COMPOSITIONS OF CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE A AND IMPROVED DRYING PROCESSES

Abstract

Provided herein are 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. Further provided herein are methods for preparing solid compositions comprising BoNT / A and HSA by drying a BoNT / A-containing solution such that the solution does not freeze during drying.

Description

REFERENCE TO THE ELECTRONICALLY TRANSMITTED SEQUENCE RECORD

[0001] This application incorporates by reference a sequence listing filed with this application as an xml file entitled “13371-306-888_SEQLISTING.xml”, created on January 3, 2024, and having a size of 6,507 bytes. 1. AREA

[0002] Provided herein are 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. Further provided herein are methods for preparing solid compositions comprising BoNT / A and HSA by drying a BoNT / A-containing solution such that the solution does not freeze during drying. 2. STATE OF THE ART

[0003] Clostridium botulinum neurotoxin serotype A (BoNT / A) is a potent toxin that causes muscle relaxation by inhibiting synaptic vesicle docking and fusion, thereby blocking the release of acetylcholine at neuromuscular junctions. BoNT / A is produced by Clostridium botulinum type A strains that synthesize a complex of a 150 kDa neurotoxin with a group of nontoxic neurotoxin-associated proteins (NAPs). ® , or onabotulinumtoxinA, is a BoNT / A product approved by the U.S. Food and Drug Administration (FDA) in 1989 for a variety of therapeutic and cosmetic indications.

[0004] The production of protein-based pharmaceutical compositions for biopharmaceutical applications involves several steps. Certain parameters of some manufacturing steps can influence the properties or functionalities of the proteins.

[0005] To date, there is a need to develop pharmaceutical compositions of botulinum toxins with improved properties.

[0006] The citation of a reference herein is not to be construed as an admission that it is a field of art which corresponds to the prior art of the present disclosure. 3. SUMMARY OF THE INVENTION

[0007] The present disclosure provides novel solid compositions comprising Clostridium botulinum neurotoxin serotype A (BoNT / A) having advantageous properties as well as novel methods for preparing such compositions.

[0008] More specifically, the present disclosure provides 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 disclosure also provides methods for preparing solid compositions comprising BoNT / A and HSA, the methods comprising a step of drying a solution comprising BoNT / A and HSA to prepare the solid composition, wherein the solution does not freeze during the drying step. The present disclosure further provides solid compositions prepared by such a method.

[0009] HSA is used as an excipient in therapeutic protein formulations to improve stability and resistance to external stresses, for example, by preventing aggregation, oxidation, and surface adsorption. HSA serves as a particularly important stabilizer for the formulation of BOTOX. ®with low fill volume and high potency, as it can reduce the unfolding and aggregation of BoNT / A due in part to its surface-covering property. Without wishing to be bound by any particular theory, the present disclosure describes that freezing during drying unexpectedly results in 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 taken to avoid freezing during vacuum drying of BoNT / A compositions, and that solid BoNT / A compositions dried without freezing exhibit advantageous properties such as consistent visual product quality and a much slower potency decay during storage, e.g., at room temperature or higher.Accordingly, the present invention provides novel solid compositions comprising BoNT / A and a small amount of HSA aggregates, as well as manufacturing methods with an improved drying process to obtain such solid compositions.

[0010] In one aspect, a solid composition comprising BoNT / A and HSA is provided herein, wherein the percentage of HSA aggregates, HSA polymer, HSA oligomer, or HSA dimer in soluble HSA contained in the solid composition is reduced. Specifically, a solid composition comprising a 900 kDa BoNT / A complex, HSA, and sodium chloride (NaCl), wherein the HSA comprises 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.

[0011] Also provided herein is a method for preparing a solid composition comprising a 900 kDa BoNT / A complex, HSA, and NaCl, the method comprising a step of drying a solution comprising BoNT / A, HSA, and NaCl to prepare the solid composition, wherein the solution does not freeze during the drying step.

[0012] In a further aspect, there is provided herein a solid composition prepared by a process described herein.

[0013] In another aspect, there is provided herein a method of treating a patient in need comprising administering a composition as described herein. 4. BRIEF DESCRIPTION OF THE CHARACTERS Fig. 1. A copy of a portion of the Certificate of Analysis for an HSA batch, CSL Bering. Fig.Figure 2A. Representative data from the vacuum drying cycle. The temperature of the product (Batch No. 4) was measured using Ellab temperature probes. Fig. Figure 2B. Representative data on freeze-drying cycles. The temperature of the product (Batch No. 9) was measured using Ellab temperature probes. Fig. 3A-3C. Representative SEC curves for HSA, recorded with different detection methods: Fluorescence (excitation 280 nm / emission 350 nm) ( Fig. 3A), UV 280 nm ( Fig. 3B) and UV 220 nm ( Fig. 3C). Fig.Figure 4A. Representative SEC curves for reconstituted products prepared by vacuum drying (Process 1), freeze-drying (Process 2), and freeze-drying with holding before and after filtration (Process 3) (UV 220 nm detector). The inset shows a graph of the percentage aggregation in the form of HSA polymer (left bars) and HSA dimer (right bars) in three drug product samples (Batch Nos. 7, 9, and 4) and in the "neat" HSA (Batch No. 1). Fig. Figure 4B. Representative SEC curves for reconstituted products prepared by vacuum drying (Process 1), freeze-drying (Process 2), and freeze-drying with holding before and after filtration (Process 3) (UV 280 nm detector). The inset shows a plot of the percentage aggregation in the form of HSA polymer (left bars) and HSA dimer (right bars) in three drug product samples (Batch Nos. 7, 9, and 4) and in the "neat" HSA (Batch No. 1). Fig.Figure 4C. Representative SEC curves for reconstituted products prepared by vacuum drying (Process 1), freeze-drying (Process 2), and freeze-drying with holding before and after filtration (Process 3) (fluorescence detector). The inset shows a graph of the percentage aggregation of HSA polymer (left bars), HSA oligomer (middle bars), and HSA dimer (right bars) in three drug product samples (Batches 7, 9, and 4) and the "neat" HSA (Batch 1). Fig. 5A-5G. Total HSA aggregation in the drug product (DP) batches manufactured using three production processes (vacuum-dried (i.e., "baseline, no hold, no freeze"), freeze-dried (i.e., "stress, freeze only"), and freeze-dried with solution hold prior to drying (i.e., "stress, hold during RT, and freeze"), determined by SEC. Data from the pure HSA batches are also included. Fig. 5A, Fig. 5C and Fig. 5E shows diagrams for UV-220 nm detector, UV-280 nm detector and fluorescence detector, respectively. Fig. 5B, Fig. 5D and Fig. 5F shows the corresponding data values for the UV-220 nm detector, UV-280 nm detector, and fluorescence detector. Data for specific batches are shown in Fig. 5G shown. Fig. 6A-6B. Particle count results for three placebo batches produced using different manufacturing processes. Fig. Figure 6A: Shown is the mean number of particles per image. Saline (left, n=1), baseline (i.e., vacuum-dried, second from left, n=5, batch 6), stressed (room temperature and freezing) (second from right, n=4, batch 11), stressed (freezing only) (right, n=4, batch 8). Fig.Figure 6B: Particle size distributions (average results) are shown. Saline, baseline (i.e., vacuum-dried, Batch No. 6), stressed (room temperature holding and freezing) (Batch No. 11), and stressed (freezing only) (Batch No. 8). Fig. 7A. pH and temperature changes during freezing and evaporative drying of the placebo solution. The blue arrow in this graph indicates the temperature of NaCl*2H2O+water crystallization, as demonstrated in a separate WAXS experiment. Fig. 7B. pH changes during evaporation and drying of the placebo solution. Fig. 8A. WAXS pattern during cooling of an aqueous 0.9% NaCl / 0.5% HSA placebo solution. Arrow: curve of NaCl*2H2O. Fig. 8B. WAXS pattern during cooling of aqueous 0.9% NaCl placebo solution. Fig. 8C. SAXS pattern during cooling of 0.9% NaCl / 0.5% HSA solution. Fig.9. Representative SEM images obtained at two magnifications. Dark areas represent HSA, and lighter areas correspond to NaCl or NaCl with HSA. From left to right: vacuum-dried (Process 1), freeze-dried (Process 2), and freeze-dried with holding solution before drying (Process 3). Fig. 10. X-ray diffraction patterns of dried placebo batches. Lower curve: vacuum-dried (Process 1, Batch No. 6); upper curve: freeze-dried with hold before drying (Process 3, No. 11). Peak positions of the placebo batches and NaCl (USP, JT Baker, Batch 0000207378) are provided in the inset. Fig. 11. Potency stability data tested at storage conditions of 25°C. Upper curve in each view: Process 1, vacuum-dried. Lower curve in each view: Process 3, freeze-dried plus hold. Top view: merged view. Bottom view: split view. 5. DETAILED DESCRIPTION

[0014] The present invention provides solid compositions comprising BoNT / A and HSA with improved properties, as well as manufacturing methods with an improved drying process for obtaining such solid compositions. Further advantages of the present disclosure will be apparent to those skilled in the art. The embodiments and aspects described in this disclosure are illustrative of the invention and are not intended to limit the scope of the invention. 5.1. Definitions

[0015] The singular forms “ein”, “eine”, “ein” and “der”, “die”, “das” include the plural unless the context clearly indicates otherwise.

[0016] The term "and / or" as used herein in a phrase such as "A and / or B" shall mean "A and B", "A or B", "A" or "B".

[0017] The terms "approximately" and "about" generally refer to a range of numbers that one of ordinary skill in the art would consider equivalent to the stated value (i.e., having the same function or result). In many cases, the terms "approximately" and "about" may include numbers rounded to the nearest significant figure. In specific embodiments, the terms "approximately" and "about" are intended to be interpreted to allow for what one of ordinary skill in the art would consider a normal deviation, such as a deviation within 20%, 10%, or 5%. In specific embodiments, the terms "approximately" and "about" encompass the exact value stated.

[0018] "Animal product-free" ("APF") or "substantially animal product-free" encompasses the absence or substantial absence of blood-derived, blood-pooled, 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 a botulinum toxin or a Clostridium botulinum bacterium. For example, an APF process or a substantially APF process means a process that is either substantially free, predominantly free, or entirely free of animal-derived proteins, such as immunoglobulins, meat digests, meat by-products, and milk or dairy products or digests.

[0019] "Clostridium botulinum neurotoxin serotype A" or "BoNT / A" refers to a neurotoxin produced by Clostridium botulinum type A strains. One such Clostridium botulinum type A strain is the type A Hall strain, for example, the type A Hall (Allergan) strain. Zhang et al. (2003) Gene 315:21, incorporated herein by reference in its entirety. BoNT / A includes both a BoNT / A complex (e.g., the 300, 500, 760, and 900 kDa complexes) and pure BoNT / A toxin (i.e., the neurotoxic molecule of approximately 150 kDa).

[0020] "BoNT / A complexes" means Clostridium botulinum serotype A neurotoxin complexes comprising a BoNT / A molecule (the neurotoxic component) and one or more hemagglutinin (HA) proteins and / or non-toxin non-hemagglutinin (NTNH) proteins. The BoNT / A complexes may be in the form of, for example, about 900 kDa, 760 kDa, 500 kDa, or 300 kDa. In one embodiment, the BoNT / A complex is in the form of about 900 kDa, comprising a BoNT / A molecule of about 150 kDa, hemagglutinin HA70, hemagglutinin HA34, hemagglutinin HA17, and non-toxic non-hemagglutinin (NTNH) proteins. In one embodiment, the BoNT / A complex is a substantially complete form of the 900 kDa BoNT / A complex. In one embodiment, the BoNT / A complex is onabotulinumtoxinA.

