US20230355758A1

US20230355758A1 - Pharmaceutical Formulations

Info

Publication number: US20230355758A1
Application number: US18/152,872
Authority: US
Inventors: Andrew Jeffrey Yates; James Gregory Clipstone
Original assignee: UCB Biopharma SRL
Current assignee: UCB Biopharma SRL
Priority date: 2016-03-10
Filing date: 2023-01-11
Publication date: 2023-11-09
Also published as: CN114767849A; JP2019512540A; CA3016117A1; LT3426294T; US20190083617A1; RS64337B1; EP3426294A1; WO2017153541A1; PL3426294T3; EP3426294B1; HRP20230733T1; DK3426294T3; CN108883175A; GB201604124D0; FI3426294T3; US11576970B2; PT3426294T; MA43705A; JP2022062014A; EP4226915A1

Abstract

The invention relates to a liquid pharmaceutical formulation comprising an antibody or antigen-binding fragment thereof, cyclodextrin and methionine. In addition, it relates to a method for reducing precipitation of an antibody or an antigen-bindmg fragment thereof in a liquid pharmaceutical formulation through addition of methionine and cyclodextrin. In particular, the liquid pharmaceutical formulations comprises an anti-sclerostin antibody, hydroxypropyl-gamma cyclodextrin and methionine and may be used in the treatment of a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength, and especially for treating osteoporosis. Furthermore, the invention relates to a method of reducing inflammation at injection site in a mammalian subject comprising administering a therapeutically effective amount of a liquid pharmaceutical formulation comprising an antibody or antigen-binding fragment thereof, cyclo dextrin and methionine.

Description

FIELD OF THE INVENTION

The present invention belongs to the field of pharmaceutical formulations. More specifically, it relates to a pharmaceutical formulation comprising an antibody, cyclodextrin and methionine.

INCORPORATION BY REFERENCE OF MATERIALS SUBMITTED ELECTRONICALLY

This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 58045B_SeqListing.xml; Size: 17,137 bytes; Created Jan. 10, 2023), which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Antibodies are large and complex molecules and are inherently chemically and physically instable when stored over a period of time at high concentration. Typical chemical instability may result in deamidation, hydrolysis, oxidation, beta-elimination or disulfide exchanges. Physical instability can result in denaturation, aggregation or precipitation.
Antibodies can be formulated in freeze-dried, i.e. lyophilized form for reconstitution in a solvent shortly before administration. Freeze-dried formulations of antibodies tend to be more stable than liquid water-based formulation as water is either a reactant or facilitates the transfer of reactants and is thus critical to chemical degradation and protein instability. Despite being less stable, interest has recently focused on liquid formulations of antibodies. These formulations are easier and more convenient for the patient and the healthcare professional to handle and administer in comparison to freeze-dried formulations. Liquid formulations do not need to be reconstituted and can be administered with minimal preparation. However, the stabilization of proteins in liquid formulations to avoid or minimize aggregation, precipitation or degradation remains a particular challenge. Aggregation, with the formation of insoluble matter or precipitate, is a particular problem. This may cause various problems. Firstly aggregate formation may result in the antibody being less or no longer active; aggregate may provoke undesired and unexpected immunological reactions upon administration and finally aggregates may prevent proper administration of the pharmaceutical formulation e.g. by blocking syringes or pumps. These problems are even more evident in liquid formulations for subcutaneous administration where antibodies are present at high concentrations in much smaller volumes than intravenous infusions.
A typical strategy for preventing aggregation is to add stabilizers to the antibody formulation. Commonly used stabilizers include sugars, salts, free amino acids, polyols, polyethylene glycols (PEGs), and other polymers, such as polysorbates or poloxamers that may reduce protein-protein interactions.
Cyclodextrins (sometimes called cycloamyloses) are a family of compounds made up of sugar molecules bound together in a ring (cyclic oligosaccharides). Cyclodextrins are produced from starch by means of enzymatic conversion. They are considered safe and are widely used in food, pharmaceutical drug delivery, and chemical industries, as well as agriculture and environmental engineering. Cyclodextrins are composed of 5 or more α-D-glucopyranoside units linked 1->4, as in amylose. Typical cyclodextrins contain 6 (alpha-cyclodextrin), 7 (beta-cyclodextrin) or 8 (gamma-cyclodextrin) glucose monomers units in a ring, creating a cone shape where the interior is hydrophobic and the exterior is hydrophilic. The hydrophobic cavity provides an environment into which non-polar compounds, e.g. non-polar amino acids can be included and form complexes.
WO2010057107 describes using cyclodextrins (CDs) for reducing aggregation under physiological conditions. Furthermore, cyclodextrins and its derivatives have been used as solubilizers for poorly water soluble drugs. Currently available drug products that contain cyclodextrin in liquid formulations include Sporanox™ (Janssen, Belgium), Prostavasin (Ono, Japan; Schwarz, Germany), Prostandin 500 (Ono, Japan), Geodon™ (Pfizer, USA), VFEND™ (Pfizer, USA), MitoExtra Mitozytrex™ (Novartis, Switzerland), and Voltaren Optha™ (Novartis, Switzerland) (Szejtli J., Pure Appl. Chem. 76:1825-1845 (2004); Loftsson T. et al, Expert Opin. Drug Deliv. 2, 335-351 (2005)). Most of these formulations are for small molecule compounds. Maximum benefit is usually obtained at low cyclodextrin concentrations, and the benefits are often only partially concentration dependent. For example, aggregation of IL-2 was optimally inhibited by 0.5% HP-beta-cyclodextrin. (Loftsson T. and Brewster M. E., J Pharm Sci 85: 1017-1025 (1996)). The solubility of human growth hormone was improved by CDs present at about 2-6%, with alpha and gamma cyclodextrins found to be several-fold less effective than beta cyclodextrins. (Otzen, D. E. et al., Protein Sci. 11:1779-1787 (2002)).
Whilst the primary sequence of an antibody define its pl and it's intrinsically responsible of its tendency to precipitate upon administration (e.g. pH shift), the formulation composition will additionally affect this behavior and also influence other properties, including viscosity and osmolality. Altered interactions between the excipients and the active ingredients in the formulation may affect its long term stability. This is particularly relevant for antibody formulations developed for subcutaneous administration, where a high protein concentration meets a lower administration volume and protein aggregation and precipitation become even more apparent and potentially responsible for injection side effects.
Given the above, there remains a need in the art to provide further improved liquid pharmaceutical formulations of antibodies with reduced protein aggregation and precipitation, especially for formulations for subcutaneous route of administration.

SUMMARY OF THE INVENTION

The present invention addresses the above-identified need by providing a novel liquid formulation comprising an antibody or an antigen-binding fragment as active ingredient. A synergistic stabilizing effect has now been surprisingly observed between stabilizers with respect to the appearance of aggregates (precipitates) in a liquid pharmaceutical formulation.
Therefore, in a first aspect, the invention provides for a liquid pharmaceutical formulation comprising an antibody or an antigen-binding fragment thereof, as an active ingredient, cyclodextrin and methionine.
In a first embodiment of this aspect the liquid pharmaceutical formulation comprises methionine at a concentration of from 7.5 mM to 200 mM.
In a second embodiment the liquid pharmaceutical formulation according to the first aspect and embodiment comprises cyclodextrin at a concentration of from 7.5 mM to 250 mM.
In a third embodiment the liquid pharmaceutical formulation according to the first aspect and its embodiments comprises an antibody or an antigen-binding fragment thereof at a concentration of from 1 to 200 mg/ml, preferably from 90 to 180 mg/ml. In a fourth embodiment the liquid pharmaceutical formulation according to the first aspect and its embodiments comprises an antibody or an antigen-binding fragment thereof which specifically binds to human sclerostin and/or is a human or humanized antibody.
In a fifth embodiment the liquid pharmaceutical formulation according to the first aspect and its embodiments comprises:

- 1. an antibody or antigen-binding fragment thereof which
  - a. comprises CDR-H1 having the sequence as defined in SEQ ID NO:1; CDR-H2 having the sequence as defined in SEQ ID NO:2; CDR-H3 having the sequence as defined in SEQ ID NO:3; CDR-L1 having the sequence as defined in SEQ ID NO:4; CDR-L2 having the sequence as defined in SEQ ID NO:5 and CDR-L3 having the sequence as defined in SEQ ID NO:6; or
  - b. has a light variable region having the sequence as defined in SEQ ID NO: 7 and a heavy variable region having the sequence as defined in SEQ ID NO: 8; or
- 2. an antibody which has
  - a. a light chain having the sequence as defined in SEQ ID NO: 9 and a heavy chain having the sequence as defined in SEQ ID NO: 10; or
  - b. a light chain having the sequence as defined in SEQ ID NO: 11 and a heavy chain having the sequence as defined in SEQ ID NO: 12.

In a sixth embodiment the liquid pharmaceutical formulation according to the first aspect and its embodiments comprises an antibody which is romosozumab.
In a seventh embodiment, liquid formulation according to the first aspect and its embodiments has pH of from 4.0 to 7.5.
In an eight embodiment the liquid formulation according to the first aspect and its embodiments comprises a cyclodextrin selected from alpha-cyclodextrin, beta-cyclodextrin, dimethyl-beta-cyclodextrin, trimethyl-beta-cyclodextrin, randomly methylated beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin or 2,3-dihydroxypropyl-beta-cyclodextrin), hydroxyisobutyl-beta-cyclodextrin sulfobutylether beta-cyclodextrin, glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-gamma-cyclodextrin (i.e. 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin or 2,3-dihydroxypropyl-gamma-cyclodextrin), cyclodextrin-containing polymers or combinations thereof. Preferably, the cyclodextrin is selected from hydroxypropyl-gamma-cyclodextrin.
In an ninth embodiment the liquid formulation according to the first aspect and its embodiments comprises from 1 mg/ml to 200 mg/ml of antibody, from 10 mM to 55 mM sodium acetate, from 0 to 14 mM calcium acetate, from 0 to 6% sucrose, from 0 to 0.006% polysorbate 20, from 55 mM to 80 mM hydroxypropyl-gamma-cyclodextrin, from 55 mM to 160 mM methionine at pH 5.2; preferably form 90 mg/ml to 180 mg/ml, more preferably from 120 mg/ml to 180 mg/ml of romosozumab.
In a second aspect the present invention provides for the liquid formulation according to the first aspect and its embodiments for use in the treatment of a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength. Alternatively, the present invention provides for the use of the liquid formulation according to the first aspect and its embodiments in the manufacture of a medicament for the treatment of a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength.
Furthermore, in a third aspect the present invention provides for a method for treating a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength in a mammalian subject comprising administering a therapeutically effective amount of the formulation according to the first aspect and its embodiments.
Preferably, in embodiments of the second and third aspects of the invention, the bone disorder is osteoporosis.
In a fourth aspect the invention provides for a method for reducing precipitation of an antibody or an antigen-binding fragment thereof in a liquid pharmaceutical formulation, the method comprising providing a liquid formulation and adding to the formulation methionine and cyclodextrin, preferably hydroxylpropyl-gamma-cyclodextrin.
Finally, in a fifth aspect, the present invention provides for a method of reducing inflammation at injection site in a mammalian subject comprising administering a therapeutically effective amount of the formulation according to the first aspect and its embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence of the light chain of romosozumab (SEQ ID NO: 11). The amino acid sequence is presented using three-letter symbols with the corresponding one-letter symbol beneath each amino acid. Intrachain disulfide bonds are indicated by lines between linked cysteines. Cysteines forming interchain bonds are indicated in boxes.

