Clostridium botulinum neurotoxin serotype a compositions and improved filtration processes

Abstract

Provided herein are compositions comprising Clostridium botulinum neurotoxin serotype A (BoNT / A) with improved stability. Such BoNT / A compositions are produced by a process comprising filtering a solution comprising BoNT / A with a filter that is not gamma-irradiated or a filter that contains no free radicals or a low amount of free radicals. Further provided herein are methods of producing compositions comprising BoNT / A by filtering a solution comprising BoNT / A with a filter that is not gamma-irradiated or a filter that contains no free radicals or a low amount of free radicals.

Description

Attorney Docket No.13371-304-228 CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE A COMPOSITIONS AND IMPROVED FILTRATION PROCESSES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 601,087, filed on November 20, 2023, which is incorporated by reference herein in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application incorporates by reference a Sequence Listing submitted with this application as an xml file entitled “Seqlisting_13371-304-228.xml” created on November 12, 2024 and having a size of 6,742 bytes. 1. FIELD

[0003] Provided herein are compositions comprising Clostridium botulinum neurotoxin serotype A (BoNT / A) with improved stability. Such BoNT / A compositions are produced by a process comprising filtering a solution comprising BoNT / A with a filter that is not gamma- irradiated or a filter that contains no free radicals or a low amount of free radicals. Further provided herein are methods of producing compositions comprising BoNT / A by filtering a solution comprising BoNT / A with a filter that is not gamma-irradiated or a filter that contains no free radicals or a low amount of free radicals. 2. BACKGROUND

[0004] Clostridium botulinum neurotoxin serotype A (BoNT / A) is a highly potent toxin that causes muscle relaxation by inhibition of synaptic vesicle docking and fusion, thereby blocking acetylcholine release at neuromuscular junctions. BoNT / A is produced by Clostridium botulinum Type A strains, which synthesize a complex of a 150 kDa neurotoxin, along with a group of non- toxic neurotoxin-associated proteins (NAPs). BOTOX®, or onabotulinumtoxinA, is a BoNT / A product approved by the United States Food and Drug Administration (FDA) in 1989 for a variety of therapeutic and cosmetic indications.

[0005] Manufacturing protein-based pharmaceutical compositions for biopharmaceutical applications involves multiple steps. Certain parameters of some manufacturing steps may affect the properties or functionalities of the proteins. To date, there remains a need for developing botulinum toxins pharmaceutical compositions with improved biological activities and enhanced NAI-1541834789v1 1Attorney Docket No.13371-304-228 stabilities.

[0006] Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure. 3. SUMMARY OF THE INVENTION

[0007] In one aspect, provided herein is a liquid composition comprising Clostridium botulinum neurotoxin serotype A (BoNT / A), wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter that is not gamma-irradiated.

[0008] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein no free radicals are detectable on the filter. In certain embodiments, the amount of free radicals on the filter is determined using ESR.

[0009] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of free radicals on the filter is less than that on a gamma-irradiated filter of the same type. In certain embodiments, the gamma- irradiated filter has a pore size of about 0.22 µm. In certain embodiments, the amount of free radicals on the gamma-irradiated filter is determined using Electron Spin Resonance (ESR). In certain embodiments, the amount of free radicals on the filter is determined using ESR.

[0010] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, said filtration is performed with a filter, and wherein the amount of free radicals on the filter is less than 250 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 100 x 1010spins / mg as determined using ESR. In specific embodiments, no free radicals are detectable on the filter as determined using ESR.

[0011] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, said filtration is performed with a filter, and wherein no Carbon-centered radicals are detectable in an aqueous solution containing a spin-trap molecule following exposure to the filter. In certain embodiments, the spin-trap molecule is N-tert-butyl-α-phenylnitrone (PBN). In certain NAI-1541834789v1 2Attorney Docket No.13371-304-228 embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 15 hours.

[0012] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, said filtration is performed with a filter, wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule following exposure to the filter is less than the amount of Carbon-centered radicals in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the gamma-irradiated filter has a pore size of about 0.22 µm. In certain embodiments, the amount of Carbon-centered radicals in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 15 hours.

[0013] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR.

[0014] In another aspect, provided herein is a liquid composition comprising BoNT / A, NAI-1541834789v1 3Attorney Docket No.13371-304-228 wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR.

[0015] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein no degradation product of a spin-trap molecule is detectable in an aqueous solution containing the spin-trap molecule following exposure to the filter. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 15 hours.

[0016] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule following exposure to the filter is less than the amount of degradation product of the spin-trap molecule in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the gamma-irradiated filter has a pore size of about 0.22 µm. In certain embodiments, the amount of degradation product of the spin-trap molecule in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated NAI-1541834789v1 4Attorney Docket No.13371-304-228 filter is determined using ESR. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 15 hours.

[0017] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR.

[0018] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR.

[0019] In another aspect, provided herein is a liquid composition comprising BoNT / A, NAI-1541834789v1 5Attorney Docket No.13371-304-228 wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein potency of the liquid composition does not decrease after 3 months of storage or decreases less than 15% after 3 months of storage.

[0020] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein potency of the liquid composition does not decrease after 18 months of storage or decreases less than 35% after 18 months of storage.

[0021] In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 3 months of storage or decreases less than 15% after 3 months of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 3 months of storage or decreases less than 10% after 3 months of storage.

[0022] In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 18 months of storage or decreases less than 35% after 18 months of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 18 months of storage or decreases less than 30% after 18 months of storage.

[0023] In various embodiments and aspects, potency of a liquid composition described herein is determined using a cell-based potency assay. In various embodiments and aspects, potency of a liquid composition described herein is determined using a mouse 50% lethal dose (MLD50) assay.

[0024] In various embodiments and aspects, the filter used to produce a liquid composition described herein has a pore size of about 0.22 µm.

[0025] In various embodiments and aspects, the filter used to produce a liquid composition described herein is autoclaved.

[0026] In various embodiments and aspects, the BoNT / A is a 900 kDa BoNT / A complex.

[0027] In various embodiments and aspects, the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum (e.g., the Type A Hall strain of Clostridium botulinum).

[0028] In various embodiments and aspects, the BoNT / A is onabotulinumtoxinA.

[0029] In various embodiments and aspects, a liquid composition described herein is animal product free.

[0030] In various embodiments and aspects, a liquid composition described herein further NAI-1541834789v1 6Attorney Docket No.13371-304-228 comprises one or more pharmaceutically acceptable carriers. In specific embodiments, the one or more pharmaceutically acceptable carriers comprise human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of BoNT / A) and sodium chloride (e.g., about 0.9 mg of sodium chloride per 100 units of BoNT / A). In certain embodiments, the human serum albumin is recombinant human serum albumin.

[0031] In various embodiments and aspects, a liquid composition described herein comprises about 50 units, about 100 units or about 200 units of BoNT / A.

[0032] In various embodiments and aspects, a liquid composition described herein has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

[0033] In various embodiments and aspects, the BoNT / A is produced by a process that comprises one or more steps of column chromatography (e.g., one or more steps of column chromatography performed during purification of the BoNT / A). In preferred embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A) comprise hydrophobic interaction chromatography. In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A) further comprise anion exchange chromatography. In specific embodiments, the one or more steps of column chromatography (e.g., during purification of the BoNT / A) further comprise cation exchange chromatography.

[0034] In various embodiments and aspects, the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate (e.g., does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the BoNT / A).

[0035] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter that is not gamma-irradiated to produce said liquid composition.

[0036] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein no free radicals are detectable on the filter. In certain embodiments, the amount of free radicals on the filter is determined using ESR.

[0037] In another aspect, provided herein is method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a NAI-1541834789v1 7Attorney Docket No.13371-304-228 filter to produce said liquid composition, and wherein the amount of free radicals on the filter is less than that on a gamma-irradiated filter of the same type. In certain embodiments, the gamma- irradiated filter has a pore size of about 0.22 µm. In certain embodiments, the amount of free radicals on the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of free radicals on the filter is determined using ESR.

