NZ714292B2 - HIGHLY CONCENTRATED FORMULATIONS OF SOLUBLE Fc RECEPTORS - Google Patents

Abstract

The invention relates to novel high concentration formulations of human soluble Fc?RIIB comprising NaCl and either a citrate or histidien buffer at specific pH. The invention further relates to the use of such formulations as pharmaceutical compositions for the treatment of autoimmune diseases, infections and other conditions where the immune system is involved. ctions and other conditions where the immune system is involved.

Description

[Annotation] KEB Highly trated Formulations of soluble Fc receptors Description The present invention relates to novel formulations of soluble Fc receptors and especially to ations containing high concentrations of a soluble FolelB receptor. The invention further relates to the use of such ations as ceutical compositions for the treatment of autoimmune diseases, infections, tumors and other conditions where the immune system is involved.

Human soluble FolelB is a promising candidate substance for treatment of Idiopathic Thrombocytopenic Purpura, Systemic Lupus Erythematosus and other autoimmune diseases. It is one ofa plurality of soluble antibody receptors which have been developed over the past 10 to 15 years.

WO 00/32767 describes soluble Fc receptors (chRs) which are composed of only the extracellular part of the receptor and are not glycosylated. Due to the absence of the transmembrane domain and the signal peptide, these proteins are present in a soluble form and not bound to cells as is normally the case for Fc receptors (FcRs). Furthermore the chRs described in WO 00/32767 can be ed recombinantly and have been ted for the treatment of autoimmune diseases due to their ability to bind the Fc part of antibodies without interfering with other ents of the immune system. WO 00/32767 additionally describes the crystal structure of certain chRs and the possibility of developing substances that inhibit the interaction of lgG with chRs with the aid of these crystal structures. The elucidation of the crystal structure enables finding such tors by eg. screening the databases using available computer programs or by computer-aided drug design.

The invention which was defined in WC 648 further developed the findings of WO 00/32767 and provides treatment methods especially for [Annotation] KEB diseases like multiple sis (MS), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) and also for es with an elevated level of natural killer (NK) cells. Even if said receptors were produced recombinantly in prokaryotes and ore were unglycosylated, the inventors of WO 648 surprisingly found that although the unglycosylated proteins were expected to be poorly e, the receptors could be purified with relatively high concentrations of up to 50 mg/ml of stR in a soluble form.

WO 00/32767, WO 03/043648 and other publications imply an important role for FcRs in defense reactions of the immune system. When pathogens have entered the blood circulation they are bound by globulins, also known as antibodies. Since the immune response to a pathogen is polyclonal, a multitude of antibodies are produced and bind to a pathogen, leading to the formation of an immune-complex (lC). le are subsequently ytised by specialized effector cells (e.g. phagocytes or macrophages) of the immune system and thus removed from the circulation. The phagocytosis is mediated by the binding of the t of the antibodies, which, together with the pathogen, form the ICs, to FcRs on the aforementioned effector cells. Other effector cells of the immune system, such as natural killer cells, phils and mast cells also carry FcRs on their surface which upon binding of ICs release stored mediators such as growth factors or toxins that support the immune response.

The FcRs of these effector cells also function as signal-transducing molecules that cally bind immunoglobulins of various isotypes during the humoral immune response. In addition, FcRs expressed on natural killer cells play a fundamental role in the destruction of antibody-coated target cells ("antibody- dependent cell-mediated cytotoxicity", ADCC).

However, in addition to the positive effects of FcRs in the defense against pathogens, overshooting reactions caused by the presence of auto-antibodies in patients may also occur which result in an undesired stimulation of the [Annotation] KEB immune system which manifests itself especially as inflammatory or autoimmune diseases. Such immune reactions ed against the body's own substances remain a major medical problem and although there are ches for treating them, these approaches are not y effective in every patient.

All members of the FoyR-family, i.e. FcRs which are specific for antibodies of the lgG type, are integral membrane glycoproteins, possessing extracellular domains related to a C2-set of globulin-related domains, having a single membrane spanning domain and an intracytoplasmic domain of variable length. There are three known FcY receptor forms, designated Fole (CD64), Folel (CD32), and Folell (CD16). This invention in preferred embodiments specifically focuses on Folel (CD32).

Folel proteins are 40KDa integral membrane glycoproteins which only bind the complexed lgG in the le. These receptors are the most widely expressed FoyRs, present on all hematopoietic cells, including monocytes, macrophages, B cells, NK cells, neutrophils, mast cells, and platelets. There are three human Folel genes (FcYRll-a, Folel-b, Folel-c), all of which bind lgG in aggregates or immune complexes.

Inflammation is a s by which the body's white blood cells react to infection by foreign substances, such as bacteria and s. It is usually characterized by pain, swelling, warmth and redness of the affected tissue.

Effector substances known as cytokines and prostaglandins l this process, and are released in an ordered and self-limiting cascade into the blood or affected tissues. The release of such effector substances increases the blood flow to the area of injury or ion. Some of the effector substances cause a leak of fluid into the tissues, resulting in swelling. This protective process may stimulate nerves and cause pain. These changes, when occurring for a limited period in the relevant area, work to the benefit of the body.

[Annotation] KEB In mune diseases the patient’s immune system has lost the ability to discriminate between body-own (“self") and foreign proteins. In consequence, antibodies are generated that recognize “self‘-proteins and form immune complexes which continuously activate the immune system because the “self”- protein is permanently produced and ized as foreign. This chronic condition can persist for years leading in the end to severe organ damage and possibly to the death of the patient. There are many different autoimmune disorders which affect the body in various ways. For example, the brain is affected in individuals with multiple sclerosis, the gut is affected in individuals having Crohn's e, and the synovium, bone and cartilage of various joints are affected in individuals suffering from rheumatoid arthritis. As autoimmune disorders progress, destruction of one or more types of body tissues, abnormal growth of an organ, or changes in organ function may result. The autoimmune disorder may affect a single organ or tissue type or may affect multiple organs and tissues. Organs and tissues commonly affected by autoimmune ers include red blood cells, blood vessels, connective tissues, endocrine glands (e.g. the thyroid or pancreas), muscles, joints, and the skin.

Examples of inflammatory and/or mune ers include, but are not limited to, primary immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune haemolytic anaemia (AIHA), diabetes, gus vulgaris, Hashimoto's thyroiditis, autoimmune inner ear disease myasthenia gravis, pernicious anemia, Addison's disease, dermatomyositis, Sjogren's syndrome, dermatomyositis, multiple sclerosis, 's syndrome, Graves disease, autoimmune hepatitis, familial adenomatous polyposis and tive colitis.

The FoyRs can be d into two general classes according to their function which may be an activating or inhibitory one. The activating ors are associated with a cytoplasmic 16 amino acid immunoreceptor tyrosine-based activation motif (ITAM) having the consensus sequence Y-Xz-L/l-sz-Y-Xz—l/L [Annotation] KEB (Barrow and Trowsdale, Equ, 2006, 36: 1646-1653). This motif can be found, for example, in . The other class of FcRs are inhibitory ors which contain a 6-amino acid tory motif (ITIM) in the cytoplasmic part of the receptor having the consensus sequence S/l/V/L-X-Y-Xz-l/V/L (Barrow and Trowsdale, Equ, 2006, 36: 1646-1653). An example of such an inhibitory FcR is FolelB.

FolelB (FoyRIIB) has two inhibitory activities. One of them is dependent on the lTlM-motif and occurs when FolelB is ligated to an lTAM-carrying receptor (e.g. FoyRIIA) resulting in the inhibition of ITAM-triggered calcium mobilization and cellular proliferation. The second inhibitory action of FClelB involves ggregation of the receptor (FolelB clustering) which delivers a pro- apoptotic signal into the cytoplasm. The pro-apoptotic signal has only been reported in B-cells and can be blocked by ligation of FolelB to the B-cell receptor (BCR) (JV Ravetch, S. Bolland, Annu Rev. Immunol. 2001; - As mentioned above, in WC 03/043648, chlelB has already been described for use in pharmaceutical preparations where relatively high amounts of ch receptors can be included in a reasonable volume of a treatment solution for eg. injection into a patient. Soluble Fey receptors and especially chlelB has been suggested for the treatment of autoimmune diseases since they can bind to antibodies but do not affect other components of the immune system. The soluble Fc receptors therefore are able to lize antibodies in the blood stream which has an attenuating effect especially on autoimmune processes.

Possible indications that are already mentioned in WC 03/043648 include soluble ch receptors for ent of multiple sclerosis (MS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and also for es with an elevated level of NK cells to avoid the antages of the usly used treatment methods for such diseases.

[Annotation] KEB The teaching of WO 648 focuses on the therein discovered fact that soluble Fc receptors can be used to form aqueous solutions with a tration of up to 50 mg/ml of soluble receptor. For certain applications such concentrations of active agent do suffice. In recent s, however, a therapeutic dose of chRs like sF0lelB of cantly more than 1mg/kg body weight of the patient has been established to be beneficial or even necessary for a successful treatment of autoimmune diseases.

Subcutaneous administration of pharmaceuticals is ered an effective and relatively uncomplicated and non-onerous method of delivering an active agent to a patient. Compared to intravenous infusion, which requires a more extensive medical equipment and in most cases administration at a doctor’s office or a , subcutaneous administration can easily be applied even by the patient himself. aneous administration will also result in a retarded onset of action, i.e. an increased half-life and time to maximal concentration.

