WO2012028683A1

WO2012028683A1 - Antibody gel system for sustained drug delivery

Info

Publication number: WO2012028683A1
Application number: PCT/EP2011/065096
Authority: WO
Inventors: Margarida Rodrigues; Karin Schoenhammer
Original assignee: Novartis Ag
Priority date: 2010-09-02
Filing date: 2011-09-01
Publication date: 2012-03-08

Abstract

Antibody formulations, and methods for their manufacture, which (i) are capable of preserving an antibody in a native and therapeutically active state while achieving a depot effect without the use of polymers or other complex formulation reagents and (ii) provide very high loading capacity, thus offering the potential for increased dose per application to patient. These formulations take the form of a gel and are based on the surprising finding that some monoclonal antibodies have a gelation property under appropriate conditions.

Description

ANTIBODY GEL SYSTEM FOR SUSTAINED DRUG DELIVERY

TECHNICAL FIELD OF THE DISCLOSURE This disclosure is in the field of monoclonal antibody pharmaceutical formulation. BACKGROUND OF THE DISCLOSURE

Monoclonal antibodies (mAbs) are typically formulated either in aqueous form ready for parenteral administration or as lyophilisates for reconstitution with a suitable buffer prior to administration. Such formulations provide immediate release of the mAb after administration; release is not sustained and the mAb is rapidly cleared from the site of administration.

In order to reduce the number of doses administered to a patient, it is desirable to provide formulations that are capable of sustaining mAb release over longer periods. For example, reference 1 discloses the encapsulation of antibodies inside biodegradable poly(lactic-glycolic) acid microspheres. These are injected subcutaneously and provide sustained release. Example 4 of reference 2 discloses a surfactant/solvent gel formulation for sustained delivery of particulate mAbs.

In addition to sustained release, it is also helpful to keep a mAb local (e.g. within a fracture or at a site of inflammation) by using a depot system. For example, references 3 and 4 disclose topical delivery systems in which antibodies were dispersed within a poly(ethylene-co-vinyl acetate) matrix shaped as disks. After insertion into the body the disks release encapsulated antibody to local tissue over a sustained period. Reference 5 discloses an intratumoral injectable gel drug delivery system for local delivery of radiolabelled immunotherapeutic mAbs. A gel formulation of polyclonal antibodies for local delivery, using carboxymethylcellulose, is disclosed in reference 6

Current depot systems for mAbs use polymers, additives or excipients to control antibody release and prevent clearance. Such systems have several disadvantages that limit their therapeutic potential and clinical use. A major drawback is that their manufacture is complex (e.g. the use of a vacuum in reference 2) and can lead to degradation of mAbs, thus rendering them inactive. A second drawback is that, even when mAbs can be maintained in active form, the capacity of such systems (the amount of antibody that can be loaded) is quite low, which limits the amount of mAb that can be delivered in a single application, thus restricting therapeutic potential and clinical use.

Reference 62 discloses an antibody gel formulation of an anti-sclerostin antibody. The gel formulation therein was derived from lyophilized drug substance that was resuspended in a sodium phosphate buffer (PBS) to form a 150 mg/ml antibody-containing solution having a pH of 6.6. The reconstituted lyophlisate formed a gel structure having a turbidity of 1350 NTU, which was capable of sustained release of active monomeric antibody in vitro. However, the time required to achieve a turbidity of 1350 NTU was at least 20 minutes. Slow gellification of the mAb could allow dispersion of the antibody into underlying tissue and blood, resulting in reduced antibody concentration at the site of administration.

Thus there remains a need for further and improved rapidly gelling formulations for providing sustained local in vivo release of antibody. The advantage of a faster gellification of the mAb is expected to result in reduced initial release during application of the gelling system. The following Disclosure provides such improved gel formulations for anti-sclerositin antibodies, e.g., ANTIBODY 1 , and methods for their preparation.

BRIEF SUMMARY OF THE DISCLOSURE

The present invention describes antibody formulations, e.g., anti-sclerostin antibody formulations, such as formulations of ANTIBODY 1, and methods for their manufacture, which (i) are capable of rapidly gelling and preserving an antibody in a native and therapeutically active state while achieving a depot effect without the use of polymers or other complex formulation reagents and (ii) provide high loading capacity, thus offering the potential for increased dose per application to patient. These formulations take the form of a gel and are based on the surprising finding that rapid gellificaiton of certain monoclonal antibodies may be induced by using: 1) specific buffers to reconstitute a lyophilisate of the antibody or 2) specific buffers in combination with an aqueous antibody formulation that has not previously been lyophilized. Due to the rapid gellificaiton of the antibody, the resultant antibody gel formulations can be immediately administered to a patient as a depot (gel) system that releases active mAb over a sustained period of time without the use of current depot excipients.

Thus, provided herein is a process for preparing a gel formulation of ANTIBODY 1, comprising lyophilising a first aqueous formulation of ANTIBODY 1 at a first pH to give a ANTIBODY 1 lyophilisate; and reconstituting the lyophilisate with a MOPS or a HEPES buffer to provide a second aqueous formulation of ANTIBODY 1 having a second pH, the second pH being different from the first pH.

Provided herein is also a process for preparing a gel formulation of ANTIBODY 1, comprising lyophilising a first aqueous formulation of ANTIBODY 1 at a first pH to give a ANTIBODY 1 lyophilisate; and reconstituting the lyophilisate with a Tris buffer having a pH between about 7.8 and about 8.6 to provide a second aqueous formulation of ANTIBODY 1 having a second pH, the second pH being different from the first pH.

In some embodiments of the aforementioned processes, the first pH is lower than the second pH. In some embodiments of the aforementioned processes, the first pH is less than about 7.0. In some embodiments of the aforementioned processes, the first pH is in the range about 5.0 to about 6.0. In some embodiments of the aforementioned processes, the second pH and the first pH differ by at least one pH unit. In some embodiments of the aforementioned processes, the second pH is in the range about 6.0- about 8.0.

Provided herein is also a process for preparing a gel formulation of ANTIBODY 1, comprising reconstituting a ANTIBODY 1 lyophilisate with a MOPS or a HEPES buffer to provide an aqueous formulation of the ANTIBODY 1 antibody having a pH of about > 5.5 to about < 9.0

Provided herein is also a process for preparing a gel formulation of ANTIBODY 1, comprising reconstituting a ANTIBODY 1 lyophilisate with a Tris buffer having a pH between about 7.8 and 8.6 to provide a gel formulation of ANTIBODY 1 having a pH of about > 5.5 to about < 9.0.

Provided herein is also a process for preparing a gel formulation of ANTIBODY 1, comprising reconstituting a ANTIBODY 1 lyophilisate with a MOPS or a HEPES buffer, wherein the a MOPS buffer has a pH between about 6.6 and about 7.4 and the HEPES buffer has a pH between about 7.0 and about 7.8; and allowing the reconstituted lyophilisate to form the gel formulation or changing the pH of the reconstituted lyophilisate to cause formation of the gel formulation.

In some embodiments of the aforementioned processes, gel formation occurs less than about 30 minutes after reconstituting. In some embodiments of the aforementioned processes, the step of reconstituting is carried out at a temperature between room temperature and 37°C.

Provided herein is also a kit comprising, a ANTIBODY 1 lyophilisate at a first pH value; and a MOPS or a HEPES buffer at a second pH, wherein said MOPS or HEPES buffer, when combined with said ANTIBODY 1 lyophilisate, is capable of providing a formulation of ANTIBODY 1 that spontaneously forms a gel.

Provided herein is also a kit comprising, a ANTIBODY 1 lyophilisate; and a Tris buffer having a pH between about 7.8 and 8.6, wherein mixing of the lyophilisate and the Tris buffer gives an aqueous formulation of ANTIBODY 1 which either spontaneously forms a gel, or is not a gel but will form a gel in vivo. In some embodiments of the aforementioned processes or kits, the lyophilisate includes one or more lyophilization stabilizers selected from the group consisting of: sugars, amino sugars, amino acids and/or surfactants.

Provided herein is also a gel formulation prepared by the aforementioned processes. In some embodiments, the gel formulation can release antibody (e.g., ANTIBODY 1) in vivo for more than 7 days. In other embodiments, the gel formulation has a turbidity of about 1500 NTU to about 4000 NTU as measured by a HACH Tubidimeter 2100 AN. In other embodiments, the formulation has a turbidity of about 2000 NTU to about 3000 NTU. Except for the antibody, gel formulations of the invention, and formulations made using the methods and kits of the invention, typically do not include a gelling polymer.

Provided herein are also gel formulations comprising, a sclerostin antibody (e.g., ANTIBODY 1) in about 80-90 mM, e.g., about 83mM, MOPS buffer, a sclerostin antibody (e.g., ANTIBODY 1) in about 80-90 mM, e.g., about 83mM, HEPES buffer and a sclerostin antibody (e.g., ANTIBODY 1) in about 80-90 mM, e.g., about 83mM, TRIS buffer. In some embodiments, the gel formulation of the sclerostin antibody (e.g., ANTIBODY 1) is used in therapy, e.g., for use (a) in the treatment of bone injuries such as a bone fracture, or (b) in promoting osseointegration of a bone plate, pin, screw, prosthetic joint or dental implant. In other embodiments, the gel formulation of the sclerostin antibody (e.g., ANTIBODY 1) is used in the manufacture of a medicament for (a) the treatment of bone injuries such as a bone fracture, or (b) promoting osseointegration of a bone plate, pin, screw, prosthetic joint or dental implant. In other embodiments, the gel formulation reduces recovery time following injury or surgery.

Provided herein is also a process for preparing a gel formulation of AANTIBODY 1, comprising adjusting a first aqueous formulation of ANTIBODY 1 at a first pH with a MOPS buffer to provide a second aqueous formulation of ANTIBODY 1 having a second pH, the second pH being different from the first pH.

