Improved Clostridium botulinum serotype A neurotoxin compositions and drying processes

Abstract

The present invention relates to solid compositions comprising Clostridium botulinum neurotoxin serotype A (BoNT / A) and human serum albumin (HSA), wherein the solid compositions comprise a small amount of HSA aggregates. The present invention further relates to methods of producing solid compositions comprising BoNT / A and HSA by drying a solution comprising BoNT / A such that the solution does not undergo freezing upon drying. Figure for abstract: 2A

Description

Title of the invention: Clostridium botulinum serotype A neurotoxin compositions and improved drying processes CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of US Provisional Application No. 63 / 623,130, filed on January 19, 2024.

[0002] REFERENCE TO AN ELECTRONICALLY SUBMITTED SEQUENCE LIST

[0003] The present application incorporates by reference a sequence listing filed with the application as an XML file titled "ABV21538 FR L2_SEQLISTING.xml", created on January 3, 2024 and having a size of 7,270 bytes. 1. DOMAIN

[0004] The present invention relates to solid compositions comprising Clostridium botulinum neurotoxin serotype A (BoNT / A) and human serum albumin (HSA), the solid compositions comprising a small amount of HSA aggregates. The present invention further relates to methods of producing solid compositions comprising BoNT / A and HSA by drying a solution comprising BoNT / A such that the solution does not undergo freezing upon drying. 2. BACKGROUND

[0005] Clostridium botulinum neurotoxin serotype A (BoNT / A) is a highly potent toxin that causes muscle relaxation by inhibiting the docking and fusion of synaptic vesicles, thereby blocking the release of acetylcholine at neuromuscular junctions. BoNT / A is produced by strains of Clostridium botulinum type A by synthesizing a complex of a 150 kDa neurotoxin together with a group of nontoxic associated proteins (NAPs). BOTOX®, or onabotulinumtoxinA, is a BoNT / A product that was approved in 1989 in the United States by the Food and Drug Administration (FDA) for a variety of therapeutic and cosmetic indications.

[0006] Many steps are implemented during the manufacture of protein-based pharmaceutical compositions intended for biopharmaceutical applications. Parameters involved in certain manufacturing steps can have consequences on the properties or functionalities of the proteins.

[0007] There is still a need today for the development of pharmaceutical compositions of botulinum toxins having improved properties.

[0008] The citation of a reference herein shall not be construed as an acknowledgment that such reference is prior to the present disclosure. 3. SUMMARY OF THE INVENTION

[0009] The present disclosure relates to novel solid compositions comprising Clostridium botulinum neurotoxin serotype A (BoNT / A) having advantageous properties, as well as novel methods for producing such compositions.

[0010] More specifically, the present disclosure relates to solid compositions comprising Clostridium botulinum neurotoxin serotype A (BoNT / A) and human serum albumin (HSA), the solid compositions comprising a small amount of HSA aggregates. The present disclosure also relates to methods of producing solid compositions comprising BoNT / A and HSA, wherein the methods comprise a step of drying a solution comprising BoNT / A and HSA to produce said solid composition, wherein the solution does not undergo freezing during the drying step. The present disclosure further relates to solid compositions which are produced by such a method.

[0011] HSA has been used as an excipient in protein-based therapeutic formulations to improve stability and resistance to external stresses, for example, by preventing aggregation, oxidation, and surface adsorption. HSA is a particularly important stabilizer in the high-dosage, low-fill-volume formulation of BOTOX®, given its ability to potentially reduce misfolding and aggregation of BoNT / A in part due to its surface-coating property.Apart from any connection with any theory, the present disclosure teaches that freezing during drying unexpectedly leads to a significant increase in the amount of HSA aggregation in a solid BoNT / A composition compared to drying without freezing, that certain measures can be used to avoid freezing during vacuum drying of BoNT / A compositions, and that solid BoNT / A compositions dried without undergoing freezing exhibit advantageous properties such as consistent visual quality of the product and a much slower decline in activity during storage, for example, at room temperature or higher. Accordingly, the present disclosure provides novel solid compositions comprising a BoNT / A and a low amount of HSA aggregates as well as methods of manufacture with an improved drying process for obtaining such solid compositions.

[0012] According to one aspect, there is provided a solid composition comprising a BoNT / A and HSA, wherein the percentage of HSA aggregates, HSA polymer, of HSA oligomer or HSA dimer in the soluble HSA contained in the solid composition is reduced. Specifically provided is a solid composition comprising a 900 kDa BoNT / A complex, HSA and sodium chloride (NaCl), wherein the HSA comprises an HSA monomer and HSA aggregates, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein less than 34% of the soluble HSA contained in the solid composition is HSA aggregates.

[0013] Also provided is a method of producing a solid composition comprising a 900 kDa BoNT / A complex, HSA and NaCl, said method comprising a step of drying a solution comprising a BoNT / A, HSA and NaCl to produce said solid composition, wherein the solution does not undergo freezing during the drying step.

[0014] According to another aspect, there is provided a solid composition produced by a method described herein.

[0015] According to another aspect, there is provided a method of treating a patient in need thereof, comprising administering a solid composition described herein. 4. BRIEF DESCRIPTION OF THE FIGURES

[0016] [Fig-1] Copy of part of the certificate of analysis of a batch of HSA, CSL Behring.

[0017] [Fig.2A] Representative data from a vacuum drying cycle. The temperature of the product (batch no. 4) was measured by Ellab temperature probes

[0018] [Fig.2B] Representative data from freeze-drying cycles. The temperature of the product (batch no. 9) was measured by Ellab temperature probes.

[0019] [Fig.3A], [Fig.3B], [Fig.3C] Representative SEC chromatograms of HSA with different detection methods: fluorescence (excitation 280 nm / emission 350 nm) ([Fig.3A]), UV 280 nm ([Fig.3B]) and UV 220 nm ([Fig.3C]).

[0020] [Fig.4A] Representative SEC chromatograms of reconstituted products produced by vacuum drying (Process 1), by freeze-drying (Process 2) and by freeze-drying with pre-filtration and post-filtration storage (Process 3) (UV detector 220 nm). The inset shows a graph representing the percentage of aggregation in the form of HSA polymer (left bars) and HSA dimer (right bars) in three finished product samples (Batch No. 7, No. 9 and No. 4) and in the "pure" HSA (Batch No. 1).

[0021] [Fig.4B] Representative SEC chromatograms of reconstituted products produced by vacuum drying (Process 1), by freeze-drying (Process 2) and by freeze-drying with pre-filtration and post-filtration storage (Process 3) (UV detector 280 nm). The inset shows a graph representing the percentage of aggregation in the form of HSA polymer (left bars) and HSA dimer (right bars) in three finished product samples (batches #7, #9 and #4) and in “pure” HSA (batch #1).

[0022] [Fig.4C] Representative SEC chromatograms of reconstituted products produced by vacuum drying (Process 1), by freeze-drying (Process 2) and by freeze-drying with pre-filtration and post-filtration storage (Process 3) (fluorescence detector). The inset shows a graph representing the percentage of aggregation in the form of HSA polymer (left bars), HSA oligomer (middle bars) and HSA dimer (right bars) in three finished product samples (Batch No. 7, No. 9 and No. 4) and in "pure" HSA (Batch No. 1).

[0023] [Fig.5A], [Fig.5B], [Fig.5C], [Fig.5D], [Fig.5E], [Fig.5F], [Fig.5G] Aggregation of total HSA determined by SEC in finished product batches manufactured with 3 production processes (by vacuum drying (i.e. “reference condition, no intermediate storage, no freezing”), by freeze-drying (i.e. “stress, freezing only”), and by freeze-drying with storage of the solution prior to drying (i.e. “stress, intermediate storage at room temperature and freezing”)). Data for pure HSA batches are also included. Figures 5A, 5C and 5E present the graphs obtained with a 220 nm UV detector, a 280 nm UV detector, and a fluorescence detector, respectively. Figures 5B, 5D and 5F present the corresponding data values obtained with a 220 nm UV detector, a 280 nm UV detector, and a fluorescence detector, respectively. Data for some batches are shown in [Fig.5G].

[0024] [Fig.ôA], [Fig.ôB] Particle counting results for three placebo batches produced using different manufacturing processes. [Fig.ôA]: An average number of particles per image is shown. Saline solution (left, n = 1), reference condition (i.e., vacuum drying, second from the left, n = 5, batch no. 6), after stress (intermediate storage at room temperature and freezing) (second from the right, n = 4, batch no. 11), after stress (freezing only) (right, n = 4, batch no. 8). [Fig.ôB]: Representation of particle size distributions (average results). Saline solution, reference condition (i.e., vacuum drying, batch no. 6), after stress (storage at room temperature and freezing) (batch no. 11), after stress (freezing only) (batch no. 8).

[0025] [Fig.7A] Changes in pH and temperature during freezing and evaporative drying of the placebo solution. The blue arrow in this graph indicates the temperature of NaCl*2 H2O + water crystallization as observed in a wide-angle X-ray scattering (WAXS) experiment conducted separately.

[0026] [Fig.7B] pH changes during evaporative drying of the placebo solution.

[0027] [Fig.8A] WAXS diffraction patterns during cooling of an aqueous placebo solution NaCl 0.9% / HSA 0.5%. Arrow: trace of NaCl*2 H2O.

[0028] [Fig.8B] WAXS diffraction patterns during cooling of a 0.9% NaCl aqueous placebo solution.

[0029] [Fig.8C] SAXS diffraction images during cooling of a 0.9% NaCl / 0.5% HSA solution.

[0030] [Fig.9] Representative images by SEM obtained at two magnifications. The areas Dark areas represent HSA, and lighter areas correspond to NaCl or NaCl + HSA. From left to right: vacuum drying (process 1), freeze-drying (process 2), and freeze-drying with pre-drying storage (process 3).

[0031] [Fig. 10] X-ray diffraction patterns of dried placebo batches. Curve of Below: Vacuum drying (Process 1, Batch No. 6), Top curve: Freeze drying with pre-drying storage (Process 3, Batch No. 11). Peak positions of placebo and NaCl (USP, JT Baker, Batch 0000207378) batches are provided in the insert.

[0032] [Fig. 11] Stability data relating to the activity test under conditions of Storage at 25°C. Upper curve in each view: Process 1, vacuum drying. Lower curve in each view: Process 3, freeze-drying + intermediate storage. Top view: merged view. Bottom view: exploded view. 5. DETAILED DESCRIPTION

[0033] The present invention provides solid compositions comprising BoNT / A and HSA having improved properties as well as manufacturing methods with an improved drying process for obtaining such solid compositions. Other advantages of the present disclosure will be apparent to those skilled in the art. The embodiments and aspects presented in the present disclosure are intended to illustrate the invention and should not be construed as limiting the scope of the invention. 5.1. Definitions

[0034] The term "and / or" as used in an expression such as "A and / or B" is herein intended to mean "A and B", "A or B", "A" or "B".

[0035] The terms "about" and "approximately" generally refer to a range of numbers that the skilled person would consider equivalent to the quoted value (i.e., having the same function or result). In many cases, the terms "about" and "approximately" may include numbers that are rounded. to the nearest significant figure. In specific embodiments, the terms "about" and "approximately" should be interpreted to allow for variation deemed normal by a person skilled in the art, such as, for example, a variation of the order of 20% or 10% or 5%. In specific embodiments, the terms "about" and "approximately" encompass the exact value stated.

[0036] The terms "animal product free" ("APF") or "substantially free of animal products" encompass, respectively, the absence or substantial absence of blood-derived products or compounds, blood pool products, and other animal-derived products or compounds. The term "animal" excludes microorganisms, such as bacteria. Thus, an APF medium or process or a substantially APF medium or process within the scope of the present invention may include botulinum toxin or Clostridium botulinum bacteria. For example, an APF process or a substantially APF process refers to a process that is either substantially free, predominantly free, or entirely free of animal-derived proteins, such as: immunoglobulins, meat digestate, meat by-products, and milk, dairy products, or digestates.

[0037] By "Clostridium botulinum neurotoxin serotype A" or "BoNT / A" is meant a neurotoxin produced by type A strains of Clostridium botulinum. One such type A strain of Clostridium botulinum is the type A strain named Hall, e.g., Hall type A strain (Allergan). cf. Zhang et al. (2003) Gene 315:21. BoNT / A encompasses both a BoNT / A complex (e.g., the 300, 500, 760, and 900 kDa complexes) as well as pure BoNT / A toxin (i.e., the approximately 150 kDa neurotoxic molecule).

[0038] By "BoNT / A complexes" is meant Clostridium botulinum serotype A neurotoxin complexes comprising a BoNT / A molecule (the neurotoxic component) and one or more hemagglutinating (HA) proteins and / or non-toxic, non-hemagglutinating (NTNH) proteins. The BoNT / A complexes may be, for example, in the following forms: complexes of about 900 kDa, 760 kDa, 500 kDa or 300 kDa. In one embodiment, the BoNT / A complex is in the form of a complex of about 900 kDa comprising a BoNT / A molecule of about 150 kDa, the hemagglutinating proteins HA70, HA34 and HA17, and non-toxic, non-hemagglutinating (NTNH) proteins. In one embodiment, the BoNT / A complex is a substantially full-length form of the 900 kDa BoNT / A complex. In one embodiment, the BoNT / A complex is onabotulinumtoxinA.