Also, it was found that the maximum plasma concentration is reduced in case of subcutaneous delivery. These effects are based on the administration beneath the skin of a patient from where the active agent is orted to the tream. Proteins larger than about 16-20 kDa are generally regarded to be taken up ily by the lymphatic system, which might be an advantage for FoyRs as the target B cell population matures and resides in the lymphatics, too (Porter, C.J.H. and Charman, SA. (2000), J.Pharm Sci. 89, 297-310).

The subcutaneous administration route, however, is preferably limited by an injection volume of 1.0 ml up to perhaps 1.5 ml per application (Gatlin, LA. and Gatlin, C.A.B, (1999), Gapta, P.K. & Brazeau, G.A., eds., lnterpharm Press, Denver, pp. 401-425). Thus, aqueous chYR solutions as known from 43648 could not have been considered for subcutaneous administration. Rather, for sufficiently high concentrations of or, precipitation and formation of undesirably large crystals of the receptors in the aqueous solution and accordingly clogging of the needles and/or pain at the injection locus had to be expected from the teaching of this document.

[Annotation] KEB Therefore, in spite of all the anticipated advantages, subcutaneous administration didn’t seem a promising route of treatment and rather intravenous ion or infusion seemed to be the only viable delivery method.

Accordingly, it was one object of the present invention to provide means and conditions for less complicated and burdensome ad ministration of Fc receptors to a patient and for aqueous formulations of soluble Fc receptors, especially of soluble FolelB, which contain the receptor in a sufficiently high concentration to enable a aneous ent regimen for autoimmune diseases.

It was a further object of the present invention to provide such aqueous formulations of highly concentrated soluble Fc receptor in a ready-to-use form which is stable under usual storage conditions for pharmaceuticals for more than 24 months or, alternatively, in a form which allows for long-term storage and easy and straightfon/vard adjustment and reconstitution prior to use in subcutaneous applications.

These objects were solved ing to the present invention by formulations containing a soluble Fc receptor (chR) in an aqueous buffered solution, wherein the concentration of the Fc or is greater than 50 mg/ml and wherein it contains a physiologically acceptable buffer substance.

During the research which led to the present invention, it was surprisingly found that, contrary to the previous expectations, it is possible to provide soluble Fc receptors dissolved in suitable buffer solutions with a concentration of even much higher than 70 mg/ml and preferably more than 150 mg/ml, by means of which it is for the first time le to provide chRs also in the form of a pharmaceutical ition for the subcutaneous application. The subcutaneous ation as parenteral mode of application is simpler and faster able than an intravenous mode of application. As mentioned above, even the patient himself can carry out a subcutaneous application.

[Annotation] KEB A short and usually thin cannula is required for subcutaneous administration.

The highly concentrated formulation of the present invention provides the chRs in a completely dissolved and homogenous form and at acceptable viscosity that permits the use of these thin cannulas or as crystal suspension containing crystals that are small enough to pass these thin cannulas. Thus, the ts' convenience is considerably increased and an administration of the receptors can be effected by the aneous route.

The inventive formulations as described in the following enable the provision of e FcRs and especially of soluble FcYRllB in concentrations of up to a maximum value which is limited mainly by the increase in viscosity of the solution due to the high concentration of the Fc receptor. Upon selection of riate buffer nces and adjustment of suitable osmolalities, a stabilization of 220 mg/ml chYRIIB and more at a physiological pH is enabled within the framework of the present invention.

The physiologically acceptable buffer substances, which are used according to the present invention are commonly used buffers for the pharmaceutical application of suitable solutions. Within the framework of the present invention, r, it was found that ing on the buffer substance used, the pH value of the solution has a erable influence on the solubility of the Fc ors. Depending on the buffer chosen, the pH value has to be adjusted within a determined range and especially to a particular optimum for achieving the desired solubility of the Fc receptors, especially if the purpose is to provide a ation which does not contain any crystalline chR.

It was further found that the protein itself is able to sufficiently act as a buffer due to the presence of positively and negatively charged amino acids. Thus, e.g. by selecting an appropriate pH value based on the pK-value of the proteins amino acid side , it is possible to forgo the on of a separate buffer substance as long as a suitable amount of protein is present in the solution to act as the physiologically acceptable buffer substance.

[Annotation] KEB In preferred embodiments of the present ion, as a buffer substance, the formulation contains one of a histidine buffer, e buffer or ate buffer.

However, it is particularly preferred to prepare the formulation with either a histidine or a citrate buffer. In both cases, it was found that the adjustment of the pH value in these ed solutions permits an adaption of the solubility of the Fc receptors by means of which high concentrations of Fc receptor can be dissolved, however, can also be caused to crystalllize by means of increasing or decreasing the pH value depending on the buffer substance used. This possibility of changing between soluble and crystalline forms of chR by the mere adaption of the pH value implies considerable advantages in view of adaptations with regard to the applicability, preservation and storage stability of pharmaceutical compositions.

For example, during marketing authorization procedures, sufficient storage stabilities of ceuticals have to be . In this context, it is essential that stability data of a storage of at least 12 months at a temperature of 5°C be included by the party requesting the marketing authorization. However, it is desirable and advantageous that a e stability for more than 24 months under corresponding conditions is achieved, whereby in view of the mentioned e conditions, a substantial amount, preferably 90% of the pharmaceutical agent still needs to be present in an active form after the expiration of the time.

In this context, the inventive formulations show particular advantages. For instance, they allow to offer highly concentrated chR formulations in liquid form which exclusively n dissolved Fc receptor and which show a high storage stability.

Moreover, the ive formulations can be subjected to Iyophilization to provide a solid storage form. Such solid forms might even show improved [Annotation] KEB storage stability as compared to the liquid ations. Such formulations are therefore a further subject matter of the present invention.

Lyophilisation can be performed in any suitable manner known to the skilled person for lising proteins. Preferably, conditions as mild as possible are used to avoid protein degradation.

From the solid form, ready-to-use liquid formulations can easily be restored by the addition of water for injection, saline or buffered aequous solutions to provide the Fc receptor again in completely dissolved form or ediate steps can be chosen in which the solubility is adapted as desired.

Also trated forms of inventive formulations are a further subject matter of the present invention. Such concentrated forms can be obtained by eg. removing part of the liquid to below the solubility limit which results in formulations containing at least some crystalline receptors. Also from such formulations, the chR can be reconstituted to a ready-to-use liquid . formulation by adding water for injection, saline or ed aequous solutions to the desired concentration of active chR and especially to concentrations in which the chR is present entirely in dissolved form.

Due to the pH dependency of the solubility of Fc receptor in the formulations according to the present invention, the ations provide the additional advantage that the Fc receptor can practically be caused to crystallize entirely by means of adapting the pH and can be obtained (eg. after sedimentation or centrifugation) as concentrated crystal Suspension. The crystals able y can be very small crystals), especially depending on the crystallisation ions. The faster the crystallisation, the smaller the crystals are. Contrary to prior art, the present invention permits the fast and nearly quantitative transformation of solubilised chR into protein microcrystals and vice versa and thus allows to tailor the solubility properties of respective solutions and formulations. This is a further ility to ensure excellent [Annotation] KEB storage stability and in particular the need for only little storage space for an active agent, which can then be converted into an entirely or mainly soluble form by means of dissolution in a suitable buffered s solution having a suitable pH value. This can, for instance, be done immediately prior to its application as a pharmaceutical by means of admixing concentrated crystal suspension with a suitable buffer. Alternatively a microcrystalline suspension or formulation might be directly administered by subcutaneous application as the present invention provides means to transform the receptor into rystals, i.e. crystals that are small enough to pass a cannula or thin needle. Compared to highly concentrated liquid formulations, the viscosity of such microcrystal suspensions is much lower and does not rise ntially with sing protein concentration.

For the purposes of the present invention and as used above in the context of describing the present invention, Fc receptors are ered as “crystalline” when crystals have an average size of more than 500 pm in diameter, whereas microcrystalline forms contain microcrystals with a size of equal to or less than 500 pm in diameter.

The present invention enables in an unprecedented way to provide soluble Fc receptors in high concentrations in different forms suitable for immediate or future pharmaceutical use. As rated above, this can be effected in a ready- to-use dissolved form or in a solid, e.g. lyophilized form ed from such solution or in microcrystalline form precipitated by pH value adaption, which can then be reconstituted by resolubilization so that a ation results in which the Fc receptor is contained in high concentration in a suitable buffer solution at the desired pH value.

In preferred embodiments of the present invention, the inventive ation contains as an Fc receptor a SFCY receptor. Regarding the possibility of treating autoimmune diseases, the chYRII ors and especially chYRllB have to be considered. A particularly preferred inventive ation thus contains the [Annotation] KEB soluble FClelB receptor in a pharmaceutically applicable solution with a suitable buffer substance.

For the purposes of the present invention, the FolelB receptor has a sequence as described in the prior art, ally WO 00/32767 and WO 03/043648 or other documents referring especially to FClelB and especially chvRIIB. r, the term is meant to encompass forms ofthe receptor which can differ especially in their N-terminal parts. An especially preferred chYRIIB protein is shown in SEQ ID NO:1. This sequence contains a methionine residue at the inus, which is 6.9. required for prokaryotic expression, however is cleaved off in a major part of the produced proteins by bacterial isms lateron. Therefore proteins according to SEQ ID NO:1 lacking the N-terminal methionine are also assed within the present invention as well as mixtures of proteins with and without the N-terminal Met. Further, ing on the production process and the condition of the bacterial production , additional changes in the N-terminal five amino acids can occur. E.g. in addition to methionine also the following residues can be cleaved off or methionine could be ged for another amino acid like norleucine.

Therefore also mixtures of all these proteins differing at the N-terminus and originating from production processes using a DNA sequence which encodes for the amino acid sequence of SEQ ID NO:1 are encompassed by the current definition of sFClelB and especially by the term elB.

