WO2010100179A2 - Self-forming gel system for sustained drug delivery - Google Patents

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

SELF-FORMING GEL SYSTEM FOR SUSTAINED DRUG DELIVERY

TECHNICAL FIELD

This invention is in the field of monoclonal antibody pharmaceutical formulation.

BACKGROUND Monoclonal antibodies (mAbs) are typically formulated either in aqueous form ready for parenteral administration or as ryophilisates for rcconstitution with a suitable diluent 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 I discloses the encapsulation of antibodies inside biodegradable polyOactk-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 S discloses an intratumoral injectable gel drug delivery system for local delivery of radiolabeled immunotheπφeυtic 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.

Thus there remains a need for further and improved formulations for providing sustained local in vivo release of antibody.

DISCLOSURE OF THE INVENTION

The present invention describes 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 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, and that antibodies are released from the gel in active form. Thus the gel can either be administered to a patient, or may form in vivo in the patient, and can form a depot (gel) system which releases active mAb over a sustained period of time without the use of current depot excipients.

Thus the invention provides a process for preparing a gel formulation of a monoclonal antibody, comprising steps of: (i) providing a first aqueous formulation of the monoclonal antibody at a first pH; (ii) lyophilising the first aqueous formulation to give a lyophilisate; (Hi) reconstituting the lyophilisate with an aqueous reconstituent to provide a second aqueous formulation of the monoclonal antibody having a second pH; and (iv) allowing the second aqueous formulation to form the gel formulation. The first pH and second pH arc different, and the first pH will generally be lower than the second pH.

The invention provides a process for preparing a gel formulation of a monoclonal antibody, comprising steps of: (i) providing a First aqueous formulation of the monoclonal antibody at a first pH; (ii) lyophilising the first aqueous formulation to give a lyophilisate; (iti) reconstituting the lyophilisate with an aqueous reconstituent to provide a second aqueous formulation of the monoclonal antibody, and (iv) changing the pH of the second aqueous formulation, thereby causing formation of the gel formulation. Step (iv) may occur in vivo or in vitro.

The invention also provides a process for preparing a get formulation of a monoclonal antibody, comprising steps of: (i) reconstituting a monoclonal antibody lyophilisate with an aqueous reconstituent, wherein the aqueous reconstituent has a pH below about 6.8 or above about 7.2; and (ii) allowing the reconstituted material to form the gel formulation.

The invention also provides a process for preparing a gel formulation of a monoclonal antibody, comprising steps of: (i) reconstituting a monoclonal antibody lyophilisate with an aqueous reconstituent; and (ii) changing the pH of the reconstituted aqueous material to cause formation of the gel formulation. Step (ii) may occur in vivo or in vitro. The invention also provides a monoclonal antibody lyophilisate which, when reconstituted with an aqueous reconstituent, gives a gel formulation of the monoclonal antibody.

The invention also provides a gel formulation of a monoclonal antibody wherein, except for the antibody, the formulation does not include a gelling polymer. The gel formulation can be used as a sustained release formulation of the monoclonal antibody. The invention also provides an aqueous monoclonal antibody formulation, wherein the formulation is not a gel but will form a gel if incubated at room temperature for less than about I hour (e.g. < about 45 minutes, < about 30 minutes, < about 20 minutes, < about 10 minutes, < about 5 minutes, < about 2 minutes, etc.).

The invention also provides an aqueous monoclonal antibody formulation, wherein the formulation is not a gel but will form a gel if its pH is changed. The invention also provides an in vitro aqueous monoclonal antibody formulation, wherein the formulation is not a gel but will form a gel in vivo. This formulation is appropriate for administration to a patient.

The invention also provides a kit comprising (i) a monoclonal antibody lyophilisate and (ii) a reconstituent, wherein mixing of the lyophilisate and the reconstrtuent gives an aqueous formulation which spontaneously forms a gel.

The invention also provides a kit comprising (i) a monoclonal antibody lyophilisate and (ii) a reconstituent, wherein mixing of the lyophilisate and the reconstituent gives an aqueous formulation which is not a gel but will form a gel in vivo. Except for the mAb, formulations of the invention, and formulations made using the methods and kits of the invention, typically do not include a gelling polymer.

Lyophilisaϊes

Because antibodies useful with the invention have a gelation property under certain aqueous conditions, they are ideally prepared and stored in lyophiliscd form. This procedure also facilitates a change in their formulation pH, which can initiate the gelation process, by using a different pH for rcconstitution than the pre-lyophilisation pH.

