RU2773172C2 - Stable compositions of immunoglobulin single variable domain and their use - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine; it is intended for the treatment or prevention of Willebrand factor (hereinafter – WF)-related diseases. A composition for the treatment or prevention of WF-related diseases contains a Willebrand factor (WF)-binding agent, which contains at least one of SEQ ID NO: 1-19, citrate buffer, auxiliary substance and Twin-80. A method for the preparation of this composition includes stages of: expression of the WF-binding agent in a cell culture; purification of the WF-binding agent by chromatographic purification and ultrafiltration/diafiltration; as well as bringing to the required concentration of the WF-binding agent in the composition. In addition, this group of inventions relates to a method for the preparation of a restored composition; and a kit for the treatment or prevention of WF-related diseases.

EFFECT: implementation of the invention provides compositions that are stable during storage for long periods of time and over a wide temperature range; compositions of the present invention provide high stability of polypeptide, allowing for multiple freeze-thaw cycles without chemical or physical damage, and provide stability against mechanical stress, such as stress during shaking, shifting or mixing.

13 cl, 32 tbl, 11 dwg

Description

1. The field of technology to which the invention belongs

The present invention relates to stable compositions of polypeptides, for example, immunoglobulin single variable domains, in particular, immunoglobulin single variable domains directed to von Willebrand factor (VWF), such as immunoglobulin single variable domains according to SEQ ID NO: 1-19, in particular , SEQ ID NO: 1, i. e. nanobody ALX-008.

The invention provides compositions that are stable during storage for long periods of time and over a wide range of temperatures. The compositions of the present invention ensure high stability of the polypeptide, allowing for multiple freeze-thaw cycles without chemical or physical disruption, and provide resistance to mechanical stress such as shaking, shearing or agitation. They are suitable for pharmaceutical and diagnostic applications and are compatible with pharmaceutically acceptable diluents such as saline, Ringer's solution or glucose/dextrose solution.

The present invention also relates to methods for preparing, storing and using the compositions. The invention also relates to dosage forms, kits and medical use of the compositions.

2. Prior Art

Immunoglobulin single variable domains, such as camel VHH domains, camelized VH domains, or humanized VHH domains, represent a rapidly growing class of antibody-based therapeutic agents. For example, anti-VWF immunoglobulin single variable domains are described in WO2004/015425 [P02-008], WO2004/062551 [P03-001], WO2006/074947 [P05-001], WO2006/122825 [P05-003], WO2009/115614 [ P08-013], and WO2011/067160.

Proteins such as immunoglobulin single variable domains (IVDs) typically require storage and transport from their original site of manufacture prior to use, eg, administration to a patient. The processes of transportation, production, storage and delivery can have a variety of stressful effects on immunoglobulin single variable domains, such as chemical and physical stress. During storage, chemical modification may occur, such as, for example, deamidation, racemization, hydrolysis, oxidation, isomerization, beta elimination, or disulfide exchange. Physical stress can cause denaturation and unfolding, aggregation, particle formation, precipitation, opalescence, or adsorption.

There is a need to provide compositions of immunoglobulin single variable domains, for example, as defined in this application, with increased stability, protecting the active agent from chemical or mechanical stress, and thus allowing storage and temperature changes without significant physical or chemical damage, maintaining stability in over a long period of time, and/or good tolerance by the patient, for example, when the active agent is dissolved in high concentration.

3. Brief summary of the invention

It is known that the aforementioned stress can affect the physicochemical integrity of protein drugs, such as antibody-based drugs. For example, aggregation, deamidation and oxidation have been described as the most common causes of antibody degradation (Cleland et al., 1993, Crit. Rev. Ther. Drug Carrier Systems 10, 307-377). At the same time, it is important that compositions are provided that maintain the chemical and physical integrity of the immunoglobulin single variable domains. Chemical and physical integrity is essential for use as a therapeutic agent, for example, and is typically also associated with biological activity. While our knowledge of protein stability is increasing, optimizing formulation conditions to completely suppress or minimize a variety of stresses and ensure long shelf life remains a major challenge.

There is little data on suitable compositions of immunoglobulin single variable domains. WO2010/077422 describes a TNF-binding nanobody composition containing a lyoprotectant, a surfactant and a buffering agent selected from histidine buffer and Tris-HCl at pH 5.0 to 7.5.

Specific VWF binding agents, and in particular immunoglobulin single variable domains with high affinity for VWF, such as ALX-0081 [INN: caplacizumab], have been tested as adjuvant therapy for acute coronary syndrome (ACS) patients undergoing percutaneous coronary artery angioplasty (TCA), and developed for the treatment of thrombotic thrombocytopenic purpura (TTP). Phase I clinical trials have been successfully completed and phase II trials are currently underway. So far, ALX-0081 has been presented as a phosphate-based liquid formulation containing 5 mg/ml active pharmaceutical ingredient (API) in D-PBS, 200 mM glycine, and 0.02% Tween-80 (v/v).

Although this composition has been proven to be active, it can be improved in various ways. First, at current concentrations, many subcutaneous injections are likely to be necessary (given that the volume for subcutaneous injection is limited to about 1 ml), thus reducing patient tolerance and compliance. Secondly, the storage stability and shelf life of the currently available composition ALX-0081 (hereinafter referred to as the current composition ALX-0081) can be improved at elevated temperatures. Stability in modern compositions at high temperatures is determined mainly by chemical modifications of the polypeptide. Chemical modifications may be associated with loss of activity. Although a practical shelf life can be achieved by storing the product at -20°C, however, this is not considered a favorable option for most practical purposes.

Lyophilization is a commonly used protein preservation technique to remove water from a protein preparation of interest. Lyophilization, or freeze drying, is a method in which the material to be dried is first frozen, and then ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient may be included in the compositions prior to lyophilization to improve stability during the lyophilization process and/or to improve the storage stability of the lyophilized product (Arakawa et al. Pharm. Res. 8(3):285-291 (1991)).

The present invention relates to a composition comprising a von Willebrand factor (VWF) binding agent and a citrate or phosphate buffer, preferably a citrate buffer, with a pH in the range of 5.0 to 7.5. In particular, the present invention relates to a composition as described herein, wherein said VWF binding agent comprises at least one immunoglobulin single variable domain that binds to SEQ ID NO: 20.

Said immunoglobulin single variable domain comprises or essentially consists of an immunoglobulin single variable domain which is a heavy chain variable domain sequence, in particular an immunoglobulin single variable domain which is a heavy chain variable domain sequence derived from a conventional four chain antibody, or a variable domain sequence a heavy chain derived from an antibody heavy chain, or a nanobody (including the VHH sequence), preferably, but not limited to, a nanobody.

In addition, the present invention relates to a composition as described in this application, where the specified agent that binds VWF, includes at least one of SEQ ID NO: 1-19. Further, the present invention relates to a composition as described herein, wherein said VWF binding agent is a single chain polypeptide comprising one or more immunoglobulin single variable domains, preferably wherein said VWF binding agent is monovalent or polyvalent, wherein said the VWF binding agent is monospecific or polyspecific, and/or one or more immunoglobulin single variable domains are CDR-grafted. humanized, camelized, deimmunized, and/or created in vitro (eg, selected by phage display). The present invention also relates to a composition as described herein, wherein said VWF binding agent comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1. The present invention also relates to a composition as described herein, where the specified agent that binds VWF has a concentration in the range from 0.1 to 80 mg/ml, and/or where the specified buffer has a concentration in the range of 5-200 mm.

In addition, the present invention relates to a composition as described in this application, further comprising a filler, preferably, where the specified filler has a concentration in the range of 10-500 mm, more preferably, where the specified filler is selected from the list including sucrose, glycine, mannitol, trehalose and NaCl, even more preferably, wherein said sucrose has a concentration in the range of 1-15%, preferably 2-12%, preferably 4-10%. for example 4, 5, 6, 7, 8 or 9% (w/v), most preferably 7%.

The present invention also relates to a composition as described in this application, selected from a citrate buffer, preferably, where the specified citrate buffer has a pH of from 6.0 to 7.0, more preferably 6.5; and a phosphate buffer, preferably wherein said buffer has a pH in the range of 6.5 to 7.5, preferably 7.1.

In addition, the present invention relates to a composition as described in this application, additionally containing a non-ionic detergent, such as Tween-80, preferably at a concentration of from 0.001 to 0.5% (v/v), more preferably 0.01-0 .02% (v/v).

Further, the present invention relates to compositions as described in this application, where the specified buffer is a citrate buffer at pH 6.5±0.5, for example, 6.2; 6.3; 6.4; 6.5; 6.6; 6.7 or 6.8, in particular 6.5, and wherein said composition preferably comprises sucrose having a concentration in the range of 1-15%, preferably 2-12%, preferably 4-10%, e.g. 4, 5, 6, 7, 8 or 9% (w/v), most preferably 7%, and preferably includes a non-ionic detergent such as Tween-80, preferably at a concentration of 0.01% (v/v).

Further, the present invention relates to a composition as described herein, wherein said composition has an osmolality in the range of 290±60 mOsm/kg, more preferably in the range of 290±20 mOsm/kg.

Further, the present invention relates to a composition comprising:

(a) a VWF binding agent at a concentration of from about 0.1 mg/ml to about 80 mg/ml;

(b) an excipient selected from sucrose, glycine, mannitol, trehalose, or NaCl at a concentration of from about 1% to about 15% (w/v);

(c) Tween-80 at about 0.001% to about 0.5% (v/v); and

(d) a buffer selected from citrate buffer at a concentration of from about 5 mM to 200 mM, so that the pH of the composition is from about 6.0 to 7.0, and phosphate buffer at a concentration of from about 10 mM to 50 mM, so that the pH composition is from about 6.5 to 7.5, where the VWF binding agent in the composition retains at least 80% activity when stored for at least 12 months at 5°C or even 24 months at 5°C.

The invention also relates to a composition that contains less than 5% high molecular weight species (HMW) after storage for at least 12 months at 5°C, or even 24 months at 5°C; and/or less than 5% low molecular weight species (LMW) after storage for at least 12 months at 5°C or even 24 months at 5°C.

The invention also relates to a composition in which at least 80%, preferably at least 90%, more preferably at least 95%, or even at least 99% of the VW binder retains binding activity after storage, according to compared with the binding activity before storage, where the specified binding activity is measured by ELISA and/or Biacore.

In addition, the invention relates to a composition as described in this application, where the specified composition is in liquid, lyophilized, freeze-dried, reconstituted lyophilized or frozen form; in particular, the invention relates to a liquid or reconstituted lyophilized composition comprising:

(a) a VWF binding agent at a concentration of from about 0.1 mg/ml to about 80 mg/ml;

(b) sucrose at a concentration of about 15 to 15% (w/v);

(c) Tween-80 at about 0.001%-0.5% (v/v); and

(d) citrate buffer at a concentration of about 5 mM to 200 mM such that the pH of the composition is about 6.0 to 7.0.

The lyophilized composition can then be reconstituted, if necessary, by mixing the lyophilized form with a suitable solvent (eg water) to resolubilize the components of the original composition to the desired concentration.

The present invention also relates to a composition as described herein, which is a bulk storage composition comprising:

(a) a VWF binding agent at a concentration of from about 0.1 mg/ml to about 80 mg/ml;

(b) sucrose at a concentration of about 15 to 15% (w/v);

(c) Tween-80 at about 0.001%-0.5% (v/v); and

(d) citrate buffer at a concentration of about 5 mM to 200 mM such that the pH of the composition is about 6.0 to 7.0, where at least 100 liters of the composition is stored below freezing.

In addition, the present invention relates to a composition suitable for parenteral use in a subject, for example, in a human subject (eg, a patient having a VWF-dependent disease). The composition can be applied to the subject by injection (eg, intravenous, subcutaneous, intramuscular, or intraperitoneal).

In addition, the present invention provides a composition as described herein for use in a method of treating a human or animal subject, preferably for use in the treatment of VWF dependent diseases such as, for example, acute coronary syndrome (ACS), transient cerebrovascular accident , unstable or stable angina, stroke, myocardial infarction or thrombotic thrombocytopenic purpura (TTP), most preferably for use in the treatment of TTP or ACS. Further, the present invention relates to a method or process for preparing a composition as described in this application. The method or process includes expressing a VWF binding agent in cell culture; purifying the VW-binding agent, for example, by passing the VW-binding agent through at least one of the chromatographic purification step, ultrafiltration/diafiltration steps; regulation of the concentration of the agent that binds VWF, for example, up to about 0.1-80 mg/ml in a composition containing lyoprotectant, surfactant and buffer, as described in this application, for example, sucrose at a concentration of from about 1% to 15%; Tween-80 at about 0.001% to about 0.5% (w/v); citrate buffer at a concentration of from about 5 mm to 200 mm, so that the pH of the composition was from about 6.0 to 7.0; and optionally includes the step of obtaining a finished dosage form from the composition.

The invention also relates to a method or process for preparing a reconstituted composition containing a VW binder, such as ALX-0081, as described in this application. The method includes: lyophilization of a mixture of VW binder, lyoprotectant, surfactant and buffer to obtain a lyophilized mixture; and recovery of the lyophilized mixture in a diluent, with the preparation of the composition, as described in this application. In particular, the composition comprises: (a) a VWF binding agent, for example, ALX-0081 at a concentration of from about 0.1 to 80 mg/ml; (b) sucrose at a concentration of from about 1% to about 15% (w/v); (c) Tween-80 at about 0.001% to about 0.5% (v/v); and (d) citrate buffer at a concentration of from about 5 to 200 mm, so that the pH of the composition is from about 6 to 7.0; and optionally includes the step of preparing a finished dosage form from the composition.

The present invention also relates to a method of stabilizing a VWF binding agent, preferably a polypeptide, comprising at least one of SEQ ID NOs: 1-19, for storage, comprising preparing a composition as described in this application.

In addition, the invention relates to a method for storing a VWF binding agent, preferably a polypeptide, comprising at least one of SEQ ID NOs: 1-19, including preparing a composition as described in this application.

Also provided are pharmaceutical or diagnostic compositions, including any of the compositions described in this application or obtained by the methods described in this application.

Further, the present invention relates to a method for analyzing a product or process, such as a manufacturing process. The method includes providing a composition of a VWF binding agent, such as ALX-0081, as described herein, and assessing a composition parameter such as color, clarity, viscosity, or the amount of one or more HMW, LMW species, as described herein. The evaluation may include determining one or more parameters, such as determining whether a parameter meets pre-selected criteria, such as determining whether a pre-selected criterion exists or whether it meets a predetermined range, thus analyzing the process. For example, process evaluation includes determining the stability of the VWF binding agent composition. The stability of the ALX-0081 composition can be assessed, for example, by the formation of aggregates, which are quantified, for example, using size exclusion high performance liquid chromatography (E-HPLC), by color, transparency, or viscosity, as described in this application.

In addition, the method may further include comparing two or more exemplary compositions in a method for monitoring or controlling variation between batches, comparing the product with a reference material, classifying, selecting, accepting or rejecting, releasing or suspending, processing into a drug product, shipping, moving to another plot, drug formulation, labeling, or packaging of the composition, based on comparison. In addition, the method may further include providing a protocol including data related to the evaluated composition parameter and optionally including a composition batch identifier; transfer of the said protocol to the person making the decision; optionally, receiving a message from said decision maker; optionally, making a decision to release or sell a series of compositions based on a communication from the decision maker.

Kits or products of manufacture are also provided, including the composition of the present invention and instructions for their use, for example, by a healthcare professional. Kits or products of manufacture may include a vial or syringe containing a composition of the present invention, as described in this application. Preferably, the vial or syringe is constructed from glass, plastic, or a polymer material selected from a cyclic olefin polymer or copolymer. Further, the composition may also be present in an injection device (eg, an injection syringe, eg, a pre-filled injection syringe).

The invention also provides pharmaceutical unit dosage forms comprising unitary compositions of the present invention suitable for parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous) compositions of the invention to a human patient.

Further, the compositions of the present invention can be used to store a VWF binding agent, preferably a polypeptide comprising at least one of SEQ ID NOs: 1-19, such as ALX-0081, as described herein, where said storage is 1 -36 months, such as 1; 1.5; 3, 6, 9, 12, 18, 24, 30 or 36 months, preferably at least 12 months, e.g. -70°C to +40°C, such as -70°C, -20° C, +5°C, +25°C or +40°C, preferably between -70°C and +25°C.

The present invention also relates to a method of treating or preventing a VWF-dependent disease, such as, for example, acute coronary syndrome (ACS), transient cerebral ischemic attack, unstable or stable angina, stroke, myocardial infarction, or thrombotic thrombocytopenic purpura (TTP); wherein said method comprises administering to a subject a pharmaceutical composition comprising a composition of the invention, thereby reducing one or more symptoms associated with said VWF dependent disease. In particular, said VWV-dependent disease is TTP.

4. Brief description of the figures

Fig. 1. A flowchart representing the various steps in a standard 65 hour lyophilization program run on ALX-0081.

Fig. 2A. Relevant part of RP-HPLC chromatograms of ALX-0081 after 1 and 2 months of storage at 70°C, +5°C and +25°C; mAU: milli - absorption units.

Fig. 2B. The increase in the relevant part of the RP-HPLC chromatograms of ALX-0081 after 0, 4 and 8 weeks of incubation at 37°C. Splitting of the main peak of RP-HPLC is observed as a result of prolonged incubation at 37°C (0, 4, 8 weeks); mAU: milli - absorbance units.

Fig. 3A. Overlay of E-HPLC profiles of citrate control buffer (bcb) and ALX-0081 in 20 mM citrate buffer, pH 7.0 at 55.9 mg/mL before (a) and after 10 freeze-thaw (30) cycles at -20° C (c) and -70°C (b) (λ = 280 nm). The minimum citrate peak is observed for samples pre-diluted in separation buffer; mAU: milli - absorbance units.

Fig. 3B. Overlay of E-HPLC profiles of citrate control buffer (bcb) and ALX-0081 in 20 mM citrate buffer, pH 7.0 at 55.9 mg/mL, after approximately 1 week of storage at +4°C (λ = 280 nm). ALX-0081 gave on separation one major peak (97%) corresponding to intact, unmodified ALX-0081 and minor preliminary peaks representing only 3% of the total peak area. A minor citrate peak is observed for samples previously diluted in separation buffer; mAU: milli - absorbance units.

Fig. 4A. Scattering intensity of stirred ALX-0081 samples in 50 mM citrate buffer, pH 6.0; 50 mM citrate buffer, pH 6.0 + 0.01% Tween-80 (v/v) and 50 mM citrate buffer, pH 6.0 + 0.02% Tween-80 (v/v) at +25°C . "+" represents samples in 50 mM citrate buffer, pH 6.0 (y=0.0044x+3.5962, R²=0.9549); "o" represents samples in 50 mM citrate buffer, pH 6.0+0.02% Tween-80 (v/v) (y=0.0002x+1.0447, R²=0.4673); "x" represents samples in 50 mM citrate buffer, pH 6.0+0.01% Tween-80 (v/v) (y=0.0004x+0.5125, R²=0.6804); (x-axis = time in seconds; y-axis = scattering intensity).

Fig. 4B. Scattering intensity of stirred ALX-0081 samples in 50 mM citrate buffer, pH 6.0; 50 mM citrate buffer, pH 6.5 + 0.01% Tween-80 (v/v) and 50 mM citrate buffer, pH 6.5 + 0.02% Tween-80 (v/v) at +25°C . "+" represents samples in 50 mM citrate buffer, pH 6.5 (y=0.0041x+4.7667, R²=0.9431); "o" represents samples in 50 mM citrate buffer, pH 6.5 + 0.02% Tween-80 (v/v) (y=0.0004x - 0.0208, R²=0.9391); "x" represents samples in 50 mM citrate buffer, pH 6.5 + 0.01% Tween-80 (v/v) (y=0.0001x - 1.8853, R²=0.0376); (x-axis = time in seconds; y-axis = scattering intensity).

Fig. 5. Overlay of capillary isoelectric focusing profiles of ALX-0081 at 5 mg/ml in D-PBS + 200 mM glycine + 0.01% Tween-80 after 1 month of storage at +40°C (a) and -70°C (b) ; (λ = 280 nm). AU: absorption units; pxlpos: position of pixels.

Fig. 6. Image of lyophilized formulations of ALX-0081 (form 3 = citrate/sucrose, pH 6.0; form 7 = citrate/sucrose, pH 6.5; form 17 = D-PBS/glycine) before (panel A) and after reconstitution Milli-Q water (panel B).

Fig. 7. Image of lyophilized compositions based on ALX-0081 citrate/sucrose.

Fig. 8. Images of liquid compositions ALX-0081, 28 mg/ml containing 15, 20, 25, 30, 40 and 50 mM citrate buffer, pH 6.5 after 4 days of storage at +25°C (panel A) or +5 °C (panel B). Pure citrate buffer (50 mM) is included as a control.

Fig. 9. Images of liquid compositions of ALX-0081, 20 mg/ml, containing 15 mM citrate buffer, pH 6.5, and various amounts of sucrose and Tween-80 after 4 days of storage at +25°C (panel A) or +5° C (panel B). Pure citrate buffer (50 mM) is included as a control.

Fig. 10. Prediction of the content of pyroglutamate in the lyophilized drug product ALX-0081 depending on time when stored at +5°C.

Fig. 11. Prediction of the content of pyroglutamate in the lyophilized drug product ALX-0081 depending on time when stored at +25°C.

