disease conditions. Thus, there is a need for a stable, highly concentrated, low-viscosity anti-MASP-2 monoclonal antibody preparation suitable for parenteral (eg, subcutaneous) administration for the treatment of subjects suffering from diseases and disorders associated with the MASP-2-dependent complement pathway.

The essence of the invention

In one aspect, the present invention relates to a stable pharmaceutical preparation suitable for parenteral administration to a mammalian subject, comprising (a) an aqueous solution containing a buffer system having a pH of 5.0-7.0; and (b) a monoclonal antibody or fragment thereof that specifically binds human MASP-2, at a concentration of from about 50 mg/ml to about 250 mg/ml, wherein said antibody or fragment thereof comprises (i) a heavy chain variable region containing CDR-H1, CDR-H2 and CDR-H3 in SEQ ID NO: 2, and (ii) a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 in SEQ ID NO: 3; or a variant thereof comprising a heavy chain variable region having at least 95% identity with SEQ ID NO: 2 and a light chain variable region having at least 95% identity with SEQ ID NO: 3;

wherein the preparation has a viscosity of 2 to 50 centipoise (cP), and the preparation is stable when stored at a temperature of 2 to 8°C for at least one month.

In some embodiments, the concentration of antibody in the formulation is from about 150 mg/ml to about 200 mg/ml. In some embodiments, the viscosity of the formulation is less than 25 cP. In some embodiments, the buffer system contains histidine. In some embodiments, the buffer system contains citrate. In some embodiments, the formulation also contains an excipient, such as a tonicity-modifying agent, in an amount sufficient to cause the formulation to be hypertonic. In some embodiments, the formulation also contains a surfactant. In some embodiments, the formulation also contains the enzyme hyaluronidase in an amount effective to increase dispersion and/or absorption of the antibody after subcutaneous administration.

In another aspect, the drug is contained in a subcutaneous administration device, such as a prefilled syringe.

In another aspect, the present invention relates to a kit including a pre-filled container containing a drug.

In another aspect, the present invention provides a pharmaceutical composition for use in the treatment of a patient suffering from or at risk of developing a MASP-2-dependent disease or condition, which is a sterile single-use dosage form containing from about 350 mg to about 400 mg (t i.e. 350, 360, 370, 380, 390 or 400 mg) of a MASP-2 inhibitory antibody, wherein the composition contains from about 1.8 ml to about 2.2 ml (i.e. 1.8, 1.9 , 2.0, 2.1, or 2.2 ml) of a 185 mg/ml antibody preparation such as disclosed herein, wherein said antibody or fragment thereof comprises (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3, wherein the preparation is stable when stored at a temperature of 2 to 8° C. for at least six months.

In some embodiments, the MASP-2 dependent disease or condition is selected from the group consisting of aHUS, HSCT-TMA, IgA-H, and lupus nephritis (LN).

In another aspect, the present invention provides a method of treating a subject suffering from a disease or disorder that is treatable with a MASP-2 inhibitory antibody, comprising administering a preparation containing an anti-MASP-2 antibody disclosed herein.

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Description of drawings

The foregoing aspects and many additional advantages of the present invention will be more appreciated and understood by reading the following detailed description in conjunction with the accompanying drawings.

Fig. 1A graphically illustrates the extent of lectin pathway-dependent membrane attack complex (MAC) deposition in the presence of varying amounts of human anti-MASP-2 monoclonal antibody (OMS646), showing that OMS646 inhibits lectin-mediated MAC deposition with an _IC50 value of approximately 1 nM. as described in example 1.

Fig. 1B graphically illustrates the extent of classical pathway-dependent MAC deposition in the presence of varying amounts of human anti-MASP-2 monoclonal antibody (OMS646), indicating that OMS646 does not inhibit classical pathway-mediated MAC deposition as described in Example 1.

Fig. 1C graphically illustrates the extent of alternative pathway-dependent MAC deposition in the presence of human anti-MASP-2 monoclonal antibody (OMS646), which shows that OMS646 does not inhibit alternative pathway-mediated MAC deposition as described in Example 1.

Fig. 2A graphically illustrates the results of a dynamic light scattering (DLS) analysis for screening excipients of the OMS646 formulation, which shows the particle outer diameter observed in formulations containing different potential excipients as described in Example 2.

Fig. 2B graphically illustrates the results of the DLS analysis for screening excipients of the OMS646 drug, which shows the overall polydispersity observed in preparations containing different potential excipients, as described in Example 2.

Fig. Figure 3 graphically illustrates the results of viscosity analysis over a range of OMS646 concentrations in different formulations when measured at pH 5.0 and 6.0, as described in Example 2.

Fig. Figure 4 graphically illustrates the percent protein recovery after buffer exchange in the OMS646 solubility/viscosity study conducted with different drug candidates as described in Example 2.

Fig. Figure 5 graphically illustrates the dependence of viscosity (determined by exponential fitting of viscosity data) versus protein concentration in the OMS646 solubility/viscosity study conducted with different drug candidates as described in Example 2.

Fig. 6 graphically illustrates viscosity data normalized to protein concentration in a viscosity study conducted with different OMS646 drug candidates as described in Example 2.

Fig. 7A graphically illustrates the average load (lbf) for three OMS646 drug candidates in syringe feasibility studies using 27 GA (1.25), 25 GA (1), and 25 GA thin-walled (1) needles as described in Example 3.

Fig. 7B graphically illustrates the maximum load (lbf) for three OMS646 drug candidates in syringe feasibility studies using 27 GA (1.25), 25 GA (1), and 25 GA thin-walled (1) needles as described in Example 3.

Description of the sequence list

SEQ ID NO: 1 human MASP-2 protein (mature).

SEQ ID NO: 2: OMS646 heavy chain variable region (VH) polypeptide.

SEQ ID NO: 3: OMS646 light chain variable region (VL) polypeptide.

SEQ ID NO: 4: full length mutant IgG4 heavy chain polypeptide OMS646.

SEQ ID NO: 5: Full length OMS646 light chain polypeptide.

SEQ ID NO: 6: DNA encoding full-length heavy chain polypeptide OMS646.

SEQ ID NO: 7: DNA encoding the full-length OMS646 light chain polypeptide.

Detailed description

I. Definitions.

Unless specifically stated otherwise herein, all terms used herein have the meaning commonly given to them by those skilled in the art to which this invention relates. The following definitions are provided for clarity of terms used in the specification and claims to describe the present invention.

Standard methods can be used for the production of recombinant DNA, oligonucleotide synthesis, and for tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification methods can be used in accordance with the manufacturer's instructions, as is generally accepted in the art or as described herein. These and related methods and procedures generally can be applied in a manner generally known in the art and as described in the various general and specialized literature cited and discussed throughout this specification. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Edited by:

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John E. ColIgA-H, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober, 2001, John Wiley & Sons, NY, NY); or other relevant Current Protocol publications and similar literature. Unless specific definitions are provided, the nomenclature used in connection with the description of laboratory procedures and methods of molecular biology, analytical chemistry, organic synthesis chemistry, and medicinal and pharmaceutical chemistry described herein is well known and used in the art. Standard methods can be used for recombinant DNA technology, molecular biology techniques, microbiology, chemical synthesis, chemical assays, pharmaceutical preparation, formulation and delivery, and patient treatment.

The term pharmaceutical drug means a drug that is in a form that allows the biological activity of the active agent (eg, a MASP2 inhibitory antibody) to be used for effective treatment, and does not contain additional components that are unacceptably toxic to the subject to whom the drug will be administered. Such preparations are sterile. In one embodiment, the pharmaceutical preparation is suitable for parenteral administration, for example, subcutaneous administration.

The term MASP-2 stands for mannan-binding lectin-associated serine protease-2. The protein sequence of (mature) human MASP-2 is given in SEQ ID NO: 1.

The term MASP-2-dependent complement activation refers to MASP-2-dependent activation of the lectin pathway that occurs under physiological conditions (i.e., in the presence of Ca++), leading to the formation of the C3 convertase C4b2a of the lectin pathway and subsequently, after accumulation of the product cleavage of C3, C3b, to the formation of C5 convertase C4b2a(C3b)n.

The term lectin pathway refers to complement activation that occurs through the specific binding of whey and non-whey carbohydrate-binding proteins, including mannan-binding lectin (MBL), CL-11, and ficolins (H-ficolin, M-ficolin, or L-ficolin).

The term classical pathway refers to complement activation, which is triggered by an antibody bound to a foreign particle and requires binding of the recognition molecule C1q.

The term MASP-2 inhibitory antibody means an antibody or antigen binding fragment thereof that binds MASP-2 and effectively inhibits MASP-2-dependent complement activation (eg, OMS646). MASP-2 inhibitory antibodies that can be used in the method of the invention can reduce MASP-2-dependent complement activation by more than 20%, for example more than 30%, or more than 40%, or more than 50%, or more more than 60%, or more than 70%, or more than 80%, or more than 90%, or more than 95%.

The term monoclonal antibody OMS646 means a monoclonal antibody containing CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 2, and containing CDR-L1, CDR-L2 and CDR-L3 of the variable region light chain region with the amino acid sequence set forth in SEQ ID NO: 3. This particular antibody is an example of a MASP-2 inhibitory antibody that specifically binds MASP-2 and inhibits MASP-2-dependent complement activation.

The term monoclonal antibody means a homogeneous population of antibodies in which the monoclonal antibody is composed of amino acids (natural or non-natural) involved in the selective binding of an epitope. Monoclonal antibodies are highly specific for the target antigen. The term monoclonal antibody covers not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain antibodies (scFv), variants thereof, fusion proteins containing antigen-binding fragment, humanized monoclonal antibodies, chimeric monoclonal antibodies and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding fragment (epitope recognition site) with the necessary specificity and ability to bind to the epitope. The term should not be limited as to the source of the antibodies or the method of their production (eg, using hybridomas, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments, etc., as defined above in the definition of the term antibody.

The term antibody fragment means a fragment derived from or derived from a full-length antibody, such as, for example, a MASP-2 inhibitory antibody, typically containing its antigen-binding or variable region. Illustrative examples of antibody fragments include Fab, Fab', F(ab) ₂ , F(ab') ₂ and Fv fragments, scFv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

As used herein, the term single chain Fv or scFv antibody fragment comprises the V _H and V _L domains of the antibody, these domains being on the same polypeptide chain. Typically, the Fv polypeptide also contains a polypeptide linker between the VH and VL domains, which allows the scFv to adopt the desired structure for antigen binding.

The term CDR region or CDR refers to the hypervariable regions of the immunoglobulin heavy and light chains as defined by Kabat et al., 1991 (Kabat, E.A. et al. (1991), Sequences of

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Proteins of Immunological Interest, 5th edition and later editions). The antibody typically contains a heavy chain CDR region and 3 light chain CDR regions. The term CDR or CDR regions is used herein to refer, as appropriate, to one or more of these regions, or even to the whole of those regions, which contain the majority of the amino acid residues responsible for binding with the affinity of the antibody for the antigen epitope that it recognizes.

The term specific binding refers to the ability of an antibody to preferentially bind to a specific analyte that is in a homogeneous mixture with different analytes. In specific embodiments, the specific binding interaction allows discrimination between desired and undesired analytes in a sample, in some embodiments, with greater than about 10- to 100-fold or greater (eg, greater than about 1000- or 10000-fold) affinity. In specific embodiments, the affinity between the capture agent and the analyte, when specifically bound in a capture agent/analyte complex, is characterized by an RD value of less than about 100 nM, or less than about 50 nM, or less than about 25 nM, or less about 10 nM, or less than about 5 nM, or less than about 1 nM.

The term isolated antibody means an antibody that has been identified and separated and/or extracted and/or purified from components of its natural environment or expression system in cell culture. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of the antibody, and most preferably greater than 99% by weight; when determining protein concentration by an appropriate measurement method, such as, for example, the Lowry method or absorbance at OD ₂₈₀ ;

(2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotating beaker sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue or preferably silver staining.

Typically, the isolated antibody used in the formulations disclosed herein will be prepared using at least one purification step.

In this document, amino acid residues are designated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), series (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

Broadly speaking, naturally occurring amino acids can be divided into groups based on the chemical characteristics of the side chains of the corresponding amino acids. The hydrophobic amino acid is Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. A hydrophilic amino acid is Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. These groups of amino acids can be further subclassified as follows. An uncharged hydrophilic amino acid is Ser, Thr, Asn or Gln. The acidic amino acid is Glu or Asp. The main amino acid is Lys, Arg or His.

As used herein, the term conservative amino acid substitution means a substitution between amino acids within each of the following groups:

(1) glycine, alanine, valine, leucine and isoleucine;

(2) phenylalanine, tyrosine and tryptophan;

(3) serine and threonine;

(4) aspartate and glutamate;

(5) glutamine and asparagine; and (6) lysine, arginine and histidine.

As used herein, the term subject includes all mammals, including, without limitation, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

The term pharmaceutically acceptable, when applied to an excipient in a pharmaceutical preparation, means that the excipient is suitable for administration to a human subject.

The term subcutaneous administration means administration of the drug under all layers of the subject's skin.

The term buffer means a buffer solution that is resistant to changes in pH due to the action of its associated acid-base components. The buffer of this invention has a pH value in the range of about 4 to about 8, preferably from about 5 to about 7, and most preferably has a pH value in the range of about 5.5 to about 6.5. Examples of buffers that will control pH within this range include acetate (eg, sodium acetate), succinate (eg, sodium succinate), gluconate, histidine, citrate, and other organic acid-based buffers. A buffer agent is a compound that is used to prepare buffer solutions.

The term histidine specifically means L-histidine unless otherwise noted.

The term isotonic refers to a drug that has essentially the same osmotic pressure as human blood. Isotonic drugs will generally have an osmotic pressure of about 250 mOsmol/kg to about 350 mOsmol/kg H ₂ O. Isotonicity can be determined using a headspace osmometer or a freezing point depression osmometer, for example.

The term hypertonic refers to a drug with an osmotic pressure higher than that in the human body (ie, higher than 350 mOsmol/kg H ₂ O).

The term tonicity-modifying agent means a pharmaceutically acceptable agent suitable for producing an isotonic or, in some embodiments, a hypertonic drug.

The term sterile refers to a pharmaceutical preparation that is aseptic or does not contain viable bacteria, fungi or other microorganisms, which can be achieved by any suitable method, such as, for example, aseptically processing the preparation and dispensing it into a container or filtering it through filtration sterilization membranes until or after receiving the drug and adding it to the container.

The term stable drug means a drug that maintains its original level of purity over a period of time. In other words, if the drug is at least 95% pure, such as at least 96%, at least 97%, at least 98%, or at least 99% pure, in terms of the particular type of antibody (e.g., MASP-inhibiting 2 antibodies) at time 0, stability is a measure of how well and for how long the drug maintains essentially a given level of purity (e.g., without forming other compounds such as fragmented moieties (FMM) or pure molecule aggregates (PMA)). The drug is stable if the purity level does not decrease substantially when stored at a temperature of about 2-8°C for a specified period of time, for example at least 6 months, at least 9 months, at least 12 months or at least 24 months . The expression substantially unchanged means that the purity level of the drug changes by less than 5%, such as less than 4%, or less than 3%, or less than 2%, or less than 1%, over a period of time (for example, within 6 months, within 9 months, or within 12 months, or within 24 months). In one embodiment, the stable preparation is stable at a temperature of 2-8°C for a period of time of at least six months. In a preferred embodiment, the stable preparation is stable at a temperature of 2-8°C for a period of at least one year or for a period of at least two years. In one embodiment, the drug is stable if the MASP-2 inhibitory antibody remains at least 95% monomeric when stored at 2 to 8°C for at least one month or for at least six months or for at least 12 months when determined by EC-HPLC.

The term preservative means a compound that can be included in a drug to significantly reduce bacterial growth or contamination. Non-limiting examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethetonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

The term excipient means an inert substance in a formulation that imparts a beneficial physical property to the formulation, such as increased protein stability and/or reduced viscosity. Examples of suitable excipients include, but are not limited to, proteins (eg, serum albumin), amino acids (eg, aspartic acid, glutamic acid, lysine, arginine, glycine and histidine), saccharides (eg, glucose, sucrose, maltose and trehalose), polyols ( eg mannitol and sorbitol), fatty acids and phospholipids (eg alkyl sulfonates and caprylate).

The term substantially absent means that the substance is either absent or present only in minimal, trace amounts that do not have a significant effect on the properties of the composition. If a substance is stated to be missing, it should be understood that there is no detectable quantity present.

The term viscosity refers to a measure of the resistance of a fluid to being deformed by either shear stress or tensile stress; it can be assessed using a viscometer (eg rolling ball viscometer) or a rheometer. Unless otherwise specified, viscosity (centipoise, cP) measurements are made at approximately 25°C with a shear rate ranging from 100,000 to 250,000 1/s.

The term parenteral administration means a route of administration other than the digestive system and includes the injection of a dosage form into the body using a syringe or other mechanical device such as an infusion pump. Parenteral routes of administration may include intravenous, intramuscular, subcutaneous and intraperitoneal routes. Subcutaneous injection

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The term treatment means therapeutic treatment and/or prophylactic or preventive measures. Those in need of treatment include subjects already having the disease as well as those in whom the disease is to be prevented. Thus, herein, the patient who will receive treatment may be a patient who has already been diagnosed with the disease, or who may be predisposed or susceptible to the disease.

The term effective amount means the amount of a substance that produces the desired effect. In the case of a pharmaceutical drug substance, it represents the amount of the active ingredient effective to treat a disease in a patient. In the case of a formulation ingredient, for example, hyaluronidase enzyme, the effective amount is the amount necessary to increase the dispersion or absorption of the co-administered MASP-2 inhibitory antibody such that the MASP-2 inhibitory antibody can act in a therapeutically effective manner as described above.

As used herein, the term should approximately indicate that the particular value reported may vary to some extent, for example, variations within ±10%, preferably ±5%, most preferably ±2% are included in the specific value. For example, the expression pharmaceutical preparation containing about 200 mg/ml of a MASP-2 inhibitory antibody should be understood to mean that the preparation may contain from about 180 to 220 mg/ml of a MASP-2 inhibitory antibody (eg, OMS646). Where ranges are specified, the end values are included in the range unless otherwise indicated or the context clearly indicates.

As used herein, terms in the singular include the corresponding plural terms unless the context clearly requires otherwise. Thus, for example, as used herein, an excipient also includes a variety of such excipients and their equivalents known to those skilled in the art, the agent includes one agent as well as two or more agents; antibody includes a plurality of such antibodies and framework region means one or more framework regions and equivalents thereof known to those skilled in the art, etc.

Each embodiment in this specification shall apply, mutatis mutandis, to every other embodiment unless specifically stated otherwise. It is intended that any embodiment described in this specification may be implemented in connection with any method, kit, reagent or composition of the invention, and vice versa. Moreover, the compositions of the invention can be used to carry out the methods of the invention.

II. Overview of the invention.

The present invention relates to stable, highly concentrated, low-viscosity pharmaceutical preparations of a MASP-2 inhibitory antibody suitable for parenteral administration (eg, subcutaneous administration) and also suitable for dilution prior to intravenous administration. Highly concentrated pharmaceutical preparations of the therapeutic antibody are desirable because they allow for a smaller volume and/or fewer administrations, which therefore means less discomfort for the patient. Additionally, smaller volumes allow therapeutic doses of the MASP-2 inhibitory antibody to be packaged in individual single-dose prefilled syringes or vials for self-administration. The highly concentrated, low-viscosity formulations of the present invention are an aqueous solution containing a buffer system having a pH of 4.0 to 8.0, more preferably having a pH of from about 5.0 to about 7.0, and a MASP-2 inhibitory monoclonal antibody (eg , OMS646), or an antigen-binding fragment thereof, at a concentration of from about 50 mg/ml to about 250 mg/ml. In preferred embodiments, the MASP-2 inhibitory antibody (eg, OMS646) is present in highly concentrated formulations suitable for subcutaneous administration at a concentration of from about 100 mg/ml to about 250 mg/ml. In specific embodiments, the MASP-2 inhibitory antibody (e.g., OMS646) is present in high-strength formulations at a concentration of about 150 mg/mL to about 200 mg/mL, such as about 175 mg/mL to about 195 mg/mL, such as about 185 mg/ml.

In various embodiments, the pharmaceutical preparations also contain, in addition to a high concentration of MASP-2 inhibitory antibody and a buffer system, one or more excipients, such as a tonicity modifying agent (eg, an amino acid with a charged side chain) and optionally a nonionic surfactant. In some embodiments, the pharmaceutical preparations of this invention also contain the enzyme hyaluronidase.

A significant advantage of the highly concentrated MASP-2 inhibitory antibody pharmaceutical preparations of the present invention is their low viscosity at high protein concentrations. As is known to those skilled in the art, the high viscosity of monoclonal antibody pharmaceutical preparations at concentrations >100 mg/ml may prevent their development as preparations suitable for subcutaneous and/or intravenous delivery. Thus, pharmaceuticals having a lower viscosity are highly desirable due to their ease of

- 7 043928 production, for example, but not limited to, processing, filtering and filling into containers. As described in Examples 2 and 3 herein, formulations of the present invention containing the MASP-2 inhibitory antibody OMS646 at a concentration of 100 to 200 mg/ml have a surprisingly low viscosity, such as a viscosity of less than about 50 cP, such as 2 to 50 cP , for example from 2 to 40 cP, for example from 2 to 30 cP, or from 2 to 25 cP, or from 2 to 20 cP, or from 2 to 18 cP.

In addition, the low-viscosity, high-concentration MASP-2 inhibitory antibody pharmaceutical preparations of the present invention are capable of administration using standard syringes and needles, auto-injection devices, and microinfusion devices known in the art. As described in Example 3, the highly concentrated, low-viscosity MASP-2 inhibitory antibody pharmaceutical preparations disclosed herein are shown to have syringable and needle-able properties that make them suitable for subcutaneous administration. Syringability and ability to pass through a needle are key parameters for the suitability of a pharmaceutical drug for any parenteral administration, such as intramuscular or subcutaneous, and allow such drugs to be administered by intramuscular or subcutaneous injection through small bore needles typically used for such injections, e.g. 29 GA, regular or thin wall, 27 GA (1.25), regular or thin wall, or 25 GA (1), regular or thin wall needles. In some cases, the low viscosity of the MASP-2 inhibitory antibody pharmaceutical formulations disclosed herein allows for a reasonable (eg, 1-3 ^cc ) injection volume to be administered while delivering an effective amount of the MASP-2 inhibitory antibody OMS646 with a single injection to a single site. injections.