Techniques for lyophilisation of mAbs are well known in the art e.g. sec references 7 to 15. For example, monoclonal antibody products SYNAGIS™, REMICADE™, NEUTROSPEC™, RAPTTV A™, SIMULECT™ XOLAIR™ and HERCEPTIN™ are supplied as lyophiljsates. The lyophilisate may include, in addition to the mAb, iyophilisation 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, hydroxycthyl 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, argininc and polysorbatc 80 has been shown to be suitable for lyophilisation of antibody BPS804.

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 some embodiments the pH of an aqueous mAb formulation prior to lyophilisation is different from its pH prior to gelation (post-reconstitution). The pre-lyophilisation pH should ideally be selected or controlled to ensure that gelation does not occur prior to lyophilisation. Gelation is easily detected and so it is simple to select appropriate pH conditions for any particular mAb. The pH should not be so extreme as to irreversibly denature the mAb, though. Such dcnaturation is also easily detected, and so an appropriate pH window can readily be identified to avoid both gelation and irreversible denaturation. For example, in some embodiments the prc-lyophilisation pH 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 prc-lyophilisation pH of 5.3+0.1 (via histidine buffer) is suitable for antibody BPS804.

Aqueous reconstltution

Before a lyophilisate can be administered to a patient it should be reconstituted with an aqueous reconstituent. This step permits antibody in the lyophilisate to re-dissolve.

Typical reconstituents for lyophilised mAbs include sterile water or buffer, optionally containing a preservative. Rather than reconstitute the lyophilisate with water, however, it is typical with the invention to use a buffer. Buffered reconstituents are helpful in adjusting the pH of the formulation, to give a final pH that differs from the pre-lyophilisation pH, and this pH change can be used to initiate gelation. Suitable reconstituent buffers include a Tris buffer, a citrate buffer, a phosphate buffer, a succinate buffer, or a histidine buffer. 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.

The post-reconstitution pH typically differs from the pre-lyophilisation pH by at least one pH unit e.g. a difference of >1.5, >2, >2.5, etc. The post-reconstituϋon pH may be higher than the pre- lyophilisation pH or lower than the pre-lyophilisation pH. After reconstitution the mAb 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 changed (for example by at least one pH unit e.g. a difference of >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 10 minutes, < about 5 minutes, < about 2 minutes, 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 10 minutes, < about 5 minutes, < about 2 minutes, etc.).

Gelation is easily detected and so it is simple to select appropriate pH conditions, whether post- reconstitution or post-administration, for any particular mAb. The 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 can readily be identified which ensures appropriate gelation while avoiding irreversible dcnaturation. For example, in some embodiments the post-lyophilisation pH will 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. For example, a post- lyophilisation pH of 6.6+0.1 is suitable for initiating gelation of antibody BPS804, and this can be achieved by reconstitution with phosphate-buffered saline (PBS), pH 7.4, causing gelation to occur about 3- about 5 minutes after reconstitution.

The final pH of the formulation will depend on the pre-lyophilisation pH and on the pH of the aqueous rcconstituenU Appropriate pH values can be selected 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 12- about 8.5 or about 7.4 to about 8.0). For general guidance, if the pre-lyophilisation pH was below pH 7.0 then a reconstituent with pH above about 7.0 will be used, and vice versa. 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.

Non-lyophilised embodiments

As an alternative to using lyophilisation, the invention can be used with an aqueous antibody formulation without lyophilisation.

Thus the pH of an aqueous antibody formulation, which is not already a gel, can be changed to initiate gelation. The pH may be changed by at least one pH unit e.g. a difference of > about 1.5, > about 2, > about 2.5, etc. The final pi 1 may be higher than the starting pH or lower than the starting pH. The pH change can be achieved by adding acid or base to an aqueous formulation.

After the pH change gelation may occur 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 such administration. 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 shortly after the pH change (e.g. < about 60 minutes after, such as < about 45 minutes, < about 30 minutes, < about 20 minutes, < about 10 minutes, < about 5 minutes, < about 2 minutes, etc.). Gelation is easily detected and so it is simple to select appropriate gelation-causing pH changes for any particular aqueous mAb formulation. The pH change should not be so extreme as to irreversibly denature the mAb, though. Such denaturation is also easily detected, and so an appropriate pH window can readily be identified which ensures appropriate gelation while avoiding irreversible denaturatioft. For example, m some embodiments the final pH will 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.