5. Detailed description of the invention

Unless otherwise indicated, all methods, steps, techniques and manipulations that are not specifically described in detail can be carried out, and are carried out in a known manner, as understood by a person skilled in the art. Links, for example, can be made to the standard reference books and the general state of the art mentioned in this application, and to additional references given therein; and also, for example, the following reviews: Presta, Adv. drug deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1): 49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et al., Tumor Biol., 2005, 26(1), 31-43, describing protein engineering techniques such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

It has surprisingly been found that VWF binding agents, and in particular ALX-0081 (SEQ ID NO:1), can be used in specific dosing regimens in humans. VWF binding agents, and in particular ALX-0081, provide a pharmacodynamic effect, with a rapid onset of effect immediately at the end of a dose and persistence of activity for about 12-24 hours. In addition, VWF binders, and in particular ALX-0081 (SEQ ID NO:1), were found to be well tolerated and safe in healthy male volunteers. These results indicate that VWF binders, and in particular ALX-0081 (SEQ ID NO:1), are useful for the short-term treatment of patients with stable angina undergoing percutaneous coronary angioplasty (hereinafter also "PCA") and the treatment of patients with thrombotic thrombocytopenic purpura (hereinafter also "TTP").

However, current formulations of VWF binding agents, and in particular ALX-0081 (SEQ ID NO:1), which have been used in human recipients, can be improved.

The re-formulation of VWF binders, and in particular ALX-0081 described in this application, provides a new citrate/sucrose based formulation with increased solubility (up to 80 mg/mL) and significantly improved liquid storage stability ( for example, less oxidation occurs when the new composition is stored, compared to the original composition, in the liquid state). In addition, in the lyophilized form, there is essentially no oxidation or asp isomerization, which could be detected after 12 months of storage at +40°C, or even after 24 months of storage at +40°C. Residual formation of small amounts of pyroglutamate may still be observed. Further optimization of the citrate and sucrose concentration results in a reduction in the moisture content of the lyophilized product, thus minimizing the level of residual pyroglutamate formation.

Accordingly, the present invention provides stable liquid or lyophilized compositions of VWF binding agents (eg, ALX-0081) and their use in the treatment or prevention of VWF dependent diseases.

5.1. Polypeptide(s) of the invention

VWF binding agents used in the present invention are typically proteins or peptides that bind to human von Willebrand factor (VWF, SEQ ID NO: 20). Preferably, the VWF binding agents are proteins or polypeptides comprising or consisting of at least one immunoglobulin sequence, such as an immunoglobulin single variable domain (IVD). Even more preferably, the VWF binding agents of the present invention are proteins or polypeptides comprising or consisting of SEQ ID NOs: 1-19, and most preferably SEQ ID NOs: 1. VWF binding agents can be used as an adjuvant therapy for patients with ACS undergoing PCI, or as a treatment for thrombotic thrombocytopenic purpura (TTP). The terms "protein", "polypeptide" and "amino acid sequence" are used interchangeably in this application. Thus, the amino acid sequence of the present invention is a VWF binding agent.

Thus, suitable VWF binding agents for use in the present invention may include a compound from Table A-1, for example, SEQ ID NOs: 1-19, or a compound having 80% or more, more preferably 85% or more, more preferably 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with the compound of Table A-1 (see "sequence identity" in the "Definitions" section).

Preferably, VWF binding agents for use in the present invention are compounds like 12A02H1. For the purposes of the present invention, compounds like 12A02H1 are a compound comprising 12A02H1 (i.e. SEQ ID NO: 19) or a compound having 80% or more, more preferably 85% or more, most preferably 90%, 95%, 96 %, 97%, 98%, 99% or more amino acid sequence identity (as defined in this application) with 12A02H1 (SEQ ID NO: 19). A particularly preferred VWF binder is ALX-0081 (SEQ ID NO: 1).

All vWF binding agents are well known in the literature. This includes their production (see in particular eg WO2006/122825 and also WO2004/062551). For example, ALX-0081 is prepared as described in WO 2006/122825 or WO 2009/115614 [P08-013].

Unless otherwise indicated, the term "immunoglobulin sequence" used to refer to the heavy chains of an antibody or a conventional four-chain antibody is used as a general term to include both the full-length antibody, its individual chains, and all of its parts, domains or fragments (including antigen-binding domains or fragments such as V _HH domains or V _H /V _L domains, respectively, but not limited to). The terms "antigen-binding molecule" or "antigen-binding protein" are used interchangeably with an immunoglobulin sequence, and include an immunoglobulin single variable domain such as Nanobodies®.

Embodiments of the present invention relate to immunoglobulin sequences that are immunoglobulin single variable domains, such as light chain variable domain sequences (eg V _L sequence) or heavy chain variable domain sequences (eg V _H sequence); in particular, heavy chain variable domain sequences that are derived from a conventional four chain antibody, or heavy chain variable domain sequences that are derived from antibody heavy chains (eg, V _HH sequence).

The term "immunoglobulin single variable domain" defines molecules in which an antigen-binding site is present on or formed from a single immunoglobulin domain or suitable fragments thereof. This separates immunoglobulin single variable domains from "regular" immunoglobulins or fragments thereof, where two immunoglobulin domains, in particular two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V _H ) and the light chain variable domain (V _L ) interact to form an antigen binding site. In this case, the hypervariable regions (CDRs) of both V _H and V _L contribute to the antigen binding site, ie. a total of 6 CDRs are involved in the formation of the antigen-binding site.

In contrast, an antigen binding site from an immunoglobulin single variable domain is formed by a single V _H or V _L domain. Thus, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs, for example one, two or three CDRs.

Thus, the term "immunoglobulin single variable domain" does not include conventional immunoglobulins or fragments thereof that require the interaction of at least two variable domains to form an antigen-binding site. This also applies to embodiments of the invention "comprising" or "comprising" an immunoglobulin single variable domain. In the context of the present invention, such embodiments exclude conventional immunoglobulins or fragments thereof. Thus, a composition "comprising" or "comprising" an immunoglobulin single variable domain may refer to, for example, constructs comprising more than one immunoglobulin single variable domain. Alternatively, they may contain additional components other than immunoglobulin single variable domains, such as auxiliary agents of various kinds, protein labels, dyes, pigments, etc. However, these terms include fragments of conventional immunoglobulins, where the antigen-binding site is formed by a single variable domain.

In accordance with the present invention, a polypeptide of the invention, in particular immunoglobulin sequences, may consist of, or include one or more of the following: domain antibodies, or amino acid sequences suitable for use as domain antibodies, single domain antibodies, or amino acid sequences suitable for use as single domain antibodies, "dAbs", or amino acid sequences suitable for use as dAbs, or Nanobodies® comprising V _HH sequences such as humanized V _HH sequences or camelized V _H sequences, and preferably being Nanobodies®, but not limited to them.

The present invention encompasses suitable fragments of immunoglobulin single variable domains. "Suitable fragments" of immunoglobulin single variable domains refer to polypeptides containing fewer amino acids than native immunoglobulin single variable domain but still exhibiting antigen-binding activity (which typically contain at least some of the amino acid residues forming at least one of the CDRs, such as described later in this application). Such immunoglobulin single variable domains and fragments most preferably comprise an immunoglobulin chain fold, or are capable of forming an immunoglobulin chain fold under suitable conditions. In particular, immunoglobulin single variable domains and fragments thereof are capable of binding to a target antigen. As such, an immunoglobulin single variable domain may, for example, comprise a light chain variable domain sequence (eg, a V _L sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (eg, V _H sequence or V _HH sequence), or a suitable fragment thereof; as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that essentially consists of an immunoglobulin single variable domain, such that interaction of a single antigen-binding domain with another variable domain is not required to form an antigen-binding unit, such as in the case of variable domains present, for example, in conventional antibodies, and scFv fragments that require interaction with another variable domain, for example, via a V _H /V _L interaction, to form a functional antigen-binding domain).

The immunoglobulin sequences of the present invention are preferably in substantially isolated form. The immunoglobulin sequences of the present invention may also form part of a protein or polypeptide of the present invention (as described herein) which may include or essentially consist of one or more amino acid sequences of the present invention, and which may optionally contain one or more additional amino acids. sequences (all optionally linked to one or more suitable linkers). For example, and without limitation, one or more amino acid sequences of the present invention can be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more additional amino acid sequences serving as a binding unit, to provide a monovalent, polyvalent or polyspecific polypeptide of the present invention, respectively, as described in this application. Such a protein or polypeptide may also be in substantially isolated form.

The present invention relates to immunoglobulin sequences of various origins, including mouse, rat, rabbit, donkey, human and camel immunoglobulin sequences. The invention also includes fully human, humanized or chimeric immunoglobulin sequences. For example, the invention includes camel immunoglobulin sequences and humanized camel immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs, as described by Ward et al. (See, for example, WO 94/04678 and Davies and Riechmann (1994 and 1996)). Further, the invention includes fused immunoglobulin sequences, for example, forming a polyvalent and/or polyspecific construct (for polyvalent and polyspecific polypeptides containing one or more V _HH domains, and their preparations, reference can also be made to Conrath et al., J. Biol. Chem ., Vol. ., which are derived from the immunoglobulin sequences of the present invention. Immunoglobulin single variable domains have also been described in the shark (also referred to as "IgNAR", as described, for example, in WO03/014161 or Streltsov, 2005).

In a particular embodiment, the immunoglobulin single variable domains of the present invention are Nanobodies®, in particular camel V _HH domains, humanized V _HH domains, or camelized V _H domains. The person skilled in the art will be familiar with humanization of V _HH and/or camellization of V _H domains.

The amino acid sequence and structure of an immunoglobulin sequence, in particular, Nanotela® can be considered, without any limitation, as including four framework regions or "FR", which are referred to in the art and in this application as "Framework region 1" or " FR1"; as "Frame Section 2" or "FR2"; as "Frame Section 3" or "FR3"; and as "Frame Section 4" or "FR4", respectively; where the framework regions are interspersed with three hypervariable regions or "CDRs", which are referred to in the art as "Hypervariable Region 1" or "CDR1"; as "Hypervariable region 2" or "CDR2", as "Hypervariable region 3" or "CDR3", respectively.

The total number of amino acid residues in the Nanobody® may be 110-120, preferably 112-115, and most preferably 113. However, it should be noted that parts, fragments, analogs or derivatives (as further described herein) of the Nanobody® are not specifically limited to their length. and/or size, if these parts, fragments, analogues or derivatives meet the additional requirements set forth in this application, and also preferably are suitable for the purposes described in this application.

Thus, as a rule, immunoglobulin variable domains are amino acid sequences consisting, or essentially consisting of four framework regions (FR1 - FR4, respectively), and 3 hypervariable regions (CDR1 - CDR3, respectively). "Essentially consists" in the present context means that additional elements may be present, such as labels used for purification or labelling, but such additional elements are small compared to the immunoglobulin single variable domain per se and do not interfere with antigen-binding activity. immunoglobulin single variable domain.

As used herein, the term "immunoglobulin sequences" or "immunoglobulin single variable domains" refers to both the nucleic acid sequences encoding a polypeptide and the polypeptide itself. Any more restrictive meaning will become clear from the specific context.

In particular, the amino acid sequence of the present invention may be Nanotel® or a suitable fragment thereof. For a further description of V _HH and Nanotel reference is made to a review article by Muyldermans in Reviews in Molecular Biotechnology 74(2001), 277-302; as well as the following patent applications, which are mentioned in the general state of the art: WO94/04678, WO95/04079 and WO96/34103 from Vrije Universiteit Brussel; WO94/25591, WO99/37681, WO00/40968, WO00/43507, WO00/65057, WO01/40310, WO01/44301, EP1134231 and WO02/48193 from Unilever; WO97/49805, WO01/21817, WO03/035694, WO03/054016 and WO03/055527 from Vlaams Instituut voor Biotechnologie (VIB); WO03/050531 from Algonomics NV and Ablynx NV; WO 01/90190 from the National Research Council of Canada; WO03/025020 (= EP1433793) from the Institute of Antibodies; as well as WO04/041867, WO04/041862, WO04/041865, WO04/041863, WO04/062551, WO05/044858, WO06/40153, WO06/079372, WO06/122786, WO06/122787 and WO06/122787 and WO06/122787 and WO06/122787 and WO06/122787 and WO06/122787 and other published patent applications from Ablynx NV References are also made to the prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of International Application WO06/040153, of which the list and references are hereby incorporated by reference. . As described in these references, Nanobodies (particularly V _HH sequences, and partially humanized nanobodies) can specifically be characterized by the presence of one or more "characteristic residues" in one or more of the framework regions. Additional description of nanobodies, including humanization and/or camellization of nanobodies, as well as other modifications, parts or fragments, derivatives or "hybrids of nanobodies", polyvalent constructs (including non-limiting examples of linker sequences), and various modifications to increase the shelf life of nanobodies and their preparations can be found, for example, in WO07/104529.

The immunoglobulin single variable domains provided by the present invention are preferably in isolated form or substantially isolated form. The immunoglobulin sequences of the present invention may also form a portion of a protein or polypeptide of the present invention which may include or essentially consist of one or more immunoglobulin single variable domains, and which may optionally further comprise one or more additional amino acid sequences (all optionally linked by one or several suitable linkers). For example, and without limitation, one or more immunoglobulin single variable domains can be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more additional amino acid sequences that can serve as a binding unit, so as to provide a monovalent, polyvalent or polyspecific polypeptide of the present invention, respectively, as described in this application. Such a protein or polypeptide may also be in isolated or substantially isolated form. Thus, in accordance with the present invention, immunoglobulin single variable domains include constructs containing two or more antigen-binding units in the form of single domains, as described above. For example, two (or more) immunoglobulin single variable domains with the same or different antigenic specificity can be linked to form, for example, a bivalent, trivalent, or polyvalent construct. By combining immunoglobulin single variable domains from two or more specificities, bispecific, trispecific, etc. can be formed. designs. For example, a polypeptide of the present invention may contain two immunoglobulin single variable domains directed against target A and one immunoglobulin single variable domain directed against target B, making it bivalent for A and monovalent for B. Such constructs and modifications thereof, which are easily can provide a person skilled in the art, all covered by the present invention. In particular embodiments, the invention relates to bi-paratope constructs containing at least two immunoglobulin single variable domains directed to different epitopes within the same target antigen.

All of these molecules are also referred to as "polypeptide of the present invention", which is synonymous with "immunoglobulin sequences" or "immunoglobulin single variable domains" of the present invention.

In addition, the term "sequence", as used in this application (for example, in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain sequence," V _HH -sequence "or" protein sequence "), is necessary generally understood to include both the relevant amino acid sequence and the nucleic acid sequence, or the nucleotide sequence encoding it, unless the context requires a more limited interpretation.

In accordance with one non-limiting embodiment of the present invention, the immunoglobulin sequences, Nanotelo® or polypeptide of the present invention are glycosylated. According to another non-limiting embodiment of the invention, the immunoglobulin sequences, Nanotelo® or polypeptide of the present invention are non-glycosylated.

5.2. "Binding" to an antigen

The present invention relates to immunoglobulin sequences that can bind and/or have affinity for an antigen as defined herein, for example, von Willebrand factor. In the context of the present invention, the term "binding and/or having affinity" for a specific antigen has its usual meaning in the art, as is meant, for example, in the context of antibodies and their respective antigens.

In particular embodiments of the present invention, the term "binding and/or having affinity" means that an immunoglobulin sequence interacts specifically with an antigen, and is used interchangeably with immunoglobulin sequences "against" said antigen.

The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular immunoglobulin sequence, antigen-binding molecule, or antigen-binding protein (such as an immunoglobulin single variable domain, Nanotelo®, or polypeptide of the present invention) can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity. Affinity, represented by the equilibrium constant for the dissociation of an antigen and an antigen-binding protein (KD), is a measure of the strength of the binding between the antigen determinant and the antigen-binding site on the antigen-binding protein: the lower the KD value, the higher the strength of the binding between the antigen determinant and the antigen-binding molecule (alternatively, affinity can also be expressed as an affinity constant (KA), which is equal to 1/KD). As will be understood by one of skill in the art (eg, based on the following description), affinity can be defined as such, depending on the specific antigen of interest. Avidity is a measure of the strength of the binding between an antigen-binding molecule (such as an immunoglobulin single variable domain, Nanotelo® or a polypeptide of the present invention) and the corresponding antigen. Avidity refers both to the affinity between an antigenic determinant and its antigen-binding site on an antigen-binding molecule, and to the number of corresponding binding sites present on an antigen-binding molecule.

Typically, immunoglobulin sequences of the present invention (such as amino acid sequences, immunoglobulin single variable domains, Nanobodies® and/or polypeptides of the present invention) bind to an antigen with a dissociation constant (KD) of 10 ^-5 to 10 ^-12 mol/liter or less, and preferably 10 ^-7 to 10 ^-12 mol/liter or less, and more preferably 10 ^-8 to 10 ^-12 mol/liter (i.e., with an association constant (CA) of 10 ⁵ to 10 ¹² mol/liter or more, and preferably 10 ⁷ to 10 ¹² mol/liter or more, and more preferably 10 ⁸ to 10 ¹² mol/liter), and/or binding to their antigen is defined as a K _on rate of 10 ² M ^-1 s ^-1 to about 10 ⁷ M ^-1 s ^-1 , preferably from 10 ³ M ^-1 s ^-1 to 10 ⁷ M ^-1 s ^-1 , more preferably from 10 ⁴ M ^-1 s ^-1 to 10 ⁷ M ^-1 s ^-1 , such as from 10 ⁵ M ^-1 s ^-1 to 10 ⁷ M ^-1 s ^-1 ; and / or binding to their antigen, as defined in this application, K _off at a rate of from 1 s ^-1 (t1 / 2 = 0.69 sec) to 10 ^-6 s ^-1 (providing an almost irreversible complex with t1 / 2 from many days), preferably from 10 ^-2 s ^-1 to 10 ^-6 s ^-1 , more preferably from 10 ^-3 s ^-1 to 10 ^-6 s ^-1 , such as from 10 ^-4 s ^-1 to 10 ^-6 with ^-1 .

Any KD value greater than 10 ^-4 M (or any CA value below 10 ⁴ M ^-1 ) is generally considered to be indicative of non-specific binding.

Preferably, the monovalent immunoglobulin sequence of the present invention binds to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

The specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined by any suitable method known, e.g., the Scatchard assay and/or competitive binding assays such as radioimmunoassays (RIAs), enzymatic immunoassays (FIAs), and competitive sandwich assays, and various thereof. options known in the art; as well as other techniques mentioned in this application.

The dissociation constant (KD) may be a true or apparent dissociation constant, as understood by one of skill in the art. Methods for determining the dissociation constant are known to the person skilled in the art, and include, for example, the methods mentioned in this application. In this regard, it should also be clear that it is not possible to measure dissociation constants greater than 10 ^-4 mol/liter or 10 ^-3 mol/liter (for example, 10 ^-2 mol/liter). Optionally, one skilled in the art will appreciate that the (true or apparent) dissociation constant can be calculated based on the (true or apparent) association constant (KA) in relation to [KD = 1/KA].

Affinity means the strength or stability of a molecular interaction. Affinity is usually determined by the CD, or dissociation constant, which is measured in mol/liter (or M). Affinity can also be expressed by the association constant, KA, which is equal to 1/KD and is measured in (mol/liter) ^-1 (or M ^-1 ). In the present specification, the stability of an interaction between two molecules (such as an amino acid sequence, an immunoglobulin sequence, an immunoglobulin single variable domain, a Nanotelo® or a polypeptide of the present invention, and its designated target) is primarily expressed in terms of the KD value of their interaction; one skilled in the art will appreciate that given the ratio KA=1/KD, the expression of molecular interaction strength in terms of KD value can also be used to calculate the corresponding KA value. The KD value characterizes the strength of the molecular interaction also in a thermodynamic sense, since it is related to the free energy (DG) of binding by the well-known relationship DG=RT.ln(KD) (equivalent to DG=-RT.ln(KA)), where R is equal to the gas constant , T is the absolute temperature, and ln is the natural logarithm.

KD for biological interactions, such as the binding of immunoglobulin sequences of the present invention with VWF, as defined in this application, which is significant (for example, specific), as a rule, is in the range from 10 ^-10 M (0.1 nm) to 10 ^-5 (10000 nM). The stronger the interaction, the lower the CD.

KD can also be expressed as the ratio of the dissociation rate constant of the complex, denoted k _off , to the association rate, denoted k _on (so KD =k _off /k _on and KA = k _on /k _off ). The dissociation rate k _off is expressed in units of s ^-1 (where "s" in the C system means seconds). The rate of association is expressed in units of M ^-1 s ^-1 .

With respect to the immunoglobulin sequences of the present invention, the association rate can vary from 10 ² M ^-1 s ^-1 to 10 ⁷ M ^-1 s ^-1 , reaching a diffusion-limited association rate constant for bimolecular interactions. The dissociation rate is related to the half-life of a certain molecular interaction by the ratio t1/2=ln(2)/k _off . The rate of dissociation of the immunoglobulin sequences of the present invention can vary from 10 ^-6 s ^-1 (almost irreversible complex with t1/2 of multiple days) to 1 s ^-1 (t1/2=0.69 s).

The affinity of the molecular interaction between two molecules can be measured by various known techniques, such as the well-known surface plasmon resonance biosensor technique (see, for example, Ober et al., Intern. Immunology, 13, 1551-1559, 2001), where one molecule is immobilized on a biosensor chip, and another molecule is passed over the immobilized molecule under flow conditions that provide measurements of k _on , k _off , and thus, CD (or KA) values. This can be done, for example, with the well-known Biacore tools.