An additional important advantage of the formulations of the present invention is that highly concentrated, low-viscosity MASP-2 inhibitory antibody formulations (i.e., at concentrations of >100 to 200 mg/mL) are stable when stored at 2 to 8°C for up to at least 30 days, up to at least 9 months, or up to at least 12 months, or longer, as determined in the stability studies described in Examples 2 and 4.

The present invention also relates to a method for producing highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations, containers containing such preparations, therapeutic kits including the preparations, as well as therapeutic methods of using such preparation, containers and kits for treating a subject suffering from or at risk of developing , a disease or condition associated with MASP-2-dependent complement activation.

MASP-2 inhibitory antibody.

As described in detail herein, the present invention relates to preparations containing monoclonal antibodies and antigen-binding fragments thereof that specifically bind MASP-2 and inhibit MASP-2-dependent complement activation. In specific embodiments, the MASP-2 inhibitory antibody or antigen binding fragment thereof for use in the subject formulations is a MASP-2 inhibitory antibody called OMS646, described in WO2012/151481 (the contents of which are incorporated herein by reference), which contains a heavy chain polypeptide , containing the amino acid sequence of SEQ ID NO: 2, and a light chain polypeptide containing the amino acid sequence of SEQ ID NO: 3. As described in WO2012/151481 and described in Example 1, OMS646 specifically binds human MASP-2 with high affinity and has the ability to block lectin pathway of complement activation. In specific embodiments, the MASP-2 inhibitory antibody or antigen binding fragment thereof for use in the claimed formulations is a MASP-2 inhibitory antibody comprising (a) a heavy chain variable region comprising (i) a CDR-H1 comprising the sequence of amino acids 31-35 SEQ ID NO: 2, (ii) CDR-H2 containing the sequence of amino acids 50-65 in SEQ ID NO: 2, and iii) CDR-H3 containing the sequence of amino acids 95-107 in SEQ ID NO: 2; and (b) a light chain variable region containing

i) CDR-L1 containing the sequence of amino acids 24-34 in SEQ ID NO: 3, ii) CDR-L2 containing the sequence of amino acids 50-56 in SEQ ID NO: 3, and iii) CDR-L3 containing the sequence of amino acids 89- 97 at SEQ ID NO: 3.

In some embodiments, the MASP-2 inhibitory antibody for use in the subject formulations is an OMS646 variant comprising a heavy chain variable region having at least 95% identity with SEQ ID NO: 2 and comprising a light chain variable region having at least 95% identity to SEQ ID NO: 3. In some embodiments, the MASP-2 inhibitory antibody for use in the subject formulations is (a) an OMS646 variant comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 2, wherein residue 31 is R, residue 32 is G, residue 33 is K, residue 34 is M, residue 35 is

- 8 043928 is G, residue 36 is V, residue 37 is S, residue 50 is L, residue 51 is A, residue 52 is H, residue 53 is I, residue 54 is F, residue 55 is S, residue 56 is S, residue 57 is D, residue 58 is E, residue 59 is K, residue 60 is S, residue 61 is Y, residue 62 is R, residue 63 is T, residue 64 is S, residue 65 is L, residue 66 is K, residue 67 is S, residue 95 is Y, residue 96 is Y, residue 97 is C, residue 98 is A, residue 99 is R, residue 100 is I, residue 101 is R, residue 102 is R or A, residue 103 is G, residue 104 is G, residue 105 is I, residue 106 is D, and residue 107 is Y; And

b) a light chain variable region containing an amino acid sequence having at least 95% identity with SEQ ID NO: 3, wherein residue 23 is S, residue 24 is G, residue 25 is E or D, residue 26 is is K, residue 27 is L, residue 28 is G, residue 29 is D, residue 30 is K, residue 31 is Y or F, residue 32 is A, residue 33 is Y, residue 49 is is Q, residue 50 is D, residue 51 is K or N, residue 52 is Q or K, residue 53 is R, residue 54 is P, residue 55 is S, residue 56 is G, residue 88 is Q, residue 89 is A, residue 90 is W, residue 91 is D, residue 92 is S, residue 93 is S, residue 94 is T, residue 95 is A, residue 96 is is V and residue 97 is F.

In some embodiments, the MASP-2 inhibitory monoclonal antibody (eg, OMS646 or a variant thereof) for use in the subject formulations is a full-length monoclonal antibody. In some embodiments, the monoclonal MASP-2 inhibitory antibody is a human full-length IgG4 antibody. In some embodiments, IgG4 has a point mutation in the hinge region to improve the stability of the antibody.

In some embodiments, a MASP-2 inhibitory antibody (e.g., OMS646 or a variant thereof) consists of human antibody-derived variable regions fused to human IgG4 lambda heavy chain and light chain constant regions, wherein the heavy chain has a point mutation in the hinge region (for example, the IgG4 molecule has the S228P mutation) to increase the stability of the antibody. In some embodiments, the MASP-2 inhibitory antibody is a tetramer consisting of two identical heavy chains having the amino acid sequence set forth in SEQ ID NO: 4 and two identical light chains having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the concentration of the MASP-2 inhibitory antibody in the formulation is from about 100 mg/ml to about 250 mg/ml, such as from about 150 mg/ml to about 220 mg/ml, such as from about 175 mg/ml to about 200 mg/ml or from about 175 mg/ml to about 195 mg/ml. In specific embodiments, the MASP-2 inhibitory antibody is present in the formulation at a concentration of from about 175 mg/ml to about 195 mg/ml, such as from about 180 mg/ml to about 190 mg/ml, such as about 175 mg/ml, such as about 180 mg/ml, approximately 181 mg/ml, approximately 182 mg/ml, approximately 183 mg/ml, approximately 184 mg/ml, approximately 185 mg/ml, approximately 186 mg/ml, approximately 187 mg/ml, approximately 188 mg /ml, about 189 mg/ml or, for example, about 190 mg/ml.

In some embodiments, minor variations in the amino acid sequences of MASP-2 inhibitory antibodies or fragments thereof are also provided to be part of the claimed formulations, provided that across the variations, the amino acid sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the MASP-2 inhibitory antibodies or antigen binding fragments thereof described herein (i.e., at least 90%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 and/or at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3), and retains the ability to inhibit MASP-2-dependent complement activation.

It is understood that MASP-2 inhibitory antibodies or antigen binding fragments thereof formulated in the context of the present invention can be produced using methods well known in the art (e.g., recombinant DNA technologies, phage display technologies, synthesis technologies, or combinations of such technologies or other technologies known in the art

- 9 043928 region). Methods for preparing and purifying antibodies and antigen-binding fragments are well known in the art and are described, for example, in Harlow & Lane (1988) Antibodies, A Laboratory Manual,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15.

For example, MASP-2 inhibitory antibodies, such as OMS646, can be expressed in appropriate mammalian cell lines. Sequences encoding the heavy chain variable region and the light chain variable region of a particular antibody of interest, such as OMS646 (eg, SEQ ID NO: 6 and SEQ ID NO: 7) can be used to transform a suitable mammalian host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into nuclei.

Mammalian cell lines suitable as expression hosts are well known in the art and include a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, newborn hamster kidney (BNK), monkey kidney (COS) cells, human hepatocellular carcinoma cells (eg, HepG2), human embryonic kidney 293 (HEK293) cells, and a variety of other cell lines.

Following the protein production phase of the cell culture process, MASP-2 inhibitory antibodies are recovered from the cell culture medium using methods known to those skilled in the art. Specifically, in some embodiments, the MASP-2 inhibitory antibody heavy and light chain polypeptides are recovered from the culture medium as secreted polypeptides.

MASP-2 inhibitory antibodies can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, as well as any combination of known or future developed purification methods, including, but not limited to, protein A chromatography, ion exchange fractionation column, ethanol precipitation, reverse phase HPLC, silica gel chromatography, SEPHAROSET® heparin column chromatography, anion exchange or cation exchange resin chromatography (e.g. polyaspartic acid column), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation. The purification method may also include additional steps resulting in the inactivation and/or removal of viruses and/or retroviruses that may potentially be present in the mammalian cell lineage culture medium. Many different steps can be used to purify viruses, including, but not limited to, treatment with uncoupling reagents such as urea or guanidine, detergents, additional ultrafiltration/diafiltration steps, conventional separation methods such as ion exchange or size exclusion chromatography, exposure to pH extremes, heat , the use of proteases, organic solvents, or any combination of the above.

Purified MASP-2 inhibitory antibodies typically require concentration changes and buffer changes before storage or further processing. As a non-limiting example, a flow-through filtration (TFF) system can be used to concentrate and replace the elution buffer from a previously used purification column with a final buffer designed for the drug substance.

The monoclonal MASP-2 inhibitory antibody formulated in accordance with the present invention is preferably substantially pure and desirably substantially homogeneous (ie, free of contaminant proteins, etc.). The term substantially pure antibody means a composition containing at least 90% by weight of the antibody based on the total weight of the composition, preferably at least 95% by weight. The term substantially homogeneous antibody means a composition containing at least about 99% by weight of the antibody based on the total weight of the composition.

Aqueous solutions.

The highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention is an aqueous solution containing a buffer system having a pH of from 4.0 to 8.0 (e.g., having a pH of from about 5.0 to about 7.0, or having a pH of about 5.5 to about 6.5), and a MASP-2 inhibitory antibody (e.g., OMS646 or a variant thereof) or an antigen binding fragment thereof at a concentration of from about 50 mg/ml to about 250 mg/ml (e.g., from about 100 mg/ml ml to approximately 250 mg/ml). The aqueous solution for use in the preparations of the present invention is a pharmaceutically acceptable solution (safe and non-toxic for administration to humans) which is used to prepare a liquid preparation. In some embodiments, the aqueous solution is water, such as sterile water for injection (WFI), which is sterile, solute-free distilled water. Alternatively, other aqueous solutions that are suitable for therapeutic administration and that will not adversely affect the stability of the drug, such as deionized water, can be used. Other suitable aqueous solutions include bacteriostatic water for injection (BWFI), sterile saline, Ringer's solution, or other similar aqueous solutions used to prepare pharmaceutical solutions.

Buffer systems.

The highly concentrated, low viscosity MASP-2 inhibitory antibody preparation of the present invention is pH adjusted to a value of 4.0-8.0, preferably to a pH of 5.0-7.0. The required pH value is maintained through the use of a buffer system. In some embodiments, the buffer system contains at least one pharmaceutically acceptable buffer agent with an acid dissociation constant within 2 pH units of the pH of the formulation. The buffer system used in the formulations of the present invention has a pH in the range of about 4.0 to about 8.0. Various buffering agents are known to those skilled in the art. Examples of buffers that will control the pH within this range include acetate, succinate, gluconate, histidine, citrate and other organic acid based buffers. In some embodiments, the buffering agent is selected from the group consisting of succinate, histidine, and citrate. In some embodiments, the pharmaceutical preparations contain a buffer system with a buffer agent at a concentration of 1 to 50 mM, such as 10-40 mM, or, for example, 10-30 mM, or 20-30 mM, or about 20 mM.

In some embodiments, the buffering agent is a histidine buffer. A histidine buffer is a buffer containing the amino acid histidine. Examples of histidine buffers include buffers containing histidine or any histidine salts, including histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, including combinations of any of these salts with or without histidine. In one embodiment, the buffer system is a histidine hydrochloride (L-histidine/HCl) buffer solution. Such a histidine hydrochloride buffer solution can be prepared by titrating L-histidine (free base, solid) with dilute hydrochloric acid or using an appropriate mixture of histidine and histidine hydrochloride. In some embodiments, the pH of the L-histidine/HCl buffer is from about 5.0 to about 7.0, such as from about 5.5 to about 6.0, such as about 5.8 or about 5.9.

In some embodiments, the buffering agent is a citrate buffer. Such a citrate buffer can be prepared by titrating citric acid, monosodium citric acid and/or disodium citric acid with a dilute sodium hydroxide solution to the appropriate pH value, or using an appropriate mixture of citric acid and salt(s) to achieve the same pH value. In another embodiment, the citrate buffer can be prepared by titrating a solution of trisodium citrate with a dilute solution of hydrochloric acid to the appropriate pH value. In this case, the ionic strength may be slightly higher than in the case of citric acid due to the formation of additional sodium and chloride ions in the solution. In specific embodiments, the pH of the citrate buffer is from about 5.0 to about 7.0, such as from about 5.5 to about 6.0, such as about 5.8 or about 5.9. In some embodiments, the buffering agent is a succinate buffer. In specific embodiments, the pH of the succinate buffer is from about 5.5 to about 6.0, such as about 5.8 or about 5.9.

In some embodiments, the buffering agent is a sodium citrate buffer solution, wherein the sodium citrate is present in the formulation at a concentration of from about 10 mM to about 50 mM, such as from about 10 mM to about 25 mM, such as about 20 mM. In some embodiments, the buffering agent is an L-histidine buffer, wherein the L-histidine is present in the formulation at a concentration of from about 10 mM to about 50 mM, such as from about 10 mM to about 25 mM, such as about 20 mM. In some embodiments, the formulation contains about 20 mM sodium citrate and has a pH of from about 5.0 to about 7.0. In some embodiments, the formulation contains about 20 mM L-histidine and has a pH of from about 5.0 to about 7.0.

Excipients.

In some embodiments, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention also contains at least one excipient. Examples of suitable excipients include, but are not limited to, proteins (eg, serum albumin), amino acids (eg, aspartic acid, glutamic acid, lysine, arginine, glycine and histidine), saccharides (eg, glucose, sucrose, maltose and trehalose), polyols ( eg mannitol and sorbitol), fatty acids and phospholipids (eg alkyl sulfonates and caprylate).

In some embodiments, the formulation contains an excipient selected from the group consisting of an amino acid with a charged side chain, a sugar or other polyol, and a salt. In some embodiments, the formulation contains sugar or other polyol, such as, for example, sucrose, trehalose, mannitol, or sorbitol. In some embodiments, the formulation contains a salt, such as, for example, NaCl or an amino acid salt.

In some embodiments, the formulation contains an excipient that is a tonicity-modifying agent. In some embodiments, the modifying

- 11 043928 tonicity The agent is included in the preparation in a concentration suitable for obtaining an isotonic preparation. In some embodiments, the tonicity-modifying agent is included in the formulation at a concentration suitable to formulate a hypertonic medication. In some embodiments, the tonicity modifying agent for use in the formulation is selected from the group consisting of a side chain charged amino acid, a sugar or other polyol, and a salt. In some embodiments, the tonicity-modifying agent is an amino acid with a charged side chain (ie, a negatively charged side chain or a positively charged side chain) at a concentration of from about 50 mM to about 300 mM. In some embodiments, the tonicity-modifying agent is an amino acid with a negatively charged side chain, such as glutamate. In some embodiments, the formulation contains glutamate at a concentration of from about 50 mM to about 300 mM. In some embodiments, the tonicity-modifying agent is an amino acid with a positively charged side chain, such as arginine. In some embodiments, the formulation contains arginine (eg, arginine/HCl) at a concentration of from about 50 mM to about 300 mM, such as from about 150 mM to about 225 mM.

Preferably, the pharmaceutical preparations disclosed herein are hypertonic (ie, have a higher osmotic pressure than human blood). As described herein, it has been surprisingly found that hypertonicity leads to a decrease in sample viscosity, which was achieved, for example, with a moderate increase in arginine concentration. As described in Example 2, it has surprisingly been found that low viscosity (eg, less than 25 cP) can be achieved for highly concentrated MASP-2 inhibitory antibody preparations in a citrate/arginine and histidine/arginine solution containing arginine at a concentration of 200 mM or higher , in the absence of CaCl ₂ . Accordingly, in some embodiments, the formulation contains arginine (eg, arginine-HCl) at a hypertonic level of about 200 mM to about 300 mM.

As further described in Example 2, it has also been found that formulations containing divalent cations (CaCl ₂ or MgCl ₂ ) have an increased content of high molecular weight material compared to formulations that do not contain added CaCl ₂ or MgCl ₂ . Accordingly, in one embodiment, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention contains substantially no added CaCl ₂ . In one embodiment, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention contains substantially no MgCl ₂ additive.

As further described in Example 2, it was found for high-concentration anti-MASP-2 antibody preparations that inclusion of sucrose was associated with increased polydispersity in all buffer systems tested. Accordingly, in one embodiment, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention contains substantially no sucrose.

As described in Example 2, it was also found for high-concentration anti-MASP-2 antibody preparations that the inclusion of sorbitol was associated with increased polydispersity in all buffer systems tested. Accordingly, in one embodiment, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention contains substantially no sorbitol.

Surfactants.

Optionally, in some embodiments, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention also contains a pharmaceutically acceptable surfactant. Non-limiting examples of suitable pharmaceutically acceptable surfactants include polyoxyethylene sorbitan fatty acid esters (eg, Tween), polyethylene polypropylene glycols, polyoxyethylene stearates, polyoxyethylene alkyl ethers (eg, polyoxyethylene monolauryl ether), alkylphenyl polyoxyethylene ethers (eg, Triton-X), polyoxyethylene copolymer. polyoxypropylene (such as poloxamer and Pluronic) and sodium dodecyl sulfate (SDS). In specific embodiments, the pharmaceutically acceptable surfactant is a polyoxyethylene sorbitan fatty acid ester (polysorbate), such as polysorbate 20 (sold under the trade name Tween 20™) and polysorbate 80 (sold under the trade name Tween 80™). In some embodiments, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention contains a nonionic surfactant. The nonionic surfactant may be a polysorbate (eg, selected from the group consisting of polysorbate 20, polysorbate 80, and polyethylene-polypropylene copolymer). In some embodiments, the surfactant concentration is from about 0.001 to 0.1% (w/v), or from 0.005 to 0.1% (w/v), or from 0.01 to 0.1% (w/v). /vol.), or 0.01-0.08% (wt./vol.), or 0.025-0.075% (wt./vol.), or more specifically about 0.01% (wt./vol.) , about 0.02% (w/v), about 0.04% (w/v), or about 0.06% (w/v), or about 0.08% (w/v). vol.), or approximately 0.10% (w/v). In some embodiments, the formulation contains a nonionic surfactant (eg, polysorbate 80) in

- 12 043928 concentrations of approximately 0.001-0.1% (w/v), or from 0.005 to 0.1% (w/v), or 0.01-0.1% (w/v). ), or 0.01-0.08% (w/v), or 0.025-0.075% (w/v), or more specifically about 0.01% (w/v), about 0 .02% (w/v), about 0.04% (w/v), or about 0.06% (w/v), or about 0.08% (w/v). ), or approximately 0.10% (w/v). As described in Example 2, it was surprisingly found that inclusion of the nonionic surfactant polysorbate 80 (PS-80) resulted in a further reduction in viscosity while maintaining protein yield, allowing the use of a high concentration of the OMS646 antibody while maintaining a low viscosity. suitable for use in an injection device such as an auto-injector.

Stabilizers.

Optionally, in some embodiments, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention also contains a stabilizer. A stabilizer (the term is synonymous with the term stabilizing agent as used herein) may be a carbohydrate or saccharide or sugar recognized by regulatory authorities as a suitable additive or excipient in pharmaceutical preparations, for example, trehalose or sucrose. A typical stabilizer concentration is 15-250 or 150-250 mM or approximately 210 mM. The formulations may contain a secondary stabilizer such as methionine, for example, at a concentration of 5-25 mM or at a concentration of 5-15 mM (eg, methionine at a concentration of about 5 mM, about 10 mM, or about 15 mM).

Preservatives.

Optionally, in some embodiments, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation of the present invention also contains a preservative (eg, an antimicrobial agent). Antimicrobial agents are generally required for parenteral drugs intended for repeated use. Similarly, preservatives are added to pharmaceutical preparations aseptically packaged in single-dose vials if the active ingredient(s) are not bactericidal or bacteriostatic or stimulate the growth of microorganisms. Some typical preservatives used are benzyl alcohol (0.9 to 1.5%), methylparaben (0.18 to 0.2%), propylparaben (0.02%), benzalkonium chloride (0.01 to 0. 02%) and thimerosal (from 0.001 to 0.01%).

Possibility of administration by syringe.

For subcutaneous administration, injections must be performed using injection devices such as syringes, auto-injectors, wearable pumps, or other devices that impose limitations on the drug in terms of injected volume and solution viscosity. In addition, the drug must be suitable for use in the injection device in terms of injection force, time required to perform the injection. As used herein, the term syringability refers to the ability of an injectable therapeutic agent to readily pass through a hypodermic needle when drawn from a vial prior to injection. As used herein, the term needle patency refers to the performance of a drug during injection (see, for example, Cilurzo F., Selmin F., Minghetti P. et al., Injectability Evaluation: An Open Issue, AAPS PharmSciTech, 2011, 12( 2):604-609). Syringeability includes factors such as ease of withdrawal from the container, tendency for needle clogging and foaming, and accuracy of dose measurement. Needle patency includes the applied pressure (or force) required to achieve the injection, uniformity of flow, and absence of clogging (i.e., not clogging the syringe needle). The possibility of syringing and patency through the needle may depend on the geometric parameters of the needle, i.e. internal diameter, length, orifice shape, and surface finish of the syringe, especially in self-injection devices such as pens and auto-injectors (e.g., those equipped with 29-31 GA needles), and in prefilled hypodermic syringes ( for example, equipped with 24-27 GA needles). Injection force (or sliding friction force) is a complex factor dependent on solution viscosity, needle size (ie needle gauge) and container/closure surface tension. Smaller needles, such as smaller gauges, will cause less pain to patients. Overcashier et al. found that the viscosity-sliding friction force relationship depends on the needle gauge according to the Hagen-Poiseuille equation (Overcashier et al., Am. Pharm. Rev., 9(6):77-83 (2006)). For example, in the case of a 27-gauge thin-walled needle, the fluid viscosity must be maintained at or below 20 cP so that the sliding frictional force of 25 newtons (N) is not exceeded.

In specific embodiments, the pharmaceutical preparations of the invention have an injection friction force of about 25 N or less when injected through a 27 GA (1.25) needle at room temperature.

In specific embodiments, the pharmaceutical preparations of the invention have an injection friction force of about 20 N or less when injected through a 25 GA needle (1) at room temperature.

As described in Example 3, the highly concentrated, low viscosity MASP-2 inhibitory antibody preparations (e.g., OMS646) of the present invention are surprisingly well characterized.