[0038] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of free radicals on the filter is less than 250 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 100 x 1010spins / mg as determined using ESR. In specific embodiments, no free radicals are detectable on the filter as determined using ESR.

[0039] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein no Carbon-centered radicals are detectable in an aqueous solution containing a spin-trap molecule following exposure to the filter. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 15 hours.

[0040] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule following exposure to the filter is less than the amount of Carbon-centered radicals in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the gamma-irradiated filter has a pore size of about 0.22 µm. In certain embodiments, the amount of Carbon-centered radicals in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following NAI-1541834789v1 8Attorney Docket No.13371-304-228 exposure to the filter is determined using ESR. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 15 hours.

[0041] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR.

[0042] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR.

[0043] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein no degradation product of a spin-trap molecule is detectable in an aqueous solution containing the spin-trap molecule following exposure to the filter. In certain embodiments, the spin-trap molecule is PBN. In certain NAI-1541834789v1 9Attorney Docket No.13371-304-228 embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 15 hours.

[0044] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, wherein the amount of degradation product of a spin- trap molecule in an aqueous solution containing the spin-trap molecule following exposure to the filter is less than the amount of degradation product of the spin-trap molecule in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the gamma-irradiated filter has a pore size of about 0.22 µm. In certain embodiments, the amount of degradation product of the spin-trap molecule in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 15 hours.

[0045] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap NAI-1541834789v1 10Attorney Docket No.13371-304-228 molecule following exposure to the filter is determined using ESR.

[0046] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR.

[0047] In various embodiments and aspects, the filter used to produce a liquid composition described herein has a pore size of about 0.22 µm.

[0048] In various embodiments and aspects, the filter used to produce a liquid composition described herein is autoclaved.

[0049] In various embodiments and aspects, the BoNT / A is a 900 kDa BoNT / A complex.

[0050] In various embodiments and aspects, the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum (e.g., the Type A Hall strain of Clostridium botulinum).

[0051] In various embodiments and aspects, the BoNT / A is onabotulinumtoxinA.

[0052] In various embodiments and aspects, a liquid composition described herein is animal product free. In various embodiments and aspects, a solution described herein comprising BoNT / A is animal product free.

[0053] In various embodiments and aspects, a liquid composition described herein further comprises one or more pharmaceutically acceptable carriers. In various embodiments and aspects, a solution described herein comprising BoNT / A further comprises one or more pharmaceutically acceptable carriers. In specific embodiments, the one or more pharmaceutically acceptable carriers comprise human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of BoNT / A) and sodium chloride (e.g., about 0.9 mg of NAI-1541834789v1 11Attorney Docket No.13371-304-228 sodium chloride per 100 units of BoNT / A). In certain embodiments, the human serum albumin is recombinant human serum albumin.

[0054] In various aspects and embodiments, a liquid composition described herein comprises about 50 units, about 100 units or about 200 units of BoNT / A.

[0055] In various embodiments and aspects, a liquid composition described herein has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

[0056] In various aspects and embodiments, the BoNT / A is produced by a process that comprises one or more steps of column chromatography (e.g., one or more steps of column chromatography performed during purification of the BoNT / A). In preferred embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A) comprise hydrophobic interaction chromatography. In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A) further comprise anion exchange chromatography. In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A) further comprise cation exchange chromatography.

[0057] In various aspects and embodiments, the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate (e.g., does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the BoNT / A).

[0058] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is produced by a method described herein of producing a liquid composition comprising BoNT / A.

[0059] In another aspect, provided herein is a solid composition, which is a dried product (e.g., a vacuum-dried, freeze-dried, or lyophilized product) of a liquid composition described herein. In certain embodiments, the solid composition comprises about 50 units, about 100 units or about 200 units of BoNT / A. In certain embodiments, the solid composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

[0060] In another aspect, provided herein is a powdered pharmaceutical composition comprising a 900 kDa BoNT / A complex (e.g., a powdered pharmaceutical composition comprising about 50 units, about 100 units or about 200 units of a 900 kDa BoNT / A complex), human serum albumin, and sodium chloride, wherein the powdered pharmaceutical composition NAI-1541834789v1 12Attorney Docket No.13371-304-228 is a dried product (e.g., a vacuum-dried, freeze-dried, or lyophilized product) of a liquid composition comprising the 900 kDa BoNT / A, wherein the liquid composition is a filtration product of a solution comprising the 900 kDa BoNT / A complex, and said filtration is performed with a filter that is not gamma-irradiated. In certain embodiments, the 900 kDa BoNT / A complex is produce by a process that comprises one or more steps of column chromatography. In specific embodiments, the one or more steps of column chromatography are performed during purification of the 900 kDa BoNT / A complex. In specific embodiments, the one or more steps of column chromatography comprise hydrophobic interaction chromatography. In specific embodiments, the one or more steps of column chromatography further comprise anion exchange chromatography. In specific embodiments, the one or more steps of column chromatography further comprise cation exchange chromatography. In certain embodiments, the 900 kDa BoNT / A complex is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate (e.g., during purification of the 900 kDa BoNT / A complex). In specific embodiments, the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum (e.g., the Type A Hall strain of Clostridium botulinum). In specific embodiments, the 900 kDa BoNT / A complex is onabotulinumtoxin A. In specific embodiments, the powdered pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers. In a specific embodiment, the one or more pharmaceutically acceptable carriers comprise human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of the 900 kDa BoNT / A complex) and sodium chloride (e.g., about 0.9 mg of sodium chloride per 100 units of the 900 kDa BoNT / A complex). In specific embodiments, the human serum albumin is recombinant human serum albumin. In specific embodiments, the powdered pharmaceutical composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

[0061] In another aspect, provided herein is a method of producing a powdered pharmaceutical composition comprising a 900 kDa BoNT / A complex (e.g., a powdered pharmaceutical composition comprising about 50 units, about 100 units or about 200 units of a 900 kDa BoNT / A complex), human serum albumin, and sodium chloride, said method comprising filtering a solution comprising the 900 kDa BoNT / A complex with a filter that is not gamma-irradiated to produce a liquid composition comprising the 900 kDa BoNT / A complex, and drying (e.g., vacuum-drying, freeze-drying, or lyophilizing) the liquid composition to NAI-1541834789v1 13Attorney Docket No.13371-304-228 produce the powdered pharmaceutical composition. In certain embodiments, the method comprises producing the 900 kDa BoNT / A complex by a process that comprises one or more steps of column chromatography. In specific embodiments, the one or more steps of column chromatography are performed during purification of the 900 kDa BoNT / A complex. In specific embodiments, the one or more steps of column chromatography comprise hydrophobic interaction chromatography. In specific embodiments, the one or more steps of column chromatography further comprise anion exchange chromatography. In specific embodiments, the one or more steps of column chromatography further comprise cation exchange chromatography. In certain embodiments, the method comprises producing the 900 kDa BoNT / A complex by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate (e.g., during purification of the 900 kDa BoNT / A complex). In specific embodiments, the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum (e.g., the Type A Hall strain of Clostridium botulinum). In specific embodiments, the 900 kDa BoNT / A complex is onabotulinumtoxin A. In specific embodiments, the powdered pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers. In a specific embodiment, the one or more pharmaceutically acceptable carriers comprise human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of the 900 kDa BoNT / A complex) and sodium chloride (e.g., about 0.9 mg of sodium chloride per 100 units of the 900 kDa BoNT / A complex). In specific embodiments, the human serum albumin is recombinant human serum albumin. In specific embodiments, the powdered pharmaceutical composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

[0062] In various embodiments and aspects, a method described herein does not involve using a protease inhibitor. In certain embodiments, a method described herein does not involve using benzamidine hydrocholoride.