One skilled in the art will also appreciate that the measured CD may correspond to the true CD if the measurement process in any way affects the objective binding affinity of the putative molecules, such as through anomalies associated with applying a single molecule to the biosensor. In addition, true CD can be measured if one molecule contains more than one recognition site for another molecule. In such a situation, the measured affinity can be affected by the avidity of the interaction of two molecules.

Another approach that can be used to assess affinity is the 2-step ELISA procedure (enzyme-linked immunosorbent assay) by Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This method provides a measure of the equilibrium of binding in the dissolved phase and eliminates possible anomalies associated with the adsorption of one of the molecules on a substrate such as plastic.

However, accurate measurement of CD can be quite laborious, and as a result, apparent CD values are often determined to estimate the binding strength of two molecules. It should be noted that since all measurements are performed in a sequential manner (for example, keeping the analysis conditions unchanged), the definition of apparent CD can be applied as an approximation to the true CD, and thus, in this document, CD and apparent CD are applied with equal significance or relevance.

Finally, it should be noted that in many cases the experienced scientist may find it convenient to determine the binding affinity for some reference molecule. For example, to assess the strength of the binding between molecules A and B, one can, for example, use a reference molecule C, which is known to bind to B, and which is suitably labeled with a fluorophore or chromophore group or other chemical moiety such as biotin to simplify detection in ELISA or FACS (Fluorescence Activated Cell Sorting) or other format (fluorophore for fluorescent detection, chromophore for light absorption detection, biotin for streptavidin-mediated ELISA detection). Typically, the control molecule C is kept at a fixed concentration, and the concentration of A varies for a certain concentration or amount of B. The result is an IC50 value corresponding to the concentration of A, in which the signal measured for C in the absence of A is divided by 2. With the condition, what KDref is known, the KD of the reference molecule, and the total concentration c _ref of the reference molecule, the apparent KD for the A-B interaction can be obtained from the following formula: KD =IC50/(1+c _ref / KDref). We note that if c _ref << KDref, KD ≈ IC50. Provided that IC50 measurements are made in a consistent manner (e.g., keeping c _ref fixed) for the binding agents being compared, the strength or stability of the molecular interaction can be estimated by IC50, and this measurement is considered to be equivalent to CD or apparent CD throughout this text.

5.3. Target antigen

The immunoglobulin single variable domains of the present invention bind and/or have affinity for VWF. In the context of the present invention, "VW" includes, but is not limited to, cynomolgus monkey, baboon, pig, guinea pig, mouse and/or human VW, and most preferably human VW, i. SEQ ID NO: 20 or GenBank entry: NP_000543.

5.4. Specific embodiments of immunoglobulin sequences

The present invention relates to immunoglobulin single variable domains as described or obtained by the methods disclosed in WO2004/015425 [P02-008], WO2004/062551 [P03-001], WO2006/074947 [P05-001], WO2006/122825 [P05 -003], WO2009/115614 [P08-013], or WO2011/067160, all on behalf of the author of this application.

The present invention also covers optimized variants of these amino acid sequences. Typically, an "optimized version" of an amino acid sequence according to the present invention is one that includes one or more useful substitutions, such as substitutions that include (i) a degree of "humanization"; (ii) chemical stability, and/or (iii) expression level; while activity (measured, for example, by activity assay as described in the experimental part of WO2006/122825) remains comparable (i.e., within 10% deviation) to wild type [P05-00312A02 (as defined in WO2006/ 122825), or comparable to variant 12A02H1 (SEQ ID NO: 19), also as defined in WO2006/122825. Preferably, compared to the wild-type sequence 12A02, the amino acid sequence of the present invention contains at least one substitution, and preferably at least two such substitutions, and preferably at least three humanizing substitutions, and preferably at least 10 such substitutions. humanizing substitutions.

In a particular aspect, the amino acid sequences of the present invention have a total of 1 to 15, preferably 2 to 14, such as 9 to 13, such as 10, 11, or 12 amino acid substitutions, compared to the wild-type sequence 12A02. As mentioned, these differences preferably include at least one, and preferably at least two, such as three, four, or five, or ten humanizing substitutions, and may optionally include one or more additional substitutions (such as any of the above, or any suitable combination of two or more additional substitutions (a)-(c) as mentioned in this application). Again, based on the present disclosure, and optionally after a limited degree of trial and error, one or more such suitable humanizing and/or complementary substitutions may be selected by one skilled in the art (as appropriate combination).

The present invention encompasses polypeptide sequences that closely resemble any of the specific examples provided herein or any of the specific examples defined by reference above. "Strongly similar" means at least 90% amino acid sequence identity, such as 95%, 97%, 98% or 99%. Strongly similar polypeptide sequences have the same function as the sequence from which they are derived, ie. they bind to VWF, in particular, bind to and inhibit the interaction between VWF and platelets.

In a particular embodiment, the invention relates to sequences with high similarity to any of SEQ ID NO: 1-19, in particular, SEQ ID NO: 1. However, for each sequence variant, it is necessary to evaluate the stability in the composition, as defined in this application, so that the present invention particularly relates to sequence variants with high similarity, stable in compositions, as defined in this application.

Methods for generating polypeptide sequences of the present invention are widely known and include, for example, recombinant expression or synthesis. The person skilled in the art is well aware of suitable expression technology, eg suitable recombinant vectors and host cells, eg bacterial or yeast host cells. One skilled in the art is also well aware of suitable purification techniques and protocols.

5.5. Compositions of the present invention

The present invention provides polypeptide compositions directed against VWF, for example, immunoglobulin single variable domains (IVDs) or polypeptides containing at least one immunoglobulin single variable domain, which are stable, and preferably suitable for pharmaceutical use, including the preparation of medicaments.

The composition of an agent that binds VWF, for example, IOVD, includes OVD, a compound serving as a cryoprotectant and/or lioprotector, and a buffer. The pH value of the composition, as a rule, is 5-7.5. In some embodiments, the composition is stored as a liquid. In other embodiments, the composition is formulated as a liquid and then dried, such as by lyophilization or spray drying, prior to storage. The dry composition (i.e., lyophilisate) can be used as a dry compound, for example, as an aerosol or powder, or reconstituted to the original or other concentration, for example, using water, a buffer, or other suitable liquid (diluent).

The VW binder purification process is designed to transfer the VW binder into a composition suitable for long term storage, eg as a frozen liquid and/or subsequently lyophilized (eg using a citrate/sucrose composition). The composition is lyophilized with a protein, such as a VWF binding agent, at a specific concentration. The lyophilized composition may then be reconstituted, if necessary, with a suitable solvent (eg water) to re-dissolve the components of the original composition to the desired concentration, typically the same or higher concentration than the concentration prior to lyophilization. The lyophilized composition can be reconstituted to obtain a composition having a concentration different from the original concentration (ie, before lyophilization), depending on the amount of solvent added to the lyophilisate, relative to the volume of liquid that was originally lyophilized. Suitable compositions can be identified by quantitative analysis of one or more VWF binding agent integrity parameters. The parameters determined are typically the percentage of high molecular weight species (HMW) or the percentage of low molecular weight species (LMW) detected by size exclusion HPLC (S-HPLC).

Accordingly, the present invention provides compositions of stable purity and at suitable concentrations as needed, for example, for pharmaceutical purposes. The compositions provide polypeptides, for example, immunoglobulin single variable domains or polypeptides containing at least one immunoglobulin single variable domain, as defined in this application, in stable form over a wide range of concentrations, and under a wide range of storage conditions, for example, temperatures, including stress conditions such as elevated temperatures (eg +25°C or higher), lyophilization, shaking, or other forms of physical stress.

The composition contains an aqueous carrier. The aqueous carrier is in particular a buffer.

However, the present invention also covers a product obtained by further processing of a liquid composition, such as a frozen, freeze-dried or spray-dried product. When reconstituted, these solid products can become liquid compositions, as described in this application (but not limited to). In its broadest sense, the term "composition" encompasses both liquid and solid compositions. However, solid compositions are meant to be obtained from liquid compositions (eg, by freezing, lyophilization or spray drying), and thus have different characteristics, which are determined by the features specified in this application for liquid compositions. The invention does not exclude reconstitution which results in a composition deviating from the original composition, for example prior to lyophilization or spray drying.

Compositions of the present invention include at least one VWF binding agent, in particular immunoglobulin single variable domains or polypeptides comprising at least one immunoglobulin single variable domain as defined herein. In particular embodiments, the composition contains one or more polypeptides selected from SEQ ID NO: 1-19, preferably SEQ ID NO: 1. The polypeptides can also be increased in half-life, for example, by including a peptide or domain that binds serum albumin that is capable of increasing the half-life of the construct (compared to the same construct without a serum albumin binding peptide or domain), and may be, in particular, serum albumin binding peptides as described in WO2008/068280 by the present applicant ( and in particular, WO2009/127691 and WO2011/095545, both from the present applicant), or an immunoglobulin single variable domain that binds serum albumin (such as a nanobody that binds serum albumin; for example, Alb-1 or a humanized version of Alb-1, such like Alb-8, for which reference can be made to, for example, WO06/122787). Alternative means of extending half-life, which are also covered by the present invention, include, for example, pegylation (PEG), as is well known in the art, including site-specific or random pegylation, preferably site-specific pegylation. PEG can be used with a molecular weight above 5000, for example from 10,000 to 200,000, preferably in the range of 20,000 and 100,000. In any aspect of increased half-life, it is understood that the activity of the polypeptide as defined herein is not impaired, e.g., at least 75%, 80%, 85%, 90%, or 95% of the activity of the same polypeptide is retained without extending the half-life. The activity may be associated, for example, with binding to the target antigen, and/or activity in the bioassay. One skilled in the art will also appreciate that the selected half-life enhancement technology is useful because it does not increase or even decrease immunogenicity.

5.5.1. Buffer

The composition of the present invention contains a buffer selected from at least a citrate or phosphate buffer, preferably a citrate buffer. In a particular embodiment, the citrate buffer is prepared using citric acid monohydrate and anhydrous trisodium citrate, for example 0.2154 g/l citric acid monohydrate and 5.5805 g/l anhydrous trisodium citrate. As determined by measuring melting points in a non-limiting example, these buffers increase the stability of VW binders compared to other buffers tested.

The composition in accordance with the present invention contains a citrate buffer in a concentration in the range of 5-200 mm, for example, 5; 7.5; 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mM, preferably 5-100 mM , more preferably 7.5-80 mM, even more preferably 10-50, such as 10, 15, 20, 25 or 30 mM, and most preferably 20 mM, where each value is meant to optionally cover the range of ±5 mM. The composition in accordance with the present invention may contain a phosphate buffer in a concentration in the range of 5-200 mm, for example, 5; 7.5; 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mM, preferably 5-80 mM , more preferably 7.5-60 mM, even more preferably 10-40, eg 10, 15, 20, 25 or 30 mM, and most preferably 10 mM. where each value is intended to optionally span the range of ±5 mM. It must be understood that lower buffer concentrations have an impact on the final osmolality, and thus additional solutes that may need to be added.

The pH value of the composition of the present invention is in the range from 5.0 to 7.5, where each value is meant to cover the range of ±0.2. Specific examples of preferred pH values for compositions of the present invention may be selected from a non-limiting list including pH 5.0; 5.5; 5.8; 6.0; 6.2; 6.5; 6.7; 7.0; 7.1; 7.2 or 7.5, preferably 6.0 to 7.0, more preferably 6.1; 6.2; 6.3; 6.4; 6.5; 6.6; 6.7; 6.8 or 6.9, such as 6.5, where each value is intended to optionally span the range of ±0.2.

Surprisingly, citrate and phosphate buffers have overlapping pH ranges, eg with histidine and Tris-HCl buffers, also contributing to stability.

The most preferred pH value depends on the buffer contained in the composition. Therefore, the invention relates in particular to a composition containing a phosphate buffer, which preferably has a pH in the range from 6.5 to 7.5, preferably 6.9; 7.0; 7.1, for example, 7.1.

It was shown that the composition, including citrate buffer, was particularly suitable for storage and use. However, in contrast to conventional experience, liquid compositions containing citrate buffer were most stable at a pH of about 6.0, while lyophilized compositions containing citrate buffer were most stable at a pH of about 6.5. Therefore, the present invention relates to a composition containing a citrate buffer, preferably having a pH of 6.0 to 7.0, more preferably 6.1; 6.2; 6.3; 6.4; 6.5; 6.6; 6.7; 6.8 or 6.9, such as 6.5, where each value is intended to optionally span the range of ±0.2.

5.5.2. Concentration

Compositions of the present invention preferably include VWF binding agents as described herein, in particular immunoglobulin single variable domains or polypeptides comprising at least one immunoglobulin single variable domain at a concentration suitable for clinical purposes, which includes concentrations used in stock solutions for dilution before administration to the patient. In addition to improved stabilization, the compositions of the present invention provide higher concentrations of VWF binding agents, eg, OVD or polypeptides. In particular, the compositions of the present invention remain physically stable, i. no turbidity and/or formation of small particles, as confirmed by visual analysis, microscopy, E-HPLC and DLS (dynamic light scattering). Storage at elevated temperatures for extended periods of time and repeated freeze-thaw cycles do not clearly affect the physical stability of the VW binders in these compositions.

Typical concentrations of the active agent, for example, polypeptide binding agents of the present invention, in the compositions of the present invention include non-limiting examples of concentrations in the range from 0.1 to 80 mg/ml, preferably 1-70 mg/ml, 5-60 mg/ ml, 7.5-50 mg/ml, or 10-40 mg/ml, such as 5; 7.5; ten; 12.5; fifteen; 17.5; 20, 25, 30, 35, 40, 45, 50, or 60 mg/mL, preferably 12.5 mg/mL or 10 mg/mL, where each value is intended to optionally cover a range of ±20% (e.g., a value of 10 optionally covers range 8 to 12 mg/mL).

5.5.3. Excipients

Compositions in accordance with the present invention may also optionally include one or more excipients. The term "adjuvant" as used in this application means an inert substance, which is usually used as a diluent, solvent, preservative, lyoprotectant, binder or stabilizing agent for compounds, which provides a favorable physical property of the composition. The person skilled in the art is aware of excipients suitable for pharmaceutical purposes, which can perform specific functions in the composition, such as lyoprotection, stabilization, preservation, etc. Commonly used stabilizers and preservatives are well known to the person skilled in the art (see, for example, WO 2010/077422). Pharmaceutically acceptable carriers that can be used in these compositions include ion exchangers, alumina, aluminum stearate, lecithin, whey proteins such as human serum albumin; buffer substances such as phosphates; glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose base, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block copolymers, polyethylene glycol, and lanolin. In preferred embodiments, the excipient may be one or more selected from the list consisting of NaCl, trehalose, sucrose, mannitol, or glycine.

In evaluating the present invention, a person skilled in the art can easily determine the appropriate concentrations of excipients to be added to the compositions. In exemplary embodiments, NaCl has a concentration in the range of 10-500 mM, such as 25, 30, 40, 50, 60, 70, 100, 150, 250, or 500 mM, preferably 50-150 mM, such as 75 or 140 mM, where each value is meant to optionally cover the range of ±5 mM; and/or the mannitol has a concentration of 1-10%, preferably 2-4%, such as 2, 3 or 4% (m/m), where each value is meant to optionally cover the range of ±0.5%; and/or sucrose has a concentration of 1-15%, preferably 2-12% or 4-10%, for example 4, 5, 6, 7, 8 or 9% (m/m) and most preferably 7%, where each value implied as optionally covering the range of ±0.5%; and/or glycine has a concentration in the range of 10-500 mM such as 25, 30, 40, 50, 60, 70, 75, 100, 150, 250 or 500 mM, preferably 50-400 mM, 75-300 mM, 100 -250 mM, such as 140 or 200 mM, where each value is intended to optionally cover the range of ±5 mM; and/or trehalose has a concentration in the range of 10-500 mM, such as 25, 30, 40, 50, 60, 70, 75, 100, 150, 250 or 500 mM, preferably 100-300 mM, 150-280 mM, e.g. , 160 mM or 260 mM, where each value is intended to optionally cover the range of ±5 mM.

In a preferred embodiment, the compositions in accordance with any aspect of the present invention are isotonic with respect to human blood. Isotonic solutions provide the same osmotic pressure as blood plasma and thus can be administered intravenously to a subject without changing the osmotic pressure of the subject's blood plasma. Tonicity may be expressed as osmolality, which may be a theoretical osmolality, or preferably an experimentally determined osmolality. Typically, the osmolality is in the range of 290±60 mOsm/kg, preferably 290±20 mOsm/kg.

Thus, in selecting excipients (if present), one skilled in the art will take into account the buffer concentration and the concentrations of one or more excipients, and preferably achieve a composition with osmolality in the ranges indicated above. The person skilled in the art is familiar with calculations to obtain osmolality (see, for example, WO2010/077422). If desired, one of skill in the art may also further include a compound to control the osmolality of the composition. Exemplary compounds include, but are not limited to, the aforementioned excipients and/or one or more of sorbitol, methionine, dextrose, inositol, arginine, or arginine hydrochloride.

It was shown that the composition containing sucrose was particularly suitable for maintaining physical stability, for example, during storage and freeze-thaw of polypeptides. Accordingly, the present invention relates to compositions containing about 5-9%, more preferably 6-8%, and even more preferably 7% sucrose, where each value is meant to optionally cover the range of ±0.5%.

The compositions of the present invention may also contain compounds that are particularly useful for protecting the polypeptide of the present invention during lyophilization. Such compounds are also known as lyoprotectants, and are well known to the person skilled in the art. Specific examples include sugars such as sucrose, sorbitol or trehalose; amino acids such as glutamate, in particular monosodium glutamate or histidine; betaine, magnesium sulfate, sugar alcohols, propylene glycol, polyethylene glycols, and combinations thereof, but are not limited to. In evaluating the present invention, the required amount of such a compound to be added can be easily determined by one skilled in the art, taking into account the stability of the composition in liquid form and during lyophilization. Compositions particularly suitable for lyophilization may additionally contain bulking agents. Suitable agents are well known to the person skilled in the art. It has been shown that the composition containing sucrose was not only particularly suitable for maintaining physical stability, for example during storage and freeze-thaw of VW binders, but also as a lyoprotectant.

5.5.4. Detergent

In another embodiment of the present invention, the composition in accordance with any aspect of the present invention may further contain a detergent or surfactant. Suitable detergents or surfactants for use in the present invention include, but are not limited to, polyoxyethylene sorbitan fatty acid esters such as polysorbate -20, -40, -60, -65, -80, or -85. Common brand names for polysorbates include Alkest, Canarcel, and Tween. The person skilled in the art is aware of other non-limiting examples of detergents, such as those listed, for example, in WO2010/077422. In a preferred embodiment, the detergent is a non-ionic detergent. In particular, the detergent is polysorbate-80, also referred to hereinafter as Tween-80. A person skilled in the art can easily determine the appropriate concentration of detergent for the composition of the present invention. Typically, the concentration is as low as possible while retaining the beneficial effects of detergents, such as stabilizing effects under shear stress conditions, such as agitation, which reduce aggregation of the composition's VW binders. In exemplary, non-limiting embodiments, the detergent concentration may be in the range of 0.001 to 0.5%, such as 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.01%, 0.015%, 0.02% , 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%, preferably at a concentration of 0.01 to 0.05%, more preferably 0.01 to 0.02%. for example, 0.01% (v/v).

5,5,5. Combinations

Various embodiments, as described above in sections 5.5.1-5.5.4, can be combined in the compositions of the present invention without limitation. For example, ranges of values using combinations of any of the above values, such as upper and/or lower limits, are intended to be included. However, preferred non-limiting examples of compositions include compositions wherein the buffer is citrate buffer at a pH of about 6.5, preferably at a concentration of 20 mM, and the composition further comprises sucrose, preferably at a concentration of about 7% (w/v), and optionally further includes a non-ionic detergent such as Tween-80, preferably at a concentration of 0.01% (v/v).

5.6. Additional processing

As described, any of the above compositions can be further processed, for example, by lyophilization, spray drying or freezing, for example, bulk freezing. The resulting processed product has characteristics derived from the liquid feed composition as defined above. When necessary, additional post-treatment agents may be included, such as lyoprotectants, etc., for example.

5.6.1. Freezing

In some instances, compositions containing VWF binders are frozen for storage. Accordingly, it is necessary that the composition be relatively stable under conditions such as freeze-thaw (30) cycles. One way to determine the suitability of a composition is to treat a sample of the composition with at least two, for example, three, four, five, eight, ten or more cycles of freeze (for example, at -20°C or -70°C) and thaw (for example, rapid thawing in a water bath at 25°C or slow thawing at a temperature of +2°C to +8°C), with the determination of the mass yield of the initial product and the presence and/or number of LMW species and/or HMW species accumulating after 3D cycles , and comparing them with the number of LMW species or HMW species present in the sample prior to the 3D procedure, for example, E-HPLC. An increase in LMW or HMW species indicates a decrease in stability.