[0021] "150 kDa Clostridium botulinum serotype A neurotoxin" or "150 kDa BoNT / A" means a neurotoxin of approximately 150 kDa produced from a culture of Clostridium botulinum type A strain (e.g., the Hall strain of Clostridium botulinum). The preferred sequences of 150 kDa botulinum toxin type A (BoNT / A) used in the context of the present disclosure are listed in Table 1. In one embodiment, the 150 kDa BoNT / A used in the context of the present disclosure comprises, for example, (e.g., consists of) a light chain having an amino acid sequence set forth in SEQ ID NO. 2 and a heavy chain having an amino acid sequence set forth in SEQ ID NO. 3, wherein the disulfide bridges are located between positions 429 and 453 and between positions 1234 and 1279.

[0022] The term "BoNT / A composition" refers to any composition comprising BoNT / A and includes both solid and liquid compositions. In certain embodiments, a BoNT / A composition described herein (e.g., a solid composition or a liquid composition) is a pharmaceutical composition. In certain embodiments, a BoNT / A composition described herein (e.g., a solid composition or a liquid composition) is a medicament (e.g., a finished dosage form). In a preferred embodiment, a BoNT / A composition described herein is in powder form (e.g., vacuum-dried powder).

[0023] The term “carrier” used in connection with a pharmaceutical excipient refers to all solvents, dispersants, preservatives, coatings, isotonic and absorption-delaying agents and the like that are compatible with pharmaceutical administration.

[0024] The terms “patient,” “subject,” “individual,” and the like refer to human beings.

[0025] “Pharmaceutical composition” means a formulation in which an active ingredient may be a BoNT / A. The word “formulation” means that in addition to a BoNT / A active ingredient, at least one additional active ingredient (such as, but not limited to, an albumin (such as human serum albumin (HSA) or recombinant human albumin) and / or sodium chloride) is present in the pharmaceutical composition. The human serum albumin excipient may be derived from human plasma or produced recombinantly. A pharmaceutical composition is therefore a formulation suitable for diagnostic, therapeutic, and / or cosmetic administration (e.g., by intramuscular or subcutaneous injection, or by insertion of a depot or implant) to a subject, such as a human patient. In one embodiment, the active ingredient is onabotulinumtoxinA.Exemplary methods for formulating a pharmaceutical composition containing BoNT / A active ingredient are disclosed in U.S. Patent Application No. 2003 / 0118598, filed November 5, 2002, which is incorporated herein by reference in its entirety. In a preferred embodiment, a pharmaceutical composition described herein is in a dried form (e.g., a vacuum-dried form). The pharmaceutical compositions can be vacuum-dried and are suitable for administration by injection, either subcutaneously or intramuscularly, 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 HSA and 0.9 mg sodium chloride per 100 units of BoNT / A. Most preferably, such pharmaceutical compositions comprise 50, 100 or 200 units of BoNT / A.

[0026] “Unit” or “U” refers to the LD 50 -dose or the dose determined by a cell-based potency assay (CBPA). The LD 50 Dose is defined as the amount of BoNT / A that kills 50% of the mice injected with BoNT / A. The CBPA dose is determined as described in U.S. Patent Nos. 8,618,261; 8,198,034; 9,249,216; 10,703,806; 11,261,240; and 11,332,518; the assay details of which are incorporated herein by reference.

[0027] Unless the context requires otherwise, the terms "comprise," "comprises," and "comprising" are used on the basis and with the clear understanding that they are to be interpreted inclusively, not exclusively, so as to indicate the inclusion of the stated feature without, however, excluding one or more other such features. However, it is to be understood that wherever aspects and embodiments are described herein using the phrase "comprise" (or "comprises" or "comprising"), otherwise analogous aspects described using terms such as "consist of" (or "consists of" or "consisting of") and / or "consist mainly of" (or "consists mainly of" or "consisting mainly of") are also provided. 5.2. Clostridium botulinum neurotoxin serotype A (BoNT / A)

[0028] Clostridial bacteria can produce botulinum toxin type A complexes in various forms, including, but not limited to, 900 kDa, 760 kDa, 500 kDa, and 300 kDa complexes (approximate molecular weights).

[0029] In one embodiment, the BoNT / A described herein is in the form of a 900 kDa BoNT / A complex. In one embodiment, the BoNT / A described herein is in the form of a 900 kDa BoNT / A complex made from the 150 kDa BoNT / A molecule and the proteins hemagglutinin HA70, hemagglutinin HA34, hemagglutinin HA17, and non-toxic non-hemagglutinin (NTNH). In a specific embodiment, the BoNT / A described herein (e.g., the 900 kDa BoNT / A complex) is produced in a Clostridium botulinum type A strain. In a specific embodiment, the BoNT / A described herein (e.g., the 900 kDa BoNT / A complex) is produced in a Clostridium botulinum type A Hall strain. In a preferred embodiment, the BoNT / A described herein is onabotulinumtoxinA.

[0030] In one embodiment, the 150 kDa BoNT / A molecule useful in the present disclosure has the sequence shown in Table 1. In one embodiment, the 150 kDa BoNT / A molecule comprises a light chain (LC: residues 2-438, approximately 50 kDa) and a heavy chain (HC: residues 449-1296, approximately 100 kDa). Residues 439-448 are the nick site and are in italics. 5.3. Production of BoNT / A and production of BoNT / A compositions

[0031] Botulinum toxin type A has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of essential blepharospasm, strabismus, and hemifacial spasm in patients over the age of 12, cervical dystonia, glabellar lines (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) and is commercially available from Allergan, an AbbVie company, North Chicago, Illinois, USA. BOTOX ® Contains a purified 900 kDa botulinum toxin type A complex, human serum albumin, and sodium chloride, packaged in a sterile, vacuum-dried container. 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., non-APF process). It is purified from the culture broth through a series of precipitation steps (including acid precipitation) to yield a crystalline complex consisting of the active high-molecular-weight toxin protein and an associated hemagglutinin protein. The crystalline complex is redissolved in a solution containing saline and albumin and sterile filtered through a gamma-irradiated filter (0.2 micron) prior to vacuum drying. BOTOX ® can be reconstituted with sterile, non-preserved saline prior to intramuscular injection. Each vial contains 100 units of BOTOX ®consists of approximately 5 ng of purified botulinum toxin type A complex, 0.5 mg of human serum albumin and 0.9 mg of sodium chloride in vacuum-dried form and is intended for reconstitution with sterile normal saline solution without preservatives (0.9% sodium chloride injection).

[0032] Generally, the preparation of a BoNT / A pharmaceutical composition involves first preparing the BoNT / A drug substance and then mixing the BoNT / A drug substance with one or more excipients. At the end of the manufacturing process, a drying step is often performed to produce a dried form of the pharmaceutical composition that can be more easily stored and / or transported. This disclosure describes steps and measures that can be taken to ensure that the drug product does not freeze during the drying process, as freezing during drying unexpectedly leads to a significant increase in HSA aggregation in BoNT / A compositions.

[0033] Several steps are required to produce BoNT / A drugs (i.e., to obtain purified BoNT / A). A first step may involve culturing a clostridial bacterium (e.g., the Hall strain of Clostridium botulinum), usually on agar plates in an environment conducive to bacterial growth, such as a warm anaerobic atmosphere. This culture step allows for the obtaining of clostridial colonies with the desired morphology and other properties. The culture step can also be performed with bacteria from a bioproduct-free working cell bank. In a second step, selected cultured clostridial colonies can be fermented in a suitable medium. After a period of fermentation, the clostridia typically lyse and release clostridial toxin (e.g., BoNT / A) into the medium.Third, the toxin can be purified from the culture medium to yield a bulk or crude BoNT / A toxin drug compound. Preferably, the BoNT / A toxin drug compound will not have been subjected to precipitation (e.g., precipitation with cold ethanol, hydrochloric acid, and / or ammonium sulfate) during purification. In certain aspects of the invention, the BoNT / A toxin drug compound 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 the BoNT / A, the toxin is purified using a process that utilizes an HIC column prior to any other chromatography columns.The BoNT / A drug substance should preferably be manufactured using a process that can be used in accordance with the Good Manufacturing Practice regulations issued by the U.S. Food and Drug Administration.

[0034] In some embodiments, the BoNT / A drug is determined using a substantially, predominantly, or entirely animal protein-free (APF) process. The process may include APF or substantially APF culture and fermentation processes. An APF or substantially APF chromatography system and process may be used to purify a clarified culture of Clostridium botulinum obtained from the APF or substantially APF culture and fermentation process. In some embodiments, the chromatography-based purification process is as disclosed in U.S. Pat. No. 8,129,139, which is incorporated herein by reference in its entirety.

[0035] The BoNT / A drug can be prepared by exemplary methods as described in Example 1 of U.S. Pat. No. 8,129,139, Example 2 of U.S. Pat. No. 8,129,139, or Example 1 of U.S. Pat. No. 9,469,849 (which is incorporated herein by reference in its entirety), or using one or more steps as described in one or more of these examples.

[0036] In certain embodiments, the BoNT / A drug is produced by a process that includes one or more column chromatography steps (e.g., one or more column chromatography steps performed during the purification of the BoNT / A drug).

[0037] In preferred embodiments, the one or more column chromatography steps (e.g., performed during purification of the BoNT / A drug) comprise hydrophobic interaction chromatography, anion exchange chromatography, and / or cation exchange chromatography, preferably hydrophobic interaction chromatography being performed before anion exchange chromatography and / or cation exchange chromatography.

[0038] In certain embodiments, the BoNT / A drug substance is prepared by a process that does not include a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate (e.g., during purification of the BoNT / A drug substance).

[0039] In certain embodiments, the BoNT / A drug substance is prepared by a process that does not utilize a protease inhibitor. In certain embodiments, the BoNT / A drug substance is prepared by a process that does not utilize benzamidine hydrochloride.

[0040] After preparing the BoNT / A drug substance, it can be stabilized in a suitable solution. The BoNT / A drug substance can be combined with one or more excipients (e.g., human serum albumin such as recombinant human serum albumin and sodium chloride) and further sterile filtered to produce a pharmaceutical composition suitable for administration to a human. The pharmaceutical compositions can be converted into a solid form (e.g., powder) by drying (e.g., vacuum drying). Solid pharmaceutical compositions can be stored and reconstituted prior to injection. The BoNT / A pharmaceutical compositions described herein can comprise a 900 kDa BoNT / A complex as the active pharmaceutical ingredient. The pharmaceutical composition can also include one or more excipients, buffers, carriers, stabilizers, preservatives, and / or fillers.Such pharmaceutical compositions are preferably chemically and physically stable, so that the BoNT / A active pharmaceutical ingredients can continue to be used as a pharmaceutical product after storage. BoNT / A products can be stored at room temperature, refrigerated, or below 0°C. Preferably, the BoNT / A remains stable during storage for at least about 12 months, preferably at least about 18 months.

[0041] In various aspects and embodiments, a solid BoNT / A composition described herein is dried from a solution comprising a BoNT / A drug (e.g., a BoNT / A drug prepared by a method described herein) and HSA. More specifically, the present invention relates to a method for preparing a solid composition comprising BoNT / A and HSA, the method comprising a step of drying a solution comprising BoNT / A and HSA to prepare the solid composition, wherein the solution does not freeze during the drying step. In specific embodiments, the method produces a solid composition described herein.

[0042] Whether a solution (or reference solution) described herein freezes during drying can be determined by monitoring its temperature during drying (for example, as described in Example 1). Monitoring can be performed by inserting a temperature probe into the solution (or reference solution). Monitoring can be performed using a Pirani meter or an Ellab temperature probe. If multiple vials of the solution (or reference solution) are dried together (e.g., in the same drying chamber or on the same drying tray), monitoring can be performed for one or more (e.g., one or two) representative vials and need not be performed for all vials. In certain embodiments, a drying process in which a solution does not freeze comprises maintaining the temperature of the solution above its freezing point during drying.In certain embodiments, a drying process in which a solution does not freeze comprises maintaining the temperature of the solution above 0°C during drying.