In further preferred embodiments, FolelB proteins are ered as encompassed by the present invention as long as they have an at least 90 % identity to the protein of SEQ ID NO:1. For the determination of sequence identity a comparison is made by aligning the sequences in a manner to provide the m correspondence of amino acids. It is especially preferred that differences in the claimed proteins occur only within the first ten and most preferably within the first five amino acids. It is especially preferred that the proteins have an amino acid ty of at least 95 % with differences occurring [Annotation] KEB within the first five amino acids of SEQ ID N021, wherein the differences in the amino acids are based on at least one of deletions, tutions and additions.

In a particularly preferred embodiment, this formulation according to the present invention contains the ch receptor and especially the sF0yRIIB receptor in concentrations of greater than 60 mg/ml, more preferably greater than 60-80 mg/ml, still more preferably greater than 80 mg/ml, even more preferably greater than 100 mg/ml and ularly preferred greater than 150 mg/ml and most preferably even greater than 200 mg/ml.

The inventive ations optionally contain further pharmaceutically acceptable substances, which are for example used for the adjustment of the ionic th of the solution and/or promote the solubility and stability of the receptor protein contained therein. Such substances are known to the skilled person. For the adjustment of the ionic strength the inventive formulation optionally contains a salt and ably NaCl. For the stabilization of the protein, polyols and especially sugars and sugar ls like sucrose or mannitol can be used. Further, the ive formulation preferably contains detergents which are suitable for pharmaceutical applications, as for example rbates.

Buffer substances are preferably contained in the formulation of the present invention in an amount of 0.1 uM to 300 mM. In more preferred embodiments, the physiologically acceptable buffer is present in an amount of0.1 to 150 mM and especially 1 to 50 mM.

For the adjustment of the lity (isotonicity), salts like sodium chloride are suitable in such amounts which adjust a ably physiological osmolality, at least as far as the formulation is intended for the direct administration to a patient. The osmolality of the on can be adjusted over a wide range and can be set to between 10 mOsm/kg and greater than 600 mOsm/kg without having a considerable effect on the solubility of the Fc receptor.

[Annotation] KEB Salt, preferably NaCl, is present in the formulation in a concentration of about 0 to 250 mM, preferably 5 to 200 mM and most preferably 10 to 50 mM.

Polyols like sucrose are not necessarily contained in the inventive formulations, however in red ments they are present in an amount of at least 1.0 % and more preferred at least 2.0 %. The preferred upper limit of the amount of polyols is approximately 25%, more preferably % and most preferably 8%. Sugars are known to stabilize proteins in solution.

Salts and sugars need to be balanced to adapt the osmolality of the formulation, preferably to be isotonic. The more sugar is contained in the formulation, the less salt can be added and vice versa.

Suitable amounts of detergents, which are preferably used within the context of the invention, are 0.001-0.1%, particularly 0.005-0.05%.

As already mentioned above, the findings, which have been obtained within the framework of the t ion, allow the adaption of the solubility conditions for soluble Fc receptors and especially chYRllB in such a way that for a content of Fc receptor of greater than 50 mg/ml, a predetermined provision of the receptors is made possible in either completely dissolved form or in a microcrystalline form for administration to a patient. As also mentioned above, it is often advantageous for an stration to a t to provide as high a concentration as possible in a completely dissolved form or at least transformable into a dissolved form.

For formulations ning microcrystalline chRs, an administration to the patient may also be le, whereby the microcrystals completely dissolve after administration and the active substance is available with its physiologic effect. For other administrations and also for storage, it can be of advantage [Annotation] KEB to rather keep the or in lline form, wherein it is especially stable against degradation and thus loss of activity.

Accordingly, preferred embodiments of the present invention are formulations, which are completely liquid and wherein the receptor is present in solubilized form or in suitable microcrystalline form An especially preferred formulation of the present invention contains the soluble FolelB receptor in a citrate buffered solution and possesses a pH value of equal to or greater than 6. The pH value is preferably ed within a range of 6.0-7.5. In such citrate buffered solutions with a physiological pH value, the FCyRIIB or is soluble in concentrations of greater than 140 mg/ml. Due to the physiological pH value, such formulation also has the advantage that it can directly be administered to a t without g side effects like pain at the site of administration.

In another red embodiment, the soluble FolelB receptor is contained in a histidine ed solution with a pH value of 5.2-5.9. When using a histidine buffer, sF0yRIIB is soluble in concentrations of more than 100 mg/ml.

At a pH of approximately 6.0, the solubility of the receptor is still relatively high, however, crystalline precipitates are ing to form whereas at a higher pH, only a substantially lower solubility of the receptor is observed.

Both formulations described above enable a high concentration of solubilized sFCyRIIB receptor, y this could be shown for both the mentioned citrate buffered formulation and for the histidine buffered formulation up to the viscosity limited regimen of approximately 220 mg/ml (see enclosed examples).

[Annotation] KEB The inventive formulations further have excellent freeze/thaw stability properties and also excellent stabilities at reduced atures of 2°C-8°C.

Even the stability at room temperature is ent for these solutions.

The usability of both solutions is both given for a direct administration to the patient and in the production of a lyophilized or highly concentrated ation that can contain crystals and is convenient for storing or generation of injection solutions which are directly reconstitutable by the patient.

For the direct administration, as already mentioned above, the citrate buffered solution with a physiological pH value is particularly preferred.

A particularly preferred further subject of the invention is a formulation which contains the receptor in crystalline form. Such formulations are preferably embodied as a citrate buffered suspension with a pH value of 5.2-5.9 or alternatively as a histidine buffered suspension at a pH value of 5. Such suspensions can for example preferably be used as storage-stable forms which, for the administration to the patient, can be transformed to a formulation containing high trations of the solubilized receptor by means of pH ment. Additionally, the same can also be concentrated or the receptor be obtained from them by separation of the solution in order to obtain a highly trated crystal suspension. The receptor can be recovered in completely dissolved form by reconstitution in a suitable buffer at a suitable pH value.

The described inventive formulations and the finding that, using certain buffer nces, depending on the pH value different solubility levels of Fc receptors can be realized enable on the one hand the provision of to- use ions for the subcutaneous administration to a patient, or on the other hand the provision of particularly storage-stable variants containing crystalline Fc receptor. Even lyophilized or ise solid forms of the receptor are ed which can be transformed by a simple addition of the suitable [Annotation] KEB solutions into to-use forms ning high trations of soluble receptor.

A further subject of the present invention is thus a pharmaceutical composition, which comprises a formulation according to the present invention as described above and in which further pharmaceutically acceptable excipients and/or adjuvants and/or rs can be present. In particularly preferred embodiments these pharmaceutical compositions are directly applicable for the subcutaneous injection of an effective amount of a soluble Fc receptor and especially for the treatment of autoimmune diseases.

In one preferred embodiment, the pharmaceutical ition preferably contains a sufficient amount of completely dissolved ch receptor in a suitable buffer substance and at a physiological pH value. Such ceutical composition is a ready-to-use medicament and can be directly applied to the t. The dissolved soluble receptor can e.g. easily be absorbed into the patient's lymphatic circulation and be directly effective there or after transport within the patient's body by blood or body fluid circulation.

Alternatively, the pharmaceutical composition can contain the receptor in a highly concentrated and at least partially rystalline or crystalline form.

Diluted as necessary with suitable buffer solutions, the ceutical composition is again particularly suitable for the subcutaneous ion of an effective amount of Fc receptors.

As already explained above in the context of describing the present ion, Fc receptors are considered as “crystalline” when crystals have an average size of more than 500 pm in diameter, whereas microcrystalline forms contain crystals with a size of equal to or less than 500 pm in diameter. As far as a direct application to the patient is considered, pharmaceutical compositions containing completely dissolved Fc receptor can of course be used but also pharmaceutical compositions ning formulations with the Fc receptor [Annotation] KEB being solely or partially in microcrystalline form have merit in pharmaceutical applications. These microcrystals are small enough to not clog the needles for subcutaneous application. The use of microcrystals-containing solutions can be beneficial for e.g. d or sustained release of the active ch receptor to the t’s system and, under certain circumstances, such microcrystalline forms can even be considered as preferred pharmaceutical compositions.

Pharmaceutical compositions of the t ion containing microcrystalline or crystalline forms of Fc receptors can e.g. be obtained by concentration of the ch beyond its solubility by conventional concentration techniques like ultrafiltration. It is possible to maintain the pharmaceutical in liquid form ning a certain amount of crystals or microcrystals. Instead of using a mechanical concentration method for obtaining crystals or microcrystals, it is also possible and a preferred embodiment of the present invention to crystallize the or by adjusting the pH to a value wherein the receptor has a considerably lower solubility. The precipitated crystals or microcrystals can be separated from the solution and used for storage and subsequent reconstitution or direct administration. The solid forms of receptor formulations are especially storage stable and maintain their effectiveness for at least more than 24 months.

In such cases, the pharmaceutical composition is conveniently provided in a pharmaceutical kit format, which in addition to the solid or highly concentrated chR also comprises the suitable liquid for the reconstitution of an injectable on. A further subject matter of the present invention is therefore a pharmaceutical kit containing crystalline or Iyophilized soluble Fc or and a suitable pharmaceutically acceptable liquid like buffer solution or simply water for the reconstitution of the ion solution in suitable separate storage units.

It is especially preferred for an inventive pharmaceutical kit, if chR or and buffer on for the reconstitution of the injection on are provided [Annotation] KEB in suitable devices for simple mixing and well protected t contamination.