An aqueous formulation 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.

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 docs not have to include a gelling polymer, such as celluloses or poiyacrylates 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, etc. when measured at 25°C and atmospheric pressure. For example, a useful gel formulation of antibody BPS804 has a turbidity of about 1350 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, cither locally or remotely. GcI 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 high 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 reconstitucnt 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), RJTUXAN™ (rituximab), SYNAG1S™ (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 wilJ usually be πon-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 reconstitucnt gives a gel formulation with lower loading capacity. Reconstitution to give a mAb concentration of at least about 50 mg/mL is typical e.g. > about 100 mg/mL, > about 150 mg/mL, > about 200 mg/mL, > about 250 mg/mL, etc. These concentrations arc achievable in aqueous formulations e.g. SYNAGIS™ is provided for reconstitution to give a mAb concentration of 100 mg/mL. 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-scJerostin antibody such as BPS804) 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-sclcrostin antibody such as BPS804 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 lhe 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 (HUM1RA™) or infliximab (REMICΛDE™).

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 (RfTUXAN™ 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 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; RJTUXAN™ is administered weekly at 375 mg/m²; S YNAGIS™ 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 geJ after * 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 (H) 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. IT 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. Equiniscd, bovinised, caninised, camelised and felinised antibodies arc known in the art.

With mAb BPS804 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 hermetkally-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 HERCEPTtN™ expressed in a CHO cell line, and for the pnage-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 Kohlcr & Milstcin), 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 arc 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 (K) or lambda (λ), based on lhe 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 IgA₁ IgD, IgE, IgG, or IgM class. Classes may be further divided into subclasses or isotypes e.g. IgGl, lgG2, IgG3, IgG4, IgA, IgA2, etc. Antibodies may also be classified by allotype e.g. a γ heavy chain may have GIm allotype a, f, x or z, G2m allotype n, or G3m allotype bθ, bl, b3, b4, b5, c3, c5, gl, g5, s, t, u, or v; a K light chain may have a Km(I)₁ Km(2) or Kro(3) allotype. A native IgG antibody has two identical light chains (one constant domain C_t and one variable domain VO and two identical heavy chains (three constant domains C_HI, C_H2 & C_H3 and one variable domain Vn), 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 tc or λ light chain. Where an antibody of the invention has a heavy chain with a constant domain, it may be an α, δ, ε, 7 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 residua! ^αFc" fragment without antigen-binding activity. Pepsin treatment yields a "F(Bb¹V 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 V_L 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 V_H and V_L 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 V_H and V_t 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 JdDa (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 III 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 arc similar in nature and affinity to those of natural antibodies. The FnIIl domain has 7 or 8 beta strands which arc 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 b the boundary of the protein perpendicular to the direction of the beta strands. The FnIII 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 FnIII 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 ail 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 ¹³¹I isotopes, respectively. As a further example, an antibody may include a cytotoxic molecule e.g. MYLOTARG™ is linked to N-acetyl-γ-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 K or λ light chain or an α, S, 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, O 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); CIq 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-termina) 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 FcγRI (CD64), a FcγRIΪ (e.g. FcγRIIA, FcγRIIBl, FcγRJIB2, FcγRIIC), a FcγRIH (e.g. FcγRlllA, FcγRHIB), a FcRn, FcαR (CD89), FcδR, FcμR, a FcεRI (e.g. FceRlαβγ₂ or FCeRI(Ty₂), FcεRlI (e.g. FcεRIIA or FcεRIIB), etc. The Fc region may also or alternatively be able to bind to a complement protein, such as CIq. 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 CIq 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, FcRH, 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 lgG4 is substituted by the CH3 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 lgG4 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 CIq 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^'11 M, 10 ¹² M₁ 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. Radio- immunoassay 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 Abgcnix [46], collectively referred to herein as "human Ig mice". Phage display has also been successful [47], and led to the HUM1RA™ 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 arc humanised mAbs, CDR-graftcd 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 [48-53J. To create a humanized antibody, one or more non-CDR variable framework rcsiduc(s) is also altered. The Hl, H2 and H3 CDRs may be transferred together into an acceptor V_H domain, but it may also be adequate to transfer only one or two of them [51]. Similarly, one two or all three of the Ll , L2 and L3 CDRs may be transferred into an acceptor V_L domain. Preferred antibodies will have 1, 2, 3, 4, 5 or all 6 of the donor CDRs. Where only one CDR is transferred, h 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 Kohlcr & Milstcin 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 MOR05813 disclosed in reference 54 (the complete contents of which are incorporated herein by reference). MOR05813 (referred to herein as 'BPS804') has a V_H domain with amino acid SEQ ID NO: 1 and a V_L 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 MOR05813. 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 MOR058 I3 variable domains 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 arc 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 "abouf^* in relation Io a numerical value .x is optional and means, for example, x±] 0%.