[0043] Whether a solution (or reference solution) described herein freezes during drying can also be determined by examining the dried solid composition (or dried solid reference composition) using a scanning electron microscope (SEM) (for example, as described in Example 1). If multiple vials of the solution (or reference solution) are dried together (e.g., in the same drying chamber or on the same drying tray), the examination can be performed for one or more (e.g., one or two) representative vials of the dried solution composition and need not be performed for all vials. In certain embodiments, the absence of pores in a dried solid composition indicates that it was dried from a solution that did not freeze during drying.

[0044] In various embodiments and aspects, the drying step is at least partially controlled manually. In various embodiments and aspects, the drying step is fully controlled manually. In various embodiments and aspects, the drying step is at least partially controlled digitally. In various embodiments and aspects, the drying step is fully controlled digitally.

[0045] In various embodiments and aspects, the drying step is vacuum drying, spray drying, convection drying, microwave drying, or a combination thereof.

[0046] In various embodiments and aspects, the drying step is vacuum drying. To prevent freezing of the drug solution, a controlled depressurization / evacuation phase may be performed. Measures that may be taken during the controlled depressurization / evacuation phase to prevent freezing include: using a slower chamber depressurization rate, performing depressurization in staggered steps, monitoring the drug solution (for example, to ensure it is a certain number of degrees above freezing), and / or using a high storage temperature (for example, setting the storage temperature to approximately 20°C to 25°C). The exact chamber depressurization rates and steps may depend on the drying equipment, chamber size, condenser capacity, chamber load, and / or vacuum pump power, etc.depend on and can be determined by those skilled in the art and may require manual adjustment. In certain embodiments, the vacuum drying step can, for example, comprise a stepwise evacuation step, which can be carried out such that the temperature of a solution described herein can decrease under evacuation until it reaches a temperature that is still 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 evacuation is stopped until the temperature of the solution rises again to a higher temperature (e.g., about 20°C to 25°C), and then the evacuation is resumed. The aforementioned step can be repeated one or more times, whereby the exact temperature at which the evacuation is interrupted or resumed need not be the same for each repetition.In certain other embodiments, the vacuum drying step may include a slow evacuation step. 5.4. BoNT / A compositions

[0047] The present disclosure provides 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.

[0048] HSA contained in a BoNT / A composition can be in the form of HSA monomer or HSA aggregates. There are two types of HSA aggregates: soluble HSA aggregates and insoluble HSA aggregates. Soluble HSA aggregates can be in the form of HSA polymer, HSA oligomer, HSA dimer, or a mixture thereof. In certain embodiments, soluble HSA polymer, soluble HSA oligomer, soluble HSA dimer, and soluble HSA monomer can be separated by size exclusion chromatography (SEC). For example, peaks on an SEC chromatogram can be assigned to soluble HSA polymer, soluble HSA oligomer, soluble HSA dimer, and soluble HSA monomer, respectively, based on retention times obtained when performing a standard control (e.g.,a gel filtration standard consisting of bovine thyroglobulin (MW 670 kDa), bovine γ-globulin (MW 158 kDa), chicken albumin (MW 44 kDa), horse myoglobulin (MW 17 kDa), and vitamin B12 (MW 1350 Da), such as the Biorad gel filtration standard (Cat 151-1901)) on the same column. In a specific embodiment, peaks on an SEC chromatogram can be assigned to soluble HSA polymer, soluble HSA oligomer, soluble HSA dimer, and soluble HSA monomer, respectively, as described in Example 1.

[0049] 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 with sedimentation velocity (SV-AUC)), asymmetric fast field flow fractionation (FFF or AF4), and turbidity measurements.

[0050] Suitable methods for measuring the level of HSA aggregates, polymer, oligomer, or dimer in soluble HSA include HPLC (e.g., SEC), DLS, gel electrophoresis (e.g., SDS-PAGE), ultracentrifugation (e.g., AUC such as SV-AUC), asymmetric fast field flow fractionation (FFF or AF4), and turbidity measurements.

[0051] 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 fast field flow fractionation (FFF or AF4), microscopy (e.g., optical microscopy), microflow imaging (MFI), turbidity measurements, light turbidity (e.g., by HIAC (high-precision liquid particle counter)), and subvisual particle detection (such that the percentage of insoluble HSA aggregates equals or is represented by the percentage of subvisual particles) (for example, a non-invasive subvisual particle detection method described in Example 1 or in U.S. Patent No. 10,132,736 B2 which is hereby incorporated by reference in its entirety, optical microscopy, MFI or light shadowing (e.g.B. by HIAC)).

[0052] In certain embodiments, the percentage of HSA aggregates or the percentage of one type of HSA aggregates in a solid composition described herein is determined after at least a portion of the solid composition has been reconstituted. 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 about 0.01 mg / ml to about 100 mg / ml, about 0.1 mg / ml to about 10 mg / ml, about 0.25 mg / ml to 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 certain embodiments, the percentage of HSA aggregates or the percentage of one type of HSA aggregates in a solid composition described herein is determined using SEC after at least a portion of the solid composition has been reconstituted. Detectors that can be used to detect soluble HSA aggregates (e.g., using SEC) 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).

[0053] The present invention provides solid compositions comprising BoNT / A and HSA, wherein the solid compositions comprise a low percentage of HSA aggregates including HSA polymer, oligomer, and / or dimer. As shown in Example 1, a BoNT / A composition dried without freeze-dried not only has a low overall percentage of HSA aggregates, but also low percentages each of HSA polymer, oligomer, and dimer.

[0054] 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 soluble HSA contained in the solid composition is determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted. 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 soluble HSA contained in the solid composition is determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.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 soluble HSA contained in the solid composition is determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.

[0055] In one aspect, provided herein is a solid composition comprising BoNT / A and HSA, wherein the HSA comprises 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. In this disclosure, it is understood 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 the soluble HSA included 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 the soluble HSA included in the solid composition is HSA aggregates.

[0056] In specific embodiments, less than 19% of the soluble HSA contained in the solid composition is HSA aggregates, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted. 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 the soluble HSA contained in the solid composition is HSA aggregates, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA aggregates, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.

[0057] In specific embodiments, less than 34% of the soluble HSA contained in the solid composition is HSA aggregates, as determined using SEC with a UV detector having a wavelength of 280 nm, after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA aggregates, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion the solid composition was reconstituted.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 the soluble HSA contained in the solid composition is HSA aggregates, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.

[0058] In specific embodiments, less than 20% of the soluble HSA contained in the solid composition is HSA aggregates, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA aggregates, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA aggregates, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.

[0059] In another aspect, there is provided herein a solid composition comprising 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, wherein less than 30% of the soluble HSA contained in the solid composition is HSA polymer.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 the soluble HSA included in the solid composition is 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 the soluble HSA included in the solid composition is HSA polymer.

[0060] In specific embodiments, less than 13% of the soluble HSA contained in the solid composition is HSA polymer, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted. 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 the soluble HSA contained in the solid composition is HSA polymer, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA included in the solid composition is HSA polymer, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.

[0061] In specific embodiments, less than 30% of the soluble HSA contained in the solid composition is HSA polymer, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA polymer, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA included in the solid composition is HSA polymer, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.

[0062] In specific embodiments, less than 13% of the soluble HSA contained in the solid composition is HSA polymer, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted. 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 the soluble HSA contained in the solid composition is HSA polymer, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA included in the solid composition is HSA polymer, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.

[0063] In another aspect, a solid composition comprising BoNT / A and HSA is provided herein, 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, wherein less than 0.8% of the soluble HSA contained in the solid composition is 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 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 the soluble HSA included in the solid composition is HSA oligomer.

[0064] In specific embodiments, less than 0.8% of the soluble HSA contained in the solid composition is HSA oligomer, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted. 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 HSA oligomer, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA oligomer, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.

[0065] In specific embodiments, less than 0.8% of the soluble HSA contained in the solid composition is HSA oligomer, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted. 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 HSA oligomer, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA oligomer, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.

[0066] In specific embodiments, less than 0.8% of the soluble HSA contained in the solid composition is HSA oligomer, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted. 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 HSA oligomer, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA oligomer, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.

[0067] In another aspect, a solid composition comprising BoNT / A and HSA is provided herein, 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, wherein less than 4.8% of the soluble HSA contained in the solid composition is 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 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 the soluble HSA included in the solid composition is HSA dimer.

[0068] In specific embodiments, less than 4.6% of the soluble HSA contained in the solid composition is HSA dimer, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted. 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 HSA dimer, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA dimer, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.

[0069] In specific embodiments, less than 3.7% of the soluble HSA contained in the solid composition is HSA dimer, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted. 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 the soluble HSA contained in the solid composition is HSA dimer, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA contained in the solid composition is HSA dimer, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.

[0070] In specific embodiments, less than 4.8% of the soluble HSA contained in the solid composition is HSA dimer, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted. 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 HSA dimer, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.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 the soluble HSA included in the solid composition is HSA dimer, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.

[0071] The present invention also provides a solid composition comprising BoNT / A and HSA, wherein the HSA is incorporated from a starting material into the solid composition. As shown in Example 1, a BoNT / A composition dried without freeze-drying not only exhibits a smaller increase in the total percentage of HSA aggregates over the HSA starting material, but also a smaller increase in the percentages of HSA polymer, oligomer, and dimer over the HSA starting material, compared to a BoNT / A composition dried using a process involving freezing. In certain embodiments, the starting material is a commercially available HSA product. In some embodiments, the starting material is in liquid form (e.g., in the form of a 25% solution). In other embodiments, the starting material is in solid form.

[0072] The percentage of HSA aggregates, polymer, oligomer, or dimer in soluble HSA contained in a solid composition described herein and the percentage of HSA aggregates, polymer, oligomer, or dimer in soluble HSA contained in the corresponding starting material are preferably determined using the same method. When the HSA starting material is in solid form, in certain embodiments, the percentage of HSA aggregates, polymer, oligomer, or dimer in soluble HSA contained in the starting material is determined after at least a portion of the starting material has been reconstituted, and the reconstitution can be performed as described above.

[0073] In one aspect, there is provided herein a solid composition comprising BoNT / A and HSA, wherein the HSA is incorporated into the solid composition from a starting material and comprises 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 soluble HSA contained in the solid composition is no more than 6.8 times the percentage of HSA aggregates in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is not more than 6.5 times, not more than 6 times, not more than 5.5 times, not more than 5 times, not more than 4.5 times, not more than 4 times, not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times, not more than 1.6 times, not more than 1.5 times, not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage of HSA aggregates in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA aggregates in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, not more than 3.5 to 4 times, not more than 4 to 4.5 times, not more than 4.5 up to 5 times, not more than 5 to 5.5 times, not more than 5.5 to 6 times, not more than 6 to 6.5 times, or not more than 6.5 to 6.8 times the percentage of HSA aggregates in soluble HSA contained in the starting material.

[0074] In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is no more than 4.9 times the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is not more than 4.5 times, not more than 4 times, not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times, not more than 1.6 times, not more than 1.5 times, not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a UV detector with a wavelength of 220 nm after at least part of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, not more than 3.5 to 4 times, not more than 4 to 4.5 times, or not more than 4.5 to 4.9 times the percentage of HSA aggregates in soluble HSA contained in the starting material as determined using SEC through a UV detector with a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.

[0075] In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is no more than 6.8 times the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted. In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is not more than 6.5 times, not more than 6 times, not more than 5.5 times, not more than 5 times, not more than 4.5 times, not more than 4 times, not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times,not more than 1.6 times, not more than 1.5 times, not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted. In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted. In specific embodiments, the percentage of HSA aggregates in soluble HSA iswhich is contained in the solid composition, not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, not more than 3.5 to 4 times, not more than 4 to 4.5 times, not more than 4.5 to 5 times, not more than 5 to 5.5 times, not more than 5.5 to 6 times, not more than 6 to 6.5 times, or not more than 6.5 to 6.8 times the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a UV detector with a wavelength of 280 nm,after at least part of the solid composition has been reconstituted.

