Liquid botulinum toxin preparations and uses thereof

Abstract

The present invention relates to a liquid formulation comprising (i) a botulinum toxin; (ii) human serum albumin (HSA); and, optionally, (iii) a tonicity agent and / or (iv) a buffering agent. In one embodiment, the liquid formulation is characterized by a very low concentration of iron ions. In another embodiment, the liquid formulation is characterized by the absence or very low concentration of tryptophan and / or N-acetyltryptophan. According to the present invention, the liquid formulation can be prepared by a method comprising the steps of contacting human serum albumin with a chelating agent to obtain a mixture and removing the chelating agent from the mixture. Furthermore, the present invention relates to the use of the liquid formulation in the treatment of therapeutic indications and cosmetic conditions.

Description

[Technical field]

[0001] The present invention relates to a liquid formulation comprising (i) a botulinum toxin; (ii) human serum albumin (HSA); and, optionally, (iii) a tonicity agent and / or (iv) a buffering agent. The liquid formulation is characterized by an extremely low iron concentration and / or the absence or extremely low concentration of tryptophan and / or N-acetyltryptophan. The present invention also relates to a liquid formulation prepared by a method comprising contacting human serum albumin with a chelating agent to obtain a mixture and removing the chelating agent from the mixture. Furthermore, the present invention relates to the use of the liquid formulation in the treatment of therapeutic indications and cosmetic conditions. [Background technology]

[0002] Botulinum neurotoxins (BoNT; or botulinum toxins (BT)) are a type of bacterial neurotoxin that has come to be widely used to treat a variety of neurological, medical, and cosmetic conditions. There are eight widely recognized "classical" BoNT serotypes, designated BoNT / A-H. Currently, two serotypes, type A (BoNT / A) and type B (BoNT / B), are in clinical use. BoNTs are produced in the form of high molecular weight (up to approximately 900 kDa) complexes by Clostridium species, particularly Clostridium botulinum. These toxin complexes consist of an active 150 kDa neurotoxin and several complexing proteins (non-toxic neurotoxin associated proteins, NAPs).

[0003] The 150 kDa neurotoxin is synthesized as an inactive single polypeptide chain (~150 kDa) that is proteolytically cleaved to a light chain (LC, ~50 kDa) and a heavy chain (HC, ~100 kDa) connected by interchain disulfide bonds. The HC contains a C-terminal domain that mediates receptor binding and an N-terminal domain that mediates translocation of the LC across the endosomal membrane. The LC acts as a protease in neurons, cleaving neuronal SNARE proteins, which inhibits fusion of synaptic vesicles with the plasma membrane and suppresses neurotransmitter release from certain neurons.

[0004] Formulation of BoNT is very challenging due to its complex structure and the low product concentrations used. BoNT is highly sensitive to a variety of conditions, including heat and alkaline pH. Thus, since BoNT is only functional if its structure is intact, the challenge in preparing a medical dosage form of BoNT is to formulate a composition that protects BoNT from inactivation or partial loss of biological activity during manufacture, storage, or use of the product. Another problem is that pharmaceutical formulations of BoNT contain extremely small amounts of the potent toxin (lethal dose in humans is about 0.1-1 ng / kg), ranging from only about 1 ng per vial. This further exacerbates the problem of loss of toxin activity due to surface denaturation. Furthermore, formulations of BoNT for a wide variety of applications must be compatible for injection into widely different types of tissues, such as muscle, layers of the skin (dermis, subcutaneous tissue, etc.), or glands (e.g., salivary glands).

[0005] In view of the above, the main BoNT products currently available are provided as lyophilized powders, i.e. in a form that is stable for long periods even when stored at 2-8°C or at room temperature (e.g., Xeomin®). Lyophilized forms of the BoNT / A complex were first introduced to the market in 1989 (Botox®, Allergan) and 1991 (Dysport®, Ipsen). In 2005, the first stable formulation of the pure 150 kDa BoNT / A neurotoxin without complexing proteins was approved (Xeomin®; Merz Pharmaceuticals). However, these lyophilized products need to be reconstituted before use, a process that can lead to administration errors and sterility problems. Therefore, great efforts have been made to develop liquid formulations of BoNT that are convenient to use and can be easily administered.

[0006] The first liquid formulation of BoNT was approved in 2000 and launched in Europe in 2001 as Neurobloc®. Neurobloc® (Eisai) is a sterile solution of the BoNT / B complex in a buffer containing disodium succinate, sodium chloride, human serum albumin (HSA), sodium caprylate, and sodium N-acetyltryptophan. However, the product has an acidic pH, which can make it painful when injected. Only two liquid formulations of BoNT / A have been approved for sale to date: Innotox® (Medytox), limited to domestic Asian markets (e.g., South Korea, approved in 2013), and Alluzience® (Ipsen / Galderma), approved for use in Europe in 2021. All of these liquid formulations contain, in addition to the BoNT / A toxin complex, water, sodium chloride, surfactants (Innotox®: polysorbate 20; Alluzience®: polysorbate 80), amino acids (Innotox®: methionine; Alluzience®: histidine), and excipients (Innotox®: sodium phosphate as a buffer; Alluzience®: sucrose).

[0007] Despite these advances in the preparation of liquid botulinum toxin formulations, there remains an ongoing need for stable liquid formulations of botulinum toxin, and in particular, there remains a demand for new options for developing ready-to-use liquid formulations of botulinum toxin that are stable during shipping and storage.

[0008] Object of the invention It is therefore an object of the present invention to provide a stable liquid formulation of a botulinum toxin suitable for use in cosmetic and therapeutic applications. Summary of the Invention

[0009] The present invention is based on the surprising discovery that human serum albumin (HSA), pretreated by incubation with a chelating agent such as EDTA and subsequent removal of the chelating agent, allows for the preparation of liquid formulations of botulinum toxin that exhibit not only good storage stability but also improved photostability. Furthermore, the present invention provides a method for the preparation of liquid formulations of botulinum toxin that exhibit good storage stability as well as improved photostability. 3+ This is based on the surprising discovery that ions have a detrimental effect on the photostability of liquid botulinum toxin formulations containing HSA.

[0010] Furthermore, the present invention is based on the surprising discovery that tryptophan and N-acetyltryptophan reduce the photostability of liquid botulinum toxin formulations containing human serum albumin (HSA). This discovery is particularly surprising because, in addition to water and sodium chloride, commercially available HSA products typically contain sodium caprylate and N-acetyltryptophan to stabilize HSA at high temperatures, which leads to contamination of the botulinum toxin formulation. What is also surprising is that tryptophan has been described as a stabilizing additive for botulinum toxin (see, for example, EP 3679946).

[0011] That is, in a first aspect, the present invention provides a method for producing a composition comprising the steps of: (i) a botulinum toxin; and (ii) human serum albumin (HSA); A liquid formulation comprising: Fe 3+ ions at a concentration of less than 1 μM, preferably less than 500 nM, more preferably less than 250 nM, and most preferably less than 100 nM; A liquid formulation is provided.

[0012] Fe 3+ Liquid botulinum toxin formulations having a low concentration, such as less than 1 μM, can be prepared by a method comprising the steps of: (a) contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; and (b) removing the chelating agent from the mixture.

[0013] In a second aspect, the present invention provides a method for producing a composition comprising the steps of: (i) a botulinum toxin; and (ii) human serum albumin (HSA); A liquid formulation comprising: The liquid formulation does not contain tryptophan and N-acetyltryptophan or contains tryptophan and N-acetyltryptophan at 50 μM or less, preferably 20 μM or less, more preferably 10 μM or less, even more preferably 1 μM or less, and most preferably 0 μM (total concentration of both Trp and N-AcTrp); A liquid formulation is provided.

[0014] Liquid formulations according to the first and second aspects of the invention preferably further comprise: (iii) a tonicity agent, particularly sodium chloride; and (iv) a buffer, particularly a histidine buffer, a phosphate buffer, or a mixture thereof. Liquid botulinum toxin formulations that are free of, or contain only very low concentrations of, tryptophan and / or N-acetyltryptophan can be prepared by purifying an HSA starting material by methods such as dialysis and using the purified HSA material in formulating the liquid formulations of the invention.

[0015] In a third aspect, the present invention provides a method for producing a composition comprising the steps of: (i) a botulinum toxin; and (ii) human serum albumin (HSA); A liquid formulation comprising: The human serum albumin is (a) contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; and (b) removing the chelating agent from the mixture; prepared by a process comprising: A liquid formulation is provided.

[0016] The contacting step (a) is preferably carried out by adding a chelating agent (e.g., EDTA) to a composition comprising HSA (e.g., an HSA solution), incubating the resulting mixture for a period of time, and then optionally dialyzing against a buffer containing the chelating agent (e.g., EDTA). In step (b), the chelating agent is removed by any suitable method, for example by dialysis.

[0017] Furthermore, the liquid formulation according to the third aspect of the present invention preferably further comprises: (iii) an isotonicity agent, in particular sodium chloride; and / or (vi) a buffering agent, in particular a histidine buffer, a phosphate buffer, or a mixture thereof.

[0018] In a fourth aspect, the present invention relates to a method for preparing a liquid formulation according to the second aspect of the invention, the method comprising the steps of: - purifying the human serum albumin starting material by dialysis, diafiltration, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, or field-flow fractionation, preferably dialysis, to obtain a purified human serum albumin material; - mixing the obtained purified human serum albumin with a botulinum toxin and, optionally, further ingredients, to obtain a liquid formulation; Includes.

[0019] Another method according to the fourth aspect of the present invention comprises the steps of: - purifying a liquid composition comprising the botulinum toxin and human serum albumin by subjecting said liquid composition to dialysis, diafiltration, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, or field-flow fractionation, preferably dialysis; - mixing the resulting purified liquid composition with further ingredients to obtain said liquid formulation; Includes.

[0020] In a fifth aspect, the present invention relates to a liquid formulation of the invention for use in therapy, in particular for use in the treatment of neuromuscular disorders, pain, sialorrhea, hyperhidrosis, urological disorders, and neurological disorders.

[0021] In a sixth aspect, the present invention relates to the cosmetic (aesthetic) use of a liquid formulation of the present invention for the treatment of a cosmetic condition, preferably a skin condition, in particular fine lines, folds or wrinkles of the skin.

[0022] In a seventh aspect, the present invention provides a method of treating a disease or condition comprising administering to an individual in need thereof an effective amount of a liquid formulation of the present invention.

[0023] Preferred embodiments of the liquid formulation, its uses and methods of use according to the present invention are set out in the accompanying claims.

[0024] The present invention will be more readily understood by reference to the following detailed description of the invention and the examples included therein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention is based on the unexpected discovery that the use of pretreated human serum albumin (HSA) in the formulation of liquid botulinum toxin formulations reduces photosensitivity and results in improved photostability. Furthermore, it was unexpectedly found that the use of pretreated HSA reduces photosensitivity and results in improved photostability after chelating agent treatment and subsequent dialysis. 3+ It has been found that iron (iron) dramatically increases the photosensitivity of liquid botulinum toxin formulations containing human serum albumin (HSA). This is surprising because iron is a relatively common and ubiquitous metal, and furthermore, other metal ions such as copper, cobalt, or nickel have not been found to exhibit such destabilizing effects.

[0026] Moreover, the present invention is based on the unexpected discovery that tryptophan and / or N-acetyltryptophan impair the photostability of liquid human serum albumin (HSA)-containing botulinum toxin formulations. In particular, it has been found that removal of N-acetyltryptophan from conventional HSA preparations by treatment with a chelating agent followed by dialysis results in liquid HSA-containing formulations of botulinum toxin with improved photostability.

[0027] The liquid formulation of the present invention does not need to be reconstituted before injection, and can be used immediately, and can be used in forms such as prefilled syringes, which provides safety and dosage accuracy compared to powdered freeze-dried botulinum toxin formulations.In addition, the excellent stability of liquid formulations makes transportation and storage, as well as handling by physicians, easier and simpler.In particular, the excellent light stability of the liquid formulation of the present invention simplifies the manufacturing process, eliminates the need for extensive light protection during product filling and packaging, and also reduces the possibility of loss of activity when exposed to light during storage in the physician's environment before use.In addition, the liquid formulation of the present invention does not contain any substance that enhances the pain of injection, which makes the formulation more acceptable to physicians and patients, especially in the aesthetic field.

[0028] Without wishing to be bound by theory, it is believed that the photosensitivity of the formulations of the present invention is due to the presence of N-acetyltryptophan and Fe, which are contained in the HSA used to prepare the liquid botulinum toxin formulation. 3+ However, the exact mechanism by which the removal of these substances enhances the ability of HSA to light-stabilize botulinum toxin remains unclear.

[0029] The term "comprising", as well as the terms "including" and "containing", and any of their variations, such as "comprise", "include" and "contain", are intended to refer to a non-exclusive inclusion, and a process, method, product-by-process, composition, or formulation that comprises, includes, or contains an element or list of elements may include not only those elements, but also other elements not explicitly listed for such process, method, product-by-process, composition, or formulation. In addition, within the framework of the present invention, it is intended that "comprise", "comprising", "include", "including", "contain", "containing", and any of their variations may be replaced with "consist", "consisting", or any of their variations (e.g., "consist essentially of"), which are understood to refer to an exclusive inclusion of the indicated elements.

[0030] As used in the context of the present invention, the terms "a," "an," and "the," and similar references are to be construed to encompass both the singular and the plural, and thus may relate to "at least one" or "more than one," unless otherwise indicated herein or clearly contradicted by context.

[0031] The term "liquid formulation" or "liquid botulinum toxin formulation" as used herein generally refers to an aqueous formulation, typically an aqueous solution. Here, the term "liquid formulation" may be used interchangeably with "liquid composition". The above liquid formulation is generally a pharma- ceutically acceptable liquid formulation, i.e., a liquid pharmaceutical formulation. The liquid formulation of the present invention typically has a pH in the range of 5.0 to 8.0, particularly in the range of 5.5 to 7.5, preferably in the range of 5.5 to 7.0 or 6.0 to 7.5, more preferably in the range of 6.0 to 7.0, and most preferably in the range of 6.0 to 6.5.