- 13 043928 with the possibility of administration with a syringe and passability through a needle. The highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations disclosed herein can be administered by intramuscular or subcutaneous injection through small-gauge needles typically used for such injections, e.g., 27 G (1.25), 27 G thin-walled, 25 G thin-walled (1) or 25 G (1). In some cases, the low viscosity of the MASP-2 inhibitory antibody preparations disclosed herein allows for a tolerable injection volume (eg, 1-3 ^cc ) to deliver an effective amount of MASP-2 inhibitory antibody in a single injection to a single injection site.

Stability.

For any of the foregoing aspects, it should be noted that the MASP-2 inhibitory antibody, or antigen binding fragment thereof, in the formulation retains the ability to inhibit MASP-2 dependent complement activation. For example, a MASP-2 inhibitory antibody retains the ability to bind MASP-2 and inhibit the lectin pathway activation, as described in Example 1, or in another lectin complement activation pathway assay, for example, as described in WO 2012/151481. In addition to potency assays, a variety of other physicochemical assays can be used to assess stability, including isoelectric focusing, polyacrylamide gel electrophoresis, size exclusion chromatography, and visible and non-visible particulate matter.

In specific embodiments, the formulations of the present invention exhibit stability in the temperature range of -20 to 8°C for at least 30 days, up to at least 9 months or longer, or up to at least 12 months or longer, wherein as evidenced by the results of the stability studies in Examples 2 and 4. Additionally or alternatively, in certain embodiments, the formulations are stable at a temperature of -20 to 8°C, such as 2 to 8°C, for at least 6 months, at least 1 years, or at least 2 years, or longer. In certain embodiments, stability can be assessed, for example, by maintaining a level of purity over time. For example, in specific embodiments, the formulations of the present invention exhibit a less than 5% reduction, such as a less than 4% reduction, such as a less than 3% reduction, such as a less than 2% reduction, such as a less than 1% reduction, in purity through month, 6 months, 9 months or 1 year when stored at a temperature of 2 to 8 ° C, which is determined by size exclusion chromatography (SEC), which allows you to detect the presence or absence of fragments (FMM) and/or aggregates (AGM) of molecules.

In specific embodiments, the formulations of the present invention have low or undetectable levels of aggregation and/or fragmentation and remain active after storage for a certain period of time. In other words, the formulations disclosed herein are capable of maintaining the structural integrity of the MASP-2 inhibitory antibody OMS646 present at high concentrations in solution, for example, at concentrations greater than 150 mg/ml, or greater than 175 mg/ml, or at least 185 mg/ml, such that the MASP-2 inhibitory antibody may remain predominantly monomeric (ie, at least 95% or more) after storage for a certain period of time at a temperature of from about 2 to 8°C. Preferably, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, and most preferably no more than 0.5% of the antibodies form fragmented (FMM) or aggregated (AMM) forms, as determined by the method SEC after storage for a certain period of time at a temperature of approximately 2 to 8°C.

As described in Example 4 herein, we provide formulations suitable for maintaining the MASP-2 inhibitory antibody, OMS646, at a concentration of about 185 mg/ml in a predominantly monomeric form for at least 12 months at a temperature of about 2 to 8 °C.

Tissue permeability modifier.

In another embodiment, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations of the present invention also contain a tissue permeability modifier that helps increase the absorption or dispersion of the MASP-2 inhibitory antibody following parenteral administration (eg, subcutaneous injection). In some embodiments, the tissue permeability modifier is a hyaluronidase enzyme that acts as a tissue permeability modifier and helps to increase the dispersion and absorption of the injected MASP-2 inhibitory antibody. A particularly useful tissue permeability modifier is hyaluronidase (eg, recombinant human hyaluronidase). Hyaluronidases act as tissue permeability modifiers by temporarily disrupting the hyaluronan barrier and allowing access to lymphatic and capillary vessels, allowing injected drugs and fluids to be rapidly absorbed into the circulatory system. Hyaluronan is naturally reduced and the barrier is completely restored, for example, within 48 hours. The addition of hyaluronidase to injectable pharmaceutical preparations helps to increase the bioavailability of the MASP-2 inhibitory antibody after parenteral administration, in particular subcutaneous administration. It also allows larger volumes (ie, greater than 1 mL) to be injected into the injection site with less pain and discomfort, and also minimizes the incidence of injection site reactions (eg, reduces swelling at the injection site).

In some embodiments, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation (eg, OMS646) of the present invention contains from about 100 U/ml to about 20,000 U/ml of hyaluronidase enzyme. The actual concentration of the hyaluronidase enzyme depends on the type of hyaluronidase enzyme used in the preparation of the MASP-2 inhibitory antibody preparations of the present invention. The effective amount of hyaluronidase can be determined by one skilled in the art. The enzyme must be provided in sufficient quantity to allow increased dispersion and absorption of the co- or sequentially administered MASP-2 inhibitory antibody. The minimum amount of hyaluronidase enzyme exceeds 100 units/ml. More specifically, the effective amount of the hyaluronidase enzyme is from about 150 U/ml to about 20,000 U/ml, which amount corresponds to from about 0.01 mg to about 0.16 mg of protein based on an assumed specific activity of 100,000 U/mg. In some embodiments, the pharmaceutical preparations contain hyaluronidase at a concentration of from about 1000 to about 20,000 U/ml, such as from about 1,000 to about 16,000 U/ml. Alternatively, the concentration of hyaluronidase is from about 1500 to about 12,000 U/ml, or more particularly from about 2,000 U/ml to about 12,000 U/ml. The amounts specified herein correspond to the amount of hyaluronidase initially added to the pharmaceutical preparation. In some embodiments, the ratio (by weight) of hyaluronidase to MASP-2 inhibitory antibody is in the range of 1:1000 to 1:8000, or in the range of 1:4000 to 1:6000, or in the range of about 1:4000 to 1 :5000.

Hyaluronidase may be present as a component of the highly concentrated, low viscosity MASP-2 inhibitory antibody preparation of the present invention, or it may be provided as a separate solution in the kit. Thus, in one embodiment, the MASP-2 inhibitory antibody is co-formulated with hyaluronidase. In another embodiment, the MASP-2 inhibitory antibody and hyaluronidase are formulated separately and mixed immediately prior to subcutaneous administration. In another embodiment, both the MASP-2 inhibitory antibody and the hyaluronidase are formulated and administered separately, eg, the hyaluronidase is administered by a separate injection immediately before or after administration of a preparation containing the MASP-2 inhibitory antibody. In some cases, the hyaluronidase is administered subcutaneously earlier, from about 5 seconds to about 30 minutes, than the pharmaceutical preparation containing the MASP-2 inhibitory antibody of the present invention is injected into the same injection site. In specific embodiments, the MASP-2 inhibitory antibody pharmaceutical preparation and the hyaluronidase solution are contained in separate chambers of the pharmaceutical device, which automatically delivers either simultaneously (eg, using a double-barreled syringe) or sequentially.

Pre-filled containers.

In another aspect of the present invention, the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation disclosed herein is contained in a prefilled sealed container in an amount sufficient to be administered to a mammalian subject. Thus, a sufficient amount of the drug composition formulated in accordance with the present invention that is equal to or slightly greater (i.e., no more than 25% in excess, e.g., no more than 10% in excess) is the MASP-2 inhibitory amount the antibody to be administered to a mammalian subject is contained in a pre-filled container to facilitate dispensing of the antibody preparation for parenteral administration (ie, injection or infusion). In some embodiments, the prefilled container contains at least one pharmaceutical unit dosage form of a MASP-2 inhibitory antibody.

For example, a single-use amount of a highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation may be packaged in a prefilled container, such as, for example, a glass vial, closed with a stopper or other cap containing a septum through which a hypodermic needle can be inserted for drug selection, or may be packaged in a prefilled syringe or other prefilled container suitable for injection (eg, subcutaneous injection) or infusion. Examples of such containers include, but are not limited to, vials, syringes, ampoules, bottles, cartridges and bags. Preferably, the containers are individual pre-filled disposable syringes, which may preferably be made of glass or a polymeric material such as cycloolefin polymers or acrylonitrile butadiene styrene (ABS), polycarbonate (PC), polyoxymethylene (POM), polystyrene (PS), polybutylene terephthalate (PBT) ), polypropylene (PP), polyethylene (PE), polyamide (PA), thermoplastic elastomer (TPE), as well as their combinations. The syringe barrel contains an elastomeric piston that can move along the barrel to force liquid contents through a needle attached to the barrel. In some embodiments, each syringe has a needle firmly attached.

In some embodiments, a highly concentrated, low-viscosity MASP-2 inhibitory antibody preparation disclosed herein is contained in a prefilled container selected from the group consisting of a syringe (e.g., a single-barreled or double-barreled syringe), a pen, a sealed vial (e.g., a dual-barrel vial), autoinjector, cassette, and pumping device (eg, body-worn patch pump, tethered pump, or osmotic pump). For subcutaneous delivery, the drug may be contained in a prefilled device suitable for subcutaneous delivery, such as, for example, a prefilled syringe, auto-injector, injection device (eg, INJECTEASE™ or GENJECT™ device), pen (such as GENPEN™), or other device suitable for subcutaneous administration.

The formulations of the present invention may be formulated in unit dosage forms in a pre-filled container, which may be particularly suitable for self-administration. For example, a unit dose per vial, cartridge, or other prefilled container (eg, prefilled syringe or disposable pen) may contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0 ,6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1 ,9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5 , 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 ml, or about 10.0 mL, or a greater volume, of a highly concentrated formulation containing a MASP-2 inhibitory antibody (e.g., OMS646) at varying concentrations ranging from about 100 mg/mL to about 250 mg/mL, from about 150 mg/mL to about 200 mg/ml, from about 175 mg/ml to about 200 mg/ml, such as about 185 mg/ml, which corresponds to a total unit dose of OMS646 per container ranging from about 20 mg to about 1000 mg or higher.

In some embodiments, the drug of the present invention is formulated as a unit dosage form in a prefilled container, such as a vial or syringe, with a unit dose of about 350 mg to 400 mg, such as about 350 mg, about 360 mg, about 370 mg, about 380 mg, approximately 390 mg, or approximately 400 mg.

In some embodiments, the formulations of the present invention are formulated in a unit dosage form in a pre-filled syringe with a volume of from 0.1 to 3.0 mL, such as about 0.1, 0.2, 0.3, 0.4, 0. 5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1, 8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 ml or approximately 3, 0 ml containing approximately 20 to 750 mg of a MASP-2 inhibitory antibody (eg, OMS646). As described herein, stable formulations formulated in unit doses may be administered directly to a subject (eg, by subcutaneous injection) or alternatively they are prepared for reconstitution prior to intravenous administration.

The preparations of the present invention can be sterilized by various sterilization methods suitable for antibody preparations, such as filtration sterilization. In certain embodiments, the antibody preparation is sterilized by passage through a filter, such as a pre-sterilized filter with a pore diameter of 0.2 microns. Sterilized preparations of the present invention can be administered to a subject to prevent, treat, or alleviate a disease or disorder associated with MASP-2-dependent complement activation.

In a related aspect, the present invention provides a method of making an article comprising filling a container with a highly concentrated MASP-2 inhibitory antibody preparation of the present invention.

In one embodiment, the present invention provides a pharmaceutical composition for use in the treatment of a patient suffering from or at risk of developing a MASP-2-related disease or condition, which is a sterile single-use dosage form containing from about 350 mg to about 400 mg (t i.e. 350, 360, 370, 380, 390 or 400 mg) of a MASP-2 inhibitory antibody, wherein the composition contains from about 1.8 ml to about 2.2 ml (i.e. 1.8, 1.9 , 2.0, 2.1 or 2.2 ml) of a 185 mg/ml antibody preparation disclosed herein, wherein said antibody or fragment thereof comprises (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3, wherein the preparation is stable when stored at a temperature of 2 to 8° C. for at least six months.

In some embodiments, the MASP-2 dependent disease or condition is selected from the group consisting of aHUS, HSCT-TMA, IgA-H, and lupus nephritis (LN).

Kits containing highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations.

The present invention also relates to therapeutic kits comprising at least one container containing a highly concentrated, low-viscosity inhibitory drug.

- 16 043928

MASP-2 antibodies disclosed herein.

In some embodiments, the present invention provides a kit comprising (i) a container containing any of the MASP-2 inhibitory antibody preparations described herein; and (ii) a suitable means for delivering the drug to the patient who needs it.

In some embodiments of any of the kits described herein, the agent is suitable for subcutaneous delivery of the drug to a patient.

Various types of containers are suitable for containing the pharmaceutical preparations of the MASP-2 inhibitory antibody included in the kits of the present invention. In specific embodiments of the kits of the present invention, the container is a prefilled syringe (eg, a single-barreled or double-barrel syringe) or a prefilled sealed vial.

In some embodiments, the container containing the MASP-2 inhibitory antibody drug is a prefilled container selected from the group consisting of a syringe (e.g., a single-barreled or double-barreled syringe), a pen, a sealed vial (e.g., a dual-chamber vial) , autoinjector, cassette, and pumping device (eg, body-worn patch pump, tethered pump, or osmotic pump). For subcutaneous delivery, the drug may be contained in a prefilled device suitable for subcutaneous delivery, such as, for example, a prefilled syringe, auto-injector, injection device (eg, INJECT-EASE™ or GENJECT™ device), pen (such as GENPEN™ ) or other device suitable for subcutaneous administration.

In addition to a container pre-filled with a single dose of pharmaceutical preparation, the kit of the present invention may also include an outer container in which the pre-filled container is placed. For example, the outer container may include a plastic or cardboard tray with a formed recess into which the prefilled container is placed and held stationary during transport and handling prior to use. In some embodiments, the outer container is preferably opaque and protects the prefilled container from light, preventing light-induced degradation of the pharmaceutical components. For example, a plastic or cardboard tray containing a pre-filled container may be additionally packaged in a cardboard box that provides protection from light. The kit of the present invention may also include a set of instructions for administration and use of the MASP-2 inhibitory antibody preparations of the present invention, which may be printed on the outer container or printed on a sheet of paper contained within the outer container.

In some embodiments, the kits include a second container (eg, a prefilled syringe) containing an effective dose of hyaluronidase.

The kit may also include other materials required from a commercial and user point of view, including needles, syringes, package inserts, etc.

Illustrative preparations.

As described above, the stable, high-concentration, low-viscosity MASP-2 inhibitory antibody preparations of the present invention contain a MASP-2 inhibitory antibody at a concentration of 50 to 250 mg/ml in an aqueous solution containing a buffer having a pH of 4.0 to 8.0.

A buffer system, such as histidine, citrate or succinate, is preferably included at a concentration of from about 10 mM to about 50 mM, and preferably about 20 mM. In some preferred embodiments, the formulation also contains a charged side chain amino acid at a concentration of 50 to 300 mM. In some embodiments, the formulation contains a positively charged side chain amino acid, such as arginine, at a concentration of 50 to 300 mM. In some preferred embodiments, the formulation also contains a nonionic surfactant, such as polysorbate 80, in an amount of from 0.001 to 0.1% (w/v), such as from about 0.05% (w/v) to about 0 .1% (w/v). In some embodiments, the formulation also contains the enzyme hyaluronidase in an amount effective to increase dispersion and/or absorption of the MASP-2 inhibitory antibody following subcutaneous administration.

In some embodiments, the stable, highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations of the present invention contain, consist of, or consist primarily of the following compositions:

a) 100-200 mg/ml MASP-2 inhibitory antibody, 10-50 mM histidine buffer pH from about 5.0 to about 7.0, 100-225 mM arginine, and optionally 100-20,000 U/ml hyaluronidase;

b) 100-200 mg/ml MASP-2 inhibitory antibody, 10-50 mM histidine buffer pH from about 5.0 to about 7.0; 100-225 mM arginine, about 0.01 to 0.08% (w/v) non-ionic surfactant, and optionally 100-20,000 U/ml hyaluronidase;

c) 100-200 mg/ml MASP-2 inhibitory antibody, 10-50 mM citrate buffer with a pH of ca.

- 17 043928

5.0 to about 7.0, 100-225 mM arginine and optionally 100-20,000 U/ml hyaluronidase;

d) 100-200 mg/ml MASP-2 inhibitory antibody, 10-50 mM citrate buffer with a pH of ca.

5.0 to about 7.0, 100 to 225 mM arginine, about 0.01 to 0.08% (w/v) nonionic surfactant, and optionally 100 to 20,000 U/mL hyaluronidase;

e) 100-200 mg/ml MASP-2 inhibitory antibody, 10-50 mM succinate buffer pH from about 5.0 to about 7.0, 100-225 mM arginine, and optionally 100-20,000 U/ml hyaluronidase;

f) 100-200 mg/ml MASP-2 inhibitory antibody, 10-50 mM succinate buffer pH about 5.0 to about 7.0, 100-225 mM arginine, about 0.01 to 0.08% ( w/v) non-ionic surfactant and optionally 100-20,000 units/ml hyaluronidase.

In some embodiments, the stable, highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations of the present invention contain, consist of, or consist primarily of the following compositions:

g) 185 ± 18.5 mg/ml MASP-2 inhibitory antibody, 20 ± 2 mM citrate buffer pH approximately 5.8, 200 ± 20 mM arginine and optionally 100-20,000 U/ml hyaluronidase;

h) 185 ± 18.5 mg/ml MASP-2 inhibitory antibody, 20 ± 2 mM citrate buffer pH approximately 5.8, 200 ± 20 mM arginine, approximately 0.01% (w/v) polysorbate 80 and optionally 100-20000 units/ml hyaluronidase;

i) 185 ± 18.5 mg/ml MASP-2 inhibitory antibody, 20 ± 2 mM histidine buffer pH approximately 5.9, 200 ± 20 mM arginine and optionally 100-20,000 U/ml hyaluronidase;

j) 185 ± 18.5 mg/ml MASP-2 inhibitory antibody, 20 ± 2 mM histidine buffer pH about 5.9, 200 ± 20 mM arginine, about 0.01% polysorbate 80 and optionally 100-20,000 U/ml hyaluronidase.

Methods for producing highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations

In another aspect, the present invention relates to a method of producing a preparation containing 100 mg/ml or more of a MASP-2 inhibitory antibody, comprising (a) preparing a first pharmaceutical preparation containing purified OMS646, wherein the first pharmaceutical preparation has a first composition and contains no more than 50 mg/ml OMS646 protein;

(b) filtering the first pharmaceutical preparation to obtain a second pharmaceutical preparation, wherein the second pharmaceutical preparation has a second composition as a result of the filtration; and (c) concentrating the second pharmaceutical preparation to obtain a concentrated antibody solution containing 100 mg/ml or more OMS646.

The formulated bulk solution usually has a fixed protein concentration so that the desired volume dispensed into the container remains constant. A method for producing a liquid drug formulation typically involves mixing a MASP-2 inhibitory antibody with a buffer system, excipients, and optionally a surfactant, followed by aseptic filtration and dispensing into vials (or other containers such as syringes) and sealing (e.g., stoppering or lid or the like).

Table 1

Illustrative preparation 1

Ingredient (USP) added to water for injection Concentration OMS646 antibody 185 mg/ml Sodium citrate 20 mM L-arginine-NS! 200 mM Polysorbate 80 0.01%

table 2

Illustrative preparation 2

Ingredient (USP) added to water for injection Concentration OMS646 antibody 185 mg/ml L-Histidine 20 mM L-arginine-NS! 200 mM Polysorbate 80 0.01%

Methods of treatment.

In another aspect, the present invention relates to a method of treating a patient suffering from or at risk of developing a MASP-2-dependent complement-related disease or disorder,

- 18 043928 comprising the administration of a highly concentrated, low viscosity preparation containing a MASP-2 inhibitory antibody (eg, OMS646) disclosed herein.

As described in US Patent No. 7919094, US Patent No. 8840893, US Patent No. 8652477, US Patent No. 8951522, US Patent No. 9011860, US Patent No. 9644035, US Patent Application Publications No. US2013/0344073, US2013/0266560, US2015/ 0166675, US2017/0137537, US2017/0189525 and simultaneously pending US Patent Applications Serial Numbers 15/476154, 15/347434, 15/470647, 62/315857, 62/275025 and 62/527926 (assigned in each case to Omeros Corporation, successor to the present application, all referenced documents are incorporated herein by reference), MASP-2-dependent complement activation contributes to the pathogenesis of numerous acute and chronic disease conditions. For example, as described in US Pat. No. 8,951,522, the primary function of the complement system, part of the innate immune system, is to protect the host from infectious agents, but inadequate or excessive activation of the complement system can lead to serious disease such as thrombotic microangiopathies (TMAs, including aHUS). , TTP and HUS), in which endothelial damage and fibrin- and platelet-rich thrombi in the microvasculature lead to organ damage. The lectin pathway plays a dominant role in complement activation under conditions of endothelial cell stress or injury, and preventing activation of MASP-2 and the lectin pathway stops the sequence of enzymatic reactions leading to membrane attack complex formation, platelet activation, and lymphocyte recruitment. As described in US Pat. No. 8,652,477, in addition to initiating the lectin pathway, MASP-2 can also activate the blood coagulation system and can cleave prothrombin to thrombin.

As described in Example 1 and US Pat. No. 9,011,860, OMS646 is a potent inhibitor of lectin-dependent complement activation. This antibody does not bind significantly (with at least 5000-fold lower affinity) to the other serine proteases of the complement pathway, C1r, C1s, MASP-1 and MASP-3, and does not inhibit the classical complement pathway.

Accordingly, in some embodiments, the method comprises administering to a patient suffering from or at risk of developing a MASP-2-dependent complement-related disease or disorder any of the highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations disclosed herein in an amount sufficient to inhibit MASP-2 dependent activation of complement in a specified mammalian subject and thereby treating a disease or disorder. In some embodiments, the methods may use any of the kits or prefilled containers (eg, prefilled syringes or vials) described herein. In some embodiments, the method may further include, prior to administering the drug to a patient, determining that the patient has a disease or disorder associated with the lectin pathway of complement activation. In some embodiments, the method further comprises administering a tissue permeability modifier (eg, hyaluronidase) that enhances the absorption or dispersion of the MASP-2 inhibitory antibody following parenteral administration. The tissue permeability modifier can be administered concomitantly with the MASP-2 inhibitory antibody preparation or administered sequentially (eg, within 5 minutes of administration of the MASP-2 inhibitory antibody preparation at the same site or adjacent to the site of administration).