[0076] In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is no more than 1.9 times the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is no more than 1.8 times, no more than 1.7 times, no more than 1.6 times, no more than 1.5 times, no more than 1.4 times, no more than 1.3 times, no more than 1.2 times, or no more than 1.1 times the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA aggregates in soluble HSA contained in the solid composition is no more than 1.1 to 1.3 times, no more than 1.3 to 1.5 times, no more than 1.5 to 1.7 times, or no more than 1.7 to 1.9 times the percentage of HSA aggregates in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.

[0077] In another aspect, there is provided herein a solid composition comprising BoNT / A and HSA, wherein the HSA is incorporated 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 soluble HSA contained in the solid composition is no more than 6.4 times the percentage of HSA polymer in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is not more than 6 times, not more than 5.5 times, not more than 5 times, not more than 4.5 times, not more than 4 times, not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times, not more than 1.6 times, not more than 1.5 times, not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage Proportion of HSA polymer in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA polymer in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, not more than 3.5 to 4 times, not more than 4 to 4.5 times, not more than 4.5 to 5 times, not more than 5 to 5.5 times, not more than 5.5 to 6 times, or not more than 6 to 6.4 times the percentage of HSA polymer in soluble HSA contained in the starting material.

[0078] In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is no more than 5.2 times the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In certain embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is not more than 5 times, not more than 4.5 times, not more than 4 times, not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times, not more than 1.6 times, not more than 1.5 times, not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage of HSA polymer in soluble HSA contained in the starting material, such as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, not more than 3.5 to 4 times, not more than 4 to 4.5 times, not more than 4.5 to 5 times, or not more than 5 to 5.2 times the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.

[0079] In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is no more than 6.4 times the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted. In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is not more than 6 times, not more than 5.5 times, not more than 5 times, not more than 4.5 times, not more than 4 times, not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times, not more than 1.6 times, not more than 1.5 times,not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted. In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted. In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, not more than 3.5 to 4 times, not more than 4 to 4.5 times, not more than 4.5 to 5 times, not more than 5 to 5.5 times, not more than 5.5 to 6 times, or not more than 6 to 6.4 times the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.

[0080] In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is no more than 1.6 times the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is no more than 1.5 times, no more than 1.4 times, no more than 1.3 times, no more than 1.2 times, or no more than 1.1 times the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA polymer in soluble HSA contained in the solid composition is no more than 1.1 to 1.3 times, no more than 1.3 to 1.5 times, or no more than 1.5 to 1.6 times the percentage of HSA polymer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.

[0081] In another aspect, there is provided herein a solid composition comprising BoNT / A and HSA, wherein the HSA is incorporated 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 soluble HSA contained in the solid composition is no more than 3.9 times the percentage of HSA oligomer in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is no more than 3.5 times, no more than 3 times, no more than 2.5 times, no more than 2 times, no more than 1.9 times, no more than 1.8 times, no more than 1.7 times, no more than 1.6 times, no more than 1.5 times, no more than 1.4 times, no more than 1.3 times, no more than 1.2 times, or no more than 1.1 times the percentage of HSA oligomer in soluble HSA contained in the starting material. In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA oligomer in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, or not more than 3.5 to 3.9 times the percentage of HSA oligomer in soluble HSA contained in the starting material.

[0082] In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is no more than 3.9 times the percentage of HSA oligomer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times, not more than 1.6 times, not more than 1.5 times, not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage of HSA oligomer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition was reconstituted.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA oligomer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, or not more than 3.5 to 3.9 times the Percentage of HSA oligomer in soluble HSA contained in the starting material as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.

[0083] In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is no more than 3.9 times the percentage of HSA oligomer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times, not more than 1.6 times, not more than 1.5 times, not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage of HSA oligomer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition was reconstituted.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA oligomer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, or not more than 3.5 to 3.9 times the Percentage of HSA oligomer in soluble HSA contained in the starting material as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.

[0084] In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is no more than 3.9 times the percentage of HSA oligomer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times, not more than 1.6 times, not more than 1.5 times, not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage of HSA oligomer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least part of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA oligomer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA oligomer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, or not more than 3.5 to 3.9 times the Percentage of HSA oligomer in soluble HSA contained in the starting material as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.

[0085] In another aspect, there is provided herein a solid composition comprising BoNT / A and HSA, wherein the HSA is incorporated 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 soluble HSA contained in the solid composition is no more than 4.6 times the percentage of HSA dimer in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is no more than 4.5 times, no more than 4 times, no more than 3.5 times, no more than 3 times, no more than 2.5 times, no more than 2 times, no more than 1.9 times, no more than 1.8 times, no more than 1.7 times, no more than 1.6 times, no more than 1.5 times, no more than 1.4 times, no more than 1.3 times, no more than 1.2 times, or no more than 1.1 times the percentage of HSA dimer in soluble HSA contained in the starting material. In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA dimer in soluble HSA contained in the starting material.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, not more than 3.5 to 4 times, not more than 4 to 4.5 times or not more than 4.5 to 4.6 times the percentage of HSA dimer in soluble HSA contained in the starting material.

[0086] In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is no more than 3.5 times the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is no more than 3 times, no more than 2.5 times, no more than 2 times, no more than 1.9 times, no more than 1.8 times, no more than 1.7 times, no more than 1.6 times, no more than 1.5 times, no more than 1.4 times, no more than 1.3 times, no more than 1.2 times, or no more than 1.1 times the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, or not more than 3 to 3.5 times the percentage of HSA dimer in soluble HSA contained in the starting material as determined using SEC with a UV detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted.

[0087] In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is no more than 4.6 times the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is not more than 4.5 times, not more than 4 times, not more than 3.5 times, not more than 3 times, not more than 2.5 times, not more than 2 times, not more than 1.9 times, not more than 1.8 times, not more than 1.7 times, not more than 1.6 times, not more than 1.5 times, not more than 1.4 times, not more than 1.3 times, not more than 1.2 times, or not more than 1.1 times the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a UV detector with a wavelength of 280 nm after at least part of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, not more than 2.5 to 2.6 times, not more than 2.5 to 3 times, not more than 3 to 3.5 times, not more than 3.5 to 4 times, not more than 4 to 4.5 times or not more than 4.5 to 4.6 times the percentage of HSA dimer in soluble HSA contained in the starting material as determined using SEC with a UV detector having a wavelength of 280 nm after at least a portion of the solid composition has been reconstituted.

[0088] In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is no more than 2.6 times the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is no more than 2.5 times, no more than 2 times, no more than 1.9 times, no more than 1.8 times, no more than 1.7 times, no more than 1.6 times, no more than 1.5 times, no more than 1.4 times, no more than 1.3 times, no more than 1.2 times, or no more than 1.1 times the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is equal to or less than the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector having an excitation wavelength of 280 nm and an emission wavelength of 350 nm after at least a portion of the solid composition has been reconstituted.In specific embodiments, the percentage of HSA dimer in soluble HSA contained in the solid composition is not more than 1.1 to 1.3 times, not more than 1.3 to 1.5 times, not more than 1.5 to 1.6 times, not more than 1.5 to 1.7 times, not more than 1.7 to 1.9 times, not more than 1.1 to 1.5 times, not more than 1.5 to 2 times, not more than 2 to 2.5 times, or not more than 2.5 to 2.6 times the percentage of HSA dimer in soluble HSA contained in the starting material, as determined using SEC with a fluorescence detector with an excitation wavelength of 280 nm and a Emission wavelength of 350 nm after at least part of the solid composition has been reconstituted.

[0089] In various embodiments and aspects, there is provided herein a solid composition comprising BoNT / A and HSA, wherein the solid composition has one, two, three, four, five or more of the properties described above.

[0090] In various embodiments and aspects, there is provided herein a solid composition comprising BoNT / A and HSA, wherein the solid composition is prepared by a method described in Section 5.3.

[0091] In various embodiments and aspects, a solid composition described herein is bioproduct-free. In various embodiments and aspects, a solid composition described herein is dried from a solution comprising BoNT / A and HSA, wherein the solution is bioproduct-free. In various embodiments and aspects, a solid composition described herein can be reconstituted, for example, with water or saline, into a solution comprising BoNT / A and HSA, wherein the reconstituted solution is bioproduct-free. In various embodiments and aspects, a solid composition described herein does not contain a protease inhibitor. In certain embodiments, a solid composition described herein does not contain benzamidine hydrochloride.In various embodiments and aspects, a solution described herein comprising BoNT / A and HSA does not contain a protease inhibitor. In certain embodiments, a solution described herein comprising BoNT / A and HSA does not contain benzamidine hydrochloride. In various embodiments and aspects, both the solid composition and the solution comprising BoNT / A and HSA do not contain a protease inhibitor. In certain embodiments, both the solid composition and the solution comprising BoNT / A and HSA do not contain benzamidine hydrochloride.

[0092] In various embodiments and aspects, the HSA described herein is recombinant HSA. In a specific embodiment, the recombinant HSA is animal product-free. In a specific embodiment, the recombinant HSA is not produced by any living organism. In a specific embodiment, the recombinant HSA is produced from a microorganism such as bacteria. 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 HSA derived from human plasma. In a specific embodiment, the HSA content in a solid composition described herein is about 0.5 mg per 100 units of BoNT / A.

[0093] In various embodiments and aspects, the solid composition described herein further comprises one or more pharmaceutically acceptable carriers. In various embodiments and aspects, a solution described herein comprising 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 sodium chloride per 100 units of BoNT / A). In specific embodiments, the solid composition described herein comprises about 0.9 mg sodium chloride per 100 units of BoNT / A.

[0094] In various embodiments and aspects, a solid composition of a solution comprising BoNT / A and HSA described herein is dried such that the solution does not freeze during drying.

[0095] In various embodiments and aspects, it is determined whether a solution described herein (or a reference solution described herein) freezes during drying as described in Section 5.3.

[0096] In various aspects and embodiments, a solid composition described herein comprises about 50 units, about 100 units, or about 200 units of BoNT / A.

[0097] In various embodiments and aspects, a solid composition described herein has a potency of about 2.4 × 10 7 Units / mg to about 6.0 × 10 7 Units / mg

[0098] The present invention provides a solid composition comprising BoNT / A and HSA that is dried without freezing. Such a solid composition has advantageous properties such as consistent visual product quality and a much slower loss of potency during storage, e.g., at room temperature or higher, compared to a solid reference composition stored under the same conditions. A solid reference composition is dried from the same liquid composition as the solid composition under investigation, with the two compositions differing only in the drying process: The solid reference composition is exposed to freezing conditions during the drying process.In other words, a solid reference composition has a higher percentage of HSA aggregates in HSA, a higher percentage of HSA aggregates, polymer, oligomer, or dimer in soluble HSA, and / or a higher percentage of insoluble HSA aggregates in HSA than the solid composition under study, but is otherwise essentially identical to the solid composition under study. The term "essentially identical" is used in this context or the like to describe that the solid reference composition is identical to the solid composition or sufficiently similar to it (for example, with respect to its ingredients and the respective concentrations of the ingredients) so that the solid reference composition can serve as a suitable comparative control.The potency of a solid composition dried without freezing reaches a steady state after a certain storage period, and the steady-state potency of the solid composition is greater than the steady-state potency of a solid reference composition stored under the same conditions. In certain embodiments, both the solid composition and the solid reference composition are stored at about 5°C, at about 25°C (and optionally at about 60% relative humidity), or at about 40°C (and optionally at about 75% relative humidity).