FIG. 2 shows the sequence of the heavy chain of romosozumab (SEQ ID NO: 12). The amino acid sequence is presented using three-letter symbols with the corresponding one-letter symbol beneath each amino acid. Intrachain disulfide bonds are indicated by lines between linked cysteines. Cysteines forming interchain bonds are indicated in boxes.

DETAILED DESCRIPTION OF THE INVENTION

The liquid pharmaceutical formulation according to the invention comprises an antibody or an antigen-binding fragment thereof as active ingredient, and the combination of two stabilizers, methionine and cyclodextrin.
The synergistic combination of methionine and cyclodextrin provides the additional advantages of reducing the rate of precipitation of the antibody or the antigen-binding fragment thereof in the form of aggregates over time thus potentially reducing the occurrence of injection side reactions. Therefore, the present invention also provides for a method for reducing precipitation of an antibody or antigen-binding fragment thereof in a liquid pharmaceutical formulation, the method comprising providing a liquid formulation and adding to the formulation methionine and cyclodextrin.
The concentration of cyclodextrin in the liquid pharmaceutical formulation may be from 7.5 mM to 250 mM, or from 16 mM to 25 0mM, or from 32 mM to 200 mM, or from 63 mM to 160 mM or preferably from 55 mM to 80 mM.
The concentration of methionine in the liquid pharmaceutical formulation may be from 7.5 mM to 200 mM or from 15 mM to 200 mM or from 30 mM to 200 mM or from 55 mM to 160 mM or preferably from 55 mM to 125 mM.
In one embodiment, the liquid pharmaceutical formulation according to the invention comprises an antibody or antigen binding fragment thereof as active ingredient and a ratio of from 1:1 to 1:3, preferably a ratio of 1:2 of cyclodextrin to methionine.
In one preferred embodiment, the liquid pharmaceutical formulation according to the invention comprises an antibody or an antigen-binding fragment thereof as active ingredient, from 55 mM to 160 mM of methionine and from 55 mM to 100 mM of cyclodextrin.
In particular, the cyclodextrin may be selected from alpha-cyclodextrin, beta-cyclodextrin, dimethyl-beta-cyclodextrin, trimethyl-beta-cyclodextrin, randomly methylated beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin or 2,3-dihydroxypropyl-beta-cyclodextrin), hydroxyisobutyl-beta-cyclodextrin sulfobutylether beta-cyclodextrin, glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-gamma-cyclodextrin (i.e. 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin or 2,3-dihydroxypropyl-gamma-cyclodextrin), cyclodextrin-containing polymers or combinations thereof.
The most common natural cyclodextrins are alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin, all of which are hydrophilic. Substitutions of the hydroxyl groups further enhance the cyclodextrin solubility (Loftsson T. et al, Expert Opin. Drug Deliv. 2, 335-351 (2005); Davis M.E. & Brewster M.E., Nature Reviews — Drug discovery 3, 1023-1035 (2004)).
Chemically modified (i.e. substituted at the hydroxyl groups) cyclodextrins of interest are mono, di or trimethyl-beta-cyclodextrin, randomly methylated beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin or 2,3-dihydroxypropyl-beta-cyclodextrin), hydroxyisobutyl-beta-cyclodextrin sulfobutylether beta-cyclodextrin, glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin (i.e. 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin or 2,3-dihydroxypropyl-gamma-cyclodextrin) or combination thereof.
Hence, the liquid pharmaceutical formulation according to the invention comprises an antibody or an antigen-binding fragment thereof as active ingredient, and the combination of methionine and a chemically modified cyclodextrin, preferably selected from mono, di or trimethyl-beta-cyclodextrin, randomly methylated beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin or 2,3-dihydroxypropyl-beta-cyclodextrin), hydroxyisobutyl-beta-cyclodextrin sulfobutylether beta-cyclodextrin, glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin (i.e. 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin or 2,3-dihydroxypropyl-gamma-cyclodextrin) or combination thereof.
The concentration of the chemically modified cyclodextrin in the liquid pharmaceutical formulation may be from 7.5 mM to 250 mM, or from 16 mM to 250 mM, or from 32 mM to 200 mM, or from 63 mM to 160 mM or preferably from 55 mM to 80 mM.
In one embodiment, the liquid pharmaceutical formulation according to the invention comprises an antibody or antigen binding fragment thereof as active ingredient and a ratio of from 1:1 to 1:3, preferably a ratio of 1:2 of chemically modified cyclodextrin to methionine.
In one preferred embodiment, the liquid pharmaceutical formulation according to the invention comprises an antibody or an antigen-binding fragment thereof as active ingredient, from 55 mM to 160 mM of methionine and from 55 mM to 100 mM of a chemically modified cyclodextrin.
The preferred cyclodextrin according to the present invention is hydroxypropyl-gamma-cyclodextrin such as 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin, 2,3-dihydroxypropyl-gamma-cyclodextrin or combinations thereof.
Hence, in one preferred embodiment, the liquid pharmaceutical formulation according to the invention comprises an antibody or an antigen-binding fragment thereof as active ingredient, and the combination of methionine and hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin, 2,3-dihydroxypropyl-beta-cyclodextrin or combinations thereof).
The concentration of hydroxypropyl-gamma-cyclodextrin in the liquid pharmaceutical formulation may be from 7.5 mM to 250 mM, or from 16 mM to 250 mM, or from 32 mM to 200 mM, or from 63 mM to 160 mM or preferably from 55 mM to 80 mM.
In another embodiment, the liquid pharmaceutical formulation according to the invention comprises an antibody or antigen binding fragment thereof as active ingredient and a ratio of from 1:1 to 1:3, preferably a ratio of 1:2 of hydroxypropyl-gamma-cyclodextrin to methionine.
In another embodiment, the liquid pharmaceutical formulation according to the invention comprises an antibody or an antigen-binding fragment thereof as active ingredient, from 55 mM to 160 mM of methionine and from 55 mM to 100 mM of hydroxypropyl-gamma-cyclodextrin.
The concentration of antibody or antigen-binding fragment thereof in the liquid formulation according to the present invention may be from 1 to 200 mg/ml, or from to 200 mg/ml, or from to 200mg/ml or preferably from 90 to 180 mg/ml. In another embodiment, the liquid pharmaceutical formulation according to the invention comprises from 90 mg/ml to 180 mg/ml of an antibody as active ingredient, from 55 mM to 160 mM of methionine and from 55 mM to 100 mM of cyclodextrin.
In particular, the cyclodextrin may be selected from beta-cyclodextrin, dimethyl-beta-cyclodextrin, trimethyl-beta-cyclodextrin, randomly methylated beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin or 2,3-dihydroxypropyl-beta-cyclodextrin), hydroxyisobutyl-beta-cyclodextrin sulfobutylether beta-cyclodextrin, glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-gamma-cyclodextrin (i.e. 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin or 2,3-dihydroxypropyl-gamma-cyclodextrin), cyclodextrin-containing polymers or combinations thereof.
In another embodiment, the liquid pharmaceutical formulation according to the invention comprises from 90 mg/ml to 180 mg/ml of an antibody as active ingredient, from 55 mM to 160 mM of methionine and from 55 mM to 100 mM of a chemically modified cyclodextrin, preferably selected from mono, di or trimethyl-beta-cyclodextrin, randomly methylated beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin or 2,3-dihydroxypropyl-beta-cyclodextrin), hydroxyisobutyl-beta-cyclodextrin sulfobutylether beta-cyclodextrin, glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin (i.e. 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin or 2,3-dihydroxypropyl-gamma-cyclodextrin) or combinations thereof.
In one preferred embodiment, the liquid pharmaceutical formulation according to the invention comprises from 90 mg/ml to 180 mg/ml of an antibody as active ingredient, from 55 mM to 160 mM of methionine and from 55 mM to 100 mM of hydroxypropyl-gamma-cyclodextrin such as 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin, 2,3-dihydroxypropyl-gamma-cyclodextrin or combinations thereof.
The active ingredient comprised in the liquid pharmaceutical formulation according to the invention is preferably an antibody. Preferably the antibody has an isoelectric point (pl) lower than the physiological pH (−7.0) but higher than the formulation pH. In one embodiment the liquid formulation according to the present invention and its embodiments comprises from 1 to 200 mg/ml of an antibody, wherein the antibody has a pl of from 5.0 to 6.9, preferably from 5.3 to 6.9.
Preferably the antibody comprised in the liquid pharmaceutical formulation according to the invention specifically binds to human sclerostin.
The term “specifically binds to human sclerostin”, “specifically binding to human sclerostin”, and equivalents as used herein when referring to an antibody means the antibody will bind to human sclerostin with sufficient affinity and specificity to achieve a biologically meaningful effect. The antibody selected will normally have a binding affinity for human sclerostin, for example, the antibody may bind human sclerostin with a Kd value of between 100 nM and 1 pM. Antibody affinities may be determined by a surface plasmon resonance bases assay, such as the BlAcore assay; enzyme-linked immunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), for example. Within the meaning of the present invention an antibody specifically binding to human sclerostin may also bind to another molecule, e.g. mouse sclerostin or such as by way of a non-limiting example in the case of a bispecific antibody.
Sclerostin, a protein encoded by the SOST gene, is a glycoprotein secreted by osteocytes and is a potent inhibitor of osteoblastogenesis. Loss-of-function mutations in SOST are associated with an autosomal-recessive disorder, sclerosteosis, which causes progressive bone overgrowth. A deletion downstream of the SOST gene, which results in reduced sclerostin expression, is associated with a milder form of the disease called van Buchem disease. Furthermore, SOST-null mice have a high-bone-mass phenotype.
The antibody or antigen-binding fragment thereof binding specifically to human sclerostin, preferably also neutralizes human sclerostin.
The term “neutralizes” as used herein refers to an antibody that inhibits or substantially reduces the biological effect of the molecule to which it specifically binds. Therefore, the expression “the antibody neutralizes human sclerostin” refers to an antibody that specifically binds to human sclerostin and inhibits or substantially reduces the biological effect thereof.
The term “antibody” or “antibodies” as used herein refers to monoclonal or polyclonal antibodies. The term “antibody” or “antibodies” as used herein includes but is not limited to recombinant antibodies that are generated by recombinant technologies as known in the art.
Preferably, the antibody comprised in the liquid formulation according to the invention is a monoclonal antibody which specifically binds human sclerostin.
“Antibody” or “antibodies” include antibodies' of any species, in particular of mammalian species, having two essentially complete heavy and two essentially complete light chains, human antibodies of any isotype, including IgA₁, IgA₂, IgD, IgG₁, IgG_2a, IgG_2b, IgG₃, IgG₄IgE and IgM and modified variants thereof, non-human primate antibodies, e.g. from chimpanzee, baboon, rhesus or cynomolgus monkey, rodent antibodies, e.g. from mouse, rat or rabbit; goat or horse antibodies, and camelid antibodies (e.g. from camels or llamas such as Nanobodies™) and derivatives thereof, or of bird species such as chicken antibodies or of fish species such as shark antibodies. The term “antibody” or “antibodies” also refers to “chimeric” antibodies in which a first portion of at least one heavy and/or light chain antibody sequence is from a first species and a second portion of the heavy and/or light chain antibody sequence is from a second species. Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences. “Humanized” antibodies are chimeric antibodies that contain a sequence derived from non-human antibodies. For the most part, humanized antibodies are human antibodies (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region [or complementarity determining region (CDR)] of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken or non-human primate, having the desired specificity, affinity, and activity. In most instances residues of the human (recipient) antibody outside of the CDR; i.e. in the framework region (FR), are additionally replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. Humanization reduces the immunogenicity of non-human antibodies in humans, thus facilitating the application of antibodies to the treatment of human diseases. Humanized antibodies and several different technologies to generate them are well known in the art. The term “antibody” or “antibodies” also refers to human antibodies, which can be generated as an alternative to humanization. For example, it is possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of production of endogenous murine antibodies. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies with specificity against a particular antigen upon immunization of the transgenic animal carrying the human germ-line immunoglobulin genes with said antigen. Technologies for producing such transgenic animals and technologies for isolating and producing the human antibodies from such transgenic animals are known in the art. Alternatively, in the transgenic animal; e.g. mouse, only the immunoglobulin genes coding for the variable regions of the mouse antibody are replaced with corresponding human variable immunoglobulin gene sequences. The mouse germline immunoglobulin genes coding for the antibody constant regions remain unchanged. In this way, the antibody effector functions in the immune system of the transgenic mouse and consequently the B cell development are essentially unchanged, which may lead to an improved antibody response upon antigenic challenge in vivo. Once the genes coding for a particular antibody of interest have been isolated from such transgenic animals the genes coding for the constant regions can be replaced with human constant region genes in order to obtain a fully human antibody. The term “antibody” or “antibodies” as used herein, also refers to an aglycosylated antibody.
The term “antigen-binding fragment thereof” or grammatical variations thereof as used herein refers to an antibody fragment. A fragment of an antibody comprises at least one heavy or light chain immunoglobulin domain as known in the art and binds to one or more antigen(s). Examples of antibody fragments according to the invention include Fab, Fab′, F(ab′)₂, and Fv and scFv fragments; as well as diabodies, triabodies, tetrabodies, minibodies, domain antibodies(dAbs), such as sdAbs, VHH and VNAR fragments, single-chain antibodies, bispecific, trispecific, tetraspecific or multispecific antibodies formed from antibody fragments or antibodies, including but not limited to Fab-Fv or Fab-Fv-Fv constructs. Antibody fragments as defined above are known in the art.
The antibody or antigen-binding fragment thereof comprised as active ingredient in the liquid pharmaceutical formulation according to the invention may be human or humanized antibody, preferably a humanized monoclonal antibody which specifically binds human sclerostin.