In some embodiments, the method includes administering to a subject in need thereof an injection from a first prefilled syringe containing a highly concentrated, low-viscosity preparation containing a MASP-2 inhibitory antibody (eg, OMS646) to inhibit MASP-2-dependent complement activation. In some embodiments, the method further comprises administering to the subject an injection from a second prefilled syringe containing a tissue permeation modifier, wherein the injection is performed at or adjacent to a site at which the MASP-2 inhibitory antibody was injected.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is a thrombotic microangiopathy (TMA), including thrombotic thrombocytopenic purpura (TTP), refractory TTP, Upshaw-Schulman syndrome (ASS), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), independent of factor H atypical hemolytic uremic syndrome, aHUS caused by infection, plasma therapy-resistant aHUS, TMA caused by cancer, TMA caused by chemotherapy, TMA caused by transplantation, or TMA associated with hematopoietic stem transplantation cells.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is a kidney disease, including, but not limited to, mesangioproliferative glomerulonephritis, membranous glomerulonephritis, membranous proliferative glomerulonephritis (mesangiocapillary glomerulonephritis), acute postinfectious glomerulonephritis (poststreptococcal glomerulonephritis), C3-glomerulopathy, cryoglobulinemic glomerulonephritis, weakly immune necrotizing crescentic glomerulonephritis, lupus nephritis, purpura

- 19 043928

Henoch-Schönlein and IgA nephropathy.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is renal fibrosis (eg, tubulointerstitial fibrosis) and/or proteinuria in a subject suffering from or at risk of developing chronic renal failure, chronic renal failure, glomerular disease (eg , focal segmental glomerulosclerosis), immune complex disease (eg, IgA nephropathy, membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial damage and glomerulonephritis (eg, C3 glomerulopathy), or a disease or condition associated with proteinuria, including but not limited to nephrotic syndrome, preeclampsia, eclampsia, toxic kidney disease, amyloidosis, vascular collagenosis (eg, systemic lupus erythematosus), dehydration, glomerular diseases (eg, membranous glomerulonephritis, focal segmental glomerulonephritis, C3 glomerulopathy, minimal changes, lipoid nephrosis), conditions during intense physical activity, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IgAnephropathy (i.e. Berger's disease), IgM nephropathy, membranous proliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport syndrome, diabetes mellitus (diabetic nephropathy), drug toxicity (eg, caused by NSAIDs, nicotine, penicillamine, lithium carbonate, gold and others heavy metals, ACE inhibitors, antibiotics (eg adriamycin) or opiates (eg heroin), or other nephrotoxins); Fabry disease, infections (eg, HIV, syphilis, hepatitis A, B or C, post-streptococcal infection, genitourinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell anemia, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (eg, kidney transplant rejection), Ebola hemorrhagic fever, nail-patella syndrome, familial Mediterranean fever, HELLP syndrome, systemic red lupus, Wegener's granulomatosis, rheumatoid arthritis, glycogen storage disease type 1, Goodpasture's syndrome, Henoch-Schönlein purpura, urinary tract infection spreading to the kidneys, Sjögren's syndrome and post-infectious glomerulonephritis.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is an inflammatory response resulting from tissue or organ transplantation, including, but not limited to, allotransplantation or xenotransplantation of whole organs (e.g., kidney, heart, liver, pancreas, lung, corneas, etc.) or tissue transplants (for example, valves, tendons, bone marrow, etc.).

In some embodiments, the MASP-2-dependent complement-related disease is ischemic and reperfusion injury (I/R), including, but not limited to, myocardial I/R, gastrointestinal I/R, renal I/R, and I/R. R after repair of an aortic aneurysm, I/R associated with the use of cardiopulmonary bypass, I/R of the brain after a stroke, after organ transplantation or reattachment of separated or injured limbs or fingers; due to revascularization of grafts and/or replants, as well as hemodynamic restoration after shock and/or surgical procedures.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is a complication associated with non-obese diabetes (type 1 diabetes or insulin-dependent diabetes mellitus) and/or complications associated with type 1 or type 2 diabetes (developing in adults), including but not limited to diabetic angiopathy, diabetic neuropathy, diabetic retinopathy or diabetic macular edema.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is a cardiovascular disease or disorder, including, but not limited to, Henoch-Schönlein purpura, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis ( also called malignant rheumatoid arthritis), hemorrhagic vasculitis and Takayasu syndrome; dilated cardiomyopathy; diabetic angiopathy; Kawasaki disease (arteritis); venous gas embolism (VGE) and suppression of restenosis after stent placement, rotational atherectomy and/or percutaneous transluminal coronary angioplasty (PTCA).

In some embodiments, the MASP-2-dependent complement-related disease or disorder is an inflammatory gastrointestinal disease, including, but not limited to, pancreatitis, diverticulitis, and bowel diseases, including Crohn's disease, ulcerative colitis, irritable bowel syndrome, and inflammatory bowel disease ( IBD).

In some embodiments, the MASP-2-dependent complement-related disease or disorder is a pulmonary disease, including, but not limited to, acute respiratory distress syndrome, transfusion-associated acute lung injury, ischemia/reperfusion acute lung injury, chronic obstructive pulmonary disease , asthma, Wegener's granulomatosis, disease with the formation of antibodies to the glomerular basement membrane (Goodpasture's syndrome), meconium aspiration syndrome, aspiration pneumonia, bronchiolitis obliterans, idiopathic pulmonary fibrosis, acute lung injury after a burn, non-cardiogenic pulmonary edema, transfusion-related blood respiratory depression and emphysema.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is an extracorporeal procedure-induced inflammatory response, and the method includes treating a subject undergoing an extracorporeal circulation procedure, including, but not limited to, hemodialysis, plasmapheresis, leukapheresis, extracorporeal membrane oxygenation (ECMO). , heparin-induced extracorporeal LDL precipitation (HELP) and cardiopulmonary bypass (CPB).

In some embodiments, the MASP-2-dependent complement-related disease or disorder is inflammatory or non-inflammatory arthritis and other musculoskeletal diseases, including, but not limited to, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis , ankylosing spondylitis or other spondyloarthropathy and crystalline arthropathy, muscular dystrophy and systemic lupus erythematosus (SLE).

In some embodiments, the MASP-2-dependent complement-related disease or disorder is a skin disease, including, but not limited to, psoriasis, autoimmune bullous dermatoses, eosinophilic spongiosis, bullous pemphigoid, epidermolysis bullosa acquisita, atopic dermatitis, herpes gravidarum, and other skin diseases , as well as thermal and chemical burns, including the capillary leak syndrome they cause.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is a disease or injury of the peripheral nervous system (PNS) and/or central nervous system (CNS), including, but not limited to, multiple sclerosis (MS), myasthenia gravis ( MG), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Guillain-Barre syndrome, reperfusion after stroke, osteochondrosis, brain injury, Parkinson's disease (PD), Alzheimer's disease (AD), Miller-Fisher syndrome, trauma and /or bleeding in the brain, traumatic brain injury, demyelination and meningitis.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is sepsis or a condition resulting from sepsis, including, without limitation, severe sepsis, septic shock, acute respiratory distress syndrome due to sepsis, hemolytic anemia, systemic inflammatory reaction or hemorrhagic shock.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is a genitourinary disease, including, but not limited to, painful bladder syndrome, overactive bladder syndrome, chronic nonbacterial cystitis and interstitial cystitis, male and female infertility, placental dysfunction , as well as spontaneous abortion and preeclampsia.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is an inflammatory response in a subject receiving chemotherapeutic agents and/or radiation therapy, including, without limitation, for the treatment of cancer conditions.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is an angiogenesis-dependent cancer, including, but not limited to, solid tumor(s), hematopoietic tumor(s), carcinoid tumors with high malignant potential, and tumor metastases.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is an angiogenesis-dependent benign tumor, including, but not limited to, hemangiomas, acoustic neuromas, neurofibromas, trachomas, carcinoid tumors, and pyogenic granulomas.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is an endocrine disorder, including, but not limited to, Hashimoto's thyroiditis, stress, anxiety, and other potential hormonal disorders involving controlled release of prolactin, growth factor, or insulin-like growth factor, and also adrenocorticotropin from the pituitary gland.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is an ophthalmic disease or disorder, including, but not limited to, age-related macular degeneration, glaucoma, and endophthalmitis.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is an angiogenic ocular disease or condition, including, but not limited to, age-related macular degeneration, uveitis, ocular melanoma, corneal neovascularization, primary pterygium, stromal herpetic keratitis, HSV-1 -induced corneal lymphangiogenesis, proliferative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, retinal vein occlusion, corneal graft rejection, neovascular

- 21 043928 glaucoma, vitreous hemorrhage due to proliferative diabetic retinopathy, optic neuromyelitis and redness of the iris.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is disseminated intravascular coagulation (DIC) or other complement-mediated bleeding disorder, including DIC due to sepsis, severe trauma, including neurological trauma (eg, acute trauma head, see Kumura et al., Acta. Neurochirurgica, 85:23-28 (1987), infection (bacterial, viral, fungal, parasitic), cancer, obstetric complications, liver disease, severe toxic reaction (eg, snake bite , insect bite, blood transfusion reaction), shock, heat stroke, transplant rejection, vascular aneurysm, liver failure, chemotherapy or radiation therapy for cancer, burn or accidental radiation.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is selected from the group consisting of acute radiation syndrome, dense sediment disease, Degos disease, catastrophic antiphospholipid syndrome (CAPS), Behçet's disease, cryoglobulinemia; paroxysmal nocturnal hemoglobinuria (PNH) and cold agglutinin disease.

In some embodiments, the MASP-2-dependent complement-related disease or disorder is selected from the group consisting of aHUS, HSCT-TMA, IgA-H, and lupus nephritis (LN).

Atypical hemolytic uremic syndrome (aHUS).

Atypical hemolytic uremic syndrome (aHUS) belongs to a group of conditions called thrombotic microangiopathies. In atypical HUS (aHUS), the disease is associated with defective complement regulation and can be either sporadic or familial. Familial cases of aHUS are associated with mutations in genes encoding complement activation or complement regulation proteins, including the proteins factor H, factor I, factor B, membrane complement cofactor CD46, and complement factor H-related protein 1 (CFHR1) and complement factor H protein 3 (CFHR3) (Zipfel, P.F. et al., PloS Genetics, 3(3):e41 (2007)). The unifying feature of this diverse array of genetic mutations associated with aHUS is a predisposition to increased complement activation at cell or tissue surfaces. A subject is at risk of developing aHUS following the onset of at least one or more symptoms consistent with aHUS (eg, anemia, thrombocytopenia, and/or renal failure) and/or the presence of thrombotic microangiopathy in a biopsy specimen obtained from the subject. Determining whether a subject is at risk for developing aHUS involves determining whether the subject has a genetic predisposition to developing aHUS, which can be determined by assessing genetic information (for example, from a database containing information about the subject's genotype) or by performing at least one genetic test. screening test for a subject to determine the presence or absence of a genetic marker associated with aHUS (i.e., determining the presence or absence of a genetic mutation associated with aHUS in the genes encoding factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactor CD46, complement component C3, complement factor H-related protein 1 (CFHR1), or the THBD gene (encoding the anticoagulant protein thrombomodulin), or genes encoding complement factor H-related protein 3 (CFHR3), or complement factor H-related protein 4 (CFHR4)), either by genome sequencing, gene-specific analysis (eg, PCR analysis), and/or determining whether the subject has a family history of aHUS. Methods for genetic screening for the presence or absence of a genetic mutation associated with aHUS are well known, for example, see Noris M. et al., Atypical Hemolytic-Uremic Syndrome, 2007 Nov 16 [modified 2011 Mar 10]; B: Pagon R.A., Bird T.D., Dolan C.R. et al., editors. GeneReviews™, Seattle (WA): University of Washington, Seattle.

As described in U S2015/0166675, in an ex vivo human experimental model of thrombotic microangiopathy (TMA), antibody OMS646 inhibited complement activation and thrombus formation on microvascular endothelial cells exposed to serum samples from patients with aHUS, both acutely and during remission. As further described in US 2017/0137537, data obtained from a phase 2 open-label clinical trial (IV dose of 2-4 mg/kg MASP-2 inhibitory antibody OMS646 once weekly for 4 consecutive weeks) showed that treatment with OMS646 was effective in patients with aHUS. Platelet counts in all three patients with aHUS in the medium- and high-dose groups (two in the medium-dose group and one in the high-dose group) returned to normal with a statistically significant mean increase from baseline values of approximately 68,000 platelets/mL (p=0. 0055).

Hematopoietic stem cell transplantation-associated TMA (HSCT-TMA).

Hematopoietic stem cell transplantation-associated TMA (HSCT-TMA) is a life-threatening complication caused by endothelial damage. The kidney is the most commonly affected organ, although HSCT-TMA can be a multisystem disease also affecting the lungs, intestines, heart, and brain. The development of even mild TMA is associated with long-term renal failure. The incidence of TMA associated with allogeneic HSCT varies depending on varying diagnostic criteria and pre-transplantation procedures, and

- 22 043928 also schemes for the prevention of graft-versus-host disease, with the most commonly used drugs being calcineurin inhibitors (But V.T. et al., Biol. Blood Marrow. Transplant, 11(8):571-5, 2005).

As described in US 2017/0137537, in a phase 2 clinical trial (IV dose of 4 mg/kg MASP-2 inhibitory antibody OMS646 once weekly for 4-8 consecutive weeks), administration of OMS646 resulted in improved TMA scores in patients patients suffering from HSCT-TMA, including statistically significant improvements in LDH and haptoglobin levels. HSCT-TMA patients treated with OMS646 are among the most difficult to treat, i.e. Clinical evidence of the therapeutic effect of OMS646 in patients with HSCT-TMA was obtained.

Immunoglobulin A - nephropathy (IqA-H).

Immunoglobulin A nephropathy (IgA-H) is an autoimmune kidney disease leading to intrarenal inflammation and kidney damage. IgA-H is the most common primary glomerulopathy worldwide. With an annual incidence rate of 2.5 per 100,000, the estimated incidence of IgA-H in the United States is 1 in 1,400 people. Approximately 40% of patients with IgA-H will develop end-stage renal disease (ESRD). Patients typically have microscopic hematuria with mild to moderate proteinuria and varying degrees of renal impairment (Wyatt R. J. et al., N. Engl. J. Med., 368(25):2402-14, 2013). Clinical indicators such as renal dysfunction, persistent hypertension and severe proteinuria (more than 1 g per day) are associated with a poor prognosis (Goto M. et al., Nephrol. Dial. Transplant, 24(10):3068-74, 2009 ; Berthoux F. et al., J. Am. Soc. Nephrol., 22(4):752-61,20 11). Proteinuria is the strongest prognostic factor, independent of other risk factors, in numerous large observational studies and prospective studies (Sorro R. et al., J. Nephrol., 18(5):503-12, 2005; Reich H.N. et al., J Am Soc Nephrol, 18(12):3177–83, 2007). It is estimated that 15-20% of patients achieve CRF within 10 years of disease onset if left untreated (D'Amico G., Am. J. Kidney Dis., 36(2):227-37, 2000). The diagnostic hallmark of IgA-H is abundant deposits of IgA alone or with IgG, IgM, or both in the glomerular mesangium.

As described in US2017/0189525, in a phase 2 open-label clinical trial for kidney disease (IV dose of 4 mg/kg MASP-2 inhibitory antibody OMS646 once weekly for 12 consecutive weeks), in patients with IgA nephropathy, treated with OMS646, there was a clinically significant and statistically significant decrease in urine albumin/creatinine ratio (uACR) throughout the trial and a decrease in 24-hour urine protein levels from baseline to the end of treatment.

Lupus nephritis (LN).

The main complication of systemic lupus erythematosus (SLE) is nephritis, also known as lupus nephritis, which is classified as a secondary form of glomerulonephritis. Up to 60% of adults with SLE have some degree of kidney disease in the later stages of the disease (Koda-Kimble et al., Koda-Kimble & Young's Applied Therapeutics: the clinical use of drugs, 10th ed, Lippincott Williams & Wilkins , pp. 792-9, 2012) with an incidence of 20-70 cases per 100,000 people in the United States. Lupus nephritis often occurs in patients with other symptoms of active SLE, including fatigue, fever, rash, arthritis, serositis, or central nervous system disease (Pisetsky D.S. et al., Med. Clin. North. Am., 81(1):113 -28, 1997). Some patients have asymptomatic lupus nephritis; however, with regular evaluation, abnormal laboratory results such as elevated serum creatinine levels, low albumin levels, or protein or sediment in the urine indicate active lupus nephritis.

As described in US Patent Application No. 15/470647, in a phase 2 open-label clinical trial for kidney disease (IV dose of 4 mg/kg MASP-2 inhibitory antibody OMS646 once weekly for 12 consecutive weeks) in 4 of 5 Patients with lupus nephritis (LN) treated with an anti-MASP-2 antibody were found to have a clinically significant decrease in 24-hour urine protein levels from baseline to the end of treatment.

Introduction.

The highly concentrated, low-viscosity MASP-2 inhibitory antibody preparations described herein can be administered to a subject in need of treatment by methods known in the art, such as single or multiple injections or infusions over a period of time, performed appropriately, e.g. subcutaneous, intravenous, intraperitoneal, intramuscular injection or infusion. As described herein, parenteral preparations may be formulated in a unit dosage form for ease of administration and uniformity of dosage. As used herein, the term unit dosage form means physically discrete units suitable as unitary dosages for a subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect, in combination with a selected pharmaceutical aqueous solution .

For the prevention or treatment of diseases, the appropriate dose of a MASP-2 inhibitory antibody will depend on the type of disease being treated, the severity and course of the disease. The antibody is preferably administered to the patient once or in a series of administrations. Depending on the type and severity of the disease, the MASP-2 inhibitory antibody can be administered in a fixed dose or in a dose calculated in milligrams per kilogram of body weight (mg/kg). Exemplary doses of MASP-2 inhibitory antibody contained in the formulations described herein include, for example, from about 0.05 mg/kg to about 20 mg/kg, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg, they can be administered daily, twice a week, once a week, once every two weeks or once a month.

Exemplary fixed doses of a MASP-2 inhibitory antibody, such as those described herein, include, for example, from about 10 mg to about 1000 mg, for example from about 50 mg to about 750 mg, for example from about 100 mg to about 500 mg, for example from about 200 mg to about 400 mg, for example about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 32 5 mg, about 350 mg, about 375 mg or about 400 mg, they can be administer daily, twice a week, once a week, once every two weeks or once a month.

With regard to delivery volume for drugs, the concentration of the antibody in the drug used therapeutically is determined by providing the antibody in a dose and volume that is tolerated and provides therapeutic benefit to the patient. In the case of an antibody therapeutic drug administered by injection, the concentration of the antibody will depend on the volume of injection (typically 0.5 to 3 ml). Antibody-based therapy may require dosages of several mg/kg once daily, weekly, monthly, or over several months. Accordingly, if a MASP-2 inhibitory antibody is to be administered at a dose of 1 to 5 mg/kg (e.g., 1, 2, 3, 4, or 5 mg/kg) of the patient's body weight, and the average patient's body weight is 75 kg, then from 75 to 375 mg of antibody will need to be administered in an injectable volume of 0.5 to 3.0 ml. Alternatively, the drug is administered at a concentration suitable for delivery to more than one injection site in each procedure.

In a preferred embodiment in which the concentration of OMS646 antibody in the formulation is about 185 mg/mL, to achieve a dose of 1 to 5 mg/kg of patient body weight (assumed to be 75 kg), the formulation must be delivered subcutaneously in an injectable volume of about 0 .40 ml to approximately 2.0 ml.

As described herein, the formulations of the present invention are suitable for both intravenous (IV) and subcutaneous (SC) administration.

Depending on the type and severity of the disease, a MASP-2 inhibitory antibody may be administered intravenously in a fixed dose or in a milligram per kilogram (mg/kg) dose. Exemplary doses of the MASP-2 inhibitory antibody contained in the formulations described herein may be delivered intravenously by diluting an appropriate amount of the highly concentrated formulation described herein with a pharmaceutically acceptable diluent prior to administration such that the MASP-2 inhibitory antibody is administered to the human subject at dose, for example from about 0.05 mg/kg to about 20 mg/kg, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, or 20 mg/kg, which can be administered daily, twice a week, once a week, once every two weeks, or once a month.

The MASP-2 inhibitory antibody can also be administered intravenously at a fixed dose by diluting an appropriate amount of the highly concentrated formulation described herein with a pharmaceutically acceptable diluent prior to administration such that the MASP-2 inhibitory antibody is administered to a human subject at a dose of, for example, about 10 mg to about 1000 mg, for example from about 50 mg to about 750 mg, for example from about 100 mg to about 500 mg, for example from about 200 mg to about 400 mg, for example about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, for example from about 300 mg to about 400 mg, for example about 310 mg, about 320 mg, about 325 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 375 mg, about 380 mg, about 390 mg, or about 400 mg, which can be administered daily, twice a week, once a week, once every two weeks, or once a month.

In some embodiments, the MASP-2 inhibitory antibody formulation is reconstituted in a pharmaceutically acceptable diluent prior to systemic (eg, intravenous) administration. Exemplary diluents that may be used include water for injection, 5% dextrose, 0.9% saline, Ringer's solution, and other pharmaceutically acceptable diluents suitable for intravenous administration. Without any limitation, exemplary doses of a MASP-2 inhibitory antibody administered intravenously to treat a subject suffering from a MASP-2-dependent complement-related disease or disorder include, for example, from about 0.05 mg/kg to about 20 mg /kg, for example approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg, they can be administer daily, twice a week, once a week, once every two weeks or once a month. Exemplary fixed doses of a MASP-2 inhibitory antibody administered intravenously to treat a subject suffering from a MASP-2-dependent complement-related disease or disorder include, for example, from about 10 mg to about

- 24 043928

1000 mg, for example from about 50 mg to about 750 mg, for example from about 100 mg to about

500 mg, e.g. about 200 mg to about 400 mg, e.g. about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about

375 mg or approximately 400 mg, they can be administered daily, twice a week, once a week, once every two weeks or once a month.