[0032] As used herein, the term "pharmaceutical acceptable" means that the liquid formulation will not cause unacceptable adverse side effects when administered to a human patient or subject, i.e., the liquid formulation is suitable for human use. The aqueous solution may be a buffered solution, with or without saline, or may be a saline solution, such as a buffered (e.g., phosphate and / or histidine buffered) saline solution.

[0033] It is contemplated herein that the liquid formulation of the present invention can be stored in any suitable container system.The container system suitable for storing the liquid formulation of the present invention is any device that can be sealed or is sealed and has a partially or entirely enclosed space that can be used to contain, store, and / or transport liquid.The container system is preferably a closed (or sealed) container made of glass or plastic (such as organic polymers), or partially or mainly made of glass or plastic, and examples include containers in the form of (i) syringe, (ii) vial, (iii) carpoule, or (iv) ampoule.In a preferred embodiment of the present invention, the liquid formulation is stored in syringe in the form of a prefilled syringe, as known in the art.

[0034] The term "botulinum toxin" as used herein is not particularly limited and includes, for example, any serotype of botulinum toxin (e.g., BoNT / A-G). For example, the botulinum toxin may be serotype A or B botulinum toxin (BoNT / A, BoNT / B). Preferably, the botulinum toxin is serotype A, more preferably serotype A1 (BoNT / A1), and most preferably BoNT / A1 produced by the Clostridium botulinum strain Hall. Furthermore, the botulinum toxin may be a naturally occurring neurotoxin obtained from the bacterium Clostridium botulinum or any other botulinum toxin, such as a botulinum toxin obtained from an alternative source, such as by recombinant technology, and genetic or chemical modification.

[0035] Furthermore, the term "botulinum toxin" ("BT") as used herein, and its synonym "botulinum neurotoxin" ("BoNT"), unless otherwise specified or the context indicates otherwise, are intended to refer to pure botulinum neurotoxin and / or any complex thereof, i.e. any complex of pure botulinum neurotoxin with complexing proteins (referred to as "toxin complex"). Preferably, in the framework of the present invention, the botulinum toxin is a botulinum neurotoxin without complexing proteins, more preferably a botulinum neurotoxin of serotype A without complexing proteins.

[0036] The term "pure botulinum neurotoxin" as used herein refers to botulinum neurotoxin free of complexing proteins (sometimes also referred to as "neurotoxic component"), more precisely, botulinum neurotoxin free of neurotoxin-associated complexing proteins (NAPs). Pure botulinum neurotoxin is the (active) neurotoxic polypeptide that ultimately inhibits acetylcholine release. It is a two-chain protein with a light chain (LC; about 50 kDa) and a heavy chain (HC; about 100 kDa) linked together by a disulfide bond. Thus, the active neurotoxic polypeptide may also be referred to herein as "150 kDa neurotoxin", "botulinum neurotoxin (150 kD)", or "neurotoxic component".

[0037] The term "toxin complex" as used herein refers to a macromolecular complex of a neurotoxic component and a set of complexing proteins (NAPs). In particular, the term "toxin complex" includes the 900 kDa, 500 kDa, and 300 kDa Clostridium botulinum type A toxin complexes. The complexing proteins are the non-toxic non-hemagglutinin (NTNHA) and, in strains of serotypes A-D, the various hemagglutinins (HA). For example, onabotulinumtoxinA (Botox® / Vistabel®, Allergan, Inc., Irvine, Calif., USA) contains the 900 kDa complex. Also, Dysport® (Azzalure®, Ipsen, Paris, France), Alluzience® (Ipsen / Galderma), and Innotox® (Medytox) contain the toxin complex as the active agent. Thus, the toxin complex contained in Botox®, Dysport®, Alluzience® and Innotox® is a toxin complex within the meaning of the present invention and therefore a "botulinum toxin" according to the present invention. According to the present invention, the botulinum toxin may be present in the liquid formulation of the present invention in the range of 1-1000 U / ml, preferably in the range of 10-200 U / ml, more preferably in the range of 20-150 U / ml, most preferably in the range of 50 U / ml to 100 U / ml.

[0038] As used herein, the term "unit" or "U" refers to the biological activity (biological potency) of a toxin and is the dose that is lethal to 50% of the mice tested (LD 50 More specifically, in the context of the present invention, LD 50 is measured using the Mouse Bioassay (MBA) unless otherwise stated. The MBA measures the mean lethal dose (LD) of a toxin / neurotoxin following intraperitoneal injection into mice. 50 ), i.e., the amount of toxin / neurotoxin that can cause 50% death in a group of mice. Based on this, one unit (U) of toxin / neurotoxin as used herein is defined as 1 mouse LD50 (1.0 LD 50 = 1.0U).

[0039] LD 50 The mouse bioassay is the gold standard among various biological, chemical, or immunological detection methods for botulinum toxin and is known to those of skill in the art (see, e.g., Pearce, LB; Borodic, GE; First, ER; MacCallum, RD Measurement of botulinum toxin activity: Evaluation of the lethality assay. Toxicol. Appl. Pharmacol. 1994, 128:69-77). Those of skill in the art will be able to determine the appropriate botulinum toxin concentration depending on the serotype and intended use. Alternatively, botulinum toxin activity can be measured using a cell-based assay, as described in WO 2009 / 114748, WO 2013 / 049508, or WO 2014 / 207109. Those of skill in the art will be able to determine the botulinum toxin activity resulting from the cell-based assay, as well as the LD 50 Calibration with standard samples revealed that mouse LD 50 It will be possible to correlate this with the results obtained in the assay.

[0040] LD used by manufacturers of commercial botulinum toxin preparations 50Because the tests vary, unit potencies provided by manufacturers of commercial botulinum toxin preparations are proprietary and not easily comparable. Thus, within the framework of the present invention, incobotulinumtoxinA ("INCO"; Xeomin®, Bocouture®; botulinum toxin serotype A without complexing proteins; Merz Pharmaceuticals), onabotulinumtoxinA ("ONA"; Botox®, Vistabel®; botulinum toxin complex of serotype A; Allergan), abobotulinumtoxinA ("ABO"; Dysport®, Azzalure®; botulinum toxin complex of serotype A; Medicis Pharmaceutical, Galderma Lab), rimabotulinumtoxinB ("RIM"; Myobloc®, NeuroBloc®; botulinum toxin serotype B; Solstice Neurosciences), and PurTox® ("TBD"; botulinum toxin serotype A; Mentor The relative potencies of ONA and INCO (Worldwide, Inc.) are determined using the conversion ratios provided below. As used herein, the conversion ratio of ONA to INCO is 1:1. The conversion ratio of ONA / INCO:ABO is 1:2.5. The conversion ratio of ONA / INCO:RIM is 1:50, and the conversion ratio of ONA / INCO:TBD is 1:1.5. Furthermore, in the context of the present invention, 1 U of INCO (Xeomin®) and 1 U of onabotulinumtoxin A ("ONA"; Botox®) shall be considered to be equivalent to 1 mouse LD50 (1.0 LD50), i.e., 1 U, as determined above.

[0041] The term "human serum albumin" as used herein, or its abbreviation "HSA", is intended to refer to donor HSA (HSA derived from human blood, or more precisely, from human plasma) and recombinant HSA. Preferably, the human serum albumin is donor HSA. In the present invention, human serum albumin (HSA) acts as a stabilizing protein. The term "stabilizing protein" as used herein generally refers to a polypeptide that provides increased stability of botulinum toxin. According to the present invention, HSA may be present in the liquid formulation in an amount of 0.001-2.0% (w / v), preferably in an amount of 0.001-1.00% (w / v), more preferably in an amount of 0.01-0.5% (w / v), even more preferably in an amount of 0.02-0.3% (w / v), and most preferably in an amount of 0.03-0.15% (w / v).

[0042] In a first aspect, the present invention provides a method for producing a composition comprising the steps of: (i) a botulinum toxin; and (ii) human serum albumin; A liquid formulation comprising: Fe 3+ ions at a concentration of less than 1 μM, Concerning liquid formulations.

[0043] Preferably, Fe in the liquid formulation 3+ The concentration is less than 1000 nM, less than 750 nM, less than 500 nM, or less than 250 nM, more preferably less than 1000 nM, more preferably ... 3+ The concentration is less than 200 nM, less than 150 nM, or less than 100 nM, and particularly preferred is Fe in the liquid formulation. 3+ The concentration is less than 50 nM or less than 10 nM, most preferably less than 10 nM Fe in the liquid formulation. 3+ The concentration is less than 1 nM, less than 100 pM, less than 10 pM, or less than 1 pM.

[0044] Metal ions (e.g., Ca 2+ , Co 2+ , Cu 2+ , Ni 2+ , or Fe 3+The concentration of metal ions may be determined by measurement methods known to those skilled in the art, such as atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS), and inductively coupled plasma atomic emission spectrometry (ICP-AES), also called inductively coupled plasma optical emission spectrometry (ICP-OES). Preferably, ICP-MS or ICP-OES, in particular ICP-OES, may be used to measure the concentration of metal ions (see USP38-NF33, Second Supplement, Chemical Testing / <233> See also Elemental Impurities - Procedure 1 (ICP-OES) and Procedure 2 (ICP-MS), 2015).

[0045] Without being limited by theory, it is believed that the HSA material used to prepare the liquid formulations of the present invention, particularly donor HSA derived from human blood, which contains high amounts of iron ions, is rich in Fe, which is at least partially responsible for the light sensitivity-inducing properties of unpurified (unprocessed) HSA. 3+ It is believed to contain a significant amount of ions.

[0046] In addition to the advantages mentioned above, this aspect of the invention also has advantages in manufacturing processes and in the development of new formulations: for example, equipment (such as containers) and materials (such as excipients) can be selected to ensure that the final product is as free of iron as possible.

[0047] Fe 3+ Low concentration liquid botulinum toxin formulations of the invention may be prepared by a process comprising the steps of: (a) contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; and (b) removing the chelating agent from the mixture.

[0048] Preferably, the method for preparing the liquid formulation according to the first aspect of the invention is the same as that described in relation to the third aspect of the invention. Also, the liquid formulation according to the first aspect of the invention may have the same composition as the liquid formulation according to the third aspect of the invention. Accordingly, all explanations, comments, disclosures, definitions and the like given for the liquid formulation according to the third aspect of the invention apply equally to the liquid formulation according to the first aspect of the invention, unless expressly stated otherwise.

[0049] In a second aspect, the present invention provides a method for producing a composition comprising: (i) a botulinum toxin; and (ii) human serum albumin; A liquid formulation comprising: Contains no tryptophan or N-acetyltryptophan or less than 50 μM Concerning liquid formulations.

[0050] As used herein, the term "free" of tryptophan and N-acetyltryptophan means that the liquid formulation does not contain tryptophan and N-acetyltryptophan or does not contain any tryptophan or N-acetyltryptophan. Specifically, it means that tryptophan and N-acetyltryptophan are not added to the formulation. Alternatively, it means that the concentration of tryptophan and N-acetyltryptophan in the liquid formulation is 0 μM (i.e., <0.5 μM, according to the general rule of rounding), particularly ≦0.1 μM, ≦0.01 μM, or ≦0.001 μM, and more particularly 0 nM (i.e., <0.5 nM, according to the general rule of rounding). As used herein, the term "comprises less than or equal to" tryptophan and N-acetyltryptophan means that the total amount of tryptophan and N-acetyltryptophan may be ≦50 μM, preferably ≦20 μM, more preferably ≦10 μM, even more preferably ≦1 μM, even more preferably ≦0.1 μM, and most preferably ≦0.01 μM or ≦0.001 μM. Thus, as used herein, the term "free or contains less than or equal to" means that the total amount of tryptophan and N-acetyltryptophan in the liquid formulation is between 0 μM and X, or between 0 nM and X, where X is 50 μM, 20 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM.

[0051] The concentration of the amino acids tryptophan and N-acetyltryptophan can be measured by various techniques (e.g., DC, HPLC, LC-MS, GC-MS, CE, etc.) as known to those skilled in the art. For example, N-acetyltryptophan can be measured by separation using liquid chromatography on a reversed-phase column and UV detection at 280 nm as described by Nelis et al. (Nelis et al., J. Chromatogr., 1985, 333(2):381-387), or by a method based on UV spectroscopy of the acid-soluble fraction remaining after protein precipitation as described by Yu and Finlayson (Yu, MW and Finlayson, JS, J. Pharm. Sci., 1984, 73(1):82-86). Tryptophan can be quantitatively measured using a liquid chromatography / tandem mass spectrometry method, for example, as described by Wentao et al. (Wentao et al., Analytical and Bioanalytical Chemistry, 2011, 401:3249-3261).

[0052] As used herein, the term "tryptophan" refers to L-tryptophan, D-tryptophan, or a mixture of L-tryptophan and D-tryptophan (D / L-tryptophan). Similarly, as used herein, the term "N-acetyltryptophan" refers to N-acetyl-L-tryptophan, N-acetyl-D-tryptophan, or a mixture of N-acetyl-L-tryptophan and N-acetyl-D-tryptophan (N-acetyl-D / L-tryptophan).

[0053] The human serum albumin used in preparing the liquid formulation is preferably a human serum albumin material containing no more than 50 mM, more preferably no more than 20 mM or no more than 10 mM, even more preferably no more than 1 mM or no more than 0.1 mM, and most preferably no more than 0.01 nM or no more than 0 mM tryptophan and N-acetyltryptophan.

[0054] It has been noted that all commercially available donor HSA products contain significant amounts (>10 mM) of N-acetyltryptophan. Therefore, such products must be purified to reduce the amount of N-acetyltryptophan to the desired level before being used to formulate the liquid formulations of the present invention. A suitable method for preparing "purified HSA" is detailed below in connection with the fourth aspect of the present invention. Briefly, human serum albumin that is free of tryptophan and N-acetyltryptophan or contains only low amounts of tryptophan (also referred to herein as "purified HSA") can be obtained by removing tryptophan and / or N-acetyltryptophan from the human serum albumin starting material by dialysis, diafiltration, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, field-flow fractionation, or precipitation methods (e.g., salting out or ethanol precipitation).

[0055] According to the invention, the liquid formulations of the invention, such as those according to the first, second and / or third aspects of the invention, may further comprise one or both of: (iii) a tonicity agent; and (iv) a buffering agent; as described in more detail below. This is particularly relevant for liquid formulations according to the third aspect of the invention.