- 1) More preferably the liquid pharmaceutical formulation according to the invention comprises: an antibody or antigen-binding fragment thereof which
  - a. comprises CDR-H1 having the sequence as defined in SEQ ID NO:1; CDR-H2 having the sequence as defined in SEQ ID NO:2; CDR-H3 having the sequence as defined in SEQ ID NO:3; CDR-L1 having the sequence as defined in SEQ ID NO:4; CDR-L2 having the sequence as defined in SEQ ID NO:5 and CDR-L3 having the sequence as defined in SEQ ID NO:6; or
  - b. has a light variable region having the sequence as defined in SEQ ID NO: 7 and a heavy variable region having the sequence as defined in SEQ ID NO: 8; or
- 2) an antibody which has
  - a. a light chain having the sequence as defined in SEQ ID NO: 9 and a heavy chain having the sequence as defined in SEQ ID NO: 10; or
  - b. a light chain having the sequence as defined in SEQ ID NO: 11 and a heavy chain having the sequence as defined in SEQ ID NO: 12.

Throughout this specification, complementarity determining regions (“CDR”) are defined according to the Kabat definition. The Kabat definition is a standard for numbering the residues in an antibody and it is typically used to identify CDR regions (Kabat et al., (1991), 5th edition, NIH publication No. 91-3242).
The sequences are shown in Table 1 below and in FIGS. 1 and 2 .

TABLE 1

Region and SEQ
ID identifier	Amino acid sequence

CDR-H1	DYNMH
SEQ ID NO: 1

CDR-H2	EINPNSGGAGYNQKFKG
SEQ ID NO: 2

CDR-H3	LGYDDIYDDWYFDV
SEQ ID NO: 3

CDR-L1	RASQDISNYLN
SEQ ID NO: 4

CDR-L2	YTSRLLS
SEQ ID NO: 5

CDR-L3	QQGDTLPYT
SEQ ID NO: 6

Light variable	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLLSG
region	VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDTLPYTFGGGTKVEIK
SEQ ID NO: 7

Heavy variable	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQGLEWMGEINPN
region	SGGAGYNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARLGYDDIYDDWY
SEQ ID NO: 8	FDVWGQGTTVTVSS

Light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS
SEQ ID NO: 9	RLLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDTLPYTFGGGTKVEIKRT
	VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
	QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy chain	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQGLEWMGEINPN
SEQ ID NO: 10	SGGAGYNQKPKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARLGYDDIYDDWY
	FDVWGQGTTVTYSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW
	NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKT
	VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
	FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGL
	PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
	QPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK
	SLSLSPGK