The word "substantially" docs 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.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows BPS804 release (%, as measured by SEC) over time (hours) from a gel. Figure 2 shows the % of released material, measured by SEC, which was an aggregation product (AP; filled circles) or a degradation product (DP; open circles), over 350 hours.

Figure 3 shows a SEC chromatogram of released BPS, at 20 hours. The main peak is active BPS804. The small left-hand peak (025%) is AP. The small right-hand peak (0.14%) is DP. Figure 4 shows the activity (%) of BPS804 released over time (hours). The dotted line is the mean. Figure 5 shows BPS804 release (%, as measured by SEQ over time (hours) from a gel.

Figure 6 shows BPS804 release (%, as measured by SEC) over time (hours) from a gel, with buffer exchange.

Figure 7 shows the % of released material, measured by SEC, which was an aggregation product. Figure 8 shows the % of released material, measured by SEC, which was a degredation product

MODES FOR CARRYING OUT THE INVENTION

Antibody 'BPS804' recognises sclerostin and is disclosed as 'MOR05813' in reference 54. It is a human IgG2λ mAb obtained via phage display. Its heavy and light chains are SEQ ID NOs: 9 and 10.

During studies with BPS 804 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 monomelic active BPS804 under simulated physiological conditions for a period of two weeks.

pH and antibody aggregation Three BPS804 solutions were provided at a concentration of 50 mg/ml mAb in the presence of 16.6 mM histidinc and 0.02% (w/v) polysorbate 80. The three solutions had pH 4.0 (I), 5.0 (II) or 6.0 (111). Formulations I and Il were clear liquid solutions without visible particles. During stirring at room temperature of formulation HI, however, the solution rums turbid (milky, white). Slowly adding I M HCl to give pH 5.48 turns formulation III into an opalescent, turbid solution. Further adding of HCI to pH 5.18 turns the formulation III to a clear liquid solution without visible particles. Thus aggregation of BPS804 is pH-dcpendent above pH 5.3. The pH-dependent aggregation seems to be a non-covalcnt aggregation, due to its reversibility.

Formulation Il was adjusted to pH 4.6 and lyophilised. Samples of the lyophilisatc were reconstituted in different solutions to achieve different compositions with different pH. Reconstituents included: (i) 27OmM mannitol solution; (ii) 27OmM mannitol solution, adjusted to pH 4.0; (iii) 27OmM mannitol solution, adjusted to pH 4.6; (iv) 27OmM mannitol solution, adjusted to pl l 5.2; (v) 15OmM NaCI solution; (vi) 15OmM NaCl solution, adjusted to pH 4.0; (vii) 15OmM NaCI solution, adjusted to pH 4.6; (viii) 15OmM NaCI solution, adjusted to pH 52; (ix) 15OmM Arginine-HCI solution; (x) 15OmM Arginine-HCl solution, adjusted to pH 4.0; (xi) 15OmM Arginine-HCl solution, adjusted to pH 4.6; and (xiϊ) 15OmM Argininc-HCl solution, adjusted to pH 5.2.

At low pH all formulations showed lowest turbidity values, even after prolonged incubation at 2-8°C. At higher pH the formulation showed higher turbidity. Mannitol formulations showed the lowest turbidity. Storage at 2-8°C led to aggregation (visible by increasing turbidity), with higher pH giving more rapid aggregation. NaCI formulations formed aggregates most rapidly.

Gd formation

Bulk BPS804 was provided in 10 mM histidine buffer. pH 5.3. The low pH of this solution means that it does not spontaneously form a gel under normal conditions. The solution was tyophilized to generate a formulation containing 150 mg BPS804, 30 mM histidine, 27O mM sucrose, 51 mM arginine, and 0.06% polysorbate 80.