5.6.2. Lyophilization

The compositions can be stored after lyophilization. Thus, testing the stability of the polypeptide component of the composition after lyophilization is useful to determine the suitability of the composition. A suitable method for this is described above for freezing, except that the sample composition was lyophilized instead of frozen, reconstituted to its original volume, and the presence of LMW species and/or HMW species was determined. The lyophilized sample of the composition is compared with a corresponding sample of the composition that has not been lyophilized. An increase in LMW or HMW species in the freeze-dried sample, compared to the corresponding sample, indicates a decrease in stability in the freeze-dried sample. Typically, the lyophilization protocol includes loading the sample into a lyophilizer or freezer, pre-cooling period, freezing, starting a vacuum, going to the primary drying temperature, primary drying, going to the secondary drying temperature, secondary drying, plugging the sample. Although the lyophilization method is well known in the art, various factors determine the lyophilization characteristics of a sample, including the glass transition temperature (Tg') and compression temperature (Tc). Additional parameters that may be selected for the lyophilization protocol include vacuum (eg, in microns) and condenser temperature.

Suitable transition rates for temperatures are from about 0.1°C/min to 2°C/min, for example, from 0.1°C/min to 1.0°C/min, from 0.1°C/min to 0.5°C/min, 0.2°C/min to 0.5°C/min, 0.1°C/min, 0.2°C/min, 0.3°C/min, 0 .4°C/min, 0.5°C/min, 0.6°C/min, 0.7°C/min, 0.8°C/min, 0.9°C/min, and 1, 0°C/min Suitable shelf temperatures during freezing for a lyophilization cycle are typically about -55°C to -5°C, -25°C to -5°C, -20°C to -5°C, - 15°C to -5°C, -10°C to -5°C, -10°C, -11°C, -12°C, -13°C, -14°C, -15°C, -16°C, -17°C, -18°C, -19°C, -20°C, -21°C, -22°C, -23°C, -24°C, or -25°C . Shelf temperatures may be different for primary drying and secondary drying, for example primary drying may be carried out at a lower temperature than secondary drying. In a non-limiting example, primary drying can be carried out at 0°C, or alternatively, at +5°C, and secondary drying at +25°C. In some cases, the hardening protocol is applied during freezing and before running the vacuum. In some cases, the hardening time must be chosen, and the temperature, as a rule, exceeds the glass transition temperature of the composition. Typically, the hardening time is about 2 to 20 hours, about 3 to 19 hours, about 2 to 10 hours, about 3 to 5 hours, about 3 to 4 hours, about 2 hours, about 3 hours, about 5 hours, about 8 hours, about 10 hours, about 12 hours, about 15 hours, or about 19 hours. The tempering temperature is typically about -35°C to -5°C, for example, about -25°C to -8°C, about -20°C to -10°C, about -25° C, about -20°C, about -15°C, about 0°C, or about -5°C. In some cases, the tempering temperature is generally -35°C to +5°C, such as -25°C to -8°C, -20°C to -10°C, -25°C, -20°C, -15°C, 0°C, +5°C.

The stability of the compositions described in this application can be determined using various lyophilization parameters, including the temperature of the shelves during primary drying from -25°C to +30°C, and the duration of the secondary drying for 2 hours to 33 hours from 0°C to +30°C. The secondary drying temperature should be as high as possible without causing degradation of the active pharmaceutical ingredient.

The excipient for use in the composition of the present invention should preferably satisfy one or more of the following parameters: be pharmacologically inert; be compatible with processing requirements; well tolerated by the patient; not damage the active material; provide a soluble, absorbable product; provide a shelf-stable product; and provide a commercially viable product.

In one embodiment, the composition of the present invention is prepared by lyophilization, for example, as shown in Figure 1 or Table 14.

It has been shown that lyophilization of citrate/sucrose formulations dramatically improves the stability of VW binders. In particular, citrate/sucrose formulations essentially prevent chemical modifications that occur in liquid form, except for the formation of small amounts of pyroglutamate. Surprisingly, lowering the concentration of citrate, and at the same time increasing the concentration of sucrose, increased the chemical stability, for example, reduced the formation of pyroglutamate. The VWF binding agent was shown to be resistant after lyophilization to temperature extremes. Indeed, the stability profile was identical for material prepared using multiple freeze-thaw cycles.

Typically, a lyophilization cycle can take from 10 hours to 100 hours, e.g., 20 hours to 80 hours, 30 hours to 70 hours, 40 hours to 60 hours, 45 hours to 50 hours, 50 hours to 66 hours .

In a non-limiting example, a composition of 20 mM citrate, 7% sucrose, 0.01% Tween-80, pH 6.5, with a protein concentration of 12.5 mg/ml VW binder was prepared in series and lyophilized.

Non-limiting examples of the temperature range for storing the composition of the present invention are from about -20°C to +50°C, for example, from about -15°C to +40°C, from about -15°C to +30°C, approx. -15°C to +20°C, approx. +5°C to +25°C, approx. +5°C to +20°C, approx. +5°C to +15°C, approx. +2°C to +12°C, approx. +2°C to +10°C, approx. +2°C to +8°C, approx. +2°C to +6°C, or approx. +2 °C, +3°C, +4°C, +5°C, +6°C, +7°C, +8°C, +10°C, +15°C, +25°C, +30 °C or +40°C. Not limited to storage temperatures, in some instances, samples are stable with temperature changes that may temporarily occur under the storage and transport conditions that are expected for such compositions.

It has been found that when working with the compositions of the present invention, as defined in this application, it is possible to obtain a final dry powder having a particle size suitable for convenient storage and rapid dissolution of the active material. The dry composition in accordance with the present invention includes particles that remain stable and uniform during processing, finishing, storage and distribution. The composition is storage stable and free flowing, presents no problems when poured into the final container, and is easy to administer to the patient.

5.6.3. Spray drying

In some cases, the composition is spray dried and then stored. Spray drying is carried out using methods known in the art and can be modified to use liquid or freeze spray drying (for example, using methods such as from Niro Inc. (Madison, Wisconsin), Upperton Particle Technologies ( Nottingham, England), or from US Patent Nos. 2003/0072718 and 2003/0082276), or from Buchi (Brinkman Instruments Inc., Westbury, NY).

5.6.4. Solvent

Lyophilized compositions as disclosed herein may be reconstituted, if necessary, by mixing the lyophilized form with a stable solvent to re-dissolve the components of the original composition to the desired concentration. The term "solvent", as used in this application, refers to a pharmacologically suitable (safe and non-toxic for human use) solvent to change or achieve a suitable concentration, as described in this application. Exemplary solvents include, but are not limited to, sterile water (eg, water for injection, Milli-Q water), saline, dextrose solution, Ringer's solution, and aqueous buffer solutions.

5.7. Pharmaceutical compositions

The compositions of the present invention are preferably suitable for use in methods of treating an animal or human body. Thus, the present invention relates to pharmaceutical or diagnostic compositions containing the composition of the polypeptide in accordance with any aspect of the invention, or obtained by any method of the present invention.

The compositions of the present invention are preferably pharmaceutical compositions. In particular, the compositions are suitable for parenteral administration in humans, eg subcutaneous, intravenous, intramuscular, intradermal or intraperitoneal administration, preferably intravenous or subcutaneous administration. The use encompasses any means of administering the liquid composition, in particular injection. Other forms of systemic administration, eg via implantable devices, micro-infusion pumps (optionally implantable), and/or (implantable) sustained release formulations, eg depot formulations, gels, biodegradable polymer formulations, are also within the scope of the present invention. The pharmacological compositions are sterile and stable during manufacture and storage, since derivatives/degradants of VWF binding agents are undesirable in clinical use. The composition must also be of high purity, eg free of bacterial products such as LPS. The compositions may be sterilized by any suitable means, for example sterilizing filtration, irradiation, and combinations thereof. Preferably, the pharmaceutical compositions are adapted for parenteral (especially intravenous, intraarterial or transdermal) administration. Intravenous administration is considered especially important. Preferably, the VWF binding agent is in parenteral form, most preferably intravenously and subcutaneously.

To ensure suitability as a pharmaceutical composition, the composition of the present invention typically contains the polypeptide of the present invention (ie active agent) in a suitable ratio to volume. For example, for subcutaneous injections, the concentration of the active agent may be higher to provide the required pharmaceutical dosage for small volume administration, compared to an intravenous formulation. However, in some embodiments, the implementation of the concentration of the active agent is identical for subcutaneous or intravenous administration, and may be in the approximate ranges as defined in this application.

In some embodiments, the compositions of the present invention may contain additional agents, for example, additional active agents, excipients, stabilizers, preservatives such as antimicrobial agents, etc.

The compositions of the present invention are preferably in the dosage administered to the patient in need thereof. However, the specific regimen and dosage may be selected by the attending physician taking into account the specific characteristics of the patient, especially age, body weight, lifestyle, activity level, and general condition, as appropriate. In particular, ALX-0081 is administered intravenously or subcutaneously at 24 hour intervals. Even more preferably, ALX-0081 is administered intravenously or subcutaneously at 24 hour intervals based on aggregation activity, e.g. analysis of platelet agglutination induced by ristocetin cofactor (Howard MA, Firkin BG. “Ristocetin – a new tool in the investigation of platelet aggregation”. Thrombosis et Diathesis Haemorrhagica 1971; 26: 362-9 (“Ristocetin – a new tool in the investigation of platelet aggregation” )). For example, the next dose is not applied if the aggregation activity remains below 10% as measured by RIPA or below 20% as measured by RICO for the next 6 hours (clinically significant inhibition).

However, in general, the dosage of VWF binding agents may depend on various factors such as the potency and duration of action of the active ingredient, the species of warm-blooded animal, and/or the sex, age, body weight, and individual condition of the warm-blooded animal.

Typically, the dosage is such that a single dose of the VWF binding agent, for example, is set based on the results of a dose escalation study for subchronic toxicity analysis in cynomolgus monkeys. Based on this set of preclinical studies, the initial and subsequent escalating dose for the VWF binding agent can be determined. For example, the dose may be 0.5-50 mg, especially 1-30 mg, and it is used in a warm-blooded animal with a body weight of about 75 (±30) kg (but may also differ from this norm). If necessary, this dose may consist of several, optionally equal, partial doses ("mg" means mg of drug per mammal, including the human being treated).

The dose mentioned above, administered as a single dose (which is one embodiment) or as multiple partial doses, may be repeated as mentioned above, for example, every six hours, every 12 hours, or every day. In other words, the pharmaceutical compositions can be used in regimens ranging from continuous therapy every 6 hours to therapy with longer dosing intervals.

Preferably, VWF binding agents are used at doses that are in the same order of magnitude as that used in adjuvant therapy for patients requiring PCI, as contemplated herein for ALX-0081. For example, for preferred 12A02H1-containing VWF binders, e.g., ALX-0081 and functional variants thereof, doses of VWB binders in the range of about 0.5 to 40 mg, preferably about 1 to 35 mg, may be used, or about 2 to 30 mg, even more preferably about 3 to 25 mg, or about 4 to 20 mg, or about 5 to 17.5 mg, or even about 6 to 16 mg, or about 7.5 to 15 mg, or even about 10 to 14 mg, more preferably about 10, about 12.5 or about 13.8 mg, for the acute treatment of human patients.

Compositions in a single dose dosage form preferably contain about 0.5 to 40 mg, preferably about 1 to 35 mg, or about 2 to 30 mg, even more preferably about 3 to 25 mg, or about 4 to 20 mg, or about 5 to 17.5 mg, or even about 6 to 16 mg, or about 7.5 to 15 mg, or even about 10 to 14 mg, more preferably about 10, about 12.5, or about 13 .8 mg, and compositions not in a single dose dosage form preferably contain from about 0.5 to 40 mg, preferably from about 1 to 35 mg, or from about 2 to 30 mg, even more preferably from about 3 to 25 mg , or about 4 to 20 mg, or about 5 to 17.5 mg, or even about 6 to 16 mg, or 7.5 to 15 mg, or even about 10 to 14 mg, more preferably about 10 , about 12.5 or about 13.8 mg of the active ingredient.

Pharmaceutical preparations for parenteral use are, for example, in dosage forms such as ampoules. They are prepared in a manner known per se, for example by conventional mixing, dissolving or lyophilization methods.

Parenteral compositions are especially injectable liquids administered by various routes, such as at the site of the ICA, intra-arterially, intramuscularly, intraperitoneally, intranasally, intradermally, subcutaneously, or preferably intravenously. Such liquids are preferably isotonic aqueous solutions or suspensions, which can be prepared before use, for example, from lyophilized preparations or a concentrate, which contain the active ingredient, alone or together with a pharmaceutically acceptable carrier. Pharmaceutical preparations may be sterilized and/or contain additives such as preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, osmotic salts and/or buffers.

Suitable compositions for transdermal use include an effective amount of the active ingredient with a carrier. Preferred carriers include absorbable pharmacologically acceptable solvents to allow passage through the skin of the carrier. Typically, transdermal devices are in the form of a band containing a support member, a reservoir containing a compound, optionally with carriers, optionally a rate controlling barrier, for delivering the active ingredient to the host's skin at a controlled and predetermined rate over an extended period of time, and means for attaching the device to the skin.

The following table lists some non-limiting examples of compositions of the present invention based on citrate and phosphate buffer. In all compositions, the osmolality can be adjusted up to 290±60 mOsm/kg by adding a suitable excipient, if necessary. The compositions may contain one or more of any of the polypeptides of the present invention, for example, SEQ ID NO: 1-19, in particular SEQ ID NO: 1.

Buffer Buffer concentration (mM) pH Buffer Buffer concentration (mM) pH citrate ten 6.0 Phosphate ten 6.5 citrate ten 6.5 Phosphate ten 7.0 citrate ten 7.0 Phosphate ten 7.5 citrate twenty 6.0 Phosphate twenty 6.5 citrate twenty 6.5 Phosphate twenty 7.0 citrate twenty 7.0 Phosphate twenty 7.5 citrate thirty 6.0 Phosphate thirty 6.5 citrate thirty 6.5 Phosphate thirty 7.0 citrate thirty 7.0 Phosphate thirty 7.5 citrate 40 6.0 Phosphate 40 6.5 citrate 40 6.5 Phosphate 40 7.0 citrate 40 7.0 Phosphate 40 7.5 citrate fifty 6.0 Phosphate fifty 6.5 citrate fifty 6.5 Phosphate fifty 7.0 citrate fifty 7.0 Phosphate fifty 7.5

Buffer concentrations in this table are to be understood as optionally spanning ±5 mM. The pH value is to be understood as optionally encompassing ±0.2. Each of the above buffers can be combined with one or more excipients selected from, for example, NaCl at a concentration of, for example, 25, 30, 40, 50, 60, 70, 100, 150, 250 or 500 mM; mannitol at a concentration of, for example, 2, 3 or 4% (w/v); glycine at a concentration of, for example, 25, 30, 40, 50, 60, 70, 100, 150, 250 or 500 mM; trehalose at a concentration of, for example, 25, 30, 40, 50, 60, 70, 100, 150, 250 or 500 mM, and sucrose at a concentration of, for example, 4, 5, 6, 7, 8 or 9% (w/o ), and / or a surfactant, for example, Tween-80 at a concentration of 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5% (v/v).

5.8. Effects achieved by the present invention

The invention provides stable compositions of VWF binding agents, for example, immunoglobulin single variable domains, as described in this application, for example, SEQ ID NO: 1-19, in particular, SEQ ID NO: 1. "Stable", as a rule, means that immunoglobulin single variable domains do not experience significant physical or chemical changes when stored for long periods of time, for example, from 1 to 36 months, even when exposed to one or more chemical or physical stresses, such as elevated temperatures (equal to or greater than +25° C), or physical stress such as shaking or stirring. In particular, "stable" means that when stored for long periods of time (as defined) under conditions (as defined), there is only limited formation (as defined) of one or more degradation products, e.g., low molecular weight (LMW) derivatives (fragments) of polypeptides of the present invention; and/or chemical derivatives or modifications such as, for example, pyroglutamate variants; and/or high molecular weight (HMW) derivatives (oligomers or polymers) formed, for example, by aggregation.

Those skilled in the art are well aware of techniques for assessing the size of proteins, such as size exclusion chromatography - HPLC, or assessing the formation of chemical derivatives, such as reverse phase HPLC. One of skill in the art will also be familiar with commonly used apparatus and software for performing such analyses. For example, one of ordinary skill in the art will be familiar with commonly used software for analyzing chromatograms, for example by peak area ratio. Examples include an Agilent 1200 HPLC system equipped with ChemStation software (Agilent Technologies, Palo Alto, USA, Rev B) or a Dionex Ultimate 3000 HPLC system equipped with Chromeleon software (Dionex Corporation, Sunnyvale, CA, USA, V6.8), but not limited to them.

General techniques that can be used to evaluate the stability of a protein, for example, an immunoglobulin single variable domain, include static light scattering, tangential flow filtering, Fourier transform infrared spectroscopy, circular dichroism, urea-induced protein unfolding, tryptophan intrinsic fluorescence, and/or protein binding to 1-aniline-8-naphthalenesulfonic acid. In addition, the composition of the present invention shows little or no loss of activity/biological activity during storage and/or under the influence of one or more stresses, as defined in this application. Biological activity and/or activity can be determined, for example, as described in WO2006/122825.

5.8.1. thermal stability

Compositions of the present invention are characterized by providing high thermal stability of VWF binding agents, for example immunoglobulin single variable domains as defined herein. Thermal stability can be assessed, for example, by determining the melting point (Tm). Suitable techniques for determining the melting point are known, and include, for example, thermal shift analysis (TSA), for example, as described in this application. In particular, the compositions of the present invention result in an increase in the Tp of immunoglobulin single variable domains, as determined by the TSA, compared to other compositions. This effect is shown in Table 1 from the section on experiments.

As seen in the experimental section, high thermal stability, such as high Tm, can be taken as an indication of storage stability.

In accordance with the present invention, the compositions of the invention have a positive effect on Tp over a wide range of pH values, for example, from 6.0 to 7.0 for citrate buffer, and from 6.5 to 7.5 for phosphate buffer. The most preferable effect on Tp can be observed for citrate buffer at pH 6-7, and in particular for pH 6.5 ± 0.2, and for phosphate buffer for pH from 6.5 to 7.5, in particular pH 7.1±0.2.

The addition of excipients may have an additional negative or positive effect on Tp (table 1). For example, trehalose can increase Tp (in the context of a specific buffer), for example, from 150 mM to 300 mM. Further, mannitol or sucrose have a clear positive effect on Tp. These excipients may find use in particular embodiments of the present invention, for example in compositions where a bulking agent or lyoprotectants are preferred. These exemplary embodiments do not exclude the use of other known lyoprotectants or bulking agents, alone or in combination with mannitol or sucrose.

As seen in the experimental section of this specification, Tp, as determined by TSA, is a valuable indicator of the stability of VWF binding agents, eg, the immunoglobulin single variable domains of the present invention.

5.8.2. Stability against mechanical stress

The compositions of the present invention are highly resistant to mechanical stress such as agitation, agitation or shear stress. A possible assay for evaluating resistance to mechanical stress is to monitor the 500 nm scatter signal in a spectrofluorimeter, or by UV spectrophotometry, for example at 340 nm. An increase in scattering or UV absorption reflects the formation of aggregates. When aggregates (HMW) are formed, the increase over time forms a linear curve for which the tangent of the slope (scattering intensity/absorption time, units/sec) can be determined. Preferably, the compositions of the present invention are characterized by a slope angle of less than 0.0006, for example, less than 0.0005, for example, from 0 to 0.0004 (see figures 4A and 4B).

Compositions containing citrate buffer are particularly preferred and have a positive effect on the yield of proteins, for example after mixing, as defined above. For example, the mass yield is at least 90%, 95%, 98%, or 100%. The protein yield is determined in comparison to the total protein content before the sample is stressed, for example by mixing. Compositions containing phosphate buffers provide a yield of at least 75%, 80%, 85% or more after mixing, as defined above.

At an approximate, non-limiting concentration of 5 mg/mL, the compositions of the present invention form only reversible aggregates in response to agitation in the absence of Tween. Thus, the compositions of the present invention prevent the formation of irreversible aggregates under mechanical stress. Accordingly, in another embodiment of the invention, the compositions of the present invention may contain a non-ionic detergent as defined above, for example, Tween-80, for example, at a concentration as defined above, for example, from 0.01% to 0.02% (o/ about). The addition of a detergent may further improve the physical stability of the composition. For example, at a non-limiting exemplary concentration of 5 mg/ml, addition of detergent can prevent the formation of aggregates (reversible and irreversible) as determined, for example, by monitoring the scattering signal at 500 nm in a spectrofluorimeter, or by UV spectrophotometry (340 nm) (Figures 4A and 4B).

The physical stability of the compositions of the present invention can also be demonstrated by e-HPLC. Various non-limiting compositions of immunoglobulin single variable domains of the present invention can withstand mechanical stress, such as agitation stress, without the formation of oligomers (HMW) or degradation products (HMW). The compositions of the present invention remain stable without degradation or oligomerization, for example, after 1.5 hours of mixing, according to the results of E-HPLC analysis.

No oligomerization or degradation (eg, by RP-HPLC (degradation only) or E-HPLC profiling) was detected in any of the compositions. Thus, in accordance with a preferred embodiment of the present invention, the compositions contain a citrate buffer, and show a yield of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even about 100%, for example, under conditions as described above, where the yield is determined, for example, by RP-HPLC or E-HPLC, compared to an unstressed sample. Preferably, the excipient in the context of the citrate buffer may be sucrose and the yield as defined above is at least 80%, 85%, 90%, 95%, 98%, or even about 100%.

5.8.3. Analysis of the stability of liquid compositions

5.8.3.1. Storage stability

Liquid compositions of the present invention provide good storage stability, for example at -70°C, -20°C, +5°C, +25°C or +40°C, for example, within 1-36 months, such time as 1; 1.5; 3, 6, 9, 12, 18, 24, 30 or 36 months. Most preferred results can be obtained with citrate buffer formulations as indicated in the examples in Table 5.