Even more preferably, the antibody comprised as active ingredient in the liquid pharmaceutical formulation according to the invention is romosozumab, an anti-human sclerostin antibody as described in WO2006119107 and WO2011143307 (incorporated herein by reference).
In particular, the present invention also provides for a liquid pharmaceutical formulation comprising romosozumab and a cyclodextrin, preferably selected from alpha-cyclodextrin, beta-cyclodextrin, dimethyl-beta-cyclodextrin, trimethyl-beta-cyclodextrin, randomly methylated beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin or 2,3-dihydroxypropyl-beta-cyclodextrin), hydroxyisobutyl-beta-cyclodextrin sulfobutylether beta-cyclodextrin, glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-gamma-cyclodextrin (i.e. 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin or 2,3-dihydroxypropyl-gamma-cyclodextrin), cyclodextrin-containing polymers or combinations thereof, more preferably the liquid pharmaceutical formulation comprising romosozumab and hydroxypropyl-gamma-cyclodextrin.
The liquid pharmaceutical formulation according to any of the embodiments of the invention may contain a buffering agent to maintain a constant pH during storage and administration. There are many buffering agents used in the field of liquid pharmaceutical formulations, such as, but not limited to, citrate, phosphate, lactate, histidine, glutamate, maleate, tartrate, or succinate. A preferred buffer species is typically selected amongst those having a pKa that is close (±1 pH unit) to the preferred pH for optimal protein stability in order to maintain high buffering capacity, and is associated with the maximal demonstrated stability observed for a particular protein when placed in a series of varied buffer species. The adequate pH ranges of a formulation are generally chosen from those associated with the maximal demonstrated stability observed for a particular protein when placed in a series of varied pH formulations.
In a particular embodiment of the invention, the liquid pharmaceutical formulation according to any of the embodiments of the invention comprises sodium acetate, preferably at a concentration of 10 mM to 100 mM, 10 mM to 80 mM, 10 mM to 60 mM, 25 mM to 60 mM, preferably 40 mM to 60 mM, or 50 mM. The liquid pharmaceutical formulation according to the invention may also comprise calcium acetate, preferably at a concentration of 0.5 mM to 50 mM, 5 mM to 40 mM, 10 mM to 30 mM or 14 mM. More preferably, the liquid pharmaceutical formulation according to the invention comprises 50 mM sodium acetate and 14 mM calcium acetate.
In a further embodiment, the liquid pharmaceutical formulation according to any of the embodiments of the invention may have a pH of 4 to 7.5, preferably 4 to 6, preferably 4.5 to 5.5, or 5.2.
In a further embodiment the liquid pharmaceutical formulation according to any of the embodiments of the invention additionally comprises a surfactant. The skilled artisan is aware of the choice of surfactants available for use in the liquid formulation according to any of the embodiments of the invention such as but not limited to polysorbate 80, polysorbate 20, lecithin, poloxamer (e.g. poloxamer 188), sodium dodecyl sulfate (SDS), sodium laurel sulfate, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl- or stearyl-sarcosine, linoleyl-, myristyl-, or cetyl-betaine, lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl-, or disodium methyl oleyl-taurate, polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol. In a further embodiment of the invention, the liquid pharmaceutical formulation may comprise polysorbate 20.
The liquid formulation of the invention may comprise from 0.001% to 10% Polysorbate 20, from 0.001% to 5% Polysorbate 20, from 0.001 to 1% Polysorbate 20, from 0.001 to 0.006% Polysorbate 20, or 0.006% Polysorbate 20.
The liquid formulation according to the embodiments of the invention may further comprise additional excipients commonly used in the preparation of liquid pharmaceutical formulations including but not limited to sugars (e.g. sucrose or treahalose), poly-ethylene glycols (e.g. PEG100, PEG300, PEG600, PEG1500, PEG2000, PEG3000, PEG3350, PEG4000, PEG6000, PEG8000 or PEG20000), polyols (e.g. mannitol, sorbitol or lactitol), other amino acids than methionine, polyvinylpyrrolidone, trimethylamine N-oxide, trimethylglycine or combinations thereof.
In particular, the liquid formulation according to the embodiments of the invention may further comprise sucrose, from 0 to 6% sucrose, more preferably 6% sucrose.
In an another embodiment the liquid pharmaceutical formulation according to any of the embodiments of the invention comprises from 1 mg/ml to 200 mg/ml of antibody, from 10 mM to 55 mM sodium acetate, from 0 to 14 mM calcium acetate, from 0 to 6% sucrose, from 0 to 0.006% polysorbate 20, from 55 mM to 80 mM of a chemically modified cyclodextrin, from 55 mM to 160 mM methionine at pH 5.2; more preferably form 90 mg/ml to 180 mg/ml and even more preferably from 120 mg/ml to 180 mg/ml of antibody, wherein the chemically modified cyclodextrin is preferably selected from mono, di or trimethyl-beta-cyclodextrin, randomly methylated beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin or 2,3-dihydroxypropyl-beta-cyclodextrin), hydroxyisobutyl-beta-cyclodextrin sulfobutylether beta-cyclodextrin, glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin (i.e. 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin or 2,3-dihydroxypropyl-gamma-cyclodextrin) or combinations thereof.
In a further embodiment the liquid pharmaceutical formulation according to any of the embodiments of the invention comprises from 1 mg/ml to 200 mg/ml of antibody, from 10 mM to 55 mM sodium acetate, from 0 to 14 mM calcium acetate, from 0 to 6% sucrose, from 0 to 0.006% polysorbate 20, from 55 mM to 80 mM hydroxypropyl-gamma-cyclodextrin, from 55 mM to 160 mM methionine at pH 5.2; more preferably form 90 mg/ml to 180 mg/ml and even more preferably from 120 mg/ml to 180 mg/ml of antibody.
In a preferred embodiment, the liquid pharmaceutical formulation according to the invention comprises at least 90 mg/ml antibody, 55 mM hydroxypropyl-gamma-cyclodextrin, 125 mM methionine 55 mM sodium acetate buffer at pH 5, 14 mM calcium acetate, 6% sucrose and 0.006% polysorbate 20, wherein the antibody is an anti-human sclerostin antibody. Even more preferably the liquid pharmaceutical formulation according to the invention comprises at least 90 mg/ml antibody, 55 mM hydroxypropyl-gamma-cyclodextrin, 125 mM methionine 55 mM sodium acetate buffer at pH 5, 14 mM calcium acetate, 6% sucrose and 0.006% polysorbate 20, wherein the antibody is an anti-human sclerostin antibody and the formulation is for subcutaneous administration.
In another embodiment the liquid pharmaceutical formulation according to any of the embodiments of the invention exhibits an osmolality of 800 mOsm/l or less and/or a viscosity of 80 cP or less.
In an embodiment the liquid formulation is not a hydrogel. In an embodiment the liquid formulation comprises at least 2%, at least 5%, at least 10%, at least 25%, between 2% and 90%, between 2% and 75%, or between 2% and 50% water.
The liquid pharmaceutical formulation according to any of the embodiments of the invention may be used in the treatment of a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength. In addition, the invention provides for a method of treating a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strengthin a mammalian subject comprising administering a therapeutically effective amount of the liquid pharmaceutical formulation according to any of the embodiments of the invention. Furthermore, the invention provides for the use of the liquid formulation according to any one of the embodiments of the invention for the manufacture of a medicament for the treatment of a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength.
The mammalian subject may be selected from the group of rodents, cats, dogs, horses, bovine, ovine, non-human primates and human subjects. Preferably, the mammalian subject is a human subject.
The invention is typically used to treat or help prevent a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength. The bone disorder is characterized by an altered function of sclerostin or by an increased/decreased expression of sclerostin protein in relevant tissue (e.g. bone, cartilage). The invention may be, for example, employed to increase at least one of bone formation, bone mineral density, bone mineral content, bone mass, bone quality and bone strength.
The bone disorder may be associated with abnormal osteoblast or osteoclast activity, with decreased bone density, increased bone resorption and/or bone-related disorders, i.e. osteoporosis. In particular, the bone disorder may be selected from the group of achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher's Disease, hypophosphatemia rickets, Marfan's syndrome, multiple hereditary exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions, pseudoarthrosis, pyogenic osteomyelitis, periodontal disease, anti-epileptic drug induced bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism syndromes, weightlessness induced bone loss, osteoporosis in men, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative disorders of bone, oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis, metabolic bone diseases, mastocytosis, sickle cell anemia/disease, organ transplant related bone loss, kidney transplant related bone loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritides, thalassemia, mucopolysaccharidoses, Fabry Disease, Turner Syndrome, Down Syndrome,
Klinefelter Syndrome, leprosy, Perthes' Disease, adolescent idiopathic scoliosis, infantile onset multi-system inflammatory disease, Winchester Syndrome, Menkes Disease, Wilson's Disease, ischemic bone disease (such as Legg-Calve-Perthes disease, regional migratory osteoporosis), anemic states, conditions caused by steroids, glucocorticoid-induced bone loss, heparin-induced bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, osteoporosis, osteopenia, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis, osteomalacia, bone gap defect, alveolar bone loss, bone loss associated with joint replacement, HIV associated bone loss, bone loss associated with loss of growth hormone, bone loss associated with cystic fibrosis, chemotherapy associated bone loss, tumor induced bone loss, cancer-related bone loss, hormone ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa, disease associated facial bone loss, disease associated cranial bone loss, disease associated bone loss of the jaw, disease associated bone loss of the skull, bone loss associated with aging, facial bone loss associated with aging, cranial bone loss associated with aging, jaw bone loss associated with aging, skull bone loss associated with aging and bone loss associated with space travel and combinations thereof.
Bone loss, decreased bone mineral density, decreased bone volume, and/or decreased bone mineral content associated with these disorders may be treated in the context of the invention. In one instance, the subject to be treated may be pregnant. For instance, the invention may be employed to help in pregnancy-related bone loss. The invention may be used to slow, or reverse, bone loss in general.
In one preferred embodiment, the bone disorder to be treated is osteoporosis or osteopenia. In one instance, the mammalian subject to be treated is a postmenopausal woman, for instance, one with osteoporosis, particularly such a subject who is at increased, or high risk, for fracture, or has failed or is intolerant to other available osteoporosis therapy. In further instances, the invention may be employed in improving the outcome in a mammal undergoing one or more of an orthopedic procedure, dental procedure, implant surgery, joint replacement, bone grafting, bone cosmetic surgery and bone repair such as fracture healing, nonunion healing, delayed union healing and facial reconstruction.
In another preferred embodiment, the subject has, or is thought at risk of, a bone fracture. For instance, it may be that the subject has a bone mineral density that identifies them at risk of bone fracture. It may be that the subject has recently been diagnosed with a bone disorder as a result of a bone fracture. In some instances, it may be that the subject has a fracture of the wrist, arm, leg, hip or vertebrae and in some cases it may be that the fracture has resulted in the diagnosis of the bone disorder.
Preferably, the bone disorder is selected from osteoporosis, bone gap defect, alveolar bone loss, inhibited bone resorption or combinations thereof.
The present invention also provides for a method for reducing inflammation at injection site in a mammalian subject comprising administering a therapeutically effective amount of the liquid pharmaceutical formulation according to any of the embodiments of the invention.
The liquid pharmaceutical formulations according to the invention are administered in a therapeutically effective amount. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent (i.e. active ingredient) needed to treat, ameliorate or prevent a targeted disease, disorder or condition, or to exhibit a detectable therapeutic, pharmacological or preventative effect. For any antibody or antigen-binding fragments thereof, the therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
The precise therapeutically effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, a therapeutically effective amount of antibody will be from 0.01 mg/kg to 500 mg/kg, for example 0.1 mg/kg to 200 mg/kg, such as 100 mg/kg.
The liquid pharmaceutical formulation may be conveniently presented in unit dose forms containing a predetermined amount of an active agent of the invention per dose.
For the treatment of the above diseases and/or disorders, the appropriate dosage will vary depending upon, for example, the particular antibody to be employed, the subject treated, the mode of administration and the nature and severity of the condition being treated. In a particular embodiment the liquid pharmaceutical formulation of the invention is administered by intravenous or subcutaneous route. When administered via intravenous injection, it may be administered as a bolus injection or as a continuous infusion. The liquid pharmaceutical formulation according to any of the embodiments of the invention may also be administered by intramuscular injection. The liquid pharmaceutical formulation may be injected using a syringe, an injection device such as an autoinjector, a needleless device, an implant and a patch. The liquid pharmaceutical formulation of the invention may also be administered via inhalation using an inhalation device containing said formulation for such delivery, for example using a nebulizer or liquid inhaler.