In some embodiments of the aforementioned processes, the first pH is lower than the second pH. In some embodiments of the aforementioned processes, the first pH is < about 7.0. In some embodiments of the aforementioned processes, the first pH is in the range of about 5.0 to about 6.0. In some embodiments of the aforementioned processes, the second pH and the first pH differ by at least one pH unit. In some embodiments of the aforementioned processes, the second pH is in the range of about 6.0 to about 8.0. In some embodiments of the aforementioned processes, the first aqueous formulation of ANTIBODY 1 is an aqueous ANTIBODY 1 formulation that has not previously been lyophilized. In some embodiments of the aforementioned processes, gel formation occurs less than about 30 minutes after reconstituting. In some embodiments of the aforementioned processes, the adjusting is carried out at a temperature between room temperature and 37°C.

Provided herein is also a kit comprising, an aqueous formulation of ANTIBODY 1 at a first pH; and a MOPS buffer at a second H, wherein said MOPS buffer, when combined with said aqueous formulation of ANTIBODY 1, is capable of providing a formulation of ANTIBODY 1 that spontaneously forms a gel.

Provided herein is also a gel formulation prepared by the aforementioned processes. In some embodiments, the gel formulation can release antibody (e.g., ANTIBODY 1) in vivo for more than 7 days. In other embodiments, the gel formulation has a turbidity of about 1500 NTU to about 4000 NTU as measured by a HACH Tubidimeter 2100 AN. In other embodiments, the gel formulation has a turbidity of about 2000 NTU to about 3000 NTU.

Provided herein is also a gel formulation comprising, a sclerostin antibody in about 20-40 mM, e.g., 26mM to about 34mM, MOPS buffer. In some embodiments, the sclerostin antibody is the ANTIBODY 1 antibody. Except for the antibody, gel formulations of the invention, and formulations made using the methods and kits of the invention, typically do not include a gelling polymer.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows ANTIBODY 1 release (%, as measured by SEC) in triplicate (vial 1-3) over time (hours) from a gel formed from ANTIBODY 1 lyophilisate (DP) reconstituted with a PBS buffer. The release experiments were performed at 37°C in 0.1M PBS buffer.

Figure 2 shows the % of released aggregation products from a gel formed from lyophilisate (DP) reconstitued with a PBS buffer, measured by SEC. Figure 3 shows the % of released degradation products from a gel formed from lyophilisate (DP) reconstitued with a PBS buffer, measured by SEC

Figure 4 shows the activity (%) at two timepoints of released ANTIBODY 1 from a gel formed of a lyophilisate (DP) reconstitued with a PBS buffer. At both timepoints the released mAb from the gelling system showed high bioactivity. The dotted line is the mean. Figure 5 shows the turbidity (NTU) increase over time (minutes) of ANTIBODY 1 lyophilisate (DP) reconstitued with different buffers at different pH levels. Note: the upper limit of the turbidimeter measurements is 4000NTU.

Figure 6 shows the effect of adding MOPS buffer pH 7.0 to ANTIBODY 1 drug substrate (DS). The first two images are 1 mL of ANTIBODY 1 drug substance at two different concentrations in 6R vials. The second two images are ANTIBODY 1 drug substance 1 min after addition of 100 mM

MOPS pH 7.0. ANTIBODY 1 concentrations are specified.

DETAILED DESCRIPTION OF THE DISCLOSURE

Non-lyophilised embodiments In certain embodiments, provided is an antibody gel formulation prepared from a liquid (e.g., aqueous) antibody formulation that has not previously been lyophilized (referred to herein as an antibody "drug substance" or "DS"), e.g., a liquid (e.g., aqueous) ANTIBODY 1 drug substance. The novel antibody drug substances have the desirable property of rapidly gellifying following a pH change. The pH of an antibody drug substance can be changed to initiate gelation. The pH change can be achieved by adding acid or base to the antibody drug substance, or by adding a diluent, e.g., a buffer (such as a MOPS buffer). The pH may be changed by at least one pH unit e.g. a difference of > about 1.0, > about 1.5, > about 2, > about 2.5, etc. The post-dilution pH (e.g., following addition of the buffer, e.g., a MOPS buffer, acid or base) may be higher than the starting pH or lower than the starting pH, preferably higher than the starting pH. The MOPS buffer may have a lower (e.g., pH 5.0) or higher (e.g., pH 7.0) pH than that of the aqueous antibody drug substance.

In one embodiment, the ANTIBODY 1 drug substance is in a Histidine buffer (e.g., 6.4 mM Histidine buffer). In one embodiment the ANTIBODY 1 drug substance is in a Histidine buffer having a pH of > 5.0, e.g., 5.3+0.1.

In one embodiment, the pH change of the antibody (e.g., ANTIBODY 1) drug substance is achieved by adding a MOPS buffer (e.g., about 1 mM to about 1 M MOPS buffer, preferably about 100 mM) having a pH of about 7.0.

In one embodiment, the concentration of the MOPS buffer following addition to the antibody (e.g., ANTIBODY 1) drug substance is about 20mM to about 30mM, e.g., about 26mM, for lOOmg/ml protein concentration. In another embodiment, the concentration of the MOPS buffer following addition to the antibody (e.g., ANTIBODY 1) drug substance is about 30mM to about 40mM, e.g., about 34mM, for 50mg/ml protein concentration.

After the pH change, e.g., caused by adding MOPS buffer to a ANTIBODY 1 drug substance, gelation ideally occurs spontaneously. In some embodiments, however, the pH change does not cause gelation, but is used to provide a formulation that is less painful for patient administration, with gelation being initiated after administration. In other embodiments, the pH change does not immediately cause gelation, but is used to provide a formulation that may be induced to form a gel

(e.g., by inducing further pH change) just prior to administration to a patient.

The kinetics of gelation caused by pH change may vary and gelation may occur quickly or slowly. For example, in some embodiments it may be substantially simultaneous with the the pH change. In other embodiments it may occur rapidly after the pH change (e.g. < about 60 minutes after, such as < about 45 minutes, < about 30 minutes, < about 20 minutes, < about 15 minutes, < about 14 minutes,

< about 13 minutes, < about 12 minutes, < about 11 minutes, ^about 10 minutes, < about 5 minutes,

< about 2 minutes, < about 1 minute etc.).

Gelation is easily detected and so it is simple to select appropriate gelation-causing pH changes for any particular aqueous mAb formulation. However, the pH change should not be so extreme as to irreversibly denature the mAb. Such denaturation is also easily detected, and so an appropriate pH window can readily be identified which ensures appropriate gelation while avoiding irreversible denaturation. For example, following addition of the diluent (e.g., a MOPS buffer) to the antibody (e.g., anti-sclerostin antibody, e.g., ANTIBODY 1) drug substance, the pH may be > about 5.5 (e.g. > about 6.0, > about 6.5, etc.), while typically not being above about 9.0 e.g. in the range about 5.5- about 9.0, or about 6.0 to about 8.0, or about 6.5 to about 7.5. The aqueous formulation formed in this manner may include active ingredients in addition to the mAb. For instance, further pharmacological agents may be included, such as chemotherapeutic compounds. These can be incorporated into a gel during its formation, facilitating co-delivery together with the mAb. In one embodiment, the pH following addition of the diluent, e.g., a MOPS buffer, to the antibody (e.g., ANTIBODY 1) drug substance is > 5.3, e.g., > 5.5 to < 8.0.

Provided herein is also a process for preparing a gel formulation of ANTIBODY 1, comprising adjusting a first aqueous formulation of ANTIBODY 1 at a first pH with a MOPS buffer to provide a second aqueous formulation of ANTIBODY 1 having a second pH, the second pH being different from the first pH.

In some embodiments of the aforementioned processes, the first pH is lower than the second pH. In some embodiments of the aforementioned processes, the first pH is < about 7.0. In some embodiments of the aforementioned processes, the first pH is in the range of about 5.0- about 6.0. In some embodiments of the aforementioned processes, the second pH and the first pH differ by at least one pH unit. In some embodiments of the aforementioned processes, the second pH is in the range of about 6.0 to about 8.0. In some embodiments of the aforementioned processes, the first aqueous formulation of ANTIBODY 1 is an aqueous ANTIBODY 1 formulation that has not previously been lyophilized. In some embodiments of the aforementioned processes, gel formation occurs less than about 30 minutes after reconstituting. In some embodiments of the aforementioned processes, the adjusting is carried out at a temperature between room temperature and 37°C.

Provided herein is also a kit comprising, an aqueous formulation of ANTIBODY 1 at a first pH; and a MOPS buffer at a second pH, wherein said MOPS buffer, when combined with said aqueous formulation of ANTIBODY 1, is capable of providing a formulation of ANTIBODY 1 that spontaneously forms a gel.

Provided herein is also a gel formulation prepared by the aforementioned processes. As used herein, the term "gel" refers to a substance (e.g., a solution or formulation) having a turbidity of at least about 1000 NTU, e.g., about 1200 NTU, 1400 NTU, 1600 NTU, 1800 NTU, 2000 NTU, 2200 NTU, 2400 NTU, 2600 NTU, 2800 NTU, 3000 NTU, 3200 NTU, 3400 NTU, 3600 NTU, 3800 NTU, 3800 NTU, 4000 NTU, 4200 NTU, 4400 NTU, 4600 NTU, 4800 NTU, 5000 NTU, 5200 NTU. In some embodiments, a gel solution of the disclosure has a turbidity of at least about 1000 NTU to about 4000 NTU, e.g., at least about 1000 NTU, at least about 1500 NTU, at least about 2000 NTU, at least about 3000 NTU, at least about 4000 NTU, etc. to . In other embodiments, the gel formulation has a turbidity of about 1500 NTU to about 4000 NTU as measured by a HACH Tubidimeter 2100 AN. In other embodiments, the gel formulation has a turbidity of about 2000 NTU to about 3000 NTU. The term "gellify" is used to refer to the process whereby a formulation takes on gel properties. In some embodiments, the gel formulation can release antibody (e.g., ANTIBODY 1) in vivo for more than 7 days.

Provided herein is also a gel formulation comprising, a sclerostin antibody in about 20 mM to about 40 mM, e.g., about 26mM to about 34mM, MOPS buffer. In some embodiments, the sclerostin antibody is the ANTIBODY 1 antibody. Except for the antibody, gel formulations of the invention, and formulations made using the methods and kits of the invention, typically do not include a gelling polymer.