Preferably, the kit ns a buffer solution based on a phosphate buffer, a histidine buffer or a citrate buffer. It is further red for the buffer solutions that the pH is adapted to provide optimum solubility for the Fc receptor. In especially preferred embodiments of the present invention, the buffer solution is a citrate buffered solution with a pH of above 6, especially 6.1 to 7.5, or a histidine buffered solution with a pH of below 6.0, especially 5.2 to 5.9. The e buffered solution is the most preferred buffer contained in a pharmaceutical kit according to the present invention.

The amount of buffered solution contained within the pharmaceutical kit is adapted to the amount of solid or concentrated chR in the kit. Depending on whether a complete dissolution of the ch receptor or maintaining some amount of microcrystals is desired, a corresponding buffer is selected in a suitable amount of liquid and also the pH of the solution is adapted according to the teaching ning chR solubility as provided .

A further subject matter of the present ion is the use of the formulations, the pharmaceutical compositions and pharmaceutical kits of the present invention for the prevention or treatment of autoimmune diseases. More particularly, the present invention is intended for the use within the framework of the tion or treatment of multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, primary immune thrombocytopenia and autoimmune haemolytic anemia (AIHA). Further, the ations, ceutical agents and ceutical kits can be used for the treatment of inflammatory disorders. The inventive subject matter enable the application of Fc receptors for all indications for which these have already been described or for which they are considered to be suitable in the future. The inventive formulations and ceutical compositions and kits allow especially the subcutaneous administration which is very efficient and easily applicable to (or by) a patient. The ility of administering especially high amounts and [Annotation] KEB concentrations of Fc ors is a particular advantage of the present invenfion.

The following examples shall further explain the invention and its advantageous effects and ments.

Example: Development of high concentrated liguid huchlelb formulations suitable for subcutaneous m: 1. Materials The following solutions of huchYRllB (soluble human FelelB receptor having the amino acid sequence as shown in SEQ ID NO. 1) were used as parent material for all experiments: [Annotation] KEB a) husFClelB 5 mg/ml concentrate for solution for infusion mg/mL husFClelB in 5.3 mM NaHzPO4, 1.94 mM KH2P04, 150 mM NaCl, 2%(w/v) mannitol, 0.005% rbate 20 pH 6.5 b) husF0lelB 20 mg/mL concentrate for solution for infusion mg/mL hUSFCyRIIB in 20 mM histidine, 150 mM NaCl, 2%(w/v) sucrose, 1% (w/v) mannitol, 0.005% polysorbate 20 pH 6.5.

The following chemicals of at least the indicated grades were used: Name Purity Supplier Citric acid monohydrate p.a. Merck Sodium hydroxide Ph. Eur. (2 98%) Carl Roth Sodium de Ph. Eur. (2 99%) Carl Roth Ethanol, abs. Ph. Eur. (2 99.8%) Carl Roth Histidine Ph. Eur. (2 98.5%) Carl Roth Hydrochloric acid, 37% pa. Carl Roth Sucrose Ph. Eur. (2 99%) Carl Roth Mannitol ACS reagent (2 99%) Fluka Polysorbate 20 (Tween 20) cell e tested Sigma Aldrich Trehalose for biochemistry Merck [Annotation] KEB 2. Methods a) lB content by UVNis oscopy The sample was transferred to a UV micro-cuvette (UV cuvette micro, Plastibrand, Brand) and the ance was measured with a Spectrophotometer (Cary 100, Varian) using the respective buffer as blank.

The husFClelB concentration was calculated by the following equation: husFCyRIchonc. [mg/mL] = (A280-A320) x DF x 0.64 DF 5 dilution factor b) huchlelB buffer exchange by cation exchange chromatography 1000 - 1800 mg huchYRllB (husF0lelB 5 mg/mL concentrate for solution for on) was carefully diluted with 10 mM citrate/NaOH pH 6.5 until the conductivity reached 50:01 mS/cm. The diluted protein was filtered (0.2 pm Durapore membrane PVDF hydrophil, 47 mm, Millipore) and loaded at .5 mL/min onto a 57 mL SP Sepharose HP cation ge (CEX) resin (26 x 107 mm, GE Healthcare; equals 17.5 - 31.6 mg husF0lelB/mL resin) equilibrated in 10 mM citrate/NaOH, 20 mM NaCl pH 6.5. Bound protein was washed at 5.5 mL/min with 200 mL 10 mM citrate/NaOH, 20 mM NaCl pH 6.5 and eluted with a 300 mL linear gradient ranging from 20 mM to 600 mM NaCI in 10 mM citrate/NaOH pH 6.5. Eluate tion was started after ODzao exceeded 250 mAU (AU: adsorbance units) and was stopped after it dropped below 200 mAU (1 cm path length, Akta Explorer 100, GE Healthcare). The column was regenerated with 100 mL 1 M NaCI, washed with 150 mL MilliQ H20 (ultrapure water, ore Corp.) and stored until further use in 20% ethanol. After huch‘lelB content measurement by UV/Vis spectroscopy the eluate was filtered x 33 mm, 0.2 pm Durapore PVDF (polyvinyliden fluoride) hydrophil, both from Millipore Corp), aliquoted, snap-frozen in liquid nitrogen and stored at S-70°C until use.

[Annotation] KEB The NaCl content was calculated by correlation of the mean conductivity of the eluate to the measured tivity at 10 mM citrate/NaOH, 20 mM NaCI and mM citrate/NaOH, 600 mM NaCl.

Histidine buffered huchYRIIB was prepared similarly using 10 mM histidine/HCI as buffering species. c) pH stability screen A concentrated husFCyRIIB solution in 20 mM histidine, 324 mM NaCl pH 6.5 was adjusted to 0.5 mg/mL RIIB, 20 mM histidine,150 mM NaCI, 2.5X Sypro Orange (5000X in DMSO, Molecular Probes“, ogen) using suitable stock solutions. The pH of the solution was adjusted between pH 4 and pH 12 based on an experimental titration curve with 0.2 M HCI or 0.2 M NaOH. 40 uL of the solution were incubated in a sealed 96 well half area well plate (uclear , black, medium binding; Greiner BioOne) for 3h at 25°C and assayed for Sypro Orange fluorescence (Excitation 485 nm, Emission 590 nm, gain 60, lag time 0 ps, integration time 40 us; TECAN Spectrofluor plus). d) Preparation of high concentrated formulations The required NaCl content of histidine or citrate buffered husFClelB (initial concentration . 200 - 300 mM NaCl) was ed by dilution with the appropriate buffer (10 mM e or 10 mM histidine pH 6.5) and subsequent ultrafiltration pin 20, 5 kDa MWCO, Sartorius). In order to keep the processed volumes small, the procedure was repeated for up to 3 cycles in total. After final dilution, the pH was measured (pH-meter Hl8314, pH- electrode HI1217, Hanna instruments), adjusted with 0.2 M NaOH or 0.2 M HCI in the riate buffer and the protein solution was concentrated. The huchYRllB content was measured by means of UV/Vis spectroscopy in triplicate, the huchleIB concentration was ed with the appropriate buffer and the solution was filtered (Ultrafree MC, 0.2 pm Durapore PVDF hydrophil, Millipore).

[Annotation] KEB e) huchlelB solubility screens in 384 well format The solubility of high concentrated huchYRllB solutions in relation to buffering species, pH, salt concentration and sugar/polyol tration was assessed in 384 well format (uclear, white, nding, Greiner BioOne) using 30 uL per well. The final formulations were prepared by direct addition of filtered stock solutions to each well. The following stock solutions were used: 100 - 195 mg/mL elB, 1.5 M NaCl and 30% (w/v) sucrose + 15% (w/v) mannitol in either 10 mM citrate pH 7.0 or 10 mM histidine pH 5.5. Each solution was supplied with 0.01% (w/w) rbat 20 and depending on the screen with 10 - 50 mM NaCl.

The pH of each well was adjusted with 0.5 M - 0.75 M HCI or NaOH. The ed amount of acid or base was calculated based on theoretical titration curves assuming that all nine histidine residues of huchYRllB (pKa = 6.00) provide additional buffering capacity. The plate was centrifuged (500-9, 1 min), sealed with adhesive tape (microtest tape, permacel, neo-lab) and stored at 5i3°C in the dark.