The person skilled in the art is aware that storage at +25°C, and even more so at +40°C, presents stressful storage conditions. Such conditions are expected to enhance and hasten the onset of any signs of instability, such as chemical or physical instability. Thus, a relatively short storage period, eg at +25°C or +40°C, provides a good stability index for long-term storage under milder conditions (eg, at +5°C or frozen).

5.8.3.2. Storage stability in terms of protein yield

For example, compositions of the present invention provide a protein yield of at least 95%, such as at least 96%, 97%, 98%, 99%, or even about 100%, after storage at -70°C to +40°C. The yield of proteins can be determined by any known means of quantitative analysis of proteins, for example, by RP-HPLC or E-HPLC, as shown in table 5, compared with a control image stored at -70°C. These results can be observed, for example, after storage at the specified temperature for 1 month, 1.5 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, or even 36 months.

5.8.3.3. Storage stability in terms of chemical derivatives/degradation products

Further, the compositions of the present invention minimize the formation of chemical derivatives, such as pyroglutamate variants, to less than 5.0% as measured by peak size in, for example, RP-HPLC (see Table 5). In this type of analysis, the area of a given peak is compared with the total area of the chromatogram, and the relative area for each peak is determined. Suitable analysis tools are known to one skilled in the art, such as suitable software for analyzing chromatograms (specific non-limiting examples include an Agilent 1200 HPLC system equipped with ChemStation software (Agilent Technologies, Palo Alto, USA, Rev B) or a Dionex HPLC system Ultimate 3000 equipped with Chromeleon software (Dionex Corporation, Sunnyvale, CA, USA V6.8) Thus preferably the pyroglutamate variants have a peak area of less than 5%, preferably less than 4.6%, e.g. 4.5%, 4.3%, 4.2%, 4.0%, or even less than 3.8% by RP-HPLC when stored at -70°C to +40°C, e.g. +40°C, eg after storage for the time defined above, eg 1 month.

The compositions of the present invention also minimize oxidation, such as the formation of oxidized products (as determined by e.g. RP-HPLC) over a storage period as defined above, e.g. 1 month at -70°C to +40°C. (see table 5). Thus, the compositions of the present invention lead to the formation of oxidative variants with a peak area of less than 3%, preferably less than 2.7%, preferably less than 2.5%, for example, less than 2.3%, 2.2%, for example, 2, 0%, or even less than 1.7% or 1.5%, when stored at temperatures from 70°C to +40°C, for example +40°C, for example, after storage for a time as defined above, for example , 1 month (according to the results, for example, RP-HPLC).

5.8.3.4. Storage stability in terms of oligomerization

The compositions of the present invention also provide storage stability such that no apparent soluble oligomeric material is formed (as measured by e.g. E-HPLC) when stored at temperatures between -70°C and +40°C, at storage times as defined above. eg within 1 month; or less than 1%, preferably less than 0.5%, e.g. 0.3% soluble oligomeric material is formed (as determined by e.g. E-HPLC) at storage temperatures from -70°C to +40°C, e.g. +40 °C, for storage times as defined above, eg 1 month.

The present invention also has the effect of providing an aggregation index, as determined by absorbance values [(100×A340)/(A280-A340)], which remains below 0.15, preferably below 0.1 after storage at 70°C to +40°C when stored for the time as defined above, eg 1 month.

5.8.3.5. Storage stability as reflected by the yield of the main product

The compositions of the present invention have an effect on the peak area of the main product, as determined by e.g. RP-HPLC (see Table 5), of about 90% after storage from -70°C to +40°C at storage times as above , for example, 1 month; or on the peak area of the main product, according to the results, for example, RP-HPLC (see table 5), constituting at least about 85% or more, such as 86%, 87% or 88%. More preferably, the main peak is 90%, 92% or 95%, such as at least 97%, more preferably 100%, after storage at -70°C to +40°C, such as +40°C, at storage duration as above, eg 1 month; or the peak of the main product according to e.g. E-HPLC is at least 85%, or at least 90%, preferably at least 95%, e.g. at least 98%, or even about 100%, after storage at -70°C to +40°C, eg +40°C, for storage times as above, eg 1 month.

The compositions according to the present invention also affect the area of the main peak in RP-HPLC after storage, for example, at a concentration of up to 20 mg/or at a temperature of from -70°C to +25°C for 1 to 3 months, remaining unchanged compared to the composition prior to storage, and representing at least 90%, more preferably at least 95% of the total peak area, where the control sample has the main peak, for example, 95%. When stored at +40°C for 1 month, the composition of the present invention retains the main peak on the results of RP-HPLC at least 80%, 85% or 90%; after storage for 2 months at least 80% or 85%, and after storage for 3 months at least 75% or 80%.

Further, according to the results of capillary isoelectric focusing (cIEF), the composition of the present invention has an effect on ensuring the yield of the main product after storage at a concentration of, for example, up to 20 mg/ml, for 1-3 months at temperatures from -70°C to +40 °C comparable to a control sample (composition without storage, with a main peak of at least 98%), for example, the main peak is at least 85% or more, such as 86%, 87% or 88%. More preferably, the main peak is 90%, 92% or 95%, such as at least 97%, more preferably 100%, after storage at -70°C to +40°C.

5.8.3.6. Stability under freeze-thaw conditions

In addition to ensuring the stability of the compositions under storage conditions that remain constant over time (for example, storage at +5°C), or involving a single cycle of 30 (for example, when stored at -20°C or -70°C), an additional effect of the present invention is the stability under the conditions of repeated cycles of SO. Each transition from a frozen to a liquid state and vice versa is a particularly stressful state for immunoglobulin single variable domains.

The compositions of the present invention also have the effect of providing good stability under 3D conditions. For example, the compositions of the present invention can be subjected to, for example, 10 30 cycles from -70°C to room temperature (eg +25°C), or from -20°C to room temperature. The immunoglobulin single variable domains included in the compositions withstand these conditions without significant impairment as measured by, for example, RP-HPLC or E-HPLC. The effect of repeated 30 cycles on various non-limiting embodiments of the compositions of the present invention was evaluated, and it was found that in all cases, the chemical and physical integrity of VWF binding agents, eg, immunoglobulin single variable domains, was maintained. The overall yield ranged from 95% to 100%, preferably at least 95%, 98% or 99%. The relative proportion of different peaks remains unchanged, compared to the control subjected to only one cycle of 3D.

In particular, at concentrations between 5 mg/or and 20 mg/mL, 10 cycles of 3D result in a yield (as determined based on, for example, total peak area, i.e., the AU of the polypeptide as determined by RP-HPLC or E-HPLC is at least 90%, 95%, 89%, or 100%, wherein in a particular embodiment, RP-HPLC or E-HPLC does not change the profile compared to the control (1 30 cycle).

5.8.3.7. Stability in terms of activity

The person skilled in the art is aware of various methods for determining the activity of VWF binding agents, in particular immunoglobulin single variable domains, in particular polypeptides according to any of SEQ ID NOs: 1-19, for example, SEQ ID NO: 1 (see , for example, the section on experiments from WO2006/122825 [P05-003], for example, examples 3-6, 18 and 19, or the section on experiments from WO2009/115614 [P08-013]).

In one embodiment, the activity of a polypeptide of the present invention can be determined by binding to its antigen by conventional assay methods, eg, ELISA, Biacore, RIA, FACS, etc.

The activity of the VWF binding agents remains acceptable in the compositions of the present invention as analyzed under stress conditions, ie. after 4 weeks of storage at +40°C.

5.8.3.8. Stability in terms of compatibility

The compositions of the present invention are also compatible with a wide range of different solvents. For example, the compositions can be mixed/diluted with such solvents without affecting the chemical or physical stability of the immunoglobulin single variable domains.

Thus, the compositions of the present invention also provide stability over a wide range of concentrations, as defined in this application.

5.8.3.9. Generalization of stabilizing effects

The compositions of the present invention have an impact on maintaining the chemical and physical integrity of the polypeptides of the present invention even after prolonged storage, for example, during the time defined above, at temperatures from -70°C to +25°C.

Storage of immunoglobulin single variable domains as defined in this application, in particular ALX-0081 at -70°C for 1 month does not affect their physico-chemical characteristics for any of the compositions of the invention, in particular for non-limiting examples of buffers tested in the experiments section. Storage had no significant effect on RP-HPLC, E-HPLC, or cIEF profiles.

5.8.4. Stability Analysis of Lyophilized Compositions

In addition, the invention provides stable compositions of VWF binding agents, eg immunoglobulin single variable domains as defined herein, eg SEQ ID NOs: 1-19, preferably SEQ ID NOs: 1, which are particularly suitable for lyophilization. The compositions of the present invention lead to improved stability and improved storage stability after lyophilization.

5.8.4.1. Storage stability

The compositions of the present invention provide good stability after lyophilization during storage, for example at -70°C, -20°C, +5°C, +25°C or +40°C, for example, within 1-36 months , like 1; 1.5; 3, 6, 9, 12, 18, 24, 30 or 36 months. Most preferred results can be obtained with citrate buffer compositions, eg compositions 3 and 7, as shown in the examples in the experimentation section (eg good precipitate formation with no visual signs of decay, figure 6). One of skill in the art will appreciate that in the discussion below, preferred values reflect citrate buffer compositions, for example, as shown in Table 8.

The person skilled in the art will also appreciate that storage at +25°C, and in particular at +40°C, presents stressful storage conditions. It is expected that such conditions increase and accelerate the appearance of any signs of instability, such as chemical or physical instability. Therefore, a relatively short storage, eg at +25°C or +40°C, is a good indicator of long-term storage stability under milder conditions (eg, at +5°C or frozen).

5.8.4.2. Storage stability in terms of protein yield

For example, compositions of the present invention provide a protein yield after lyophilization of at least 95%, such as at least 96%, 97%, 98%, 99% or even about 100% after storage at -70°C to +40°C. Protein yield can be determined by any known means of protein quantification, for example by content, RP-HPLC or E-HPLC. These results can be observed, for example, after storage at the specified temperature for 1-36 months, as within 1; 1.5; 3, 6, 9, 12, 18, 24, 30 or 36 months.

5.8.4.3. Storage stability in terms of chemical derivatives/degradation products

Further, the compositions of the present invention can prevent and minimize the formation of chemical derivatives after lyophilization, as confirmed, for example, e-HPLC.

5.8.4.4. Storage stability in terms of oligomerization

The compositions of the invention may also provide storage stability after lyophilization such that no apparent soluble oligomeric material (as determined by e.g. E-HPLC) is formed at storage temperatures from -70°C to +40°C, after storage for periods of time , as defined above, for example, 1 month; or less than 1%, preferably less than 0.5%, e.g. 0.3% soluble oligomeric material is formed (as determined by e.g. E-HPLC) at temperatures from -70°C to +40°C, e.g. +40° C, after storage for periods of time as defined above, for example, 1-36 months, as within 1; 1.5; 3, 6, 9, 12, 18, 24, 30 or 36 months.

5.8.4.5. Storage stability reflected by the yield of the main product

The compositions of the present invention may also provide, after lyophilization, a major product peak, as measured by e.g. at the storage duration indicated above, for example, 1, 3, 6, 9, 12, 18 or 24 months; or the main peak of the product, according to the results, for example, E-HPLC (see table 18 and tables 27-29) at least 85% or more, such as 86%, 87% or 88%. More preferably, the main peak is 90%, 92% or 95%, such as at least 97%, more preferably 100%, after storage -70°C to +40°C, such as +25°C, after storage within the time specified above, for example, 1, 3, 6, 9, 12, 18 or 24 months; or the main peak of the product, as determined by e.g. e-HPLC, is at least 85%, at least 90%, preferably at least 95%, e.g. at least 98%, or even about 100% after storage from -70°C to +40°C, eg +40°C, after storage for a time as above, eg 1, 3, 6, 9, 12, 18 or 24 months.

The compositions according to the present invention also provide, after lyophilization, no change in the main peak according to the results of RT-HPLC after storage, for example, at a concentration of 12.5 mg/ml at a temperature of from -70°C to +40°C from 1 to 12 months, compared to the composition before storage, and representing at least 90%, more preferably at least 93% of all peaks, where the control sample has a main peak, for example, 93% (see table 15). When stored after lyophilization at +40°C for up to 12 months, the composition of the present invention retains the main peak on the results of RP-HPLC at least 91%, 92% or 93%.

Further, according to the results of cIEF (see tables 27-29), the compositions of the present invention provide after lyophilization the yield of the main product after storage at a concentration of, for example, 12.7 mg/ml for 1-24 months at a temperature of -70°C up to +40°C, comparable to the control sample (composition without storage, the main peak is at least 96%), for example, the main peak is at least 85% or more, like 86%, 875 or 88%. More preferably, the main peak is 90%, 92%, 93%, 94%, 95% or 96%, such as at least 97%, more preferably 100%, after storage at -70°C to +40°C .

5.8.4.6. Stability under freeze-thaw conditions

The compositions of the present invention also provide good stability after freeze-drying under 3D conditions. For example, the compositions of the present invention can be subjected to, for example, 5 cycles of 30 from -20°C to room temperature (for example, +25°C). The immunoglobulin single variable domains contained in the compositions withstand these conditions without significant impairment as measured by, for example, RP-HPLC or E-HPLC. In all cases, the chemical and physical integrity of VWF binding agents, such as immunoglobulin single variable domains, is maintained. The overall yield is in the range of 95 to 100%, preferably at least 95, 98 or 99%, compared to a liquid control stored at -70°C.

In particular, at a concentration of 16 mg/mL, 5 cycles of 300 result in a yield (as determined based on e.g. total area, i.e. AU) of the polypeptide, as determined by RP-HPLC or E-HPLC, of at least 90%, 95%, 98%, 99% or 100%; where in a particular embodiment, the RP-HPLC or E-HPLC profile does not change, compared to the control sample (liquid control sample stored at -70°C) (see table 12).

5.8.4.7. Stability in terms of activity

The person skilled in the art is aware of various methods for determining the activity of VWF binding agents, in particular immunoglobulin single variable domains, in particular polypeptides according to any of SEQ ID NOs: 1-19, for example, SEQ ID NO: 1 (see eg the experiment section of WO2006/122825 [P05-003] eg examples 3-6, 18 and 19 or the experiment section of WO2009/115614 [P08-013]). The activity of VWF binding agents after lyophilization was not affected by repeated cycles of the 30 compositions. In particular, the activity of the VWF binding agents remained stable in the compositions of the invention tested under stress conditions, ie. up to 12 months storage at +40°C (Table 23) and even up to 24 months when stored at +40°C (Table 29). In one embodiment, the activity of a polypeptide of the present invention after lyophilization can be determined by binding to its antigen by conventional assay methods, eg, ELISA, Biacore, RIA, FACS, etc. In particular, in the compositions of the present invention, at least 80%, preferably at least 90%, more preferably at least 95%, or even at least 99% of the VWF binders retain their binding activity after storage under the aforementioned stress conditions. conditions, compared with the binding activity before storage.

In another aspect, the compositions of the present invention substantially show no loss of biological activity when comparing the ALX-0081 liquid composition with the lyophilized composition in various immunological assays, including the Biacore assay, enzyme immunoassay (ELISA), ristocetin-induced cofactor activity (RICO) assay. ) and/or Gyrolab-based analysis (see Section 7.13 and Table 24).

5.8.4.8. Generalization of stabilizing effects

The compositions of the present invention have an effect on maintaining, after lyophilization, the chemical and physical integrity of the polypeptides of the present invention, ALX-0081, i.e. even after extended storage, for example, for the time defined above, at temperatures from -70°C to +40°C, the purity/impurity profile of the product is essentially unchanged. For example, prolonged storage after lyophilization did not significantly affect RP-HPLC, E-HPLC, or sIEF profiles, as confirmed in the experimental section.

5.9. Methods of the present invention

VWF binding agents of the present invention may be prepared by any conventional method. Representative examples include recombinant expression in a suitable carrier system, such as bacteria or yeast. VWF binding agents are subjected to a suitable purification regimen prior to preparing compositions in accordance with the present invention.

The present invention covers methods for preparing the compositions described in this application.

The purification and formulation steps may overlap, for example, when the VWF binders of the present invention are eluted from the column using a buffer of the present invention. Alternatively, the compositions of the present invention may be prepared by buffer exchange by any suitable means, for example, means commonly used in the art such as dialysis, ultrafiltration, etc.

In some embodiments, the process for preparing a composition of the present invention may also refer to reconstituting a lyophilized or spray-dried composition, for example by adding water or a suitable buffer (which may optionally contain additional excipients).

Methods for preparing a composition in accordance with the present invention may include additional steps such as filling into vials suitable for clinical use, such as sealed containers, and/or final processing to obtain a finished dosage form. The methods may also include additional steps such as spray drying, lyophilization or freezing, eg bulk freezing. The invention also includes containers, dosage forms or other products obtained by any of the methods described in this application. Compositions of the present invention can be used to store agents that bind vWF, for example, OVD, as defined in this application. Thus, the invention encompasses a method of storing a VW binder as used herein, characterized by the use of a composition as defined herein. In particular, the invention encompasses methods for stabilizing a VW binder as defined herein for storage, including, for example, preparing a composition as described herein. Storage can be carried out for 1-36 months, such time as 1; 1.5; 3, 6, 9, 12, 18, 24, 30 or 36 months, e.g. at least 12 or even 24 months, optionally at -70°C to +40°C, such as -70°C, -20°C, +5°C, +25°C or +40°C, preferably at -70°C to +25°C, more preferably at -20°C to +5°C. Thus, storage may include freezing, freeze-drying (lyophilization), and/or spray drying. Storage methods may further include assessing the physical and chemical integrity of VW binders as defined herein.

The present invention also relates to methods for analyzing compositions containing at least one of the VWF binding agents as defined in this application. Compositions can be analyzed to determine any signs of chemical or physical instability of agents that bind VWF, as defined in this application. For example, the presence of degradation products can be assessed in the compositions, for example, low molecular weight derivatives such as proteolytic fragments; and/or chemical derivatives, eg pyroglutamate variants; and/or high molecular weight derivatives such as aggregates, agglomerates, etc. The composition can also evaluate the total protein content and/or activity. Each of the various analysis methods as defined in this application can be used in the analysis method of the present invention.

Thus, the present invention also relates to a method for monitoring and/or evaluating the quality and/or stability of a composition, for example, during one or more of production, storage and use. The invention also relates to a method for controlling the quality of a composition, such as evaluating the conformity of a composition to product specifications, as described below. The invention in any of these aspects includes one or more selected from comparison with one or more reference samples, analysis of batch-to-batch variation, and constant monitoring of the manufacturing process.

The present invention relates to any product associated with the compositions of the present invention, for example, including them, or necessary for their production or obtaining a finished product, without any limitation.

For example, the present invention relates to an article of manufacture, for example, a sealed container containing one or more compositions in accordance with the present invention. The invention also relates to a pharmaceutical dosage form, for example, a dosage form suitable for parenteral use in a patient, preferably a human patient, comprising one or more of the compositions in accordance with any embodiment described in this application. The dosage form may be, for example, in the form of a pre-filled syringe, ampoule, cartridge or vial. The syringe, ampoule, cartridge or vial may be made from any suitable material such as glass or plastic and may include rubber materials such as rubber stoppers for vials and rubber pistons and rubber seals for syringes and cartridges. The invention also relates to a kit containing one or more of the compositions in accordance with the present invention. The kit may further contain instructions for use and/or annotation to the medicinal product. In any embodiment of the products as defined in this application, the invention also covers the presence of packaging material, instructions for use, and/or annotations to the medicinal product, for example, depending on the particular regulation.

5.10. Definitions

5.10.1. Identity

For the purpose of comparing two or more amino acid sequences, the percentage of "sequence identity" between the first amino acid sequence and the second amino acid sequence (also referred to herein as "amino acid identity") can be calculated by dividing the [number of amino acid residues in the first amino acid sequence that are identical to amino acid residues at the corresponding positions in the second amino acid sequence] by [total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], where each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence, compared to the first amino acid sequence , is considered the difference between a single amino acid residue (position), i.e. "amino acid difference", as defined in this application.

Alternatively, the degree of sequence identity between two or more amino acid sequences can be calculated using a known computer algorithm for sequence alignment, such as NCBI Blast v2.0., using standard settings.

Some other techniques, computer algorithms and setups for determining sequence identity are described in, for example, WO04/037999, EP0967284, EP1085089, WO00/55318, WO00/78972, WO98/49185 and GB2357768-A.

Generally, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the highest number of amino acid residues is taken as the "first" amino acid sequence and the other amino acid sequence is taken as the "second" amino acid sequence. .