Lyophilisates

Because antibodies useful with the invention have a gelation property under certain aqueous conditions, in certain embodiments of the invention, liquid antibody formulations (i.e., antibody drug substances) are prepared and stored in a lyophilised form (referred to herein as "lyophilisate", "drug product" or "DP"). Techniques for lyophilisation of mAbs are well known in the art e.g. see references 7 to 15. For example, monoclonal antibody products SYNAGIS™, REMICADE™,

NEUTROSPEC™, RAPTIVA™, SIMULECT™, XOLAIR™ and HERCEPTIN™ are supplied as lyophilisates.

The pH of the antibody drug substance to be lyophilized (i.e., the pre-lyophilisation pH) should be selected or controlled to ensure that gelation does not occur prior to lyophilization, unless such is specifically desired. Gelation is easily detected and so it is simple to select appropriate pH conditions for any particular mAb. For example, in some embodiments the pre-lyophilisation pH of the drug substance will be < about 7.0 (e.g. <6.5, <6.0, <5.5, etc.), while typically not being below about 4.5 e.g. in the range about 4.5- about 6.5 or about 5.0- about 6.0. For example, a pre-lyophilisation pH of 5.3+0.1 (via histidine buffer) is suitable for antibody ANTIBODY 1.

The lyophilisate may include, in addition to the mAb, lyophilisation stabilisers such as sugars, amino sugars, amino acids and/or surfactants. For instance, the lyophilisate may include one or more of: glycine, mannitol, sucrose, trehalose, hydroxyethyl starch and/or polyethylene glycol. These components will be present in the pre-lyophilisation aqueous formulation. Further components which may be present in the pre-lyophilisation aqueous formulation include buffers, salts, etc. A formulation containing sucrose, arginine and polysorbate 80 has been shown to be suitable for lyophilisation of antibody ANTIBODY 1.

The lyophilisate may include active ingredients in addition to the mAb. For instance, further pharmacological agents may be included, such as chemotherapeutic compounds. For instance, methotrexate may be included, and it is known to include methotrexate sodium in lyophilisates.

In one embodiment, the ANTIBODY 1 lyophilisate is 150 mg/ml ANTIBODY 1, 270 mM Sucrose, 51 mM Arg-HCl, 30 mM Histidine, 0.06% Tween 80, pH 5.3.

Aqueous reconstitution

Before a lyophilisate can be administered to a patient it should be reconstituted with an aqueous reconstituent. This step permits the antibody in the lyophilisate to re-dissolve. Reconstitution of the lyophilisate using an aqueous reconstituent (e.g., an aqueous solution such as a buffer) provides an aqueous antibody solution (hereinafter referred to as an antibody "reconstituate"). The process of reconstitution can facilitates a change in the formulation pH, which can initiate the gelation process. This may be achieved by employing a reconstituent having a different pH than the lyophilisate pH. Typical reconstituents for lyophilised mAbs include sterile water or buffer, optionally containing a preservative. Rather than reconstitute the anti-sclerositin (e.g., ANTIBODY 1) lyophilisate with water, however, the instant disclosure provides specific buffers as reconstituents. Buffered reconstituents are helpful in adjusting the pH of the antibody formulation to give a post- reconstitution pH that differs from the pre-lyophilisation pH. Suitable reconstituent buffers include TRIS, MOPS, HEPES, ACES, PIPES, MOPSO, TES, DIPSO, BES, TAPSO, MES, citrate, maleate, histidine, carbonate, and phosphate buffer.

In certain embodiments, the buffer used for reconstitution of the lyophilisate is at a concentration of from about 1 mM to about 1 M, e.g., about 10 mM, about 100 mM, etc. In one embodiment, the buffer concentration, e.g., MOPS, HEPES or Tris buffer concentration, is about 100 mM.

In certain embodiments, the final concentration of the buffer in the reconstitute is about 80-90 mM, e.g., 83mM.

In certain embodiments, the pH of the buffer used for reconstitution of the antibody lyophilisate is from about 6.8 to about 8.6, e.g., Tris buffer at about pH 7.8 to about pH 8.6, HEPES buffer at about pH 7.0 to about pH 7.8, and MOPS buffer at about pH 6.8 to about pH 7.4.

The aqueous reconstituent may include pharmacological agents, such as chemotherapeutic compounds, which can be incorporated into the gel during its formation, facilitating co-delivery together with the mAb.

In some embodiments, the pH of the antibody (e.g, ANTIBODY 1) drug substance (i.e., the pre- lyophilization pH) is different from the pH of the antibody (e.g., ANTIBODY 1) reconstituate (i.e., the post-reconstitution pH). The pre-lyophilisation pH should ideally be selected or controlled to ensure that gelation does not occur prior to lyophilization, unless such is specifically desired. Gelation is easily detected and so it is simple to select appropriate pH conditions for any particular mAb. The post-reconstitution pH should not be so extreme as to irreversibly denature the mAb, though. Such denaturation is also easily detected, and so an appropriate pH window for the pre- lyophilisate can readily be identified to avoid both gelation and irreversible denaturation.

The post-reconstitution pH of the antibody reconstituate typically differs from the pre-lyophilisation pH of the antibody drug substance by at least one pH unit e.g. a difference of >1.0, > 1.5, >2, >2.5, etc. The post-reconstitution pH may be higher than the pre-lyophilisation pH or lower than the pre- lyophilisation pH, preferably it is higher than the pre-lyophilisation pH. In some embodiments the post-reconstitution pH of the reconstituate will be > about 5.4 (e.g._> about 5.5, > about 6.0, > about

6.5, etc.), while typically not being above about 9.0, e.g., in the range about 5.5- about 9.0 or about 6.0 to about 8.0 or about 6.5 to about 7.5. In some embodiments, the pre-lyophilisation pH of the drug substance will be < about 7.0 (e.g. <6.5, <6.0, <5.5, etc.), while typically not being below about 4.5 e.g. in the range about 4.5- about 6.5 or about 5.0- about 6.0. For example, a pre-lyophilisation pH of 5.3+0.1 (via histidine buffer) is suitable for antibody ANTIBODY 1 drug substance.

In one embodiment, a pH of about 6.6+0.1 is provided for an antibody reconstituate, e.g., a ANTIBODY1 reconstituate. In some embodiments, this is achieved by reconstitution of the lyophilisate (e.g, a ANTIBODY 1 lyophilisate) with Tris buffer at about pH 7.8 to about pH 8.6, HEPES buffer at about pH 7.0 to about pH 7.8, or MOPS buffer at about pH 6.8 to about pH 7.4, which causes gelation to occur rapidly after reconstitution.

It will be understood that the post-reconstitution pH of the reconstituate will depend on the pre- lyophilisation pH of the drug substance and the pH of the aqueous reconstituent. Appropriate pH values can be selected for the drug substance and reconstituent according to the pH-related gelation properties of the antibody in question. For example, the reconstituent may have a pH below about 7.0 (e.g. below about 6.8, such as in the range about 5.0- about 6.8 or about 5.4- about 6.4) or a pH above about 7.0 (e.g. above about 7.2, such as in the range about 7.2- about 8.5 or about 7.4 to about 8.0). For general guidance, if the pre-lyophilisation pH is below pH 7.0 then a reconstituent with pH above about 7.0 will be used, and vice versa. After reconstitution, the anti-sclerositin antibody (e.g., ANTIBODY 1) reconstituate is suitable for gelation. The gelation may occur spontaneously after reconstitution or its initiation may require further alteration of the reconstituted formulation. For instance, the post-reconstitution pH may be further changed (for example by at least one pH unit e.g. a difference of >1.0,_>1.5, >2, >2.5, etc.) by addition of acid or base. The post-reconstitution pH may also be modified by administration of the formulation to a mammal, with the pH altering in vivo.

Whether gelation is initiated spontaneously after reconstitution, or requires further alteration of the reconstituted formulation, the kinetics of gelation may vary. Thus gelation may occur quickly or slowly. For example, in some embodiments it may be substantially simultaneous with reconstitution.

In other embodiments it may occur shortly after reconstitution (e.g. < about 60 minutes after reconstitution, such as < about 45 minutes, < about 30 minutes, < about 20 minutes, < about 15 minutes, < about 14 minutes, < about 13 minutes, < about 12 minutes, < about 11 minutes, < about

10 minutes, < about 5 minutes, < about 2 minutes, < about 1 minute etc.) or shortly after administration to a mammal (e.g. < about 60 minutes after administration, such as < about 45 minutes, < about 30 minutes, < about 20 minutes, < about 15 minutes, ^about 14 minutes, < about 13 minutes, < about 12 minutes, < about 11 minutes, < about 10 minutes, < about 5 minutes, < about 2 minutes, < about 1 minute etc.). Gelation may not occur if the antibody concentration is too low after reconstitution. Moreover, even when a gel is formed, excessive dilution leads to reduced loading capacity of the gel formulation. Thus reconstitution should be performed with an appropriate volume of reconstituent. Again, because gelation is easily detected it is simple to select appropriate post-reconstitution concentrations for any particular mAb. Thus, provided herein is a process for preparing a gel formulation of ANTIBODY 1, comprising lyophilising a first aqueous formulation of ANTIBODY 1 at a first pH to give a ANTIBODY 1 lyophilisate; and reconstituting the lyophilisate with a MOPS or a HEPES buffer to provide a second aqueous formulation of ANTIBODY 1 having a second pH, the second pH being different from the first pH. Provided herein is also a process for preparing a gel formulation of ANTIBODY 1, comprising lyophilising a first aqueous formulation of ANTIBODY 1 at a first pH to give a ANTIBODY 1 lyophilisate; and reconstituting the lyophilisate with a Tris buffer having a pH between about 7.8 and about 8.6 to provide a second aqueous formulation of ANTIBODY 1 having a second pH, the second pH being different from the first pH.

Provided herein is also a process for preparing a gel formulation of ANTIBODY 1, comprising reconstituting a ANTIBODY 1 lyophilisate with a MOPS or a HEPES buffer, wherein the a MOPS buffer has a pH between about 6.6 and about 7.4 and the HEPES buffer has a pH between about 7.0 and about 7.8; and allowing the reconstituted lyophilisate from step a) to form the gel formulation or changing the pH of the reconstituted lyophilisate from step a) to cause formation of the gel formulation.