[0099] In various embodiments and aspects, the potency of a solid composition described herein reaches a steady state after a certain storage time, and wherein the steady state potency 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 potency of a reference solid composition comprising BoNT / A and HSA under the same storage conditions. In various embodiments and aspects, the potency of a solid composition described herein reaches a steady state after a certain storage time, and wherein the loss of potency of the solid composition in the steady state compared to the beginning of storage is less than (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%) of the loss of potency of a fixed reference composition.

[0100] Whether the potency of a solid composition has reached a steady state can be determined by experts. For example, the potency of a solid composition can be considered to have reached a steady state if the potency of the solid composition does not decrease by more than 20% after 3 months of storage.

[0101] In various embodiments and aspects, a solid composition described herein is a powdered pharmaceutical composition.

[0102] In another aspect, there is provided herein a liquid composition comprising BoNT / A and HSA, which is obtained from a solid composition described herein, for example, a dissolved or reconstituted form of a solid composition described herein.

[0103] In another aspect, there is provided herein a method of treating a patient in need (preferably a human patient) comprising administering a composition as described herein. 5.4.1 Characterization of BoNT / A compositions

[0104] In one embodiment, the BoNT / A compositions described herein have a potency of at least about 1.5×10 7 Units / mg, e.g., about 1.5×10 7 up to about 6.0×107 units / mg, about 2.0×10 7 up to about 6.0×10 7 Units / mg, about 2.4×10 7 up to about 6.0 ×10 7Units / mg, about 2.4×10 7 up to about 5.9×10 7 Units / mg, about 2.4×10 7 up to about 5.8 × 10 7 Units / mg, about 2.4 × 10 7 up to about 5.7 × 10 7 Units / mg, about 2.4 × 10 7 up to about 5.6 × 10 7 Units / mg, about 2.4 × 10 7 up to about 5.5 × 10 7 Units / mg, about 2.4×10 7 up to about 5.4×10 7 Units / mg, about 2.5×10 7 up to about 6.0×10 7 Units / mg, about 2.6×10 7 up to about 6.0×10 7 Units / mg, about 2.7×10 7 up to about 6.0×10 7 Units / mg, about 2.8×10 7 up to about 6.0×10 7 Units / mg, about 2.9×10 7 up to about 6.0×10 7 Units / mg, about 3.0×10 7 up to about 6.0×10 7 Units / mg or any values between these ranges.

[0105] In one embodiment, the BoNT / A compositions described herein have a potency of about 2.4 × 10 7 Units / mg to about 5.4 × 10 7 units / mg. In preferred embodiments, the term “unit” as used herein refers to the LD 50 -Dose.

[0106] In various embodiments and aspects, a solid composition described herein is a dried product of a solution comprising BoNT / A and HSA and has a higher potency than a solid reference composition, wherein the solid reference composition is prepared from a reference solution comprising BoNT / A and HSA by a drying process conducted such that the reference solution freezes during drying, and wherein the reference solution is substantially identical to the solution. The term "substantially identical" is to be understood as described above. A further description of a solid reference composition is provided above in Section 5.3.In certain embodiments, the solid composition described herein has a potency 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 higher than the potency of the reference solid composition. In certain embodiments, the solid composition described herein has a potency that is at least 0.1 × 10 7 Units / mg, at least 0.2×10 7 Units / mg, at least 0.3×10 7 Units / mg, at least 0.4×10 7 Units / mg, at least 0.5×10 7 Units / mg, at least 0.6×10 7 Units / mg, at least 0.7×10 7 Units / mg, at least 0.8× 10 7 Units / mg, at least 0.9×10 7 Units / mg, at least 1 × 10 7 Units / mg, at least 1.5 ×10 7 Units / mg, at least 2 ×10 7Units / mg, at least 3 × 10 7 units / mg or at least 4 × 10 7 units / mg higher than the potency of the solid reference composition.

[0107] In various embodiments and aspects, a solid composition described herein is a dried product of a solution comprising BoNT / A and HSA and exhibits a slower potency decay than a solid reference composition, wherein the solid reference composition is prepared from a reference solution comprising BoNT / A and HSA by a drying process conducted such that the reference solution freezes during drying, and wherein the reference solution is substantially identical to the solution. The term "substantially identical" is to be understood as described above. A further description of a solid reference composition is provided above in Section 5.3.In certain embodiments, the solid composition described herein has a potency decay 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%, or at least 95% slower compared to the reference solid composition.

[0108] The potency of the BoNT / A compositions described herein can be determined by methods known in the art, including, but not limited to, Light-Chain Activity High-Performance Liquid Chromatography assay (LCA-HPLC assay), Mouse 50% lethal dose assay (MLD 50 assay), Mouse Digit Abduction Score assay (DAS assay), SNAP-25 assay, cell-based potency assay (CBPA), etc.

[0109] The LCA-HPLC assay measures the specificity of SNAP-25 cleavage. Samples react with a commercially available BoNT / A fluorescent substrate derived from the SNAP-25 sequence. The fluorescently labeled cleavage products are separated and detected using a reverse-phase HPLC (RP-HPLC) method. Further descriptions 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, each of which is incorporated herein by reference in its entirety.

[0110] In one embodiment, potency is determined using a 50% lethal dose in mice (MLD50) assay. The 50% lethal dose in mice (MLD50) assay is described in, e.g., Schantz and Kautter (1978) Journal of the AOAC 61(1):96-99, Hunt and Kenneth (2009) Clinical Neuropharmacology 32(1):28-31, U.S. Patent No. 7,160,699, and U.S. Patent No. 9,725,705, each of which is incorporated herein by reference in its entirety. The mouse 50% lethal dose assay (MLD50 assay) is a method for measuring the potency of a botulinum toxin by intraperitoneal injection of the botulinum toxin into female mice (approximately four weeks old) that each weighed 17-22 grams at the start of the assay.Each mouse is placed in a supine position with its head tilted downward and injected intraperitoneally into the right lower abdomen at an angle of approximately 30 degrees using a 25 to 27 gauge, 3 / 8 inch to 5 / 8 inch needle with one of several serial dilutions of botulinum toxin in saline. Death rates over the following 72 hours for each dilution are recorded. Dilutions are prepared so that the most concentrated dilution results in a death rate of at least 80% of the injected mice and the least concentrated dilution results in a death rate of no more than 20% of the injected mice. There must be at least four dilutions that fall within the monotonically decreasing range of death rates. The monotonically decreasing range begins at a death rate of at least 80%. Within the four or more monotonically decreasing rates, the two largest and the two smallest rates must be decreasing (i.e., not equivalent).The dilution at which 50% of mice die within the observation period of three days after injection is defined as a dilution containing one unit (1 U) of botulinum toxin.

[0111] The mouse Digit Abduction Score (DAS) assay 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 assay can be used to assess the potency of BoNT / A compositions on muscle paralysis, as well as the duration of the effect. Detailed protocols for the DAS assay are disclosed in Aoki et al. (1999) Eur. J. Neurol. 6:s3-s10, Aoki (2001) Toxicon 39:1815-1820, Broide et al. (2013) Toxicon 71:18-24, and Rupp et al. (2020) Toxine 12(6):393, each of which is incorporated by reference in its entirety. For example, the DAS assay can be performed by injecting a BoNT / A composition described herein into the mouse gastrocnemius / soleus complex, followed by assessment of the digital abduction score according to the method of Aoki (2001) Toxicon 39: 1815-1820.In the DAS assay, mice are briefly suspended by the tail to elicit a characteristic startle response in which the mouse extends its hind limbs and abducts its hind digits. Following injection of the BoNT / A composition, the various degrees of digit abduction are scored on a five-point scale (0 = normal to 4 = maximal reduction in digit abduction and leg extension). The safety ratio, the ratio between the amount of toxin required to produce a 10% decrease in body weight (measured at peak effect within the first seven days after administration to a mouse) and the amount of toxin required for a DAS score of 2, can also be determined to evaluate the therapeutic index of the BoNT / A composition described herein, as described in U.S. Patent No. 9,920,310, which is incorporated herein by reference in its entirety.A high safety ratio score is therefore desirable and indicates a toxin capable of effectively paralyzing a target muscle without causing adverse off-target effects.

[0112] The SNAP-25 assay is an ELISA-based method for measuring the proteolytic activity of the botulinum toxin SNAP-25. The assay uses a truncated SNAP-25 protein (the 206-amino acid residue peptide) bound to 96-well polystyrene microtiter plates and a monoclonal antibody that recognizes the cleaved product (a 197-amino acid residue peptide) formed by enzymatic hydrolysis between amino acids 197 and 198 of SNAP-25 by reduced botulinum toxin type A. The monoclonal antibody bound to the cleaved product is then detected with a secondary antibody (goat anti-mouse IgG conjugated to horseradish peroxidase (HRP), which produces a color change in the presence of a chromogenic substrate (TMB). Example SNAP-25 methods are described in Ekong et al. (1997) Microbiology 143:3337-3347 and US Patent 7,160,699, each of which is incorporated herein by reference.

[0113] The cell-based potency assay (CBPA) has been described in, e.g., Fernandez-Salas et al. (2012) PLOS ONE 7(11):e49516, Rupp et al. (2020) Toxins 12(6):393, WO 2010 / 105234, and WO 2009 / 114748, each of which is incorporated herein by reference. In one embodiment, the SNAP-25 197 SiMa H1 electrochemiluminescence (ECL) CBPA was used to determine the potency of the BoNT / A compositions described herein. The SNAP-25 197SiMa H1 electrochemiluminescence (ECL) CBPA is a cell-based in vitro assay that measures the key steps of BoNT / A intoxication: receptor-mediated cell binding and internalization, translocation of the protease domain (light chain) into the cytosol, and proteolytic cleavage of SNAP-25, allowing a direct comparison of the biological activity of BoNT / A products in vitro (Fernandez-Salas et al. (2012) PLOS ONE 7(11):e49516; Rupp et al. (2020) Toxins 12(6):393). Briefly, human neuroblastoma SiMa H1 cells are cultured on poly-D-lysine (PDL) 96-well plates in serum-free medium (SFM) containing 25 µg / ml GT. 1b plated for three days and treated with toxin samples for 24 hours. After treatment, the toxins are removed, the cells are lysed, and the lysates are transferred to MSD High Bind plates labeled with the monoclonal antibody (mAb) 2E2A6 against SNAP-25. 197The plates are then washed and incubated with a SULFO-TAG NHS ester-labeled polyclonal anti-SNAP-25 antibody (pAb) for detection. The captured SNAP-25, cleaved by BoNT / A toxin, is then quantified using an MSD plate reader.

[0114] In various embodiments and aspects, a solid composition described herein is a dried product of a solution comprising BoNT / A and HSA and has a higher visual product quality (e.g., less or no fluffy product) compared to a solid reference composition, wherein the solid reference composition is prepared from a reference solution comprising BoNT / A and HSA through a drying process performed such that the reference solution freezes during 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 BoNT / A and HSA and has a more consistent visual product quality (e.g., more consistently exhibits less fluffy or no fluffy product) compared to a solid reference composition.The term "essentially identical" is to be understood as described above. A further description of a fixed reference composition is provided above in Section 5.3.

[0115] In various embodiments and aspects, a solid composition described herein is a dried product of a solution comprising BoNT / A and HSA and has lower immunogenicity than a solid reference composition, wherein the solid reference composition is prepared from a reference solution comprising BoNT / A and HSA by a drying process conducted such that the reference solution freezes during drying, and wherein the reference solution is substantially identical to the solution. The term "substantially identical" is to be understood as described above. A further description of a solid reference composition is provided above in Section 5.3.