[0063] In various embodiments and aspects, a liquid composition described herein does not contain a protease inhibitor. In certain embodiments, a liquid composition described herein does not contain benzamidine hydrochloride.

[0064] 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. NAI-1541834789v1 14Attorney Docket No.13371-304-228

[0065] In various embodiments and aspects, a powdered pharmaceutical composition described herein does not contain a protease inhibitor. In certain embodiments, a powdered pharmaceutical composition described herein does not contain benzamidine hydrochloride. 3.1. Illustrative Embodiments 1. A liquid composition comprising Clostridium botulinum neurotoxin serotype A (BoNT / A), wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter that is not gamma-irradiated. 2. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein no free radicals are detectable on the filter. 3. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of free radicals on the filter is less than that on a gamma- irradiated filter of the same type. 4. The liquid composition of embodiment 3, wherein the gamma-irradiated filter has a pore size of about 0.22 µm. 5. The liquid composition of embodiment 3 or 4, wherein the amount of free radicals on the gamma-irradiated filter is determined using Electron Spin Resonance (ESR). 6. The liquid composition of any one of embodiments 2-5, wherein the amount of free radicals on the filter is determined using ESR. 7. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of free radicals on the filter is less than 250 x 1010spins / mg as determined using ESR. 8. The liquid composition of embodiment 7, wherein the amount of free radicals on the filter is less than 100 x 1010spins / mg as determined using ESR. 9. The liquid composition of embodiment 7 or 8, wherein no free radicals are detectable on the filter as determined using ESR. 10. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, NAI-1541834789v1 15Attorney Docket No.13371-304-228 and wherein no Carbon-centered radicals are detectable in an aqueous solution containing a spin-trap molecule following exposure to the filter. 11. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin- trap molecule following exposure to the filter is less than the amount of Carbon-centered radicals in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. 12. The liquid composition of embodiment 11, wherein the gamma-irradiated filter has a pore size of about 0.22 µm. 13. The liquid composition of embodiment 11 or 12, wherein the amount of Carbon-centered radicals in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. 14. The liquid composition of any one of embodiments 10-13, wherein the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. 15. The liquid composition of any one of embodiments 10-14, wherein the exposure is for about 30 minutes. 16. The liquid composition of any one of embodiments 10-14, wherein the exposure is for about 15 hours. 17. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. 18. The liquid composition of embodiment 17, wherein the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. NAI-1541834789v1 16Attorney Docket No.13371-304-228 19. The liquid composition of embodiment 17 or 18, wherein no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. 20. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. 21. The liquid composition of embodiment 20, wherein the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. 22. The liquid composition of embodiment 20 or 21, wherein no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. 23. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein no degradation product of a spin-trap molecule is detectable in an aqueous solution containing the spin-trap molecule following exposure to the filter. 24. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule following exposure to the filter is less than the amount of degradation product of the spin-trap molecule in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. 25. The liquid composition of embodiment 24, wherein the gamma-irradiated filter has a pore size of about 0.22 µm. 26. The liquid composition of embodiment 24 or 25, wherein the amount of degradation product of the spin-trap molecule in the reference aqueous solution containing the spin- trap molecule following exposure to the gamma-irradiated filter is determined using ESR. NAI-1541834789v1 17Attorney Docket No.13371-304-228 27. The liquid composition of any one of embodiments 23-26, wherein the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin- trap molecule following exposure to the filter is determined using ESR. 28. The liquid composition of any one of embodiments 23-27, wherein the exposure is for about 30 minutes. 29. The liquid composition of any one of embodiments 23-27, wherein the exposure is for about 15 hours. 30. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. 31. The liquid composition of embodiment 30, wherein the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. 32. The liquid composition of embodiment 30 or 31, wherein no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. 33. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. 34. The liquid composition of embodiment 33, wherein the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. 35. The liquid composition of embodiment 33 or 34, wherein no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. NAI-1541834789v1 18Attorney Docket No.13371-304-228 36. The liquid composition of any one of embodiments 10-35, wherein the spin-trap molecule is N-tert-butyl-α-phenylnitrone (PBN). 37. The liquid composition of any one of embodiments 1-36, wherein potency of the liquid composition does not decrease after 3 months of storage or decreases less than 15% after 3 months of storage. 38. The liquid composition of any one of embodiments 1-36, wherein potency of the liquid composition does not decrease after 3 months of storage or decreases less than 10% after 3 months of storage. 39. The liquid composition of any one of embodiments 1-38, wherein potency of the liquid composition does not decrease after 18 months of storage or decreases less than 35% after 18 months of storage. 40. The liquid composition of any one of embodiments 1-38, wherein potency of the liquid composition does not decrease after 18 months of storage or decreases less than 30% after 18 months of storage. 41. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein potency of the liquid composition does not decrease after 3 months of storage or decreases less than 15% after 3 months of storage. 42. The liquid composition of embodiment 41, wherein potency of the liquid composition does not decrease after 3 months of storage or decreases less than 10% after 3 months of storage. 43. The liquid composition of embodiment 41 or 42, wherein potency of the liquid composition does not decrease after 18 months of storage or decreases less than 35% after 18 months of storage. 44. The liquid composition of any one of embodiments 41-43, wherein potency of the liquid composition does not decrease after 18 months of storage or decreases less than 30% after 18 months of storage. 45. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein potency of the liquid composition does not decrease after 18 months of storage or decreases less than 35% after 18 months of storage. NAI-1541834789v1 19Attorney Docket No.13371-304-228 46. The liquid composition of embodiment 45, wherein potency of the liquid composition does not decrease after 18 months of storage or decreases less than 30% after 18 months of storage. 47. The liquid composition of any one of embodiments 37-46, wherein potency of the liquid composition is determined using a cell-based potency assay. 48. The liquid composition of any one of embodiments 37-46, wherein potency of the liquid composition is determined using a mouse 50% lethal dose (MLD50) assay. 49. The liquid composition of any one of embodiments 1-48, wherein the filter has a pore size of about 0.22 µm. 50. The liquid composition of any one of embodiments 1-49, wherein the filter is autoclaved. 51. The liquid composition of any one of embodiments 1-50, wherein the BoNT / A is a 900 kDa BoNT / A complex. 52. The liquid composition of embodiment 51, wherein the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum. 53. The liquid composition of 52, wherein the Type A strain of Clostridium botulinum is the Type A Hall strain of Clostridium botulinum. 54. The liquid composition of any one of embodiments 1-53, wherein the BoNT / A is onabotulinumtoxinA. 55. The liquid composition of any one of embodiments 1-54, which is animal product free. 56. The liquid composition of any one of embodiments 1-55, further comprising one or more pharmaceutically acceptable carriers. 57. The liquid composition of embodiment 56, wherein the one or more pharmaceutically acceptable carriers comprise human serum albumin and sodium chloride. 58. The liquid composition of embodiment 57, wherein the one or more pharmaceutically acceptable carriers comprise about 0.5 mg of human serum albumin per 100 units of BoNT / A. 59. The liquid composition of embodiment 57 or 58, wherein the one or more pharmaceutically acceptable carriers comprise about 0.9 mg of sodium chloride per 100 units of BoNT / A. 60. The liquid composition of any one of embodiments 57-59, wherein the human serum albumin is recombinant human serum albumin. NAI-1541834789v1 20Attorney Docket No.13371-304-228 61. The liquid composition of any one of embodiments 1-60, which comprises about 50 units, about 100 units or about 200 units of BoNT / A. 62. The liquid composition of any one of embodiments 1-61, which has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg. 63. The liquid composition of any one of embodiments 1-62, wherein the BoNT / A is produced by a process that comprises one or more steps of column chromatography. 64. The liquid composition of embodiment 63, wherein the one or more steps of column chromatography are performed during purification of the BoNT / A. 65. The liquid composition of embodiment 63 or 64, wherein the one or more steps of column chromatography comprise hydrophobic interaction chromatography. 66. The liquid composition of embodiment 65, wherein the one or more steps of column chromatography further comprise anion exchange chromatography. 67. The liquid composition of embodiment 65 or 66, wherein the one or more steps of column chromatography further comprise cation exchange chromatography. 68. The liquid composition of any one of embodiments 1-67, wherein the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate. 69. The liquid composition of embodiment 68, wherein the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the BoNT / A. 70. The liquid composition of any one of embodiments 1-69, which does not contain a protease inhibitor. 71. The liquid composition of any one of embodiments 1-70, which does not contain benzamidine hydrochloride. 72. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter that is not gamma- irradiated to produce said liquid composition. 73. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein no free radicals are detectable on the filter. NAI-1541834789v1 21Attorney Docket No.13371-304-228 74. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of free radicals on the filter is less than that on a gamma-irradiated filter of the same type. 75. The method of embodiment 74, wherein the gamma-irradiated filter has a pore size of about 0.22 µm. 76. The method of embodiment 74 or 75, wherein the amount of free radicals on the gamma- irradiated filter is determined using ESR. 77. The method of any one of embodiments 73-76, wherein the amount of free radicals on the filter is determined using ESR. 78. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of free radicals on the filter is less than 250 x 1010spins / mg as determined using ESR. 79. The method of embodiment 78, wherein the amount of free radicals on the filter is less than 100 x 1010spins / mg as determined using ESR. 80. The method of embodiment 78 or 79, wherein no free radicals are detectable on the filter as determined using ESR. 81. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein no Carbon-centered radicals are detectable in an aqueous solution containing a spin-trap molecule following exposure to the filter. 82. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule following exposure to the filter is less than the amount of Carbon-centered radicals in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. 83. The method of embodiment 82, wherein the gamma-irradiated filter has a pore size of about 0.22 µm. NAI-1541834789v1 22Attorney Docket No.13371-304-228 84. The method of embodiment 82 or 83, wherein the amount of Carbon-centered radicals in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. 85. The method of any one of embodiments 81-84, wherein the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. 86. The method of any one of embodiments 81-85, wherein the exposure is for about 30 minutes. 87. The method of any one of embodiments 81-85, wherein the exposure is for about 15 hours. 88. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. 89. The method of embodiment 88, wherein the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. 90. The method of embodiment 88 or 89, wherein no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. 91. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. 92. The method of embodiment 91, wherein the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. NAI-1541834789v1 23Attorney Docket No.13371-304-228 93. The method of embodiment 91 or 92, wherein no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. 94. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein no degradation product of a spin-trap molecule is detectable in an aqueous solution containing the spin-trap molecule following exposure to the filter. 95. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule following exposure to the filter is less than the amount of degradation product of the spin-trap molecule in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. 96. The method of embodiment 95, wherein the gamma-irradiated filter has a pore size of about 0.22 µm. 97. The method of embodiment 95 or 96, wherein the amount of degradation product of the spin-trap molecule in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. 98. The method of any one of embodiments 94-97, wherein the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR. 99. The method of any one of embodiments 94-98, wherein the exposure is for about 30 minutes. 100. The method of any one of embodiments 94-98, wherein the exposure is for about 15 hours. 101. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of degradation product of a spin-trap molecule in NAI-1541834789v1 24Attorney Docket No.13371-304-228 an aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. 102. The method of embodiment 101, wherein the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. 103. The method of embodiment 101 or 102, wherein no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. 104. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. 105. The method of embodiment 104, wherein the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. 106. The method of embodiment 104 or 105, wherein no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. 107. The method of any one of embodiments 81-106, wherein the spin-trap molecule is PBN. 108. The method of any one of embodiments 72-107, wherein the filter has a pore size of about 0.22 µm. 109. The method of any one of embodiments 72-108, wherein the filter is autoclaved. 110. The method of any one of embodiments 72-109, wherein the BoNT / A is a 900 kDa BoNT / A complex. 111. The method of embodiment 110, wherein the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum. NAI-1541834789v1 25Attorney Docket No.13371-304-228 112. The method of 111, wherein the Type A strain of Clostridium botulinum is the Type A Hall strain of Clostridium botulinum. 113. The method of any one of embodiments 72-112, wherein the BoNT / A is onabotulinumtoxinA. 114. The method of any one of embodiments 72-113, wherein the solution comprising BoNT / A is animal product free. 115. The method of any one of embodiments 72-114, wherein the liquid composition is animal product free. 116. The method of any one of embodiments 72-115, wherein the solution comprising BoNT / A comprises one or more pharmaceutically acceptable carriers. 117. The method of any one of embodiments 72-116, wherein the liquid composition comprises one or more pharmaceutically acceptable carriers. 118. The method of embodiment 116 or 117, wherein the one or more pharmaceutically acceptable carriers comprise human serum albumin and sodium chloride. 119. The method of embodiment 118, wherein the one or more pharmaceutically acceptable carriers comprise about 0.5 mg of human serum albumin per 100 units of BoNT / A. 120. The method of embodiment 118 or 119, wherein the one or more pharmaceutically acceptable carriers comprise about 0.9 mg of sodium chloride per 100 units of BoNT / A. 121. The method of any one of embodiments 118-120, wherein the human serum albumin is recombinant human serum albumin. 122. The method of any one of embodiments 72-121, wherein the liquid composition comprises about 50 units, about 100 units or about 200 units of BoNT / A. 123. The method of any one of embodiments 72-122, wherein the liquid composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg. 124. The method of any one of embodiments 72-123, wherein the BoNT / A is produced by a process that comprises one or more steps of column chromatography. 125. The method of embodiment 124, wherein the one or more steps of column chromatography are performed during purification of the BoNT / A. NAI-1541834789v1 26Attorney Docket No.13371-304-228 126. The method of embodiment 124 or 125, wherein the one or more steps of column chromatography comprise hydrophobic interaction chromatography. 127. The method of embodiment 125 or 126, wherein the one or more steps of column chromatography further comprise anion exchange chromatography. 128. The method of embodiment 126 or 127, wherein the one or more steps of column chromatography further comprise cation exchange chromatography. 129. The method of any one of embodiments 72-128, wherein the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate. 130. The method of embodiment 129, wherein the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the BoNT / A. 131. The method of any one of embodiments 72-130, wherein the method does not involve using a protease inhibitor. 132. The method of any one of embodiments 72-131, wherein the method does not involve using benzamidine hydrocholoride. 133. A liquid composition comprising BoNT / A, wherein the liquid composition is produced by the method of any one of embodiments 72-133. 134. A solid composition, which is a dried product of the liquid composition of any one of embodiments 1-71 and 133. 135. The solid composition of embodiment 134, which is a vacuum-dried product of the liquid composition. 136. The solid composition of embodiment 134, which is a freeze-dried product of the liquid composition. 137. The solid composition of embodiment 134, which is a lyophilized product of the liquid composition. 138. The solid composition of any one of embodiments 134-137, which does not contain a protease inhibitor. 139. The solid composition of any one of embodiments 134-138, which does not contain benzamidine hydrochloride. NAI-1541834789v1 27Attorney Docket No.13371-304-228 140. The solid composition of any one of embodiments 134-139, which comprises about 50 units, about 100 units or about 200 units of BoNT / A. 141. The solid composition of any one of embodiments 134-140, which has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg. 142. A powdered pharmaceutical composition comprising a 900 kDa BoNT / A complex, human serum albumin, and sodium chloride, wherein the powdered pharmaceutical composition is a dried product of a liquid composition comprising the 900 kDa BoNT / A, wherein the liquid composition is a filtration product of a solution comprising the 900 kDa BoNT / A complex, and said filtration is performed with a filter that is not gamma-irradiated. 143. The powdered pharmaceutical composition of embodiment 142, which is a vacuum-dried product of the liquid composition. 144. The powdered pharmaceutical composition of embodiment 142, which is a freeze- dried product of the liquid composition. 145. The powdered pharmaceutical composition of embodiment 142, which is a lyophilized product of the liquid composition. 146. The powdered pharmaceutical composition of any one of embodiments 142-145, wherein the 900 kDa BoNT / A complex is produced by a process that comprises one or more steps of column chromatography. 147. The powdered pharmaceutical composition of embodiment 146, wherein the one or more steps of column chromatography are performed during purification of the 900 kDa BoNT / A complex. 148. The powdered pharmaceutical composition of embodiment 146 or 147, wherein the one or more steps of column chromatography comprise hydrophobic interaction chromatography. 149. The powdered pharmaceutical composition of embodiment 148, wherein the one or more steps of column chromatography further comprise anion exchange chromatography. 150. The powdered pharmaceutical composition of embodiment 148 or 149, wherein the one or more steps of column chromatography further comprise cation exchange chromatography. NAI-1541834789v1 28Attorney Docket No.13371-304-228 151. The powdered pharmaceutical composition of any one of embodiments 142-150, wherein the 900 kDa BoNT / A complex is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate. 152. The powdered pharmaceutical composition of embodiment 151, wherein the 900 kDa BoNT / A complex is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the 900 kDa BoNT / A complex. 153. The powdered pharmaceutical composition of any one of embodiments 142-152, wherein the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum. 154. The powdered pharmaceutical composition of 153, wherein the Type A strain of Clostridium botulinum is the Type A Hall strain of Clostridium botulinum. 155. The powdered pharmaceutical composition of any one of embodiments 142-154, wherein the 900 kDa BoNT / A complex is onabotulinumtoxin A. 156. The powdered pharmaceutical composition of any one of embodiments 142-155, which further comprises one or more pharmaceutically acceptable carriers. 157. The powdered pharmaceutical composition of embodiment 156, wherein the one or more pharmaceutically acceptable carriers comprise human serum albumin and sodium chloride. 158. The powdered pharmaceutical composition of embodiment 157, wherein the one or more pharmaceutically acceptable carriers comprise about 0.5 mg of human serum albumin per 100 units of BoNT / A. 159. The powdered pharmaceutical composition of embodiment 157 or 158, wherein the one or more pharmaceutically acceptable carriers comprise about 0.9 mg of sodium chloride per 100 units of BoNT / A. 160. The powdered pharmaceutical composition of any one of embodiments 157-159, wherein the human serum albumin is recombinant human serum albumin. 161. The powdered pharmaceutical composition of any one of embodiments 142-160, which comprises about 50 units, about 100 units or about 200 units of the 900 kDa BoNT / A complex. NAI-1541834789v1 29Attorney Docket No.13371-304-228 162. The powdered pharmaceutical composition of any one of embodiments 142-161, which has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg. 163. A method of producing a powdered pharmaceutical composition comprising a 900 kDa BoNT / A complex, human serum albumin, and sodium chloride, said method comprising filtering a solution comprising the 900 kDa BoNT / A complex with a filter that is not gamma-irradiated to produce a liquid composition comprising the 900 kDa BoNT / A complex, and drying the liquid composition to produce the powdered pharmaceutical composition. 164. The method of embodiment 163, wherein the step of drying is vacuum-drying. 165. The method of embodiment 163, wherein the step of drying is freeze-drying. 166. The method of embodiment 163, wherein the step of drying is lyophilizing. 167. The method of any one of embodiments 163-166, said method comprising producing the 900 kDa BoNT / A complex by a process that comprises one or more steps of column chromatography. 168. The method of embodiment 167, wherein the one or more steps of column chromatography are performed during purification of the 900 kDa BoNT / A complex. 169. The method of embodiment 167 or 168, wherein the one or more steps of column chromatography comprise hydrophobic interaction chromatography. 170. The method of embodiment 169, wherein the one or more steps of column chromatography further comprise anion exchange chromatography. 171. The method of embodiment 169 or 170, wherein the one or more steps of column chromatography further comprise cation exchange chromatography. 172. The method of any one of embodiments 163-171, said method comprising producing the 900 kDa BoNT / A complex by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate. 173. The method of embodiment 172, said method comprising producing the 900 kDa BoNT / A complex by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the 900 kDa BoNT / A complex. 174. The method of any one of embodiments 163-173, wherein the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum. NAI-1541834789v1 30Attorney Docket No.13371-304-228 175. The method of embodiment 174, wherein the Type A strain of Clostridium botulinum is the Type A Hall strain of Clostridium botulinum. 176. The method of any one of embodiments 163-175, wherein the 900 kDa BoNT / A complex is onabotulinumtoxin A. 177. The method of any one of embodiments 163-176, wherein the powdered pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers. 178. The method of embodiment 177, wherein the one or more pharmaceutically acceptable carriers comprise human serum albumin and sodium chloride. 179. The method of embodiment 178, wherein the one or more pharmaceutically acceptable carriers comprise about 0.5 mg of human serum albumin per 100 units of BoNT / A. 180. The method of embodiment 178 or 179, wherein the one or more pharmaceutically acceptable carriers comprise about 0.9 mg of sodium chloride per 100 units of BoNT / A. 181. The method of any one of embodiments 178-180, wherein the human serum albumin is recombinant human serum albumin. 182. The method of any one of embodiments 163-181, wherein the method does not involve using a protease inhibitor. 183. The method of any one of embodiments 163-182, wherein the method does not involve using benzamidine hydrocholoride. 184. The method of any one of embodiments 163-183, wherein the powdered pharmaceutical composition comprises about 50 units, about 100 units or about 200 units of the 900 kDa BoNT / A complex. 185. The method of any one of embodiments 163-184, wherein the powdered pharmaceutical composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg. 4. DETAILED DESCRIPTION