[0056] Furthermore, the liquid formulation according to the second aspect of the present invention contains Fe 3+ It is also contemplated herein that the concentration of ions is preferably less than 1000 nM. Specifically, Fe in the liquid formulation 3+ The concentration is preferably less than 750 nM, less than 500 nM, or less than 250 nM, more preferably less than 10 ... 3+ The concentration is less than 200 nM, less than 150 nM, or less than 100 nM, and particularly preferred is Fe in the liquid formulation. 3+ The concentration is less than 50 nM or less than 10 nM, most preferably less than 10 nM Fe in the liquid formulation. 3+The concentration is less than 1 nM, less than 100 pM, less than 10 pM, or less than 1 pM.

[0057] As mentioned above, Fe 3+ It has also been found that Fe ions reduce the photostability of botulinum toxins, thereby increasing the destabilizing effects of tryptophan and N-acetyltryptophan. The HSA material used to prepare the liquid formulations of the present invention, particularly donor HSA derived from human blood, contains Fe ions, which contribute to the photostability reducing properties of unpurified (unprocessed) HSA. 3+ It is believed to contain a significant amount of Fe ions. 3+ To achieve this concentration, equipment (e.g., containers) and materials (e.g., excipients) should be selected so that as little iron as possible is present in the final product, for example by selecting containers made of iron-free materials such as polypropylene or polycarbonate.

[0058] Metal ions (Fe 3+ , Ca 2+ , Co 2+ , Cu 2+ , and Ni 2+ etc.) can be measured by the measurement methods disclosed above that are known to those skilled in the art.

[0059] The low Fe 3+ The content may be achieved by the methods described in detail in relation to the fourth aspect of the invention, in particular by methods comprising contacting human serum albumin with a chelating agent and then removing the chelating agent as described herein.

[0060] According to the present invention, the liquid formulation may further comprise one or more additional pharma- ceutically acceptable excipients, unless otherwise specified or intended. For example, the liquid formulation may comprise one or more of glycerol, sucrose, lactose, mannitol, dextran, hyaluronic acid, polyvinylpyrrolidone, lactic acid, citric acid, amino acids (other than tryptophan and N-acetyltryptophan), benzyl alcohol, lidocaine, gelatin, hydroxyethyl starch (HES), polyethylene oxide, and polysorbates (e.g., polysorbate 20 and polysorbate 80). Other suitable pharma-ceutically acceptable excipients include those well known in the art. See, for example, Remington's Pharmaceutical Sciences (Mack Publishing, Easton, PA).

[0061] However, it is also contemplated herein that the liquid formulations of the present invention specifically lack certain components (i.e., compounds, materials, or substances), such as chelating agents and / or phosphates, surfactants, polysaccharides, amino acids, stabilizing peptides, and the like (including any combination thereof). As used herein, the term "detergent" is used synonymously with "surfactant" and is intended to include non-ionic surfactants and ionic surfactants. As used herein, the term "stabilizing peptide" generally refers to a peptide of 5-50 amino acids, including, for example, peptides of 10-40 amino acids or 15-30 amino acids. Thus, the term "stabilizing peptide" excludes HSA.

[0062] In one embodiment, the liquid formulation of the invention does not contain a surfactant, in particular does not contain a polysorbate, and more particularly does not contain polysorbate 20 and / or polysorbate 80. In another embodiment, the liquid formulation of the invention does not contain an alginate. In another embodiment, the liquid formulation of the invention does not contain a succinate. In another embodiment, the liquid formulation of the invention does not contain one or more (e.g., 2, 3, 4, or 5) amino acids selected from the group consisting of arginine, glutamic acid, methionine, tryptophan, and serine. In another embodiment, the liquid formulation of the invention does not contain a saccharide, such as a monosaccharide, oligosaccharide, or polysaccharide, or a mixture thereof. In particular, the liquid formulation of the invention may not contain one or more (e.g., 2, 3, or 4) of sucrose, lactose, maltose, and trehalose. In another embodiment, the liquid formulation of the invention does not contain a chelating agent, in particular a chelating agent described herein in connection with the present invention, and / or a phosphate. It is contemplated herein that the liquid formulation may lack more than one, or all, of the above-mentioned compounds.

[0063] In one embodiment, the liquid formulation of the present invention comprises: (i) a surfactant and a monosaccharide, an oligosaccharide, and a polysaccharide; (ii) a surfactant and any amino acid; or a surfactant and all amino acids except histidine; (iii) a surfactant and a stabilizing peptide; (iv) a monosaccharide, an oligosaccharide, and a polysaccharide and any amino acid; or a monosaccharide, an oligosaccharide, and a polysaccharide and all amino acids except histidine; (v) a monosaccharide, an oligosaccharide, and a polysaccharide and a stabilizing peptide; (vi) any amino acid and a stabilizing peptide; or all amino acids except histidine and a stabilizing peptide; (vii) a surfactant, a monosaccharide, an oligosaccharide, and a polysaccharide, and any amino acid; or a surfactant, a monosaccharide, (viii) surfactants, monosaccharides, oligosaccharides, and polysaccharides, and stabilizing peptides; (ix) surfactants, any amino acids, and stabilizing peptides; or surfactants, all amino acids except histidine, and stabilizing peptides; (x) monosaccharides, oligosaccharides, and polysaccharides, any amino acids, and stabilizing peptides; or monosaccharides, oligosaccharides, and polysaccharides, all amino acids except histidine, and stabilizing peptides; (xi) surfactants, monosaccharides, oligosaccharides, and polysaccharides, any amino acids, and stabilizing peptides; or surfactants, monosaccharides, oligosaccharides, and polysaccharides, all amino acids except histidine, and stabilizing peptides.

[0064] In another embodiment, the liquid formulation of the present invention does not contain any amino acids other than histidine. In another embodiment, the liquid formulation of the present invention does not contain any monosaccharides, disaccharides, or trisaccharides. In another embodiment, the liquid formulation of the present invention does not contain any stabilizing peptides or proteins other than HSA. In another embodiment, the liquid formulation of the present invention does not contain phosphate, for example in the form of phosphate buffer.

[0065] In yet other embodiments, the liquid formulations of the present invention include a mixture of: (i) succinate and a surfactant (such as a polysorbate); (ii) succinate and methionine; (iii) succinate and sucrose; (iv) a surfactant (such as a polysorbate) and methionine; (v) a surfactant (such as a polysorbate) and sucrose; (vi) methionine and sucrose; (vii) succinate, a surfactant (such as a polysorbate), and methionine; (xiii) copolysaccharide, a surfactant (such as a polysorbate), and methionine; (ix) succinate, detergent (such as polysorbate), and sucrose; (x) detergent (such as polysorbate), methionine, and sucrose, and (xi) succinate, detergent (such as polysorbate), methionine, and sucrose; (xii) detergent (such as polysorbate) and histidine; (xiii) detergent (such as polysorbate), histidine, and sucrose.

[0066] Additionally, any of the liquid formulations of the present invention that lack one or more components (i.e., compounds, materials, or substances) may further lack a chelating agent, particularly a chelating agent described herein (such as EDTA), and / or phosphate.

[0067] Furthermore, one preferred liquid formulation of the present invention contains (i) a botulinum toxin, (ii) HSA, and (iii) an isotonicity agent, and is free of tryptophan and N-acetyltryptophan or contains them at 50 μM or less. One preferred liquid formulation of the present invention contains (i) a botulinum toxin at a concentration of 10 U / ml to 200 U / ml, (ii) HSA in an amount of 0.01% (w / v) to 1.0% (w / v), and (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.01% (w / v) to 2.0% (w / v), and is free of tryptophan and N-acetyltryptophan or contains them at 50 μM or less. One preferred liquid formulation of the present invention contains (i) a botulinum toxin at a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.01% (w / v) to 0.5% (w / v); (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.1% (w / v) to 1.5% (w / v); and is free of tryptophan and N-acetyltryptophan or contains 50 μM or less. One preferred liquid formulation of the present invention contains (i) a botulinum toxin at a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.05% (w / v) to 0.25% (w / v); (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.6% (w / v) to 1.2% (w / v); and is free of tryptophan and N-acetyltryptophan or contains 50 μM or less. One preferred liquid formulation of the present invention contains (i) a botulinum toxin at a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.01% (w / v) to 0.5% (w / v); (iii) sodium chloride as an isotonicity agent in an amount of 0.9% (w / v); and is free of tryptophan and N-acetyltryptophan or contains 50 μM or less. One preferred liquid formulation of the present invention contains (i) a botulinum toxin at a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.05% (w / v) to 0.25% (w / v); (iii) sodium chloride as an isotonicity agent in an amount of 0.9% (w / v); and is free of tryptophan and N-acetyltryptophan or contains 50 μM or less.

[0068] It is also contemplated herein that in the preferred and particularly preferred liquid formulations described in the paragraphs above and below, the upper limits of the concentrations of tryptophan and N-acetyltryptophan are less than 50 μM, preferably less than 10 μM, more preferably less than 1 μM, even more preferably less than 0.1 μM, and most preferably less than 0.01 μM or less than 0.001 μM. ... 3+ It is further contemplated that the concentration of the ion may be characterized as being less than 1000 nM or less than 500 nM, more preferably less than 250 nM or less than 100 nM, and most preferably less than 10 nM or less than 1 nM.

[0069] One preferred liquid formulation of the present invention contains (i) a botulinum toxin, (ii) HSA, (iii) a tonicity agent, and (iv) a buffering agent, and is free of tryptophan and N-acetyltryptophan or contains them at 50 μM or less. One preferred liquid formulation of the present invention contains (i) a botulinum toxin at a concentration of 10 U / ml to 200 U / ml, (ii) HSA in an amount of 0.01% (w / v) to 1.0% (w / v), (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.01% (w / v) to 2.0% (w / v), and (iv) a buffering agent at a concentration of 1 mM to 100 mM, and is free of tryptophan and N-acetyltryptophan or contains them at 50 μM or less. One preferred liquid formulation of the present invention contains (i) botulinum toxin in a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.01% (w / v) to 0.5% (w / v); (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.01% (w / v) to 2.0% (w / v); and (iv) a buffering agent in a concentration of 1 mM to 100 mM; and either does not contain tryptophan and N-acetyltryptophan or contains less than 50 μM. One preferred liquid formulation of the present invention contains (i) botulinum toxin in a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.05% (w / v) to 0.25% (w / v); (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.1% (w / v) to 2.0% (w / v); and (iv) a buffering agent in a concentration of 1 mM to 100 mM; and either does not contain tryptophan and N-acetyltryptophan or contains less than 50 μM. One preferred liquid formulation of the present invention contains (i) botulinum toxin in a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.05% (w / v) to 0.25% (w / v); (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.6% (w / v) to 1.3% (w / v); and (iv) a buffering agent in a concentration of 2 mM to 50 mM; and either does not contain tryptophan and N-acetyltryptophan or contains less than 50 μM.One preferred liquid formulation of the present invention contains (i) botulinum toxin in a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.05% (w / v) to 0.25% (w / v); (iii) sodium chloride in an amount of 0.9% (w / v); and (iv) a buffer in a concentration of 5 mM to 20 mM; and either no tryptophan and N-acetyltryptophan or contains less than 50 μM.

[0070] One preferred liquid formulation of the present invention comprises: (i) a botulinum toxin; (ii) HSA; (iii) sodium chloride as a tonicity agent; and (iv) a buffer selected from histidine, phosphate, and mixtures thereof, preferably histidine as a buffer; and is free of tryptophan and N-acetyltryptophan or contains less than 50 μM.

[0071] One particularly preferred liquid formulation of the present invention contains (i) botulinum toxin at a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.01% (w / v) to 1.0% (w / v); (iii) sodium chloride in an amount of 0.01% (w / v) to 2.0% (w / v), preferably 0.9% (w / v); (iv) a buffer selected from histidine, phosphate, and mixtures thereof at a concentration of 1 mM to 100 mM, preferably histidine at a concentration of 1 mM to 100 mM; and is free of tryptophan and N-acetyltryptophan or contains less than 50 μM.

[0072] Another particularly preferred liquid formulation of the present invention contains (i) botulinum toxin at a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.01% (w / v) to 0.5% (w / v); (iii) sodium chloride in an amount of 0.9% (w / v); and (iv) a buffer selected from histidine, phosphate, and mixtures thereof at a concentration of 1 mM to 100 mM, preferably histidine at a concentration of 1 mM to 100 mM; and is free of tryptophan and N-acetyltryptophan or contains less than 50 μM.

[0073] Yet another particularly preferred liquid formulation of the present invention contains: (i) botulinum toxin at a concentration of 10 U / ml to 200 U / ml; (ii) HSA in an amount of 0.05% (w / v) to 0.25% (w / v); (iii) sodium chloride in an amount of 0.9% (w / v); (iv) a buffer selected from histidine, phosphate, and mixtures thereof at a concentration of 1 mM to 100 mM, preferably at a concentration of 2 mM to 50 mM, more preferably at a concentration of 5 mM to 20 mM, preferably histidine at a concentration of 1 mM to 100 mM, preferably at a concentration of 2 mM to 50 mM, more preferably at a concentration of 5 mM to 20 mM; and is free of tryptophan and N-acetyltryptophan or contains less than 50 μM.

[0074] Furthermore, the above-mentioned preferred and particularly preferred liquid formulations preferably have a pH in the range of 6.0 to 7.5, more preferably in the range of 6.5 to 7.0. The botulinum toxin is preferably serotype A, more preferably a neurotoxic component of serotype A.

[0075] An example of a preferred liquid formulation of the present invention is as follows: Formulation 1: 50U / ml BoNT / A 0.9% NaCl (9 mg / mL) 0.085% HSA (purified by dialysis, etc.) (0.85 mg / mL) 0.155% histidine (1.55mg / ml; approximately 10mM) Trp and N-AcTrp <50 μm pH 6.0.

[0076] The purified HSA of Formulation Example 1 may be prepared by the process described in detail in connection with the method according to the fourth aspect of the invention.

[0077] According to the present invention, the liquid formulations of the present invention are preferably prepared or obtainable by a method according to the fourth aspect of the present invention, which allows for the removal of substances such as N-acetyltryptophan contained in the HSA starting material (e.g., commercially available HSA products) that impair the photostability of liquid botulinum toxin formulations.