The visual appearance of each formulation was assessed by light copy (Axiovert 25f, Carl Zeiss) and ranked according to an arbitrary scale (0 = no crystals; 1 = some crystals, hardly visible; 2 = some crystals clearly visible; 3 = more than 30 crystals per well clearly visible; 4 = incomplete layer of many crystals (well not fully covered); 5 = full layer of many crystals (well completely Taken into account dissolved salt, sugar and protein the osmolality of each formulation was calculated according to the following equation: gm = Z Virniij [Annotation] KEB wherein v,- is the number of particles formed by the dissociation of one molecule of the 1‘” solute and m; is the molality of the f“ solute. For simplicity the molal osmotic coefficient Fm,- for each solute was d to be equal to 1. f) Small scale crystallization of huchlelB uL - 450 uL husF0yR|lB at 50 - 140 mg/mL in 10 mM histidine, 10 mM NaCI, 0.01% polysorbate 20, pH 5.5 were diluted with appropriate diluents to 40 mg/mL husF0lelB in 10 mM histidine, 10 mM NaCl, 0.01% polysorbate 20 in a 1.5 mL polypropylene on tube. The pH was adjusted to 6.5 - 7.2 by the addition of 4.38 - 6.23 Vol% (final volume after addition of diluents) 0.3 M NaOH. The required amount of acid or base was calculated based on theoretical titration curves assuming that all nine histidine residues of huchlelB (pKa = 6.00) provide additional buffering capacity. 9) Differential scanning metry 120 pL of each formulation containing 0.5 mg/mL huchlelB were prepared in a 1.5 mL test tube rly to the procedure described above in 2.e). Sypro Orange (5000X in DMSO, lar ProbesTM, Invitrogen) was added to a final concentration of 2.5X using a 200x stock in the appropriate buffer. 30 uL of each formulation were erred in triplicate to a well plate (MicroAmp 96 well optical reaction plate, Applied Biosystems) and the plate was sealed with adhesive tape (MicroAmp optical ve film, Applied Biosystems). The plate was subjected to a ature ramp from 19°C to 90°C with a slope of n and the fluorescence emission at 610 nm was recorded (7300 Real Time PCR system, Applied Biosystems). The fluorescence was differentiated with respect to time, a spline was calculated and the first detected maximum was reported as the melting temperature of husFClelB (Origin 8.0, OriginLab). h) Turbidity screen in 384 well format hUSFC‘YRIIB formulations were prepared in a 1.5 mL test tube similarly to the procedure described above in 2.3). 30 uL of each formulation were transferred in duplicate to a 384 well plate (uclear, white, non-binding, Greiner), the plate [Annotation] KEB was sealed with adhesive tape (microtest tape, el, neo-lab) and placed in an incubator. As l the tive placebo solutions were prepared.

The turbidity was measured at 360 nm (Spectrafluor, bandpass filter 360/35 nm, 3 flashes, Tecan). To avoid corrupted measurements due to the condensation of water, the plate reader was pre-heated to the assay temperature. i) Dynamic viscosity by pressure drop measurement The dynamic viscosity of huchYRllB containing formulations was determined by measuring the pressure drop as liquid flows through a flow channel (m- Vroc, Rheosense). For each measurement 100 pL containing formulation was filled with a 200 uL pipette into the cylinder of a 100 uL gastight syringe (Hamilton). The syringe was installed into the rheometer and 80 uL were injected at a flow rate of 50 uL/min and 20°C. j) Quantitative Polysorbat 20 Assay The rbate content was determined by a modified protocol which is based on the colorimetric assay first described by Brown and Hayes, (1955) Analyst 80, 7. 500 uL of the on to be analysed were extracted three times with 500 pL ethylacetate in a 1.5 mL polypropylene tube (VWR). To accelerate the phase separation, the tube was centrifuged (20'000-g, 5 min, 25°C). The organic supernatants were combined in a HPLC vial (ND9, screw threaded, with conical bottom and PTFE screw cap, VWR) and the solvent was evaporated (25°C, 10 mbar, 0.5h - 1h). The residual solids were suspended in 800 uL reagent solution (100 mM )2, 2.63 M NH4SCN in water) and were extracted with 150 uL CHCI3. 100 pL of the CHCI3 extract were transferred to a quartz UV micro cuvette (Hellma), the spectrum was measured from 200 - 800 nm (8453 diode array spectrophotometer, t) and the absorbance at 620 nm corrected by the absorbance at 530 nm was recorded.

As blank an extract of an equivalent on containing no polysorbate 20 was used. Each sample was prepared in duplicate. The polysorbate 20 content was [Annotation] KEB determined based on a standard curve from 0 to 0.006% (w/w) polysorbate 20 in the respective buffer. k) Lyophilisation 59 formulations containing 15 -120 mg/mL lB in 5 mM citrate, 10 - 25 mM NaCI, 2 - 8%(w/v) sucrose, ose or mannitol and 0.005 - 0.01%(w/v) polysorbate 20 were prepared and 400 uL were filled into 1.5 mL clear HPLC vials (32 x 11.6 mm, wide opening, VWR). The vials were subjected to a conservative lyophilisation cycle using the freeze-drier Epsilon 2-12D FD02 (Martin Christ, Osterrode, Germany). The vacuum during the freeze-drying s was controlled by a MKS Capacitance Manometer. The samples were frozen at -45°C, primary drying was performed for 15h at 45°C to 15°C, 0.12 mbar and secondary drying was performed for 10h at 15°C to 20°C, 0.12 mbar.

The lyophilisate was reconstituted in 100 - 400 uL water for injection. The on was analyzed in respect to the formation of particulates by visual inspection, turbidity measurement and fluorescence microscopy. In brief, for scence microscopic examination 50 uL of the huchlelB containing solution was placed into a 384 well plate (uclear, white, non-binding, Greiner) and mixed with 5 uL 25X Sypro Orange (5000X in DMSO, Molecular ProbesT'V', lnvitrogen) in 5 mM citrate, 10 mM NaCI pH 6.7. The plate was ted for min at 25°C, centrifuged (1000-9, 3 min) and the appearance of each formulation was assessed by fluorescence microscopy (Axiovert 25f, excitation filter 470/20 nm, dichroic 493 nm, on filter 503-530 nm, Carl Zeiss).

[Annotation] KEB - 28 _ 3. Results a) Definition of pH range and buffering species for high concentrated huchlelB solutions The huchYRllB denaturing pH range was determined using Sypro Orange as an indicator for the presence of unfolded protein. Sypro Orange is an environment sensitive dye whose fluorescence emission is strongly increased after its binding to hobic structures (Layton & Hellinga, 2010, Biochemistry 49 (51), 10831-10841). Fig. 1 shows the results of experiments to determine the ring pH range. Therefore, 0.5 mg/mL hUSFCyRIIB in 20 mM histidine, 150 mM NaCl (0) and blank buffer (+) were incubated at the respective pH for 3 h at room temperature. An increase in Sypro Orange fluorescence indicated the ce of denatured husF0yRIIB. As shown in Fig. 1, husFCyRIIB did not unfold from pH 5.2 to at least pH 11.

To prevent pain during subcutaneous administration the administered solution should have a pH in the physiologic range. Typical buffering species that buffer in this range and are lly regarded as safe include ine (pka ~6.0), citrate (pKa3 ~6.4) and phosphate (pKaz ~7.2). Due to its tendency to promote pH shifts during freeze/thaw (MacKenzie, 1977) phosphate was not included in subsequent lity screens. in an initial t to determine the limiting husFClelB concentration in respect to protein precipitation husF0lelB was concentrated by ultrafiltration in the presence of 10 mM histidine or 10 mM citrate and 10 mM NaCl until a visible precipitate was formed. As shown in Table 1 ine buffered elB showed increased solubility in the ly acidic range from pH 5.5 to 6.0 whereas citrate as buffering species provided solubility in the near neutral pH range around pH 6.5. In summary the huchYRllB solubility at various pH is largely dependent on the buffering s used. By microscopy it was shown that the precipitate is composed of crystalline needles.

[Annotation] KEB Table 1 : huch;/RIIB solubility limit (in mg/mL) in 10 mM ine or 10 mM citrate at low ionic strength from pH 5.5 to 7.5. husFCyRIIB was trated by ultrafi/tration until the solution became cloudy. Histidine buffered huchyRIlB itated at pH 6.0, 6.5 and 7.0 whereas citrate buffered husFCyRilB precipitated at pH 5.5 and pH 6.0. The sediment was identified as husFCyRIIB protein crystals. .5 6.0 6.5 7.0 Histidine >162 S174 $40 510 Citrate s40 . S158 >154 >148 b) Crystallization of huchleIB Histidine buffered llB remains soluble above 100 mg/mL at pH 5.5 and can be crystallized by neutralization at low ionic strength, on the contrary citrate buffered husF0yR||B remains soluble at neutral pH above 100 mg/mL and can be crystallized by mild acidification at low ionic strength (Table 1). In a r test, crystallisation of husF0le|B was investigated in dependence of the presence and the amount of sugar and NaCI in the buffer. husFClelB crystallisation was performed in 10 mM histidine pH 6.7 (Fig. 2a) or 10 mM citrate pH 5.5 (Fig. 2b) as a function of NaCl and sugar (2:1 sucrose:mannito|) concentration. huchYRIIB in 10 mM histidine, 10 mM NaCl pH 5.5 or 10 mM e, 10 mM NaCI pH 7.0, respectively, was concentrated to 140 mg/mL by ultrafiltration and diluted to 40 mg/mL with appropriate stock solutions. In case of histidine buffered husF0lelB, the crystal yield was ined by measuring the husF0lelB concentration in the supernatant after 3d at 2-8°C.

In case of e buffered husFCyRIIB, no crystal growth was detected until day 10. ore, the crystal yield was determined after 14d at 28°C. Each solution contained 0.01% polysorbate 20.

[Annotation] KEB Fig. 2 shows that hUSFCyRIIB crystallizes more readily and the crystal yield is much higher in the presence of 10 mM histidine, 10 mM NaCl pH 6.7 compared to 10 mM citrate, 10 mM NaCl pH 5.5. r ion of the sodium chloride concentration from 10 mM to 5 mM resulted in a marginal increase in crystal yield when using histidine as buffering species but showed a strong effect when using citrate buffer. Reduction of the salt concentration below 5 mM and/or reduction of pH might further stimulate the crystallization process in the ce of citrate. Increased NaCI concentrations or the addition of polyols, e.g. sucrose or mannitol, above 5% ted the crystallization process.

Fig. 3 shows an experiment in which huchYRllB crystallisation was med in 10 mM histidine, 10 mM NaCI as a function of pH. huchYRllB in 10 mM hisitidine, 10 mM NaCl pH 5.5 was concentrated to 140 mg/mL by ultrafiltration and diluted to 40 mg/mL with appropriate stock solutions. The crystal yield was determined by measuring the husF0lelB concentration in the supernatant after 3d at 2.8’°C. Each solution contained 0.01% polysorbate 20.