[0066] The present invention provides compositions comprising Clostridium botulinum neurotoxin serotype A (BoNT / A) with improved stability. Such compositions are produced by a NAI-1541834789v1 31Attorney Docket No.13371-304-228 process comprising filtering a solution comprising BoNT / A with a filter that is not gamma- irradiated or a filter that contains no free radicals or a low amount of free radicals. The present invention also provides compositions comprising BoNT / A that are filtration products of a solution comprising BoNT / A, wherein the liquid compositions have a slow potency decline. The present invention also provides methods of producing liquid compositions comprising BoNT / A, wherein the methods comprise filtering a solution comprising BoNT / A with a filter that is not gamma-irradiated or a filter that contains no free radicals or a low amount of free radicals. The present invention further provides liquid compositions that are produced by such a method. The present invention also provides solid compositions which are dried products of a liquid composition described herein. Without wishing to be bound by any one theory, the present disclosure describes a correlation between the use of a gamma-irradiated filter in the filtration process for manufacturing a liquid BoNT / A composition and its loss of potency during storage. Without wishing to be bound by any one theory, the present disclosure also describes that free radicals on a gamma-irradiated filter may be introduced into a BoNT / A composition during the filtration process and thereby causing its loss of potency over time. Accordingly, the present invention provides BoNT / A compositions with improved properties such as a slow potency decline, and manufacturing methods for obtaining such BoNT / A compositions with an improved filtration process.

[0067] Further benefits of the present disclosure will be apparent to one skilled in the art. The embodiments and aspects described in this disclosure are intended to illustrate the invention and should not be deemed to narrow the scope of the invention. 4.1. Definitions

[0068] The singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0069] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to mean “A and B”, “A or B”, “A” or “B”.

[0070] The terms “about” and “approximately” generally refer to a range of numbers that one of skill in the art would consider equivalent to the recited value (i.e., having the same function or result). In many instances, the terms “about” and “approximately” may include numbers that are rounded to the nearest significant figure. In specific embodiments, the terms NAI-1541834789v1 32Attorney Docket No.13371-304-228 “about” and “approximately” shall be construed so as to allow normal variation as judged by a person of ordinary skill in the art, such as, for example, a variation within 20% or 10% or 5%. In specific embodiments, the terms “about” and “approximately” encompass the exact value recited.