[0078] In a third aspect, the present invention provides a method for producing a composition comprising the steps of: (i) a botulinum toxin; and (ii) human serum albumin (HSA); A liquid formulation comprising: (a) contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; and (b) removing the chelating agent from the mixture; prepared by a process comprising: Concerning liquid formulations.

[0079] Regarding component (i) of the liquid preparation of the present invention, the botulinum toxin is not particularly limited and may be any serotype of botulinum toxin (BoNT / A to G). Preferably, the botulinum toxin is as defined above.

[0080] As defined above, with respect to component (ii) of the liquid formulation, "human serum albumin" or "HSA" is intended to refer to donor HSA and recombinant HSA, preferably donor HSA. HSA may be present in the liquid formulation in an amount of 0.001-2.0% (w / v), preferably in an amount of 0.001-1.00% (w / v), more preferably in an amount of 0.01-0.5% (w / v), even more preferably in an amount of 0.02-0.3% (w / v), and most preferably in an amount of 0.03-0.15% (w / v).

[0081] According to the present invention, the liquid formulation of the present invention is prepared by a method comprising at least steps (a) and (b), the first step being as follows: (a) contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent;

[0082] The term "contacting" as used herein is intended to be broadly interpreted as combining two or more components together. This can be accomplished by a variety of different methods, such as dissolving, mixing, suspending, blending, slurrying, stirring, flowing by, adsorption, binding, extraction, etc. Typically, the chelating agent is added to a composition containing HSA (e.g., a solution of HSA), which is then mixed to obtain a homogenous mixture, and optionally subjected to further processing steps (e.g., dialysis against a chelating agent-containing buffer).

[0083] It is therefore contemplated that "contacting" can be accomplished by, for example, loading the HSA material in the form of an aqueous solution onto an immobilized metal affinity resin (IMAC). As used herein, the term "immobilized metal affinity resin" (IMAC) includes, but is not limited to, resins that contain immobilized functional moieties (such as iminodiacetic acid) capable of binding and coordinating polyvalent cations, such as Chelating-Sepharose, Fractogel-EMD-Chelate, POROS 20MC, Matrex Cellufine Chelate, TALON, and Chelex 100 resins. Such immobilized metal affinity resins are typically used in the form of chromatographic metal affinity columns, as known to those skilled in the art. As used herein, the term "contacting" also includes batch mode binding using a suitable immobilized metal affinity resin.

[0084] Furthermore, with respect to step (a), it should be noted that the terms "human serum albumin" and "chelating agent" used in step (a) of the method for preparing the liquid formulation of the present invention are not intended to imply any limitation regarding their physical form, or to exclude the presence of other substances or compounds mixed with or contained in the HSA and chelating agent. That is, the "human serum albumin" contacted with the chelating agent in step (a) may be present in any form, such as a solid or liquid (e.g., an aqueous composition or an aqueous solution). Similarly, the "chelating agent" contacted with the human serum albumin in step (a) may be present in any form, such as a solid or liquid (e.g., an aqueous composition or an aqueous solution). Furthermore, the expression "contacting human serum albumin with a chelating agent" does not exclude that (i) the human serum albumin is in the form of a composition (e.g. a solid or liquid composition, in particular an aqueous or aqueous solution) which comprises one or more additional components that will be included in the final liquid formulation, such as a tonicity agent or a buffering agent; and / or (ii) the chelating agent is in the form of a composition (e.g. a solid or liquid composition, in particular an aqueous or aqueous solution) which comprises one or more additional components that will be included in the final liquid formulation, such as a tonicity agent or a buffering agent.

[0085] Preferably, the mixture obtained in step (a) is an aqueous mixture. This aqueous mixture can be prepared in various ways. For example, human serum albumin can be in the form of an aqueous composition, e.g., an aqueous solution, and this aqueous composition can be mixed with a chelating agent that can be in solid or liquid form, e.g., a chelating agent that can be in the form of an aqueous solution. Furthermore, human serum albumin can be in the form of a solid, such as a lyophilized material, mixed with a chelating agent and an aqueous solution, or mixed with an aqueous solution of the chelating agent. Preferably, human serum albumin is in the form of an aqueous composition, more preferably an aqueous solution, and the chelating agent is a solid or an aqueous composition, e.g., an aqueous solution.

[0086] In particular, the HSA contacted with the chelating agent in step (a) may be in the form of an aqueous solution containing at least 5% (w / v) HSA, more preferably 10-30% (w / v) HSA, and most preferably 20% (w / v) HSA. The pH of the mixture obtained in step (a) may be adjusted to a pH in the range of 6.0-9.0, preferably 6.5-8.5, more preferably 7.0-8.5, and most preferably 7.0-8.0.

[0087] Furthermore, the contacting step (a) of the method for preparing a liquid formulation according to the present invention may include several sub-steps. For example, in one embodiment, the contacting step (a) includes or consists of mixing a chelating agent (e.g., EDTA) and human serum albumin, incubating the mixture for a predetermined time, and optionally dialyzing the mixture against a buffer containing a chelating agent, preferably the same chelating agent as used in the incubation sub-step. In another embodiment, the mixture is not incubated, but is (directly) dialyzed against a buffer containing a chelating agent, which is preferably the same as used in the step of mixing a chelating agent (e.g., EDTA) and human serum albumin. In another embodiment, the chelating agent and human serum albumin are not mixed before dialysis. That is, the contacting step (a) includes or consists of dialyzing the mixture against a buffer containing a chelating agent.

[0088] Preferably, step (a) comprises or consists of adding a chelating agent to a composition comprising HSA (e.g., a solution of HSA); or mixing a chelating agent with a composition comprising HSA (e.g., a solution of HSA). The resulting mixture is then incubated for a period of time, e.g., left unstirred for a given period of time, or stirred for a given period of time.

[0089] The incubation time is not limited to a particular range, but is typically at least 0.5 hours, particularly at least 1 hour, and more particularly at least 2 hours. The upper limit of the incubation time is not critical and may be, for example, 1 hour, 2 hours, 5 hours, or 10 hours. Thus, the incubation time may be, for example, 0.5 to 5 hours, or 1 to 10 hours. Similarly, the incubation temperature is not particularly limited and may be, for example, within the range of 0°C to 60°C. Preferably, the temperature is 0°C to 30°C. That is, in the present invention, room temperature (20°C or 25°C) is a suitable temperature. As is known to those skilled in the art, the temperature affects the reaction time. In general, the incubation conditions (e.g., time and temperature) are selected so that the remaining amount of the chelating agent (e.g., EDTA) contained in the final product is 100 μM or less, preferably 10 μM or less, more preferably 1 μM or less.

[0090] Optionally, the incubation step is followed by further processing, for example, by dialysis of the incubated mixture against a buffer containing a chelating agent, which is typically the same as that used in the incubation step. The chelating agent used in this optional dialysis step is preferably contained in the dialysis buffer at a concentration of 0.1 mM to 1000 mM, more preferably 1 mM to 200 mM, and most preferably 10 mM to 100 mM. It is also preferred that the buffer used in the dialysis step (i) has a pH of 7.5 to 8.5; (ii) further comprises a buffering agent, which is preferably in accordance with the final composition; or (iii) further comprises an isotonicity agent, which is preferably 0.9% (w / v) sodium chloride; or (i) and (ii); (i) and (iii); (ii) and (iii); or (i) and (ii) and (iii);.

[0091] In the present invention, the term "chelating agent" as used herein is not particularly limited as long as it can bind metal ions. The term "chelating agent" as used herein may also be referred to as "chelator" or "sequestering agent". Chelating agents for use herein are typically metal ion-binding organic compounds. Metal ions generally form multiple coordinate bonds with organic chelating agents that act as multidentate ligands.

[0092] Chelators suitable for use herein include, but are not limited to, aminopolycarboxylic acids (e.g., aminopolycarboxylic acids having 3-6, preferably 4, carboxylic acid functional groups); and other compounds such as citrate, porphyrins, TPEN (N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine), TETA (triethylenetetramine), and mixtures thereof. Examples of aminopolycarboxylic acids include NTA (nitrilotriacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TED (ethylenediaminotriacetic acid), EDTA (ethylenediaminetetraacetic acid), EGTA (ethyleneglycol-bis(d-aminoethylether)-N,N,N',N'-tetraacetic acid), BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N',N'-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), and TTHA (triethylenetetraminehexaacetic acid).

[0093] Particularly preferred chelating agents for use herein include those having the general formula (I): (HO2CCH2)2N-RN(CH2CO2H)2(I) The R group is not particularly limited, and may contain no carboxylic acid functional groups, or may contain one or two carboxylic acid functional groups. Preferably, the R group contains no carboxylic acid functional groups or one carboxylic acid functional group. Most preferably, the R group does not contain a carboxylic acid functional group.

[0094] Examples of compounds of general formula (I) include EDTA, EGTA, BAPTA, DTPA, and TTHA. Particularly preferred for use herein are EDTA, EGTA, and DTPA, more preferred are EDTA and DTPA, and most preferred is EDTA. Mixtures of any of the above chelating agents can also be used in the present invention, for example, in solution, solid, or bound to a matrix.

[0095] In step (a), preferably, the HSA is contacted with a constant amount of chelating agent such that the chelating agent is present in the liquid formulation at a concentration of 0.1 mM to 1000 mM or 0.1 mM to 500 mM, more preferably 0.5 mM to 500 mM or 1 mM to 500 mM, and most preferably 10 mM to 100 mM.

[0096] According to the present invention, the method for preparing a liquid formulation further comprises the steps of: (b) removing said chelating agent from said mixture.

[0097] In step (b), the chelating agent may be removed by any suitable technique, such as dialysis (conventional dialysis using a dialysis bag, counterflow dialysis, etc.), reverse osmosis, filtration, cross-flow filtration, ultrafiltration, and chromatographic methods (e.g. ion exchange chromatography or gel filtration chromatography).

[0098] Preferably, the chelating agent is removed by dialysis. Dialysis is typically performed for 0.5 to 48 hours, particularly 1 to 24 hours or 1 to 12 hours, at a temperature of 0°C to 30°C, particularly 2°C to 30°C or 4°C to 25°C, for example at room temperature. Furthermore, dialysis is typically performed against a dialysis buffer in an amount 10 to 1000 times the amount of the incubated HSA / chelating agent mixture, and the dialysis buffer is usually exchanged at least once. The molecular weight cutoff of the dialysis membrane used is, for example, 10 kDa.

[0099] In general, dialysis conditions (e.g., time, temperature, amount of buffer, number of buffer exchanges) are selected so that the residual amount of chelating agent (e.g., EDTA) in the final product is at a concentration of 100 μM or less, preferably 10 μM or less, and more preferably 1 μM or less.

[0100] According to a preferred embodiment of the present invention, the HSA starting material is first pretreated by contacting it with a chelating agent, and then the chelating agent is removed. The pretreated HSA thus obtained is then used to prepare the liquid botulinum toxin formulation of the present invention. The term "human serum albumin starting material" as used herein is intended to refer to donor HSA material (human blood-derived, or more precisely human plasma-derived, HSA) or recombinant HSA material that is commercially available or conventionally available, i.e., that has not been pretreated as described herein. A mixture of donor HSA material and recombinant HSA material is also encompassed by the term "human serum albumin starting material" as used herein.

[0101] More specifically, according to this preferred embodiment, a liquid formulation comprising: (i) a botulinum toxin; and (ii) human serum albumin; (a) contacting a human serum albumin starting material with a chelating agent to obtain a mixture of said human serum albumin starting material and said chelating agent; (b) removing the chelating agent from the mixture to obtain a pretreated human serum albumin material; and (c) mixing a botulinum toxin with the pretreated human serum albumin material; It is prepared by a method comprising:

[0102] According to another preferred embodiment of the present invention, the liquid formulation is obtained by contacting the liquid pre-formulation with a chelating agent and then removing the chelating agent. As used herein, the term "liquid pre-formulation" refers to a liquid formulation that contains at least components (i) and (ii) (i.e., botulinum toxin and HSA) and preferably contains all the components and substances contained in the final liquid composition, in particular components (iii) and (iv). In the latter case, the final liquid formulation is obtained by the above-mentioned chelating agent treatment and removal.

[0103] More specifically, according to this preferred embodiment, a liquid formulation comprising: (i) a botulinum toxin; and (ii) human serum albumin; (a) contacting a liquid pre-formulation comprising a botulinum toxin and human serum albumin with a chelating agent to obtain a mixture of the liquid pre-formulation and the chelating agent; and (b) removing the chelating agent from the mixture to obtain a liquid formulation; It is prepared by a method comprising:

[0104] According to the present invention, the liquid formulation (according to any aspect described herein, i.e. according to the first, second and third aspects) may further optionally comprise: (iii) tonicity agent; may include:

[0105] The term "isotonicity agent" as used herein refers to an agent that, when added to an injection formulation, gives the formulation osmotic properties similar to those of physiological fluids. The isotonicity agent is also called an "osmotic pressure adjusting agent". The isotonicity agent is not particularly limited and may be selected from the group consisting of, for example, sugars, salts, polymers, and mixtures thereof.

[0106] Examples of tonicity agents include sucrose, glucose, sodium carbonate, amino acids, polyethylene glycol (PEG), dextran, cyclodextrin, and colloids (e.g., colloidal polysaccharides).Typically, the concentration of the tonicity agent is in the range of 0-2.0% (w / v), particularly 0.01-2.0% (w / v) or 0.1-1.5% (w / v), more particularly 0.6-1.2% (w / v).

[0107] Preferably, the isotonicity agent is sodium chloride (NaCl), which may be present in the liquid formulation of the present invention in an amount of 0.01-2.0% (w / v), preferably 0.1-1.5% (w / v), more preferably 0.5-1.2% (w / v) or 0.8-1.0% (w / v), and most preferably 0.9% (w / v).

[0108] According to the present invention, the liquid formulation (according to any aspect described herein, i.e. according to the first, second and third aspects) may further optionally comprise: (iv) a buffer; may include:

[0109] The term "buffering agent" as used herein refers to an agent that maintains the pH of a liquid formulation within an acceptable range, i.e., an agent that can adjust the pH of the formulation. A suitable buffering agent is one that does not chemically react with other ingredients and is present in an amount sufficient to provide the desired pH buffering effect. Examples of such buffering agents include amino acids, acetate, malic acid, ascorbate, citrate, tartrate, fumarate, succinate, phosphate, bicarbonate, TRIS, Bis-TRIS, ACES, MES, BES, MOPS, HEPES, TES, PIPES, tricine, and imidazole.