Provided herein is also a kit comprising, a ANTIBODY 1 lyophilisate; and a Tris buffer having a pH between about 7.8 and 8.6, wherein mixing of the lyophilisate and the Tris buffer gives an aqueous formulation of ANTIBODY 1 which either spontaneously forms a gel, or is not a gel but will form a gel in vivo.

In some embodiments of the aforementioned processes or kits, the lyophilisate includes one or more lyophilization stabilizers selected from the group consisting of: sugars, amino sugars, amino acids and/or surfactants.

Provided herein are also gel formulations comprising, a sclerostin antibody (e.g., ANTIBODY 1) in about 80-90 mM, e.g., 83mM, MOPS buffer, a sclerostin antibody (e.g., ANTIBODY 1) in about 80- 90 mM, e.g., 83mM, HEPES buffer and a sclerostin antibody (e.g., ANTIBODY 1) in about 80-90 mM, e.g., 83mM, TRIS buffer. In some embodiments, the gel formulation of the sclerostin antibody (e.g., ANTIBODY 1) is used in therapy, e.g., for use (a) in the treatment of bone injuries such as a bone fracture, or (b) in promoting osseointegration of a bone plate, pin, screw, prosthetic joint or dental implant. In other embodiments, the gel formulation of the sclerostin antibody (e.g., ANTIBODY 1) is used in the manufacture of a medicament for (a) the treatment of bone injuries such as a bone fracture, or (b) promoting osseointegration of a bone plate, pin, screw, prosthetic joint or dental implant. In other embodiments, the gel formulation reduces recovery time following injury or surgery. The gel formulation

The invention provides gel formulations of mAbs. These formulations can give sustained release of the mAb in vivo. The gel formulation is physically distinct from mere antibody precipitates and opalescent turbid antibody suspensions, both of which are known in the art {e.g. see references 16 & 17). Although the gels have not been subjected to detailed rheological analysis, once formed they are structurally stable e.g. they do not appreciably flow out of an inverted test tube, and water droplets will stay on the gel surface rather than penetrate it.

An advantage of the disclosed gel formulations is that they do not require the presence of the polymers, additives or excipients that are currently used for sustained mAb release. Thus, for instance, the formulation does not have to include a gelling polymer, such as celluloses or polyacrylates or polyvinyl alcohols. Instead, the capacity for gel formation is intrinsic in the mAb itself rather than in any non-mAb component in the formulation (including any non-mAb component which may be attached to the mAb). In some embodiments it may be useful to include such gelling polymers {e.g. to slow down release of mAb from the gel), but their absence is preferred. The absence of extrinsic gelling components reduces the potential for adverse patient reactions.

Gel formulations of the invention are typically turbid. For example, they may have a turbidity above about 500 NTU (Nephelometric Turbidity Units) e.g. > about 750 NTU, > about 1000 NTU, > about 1250 NTU, > about 1500 NTU, > about 2000 NTU, > about 2500 NTU, > about 3000 NTU, > about 3500 NTU, > about 4000 NTU etc. when measured at 25°C and atmospheric pressure. For example, a useful gel formulation of antibody ANTIBODY 1 may have a turbidity of about 1350 NTU to about 4000 NTU, about 1500 NTU to about 4000 NTU, or about 2000 NTU to about 3000 NTU, e.g., about at least 1350 NTU or about at least 1500 NTU.

An advantageous feature of gel formulations of the invention is their ability to release antibody in active form into surrounding aqueous media. Thus the gel can be contacted with an aqueous medium (whether in vitro or in vivo) and antibodies can transfer passively from the gel into the medium in active form. After release they can interact with target antigens, either locally or remotely. Gel formulations of the invention may be able to release antibody for more than about 2 days e.g. > about 3 days, > about 4 days, > about 5 days, > about 6 days, > about 7 days, > about 10 days, > about 14 days, > about 21 days, > about 28 days, etc. Release typically occurs at a higher initial rate which decreases over time. Gel formulations of the invention are pharmaceutically acceptable and are suitable for administration to a patient. In addition to mAb and water they may include further components, including those typical of pharmaceutical formulations buffers, salts, amino acids, glycerol, alcohols, preservatives, surfactants, etc. A thorough discussion of such pharmaceutical ingredients is available in reference 18. When the gel formulation has been formed from a lyophilisate and reconstituent then these pharmaceutical ingredients may originate from either source.

The use of mAbs as the active ingredient of pharmaceuticals is now widespread, including the products HERCEPTIN™ (trastuzumab), RITUXAN™ (ntuximab), SYNAGIS™ (palivizumab), etc. Techniques for purification of mAbs to a pharmaceutical grade are well known in the art. The gel formulation will usually be sterile, at least at the time of its formation. The composition will usually be non-pyrogenic e.g. containing < about 1 EU (endotoxin unit, a standard measure) per dose, and preferably < about 0.1 EU per dose. The composition is preferably gluten free.

As mentioned above, excessive volume of reconstituent gives a gel formulation with lower loading capacity. In embodiments in which a lyophilisate is reconstituted, reconstitution gives a mAb concentration of at least about 50 mg/rriL is typical e.g. > about 100 mg/rriL, > about 150 mg/rriL, > about 200 mg/rriL, > about 250 mg/rriL, etc. These concentrations are achievable in aqueous formulations e.g. SYNAGIS™ is provided for reconstitution to give a mAb concentration of 100 mg/mL. In embodiments in which the pH change is initiated by adding a specific diluent (e.g., a buffer) to an antibody (e.g., ANTIBODY 1) drug substance, the mAb concentration is at least about 50 mg/mL, e.g. > about 100 mg/mL, > about 150 mg/mL, > about 200 mg/mL, > about 250 mg/mL, etc.

Within formulations of the invention, a mAb preferably make up at least about 80% by weight {e.g. at least about 90%, about 95%, about 97%, about 98%, about 99% or more) of the total protein in the formulation. The mAb is thus in purified form. Target diseases and disorders

Gel formulations of the invention can be used to treat or prevent a variety of diseases or disorders.

For example, the gel is suitable for treatment of bone injuries. The gel can be formed at the site of the bone injury and can stay in local contact with it while releasing its active mAb ingredient. The mAb

{e.g. an anti-sclerostin antibody such as ANTIBODY 1) can be maintained at the injury site for extended time, allowing penetration into the canaliculi to reach high concentrations at osteocytes. Thus, in one embodiment, the gel may be applied at the site of a bone fracture. Such an application would reduce healing time. This embodiment would be particularly useful for the treatment of open fractures, complete fractures, spiral fractures or multi-fragmentary fractures. In another embodiment, a gel comprising an anti-sclerostin antibody such as ANTIBODY 1 may be used as a slow-release depot system for the treatment of osteoporosis.

The gel may also be applied at a site where a bone prosthesis is used, to promote osseointegration. Thus, the gel may be applied at the site where a bone plate, pin or screw is located. For example, such plates, pins or screws may be used to assist with fracture healing. The gel may be coated onto the plate, pin or screw, prior to fixation to the bone. Alternatively, the gel may be applied subsequent to fixation of the plate, pin or screw. The plates, pins and screws may be made out of various materials, or combinations of materials such as stainless steel, titanium, ceramic, collagen or plastic. Various types of plates, pins and screws used with bone and fracture healing are known in the art, and various types are summarised in reference 19.

In another embodiment, the gel may be applied at a site of joint replacement, to promote osseointegration of the prosthesis. Such joint replacements typically include hip, knee, shoulder and elbow replacements. In one embodiment, the gel may be placed into the bone marrow cavity prior to fixation of the artificial joint. In another embodiment, the gel may be used as a filler following fixation of the artificial joint.

The gel is also suitable for treatment of dental disorders and for improving the success of dental implants. About 8% of maxillar and 5% of mandibular implants fail in the normal population. To reduce this failure rate a mAb (e.g. an anti-sclerostin antibody) can be used to treat the alveolar socket and/or ridge prior to re-implantation of a tooth or implantation of a prosthetic tooth. Damage caused by drilling of the jaw bones can be minimised by administration of a gel of the invention prior to insertion of the tooth, thereby improving fixation, decreasing healing time and improving osseointegration of the tooth. The implant may be a re-implantation of a subject's own tooth (e.g. lost through trauma) or a prosthetic implant (made of, for example, plastic, ceramic, metal or from stem cells as described in WO2004/074464).

The gel is also suitable for treatment of respiratory diseases. Topical treatment of lung disease (e.g. COPD) with mAbs is known in the art, such as by delivery of an anti-inflammatory mAb. The gel is useful for treatment of osteo- or psoriatic- or rheumatoid arthritis. Thus the gel may be applied at joints. Arthritis therapy by mAbs is well known in the art e.g. using adalimumab (HUMIRA™) or infliximab (REMICADE™).

The gel is also useful for local treatment of tumours. Tumour therapy by mAbs is known in the art e.g. using trastuzumab (HERCEPTIN™), rituximab (RITUXAN™ or MABTHERA™).

The gel is also useful for topical treatment of skin to aid healing and/or regeneration. Skin treatment by mAbs is known in the art e.g. using efalizumab (RAPTIVA™).

Patient administration

As mentioned above, a gel formulation of the invention may form in vitro and then be administered to a patient, or it may form in vivo after its ingredients have been administered. Administration will typically be via a syringe.

Patients will receive an effective amount of the mAb active ingredient i.e. an amount that is sufficient to detect, treat, ameliorate, or prevent the disease or disorder in question. Therapeutic effects may also include reduction in physical symptoms. The optimum effective amount and concentration of mAb in a gel for any particular subject will depend upon various factors, including the patient's age, size, health and/or gender, the nature and extent of the condition, the activity of the particular mAb, the rate of its clearance by the body, and also on any possible further therapeutic(s) administered in combination with the mAb. The effective amount delivered for a given situation can be determined by routine experimentation and is within the judgment of a clinician. For purposes of the present invention, an effective dose may be from about 0.01 mg/kg to about 50 mg/kg, or about 0.05 mg/kg to about 30 mg/kg, e.g., 10 mg/kg. Known antibody-based pharmaceuticals provide guidance in this respect e.g. HERCEPTIN™ is administered with an initial loading dose of 4 mg/kg body weight and a weekly maintenance dose of 2 mg/kg body weight; RITUXAN™ is administered weekly at 375 mg/m²; SYNAGIS™ is administered intramuscularly at 15 mg/kg body weight; etc. The invention provides a method for delivering a monoclonal antibody to a mammal (e.g. a human), comprising a step of administering to the patient a gel formulation of the invention.