[0071] “Animal product free” (“APF”) or “substantially animal product free” encompasses, respectively, the absence or substantial absence of blood derived, blood pooled and other animal derived products or compounds. “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 can include a botulinum toxin or a Clostridial botulinum bacterium. For example, an APF process or a substantially APF process means a process which is either substantially free or essentially free or entirely free of animal-derived proteins, such as immunoglobulins, meat digest, meat by-products and milk or dairy products or digests.

[0072] “Clostridium botulinum neurotoxin serotype A” or “BoNT / A” means 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 encompasses both a BoNT / A complex (e.g., the 300, 500, 760, and 900 kDa complexes) as well as pure BoNT / A toxin (i.e. the about 150 kDa neurotoxic molecule).

[0073] “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) protein. The BoNT / A complexes can be in the forms of, e.g., 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 an about 150 kDa BoNT / A molecule, hemagglutinin HA70, hemagglutinin HA34, hemagglutinin HA17, and nontoxic-nonhemagglutinin (NTNH) proteins. In one embodiment, the BoNT / A complex is onabotulinumtoxinA.

[0074] “150 kDa Clostridium botulinum serotype A neurotoxin” or “150 kDa BoNT / A” means a neurotoxin of approximately 150 kDa made 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 shown in Table 1. For example, in one embodiment, the 150 kDa BoNT / A used in the context of the NAI-1541834789v1 33Attorney Docket No.13371-304-228 present disclosure comprises (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, with disulfide bridges located between positions 429 and 453 and between positions 1234 and 1279.

[0075] “BoNT / A composition” refers to any composition comprising BoNT / A and encompasses both solid compositions and liquid compositions. In certain embodiments, a BoNT / A composition (e.g., a liquid composition or solid composition) described herein is a pharmaceutical composition. In specific embodiments, a BoNT / A composition (e.g., a liquid composition or solid composition) described herein is a drug product (i.e., a finished dosage form). In a specific embodiment, a BoNT / A composition described herein is in the form of a solution. In another specific embodiment, a BoNT / A composition described herein is in the form of powder (e.g., vacuum-dried powder, freeze-dried or lyophilized powder).

[0076] The term “carrier” used in connection with a pharmaceutical excipient refers to any and all solvents, dispersion media, preservatives, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.

[0077] The term “patient”, “subject”, “individual” and the like refers to humans.

[0078] “Pharmaceutical composition” means a formulation in which an active ingredient can be a BoNT / A. The word “formulation” means that there is at least one additional ingredient (such as, for example and not limited to, an albumin (such as a human serum albumin (HSA) or a recombinant human albumin) and / or sodium chloride) in the pharmaceutical composition in addition to a botulinum neurotoxin active ingredient. A pharmaceutical composition is therefore a formulation which is 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. The pharmaceutical composition can be in a freeze-dried, lyophilized or vacuum-dried condition, a solution formed after reconstitution of the freeze-dried, lyophilized or vacuum-dried form of the pharmaceutical composition with, for example, saline or water, or as a solution that does not require reconstitution. In one embodiment, the active ingredient is the botulinum toxin serotype A made natively by Clostridial bacteria. In one embodiment, the active ingredient is onabotulinumtoxinA. As stated, a pharmaceutical composition can be liquid or solid, for example vacuum-dried. Exemplary methods for formulating a botulinum toxin active ingredient pharmaceutical composition are disclosed in U.S. Patent Application Publication No. NAI-1541834789v1 34Attorney Docket No.13371-304-228 2003 / 0118598, filed Nov.5, 2002, herein incorporated by reference in its entirety. The pharmaceutical compositions can be vacuum-dried and suitable for administration by injection either subcutaneously or intramuscularly upon reconstitution with normal saline, comprising 900 kDa BoNT / A, human serum albumin (HSA), and sodium chloride. Preferably such pharmaceutical compositions comprise 0.5 mg of HSA and 0.9 mg of sodium chloride per 100 Units of BoNT / A. Most preferably such pharmaceutical compositions comprise 50, 100 or 200 Units of BoNT / A.

[0079] “Unit” or “U” refers to the LD50 dose or the dose determined by a cell-based potency assay (CBPA). The LD50dose is defined as the amount of BoNT / A that killed 50% of the mice injected with the BoNT / A. The CBPA dose is determined as described in US 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 by reference herein.

[0080] Unless the context requires otherwise, the terms “comprise,” “comprises,” and “comprising” are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, such that they indicate the inclusion of the recited feature but without excluding one or more other such features. However, it is understood that wherever aspects and embodiments are described herein with the language “comprise” (or “comprises” or “comprising”), otherwise analogous aspects described in terms of “consist of” (or “consists of” or “consisting of”) and / or “consist essentially of” (or “consists essentially of” or “consisting essentially of”) are also provided. 4.2. Clostridium Botulinum Neurotoxin Serotype A (BoNT / A)

[0081] A 150kDa BoNT / A molecule is a zinc endopeptidase which can specifically hydrolyze a peptide linkage of the intracellular, vesicle-associated protein (VAMP, also called synaptobrevin) 25 kiloDalton (kDa) synaptosomal associated protein (SNAP-25). A 150 kDa BoNT / A molecule is translated as a single chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally-occurring protease. This posttranslational processing yields a di-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC) held together by a single disulfide bond and noncovalent interactions.

[0082] Clostridial bacterium can produce botulinum toxin type A complexes in various NAI-1541834789v1 35Attorney Docket No.13371-304-228 forms, which include, and are not limited to, 900 kDa, 760 kDa, 500 kDa, and 300 kDa complexes (approximate molecular weights). The complexes (i.e., molecular weight greater than about 150 kDa) comprise a 150 kDa botulinum toxin molecule (the neurotoxic component) and one or more hemagglutinin (HA) proteins and / or non-toxin non-hemagglutinin (NTNH) protein.

[0083] In one embodiment, the 150 kDa BoNT / A molecule that can be used in the context of the present disclosure has a sequence shown in Table 1. In one embodiment, the 150 kDa BoNT / A molecule comprises a light chain (LC: residues 2–438, about 50 kDa) and a heavy chain (HC: residues 449–1296, about 100 kDa) . Residues 439–448 are the nicking site and are italicized.