[0039] By "150 kDa Clostridium botulinum serotype A neurotoxin" or "150 kDa BoNT / A" is meant a neurotoxin of about 150 kDa obtained from a culture of a type A strain of Clostridium botulinum (e.g., Clostridium botulinum Hall strain). Preferred 150 kDa botulinum toxin type A (BoNT / A) sequences used herein are shown in Table 1. For example, in one embodiment, the 150 kDa BoNT / A used herein comprises (e.g., consists of) a light chain having an amino acid sequence shown in SEQ ID NO. 2 and a heavy chain having an amino acid sequence shown in SEQ ID NO. 3, with disulfide bridges located between positions 429 and 453 and between positions 1234 and 1279.

[0040] The term "BoNT / A composition" refers to any composition comprising a BoNT / A and encompasses both solid compositions and liquid compositions. In some embodiments, a BoNT / A composition (e.g., a solid composition or a liquid composition) described herein is a pharmaceutical composition. In particular embodiments, a BoNT / A composition (e.g., a solid composition or a liquid composition) described herein is a finished drug product (i.e., a finished dosage form). In a preferred embodiment, a BoNT / A composition described herein is in powder form (e.g., vacuum-dried powder).

[0041] The term "carrier" as used in connection with a pharmaceutical excipient means any solvent, dispersion medium, preservative, coating, absorption delaying agent and isotonic agent, and any similar element, which is compatible with pharmaceutical administration.

[0042] The terms "patient", "subject", "individual" and the like refer to a human being.

[0043] The term "pharmaceutical composition" refers to a formulation in which an active ingredient may be a BoNT / A. The word “formulation” means that there is at least one additional ingredient (such as, for example, but not limited to, an albumin (such as human serum albumin (HSA) or recombinant human albumin) and / or sodium chloride) in the pharmaceutical composition in addition to a BoNT / A active ingredient. The human serum albumin excipient may be derived from human plasma or recombinantly produced. A pharmaceutical composition is therefore a formulation suitable for diagnostic purposes and therapeutic and / or cosmetic administration (for example, by intramuscular or subcutaneous injection or by insertion of a depot form or implant) to a subject, such as a human patient. In one embodiment, the active ingredient is onabotulinumtoxinA. Exemplary methods for formulating a BoNT / A active pharmaceutical composition are described in the application US Patent No. 2003 / 0118598, filed November 5, 2002. In a preferred embodiment, a pharmaceutical composition described herein is in a dried form (e.g., a vacuum-dried form). The pharmaceutical compositions may be vacuum-dried and suitable for administration by either subcutaneous or intramuscular injection after reconstitution with normal saline, comprising 900 kDa BoNT / A, human serum albumin (HSA), and sodium chloride. Preferably, such pharmaceutical compositions comprise 0.5 mg of HSA and 0.9 mg of sodium chloride per 100 units of BoNT / A. More preferably, such pharmaceutical compositions comprise 50, 100, or 200 units of BoNT / A.

[0044] A "unit" or "U" refers to the lethal dose LD50 or the dose determined by a cell-based potency assay (CBPA). The lethal dose LD50 is defined as the amount of BoNT / A that causes the death of 50% of a population of mice injected with BoNT / A. The CBPA dose is determined as described in US Patents 8,618,261, 8,198,034, 9,249,216, 10,703,806, 11,261,240 and 11,332,518.

[0045] Unless the context otherwise requires, the terms "include," "comprises," and "comprising" are used with the clear understanding that they are to be interpreted inclusively, rather than exclusively, so as to indicate the inclusion of the recited feature, but not to the exclusion of one or more other such features. However, it is to be understood that wherever aspects and embodiments are described herein with the term "include" (or "includes" or "comprising"), otherwise analogous aspects described in terms of "consisting of" (or "consists of" or "consisting of") and / or "consisting primarily of" (or "consists primarily of" or "consisting primarily of") are also provided.

[0046] 5.2. Clostridium Botulinum Neurotoxin Serotype A (BoNT / A)

[0047] A bacterium of the order Clostridiales can produce botulinum toxin type A complexes in various forms, which include, but are not limited to, complexes of 900 kDa, 760 kDa, 500 kDa and 300 kDa (approximate molecular masses).

[0048] In one embodiment, the BoNT / A described herein is present as a 900 kDa BoNT / A complex. In one embodiment, the BoNT / A described herein is present as a 900 kDa BoNT / A complex formed by the 150 kDa BoNT / A molecule and the hemagglutinating proteins HA70, HA34, and HA17, and non-toxic, non-hemagglutinating (NTNH) proteins. In a particular embodiment, the BoNT / A described herein (e.g., the 900 kDa BoNT / A complex) is produced from a Clostridium botulinum type A strain. In In a particular embodiment, the BoNT / A described herein (e.g., the 900 kDa BoNT / A complex) is produced from a Hall strain of Clostridium botulinum type A. In a preferred embodiment, the BoNT / A described herein is onabotulinumtoxinA.

[0049] In one embodiment, the 150 kDa BoNT / A molecule suitable for use within the scope of the present disclosure has a sequence shown in Table 1. In one embodiment, the 150 kDa BoNT / A molecule comprises a light chain (LC: residues 2 to 438, about 50 kDa) and a heavy chain (HC: residues 449 to 1296, about 100 kDa). Residues 439 to 448 are the nick site and are in italics.

[0050] [Table 1]. Preferred sequences of BoNT / A molecules <h2 style=";text-align:left;direction:ltr">SEP ID NO. 1: Sequence of amino acids in the BoNT / A molecule of 150 kDa

[0051] MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 50<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS 100<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TDLGRMLLTS IVRGIPFWGG STIDTELKVI DTNCINVIQP DGSYRSEELN 150 LVIIGPSADIIQFECKSFGH EVLNLTRNGY GSTQYIRFSP DFTFGFEESL 200 EVDTNPLLGA GKFATDPPAVT LAHELIHAGH RLYGIAINPN RVFKVNTNAY 250<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT 350<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EDNFVKFFKV LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN 400<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FNGQNTEINN MNFTKLKNFT GLFEFYKLLC VRGIITSKTK SLDKGYNKAL 450<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE ITSDTNIEAA EENISLDLIQ 500 QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG KKYELDKYTM 550 FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA 600<h2 style=";text-align:left;direction:ltr"> AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD 650 DFVGALIFSG AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS 700 KRNEKWDEVY KYIVTNWLAK VNTQIDLIRK KMKEALENQA EATKAIINYQ 750 YNQYTEEEKN NINFNIDDLS SKLNESINKA MININKFLNQ CSVSYLMNSM 800 IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK VNNTLSTDIP 850 FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI 900 GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP 950 KYFNSISLNN EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK 1000 YSQMINISDY INRWIFVTIT NNRLNNSKIY INGRLIDQKP ISNLGNIHAS 1050 NNIMFKLDGC RDTHRYIWIK YFNLFDKELN EKEIKDLYDN QSNSGILKDF 1100 WGDYLQYDKP YYMLNLYDPN KYVDVNNVGI RGYMYLKGPR GSVMTTNIYL 1150 NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA 1200 GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI 1250 <h2 style=";text-align:left;direction:ltr">GFHQFNNIAK LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPL 1296

[0052] SEP ID NO. 2: Sequence of amino acids from the left chain (LC) of BoNT / A molécule<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0053] PFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT 50<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS 100<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TDLGRMLLTS IVRGIPFWGG STIDTELKVI DTNCINVIQP DGSYRSEELN 150 LVIIGPSADIIQFECKSFGH EVLNLTRNGY GSTQYIRFSP DFTFGFEESL 200 EVDTNPLLGA GKFATDPPAVT LAHELIHAGH RLYGIAINPN RVFKVNTNAY 250<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT 350<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EDNFVKFFKV LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN 400<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FNGQNTEINN MNFTKLKNFT GLFEFYKLLC VRGIITSK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0054] SEP ID NO. 3: Sequence of amino acids in the milk (HC) of the BoNT / A molécule<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0055] AL 450<h2 style=";text-align:left;direction:ltr"> NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE ITSDTNIEAA EENISLDLIp 500 pYYLTFNFDN EPENISIENL SSDIIGpLEL MPNIERFPNG KKYELDKYTM 550 FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA 600 AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD 650 DFVGALIFSG AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS 700 KRNEKWDEVY KYIVTNWLAK VNTQIDLIRK KMKEALENQA EATKAIINYQ 750 YNQYTEEEKN NINFNIDDLS SKLNESINKA MININKFLNQ CSVSYLMNSM 800 IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK VNNTLSTDIP 850 FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI 900 GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP 950 KYFNSISLNN EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK 1000 YSQMINISDY INRWIFVTIT NNRLNNSKIY INGRLIDQKP ISNLGNIHAS 1050 NNIMFKLDGC RDTHRYIWIK YFNLFDKELN EKEIKDLYDN QSNSGILKDF 1100 WGDYLQYDKP YYMLNLYDPN KYVDVNNVGI RGYMYLKGPR GSVMTTNIYL 1150 NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA 1200 GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI 1250 GFHQFNNIAK LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPL 1296

[0056] 5. 3. Production of BoNT / A and production of BoNT / A compositions

[0057] Botulinum toxin type A has been approved by the United States Food and Drug Administration (FDA) for the treatment of essential blepharospasm, strabismus, and hemifacial spasm in patients over the age of twelve, cervical dystonia, glabellar (facial) wrinkles, and for the treatment of hyperhidrosis. A commercially available pharmaceutical composition containing botulinum toxin type A is marketed under the trademark BOTOX® (onabotulinumtoxinA). It is commercially available from Allergan, an AbbVie company, North Chicago, Illinois, USA. BOTOX® contains a purified 900 kDa complex of botulinum toxin type A, human serum albumin, and sodium chloride packaged in a sterile, vacuum-dried form. The botulinum toxin type A complex in BOTOX® is produced from a culture of the Hall strain of Clostridium botulinum grown in a medium containing NZ amine casein and yeast extract (i.e., a non-APF process) and purified from the culture solution by a series of precipitation steps (including acid precipitation) until a crystalline complex consisting of the high molecular weight active toxin protein and an associated hemagglutinating protein is obtained. The crystalline complex is redissolved in a solution containing saline and albumin and sterile filtered through a gamma-irradiated filter (0.2 micron) before vacuum drying. BOTOX® may be reconstituted with sterile, preservative-free saline solution before intramuscular injection.Each 100-unit vial of BOTOX® consists of approximately 5 ng of botulinum toxin type A complex, 0.5 mg of human serum albumin, and 0.9 mg of sodium chloride, in vacuum-dried form and intended for reconstitution with sterile, preservative-free normal saline (0.9% sodium chloride injection).

[0058] In general, the production of a BoNT / A pharmaceutical composition involves first manufacturing the BoNT / A active substance, and then mixing the BoNT / A active substance with excipient(s). A drying step is often used at the end of the production process to produce a dried form of the pharmaceutical composition for ease of storage and / or transportation. The present disclosure sets forth steps and measures that may be implemented to ensure that the finished product does not undergo freezing during the drying process, since freezing during drying unexpectedly leads to a significant increase in the amount of HSA aggregation in BoNT / A compositions.

[0059] A number of steps are required for the manufacture of BoNT / A active substances (i.e., for obtaining purified BoNT / A). A first step may consist of culturing Clostridial bacteria (e.g., Clostridium botulinum Hall strain), typically on agar plates, in an environment favorable to bacterial growth, for example, in an anaerobic heated atmosphere. The culturing step allows Clostridial colonies to be obtained according to a desired morphology and other characteristics. The culturing step may also be carried out with bacteria from a working cell bank free of animal products. In a second step, selected cultured Clostridial colonies may be fermented in a suitable medium.After some fermentation time, Clostridial bacteria usually undergo lysis and release a Clostridial toxin (e.g., BoNT / A) into the medium. In a third step, the toxin can be purified from the culture medium to obtain a crude BoNT / A toxin active substance. or in bulk. Preferably, the BoNT / A toxin active substance has not been subjected to precipitation (e.g., cold ethanol, hydrochloric acid, and / or ammonium sulfate precipitation), for example, during purification. In certain aspects of the invention, the BoNT / A toxin active substance is purified by column chromatography including a hydrophobic interaction chromatography (HIC) column. In certain aspects of the invention, when multiple chromatography columns are used to purify BoNT / A, the toxin is purified using a process in which an HIC column is used prior to any other chromatography columns. Preferably, a BoNT / A active substance is produced by a process qualified for use under the Good Manufacturing Practice regulations promulgated by the United States Food and Drug Administration.

[0060] In some embodiments, the BoNT / A active substance is obtained using a process that is substantially, mainly, or completely free of animal-derived proteins (APF). The process may include so-called APF or substantially APF culture and fermentation processes. A so-called APF or substantially APF chromatographic process and system may be used to purify a clarified culture of Clostridium botulinum obtained from the APF or substantially APF culture and fermentation processes. In some embodiments, the chromatographic purification process is as described in U.S. Patent 8,129,139.

[0061] The active substance of BoNT / A may be produced by exemplary methods as described in Example 1 of US Patent 8,129,139, Example 2 of US Patent 8,129,139 or Example 1 of US Patent 9,469,849, or by using one or more steps as described in one or more of these examples.

[0062] In some embodiments, the BoNT / A active substance is produced by a process that includes one or more column chromatography steps (e.g., one or more column chromatography steps performed during purification of the BoNT / A active substance).

[0063] In preferred embodiments, the one or more column chromatography steps (e.g., performed during purification of the BoNT / A active substance) comprise hydrophobic interaction chromatography, anion exchange chromatography and / or cation exchange chromatography, wherein the hydrophobic interaction chromatography is preferably performed before the anion exchange chromatography and / or the cation exchange chromatography.

[0064] In some embodiments, the BoNT / A active substance is produced by a process that does not include a cold ethanol, hydrochloric acid, or ammonium sulfate precipitation step (e.g., in the purification of the BoNT / A active substance).