The invention also provides a method for delivering a monoclonal antibody to a mammal, comprising steps of: (i) preparing an aqueous formulation of the monoclonal antibody {e.g. as described above), wherein the aqueous formulation will form a gel after x minutes of its preparation; and (ii) administering the aqueous formulation to the patient within x minutes of its preparation. The invention also provides a method for delivering a monoclonal antibody to a mammal, comprising steps of: (i) preparing an aqueous formulation of the monoclonal antibody (e.g. as described above), wherein the aqueous formulation will form a gel in vivo; and (ii) administering the aqueous formulation to the patient to permit formation of the gel. The invention also provides formulations of the invention for use as medicaments e.g. for use in delivering a monoclonal antibody to a mammal.

The mammal is preferably a human but may also be, for example, a horse or a cow or a dog or a cat. The mAb will ideally be chosen to match the target species e.g. a human antibody for human administration, an equine antibody for horses, a canine antibody for dogs, etc. If native host antibodies are not available then transfer of antibody specificity from one species to another can be achieved by transfer of CDR residues (and typically, in addition, one or more framework residues) from a donor antibody into a recipient framework from the host species e.g. as in humanisation. Equinised, bovinised, caninised, camelised and felinised antibodies are known in the art.

With mAb ANTIBODY 1 these methods and uses may be for treating a bone injury. Dosage can be by a single dose schedule or a multiple dose schedule.

Ingredients for forming gels (e.g. kit components) may be supplied in hermetically-sealed containers.

The monoclonal antibody

The invention concerns the formulation of monoclonal antibodies. The term "monoclonal" as originally used in relation to antibodies referred to antibodies produced by a single clonal line of immune cells, as opposed to "polyclonal" antibodies that, while all recognizing the same target protein, were produced by different B cells and would be directed to different epitopes on that protein. As used herein, the word "monoclonal" does not imply any particular cellular origin, but refers to any population of antibodies that display a single binding specificity and affinity for a particular epitope in the same target protein. This usage is normal e.g. the product datasheets for the CDR-grafted humanised antibody SYNAGIS™ expressed in a murine myeloma NSO cell line, for the humanised antibody HERCEPTIN™ expressed in a CHO cell line, and for the phage-displayed antibody HUMIRA™ expressed in a CHO cell line, all refer to the active ingredients as "monoclonal" antibodies. Thus a mAb may be produced using any suitable protein synthesis system, including immune cells, non-immune cells, acellular systems, etc. A mAb can thus be produced by a variety of techniques, including conventional monoclonal antibody methodology {e.g. the standard somatic cell hybridization technique of Kohler & Milstein), by viral or oncogenic transformation of B lymphocytes, by combinatorial synthesis, by phage display, etc.

Antibodies used with the invention can take various forms. For instance, they may be native antibodies, as naturally found in mammals. Native antibodies are made up of heavy chains and light chains. The heavy and light chains are both divided into variable domains and constant domains. The ability of different antibodies to recognize different antigens arises from differences in their variable domains, in both the light and heavy chains. Light chains of native antibodies in vertebrate species are either kappa (κ) or lambda (λ), based on the amino acid sequences of their constant domains. The constant domain of a native antibody's heavy chains will be α, δ, ε, γ or μ, giving rise respectively to antibodies of IgA, IgD, IgE, IgG, or IgM class. Classes may be further divided into subclasses or isotypes e.g. IgGl, IgG2, IgG3, IgG4, IgA, IgA2, etc. Antibodies may also be classified by allotype e.g. a j heavy chain may have Glm allotype a, f, x or z, G2m allotype n, or G3m allotype bO, bl, b3, b4, b5, c3, c5, gl, g5, s, t, u, or v; a κ light chain may have a Km(l), Km(2) or Km(3) allotype. A native IgG antibody has two identical light chains (one constant domain C_L and one variable domain V_L) and two identical heavy chains (three constant domains C_HI , C_H2 & C_H3 and one variable domain V_H), held together by disulfide bridges. The domain and three-dimensional structures of the different classes of native antibodies are well known.

Where an antibody of the invention has a light chain with a constant domain, it may be a κ or λ light chain. Where an antibody of the invention has a heavy chain with a constant domain, it may be an a, δ, ε, γ or μ heavy chain. Heavy chains in the γ class (i.e. IgG antibodies) are preferred.

Antibodies of the invention may be fragments of native antibodies that retain antigen binding activity. For instance, papain digestion of native antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment without antigen-binding activity. Pepsin treatment yields a "F(ab')₂" fragment that has two antigen-binding sites. "Fv" is the minimum fragment of a native antibody that contains a complete antigen-binding site, consisting of a dimer of one heavy chain and one light chain variable domain. Thus an antibody of the invention may be Fab, Fab', F(ab')₂, Fv, or any other type, of fragment of a native antibody. An antibody of the invention may be a "single-chain Fv" ("scFv" or "sFv"), comprising a VH and VL domain as a single polypeptide chain [20-22]. Typically the VH and VL domains are joined by a short polypeptide linker (e.g. >12 amino acids) between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. A typical way of expressing scFv proteins, at least for initial selection, is in the context of a phage display library or other combinatorial library [23-25]. Multiple scFvs can be linked in a single polypeptide chain [26].

An antibody of the invention may be a "diabody" or "triabody" etc. [27-30], comprising multiple linked Fv (scFv) fragments. By using a linker between the VH and VL domains that is too short to allow them to pair with each other (e.g. <12 amino acids), they are forced instead to pair with the complementary domains of another Fv fragment and thus create two antigen-binding sites. These antibodies may include CH and/or CL domains.

An antibody of the invention may be a single variable domain or VHH antibody. Antibodies naturally found in camelids (e.g. camels and llamas) and in sharks contain a heavy chain but no light chain. Thus antigen recognition is determined by a single variable domain, unlike a mammalian native antibody [31-33]. The constant domain of such antibodies can be omitted while retaining antigen-binding activity. One way of expressing single variable domain antibodies, at least for initial selection, is in the context of a phage display library or other combinatorial library [34].

An antibody of the invention may be a "domain antibody" (dAb). Such dAbs are based on the variable domains of either a heavy or light chain of a human antibody and have a molecular weight of approximately 13 kDa (less than one-tenth the size of a full antibody). By pairing heavy and light chain dAbs that recognize different targets, antibodies with dual specificity can be made. dAbs are cleared from the body quickly and so benefit from a sustained release system, but can additionally be sustained in circulation by fusion to a second dAb that binds to a blood protein (e.g. to serum albumin), by conjugation to polymers (e.g. to a polyethylene glycol), or by other techniques. The antibody may have a scaffold which is based on the fibronectin type ΠΙ domain, as disclosed in reference 35 e.g. an adnectin or trinectin. The fibronectin-based scaffold is not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment. Because of this structure the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of natural antibodies. The Fnffl domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to antibody CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands. The Fnlll loops can be replaced with immunoglobulin CDRs using standard cloning techniques, and can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. The Fnlll scaffold may be based on the tenth module of fibronectin type III {i.e. 10Fn3).

Thus the term "antibody" as used herein encompasses a range of proteins having diverse structural features, but usually including at least one immunoglobulin domain, having an all-β protein fold with a 2-layer sandwich of anti-parallel β-strands arranged in two β-sheets. In all embodiments, however, the mAb has the ability to form a gel as described herein. Although not all mAbs will have this inherent gelation property, it is simple to determine if it is possessed by any particular mAb e.g. by detecting physicochemical changes after lyophilisation and reconstitution as described above.

Antibodies used with the invention may include a single antigen-binding site {e.g. as in a Fab fragment or a scFv) or multiple antigen-binding sites {e.g. as in a F(ab')₂ fragment or a diabody or a native antibody). Where an antibody has more than one antigen-binding site then advantageously it can result in cross-linking of antigens.

Where an antibody has more than one antigen-binding site, the antibody may be mono-specific {i.e. all antigen-binding sites recognize the same antigen) or it may be multi-specific {i.e. the antigen- binding sites recognise more than one antigen).

An antibody of the invention may include a non-protein substance e.g. via covalent conjugation. For example, an antibody may include a radio-isotope e.g. the ZEVALIN™ and BEXXAR™ products include ⁹⁰Y and ^{13 l}l isotopes, respectively. As a further example, an antibody may include a cytotoxic molecule e.g. MYLOTARG™ is linked to N-acetyl-y-calicheamicin, a bacterial toxin. As a further example, an antibody may include a covalently-attached polymer e.g. attachment of polyoxyethylated polyols or polyethylene glycol (PEG) has been reported to increase the circulating half-life of antibodies.

In some embodiments, an antibody can include one or more constant domains {e.g. including C_H or C_L domains). As mentioned above, the constant domains may form a κ or λ light chain or an α, δ, ε, γ or μ heavy chain. Where an antibody includes a constant domain, it may be a native constant domain or a modified constant domain. A heavy chain may include either three (as in α, γ, δ classes) or four (as in μ, ε classes) constant domains. Constant domains are not involved directly in the binding interaction between an antibody and an antigen, but they can provide various effector functions, including but not limited to: participation of the antibody in antibody-dependent cellular cytotoxicity

(ADCC); Clq binding; complement dependent cytotoxicity; Fc receptor binding; phagocytosis; and down-regulation of cell surface receptors.