[0116] The immunogenicity of a BoNT / A composition can be measured by any method described herein or known in the art that is 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

[0117] The present disclosure includes the following non-limiting illustrative 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 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 according to embodiment 1, wherein the HSA aggregates comprise HSA polymer, HSA oligomer, and HSA dimer. 3. The solid composition according to embodiment 1 and embodiment 2, wherein less than 30% of the soluble HSA contained in the solid composition is HSA polymer. 4. The solid composition according to embodiment 1 and embodiment 2, wherein less than 0.8% of the soluble HSA contained in the solid composition is HSA oligomer. 5. The solid composition according to embodiment 1 and embodiment 2, wherein less than 4.8% of soluble HSA contained in the solid composition is HSA dimer. 6. The 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 using size exclusion chromatography (SEC) after at least a portion of the solid composition has been reconstituted with water. 7. The solid composition according to any one of embodiments 1 to 6, wherein the solid composition is a powdered pharmaceutical composition. 8. The 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 using size exclusion chromatography (SEC) with a detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted with water. 9. Solid composition according to any one of embodiments 1 to 8, wherein the solid composition is a living organism product-free one. 10. The solid composition according to any one of embodiments 1 to 9, wherein the HSA is recombinant HSA. 11. The solid composition of any one of embodiments 1 to 10, wherein the solid composition comprises about 0.5 mg HSA per 100 units BoNT / A. 12. The solid composition of any one of embodiments 1 to 11, wherein the solid composition comprises about 0.9 mg NaCl per 100 units of BoNT / A. 13. The solid composition according to any one of embodiments 1 to 12, wherein the BoNT / A is onabotulinumtoxinA. 14. The solid composition according to any one of embodiments 1 to 13, wherein the BoNT / A is prepared by a column chromatography-based purification process. 15. The solid composition according to any one of embodiments 1 to 14, wherein the BoNT / A is prepared by a column chromatographic purification process, wherein the column chromatographic purification process comprises hydrophobic interaction chromatography. 16. The solid composition according to any one of embodiments 1 to 15, wherein the column chromatographic purification process further comprises anion exchange chromatography. 17. The solid composition according to any one of embodiments 1 to 16, wherein the column chromatographic purification process further comprises cation exchange chromatography. 18. The solid composition according to any one of embodiments 1 to 17, wherein the solid composition does not comprise a protease inhibitor. 19. The solid composition according to any one of embodiments 1 to 18, wherein the solid composition does not comprise benzamidine hydrochloride. 20. The solid composition according to any one of embodiments 1 to 19, wherein the solid composition has a potency of about 1.5 × 10 7 Units / mg to about 6.0 × 10 7 units / mg. 21. A method for preparing a solid composition comprising a 900 kDa BoNT / A complex, HSA and NaCl, the method comprising a step of drying a solution comprising BoNT / A, HSA and NaCl to prepare the solid composition, wherein the solution does not freeze during the drying step. 22. The method of embodiment 21, wherein the drying step is at least partially manually controlled. 23. The method of 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 stepwise evacuation step. 25. The method according to any one of embodiments 21-24, wherein the method produces a solid composition according to any one of claims 1-20. 26. A solid composition prepared by the process of any one of embodiments 21 to 25. 27. A method of treating a patient in need, comprising administering the solid composition of any one of embodiments 1-20 and 26. 28. Method substantially as described herein. 29. Composition substantially as described herein. 7. EXAMPLE

[0118] Certain embodiments provided herein are illustrated by the following non-limiting examples, which describe different methods for obtaining BoNT / A and show that freezing during drying results in 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 freezing decrease in potency more slowly during storage. 7.1. Example 1 - Chemical and physical stability of human serum albumin during the manufacture of a botulinum toxin drug product: Laboratory-scale study.7.1.1. Introduction

[0119] Most commercial neurotoxin preparations contain human serum albumin (HSA) as an excipient. Understanding HSA aggregation could be important for the development and commercialization of neurotoxin products. HSA aggregates can be introduced into the drug product (DP) from incoming HSA used in DP manufacturing. The HSA monograph for the European Pharmacopoeia specifies that the total area of peaks due to HSA polymers and aggregates should not exceed 10% (http: / / www.uspbpep.com / ep60 / human%20albumin%20solution%200255e.pdf, EUROPEAN PHARMACOPOEIA 6.0, Human Albumin Solution). Manufacturers of pharmaceutical-grade HSA may also impose stricter restrictions on the level of aggregation. For example, one HSA supplier, CSL Berring, specifies a requirement for aggregates of ≤ 8% ( Fig.1). This study investigated whether HSA aggregates can also form during DP production. As with any sterile biopharmaceutical product, the production of a BoNT / A-DP involves several steps.

[0120] In the present study, HSA aggregation was measured in laboratory-prepared BoNT / A DP batches and placebo batches, prepared under three sets of conditions using the same HSA and NaCl-containing formulation with (for BoNT / A-DP) or without (for placebo) the active ingredient BoNT / A. The main differences in the manufacturing processes include the drying process and the holding of the bulk solution prior to the drying step. Commercial BoNT / A drug products are manufactured either by lyophilization (freeze-drying) or vacuum drying. The same freeze-drying equipment can be used for both manufacturing processes, with the main difference being the freezing step used in the former. However, evaporative cooling during vacuum drying can also result in freezing.

[0121] Another potential difference in manufacturing conditions is the length of time a bulk drug product is stored before sterilization filtration begins, as well as from filtration until the vials are fully filled. While such holding times can be minimized to less than one hour during laboratory manufacturing, where dozens to several hundred vials are produced, the holding time before drying would be much longer in commercial production, where the number of vials produced runs into the thousands and tens of thousands. Additionally, a typical commercial process is validated for longer holding times to account for the possibility of unexpected delays, such as a filling line malfunction. Accordingly, this study also investigated the influence of holding the bulk drug solution before drying. The formulation contains HSA and NaCl.Batches were manufactured with the active ingredient BoNT / A (active DP batches) and without BoNT / A (placebo batches). 7.1.2. Materials and methods7.1.2.1. Materials

[0122] HSA was purchased from CSL Behring as a 25% solution. A sterile 0.9 wt% NaCl solution (USP, EP, BP, JP grade, Terumo BCT Ltd), NaCl granules (JT Baker, USP, EP, JP, BP grade), and MilliQ water were used for preparation. A concentrated solution of the BoNT / A drug substance (DS) was provided by AbbVie. Vials (Type 1 glass, 10 mL, Gerresheimer) were washed and depyrogenated. Stoppers (20 mm Lyotec West S-87-J, 4432 / 50 GRY, ready-to-sterilize) were sterilized and dried. For batches containing an active ingredient, the BoNT / A drug solution was added to the excipient solution (0.5 wt% HSA and 0.9 wt%-% NaCl) was added at a dilution factor of 23000, filtered with a gamma-irradiated Millipak-20 0.2 µm sterilizing filter, aseptically filled into 10-mL Type 1 glass vials at 0.1 mL / vial, partially capped, placed in the LyoStar2 freeze-dryer, vacuum-dried or freeze-dried, closed under vacuum at the end of the drying process, and sealed with an aluminum sleeve. The placebo batches were prepared similarly, except for the DS addition and a higher fill volume of 0.2 mL / vial. The product temperature during vacuum drying was measured in real time using temperature probes (Ellab, Denmark); temperature data were also recorded using data loggers. Three sets of manufacturing conditions were used, with key differences shown in Table 2 and further details explained in Section 7.1.2.2 below. Five drug product batches and three placebo batches were manufactured. . Fig.2A and Fig. 2B shows representative vacuum drying and freeze-drying cycles. Table 2. Main differences lie in the manufacturing conditions of the laboratory drug product batches. Manufacturing process ID Holding before / after filtration of the bulk DP 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 hours / 24 hours Freeze-drying 2 / 1 7.1.2.2. Process7.1.2.2.1. Drying process

[0123] The cycle of Process 1 (i.e., vacuum drying) is shown in Table 3. The vials are cooled by evacuating the Lophilizer. To prevent the solutions in the vials from freezing, a special step-by-step procedure was used that allowed the Lophilizer chamber to be evacuated without freezing any solutions in the vials. Specifically, before initiating the automated vacuum drying cycle in Table 3 (i.e., before the "Vacuum On" step and after the "Pre-Freeze" step), the vacuum was manually controlled as follows to minimize the risk of freezing of the solutions in the vials due to rapid evacuation of the Lophilizer sample chamber. The main reason for this step-by-step manual control is that the evacuation rate of the Lyostar2 cannot be controlled during the cycle.Instead, the vacuum was turned on, leaving the evacuation rate uncontrolled, and stopped at various intervals to allow the product temperature to rise to a higher temperature (such as 20°C) before the vacuum was turned on again. Two thermocouples were placed in two vials near the center of the tray to monitor the product temperature. The manual cycle was turned on. The storage temperature was set to 20°C. The condenser was turned on. The vacuum was then turned on and manually stopped at 20,000 mTorr. The product temperature obtained from the thermocouple measurements was 16°C. The product temperature was allowed to rise to 20°C, and the evacuation continued until the Pirani pressure measurement was 5,000 mTorr, at which point the vacuum was turned off. The product temperature was 2°C. The product temperature was again allowed to rise to 18°C.At this point, the automated cycle shown in Table 3 below was initiated. After the vacuum drying cycle was completed, the vials were fully sealed with stoppers and caps and stored in a -20°C freezer. Table 3. Cycle parameters of Process-1. Step Temperature, °C Rate of increase, °C / min . Hold time, min . Pressure, mTorr Pre-freezing 20 1 15 Vicinity Freeze N / A N / A N / A Vicinity Additional freezing time N / A N / A N / A Vicinity Vacuum "On" 20 0 15 84 Primary drying 20 1 300 84 Secondary drying N / A N / A N / A N / A Hold 5 N / A N / A 84 NA = not applicable

[0124] The cycle of Process 2 (i.e., freeze-drying) is shown in Table 4. Specifically, this cycle included a freezing cycle at -50°C for 120 minutes. The cycle of Process 2 did not include a gradual evacuation step. After the cycle was completed, the vials were completely sealed under vacuum, and the freeze-dryer was returned to ambient conditions. The vials were capped and stored at -20°C until testing. Table 4. Cycle parameters of process 2. Step Temperature, °C Rate of increase, °C / min . Hold time, min . Pressure, mTorr Pre-freezing 2 1 15 Vicinity Freeze -50 1 120 Vicinity Additional freezing time N / A N / A N / A Vicinity Vacuum "On" -50 0 15 84 Primary drying 20 1 300 84 Secondary drying N / A N / A N / A N / A Hold 5 N / A N / A 84 NA = not applicable

[0125] The cycle of Process 3 (i.e., freeze-drying plus holding at room temperature) is shown in Table 5. Specifically, this cycle included a freezing cycle at -50°C for 120 minutes. The cycle of Process 3 did not include a stepwise evacuation step. After the cycle was completed, the vials were completely sealed under vacuum, and the freeze-dryer was returned to ambient conditions. The vials were capped and stored at -20°C until testing. Before the drying cycle, the product solution was stored at room temperature for 24 hours before filtration and for another 24 hours after filtration. Table 5. Cycle parameters of process 3. Step Temperature, °C Rate of increase, °C / min . Hold time, min . Pressure, mTorr Pre-freezing 2 1 15 Vicinity Freeze -50 1 120 Vicinity Additional freezing time N / A N / A N / A Vicinity Vacuum "On" -50 0 15 84 Primary drying 20 1 300 84 Secondary drying N / A N / A N / A N / A Hold 5 N / A N / A 84 NA = not applicable 7.1.2.2.2 Analysis of HSA aggregation by SEC

[0126] The aggregation of human serum albumin (HSA) was measured by size exclusion chromatography (SEC).