[0110] Preferably, the buffer is a phosphate (i.e., phosphate buffer), an amino acid, or a mixture thereof. The term "phosphate" as used herein generally refers to the unprotonated and protonated forms, and any salts thereof. The amino acid may be selected from aspartate, glycine, glutamate, histidine, proline, taurine, methionine, serine, tyrosine, tryptophan, and mixtures thereof, and is preferably selected from histidine, proline, taurine, methionine, serine, tyrosine, and mixtures thereof. Most preferably, the amino acid is histidine. The most preferred buffer for use herein is histidine, a phosphate, or a mixture thereof.

[0111] The concentration of the buffer in the liquid formulation of the present invention is preferably 1 to 100 mM, preferably 2 to 50 mM, more preferably 5 to 20 mM. When the buffer is an amino acid (e.g., histidine), it may be present in the liquid formulation at a concentration of 1 to 100 mM, preferably 2 to 50 mM, more preferably 5 to 20 mM, most preferably 10 mM. When the buffer is a phosphate, it may be present in the liquid formulation at a concentration of 1 to 100 mM, preferably 2 to 50 mM, more preferably 5 to 20 mM, most preferably 10 mM.

[0112] In addition, the liquid formulation of the present invention may further comprise one or more additional pharma- ceutically acceptable excipients, unless otherwise specified or intended.For example, the liquid formulation may comprise one or more of glycerol, sucrose, lactose, mannitol, dextran, hyaluronic acid, polyvinylpyrrolidone, lactic acid, citric acid, amino acids, benzyl alcohol, lidocaine, gelatin, hydroxyethyl starch (HES), polyethylene oxide, and polysorbate (e.g., polysorbate 20, polysorbate 80).Other suitable pharma-ceutically acceptable excipients include those well known in the art.See, for example, Remington's Pharmaceutical Sciences (Mack Publishing, Easton, PA).

[0113] However, it is also contemplated herein that the liquid formulations of the present invention specifically lack certain components (i.e., compounds, materials, or substances), such as chelating agents, surfactants, polysaccharides, amino acids, stabilizing peptides, and the like (including any combination thereof). As used herein, the term "surfactant" is used synonymously with "surface active substance" and is intended to include non-ionic surfactants and ionic surfactants. As used herein, the term "stabilizing peptide" generally refers to a peptide consisting of 5-50 amino acids, including, for example, peptides of 10-40 amino acids or 15-30 amino acids. Thus, the term "stabilizing peptide" excludes HSA.

[0114] In one embodiment, the liquid formulation of the invention does not contain a surfactant, in particular does not contain a polysorbate, and more particularly does not contain polysorbate 20 and / or polysorbate 80. In another embodiment, the liquid formulation of the invention does not contain an alginate. In another embodiment, the liquid formulation of the invention does not contain a succinate. In another embodiment, the liquid formulation of the invention does not contain one or more (e.g., 2, 3, 4, or 5) amino acids selected from the group consisting of arginine, glutamic acid, methionine, tryptophan, and serine. In another embodiment, the liquid formulation of the invention does not contain a saccharide, such as a monosaccharide, oligosaccharide, or polysaccharide, or a mixture thereof. In particular, the liquid formulation of the invention may not contain one or more (e.g., 2, 3, or 4) of sucrose, lactose, maltose, and trehalose. In another embodiment, the liquid formulation of the invention does not contain a chelating agent, in particular a chelating agent described herein in connection with the present invention. It is contemplated herein that the liquid formulation may lack more than one, or all, of the above-mentioned compounds.

[0115] In one embodiment, the liquid formulation of the present invention comprises: (i) a surfactant and a monosaccharide, an oligosaccharide, and a polysaccharide; (ii) a surfactant and any amino acid; or a surfactant and all amino acids except histidine; (iii) a surfactant and a stabilizing peptide; (iv) a monosaccharide, an oligosaccharide, and a polysaccharide and any amino acid; or a monosaccharide, an oligosaccharide, and a polysaccharide and all amino acids except histidine; (v) a monosaccharide, an oligosaccharide, and a polysaccharide and a stabilizing peptide; (vi) any amino acid and a stabilizing peptide; or all amino acids except histidine and a stabilizing peptide; (vii) a surfactant, a monosaccharide, an oligosaccharide, and a polysaccharide, and any amino acid; or a surfactant, a monosaccharide, (viii) surfactants, monosaccharides, oligosaccharides, and polysaccharides, and stabilizing peptides; (ix) surfactants, any amino acids, and stabilizing peptides; or surfactants, all amino acids except histidine, and stabilizing peptides; (x) monosaccharides, oligosaccharides, and polysaccharides, any amino acids, and stabilizing peptides; or monosaccharides, oligosaccharides, and polysaccharides, all amino acids except histidine, and stabilizing peptides; (xi) surfactants, monosaccharides, oligosaccharides, and polysaccharides, any amino acids, and stabilizing peptides; or surfactants, monosaccharides, oligosaccharides, and polysaccharides, all amino acids except histidine, and stabilizing peptides.

[0116] In another embodiment, the liquid formulation of the present invention does not contain any amino acids other than histidine. In another embodiment, the liquid formulation of the present invention does not contain any monosaccharides, disaccharides, or trisaccharides. In another embodiment, the liquid formulation of the present invention does not contain any stabilizing peptides or proteins other than HSA. In another embodiment, the liquid formulation of the present invention does not contain phosphate, for example in the form of phosphate buffer.

[0117] In yet another embodiment, the liquid formulation of the present invention comprises: (i) succinate and a surfactant (such as a polysorbate); (ii) succinate and methionine; (iii) succinate and sucrose; (iv) a surfactant (such as a polysorbate) and methionine; (v) a surfactant (such as a polysorbate) and sucrose; (vi) methionine and sucrose; (vii) succinate, a surfactant (such as a polysorbate), and methionine; (xiii) cobalt salt, a surfactant (such as a polysorbate), and methionine; (ix) succinate, detergent (such as polysorbate), and sucrose; (x) detergent (such as polysorbate), methionine, and sucrose, and (xi) succinate, detergent (such as polysorbate), methionine, and sucrose; (xii) detergent (such as polysorbate and histidine); (xiii) detergent (such as polysorbate), histidine, and sucrose.

[0118] Additionally, any of the liquid formulations of the present invention that lack one or more components (ie, compounds, materials, or substances) may further lack a chelating agent, particularly a chelating agent described herein.

[0119] Further, one preferred liquid formulation of the present invention comprises (i) a botulinum toxin; (ii) HSA; and (iii) an isotonicity agent. One preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.01-1.0% (w / v); and (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.01-2.0% (w / v). One preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.01-0.5% (w / v); and (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.1-1.5% (w / v). One preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.05-0.25% (w / v); and (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.6-1.2% (w / v). One preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.01-0.5% (w / v); and (iii) sodium chloride as an isotonicity agent in an amount of 0.9% (w / v). One preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.05-0.25% (w / v); and (iii) sodium chloride as an isotonicity agent in an amount of 0.9% (w / v).

[0120] Also, the preferred liquid formulations described in the above and following paragraphs herein contain Fe 3+ It is further contemplated that the concentration of the ion may be characterized as being less than 1 μM, preferably less than 1000 nM or less than 500 nM, more preferably less than 250 nM or less than 100 nM, and most preferably less than 10 nM or less than 1 nM.

[0121] One preferred liquid formulation of the present invention comprises (i) a botulinum toxin; (ii) HSA; (iii) a tonicity agent; and (iv) a buffering agent. One preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.01-1.0% (w / v); (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.01-2.0% (w / v); and (iv) a buffering agent at a concentration of 1-100 mM. One preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.01-0.5% (w / v); (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.01-2.0% (w / v); and (iv) a buffering agent at a concentration of 1-100 mM. One preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.05-0.25% (w / v); (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.1-2.0% (w / v); and (iv) a buffer at a concentration of 1-100 mM. One preferred liquid formulation of the present invention comprises (i) a botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.05-0.25% (w / v); (iii) an isotonicity agent, preferably sodium chloride, in an amount of 0.6-1.3% (w / v); and (iv) a buffer at a concentration of 2-50 mM. One preferred liquid formulation of the present invention comprises: (i) botulinum toxin in a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.05-0.25% (w / v); (iii) sodium chloride in an amount of 0.9% (w / v); and (iv) a buffer in a concentration of 5-20 mM. Also herein, the preferred liquid formulation described in this paragraph comprises Fe 3+ It is further contemplated that the concentration of the ion may be characterized as being less than 1 μM, preferably less than 1000 nM or less than 500 nM, more preferably less than 250 nM or less than 100 nM, and most preferably less than 10 nM or less than 1 nM.

[0122] One preferred liquid formulation of the present invention comprises: (i) a botulinum toxin; (ii) HSA; (iii) sodium chloride as a tonicity agent; and (iv) a buffer selected from histidine, phosphate, and mixtures thereof, preferably histidine, as a buffer.

[0123] One particularly preferred liquid formulation of the present invention comprises: (i) botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.01-1.0% (w / v); (iii) sodium chloride in an amount of 0.01-2.0% (w / v), preferably 0.9% (w / v); and (iv) a buffer selected from histidine, phosphate, and mixtures thereof at a concentration of 1-100 mM, preferably histidine at a concentration of 1-100 mM.

[0124] Another particularly preferred liquid formulation of the present invention comprises: (i) botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA at an amount of 0.01-0.5% (w / v); (iii) sodium chloride at an amount of 0.9% (w / v); and (iv) a buffer selected from histidine, phosphate, and mixtures thereof at a concentration of 1-100 mM, preferably histidine at a concentration of 1-100 mM.

[0125] Yet another particularly preferred liquid formulation of the present invention comprises: (i) botulinum toxin at a concentration of 10-200 U / ml; (ii) HSA in an amount of 0.05-0.25% (w / v); (iii) sodium chloride in an amount of 0.9% (w / v); and (iv) a buffer selected from histidine, phosphate, and mixtures thereof at a concentration of 1-100 mM, preferably at a concentration of 2 mM to 50 mM, more preferably at a concentration of 5 mM to 20 mM, preferably histidine at a concentration of 1-100 mM, preferably at a concentration of 2 mM to 50 mM, more preferably at a concentration of 5 mM to 20 mM.

[0126] Furthermore, the above-mentioned preferred and particularly preferred liquid formulations preferably have a pH in the range of 6.0 to 7.5. The botulinum toxin is preferably serotype A, and more preferably a neurotoxic component of serotype A.

[0127] An example of a preferred liquid formulation of the present invention is as follows: Formulation 1: 50U / ml BoNT / A 0.9% NaCl (9 mg / mL) 0.085% HSA (EDTA-dialyzed) (0.85mg / mL) 0.155% histidine (1.55mg / ml; approximately 10mM) pH 6.0

[0128] The HSA (EDTA-dialyzed) of Preparation Example 1 is, for example, - adding 37.2 mg of Na2-EDTA per ml of 20% HSA solution under stirring and adjusting the pH to 8.0 with NaOH; - incubating for 6 hours at room temperature under stirring; - dialysis (dialysis membrane MWCO: 10 kDa) against 100 volumes of EDTA buffer (100 mM Na2-EDTA; 10 mM histidine; 0.9% NaCl, pH 7.0) with gentle stirring on a magnetic stirrer at room temperature for 16 hours; - dialysis against 100 volumes of 10 mM histidine, 0.9% NaCl, pH 6.0 at room temperature for 24 hours, followed by replacement of the dialysis buffer with fresh buffer and dialysis for another 24 hours, which is repeated two more times; It is prepared by a process comprising:

[0129] In formulation example 1, EDTA can be optionally replaced with other chelators such as EGTA, BAPTA, or DTPA. Instead of adding solid EDTA to HSA solution, concentrated EDTA solution can be used (e.g., 200 mM EDTA, pH 8.0). The first dialysis step (against EDTA buffer) can be omitted. Other methods such as cross-flow dialysis or ultrafiltration can be used instead of dialysis.

[0130] The present invention also relates to a liquid formulation comprising a botulinum toxin, wherein the toxin activity does not decrease by more than 20% relative to the initial toxin activity when the liquid formulation is stored at an elevated temperature of 40° C. for 4 weeks. The present invention also relates to a liquid formulation comprising a botulinum toxin, wherein the liquid formulation is stored at an elevated temperature of 250 W / m 2 Preferably, the liquid formulation is a liquid formulation in which the toxin activity does not decrease by more than 20% relative to the initial toxin activity even after 7 hours of exposure to a light source of 250 W / m 2 For a 7-h exposure at 1000 K, a SUNTEST CPS+ instrument (ATLAS Material Testing Technology, Inc.) can be used, equipped with a filter set providing a spectral distribution in the wavelength range of 320-800 nm, corresponding to ID65 (indirect daylight standard) according to ISO 10977, using window glass filters according to ICH Q1B.

[0131] The present invention further relates to a method for preparing a liquid botulinum toxin formulation as described herein, comprising adding one or more of the components as described herein, particularly components (i)-(ii), and optionally components (iii) and (vi), preferably components (i), (ii), (iii), and (iv). The preparation of the liquid formulations of the present invention is not particularly limited, and the respective formulation techniques are known to those skilled in the art. As mentioned above, the liquid formulation of botulinum toxin is generally an aqueous solution, preferably a saline solution, more preferably a physiological saline solution, and most preferably a buffered (e.g., phosphate or histidine buffered) physiological saline solution.

[0132] Preferably, the salt is dissolved first, then the HSA is added, the pH is adjusted if necessary, and the botulinum toxin is added. This order is not essential, but it is believed to ensure maximum specific activity of the BoNT. Preferably, the method for preparing a liquid botulinum toxin formulation does not include reconstituting a lyophilized botulinum toxin formulation in powder form.