Within a pH range of at least 0.5 units, more than 93% of histidine buffered husF0lelB can be crystallized. With a total husFClelB concentration of 40 mg/mL'the solubility limit of husFClelB in 10 mM histidine, 10 mM NaCl pH 6.7 — 7.2 is below 2.8 mg/mL. At pH 6.9 the crystallization was complete in less than one hour at 25°C. c) huchlelB solubility screens In order to define so called solubility sweet spots, i.e. ions where hUSFCyRIIB remains e above 100 mg/mL and does not crystallize, various husF0yRIIB ations were prepared in a 384 well microtiter plate and ted for at least 4 weeks at 2-8°C. Parameters included into the screen were husFClelB concentration (70, 100, 120, and 150 mg/mL), ing species (histidine or citrate), pH (5.5, 6.0, 6.5, 7.0, 7.5), NaCl concentration (10 - 225 mM) and sugar content (0 - 7.5%). The initial screens at 70 mg/mL (Table 2 and Table 5) and 100 mg/mL huchYRllB (Table 3 and [Annotation] KEB Table 6) were conducted with two sugar levels (0% or 3%) and four different NaCI concentrations (10, 50, 225 mM).

Basically these prescreens reproduced the results that were already obtained during the above mentioned concentration screen (Example 23) and llization screens (Example 2b). Histidine buffered husFCyRIIB crystallizes above pH 5.5, whereas citrate buffered huchlelB crystallizes at pH 5.5 to 6.0. Increasing NaCl or sugar concentrations reduced the crystallization process in both cases.

In all subsequent screens at 120 mg/mL and 150 mg/mL huchlelB, only formulations that are isotonic with blood, i.e. with a calculated osmolality around 308 mOsmol/kg were included. For that reason high sugar concentrations were matched with low concentrations of NaCl and vice versa. ine based formulations with 120 mg/mL huchYRllB were able to prevent crystallization of husF0lelB under neutral pH and high ionic strength but with one ion all histidine based ations at 150 mg/mL huchlelB showed strong crystal growth after 4 weeks at 2-8°C (Table 4). On the other hand, citrate based formulations were stable up to 150 mg/mL husF0lelB from pH 6.5 to 7.5 and at all salt combinations tested (Table 7). ore citrate buffered husF0yR|lB represents the best basis for the development of a high concentrated liquid formulation le for subcutaneous application, i.e. with physiologic pH and tonicity.