In some embodiments, the drug is diluted in a pharmaceutically acceptable diluent and administered to a subject in need thereof at an initial IV loading dose (e.g., from about 300 mg to about 750 mg, such as from about 400 mg to about 750 mg, such as from about 300 mg to about 500 mg, for example from about 300 mg to about 400 mg, for example about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg , about 380 mg, about 390 mg, or about 400 mg) followed by one or more subcutaneous injections of the drug at a dose of 1 to 5 mg/kg body weight or at a fixed dose of about 100 mg to about 400 mg, for example about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg. For example, an IV initial loading dose may be preferred in special cases, such as when a patient is in a hospital or clinic and is suffering from an acute condition (eg, aHUS) that requires an initial loading dose followed by maintenance doses. drug by subcutaneous injection.

Examples

The invention is further illustrated in the following examples, which should not be construed as limiting. All literature cited herein is expressly incorporated by reference.

Example 1.

This example shows that OMS646, a monoclonal antibody targeting human MASP-2, binds human MASP-2 with high affinity and blocks the lectin pathway of complement activation.

Introduction.

A fully human monoclonal antibody targeting human MASP-2 (sequence SEQ ID NO: 1), called OMS646, was prepared as described in WO 2012/151481, the contents of which are incorporated herein by reference. Monoclonal antibody OMS646 contains a heavy chain variable region (VH) with the sequence shown in SEQ ID NO: 2, and a light chain variable region (VL) with the sequence shown in SEQ ID NO: 3. OMS646 consists of variable regions from a human antibody, fused to the heavy chain and light chain constant regions of human IgG4 lambda, and is secreted as a disulfide-linked glycosylated tetramer consisting of two identical heavy chains (having the amino acid sequence given in SEQ ID NO: 4) and two identical lambda light chains (having amino acid sequence given in SEQ ID NO: 5). The asparagine (N) residue at position 295 of the heavy chain (SEQ ID NO: 4) is glycosylated and is shown in bold and underlined in the sequence.

Heavy chain variable region.

Below is the sequence of the heavy chain variable region (VH) of OMS646. Kabat CDR regions (31-35 (H1), 50-65 (H2) and 95-107 (H3)) are highlighted in bold; and Chothia CDR regions (26-32 (H1), 52-56 (H2) and 95-101 (H3)) are underlined.

Heavy chain variable region (VH) QMS646 (SEQ ID NO: 2).

OVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIROPPGKALEWLAHIFSSDEK

SYRTSLKSRLTISKDTSKNOWLMTTNMDPVDTATYYCARIRRGGIDYWGOGTLVTVSS

Light chain variable region.

Below is the sequence of the variable light chain (VL) region of OMS646. Kabat CDR regions (24-34 (L1); 50-56 (L2) and 89-97 (L3)) are underlined. These areas are the same when numbered under both the Kabat and Chothia systems.

Light chain variable region (VL) OMS646 (SEQ ID NO: 3).

OPVLTOPPSLSSVSPGOTASITCSGEKLGDKYAYWYOOKPGOSPVLVMYODKQRPSGIPE

RFSGSNSGNTATLTISGTOAMDEADYYCQAWDSSTAVFGGGTKLTVL

- 25 043928

Full length polypeptide (445 aa) of mutant IqG4 heavy chain as heavy chain

OMS646 (SEQ ID NO: 4).

QVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWLAHIFSSDEK

SRT PEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSL GK

Full length polypeptide (212 aa) light chain QMS646 (SEQ ID NO: 5).

QPVLTQPPSLSVSPGQTASITCSGEKLGDKYAYWYQQKPGQSPVLVMYQDKQRPSGIPE

RFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVLGQPKAAPSVTLFPPSSE ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTTPSKQSNNKYAASSYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPTECS

As described in WO 2012/151481, OMS646 binds MASP-2 and selectively inhibits the lectin pathway with little or no inhibition of the classical pathway (i.e., inhibits the lectin pathway while leaving the classical pathway unchanged), and also exhibits at least one or more of the following characteristics: the antibody binds human MASP-2 with a K _D value of 10 nM or less, the antibody binds an epitope in the CCP1 domain of MASP-2, the antibody inhibits C3b deposition in an in vitro assay in 1% human serum with a KD value of IC ₅₀ of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab' , F(ab) ₂ and F(ab') ₂ , the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation.

As described in WO 2012/151481, OMS646 was found to avidly bind human MASP-2 (SEQ ID NO: 1) with >5000-fold selectivity compared to C1s, C1r, MASP-1 or MASP-3. As shown in this example, OMS646 selectively binds human MASP-2 with high affinity and has the ability to block the lectin pathway of complement activation.

As shown above, OMS646 contains (a) a heavy chain variable region containing (i) CDR-H1 containing the sequence of amino acids 31-35 in SEQ ID NO: 2, ii) CDR-H2 containing the sequence of amino acids 50-65 in SEQ ID NO: 2, and iii) CDR-H3 containing the sequence of amino acids 95-107 in SEQ ID NO: 2; and (b) a light chain variable region containing

i) CDR-L1 containing the sequence of amino acids 24-34 in SEQ ID NO: 3, ii) CDR-L2 containing the sequence of amino acids 50-56 in SEQ ID NO: 3, and iii) CDR-L3 containing the sequence of amino acids 89- 97 at SEQ ID NO: 3.

As further described in WO2012/151481, an OMS646 variant is shown to have a heavy chain variable region with at least 95% identity to SEQ ID NO: 2 and a light chain variable region with at least 95% identity to SEQ ID NO: 3 , has functional activity similar to that of OMS646. The OMS646 variant described in WO 2012/151481 contains (a) a heavy chain variable region comprising: SEQ ID NO: 2 or a variant thereof having an amino acid sequence having at least 95% identity with SEQ ID NO: 2, with wherein residue 31 is R, residue 32 is G, residue 33 is K, residue 34 is M, residue 35 is G, residue 36 is V, residue 37 is S, residue 50 is L, residue 51 is A, residue 52 is H, residue 53 is I, residue 54 is F, residue 55 is S, residue 56 is S, residue 57 is D, residue 58 is E, residue 59 is is K, residue 60 is S, residue 61 is Y, residue 62 is R, residue 63 is T, residue 64 is S, residue 65 is L, residue 66 is K, residue 67 is S , residue 95 is Y, residue 96 is Y, residue 97 is C, residue 98 is A, residue 99 is R, residue 100 is I, residue 101 is R, residue 102 is R or A , residue 103 is G, residue 104 is G, residue 105 is I, residue 106 is D, and residue 107 is Y; and (b) a light chain variable region comprising: SEQ ID NO: 3 or a variant thereof, being an amino acid sequence having at least 95% identity with SEQ ID NO: 3, wherein residue 23 is S, residue 24 is is G, residue 25 is E or D, residue 26 is K, residue 27 is L, residue 28 is G, residue 29 is D, residue 30 is K, residue 31 is Y or F, residue 32 is A, residue 33 is Y, residue 49 is Q, residue 50 is D, residue 51 is K or N, residue 52 is Q or K, residue 53 is R, residue 54 is P , residue 55 is S, residue 56 is G, residue 88 is Q, residue 89 is A, residue 90 is W, residue 91 is D, residue 92 is S, residue 93 is S, residue 94 is T, residue 95 is A, residue 96 is V, and residue 97 is F.

1. OMS646 specifically blocks lectin-dependent activation of terminal complement components.

Methods.

The effect of OMS646 on membrane attack complex (MAC) deposition was analyzed using complement pathway-specific conditions for the lectin pathway, the classical pathway, and the alternative pathway. For this purpose, the Wieslab Comp300 complement screening kit (Wieslab, Lund, Sweden) was used according to the manufacturer's instructions.

Results.

Fig. 1A graphically illustrates the extent of lectin pathway-dependent MAC deposition in the presence of varying amounts of anti-human MASP-2 inhibitory antibody (OMS646). Fig. 1B graphically illustrates the extent of classical pathway-dependent MAC deposition in the presence of an inhibitory anti-human MASP-2 antibody (OMS646). Fig. 1C graphically illustrates the extent of alternative pathway-dependent MAC deposition in the presence of varying amounts of anti-human MASP-2 inhibitory antibody (OMS646). As can be seen in FIG. 1A, OMS646 blocks lectin activation pathway-mediated MAC deposition with an IC ₅₀ value of approximately 1 nM. However, OMS646 had no effect on MAC deposition resulting from the classical activation pathway (Fig. 1B) or from the alternative activation pathway (Fig. 1C).

Example 2.

Research prior to the formulation of OMS646.

Introduction/Rationale.

When creating a protein formulation with reduced viscosity, several factors must be considered, including, but not limited to, the nature of the protein, the concentration of the protein, the desired pH range, the temperature at which the protein formulation will be stored, the period of time for which the protein formulation will be stored, and how the protein formulation will be stored. will be administered to the patient. When a drug with reduced viscosity is injected, the protein concentration depends on the injected volume (usually from 1.0 to 2.25 ml). If the protein is to be administered at a dose of 2-4 mg/kg of patient body weight and the average patient's body weight is 75 kg, then 150-300 mg of protein would need to be administered in an injected volume of 1.0 to 1.62 ml. The viscosity is ideally maintained below 25 cP to allow realistic hypodermic injection of the therapeutic drug. In some embodiments, the viscosity is maintained below about 20 cP to allow delivery of the therapeutic drug by an injection device, as well as to allow the use of various biological treatments, such as flow filtration along the flow.

The primary objective of these studies was to identify formulation components that would achieve optimal chemical, physical and structural stability of the OMS646 antibody in a liquid formulation to produce a stable formulation with a viscosity of less than 25 cP, e.g. less than 20 cP, with a high concentration of OMS646 (100 mg/ ml or higher) suitable for subcutaneous injection into a human subject.

Analytical methods.

To test different combinations of buffer and excipients, a purified OMS646 antibody preparation (102 mg/ml in 20 mM sodium acetate, 50 mg/ml sorbitol, pH 5.0) was diluted to a concentration of ~1 mg/ml in selected drug solutions and 4-ml samples were placed in concentrators, previously washed with the appropriate buffer. Each sample was concentrated by centrifugation at 3200xg to a volume of ~1 ml. This procedure involved a total of three rounds of buffer replacement.

The appearance of the preparation was assessed using an Eisai Machinery inspection lamp, model MIH-DX, against water using a white and black background. Each sample of the drug was tested for color, transparency (opalescence) and the presence of small particles.

The protein content of OMS646 preparations was determined using an extinction coefficient of 1.49 mL/mgcm. Absorbance measurements at 280 nm corrected for absorbance at 320 nm were performed with

- 27 043928 using single-use UVette cuvettes and a path length of 0.2 cm. Samples were prepared in duplicate by diluting 1x with Dulbecco's phosphate-buffered saline (DPBS) to a final concentration of ~2 mg/ml. For highly concentrated samples, undiluted solutions were first diluted 1:1 in drug buffer and then diluted to a concentration of ~2 mg/mL in 1x DPBS. The results of duplicate measurements for each sample were averaged and the relative standard deviation (RSD) was calculated as a percentage. For any duplicate samples having >5% OCO, an additional set of diluted samples was prepared and measured.

Protein concentration was calculated as follows:

Adjusted A ₂₈₀ =A ₂₈₀ -A ₃₂₀

Protein concentration (mg/ml)=(adjusted A ₂₈₀ χdilution factor)/1.49 ml/mgxcm

To assess sample turbidity/light scattering, measurements were taken on 100 µL of undiluted sample at 320 nm in a 1 cm path length UVette disposable cuvette. For each sample, a blank with the appropriate protein-free replacement buffer was measured on the spectrophotometer. After measurements, samples were collected and used for pH analysis. To normalize the turbidity indicators to the sample concentration, the A320 indicator was also divided by the concentration in mg/ml and the resulting value was recorded in mEOPxml/mg.

pH measurements of all preparations and solutions were carried out at room temperature using a calibrated SevenMulti Meter (Mettler Toledo) with an automatic temperature compensation electrode.

The thermal stability of OMS646 preparations was monitored by differential scanning calorimetry (DSC). Melting temperature ( _Tm ) data for mAbs were obtained using MicroCal capillary DSC. Protein samples were diluted to a final concentration of ~2 mg/mL in the appropriate replacement buffer solution. Samples were assessed by DSC by scanning at 20-110°C at a speed of 1 or 2°C/min. The pre-scan thermostat was set to 10 min, the post-scan thermostat to 0 min, and the post-scan thermostat to 25°C. To analyze the T _m data, the buffer-scan was subtracted from the sample-buffer scan, and the thermogram was then normalized to the protein concentration (molar) using an estimated molecular mass of 150 kDa. A progressive baseline was obtained and subtracted from the data to facilitate determination of T _m . Melting points were determined using the peak selection function of the appropriate Origin® scientific data processing software.

Dynamic light scattering (DLS) measures time-dependent fluctuations in the intensity of light scattering from particles in a sample, and the Stokes-Einstein equation is used to calculate the hydrodynamic radius of the particle(s) in solution. DLS experiments for OMS646 preparations were performed with duplicate undiluted samples (30–40 μL) on a DynaPro™ Plate Reader II (Wyatt). A total of 10 individual scans were obtained at 25°C with an exposure time of 5 s. Viscosity was set to 1.019 cP phosphate-buffered saline. The resulting intensity distribution plots were compared to evaluate the effects of different drug components on the average particle size by intensity (outer diameter), the parameter of the overall width of the particle size distribution (overall percent polydispersity, or %PD), the average peak diameter of the OMS646 monomer (peak diameter 2), and parameter of the width of this peak (% PD of peak 2). The percentage polydispersity (total or peak 2) is a width parameter that reflects the heterogeneity found in the intensity distribution plot, where %PD <20% is an indicator of a nearly monodisperse solution and/or molecular conformation.

Stability against chemical denaturation was assessed using the AVIA Isothermal Chemical Denaturation System (Model 2304), which allows chemical stability to be tested under ambient conditions in an automated manner by generating a denaturant gradient by mixing constant volumes of protein formulated in drug buffer and drug buffer. containing urea. The briefly formulated protein was diluted to a concentration of 0.33 mg/mL in drug buffer. For a specific drug, a second drug buffer containing 10 M urea was also prepared. Due to solubility problems, 9 M urea solutions were prepared for preparations containing sucrose and sorbitol. After a standard incubation time (∼30 min), intrinsic protein fluorescence (i.e., tryptophan fluorescence) was measured for each data point, with chemical unfolding of the protein exposing buried tryptophan residues to the solvent, with a corresponding red shift of the fluorescence signal. For each drug, data were obtained with a total of 24 urea concentrations (0-9.0 M for 10 M urea stock solutions and 0-8.1 M for 9 M urea stock solutions), the ratio A ₃₅₀ /A ₃₃₀ was used for basic subtraction changes in fluorescence background, and used a nonlinear least squares fit to the unfolded conformational transition data using either a 2-state or 3-state model.

The viscosity of the preparations was determined using either a rolling ball viscometer or a rheometer. All viscosity measurements were carried out at 25°C with a shear rate ranging from 0.5 s ^-1 to

- 28 043928

1000 s ^-1 . For measurements using the rolling ball method, an Anton Paar AMVn viscometer was used. For rolling ball viscosity measurements, we measured the time it took for a gold ball to travel the distance in a capillary filled with a sample after tilting the capillary at a given angle (80°). The capillaries were tilted a total of three times and the results were averaged to determine the final dynamic viscosity, a value that is independent of the sample density. For rolling ball measurements, the capillary was first cleaned using d/i water and methanol. Instrument/capillary calibration was confirmed by measuring 10, 50 and/or 100 cP Brookfield viscosity standards. The capillary was re-cleaned with water and methanol before and between measurements of all samples.

Rheometer viscosity measurements were performed using a DV-III Ultra programmable rheometer, which was calibrated using Brookfield 10 and 50 standard viscosity fluids. Measurements were taken for 0.5 ml of each sample at different spindle speeds (shear rates). Samples exhibiting viscosity values (cP) of <10% RSD for all shear rates were considered Newtonian in this range, and samples whose viscosity was dependent on shear rate were considered non-Newtonian.

Density measurements were carried out using an Anton Paar DMA 4500M densitometer. Briefly, the device was washed several times with water and then with methanol. The instrument was calibrated for air and water before measuring the density of water as a sample. The device was again washed with water and methanol, and a single sample was measured on material with a concentration of ~175 mg/ml, combined from several preparations. This value was used as a sufficiently accurate approximation of the density of highly concentrated OMS646 preparations for use in gravimetric capacitance measurements.

Osmolality measurements were made using a freezing point depression osmometer (Multi-Osmette Osmometer, Precision Systems, model 2430), which measures the decrease in the freezing point of a solution as the solute concentration increases.

A Particle Liquid Counting System (Hach Model 9703, sensor model: HRLD-150) was used to determine particle size and content of OMS646 drug samples. Data for samples were obtained using one 500-μL collected sample (200 μL container volume). Briefly, the device was left to warm for ~30 min and both the syringe (1 mL) and system were rinsed with deionized water using at least 10 cycles before use. Environmental suitability was verified by demonstrating that 25 mL of deionized water contained no more than 25 particles >10 μm in size. The suitability of the system was confirmed by analyzing one 500-μl selected sample from 2, 5, 10, and 15-μm standards using appropriate channel sizes. If the determined cumulative count/mL met the specification for a particular standard, the system was considered suitable for sample testing. Before measuring the first sample, the system was washed once with 1x phosphate buffered saline (PBS) to ensure that the samples would not precipitate when in contact with deionized water. Samples were analyzed using a single 500-μl sample sample, and the cumulative counts/ml for the 2-, 5-, 10-, and 25-μm channels were determined to the nearest whole number.

Size exclusion chromatography (SEC) was used to evaluate the amount of aggregates and degradation products present in OMS646 preparations. Briefly, an Agilent 1100 HPLC system equipped with a G3000SWxl EC column (Tosoh, 7.8 x 300 mm, 5 μm particle size) was used. OMS646 samples were diluted to 2.5 mg/mL in SEC mobile phase (140 mM potassium phosphate, 75 mM potassium chloride, pH 7.0), and 20 μL of sample was injected onto the HPLC column. Chromatography was carried out at a flow rate of 0.4 ml/min, and the eluted protein was determined by absorbance at 280 nm (bandwidth 4 nm) without correction for the reference. To evaluate the suitability of the system, all samples were injected between injections of mobile phase blank and gel filtration standard, and the reference material was injected in duplicate at the beginning of the sequence. The percentages, individually and cumulatively, of high molecular weight (HMW) molecules and low molecular weight (LMW) molecules were determined in addition to the percentage of monomer and total integrated peak area.

SDS-gel capillary electrophoresis (SDS-CE) analysis under reducing conditions was performed using a Beckman Coulter PA 800 Plus capillary electrophoresis system and an FDM detector module using the SDS-MW Analysis Kit. Samples and standard were diluted to 1.0 mg/mL in SDS-MW sample buffer. To 95 μL of this working solution, 5 μL of β-mercaptoethanol and 2 μL of internal standard (10 kDa) were added. All samples were centrifuged at 300xg for 1 min, heated at 70 ± 2°C for ~10 min, transferred to a PCR tube and stored at 25°C until analysis. Separation was performed by applying 15 kV (reverse polarity) to the capillary for 30 min and using 20.0 psi inlet and outlet pressure. Data were acquired at 220 nm with a refresh rate of 4 Hz.

The reference (raw OMS646) was injected twice at the beginning of each sequence

- 29 043928 operations. Data for LC, NS and IgG were recorded.

Capillary SDS gel electrophoresis under non-reducing conditions was performed as described for CE-SDS under reducing conditions, except that a freshly prepared 250 mM iodoacetamide solution was used instead of the reducing agent, and separation was carried out for 35 min. The total electropherogram area and % IgG were recorded.

Purified antibody preparation OMS646 (102 mg/ml) was prepared by recombinant methods as described in WO 2012/151481, the contents of which are incorporated herein by reference. Briefly, the OMS646 antibody was produced in CHO cells containing expression constructs encoding the OMS646 heavy chain and light chain polypeptides and purified by standard methods.

1. Comparison of potential buffer systems.

Methods.

In pre-formulation studies, the stability of the MASP-2 inhibitory antibody OMS646 was first assessed using a panel of potential buffers, including those commonly used in antibody therapeutics (citrate, histidine, phosphate) as well as less common buffers (acetate, succinate). to cover a wide pH range (pH 4.0-8.0). For this study, the protein was converted to 20 mM succinate (pH 4.0, 5.0 and 5.5), acetate (pH 4.0, 5.0 and 5.5), citrate (pH 5.0, 6.0 and 7.0), histidine (pH 6.0 and 7.0) and phosphate (pH 6.0, 7.0 and 8.0) buffers using Amicon Ultra-4 (10 kDa HOMM) concentrators. Purified antibody preparation OMS646 (102 mg/ml in 20 mM sodium acetate, 50 mg/ml sorbitol, pH 5.0) was diluted to a concentration of ~1 mg/ml in each of 14 drug solutions and 4.0-ml samples were placed into concentrators previously washed with the appropriate buffer. Each sample was concentrated by centrifugation at 3200xg to a volume of ~1 ml. This procedure involved a total of three rounds of buffer replacement. During the final round of concentration, the protein was overconcentrated to a volume of <1 mL. The approximate volume and centrifugation time for each solution were recorded after each cycle.

Results.

Overall, the data obtained for the five buffer types were comparable in terms of buffer exchange rate, extracted protein content, differential scanning calorimetry (DSC) data, dynamic light scattering (DLS), and chemical stability (data not shown). Acetate, citrate, and histidine were selected for further evaluation based on the observed overall optimal thermal and conformational properties of OMS646 in the pH range of 5.5–6.0. Acetate was preferred over succinate at pH 5.5, mainly due to higher thermal stability, while histidine and citrate were preferred over phosphate at pH 6.0 based on DLS data.

2. Screening of excipients.

The stability of OMS646 was assessed in the presence of various excipients with known antibody stabilizing properties using buffer systems selected during the initial buffer screening (20 mM acetate, pH 5.5; citrate, pH 6.0; and histidine, pH 6.0). For this study, OMS646 was transferred into each of the potential buffers containing either 150 mM NaCl, 250 mM sorbitol, 250 mM sucrose, 150 mM L-arginine, 150 mM L-glutamate, or 250 mM L-proline using Amicon Ultra-4 concentrators ( 10 kDa NOMM). Samples were prepared as described for the buffer system comparison procedure, with the target protein concentration being 2.0 mg/mL.

Results.

In terms of protein recovery, protein recovery was estimated to be in the range of ~72-106%, which was a slight improvement over recovery in the absence of excipient. The histidine buffer seemed to be preferred for most excipients, while mixed results were obtained for the acetate and citrate buffers.