In addition, when determining the degree of sequence identity between two amino acid sequences, one skilled in the art can consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue of similar chemical structure. , and which essentially have no or little effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO04/037999, GB2357768-A, WO98/49185, WO00/46383 and WO01/09300; and (preferred) types and/or combinations of such substitutions may be selected based on the relevant teachings from WO04/037999 as well as WO98/49185, and from other references cited in this application. Such conservative substitutions are preferably substitutions in which one amino acid within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Tre , Pro and Gli; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phen, Tyr and Trp. Particularly preferred conservative substitutions are the following: Ala to Gly or to Ser; Arg on Liz; Asn on Gln or on Gis; Asp on Glu; Cys to Ser; Gln to Asn; Glu on Asp; Gli on Ala or Pro; Gis on Asn or on Gln; Ile to Lei or to Val; Lei on Ile or on Val; Liz on Arg, on Gln or on Glu; Met on Ley, Tyr or Ile; Hairdryer on Met, Lei or Tyr; Ser on Tre; Tre na Ser; Trp to Tyr; Shooting range on Trp; and/or Fen on Val, on Ile or on Lei. Any amino acid substitutions applied to the polypeptides described in this application may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species, developed by Schulz et al., "Principles of Protein Structure", Springer-Verlag, 1978 ("Principles of Protein Structure"). structures"), on structure-forming potential assays developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the hydrophobicity pattern analysis of proteins developed by Eisenberg et al., Proc. Natl. Acad. sci. USA 81: 140-144, 1984; Kyte &Doolittle; J Molec. Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986, each of which is incorporated herein by reference in its entirety. Information on the primary, secondary and tertiary structure of Nanotel® is given in the description of the present application, and in general in the prior art cited above. Also for this purpose, the crystal structure of the llama V _HH domain is given in, for example, Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Nature Structural Biology (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further information on certain amino acid residues that form the V _H /V _L interface in normal V _H domains and possible camelizing substitutions at these positions can be found in the prior art cited above.

6. Abbreviations

API Active Pharmaceutical Ingredient

KIEF Capillary isoelectric focusing

DLS Dynamic Light Scattering

DE Experiment Design

LP Medicinal product

LS Medicinal substance

ZO Freeze-Thaw

HMW High molecular weight

HMW Low molecular weight

MALS Multi-angle light scattering

RH Relative humidity

OFC Reverse Phase Chromatography

RP-HPLC Reverse Phase High Performance Chromatography

E-HPLC Size Exclusion High Performance Chromatography

SOP Standard Operating Procedure

Tp Melting point (°C)

TSA Thermal Shift Analysis

PV Willebrand Factor

WFI Water for injection

The invention is further described by means of the following non-limiting preferred aspects, examples and figures.

The entire contents of all references (including literary references, published patents, published patent applications, and simultaneously pending patent applications) cited in this application are hereby expressly incorporated by reference, in particular for the teachings given above.

7. Examples

A number of experiments have been carried out to provide an improved formulation buffer designed to satisfy a wide range of different and seemingly incompatible tasks. In particular, exemplary compositions are provided that are capable of maintaining the stability, biological activity, purity, and quality of ALX-0081 over an extended period of time, resistant to various types of stress, such as freezing, lyophilization, heating, and/or reconstitution.

The modern drug substance ALX-0081 is presented as a liquid composition containing 5 mg/ml active pharmaceutical ingredient (API) in phosphate-based buffer (D-PBS) containing 200 mM glycine and 0.02% Tween-80 (v/v ), with pH 7.1 (LS). Although this composition was used during the original clinical trials, it can be improved in various ways. First, the relatively low concentration is likely to require multiple subcutaneous injections (given that the volume for subcutaneous injections is limited to about 1 ml), thus reducing patient tolerance. Secondly, the storage stability at 3-8° C. or room temperature of the current ALX-0081 composition is limited. The limited shelf life of a modern composition is mainly determined by chemical modification (see section 7.2). Chemical modifications may be associated with loss of activity. Although an achievable shelf life can be achieved by storing the product at -20°C, however, this is not considered a favorable option for most practical purposes.

7.1. Methods

Samples were analyzed essentially in accordance with standard operating procedures for content, activity and purity, precipitation, concentration, degradation, aggregation and activity. In addition, all samples were visually assessed for turbidity or the presence of protein aggregates or sedimentation. The content of residual moisture in specific lyophilized samples was determined by titration according to Karl Fischer.

In the present study, three different lyophilization programs were used to dry ALX-0081: a standard 65 hour cycle (Figure 1), a shortened 37 hour cycle, and a longer cycle of 66 hours optimized to reduce residual moisture content as described in Table 14.

Briefly, at the beginning of the shortened 37 hour lyophilization process, the temperature of the shelves was +20°C, and it was brought to -50°C in 2 hours. Then a vacuum of 0.04 mbar was created for 1 hour. After reaching a vacuum of 0.04 mbar, the temperature of the shelves for 4 hours was maintained at -50°C. After these 4 hours, the temperature was gradually raised to 0° C. over 15 hours (ie, performing the primary drying step, removing the frozen water). The temperature of the shelves 0°C was maintained for 7 hours while maintaining a vacuum of 0.04 mbar. After 7 hours, the temperature was raised to +25°C for 3 hours, and then maintained at 25°C for 5 hours (i.e., a secondary drying step was carried out, removing defrosted water). The vials were sealed under vacuum ±0.400 mbar, after which normal pressure was restored.

The same method was used for the standard 65 hour regimen, and it only differed in the second drying step, which was extended to 28 hours at 25° C. under vacuum, resulting in a total cycle time of about 65 hours. A schematic overview of the various steps of a standard 65 hour lyophilization schedule is shown in Figure 1. During the lyophilization process, the temperatures of three vials were controlled at strategic positions. Finally, the standard 65 hour regimen was modified during operation, according to the readings of the temperature sensors, resulting in prolonged lyophilization cycles, as described in Table 14.

7.2. Chemical stability of the modern composition ALX-0081

RP-HPLC is one of the most informative methods for assessing the chemical stability of a drug substance (drug).

RP-HPLC allowed ALX-0081 to be resolved in a number of different species. In addition to the main peak, pre peaks (substance eluting before the intact unmodified material) and a number of subsequent peaks can be seen. In the series obtained to date, the pre peaks and subsequent peak 1, respectively, represent approximately 2% and 3.6% of the LS, while the other subsequent peaks are less than 1% of the LS.

However, when stored under accelerated (+5°C) or stressed (+25°C and +37°C/+40°C) conditions, the relative presence of certain variants associated with the product increases with time and temperature, as depicted in figure 2A. In addition, the main peak in RP-HPLC is separated into several different types during prolonged incubation, especially at elevated temperatures (≥+25°C) (figure 2B). These data show that some new molecular species eluted earlier are formed during storage.

The most important modifications present in ALX-0081 at the time of manufacture or during storage are: (i) prepeak 1 (oxidation); (ii) subsequent peak 1 (Nor-Lay variant); (iii) subsequent peak 2 (pyroglutamate formation), and (iv) cleavage of the main peak (isomerization). Modifications (i), (ii), and (iii) do not significantly affect activity (data not shown). In contrast, isomerization of aspartic acid residues at positions 105 and 236 of SEQ ID NO: 1, which are located in the CDR3 region, has been shown to be the predominant molecular mechanism underlying the possible loss of ALX-0081 activity (see (iv) above).

Some of the variants associated with ALX-0081 that are present during manufacture or occur during storage can also be detected by cIEF. In this case, the pyroglutamate modification appears as a subsequent peak (see (iii) above). Also, similar to what was observed in the RP-HPLC analysis, the isomerization occurring at position 105 in both 12A02H1 domains results in a broadening of the main peak, and eventually splitting of the main sIEF peak (see (iv) above).

7.3. Selection of buffers and excipients

To further develop the composition of VWF binding agents, a complex set of experiments were carried out with the evaluation of various parameters that influenced each other, including (i) different buffers, (ii) at different concentrations, (iii) with each buffer at different pH values; and (iv) combining each buffer with different excipients.

Buffer systems should have as low a buffer capacity as possible so as not to cause significant disruption of the body's buffer system upon administration. In addition, the effect of buffer type and concentration on active pharmaceutical ingredient (API) activity must be carefully evaluated.

Typically, increased levels of protein stability are associated with high melting points. Accordingly, the thermal properties of ALX-0081 were controlled in the presence of various compositions. In particular, the TSA experiment was performed with 192 different isotonic compositions for which the results were reported in the design of experiment (DE) to evaluate the effect of buffer, concentration, ionic strength, pH and excipients on the thermal stability of ALX-0081. The melting point (Tm) of ALX-0081 was read, which is an indicator of the thermal stability of the protein in various tested compositions.

Briefly, the applied thermal shift analysis (TSA) uses the signal changes of a fluorescent dye such as Sypro Orange (orange) when the protein is thermally unfolded. When Sypro Orange is added to a solution of a suitably folded protein, it cannot bind to any surface on the protein and its fluorescent signal is quenched. As the temperature rises, the protein undergoes thermal unfolding, exposing its hydrophobic core region. Sypro Orange then binds to the hydrophobic regions and is activated resulting in an increase in the fluorescent signal. The assay is performed with solutions containing various test compositions, ALX-0081 at 0.2 mg/ml and 10x Sypro Orange. The program consists of the following steps: heating up to 37°C at a rate of change of 4.4°C/sec and holding for 10 sec; heating up to 90°C at a constant rate of change of 0.02°C/sec (acquisition of data 20 times per °C); and cooling to 37° C. at a rate of change of 2.2° C./sec and holding for 10 seconds.

The following set of buffers was used with various concentrations (10-200 mM), pH values and excipients:

- citrate pH 6.0-6.5-7.0;

- histidine pH 5.5-6.0-6.5;

- phosphate pH 6.5-7.0-7.5;

- Tris-HCl pH 7.4-7.7-8.0;

- NaCl 0-140 mM concentration range;

- glycine 0-270 mM concentration range;

- mannitol 0-270 mM concentration range;

- sucrose 0-270 mM concentration range;

- trehalose 0-270 mM concentration range.

The obtained melting points (Tm) were entered into the Design Expert program to analyze the factorial screening experiment to predict 50 compositions providing the highest thermal stability (see table 1).

The highest Tp values were predicted for phosphate (pH 7.0–7.5) and citrate (pH 6.2–7.0) containing trehalose, sucrose, mannitol, or glycine. Quite unexpectedly, the results of the study suggest that Tris-HCl (pH 7.8-8.0) and Histidine-HCl (pH 6.5) buffers return significantly lower melting temperatures, although they were tentatively chosen as the buffer systems of choice for controlling the pH of an immunoglobulin single variable domain solution as described in WO2010/077422.

Accordingly, it has been concluded that phosphate and citrate formulations containing trehalose, sucrose, glycine, or mannitol are particularly good at stabilizing VWF binding agents, such as ALX-0081.

7.4. Solubility analysis

To determine if further enhancement of the solubility of ALX-0081 was possible, an initial screening of some formulations was performed. In ALX-0081, the buffer was replaced with the composition of interest (except Tween-80) and further concentrated in a stirred cell (eg, Amicon type) equipped with a 5 kDa filter. Once visible precipitation or turbidity was noted, the sample was filtered and the protein concentration measured. Table 2 summarizes the results obtained.

The concentration of buffers based on phosphate and histidine led to sample turbidity and precipitate formation at relatively low protein concentrations (<10 mg/ml). In contrast, ALX-0081 remained physically stable in citrate buffer, even after reaching a concentration of about 56 mg/ml. In addition to visual analysis, the absence of particles or HMW species was confirmed by fluorescence microscopy (with Nile red staining, by E-HPLC and DLS). Further, when the ~56 mg/ml solution was subjected to either 10 cycles of 3D at -20°C or -70°C, or storage at +4°C for about 1 week, this did not seem to affect the physical stability of the molecule from the results E-HPLC analysis (see figures 3A and 3B, respectively).

The relatively high solubility of ALX-0081 in citrate buffer was confirmed by PEG precipitation analysis (data not shown).

7.5. Twin-80

To determine if the non-ionic surfactant polysorbate, also referred to as Tween (Polyoxyethylene (N) sorbitan monolaurate; where N=20, 40, 60, 65, 80, or 85), is required for the formation of ALX-0081, several stress experiments were performed, caused by stirring, in 50 mm citrate buffer at pH 6.0 and 6.5. The effect of various concentrations of Tween-80 (no Tween-80, with 0.01% and 0.02% (v/v)) on the physical stability of ALX-0081 was evaluated at 5 mg/ml by monitoring the scattered signal at 500 nm on a spectrofluorimeter.

Twin-80 prevented an increase in the scatter signal in both buffers, demonstrating a protective effect (FIGS. 4A and 4B). There were no significant differences between samples containing 0.01% or 0.02% Tween-80 (v/v). Further, the E-HPLC profiles of the samples before and after mixing do not show any difference: 95-100% yield is achieved and no oligomerization or degradation is detected.

Based on these results, it was decided to include 0.01% Tween-80 (v/v) in the formulation of VWF binding agents, eg ALX-0081.

7.6. Twin

To determine if other members of the polysorbate series that differ in polyoxyethylene chain length and fatty acid ester component, such as Tween-20, Tween-40, Tween-60, Tween-65, and Tween-85, are needed in the composition of VWF binding agents , performed several stir stress experiments in 50 mM citrate buffer at pH 6.0 and 6.5, especially as described in section 7.5 above. The effect of different concentrations of different members of the Tween series (no Tween, with 0.01% and with 0.025 (v/v)) on the physical stability of the VWF binding agent at 5 mg/mL was assessed by monitoring the signal scattering at 500 nm on a spectrofluorimeter.

Twin-20, Twin-40, Twin-60, Twin-65 and Twin-85 provided essentially the same favorable result as Twin-80.

7.7. Analysis of the stability of liquid compositions

A more thorough study was performed to evaluate the stability of ALX-0081 in various isotonic formulations based on citrate at a concentration of 20 mg/ml. Table 3 provides an overview of the various compositions tested.

The main objective was to evaluate the effect of pH (6.0-6.5-7.0) and the type of excipient (NaCl, mannitol, sucrose or glycine) on the stability of the liquid product. For control and direct comparison purposes, the study also included the current formulation of ALX-0081 5mg/mL with D-PBS and glycine (identical to the current formulation except at lower concentrations of Tween-80), as well as the various isotonic solutions of ALX-0081 previously mentioned at based on citrate, but with a concentration of 5 mg/ml instead of 20 mg/ml. Altogether, this resulted in 17 different liquid compositions (compositions NO: 1-17) which were subjected to a long-term stability analysis. To exclude the influence of differences in the concentration of Tween, all compositions contained 0.01% Tween-80 (v/v).

7.7.1. Freeze-thaw stability

The effect of repeated 30 cycles on the stability of ALX-0081 as a liquid formulation was evaluated. Aliquots of the various compositions (0.5 ml/tube) were exposed to up to 10 cycles of 30 from -70°C or -20°C. One cycle included freezing for ±20 min followed by thawing for 5 min in a water bath at +25°C. After this treatment, all compositions remained visually transparent. RP-HPLC analyzes showed good yields (95-100%) and no significant difference in profiles was noted, suggesting that the quality of VWF binding agents such as ALX-0081 is not affected by repeated freeze-thaw in the 17 different liquid compositions.

7.7.2. Storage stability

The stability of 17 different formulations was also assessed by storing aliquots (0.5 ml per vial) under stress conditions, ie. at +40°С; long-term storage conditions at -70°C were included as controls. The analyzes were focused on RP-HPLC, as this method is generally known as a particularly informative method for detecting chemical modifications that occur during storage (see table 4). This section provides an overview of the data obtained after 1 month of storage; the results confirm observations at earlier time periods, eg 1 week and 2 weeks later.

(a) RP-HPLC

As stated in section 7.2 above, RP-HPLC analysis showed resolution of the current drug ALX-0081 (formulation with D-PB glycine) for some product related variants and impurities. Briefly, under stressful conditions (eg, +40°C), the purity (% of the main peak) decreased simultaneously with the increase in some of the existing previous/next peaks, as well as the formation of additional peaks.

The RP-HPLC data from this study are summarized in Table 5.

Overall, the results show that essentially the same modifications occur in the various citrate buffers as observed in the present formulation buffer (i.e., D-PBS/glycine), although some difference in relative peak area can be noted. In particular, the increase in the area of the previous peaks (oxidation) was lower in citrate compositions (especially at pH 6.0), compared with the composition in D-PBS/glycine. With regard to the formation of this preliminary peak, glycine seems to be the least favorable among the various excipients. The profile of various subsequent peaks after storage for 1 month at +40°C was comparable for all compositions, although the second subsequent peak (i.e. pyroglutamate variant) seemed more pronounced at pH 7.0 than at pH 6.0-6 ,5. The degree of broadening/splitting of the main peak as a result of Asp isomerization is difficult to assess due to poor resolution; the percentage area of the shoulder peak cannot be accurately determined, and thus has been included in the relative peak area noted in Table 5 for the main peak. However, the corresponding RP-HPLC chromatograms (data not shown) provided a quantification; these data suggested that the degree of isomerization is quite similar in different compositions.

(b) KIEF

Like RP-HPLC, the sIEF method provides for the detection of certain product variants that occur during storage under stress conditions (see Section 7.2 for details). Examples are shown in Figure 5, which compares electropherograms of modern ALX-0081 after storage for 1 month at -70°C and +40°C.

The sIEF data obtained in the present study (data not shown) largely confirm the conclusions provided by the RP-HPLC analysis, i.e. the same type of modification occurs to about the same extent in different citrate buffers as observed in the buffer of the present composition, represented by composition 17, as depicted in figure 5 (ie D-PBS/glycine). However, some difference in the relative area of the peaks can be observed. In particular, the subsequent peak (i.e., the pyroglutamate variant) appears more pronounced at pH 7.0 than at pH 6.0-6.5, consistent with the RP-HPLC observations as previously summarized in Table 5.

(c) E-HPLC

E-HPLC analysis was performed to assess the physical stability of ALX-0081, ie. to identify HMW species and/or degradation products that may form during storage under stressful conditions. For all formulations tested, stress conditions did not seem to have a significant effect on the E-HPLC chromatograms.

(d) Conclusion

A summary of the most important observations regarding the storage stability of various ALX-0081 liquid formulations is shown in Table 5. Only the most informative data based on RP-HPLC analysis are listed. These data suggest a higher chemical stability in 50 mM citrate at pH 6.0-6.5. With the exception of glycine, the type of excipient does not significantly affect stability. With regard to physical stability, no differences were observed between the various compositions. The latter was evidenced by approximately 100% yield observed for all samples in various HPLC assays, as well as E-HPLC chromatograms showing no aggregation/degradation.

Based on the above results, it was decided to further exploit the potential of citrate/sucrose compositions at pH 6.0-6.5.

7.8. Stability Analysis of Lyophilized Compositions

The effect of lyophilization was assessed by comparing the storage stability of ALX-0081 in liquid and lyophilized citrate/sucrose formulations (20 mg/ml API at pH 6.0-6.5). An overview of the formulations tested is shown in Table 6. Prior art formulations based on D-PBS/glycine (5 mg/mL API) were included for comparison. Liquid (i.e. before lyophilization) and lyophilized ALX-0081 composition was stored frozen (-70°C for liquid samples and -20°C for lyophilized compositions), as well as at +5°C, +25°C and +40°C, and the samples were analyzed after 2 weeks and 1.5 months of storage.

On Panel A of Fig. 6 shows an image of the vials after the lyophilization process using the standard 65 hour regimen as shown in FIG. good lyophilisate. All samples could easily be resolubilized in Milli-Q water and the solutions were clear and colorless (Figure 6, panel B).

7.8.1. Product evaluation before or after lyophilization

RP-HPLC and E-HPLC analysis showed no significant differences in terms of physicochemical characteristics between the liquid starting product (stored at ≤ -70°C) and the product after lyophilization and reconstitution of any of the compositions tested. Further, complete recovery of the sample was demonstrated for all compositions (table 7).

7.8.2. Evaluation of freeze-dried product after 1.5 months of storage

(a) Visual analysis and content.

The lyophilisate from the lyophilized samples showed no visual signs of disintegration after 1.5 months of storage at -20°C, +5°C, +25°C or +40°C.

Samples were clear and colorless after reconstitution with Milli-Q water. In addition, storage did not significantly affect the content measured after reconstitution (Table 8).

(b) RP-HPLC.

The profiles of 3 different lyophilized compositions (No. 3, 7 and 17) were compared after 1.5 months of storage at -20°C, +5°C, +25°C and +40°C, respectively. Comparison under the most active conditions (+40°C) best reveals the effect of lyophilization on chemical stability.

The corresponding results are summarized in Table 8. As can be seen, storage in frozen form does not affect ALX-0081 in any of the formulations tested in this study.

In general, the main conclusion to be drawn from the findings is that lyophilization of the citrate/sucrose composition essentially prevents chemical modifications occurring in liquid form, except for some minimal amounts of pyroglutamate modification. These lyophilized compositions showed no increase in area percentage of previous peaks, nor evidence of main peak expansion/split. In contrast, lyophilization of the D-PBS/glycine composition does not lead to a significant improvement in chemical stability. Pyroglutamate formation in the citrate/sucrose lyophilized composition appears to be slightly more pronounced at pH 6.0 than at pH 6.5. This is justified by data at +25°C, at which the rate of formation of pyroglutamate is lower, but shows the same dependence on pH. As expected, storage up to 1.5 months at -20°C or +5°C does not cause any detectable impairment of lyophilized ALX-0081 (data not shown).

Surprisingly, at +40°C, an improvement in the stability of the citrate/sucrose based compositions was obtained compared to the D-PBS/glycine based composition, the latter showing a significantly higher susceptibility to chemical modifications.

For liquid compositions, storage up to 1.5 months at -70°C, +5°C and +25°C had no significant effect on ALX-0081 (data not shown). Deterioration was observed after 1.5 months of storage at +40°C, which is roughly consistent with earlier observations (see section 7.7.2).

(с) cIEF

The results obtained by SIEF analysis are consistent with the results of RP-HPLC. Importantly, lyophilization of the citrate/sucrose composition did not completely prevent pyroglutamate modification. Indeed, storage of the lyophilized product at +40°C for 1.5 months resulted in an increase in the subsequent peak. Again, faster formation of pyroglutamate was observed in the citrate/sucrose formulation at pH 6.0 compared to pH 6.5.