[0065] In some embodiments, the BoNT / A active substance is produced by a method that does not involve the use of a protease inhibitor. In some embodiments, the BoNT / A active substance is produced by a method that does not involve the use of benzamidine hydrochloride.

[0066] After production of the BoNT / A active substance, the latter may be stabilized in a suitable solution. The bulk BoNT / A active substance may then be mixed with one or more excipients (e.g., human serum albumin, such as recombinant human serum albumin, and sodium chloride) and may be further filtered by sterile filtration to make a pharmaceutical composition suitable for administration to a human. The pharmaceutical compositions may be made into solid form (e.g., powder) by drying (e.g., vacuum drying). Solid pharmaceutical compositions may be stored and reconstituted prior to injection. The BoNT / A pharmaceutical compositions described herein may comprise a 900 kDa serotype A BoNT / A complex as the active pharmaceutical ingredient.The pharmaceutical composition may also include one or more excipients, buffers, carriers, stabilizers, preservatives and / or diluents. Such pharmaceutical compositions are preferably chemically and physically stable such that the active pharmaceutical ingredient of BoNT / A remains suitable for use as a pharmaceutical product after storage. The BoNT / A products may be stored at room temperature, under refrigerated conditions, or at a temperature below 0°C. Preferably, the BoNT / A is stable upon storage for at least about 12 months, more preferably at least about 18 months.

[0067] In various embodiments and aspects, a solid BoNT / A composition described herein is dried from a solution comprising a BoNT / A active substance (e.g., a BoNT / A active substance produced by a method described herein) and HSA. In particular, the present disclosure provides a method for producing a solid composition comprising a BoNT / A and HSA, said method comprising a step of drying a solution comprising a BoNT / A and HSA to produce said solid composition, wherein the solution does not undergo freezing during the drying step. In specific embodiments, the method produces a solid composition described herein.

[0068] To determine whether a solution described herein (or a reference solution described herein, if applicable) undergoes freezing during drying, it is possible to follow its temperature during drying (e.g., as described in Example 1). Monitoring may be accomplished by inserting a temperature probe into the solution (or reference solution, as the case may be). Monitoring may be accomplished using a Pirani gauge or an Ellab temperature probe. If a plurality of vials of the solution (or reference solution, as the case may be) are dried together (e.g., in the same drying chamber or on the same drying tray), then monitoring may be accomplished for one or more representative vials (e.g., one or two) and need not be accomplished for all vials. In some embodiments, a drying process during which a solution does not undergo freezing includes maintaining the temperature of the solution above its freezing point during drying.In some embodiments, a drying process during which a solution does not undergo freezing includes maintaining the temperature of the solution above 0°C during drying.

[0069] To determine whether a solution described herein (or a reference solution described herein, if applicable) undergoes freezing during drying, the dried solid composition (or the dried reference solid composition, as applicable) may be examined using a scanning electron microscope (SEM) (e.g., as described in Example 1). If a plurality of vials of the solution (or the reference solution, as applicable) are dried together (e.g., in the same drying chamber or on the same drying tray), then the analysis may be performed on one or more representative vials of the dried solid composition (e.g., one or two) and need not be performed for all vials. In some embodiments, the absence of pores in a dried solid composition indicates that it is dried from a solution that did not undergo freezing during drying.

[0070] In various embodiments and aspects, the drying step is at least partially manually controlled. In various embodiments and aspects, the drying step is fully manually controlled. In various embodiments and aspects, the drying step is at least partially digitally controlled. In various embodiments and aspects, the drying step is fully digitally controlled.

[0071] In various embodiments and aspects, the drying step is vacuum drying, spray drying, convection drying, microwave drying, or a combination thereof.

[0072] In various embodiments and aspects, the drying step is vacuum drying. To prevent freezing of the finished product solution, a controlled depressurization / vacuumization phase may be implemented. Measures that may be taken during the controlled depressurization / vacuumization phase to help prevent freezing include: using a depressurization rate slower chamber speed, implementing stepwise depressurization, monitoring the finished product solution (e.g., to ensure its temperature is a few degrees above freezing), and / or using a high shelf temperature (e.g., setting the shelf temperature between about 20°C and 25°C). The exact chamber depressurization rates and steps to be used may depend on the drying equipment, chamber size, condenser capacity, chamber loading and / or vacuum pump performance, etc., and can be determined by a person of ordinary skill in the art and may involve manual adjustment.For example, in some embodiments, the vacuum drying step may include a staged vacuuming step, which may be performed such that the temperature of a solution described herein is allowed to decrease under vacuum until it reaches a temperature that is always above the freezing point of the solution and preferably close to the freezing point of the solution (e.g., about 2°C to 5°C above the freezing point of the solution), then the vacuum is removed until the temperature of the solution returns to a higher temperature (e.g., about 20°C to 25°C), and then the vacuum is reapplied. The preceding step may be repeated once or more than once, while the exact temperature at which the vacuuming is stopped or resumed need not be the same for each repetition.In some other embodiments, the vacuum drying step may include a slow vacuuming step. 5.4. BoNT / A compositions

[0073] The present invention relates to solid compositions comprising Clostridium botulinum neurotoxin serotype A (BoNT / A) and human serum albumin (HSA), the solid compositions comprising a small amount of HSA aggregates.

[0074] The HSA contained in a BoNT / A composition may exist as HSA monomer or HSA aggregates. There are two types of HSA aggregates, namely soluble HSA aggregates and insoluble HSA aggregates. The soluble HSA aggregates may exist as HSA polymer, HSA oligomer, HSA dimer, or a mixture thereof. In some embodiments, a soluble HSA polymer, a soluble HSA oligomer, a soluble HSA dimer, and a soluble HSA monomer may be separated by size-exclusion chromatography (SEC). For example, peaks on an SEC chromatogram can be assigned to a soluble HSA polymer, a soluble HSA oligomer, a soluble HSA dimer, and a soluble HSA monomer, respectively, based on retention times obtained by analyzing a reference standard on the same column (e.g., example, a gel permeation standard consisting of bovine thyroglobulin (MW 670 kDa), bovine gammaglobulin (MW 158 kDa), chicken albumin (MW 44 kDa), horse myoglobulin (MW 17 kDa), and vitamin B12 (MW 1350 Da), such as the Bio-Rad Gel Permeation Standard (Cat. No. 151-1901). In a specific embodiment, peaks on an SEC chromatogram can be assigned to a soluble HSA polymer, a soluble HSA oligomer, a soluble HSA dimer, and a soluble HSA monomer, respectively, as described in Example 1.

[0075] Suitable methods for measuring the level of soluble and / or insoluble HSA aggregates include, but are not limited to, dynamic light scattering (DLS), gel electrophoresis (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)), transmission electron microscopy (TEM), ultracentrifugation (e.g., analytical ultracentrifugation (AUC), such as analytical ultracentrifugation according to the sedimentation velocity method (SV-AUC)), asymmetric flow field-flow fractionation (FFF or AF4), and turbidity measurements.

[0076] Suitable methods for measuring the level of HSA aggregates, polymer, oligomer or dimer in soluble HSA include, but are not limited to, high performance liquid chromatography HPLC (e.g., SEC), DLS, gel electrophoresis (e.g., SDS-PAGE), ultracentrifugation (e.g., AUC, such as SV-AUC), asymmetric flow-force coupled fractionation (FFF or AF4), and turbidity measurements.

[0077] Suitable methods for measuring the level of insoluble HSA aggregates include, but are not limited to, DLS, gel electrophoresis (e.g., SDS-PAGE), transmission electron microscopy (TEM), ultracentrifugation (e.g., AUC, such as SV-AUC), asymmetric flow force fractionation (FFF or AF4), microscopy (e.g., optical microscopy), micro-flow imaging (MFI), turbidity measurements, light obscuration (e.g., high accuracy liquid particle counter (HIAC)), and subvisible particle detection (such that the percentage of insoluble HSA aggregates is or is represented by the percentage of subvisible particles) (e.g., a non-invasive subvisible particle detection method (e.g.,a non-invasive subvisible particle detection method described in , Example 1 or described in US Patent 10,132,736 B2, optical microscopy, MFI or light obscuration (e.g., by HIAC)).

[0078] In some embodiments, the percentage of HSA aggregates or the percentage of one type of HSA aggregates in a solid composition described herein is determined after reconstitution of at least a portion of the solid composition. Reconstitution of the solid composition (or a portion thereof) may be accomplished by mixing the solid composition (or a portion thereof) with a liquid (e.g., water or saline). In specific embodiments, the reconstituted composition comprises between about 0.01 mg / mL and about 100 mg / mL, between about 0.1 mg / mL and about 10 mg / mL, between about 0.25 mg / mL and about 2 mg / mL, about 0.25 mg / mL, about 0.5 mg / mL, about 1 mg / mL, or about 2 mg / mL of HSA.In some embodiments, the percentage of HSA aggregates or the percentage of one type of HSA aggregates in a solid composition described herein is determined by SEC after reconstitution of at least a portion of the solid composition. Detectors that may be used to detect soluble HSA aggregates (e.g., during SEC analysis) include, but are not limited to, UV detectors (e.g., UV detectors with a wavelength of about 220 nm, UV detectors with a wavelength of about 280 nm, UV detectors with a wavelength of about 254 nm, and UV detectors with a wavelength between 220 nm and 280 nm) and fluorescence detectors (e.g., fluorescence detectors with an excitation wavelength of about 280 nm and an emission wavelength of about 350 nm).

[0079] The present invention provides solid compositions comprising a BoNT / A and HSA, the solid compositions comprising a low percentage of HSA aggregates, including HSA polymer, oligomer and / or dimer. As shown in Example 1, a non-freeze dried BoNT / A composition not only has a low overall percentage of HSA aggregates, but also has low percentages of each of HSA polymer, oligomer and dimer.

[0080] In specific embodiments, the percentage of HSA aggregates, the percentage of HSA polymer, the percentage of HSA oligomer and / or the percentage of HSA dimer, in the soluble HSA contained in the solid composition is determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA aggregates, the percentage of HSA polymer, the percentage of HSA oligomer and / or the percentage of HSA dimer, in the soluble HSA contained in the solid composition is determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA aggregates, the percentage of HSA polymer, the percentage of HSA oligomer and / or the percentage of HSA dimer, in the soluble HSA contained in the solid composition is determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.

[0081] In one aspect, there is provided a solid composition comprising a BoNT / A and HSA, wherein the HSA comprises an HSA monomer and HSA aggregates, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein less than 34% of the soluble HSA contained in the solid composition is HSA aggregates. It is understood herein that the soluble HSA aggregates described herein include all types of soluble HSA aggregates (i.e., soluble HSA dimer, soluble HSA polymer, and soluble HSA oligomer).In specific embodiments, less than 33%, less than 32%, less than 31%, less than 30%, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of soluble HSA contained in the solid composition is HSA aggregates.In specific embodiments, about 1% to about 4%, about 4% to about 6%, about 6% to about 10%, about 4% to about 10%, about 10% to about 12%, about 10% to about 15%, about 15% to about 18%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30% or about 30% to about 33% of soluble HSA contained in the solid composition are HSA aggregates.

[0082] In specific embodiments, less than 19% of soluble HSA contained in the solid composition are HSA aggregates, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of soluble HSA contained in the solid composition are HSA aggregates, as determined by SEC chromatography with a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. at least a portion of the solid composition. In specific embodiments, about 1% to about 4%, about 4% to about 6%, about 6% to about 10%, about 4% to about 10%, about 10% to about 12%, about 10% to about 15% or about 15% to about 18% of soluble HSA contained in the solid composition are HSA aggregates, as determined by SEC chromatography with a UV detector at the wavelength of 220 nm after reconstitution of at least a portion of the solid composition.

[0083] In specific embodiments, less than 34% of soluble HSA contained in the solid composition are HSA aggregates, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, less than 33%, less than 32%, less than 31%, less than 30%, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of soluble HSA contained in the solid composition are HSA aggregates, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least part of the solid composition.In specific embodiments, about 1% to about 4%, about 4% to about 6%, about 6% to about 10%, about 4% to about 10%, about 10% to about 12%, about 10% to about 15%, about 15% to about 18%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30% or about 30% to about 33% of soluble HSA contained in the solid composition are HSA aggregates, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.

[0084] In specific embodiments, less than 20% of soluble HSA contained in the solid composition are HSA aggregates, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of soluble HSA contained in the solid composition are HSA aggregates, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, about 1% to about 4%, about 4% to about 6%, about 6% to about 10%, about 4% to about 10%, about 10% to about 12%, about 10% to about 15% or about 15% to about 18% of soluble HSA contained in the solid composition are HSA aggregates, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.

[0085] According to another aspect, there is provided a solid composition comprising a BoNT / A and HSA, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein less than 30% of the soluble HSA contained in the solid composition is a polymer of HSA. In specific embodiments, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of soluble HSA contained in the solid composition is an HSA polymer.In specific embodiments, about 1% to about 4%, about 4% to about 6%, about 6% to about 10%, about 4% to about 10%, about 10% to about 12%, about 10% to about 15%, about 15% to about 18%, about 15% to about 20%, about 20% to about 25% or about 25% to about 29% of soluble HSA contained in the solid composition is a polymer of HSA.

[0086] In specific embodiments, less than 13% of soluble HSA contained in the solid composition is a polymer of HSA, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of soluble HSA contained in the solid composition is a polymer of HSA, as determined by SEC chromatography with a UV detector at the wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, about 1% to about 4%, about 4% to about 6%, about 6% to about 10%, about 4% to about 10% or about 10% to about 12% of soluble HSA contained in the solid composition is a polymer of HSA, as determined by SEC chromatography with a UV detector at the wavelength of 220 nm after reconstitution of at least a portion of the solid composition.