The lyophilized formulation was reconstituted with 1 mL PBS (0.1 M phosphate buffer saline, pH 7.4, prepared by mixing 19 g of 0.2 M Na-Dihydrogen phosphate, 81 g of 0.2 M Di-Na- Hydrogenphosphatc and adjusting to 200 g with water). The lyophilisate was fully reconstituted within 3 minutes, giving a clear, colorless, solution with pH 6.6. The turbidity of the reconstituted formulation 5 min after reconstitution was 325 NTU.

After further incubation at room temperature (22.5°C) for 5 minutes, however, the solution formed a gel structure with a turbidity of 1350 NTU. The turbidity could not be reduced by dilution with PBS (1 : 10) or by heating to 37°C. In contrast to these results, gelation was not seen during 24 hours of observation (a) when the reconstituent was a 5% glucose solution, to give a final pH 5.3 or (b) when the antibody concentration was 15-fold lower after reconstitution with the same PBS (mAb at 10 mg/ml, giving a final pH 7.4).

Release from gel To evaluate the release kinetics of BPS804 from the gel, another lyophilized formulation (same composition as described above) was reconstituted with PBS and, while still in the liquid state, was transferred to an infusion tube to allow gel formation in the tube. The gel formation process was visible by increase of turbidity within 5-10 minutes. After 20 minutes of incubation a gel had formed. This gel was transferred out of the tube into a Petri dish and covered with 10 mL 0.1 M PBS_r pH 7.4 in a water bath maintained at 37°C. The release of BPS804 from the gel was monitored using (a) UV-VIS spectroscopy to evaluate the total amount of mAb released and (b) size exclusion chromatography to characterize the aggregation state of the released mAb. After sampling the withdrawn volume of supernatant solution was replaced by same volume of PBS.

Release of BPS804 from the gel was characterized by a rapid release phase exceeding 60% of the total loading within the first 20 hours, levelling off at later time points (Figure 1). After 44 hours, debris of the gel was still present. After 92 hours, 74% of the total BPS804 loading had been released and by 165 hours 80% of total loading had been released. The released mAb existed mostly as the tnonomeric form, as confirmed by size exclusion chromatography (Figures 2 & 3) and was bioactive, as confirmed by bioassay (bioactivity > 90%, Figure 4).

Long term incubation

Further experiments were carried out to determine the kinetics of antibody release from the gel. As before, the lyophilized formulation (150mg BPS804, 270mM sucrose, 5ImM arginine, 3OmM hisridine, and 0.06% polysorbate 80) was reconstituted in phosphate buffer pH 7.40: l .Og of 0.1 M phosphate buffer (PB). After 5 minutes (to), the reconstituted solution was a clear and colorless solution. Five 30mL vials filled with 10 mL of 0.1M PB were prepared for the experiments.

No buffer exchange

Following reconstitution, the gel formation was carried out inside a silicone tube by taking up the liquid solution in a syringe, emptying the syringe into a silicone tube and allowing a solid gel formation at RT for 20 min. Finally, the gel was removed from the silicone tube by cutting the tube. The gels were then placed into the vials containing 10 mL dissolution buffer (O. I M PB, pH 7.4). A sample was taken immediately (to) with further samples being taken after 30min, 24h, 48h, 78h and 1 week (each sample being 0.3 mL). The same amount of fresh buffer (0.3 mL) was added to the vial after each sampling time, to maintain the total volume in the vial (10 mL). All the samples were frozen until analysis by size exclusion chromatography (SEC).

In vitro release of BPS804 from the gel was characterized by a rapid release phase exceeding 60% on average of the total loading within the first 24 hours, and leveled off at later time points (Figure 5). After 78 hours, 71% on average of the total BPS804 loading had been released. The released mAb existed mostly as the monomeric form, as confirmed by size exclusion chromatography (Figure 7 and 8). Incubation with buffer exchange

The gel was formed in a silicone tube and the experiment was carried out as described above for the "no buffer exchange" experiment However, in this experiment, the whole PB in the vial was replaced by fresh PB after sampling at 6 h and 24 h. For the other time points, the sampled buffer was replaced by the same volume of fresh buffer (approximately 0.3 mL).