The liquid pharmaceutical formulation of the invention is suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards; it may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the conditions as described herein before.
The antibody or antigen-binding fragment thereof may be the sole active ingredient in the liquid pharmaceutical formulation. Alternatively, the antibody or antigen-binding fragment thereof may be administered in combination, e.g. simultaneously, sequentially or separately, with one or more other therapeutically active ingredients. Active ingredient as employed herein refers to an ingredient with a pharmacological effect, such as a therapeutic effect, at a relevant dose. In some embodiments the antibody or antigen-binding fragment thereof in the liquid pharmaceutical formulation may be accompanied by other active ingredients including other antibodies or non-antibody ingredients. When the antibody in the liquid pharmaceutical formulation according to the invention is an anti-sclerostin antibody, the subject may be administered with an additional active ingredient to treat their bone disorder. The subject may be, for instance, treated with any other therapy for treating bone disorders. In one embodiment, the subject is administered an anti-resorptive, in a particularly preferred instance the additional agent is administered at a time when the subject is not being treated with the liquid pharmaceutical formulation according to the invention and its embodiments. In an alternative embodiment, such an additional active ingredient may be administered at the same time or overlapping with the administration of the liquid pharmaceutical formulation according to the invention. In any embodiment discussed herein, the additional agent may be, for instance, vitamin D. In some embodiments, the other therapeutic active ingredient may be a bone resorption inhibitor. For instance, any suitable anti-resorptive may be employed. In one preferred embodiment, the bone resorption inhibitor is a bisphosphonate, particularly a nitrogen containing bisphosphonate. Examples of bisphosphonates include, but are not limited to, Alendronate, bonefos ciodronate, etidronate, ibandronic acid, olpadronate, neridronate, risedronate sodium, skelid, and zoledronic acid. In another preferred embodiment, the bisphosphonate is zoledronic acid. Bisphosphonates which may be employed include, for instance, Actonel™, Aclasta™/Reclast™, Boniva™/Bonviva™, Fosamax™, and Zometa™. An advantage of alternating between the liquid pharmaceutical formulation comprising an anti-sclerostin antibody and bisphosphonate is that it may help avoid possible side effects arising from the subject being treated with bisphosphonates for a prolonged period. Hence, alternating helps avoid such side-effects, whilst also addressing the problem of resistance developing to the antibody. In a preferred embodiment, the additional active ingredient is an anti-resorptive and even more preferably is Alendronate.
Selected estrogen receptor modulators may also be employed as bone resorption inhibitors, for instance, arzoxifene, bazedoxifene, FC 1271, lasofoxifene, raloxifene, and Tibolone are examples of suitable SERMs. Other bone resorption inhibitors which may be used include estrogen and calcitonin, with examples of calcitonin including salmon calcitonins, such as Miacalcin™.
Strontium compounds, in particular strontium ranelate, or PTH, in particular recombinant parathyroid hormone releasing peptide, parathyroid hormone or analogs thereof (e.g., teriparatide (FORTEO®), may also be employed as the bone resorption inhibitor.
In other embodiments, the bone resorption inhibitor is a RANKL inhibitor, such as an anti-RANKL antibody. In one preferred embodiment, the bone resorption inhibitor employed may be denosumab.
In other embodiments the anti-resorptive employed is not a bisphosphonate. Examples, of such agents which may be employed include PROLIA®, calcitonin, and cathepsin K inhibitors (e.g., odanacatib).
In one case, a bone resorption inhibitor may be administered at the same time, or approximately the same time, as the antibody, or so the two therapies overlap. It may be that the bone resorption inhibitor is given to help prolong further the effect of the anti-sclerostin antibody by reducing the breakdown of bone that the antibody has stimulated and in particular where the compound is a bisphosphonate. In one preferred instance, there is no overlap in the treatment of the subject with anti-sclerostin antibody and the further treatment for the bone disorder, for instance the two treatments are alternated, but never overlap.
Antibody molecules may be typically produced by culturing a host cell containing a vector encoding the antibody sequence under conditions suitable for leading to expression of protein from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.
The antibody molecule may comprise only a heavy or light chain polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence needs to be used to transfect the host cells. For production of products comprising both heavy and light chains, the cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides.
An antibody or an antigen-binding fragment thereof that can be manufactured according to industrial scales can be produced by culturing eukaryotic host cells transfected with one or more expression vectors encoding the recombinant antibody fragment. The eukaryotic host cells are preferably mammalian cells, more preferably Chinese Hamster Ovary (CHO) cells.
Mammalian cells may be cultured in any medium that will support their growth and expression of the recombinant protein, preferably the medium is a chemically defined medium that is free of animal-derived products such as animal serum and peptone. There are different cell culture mediums available to the person skilled in the art comprising different combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose or an equivalent energy source, present at appropriate concentrations to enable cell growth and protein production. Additional cell culture media components may be included in the cell culture medium at appropriate concentrations at different times during a cell culture cycle that would be known to those skilled in the art.
Mammalian cell culture can take place in any suitable container such as a shake flask or a bioreactor, which may or may not be operated in a fed-batch mode depending on the scale of production required. These bioreactors may be either stirred-tank or air-lift reactors. Various large scale bioreactors are available with a capacity of more than 1,000 L to 50,000 L, preferably between 5,000 L and 20,000 L, or to 10,000 L. Alternatively, bioreactors of a smaller scale such as between 2 L and 100 L may also be used to manufacture an antibody or antibody fragment.
An antibody or antigen-binding fragment thereof is typically found in the supernatant of a mammalian host cell culture, typically a CHO cell culture. For CHO culture processes wherein the protein of interest such as an antibody or antigen-binding fragment thereof is secreted in the supernatant, said supernatant is collected by methods known in the art, typically by centrifugation.
Therefore, the antibody or antigen-binding fragment thereof production method comprises a step of centrifugation and supernatant recovery after cell culture and prior to protein purification. In a further particular embodiment said centrifugation is continuous centrifugation. For avoidance of doubt, supernatant denotes the liquid lying above the sedimented cells resulting from the centrifugation of the cell culture.
Alternatively, host cells are prokaryotic cells, preferably gram-negative bacteria. More preferably, the host cells are E. coli cells. Prokaryotic host cells for protein expression are well known in the art (Terpe, K. Appl Microbiol Biotechnol 72, 211-222 (2006)). The host cells are recombinant cells which have been genetically engineered to produce the protein of interest such as an antigen-binding fragment of an antibody. The recombinant E. coli host cells may be derived from any suitable E. coli strain including from MC4100, TG1, TG2, DHB4, DH5a, DH1, BL21, K12, XL1Blue and JM109. One example is E. coli strain W3110 (ATCC 27,325) a commonly used host strain for recombinant protein fermentations. Antibody fragments can also be produced by culturing modified E. coli strains, for example metabolic mutants or protease deficient E. coli strains.
An antibody fragment is typically found in either the periplasm of the E. coli host cell or in the host cell culture supernatant, depending on the nature of the protein, the scale of production and the E. coli strain used. The methods for targeting proteins to these compartments are well known in the art (Makrides, S. C.; Microbiol Rev 60, 512-538 (1996)). Examples of suitable signal sequences to direct proteins to the periplasm of E. coli include the E. coli PhoA, OmpA, OmpT, LamB and OmpF signal sequences. Proteins may be targeted to the supernatant by relying on the natural secretory pathways or by the induction of limited leakage of the outer membrane to cause protein secretion examples of which are the use of the pelB leader, the protein A leader, the co-expression of bacteriocin release protein, the mitomycin-induced bacteriocin release protein along with the addition of glycine to the culture medium and the co-expression of the kil gene for membrane permeabilization. Most preferably, the recombinant protein is expressed in the periplasm of the host E. coll.
Expression of the recombinant protein in the E. coli host cells may also be under the control of an inducible system, whereby the expression of the recombinant antibody in E. coli is under the control of an inducible promoter. Many inducible promoters suitable for use in E. coli are well known in the art and depending on the promoter expression of the recombinant protein can be induced by varying factors such as temperature or the concentration of a particular substance in the growth medium. Examples of inducible promoters include the E.coli lac, tac, and trc promoters which are inducible with lactose or the non-hydrolyzable lactose analog, isopropyl-b-D-1-thiogalactopyranoside (IPTG) and the phoA, trp and araBAD promoters which are induced by phosphate, tryptophan and L-arabinose respectively. Expression may be induced by, for example, the addition of an inducer or a change in temperature where induction is temperature dependent. Where induction of recombinant protein expression is achieved by the addition of an inducer to the culture the inducer may be added by any suitable method depending on the fermentation system and the inducer, for example, by single or multiple shot additions or by a gradual addition of inducer through a feed. It will be appreciated that there may be a delay between the addition of the inducer and the actual induction of protein expression for example where the inducer is lactose there may be a delay before induction of protein expression occurs while any pre-existing carbon source is utilized before lactose.
E. coli host cell cultures (fermentations) may be cultured in any medium that will support the growth of E. coli and expression of the recombinant protein. The medium may be any chemically defined medium such as e.g. described in Durany O, et al. (2004). Studies on the expression of recombinant fuculose-1-phosphate aldolase in Escherichia coli. Process Biochem 39, 1677-1684.
Culturing of the E. coli host cells can take place in any suitable container such as a shake flask or a fermenter depending on the scale of production required. Various large scale fermenters are available with a capacity of more than 1,000 liters up to about 100,000 liters. Preferably, fermenters of 1,000 to 50,000 liters are used, more preferably 1,000 to 25,000, 20,000, 15,000, 12,000 or 10,000 liters. Smaller scale fermenters may also be used with a capacity of between 0.5 and 1,000 liters.
Fermentation of E. coli may be performed in any suitable system, for example continuous, batch or fed-batch mode depending on the protein and the yields required. Batch mode may be used with shot additions of nutrients or inducers where required. Alternatively, a fed-batch culture may be used and the cultures grown in batch mode pre-induction at the maximum specific growth rate that can be sustained using the nutrients initially present in the fermenter and one or more nutrient feed regimes used to control the growth rate until fermentation is complete. Fed-batch mode may also be used pre-induction to control the metabolism of the E. coli host cells and to allow higher cell densities to be reached.
If desired, the host cells may be subject to collection from the fermentation medium, e.g. host cells may be collected from the sample by centrifugation, filtration or by concentration. In this case the process typically comprises a step of centrifugation and cell recovery prior to extracting the protein.
For E. coli fermentation processes wherein the protein of interest such as an antibody or antigen-binding fragment of an antibody is found in the periplasmic space of the host cell it is required to release the protein from the host cell. The release may be achieved by any suitable method such as cell lysis by mechanical or pressure treatment, freeze-thaw treatment, osmotic shock, extraction agents or heat treatment. Such extraction methods for protein release are well known in the art. Therefore in a particular embodiment, the production process comprises an additional protein extraction step prior to protein purification.
Other methods for obtaining antigen-binding fragment of a human antibody in vitro are based on display technologies such as phage display or ribosome display technology, wherein recombinant DNA libraries are used that are either generated at least in part artificially or from immunoglobulin variable (V) domain gene repertoires of donors. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies may also be generated from isolated human B cells that are ex vivo immunized with an antigen of interest and subsequently fused to generate hybridomas which can then be screened for the optimal human antibody.