[0127] The dried samples were reconstituted by adding 0.25 ml of water per vial. The 25% pure HSA solutions were diluted with water to 0.25 mg / ml prior to HPLC injection. System suitability standards were injected five times prior to sample injections and subsequently every 10 injections to verify system suitability. The HPLC parameters are listed in Table 6. The samples were run and analyzed using Empower. Five active and three placebo batches were tested using SEC. A statistical analysis of the SEC data was performed using JMP 10.0 statistical software. Table 6. SEC parameters. parameter Proceedings Injection volume 50 µl Column temperature Room temperature column Superdex 200 10 / 300 flow rate 0.5 ml / min Mobile phase 50 mm sodium phosphate + 150 mm ammonium sulfate, pH 7.0 Duration 60 minutes Sample preparation Reconstitute vials with 0.25 mg / ml water Proof (a) 280 nm (b) 220 nm (c) Fluorescence (excitation 280 nm, emission 350 nm) 7.1.2.2.3. Karl Fischer titration

[0128] Coulometric Karl Fischer titration was used to quantify the residual water content in the dried product. A KF titrator with an oven and automatic sampler was used. Samples were analyzed in triplicate. Sealed vials were heated to 115 °C in a KF oven. Nitrogen gas was used to purge the vials and carry moisture in the headspace into the reaction vessel containing the KF reagent. The reaction was continued until the rate fell below 20 µg / l. <Min. fiel. Leere Fläschchen, die die gleichen Gefriertrocknungs- oder Vakuumtrocknungszyklen durchliefen, wurden ebenfalls getestet, und der Wassergehalt im Leerwert wurde abgezogen, um den Wassergehalt im getrockneten Material zu erhalten. Vier repräsentative Chargen wurden getestet. 7.1.2.2.4. Scanning electron microscopy (SEM)

[0129] Three batches of dried placebo cakes were examined using an FEI Quanta-450 (Thermo Fisher Scientific, Hillsboro, OR) field emission SEM equipped with a backscattered electron detector. Imaging conditions were 10 kV and 10 mm working distance under low vacuum. The samples were uncoated. SEM imaging of the morphology of the dried placebo cake required breaking the glass vial to isolate the dried cake / glass bottom pieces. This was achieved by scoring the vial near the bottom rim with a diamond pinion and then breaking the glass with a sharp blow. Corresponding pieces of each sample were then mounted on an SEM stump using carbon tape. Three placebo batches were tested by SEM. 7.1.2.2.5. Test for subvisible particles

[0130] For the subvisible particle test (also referred to as the Subvisible Particle Test), three placebo batches were tested, each prepared using Process 1, 2, and 3. Vials of dried placebo were reconstituted by adding 2 ml of saline to each vial and mixed gently. Vials from each batch were tested using a laboratory-built noninvasive invisible particle imaging system as described in U.S. Patent No. 10,132,736 B2, which is hereby incorporated by reference in its entirety. During particle measurement, a reconstituted solution in the vial was partially illuminated with a laser beam focused onto 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.This system allows for the noninvasive counting of subvisible particles in syringes or vials. The software, developed in the laboratory, can count the number, size, and intensity of particles in each image in real time. The absolute number and size of particles can be calibrated using standard particle samples. 7.1.2.2.6. XRPD

[0131] The XRPD diffractograms were acquired on a MiniFlex 600 X-ray diffractometer (Rigaku, Texas, USA) using Cu Kα radiation (λ = 1.54 Å, 40 kV / 15 mA). The instrument was calibrated using a silicon standard with a reference peak at 28.44° (2θ). Fixed samples were prepared in a low-background Si holder 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° (2θ) with a continuous scan of 1° (2θ) per minute with a step size of 0.01° (2θ). Data analysis was performed using PDXL software. 7.1.2.2.7. Small-angle X-ray scattering / wide-angle X-ray scattering (SAXS / WAXS)

[0132] The SAXS / WAXS tests of two solutions (0.9% NaCl / 0.5% HSA and 0.9% NaCl in water) were performed on beamline ID02 of the European Synchrotron Radiation Facility (ESRF). The solutions were filled into 1 mm capillaries and loaded into the Linkam stage for test runs at variable temperatures. Two-dimensional SAXS and WAXS images were acquired 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 most cases. The two-dimensional images were normalized to an absolute intensity scale after applying standard detector corrections and azimuthally integrated to obtain the corresponding one-dimensional X-ray diffraction curves. The SAXS and WAXS Q-scales were calibrated using silver behenate and silicon powder, respectively. All combined SAXS / WAXS measurements were normalized. 7.1.2.2.8. Changes in apparent pH

[0133] To monitor the 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). 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 containing 50 ml of placebo solution was transferred to a lyophilizer (Lyostar III, SP Scientific). The temperature probe and the low-temperature pH electrode were placed in the beaker containing the placebo solution. The lyophilizer door was closed, and refrigeration was started in manual mode with a storage temperature set at -55°C. The pH value was recorded for each change in sample temperature by one unit. After the sample temperature reached -50°C, the storage set point was increased in 10°C increments until the sample temperature was within 2°C of the storage temperature and the sample was allowed to thaw to 20°C. The pH value was also measured during evaporation using the microelectrode InLab ®Micro (pH 0 to 14, temperature 0 °C to 80 °C, electrolyte 3 M KCl) (METTLER TOLEDO), while the sample weight was determined at regular intervals while the placebo evaporated under nitrogen overflow. The pH microelectrode was calibrated at room temperature using the same NIST-traceable buffer standards mentioned above. Approximately 7.3 g of placebo solution was placed in a glass vial and exposed to nitrogen overflow in a fume hood. The sample vial was intermittently removed from the hood to record pH and weight data. 7.1.3. Results7.1.3.1. Characterization of “pure” HSA.

[0134] Representative SEC chromatograms for an HSA solution (raw material) are shown in Fig.3A-3C. In addition to the HSA monomer peak (retention time, RT ~27 min), there were three peaks of the higher molecular weight species, which were identified as dimer (RT ~23.5 min), HSA oligomer (RT ~21 min), and HSA polymer (RT ~16 min). The assignment of the polymer, oligomer, dimer, and monomer peaks was based on the retention times obtained when running the Biorad gel filtration standard (Cat 151-1901) on the same column. The gel filtration standard consisted of bovine thyroglobulin (MW 670 kDa), bovine γ-globulin (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 chromatography was observed to be between approximately 4% and approximately 10%, with the highest values measured with a fluorescence detector and subsequently with a UV detector with a wavelength of 280 nm.Oligomers were detected by fluorescence but not by UV detection, probably due to the higher sensitivity of the fluorescence detector.

[0135] The aggregation levels were similar among the three HSA batches used in this study (Table 7-9). Table 7. SEC analysis of aggregates in HSA solution (A220 nm). HSA Batch No. . 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 Table 8. SEC analysis of aggregates in HSA solution (A280 nm). HSA Batch No. . A280 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 Table 9. SEC analysis of aggregates in HSA solution (fluorescence). fluorescence HSA Batch No. . 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. HSA aggregation in the active DP batches and placebo batches.

[0136] The aggregation of HSA in BoNT / A DP batches was characterized using SEC. Examples of SEC curves for drug batches produced using three different manufacturing processes are shown in Fig.4A-4C. A significant decrease in the peak was observed in both freeze-dried batches prepared by Process 2 and Process 3 compared to the vacuum-dried batch (Process 1). The HSA dimer and polymer peaks were stronger in the freeze-dried samples than in the vacuum-dried product. Additionally, two poorly resolved polymer peaks were observed in the freeze-dried samples, particularly during UV detection at wavelengths of 220 and 280 nm.

[0137] The inserted bar charts in Fig. Figures 4A-4C show the percentage of different types of HSA aggregates in soluble HSA subjected to SEC chromatography for three DP batches and for the “pure” HSA. Fig.The values shown in Figures 4A-4C are presented in Tables 10-12 below, respectively. The levels of all three aggregate types were similar for the “neat” HSA and the vacuum-dried DP (Manufacturing Process-1), while a significant increase in aggregation was observed for both freeze-dried DP batches. The quantitative extent of aggregation detected depended on the detector type (see Fig. 4A-4C and Table 10-12). 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 holding 15,3 0,6 6,2 0,3 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 holding 32,7 0,8 4,8 0,3 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 holding 15 0,5 1,1 0,1 6,3 0,2

[0138] The total HSA aggregation, which represents the sum of all three aggregate types in soluble HSA subjected to SEC chromatography for multiple DP batches, is shown in Fig. 5A-5G. There was no significant increase in aggregation in the DP batches produced using manufacturing process 1 (vacuum drying), whereas there was a significant increase in HSA aggregation in the DP batch produced using manufacturing process 2 (freeze drying). The longer holding time before drying (process 3) did not result in any additional significant increase in HSA aggregation.

[0139] The test for invisible particles was carried out using dried placebo batches. The particle count results are shown in Fig. 6A and Fig.6B and Table 13. The average total particle count was lower in the vacuum-dried batch, while both freeze-dried batches exhibited significantly higher levels of subvisible particles. For the two freeze-dried batches, the average particle counts were similar within experimental uncertainty. The results for subvisible particles are consistent with the SEC data for soluble HSA aggregates ( Fig. 5A-5G), in which the aggregation level was higher in the freeze-dried samples than in the vacuum-dried batches. Table 13. Results of test for subvisible particles. saline solution Baseline (Batch No. 6) Claimed (Hold during RT and Freeze) (Batch No. 11) Claimed (Freeze Only) (Batch No. 8) Average number of particles (n=1) Standard deviation (SD) Average number of particles (n=5) Standard deviation (SD) Average number of particles (n=4) Standard deviation (SD) Average number of particles (n=4) Standard deviation (SD) 2,867 40,1802 19,71 260,067 94,77 370,459 85,93 7.1.3.3. Physical characterization of the medicinal product

[0140] To gain insights into possible mechanisms leading to the increase in HSA aggregation during freeze-drying, additional physical tests were performed.

[0141] 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 Fig. 7A and Fig.7B. A relatively small but notable pH increase from an initial pH of 6.7 to 7 was observed during cooling of the solution from RT to -10 °C. The ice nucleation, which was observed as a product temperature peak at -10 °C, did not result in any notable pH change. During further cooling to -35 °C, the pH remained between 6.9 and 7.1, whereas the pH increased to approximately 8.1 during 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 did not indicate an exothermic effect due to secondary crystallization, the lack of a thermal effect could be due to the sensitivity due to the low NaCl concentration (0.9 wt%).However, it should be noted that the temperature range of the pH electrode, according to the manufacturer's specifications, only extends to -30 °C; therefore, pH results below -30 °C would need to be confirmed by an orthogonal method. In the evaporation experiment, a slight pH increase 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%.

[0142] Phase transitions in water / NaCl and water / NaCl / HSA solutions during cooling were investigated using small-angle and wide-angle X-ray scattering (SAXS / WAXS). Two aqueous placebo solutions were tested, one with 0.9% NaCl and 0.5% HSA and another with only 0.9% NaCl. The WAXS data showed the formation of hexagonal ice at either -20 °C (HSA+NaCl solution) or -15 °C (NaCl solution) ( Fig. 8A and Fig.8B). Upon further cooling, characteristic peaks of NaCl dihydrate were detected at -35 °C (HSA+NaCl solution) and at -25 °C (NaCl solution). The WAXS results indicate that HSA can inhibit the crystallization of NaCl dihydrate.

[0143] SAXS patterns are in Fig. 8C. A protein interaction peak was observed in the unfrozen solution, whereas freezing leads to the "disappearance" of the peak. In addition to changes in the protein interaction peak, freezing observed by WAXS at -20 °C resulted in increased small-angle scattering intensity, indicating the formation of additional interfaces, i.e., ice / solution and likely ice / air / solution. Further cooling to -35 °C resulted in an additional increase in small-angle scattering, indicating the formation of new interfaces; this increase in interface size coincided with the crystallization of NaCl*2H2O observed by WAXS.