[0084] Table 1. Preferred sequences of BoNT / A molecules. SEQ ID NO.1: amino acid sequence of the 150 kDa BoNT / A molecule MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT 50 FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS 100 TDLGRMLLTS IVRGIPFWGG STIDTELKVI DTNCINVIQP DGSYRSEELN 150 LVIIGPSADI IQFECKSFGH EVLNLTRNGY GSTQYIRFSP DFTFGFEESL 200 EVDTNPLLGA GKFATDPAVT LAHELIHAGH RLYGIAINPN RVFKVNTNAY 250 YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA 300 KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT 350 EDNFVKFFKV LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN 400 FNGQNTEINN MNFTKLKNFT GLFEFYKLLC VRGIITSKTK SLDKGYNK AL 450 NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE ITSDTNIEAA EENISLDLIQ 500 QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG KKYELDKYTM 550 FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA 600 AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD 650 DFVGALIFSG AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS 700 KRNEKWDEVY KYIVTNWLAK VNTQIDLIRK KMKEALENQA EATKAIINYQ 750 YNQYTEEEKN NINFNIDDLS SKLNESINKA MININKFLNQ CSVSYLMNSM 800 NAI-1541834789v1 36Attorney Docket No.13371-304-228 IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK VNNTLSTDIP 850 FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI 900 GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP 950 KYFNSISLNN EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK 1000 YSQMINISDY INRWIFVTIT NNRLNNSKIY INGRLIDQKP ISNLGNIHAS 1050 NNIMFKLDGC RDTHRYIWIK YFNLFDKELN EKEIKDLYDN QSNSGILKDF 1100 WGDYLQYDKP YYMLNLYDPN KYVDVNNVGI RGYMYLKGPR GSVMTTNIYL 1150 NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA 1200 GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI 1250 GFHQFNNIAK LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPL 1296 SEQ ID NO.2: amino acid sequence of the light chain (LC) of BoNT / A molecule PFVNKQFNY KDPVNGVDIA YIKIPNAGQM KIWVIPERDT 50FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS 100 TDLGRMLLTS IVRGIPFWGG STIDTELKVI DTNCINVIQP DGSYRSEELN 150 LVIIGPSADI IQFECKSFGH EVLNLTRNGY GSTQYIRFSP DFTFGFEESL 200 EVDTNPLLGA GKFATDPAVT LAHELIHAGH RLYGIAINPN RVFKVNTNAY 250 YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA 300 KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT 350 EDNFVKFFKV LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN 400 FNGQNTEINN MNFTKLKNFT GLFEFYKLLC VRGIITSK SEQ ID NO.3: amino acid sequence of the heavy chain (HC) of BoNT / A molecule AL 450 NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE ITSDTNIEAA EENISLDLIQ 500 QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG KKYELDKYTM 550 FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA 600 AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD 650 NAI-1541834789v1 37Attorney Docket No.13371-304-228 DFVGALIFSG AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS 700 KRNEKWDEVY KYIVTNWLAK VNTQIDLIRK KMKEALENQA EATKAIINYQ 750 YNQYTEEEKN NINFNIDDLS SKLNESINKA MININKFLNQ CSVSYLMNSM 800 IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK VNNTLSTDIP 850 FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI 900 GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP 950 KYFNSISLNN EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK 1000 YSQMINISDY INRWIFVTIT NNRLNNSKIY INGRLIDQKP ISNLGNIHAS 1050 NNIMFKLDGC RDTHRYIWIK YFNLFDKELN EKEIKDLYDN QSNSGILKDF 1100 WGDYLQYDKP YYMLNLYDPN KYVDVNNVGI RGYMYLKGPR GSVMTTNIYL 1150 NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA 1200 GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI 1250 GFHQFNNIAK LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPL 1296

[0085] In one embodiment, the BoNT / A described herein is present as a 900 kDa BoNT / A complex. In one embodiment, the BoNT / A described herein is present as a 900 kDa BoNT / A complex formed by the 150 kDa BoNT / A molecule and hemagglutinin HA70, hemagglutinin HA34, hemagglutinin HA17, and nontoxic-nonhemagglutinin (NTNH) proteins. 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.

[0086] In one embodiment, the 150 kDa BoNT / A molecule described herein comprises 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, with disulfide bridges located between positions 429 and 453 and between positions 1234 and 1279. 4.3. Production of BoNT / A Compositions

[0087] 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 NAI-1541834789v1 38Attorney Docket No.13371-304-228 over the age of twelve, cervical dystonia, glabellar line (facial) wrinkles and for treating hyperhydrosis. A commercially available botulinum toxin type A containing pharmaceutical composition is sold under the trademark BOTOX®(onabotulinumtoxinA), available commercially 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 sterile, vacuum-dried form. The botulinum toxin type A complex in BOTOX®is made from a culture of the Hall strain of Clostridium botulinum grown in a medium containing N-Z amine casein and yeast extract (i.e., non-APF process) and purified from the culture solution by a series of precipitation (including acid precipitation) steps to a crystalline complex consisting of the active high molecular weight toxin protein and an associated hemagglutinin protein. The crystalline complex is re-dissolved in a solution containing saline and albumin and sterile filtered using a gamma-irradiated filter (0.2 microns) prior to vacuum- drying. BOTOX®can be reconstituted with sterile, non-preserved saline prior to intramuscular injection. Each 100 unit vial of BOTOX®consists of about 5 ng of purified botulinum toxin type A complex, 0.5 mg human serum albumin, and 0.9 mg sodium chloride, vacuum-dried form and intended for reconstitution with sterile normal saline without a preservative (0.9% sodium chloride injection).

[0088] A number of steps are required to make the BoNT / A compositions described herein suitable for administration to a human or animal for a therapeutic, diagnostic, research or cosmetic purpose. In one embodiment, these steps can include obtaining a purified Clostridium botulinum neurotoxin serotype A (BoNT / A) and then compounding the purified BoNT / A. A first step can be to culture a Clostridial bacteria (e.g., the Hall strain of Clostridium botulinum), typically on agar plates, in an environment conducive to bacterial growth, such as in a warm anaerobic atmosphere. The culture step allows Clostridial colonies with desirable morphology and other characteristics to be obtained. In a second step, selected cultured Clostridial colonies can be fermented in a suitable medium. After a certain period of fermentation the Clostridial bacteria typically lyse and release Clostridial toxin (e.g., BoNT / A) into the medium. Thirdly, the toxin can be purified from the culture medium to obtain a bulk or raw BoNT / A toxin drug substance. Preferably, the BoNT / A toxin drug substance will not have been subjected to precipitation ( e.g., precipitation with cold ethanol, hydrochloric acid, and / or ammonium sulfate), e.g., during purification. Also preferred is BoNT / A toxin drug substance NAI-1541834789v1 39Attorney Docket No.13371-304-228 that has been purified by column chromatography, particularly BoNT / A toxin drug substance produced by purification using a hydrophobic interaction chromatography (HIC) column. When multiple chromatography columns are used to purify the BoNT / A, it is preferable that the toxin is purified using a process wherein a HIC column is used prior to all other chromatography columns. It is preferable that BoNT / A drug substance is produced by a process that qualifies for use under the Good Manufacturing Practice regulations promulgated by the U.S. Food and Drug Administration.

[0089] In some embodiments, the BoNT / A compositions described herein are obtained using a substantially, essentially or entirely animal protein free (APF) process. The process can comprise the following sequential steps: culturing Clostridium botulinum bacteria (e.g., the Hall strain of Clostridium botulinum) in a substantially APF culture medium; fermenting Clostridium botulinum bacteria from the culture medium in a substantially APF fermentation medium, harvesting the fermentation medium by removing cellular debris present in the fermentation medium using filtration or centrifugation; concentrating the harvested fermentation medium by filtration, such as by ultrafiltration (UF); diluting the concentrated fermentation medium by adding a buffer. Following dilution with the buffer, a substantially APF chromatographic process can be undertaken to obtain biologically active highly purified BoNT / A complex. In some embodiments, the APF process described herein does not involve using a protease inhibitor. In some embodiments, the APF process described herein does not involve using benzamidine hydrochloride.

[0090] The substantially APF culture and / or fermentation medium can contain a protein product obtained from yeast (e.g., yeast extract or yeast extract concentrate), or from a vegetable (e.g., wheat, soy, malt, Lupinus, corn, cottonseed, L. campestri seed, etc.). The APF culture and / or fermentation medium can further comprise a carbon source (e.g., glucose) and / or a source of salt (e.g., sodium chloride). In one embodiment, the medium used for fermentation of Clostridium botulinum is free of animal by-products and comprises approximately 10-100 g / L (e.g., 20-60 g / L) hydrolyzed soy (Hy-Soy), approximately 7.5 g / L glucose, and 5.0 g / L NaCl, as disclosed in U.S. Pat. No.7,354,740, which is incorporated by reference herein in its entirety. In one embodiment, the fermentation medium can comprise 3% w / v or 5% w / v HySoy; 1% w / v HyYeast; and 1% w / v glucose, as disclosed in U.S. Pat. No.8,129,139, which is incorporated by reference herein in its entirety. In one embodiment, the APF fermentation medium can comprise NAI-1541834789v1 40Attorney Docket No.13371-304-228 3.25 % w / v soy peptone type II, 1.2 % w / v yeast extract, 1.5 % w / v glucose, pH adjusted to 7.3 using sodium hydroxide.