EXAMPLES

Example 1: Hydroxypropyl-Gamma-Cyclodextrin (HPGCD) Screening

Stability and propensity to precipitate within a formulation is greatly affected by the concentration (steric crowding) and the excipients used within the formulation to control the interactions observed due to steric crowding.
In order to determine whether HPGCD has an impact on the precipitation profile at physiological pH, basic formulation of romosozumab (pl 6.9) at a concentration of 120 mg/mL with and without HPGCD were run through the pH shift model.
The pH shift model is designed to replicate the pH shift that occurs when a formulation comprising a drug is administered by subcutaneous injection (Elena Garcia-Fruitos, Insoluble Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1258, Chapter 18:321-330, Springer Science+Business Media New York (2015)). Biotherapeutic proteins, in particular antibodies, are often formulated below physiological pH (−pH7.0). If the pl of the protein is less than pH 7.0 but higher than the formulation pH, the protein will shift through its pl. This will reduce the solubility of the protein and cause it to precipitate. This model is designed to replicate this pH shift and aid in assessing formulations' suitability for subcutaneous injection.
The model is run over 24 hours. A tissue buffer (10 mM Sodium Bicarbonate and 150 mM Sodium Chloride) is prepared the day before and placed into the CO2 incubator at 37° C./7% CO₂on a gentle stir overnight to equilibrate. About 0.5 mL of each test sample is loaded into a slide-a-lyser cassette according to the manufacturer 'instructions (Thermo Scientific, Slide-A-Lyzer Dialysis Cassettes G2, Prod#87727, 7000 MWCO, 0.5 mL). The slide-a-lyser cassettes are gently placed into the equilibrated tissue buffer within the CO₂incubator and left overnight. The slide-a-lyser cassettes are visually assessed against a white and black background for signs of precipitation every hour for the first 4-6 hours and then after 24 hours (or longer).
Romosozumab at 120 mg/mL in 50 mM sodium acetate, 14 mM calcium acetate, 6% sucrose, 0.006% polysorbate 20 at pH 5.2 (drug solution, DS) were prepared by performing a 1:1 serial dilution. An initial solution was prepared by adding 881 mg of HPGCD to 2 ml of romosozumab at 120 mg/mL in DS to give a final HPGCD concentration of 250 mM. Half of this solution (1 mL) was mixed 1:1 with the same volume of romosozumab at 120 mg/mL in DS to achieve a sample of romosozumab at 120 mg/mL in DS with 125 mM HPGCD. This process was repeated to achieve samples of romosozumab at 120 mg/mL in DS with 62.5 mM, 31.25 mM, 15.63 mM and 7.8 mM HPGCD. Romosozumab at 120 mg/mL in DS was used as the control.
The formulations comprising 0 to 250 mM concentrations of HPGCD were buffer exchanged and concentrated using 30 kDa Sartorius Viva Spin centrifugal spin filters of varying volumes (2-20 mL). The spin tubes were centrifuged at 3000 rpm, 5° C. for 60 minutes. Samples were mixed between spin cycles using a positive displacement pipette volume exchange.
The concentration of each sample was measured in duplicate before and after dialysis (Table 2) using the C Technologies Solo VPE high concentration UV spectrophotometer linked to a Cary 50 spectrophotometer. Concentration was determined by absorbance at A280. Result reported as mean of duplicate sample readings. The pH was determined using the Hach H260G pH meter and ISFET solid state probe. All samples were stored at 5° C. after preparation.

	TABLE 2

	Replicates

Sample

	1	2	Mean	SD	% CV

Pre-dialysis

0 mM HPGCD	122.18	120.58	121.38	1.13	0.93
7.8 mM HPGCD	120.15	120.60	120.38	0.32	0.26
15.6 mM HPGCD	122.61	120.80	121.71	1.28	1.05
31.3 mM HPGCD	118.52	119.04	118.79	0.36	0.31
62.5 mM HPGCD	114.86	115.69	115.28	0.59	0.51
125 mM HPGCD	109.36	109.20	109.29	0.11	0.10
250 mM HPGCD	96.77	96.82	96.8	0.03	0.03

Post-dialysis

0 mM HPGCD	29.18	28.99	29.1	0.13	0.45
7.8 mM HPGCD	29.42	27.66	28.5	1.24	4.35
15.6 mM HPGCD	26.89	26.78	26.8	0.08	0.29
31.3 mM HPGCD	27.25	27.35	27.3	0.07	0.26
62.5 mM HPGCD	28.98	28.78	28.9	0.14	0.49
125 mM HPGCD	—	—	—	—	—
250 mM HPGCD	—	—	—	—	—

Visual assessment of the sample was performed each hour for the first five hours and after 72 hours. Results are reported in Table 3. Addition of HPGCD reduces the rate of precipitation.

	TABLE 3

	Precipitation Assessment

Time

	0 mM	7.8 mM	15.6 mM	31.3 mM	62.5 mM	125 mM	250 mM
(hr)	HPGCD	HPGCD	HPGCD	HPGCD	HPGCD	HPGCD	HPGCD

0	No	No	No	No	No	No	No
1	Light	Light	Light	Light	No	No	No
2	Moderate	Moderate	Moderate	Moderate	Light	No	No
3	Heavy	Heavy	Heavy	Heavy	Moderate	No	No
4	Heavy	Heavy	Heavy	Heavy	Moderate	Light	No
5	Heavy	Heavy	Heavy	Heavy	Heavy	Light	No
72	Heavy	Heavy	Heavy	Heavy	Heavy	Moderate	Light

Example 2: Methionine Screening

Once the impact of HPGCD on the precipitation profile of the basic romosozumab formulation of example 1 was assessed, the effect of methionine was investigated.
In order to determine whether methionine has an impact on the precipitation profile at physiological pH, basic formulations of romosozumab (pl 6.9) at a concentration of 90 mg/ml (control only) 120 mg/mL or 180 mg/ml with (55 mM and 200 mM) and without methionine were run through the pH shift model described in example 1.
Romosozumab in 50 mM sodium acetate, 14 mM calcium acetate, 6% sucrose, 0.006% polysorbate 20 at pH 5.2 (drug solution, DS) was concentrated to 125 mg/mL or to 190 mg/mL using 30 kDa Sartorius Viva Spin centrifugal spin filters. The spin tubes were centrifuged at 3000 rpm, 5° C. for 60 minutes; samples were mixed between spin cycles using a positive displacement pipette.
The required amount of methionine to produce 2.2 mL of sample with the required concentration (55 and 200 mM) was weighed (0.018 g and 0.0066 g, respectively) using an analytical balance. Methionine was dissolved in 2.2 mL of the appropriate protein solution (125 mg/mL or 190 mg/mL).
The concentration of each sample was measured in duplicate before and after dialysis (Table 4) using the C Technologies Solo VPE high concentration UV spectrophotometer linked to a Cary 50 spectrophotometer. Concentration was determined by absorbance at Å280. Result reported as mean of duplicate sample readings.

	TABLE 4

	Replicates

Sample

	1	2	Mean	SD	% CV

Pre-dialysis

120 mg/mL	122.39	124.35	123.38	1.39	1.12
55 mM Meth
120 mg/mL	121.81	124.43	123.13	1.85	1.51
200 mM Meth
180 mg/mL	188.84	187.96	188.40	0.62	0.33
55 mM Meth
180 mg/mL	186.70	186.69	186.70	0.01	0.00
200 mM Meth
90 mg/mL	90.93	91.32	91.13	0.27	0.30
Control
120 mg/mL	118.18	119.16	118.67	0.69	0.58
Control
180 mg/mL	182.90	183.31	183.11	0.29	0.16
Control

Post-dialysis

120 mg/mL	24.64	24.57	24.61	0.05	0.22
55 mM Meth
120 mg/mL	24.38	24.41	24.40	0.01	0.08
200 mM Meth
180 mg/mL	31.39	31.58	31.49	0.13	0.42
55 mM Meth
180 mg/mL	30.75	30.57	30.66	0.12	0.40
200 mM Meth
90 mg/mL	22.51	22.60	22.56	0.06	0.28
Control
120 mg/mL	23.54	23.57	23.56	0.02	0.10
Control
120 mg/mL	31.47	31.30	31.39	0.12	0.39
Control

The pH of the formulations pre-dialysis and after 24 h, measured using the Hach H260G pH meter and ISFET solid state probe, confirmed the pH shift (Table 5).

TABLE 5

pH	Viscosity	Osmolality

Sample	t0	t24	(cP)	(mOsmol)

120 mg/mL 55 mM Meth	5.14	7.80	5.21	392
120 mg/mL 200 mM Meth	5.28	7.81	5.55	573
180 mg/mL 55 mM Meth	5.23	7.75	34.36	508
180 mg/mL 200 mM Meth	5.23	7.73	29.56	712
90 mg/mL Control	5.27	7.86	2.55	321
120 mg/mL Control	5.22	7.80	4.76	320
180 mg/mL Control	5.28	7.77	27.21	398
Tissue buffer	6.99

Visual assessment of the sample was performed each hour for the first four hours. Results are reported in Table 6.

TABLE 6

	120 mg/mL	120 mg/mL	180 mg/mL	180 mg/mL
Time	55 mM	200 mM	55 mM	200 mM	90 mg/mL	120 mg/mL	180 mg/mL
(hr)	Meth	Meth	Meth	Meth	Control	Control	Control

0	No	No	No	No	No	No	No
1	Light	Light	Heavy	Heavy	Very Light	Light	Moderate
2	Heavy	Heavy	Very Heavy	Very Heavy	Moderate	Moderate	Very Heavy
3	Heavy	Heavy	Very Heavy	Very Heavy	Heavy	Heavy	Very Heavy
4	Heavy	Heavy	Very Heavy	Very Heavy	Heavy	Heavy	Very Heavy

As can be observed by the results reported in Table 6, the addition of methionine alone did not alter the rate of precipitation.