[0087] In specific embodiments, less than 30% of the soluble HSA contained in the solid composition is a polymer of HSA, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of soluble HSA contained in the solid composition is a polymer of HSA, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, about 1% to about 4%, about 4% to about 6%, about 6% to about 10%, about 4% to about 10%, about 10% to about 12%, about 10% to about 15%, about 15% to about 18%, about 15% to about 20%, about 20% to about 25% or about 25% to about 29% of soluble HSA contained in the solid composition is a polymer of HSA, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.

[0088] In specific embodiments, less than 13% of the soluble HSA contained in the solid composition is a polymer of HSA, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of soluble HSA contained in the solid composition is a polymer of HSA, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, about 1% to about 4%, about 4% to about 6%, about 6% to about 10%, about 4% to about 10%, or about 10% to about 12% of soluble HSA contained in the solid composition is a polymer of HSA, as determined by chromatography. SEC using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.

[0089] According to another aspect, there is provided a solid composition comprising a BoNT / A and HSA, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein less than 0.8% of the soluble HSA contained in the solid composition is an HSA oligomer. In specific embodiments, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% of the soluble HSA contained in the solid composition is an HSA oligomer. In specific embodiments, about 0.1% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.4% to about 0.5%, about 0.5% to about 0.6% or about 0.6% to about 0.7% of soluble HSA contained in the solid composition is an HSA oligomer.

[0090] In specific embodiments, less than 0.8% of soluble HSA contained in the solid composition is an HSA oligomer, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% of soluble HSA contained in the solid composition is an HSA oligomer, as determined by SEC chromatography with a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, about 0.1% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.4% to about 0.5%, about 0.5% to about 0.6% or about 0.6% to about 0.7% of soluble HSA contained in the solid composition is an HSA oligomer, as determined by SEC chromatography with a UV detector at the wavelength of 220 nm after reconstitution of at least a portion of the solid composition.

[0091] In specific embodiments, less than 0.8% of soluble HSA contained in the solid composition is an HSA oligomer, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% of soluble HSA contained in the solid composition is an HSA oligomer, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition. In specific embodiments specific embodiments, about 0.1% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.4% to about 0.5%, about 0.5% to about 0.6% or about 0.6% to about 0.7% of soluble HSA contained in the solid composition is an oligomer of HSA, as determined by SEC chromatography with a UV detector at the wavelength of 280 nm after reconstitution of at least a portion of the solid composition.

[0092] In specific embodiments, less than 0.8% of soluble HSA contained in the solid composition is an HSA oligomer, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% of soluble HSA contained in the solid composition is an HSA oligomer, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, about 0.1% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.4% to about 0.5%, about 0.5% to about 0.6%, or about 0.6% to about 0.7% of soluble HSA contained in the solid composition is an HSA oligomer, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.

[0093] According to another aspect, there is provided a solid composition comprising a BoNT / A and HSA, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein less than 4.8% of the soluble HSA contained in the solid composition is an HSA dimer. In specific embodiments, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5% of the soluble HSA contained in the solid composition is an HSA dimer.In specific embodiments, about 0.5% to about 1%, about 1% to about 1.5%, about 1.5% to about 2%, about 2% to about 2.5%, about 2.5% to about 3%, about 3% to about 3.5%, about 3.5% to about 3.6%, about 3.5% to about 4%, about 4% to about 4.5% or about 4.5% to about 4.7% of soluble HSA contained in the solid composition is an HSA dimer.

[0094] In specific embodiments, less than 4.6% of soluble HSA contained in the solid composition is an HSA dimer, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1% or less than 0.5% of soluble HSA contained in the solid composition is an HSA dimer, as determined by SEC chromatography with a UV detector at the wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, about 0.5% to about 1%, about 1% to about 1.5%, about 1.5% to about 2%, about 2% to about 2.5%, about 2.5% to about 3%, about 3% to about 3.5%, about 3.5% to about 3.6%, about 3.5% to about 4% or about 4% to about 4.5% of soluble HSA contained in the solid composition is HSA dimer, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.

[0095] In specific embodiments, less than 3.7% of soluble HSA contained in the solid composition is an HSA dimer, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1% or less than 0.5% of soluble HSA contained in the solid composition is an HSA dimer, as determined by SEC chromatography with a UV detector at the wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, about 0.5% to about 1%, about 1% to about 1.5%, about 1.5% to about 2%, about 2% to about 2.5%, about 2.5% to about 3%, about 3% to about 3.5% or about 3.5% to about 3.6% of soluble HSA contained in the solid composition is HSA dimer, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.

[0096] In specific embodiments, less than 4.8% of soluble HSA contained in the solid composition is HSA dimer, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1% or less than 0.5% of soluble HSA contained in the solid composition is a dimer of HSA, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, about 0.5% to about 1%, about 1% to about 1.5%, about 1.5% to about 2%, about 2% to about 2.5%, about 2.5% to about 3%, about 3% to about 3.5%, about 3.5% to about 3.6%, about 3.5% to about 4%, about 4% to about 4.5% or about 4.5% to about 4.7% of soluble HSA contained in the solid composition is HSA dimer, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.

[0097] The present invention also provides a solid composition comprising a BoNT / A and HSA, wherein the HSA is formulated into the solid composition from a starting material. As shown in Example 1, a BoNT / A composition dried without freezing not only exhibits a smaller increase in the overall percentage of HSA aggregates relative to the HSA starting material, but also exhibits a smaller increase in the percentages of each of the HSA polymer, oligomer, and dimer relative to the HSA starting material, compared to a BoNT / A composition dried using a process that involves freezing. In some embodiments, the starting material is a commercially available HSA product. In some embodiments, the starting material is in liquid form (e.g., as a 25% solution).In other embodiments, the starting material is in solid form.

[0098] The percentage of HSA aggregates, polymer, oligomer or dimer in the soluble HSA contained in a solid composition described herein and the percentage of HSA aggregates, polymer, oligomer or dimer in the soluble HSA contained in the corresponding starting material are preferably determined with the same method. When the HSA starting material is in solid form, in some embodiments, the percentage of HSA aggregates, polymer, oligomer or dimer in the soluble HSA contained in the starting material is determined after reconstitution of at least a portion of the starting material, and the reconstitution may be carried out as described below.

[0099] In one aspect, there is provided a solid composition comprising a BoNT / A and HSA, wherein the HSA is formulated into the solid composition from a starting material and comprises an HSA monomer and HSA aggregates, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most equal to 6.8 times the percentage of HSA aggregates in the soluble HSA contained in the starting material. In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most equal to 6.5 times, 6 times, 5.5 times, 5 times, 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times or 1.1 times the percentage of HSA aggregates in the soluble HSA contained in the starting material. In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA aggregates in the soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times, 6 to 6.5 times or 6.5 to 6.8 times the percentage of HSA aggregates in the soluble HSA contained in the starting material.

[0100] In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 4.9 times the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, or 1.1 times the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 3 times, 3. 3.5 times, 3.5 to 4 times, 4 to 4.5 times or 4.5 to 4.9 times the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.

[0101] In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 6.8 times the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 6.5 times, 6 times, 5.5 times, 5 times, 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, or 1.1 times the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times, 6 to 6.5 times or 6.5 to 6.8 times the percentage of HSA aggregates in the soluble HSA contained in the solid composition. in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.

[0102] In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 1.9 times the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times or 1.1 times the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA aggregates in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.7 times, or 1.7 to 1.9 times the percentage of HSA aggregates in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.

[0103] According to another aspect, there is provided a solid composition comprising a BoNT / A and HSA, wherein the HSA is formulated into the solid composition from a starting material, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 6.4 times the percentage of HSA polymer in the soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 6 times, 5.5 times, 5 times, 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, or 1.1 times the percentage of HSA polymer in the soluble HSA contained in the starting material. In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA polymer in the soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times or 6 to 6.4 times the . percentage of HSA polymer in the soluble HSA contained in the starting material.

[0104] In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 5.2 times the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 5 times, 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times or 1.1 times the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times or 5 to 5.2 times the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm. after reconstitution of at least part of the solid composition.

[0105] In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 6.4 times the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 6 times, 5.5 times, 5 times, 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times or 1.1 times the percentage of HSA polymer in the soluble HSA contained in the solid composition. in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times or 6 to 6.4 times the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography at using a UV detector at a wavelength of 280 nm after reconstitution of at least part of the solid composition.

[0106] In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 1.6 times the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 1.5 times, 1.4 times, 1.3 times, 1.2 times, or 1.1 times the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA polymer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA polymer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, or 1.5 to 1.6 times the percentage of HSA polymer in the soluble HSA contained. in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.

[0107] According to another aspect, there is provided a solid composition comprising a BoNT / A and HSA, wherein the HSA is formulated into the solid composition from a starting material, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 3.9 times the percentage of HSA oligomer in the soluble HSA contained in the starting material. In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times or 1.1 times the percentage of HSA oligomer in the soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA oligomer in the soluble HSA contained in the starting material. In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times or 3.5 to 3.9 times the percentage of HSA oligomer in the soluble HSA contained in the starting material.

[0108] In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 3.9 times the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, or 1.1 times the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is less than or equal to the percentage of oligomer. of HSA in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times, or 3.5 to 3.9 times the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.

[0109] In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 3.9 times the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, or 1.1 times the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times, or 3.5 to 3.9 times the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.

[0110] In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 3.9 times the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, or 1.1 times the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA oligomer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times or 3.5 to 3.9 times the percentage of HSA oligomer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least part of the solid composition.

[0111] According to another aspect, there is provided a solid composition comprising a BoNT / A and HSA, wherein the HSA is formulated into the solid composition from a starting material, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 4.6 times the percentage of HSA dimer in the soluble HSA contained in the starting material. In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times or 1.1 times the percentage of HSA dimer in the soluble HSA contained in the starting material.In specific embodiments, the percentage of . HSA dimer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA dimer in the soluble HSA contained in the starting material. In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times or 4.5 to 4.6 times the percentage of HSA dimer in the soluble HSA contained in the starting material.

[0112] In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 3.5 times the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, or 1.1 times the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times or 3 to 3.5 times the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition.

[0113] In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 4.6 times the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 4.5 times, 4 times, 3.5 times, 3 times, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times or 1.1 times the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition. In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 2.6 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times or 4.5 to 4.6 times the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a UV detector at a wavelength of 280 nm after reconstitution of at least less part of the solid composition.

[0114] In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 2.6 times the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, or 1.1 times the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is less than or equal to the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the composition. solid. In specific embodiments, the percentage of HSA dimer in the soluble HSA contained in the solid composition is at most 1.1 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.6 times, 1.5 to 1.7 times, 1.7 to 1.9 times, 1.1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times or 2.5 to 2.6 times the percentage of HSA dimer in the soluble HSA contained in the starting material, as determined by SEC chromatography using a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after reconstitution of at least a portion of the solid composition.

[0115] In various embodiments and aspects, there is provided a solid composition comprising a BoNT / A and HSA, wherein the solid composition has one, two, three, four, five, or more of the properties described above.

[0116] In various embodiments and aspects, there is provided a solid composition comprising a BoNT / A and HSA, wherein the solid composition is produced by a method described in Section 5.3.

[0117] In various embodiments and aspects, a composition described herein is free of animal products. In various embodiments and aspects, a solid composition described herein is dried from a solution comprising a BoNT / A and HSA, wherein the solution is free of animal products. In various embodiments and aspects, a solid composition described herein may be reconstituted, for example, with water or saline, into a solution comprising a BoNT / A and HSA, wherein the reconstituted solution is free of animal products. In various embodiments and aspects, a solid composition described herein does not contain a protease inhibitor. In some embodiments, a solid composition described herein does not contain benzamidine hydrochloride.In various embodiments and aspects, a solution described herein comprising a BoNT / A and HSA does not contain a protease inhibitor. In some embodiments, a solution described herein comprising a BoNT / A and HSA does not contain benzamidine hydrochloride. In various embodiments and aspects, both the solid composition and the solution comprising a BoNT / A and HSA do not contain a protease inhibitor. In some embodiments, both the solid composition and the solution comprising a BoNT / A and HSA do not contain benzamidine hydrochloride.

[0118] In various embodiments and aspects, the HSA described herein is a recombinant HSA. In a specific embodiment, the recombinant HSA is free of animal products. In a specific embodiment, the recombinant HSA is not produced from an animal. In a specific embodiment, the recombinant HSA is produced from a micro organism, such as a bacterium. In a specific embodiment, the recombinant HSA is produced from a plant-based expression system. In various embodiments and aspects, the HSA described herein is a human plasma-derived HSA. In a specific embodiment, the HSA contained in a solid composition described herein is about 0.5 mg per 100 BoNT / A units.

[0119] In various embodiments and aspects, a solid composition described herein further comprises one or more pharmaceutically acceptable carriers. In various embodiments and aspects, a solution described herein comprising a BoNT / A and HSA further comprises one or more pharmaceutically acceptable carriers. In specific embodiments, the one or more pharmaceutically acceptable carriers comprise sodium chloride (e.g., about 0.9 mg of sodium chloride per 100 units of BoNT / A). In specific embodiments, the solid composition described herein comprises about 0.9 mg of sodium chloride per 100 units of BoNT / A.

[0120] In various embodiments and aspects, a solid composition described herein is dried from a solution comprising a BoNT / A and HSA such that the solution does not undergo freezing upon drying.

[0121] In various embodiments and aspects, whether a solution described herein (or a reference solution described herein, as the case may be) undergoes freezing upon drying is determined as described in Section 5.3.