The constant domains can form a "Fc region", which is the C-terminal region of a native antibody's heavy chain. Where an antibody of the invention includes a Fc region, it may be a native Fc region or a modified Fc region. A Fc region is important for some antibodies' functions e.g. the activity of HERCEPTIN™ is Fc-dependent. Although the boundaries of the Fc region of a native antibody may vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226 or Pro230 to the heavy chain's C-terminus. The Fc region will typically be able to bind one or more Fc receptors, such as a FcyRI (CD64), a FcyRII {e.g. FcyRIIA, FcyRIIBl, FcyRIIB2, FcyRIIC), a FcyRIII (e.g. FcyRIIIA, FcyRIIIB), a FcRn, FcaR (CD89), Fc5R, FcμR, a FcsRI {e.g. Fc8RIa y₂ or FcsRIor^), FcsRII {e.g. FcsRIIA or FcsRIIB), etc. The Fc region may also or alternatively be able to bind to a complement protein, such as Clq. Modifications to an antibody's Fc region can be used to change its effector function(s) e.g. to increase or decrease receptor binding affinity. For instance, reference 36 reports that effector functions may be modified by mutating Fc region residues 234, 235, 236, 237, 297, 318, 320 and/or 322. Similarly, reference 37 reports that effector functions of a human IgGl can be improved by mutating Fc region residues (EU Index Kabat numbering) 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 and/or 439. Modification of Fc residues 322, 329 and/or 331 is reported in reference 38 for modifying Clq affinity of human IgG antibodies, and residues 270, 322, 326, 327, 329, 331, 333 and/or 334 are selected for modification in reference 39. Mapping of residues important for human IgG binding to FcRI, FcRII, FcRIII, and FcRn receptors is reported in reference 40, together with the design of variants with improved FcR-binding properties. Whole C_H domains can be substituted between isotypes e.g. reference 41 discloses antibodies in which the C_H3 domain (and optionally the C_H2 domain) of human IgG4 is substituted by the C_H3 domain of human IgGl to provide suppressed aggregate formation. Reference 41 also reports that mutation of arginine at position 409 (EU index Kabat) of human IgG4 to e.g. lysine shows suppressed aggregate formation. Mutation of the Fc region of available monoclonal antibodies to vary their effector functions is known e.g. reference 42 reports mutation studies for RITUXAN™ to change Clq-binding, and reference 43 reports mutation studies for NUMAX™ to change FcR-binding, with mutation of residues 252, 254 and 256 giving a 10-fold increase in FcRn-binding without affecting antigen-binding. Antibodies will typically be glycosylated. N-linked glycans attached to the C_H2 domain of a heavy chain, for instance, can influence Clq and FcR binding [40], with aglycosylated antibodies having lower affinity for these receptors. The glycan structure can also affect activity e.g. differences in complement-mediated cell death may be seen depending on the number of galactose sugars (0, 1 or 2) at the terminus of a glycan's biantennary chain. An antibody's glycans preferably do not lead to a human immunogenic response after administration.

Antibodies can be prepared in a form free from products with which they would naturally be associated. Contaminant components of an antibody's natural environment include materials such as enzymes, hormones, or other host cell proteins. Useful antibodies have nanomolar or picomolar affinity constants for their target antigens {e.g. 10^"9 M, 10^"10 M, 10^"n M, 10^"12 M, 10^"13 M or tighter). Such affinities can be determined using conventional analytical techniques e.g. using surface plasmon resonance techniques as embodied in BIAcore™ instrumentation and operated according to the manufacturer's instructions. Radioimmunoassay using radiolabeled target antigen (hemagglutinin) is another method by which binding affinity may be measured.

The monoclonal antibody used with the invention may be a human antibody, a humanized antibody, a chimeric antibody or (particularly for veterinary purposes) a non-human antibody.

In some embodiments the antibodies are human mAbs. These can be prepared by various means. For example, human B cells producing an antigen of interest can be immortalized e.g. by infection with Epstein Barr Virus (EBV), optionally in the presence of a polyclonal B cell activator [44 & 45]. Human monoclonal antibodies can also be produced in non-human hosts by replacing the host's own immune system with a functioning human immune system e.g. into Scid mice or Trimera mice. Transgenic and transchromosomic mice have been successfully used for generating human monoclonal antibodies, including the "humab mouse" from Medarex and the "xeno-mouse" from Abgenix [46], collectively referred to herein as "human Ig mice". Phage display has also been successful [47], and led to the HUMIRA™ product. Unlike non-human antibodies, human antibodies will not elicit an immune response directed against their constant domains when administered to humans. Furthermore, the variable domains of these human antibodies are fully human (in particular the framework regions of the variable domains are fully human, in addition to the complementarity determining regions [CDRs]) and so will not elicit an immune response directed against the variable domain framework regions when administered to humans (except, potentially, for any anti-idiotypic response). Human antibodies do not include any sequences that do not have a human origin. In some embodiments the antibodies are humanised mAbs, CDR-grafted mAbs or chimeric mAbs.

These can be prepared by various means. For example, they may be prepared based on the sequence of a non-human (e.g. murine) monoclonal antibody. DNA encoding the non-human heavy and light chain immunoglobulins can be obtained and engineered to contain human immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art. To create a CDR-grafted antibody, the murine CDR regions can be inserted into a human framework [48-53]. To create a humanized antibody, one or more non-CDR variable framework residue(s) is also altered. The HI, H2 and H3 CDRs may be transferred together into an acceptor VH domain, but it may also be adequate to transfer only one or two of them [51]. Similarly, one two or all three of the LI, L2 and L3 CDRs may be transferred into an acceptor VL domain. Preferred antibodies will have 1, 2, 3, 4, 5 or all 6 of the donor CDRs. Where only one CDR is transferred, it will typically not be the L2 CDR, which is usually the shortest of the six. Typically the donor CDRs will all be from the same human antibody, but it is also possible to mix them e.g. to transfer the light chain CDRs from a first antibody and the heavy chain CDRs from a second antibody.

In some embodiments the antibodies are non-human mAbs. These can be prepared by various means e.g. the original Kohler & Milstein technique for preparing murine mAbs.

In some embodiments of the invention, the antibody has a variable domain with an isoelectric point (pi) in the range of 5.0 to 8.0. A preferred antibody for use with the invention is an IgG2.

A preferred antibody for use with the invention is an anti-sclerostin antibody such as ANTIBODY 1 disclosed in reference 54 (the complete contents of which are incorporated herein by reference). ANTIBODY 1 has a VH domain with amino acid SEQ ID NO: 1 and a VL domain with amino acid SEQ ID NO: 2. Other anti-sclerostin antibodies useful with the present invention may include one or more (1, 2, 3, 4, 5 or 6) CDRs from ANTIBODY 1. The CDRs in the heavy chain are SEQ ID NOs: 3, 4 & 5. The CDRs in the light chain are SEQ ID NOs: 6, 7 & 8. The ANTIBODY 1 heavy and light chains may be expressed as SEQ ID NOs: 9 and 10 to give a functional antibody.

General

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology, pharmacy, posology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 55-61, etc.

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y. The term "about" in relation to a numerical value x is optional and means, for example, x+10%.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from a definition of the invention.

EXAMPLES ANTIBODY 1 recognises sclerostin and is disclosed in reference 54. It is a human IgGlk mAb obtained via phage display. ANTIBODY 1 heavy and light chains are shown in SEQ ID NOs: 9 and 10.

As discussed in reference 62, during studies with ANTIBODY 1 it was sometimes seen that solutions turned turbid e.g. when pH was above 5.5, and particular when >6.3. These turbid solutions were investigated and it was found that they could lead to the formation of gels without the addition of gelling polymers. Furthermore, the gels were shown to be capable of releasing monomeric active ANTIBODY 1 under simulated physiological conditions for a period of two weeks.

The following Examples provide improved gel formulations for anti-sclerositin antibodies, e.g., ANTIBODY 1, and methods for their preparation. In some embodiments, the gel formulation is prepared from an aqueous antibody formulation that has not previously been lyophilized, e.g., an aqueous formulation of ANTIBODY 1 drug substance (DS). In other embodiments, the gel formulation is prepared from an antibody lyophilisate (DP). In both cases, the novel formulations have the desirable property of rapidly gellifying following a pH change.

1. Materials ANTIBODY 1 bulk drug substance (DS) is supplied at > 70 mg/mL in histidine buffer, pH 5.3 (used in Example 1, 3 and 4). ANTIBODY 1 DS at a concentration of 135.94 mg/mL (in 6.4 mM Histidine buffer, pH 5.3) and 76.11 mg/mL (in 7.8 mM Histidine buffer, pH 5.3) were used in Example 5.

The preparation of 0.15 M Arginine-HCl solution was performed by adding 1.58g of Arginine HC1 solid powder and adjusting to 50 mL with water. The preparation of 0.1 M stock solutions at different pH levels was performed for the following buffers: MOPS 3-(N-morpholino)propanesulfonic acid (pH 6.6, 7.0, 7.4), HEPES 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (pH 7.0, 7.4, 7.8) and TRIS 2- Amino-2-hydroxymethyl-propane-l,3-diol (pH 7.8, 8.2, 8.6). Depending on the respective pH level, the necessary pH adjustments were done with either 1 M sodium hydroxide or 1 M hydrochloric acid.

A 0.1 M phosphate buffered saline (PBS) pH 7.4 solution was prepared by mixing 19 g of 0.2 M Na-dihydrogen phosphate, 81 g of 0.2 M Di-Na-Hydrogen phosphate and adjusting to 200 g with water.

A 0.1 M potassium phosphate buffer pH 7.4 solution was prepared by mixing 9.9 mL of 1 M potassium phosphate monobasic, 40.1 mL of 1 M potassium phosphate dibasic and adjusting to 500 g with water.

A 0.1 M potassium phosphate buffer pH 7.0 solution was prepared by mixing 7.7 mL of 1 M potassium phosphate monobasic, 12.3 mL of 1 M potassium phosphate dibasic and adjusting to 100 g with water. A citrate phosphate buffer pH 7.0 solution was prepared by mixing 2.45 mL of 0.1 M citric acid and 22.55 mL 0.2 M sodium phosphate dibasic.

Size exclusion chromatography (SEC) was performed using an Agilent 1100 HPLC system with a TSK gel G3000 SWXL column (Tosoh Bioscience) and 150 mM Potassium- Phosphate running buffer at pH 6.5. Turbidity measurements were performed on a HACH Tubidimeter 2100 AN. 2. Methods

Example 1 :

ANTIBODY 1 bulk drug substance was lyophilized to generate a lyophilisate containing 150 mg ANTIBODY 1, 30 mM histidine, 270 mM sucrose, 51 mM arginine, 0.06 % polysorbate 80 at pH 5.3. The lyophilisate was reconstituted with 1 mL 0.1 M PBS pH 7.4. The gelling behavior and change in turbidity was monitored.