[Annotation] KEB Table 2: Histidine based lity screen at 70 mg/mL husFCyRIIB and 2- 8°C. The visual appearance of each formulation was assessed by light copy and ranked according to an arbitrary scale (0 = no crystals; 1 = some crystals, hard/y visible; 2 = some crystals clearly visible; 3 = more than crystals per well clearly visible; 4 = layer of many crystals (well not fully covered); 5 = layer of many crystals (we/l completely covered). huchyRIIB pH Sugar NaCl Osmolality 0 d 1 d B d 14 d 29 d Sum [rug/ml! I96] [li [mew-Ike] 70 5.5 0 10 24 0 0 0 0 O 70 6.0 0 10 24 l 3 4 4 4 70 6.5 0 10 24 2 5 5 5 5 70 7.0 0 10 24 1 5 5 5 5 70 7.5 0 10 24 0 0 S 5 5 53:35:: 70 5.5 3 10 137 0 O 0 0 0 70 6.0 3 10 137 0 D 2 3 3 70 6.5 3 10 137 0 5 5 5 5 70 7.0 3 10 137 0 0 O O 5 70 7.5 3 10 137 0 O 5 5 5 5‘!ng 70 5.5 0 50 104 O O 0 0 O 70 6.0 0 50 104 0 2 3 3 3 70 6.5 0 50 104 O O O D 5 70 7.0 0 50 104 0 0 0 0 1 70 7.5 0 50 104 0 O 4 4 4 “H0150 70 5.5 3 50 217 0 0 O 0 O 70 6.0 3 50 217 0 O 0 O 2 70 6.5 3 50 217 0 O 0 O 5 70 7.0 3 50 217 D D D 0 D 70 7.5 3 50 217 0 O 1 3 5 OOUINO 70 5.5 0 225 454 D D D D l 70 6.0 0 225 454 0 0 0 0 O 70 6.5 0 225 454 0 0 0 0 0 70 7.0 0 225 454 0 O 0 0 0 70 7.5 0 225 454 O O 0 O 0 OOOOH 70 5.5 3 225 567 0 O 0 0 O 70 6.0 3 225 567 O O 0 0 O 70 6.5 3 225 567 O 0 0 0 0 70 7.0 3 225 567 0 O 0 0 O 70 7.5 3 225 567 0 O D O O 00°00 [Annotation] KEB [Annotation] KEB Table 3: Histidine based solubility screen at 100 mg/mL husFClelB and 2-8°C. The visual appearance of each formulation was assessed by light microscopy and ranked according to an arbitrary scale (0 = no crystals; 1 = some crystals, hardly visible; 2 = some crystals clearly visible; 3 = more than crystals per well y visible; 4 = layer of many ls (well not fully covered); 5 = layer of many crystals (well completely covered). huchVRllB pH Sugar NaCl Osmolality 0 d 1 d 8 d 14 d 28 d Sum [mg/9H [‘36] [my] [mom/kg] 100 5.5 0 10 25 O 0 0 0 0 100 6.0 0 10 25 5 5 5 5 5 25 100 6.5 0 10 25 0 5 5 5 5 20 100 7.0 0 10 25 O 1 4 4 5 14 100 7.5 O 10 25 0 O 0 0 0 100 5.5 3 10 138 O O 0 0 D 100 6.0 3 10 138 1 4 4 4 4 17 100 6.5 3 10 138 0 5 5 5 5 20 100 7.0 3 10 138 3 5 5 5 5 100 7.5 3 10 138 2 2 2 2 4 12 429 5.; p 50 105 o o o o 1 , , , 100 6.0 0 50 105 0 3 3 4 4 14 100 6.5 0 50 105 5 5 5 5 5 25 100 7.0 O 50 105 4 5 5 5 5 24 100 7.5 0 50 105 2 3 1 0 1 100 5.5 3 50 218 0 0 O 0 0 100 6.0 3 50 218 1 4 4 4 4 17 100 6.5 3 50 218 4 5 5 5 5 24 100 7.0 3 SD 218 0 0 0 0 4 100 7.5 3 50 218 0 0 0 O 1 199 0 5.5 225 ass, 0 o o o o ,, , 100 6.0 0 225 455 O 0 O O 0 100 6.5 0 225 455 O 0 0 O 1 100 7.0 0 225 455 D 0 0 0 0 100 7.5 0 225 455 0 0 0 D 0 DOD-‘00 100 5.5 3 225 568 D 0 0 0 0 100 6.0 3 225 568 O 0 0 0 O ° 100 6.5 3 225 568 2 2 3 3 3 100 7.0 3 225 568 0 O 0 0 0 100 7.5 3 225 568 D O 0 0 0 [Annotation] KEB Table 4: Histidine based solubility screen at 120 mg/mL / 150 mg/mL husFCyRIIB and 2-8°C. The visual ance of each formulation was assessed by light microscopy and ranked according to an arbitrary scale (0 = no crystals; 1 = some crystals, hardly visible; 2 = some crystals clearly visible; 3 = more than 30 crystals per well clearly visible; 4 = layer of many crystals (well not fully covered); 5 = layer of many crystals (well completely d). huchYRlIB pfl Sugar NaCI Osmelality 0 d 7 d 14 d 28 d Sum [mg/ml] m 1li [mew/kg] 120 5.5 6 50 332 0 O 0 O O 120 6.0 6 50 332 O O 0 3 3 120 6.5 6 50 332 0 0 0 4 4 120 7.0 6 50 332 0 0 0 0 O 120 7.5 6 50 332 0 O 1 1 2 120 5.5 4.5 75 326 0 1 1 2 4 120 6.0 4.5 75 326 0 0 1 1 2 120 6.5 4.5 75 326 O 0 4 4 8 120 7.0 4.5 75 326 0 O 0 D 0 120 7.5 4.5 75 326 O 2 3 3 8 1,29 .52“: . ___. __3 .199 2 3 3 3 1° . 3.1? 120 6.0 3 100 319 0 0 Z 3 5 120 6.5 3 100 319 0 O 0 0 O 120 7.0 3 100 319 0 0 0 O 0 120 7.5 3 100 319 0 D 0 O O 120 5.5 1 5 125 313 0 5 5 5 15 120 6.0 1 5 125 313 0 O 1 2 3 120 5.5 1 5 125 313 0 0 0 O 0 120 7.0 1.5 125 313 O 0 O O 0 120 7.5 1.5 125 313 0 O 0 O 0 m2 .2; , 6_ a9 :34 2 2 2 2 8 , , , 150 6.0 6 50 334 0 2 3 3 8 150 6.5 6 50 334 0 5 5 5 15_ 150 7.0 6 50 334 0 D O 5 5 150 7.5 6 50 334 O 0 O 5 5 150 5.5 4.5 75 327 0 1 3 5 S 150 6.0 4.5 75 327 1 3 4 4 12 150 6.5 4.5 75 327 5 5 5 5 20 150 7.0 4.5 75 327 5 5 5 5 20 150 7.5 4.5 75 327 4 5 5 5 15 159 321 o 5 5 s 15 _ 5;. _ ,3” _190 150 6.0 3 100 321 5 5 5 5 20 150 6.5 3 100 321 4 5 5 5 15 150 7.0 3 100 321 3 5 5 5 18 150 7.5 3 100 321 0 0 0 0 O 150 5.5 1.5 125 314 2 5 5 5 17 150 6.0 1.5 125 314 3 5 5 5 18 150 6.5 1.5 125 314 3 5 5 5 18 150 7.0 1.5 125 314 3 5 5 5 18 150 7.5 1.5 125 314 3 5 5 5 18 [Annotation] KEB Table 5: Citrate based solubility screen at 70 mg/mL husFCyR/IB and 2- 8°C. The visual appearance of each formulation was assessed by light microscopy and ranked according to an arbitrary scale (0 = no crystals; 1 = some crystals, hardly visible; 2 = some crystals clearly visible; 3 = more than crystals per well clearly visible; 4 = layer of many crystals (well not fully covered); 5 = layer of many ls (well tely covered). huch‘IRIIB pH Sugar Nacl Osmolality D d I d 8 d 17 d 30 d Sum Imelmu [961 (li [mOsrplkgl 70 5.5 O 10 24 O 3 5 5 5 70 6.0 0 10 24 O 1 2 3 3 70 6.5 0 10 24 O 0 0 O 0 70 7.0 0 10 24 O 0 0 O 0 70 7.5 0 10 24 O 0 0 O 0 000m“ 70 5.5 3 10 137 O 0 3 4 5 70 6.0 3 10 137 0 O 0 1 1 70 6.5 3 10 137 0 0 D 0 0 70 7.0 3 10 137 O 0 0 D O 70 7.5 3 10 137 0 0 0 O 0 OOONN 79 5:5 0 so mg 3 o 2 4 s 5 70 6.0 0 50 104 O 1 1 1 2 70 6.5 0 50 104 O O 0 0 0 70 7.0 O 50 104 O 0 0 0 0 70 7.5 O 50 104 O O D O O 000mm 70 5.5 3 50 217 O 1 2 3 4 70 6.0 3 $0 217 O 0 D 0 O 70 5.5 3 so 217 o o o o 0 70 7.0 3 50 217 O 0 0 O O 70 7.5 3 50 217 0 0 0 0 0 00000 7o 55 0 225 454 D 2 3 4 4 70 6.0 0 225 454 D 0 0 O 0 70 6.5 0 225 454 O 0 0 0 D 70 7.0 0 225 454 O 0 0 O 0 70 7.5 0 225 454 O 0 0 O 0 00°00) 70 5.5 3 225 567 O 0 0 1 1 70 6.0 3 225 567 O O 0 0 0 70 6.5 3 225 557 D 0 0 O 0 70 7.0 3 225 567 o a o o o 70 7.5 3 225 567 O 0 O O 0 OOOON [Annotation] KEB Table 6: Citrate based solubility screen at 100 mg/mL husFClelB and 2- 8°C. The visual appearance of each formulation was assessed by light microscopy and ranked according to an ary scale (0 = no crystals; 1 = some crystals, hardly visible; 2 = some crystals clearly visible; 3 = more than crystals per well clearly visible; 4 = layer of many crystals (well not fully d); 5 = layer of many crystals (well completely covered). huchleIB pH Sugar Nad Osmolalhy 0 d 4 d 7 d 15 d 28 d Sum [ms/ml] [’6] [MM] [m°$m/ksl 100 5.5 0 10 25 1 3 S 5 5 100 6.0 0 10 25 1 4 5 5 5 100 6.5 0 10 25 0 1 1 1 2 100 7.0 0 10 25 0 O O O 0 100 7.5 O 10 25 0 O D D D 100 5.5 3 10 138 0 3 4 4 5 100 6.0 3 10 138 O 1 3 4 5 100 6.5 3 10 138 0 0 0 0 0 100 7.0 3 10 138 0 O 0 0 0 100 7.5 3 10 138 0 O O 0 O 100 5.5 0 50 105 O 5 5 5 5 100 6.0 0 50 105 O 3 4 5 5 100 6.5 0 50 105 0 0 l 1 1 100 7.0 0 50 105 0 0 O O O 100 7.5 0 50 105 O 0 O D D 100 5.5 3 50 218 0 0 3 4 5 100 6.0 3 50 218 0 0 0 0 3 100 6.5 5 50 218 O O 0 O O 100 7.0 3 50 218 O O 0 0 0 100 7.5 3 50 218 0 0 0 0 0 106 5.5 o 225 45; o a 4 a a 100 6.0 0 225 455 0 1 l 2 3 100 6.5 0 225 455 O 0 0 O O 100 7.0 0 225 455 0 0 0 0 O 100 7.5 0 225 455 O 0 0 0 0 100 5.5 3 225 568 0 0 1 3 4 100 6.0 3 225 568 O 0 0 0 0 100 6.5 3 225 568 0 O O 0 O 100 7.0 3 225 568 O O 0 0 O 100 7.5 3 225 568 O 0 0 0 0 [Annotation] KEB Table 7: Citrate based lity screen at 120 mg/mL / 150 mg/mL husFCyRIIB and 2-8°C. The visual appearance of each formulation was assessed by light copy and ranked according to an arbitrary scale (0 = no crystals; 1 = some crystals, hardly visible; 2 = some crystals clearly visible; 3 = more than 30 crystals per well clearly visible; 4 = layer of many crystals (well not fully covered); 5 = layer of many crystals (well completely covered). huchyRIlB pH Sugar NaCl Osmolality 0 d 7 d 14 d 28 6! Sum [mg/ml] [96] [mM] [mOsm/kg] 120 6.0 7.5 25 339 o 1 z 3 120 6.5 7.5 25 339 o o o 0 120 7.0 7.5 25 339 o 0 0 o 120 7.5 7.5 25 339 a o a 0 00000 120 6.0 6 50 332 o 2 3 4 120 6.5 6 so 332 0 o a o 120 7.0 6 so 332 o o 0 o 120 7.5 6 50 332 o 0 o o 60000 329, 59... 35. 7s 32:: o o o o 120 6.5 4.5 75 326 o o o o 120 7.0 4.5 75 326 o o o o 120 7.5 4.5 75 326 o o o a 6000 120 6.0 3 100 319 0 z 3 3 120 6.5 3 100 319 0 o o o 120 7.0 3 100 319 o o o o 120 7.5 3 100 319 o 0 0 o OOOG 129 6.9 1.5 125 0 1 z 2 . 313 120 6.5 1.5 125 313 o 0 o o 120 7.0 1.5 125 313 o o o o 120 7.5 1.5 125 313 a o 0 o OOOVI 159 6.0 77.5 340 o 2 3 4 . 25 150 6.5 7.5 25 340 o o 0 0 150 7.0 7.5 25 340 o o o o 150 7.5 7.5 25 340 o a o 0 000W 150 6.0 6 so 334 o 1 2 4 150 6.5 6 so 334 o o o o 150 7.0 6 50 334 0 o o 0 150 7.5 6 50 334 o 0 0 o 000% 150 6.0 4.5 75 327 o 0 2 3 150 6.5 4.5 75 327 0 0 0 o 150 7.0 4.5 75 327 0 o 0 o 150 7.5 4.5 75 327 o o o o OOOUI 150 6.0 3 100 321 o 0 0 0 150 6.5 3 100 321 o o o 1 150 7.0 3 100 321 o o o o 150 7.5 3 100 321 o 0 0 o 000-30 159 6:0 15 125 Egg 0 1 2 2 150 6.5 1.5 125 314 o o o o 150 7.0 1.5 125 314 0 o o o 150 7.5 1.5 125 314 o o o 0 OOOUI [Annotation] KEB d) Thermal ity of high concentrated huchlelB ations The formation of non-native protein aggregates and particulates could pose a major obstacle for the development of a high concentrated protein formulation (Shire et al., 2010, Chapter 15. High-concentration antibody formulations. In ation and Process Development Strategies for Manufacturing Biopharmaceuticals. Jameel, F. & nson, S., eds., John Wiley, Hoboken, NJ). For that reason formulation ates (c.f. Example 20) were ranked in respect to their y to preserve husFClelB's native structure in the presence of thermal stress and hence it non-native aggregation.

At first the melting temperature Tm of citrate buffered huchYRIIB formulations was measured by Differential ng Fluorimetry. Fig. 4 shows the respective results that were ed using 0.5 mg/mL husF0lelB in 10 mM citrate, 4.5% sugar (2:1 (w/w) sucrosezmannitol), 75 mM NaCl at the indicated pH (Fig. 4 (a) and (0)), or 0.5 mg/mL husFClelB in 10 mM citrate pH 7.0 supplemented with the indicated amount of sugar (2:1 (w/w) sucrose2mannitol) and salt (Fig. 4 (b) and (d)). huchYRIIB formulations were heated in a 96 well microtiter plate at 1°C/min in the presence of Sypro Orange and the fluorescence emission at 610 nm was recorded. The fluoresence vs. temperature plots (a) and (b) and their first derivatives (c) and (d) are shown.

The first maximum in the dF/dT plots was defined as husF0lelB melting temperature.

Depending on the composition of the respective formulation candidate Tm values from 508°C to 555°C were measured. The largest influence on the melting temperature had the pH with a Tm increase by approx. 3.5°C when the pH was lowered from 7.5 to 6.5. Addition of 7.5% sugar increased the Tm by approx. 1°C (Fig. 5, showing the change of the husFClelB melting temperature as a function of pH, sugar and salt concentration in 10 mM citrate.

The average and standard ion from three independent wells are shown).