[0122] In various embodiments and aspects, a solid composition described herein comprises about 50 units, about 100 units, or about 200 units of BoNT / A.

[0123] In various embodiments and aspects, a solid composition described herein has an activity of between about 2.4 x 107 units / mg and about 6.0 x 107 units / mg.

[0124] The present invention provides a solid composition comprising a BoNT / A and HSA which is dried without undergoing freezing. Such a solid composition has advantageous properties such as a consistent visual quality of the product and a much slower decline in activity during storage, for example, at room temperature or higher, compared to a reference solid composition stored under the same conditions. A reference solid composition is dried from the same liquid composition as the solid composition under study, the only difference being the drying process: the reference solid composition undergoes freezing conditions during the drying process.In other words, a reference solid composition has a higher percentage of HSA aggregates in the HSA, a higher percentage of HSA aggregates, polymer, oligomer or dimer in the soluble HSA and / or a higher percentage of insoluble HSA aggregates in the HSA than in the solid composition under study, but is otherwise substantially identical to the solid composition under study. The term "substantially identical" is used in this . context or similar context to mean that the reference solid composition is identical or sufficiently similar to the solid composition (e.g., regarding their ingredients and the respective concentrations of the ingredients), such that the reference solid composition can serve as a suitable comparison control. The activity of a solid composition that is dried without freezing reaches a steady state after storage for a certain period of time, and the steady-state activity of the solid composition is greater than the steady-state activity of a reference solid composition stored under the same conditions. In some embodiments, both the solid composition and the reference solid composition are stored at about 5°C, about 25°C (and optionally about 60% relative humidity), or about 40°C (and optionally about 75% relative humidity).

[0125] In various embodiments and aspects, the activity of a solid composition described herein reaches a steady state after storage for a period of time, and the steady state activity of the solid composition is greater (e.g., at least 1-fold greater, at least 1.5-fold greater, at least 2-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 3.5-fold greater, at least 4-fold greater, at least 4.5-fold greater, or at least 5-fold greater) than the steady state activity of a reference solid composition comprising a BoNT / A and HSA under the same storage conditions.In various embodiments and aspects, the activity of a solid composition described herein reaches a steady state after storage for a period of time, and the loss of activity of the solid composition at the steady state relative to the start of storage is less (e.g., less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, or less than 95%) than the loss of activity of a reference solid composition.

[0126] A person skilled in the art is able to determine whether or not the activity of a solid composition has reached a steady state. For example, the activity of a solid composition may be considered to have reached a steady state if the activity of the solid composition does not decrease more than 20% after 3 months of storage.

[0127] In various embodiments and aspects, a solid composition described herein is a powdered pharmaceutical composition.

[0128] According to another aspect, there is provided a liquid composition comprising a BoNT / A and HSA, which is derived from a solid composition described herein, e.g., a dissolved or reconstituted form of a solid composition described herein.

[0129] According to another aspect, there is provided a method of treating a patient in need thereof (preferably a human patient), comprising administering a solid composition described herein. 5.4.1. Characterization of BoNT / A compositions

[0130] In one embodiment, BoNT / A compositions described herein have an activity of at least about 1.5x107 units / mg, e.g., about 1.5x107 to about 6.0x107 units / mg, about 2.0x107 to about 6.0x107 units / mg, about 2.4x107 to about 6.0x107 units / mg, about 2.4x107 to about 5.9x107 units / mg, about 2.4x107 to about 5.8x107 units / mg, about 2.4x107 to about 5.7x107 units / mg, about 2.4x107 to about 5.6x107 units / mg, about 2.4x107 to about 5.5x107 units / mg, about 2.4x107 to about 5.4x107 units / mg, about 2.5x107 to about 6.0xl07 units / mg, about 2.6xl07 to about 6.0xl07 units / mg, about 2.7xl07 to about 6.0xl07 units / mg, about 2.8xl07 to about 6.0xl07 units / mg, about 2.9xl07 to about 6.0xl07 units / mg, about 3.0xl07 to about 6.0xl07 units / mg, or any value within such ranges.

[0131] In one embodiment, BoNT / A compositions described herein have an activity of about 2.4 x 107 units / mg to about 5.4 x 107 units / mg. In preferred embodiments, the term "unit" as used herein refers to the lethal dose LD50.

[0132] In various embodiments and aspects, a solid composition described herein is a dried product of a solution comprising a BoNT / A and HSA and has greater activity than a reference solid composition, wherein the reference solid composition is produced from a reference solution comprising a BoNT / A and HSA by a drying process performed such that the reference solution undergoes freezing upon drying and wherein the reference solution is substantially identical to the solution. The term "substantially identical" may be understood as described above. Additional description regarding a reference solid composition is provided above in Section 5.3.In some embodiments, the solid composition described herein has an activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1.5 times or at least 2 times greater than the activity of the reference solid composition. In some embodiments, the solid composition described herein has an activity that is at least 0.1 x 107 units / mg, at least 0.2 x 107 units / mg, at least 0.3 x 107 units / mg, at least 0.4 x 107 units / mg, at least 0.5 x 107 units / mg, at least 0.6 x 107 units / mg, at least 0.7 x 107 units / mg, at least 0.8 x 107 units / mg, at least 0.9 x 107 units / mg, at least 1 x 107 units / mg, at least . less than 1.5 x 107 units / mg, at least 2 x 107 units / mg, at least 3 x 107 units / mg or at least 4 x 107 units / mg greater than the activity of the solid reference composition.

[0133] In various embodiments and aspects, a solid composition described herein is a dried product of a solution comprising a BoNT / A and HSA and has a lesser decrease in activity than a reference solid composition, wherein the reference solid composition is produced from a reference solution comprising a BoNT / A and HSA by a drying process performed such that the reference solution undergoes freezing upon drying and wherein the reference solution is substantially identical to the solution. The term "substantially identical" may be understood as described above. Further description regarding a reference solid composition is provided above in Section 5.3.In some embodiments, the solid composition described herein has a lesser decrease in activity of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to that of the reference solid composition.

[0134] The activity of the BoNT / A compositions described herein may be determined with methods known in the art, including, but not limited to, for example, a light-chain activity HPLC (LCA-HPLC) assay, mouse LD50 assay, mouse digit abduction score (DAS) assay, SNAP-25 protein assay, cell-based potency assay (CBPA) assay, etc.

[0135] The LCA-HPLC assay measures the specificity of SNAP-25 cleavage. Samples are reacted with a commercially available fluorescent BoNT / A substrate derived from the SNAP-25 sequence. The fluorescently labeled cleavage products are separated and detected by a reverse-phase HPLC (RP-HPLC) method. Further description of the LCA-HPLC assay can be found in Hunt et al. (2010) Toxins 2(8):2198-2212 and Rupp et al. (2020) Toxins 12(6):393.

[0136] In one embodiment, the activity is determined using a mouse LD50 test. The mouse 50% lethal dose (LD50) test has been described in, for example, Schantz and Kautter (1978) Journal of the AOAC61(V):96-99, Hunt and Kenneth (2009) Clinical Neuropharmacology 32(1):28-31, U.S. Patent 7,160,699 and U.S. Patent 9,725,705. A mouse 50% lethal dose (LD50) test is a method for measuring the activity of a botulinum toxin by intraperitoneal injection of the toxin. botulinum toxin in female mice (approximately four weeks old) weighing 17 to 22 grams each at the start of the test. Each mouse is held in a supine position with its head tilted downward and is injected intraperitoneally into the lower right abdomen at an angle of approximately 30 degrees using a 25- to 27-gauge, 3 / 8” to 5 / 8” needle with one of several serial dilutions of botulinum toxin in saline. Death rates over the next 72 hours for each dilution are recorded. Dilutions are prepared so that the most concentrated dilution produces a death rate of at least 80% of the injected mice, and the least concentrated dilution produces a death rate not exceeding 20% of the injected mice. There must be a minimum of four dilutions that fall within the range of monotonically decreasing death rates.The monotonic decay range begins with a death rate greater than or equal to 80%. Among the four or more monotonic decay rates, the two largest and two smallest rates must be decreasing (i.e., not equivalent). The dilution at which 50% of the mice die within the three-day postinjection observation period is defined as a dilution that includes one unit (1 U) of botulinum toxin.

[0137] The mouse finger abduction test (DAS) is an in vivo assessment of toxin-induced muscle paralysis following injection of BoNT / A toxin into the hindlimb muscle of a rodent. The DAS test can be used to assess the activity of BoNT / A compositions on muscle paralysis, as well as the duration of action. Detailed DAS test protocols have been described in Aoki et al. (1999) Eur. J. Neurol. 6:s3-slO, Aoki (2001), Toxicon 39: 1815-1820, Broide et al. (2013) Toxicon 71:18-24 and Rupp et al. (2020) Toxins 12(6):393. For example, the DAS test can be performed by injecting a BoNT / A composition described here into the mouse gastrocnemius-soleus complex, followed by assessment of finger abduction score using the method of Aoki (2001) Toxicon 39:1815-1820.In the DAS test, mice are suspended briefly by the tail with the objective of eliciting a characteristic startle response in which the mouse extends its hind limbs and spreads its hind digits. After injection of BoNT / A composition, the varying degrees of digit abduction are assessed on a 5-level scale (from 0 for normal abduction to 4 for maximal reduction in digit abduction and paw extension). The so-called safety ratio is the ratio of the amount of a toxin required for a 10% loss of body weight (measured at its maximum within the first seven days after administration in mice) to the amount of toxin required for a DAS score of 2. It can also be determined to assess the therapeutic index of the BoNT / A composition described herein, as described in US Patent 9,920,310. High scores of . Safety reports are therefore desired and indicate that a toxin is able to effectively paralyze a target muscle with few adverse effects outside the target.

[0138] The SNAP-25 test is an ELISA-based method for measuring the proteolytic activity of botulinum toxin against the SNAP-25 protein. The test uses a truncated SNAP-25 protein (peptide of 206 amino acid residues) fixed on 96-well polystyrene microtiter plates and a monoclonal antibody that recognizes the cleaved product (a peptide of 197 amino acid residues) which is obtained by enzymatic hydrolysis between amino acids 197 and 198 of SNAP-25 by reduced botulinum toxin type A. The monoclonal antibody fixed to the cleaved product is then detected with a secondary antibody (goat anti-mouse IgG antibody conjugated to horseradish peroxidase HRP) which produces a color change in the presence of a chromogenic substrate (TMB). Exemplary SNAP-25 methods are described in Ekong et al. (1997) Microbiology 143:3337-3347 and in US Patent 7,160,699.

[0139] A cell-based activity assay (CBPA) has been described in, for example, Femândez-Salas et al. (2012) PLOS ONE 7(ll):e49516, Rupp et al. (2020) Toxins 12(6):393, WO 2010 / 105234 and WO 2009 / 114748. In one embodiment, the SNAP-25197 SiMa H1 electrochemiluminescence (ECL) CBPA assay is used to determine the activity of the BoNT / A compositions described herein. The CBPA SNAP-25197 SiMa H1 electrochemiluminescence (ECL) assay is an in vitro cell-based assay that assesses key steps in BoNT / A intoxication: receptor-mediated cellular binding and internalization, translocation of the protease domain (light chain) into the cytosol, and proteolytic cleavage of SNAP-25, allowing for direct comparison of the in vitro biological activity of BoNT / A products (Fernandez-Salen et al. (2012) PLOS ONE 7(ll):e49516; Rupp et al. (2020) Toxins 12(6):393).In brief, human neuroblastoma SiMa H1 cells were plated on polyD-lysine (PDL)-treated 96-well plates in serum-free media (SFM) with 25 pg / mL GTi b for three days and treated with toxin samples for 24 hours. After treatment, toxins were removed, cells were lysed, and lysates were transferred to MSD High Bind plates coated with an anti-SNAP-25i97 monoclonal antibody (mAb) 2E2A6. The plates were then washed and incubated with a SULFO-TAG NHS-Ester-labeled anti-SNAP-25 polyclonal antibody (pAb) for detection. The captured BoNT / A toxin-cleaved SNAP-25 was then quantified on an MSD plate reader.

[0140] In various embodiments and aspects, a solid composition described herein is a dried product of a solution comprising a BoNT / A and HSA and has superior visual product quality (e.g., less or no flakes) compared to a reference solid composition, wherein the reference solid composition is produced from a reference solution comprising a BoNT / A and HSA by a drying process performed such that the reference solution undergoes freezing upon drying and wherein the reference solution is substantially identical to the solution. In various embodiments and aspects, a solid composition described herein is a dried product of a solution comprising a BoNT / A and HSA and has a more consistent visual product quality (e.g., with more consistently less or no flakes) compared to a reference solid composition. The term “substantially identical” may be understood as described above. Additional description regarding a reference solid composition is provided above in Section 5.3.

[0141] In various embodiments and aspects, a solid composition described herein is a dried product of a solution comprising a BoNT / A and HSA and has lower immunogenicity than a reference solid composition, wherein the reference solid composition is produced from a reference solution comprising a BoNT / A and HSA by a drying process performed such that the reference solution undergoes freezing upon drying and wherein the reference solution is substantially identical to the solution. The term "substantially identical" may be understood as described above. Additional description regarding a reference solid composition is provided above in Section 5.3.