Example 3: Combination of HPGCD and Methionine Screening

Once assessed the impact of HPGCD alone or methionine alone on the precipitation profile of the basic romosozumab formulation of example 1, the combined effect of HPGCD and methionine was investigated.
Romosozumab in 50 mM sodium acetate, 14 mM calcium acetate, 6% sucrose, 0.006% polysorbate 20 at pH 5.2 (drug solution, DS) was concentrated to 125 mg/mL or to 190 mg/mL using 30kDa Sartorius Viva Spin centrifugal spin filters. The spin tubes were centrifuged at 3000 rpm, 5° C. for 60 minutes; samples were mixed between spin cycles using a positive displacement pipette.
The required amount of methionine to produce 2.2 mL of sample with the required concentration (55 and 200 mM) was weighed (0.018 g and 0.066 g, respectively) using an analytical balance. The Methionine was dissolved in 2.2 mL of the appropriate protein solution (125 mg/mL or 190 mg/mL). The required amount of HPGCD to produce 1 mL of 30 mM HPGCD at the required concentration was weighed (0.053 g) using an analytical balance. 1 ml of the appropriate protein/methionine solution was used to dissolve the HPGCD.
The formulations were buffer exchanged and concentrated as previously described in examples 1 and 2. The concentration of each sample was measured in duplicate before and after dialysis (Table 7) as previously described in examples 1 and 2. Results are reported as mean of duplicate sample readings.

	TABLE 7

	Replicates

Sample

	1	2	Mean	SD	% CV

Pre-dialysis

120 mg/mL 55 mM	118.83	121.55	120.19	1.92	1.60
Meth + 30 mM HPGCD
120 mg/mL 200 mM	118.63	119.00	118.82	0.26	0.22
Meth + 30 mM HPGCD
180 mg/mL 55 mM	184.44	185.70	185.08	0.89	0.48
Meth + 30 mM HPGCD
180 mg/mL 200 mM	182.84	181.63	182.24	0.86	0.47
Meth + 30 mM HPGCD
90 mg/mL Control	90.93	91.32	91.13	0.27	0.30
120 mg/mL Control	118.18	119.16	118.67	0.69	0.58
180 mg/mL Control	182.90	183.31	183.11	0.29	0.16

Post-dialysis

120 mg/mL 55 mM	21.65	21.61	21.63	0.02	0.11
Meth + 30 mM HPGCD
120 mg/mL 200 mM	21.23	21.34	21.29	0.07	0.36
Meth + 30 mM HPGCD
180 mg/mL 55 mM	27.37	27.66	27.52	0.20	0.74
Meth + 30 mM HPGCD
180 mg/mL 200 mM	26.63	26.48	26.56	0.11	0.41
Meth + 30 mM HPGCD
90 mg/mL DS	22.51	22.60	22.56	0.06	0.28
120 mg/mL DS	23.54	23.57	23.56	0.02	0.10
120 mg/mL DS	31.47	31.30	31.39	0.12	0.39

The pH of the formulations pre-dialysis and after 24 h confirmed the pH shift (Table 8).

TABLE 8

pH	Viscosity	Osmolality

Sample	t0	t24	(cP)	(mOsmol)

120 mg/mL 55 mM	5.19	7.80	6.56	486
Meth + 30 mM HPGCD
120 mg/mL 200 mM	5.24	7.75	7.09	672
Meth + 30 mM HPGCD
180 mg/mL 55 mM	5.19	7.76	41.62	621
Meth + 30 mM HPGCD
180 mg/mL 200 mM	5.28	7.66	37.07	862
Meth + 30 mM HPGCD
90 mg/mL Control	5.27	7.86	2.55	321
120 mg/mL Control	5.22	7.80	4.76	320
180 mg/mL Control	5.28	7.77	27.21	398
Tissue buffer*	6.99

Visual assessment of the sample was performed each hour for the first four hours. Results are reported in Table 9.

TABLE 9

	120 mg/mL	120 mg/mL	180 mg/mL	180 mg/mL
	55 mM	200 mM	55 mM	200 mM
	Meth +	Meth +	Meth +	Meth +
Time	30 mM	30 mM	30 mM	30 mM	90 mg/mL	120 mg/mL	180 mg/mL
(hr)	HPGCD	HPGCD	HPGCD	HPGCD	Control	Control	Control

0	No	No	No	No	No	No	No
1	Light	Very Light	Heavy	Moderate	Very Light	Light	Moderate
2	Heavy	Moderate	Heavy	Heavy	Moderate	Moderate	Very Heavy
3	Heavy	Heavy	Heavy	Heavy	Heavy	Heavy	Very Heavy
4	Heavy	Heavy	Heavy	Heavy	Heavy	Heavy	Very Heavy

The addition of HPGCD and methionine improved the rate of precipitation over the basic formulation, especially at high concentrations of romosozumab (180 mg/mL).

Example 4: Extended Methionine and HPGCD Screening and Effects on Osmolality and Viscosity

Twenty different formulations comprising combinations of HPGCD and methionine with and without calcium acetate were made in order to investigate the additional concentrations of methionine and HPGCD and their effect on the pH, viscosity and osmolality.
Romosozumab at 120 mg/mL in 50 mM sodium acetate, 14 mM calcium acetate, 6% sucrose, 0.006% polysorbate 20 at pH 5.2 (drug solution, DS) was concentrated to −190 mg/mL using 30 kDa Sartorius Viva Flow 50, bench scale tangential flow filtration (TFF) cartridge. System was run using a Masterflex peristaltic pump at a pressure of ˜2.5 Bar, ≥6x volume exchange.
Romosozumab at 120 mg/mL in DS was buffer exchanged to 55 mM sodium acetate, 6% sucrose, 0.006% polysorbate 20, pH5.2 (without calcium acetate) and concentrated to ˜190 mg/mL using 30 kDa Sartorius Viva Flow 50, bench scale tangential flow filtration (TFF) cartridge. System was run using a Masterflex peristaltic pump at a pressure of ˜2.5 Bar, ≥6x volume exchange.
Methionine and HPGCD were spiked according to the experiment design (Table 10).

TABLE 10

HPGCD
mM	Weight (g)	Methionine mM	Weight (g)

30	0.0529	55	0.0098
77.5	0.1366	127.5	0.0228
125	0.2203	200	0.0358
MW	1762 g/mol	MW	149.21 g/mol
Volume
1 ml	Volume	1.2 ml

The required amount of Methionine to produce 1.2 mL of sample with the required concentration was weighed using an analytical balance. The Methionine was dissolved in 2.2 mL of the appropriate protein solution (with/without calcium acetate). The required amount of HPGCD to produce 1 mL of sample at the required concentration was weighed using an analytical balance. 1 ml of the appropriate protein/methionine solution (with/without calcium acetate) was used to dissolve the HPGCD.
Samples were run through the pH shift model. The pH and the concentrations were measure pre- and post-dialysis as described above. Pre-dialysis osmolality and viscosity were also measured in order to identify specific formulations which would meet the physiological requirements for subcutaneous administration. Table 11 shows the 20 newly designed formulations and the 6 controls run in this experiment.

TABLE 11

				Concentration
Formulation	HPGCD	Met	Ca	(mg/mL)	Viscosity	Osmolality	pH

#	(mM)	(mM)	(14 mM)	Mean	(cP)	(mOsmol)	t0	t24

1	77.5	55	No	154.1	—	773.0	5.17	8.08
2	30	200	Yes	175.8	36.3	804.0	5.03	7.99
3	77.5	127.5	Yes	171.7	51.9	930.0	5.02	8.01
4	30	127.5	No	157.6	34.1	674.0	5.04	7.97
5	77.5	127.5	Yes	169.8	51.1	896.7	4.99	7.96
6	77.5	200	No	148.7	36.0	988.3	5.15	8.02
7	77.5	55	No	151.1	41.2	752.3	5.31	8.01
8	77.5	200	No	148.9	36.3	986.0	5.3	8.01
9	125	200	Yes	160.0	66.4	1305.5	5.25	8.08
10	30	55	Yes	178.4	36.5	590.3	5.32	8.05
11	77.5	127.5	Yes	174.0	51.8	908.3	5.2	8.00
12	30	127.5	No	160.4	27.5	654.0	5.13	8.04
13	125	55	Yes	167.7	73.2	1043.0	5.17	8.03
14	125	127.5	No	146.7	50.6	1149.0	5.22	8.05
15	125	127.5	No	146.8	52.4	1165.3	5.23	8.28
16	125	127.5	Yes	167.0	69.4	1198.0	5.18	8.04
17	77.5	55	Yes	177.5	53.3	819.0	5.15	8.15
18	77.5	200	Yes	173.5	48.6	1043.3	5.2	8.15
19	30	127.5	Yes	183.2	39.6	681.3	5.14	8.08
20	77.5	127.5	No	153.0	41.8	897.0	5.27	8.11
21	0	0	Yes	90.4	2.8	328.7	5.06	8.22
22	0	0	Yes	118.8	5.4	323.7	5.18	8.19
23	0	0	Yes	184.3	32.2	388.7	5.14	8.16
24	0	0	No	93.4	2.83	322.0	5.18	8.26
25	0	0	No	123.7		336.7	5.21	8.18
26	0	0	Yes	187.4	39.5	395.0	5.37	8.13

The concentrations of the formulations pre-dialysis were measured and are shown in Table 11, fifth column; the post-dialysis concentrations were also measured and their trends were similar to those of formulations previously tested (data not shown). Pre- and post-dialysis pH, viscosity and osmolality were measured and are also illustrated in Table 11.
Visual assessment of the sample was performed each hour for the first five hours. Results are reported in Table 12.