Based on the DSC results, it was noted that the citrate buffer resulted in thermal stabilization of OMS646 for all excipients tested. Fig. 2A graphically illustrates the results of a dynamic light scattering (DLS) analysis for screening excipients of the OMS646 formulation, which shows the particle outer diameter observed in formulations containing different potential excipients. Fig. 2B graphically illustrates the results of the DLS analysis for screening excipients of the OMS646 formulation, which shows the overall polydispersity observed in formulations containing different potential excipients. As can be seen in FIG. 2A and 2B, when analyzed by DLS, most drugs showed comparable results. However, in all buffer systems, the presence of sucrose was associated with increased polydispersity and the largest outer diameters and monomer diameters. After sucrose, sorbitol was the least preferred according to DLS results, in which case larger average particle sizes and increased polydispersity were observed. The remaining drugs were generally comparable when assessed by DLS, with monomer diameters of 10-12 nm (see Fig. 2A) and polydispersity <20%, indicating monodisperse populations (see Fig. 2B). Regarding stability under chemical denaturation conditions, when evaluated using the AVIA Isothermal Chemical Denaturation System, a buffer/pH dependence was clearly observed, with formulations in acetate buffer pH 5.5 denatured at urea concentrations of ~0.5 M is lower than preparations in citrate and histidine buffers at pH 6.0 for all excipients tested. The results for citrate and histidine buffers were comparable for all excipients.

In summary, the data suggested that citrate with a pH of approximately 6.0 was the optimal buffer/pH combination, which was used further in solubility studies. Given the unsatisfactory DLS data for all types of buffers, sucrose was excluded from further consideration.

3. Solubility/viscosity screening.

First viscosity study.

Methods.

To establish conditions for maximum solubility of OMS646, 20 mM citrate (pH 5.0 and 6.0) and 20 mM succinate (pH 4.0) were used in the presence of several isotonic combinations of NaCl, sorbitol, arginine, glutamate, and proline. Buffer exchange for OMS646 was carried out using Amicon 15 concentration cells over several cycles, and in the last cycle the volume of each solution was reduced to ~1 ml. Buffer exchange rates for all drugs and the number of exchange cycles were recorded and analyzed. After buffer exchange, protein content was measured, percent recovery was calculated, and samples were stored overnight at 5°C. It was found that when the drug was stored in succinate/glutamate, a precipitate formed and such drugs were not analyzed further. The remaining drugs were added to Amicon 4 concentration cells and concentrated until a target concentration of ~200 mg/mL was achieved or until centrifugation no longer resulted in volume reduction and/or it was determined that sample viscosity could not be changed (by sample manipulation).

Results.

In terms of buffer replacement rates, the highest buffer replacement rates were clearly observed in the pH 4.0 samples, with the highest replacement rates occurring in the succinate/sorbitol system. Substitution rates at pH 5.0 and 6.0 were comparable, with formulations containing only charged amino acid excipients exhibiting higher rates than other formulations. The lowest replacement rate was observed in the case of the drug in the citrate/sorbitol system at pH 6.0. This preparation was a single sample with a pH >5.0 and an uncharged excipient component. Assuming that the rate of substitution is a surrogate for OMS646 self-assembly, it appears that charged molecules are important in attenuating this phenomenon at a more neutral pH. According to the DLS results, all highly concentrated preparations had comparable outer diameters of ~12 nm, with the exception of the succinate/arginine system at pH 4.0, in which a distribution with an increased overall size of >18 nM was observed.

The samples in the replacement buffer were concentrated to the point where difficulties arose in handling the samples due to their high viscosity. Maximum concentrations exceeding 225 mg/ml were achieved for both drugs at pH 4.0. For higher pH formulations, maximum OMS646 protein concentrations ranged from 160.5 to 207.6 mg/mL. Viscosity for most formulations was assessed using a rolling ball viscometer at shear rates ranging from 0.5 to 1000 s ^-1 as described above. Fig. Figure 3 graphically illustrates the results of a viscosity assay to test the solubility of OMS646 over a range of protein concentrations in different formulations at pH 5.0 and 6.0. As shown in FIG. 3, in the graphs depending on the protein concentration, an exponential increase in viscosity in the preparations was observed, while the highest viscosity was recorded for the citrate/arginine/glutamate system with a pH of 5.0 (161.1 cP for a solution with a concentration of 196.6 mg/ml) . At pH 6.0 and a comparable OMS646 protein concentration, the citrate/sorbitol formulation had a significantly higher viscosity than the sorbitol/glutamate or proline/glutamate formulation. The drug in the citrate/arginine/glutamate system with a pH of 6.0 (95.3 mg/ml) had approximately half the viscosity (5.8 versus 9.3 cP) compared to the sample in the citrate/NaCl system with a pH of 6.0 ( 87.5 mg/ml) with higher protein content, indicating the greater importance of charged amino acids compared to ionic excipients.

It is important to note that at a particular concentration (i.e., 125 mg/mL), the viscosity varies dramatically depending on the drug. The viscosity is ideally maintained below ~25 cP to ensure realistic syringability of the therapeutic drug for subcutaneous administration. In some embodiments of the OMS646 formulation, the viscosity is maintained below about 20 cP so that the therapeutic drug can be delivered by an injection device and further so that it can be subjected to various biological treatments, such as in-line flow filtration.

Second viscosity study.

In an attempt to reduce the viscosity of the OMS646 formulation and thus maximize the concentration of OMS646 in a particular formulation, additional research was conducted. Based on the initial results, formulations were selected that were most likely to have low viscosity at high concentrations, namely formulations containing succinate/sorbitol at pH 4.0 and formulations containing glutamate and arginine in citrate buffer at pH 4.0. 6.0. Results from previous studies have shown that charged amino acids impart several beneficial properties to the formulation at neutral pH, including increased buffer exchange rates, increased sample recovery during processing, and reduced viscosity. The effect of amino acids with a positively charged side chain (eg, arginine) or amino acids with a negatively charged side chain (eg, glutamate) was assessed over a range of concentrations (50 to 150 mM) to examine the effect of charge and concentration on viscosity for both excipients. Finally, CaCl ₂ has been used as an additive in both isotonic and hypertonic citrate/glutamate solutions because of its viscosity-reducing potential, as described in US Pat. No. 7,390,786.

Samples were buffer exchanged and concentrated as described above. After replacing the buffer, the protein content was calculated for all preparations. The exception was a preparation containing 50 mM glutamate and 50 mM CaCl ₂ , in which a precipitate formed after replacing the buffer; it was not further evaluated. This was apparently due in part to the limited solubility of citrate and divalent cations such as Ca ²⁺ .

Results.

Fig. Figure 4 graphically illustrates the percent protein recovery after buffer exchange in the OMS646 solubility/viscosity study conducted with different drug candidates. As can be seen in FIG. 4, there was a tendency for recovery to increase with increasing arginine concentration, with the highest protein recovery of 85% observed in the case of 150 mM arginine for the drug. Recovery rates for the remaining drugs were comparable and ranged from 64-75%. The samples were then concentrated as described below until they were difficult to handle manually. The viscosity of all preparations was assessed as described above, and the results are shown in the table below. 3.

Table 3

Summary of viscosity data from preliminary drug studies

Sample Buffer Excipient Additive pH Conc. (mg/ml) Viscosity (cP) 100 cP standard (97.2 cP stated) - 97.1 50 cP standard (49.2 declared) - 49.1 S1 20 mM succinate 250 mM sorbitol — 4.0 209.3 109.6 S2 20 mM citrate 150 mM arginine - 6, 0 181.2 70.5 S3 20 mM citrate 100 mM arginine - 6, 0 170.8 102.8 S4 20 mM citrate 50 mM arginine - 6, 0 158.3 140.1 S5 20 mM citrate 150 mM glutamate — 6, 0 180.3 71.2 S6 20 mM citrate 100 mM glutamate — 6, 0 170.7 74.6 S7 20 mM citrate 50 mM glutamate - 6, 0 152.7 137.0 S8 20 mM citrate 150 mM glutamate 50 mM CaCl ₂ 6, 0 202.8 73.4

As shown in the table above. 3, the viscosity of all drugs was >70 cP, and despite the wide range of final concentrations, clear trends were observed. From these preliminary data it is clear that increasing the concentration of arginine or glutamate leads to a decrease in viscosity. The viscosity of the drug in the succinate/sorbitol system seemed comparable to the viscosity of drugs with 150 mM amino acid. The inclusion of CaCl ₂ led to a decrease in viscosity, and the viscosity of this preparation was comparable to samples with 10% less protein content.

Four drugs (S1, S2, S5 and S8, shown in Table 3) were selected for more detailed viscosity analysis, with the extracted undiluted samples being stepwise diluted in drug buffer to 25 mg/mL. Fig. Figure 5 graphically illustrates the dependence of viscosity (determined by exponential fitting of viscosity data) on protein concentration in the OMS646 solubility/viscosity study conducted with different drug candidates. Exponential fitting of viscosity data was performed using methods described in Connolly B. et al. , Biophysical Journal vol. 103:69-78, 2012. As shown in FIG. 5, the curves for preparations containing 150 mM glutamate and arginine were almost identical, demonstrating the highest viscosity per unit concentration - a viscosity of 25 cP corresponded to a concentration of ~150 mg/ml OMS646. Performance for the succinate and sorbitol formulation was slightly better, with a viscosity of 5 cP estimated to correspond to the concentration

- 32 043928

OMS646 ~160 mg/ml. In general, the lowest viscosity was observed in the case of CaCl ₂ -containing preparation, in which an estimated viscosity of 25 cP corresponded to a concentration of ~175 mg/ml. The most intriguing result of this analysis was that in a hypertonic preparation containing 150 mM glutamate and 50 mM CaCl ₂ , the viscosity was dramatically reduced. With the intention of obtaining a liquid formulation with the highest concentration possible, the use of divalent cations and hypertonicity to reduce viscosity was further used in additional viscosity analysis.

Third viscosity study.

Building on the results of the initial viscosity studies described above, additional research was conducted to determine whether the apparent viscosity-reducing ability of CaCl ₂ was due to divalent Ca ²⁺ ions or to hypertonicity. The change of the predominant excipient from glutamate to arginine was made based on the increased rates of buffer exchange observed with arginine-containing preparations. Histidine was included due to the potential for Ca ²⁺ chelation with citrate, which could lead to precipitation. A set of samples was also used to evaluate the effect of pH and surfactant on sample viscosity, as well as the effect of CaCl ₂ and hypertonicity on the drug in a succinate/sorbitol system at pH 4.0. Samples were buffer exchanged and concentrated as described for previous viscosity studies. The viscosity of all formulations was measured using a rolling ball apparatus as described above. Viscosity data were normalized to a protein concentration of 170 mg/mL in the sample. This was accomplished by first calculating the theoretical viscosity from the measured protein content, applying exponential regression to the previously calculated viscosity/solubility data for the drug in the citrate/arginine system at pH 6.0 (y=0.0917 ^e0.0361x ). Normalized viscosity was calculated by multiplying the theoretical viscosity value for the citrate/arginine system with a pH of 6.0 at a protein concentration of 170 mg/ml (42.4 cP) by the measured viscosity/theoretical viscosity ratio (see Table 4, note b). The resulting normalized viscosity values show the trend more clearly by smoothing out the concentration-related variability (see Table 4 and Fig. 6).

Table 4

Viscosity summary for OMS646 (170 mg/ml) formulations

Form # Buffer/ pH Excipi ent Additive PS-80 Viscosity (cP) Average normal. END (mg/ml) Theor. viscosity (cP) ^a Approx. normal viscosity at 170 mg/ml (cP) ^b 100 cP standard (97.2 cP stated) 96, 9 — 1A 20 mM citrate pH 6.0 112.5 mM arginine 25 mM CaCl ₂ 38.8 165.5 36, 0 45.7 1B 112.5 mM arginine 25 mM CaCl ₂ 0.05% 41.7 168.5 40.2 44.0 2 150 mM arginine — — 20.8 155.7 25.3 34.9 3 150 mM arginine 25 mM CaCl ₂ — 20.1 157.0 26.5 32.2 4 200 mM arginine — — 22.3 169.1 41.0 23.1 5 225 mM arginine — — 20.2 169.0 40.9 20.9 6A 20 mM 112.5 25 mM - 34.1 165.4 35.9 40.4

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citrate pH 5.0 mm arginine CaC1 ₂ 6V 112.5 mM arginine 25 mM CaCl ₂ 0.05% 31.0 170.0 42.4 31.1 7 150 mM arginine — — 22.1 158.9 28.4 33.0 8 150 mM arginine 25 mM CaCl ₂ — 17.4 153.9 23.7 31.1 9 20 mM histidine pH 6.0 7 5 mM arginine 50 mM CaCl ₂ — 19.9 174.5 49.9 16, 9 10A 112.5 mM arginine 25 mM CaCl ₂ 27.9 169.6 41.8 28.4 10V 112.5 mM arginine 25 mM CaCl ₂ 0.05% 28.1 184.6 71.8 16, 6 eleven 135 mM arginine 10 mM CaCl ₂ — 34.1 167.1 38.2 37.9 12 150 mM arginine — — 35.5 156, 6 26, 1 57.7 13 200 mM arginine — — 20.2 167.2 38.3 22.3 14 225 mM arginine — — 16, 4 161.9 31.6 22.0 15 150 mM arginine 50 mM CaCl ₂ — 15.9 164.9 35.2 19.1 16A 20 mM succinate pH 4.0 125 mM sorbitol 50 mM CaCl ₂ — 19.5 172.7 46, 7 17.7 16V 125 mM sorbitol 50 mM CaCl ₂ 0.05% 18.1 168.7 40.4 19.0 17 250 mM sorbitol 50 mM CaCl ₂ — 15.5 157.2 26, 8 24.6 18 250 mM sorbitol — 16, 8 161.3 31.0 23.0

^a Theoretical viscosity was calculated using curve regression of the measured viscosity values in the citrate/arginine system at pH 6.0 (y=0.0917 ^e0.0361x ).

^b Theoretical viscosity at 170 mg/mL protein concentration in a citrate/arginine system at pH 6.0 (42.4 cP)x(measured viscosity/theoretical viscosity).

Fig. Figure 6 graphically illustrates viscosity data normalized to protein concentration in a viscosity study conducted with different OMS646 drug candidates based on the data in Table 1. 4. As shown in FIG. 6 in table. 4, for the citrate and histidine preparations, the normalized data set clearly shows that hypertonicity leads to a decrease in sample viscosity, with the main effect observed with only a small increase in arginine concentration. For example, the normalized viscosity of formulation 12 (20 mM histidine and 150 mM arginine) is 57.7 cP, compared with viscosities of 22.3 and 22.0 cP for histidine formulations containing 200 and 225 mM arginine, respectively. A similar trend was observed for drugs in the citrate/arginine system. There were no obvious benefits of including CaCl ₂ . Rather, it was surprising to find that, in the absence of CaCl _{2 ,} low viscosities (eg, less than 25 cP) were achieved for citrate/arginine and histidine/arginine formulations with arginine concentrations of 200 mM or more. Incorporation of 0.05% PS-80 resulted in a significant decrease in viscosity in two of the three formulations evaluated at pH >5.0. Finally, it appears that the viscosity values

- 34 043928 at pH 5.0 were slightly lower than those for comparable products at pH 6.0.

Taking into account the results obtained in the viscosity studies, hypertonicity due to arginine, the effect of the presence or absence of divalent cations, and drugs in the succinate/sorbitol system at pH 4.0 were further studied in surfactant screening studies to further evaluate the effect on physical, conformational and chemical stability of OMS646.

1. Surfactant screening.

The effect of surfactant on the stability of OMS646 was assessed for drug candidates selected in previous studies described herein. The following six drugs were analyzed in surfactant screening studies:

mM citrate, 200 mM arginine with pH 5.0;

mM citrate, 200 mM arginine with pH 6.0;

mM succinate, 250 mM sorbitol with pH 4.0;

mM histidine, 200 mM arginine with pH 6.0;

mM histidine, 75 mM arginine/50 mM CaCl ₂ with pH 6.0;

mM histidine, 75 mM arginine/50 mM MgCl ₂ with pH 6.0.

Each of the six drugs listed above was evaluated either without surfactant or in the presence of 0.01% PS-80, for a total of twelve unique drug variants. For each preparation, OMS646 was transferred to exchange buffer solutions (without PS-80), concentrated, quantified, and samples normalized to a protein concentration of 175 mg/mL. Each preparation was then separated and PS-80 was added to the corresponding samples to a final concentration of 0.01% (w/v).

Each of the formulated samples was subjected to mechanical stress by mixing, as well as freeze/thaw cycles. To test both types of stress, 0.5 mL of sample was transferred into four Type 1 borosilicate glass vials (2.0 mL) and the vials were sealed with FluroTec® stoppers. For stress by mixing, samples were placed in a microplate shaker at 600 rpm for ~60 h at room temperature. The control samples for the stirred samples were kept close to the shaker throughout the entire stress period. For freeze/thaw cycles, samples were frozen at −80°C for >60 min and then allowed to thaw at room temperature for a total of 5 freeze/thaw cycles. Stressed samples were stored at 2-8°C until analysis. The remaining sample was stored at 2-8°C as an unstressed control. External inspection, absorbance measurements at A ₂₈₀ / DLS and SEC were performed to evaluate the effect of surfactant on the aggregation and stability of OMS646.

Results.

After six OMS646 preparations were subjected to stress, drug-associated particles were not detected in any of the samples. The protein content was almost constant in all samples of a particular drug. Analysis of DLS data for samples subjected to freeze/thaw and stirring revealed only minor differences between preparations and results under different types of stress, with no clear overall trends associated with the inclusion of PS-80. The only exception was the pH 4.0 succinate/sorbitol formulation, in which inclusion of PS-80 resulted in high overall polydispersity (i.e., multimodal formulation) for frozen/thawed and stored at 5°C controls. This acidic preparation also showed signs of aggregation/self-assembly by DLS in the absence of PS-80 upon stirring.

SEC data were analyzed to evaluate any aggregation and/or degradation products occurring in the stressed preparations. Summary results are given in table. 5A-5D.

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Table 5A

EC summary data for OMS646 surfactant screening (2-8°C)

Form Buffer Excipient T Additive rn PS- 80 (%) Wednesdays, total number VMM (%) Avg. number of monomers (%) Avg. total quantity NMM (%) Untreated reference sample, average 3.7 96, 3 - 1 20 mM citrate 200 mM arginine — 5, 0 - 3.0 96, 3 - 2 0.01 3.1 96, 9 - 3 20 mM citrate 200 mM arginine — 6, 0 - 3.2 96, 8 - 4 0.01 3.3 96, 7 - 5 20 mM histide in 200 mM arginine 6, 0 - 3.3 96, 7 - 6 0.01 3.4 96, 6 — 7 20 mM succinate 250 mM sorbitol 4, 0 - 3.2 96, 6 0.2 8 0.01 3.2 96.5 0.2 9 20 mM HISTIDE in 7 5 mM arginine 50 mM CaCl ₂ 6, 0 - 3.3 96, 7 - 10 0.01 3.4 96, 6 — eleven 20 mM HISTIDE in 7 5 mM arginine 50 mM MdS1 ₂ 6, 0 - 3.4 96, 6 - 12 0.01 3.5 96.5 —

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Table 5 B

SEC summary data for OMS646 surfactant screening (freeze/thaw)

Form Buffer Excipient T Additive rn PS- 80 (%) Wednesdays, total number VMM (%) Avg. quantity of monomer a (%) Wednesdays, total number NMM (%) Untreated reference sample, average 3.7 96, 3 - 1 20 mM citrate 200 mM arginine 5, 0 - 3.1 96, 9 - 2 0.01 3.2 96, 8 - 3 20 mM citrate 200 mM arginine 6, 0 - 3.3 96, 7 - 4 0.01 3.3 96, 7 - 5 20 mM histide in 200 mM arginine 6, 0 - 3.3 96, 7 - 6 0.01 3.4 96, 6 — 7 20 mM succinate 250 mM sorbitol 4, 0 - 3.2 96, 6 0.2 8 0.01 3.2 96, 6 0.2 9 20 mM HISTIDE in 7 5 mM arginine 50 mM CaCl ₂ 6, 0 - 3.4 96, 6 - 10 0.01 3.4 96, 6 — eleven 20 mM HISTIDE in 7 5 mM arginine 50 mM MdS1 ₂ 6, 0 - 3.5 96, 6 - 12 0.01 3.5 96, 6 —

Table 5C

SEC summary data for OMS646 surfactant screening (25°C)

Form Buffer Excipient Additive rn PS- 80 (%) Wednesdays, total number VMM (%) Avg. quantity monomer (%) Wednesdays, total number NMM (%) Untreated reference sample, average 3.7 96, 3 - 1 20 mM citrate 200 mM arginine — 5, 0 - 3.1 96, 9 - 2 0.01 3.2 96, 8 - 3 20 mM citrate 200 mM arginine — 6, 0 - 3.3 96.7 - 4 0.01 3.4 96, 6 - 5 20 mM HISTIDE 200 mM arginine — 6, 0 - 3.3 96, 7 - 6 0.01 3.4 96, 6 - in 7 20 mM succinate 250 mM sorbitol 4, 0 - 3.3 96.5 0.2 8 0.01 3.3 96.5 0.2 9 20 mM HISTIDE in 7 5 mM arginine 50 mM CaCl ₂ 6, 0 - 3.4 96, 6 - 10 0.01 3.5 96.5 — eleven 20 mM HISTIDE in 7 5 mM arginine 50 mM MdS1 ₂ 6, 0 - 3.5 96.5 - 12 0.01 3.5 96.5 —

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Table 5D

SEC summary data for OMS646 surfactant screening (mixing)

Form Buffer Excipi ent Additive rn PS- 80 (%) Wednesdays, total number VMM (%) Medium, amount of monomer (%) Wednesdays, total number NMM (%) Untreated reference sample, average 3.7 96, 3 — 1 20 mM citra t 200 mM arginine 5, 0 - 3.0 97.0 - 2 0.01 3.2 96, 8 — 3 20 mM citra t 200 mM arginine 6, 0 - 3.3 96, 7 - 4 0.01 3.4 96, 6 — 5 20 mM histi DIN 200 mM arginine 6, 0 - 3.3 96, 7 - 6 0.01 3.4 96, 6 — 7 20 mM succinate 250 mM sorbitol 4, 0 - 2.8 97.0 0.2 8 0.01 3.3 96.5 0.2 9 20 mM GISTI DIN 7 5 mM arginine 50 mM CaCl ₂ 6, 0 - 3.4 96, 3 0.3 10 0.01 3.5 96.5 — eleven 20 mM GISTI DIN 7 5 mM arginine 50 mM MdS1 ₂ 6, 0 - 3.4 96, 6 - 12 0.01 3.6 96.5 —

As can be seen from table. 5A-5D, in general, the SEC data indicate that the OMS646 molecule generally does not respond to the inclusion of PS-80, as well as to stress in the form of freezing/thawing (Table 5B) and stirring (Table 5D), regardless of the presence surfactant. It was found that the worst performing OMS646 preparations were those containing added divalent cations (CaCl ₂ and MgCl ₂ ), while the content of high molecular weight material (HMW) in these samples was clearly increased compared to other samples and the lowest monomer levels were observed.