Example 2:

ANTIBODY 1 bulk drug substance was lyophilised to generate a lyophilisate containing 150 mg ANTIBODY 1, 30 mM histidine, 270 mM sucrose, 51 mM arginine, 0.06 % polysorbate 80 at pH 5.3. The lyophilisate was reconstituted with 1 mL 0.1 M PBS pH 7.4 (Case #1); and mixtures of 0.1 M PBS pH 7.4 and 0.15 M Arginine-HCl solutions (Cases #2, #3 and #4 - see details in Table 1). The gelling behaviour and change in turbidity was monitored. Table 1 - Set-up of the reconstitution experiments.

Case #1 Case #2 Case #3 Case #4

Final Antibody 1 DS Cone. (mg/mL) 150.0 150.0 150.0 150.0

Volume 0.1 M PBS pH 7.4 added (uL) 1000 750 500 250 Volume 0.15 M Arg. HCI added (uL) 0 250 500 750 Final Cone Arg. HCI (mM) 0 37.5 75 1 12.5 pH (after 2h at RT) 6.6 6.5 6.3 5.9

Example 3 :

ANTIBODY 1 lyophilizate as described in Example 1 was reconstituted with 0.1 M sodium phosphate buffer (PBS) pH 7.4 to form a gel as follows. Reconstituted liquid solution was transferred into a Silicon (Platinum cured) tube, which was sealed at one end. After the gellification of the solution in the tube, the gel formulation was transferred into a 30 mL vial filled with 10 mL 0.1 M PBS, which was equilibrated to 37°C in a water bath. The release of the mAb was monitored over a time period of 168h. Samples were withdrawn out of the 10 ml 0.1 M PBS release buffer at time points = 0, 0.5, 6, 24 48, 78 and 168h and analyzed by SEC. The release buffer was replaced with fresh 0.1 M PBS to 10 mL following withdrawal of each sample. The bioactivity of the mAb was tested for two gelled samples: t= 48h and t= 168h.

The bioassay method determines the potency and specificity (identity) of a ANTIBODY 1 sample is in a fluorescence resonance energy transfer (FRET) assay that measures the capacity of ANTIBODY 1 to inhibit binding of recombinant human SOST to soluble recombinant human LRP6-IgG(Fc) chimera (LRP6(Fc)). The assay uses LRP6(Fc) labeled with a Eu3+ chelate as the donor fluorophore and SOST labeled with Cy5 as the acceptor fluorophore. The Eu3+ chelate is excited at 340 nm and emits light at 615 nm. If Cy5 is in close proximity to the Eu3+-chelate, which happens when SOST is bound to LRP6(Fc), fluorescence resonance energy transfer (FRET) to Cy5 occurs, which emits light at 665 nm. ANTIBODY 1 competes with the binding of Cy5-SOST to Eu3+-LRP6(Fc) and thus reduces the fluorescence emission at 665 nm in a concentration-dependent manner. The potency of a ANTIBODY 1 test sample is quantified by comparing its ability to inhibit binding of SOST to LRP6 to that of a reference standard.

Example 4:

ANTIBODY 1 lyophilizate as described in Example 1 was reconstituted with 1 mL of various buffers having different pH levels: MOPS (pH 6.6, 7.0, 7.4); HEPES (pH 7.0, 7.4, 7.8); and TRIS (pH 7.8, 8.2, 8.6). Potassium phosphate buffer, sodium phosphate buffer (PBS) and citrate phosphate buffer at pH 7.4 were used as a reference for gellification. The time for the solution to gellify at room temperature was monitored visually and by turbidimetry. A visual check of the time for gellification of the solutions at 37°C was also performed.

Example 5:

ANTIBODY 1 DS at a concentration of 135.94 mg/ml (in 6.4 mM Histidine buffer, pH 5.3) and 76.11 mg/ml (in 7.8 mM Histidine buffer, pH 5.3) were diluted with 100 mM MOPS buffer pH 7.0 to a concentration of 100 mg/ml and 50 mg/ml, respectively. The time for gellification of the diluted DS solution was monitored visually. Example 6:

ANTIBODY 1 DS at a concentration of 73.0 mg/ml (in water) was diluted with different combinations of buffers plus excipients (either NaCl or Arginine-HCl) to a concentration of 15 mg/ml. The set up of the different combinations tested is listed in the Table 2. The turbidity of these combinations along time was monitored at 25°C.

Table 2 - Set-up of the experiments.

Case

#1 Case #2 Case #3 Case #4 Case #5 Case #6

Final ANTIBODY 1 DS Cone. (mg/mL) 15.0 15.0 15.0 15.0 15.0 15.0

Volume Phosphate Buffer added (uL) 775 775 1550 775 775 1550

Final cone. Phosphate Buffer (mM) 15 15 15 150 150 150

pH (after 3h at RT) 6.6 6.5 7.0 6.7 6.6 7.0

Example 7:

ANTIBODY 1 DS was diluted to a concentration of 15 mg/ml with different buffer/pH combinations. The viscosity and the zeta potential of these combinations were determined at 25°C. The theoretical ionic strength (IS) of the buffers was accurately adjusted to 15mM and 150mM using NaCl. The set up of the different combinations tested is listed in the Table 3.

Table 3 - Setup of the experiment.

Theoretical Ionic

Final [DS]

Combination # Buffer type PH strength of the buffer mg/mL

(mM)

1 15.0 Acetate 5.0 15

2 15.0 Histidine 6.0 15

3 15.0 Phosphate 7.0 15

4 15.0 Phosphate 7.5 15

5 15.0 Acetate 5.0 150

6 15.0 Phosphate 7.0 150

7 15.0 Phosphate 7.5 150 3. Results

Example 1 :

The ANTIBODY 1 formulation reconstituted within 3 minutes at RT, resulting in a clear, colorless, liquid solution having a pH 6.6. The turbidity of the reconstituted formulation 5 min after reconstitution was 325 NTU. After further incubation at 22.5 °C (room temperature) for 5 minutes, the solution turned into a solid gel structure with a turbidity of 1350 NTU.

Example 2:

For case #1, the ANTIBODY 1 formulation reconstituted within 3 minutes at RT, resulting in a clear, colourless, liquid solution having a pH 6.6. After further incubation at 22.5 °C (room temperature), the solution turned into a solid gel structure. The turbidity of the reconstituted formulation increased very fast to the upper detection limit of 4000 NTU of the turbidimeter. When adding increasing concentrations of Arginine-HCl the turbidity increase was less pronounced as well as the gellification process (Figure 1). Note that, for case #4 when 112.5 mM Arginine-HCl are present the turbidity reached a maximum of approximately 1000 NTU after incubation at RT for 21h (see Figure 1).

Example 3 :

A controlled release of the mAb within 168h could be observed. More than 70% of the mAb was released over the observed timeframe (Figure 2). The amount of aggregation and degradation products after 168h is below 2% and is shown in Figure 3 and 4, respectively. After 168h, the released mAb continued to display bioactivity (Figure 5).

Example 4: The time to gellify at room temperature for lyophilisates reconstituted in various buffers is shown in Table 4. The reconstituted MOPS and HEPES-based solutions, including lyophilisates reconstituted with MOPS buffer at pH 7.0 and HEPES buffer at pH 7.4, showed rapid gelling behavior. All lyhophilisates reconstituted with phophate buffered salts had slower gelling behavior in comparison to the lyhophilisates reconstituted in MOPS and HEPES buffer.

An increase in turbidity with time was observed for all solutions with different buffers. A strong increase was monitored for the ANTIBODY 1 lyophilisates reconstituted with MOPS buffer at pH 7.0 and HEPES buffer at pH 7.4 (Figure 6). The lyophilisates reconstituted with phosphate- based buffers showed slower increase in turbidity, which corresponds well to the observed gellification time shown in Table 4.

Table 4 - Time for the reconstituted lyophilized solution to gellify at room temperature.

Ionic Strength Actual pH of Time for the solution to

Buffers Buffer pH

(ni ) the gel gellify at RT (min)

6.6 18 6.0 50

0.1 M MOPS 7.0 36 6.4 12

7.4 60 6.8 18

7.0 20 6.1 25

0.1 M HEPES 7.4 39 6.7 15

7.8 63 7.1 18

7.8 69 6.4 25

0.1 M TRIS 8.2 46 7.1 30

8.6 25 7.6 120

0.1 M Potassium

7.4 262 6.5 30

Phosphate Buffer

0.1 M Sodium Phosphate

7.4 262 6.6 25

Buffer (PBS)

0.1 M Citrate Phosphate

7.4 - 25

buffer

The gelling behavior of the lyophilisates reconstituted with different buffers was repeated at 37°C (Table 5). The time for all formulations to gellify was increased when compared to the time to gellification at room temperature. A gelling time of 45 min for MOPS buffer at pH 7.0 and 38 min for HEPES buffer at pH 7.4 was observed. As with the experiments performed at room temperature, lyophilisates reconstituted with phosphate-based buffers had a slower increase in turbidity (i.e., took longer to gellify) at 37°C. Table 5 - Time for the reconstituted lyophilized solution to gellify at 37°C

Buffers PH Time for the solution to gellify at 37°C (min)

6.6 Did not gelify

0.1 M MOPS 7.0 45

7.4 50

7.0 more than 3h less than 16h

0.1 M HEPES 7.4 38

7.8 42

7.8 more than 3h less than 16h

0.1 M TRIS 8.2 more than 3h less than 16h

8.6 more than 3h less than 16h

0.1 M Potassium Phosphate Buffer 7.4 1 h20

0.1 M Sodium Phosphate Buffer 7.4 1 h20

0.1 M Citrate Phosphate buffer 7.4 Not done

Example 5:

ANTIBODY 1 antibody drug substance, when diluted with MOPS buffer pH 7.0 (to a concentration of 50 mg/mL or 100 mg/mL) gellified rapidly within 1 minute at room temperature. This rapid gellification was independent of the concentration of the antibody formulation prior to dilution. However, the effect was more immediate for the 100 mg/mL sample (Figure 7).