Under these buffer exchange conditions, a mean of 54% of the total loading was released within the first 24 hours. Antibody continued to be released after that, but at a slower rate (Figure 6). After 78 hours, 77% of the total BPS804 loading had been released and by 165 hours 87% on average of total loading had been released. The released mAb existed mostly as the monomerie form, as confirmed by size exclusion chromatography (Figure 7 and 8). Thus BPS804 can be reconstituted in PBS, to give an aqueous formulation at which the antibody spontaneously forms gels. Before the gel forms, however, the formulation can be injected in liquid form after which it can rapidly form a gel in vivo, from which a sustained release of active antibody can be expected for two weeks or more.

It will be understood that the invention will be described by way of example only, and that modifications may be made whilst remaining within the scope and spirit of the invention.

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Claims

CLAIMS 1. A process for preparing a gel formulation of a monoclonal antibody, comprising steps of: (i) providing a first aqueous formulation of the monoclonal antibody at a first pH; (ϋ) lyophilising the first aqueous formulation to give a lyophilisate; (iii) reconstituting the lyophilisate with an aqueous reconstituent to provide a second aqueous formulation of the monoclonal antibody having a second pH, different from the first pH; and (iv) allowing the second aqueous formulation to form the gel formulation.

2. The process of claim 1, wherein the first pH is lower than the second pH.

3. A process for preparing a gel formulation of a monoclonal antibody, comprising steps of: (i) providing a first aqueous formulation of the monoclonal antibody at a first pH;

(ii) lyophilising the first aqueous formulation to give a lyophilisate; (iii) reconstituting the lyophilisate with an aqueous reconstituent to provide a second aqueous formulation of the monoclonal antibody; and (iv) changing the pH of the second aqueous formulation, to a second pH, thereby causing formation of the gel formulation.

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

5. The process of claim 4, wherein the first pH is in the range about 5.0- 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 a monoclonal antibody, comprising steps of: (i) reconstituting a monoclonal antibody lyophilisate with an aqueous reconstituent, wherein the aqueous reconstituent has a pH below about 6.

8 or above about 7.2; and then either (iiA) allowing the reconstituted material to form the gel formulation or (iiB) changing the pH of the reconstituted aqueous material to cause formation of the gel formulation.

9. The process of any preceding claim, wherein gel formation occurs < about 30 minutes after reconstitution.

10. A monoclonal antibody lyophilisate which, when reconstituted with an aqueous reconstituent, gives a gel formulation of the monoclonal antibody.

1 1. A kit comprising (i) a monoclonal antibody lyophilisate and (ii) a reconstituent, wherein mixing of the lyophilisate and the reconstituent gives an aqueous formulation which either

(a) spontaneously forms a gel, or (b) is not a gel but will form a gel in vivo.

12. The process, antibody or kit of any preceding claim, wherein the lyophilisate includes one or more ryophilisation stabilisers selected from the group consisting of: sugars, amino sugars, amino acids and/or surfactants.

13. The process, antibody or kit of any preceding claim, wherein the reconstituent is buffered.

14. A gel formulation prepared by the process of any one of claims 1 to 9.

15. A gel formulation of a monoclonal antibody wherein, except for the antibody, the formulation does not include a gelling polymer.

16. An aqueous monoclonal antibody formulation, wherein the formulation is not a gel but will form a gel (a) if incubated at room temperature for less than 1 hour, or (b) if its pH is changed, or (c) when administered in vivo.

17. The formulation of claim 16, wherein gel formation occurs if the formulation's pH is changed by at feast one pH unit

18. The gel formulation of claim 14 or claim 15, or the gel formulation formed from the formulation of claim 16 or claim 17. wherein the gel formulation can release antibody in vivo for more than 7 days.

19. A gel formulation according to any one of claims 14, 15, or 18, wherein the antibody is an arrti- sclerostin antibody

20. A gel formulation according to claim 19, wherein the anti-sclerostin antibody is BPS804.

21. A gel formulation according to any one of claims 14, 15, or 18-20 for use in therapy.

22. A gel formulation according to claim 19 or claim 20 for use (i) in the treatment of bone injuries such as a bone fracture, or (ii) in promoting osseointegration of a bone plate, pin, screw, prosthetic joint or dental implant.

23. Use of a gel formulation according to claim 19 or claim 20 in the manufacture of a medicament for (i) the treatment of bone injuries such as a bone fracture, or (ii) promoting osseointegration of a bone plate, pin, screw, prosthetic joint or dental implant.

24. The use according to any one of claims 21-23, wherein the gel reduces recovery time following injury or surgery.