(d) E-HPLC/MALS/DLS.

Storage up to 1.5 months at -70°C/-20°C, +5°C and +25°C had no effect on the E-HPLC profiles of ALX-0081 lyophilized or liquid formulations (data not shown). However, at +40° C., peak expansion and shoulder peak formation could be observed in all liquid compositions. MALS analysis showed these shoulder peaks to be consistent with monomeric ALX-0081 (data not shown). These data indicate a conformational change in the ALX-0081 subpopulation as a result of storage under stress conditions. Surprisingly, the E-HPLC profile of the citrate/sucrose lyophilized compositions was not affected by stress analysis at +40°C, indicating that these lyophilized compositions also improve the physical stability of ALX-0081. However, this was not observed in the case of the lyophilized composition based on D-PBS/glycine; stressing this composition at +40° C. resulted not only in a shoulder peak, but also in some macromolecular species observed as a broad pre-peak (Table 8). DLS analysis did not reveal any large oligomeric species in any of the formulations (data not shown).

(e) Conclusion

A summary of the most important observations regarding the storage stability of the ALX-0081 lyophilized formulations tested is shown in Table 8. Overall, only limited stability differences were observed between the citrate/sucrose based formulations, although surprisingly ALX-0081 appeared to be less prone to pyroglutamate formation at pH 6.5, compared to pH 6.0. Thus, further research on changing the composition of ALX-0081 has been focused on compositions based on citrate/sucrose at pH 6.5.

7.9. Additional optimization of the composition based on citrate/sucrose

Data collected to date indicates that the citrate/sucrose formulation improves solubility, and that lyophilization of the formulation dramatically improves the storage stability of ALX-0081. However, storage of lyophilized ALX-0081 at higher temperatures, although limited, still results in the formation of pyroglutamate. It is reasonable to assume that this modification may limit the shelf life of the lyophilized product (even when stored at +5°C). It is necessary to find out why freeze-drying was unable to prevent this modification.

It has been suggested that the water remaining in the lyophilized product plays a key role.

If this hypothesis is correct, then remaining water can be minimized by optimizing physical parameters such as drying time, temperature, vacuum, etc. as listed above, but at the same time other parameters of the VW binder will stay constant. Another approach is to modify the composition, but again, at the same time, other parameters of the VWF binding agent must remain constant. In addition, adjustment of physical lyophilization parameters in combination with composition modification can be used.

7.9.1. Optimized lyophilization parameters

The optimization of the physical parameters of lyophilization, including (i) drying time, (ii) temperatures of the various stages, (iii) vacuum, and combination (i) - (iii), is not satisfactory, i.e. there is no or inadequate influence on the residual moisture content or influence on the parameters of agents that bind VWF.

7.9.2. Composition optimization for lyophilization

The effect of moisture content on the chemical stability of the lyophilized product was investigated by adjusting the concentrations of citrate buffer and sucrose excipient. In addition, the secondary drying time during the lyophilization program was examined.

7.10. Effect of Moisture Content on the Stability of a Freeze-Dried Product

Three different isotonic formulations of ALX-0081 with different concentrations of citrate and sucrose (all three at pH 6.5) were subjected to two different lyophilization programs: on the one hand, the standard 65 hour regimen, and on the other hand, a shortened 37 hour regimen. An overview of the compositions tested is shown in Table 9. Figure 7 shows the vials obtained after lyophilization. Lyophilization resulted in a good lyophilisate for all formulations.

Lyophilized samples of ALX-0081 were analyzed after 2 and 4 weeks of storage at -20°C and +40°C. In this experiment, it was decided to conduct an exhaustive analysis to further substantiate the suitability of the compositions.

First, when stored at +40° C. for 4 weeks, the lyophilizate from the lyophilized samples remained intact and the reconstitution gave clear solutions. The lyophilization cycle does not seem to have a significant effect on content (measured spectrophotometrically at 277 nm) or osmolality. In line with earlier experiments, 4 weeks of storage at +40°C did not affect the physical stability of ALX-0081 based on the results of E-HPLC, MALS and DLS analyzes (data not shown). In addition, the activity of ALX-0081 as determined by the Biacore-based assay was not affected by the lyophilization process and subsequent storage (data not shown). However, RP-HPLC analysis showed that storage again led to the formation of the pyroglutamate variant, albeit in a minimal amount. This was slightly more pronounced for the composition containing the highest concentration of citrate and the lowest concentration of sucrose (table 10). In addition, for each lyophilized composition, the total moisture content was determined by the Karl Fischer titration method. A summary of these data, together with the amount of pyroglutamate detected in the respective stressed samples, is shown in Table 10. From the data obtained for each lyophilization program individually, it can be seen that a higher moisture content leads to a higher susceptibility to the formation of pyroglutamate. This suggests that the residual water present in the lyophilized product stimulates chemical modifications. In conclusion, the results show that reducing the moisture content of lyophilized VW binders, such as ALX-0081, is beneficial to their chemical stability.

7.11. Effect of Decreasing Buffer Strength and Increasing Sucrose Content

The data obtained in the previous section show that a decrease in the concentration of citrate with an increase in the concentration of sucrose (thus maintaining an isotonic solution) is beneficial for the stability of the lyophilized product. At the same time, evidence was obtained that ALX-0081 required a sufficiently high concentration of citrate to obtain improved stability. Thus, it was decided to evaluate the effect of citrate and sucrose concentrations on the appearance of the solution during storage at +5°C and +25°C, and re-evaluate freeze-thaw stability in the presence of reduced citrate concentrations.

7.11.1. Evaluation of the effect of citrate/sucrose concentration

In the first experiment, 12 different compositions of ALX-0081 were stored at +5°C and +25°C for 4 days. The samples were checked for turbidity or sediment on a regular basis. Images of the samples obtained after 4 days of storage are shown in Figures 8 and 9. An overview of the various compositions and corresponding results is presented in Table 11. After 4 days of storage at +25°C, all samples remained clear and colorless (Figure 8, panel A) . It can be seen that the degree of turbidity is inversely proportional to the concentration of citrate, while the composition with 50 mm citrate remains transparent. In addition, the yield for the sample containing 15 mM citrate was 68% (based on A277 after 20 hours of storage), while in other cases the yield varied from 90 to 100% (data not shown). The addition of sucrose to the 15 mM citrate formulation prevented sample turbidity, although the lowest sucrose concentration (ie 5%) showed some minimal turbidity at +5°C (FIG. 9, panel B).

Observations have confirmed the importance of a high enough citrate concentration to maintain the solubility of ALX-0081, especially at low temperatures. However, increasing the concentration of citrate led to an increase in moisture content. Surprisingly, the decrease in citrate concentration could be compensated for by the addition of sucrose. No effect of Tween-80 on solubility was observed.

7.11.2. Evaluation of resistance to SO

The next experiment was focused on the resistance to 3D of some compositions based on citrate/sucrose. Nine different formulations of ALX-0081 were subjected to 5 consecutive 30 cycles at -20°C. An overview of the compositions tested and corresponding results is shown in Table 12. All samples remained clear and 30 cycles did not affect the physical stability of VWF binding agents, eg ALX-0081, as measured by content analysis and E-HPLC data.

7.11.3. Optimization of sucrose and citrate concentration in terms of isotonicity

Based on the aforementioned storage and 3D results, 20 mM was chosen as the optimal concentration of citrate buffer. The final experiment was carried out with three compositions differing in sucrose concentration. The aim of this experiment was to establish the optimal concentration of sucrose to achieve an isotonic composition and confirm the resistance to ALX-0081 3D at 20 mg/ml. In addition to the 5 subsequent cycles, each formulation was subjected to 1 OR cycle followed by 24 hours storage at +25° C. and an additional OR cycle to simulate processing steps during manufacture.

A summary of the results is shown in Table 13. All formulations tested were clear and the different treatments had no effect on content/yield or osmolality. Based on the osmolality values, it seems that a sucrose concentration of 7% is optimal to obtain an isotonic solution.

7.12. Stability Study of ALX-0081 Lyophilized Compositions Stored at Various Temperatures for 12 Months

ALX-0081 was formulated at 12.5 mg/mL in 20 mM citrate buffer, pH 6.5, with 7% sucrose (w/v) and 0.01% Tween-80 (v/v); it was lyophilized according to the conditions shown in Table 14. Samples were sequentially stored at 20°C (±5°C), +5°C (±3°C), +25°C (±2°C/60±5 % RH) and +40°C (±2°C/75±5% RH).

The stability of the lyophilized compositions was evaluated at various time points, i. initially, after 1 month, 3 months, 6 months, 9 months and 12 months, and determined their purity, appearance, physico-chemical properties and activity.

Detailed data on the characterization of the sample are given in tables 15-23.

The purity of the samples was assessed by RP-HPLC, which determined the percentage area of the main peak, as well as the percentage area of the preliminary and subsequent peaks. The protein concentration was determined by absorbance in the ultraviolet region.

Next, the lyophilized samples were visually evaluated, reconstituted, and the reconstituted composition was visually evaluated. The pH of the samples after reconstitution was measured and the moisture content of the lyophilized powder was determined by coulometric titration (according to Karl Fischer). The content of mechanical impurities was determined to count particles ≥10 µm and ≥25 µm. The samples were further determined for biological function using a Biacore-based assay. Activity was expressed as a percentage relative to the activity of the reference material.

The stability data obtained showed that the performance of the freeze-dried ALX-0081 product was essentially unaffected by storage for 12 months at -20°C or +5°C. It was found that the data collected during the stability study at these temperatures were comparable to the data obtained at the initial time point.

Some minimal changes were observed for samples stored at +25°C or +40°C, which were associated with accelerated or stress storage conditions. The main observations were:

- at +25°C and at +40°C, an increase in the subsequent peak 2 on RP-HPLC was observed during 12 months of storage, which corresponded to the formation of the pyroglutamate variant from 0.7% to 1.1% or 2.4%, respectively;

- at +40°C there was an increase in moisture content from 0.7% to 2.15 (m/m) after 12 months of storage. This may have been due to absorption of moisture from the storage environment (ie 75% RH) by the cork, followed by gradual diffusion into the product.

Results obtained under stress conditions suggest a correlation between moisture content and product chemical stability; this is consistent with the data previously given in section 7.10.

Thus, these data indicate the importance of controlling the moisture content of the drug product during storage.

Given that storage at +40°C can be considered predictive of long-term stability at +25°C, the 12-month stability data included in this application provide good indications for long-term storage stability at room temperature (such as for 18, 24, 30 or 36 months) and even prolonged storage stability under milder conditions (eg at +5°C or frozen).

7.13. Comparative in vitro study of the biological activity of a liquid and lyophilized composition of a drug product with anti-VWF nanobody caplacizumab (ALX-0081)

7.13.1. A task

A series of analyzes were performed to assess the in vitro comparability of the current drug ALX-0081 (liquid formulation containing 5 mg/ml active pharmaceutical ingredient (API) in phosphate-based buffer (D-PBS) containing 200 mM glycine and 0.02% Tween- 80 (v/v), pH 7.1) and lyophilized LP formulation ALX-0081 as described above (with 12.5 mg/ml in 20 mM citrate buffer, pH 6.5, with 7% sucrose (m /o) and 0.01% Tween-80 (o/o)) in relation to biological activity and target binding:

(a) Activity analysis based on Biacore;

(b) ELISA-based activity assay;

(c) Pharmacodynamic biomarker analysis of ristocetin-induced cofactor activity (RICO);

(d) Gyrolab based affinity determination.

These assays allowed for a visual comparison of the liquid and lyophilized drug product ALX-0081 (caplacizumab). Predefined comparability criteria were used to assess comparability for each analysis, which are listed in Table 24.

7.13.2. Methods

(a) The Biacore assay is based on Surface Plasmon Resonance (SPR) technology and measures the avid binding of ALX-0081 to the human VW Al domain immobilized on a sensor chip. The assay was chosen to determine release activity and evaluate stability.

(b) The ELISA-based activity assay is an orthogonal assay for the activity of ALX-0081 that was developed to further characterize the targeted neutralizing ability of caplacizumab. This assay measures the inhibition of ristocetin-induced binding of von Willebrand factor (VWF) to platelets by caplacizumab.

(c) The RICO assay is used as a pharmacodynamic marker for the pharmacological activity of caplacizumab. The assay measures the rate and extent to which human lyophilized platelets form aggregates after the addition of the antibiotic ristocetin, which mimics shear-induced VW activation.

(d) Gyrolab-based analysis determines the kinetic interactions of caplacizumab with multidimensional VWF target, and determines the caplacizumab affinity constant for human multidimensional VWF.

Briefly, the affinity determination on the Gyrolab platform was performed as follows: Gyrolab Bioaffy 1000 CD was used. As a capture tool, 3000 nM native biotinylated purified VWF (purified by HaemateP using size exclusion chromatography) was applied to columns prepackaged with streptavidin coated beads. A series of purified HaemateP VWF diluted 1/3 was pre-incubated for 24 hours at room temperature (+20°C) in a 96-well plate on a rotor at 600 rpm with a fixed concentration of caplacizumab (5 pM) in AD1 buffer (buffer for dilutions in the quantitation for the dose-response curve). After 24 hours, the plate was centrifuged for 1 minute at 200 g. 70 μl of the pre-incubation mixture containing free molecules of caplacizumab was transferred to a deep well PCR plate. The mixture was then passed through the column so that free caplacizumab could bind to the biotinylated VWF immobilized on the column. The Gyrolab system automatically transferred the mixture in triplicate to CD. Free caplacizumab was detected with 50 nM AlexaFluor647-labeled anti-caplacizumab monoclonal antibody diluted in Rexxip F buffer (commercial detection buffer). Three independent experiments were performed to determine the final KD. Fluorochromes were excited with a red laser so that fluorescent signals were obtained and amplified by a photomultiplier (PMT). The level of amplification in this assay was 1% PMT. An unknown ligand assay model was used to determine the caplacizumab CD. The analysis was performed with XL customized Gyrolab workstation software.

7.13.3. results

(a) The relative potency of liquid and lyophilized ALX-0081 prototypes was determined in the Biacore potency assay, relative to the ALX-0081 reference material used in the potency assay, also referred to as master standard 2 (MRS-2). The relative activity was 102.8% and 102.9%, respectively, indicating full comparability with respect to the biological activity determined by Biacore (see table 24).

(b) The relative potency of liquid and lyophilized ALX-0081 prototypes was determined in an ELISA based potency assay relative to MRS-2. Relative potency values were 99.4% and 109.5%, respectively, which met the comparability criteria well (see Table 24). Thus, these results show that both formulations are comparable in terms of potency as determined by ELISA.

(c) RICO activity of liquid and lyophilized ALX-0081 prototypes was measured in side-by-side comparison, and the concentration completely blocking RICO activity (<20%) was determined. The concentration completely blocking RICO activity (<20%) was ≤0.4 μg/ml for both compositions. These results are in good agreement with the criteria for comparability (see table 24), and indicate full comparability in terms of pharmacodynamic activity of both compositions.

(d) The affinity constant (KD value) of the liquid and lyophilized ALX-0081 prototypes was also determined in side-by-side comparison by Gyrolab-based assay. The CD values were 6.84 pM and 4.46 pM, respectively, with overlapping confidence intervals. Thus, these results show complete comparability of both formulations in terms of affinity for the multimeric target VWF (see Table 24).

7.13.4. Conclusion

The aim of this study was to evaluate the in vitro comparability of liquid and lyophilized drug product ALX-0081 (caplacizumab) through four assays capable of evaluating in vitro biological activity and target binding:

(a) Activity analysis based on Biacore;

(b) ELISA-based activity assay;

(c) Pharmacodynamic biomarker analysis of ristocetin-induced cofactor activity (RICO);

(d) Gyrolab based affinity determination.

All in vitro assays met predetermined compliance criteria and showed that both formulations of ALX-0081 are comparable in terms of biological activity and target binding (see Table 24). The tested liquid and lyophilized compositions of LP ALX-0081 showed:

- similar relative activity as determined by Biacore and ELISA assays;

- comparable in vitro pharmacodynamic activity (target neutralization) in the RICO assay;

- comparable target affinity in the Gyrolab assay.

7.14. Accelerated and long-term stability analysis of liquid and lyophilized formulations ALX-0081

In addition to Example 7.12, independent stability experiments were performed using another batch of ALX-0081 of the same composition (20 mM citrate buffer, pH 6.5; with 7% sucrose (w/v) and 0.01% Tween- 80 (o/o)).

The stability of the lyophilized and liquid composition was tested at different temperatures:

- Liquid composition with 13.8 mg/ml ALX-0081 in 20 mm citrate buffer, pH 6.5; with 7% sucrose (w/v) and 0.01% Tween-80 (v/v) were stored at ≤-60°C and +5°C (±3°C) and analyzed for stability at various time points, those. at baseline, at 9 months, 12 months, 18 months, and 24 months.

- Lyophilized composition with 12.7 mg/ml ALX-0081 in 20 mm citrate buffer, pH 6.5; with 7% sucrose (m/o) and 0.01% Tween-80 (o/o) were stored at +5°C (±3°C), +25°C (±2°C/60±5% RH) and +40°C (±2°C/75±5% RH). Like the liquid composition, the stability of the lyophilized composition was determined at baseline and after 9, 12, 18 and 24 months.

At each time point, the chemical and physical stability of the samples was monitored using a variety of analytical techniques, including cIEP, RP-HPLC, E-HPLC, visual analysis, pH determination, and UV absorbance.

The moisture content of the lyophilized sample was determined by coulometric titration. The relative activity of liquid and lyophilized samples was measured in Biacore relative to the proprietary standard sample ALX-0081.

Detailed data on the characterization of liquid and lyophilized samples are given in tables 25-26 and 27-29, respectively. Samples meeting the criteria set out in column 2 of each of the above tables were considered to meet the product specifications.

The obtained data showed that the composition in accordance with the present invention is highly stable for at least 24 months. The physicochemical characteristics as well as the biological activity of lyophilized ALX-0081 were not significantly affected by storage for 24 months at +5°C or +25°C. When ALX-0081 was stressed for 24 months at +40°C, an increase in the subsequent peak 2 was observed, which corresponded to the formation of the pyroglutamate variant from 1.1% in the starting material to 2.8%, 3.2%, 4.2% and 6.2% after 9, 12, 18 and 24 months, respectively.

Storage of liquid formulations of ALX-0081 for at least 24 months at temperatures ≤ -60°C or at +5°C did not significantly affect their physico-chemical stability: the content values were stable, the samples remained transparent, and the cIEF profiles, RP-HPLC and E-HPLC of the starting material were comparable to the sample profiles in the stability analysis.

The changes noted for lyophilized samples stored at +40°C may be associated with stressful storage conditions, and provide a good indicator of stability during long-term storage under milder conditions.

Forecast of long-term stability

Modern drug product specifications state that the allowable content of pyroglutamate is ≤4%. Based on these specifications and current stability data, the Arrhenius equation was used to predict the shelf life of the lyophilized drug product at +5°C and +25°C. The Arrhenius equation is an exact formula describing the temperature dependence of a reaction rate that is commonly used in the pharmaceutical industry. As shown in Figures 10 and 11, the lyophilized drug product was expected to remain within specifications for at least 500 months when stored at +5°C and for at least 60 months when stored at +25°C.

7.15. General conclusion

The invention of changing the composition of VW binders, and especially ALX-0081 described in this application, provides a new composition based on citrate/sucrose with improved solubility (up to 80 mg/ml) and significantly improved storage stability in liquid form (for example, less oxidation compared to the original composition). In addition, essentially no oxidation or asp isomerization was observed in the lyophilized form after 12 or even 24 months of storage at +40°C. Further optimization of the citrate and sucrose concentration resulted in a reduction in the moisture content of the lyophilized product, thus minimizing the rate of pyroglutamate formation. Each physicochemical characteristic of a VW binder has been shown to be significantly affected by various compositional factors, physical and chemical, such as buffer selection, pH, concentration, excipients, etc. Various compositions are provided that are optimized to eliminate or prevent various types of chemical and/or physical stress.

One formulation buffer was formulated to meet most of the critical criteria: 20 mM citrate, pH 6.5 + 7.0% sucrose (w/v) + 0.01% Tween-80 (v/v). Using this formulation, ALX-0081 has been shown to be stable for at least 12 or even 24 months at -20°C, +5°C, +25°C and +40°C. These data clearly indicate a significantly extended shelf life at +5°C compared to the current liquid formulation.

In addition, the present inventors have comprehensively demonstrated that the current ALX-0081 formulation that has been used in clinical studies to date is comparable to the novel, optimized ALX-0081 lyophilized formulation presented in this application in terms of biological activity and target binding. in vitro.

Table A-1. Examples of agents that bind to VWF

Name SEQID NO Subsequence 12A02H1-3a-12A02H1
(ALX-0081) one EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02-3a-12A02 2 QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02-GS9-12A02 3 QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02-GS30-12A02 four QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEGTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A05-3a-12A05 5 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSS 12A05-GS9-12A05 6 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSS 12A05-GS30-12A05 7 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSS 12B06-3a-12B06 eight QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSAAAEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12B06-GS9-12B06 9 QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12B06-GS30-12B06 ten QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12A02H4-3a-12A02H4 eleven EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12B06H2-3a-12B06H2 12 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12A02H1-GS9-12A02H1 13 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02H4-GS9-12A02H4 fourteen EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12B06H2-GS9-12B06H2 fifteen EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12A02H1-GS30-12A02H1 16 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02H4-GS30-12A02H4 17 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12B06H2-GS30-12B06H2 eighteen EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12A02H1 19 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS

Table A-2

Name SEQID NO Subsequence Human PV twenty MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYLLAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDIQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFD GIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPHMAQVTVGPGLRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKH SALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK

Table 1. Overview of 50 different buffer/auxiliary combinations predicted by Design Expert to produce the highest melting points for ALX-0081. The buffer/auxiliary combinations are ordered according to Tp value. Different types of buffers are shown in different shades of gray.