[0142] The immunogenicity of a BoNT / a composition may be measured by any method described herein or known in the art and suitable for measuring the immunogenicity of a BoNT / A composition, such as, but not limited to, the detection of neutralizing antibodies (e.g., anti-HSA antibodies and / or anti-BoNT / a antibodies). 6. ILLUSTRATIVE EMBODIMENTS

[0143] The present description includes the following illustrative and non-limiting embodiments: 1. A solid composition comprising a 900 kDa Clostridium botulinum neurotoxin serotype A (BoNT / A) complex, human serum albumin (HSA), and sodium chloride (NaCl), wherein the HSA comprises an HSA monomer and HSA aggregates, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein less than 34% of the soluble HSA in the solid composition is HSA aggregates. 2. A solid composition according to embodiment 1, wherein the HSA aggregates comprise an HSA polymer, an HSA oligomer and an HSA dimer. 3. A solid composition according to embodiment 1 and embodiment 2, wherein less than 30% of the soluble HSA contained in the solid composition is an HSA polymer. 4. A solid composition according to embodiment 1 and embodiment 2, wherein less than 0.8% of the soluble HSA contained in the solid composition is an HSA oligomer. 5. A solid composition according to embodiment 1 and embodiment 2, wherein less than 4.8% of the soluble HSA contained in the solid composition is an HSA dimer. 6. A solid composition according to any one of embodiments 1 to 5, wherein the percentage of HSA aggregates, HSA polymer, HSA oligomer and / or HSA dimer in the soluble HSA contained in the solid composition is determined by gel permeation chromatography (SEC) after reconstitution of at least a portion of the solid composition with water. 7. Solid composition according to any one of embodiments 1 to 6, wherein the solid composition is a powdered pharmaceutical composition. 8. A solid composition according to any one of embodiments 1 to 7, wherein the percentage of HSA aggregates, HSA polymer, HSA oligomer and / or HSA dimer in the soluble HSA contained in the solid composition is determined by gel permeation chromatography (SEC) using a UV detector at a wavelength of 220 nm after reconstitution of at least a portion of the solid composition with water. 9. Solid composition according to any one of embodiments 1 to 8, wherein the solid composition is free from products of animal origin. 10. A solid composition according to any one of embodiments 1 to 9, wherein the HSA is a recombinant HSA. 11. A solid composition according to any one of embodiments 1 to 10, wherein the solid composition comprises about 0.5 mg of HSA per 100 units of BoNT / A. 12. A solid composition according to any one of embodiments 1 to 11, wherein the solid composition comprises about 0.9 mg of NaCl per 100 units of BoNT / A. 13. A solid composition according to any one of embodiments 1 to 12, wherein the BoNT / A is onabotulinumtoxinA. 14. A solid composition according to any one of embodiments 1 to 13, wherein the BoNT / A is produced via a column chromatography purification process. 15. A solid composition according to any one of embodiments 1 to 14, wherein the BoNT / A is produced via a column chromatography purification process, wherein the column chromatography purification process comprises hydrophobic interaction chromatography. 16. A solid composition according to any one of embodiments 1 to 15, wherein the column chromatography purification process further comprises anion exchange chromatography. 17. A solid composition according to any one of embodiments 1 to 16, wherein the column chromatography purification process further comprises cation exchange chromatography. 18. A solid composition according to any one of embodiments 1 to 17, wherein the solid composition does not comprise a protease inhibitor. 19. Solid composition according to any one of embodiments 1 to 18, wherein the solid composition does not comprise benzamidine hydrochloride. 20. A solid composition according to any one of embodiments 1 to 19, wherein the solid composition has an activity of between about 1.5 x 107 units / mg and about 6.0 x 107 units / mg. 21. A method of producing a solid composition comprising a 900 kDa BoNT / A complex, HSA and NaCl, said method comprising a step of drying a solution comprising a BoNT / A, HSA and NaCl to produce said solid composition, wherein the solution does not undergo freezing during the drying step. 22. Method according to embodiment 21, in which the drying step is at least partially manually regulated. 23. A method according to embodiment 21 or embodiment 22, wherein the drying step is vacuum drying. 24. The method of embodiment 23, wherein the vacuum drying step comprises a staged vacuuming step. 25. A method according to any one of embodiments 21 to 24, wherein the method produces a solid composition according to any one of embodiments 1 to 20. 26. Solid composition produced by the method according to any one of embodiments 21 to 25. 27. A method of treating a patient in need thereof, comprising administering the solid composition according to any one of embodiments 1 to 20 and 26. 28. Method substantially as described herein. 29. Composition substantially as described herein. 7. EXAMPLE

[0144] Certain embodiments of the present invention are illustrated by the following non-limiting examples, which describe different methods of obtaining BoNT / A and demonstrate that freezing during drying leads to a significant increase in the amount of HSA aggregation in a solid BoNT / A composition, compared to drying without freezing, and that solid BoNT / A compositions dried without undergoing freezing have a slower decline in activity during storage.

[0145] 7 .1. Example 1 — Chemical and physical stability of serum albumin human during the manufacture of finished botulinum toxin product: a laboratory-scale study. 7.1.1. Introduction

[0146] The majority of commercially available finished neurotoxin products contain human serum albumin (HSA) as an excipient. Understanding the HSA aggregation phenomenon can be important for the development and commercialization of neurotoxin products. HSA aggregates can be introduced into a finished product (DP) from incoming HSA that is used in the manufacture of the DP. The European Pharmacopoeia monograph for HSA states that the total peak area due to HSA polymers and aggregates should be a maximum of 10% of the total area (http: / / www.uspbpep.com / ep60 / human%20albumin%20solution%200255e.pdf, EUROPEAN PHARMACOPOEIA 6.0, Human Albumin Solution). Pharmaceutical-grade HSA manufacturers may also impose stricter standards on the level of aggregation.For example, CSL Behring — one of the HSA suppliers — specifies a standard of < 8% for aggregates ([Fig. 1]). This study seeks to determine whether HSA aggregates can also be produced during the manufacturing of finished products (DP). As with any sterile biopharmaceutical product manufacturing, there are several steps in the manufacturing of a BoNT / A DP.

[0147] In the present study, HSA aggregation was measured in laboratory-produced BoNT / A DP batches and in placebo batches that were manufactured under all three sets of conditions based on the same formulation, containing HSA and NaCl, with (for the BoNT / A DP batches) and without (for the placebo batches) BoNT / A, the active ingredient. The main differences in the manufacturing processes concern the drying process and the storage of the bulk solution before the drying step. Commercially available finished BoNT / A products are produced either with freeze-drying or with vacuum drying. Both manufacturing processes can implement the same freeze-drying equipment, while the main difference concerns the freezing step used in the latter. Evaporative cooling during vacuum drying, however, can also lead to freezing.

[0148] Another possible difference in manufacturing conditions concerns the times for which a finished bulk product is stored before starting sterilizing filtration, as well as between filtration and the end of filling into vials. Such storage times for laboratory manufacturing, where a few tens to a few hundred vials are produced, can be reduced to less than one hour. Conversely, for commercial-scale production, with a number of vials produced amounting to several thousand or even tens of thousands, a pre-drying storage time would be much longer. In addition, a commercial process is generally validated for extended storage times to allow for the possibility of unforeseen delays, for example, malfunction of the filling equipment. Therefore, the consequences of pre-drying storage of the finished bulk product were also evaluated in the present study.The formulation contains HSA and NaCl. Batches were produced with the active ingredient, namely BoNT / A (active DP batches) and without BoNT / A (placebo batches). 7.1.2. Materials and processes 7.1.2.1 Materials

[0149] HSA was obtained from CSL Behring and is supplied as a 25% solution. Sterile 0.9% NaCl solution by weight (USP, EP, BP, JP grade, Terumo BCT Ltd), NaCl granules (Baker JT, USP, EP, JP, BP grade) and MilliQ water were used for manufacturing. BoNT / A active substance (DS) concentrated solution was supplied by AbbVie. Vials (Type 1 glass, 10 mL, Gerresheimer) were washed and depyrogenated. Caps (20 mm Lyotec West S-87-J, 4432 / 50 GRY, ready to sterilize) were sterilized and dried. For batches with an active ingredient, an active substance solution of BoNT / A was added to the excipient solution (0.5 wt% HSA and 0.9 wt% NaCl) using a dilution factor of 23,000, filtered through a 0.2 pm Millipak-20 gamma-irradiated sterilizing filter, aseptically filled into 10 mL Type 1 glass vials at 0.1 mL / vial, which were partially capped, loaded into a LyoStar2 freeze dryer, subjected to vacuum drying or freeze-drying, vacuum-capped at the end of the drying process, and sealed with an aluminum cap. Placebo batches were manufactured in the same manner except for the addition of DS and a larger fill volume of 0.2 mL / vial. The product temperature during vacuum drying was measured with temperature probes (Ellab, Denmark) in real time. Temperature data were also recorded with data loggers.Three sets of manufacturing conditions were used: the main differences are shown in Table 2 and more detailed information is provided in Section 7.1.2.2 below. Five finished product batches and three placebo batches were manufactured. Figures 2A and 2B show representative cycles for vacuum drying and for freeze-drying.

[0150] [Tables2] Manufacturing process reference Storage pre-filtration / post-filtration of finished product (DP) in bulk Drying process Number of batches produced Active / Placebo 1 <15 min / <15 min Vacuum drying 2 / 1 2 <15 min / <15 min Freeze-drying 1 / 1 3 24 h / 24 h Freeze-drying 2 / 1 7.1.2.2. Procedures 7.1.2.2.1. Drying procedures

[0151] The cycle of Process 1 (i.e., vacuum drying) is shown in Table 3. The evacuation of the freeze dryer results in the cooling of the vials. To prevent the freezing of the solutions in the vials, a specific staged procedure was adopted allowing the freeze dryer chamber to be evacuated without causing the freezing of the solutions in the vials. Specifically, before starting the automated vacuum drying cycle of Table 3 (i.e., before the evacuation step and after the pre-freezing step), the vacuum was manually regulated, as described below, in order to reduce the risk of freezing for the solutions in the vials due to too rapid evacuation in the sample chamber of the freeze dryer. The main reason for this manual staged regulation is that the LyoStar 2 does not allow the vacuum rate to be controlled during the cycle. Instead, vacuum was applied, with the vacuum rate uncontrolled, and it was stopped at various intervals to allow the product temperature to return to a higher temperature (such as 20 °C) before being reapplied. Two temperature thermocouples were inserted into two flasks near the middle of the shelf to monitor the product temperature. The manual cycle was started. The shelf temperature was set to 20 °C. The condenser was turned on. Vacuum was then manually applied and stopped at 20,000 mTorr. The product temperature, obtained from the thermocouple readings, was 16 °C. The product temperature was allowed to rise to 20 °C and vacuum was reapplied until the Pirani pressure reading reached 5,000 mTorr, at which point the vacuum was stopped. The product temperature was 2°C.Again, the product temperature was allowed to rise to 18°C. At this point, the automated cycle shown in Table 3 below was initiated. Once the vacuum drying cycle was completed, the vials were fully capped, sealed, and stored in a -20°C freezer.

[0152] [Table 3] Cycle Parameter — Process 1 Step Temperature °C Temperature rise °C / min Intermediate storage time min Pressure mTorr Pre-freezing 20 1 15 ambient Freezing NA NA NA ambient Further freezing NA NA NA ambient Vacuum packaging 20 0 15 84 Primary drying 20 1 300 84 Secondary drying NA NA NA NA Intermediate storage 5 NA NA 84 NA = not applicable

[0153] The cycle of Process 2 (i.e., freeze-drying) is shown in Table 4. Specifically, a freezing cycle at -50°C, for 120 minutes, was included in this cycle. The cycle of Process 2 did not include a staged vacuum step. After the cycle was completed, the vials were completely vacuum-capped and the freeze-dryer was returned to ambient conditions. The vials were capped and stored at -20°C until analysis.

[0154] [Table 4] Cycle Parameter — Process 2 Step Temperature °C Temperature rise °C / min Intermediate storage time min Pressure mTorr Pre-freezing 2 1 15 ambient Freezing -50 1 120 ambient Further freezing NA NA NA ambient Vacuum sealing -50 0 15 84 Primary drying 20 1 300 84 Secondary drying NA NA NA NA Intermediate storage 5 NA NA 84 NA = not applicable

[0155] The cycle of process 3 (i.e., freeze-drying + intermediate storage at room temperature) is shown in Table 5. Specifically, a freezing cycle at -50°C, for 120 minutes, was included in this cycle. The cycle of process 3 did not include a staged vacuum step. After the cycle was completed, the vials were completely vacuum-capped and the freeze-dryer was returned to ambient conditions. The vials were capped and stored at -20°C until analysis. Prior to the drying cycle, the product solution was stored at room temperature for 24 hours before filtration and again for 24 hours after filtration.

[0156] [Table 5] Cycle Parameter — Process 3 Step Temperature °C Temperature rise °C / min Intermediate storage time min Pressure mTorr Pre-freezing 2 1 15 ambient Freezing -50 1 120 ambient Further freezing NA NA NA ambient Vacuum sealing -50 0 15 84 Primary drying 20 1 300 84 Secondary drying NA NA NA NA Intermediate storage 5 NA NA 84 NA = not applicable 7.1.2.2.2. Analysis of HSA aggregation by SEC

[0157] Human serum albumin (HSA) aggregation was measured by gel permeation chromatography (SEC).

[0158] Dried samples were reconstituted by adding 0.25 mL of water per vial. 25% pure HSA solutions were diluted with water to 0.25 mg / mL before HPLC injection. System compliance verification standards were injected 5 times before sample injections and then every 10 injections to verify system compliance. HPLC parameters are shown in Table 6. Samples were run and analyzed with Empower. Five active batches and three placebo batches were tested by SEC. Statistical analysis of SEC data was performed using JMP 10.0 statistical software.