Example 6:

The evolution of turbidity of ANTIBODY 1 DS diluted with different combinations of buffers plus excipients along time is shown in Figure 8. The combinations where Arginine- HC1 was added as excipient (Case # 2 and #5) the turbidity increase is less significant and the gel formation takes more time.

Example 7:

When ANTIBODY 1 DS was diluted with different buffers to a concentration of 15 mg/mL, no significant change in viscosity was observed for any of the combinations tested (see Figure 9). However the overall surface charge of ANTIBODY 1 determined by zeta potential was strongly pH dependent even at low protein concentrations as well as low ionic strengths (see Figure 10). The impact of higher ionic strength could not be proven with the analytical method used. REFERENCES

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[62] PCT/EP 10/052665

SEQUENCE LISTING

<110> Schoenhammer, Karin

Rodrigues, Margarida

<120> ANTIBODY GEL SYSTEM FOR SUSTAINED DRUG DELIVERY

<130> 54354

<140> HEREWITH

<141> HEREWITH

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<170> Patentln version 3.3

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser His

20 25 30

Trp Leu Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asn lie Asn Tyr Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Asp Thr Tyr Leu His Phe Asp Tyr Trp Gly Gin Gly Thr Leu

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Val Thr Val Ser Ser

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<210> 2 <211> 113

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35 40 45

Met He Tyr Asp Val Asn Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr He Ser Gly Leu

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Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gin Ser Tyr Ala Gly Ser

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Gin

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20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Arg Ser His Trp Leu Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 50 55 60

Glu Trp Val Ser Asn lie Asn Tyr Asp Gly Ser Ser Thr Tyr Tyr Ala

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Asp Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn

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Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

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Tyr Tyr Cys Ala Arg Asp Thr Tyr Leu His Phe Asp Tyr Trp Gly Gin

115 120 125

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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys 210 215 220

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Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe

245 250 255

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro

260 265 270

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

275 280 285

Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300

Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val 305 310 315 320

Leu Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

325 330 335

Lys Val Ser Asn Lys Gly Leu Pro Ala Pro He Glu Lys Thr He Ser

340 345 350

Lys Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro

355 360 365

Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val

370 375 380

Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400

Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp

405 410 415

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

420 425 430

Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445

Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460

<210> 10

<211> 237

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Gly Thr Arg Cys Asp lie Ala Leu Thr Gin Pro Ala Ser Val Ser Gly

20 25 30

Ser Pro Gly Gin Ser lie Thr lie Ser Cys Thr Gly Thr Ser Ser Asp

35 40 45

Val Gly Asp lie Asn Asp Val Ser Trp Tyr Gin Gin His Pro Gly Lys 50 55 60

Ala Pro Lys Leu Met lie Tyr Asp Val Asn Asn Arg Pro Ser Gly Val

65 70 75 80

Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr

85 90 95 lie Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gin Ser

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Thr Val Leu Gly Gin Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro 130 135 140

Pro Ser Ser Glu Glu Leu Gin Ala Asn Lys Ala Thr Leu Val Cys Leu 145 150 155 160 lie Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp

165 170 175 Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gin 180 185 190

Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu

195 200 205

Gin Trp Lys Ser His Arg Ser Tyr Ser Cys Gin Val Thr His Glu Gly 210 215 220

Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

225 230 235

Claims

1. A process for preparing a gel formulation of ANTIBODY 1, comprising

a) lyophilising a first aqueous formulation of ANTIBODY 1 at a first pH to give a ANTIBODY 1 lyophilisate; and

b) reconstituting the lyophilisate with a MOPS or a HEPES buffer to provide a second aqueous formulation of ANTIBODY 1 having a second pH, the second pH being different from the first pH.

2. A process for preparing a gel formulation of ANTIBODY 1, comprising

b) reconstituting the lyophilisate with a Tris buffer having a pH between about 7.8 and about 8.6 to provide a second aqueous formulation of ANTIBODY 1 having a second pH, the second pH being different from the first pH.

3. The process of any preceding claim, wherein the first pH is lower than the second pH.

4. The process of any preceding claim, wherein the first pH is less than about 7.0.

5. The process of claim 4, wherein the first pH is in the range about 5.0 to about 6.0.

6. The process of any preceding claim, wherein the second pH and the first pH differ by at least one pH unit.

7. The process of any preceding claim, wherein the second pH is in the range about 6.0- about 8.0.

8. A process for preparing a gel formulation of ANTIBODY 1, comprising reconstituting a ANTIBODY 1 lyophilisate with a MOPS or a HEPES buffer to provide an aqueous formulation of the ANTIBODY 1 antibody having a pH of about > 5.5 to about < 9.0

9. A process for preparing a gel formulation of ANTIBODY 1, comprising reconstituting a ANTIBODY 1 lyophilisate with a Tris buffer having a pH between about 7.8 and 8.6 to provide a gel formulation of ANTIBODY 1 having a pH of about > 5.5 to about < 9.0.

10. A process for preparing a gel formulation of ANTIBODY 1, comprising:

a) reconstituting a ANTIBODY 1 lyophilisate with a MOPS or a HEPES buffer, wherein the a MOPS buffer has a pH between about 6.6 and about 7.4 and the HEPES buffer has a pH between about 7.0 and about 7.8; and b) allowing the reconstituted lyophilisate from step a) to form the gel formulation or changing the pH of the reconstituted lyophilisate from step a) to cause formation of the gel formulation.

11. The process of any preceding claim, wherein gel formation occurs less than about 30 minutes after reconstituting.

12. The process according to any preceding claim, wherein said step of reconstituting is carried out at a temperature between room temperature and 37°C.

13. A kit comprising,

a) a ANTIBODY 1 lyophilisate at a first pH value; and

b) a MOPS or a HEPES buffer at a second pH, wherein said MOPS or HEPES buffer, when combined with said ANTIBODY 1 lyophilisate, is capable of providing a formulation of ANTIBODY 1 that spontaneously forms a gel.

14. A kit comprising,

a) a ANTIBODY 1 lyophilisate; and

b) a Tris buffer having a pH between about 7.8 and 8.6, wherein mixing of the lyophilisate and the Tris buffer gives an aqueous formulation of ANTIBODY 1 which either spontaneously forms a gel, or is not a gel but will form a gel in vivo.

15. The process or kit of any preceding claim, wherein the lyophilisate includes one or more lyophilization stabilizers selected from the group consisting of: sugars, amino sugars, amino acids and/or surfactants.

16. A gel formulation prepared by the process of any one of claims 1 to 12 or 15.

17. The gel formulation of claim 16, wherein the gel formulation can release antibody in vivo for more than 7 days.

18. The gel formulation according to any one of claims 16-17, wherein said gel formulation has a turbidity of about 1500 NTU to about 4000 NTU as measured by a HACH Tubidimeter 2100AN.

19. The gel formulation according to claim 18, wherein said gel formulation has a turbidity of about 2000 NTU to about 3000 NTU.

20. A gel formulation comprising, a sclerostin antibody in about 83mM MOPS buffer.

21. A gel formulation comprising, a sclerostin antibody in about 83mM HEPES buffer.

22. A gel formulation comprising, a sclerostin antibody in about 83mM TRIS buffer.

23. The gel formulation according to any one of claims 16-22, wherein the sclerostin antibody is the ANTIBODY 1 antibody.

24. The gel formulation according to any one of claims 16-23 for use in therapy.

25. The gel formulation according to any one of claims 16-23 for use (a) in the treatment of bone injuries such as a bone fracture, or (b) in promoting osseointegration of a bone plate, pin, screw, prosthetic joint or dental implant.

26. Use of a gel formulation according to any one of claims 16-23 in the manufacture of a medicament for (a) the treatment of bone injuries such as a bone fracture, or (b) promoting osseointegration of a bone plate, pin, screw, prosthetic joint or dental implant.

27. The use according to any one of claim 24-26, wherein the gel formulation reduces recovery time following injury or surgery.

28. A process for preparing a gel formulation of ANTIBODY 1, comprising adjusting a first aqueous formulation of ANTIBODY 1 at a first pH with a MOPS buffer to provide a second aqueous formulation of ANTIBODY 1 having a second pH, the second pH being different from the first pH.

29. The process according to claim 28, wherein the first pH is lower than the second pH.

30. The process according to any one of claims 28-29, wherein the first pH is < about 7.0.

31. The process according to any one of claims 29-30, wherein the first pH is in the range of about 5.0- about 6.0.

32. The process according to any one of claims 28-31, wherein the second pH and the first pH differ by at least one pH unit.

33. The process according to any one of claims 28-32, wherein the second pH is in the range of about 6.0- about 8.0.

34. The process according to any one of claim 28-33, wherein the first aqueous formulation of ANTIBODY 1 is an aqueous ANTIBODY 1 formulation that has not previously been lyophilized.

35. The process according to any one of claim 28-34, wherein gel formation occurs less than about 30 minutes after reconstituting.

36. The process according to any one of claim 28-35, wherein the adjusting is carried out at a temperature between room temperature and 37°C.

37. A kit comprising, a) an aqueous formulation of ANTIBODY 1 at a first pH; and

b) a MOPS buffer at a second pH, wherein said MOPS buffer, when combined with said aqueous formulation of ANTIBODY 1, is capable of providing a formulation of ANTIBODY 1 that spontaneously forms a gel.

38. A gel formulation prepared by the process of any one of claims 28-36.

39. The gel formulation of claim 38, wherein the gel formulation can release antibody in vivo for more than 7 days.

40. The gel formulation according to any one of claims 38-39, wherein said gel formulation has a turbidity of about 1500 NTU to about 4000 NTU as measured by a HACH Tubidimeter 2100AN

41. The gel formulation according to any one of claims 38-40, wherein said gel formulation has a turbidity of about 2000 NTU to about 3000 NTU

42. A gel formulation comprising, a sclerostin antibody in about 26mM to about 34mM MOPS buffer.

43. The gel formulation according to claim 42, wherein the sclerostin antibody is the ANTIBODY 1 antibody.