[0133] The present invention further relates to a method of stabilizing a liquid botulinum toxin formulation, the method comprising combining a botulinum toxin with human serum albumin (HSA), the HSA comprising: (a) contacting a composition comprising human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; and (b) removing the chelating agent from the mixture; It is prepared by a method comprising:

[0134] The present invention further relates to the use of human serum albumin (HSA) to enhance the light and / or temperature stability of a liquid composition containing a botulinum toxin, the HSA comprising: (a) contacting a composition comprising human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; and (b) removing the chelating agent from the mixture; It is prepared by a method comprising:

[0135] Furthermore, the present invention relates to a method for producing a (a) contacting a composition comprising human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; and (b) removing said chelating agent from said mixture; The present invention relates to human serum albumin (HSA), which can be obtained by

[0136] In a fourth aspect, the present invention relates to a method for preparing a liquid formulation of the present invention, the method comprising the steps of: - purifying the human serum albumin starting material to obtain a purified human serum albumin material; - mixing the obtained purified human serum albumin with a botulinum toxin and, optionally, further ingredients, to obtain a liquid formulation; or, - purifying a liquid composition comprising a botulinum toxin and human serum albumin to obtain a purified liquid composition; - mixing the resulting purified liquid composition with further ingredients to obtain a liquid formulation; Includes.

[0137] The human serum albumin starting material is as defined herein above. Preferably, the human serum albumin starting material is in the form of an aqueous solution containing at least 5% (w / v) HSA, preferably 10% (w / v) to 35% (w / v) HSA, more preferably 15% (w / v) to 30% (w / v), and most preferably 20% (w / v) to 25% (w / v) HSA. The human serum albumin starting material may contain tryptophan and N-acetyltryptophan, particularly N-acetyltryptophan, in an amount of more than 5 mM, in an amount of 10 mM or more, in an amount of 20 mM or more, or in an amount of 30 mM or more. The pH of the mixture obtained in step (a) may also be adjusted to a pH in the range of 6.0 to 9.0, preferably 6.5 to 8.5, more preferably 7.0 to 8.5, and most preferably 7.0 to 8.0.

[0138] Preferably, in the first alternative of the method of the invention, the purification step results in a purified human serum albumin material that is free of tryptophan and / or N-acetyltryptophan or contains less than 5 mM, preferably less than 1 mM, more preferably less than 0.1 mM, most preferably less than 0.001 mM. In preparing the liquid formulation, the purified human serum albumin material is diluted so that the concentrations of tryptophan and N-acetyltryptophan are within the ranges described herein, for example less than 50 μM.

[0139] The purification step may be carried out by subjecting the human serum albumin starting material, or a liquid composition comprising botulinum toxin and human serum albumin, to dialysis; diafiltration (cross-flow filtration); ultrafiltration; chromatographic purification techniques such as ion exchange chromatography (IEX), affinity chromatography, and hydrophobic interaction chromatography (HIC); field-flow fractionation; or precipitation methods (e.g., salting out or ethanol precipitation).

[0140] Preferably, purification is carried out by dialysis. Dialysis is typically carried out for 0.5 to 48 hours, particularly 1 to 24 hours or 1 to 12 hours, at a temperature of 0°C to 30°C, particularly 2°C to 30°C or 4°C to 25°C, for example, at room temperature. Furthermore, dialysis is typically carried out against a dialysis buffer in an amount 10 to 1000 times the amount of the incubated HSA solution, and the dialysis buffer is usually exchanged at least once. The molecular weight cutoff of the dialysis membrane used is, for example, 10 kDa.

[0141] For example, purified serum albumin can be prepared by the following process: - Dialysis (MWCO of dialysis membrane: 10 kDa) of the commercial HSA preparation against 100 volumes of dialysis buffer (10 mM histidine, 0.9% NaCl, pH 6.0) with gentle stirring on a magnetic stirrer at room temperature for 24 hours; - Replace the buffer with fresh dialysis buffer and perform dialysis for 24 hours, repeating this process two more times; It may also be obtained by

[0142] The first option of the above method, i.e., purifying the starting material of human serum albumin and mixing the purified human serum albumin obtained with botulinum toxin and optionally further components, is preferred in the present invention. The mixing step to obtain the liquid formulation of the present invention is not particularly limited, but may include, for example, mixing components (i) to (ii), and optionally one or more of components (iii) and (vi). Preferably, the salt is dissolved first, then HSA is added, pH is adjusted if necessary, and botulinum toxin is added. This order is not essential, but it is believed to ensure that the maximum specific activity of BoNT is maintained. Preferably, the preparation method of the liquid botulinum toxin formulation does not include reconstitution of a lyophilized botulinum toxin formulation in powder form.

[0143] As described above with respect to the first aspect of the present invention, Fe 3+ Since ions also have a destabilizing effect on photostability, the Fe 3+It is also contemplated herein that the ion concentration is low. Thus, in accordance with the present invention, 3+ To remove the ions, the purification step of the method according to the invention is carried out as follows or further comprises the following steps: - contacting the human serum albumin starting material with a chelating agent to obtain a mixture of the human serum albumin starting material and the chelating agent; and - removing said chelating agent from said mixture to obtain a chelating agent-treated purified human serum albumin material; or, - contacting a liquid composition comprising a botulinum toxin and human serum albumin with a chelating agent to obtain a mixture of said liquid composition and said chelating agent; and - removing said chelating agent from said mixture to obtain a chelating-agent-treated purified liquid formulation.

[0144] In the contacting step, the human serum albumin starting material, and the liquid composition containing botulinum toxin and human serum albumin are each contacted with a certain amount of a chelating agent so that the chelating agent is present in the mixture at a concentration of 0.1 mM to 1000 mM or 0.1 mM to 500 mM, more preferably 0.5 mM to 500 mM or 1 mM to 500 mM, and most preferably 10 mM to 100 mM.

[0145] Removal of the chelating agent may be accomplished by dialysis, cross-flow filtration, or ultrafiltration.

[0146] The term "contacting" as used herein is intended to be broadly interpreted as combining two or more components together. This can be accomplished by a variety of different methods, such as dissolving, mixing, suspending, blending, slurrying, stirring, flowing by, adsorption, binding, extraction, etc. Typically, the chelating agent is added to a composition containing HSA (e.g., a solution of HSA), which is then mixed to obtain a homogenous mixture, and optionally subjected to further processing steps (e.g., dialysis against a chelating agent-containing buffer).

[0147] Thus, it is contemplated herein that "contacting" can be performed, for example, by loading the HSA material in the form of an aqueous solution onto an immobilized metal affinity resin (IMAC). The term "immobilized metal affinity resin" (IMAC) as used herein includes, but is not limited to, resins that contain immobilized functional moieties (such as iminodiacetic acid) capable of binding and coordinating polyvalent cations, such as Chelating-Sepharose, Fractogel-EMD-Chelate, POROS 20MC, Matrex Cellufine Chelate, TALON, and Chelex 100 resins. Such immobilized metal affinity resins are typically used in the form of chromatographic metal affinity columns, as known to those skilled in the art. The term "contacting" as used herein also includes binding in a batch mode using a suitable immobilized metal affinity resin.

[0148] Also, with respect to the contacting step, it should be noted that the terms "human serum albumin starting material" and "chelating agent" used in the definition of the contacting step are not intended to imply any limitation regarding their physical form or to exclude the presence of other substances or compounds mixed with or contained in the HSA and chelating agent. That is, the "human serum albumin starting material" that is contacted with the chelating agent may be present in any form, such as a solid or liquid (e.g., an aqueous composition or an aqueous solution). Similarly, the "chelating agent" that is contacted with the human serum albumin starting material may be present in any form, such as a solid or liquid (e.g., an aqueous composition or an aqueous solution).

[0149] Preferably, the mixture obtained in the contacting step is an aqueous mixture. This aqueous mixture can be prepared in various ways. For example, human serum albumin can be in the form of an aqueous composition, such as an aqueous solution, and this aqueous composition can be mixed with a chelating agent that can be in solid or liquid form, such as a chelating agent that can be in the form of an aqueous solution. Furthermore, human serum albumin can be in the form of a solid, such as a lyophilized material, mixed with a chelating agent and an aqueous solution, or mixed with an aqueous solution of the chelating agent. Preferably, human serum albumin is in the form of an aqueous composition, more preferably an aqueous solution, and the chelating agent is a solid or an aqueous composition (e.g. an aqueous solution).

[0150] In particular, the HSA contacted with the chelating agent in the contacting step may be in the form of an aqueous solution containing at least 5% (w / v) HSA, preferably 10-35% (w / v) HSA, more preferably 15-30% (w / v) HSA, and most preferably 20% (w / v)-25% (w / v) HSA. The pH of the mixture obtained in step (a) may be adjusted to a pH in the range of 6.0-9.0, preferably 6.5-8.5, more preferably 7.0-8.5, and most preferably 7.0-8.0.

[0151] Furthermore, the contacting step of the method for preparing a liquid formulation according to the present invention may include several sub-steps. For example, in one embodiment, the contacting step includes or consists of mixing a chelating agent (e.g., EDTA) and human serum albumin, incubating the mixture for a predetermined time, and optionally dialyzing the mixture against a buffer containing a chelating agent, preferably the same chelating agent as used in the incubation sub-step. In another embodiment, the mixture is not incubated and is (directly) dialyzed against a buffer containing a chelating agent, which is preferably the same as used in the step of mixing a chelating agent (e.g., EDTA) and human serum albumin. In another embodiment, the chelating agent and human serum albumin are not mixed before dialysis. That is, the contacting step (a) includes or consists of dialyzing the mixture against a buffer containing a chelating agent.

[0152] Preferably, the contacting step comprises or consists of adding the chelating agent to a composition comprising HSA (e.g., a solution of HSA); or mixing the chelating agent with a composition comprising HSA (e.g., a solution of HSA). The resulting mixture is then incubated for a period of time, e.g., left unstirred for a predetermined period of time, or stirred for a predetermined period of time.

[0153] The incubation time is not limited to a particular range, but is typically at least 0.5 hours, particularly at least 1 hour, and more particularly at least 2 hours. The upper limit of the incubation time is not critical and may be, for example, 1 hour, 2 hours, 5 hours, or 10 hours. Thus, the incubation time may be, for example, 0.5 to 5 hours, or 1 to 10 hours. Similarly, the incubation temperature is not particularly limited and may be, for example, within the range of 0°C to 60°C. Preferably, the temperature is 0°C to 30°C. That is, in the present invention, room temperature (20°C or 25°C) is a suitable temperature. As is known to those skilled in the art, the temperature affects the reaction time. In general, the incubation conditions (e.g., time and temperature) are selected so that the remaining amount of the chelating agent (e.g., EDTA) contained in the final product is 100 μM or less, preferably 10 μM or less, more preferably 1 μM or less.

[0154] Optionally, the incubation step is followed by further processing, for example, by dialysis of the incubated mixture against a buffer containing a chelating agent, which is typically the same as that used in the incubation step. The chelating agent used in this optional dialysis step is preferably contained in the dialysis buffer at a concentration of 0.1 mM to 1000 mM, more preferably 1 mM to 200 mM, and most preferably 10 mM to 100 mM. It is also preferred that the buffer used in the dialysis step (i) has a pH of 7.5 to 8.5; (ii) further comprises a buffering agent, which is preferably in accordance with the final composition; or (iii) further comprises an isotonicity agent, which is preferably 0.9% (w / v) sodium chloride; or (i) and (ii); (i) and (iii); (ii) and (iii); or (i) and (ii) and (iii);.

[0155] Chelators suitable for use herein are as defined herein above. Mixtures of any of the above chelators can also be used in the present invention, for example in solution, as a solid, or bound to a matrix.

[0156] According to the present invention, the chelating agent may be removed by any suitable technique, such as dialysis (conventional dialysis using a dialysis bag, countercurrent dialysis, etc.), reverse osmosis, filtration, cross-flow filtration, ultrafiltration, and chromatographic methods (e.g., ion exchange chromatography or gel filtration chromatography).

[0157] Preferably, the chelating agent is removed by dialysis. Dialysis is typically performed for 0.5 hours to 48 hours, particularly 1 hour to 24 hours or 1 hour to 12 hours, at a temperature of 0°C to 30°C, particularly 2°C to 30°C or 4°C to 25°C, for example, at room temperature. Furthermore, dialysis is typically performed against a dialysis buffer in an amount 10 to 1000 times the amount of the incubated HSA / chelating agent mixture, and the dialysis buffer is usually exchanged at least once. The molecular weight cutoff of the dialysis membrane used is, for example, 10 kDa.

[0158] In general, dialysis conditions (e.g., time, temperature, amount of buffer, number of buffer exchanges) are selected so that the residual amount of chelating agent (e.g., EDTA) in the final product is at a concentration of 100 μM or less, preferably 10 μM or less, and more preferably 1 μM or less.

[0159] In a fifth aspect, the present invention relates to a liquid formulation of the invention for use in therapy.

[0160] In particular, the liquid formulations of the present invention may be used in the treatment of neuromuscular disorders, pain, salivation, hyperhidrosis, urinary disorders, and neurological disorders.

[0161] Examples of neuromuscular diseases include dystonia, cervical dystonia, spasms, post-stroke spasticity, blepharospasm, tremors, hyperkinetic movement disorders, and cerebral palsy. Urological disorders include conditions characterized by detrusor overactivity, overactive bladder, neurogenic bladder and interstitial cystitis, vulvodynia and chronic pelvic pain treatment, benign prostatic hyperplasia (BPH), and detrusor-sphincter dyssynergia (DSD). Examples of neurological disorders include chronic migraine, trigeminal neuralgia, peripheral neuropathic pain, diabetic neuropathic pain, and depression.

[0162] In a sixth aspect, the present invention relates to the cosmetic use of the liquid formulation of the present invention for the treatment of a cosmetic condition.

[0163] This aspect of the invention relates to purely aesthetic uses of the liquid formulations of the invention.Preferred cosmetic conditions to be treated include skin conditions, particularly the treatment of skin wrinkles, especially facial wrinkles.