[0144] 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 testing the dried products using scanning electron microscopy (SEM). Representative product temperature data ( Fig. 2A) show that the temperature remained above 0 °C throughout the entire vacuum drying cycle; freezing in the vials would therefore not occur. To further confirm the absence of freezing during vacuum drying, SEM tests were performed on materials in vials without thermocouples. SEM images for freeze-dried products ( Fig.9) show the porous morphology of the two freeze-dried batches (right and middle images), as expected. Pores in the dried materials represent a "signature" of ice crystals that sublimated during primary drying. The absence of pores in the vacuum-dried material (left images) confirms that these materials were not frozen. Another key difference between vacuum-dried and freeze-dried samples is the presence of NaCl crystals, which appeared as cubic shapes lining the inner walls of cracks in the HSA in the vacuum-dried material. The freeze-dried batches showed no clear signs of NaCl crystals, possibly because they were much smaller than in the vacuum-dried batch. The smaller 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.

[0145] XRPD samples for two placebo batches produced using different manufacturing processes are shown in Fig. 10. Two sharp peaks of crystalline NaCl and an amorphous HSA halo were observed in both batches. The vacuum-dried batch (Process 1) exhibited stronger and sharper crystalline peaks, whereas the crystalline NaCl peaks in the freeze-dried sample (Process 3) were broader, likely due to a higher degree of disorder. This result is consistent with the SEM data showing larger NaCl crystals in the vacuum-dried batch. Additionally, anhydrous NaCl crystals were formed by dehydration of NaCl dihydrate during freeze-drying, whereas NaCl crystallized directly from solution during vacuum drying; this difference may also contribute to the higher degree of disorder of the NaCl crystals in the freeze-dried material. 7.1.3.4. Potency stability study

[0146] In a separate study, freezing during vacuum drying appeared to result in a statistically significant change in the potency of BoNT / A DP after 6 months of storage at 5°C compared to a process without freezing (data not shown). In this example's study, potency stability was further tested at room temperature and higher in addition to that at 5°C. Specifically, two DP batches, Batch No. 9 (Process 3, freeze-dried plus hold) and Batch No. 4 (Process 1, vacuum-dried), were tested for stability at 5°C, 25°C, and 40°C, respectively. Vials from each batch were collected at various time intervals between 0 and 6 months and examined for potency recovery by cell-based potency assay (CBPA). Results are shown in Table 14 below. Data from the test at a storage condition of 25°C are also included in Fig. 11 shown. Table 14. Results of the potency stability study. DP Batch No. . Drying process - tions Storage time, months Target potency restoration, U / vial Measured potency restoration, U / vial Potency recovery, % of baseline 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 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).

[0147] As shown, the potency of the DP batch prepared using Process 3 decreased slightly at 5 °C after 6 months, while at 25 °C and 40 °C it decreased dramatically after just 3 months. In contrast, the potency of the DP batch prepared using Process 1 decreased much more slowly at 25 °C and 40 °C. 7.1.4 Discussion

[0148] An increased level of HSA aggregation was observed in freeze-dried batches, whereas vacuum drying without freezing did not result in a significant increase in HSA aggregation. HSA aggregation did not increase during vacuum drying when freezing was avoided. The level of soluble aggregates increased after freeze-drying and reconstitution from the initial approximately 4% to approximately 10% in “pure” HSA to approximately 20% to approximately 35% (specific values depend on the SEC detector, see Fig.5A-5G). Insoluble aggregates also increased significantly in freeze-dried materials compared to vacuum-dried materials. The results demonstrate that by precisely controlling specific manufacturing conditions, HSA aggregation can be significantly reduced, with lower levels of both soluble and insoluble HSA aggregates detected when freezing during drying is avoided. The results also suggest that freezing during drying may lead to a more rapid decrease in potency during storage.

[0149] Protein destabilization and aggregation could potentially be related to protein concentration and ionic strength. However, the absence of HSA aggregation in the vacuum-dried product batches suggests that neither an increase in protein concentration nor ionic strength per se were the primary mechanisms of the observed HSA aggregation.

[0150] Without wishing to be bound by any particular theory, the results of this study suggest that the observed HSA aggregation may be related to NaCl*2H2O. In particular, secondary crystallization (NaCl*2H2O + water) may create favorable conditions for freezing-induced pressure, as it occurs within a rigid "shell" of ice crystals formed during the primary freezing process, thereby potentially contributing to protein destabilization during freezing. Crystallization of NaCl*2H2O may also contribute to HSA aggregation via a different mechanism. While crystallization of anhydrous NaCl during vacuum drying did not lead to HSA aggregation, a different NaCl crystal form, the dihydrate, was formed during freeze-drying. Anhydrous NaCl formed during vacuum drying may have different surface properties than the dihydrate formed during freezing.While the surface charge of NaCl and NaCl*2H2O crystals formed during vacuum drying and freeze-drying, respectively, is unknown, a significant difference in the electrostatic interaction of these two crystal types with HSA may be a factor contributing to freezing-induced HSA aggregation.

[0151] Finally, pH changes were observed during freezing in this study, which could also contribute to HSA aggregation. 7.1.5. Conclusions

[0152] While most commercial neurotoxin formulations contain HSA, the effects of the drug product manufacturing process on HSA properties are not well understood. In this study, a significant increase in HSA aggregation was observed during freeze-drying, whereas HSA aggregation in vacuum-dried DP did not increase significantly without freezing and remained similar to that of the original HSA solution. These observations suggest that the manufacturing conditions of neurotoxin formulations can have a significant impact on the properties of the final product. Therefore, additional testing of toxin DP, particularly HSA aggregation, could be beneficial in the development and commercialization of HSA-containing neurotoxin products. The results further suggest that freezing was the primary cause of HSA aggregation.

[0153] Regarding the mechanism of HSA aggregation during freeze-drying, the study suggests that freezing-induced pressure, interaction with NaCl*2H2O crystals, and freezing-induced pH changes may be contributing factors. 7.2. Example 2 - Effects of freezing on vacuum-dried botulinum toxin drug products.

[0154] In this study, BoNT / A DP batches prepared using a vacuum drying process without freezing the drug solutions were compared with BoNT / A DP batches prepared using a vacuum drying process that caused the drug solutions to freeze. The two types of BoNT / A DP batches are referred to in this example as evaporative vacuum-dried DP batches and vacuum-lyophilized DP batches, respectively. Both the pilot-scale Lyostar-3 freeze dryer and the industrial-scale Lyomax 40 freeze dryer were tested. With both freeze dryers, it was repeatedly observed that the evaporative vacuum-dried DP batches exhibited a more visually homogeneous morphology, resulting in consistent visual product quality for the patient, whereas vacuum-lyophilized DP batches tended to flak.During reconstitution of the drug product vial, product flakes may move near the product stopper, potentially resulting in the patient receiving less than the prescribed dose. Therefore, vacuum-dried DP batches are more cost-effective than vacuum-lyophilized DP batches.

[0155] Additional results from this study showed that using a slower chamber depressurization rate and a higher storage temperature (using a storage temperature of 20°C instead of 5°C) can help prevent freezing of the drug product (although the exact chamber depressurization rate to be used is determined by the drying equipment and depends on chamber size, condenser capacity, chamber load, and vacuum pump power). Results from this study showed that both a rapid chamber depressurization rate and a low storage temperature were significant factors contributing to lower product temperatures. 8. INCORPORATION BY REFERENCE

[0156] All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application were expressly and individually indicated as being incorporated by reference in its entirety for all purposes.

[0157] Many modifications and variations of this invention may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The embodiments described herein are exemplary only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] US 2003 / 0118598

[0025] US 8,618,261

[0026] US 8,198,034

[0026] US 9,249,216

[0026] US 10,703,806

[0026] US 11,261,240

[0026] US 11,332,518

[0026] US 8,129,139 [0034, 0035] US 9,469,849

[0035] US 10,132,736 B2 [0051, 0130] US 7,160,699 [0110, 0112] US 9,725,705

[0110] US 9,920,310

[0111] WO 2010 / 105234

[0113] WO 2009 / 114748

[0113] Cited non-patent literature

[0000] 13371-306-888_SEQLISTING.xml" was submitted on January 3, 2024

[0001] Zhang et al. (2003) Gene 315:21

[0019] Hunt et al. (2010) Toxins 2(8):2198-2212

[0109] Rupp et al. (2020) Toxins 12(6):393 [0109, 0113] Schantz and Kautter (1978) Journal of the AOAC 61(1):96-99

[0110] Hunt and Kenneth (2009) Clinical Neuropharmacology 32(1):28-31

[0110] Aoki et al. (1999) Eur. J. Neurol. 6:s3-s10

[0111] Aoki (2001) Toxicon 39: 1815-1820

[0111] Broide et al. (2013) Toxicon 71:18-24

[0111] Rupp et al. (2020) Toxins 12(6):393

[0111] Ekong et al. (1997) Microbiology 143:3337-3347

[0112] Fernändez-Salas et al. (2012) PLOS ONE 7(11):e49516

[0113] http: / / www.uspbpep.com / ep60 / human%20albumin%20solution%200255e.pdf, EUROPEAN PHARMACOPOEIA 6.0, Human Albumin Solution

[0119]

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 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 HSA polymer, HSA oligomer, and 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 HSA polymer. [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 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 HSA dimer. [6] A solid composition according to any one of claims 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 using size exclusion chromatography (SEC) after at least a portion of the solid composition has been reconstituted with water. [7] A solid composition according to any one of claims 1 to 6, wherein the solid composition is a powdered pharmaceutical composition. [8] A solid composition according to any one of claims 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 using size exclusion chromatography (SEC) with a detector having a wavelength of 220 nm after at least a portion of the solid composition has been reconstituted with water. [9] A solid composition according to any one of claims 1 to 8, wherein the solid composition is free of living organism products. [10] A solid composition according to any one of claims 1 to 9, wherein the HSA is recombinant HSA. [11] A solid composition according to any one of claims 1 to 10, wherein the solid composition comprises about 0.5 mg HSA per 100 units BoNT / A. [12] A solid composition according to any one of claims 1 to 11, wherein the solid composition comprises about 0.9 mg NaCl per 100 units of BoNT / A. [13] A solid composition according to any one of claims 1 to 12, wherein the BoNT / A is onabotulinumtoxinA [14] A solid composition according to any one of claims 1 to 13, wherein the BoNT / A is prepared by a column chromatographic purification process. [15] A solid composition according to any one of claims 1 to 14, wherein the BoNT / A is prepared by a column chromatographic purification process, wherein the column chromatographic purification process comprises hydrophobic interaction chromatography. [16] A solid composition according to any one of claims 1 to 15, wherein the column chromatographic purification process further comprises anion exchange chromatography. [17] A solid composition according to any one of claims 1 to 16, wherein the column chromatographic purification process further comprises cation exchange chromatography. [18] A solid composition according to any one of claims 1 to 17, wherein the solid composition does not comprise a protease inhibitor. [19] A solid composition according to any one of claims 1 to 18, wherein the solid composition does not comprise benzamidine hydrochloride. [20] A solid composition according to any one of claims 1 to 19, wherein the solid composition has a potency of about 1.5 × 10 7 Units / mg to about 6.0 × 10 7 units / mg. [21] A method for preparing a solid composition comprising a 900 kDa BoNT / A complex, HSA and NaCl, the method comprising a step of drying a solution comprising BoNT / A, HSA and NaCl to prepare the solid composition, wherein the solution does not freeze during the drying step. [22] The method of claim 21, wherein the drying step is at least partially manually controlled. [23] A method according to claim 21 or claim 22, wherein the drying step is vacuum drying. [24] The method of claim 23, wherein the vacuum drying step comprises a stepwise evacuation step. [25] A process according to any one of claims 21-24, wherein the process produces a solid composition according to any one of claims 1-20. [26] A solid composition prepared by the process according to any one of claims 21 to 25. [27] A method of treating a patient in need, comprising administering the solid composition of any one of claims 1-20 and 26. [28] Process substantially as described herein. [29] Composition substantially as described herein.

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