Cycle Buffer Filler 1 Glycine NaCl Tp Name Conc. (mM) Name Conc. (mM) Conc. (mM) Conc. (mM) °C one Phosphate, pH 6.92 17.24 Trehalose 239.25 0.00 0.00 77.2772 2 Phosphate, pH 6.98 16.29 Trehalose 242.04 0.00 0.00 77.2742 3 Phosphate, pH 6.95 9.47 Trehalose 262.12 0.00 0.00 77.2641 four Phosphate, pH 7.50 9.47 Mannitol 0.00 273.04 0.00 77.2096 5 Phosphate, pH 7.50 25.19 Mannitol 0.00 224.82 0.00 77.1483 6 Phosphate, pH 7.50 9.47 sucrose 0.00 273.04 0.00 77.1399 7 Phosphate, pH 7.50 28.79 Mannitol 0.00 213.78 0.00 77.1262 eight Phosphate, pH 7.50 30.88 Mannitol 0.00 207.39 0.00 77.1117 9 Phosphate, pH 7.50 32.96 Mannitol 0.00 201.00 0.00 77.0953 ten Citrate, pH 6.23 48.16 Trehalose 162.85 0.00 0.00 77.0307 eleven Citrate, pH 6.22 48.38 Trehalose 162.29 0.00 0.00 77.0307 12 Phosphate, pH 6.87 19.89 Mannitol 231.44 0.00 0.00 76.9832 13 Phosphate, pH 7.50 9.47 Trehalose 0.00 273.04 0.00 76.9483 fourteen Citrate, pH 7.00 60.84 sucrose 129.38 0.00 0.00 76.9338 fifteen Citrate, pH 7.00 57.67 sucrose 137.75 0.00 0.00 76.9312 16 Phosphate, pH 7.50 50.01 Mannitol 0.00 148.72 0.00 76.9295 17 Citrate, pH 7.00 84.92 sucrose 65.25 0.00 0.00 76.7979 eighteen Phosphate, pH 7.06 36.18 sucrose 183.48 0.00 0.00 76.7972 19 Citrate, pH 6.44 10.56 Trehalose 262.12 0.00 0.00 76.7449 twenty Citrate, pH 7.00 77.11 Mannitol 0.00 90.04 0.00 76.7297 21 Citrate, pH 7.00 75.42 Mannitol 0.00 94.69 0.00 76.7291 22 Citrate, pH 7.00 79.01 Mannitol 81.42 0.00 0.00 76.6192 23 Citrate, pH 6.17 53.45 Mannitol 146.67 2.90 0.00 76.5956 24 Citrate, pH 6.18 53.23 Mannitol 149.46 0.00 0.00 76.5955 25 Citrate, pH 6.18 53.23 Mannitol 149.46 0.00 0.00 76.5955 26 Citrate, pH 6.16 53.66 Mannitol 148.35 0.00 0.00 76.5955 27 Tris, pH 7.77 17.13 Trehalose 134.96 0.00 69.29 76.1017 28 Tris, pH 8.00 89.75 Mannitol 0.00 15.10 70.51 76.0374 29 Tris, pH 8.00 92.83 Mannitol 0.00 16.27 67.17 76.0343 thirty Tris, pH 8.00 93.86 Mannitol 0.00 0.00 74.76 76.0322 31 Tris, pH 7.83 17.13 sucrose 142.77 0.00 65.04 76.0321 32 Tris, pH 7.82 17.13 sucrose 142.21 0.00 65.34 76.0321 33 Tris, pH 8.00 82.56 Mannitol 0.00 31.37 68.38 76.0298 34 Tris, pH 8.00 97.63 Mannitol 0.00 0.00 71.42 76.0285 35 Tris, pH 8.00 95.23 sucrose 0.00 0.00 73.55 75.9714 36 Tris, pH 8.00 97.63 sucrose 0.00 0.00 71.42 75.97 37 Tris, pH 7.77 17.13 Mannitol 121.58 0.00 76.59 75.5801 38 Tris, pH 8.00 61.32 Trehalose 0.00 53.45 75.37 75.3047 39 Tris, pH 8.00 67.14 Trehalose 0.00 47.06 73.85 75.3027 40 Tris, pH 7.81 17.13 Trehalose 0.00 140.00 69.60 75.2196 41 Histidine, pH 6.50 20.03 sucrose 0.00 142.33 68.38 74.9111 42 Histidine, pH 6.50 20.03 sucrose 0.00 136.52 71.42 74.91 43 Histidine, pH 6.50 20.03 sucrose 0.00 124.90 77.50 74.9012 44 Histidine, pH 6.50 20.03 Mannitol 0.00 127.22 76.28 74.8582 45 Histidine, pH 6.50 20.03 Mannitol 0.00 131.29 74.16 74.8576 46 Histidine, pH 6.50 20.03 Mannitol 0.00 118.51 80.84 74.8558 47 Histidine, pH 6.50 20.03 Mannitol 0.00 136.52 71.42 74.8553 48 Histidine, pH 6.50 20.83 Mannitol 0.00 109.21 85.10 74.8397 49 Histidine, pH 6.49 20.03 Mannitol 0.00 146.98 65.95 74.8281 fifty Histidine, pH 6.50 20.03 Trehalose 0.00 144.07 67.47 74.4053

Table 2. Solubility analysis of ALX-0081 in various formulation buffers

Composition pH visual analysis Measured concentration
(mg/ml) Exit
(%) D-PBS + 200 mM
Glycine 7.4 Cloudy sample + small particles 8.1 88.6 10 mM phosphate + 200
mM glycine 7.4 Cloudy sample + small particles 8.7 88.3 20 mM phosphate 7.4 Cloudy sample + particles 4.9 96.4 20 mM histidine 6.5 Cloudy sample + particles <3.4 N.D. 20 mM citrate 7.0 transparent sample 55.9 97.6

N.D. = not defined

Table 3. Overview of ALX-0081 Liquid Formulations in Storage and 3D Tests, along with Measured pH and Osmolality

composition number Conc.
(mg/ml) Buffer Excipient Twin-80
(o/o) Measured pH Osmolality
mOsm/kg Type of pH Concentration Type of one twenty 50 mM citrate 6.0 75 mm NaCl 0.01% 5.9 281 2 2.0% mannitol 6.0 253 3 4.0% sucrose 6.0 272 four 140 mm Glycine 6.0 273 5 6.5 75 mm NaCl 6.5 288 6 2.0% mannitol 6.5 266 7 4.0% sucrose 6.6 280 eight 140 mm Glycine 6.6 280 9 7.0 75 mm NaCl 6.9 279 ten 2.0% mannitol 7.0 259 eleven 4.0% sucrose 7.1 271 12 140 mm Glycine 7.0 278 13 5 75 mm NaCl 6.9 274 fourteen 2.0% mannitol 7.0 254 fifteen 4.0% sucrose 7.0 267 16 140 mm Glycine 7.0 274 17 D-PBS 7.1 137/ 200 mm NaCl/
Glycine 7.2 470

Table 4. Overview of the storage stability study of various formulations of ALX-0081. Points of time, temperatures and storage methods are indicated

Moments in time Temperature Composition No. as indicated in Table 3 Methods +40°C RP-HPLC KIEF E-HPLC Week 1 X X 1-17 X 2 weeks X X 1-17 X 1 month X X 1-17 X X X

Table 5. Storage stability data for various ALX-0081 liquid formulations. The relative surface areas of most of the corresponding RP-HPLC peaks are shown after 1 month of storage at +40°C. Pyro = pyroglutamate, Main. = main peak (including shoulder peak when present), oxid. = total preliminary peaks. The color code indicates the relative purity of the samples: the highest purity is indicated in white, intermediate purity in gray, and the lowest purity in black. The yield was about 100% for all samples

composition number Conc.
(mg/ml) Buffer Excipient Twin-80
(o/o) % peak area RP-HPLC Type of pH Conc. Type of Oxidized Main Pyro one twenty 50 mM citrate 6.0 75 mm NaCl 0.01% 1.5 88.6 3.7 2 2.0% mannitol 1.7 88.1 4.3 3 4.0% sucrose 1.7 88.1 4.6 four 140 mm glycine 2.2 88.3 3.9 5 6.5 75 mm NaCl 2.5 88.5 3.6 6 2.0% mannitol 2.7 87.5 4.5 7 4.0% sucrose 2.3 88.0 4.2 eight 140 mm glycine 4.6 85.2 4.3 9 7.0 75 mm NaCl 2.4 86.8 5.2 ten 2.0% mannitol 2.6 86.0 5.9 eleven 4.0% sucrose 2.8 85.9 6.1 12 140 mm Glycine 4.7 82.8 6.2 13 5 75 mm NaCl 2.3 86.8 5.2 fourteen 2.0% Mannitol 2.4 86.5 5.8 fifteen 4.0% sucrose 2.1 87.7 5.2 16 140 mm glycine 5.0 83.1 6.4 17 D-PBS 7.1 137/200 mm NaCl/
glycine 9.0 75.2 6.4

Table 6 Overview of ALX-0081 Lyophilized/Liquid Formulations Evaluated in the Storage Stability Test

Table 7 Yield of ALX-0081 in various formulations after lyophilization and reconstitution, based on total area, by RP-HPLC and E-HPLC

Yield after lyophilization/reconstitution (%) Citrate, pH 6.0 + sucrose
(track 3) Citrate, pH 6.5 + sucrose
(track 7) D-PBS + glycine
(track 17) RP-HPLC 104.3 105.4 103.2 E-HPLC 101.3 99.6 102.8

Table 8. Overview of storage stability data for various ALX-0081 lyophilized formulations (1.5 months storage at -20°C, +5°C, +25°C and +40°C). The color code represents a qualitative assessment of sample stability ranging from white (highest stability) through gray to black (least stable)

N.T. = not tested (* compared to a liquid control stored at -70°C).

Table 9 Overview of ALX-0081 Compositions Evaluated in the Storage Stability Test

Table 10 Moisture content of ALX-0081 lyophilized samples and relative amount of pyroglutamate detected by RP-HPLC after 4 weeks storage at +40°C

Table 11. Results of visual analysis of the composition ALX-0081 during storage at +5°C and +25°C

"+" = transparent; "+/-" = slightly hazy; "-" = hazy; "h" = hours; "d" = days.

Composition Storage at +5°C Storage at +25°C Conc.
(mg/ml) Citrate pH 6.5 (mM) Sucrose m/o (%) Twin-80 o/o (%) 1h 2h 19h 24h 4d 1h 2h 19h 24h 4d 28 fifteen ---- ---- - - - - - + + + + + twenty ---- ---- + + + - - + + + + + 25 ---- ---- + + + - - + + + + + thirty ---- ---- + + + - - + + + + + 40 ---- ---- + + + - - + + + + + fifty ---- ---- + + + + + + + + + + twenty fifteen 5.0 ---- + + + + +/- + + + + + fifteen 6.0 ---- + + + + + + + + + + fifteen 7.0 ---- + + + + + + + + + + fifteen 5.0 0.01 + + + + +/- + + + + + fifteen 6.0 0.01 + + + + + + + + + + fifteen 7.0 0.01 + + + + + + + + + +

Table 12. Results of visual analysis, content determination, and E-HPLC analysis of ALX-0081 compositions after 5 consecutive 30 cycles at -20°C (*compared to liquid control stored at ≤-70°C)

Composition 5 3D cycles at -20°C Conc.
(mg/ml) Citrate, pH 6.5 (mM) Sucrose m/o (%) Twin-80 (v/v) (%) Visually Content E-HPLC Exit (%)* Profile Exit (%)* 16 twenty 5.0 0.01 Transparent 100 no influence 105.3 6.0 102 99.4 7.0 99.0 103.4 25 5.0 101 103.9 6.0 102 98.5 7.0 100 98.4 thirty 5.0 97.2 98.8 6.0 103 99.3 7.0 97.5 102.8

Table 13. Visual, Yield, and Osmolality Results of ALX-0081 Compositions after 5 consecutive SR cycles at -20°C or after 1 SR cycle + 24 hours storage + 1 SR cycle (*compared to liquid control stored at ≤ -70°C)

Table 14. Lyophilization parameters

stage number Description Temperature (°C) Pressure Time (hours:min) one Loading twenty atmospheric N.A. 2 Freezing 20 → -50 atmospheric 02:00 3 Freezing -fifty atmospheric 02:00 four vacuuming -fifty 0.130 mbar 00:10 5 Primary drying -50 → -20 0.130 mbar 1:00 6 Primary drying -twenty 0.130 mbar 19:00 7 Primary drying -20 → 5 0.130 mbar 00:50 eight Primary drying 5 0.130 mbar 05:00 7 Primary drying 5 → 25 0.130 mbar 03:00 7 Secondary drying 25 0.130 mbar 33:00 9 Pre-aeration with nitrogen fifteen 0.8 bar N.A. ten capping fifteen 0.8 bar N.A. eleven Aeration with nitrogen fifteen atmospheric N.A. Total duration (without capping) 66:00

Table 15. RP-HPLC analysis of the main peak (purity) of the composition ALX-0081 (12.5 mg/ml API, 0.01% Tween-80 (v/v) and 7% sucrose (w/v) in 20 mM citrate buffer at pH 6.5)

Points in time (months) Storage conditions Average purity
(% main peak area) Initially - 93.3 one 93.0 +5°C 93.0 +25°C/60%RH 93.0 +40°C/75%RH 92.7 3 93.3 +5°C 93.3 +25°C/60% RH 93.1 +40°C/75% RH 92.6 6 93.2 +5°C 93.3 +25°C/60% RH 93.0 +40°C/75% RH 92.4 9 93.4 +5°C 93.3 +25°C/60% RH 93.1 +40°C/75% RH 91.8 12 93.2 +5°C 93.1 +25°C/60% RH 92.8 +40°C/75% RH 91.3

Table 17 UV protein concentration results for formulation ALX-0081 (12.5 mg/ml API, 0.01% Tween-80 (v/v), and 7% sucrose (w/v) in 20 mM citrate buffer at pH 6.5)

Points in time (months) Storage conditions Average concentration of the diluted sample (mg/ml) Average concentration corrected for dilution factor (mg/vial) Initially - 0.534 13.4 one 0.532 13.3 +5°C 0.530 13.3 +25°C/60% RH 0.525 13.1 +40°C/75% RH 0.516 12.9 3 0.501 12.5 +5°C 0.524 13.1 +25°C/60% RH 0.530 13.3 +40°C/75% RH 0.534 13.4 6 0.530 13.3 +5°C 0.528 13.2 +25°C/60% RH 0.531 13.3 +40°C/75% RH 0.523 13.1 9 0.505 12.6 +5°C 0.504 12.6 +25°C/60% RH 0.511 12.8 +40°C/75% RH 0.519 13.0 12 0.504 12.6 +5°C 0.505 12.6 +25°C/60% RH 0.497 12.4 +40°C/75% RH 0.510 12.7

Table 18. E-HPLC analysis of ALX-0081 formulation (12.5 mg/ml API, 0.01% Tween-80 (v/v), and 7% sucrose (w/v) in 20 mM citrate buffer at pH 6 ,5)

Points in time (months) Storage conditions Average of preliminary peaks (% area) Average of main peak
(% area) Initially - 0.55 99.5 one 0.51 99.5 +5°C 0.53 99.5 +25°C/60% RH 0.55 99.4 +40°C/75% RH 0.56 99.5 3 0.47 99.6 +5°C 0.47 99.5 +25°C/60% RH 0.47 99.5 +40°C/75% RH 0.48 99.5 6 0.60 99.4 +5°C 0.63 99.4 +25°C/60% RH 0.65 99.4 +40°C/75% RH 0.68 99.3 12 0.66 99.4 +5°C 0.68 99.3 +25°C/60% RH 0.67 99.3 +40°C/75% RH 0.71 99.3

Table 19. Results of physical analyzes of lyophilized ALX-0081 stored at -20°C (12.5 mg/ml API, 0.01% Tween-80 (v/v), and 7% sucrose (w/v) at 20 mM citrate buffer at pH 6.5)

Equivalents

The above written description is considered sufficient to enable the implementation of the present invention by a person skilled in the art. The present invention is not limited by the scope of the examples provided, as the examples are only intended to illustrate one aspect of the invention, and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention, in addition to those shown and described in this application, will become clear to a person skilled in the art from the previous description, and fall within the scope of the claims. The advantages and objects of the present invention are not necessarily covered by every embodiment of the invention.

The contents of all references, patents and published patent applications cited throughout this application are incorporated by reference in their entirety, in particular for the application or subject matter mentioned in this application.

Claims (40)

1. Composition for the treatment or prevention of VWF-dependent diseases, containing an agent that binds von Willebrand factor (VWF), a citrate buffer, an excipient and Tween-80, where: (a) the VWF binding agent has a concentration of from about 0.1 mg/mL to 80 mg/mL; (b) the excipient is sucrose at a concentration of about 5% to about 15% (w/v); (c) Tween-80 has a concentration of about 0.001% to 0.5% (v/v); and (d) the citrate buffer has a concentration of about 5 mM to 200 mM such that the pH of the composition is about 6.0 to 7.0; and where the specified agent that binds VWF, contains at least one of SEQ ID NO: 1-19. 2. The composition of claim 1, wherein said VWF binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, preferably said amino acid sequence is identical to SEQ ID NO: 1. 3. Composition according to any one of claims 1, 2, where: (a) the VWF binding agent has a concentration of 12.5 mg/ml; (b) sucrose has a concentration of 7% (w/v); (c) Tween-80 has a concentration of 0.01% (v/v); (d) citrate buffer has a concentration of 20 mm; and (e) the composition has a pH of 6.5. 4. Composition according to any one of claims 1-3, containing: (i) less than 5% high molecular weight (HMW) compounds, which are oligomers, polymers and aggregates of the agent that binds VWF, after storage for at least 12 months at 5°C; and/or (ii) less than 5% low molecular weight (LMW) compounds, which are fragments of the agent that binds VWF, after storage for at least 12 months at 5°C. 5. A composition according to any one of claims 1 to 4, wherein the VWF binding agent in the composition retains at least about 80% of its stability after storage for at least 12 months at 5°C. 6. Composition according to any one of claims 1 to 5, wherein at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% of the VWB binder retains its binding activity after storage compared to the binding activity before storage, where the specified binding activity is measured by ELISA and/or Biacore. 7. The composition according to any one of claims 1 to 6, which is a liquid or reconstituted lyophilized composition, comprising: (a) a VWF binding agent at a concentration of from about 0.1 mg/ml to 80 mg/ml, preferably 12.5 mg/ml; (b) sucrose at a concentration of from about 5% to 15% (w/v), preferably 7% (w/v); (c) Tween-80 at about 0.001% to about 0.5% (v/v), preferably 0.01% (v/v); and (d) citrate buffer at a concentration of about 5 mM to 200 mM, preferably 20 mM, such that the pH of the composition is about 6.0 to 7.0, preferably 6.5. 8. Composition according to any one of claims 1-6, where at least 100 liters of the composition is stored under conditions below freezing. 9. A composition according to any one of claims 1 to 8 which is in liquid, lyophilized, spray dried, reconstituted lyophilized, or frozen form. 10. A method for preparing a composition according to any one of claims 1 to 9, including the steps: - expression of an agent that binds VWF in cell culture; - purifying the VW binder by passing the VW binder through at least one chromatographic purification step and an ultrafiltration/diafiltration step; - bringing the concentration of the VWF binding agent to about 0.1-80 mg/ml, preferably 12.5 mg/ml, in a composition containing: (i) sucrose at a concentration of from about 5% to 15% (w/v), preferably 7% (w/v); (ii) Tween-80 at about 0.001%-0.5% (v/v), preferably 0.01% (v/v); and (iii) citrate buffer at a concentration of about 5 mM to 200 mM, preferably 20 mM, such that the pH of the composition is about 6.0 to 7.0, preferably 6.5. 11. A method for preparing a reconstituted composition according to any one of claims 1 to 9, comprising the steps of: (i) lyophilizing a mixture of VW binder, lyoprotectant, surfactant and buffer to obtain a lyophilized mixture; and (ii) reconstituting the lyophilized mixture in a solvent to form a composition, wherein the reconstituted composition comprises: (a) a VWF binding agent at a concentration of from about 0.1 mg/ml to 80 mg/ml, preferably 12.5 mg/ml; (b) sucrose at a concentration of from about 5% to 15% (w/v), preferably 7% (w/v); (c) Tween-80 at about 0.001% to about 0.5% (v/v), preferably 0.01% (v/v); and (d) citrate buffer at a concentration of about 5 mM to 200 mM, preferably 20 mM, such that the pH of the composition is about 6.0 to 7.0, preferably 6.5. 12. A kit for the treatment or prevention of vWF-dependent diseases, including a container containing a composition according to any one of claims 1 to 9, and instructions for use. 13. The kit according to claim 12, wherein the composition is present in a vial or syringe for injection.

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