[0164] The term "wrinkles" as used herein shall be broadly interpreted to include not only wrinkles, but also lines, rhytids, creases, furrows, and folds. The terms "fine lines", "wrinkles", "fine lines", "furrows", and "folds" share similar definitions and are therefore often used interchangeably. In the present invention, "fine lines" are generally interchangeable with "wrinkles", but may preferably refer to shallower skin depressions than "wrinkles". "Folds" are interchangeable with wrinkles and fine lines, and are preferably linear depressions. "Fine lines" are interchangeable with wrinkles, fine lines, and folds. This term preferably refers to milder forms of wrinkles and may describe specific wrinkles in specific locations. "Fine lines" as used herein essentially have the same meaning as wrinkles. However, "shallow wrinkles" preferably refer to skin structures formed by irregular collections of fine wrinkles. "Deep wrinkles" are deep folds or deep fine lines in the skin.

[0165] Preferably, the wrinkles treated in accordance with the present invention are facial wrinkles, examples of which include horizontal forehead wrinkles, glabellar wrinkles, wrinkles around the eyes, crow's feet wrinkles, wrinkles at the base of the nose (i.e., wrinkles that extend downward on either side of the nose), nasolabial folds, radial wrinkles of the upper lip, radial wrinkles of the lower lip, wrinkles around the corners of the mouth, marionette lines, wrinkles around the mouth, corners of the mouth, menthol-labial folds, and chin wrinkles.

[0166] To treat the facial wrinkles listed above, botulinum toxin is typically administered by intramuscular injection into the following muscles: frontalis (horizontal forehead wrinkles), proximal and corrugator supercilii (frown lines), lateral orbicularis oculi (crow's feet / eye wrinkles), transverse nasal muscles, proximal and inferior nasal muscles (wrinkles at the base of the nose), levator labii superioris (nasolabial folds), orbicularis oris (radial wrinkles of upper and lower lips), depressor anguli oris (wrinkles around the corners of the mouth, marionette lines, corners of the mouth, mentolipal sulcus), and mentalis (wrinkles around the mouth, chin wrinkles).

[0167] One further preferred cosmetic use of the liquid formulation of the present invention relates to its use for cosmetic applications involving face and / or body rejuvenation and / or improvement of skin texture.

[0168] In a seventh aspect, the present invention relates to a method of treating a disease or condition comprising administering to an individual in need thereof an effective amount of a liquid formulation of the present invention.

[0169] The disease or condition may be any one of the diseases and conditions described herein above, regardless of whether it is a therapeutic or cosmetic indication.Accordingly, in one embodiment, the present invention relates to a (non-therapeutic) method of treating a cosmetic (aesthetic) condition, preferably a skin condition, comprising administering an effective amount of a liquid formulation of the present invention to an individual in need thereof.In another embodiment, the present invention relates to a (non-therapeutic) method of treating a cosmetic (aesthetic) condition, preferably a skin condition, comprising injecting an effective amount of a liquid formulation of the present invention to an individual in need thereof.

[0170] A further preferred method of the present invention relates to a method for rejuvenating and / or improving skin quality of the face and / or body, comprising administering to an individual in need thereof an effective amount of a liquid formulation of the present invention.

[0171] The subject of treatment is not particularly limited, so long as it has a disease or condition that can be treated according to the present invention. Those skilled in the art will be able to determine the appropriate administration regimen for the treatment of a given therapeutic or cosmetic indication. In particular, the injection may be intradermal, subcutaneous, or intramuscular, depending on the disease or condition to be treated.

[0172] Apart from the above, the present invention further relates to a method for stabilizing a liquid botulinum toxin formulation, the method comprising combining a botulinum toxin with human serum albumin (HSA), the HSA being prepared by purifying a human serum albumin starting material to obtain a purified human serum albumin material as described in relation to the fourth aspect of the invention.

[0173] The present invention further relates to the use of human serum albumin (HSA) to provide a light- and temperature-stable liquid composition comprising a botulinum toxin, the HSA being prepared by purifying a human serum albumin starting material to obtain a purified human serum albumin material as described in relation to the fourth aspect of the invention. EXAMPLES

[0174] I. Examples 1 to 3 The following examples illustrate liquid botulinum toxin formulations according to the present invention and methods for their preparation. Percentages are weight / volume (w / v) unless otherwise specified.

[0175] The biological activity of botulinum toxins was determined using a cell-based potency assay (CBA) as described in WO 2014 / 207109. Briefly, neuronal cells were incubated with neurotoxin-containing samples and standards of known potency.

[0176] After the incubation period, the cells were lysed and the amount of cleaved SNAP25 protein was measured by immunoassay, and the biological activity of the samples was calculated by comparing the cleavage rate of the cells treated with the samples with that of the standard samples.

[0177] Example 1 Light and storage stability of liquid botulinum toxin formulations (not according to the invention) containing HSA pretreated by conventional dialysis In initial experiments, it was found that the biological activity of a liquid formulation of 150 kDa botulinum neurotoxin type A (also referred to herein as "150 kDa BoNT / A") without complexing proteins was significantly reduced after exposure to light (daylight or room light) and that photosensitivity increased with increasing human serum albumin (HSA) concentrations. In further experiments, the inventors unexpectedly discovered that the light stability and storage stability of liquid botulinum toxin formulations at 40°C can be significantly increased by adding a complexing agent such as EDTA, even when EDTA was present in the form of its magnesium, calcium, or zinc complexes (results not shown).

[0178] In view of these results, it was speculated that an unknown component contained in HSA caused the photosensitivity of botulinum toxin, and that this component could be inactivated or masked in some way by a chelating agent such as EDTA. However, further experiments conducted by the present inventors surprisingly revealed that EDTA intensifies the pain of injection. Therefore, in order to dispense with a chelating agent such as EDTA, the present inventors attempted to remove the unknown component in HSA that causes photoinstability by dialysis.

[0179] For this purpose, a concentrated stock solution of HSA (e.g., 20%) was thoroughly dialyzed against a buffer of 10 mM histidine, 0.9% NaCl (pH 6.0) for 4 x 12 hours (3 buffer changes, 100 times the volume of the sample) at room temperature to obtain "dialyzed HSA". The following liquid formulations of botulinum toxin (150 kDa BoNT / A without complexed proteins) were then prepared using the dialyzed and non-dialyzed HSA: Formulation 1: 65 U / mL botulinum toxin, 0.1% HSA (undialyzed), 10 mM histidine, 0.9% NaCl (pH 6.0) Formulation 2: 65 U / mL botulinum toxin, 0.1% HSA (dialyzed), 10 mM histidine, 0.9% NaCl (pH 6.0)

[0180] The photostability of these two formulations was evaluated at 250 W / m 2 The relative botulinum toxin activity was determined by measuring the activity after 7 hours of exposure to 250 W / m light in comparison to a control sample kept in the dark. 2 For the 7-h exposure to light, a SUNTEST CPS+ instrument (ATLAS Material Testing Technology, Inc.) was used, equipped with a filter set providing a spectral distribution in the wavelength range of 320–800 nm, corresponding to ID65 (indirect daylight standard) according to ISO 10977, using window glass filters according to ICH Q1B. The results are shown in Table 1. [Table 1]

[0181] As can be seen from Table 1, limited effects on photostability were observed for formulation 2 containing dialyzed HSA compared to formulation 1 containing non-dialyzed HSA.

[0182] In addition, the storage stability of Formulations 1 and 2 was determined by measuring the relative botulinum toxin activity after 2 and 4 weeks of storage at 40° C. in comparison with the TO control sample (measured immediately after preparation of Formulations 1 and 2). The results are shown in Table 2. [Table 2]

[0183] It can be seen that dialysis does not affect the storage stability of the botulinum toxin.

[0184] Example 2 Light and storage stability of liquid botulinum toxin formulations containing complexing agents and HSA pretreated by dialysis Despite the results of Example 1 (i.e., the requirement of EDTA or an EDTA complex for photostability; dialyzed HSA does not provide significant improvement in photostability or storage stability), the inventors continued to search for liquid botulinum toxin formulations that are light- and storage-stable without the inclusion of a chelating agent such as EDTA.

[0185] First, EDTA was added to a concentrated stock solution of HSA (e.g., 20%) (100 mM = 37.2 mg Na2-EDTA per ml of HSA solution, pH 8.0) and incubated at room temperature for 6 h under stirring. The incubated mixture was then dialyzed against a buffer solution containing EDTA (100 mM EDTA, 10 mM histidine, 0.9% NaCl, pH 7.0) at room temperature for 16 h. The dialysis membrane used had a MWCO of 10 kDa and a loading volume of 1 cm of dialysis membrane. 2 The volume of the dialysis buffer was about 100 times the volume of the sample.

[0186] The EDTA was then removed by dialysis against EDTA-free dialysis buffer (10 mM histidine, 0.9% NaCl (pH 6.0)) at room temperature for 24 hours in an amount approximately 100 times the volume of the sample. The dialysis buffer was then replaced with fresh dialysis buffer, and dialysis was again performed for 24 hours. This was repeated twice to obtain pretreated HSA ("EDTA-dialyzed HSA").

[0187] This pretreated HSA was then used to prepare liquid formulation 3 of botulinum toxin (150 kDa BoNT / A without complexed proteins): Formulation 1: Same as formulation 1 in Example 2 (65 U / mL botulinum toxin, 0.1% HSA (undialyzed), 10 mM histidine, 0.9% NaCl (pH 6.0)) Formulation 3: 65 U / mL botulinum toxin, 0.085% HSA (EDTA-dialyzed), 10 mM histidine, 0.9% NaCl (pH 6.0)

[0188] Photostability of Formulations 1 and 3 at 250 W / m 2 The relative botulinum toxin activity after 7 hours of exposure to light was determined by measuring the activity in comparison to a control sample kept in the dark. The results are shown in Table 3. [Table 3]

[0189] As can be seen from Table 3, formulation 3 containing EDTA-dialyzed HSA showed a significant improvement in photostability compared to formulation 1 containing non-pretreated HSA.

[0190] Additionally, storage stability was determined by measuring the relative botulinum toxin activity after 2 and 4 weeks of storage at 40° C. in comparison with the TO control samples (measured immediately after preparation of Formulations 1 and 3). The results are shown in Table 4. [Table 4]

[0191] The results showed that the use of EDTA-treated and dialyzed HSA resulted in significantly higher biological toxin activity compared to formulation 1, which contained untreated (non-dialyzed) HSA.

[0192] Overall, this example demonstrates that pre-treating HSA with a chelating agent and then removing the chelating agent, e.g., by dialysis, can unexpectedly eliminate the need for a chelating agent (e.g., EDTA) in the liquid formulation. The resulting formulation is not only light and temperature (storage) stable, but also has reduced pain upon injection compared to liquid formulations containing a chelating agent such as EDTA.

[0193] Example 3 Effect of iron ions on the photostability of liquid botulinum toxin formulations. Furthermore, we carried out experiments to examine the effect of metal ions on photosensitivity. From this viewpoint, calcium (1 mM Ca 2+ ), cobalt (1mM Co 2+ ), copper (1mM Cu 2+ ), Nickel (1mM Ni 2+ ), or iron (1mM Fe 3+ ) was added to a liquid formulation (pH 6.0) containing 50 U / ml of 150 kDa BoNT / A, 0.085% pretreated HSA (i.e., EDTA-treated and dialyzed HSA) prepared according to Example 2, 10 mM histidine, 0.9% NaCl. The liquid formulation was prepared using high purity water, histidine, and NaCl, without the use of iron or steel equipment. The resulting formulation was exposed to light (250 W / m 2 for 7 hours) and compared with a control formulation stored in the dark.

[0194] When compared with a formulation without calcium or cobalt (79% residual toxic activity compared to the dark-stored control formulation), calcium (1 mM Ca 2+ ) and cobalt (1 mM Co 2+ The addition of copper (1 mM Cu) was found to have no effect on photostability (74% and 77% residual toxin activity compared to the dark-stored control formulation). 2+ ) and nickel (1mM Ni 2+ ) slightly decreased the photostability. In contrast, the addition of iron (1 mM Fe 3+ ) significantly reduced the photostability, and exposure to light (250 W / m 2 After incubation at RT for 7 h, residual toxin activity was only 1% compared to the dark-stored control preparation (results not shown).

[0195] Based on these results, the photosensitivity of liquid BoNT / A preparations to Fe 3+The effect of ions was investigated in more detail in the concentration range from 10 pM to 100 μM. As in the above experiment, the same formulation containing pretreated HSA (50 U / ml botulinum toxin, 0.085% pretreated HSA, 10 mM histidine, 0.9% NaCl (pH 6.0)) was used and the stock solution (50 mM Fe(NO3)3) was used to irradiate the cells with Fe(NO3). 3+ The concentration was adjusted to the desired final concentration. 2 The biological activity measured after 7 hours of exposure to light is expressed as a percentage of the biological activity measured for each sample stored in the dark and is shown in Table 5. [Table 5]

[0196] As can be seen from Table 4, at a concentration of 316 nM, Fe 3+ The addition of Fe ions significantly affected the stability of BonT / A, decreasing the stability by more than 15%. The photostability decreased significantly at a concentration of 1 μM, but was still higher than at concentrations above 1 μM. At concentrations above 1 μM, the addition of Fe 3+ Ions have a significant adverse effect on the stability of BoNT / A in liquid formulations under the influence of light.

[0197] In another experiment, a liquid botulinum toxin formulation containing pretreated HSA (i.e., 50 U / ml botulinum toxin, 0.085% pretreated HSA, 10 mM histidine, 0.9% NaCl (pH 6.0)) was stirred in a stainless steel beaker for 48 hours and showed no significant difference in the botulinum toxin concentration when exposed to light (250 W / m 2 After 7 hours in a polypropylene container, the relative BoNT / A activity was only about 2%, in contrast to 78% for the same sample stirred for 48 hours in a polypropylene container. This was a highly surprising finding, as traces of Fe were not detected in the liquid botulinum toxin preparation from the stainless steel beaker. 3+ This is presumably due to the dissolution of iron ions, indicating that iron ions are an important factor to consider when preparing liquid botulinum toxin formulations.

[0198] II. Examples 4 to 6 The photostability of liquid botulinum toxin formulations according to the invention is further demonstrated in the following Examples 4 to 6. To this end, several liquid botulinum toxin formulations were prepared (Example 4) and samples of each of the prepared formulations were exposed to a 250 W / m 2 The samples were exposed to 1000 mg of light for 7 hours or kept in the dark for 7 hours as a control. The botulinum toxin activity of the light-exposed samples and the dark-stored examples was then measured by cell-based potency assay (CBA). Light stability is expressed as a percentage of the average botulinum toxin activity measured for the light-exposed samples relative to the average botulinum toxin activity measured for the dark-stored samples.