[0159] [Table 6] SEC parameters Parameter Method Injection Volume 50 pL Column Temperature Room Temperature Column Superdex 200 10 / 300 Flow Rate 0.5 mL / min Mobile Phase 50 mM Potassium Phosphate + 150 mM NaCl, pH 7.0 Run Time 60 minutes Sample Preparation Vials reconstituted at 0.25 mg / mL in water Detection Fluorescence (excitation 280 nm, emission 350 nm) a. 280 nm b. 220 nm 7.1.2.2.3. Karl Fischer assay

[0160] The coulometric Karl Fischer (KF) assay was used to quantify the residual water content in the dried product. A KF titrator with oven and autosampler was used. Samples were analyzed in triplicate. Sealed vials were heated in the KF oven to 115 °C. Nitrogen gas was used to purge the vials and carry any moisture in the headspace to the reaction vessel, containing the KF reagent. The reaction proceeded until the rate decreased below 20 pg / min. Empty vials, which underwent the same freeze-drying or vacuum drying cycles, were also tested, and the water content of the controls was subtracted to obtain the water content in the dried material. Four representative batches were tested.

[0161] 7.1.2.2.4. Scanning electron microscopy (SEM)

[0162] Three placebo dried cake batches were examined using an FEI Quanta-450 field emission SEM (Thermo Fisher Scientific, Hillsboro, OR) with a backscattered electron detector. Imaging conditions were 10 kV and 10 mm working distance under low vacuum. Samples were uncoated. SEM imaging of placebo dried cake morphology required breaking the glass vial to isolate the dried cake / glass portions. This was accomplished by scoring the vial near the heel with a diamond tip, and then snapping the glass. Appropriate portions of each sample were then mounted on a SEM sample holder using carbon tape. Three placebo batches were tested by SEM. 7.1.2.2.5. Analysis of subvisible particles

[0163] For the subvisible particle test, three placebo batches, produced by methods 1, 2, and 3, respectively, were tested. The vials containing the dried placebo were reconstituted by adding 2 mL of saline per vial and gently mixed. Vials from each batch were tested using a laboratory-based non-invasive subvisible particle imaging system, as described in U.S. Patent 10,132,736 B2. In measuring particles, a reconstituted solution in a vial was partially illuminated by a laser beam focused into a thin sheet of light. Video images were recorded using a sensitive digital video camera with a specially designed lens system to compensate for image distortion caused by the cylindrical shape of the container. Use of this system allows for non-invasive counting of subvisible particles in syringes or vials.The software developed in the laboratory can measure the number of particles, their size and intensity in each image in real time. The absolute number of particles and their size can be calibrated using standard particle samples. 7.1.2.2.6. X-ray diffraction on XRPD

[0164] XRPD diffractograms were obtained with a MiniFlex 600 X-ray diffractometer (Rigaku, Texas, USA) with Cu Ka radiation (X = 1.54 Å, 40 kV / 15 mA). The instrument was calibrated with a silicon standard with a reference peak at 28.44° (20). Solid samples were prepared on a low-background Si support by applying light pressure to keep the sample surface flat and level with the reference surface of the sample holder. Each sample was analyzed from 3 to 45° (20) using a continuous scan of 1° (20) per minute with a step size of 0.01° (20). Data analysis was performed using PDXL software.

[0165] 7.1.2.2.7. Small angle X-ray scattering / small angle X-ray scattering wide angles (SAXS / WAXS)

[0166] SAXS / WAXS analyses of two solutions (0.9% NaCl / 0.5% HSA and 0.9% NaCl in water) were performed at the ID02 beamline of the European Synchrotron Radiation Facility (ESRF). The solutions were filled into 1 mm capillaries, and loaded onto a Linkam stage for analyses at variable temperatures. Two-dimensional SAXS and WAXS images were obtained using two different detectors. The exposure time was adjusted to utilize the maximum dynamic range of the detectors for each sample and was less than 1 second in the majority of cases. The two-dimensional images were normalized to an absolute intensity scale. after implementing standard and azimuthally integrated detector corrections to obtain the corresponding one-dimensional X-ray diffraction curves. The q calibrations for SAXS and WAXS analyses were performed with silver and silicon behenate powders, respectively. The combined SAXS / WAXS measurements were all normalized. 7.1.2.2.8. Changes in apparent pH

[0167] To monitor changes in apparent pH during freezing of the formulation, a low-temperature pH electrode, InLab®Cool (pH 1 to 11, temperature -30 °C to 80 °C, electrolyte PRISCOLYT-B®) (METTLER TOLEDO), was used. The pH electrode was calibrated at room temperature using NIST-traceable buffer standards—pH 4.01, pH 7.00, and pH 10.00 (Aqua solutions). A glass beaker with 50 mL of placebo solution was transferred to a freeze dryer (Lyostar III, SP Scientific). The temperature probe and the low-temperature pH electrode were placed in the beaker of placebo solution. The freeze dryer door was closed, and cooling was initiated in manual mode with a shelf temperature set at -55 °C. The pH reading was recorded at each unit change in sample temperature.Once the sample temperature was at -50 °C, the shelf setpoint was increased in 10 °C increments until the sample temperature was within 2 °C of the shelf temperature, allowing the sample to thaw to 20 °C. pH was also measured during evaporation, using an InLab®Micro microelectrode (pH 0 to 14, temperature 0 °C to 80 °C, electrolyte 3 M KCl) (METTLER TOLEDO), while sample mass was determined at regular intervals with the placebo under evaporation under nitrogen flow. The pH microelectrode was calibrated at room temperature using the same NIST-traceable buffer standards cited previously. Approximately 7.3 g of placebo solution was placed in a glass vial and subjected to nitrogen flow in a fume hood. The sample bottle was removed from the hood intermittently to record pH and mass data. 7.1.3. Results 7.1.3.1. Characterization of “pure” HSA.

[0168] Representative SEC chromatograms for HSA solution (raw material) are shown in Figures 3A-3C. Besides the HSA monomer peak (retention time, RT ~ 27 min), three higher molecular weight species peaks were identified as dimer (RT ~ 23.5 min), HSA oligomer (RT ~ 21 min), and HSA polymer (RT ~ 16 min). The assignment of polymer, oligomer, dimer, and monomer peaks was made with reference to the retention times obtained when analyzing the Bio-Rad standard for gel permeation (ref. 151-1901) on the same column. The gel permeation standard consists of bovine thyroglobulin (MW 670 kDa), bovine gammaglobulin (MW 158 kDa), chicken albumin (MW 44 kDa), horse myoglobulin (MW 17 kDa) and vitamin B12 (MW 1350 Da). Depending on the detection method, total aggregation in soluble HSA subjected to SEC chromatographic analysis ranging from about 4% to about 10% was observed, with the highest values being obtained by fluorescence detection followed by UV detection at a wavelength of 280 nm. Oligomers were detected by fluorescence, but not by the UV detector, probably related to the better sensitivity of the fluorescence detector.

[0169] Among the three batches of HSA used in this study, the aggregation levels were similar (Tables 7-9).

[0170] [Table 7]: SEC analysis of aggregates in HSA solution (A220 nm) Batch No. H SA A220 nm Polymer (%) Oligomer (%) Dimer (%) Total Aggregates (%) 1 2.6 + 0.5 0.0 + 0.0 1.3 + 0.2 3.83 + 0.06 2 2.4 + 0.5 0.0 + 0.0 1.8 + 0.2 4.2 + 0.0 3 2.9 + 0.0 0.0 + 0.0 1.3 + 0.0 4.2 + 0.0

[0171] [Table 8]: SEC analysis of aggregates in HSA solution (A280 nm) Batch No. H SA A220 nm Polymer (%) Oligomer (%) Dimer (%) Total Aggregates (%) 1 4.8 + 0.9 0.0 + 0.0 0.8 + 0.3 5.67 + 0.15 2 4.5 + 0.8 0.0 + 0.0 1.4 + 0.7 5.9 + 0.21 3 5.4 + 0.1 0.0 + 0.0 0.0 + 0.0 5.33 + 0.12

[0172] [Table 9]: SEC analysis of aggregates in HSA solution (fluorescence) Batch No. H SA Fluorescence Polymer (%) Oligomer (%) Dimer (%) Total Aggregates (%) 1 8.5+1.4 0.2 + 0.1 1.8 + 0.1 10.4 + 0.1 2 7.8+1.0 0.2 + 0.1 1.9+1 9.9 + 0.07 3 9.7 + 0.1 0.2 + 0.0 1.8 + 0.0 11.63 + 0.06 7.1.3.2. Aggregation of HSA in active batches of DP and in placebo batches.

[0173] HSA aggregation in BoNT / A DP batches was characterized by SEC chromatography. Examples of SEC chromatograms for finished product batches produced with three different manufacturing processes are shown in Figures 4A-4C. A significant decrease in the peak was observed in the lyophilized batches produced with both Process 2 and Process 3, compared to the vacuum-dried batch (Process 1). The HSA dimer and polymer peaks were more prominent in the lyophilized samples than in the vacuum-dried product. Furthermore, two low-resolution polymer peaks were observed in the lyophilized samples, particularly with UV detection at both wavelengths of 220 nm and 280 nm.

[0174] The inset bar charts in Figures 4A-4C show the percentage of different types of HSA aggregates in soluble HSA subjected to SEC chromatographic analysis for three batches of DP as well as for "pure" HSA. The values in Figures 4A-4C are presented in Tables 10-12 below, respectively. The levels of the three types of aggregates were similar between "pure" HSA and the vacuum-dried (manufacturing process 1) batches of DP, while a significant increase in aggregation was observed for both batches of lyophilized DP. The quantification of the detected aggregation was a function of the detector type (see Figures 4A-4C and Tables 10-12).

[0175] [Table 10]: Percentage of different types of HSA aggregation (A220 nm) polymer % polymer % error (SD) dimer % dimer % error (SD) Pure HSA 2.6 0.5 1.3 0.2 Vacuum drying 2.3 0.1 2 0.1 Freeze-drying 13.7 0.6 4.6 0.5 Freeze-drying with intermediate storage 15.3 0.6 6.2 0.3

[0176] [Table 11]: Percentage of different types of HSA aggregation (A280 nm) polymer % polymer % error (SD) dimer % dimer % error (SD) Pure HSA 4.8 0.9 0.8 0.3 Vacuum drying 4.3 0.1 1.5 0.1 Freeze-drying 30.8 0.7 3.7 0.3 Freeze-drying with intermediate storage 32.7 0.8 4.8 0.3

[0177] [Table 12]: Percentage of different types of HSA aggregation (fluorescence) polymer % polymer % error (SD) oligomer % oligomer % error (SD) dimer % dimer % error (SD) Pure HSA 8.5 1.4 0.2 0.1 1.8 0.1 Vacuum drying 8 0.3 0.2 0.1 2.4 0.1 Freeze-drying 13.8 0.4 0.8 0.1 4.8 0.4 Freeze-drying with intermediate storage 15 0.5 1.1 0.1 6.3 0.2

[0178] Total HSA aggregation representing the sum of the three types of aggregates in soluble HSA subjected to SEC chromatographic analysis for a plurality of DP batches is shown in Figures 5A-5G. There was no significant increase in aggregation of the DP batches produced with manufacturing method 1 (vacuum drying), while the DP batch produced with manufacturing method 2 (freeze drying) showed a significant increase in HSA aggregation. The longer pre-drying storage time (method 3) did not result in a significant further increase in HSA aggregation.

[0179] The subvisible particle test was performed with dried placebo batches. The particle count results are shown in Figures 6A and 6B and Table 13. The average total particle count was lower in the vacuum-dried batch, while both freeze-dried batches had higher levels of subvisible particles. Between the two freeze-dried batches, the average particle numbers were similar and within the experimental variation. The results on subvisible particles were consistent with the data obtained by SEC analysis for soluble HSA aggregates (Figures 5A to 5G), where a higher level of aggregation was observed in the freeze-dried samples compared to the vacuum-dried batches.

[0180] [Table 13]: Results of the analysis of subvisible particles Saline Solution Reference (Lot No. 6) Stress (Room Temperature Storage and Freezing) (Lot No. 11) Stress (Freezing Only) (Lot No. 8) Number Mean Standard Deviation (Me ... 7.1.3.3. Physical characterization of the finished product

[0181] In order to understand the possible mechanisms leading to the increase in HSA aggregation during lyophilization, additional physical analyses were conducted.

[0182] The apparent pH during freezing was measured using low temperature pH electrodes, InLab®Cool (pH 1 to 11, temperature -30°C to 80°C, electrolyte PRISCOLYT-B®) (METTLER TOLEDO). The results are shown in Figures 7A to 7B. A relatively small, but nevertheless noticeable increase in pH was observed from an initial pH of 6.7 to pH 7 upon cooling the solution from room temperature to -10°C. Ice nucleation, which was detected at -10°C, at the peak product temperature, did not result in a noticeable pH change. Upon further cooling to -35°C, the pH remained between 6.9 and 7.1, while the pH increased to about 8.1 upon cooling from -35°C to -50°C. This increase in pH may be related to the secondary crystallization of NaCl*2H2O + water, which was detected at -35 °C in a separate WAXS experiment.While the product temperature indicated no evidence of exothermic effect due to secondary crystallization, the absence of the thermal effect could be explained by a notion of sensitivity related to low . NaCl concentration (0.9 wt%). It should be noted, however, that the temperature range of the pH electrode, according to the manufacturer's specifications, only extends down to -30°C; therefore, pH results below -30°C should be confirmed by an orthogonal method. In the vacuum experiment, a slight increase in pH, from 6.8 to 7.1, was observed, with the total solute concentration increasing from 1.4 wt% in the initial solution to approximately 8 wt%.