Although the NaCl concentration was lowered in parallel the increased thermal [Annotation] KEB stability is clearly a function of the sed sugar concentration and not the decreased salt content, as shown in earlier experiments (data not shown) and according to the theory of preferential exclusion (Timasheff, 1992, Chapter 9.

Stabilization of Protein Structure. In Stability of Protein Pharmaceuticals, Part B: In vivo pathways of ation and strategies for protein stabilization.

Ahern, T.J. & Manning, M.C., eds., Plenum Press, New York, pp. 265-285).

This is also exemplified by raising the sugar concentration from 7.5% to 40% which resulted in a Tm increase by 6.2°C.

Next it was determined to what extent the stabilization of husF0lelB's secondary and tertiary ure, as indicated by a high g ature, would inhibit the formation of insoluble protein aggregates. Therefore the turbidity of citrate buffered husF0lelB ations was measured after incubation at 37°C, a ature well below the measured Tm. To this purpose, the accelerated stability of citrate ed husF0yR|lB formulations at 37°C were determined. The optical density at 360 nm was measured as a function of huchYRllB concentration and sucrose concentration after 1h (a), 12h (b) and 7d (c). All formulations contained 10 mM citrate pH 7.0, 25 mM NaCl. The optical density of a buffer control was subtracted. At 40% e, the highest concentrated formulation contained only 60 mg/mL husF0lelB and not 80 mg/mL.

The results are shown in Fig. 6. With increasing sucrose tration (increasing concentrations from back to front on z-axis in all diagrams of Figure 6), i.e. increasing melting temperature of the protein, the rise in turbidity is ed. With no added sucrose all ations with a strength 210 mg/mL husFCyRIIB became already turbid after one hour at 37°C, whereas at 40% sucrose no significant increase in turbidity up to 60 mg/mL huchYRllB was observed even after seven days at 37°C. Above a certain protein concentration the absolute turbidity increases linearly with increasing huchYRllB concentration but below that threshold the formation of insoluble protein aggregates is extremely slow or even inhibited. With sing sucrose [Annotation] KEB concentrations this threshold is shifted to higher hUSFCyRIIB concentrations, but from the physiologic point of view unacceptable high e concentrations will be needed to stabilize husF0lelB at 37°C and a concentration exceeding 60 mg/mL.

A further test was conducted regarding the accelerated stability of citrate buffered lB ations at 40°C. The results are shown in Fig. 7.

The se in optical density at 360 nm was measured at 10 mg/mL huchYRllB in 10 mM citrate, 150 mM NaCl pH 7.0 supplemented with 10%/ 292 mM sucrose (a, A), 10% / 292 mM trehalose (a, El), 5% / 274 mM mannitol (a, O), 30% / 876 mM sucrose (b, A), 30% / 876 mM ose (b, El), 15% / 822 mM mannitol (b, O). The buffer control supplemented with 20% sucrose is also shown (0).

Based on these observations different sugars and sugar alcohols were ranked in respect to their ability to suppress the formation of insoluble n aggregates. As shown in Fig. 7, the most efficient stabilizer is sucrose. e) Definition of required detergent concentration Turbidity assays (data not shown) indicated in accordance with published data (Timasheff, 1992, r 9. Stabilization of Protein Structure. In Stability of Protein Pharmaceuticals, Part B: In vivo pathways of degradation and strategies for protein stabilization. Ahern, T.J. & Manning, M.C., eds., Plenum Press, New York, pp. 265-285) that increasing detergent concentrations destabilize husFClelB. Also it is speculated that the use of high detergent concentrations may lead to increased immunogenicity (Hermeling et al., 2003, Pharm. Res. 20, 1903-1907). For that reason it would be mandatory to keep the detergent concentration as low as possible without compromising its izing effect towards surface stress.

Ideally, the polysorbate 20 concentration would be fixed at , the concentration which is used for already established liquid husFClelB [Annotation] KEB formulations containing 5 - 20 mg/mL huchYRllB. But since the strength of the newly developed formulation will be above 50 mg/mL and polysorbate 20 may bind to the n, it was questioned r the detergent concentration must be raised above 0.005%.

In order to test the hypothesis of unspecific detergent binding by the protein a citrate buffered formulation candidate containing 0.005% polysorbate 20 was supplied with increasing concentrations of husFClelB and the solutions were incubated for one hour at room temperature and 60°C, a temperature well above the ined Tm of husFClelB. After the protein and etically bound polysorbate 20 were removed by cation exchange chromatography the amount of free polysorbate was measured. In Fig. 8 an experiment is shown that determines free polysorbate 20 in the presence of husF0lelB. hustRllB in 10 mM citrate, 25 mM NaCl, 3% sucrose, 1.5% mannitol, 0.005% polysorbate 20, pH 6.7 was ted for 1 h at 25°C (native husF0lelB) and 60°C (denatured hUSFC’yRIIB). After llB was removed by cation exchange chromatography (CEX), the amount of polysorbate 20 was measured (a). The blank represents buffer without added elB or detergent. The system ility tests (SST) represent buffer with 0.005% polysorbate 20, SST2 was CEX treated and SST1 not. The measured polysorbate content at 0.08 — 0.72 mg/mL hUSFC‘yRIIB was extrapolated to husF0lelB concentrations above 10 mg/mL by linear regression (R2>0.998) of the polysorbate concentration versus the thmic huchYRllB concentration (b).

As shown in Fig. 8, a linear onship between the free polysorbate content and the logarithmic huchYRllB concentration was established. Therefore it is expected that the concentration of free polysorbate will only change marginally, by approx. 0.0001 - 0.0002%, in case that the strength of the formulation is increased from 20 to 100 mg/mL. The significantly lower polysorbate concentration in the samples compared to the control at 0.005% is largely attributed to the fact the samples were diluted with CEX equilibration ation] KEB buffer, which was prepared without polysorbate 20, during column loading (of.

Fig. 8 SST 1 vs. SST2). f) Viscosity of high concentrated huchlelB formulations Highly concentrated protein formulations are characterized by a high viscosity (Shire et al., 2010, supra). ore the manufacturability of a highly concentrated protein formulation might be hampered by its viscosity as the concentration process by tangential flow filtration may become unacceptable slow. In a further experiment, the solution viscosity of husFClelB in 10 mM citrate, 25 mM NaCl, pH 7.0 was measured at 20°C. The experimental data as shown in Fig. 9 (O) was fitted to an exponential growth function (-, R2>0.992) and found that the solution viscosity of the formulation is low enough to run economic TFF ses up to at least 210 mg/mL. g) lisation of huchlelB containing formulations 59 formulations with varying husFCyRIIB, sugar, salt and detergent content were subjected to a conservative lyophilisation cycle. The solids were reconstituted with a volume of water for injection that was equal to or less than the original volume prior to the lyophilisation s. In doing so the husF0lelB content after reconstitution was adjusted to a nominal content of 60 - 180 mg/mL. The suitability of the various formulations for lyophilisation was evaluated based on the reconstitution time and the particulate contamination after reconstitution. l formulations were identified that could be tituted in less than 2 min and neither showed an increased turbidity nor the formation of particulate in the'visible and sub-visible range.

Based on the above bed lyophilisation screen it was shown that ideal formulations contained low amounts of husFClelB (eg. 15 - 60 mg/mL) prior to lyophilisation and were reconstituted with low volumes of water for injection, y increasing the final detergent concentration. The particulate load of selected formulations as ined by fluorescence microscopy is shown in Fig. 10. All formulations contained 5 mM citrate pH 6.7 and the indicated [Annotation] KEB amount of salt, sugar and detergent. The formulations were lyophilized and tituted to the indicated husF0lelB content.

Claims (7)

Claims 1.

1. A ation containing a soluble human Fcγ receptor IIB (husFcγRIIB) in an aqueous buffered solution, wherein the concentration of the Fc receptor is greater than 60 mg/ml and up to 150 mg/ml and wherein it contains a physiologically able buffer nce, wherein the formulation ns 10 mM to 225 mM NaCl, wherein the receptor is present in dissolved form, and wherein the formulation is: (a) a citrate buffered solution and the pH of the formulation is adjusted to from 6.0 to 7.5; or (b) a histidine buffered solution and the pH is adjusted to from 5.2 to 5.9.

2. A formulation containing a soluble human Fcγ receptor IIB (husFcγRIIB) in an aqueous buffered solution, wherein the concentration of the Fc receptor is greater than 60 mg/ml and up to 150 mg/ml and wherein it contains a physiologically acceptable buffer substance, wherein the formulation ns 10 mM to 225 mM NaCl, wherein the ation contains the receptor in a crystalline or microcrystalline form, and wherein the formulation is: (a) a citrate buffered solution and the pH of the ation is adjusted to from 5.2 to 5.9; or (b) a histidine buffered solution and the pH of the formulation is ed to from 6.0 to 7.5.

3. The formulation according to claim 1 or 2, wherein the concentration of the Fc receptor is greater than 80 mg/ml, preferably greater than 100 mg/ml.

4. The formulation according to any one of the previous claims, wherein it contains further substances ed from sugars, salts and detergents.

5. A pharmaceutical composition comprising the formulation according to any one of claims 1 to 4, optionally in combination with pharmaceutically acceptable excipients and/or adjuvants and/or carriers.

6. The pharmaceutical ition according to claim 5 for the subcutaneous injection of an ive amount of an Fc receptor for the tion or treatment of autoimmune diseases or inflammatory diseases.

7. Use of the formulation according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 or 6 in the preparation of a medicament for treating or preventing autoimmune diseases, in particular multiple sis, systemic lupus erythematosus, rheumatoid arthritis, primary immune thrombocytopenia or autoimmune hematolytic anemia. ation] KEB - 1 /10 -