[0199] The biological activity of botulinum toxin was determined using a cell-based potency assay (CBA) as described in WO 2014 / 207109. Briefly, neuronal cells were incubated with a neurotoxin-containing sample and a standard sample of known potency. After incubation, the cells were lysed and the amount of cleaved SNAP25 protein was measured by immunoassay. The biological activity of the sample was then calculated by comparing the cleavage rate of cells treated with the sample to that of cells treated with the standard sample.

[0200] The above 250W / m 2 For the 7-h exposure to light, a SUNTEST CPS+ instrument (ATLAS Material Testing Technology, Inc.) was used, equipped with a filter set providing a spectral distribution in the wavelength range of 320–800 nm, corresponding to ID65 (indirect daylight standard) according to ISO 10977, using a window glass filter according to ICH Q1B.

[0201] Example 4 Preparation of various botulinum toxin preparations The compositions of the formulations analyzed for photostability are shown in Tables 6 and 7. The botulinum toxin (BoNT) used was NT101. [Table 6] [Table 7]

[0202] Preparation of "HSA-Dialyzed". 10 ml of 25% HSA (CSL Behring) was transferred to a dialysis cassette (Slide-A-Lyzer™, 10 kDa MWCO, 12-30 ml, Thermo Scientific / Pierce). The dialysis cassette was placed in 2 liters of a solution of 1.55 g / l histidine, 9 g / l (pH 6.0) and dialyzed for 24 hours with stirring (150 rpm, magnetic stirrer) under light protection. The dialysis buffer was then exchanged for 2 liters of fresh dialysis buffer (1.55 g / l histidine, 9 g / l NaCl (pH 6.0)) and the procedure was repeated three more times, each for approximately 12 hours. The resulting contents of the dialysis cassette are referred to as dialyzed HSA or "HSA-Dialyzed". Concentrations were measured as follows:

[0203] Preparation of HSA-EDTA-Dialyzed. 18 ml of 25% HSA (CSL Behring) was mixed with 2 ml of 1 M Na2EDTA (pH 8.0). The resulting solution was stirred for approximately 6 hours. Afterwards, 15 ml of the solution was transferred to a dialysis cassette (Slide-A-Lyzer™, 10 kDa MWCO, 12-30 ml, Thermo Scientific / Pierce). The dialysis cassette was placed in 2 liters of a solution of 1.55 g / l histidine, 9 g / l NaCl, 37.2 g / l Na2EDTA (pH 7.0) and dialyzed for 24 hours with stirring (150 rpm, magnetic stirrer) under light protection.

[0204] The dialysis buffer was then exchanged into 2 liters of 1.55 g / l histidine, 9 g / l NaCl, pH 7.0, followed by three cycles of approximately 12 hours of dialysis and exchange into 2 liters of fresh dialysis buffer (first buffer exchange: His / NaCl, pH 6.5, second and third buffer exchanges: His / NaCl, pH 6.0). The resulting contents of the dialysis cassette are referred to as EDTA-dialyzed HSA material (or "HSA-EDTA-dialyzed").

[0205] The HSA concentration after dialysis is generally significantly lower than the starting HSA solution concentration (25% (w / v)) due to the volume increase during dialysis. Since the protein concentration is high and no significant loss is expected, the absolute amount of HSA is not expected to change, and the concentration was calculated based on the volume change as known to those skilled in the art. The HSA concentration after dialysis was determined to be approximately 8.1% (w / v).

[0206] Preparation of placebo bulk solution. To prepare a formulation identical to that shown in Tables 6 and 7 except that it does not contain an active agent (BoNT) (referred to herein as "placebo bulk solution"), the starting materials HSA, NaCl, and histidine were weighed and dissolved in water (Milli-Q). 50 mM stock solutions of tryptophan (Trp) and N-acetyltryptophan (N-AcTrp) were prepared (10.212 g / l and 12.313 g / l, respectively, in warm water) and added according to the desired final Trp and N-AcTrp concentrations. The pH was then adjusted to 6.0 by adding 1M NaOH or 1M HCl, and water was added to the final volume, after which the solution was sterile filtered through a 0.22 μm filter.

[0207] Preparation of BoNT Pre-Dilution: 13.44 mg of NT101 was weighed into a 50 ml sterile low protein binding tube and mixed with 20.123 g of sterile filtered Formulation 7 placebo bulk solution to obtain a pre-dilution containing 25,000 U / ml of BoNT.

[0208] Sample preparation. 20.0 μl of BoNT pre-dilution was placed into a 15 ml sterile protein low-binding tube and 9.98 ml of the respective placebo bulk solution was added. The solutions were mixed by gentle inversion 10 times. Then, 3×1 ml of each solution was placed into a 1.5 ml low-binding tube and these were heated at 250 W / m 2 The remainder of each sample solution was covered in aluminum foil and stored in bulk at 4° C. until measurements were taken (the “dark control” samples).

[0209] Example 5 Comparison of photostability of various liquid BoNT formulations 250W / m 2 The botulinum toxin activity of samples of Formulations I-IV in Table 6 exposed to 1000 uL light for 7 hours or stored in the dark for 7 hours was measured (in duplicate) using CBA. The results are shown in Table 8 as percent activity of the light-exposed samples relative to the dark-stored control samples. [Table 8]

[0210] As can be seen from Table 8, dialysis of HSA somewhat increases the stability of BoNT against light exposure (compare formulation II with formulation I). Addition of EDTA to untreated HSA significantly increases efficacy (see formulation III). However, this addition of EDTA does not provide complete protection from the destabilizing effects of light. The best photostability is achieved by preincubation with EDTA followed by dialysis against EDTA-free histidine buffer (see formulation IV). This indicates that the observed photostability is not due to metal ions alone but may be due to other unknown compounds.

[0211] Example 6 Photostability of liquid BoNT formulations in the presence and absence of tryptophan and N-acetyltryptophan 250W / m 2 The botulinum toxin activity of samples of Formulations 1-7 in Table 7 exposed to light for 7 hours at 40° C. or stored in the dark for 7 hours was measured (in duplicate) using CBA. The results are shown in Table 9 as percent activity of the light-exposed samples relative to the dark-stored control samples. [Table 9]

[0212] Both tryptophan and N-acetyltryptophan significantly and negatively affect the light stability of BoNT liquid formulations in a dose-dependent manner. At higher concentrations (10 mM and 2 mM, respectively), the effect of decreasing light stability was significant, with very low relative biological activity (<10%). At concentrations as low as 80 μM of Trp or N-AcTrp, the decrease in biological activity of BoNT after light exposure is about 10%, compared to control formulations without Trp or N-AcTrp (compare formulations 4 and 6 with formulation 7).

[0213] The 80 μM N-acetyltryptophan concentration corresponds to dilution of 25% (w / v) HSA starting material (containing 20 mM N-AcTrp) to 0.1% (w / v), i.e., 1 mg / ml HSA. Because 1 mg / ml of HSA is within the range of concentrations typically used in botulinum toxin formulations, the use of such an HSA starting material results in the N-acetyltryptophan concentration in the BoNT / A formulation increasing the photosensitivity of the formulation. Thus, the concentration range in which tryptophan and N-acetyltryptophan adversely affect photostability is of great practical importance.

Claims

1. (i) botulinum toxin; and (ii) human serum albumin; A liquid formulation comprising: Fe ３＋ ions at a concentration of less than 1 μM, preferably less than 500 nM, more preferably less than 250 nM; Liquid formulation.

2. (i) botulinum toxin; and (ii) human serum albumin; A liquid formulation comprising: containing no tryptophan and N-acetyltryptophan or less than 50 μM; Liquid formulation.

3. (i) botulinum toxin; and (ii) human serum albumin; A liquid formulation comprising: (a) contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; and (b) removing the chelating agent from the mixture; prepared by a process comprising: Liquid formulation.

4. (a) contacting human serum albumin with a chelating agent to obtain a mixture of human serum albumin and the chelating agent; and (b) removing the chelating agent from the mixture; prepared by a process comprising: The liquid formulation of claim 1.

5. In step (a), a human serum albumin starting material is contacted with a chelating agent to obtain a mixture of the human serum albumin starting material and the chelating agent; in step (b), the chelating agent is removed from the mixture to obtain a pre-treated human serum albumin material; and in a further step (c), a botulinum toxin is mixed with the pre-treated human serum albumin material; and the removal of the chelating agent is preferably achieved by dialysis, filtration, cross-flow filtration, or ultrafiltration. or, In step (a), a liquid pre-formulation containing a botulinum toxin and human serum albumin is contacted with the chelating agent to obtain a mixture of the liquid pre-formulation and the chelating agent; in step (b), the chelating agent is removed from the mixture to obtain the liquid formulation; the removal of the chelating agent is preferably carried out by dialysis, filtration, cross-flow filtration, or ultrafiltration.

5. The liquid formulation according to claim 3 or 4.

6. 5. The liquid formulation of claim 1, wherein the human serum albumin is present in the liquid formulation in an amount of 0.001 to 1.00% (w / v).

7. Fe ３＋ 3. The liquid formulation of claim 2, comprising an ion at a concentration of less than 1000 nM, preferably less than 500 nM, more preferably less than 250 nM.

8. The liquid formulation further comprises: (iii) comprises a tonicity agent; or The liquid formulation further comprises: (iii) comprises a tonicity agent, and the tonicity agent is present in the liquid formulation in an amount of 0.01 to 2.0% (w / v), or the tonicity agent is sodium chloride, or the tonicity agent is sodium chloride and is present in the liquid formulation in an amount of 0.01 to 2.0% (w / v); The liquid formulation according to any one of claims 1 to 4.

9. The liquid formulation further comprises: (iv) containing a buffer; or The liquid formulation further comprises: (iv) comprising a buffering agent, wherein the buffering agent is present in the liquid formulation at a concentration of 1 to 100 mM, the buffering agent is an amino acid, a phosphate, or a mixture thereof, the buffering agent is histidine, or the buffering agent is histidine, a phosphate, or a mixture thereof, and the histidine and phosphate are present in the liquid formulation at a concentration of 1 to 100 mM; The liquid formulation according to any one of claims 1 to 4.

10. - purifying the human serum albumin starting material to obtain a purified human serum albumin material; - mixing the obtained purified human serum albumin with a botulinum toxin and, optionally, further ingredients, to obtain the liquid formulation; or, - purifying a liquid composition comprising botulinum toxin and human serum albumin to obtain a purified liquid composition; - mixing the obtained purified liquid composition with further ingredients to obtain the liquid formulation; Including, The purification step is preferably carried out by subjecting the human serum albumin starting material or the liquid composition comprising botulinum toxin and human serum albumin to dialysis, diafiltration, ultrafiltration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, field-flow fractionation, or precipitation (e.g., salting out or ethanol precipitation).

10. A method for preparing a liquid formulation according to claim 2 or 7.

11. The purification step is carried out as follows, or the purification step further comprises the steps of: - contacting the human serum albumin starting material with a chelating agent to obtain a mixture of said human serum albumin starting material and said chelating agent; and - removing the chelating agent from the mixture to obtain a chelating agent-treated purified human serum albumin material; or, - contacting a liquid composition comprising a botulinum toxin and human serum albumin with the chelating agent to obtain a mixture of the liquid composition and the chelating agent; and, - removing the chelating agent from the mixture to obtain a chelating agent-treated purified liquid formulation; The contacting step in methods (i) and (ii) is preferably carried out so that the concentration of the chelating agent in the mixture is between 1 mM and 500 mM, and / or the removal of the chelating agent is carried out by dialysis, cross-flow filtration, or ultrafiltration. The method of claim 10.

12. the chelating agent is selected from the group consisting of aminopolycarboxylic acids having 3 to 6 carboxylic acid functional groups, citrate salts, porphyrins, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), triethylenetetramine (TETA), and mixtures thereof; Preferably, the chelating agent has the general formula (I): (HO ２ CCH ２ ) ２ N-R-N(CH ２ CO ２ H) ２ (I) wherein R contains zero or one or two carboxylic acid groups, said aminopolycarboxylic acid being preferably selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), and mixtures thereof; 5. The liquid formulation according to claim 3 or 4.

13. The chelating agent is selected from the group consisting of an aminopolycarboxylic acid having 3 to 6 carboxylic acid functional groups, a citrate salt, a porphyrin, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), triethylenetetramine (TETA), and mixtures thereof; Preferably, the chelating agent has the general formula (I): (HO 2 CCH 2 ) 2 N-RN(CH 2 CO 2 H) 2 (I) wherein R contains zero or one or two carboxylic acid groups, said aminopolycarboxylic acid being preferably selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), and mixtures thereof; The method of claim 10.

14. 5. The liquid formulation of claim 1, wherein the botulinum toxin is a botulinum neurotoxin complex, the botulinum toxin is a botulinum neurotoxin without complexing proteins, the botulinum toxin is of serotype A, the botulinum toxin is a botulinum neurotoxin of serotype A without complexing proteins, the botulinum toxin is of serotype A and is present at a concentration of 1 to 1000 U / ml, the botulinum toxin is a botulinum neurotoxin of serotype A without complexing proteins and is present at a concentration of 1 to 1000 U / ml, or the liquid formulation has a pH in the range of 5.0 to 8.

0.

15. The botulinum toxin is a botulinum neurotoxin complex, or the botulinum toxin is a botulinum neurotoxin that does not contain a complexing protein, or the botulinum toxin is of serotype A, or the botulinum toxin is a botulinum neurotoxin of serotype A that does not contain a complexing protein, or the botulinum toxin is of serotype A and is present at a concentration of 1 to 1000 U / ml, or the botulinum toxin is a botulinum neurotoxin of serotype A that does not contain a complexing protein and is present at a concentration of 1 to 1000 U / ml, or the pH of the liquid formulation is within the range of 5.0 to 8.

0. The method of claim 10.

16. A liquid formulation according to any one of claims 1 to 4 for use in therapy, in particular for use in the treatment of neuromuscular disorders, pain, salivation, hyperhidrosis, urinary disorders and neurological disorders.

17. Cosmetic use of a liquid formulation according to any one of claims 1 to 4 for the treatment of a cosmetic condition.