2. Stability analysis under stress and no stress conditions over 28 days.

After narrowing the ranges of potential buffer, excipient and surfactant combinations in the preliminary drug studies described above, formulations based on citrate and histidine buffers were formulated containing 200 mM arginine, with a pH range of 5.5-6.5, at high concentrations of 175 and 200 mg/ml OMS646 to identify the most suitable drug under both stress (40°C) and non-stress (5°C) conditions. Arginine was included at hypertonic levels (200 mM) due to its viscosity-lowering properties at this elevated concentration. Based on the results of statistical numerical optimization of data for the preliminary drug, it was found that the most suitable drug OMS646 was the drug in the 20 mM citrate and 200 mM arginine system. A panel of samples was also prepared to evaluate the effect of 0.01% PS-80 on citrate and histidine formulations.

Buffer exchange was performed as described above, and samples were concentrated and diluted to achieve target concentrations of 175 or 200 mg/mL OMS646. During this final standardization process, PS-80 was added to the respective preparations to 0.01%. Slides were sterilized by filtration using 0.22-μm sterile Millipore Ultrafree-CL GV concentrators. One vial of each drug was stored at 5°C and one at 40°C for a 28-day incubation period. Samples were analyzed at T ₀ and 28 days for concentration, appearance, turbidity, osmolality, pH, DLS, DSC, and viscosity. After a 28-day incubation, both 175 and 200 mg/mL succinate/sorbitol formulations of OMS646 stored at 40°C were found to have a gel-like consistency and were therefore excluded from the analysis.

Results.

Regarding the stability analysis, the pH values remained stable throughout the study, regardless of the drug and storage conditions. After 28 days, analysis by both SEC and SDS-CE showed a significant increase in the content of NMM in acidic preparations with pH 5.0 and 4.0, as a result of which these preparations were excluded from further consideration. For the pH 6.0 citrate/arginine and histidine/arginine formulations formulated with 0.01% PS-80, most values were nearly indistinguishable from the corresponding surfactant-free samples. However, the results of EC showed a decrease in the content of VMM by 0.2-0.6% in comparison with similar preparations not containing surfactant. Given the apparent viscosity-reducing properties of the surfactant, it was decided to include polysorbate-80 (PS-80) in the formulations for further study.

The concentration and viscosity values of a total of 10 drugs were tested after 28 days of storage at 5°C. Representative results are shown in table. 6.

Table 6

Viscosity of drugs after 28 days of storage at 5°C

Sample A drug Concentration, 2 8 days at 5°C (mg/ml) Viscosity (sp) 1 20 mM citrate, 200 mM arginine, pH 6.0, 175 mg/ml OMS646 153.4 10.6 2 20 mM histidine, 200 mM arginine, pH 6.0, 175 mg/ml OMS646 151.3 12.7 3 20 mM citrate, 200 mM arginine, pH 6.0, 200 mg/ml OMS646 170.5 27.4 4 20 mM histidine, 200 mM arginine, pH 6.0, 200 mg/ml OMS646 184.2 18.1 5 20 mM citrate, 200 mM arginine, 0.01% PS-80, pH 6.0, 175 mg/ml OMS646 159.2 9.0 6 20 mM histidine, 200 mM arginine, 0.01% PS-80, pH 6.0, 175 mg/ml OMS646 156.0 7.8 7 20 mM citrate, 200 mM arginine, pH 5.0, 175 mg/ml OMS646 143.2 9.8 8 20 mM histidine, 200 mM arginine, pH 5.0, 200 mg/ml OMS646 182.4 15.9 9 20 mM succinate, 250 mM sorbitol, pH 4.0, 175 mg/ml OMS646 150.6 14.5 10 20 mM succinate, 250 mM sorbitol, pH 4.0, 200 mg/ml 184.3 18.0

As can be seen from the table above. 6, drugs with higher concentrations had higher viscosity. Of particular interest was the observation that the inclusion of PS-80 led to a decrease in the viscosity of the preparations in both citrate (10.6 vs. 9.0 cP) and histidine (12.7 vs. 7.8 cP) buffers, while also maintaining protein extraction. This reduction in viscosity with the inclusion of PS-80 was beneficial in allowing higher concentrations of OMS646 to be achieved while maintaining a low viscosity, making it suitable for syringe administration in a clinical setting and also suitable for use in autoinjectors and other injection devices.

Summary of results.

The primary objective of these studies was to identify formulation components that would result in optimal chemical, physical and structural stability of the highly concentrated OMS646 antibody in liquid formulations. In addition, some viscosity experiments were conducted to obtain a final formulation with the highest concentration of OMS646 antibody that could be delivered by subcutaneous injection.

Several types of buffers, pH conditions, excipient and surfactant concentrations were evaluated in an iterative manner in studies aimed at evaluating buffer systems, excipients, solubility, viscosity, and surfactant testing. An initial baseline buffer evaluation study tested five different types of buffers (acetate, citrate, succinate, histidine, and phosphate) in the pH range of 4.0–8.0. Analysis by DSC, DLS, and also using a chemical denaturation system

- 39 043928

AVIA showed that more acidic and basic conditions are less suitable for the stability of the OMS646 antibody. Based on these results, acetate, citrate, and histidine buffer systems were selected for further evaluation.

Screening of excipients allowed us to evaluate the effect of NaCl, L-arginine, L-glutamate, L-proline, sucrose and sorbitol on the stability of the OMS646 antibody in each of the three selected buffer systems. Only citrate (pH 6.0) was selected for subsequent studies to maximize opportunities for further evaluation of excipients. Only sucrose was excluded as a potential excipient due to unsatisfactory light scattering data. Solubility studies revealed the ability of preparations in citrate buffer (pH 5.0 and 6.0), containing an isotonic combination of NaCl, sorbitol, arginine, glutamate and proline, to maintain high concentrations of the OMS646 antibody in solution. All formulations were concentrated to greater than 150 mg/mL OMS646 with no evidence of aggregation. However, the succinate/arginine and succinate/glutamate formulations showed signs of sedimentation/aggregation after short-term storage and were excluded from subsequent studies. Biophysical analysis of preparations in citrate buffer showed only minor differences in the case of different excipients at pH 6.0 and only a slight decrease in the content of BMM in similar preparations at pH 5.0.

Interesting data were obtained when measuring viscosity in this subset of samples, which indicated that the citrate/glutamate and succinate/sorbitol systems provided the lowest viscosity. Given the similar biophysical stability observed with the different excipients and the importance of obtaining a formulation with maximum OMS646 content, additional viscosity studies were performed. These viscosity studies identified divalent cations and/or mild hypertonicity as important factors in reducing the viscosity of the OMS646 antibody formulation at a more neutral pH. Both citrate (pH 5.0 and 6.0) and histidine (pH 6.0) were assessed in the presence of 200 mM arginine. Histidine pH 6.0 was also assessed in the presence of 75 mM arginine and either 50 mM CaCl ₂ or 50 mM MgCl ₂ .

Finally, the succinate/sorbitol system was tested at pH 4.0. All buffer/excipient combinations were tested in either the absence or presence of 0.01% PS-80 to determine whether the surfactant contributed to the stability of the OMS646 antibody under agitated and freeze/thaw conditions. All drugs appeared stable under the stress conditions used, regardless of surfactant. One striking result was the increase in OMS646 BMM content observed by SEC in preparations containing divalent cations. As a result, CaCl ₂ and MgCl ₂ were excluded from further consideration as excipients. A decrease in the purity of the OMS646 antibody was also observed in the succinate/sorbitol system, which was mainly due to the apparent increase in VMM impurities. While the differences between the preparation containing and not containing 0.01% PS-80 were minor, samples containing surfactant appeared to have slightly increased content of TMM (~0.1%) compared to their non-surfactant counterparts.

Example 3.

This Example describes a study in which three potential high-concentration, low-viscosity OMS646 formulations, selected based on the results of the preliminary formulation studies described in Example 2, were compared for syringability.

Introduction/Rationale.

The time and effort required to administer a manual injection (or the time required to administer an injection using an auto-injector) are important factors and may affect end users' ease of administration and subsequent compliance. The force required to inject a solution at a specific injection rate through a needle of a predetermined gauge and length is called syringe capability (see, e.g., Burckbuchler, V. et al., Eur. J. Pharm. Biopharm., 76(3), 351-356, 2010. Regarding syringeability, when injecting into a human subject, it is generally not advisable to exceed a force of 25 N (although higher viscosity formulations are available on the market).A 27 GA needle or a 27 GA thin wall needle is generally are considered standard needles for subcutaneous injection of monoclonal antibodies.A thin-walled 27 GA needle has an ID approximately the same as a 25 GA needle (smaller G number corresponds to a larger diameter).

The following study was conducted to determine the feasibility of syringe delivery of three potential high-concentration, low-viscosity OMS646 formulations.

Methods.

Based on the results of the preliminary drug studies described in Example 2, the following three potential high-strength OMS646 drugs were selected and further studied, as shown in Table 1. 7. In this example, preparations were prepared using arginine hydrochloride, polysorbate 80, if indicated, and either trisodium citrate or histidine with the pH adjusted to about 5.8-6.0 with hydrochloric acid.

- 40 043928

Table 7

Potential highly concentrated drugs OMS646

A drug Buffer/Excipients/Surfactant/ pH Concentration OMS 646 Protein content 1 20 mM citrate, 200 mM arginine, 0.01% PS-80, pH 5.8 185 mg/ml 187.1 2 20 mM histidine, 200 mM arginine, 0.01% PS-80, pH 5.9 185 mg/ml 188.2 3 20 mM citrate, 200 mM arginine, pH 5.8 185 mg/ml 193.3

1. Osmolality and viscosity of QMS646 drug candidates.

Osmolality and viscosity of three potential drugs prepared as shown in Table. 7 were determined by the methods described in example 2. The liquid of the drug was considered non-Newtonian in the case of % RSD>10 at the tested shear rates. The results are shown in table. 8.

Table 8

Osmolality and viscosity

A drug Buffer/Excipients/Surfactant/pH Concentration AND I Osmolality (mOsmol/kg) Viscosity (cP) Nature of the liquid 1 20 mM citrate, 200 mM arginine, 0.01% PS-80, pH 5.8 185 mg/ml 473 16, 1 Newtonian Skye 2 20 mM histidine, 200 mM arginine, 0.01% PS-80, pH 5.9 185 mg/ml 440 15.9 Newtonian okaya 3 20 mM citrate, 200 mM arginine, pH 5.8 185 mg/ml 468 21.3 Newtonian okaya

2. Possibility of syringe injection of potential OMS 646 drugs. Methods.

Syringeability analysis of the three OMS646 drugs was performed on average load and maximum load using 27 GA (1.25), 25 GA (1), and 25 GA thin-walled (1) needles. Each of the three replicates of each drug was administered once. Syringability results for samples are the average of three replicates.

Results.

The three drugs listed in table. 7 (containing OMS646 at a concentration of 185 mg/ml) were evaluated for syringeability using 27 GA (1.25), 25 GA thin-walled (1) and 25 GA (1) needles. The results shown are the average of three replicates. The results are shown in table. 9 and graphically illustrated in FIG. 7A and 7B. Fig. 7A graphically illustrates the average load (lbf) for three OMS646 drug candidates using 27 GA, 25 GA and 25 GA thin-wall needles. fig. 7B graphically illustrates the maximum load (lbf) for three OMS646 drug candidates using 2 7 GA, 2 5 GA and 25 GA thin wall needles.

- 41 043928

Table 9

Possibility of syringe injection of potential highly concentrated drugs OMS646

A drug Condition Average load (lbf) Maximum i load (lbf) Average load (N) Maximum load (N) 1 2 7 GA 4.72 5.07 20.99 22.55 2 5 GA 1.88 2.03 8.36 9.03 2 5 GA (thin-walled) 1.27 1.36 5.65 6, 05 2 27 GA 4.51 4.85 20.06 21.57 2 5 GA 1.84 1.99 8.18 8.85 25 GA (thin-walled) 1.26 1.32 5.60 5.80 3 2 7 GA 5.58 5.83 24.82 25.93 2 5 GA 2.29 2.51 10.18 11.16 2 5 GA (thin-walled) 1.50 1.60 6, 67 7.11

As described above, regarding the possibility of syringe administration, in the case of injection into a human subject, it is generally not advisable to exceed a force of 25 N. As can be seen from the table above. 9, all three high-strength OMS646 drug candidates had acceptable syringability (ie, force less than or equal to 25 N) when administered through 25 GA or 25 GA thin-wall needles. Drug #2 also had acceptable syringeability when administered through a 27 G needle. Addition of PS-80 at a concentration of 0.01% resulted in an unexpected improvement in syringability.

3. SEC analysis of potential QMS646 drugs after injection.

Size exclusion chromatography (SEC) was used to evaluate the amount of aggregates and degradation products present in three potential OMS646 formulations after injection. Briefly, an Agilent 1100 HPLC system equipped with a G3000SWxl EC column (Tosoh, 7.8 x 300 mm, 5 μm particle size) was used. OMS646 samples were diluted to a concentration of 2.5 mg/mL in SEC mobile phase (140 mM potassium phosphate, 75 mM potassium chloride, pH 7.0), and 20 μL of sample was injected onto the HPLC column. Chromatography was carried out at a flow rate of 0.4 ml/min, and the eluted protein was determined by absorbance at 280 nm (bandwidth 4 nm) without correction for the reference. To evaluate the suitability of the system, all samples were injected between injections of mobile phase blank and gel filtration standard, and the reference material was injected in duplicate at the beginning of the sequence. The percentages, individually and cumulatively, of high molecular weight (HMW) molecules and low molecular weight (LMW) molecules were determined, in addition to the percentage of monomer and total integrated peak area.

- 42 043928

Results.

The results of SEC analysis of highly concentrated OMS646 drug candidates after injection are shown in Table. 10.

Table 10

SEC analysis of highly concentrated OMS646 preparations after injection

A drug Condition % Purity % VII % BAT 1 Control 96.5 3.3 0.1 2 7 GA 96, 4 3.5 0.2 2 5 GA 96, 4 3.4 0.2 25 GA (thin wall) 96, 4 3.4 0.2 2 Control 96, 6 3.4 Not detected 2 7 GA 96.5 3.5 Not detected 2 5 GA 96.5 3.5 Not detected 25 GA (thin wall) 96.5 3.5 Not detected 3 Control 96.5 3.4 0.2 27 GA 96, 3 3.5 0.2 2 5 GA 96, 4 3.5 0.2 25 GA (thin wall) 96, 3 3.5 0.2

These results show little or no change in purity as determined by SEC after ejection of the drug through the needle.

Summary of results.

Results from the syringeability study indicated that all three potential high-strength formulations of OMS646 had acceptable syringability when tested using needles suitable for subcutaneous administration, and there was little or no change in purity for the OMS646 formulation after expulsion through the needle. The addition of PS-80 at a concentration of 0.01% led to an unexpected improvement in the ability to syringe a drug containing citrate and arginine.

Example 4.

This example describes a study that was conducted to evaluate the stability of potential high-concentration, low-viscosity OMS646 antibody formulations during long-term storage.

Methods.

This study was conducted to evaluate the stability of high-concentration subcutaneous injection preparations of the OMS646 antibody after long-term storage.

The following two potential drugs were evaluated:

a) 20 mM citrate, 200 mM arginine, 0.01% PS-80, pH 5.8 (185 mg/ml OMS646);

b) 20 mM histidine, 200 mM arginine, 0.01% PS-80, pH 5.9 (185 mg/ml OMS646).

Samples were placed in 13-mm, 2-mL USP Type I Schott glass tubes (West Pharmaceuticals), dispensing 1.0 mL of sample, sealed with 13-mm Fluorotec stoppers (West Pharmaceuticals), and sealed with 13FO aluminum snap caps (West Pharmaceuticals or equivalent). Sample tubes were stored at controlled temperatures in refrigerators to determine stability at -75±10, -20±5, 5±3°C, 25±2°C/60±5% RH and 40±2°C/75± 5% RH. A target quantity of at least 40 sample tubes for each drug was stored for this study. Samples stored as liquid were stored upside down, while frozen samples were stored upright. The required number of tubes were collected at the appropriate time points from the appropriate conditions, and the samples were characterized by the following parameters: appearance by visual inspection, protein content by A ₂₈₀ index, osmolality, HPLC-EC, pH and ELISA results for MASP-2. Illustrative SEC-HPLC data are given in table. 11 and show that the OMS646 antibody maintained its integrity after storage at 5°C for 6, 9 and 12 months. ELISA data confirmed that the antibody retained its functional properties after storage at 5°C for 6, 9 and 12 months.

- 43 043928

Results.

The results of this study are shown in table. 11 below.

Table 11

Stability of drugs when analyzed by SEC

A drug Time point Condition Total VMM (oligomer) (%) Main peak (monomer) (%) Total BAT (%) 185 mg/ml OMS646 20 mM citrate 200 mM arginine 0.01% polysorbate 80 pH 5.8 That n/a 3.9 96.1 - 1 month -20°С 2.5 97.5 - 5°С 2.6 97.4 - 25°C/60% RH 2.7 97.3 2 months -20°С 2.9 97.1 - 5°С 3.1 96, 9 - 25°C/60% RH 3.4 96, 6 3 months -20°С 2.8 97.2 - 5°С 2.9 97.1 - 25°C/60% RH 3.3 96, 0 0.7 6 months -20°С 1.7 98.3 - 5°С 1.9 98.1 - 25°C/60% RH 2.0 98.0 9 months 5°С 3.4 96, 6 - 25°C/60% RH 4.0 95.7 0.2 12 months 5°С 3.4 96, 6 185 mg/ml OMS646 20 mM histidine 200 mM arginine 0.01% polysorbate 80 That n/a 3.8 96.2 - 1 month -20°С 2.7 97.3 - 5°С 2.7 97.3 - 25°C/60% RH 2.9 97.1 2 months -20°С 2.9 97.1 -

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pH 5.9 5°С 3.3 96, 7 - 25°C/60% RH 3.3 96, 7 3 months -20°С 2.8 97.1 0.1 5°С 3.0 96, 9 0.1 25°C/60% RH 3.1 96, 1 0.8 6 months -20°С 1.8 98.2 - 5°С 1.9 98.1 - 25°C/60% RH 2.0 98.0

As can be seen from table. 11, little or no change in purity was observed in samples stored up to 9 months at -20°C or stored up to 12 months at 5°C, the planned storage temperature. The purity of samples stored at 25°C was also maintained for 2 months, but small changes in the degree of purity at 25°C were observed when stored for more than 9 months.

Example 5.

An exemplary formulation containing the MASP-2 inhibitory antibody OMS646 at pH 5.8 was prepared by combining OMS646 (185 mg/ml) with citrate (20 mM), arginine (200 mM), and polysorbate 80 (0.01%). Sodium citrate dihydrate (4.89 mg/mL) and citric acid monohydrate (0.71 mg/mL) were used to prepare the citrate buffer, with hydrochloric acid and/or sodium hydroxide used as needed to adjust the pH.

The viscosity of this preparation was measured using a capillary viscometer, and the results are shown in table. 12. There is a slight decrease in viscosity at higher shear rates, with all values being less than 13 cP.

Table 12

Viscosity of an exemplary OMS646 preparation measured at different shear rates

A drug Temperature (°C) Shear rate (1/s) Viscosity (cP) 185 mg/ml OMS646 20 mM citrate 200 mM arginine 0.01% polysorbate 80 pH 5.8 25.0 103000 12.2 25.0 156000 11.5 25.0 211000 11.0

It was found that administration of the exemplary 185 mg/mL OMS646 formulation described in this example to human subjects (both by subcutaneous injection and intravenous administration after dilution) resulted in sustained and potent inhibition of the lectin pathway.

While preferred embodiments of the invention have been illustrated and described, it should be understood that various changes in the disclosed preparations and methods may be made without departing from the spirit and scope of the invention. Therefore, the scope of a granted patent should be limited only by the definitions of the appended claims.

In accordance with the above, the invention covers the following embodiments.

1. A stable pharmaceutical preparation suitable for parenteral administration to a mammalian subject, comprising (a) an aqueous solution containing a buffer system having a pH of 5.0-7.0; and (b) a monoclonal antibody or fragment thereof that specifically binds human MASP-2, at a concentration of from about 50 mg/ml to about 250 mg/ml, wherein said antibody or fragment thereof contains (i) a heavy chain variable region, comprising CDR-H1, CDR-H2 and CDR-H3 in SEQ ID NO: 2, and (ii) a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 in SEQ ID NO: 3; or a variant thereof comprising a heavy chain variable region having at least 95% identity with SEQ ID NO: 2 and a light chain variable region having at least 95% identity with SEQ ID NO: 3, wherein the preparation has a viscosity of 2 to 50 centipoise (cP),

- 45 043928 wherein the drug is stable when stored at a temperature of 2 to 8°C for at least one month.

2. The pharmaceutical preparation according to claim 1, wherein the concentration of the antibody is from about

100 mg/ml to approximately 225 mg/ml.

3. The pharmaceutical preparation according to claim 1, wherein the concentration of the antibody is from about 150 mg/ml to about 200 mg/ml.

4. The pharmaceutical preparation of claim 1, wherein the concentration of the antibody is from about 175 mg/ml to about 195 mg/ml.