[0183] The phase transitions of water / NaCl and water / NaCl / HSA solutions upon cooling were studied by small angle X-ray scattering / wide angle X-ray scattering (S AXS AVAXS). Two placebo aqueous solutions were tested, one containing 0.9% NaCl and 0.5% HSA, and the other containing only 0.9% NaCl. WAXS data showed the formation of hexagonal ice at both -20 °C (HSA+NaCl solution) and -15 °C (NaCl solution) (Figures 8A and 8B). After further cooling, characteristic peaks of NaCl dihydrate were detected at -35 °C (HSA+NaCl solution) and -25 °C (NaCl solution). Results obtained by WAXS diffraction indicate that HSA can hinder the crystallization of NaCl dihydrate.

[0184] SAXS diffraction patterns are shown in [Fig.8C]. A peak related to protein interaction was observed in unfrozen solution, while freezing results in the peak “disappearing”. In addition to changes related to the protein interaction peak, freezing, as observed by WAXS diffraction at -20 °C, resulted in an increase in the low-angle diffraction intensity, indicative of the formation of additional interfaces, i.e., ice / solution and probably ice / air / solution interfaces. Further cooling to -35 °C resulted in a further increase in the low-angle diffraction that indicated the formation of new interfaces. This interface increase coincided with the crystallization of NaCl*2H2O as observed by WAXS.

[0185] Two approaches were used to confirm that the material does not freeze during vacuum drying (Manufacturing Process 1): monitoring the product temperature during vacuum drying and analyzing the dried products using scanning electron microscopy (SEM). Representative product temperature data ([Fig.2A]) show that the temperature remained above 0 °C throughout the vacuum drying cycle. Therefore, freezing could not have occurred in the vials. To further confirm the absence of freezing during vacuum drying, SEM analyses were performed on products in vials without thermocouples. The SEM images obtained for the freeze-dried products ([Fig.9]) demonstrate the porous morphology of both freeze-dried batches (right and middle images), as expected. The pores in the dried products represent a “signature” related to ice crystals that have 。 were sublimated during primary drying. The absence of pores in the vacuum-dried product (left images) confirmed that this type of product had not undergone freezing. Another major difference between the vacuum-dried and freeze-dried samples concerns the appearance of the NaCl crystals, which appear as cubic shapes lining the inner walls of HSA interstices in the vacuum-dried product. The freeze-dried batches showed no obvious signs of the presence of NaCl crystals, possibly because they were smaller than in the vacuum-dried batch. The reduced crystal size is consistent with the XRPD results (below) which show broader peaks for the freeze-dried batch as expected for smaller and more disordered crystals.

[0186] X-ray diffraction (XRPD) patterns for two placebo batches, which were produced by different manufacturing processes, are shown in [Fig. 10]. Two narrow peaks of crystalline NaCl and a halo of amorphous HSA were observed in both batches. The vacuum-dried batch (Process 1) exhibits more intense and narrower crystalline peaks, while the crystalline NaCl peaks appeared broader in the freeze-dried sample (Process 3), likely reflecting a higher degree of disorder. This result is consistent with the SEM data that show more prominent NaCl crystals in the vacuum-dried batch. Furthermore, anhydrous NaCl crystals were formed by dehydration of NaCl dihydrate during freeze-drying, while NaCl crystallizes directly from solution during vacuum drying. This difference may also contribute to a higher degree of crystalline disorder of NaCl in the freeze-dried product. 7.1.3.4. Activity stability study

[0187] In a separate study, freezing during vacuum drying did not appear to result in a statistically significant change in the DP activity of BoNT / A after 6 months of storage at 5°C compared to a process without freezing (data not shown). In addition to the results at 5°C, the study in this example also tested the stability of activity at room temperature and higher temperatures. Specifically, the stability of two batches of DP—Batch 9 (Process 3, freeze-drying + intermediate storage) and Batch 4 (Process 1, vacuum drying)—was evaluated at 5°C, 25°C, and 40°C, respectively. Vials from each batch were collected at various intervals between 0 and 6 months and analyzed for activity recovery by a cell-based activity assay (CBPA). The results are shown in Table 14 below. The test data under 25°C storage conditions are also shown in [Fig. 11].

[0188] [Table 14] Results of the activity stability study. Batch No. DP Drying Process Storage Conditions Storage Time months Target Activity Recovery U / vial Measured Activity Recovery U / vial Activity Recovery % of Initial Value 9 Process 3 5C 0 100 73 100 9 Process 3 5C 6 100 44 60.27 4 Process 1 5C 0 100 153 100 4 Process 1 5C 6 100 190 124.18 9 Process 3 25°C, 60% RH 0 100 73 100 9 Process 3 25°C, 60% RH 1 100 28 38.36 9 Process 3 25°C, 60% RH % RH 3 100 20 27.4 9 Process 3 25°C, 60% RH 6 100 20 27.4 4 Process 1 25°C, 60% RH 0 100 153 100 4 Process 1 25°C, 60% RH 1 100 115 75.16 4 Process 1 25°C, 60% RH 3 100 136 88.89 4 Process 1 25°C, 60% RH 6 100 110 71.9 9 Process 3 40°C, 75% RH 0 100 73 100 9 Process 3 40°C, 75% RH 1 100 8 10.96 9 Process 3 40°C, 75% RH 3 100 6 8.22 9 Process 3 40°C, 75% RH 6 100 2 2.74 4 Process 1 40°C, 75% RH 0 100 153 100 4 Process 1 40°C, 75% RH 1 100 132 86.27 4 Process 1 40°C, 75% RH 3 100 117 76.47 4 Process 1 40°C, 75% RH 6 100 68 44.44 RH: relative humidity (intended to simulate storage under accelerated conditions).

[0189] As shown, while the activity of the DP batch produced with process 3 decreases slightly at 5 °C after 6 months, it decreases dramatically at 25 °C and 40 °C after only 3 months. Conversely, the activity of the DP batch produced with process 1 decreases much more slowly at 25 °C and 40 °C. 7.1.4. Discussion

[0190] A high level of HSA aggregation was observed in the freeze-dried batches, while vacuum drying without freezing did not result in a significant increase in HSA aggregation. HSA aggregation did not increase upon vacuum drying in the absence of freezing. The level of soluble aggregates increased from an initial content of about 4% to about 10% in "neat" HSA to a content of about 20% to about 35% (specific values depending on the SEC detector, see Figures 5A to 5G) after freeze-drying and reconstitution. Insoluble aggregates also increased significantly in the freeze-dried products compared to the vacuum-dried products.The results show that by precisely regulating specific manufacturing conditions, HSA aggregation can be significantly reduced, with lower levels of both soluble and insoluble HSA aggregates detected when freezing is avoided during drying. The results also show that freezing during drying can lead to a more rapid decline in activity during storage.

[0191] Protein destabilization and aggregation may possibly be related to protein concentration and ionic strength. However, the low aggregation of HSA in the vacuum-dried batches indicates that neither an increase in protein concentration nor ionic strength are the primary mechanisms in the observed HSA aggregation per se.

[0192] Regardless of any link to any theory, the results of the present study suggest that the observed HSA aggregation may be related to NaCl*2H2O. In particular, secondary crystallization (NaCl*2H2O + water) could create favorable freezing-induced pressure conditions, as it occurs within a rigid “enclosure” of ice crystals, which was formed during a primary freezing event, thus possibly contributing to protein destabilization upon freezing. NaCl*2H2O crystallization may also contribute to HSA aggregation through a different mechanism. While crystallization of anhydrous NaCl during vacuum drying did not result in HSA aggregation, a different crystalline form of NaCl—a dihydrate—was formed during freeze-drying.Anhydrous NaCl formed during vacuum drying may exhibit different surface properties than the dihydrate formed during freezing. While the surface charge is not known for NaCl and NaCl*2H2O crystals formed during vacuum drying and freeze-drying, respectively, a significant difference in the electrostatic interaction of these two types of crystals with HSA may be a contributing factor to freezing-induced HSA aggregation.

[0193] Finally, pH changes were observed in this study upon freezing which could also contribute to HSA aggregation. 7.1.5. Conclusions

[0194] Although the majority of neurotoxin formulations available on the market contain HSA, the consequences of the manufacturing process of the finished product on the properties of HSA are poorly understood. In the present study, a significant increase in HSA aggregation was observed during freeze-drying, while HSA aggregation in vacuum-dried DP without freezing did not increase significantly and remained similar to that of the initial HSA solution. These observations suggest that the manufacturing conditions of neurotoxin formulations may have significant consequences on the properties of the finished product. Thus, additional analyses of toxin-based DP, particularly HSA aggregation, may be beneficial during the development and commercialization of HSA-containing neurotoxin products.The results further indicate that freezing was the primary cause of HSA aggregation.

[0195] On the mechanism of HSA aggregation during freeze-drying, the study indicates that freezing-induced pressure, interaction with NaCl*2H2O crystals and freezing-induced pH changes could be contributing factors.

[0196] 7.2. Example 2 - Consequences of freezing on finished toxin products vacuum-dried botulinum

[0197] In the present study, batches of BoNT / A DP produced with a vacuum drying process that did not involve freezing of the finished product solutions were compared to batches of BoNT / A DP produced with a vacuum drying process that involved freezing of the finished product solutions. The two types of BoNT / A DP batches are referred to in this example as evaporative vacuum dried DP batches and vacuum freeze-dried DP batches, respectively. Both freeze dryers—the pilot-scale Lyostar-3 freeze dryer and the industrial-scale Lyomax 40 freeze dryer—were tested. For both freeze dryers, the evaporative vacuum dried DP batches repeatedly exhibited a visually more homogeneous morphology, resulting in consistent visual product quality for the patient, while the vacuum freeze-dried DP batches were prone to flake formation.A flaked product is likely to migrate near the cap during reconstitution of the finished product vial, resulting in a loss of product relative to the prescribed patient dose. Thus, evaporatively vacuum-dried DP batches are more advantageous than vacuum-freeze-dried DP batches.

[0198] Additional results from the present study showed that using a slower chamber depressurization rate and a higher shelf temperature (shelf temperature of 20°C instead of 5°C) can help prevent freezing of the finished product (although the exact chamber depressurization rate to be used is specific to the drying equipment and depends on the chamber size, condenser capacity, chamber loading, and vacuum pump performance). The results of this study showed that the rapid chamber depressurization rate and the low shelf temperature were significant factors contributing to the lower product temperature.

Claims

Claims

1. A solid composition comprising a 900 kDa Clostridium botulinum neurotoxin serotype A (BoNT / A) complex, human serum albumin (HSA), and sodium chloride (NaCl), wherein the HSA comprises an HSA monomer and HSA aggregates, wherein at least a portion of the HSA is in the form of soluble HSA when the solid composition is reconstituted in an aqueous medium, and wherein less than 34% of the soluble HSA contained in the solid composition is HSA aggregates.

2. The solid composition of claim 1, wherein the HSA aggregates comprise an HSA polymer, an HSA oligomer, and an HSA dimer.

3. A solid composition according to claim 1 and claim 2, wherein less than 30% of the soluble HSA contained in the solid composition is a polymer of HSA.

4. A solid composition according to claim 1 and claim 2, wherein less than 0.8% of the soluble HSA contained in the solid composition is an HSA oligomer.

5. A solid composition according to claim 1 and claim 2, wherein less than 4.8% of the soluble HSA contained in the solid composition is an HSA dimer.

6. A solid composition according to any one of claims 1 to 5, wherein the solid composition is a powdered pharmaceutical composition.

7. A solid composition according to any one of claims 1 to 6, wherein the solid composition is free from products of animal origin.

8. A solid composition according to any one of claims 1 to 7, wherein the HSA is a recombinant HSA.

9. A solid composition according to any one of claims 1 to 7, wherein the solid composition comprises about 0.5 mg of HSA per 100 units of BoNT / A.

10. A solid composition according to any one of claims 1 to 9, wherein the solid composition comprises about 0.9 mg of NaCl per 100 units of BoNT / A.

11. 67 A solid composition according to any one of claims 1 to 10, wherein the BoNT / A is onabotulinumtoxinA.

12. A solid composition according to any one of claims 1 to 11, wherein the BoNT / A is produced via a column chromatography purification process.

13. A solid composition according to any one of claims 1 to 12, wherein the BoNT / A is produced via a column chromatography purification process, wherein the column chromatography purification process comprises hydrophobic interaction chromatography.

14. A solid composition according to any one of claims 12 to 13, wherein the column chromatography purification process further comprises anion exchange chromatography.

15. A solid composition according to any one of claims 12 to 14, wherein the column chromatography purification process further comprises cation exchange chromatography.

16. A solid composition according to any one of claims 1 to 15, wherein the solid composition does not comprise a protease inhibitor.

17. A solid composition according to any one of claims 1 to 16, wherein the solid composition does not comprise benzamidine hydrochloride.

18. A solid composition according to any one of claims 1 to 17, wherein the solid composition has an activity of between about 1.5 x 107 units / mg and about 6.0 x 107 units / mg.

19. A method of producing a solid composition comprising a 900 kDa BoNT / A complex, HSA and NaCl, said method comprising a step of drying a solution comprising a BoNT / A, HSA and NaCl to produce said solid composition, wherein the solution does not undergo freezing during the drying step.

20. The method of claim 19, wherein the drying step is at least partially manually controlled.

21. A method according to claim 19 or claim 22, wherein the drying step is vacuum drying.

22.

23.

24.

25. The method of claim 20, wherein the vacuum drying step comprises a staged vacuuming step. A method according to any one of claims 19 to 22, wherein the method produces a solid composition according to any one of claims 1 to 18. A solid composition produced by the process of any one of claims 19 to 23. Composition according to any one of claims 1 to 18 and 24 for use as a medicament.

Images