TABLE 12

hr	1	2	3	4	5	6	7	8	9	10	11	12	13

0	No	No	No	No	No	No	No	No	No	No	No	No	No
1	Very	Light	Very	Very	Very	Mode	Very	Mode	Light	Heavy	Light	Very	Mode
	Heavy		Light	Heavy	Light	rate	Heavy	rate				Heavy	rate
2	Very	Mode	Mode	Very	Mode	Heavy	Very	Heavy	Mode	Very	Mode	Very	Heavy
	Heavy	rate	rate	Heavy	rate		Heavy		rate	Heavy	rate	Heavy
3	Very	Heavy	Heavy	Very	Mode	Heavy	Very	Heavy	Mode	Very	Heavy	Very	Heavy
	Heavy			Heavy	rate		Heavy		rate	Heavy		Heavy
4	Very	Heavy	Heavy	Very	Heavy	Heavy	Very	Very	Mode	Very	Very	Very	Very
	Heavy			Heavy			Heavy	Heavy	rate	Heavy	Heavy	Heavy	Heavy
5	Very	Very	Very	Very	Very	Very	Very	Very	Heavy	Very	Very	Very	Very
	Heavy	Heavy	Heavy	Heavy	Heavy	Heavy	Heavy	Heavy		Heavy	Heavy	Heavy	Heavy

hr	14	15	16	17	18	19	20	21	22	23	24	25	26

0	No	No	No	No	No	No	No	No	No	No	No	No	No
1	Very	Very	Light	Mode	Mode	Heavy	Heavy	No	Light	Very	Mode	Heavy	Very
	Heavy	Heavy		rate	rate					Heavy	rate		Heavy
2	Very	Very	Mode	Heavy	Mode	Very	Very	Light	Mode	Very	Heavy	Very	Very
	Heavy	Heavy	rate		rate	Heavy	Heavy		rate	Heavy		Heavy	Heavy
3	Very	Very	Mode	Heavy	Heavy	Very	Very	Mode	Heavy	Very	Heavy	Very	Very
	Heavy	Heavy	rate			Heavy	Heavy	rate		Heavy		Heavy	Heavy
4	Very	Very	Heavy	Very	Heavy	Very	Very	Heavy	Heavy	Very	Heavy	Very	Very
	Heavy	Heavy		Heavy		Heavy	Heavy			Heavy		Heavy	Heavy
5	Very	Very	Heavy	Very	Very	Very	Very	Heavy	Very	Very	Heavy	Very	Very
	Heavy	Heavy		Heavy	Heavy	Heavy	Heavy		Heavy	Heavy		Heavy	Heavy

All results were entered into statistical analysis package (Software SAS JMP 11, version 11.0.0; 64 bit). Four formulations (details in Table 13, second column) were selected for further testing for having predicted osmolalities (˜800 mOsml up to ˜1000 mOsmol) and viscosities (<80 cP) suitable for desired pharmaceutical formulations.
To prepare the formulation comprising HPGCD and Methionine, it was required to buffer exchange and concentrate romosozumab from ˜119 mg/mL to ˜150 mg/mL and to ˜180 mg/ml; 6 x volume buffer exchanges was performed before concentration. Concentrations were measured using the Solo VPE high concentration UV spectrophotometer. Romosozumab Extinction Coefficient used was 1.50 ml/(mg*cm). Concentration was determined from duplicate readings. Buffer exchange and concentrations were performed in a VivaSpin Spin 20, 30KD centrifugal spin filters using the centrifuge in 1 hour blocks at 3200 rpm, 5° C. The material was mixed using a positive displacement pipette between each spin. Samples were prepared in duplicate. After 20 spins all 6 volumes of buffer were added to perform the buffer exchange. Concentration of ˜150 mg/mL and ˜180 mg/mL were achieved for formulations B, C and D.
Due to issue to concentrate formulation A, it was decided to use capillary to concentrate this formulation and attain a concentration of 150 mg/mL and then one of 180 mg/ml. All material was sterile filtered using a 0.22 mm syringe filter and stored at 5° C. after preparation. Each sample was spiked with the corresponding 1% polysorbate 20 to a concentration of 0.006%. All formulations were within ±5% of target concentration (Table 13).

TABLE 13

				Predicted	Predicted
Formulation		Required	Final	Osmolality	Viscosity
#	Details	concentration	Concentration	(mOsmol)	(cP)

A	55 mM NaACE*, 14 mM	150	148.30	772.8	52.6
	CaACE^#, 6% sucrose,
	0.006 % polysorbate 20,
	96 mM HPGCD, pH 5.2	180	176.87	800.4	64.6
B	55 mM NaACE*, 14 mM	150	152.25	773.5	42.8
	CaACE^#, 6% sucrose,
	0.006% polysorbate 20,	180	182.12	801.1	54.8
	78 mM HPGCD, 55 mM
	Methionine pH 5.2
C	55 mM NaACE*, 14 mM	150	151.02	771.5	33.1
	CaACE^#, 6% sucrose,
	0.006% polysorbate 20,	180	186.24	799.1	45.1
	55 mM HPGCD, 125 mM
	Methionine pH 5.2
D	55 mM NaACE*, 14 mM	150	151.21	955.3	40.5
	CaACE^#, 6% sucrose,
	0.006% polysorbate 20,
	80 mM HPγCD, 160 mM
	Methionine pH 5.2	180	183.48	982.9	52.3

*NaACE = Sodium Acetate;
^#CaACE = Calcium Acetate

The concentrations of these formulations were measured post-dialysis and concentrations were as expected following the same trends as for formulations previously tested (data not shown). The pre- and post-dialysis pHs of each of the formulations were measured along with the viscosities and osmolalities pre-dialysis and these results are reported in Table 14. In Table 14, A150 means formulation A as of Table 12 with 150 mg/mL of romosozumab, A180 means formulation A as of Table 13 with 180 mg/mL of romosozumab etc. Control formulations comprise the basic formulation of 55 mM sodium acetate, 14mM calcium acetate, 6% sucrose, 0.006 % polysorbate 20, 96 mM HPGCD, pH 5.2 at concentration of romosozumab of 90 mg/mL, 120 mg/mL, 150 mg/mL and 180 mg/mL.

TABLE 14

pH	Viscosity	Osmolality

	Formulation	t0	t24	(cP)	(mOsmol)

A 150	5.55	7.94	164.41	1680
A 180	#	8.12	#	#
B 150	5.61	8.05	133.16	748
B 180	5.57	8.02	594.17	#
C 150	5.48	7.96	83.00	747
C 180	5.65	7.99	343.54	847
D 150	5.64	7.99	162.55	853
D 180	5.71	8.03	765.78	#
Control 90	5.28	8.03	3.19	323
Control 120	5.22	7.99	6.09	326
Control 150	5.26	7.97	13.08	354
Control 180	5.26	7.99	27.2	386
Tissue Buffer	7.17	7.15

# = not enough sample to test

In summary, the combination of HPGCD and methionine has been found to be surprisingly beneficial for stabilizing antibodies, especially at high concentrations, in liquid formulations.

Claims

1. A liquid pharmaceutical formulation comprising:

a. an antibody or an antigen-binding fragment thereof as an active ingredient;

b. cyclodextrin and

c. methionine.

2. The liquid formulation according to claim 1 comprising methionine at a concentration of from 7.5 mM to 200 mM.

3. The liquid formulation according to claim 1 comprising cyclodextrin at a concentration of from 7.5 mM to 250 mM.

4. The liquid formulation according to claim 1 comprising the antibody or the antigen-binding fragment thereof at a concentration of from 1 to 200 mg/ml, preferably from 90 to 180 mg/ml.

5. The liquid formulation according to claim 1, wherein the antibody or the antigen-binding fragment thereof specifically binds human sclerostin.

6. The liquid formulation according to claim 1 wherein the antibody is a human or humanised antibody or antigen-binding fragment thereof.

7. The liquid formulation according to claim 1, wherein:

a. the antibody or the antigen-binding fragment thereof

i. comprises CDR-H1 having the sequence as defined in SEQ ID NO:1; CDR-H2 having the sequence as defined in SEQ ID NO:2; CDR-H3 having the sequence as defined in SEQ ID NO:3; CDR-L1 having the sequence as defined in SEQ ID NO:4; CDR-L2 having the sequence as defined in SEQ ID NO:5 and CDR-L3 having the sequence as defined in SEQ ID NO:6; or

ii. has a light variable region having the sequence as defined in SEQ ID NO: 7 and a heavy variable region having the sequence as defined in SEQ ID NO: 8; or

b. the antibody has

i. a light chain having the sequence as defined in SEQ ID NO: 9 and a heavy chain having the sequence as defined in SEQ ID NO: 10; or

ii. a light chain having the sequence as defined in SEQ ID NO: 11 and a heavy chain having the sequence as defined in SEQ ID NO: 12.

8. The liquid formulation according to claim 1, wherein the antibody is romosozumab.

9. The liquid formulation according to claim 1 having a pH of from 4.0 to 7.5.

10. The liquid formulation according to claim 1 wherein the cyclodextrin is selected from alpha-cyclodextrin, beta-cyclodextrin, dimethyl-beta-cyclodextrin, trimethyl-beta-cyclodextrin, randomly methylated beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin (i.e. 2 hydroxypropyl-beta-cyclodextrin, 3-hydroxypropyl-beta-cyclodextrin or 2,3-dihydroxypropyl-beta-cyclodextrin), hydroxyisobutyl-beta-cyclodextrin sulfobutylether beta-cyclodextrin, glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-gamma-cyclodextrin (i.e. 2 hydroxypropyl-gamma-cyclodextrin, 3-hydroxypropyl-gamma-cyclodextrin or 2,3-dihydroxypropyl-gamma-cyclodextrin), cyclodextrin-containing polymers or combinations thereof.

11. The liquid formulation according to claim 10, wherein the cyclodextrin is selected from hydroxypropyl-gamma-cyclodextrin.

12. The liquid formulation according to claim 1 wherein the formulation comprises from 1 mg/ml to 200 mg/ml of antibody, from 10 mM to 55 mM sodium acetate, from 0 to 14 mM calcium acetate, from 0 to 6% sucrose, from 0 to 0.006% polysorbate 20, from 55 mM to 80 mM hydroxypropyl-gamma-cyclodextrin, from 55 mM to 160 mM methionine at pH 5.2.

13. The liquid formulation according to claim 12, wherein the formulation comprises from 90 mg/ml to 180 mg/ml, preferably from 120 mg/ml to 180 mg/ml of romosozumab.

14. The liquid formulation according to claim 1 wherein the liquid formulation is not a hydrogel.

15. The liquid formulation according to claim 1 wherein the liquid formulation comprises at least 2%, at least 5%, at least 10%, at least 25%, between 2% and 90%, between 2% and 75%, or between 2% and 50% water.

16. The liquid formulation according to claim 1 for use in the treatment of a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength.

17. A method for treating a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength in a mammalian subject comprising administering a therapeutically effective amount of the formulation of claim 1.

18. Use of the liquid formulation according to any one of claims 1 in the manufacture of a medicament for the treatment of a bone disorder associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and low bone strength.

19. (canceled)

20. A method for reducing precipitation of an antibody or an antigen-binding fragment thereof in a liquid pharmaceutical formulation, the method comprising providing a liquid formulation and adding to the formulation methionine and cyclodextrin, preferably hydroxypropyl-gamma-cyclodextrin.

21. A method of reducing inflammation at injection site in a mammalian subject comprising administering a therapeutically effective amount of the formulation of claim 1.