5. The pharmaceutical preparation according to claim 1 or 2, the viscosity of which is from about 2 cP to about 40 cP.

6. The pharmaceutical preparation according to claim 1 or 2, the viscosity of which is from about 2 cP to about 30 cP.

7. The pharmaceutical preparation according to claim 1 or 2, the viscosity of which is from about 2 cP to about 25 cP.

8. The pharmaceutical preparation according to claim 1 or 2, the viscosity of which is from about 2 cP to about 20 cP.

9. The pharmaceutical preparation according to claim 1 or 2, the viscosity of which is from about 2 cP to about 18 cP.

10. The pharmaceutical preparation according to any one of claims 1 to 9, in which the buffer system contains at least one pharmaceutically acceptable buffer agent having an acid dissociation constant within 2 pH units of the pH value of the drug.

11. The pharmaceutical preparation according to any one of claims 1 to 10, wherein the buffer system contains at least one buffer agent selected from the group consisting of succinate, histidine and citrate.

12. The pharmaceutical preparation according to claim 11, wherein at least one buffer agent is histidine or citrate.

13. The pharmaceutical preparation according to any one of claims 1 to 12, wherein at least one buffer agent is a citrate.

14. The pharmaceutical preparation according to claim 13, wherein at least one buffer agent is sodium citrate.

15. The pharmaceutical preparation according to any one of claims 1 to 12, wherein at least one buffer agent is histidine.

16. The pharmaceutical preparation according to claim 15, wherein at least one buffer agent is L-histidine.

17. The pharmaceutical preparation according to claim 13, in which the citrate is present in the solution in a concentration of 10 to 50 mM.

18. The pharmaceutical preparation according to claim 15, in which histidine is present in the solution in a concentration of 10 to 50 mM.

19. The pharmaceutical preparation according to any one of claims 1 to 18, which also contains at least one excipient selected from the group consisting of protein, amino acid, sugar, polyol, salt, fatty acid and phospholipid.

20. The pharmaceutical preparation according to claim 19, wherein the at least one excipient is a tonicity-modifying agent in an amount sufficient to cause the preparation to be hypertonic.

21. The pharmaceutical preparation according to claim 20, wherein the tonicity-modifying agent is selected from the group consisting of an amino acid with a charged side chain, a sugar or other polyol, and a salt.

22. The pharmaceutical preparation according to claim 21, wherein the tonicity modifying agent is a sugar or other polyol and is selected from the group consisting of sucrose, trehalose, mannitol and sorbitol.

23. The pharmaceutical preparation according to claim 21, wherein the tonicity-modifying agent is a salt selected from the group consisting of NaCl or an amino acid salt.

24. The pharmaceutical preparation according to claim 21, wherein the tonicity-modifying agent is an amino acid with a charged side chain.

25. The pharmaceutical preparation of claim 24, wherein the side chain charged amino acid is present at a concentration of from about 150 mM to about 300 mM.

26. The pharmaceutical preparation according to claim 24 or 25, wherein the tonicity-modifying agent is an amino acid with a negatively charged side chain.

27. The pharmaceutical preparation according to claim 24 or 25, wherein the excipient, a tonicity-modifying agent, is an amino acid with a positively charged side chain.

28. The pharmaceutical preparation according to claim 26, wherein the tonicity-modifying agent is glutamate.

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29. The pharmaceutical preparation according to claim 27, wherein the tonicity-modifying agent is arginine.

30. The pharmaceutical preparation according to claim 29, wherein the tonicity-modifying agent is L-arginine-HCl.

31. The pharmaceutical preparation according to claim 29, in which arginine is present in the solution at a hypertonic level of 200 to 300 mM.

32. The pharmaceutical preparation of claim 17, wherein the solution contains about 20 mM citrate and has a pH of from about 5.5 to about 6.5.

33. The pharmaceutical preparation according to claim 32, in which the solution also contains arginine at a concentration of approximately 200 mM.

34. The pharmaceutical preparation of claim 18, wherein the solution contains about 20 mM histidine and has a pH of from about 5.5 to about 6.5.

35. The pharmaceutical preparation according to claim 34, in which the solution also contains arginine at a concentration of approximately 200 mM.

36. The pharmaceutical preparation according to any one of claims 1 to 35, wherein the solution also contains a surfactant in a concentration of from about 0.001% (w/v) to about 0.1% (w/v).

37. The pharmaceutical preparation according to claim 36, wherein the surfactant is a non-ionic surfactant.

38. The pharmaceutical preparation according to claim 37, wherein the surfactant is a polysorbate or a poloxamer.

39. The pharmaceutical preparation according to claim 38, wherein the surfactant is polysorbate 80.

40. Pharmaceutical preparation according to any one of claims 1 to 39, which is stable when stored at a temperature of 2 to 8°C for at least 6 months.

41. Pharmaceutical preparation according to any one of claims 1 to 39, which is stable when stored at a temperature of from 2 to 8°C for at least 12 months.

42. The pharmaceutical preparation according to any one of claims 1 to 39, the viscosity of which is less than about 25 cP.

43. The pharmaceutical preparation according to any one of claims 1 to 39, the viscosity of which is less than about 20 cP.

44. The pharmaceutical preparation according to any one of claims 1 to 39, the viscosity of which is less than about 18 cP.

45. The pharmaceutical preparation according to any one of claims 1 to 39, the sliding frictional force of which during injection is about 25 Newton or less when administered through a 27 GA 1.25 needle at room temperature.

46. The pharmaceutical preparation according to any one of claims 1 to 39, the sliding friction force of which during injection is about 20 Newton or less when administered through a 25 GA 1 needle at room temperature.

47. The pharmaceutical preparation according to claim 1, containing about 20 mM sodium citrate, about 200 mM L-arginine-HCl, and the concentration of the antibody in the preparation is from about 175 mg/ml to about 195 mg/ml and the viscosity is less than approximately 25 cP.

48. The pharmaceutical preparation according to claim 47, also containing from 0.001 to 0.05% w/v. polysorbate 80.

49. The pharmaceutical preparation according to claim 1, containing about 20 mm L-histidine, about 200 mm L-arginine-HCl, and the concentration of the antibody in the drug is from about 175 mg/ml to about 195 mg/ml and the viscosity is less than about 25 cP.

50. The pharmaceutical preparation according to claim 49, also containing from 0.001 to 0.05% w/v. polysorbate 80.

51. Pharmaceutical preparation according to any one of claims 1 to 50, which is sterile.

52. The pharmaceutical preparation according to any one of claims 1 to 50, wherein the monoclonal antibody is a full-length monoclonal antibody.

53. The pharmaceutical preparation according to claim 52, wherein the antibody is a full-length human IgG4 antibody.

54. The pharmaceutical preparation according to claim 53, wherein the IgG4 has a mutation in the hinge region.

55. Pharmaceutical preparation according to any one of claims 1 to 54, not containing sucrose or sorbitol.

56. Pharmaceutical preparation according to any one of claims 1 to 54, not containing CaCl ₂ .

57. Pharmaceutical preparation according to any one of claims 1 to 54, not containing MgCl ₂ .

58. Pharmaceutical preparation according to any one of claims 1-54, not containing CaCl ₂ and not containing MgCl2.

59. Pharmaceutical preparation according to any one of claims 1-54, which does not contain an additive in the form of divalent cations.

60. The pharmaceutical preparation according to any one of claims 1 to 59, in which the concentration of the antibody is approximately 185 mg/ml.

61. Pharmaceutical preparation according to any one of claims 1 to 60, also containing the enzyme hyaluronidase in

- 47 043928 an amount effective to increase dispersion and/or absorption of the antibody after subcutaneous administration.

62. The pharmaceutical preparation according to claim 61, containing from about 100 units/ml to about 20,000 units/ml of the specified hyaluronidase enzyme.

63. The pharmaceutical preparation according to claim 1, containing (a) polysorbate 80 in a concentration of from about 0.01% w/v. to about 0.08% w/v;

(b) L-arginine-HCl at a concentration of about 150 mM to about 200 mM;

(c) sodium citrate at a concentration of from about 10 mM to about 50 mM; and (d) an antibody at a concentration of from about 150 mg/ml to about 200 mg/ml.

The pharmaceutical preparation according to claim 1, containing (a) polysorbate 80 in a concentration of from about 0.01% w/v. to about 0.08% w/v;

(b) L-arginine-HCl at a concentration of about 150 mM to about 200 mM;

(c) L-histidine at a concentration of about 10 mM to about 50 mM; and (d) an antibody at a concentration of from about 150 mg/ml to about 200 mg/ml.

65. The pharmaceutical preparation according to claim 1, containing (a) polysorbate 80 at a concentration of approximately 0.01% w/v;

(b) L-arginine-HCl at a concentration of approximately 200 mM;

(c) sodium citrate at a concentration of approximately 20 mM; and (d) an antibody at a concentration of from about 175 mg/ml to about 195 mg/ml.

66. The pharmaceutical preparation according to claim 1, containing (a) polysorbate 80 at a concentration of about 0.01% w/v;

(b) L-arginine-HCl at a concentration of approximately 200 mM;

(c) L-histidine at a concentration of approximately 20 mM; and (d) an antibody at a concentration of from about 175 mg/ml to about 195 mg/ml.

67. The pharmaceutical preparation according to claim 1, containing (a) polysorbate 80 at a concentration of approximately 0.01% w/v;

(b) L-arginine-HCl at a concentration of approximately 200 mM;

(c) sodium citrate at a concentration of approximately 20 mM; and (d) an antibody at a concentration of from about 175 mg/ml to about 195 mg/ml.

68. The pharmaceutical preparation according to claim 1, containing (a) polysorbate 80 at a concentration of about 0.01% w/v;

(b) L-arginine-HCl at a concentration of approximately 200 mM;

(c) L-histidine at a concentration of approximately 20 mM; and (d) an antibody at a concentration of from about 175 mg/ml to about 195 mg/ml.

69. The pharmaceutical preparation according to any one of claims 63-68, also containing from about 100 units/ml to about 20,000 units/ml of the enzyme hyaluronidase to effectively increase the dispersion and/or absorption of the antibody after subcutaneous administration.

70. A stable aqueous pharmaceutical preparation suitable for parenteral administration to a mammalian subject, consisting primarily of (a) polysorbate 80 at a concentration of from about 0.01% w/v. to about 0.08% w/v;

(b) L-arginine-HCl at a concentration of from about 150 mM to about 200 mM;

(c) sodium citrate at a concentration of from about 10 mM to about 50 mM; and (d) a monoclonal antibody, or fragment thereof, that specifically binds human MASP-2, at a concentration of from about 150 mg/ml to about 200 mg/ml, wherein said antibody or fragment thereof contains (i) a heavy chain variable region containing CDR-H1, CDR-H2 and CDR-H3 in SEQ ID NO: 2, and (ii) a light chain variable region containing CDR-L1, CDR-L2 and CDR-L3 in SEQ ID NO: 3; or a variant thereof comprising a heavy chain variable region having at least 95% identity with SEQ ID NO: 2 and a light chain variable region having at least 95% identity with SEQ ID NO: 3, wherein the preparation has a pH of about 5.0 to about 7.0, a viscosity of 2 to 50 centipoise (cP), and the formulation is stable when stored at 2 to 8° C. for at least one month.

71. A stable aqueous pharmaceutical preparation suitable for parenteral administration to a mammalian subject, consisting primarily of (a) polysorbate 80 at a concentration of from about 0.01% w/v. to about 0.08% w/v;

(b) L-arginine-HCl at a concentration of from about 150 mM to about 200 mM;

(c) L-histidine at a concentration of from about 10 mM to about 50 mM; and (d) a monoclonal antibody, or fragment thereof, that specifically binds human MASP-2, at a concentration of from about 150 mg/ml to about 200 mg/ml, wherein said antibody or fragment thereof contains (i) a heavy chain variable region containing CDR-H1, CDR-H2 and CDR-H3 in SEQ ID NO: 2, and

- 48 043928 (ii) a light chain variable region containing CDR-L1, CDR-L2 and CDR-L3 in SEQ ID NO: 3; or a variant thereof comprising a heavy chain variable region having at least 95% identity with SEQ ID NO: 2 and a light chain variable region having at least 95% identity with SEQ ID NO: 3, wherein the preparation has a pH of about 5.0 to about 7.0, a viscosity of 2 to 50 centipoise (cP), and the formulation is stable when stored at 2 to 8° C. for at least one month.

72. The drug according to any one of claims 1 to 71, wherein the antibody or fragment thereof contains a heavy chain variable region containing the amino acid sequence given in SEQ ID NO: 2, and a light chain variable region containing the amino acid sequence given in SEQ ID NO: 3.

73. A sealed container containing the preparation according to any one of claims 1 to 72.

74. A device for subcutaneous administration containing the drug according to any one of claims 1 to 72.

75. A kit comprising a pre-filled container containing a pharmaceutical preparation containing an anti-MASP-2 antibody according to any one of claims 1 to 72, and instructions for use of the preparation.

76. The kit of claim 75, wherein the prefilled container is selected from the group consisting of a syringe, a pen, a sealed vial, an auto-injector, and a pump device (eg, a body-worn pump or a tethered pump).

77. A kit comprising (a) a first pre-filled container containing a pharmaceutical preparation containing an anti-MASP-2 antibody according to any one of claims 1-60 or 63-72;

(b) a second prefilled container containing the hyaluronidase enzyme in an amount effective to increase dispersion and/or absorption of the anti-MASP-2 antibody after subcutaneous administration; and (c) instructions for use.

78. The kit of claim 77, wherein at least one of the first and second prefilled containers is selected from the group consisting of a syringe, a pen, a sealed vial, an auto-injector, and a pump device (e.g., a body-worn patch pump or a tethered pump).

79. Pharmaceutical unit dosage form suitable for parenteral administration to humans, containing the drug according to any one of claims 1 to 72, in a suitable container.

80. A method of treating a subject suffering from a disease or disorder that is treatable with a MASP-2 inhibitory antibody, comprising administering a drug according to any one of claims 1 to 72 to the subject in need thereof.

81. The method of claim 80, wherein the drug is administered to the subject subcutaneously.

82. The method of claim 80, further comprising administering to the subject an amount of hyaluronidase enzyme effective to increase dispersion and/or absorption of the antibody.

83. The method according to claim 82, in which the hyaluronidase enzyme is administered simultaneously with a preparation containing a MASP-2 inhibitory antibody.

84. The method according to claim 82 or 83, wherein the preparation contains a MASP-2 inhibitory antibody and the enzyme hyaluronidase.

85. The method of claim 82, wherein the hyaluronidase enzyme is administered to the subject prior to administration of the preparation containing the MASP-2 inhibitory antibody.

86. The method of claim 82, wherein the hyaluronidase enzyme is administered to the subject after administration of a preparation containing a MASP-2 inhibitory antibody.

The method of claim 80, wherein the drug is administered using a pre-filled syringe containing the drug.

88. A method of inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing a complement-related disease or disorder, comprising administering a drug according to any one of claims 1 to 72 to the subject in need thereof.

89. The method of claim 88, wherein the drug is administered to the subject subcutaneously.

90. The method of claim 89, wherein the drug is administered using a pre-filled syringe containing the drug.

91. The method of claim 88, wherein the subject suffers from or is at risk of developing a complement-related disease or disorder selected from the group consisting of thrombotic microangiopathy (TMA), renal disease, inflammatory response resulting from tissue or organ transplantation, ischemic and reperfusion injury, diabetes-related complication, cardiovascular disease or disorder, inflammatory gastrointestinal disease, pulmonary disease, ophthalmic disease or disorder, and disseminated intravascular coagulation.

92. The method according to claim 91, wherein thrombotic microangiopathy is an atypical he-

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Claims (11)

molytic uremic syndrome (aHUS). 93. The method according to claim 91, wherein thrombotic microangiopathy is associated with hematopoietic stem cell transplantation. 94. The method according to claim 91, wherein the kidney disease is IgA nephropathy. 95. The method according to claim 91, wherein the kidney disease is lupus nephritis. 96. A method of inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing a complement-related disease or disorder, comprising diluting the drug according to any one of claims 1 to 72 with a pharmaceutically acceptable diluent and administering the diluted drug systemically to the subject who needs in that. 97. The method of claim 96, wherein the diluted drug is administered to the subject intravenously. 98. The method of claim 96, wherein the subject suffers from, or is at risk of developing, a complement-related disease or disorder selected from the group consisting of thrombotic microangiopathy (TMA), kidney disease, inflammatory response resulting from tissue or organ transplantation, ischemia and reperfusion injury, diabetes-related complications, cardiovascular disease or disorder, inflammatory gastrointestinal disease, pulmonary disease, ophthalmic disease or disorder, and disseminated intravascular coagulation. 99. The method according to claim 98, wherein the thrombotic microangiopathy is atypical hemolytic uremic syndrome (aHUS). 100. The method according to claim 98, wherein thrombotic microangiopathy is associated with hematopoietic stem cell transplantation. 101. The method according to claim 98, wherein the kidney disease is IgA nephropathy. 102. The method according to claim 98, wherein the kidney disease is lupus nephritis. 103. A pharmaceutical composition for use in the treatment of a patient suffering from or at risk of developing a MASP-2-dependent complement-related disease or disorder, which is a sterile, single-use dosage form containing from about 350 mg to about 400 mg of an inhibitory MASP -2 antibodies, wherein the composition contains from about 1.8 ml to about 2.2 ml of an antibody preparation with a concentration of 185 mg/ml, wherein the antibody contains (i) a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 2, and (ii) a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 3, wherein the composition is stable when stored at a temperature of 2 to 8°C for at least six months. 104. The composition according to claim 103, containing a buffer system having a pH from 5.0 to 7.0. 105. The composition of claim 104, wherein the buffer system contains at least one pharmaceutically acceptable buffer agent selected from the group consisting of succinate, histidine and citrate. 106. The composition according to claim 103, containing from 1.8 to 2.2 ml of the MASP-2 inhibitory antibody preparation according to any one of claims 1 to 72 at a concentration of 185 mg/ml. 107. The composition according to any one of claims 103-106, wherein the MASP-2-dependent complement-related disease or disorder is aHUS. 108. The composition according to any one of claims 103-107, wherein the MASP-2-dependent complement-related disease or disorder is HSCT-TMA. 109. The composition according to any one of claims 103-107, wherein the MASP-2-dependent complement-related disease or disorder is IgA-H. 110. The composition according to any one of claims 103-107, wherein the MASP-2-dependent complement-related disease or disorder is lupus nephritis (LN). While preferred embodiments of the invention have been illustrated and described, it should be understood that various changes in the disclosed preparations and methods may be made without departing from the spirit and scope of the invention. CLAIM 1. A stable aqueous pharmaceutical preparation suitable for parenteral administration to a mammalian subject, consisting of (a) a buffer system having a pH of 5.0-7.0, wherein the buffer system contains a buffer agent selected from the group consisting of citrate in a concentration 10 to 50 mM or histidine at a concentration of 10 to 50 mM; (b) a human IgG4 monoclonal antibody that specifically binds human MASP-2 at a concentration of 50 to 200 mg/ml, wherein said antibody contains - 50 043928 (i) a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2, and (ii) a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 3; (c) a tonicity-modifying agent, wherein the tonicity-modifying agent is arginine or L-arginine-HCl at a concentration of 200 to 300 mM in an amount sufficient to make the formulation hypertonic; and (d) polysorbate 80 at a concentration of 0.01 to 0.1% (w/v), wherein the formulation has a viscosity of 2 to 50 cP when measured at about 25° C. with a shear rate ranging from 100,000 to 250,000 1/s, and the drug is stable when stored at a temperature of 2 to 8°C for at least six months. 2. The pharmaceutical preparation according to claim 1, to which at least one or more of the following applies: (a) the concentration of the antibody in the preparation is from 100 to 225 mg/ml, or from 150 to 200 mg/ml, or from 175 to 195 mg/ml; (b) the viscosity of the preparation is from 2 to 40 cP, or from 2 to 30 cP, or from 2 to 25 cP, or from 2 to 20 cP, or from 2 to 18 cP; (c) the viscosity is less than 25 cP, or, for example, less than 20 cP, or, for example, less than 18 cP; (d) the antibody concentration is 185 mg/ml; (e) the preparation is stable when stored at a temperature between 2 and 8°C for at least 12 months; (f) the sliding frictional force of the drug upon injection is 25 Newton or less when administered through a 27 GA 1.25 needle at room temperature; and/or (g) the sliding frictional force of the drug upon injection is 20 newtons or less when administered through a 25 GA 1 needle at room temperature. 3. The pharmaceutical preparation according to claim 1, wherein the buffer agent is sodium citrate. 4. The pharmaceutical preparation according to claim 1, wherein the tonicity-modifying agent is L-arginine-HCl. 5. The pharmaceutical preparation according to claim 3, in which the solution contains 20 mM citrate and has a pH from 5.5 to 6.5, optionally the solution also contains arginine at a concentration of 200 mM. 6. The pharmaceutical preparation according to claim 1, containing 20 mM sodium citrate, 200 mM L-arginine-HCl, and the concentration of the antibody in the drug is from 175 to 195 mg/ml, and the viscosity is less than 25 cP, and the drug also contains from 0.001 to 0.05% w/v. polysorbate 80. 7. Pharmaceutical preparation according to claim 1, which is sterile. 8. The pharmaceutical preparation according to claim 1, wherein the monoclonal antibody is a full-length monoclonal antibody including a mutation in the hinge region. 9. The pharmaceutical preparation according to claim 1, to which at least one or more of the following applies: (a) the preparation does not contain sucrose or sorbitol; (b) the preparation does not contain CaCl ₂ ; (c) the preparation does not contain MgCl ₂ ; (d) the preparation does not contain CaCl ₂ and does not contain MgCl ₂ ; and/or (f) the drug does not contain an additive in the form of divalent cations. 10. The pharmaceutical preparation according to claim 1, selected from the group consisting of (i) a preparation consisting of (a) polysorbate 80 at a concentration of 0.01 to 0.08% w/v, (b) L-arginine -HCl at a concentration of 150 to 200 mM, (c) a sodium citrate buffer system at a concentration of 10 to 25 mM, and (d) antibodies at a concentration of 150 to 200 mg/ml; and (ii) a preparation consisting of (a) polysorbate 80 at a concentration of 0.01% w/v, (b) L-arginine-HCl at a concentration of 200 mM, (c) a buffer system with sodium citrate at a concentration of 20 mM , and (d) antibodies at a concentration of 175 to 195 mg/ml. 11. A stable aqueous pharmaceutical preparation suitable for parenteral administration to a mammalian subject, consisting essentially of (a) polysorbate 80 at a concentration of 0.01 to 0.08% w/v; (b) L-arginine-HCl at a concentration of 150 to 200 mM; -