[0091] An animal product free or substantially animal product free chromatographic system and process can be used to purify a clarified culture of Clostridium botulinum obtained from the APF fermentation processes described herein. The chromatographic system and process can include one column, two columns or three columns. For example, the chromatographic system and process can comprise one, two, or three steps: a hydrophobic interaction chromatography (HIC), and / or an anion exchange chromatography (AEX), and / or a cation exchange chromatograph. In some embodiments, the chromatographic process comprises a first step of subjecting the BoNT / A culture obtained from the APF process to a HIC, followed by subjecting the eluent from HIC to an AEX, and then followed by subjecting the captured solution from AEX to a CEX. In some embodiments, the chromatography-based purification process further comprises processing the eluent from the columns by diafiltration (DF) and filtering the processed eluent. In some embodiments, the chromatography-based purification process is as disclosed in U.S. Pat. No.8,129,139, which is incorporated by reference herein in its entirety.

[0092] The BoNT / A complex obtained from the APF fermentation and purification processes described herein is biologically active and highly purified. In one embodiment, the BoNT / A compositions described herein comprises less than 3% (e.g., less than about 2.5%, less than about 2.0%, less than about 1.8%, less than about 1.5%, less than about 1.2%, less than about 1.0%, less than about 0.8%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1%) of host cell protein. The level of the host cell protein can be assessed using methods known in the art, e.g., using Size Exclusion High Performance Liquid Chromatography (SEC-HPLC) or SEC-HPLC in conjugation with Multi-Angle Laser Light Scattering (MALLS) as disclosed in Lietzow et al. (2008) Protein J 27:420–425, which is incorporated herein by reference in its entirety. In one embodiment, the percentage of the host cell protein in the BoNT / A compositions described herein is determined using SEC-HPLC.

[0093] After stabilization in a suitable solution, the bulk BoNT / A drug substance can be compounded with one or more excipients (e.g., human serum albumin, such as recombinant human serum albumin, and sodium chloride) and can then be sterile filtered to make a pharmaceutical composition suitable for administration to a human. An example of such a NAI-1541834789v1 41Attorney Docket No.13371-304-228 BoNT / A is onabotulinumtoxinA. The pharmaceutical compositions can be in a liquid form (e.g., as a solution). Liquid pharmaceutical compositions can be stored and used directly for injection. The pharmaceutical compositions can also be made into a solid form (e.g., as powder) by drying from a liquid form (e.g., vacuum-drying, freeze-drying or lyophilization). Solid pharmaceutical compositions can be stored and reconstituted prior to injection. The BoNT / A pharmaceutical compositions described herein can comprise a 900 kDa Clostridium botulinum neurotoxin serotype A (BoNT / A) complex as an active pharmaceutical ingredient. The pharmaceutical composition can also include one or more excipients, buffers, carriers, stabilizers, preservatives and / or bulking agents. Such pharmaceutical compositions are preferably chemically and physically stable such that the BoNT / A active pharmaceutical ingredient remains suitable for use as a pharmaceutical product following storage. BoNT / A products may be stored at room temperature, in refrigerated conditions, or below 0°C. It is preferable that the BoNT / A remains stable during storage for at least about 12 months, more preferably at least about 18 months.

[0094] In some embodiments, an animal product free composition is free of human derived human serum albumin (HSA). In some embodiments, an animal product free composition is free of animal derived nucleases. In some embodiments, an animal product free composition comprises recombinantly produced HSA. In some embodiments, the recombinantly produced HSA is commercially available or known in the art. In some embodiments, an animal product free composition is ammonium sulfate free. In some embodiments, an animal product free composition comprises chromatography resin.

[0095] In various aspects and embodiments, a liquid BoNT / A composition described herein is filtered from a solution comprising BoNT / A drug substance (for example, once the BoNT / A drug substance is produced, e.g., by a method described herein) and excipients. In various aspects and embodiments, a solid BoNT / A composition described herein is dried (e.g., vacuum- dried, freeze-dried or lyophilized) from such a liquid BoNT / A composition.

[0096] In one aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter that is not gamma-irradiated to produce said liquid composition.

[0097] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein no free radicals are detectable on the filter. NAI-1541834789v1 42Attorney Docket No.13371-304-228

[0098] The amount of free radicals on a filter can be determined using any method described herein or known in the art suitable for measuring the amount of free radicals. In certain embodiments, the amount of free radicals on the filter is determined using ESR.

[0099] In another aspect, provided herein is method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of free radicals on the filter is less than that on a gamma-irradiated filter of the same type.

[0100] The amount of free radicals on a filter can be determined using any method described herein or known in the art suitable for measuring the amount of free radicals. In certain embodiments, the amount of free radicals on the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) is determined using ESR. In certain embodiments, the amount of free radicals on the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of free radicals on the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) and the amount of free radicals on the gamma-irradiated filter are determined using the same method, e.g., ESR.

[0101] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of free radicals on the filter is less than 250 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 200 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 150 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 100 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 50 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 10 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 109spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 108spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 107spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the NAI-1541834789v1 43Attorney Docket No.13371-304-228 filter is less than 1 x 106spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 105spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 104spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 103spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 102spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 10 spins / mg as determined using ESR. In certain embodiments, the amount of free radicals on the filter is less than the stated amount described in this paragraph per 500 cm² filtration area as determined using ESR. In specific embodiments, no free radicals are detectable on the filter as determined using ESR.

[0102] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein no Carbon-centered radicals are detectable in an aqueous solution containing a spin-trap molecule following exposure to the filter. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0103] The amount of Carbon-centered radicals can be determined using any method described herein or known in the art suitable for measuring the amount of Carbon-centered radicals. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR.

[0104] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule following exposure to the filter is less than the amount of Carbon-centered radicals in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. The term “substantially identical” is used in this context to describe that the reference aqueous solution is identical or sufficiently similar to the aqueous solution (for example, in terms of their ingredients and the respective concentrations of the ingredients), such that the reference aqueous solution NAI-1541834789v1 44Attorney Docket No.13371-304-228 can serve the purpose of being an appropriate control for comparison in the opinion of a person of ordinary skill in the art. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0105] In certain embodiments, the exposure is for about 15 minutes. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 60 minutes. In certain embodiments, the exposure is for about 2 hours. In certain embodiments, the exposure is for about 5 hours. In certain embodiments, the exposure is for about 10 hours. In certain embodiments, the exposure is for about 15 hours. In certain embodiments, the exposure is for about 20 hours. In certain embodiments, the exposure is for about 30 hours. In certain embodiments, the exposure is for about 2 days. In certain embodiments, the exposure is for about 3 days. In certain embodiments, the exposure is for less than 30 minutes. In certain embodiments, the exposure is for 30 to 60 minutes. In certain embodiments, the exposure is for 1 to 5 hours. In certain embodiments, the exposure is for 5 to 10 hours. In certain embodiments, the exposure is for 10 to 15 hours. In certain embodiments, the exposure is for 15 to 20 hours. In certain embodiments, the exposure is for 20 to 30 hours. In certain embodiments, the exposure is for 1 to 2 days. In certain embodiments, the exposure is for 2 to 3 days. In certain embodiments, the exposure is for more than 3 days. In certain embodiments, a person skilled in the art would be able to pick an appropriate period of time for the exposure.

[0106] The amount of Carbon-centered radicals can be determined using any method described herein or known in the art suitable for measuring the amount of Carbon-centered radicals. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) is determined using ESR. In certain embodiments, the amount of Carbon-centered radicals in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) and the amount of Carbon-centered radicals in the reference aqueous solution containing the spin-trap molecule following exposure NAI-1541834789v1 45Attorney Docket No.13371-304-228 to the gamma-irradiated filter are determined using the same method, e.g., ESR.

[0107] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 2 x 10-7M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon- centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M as determined using ESR after being exposed to the filter for a period of time. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than the stated amount described in this paragraph per 500 cm² filtration area as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for a period of time. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0108] In certain embodiments, the period of time is about 15 minutes. In certain embodiments, the period of time is about 30 minutes. In certain embodiments, the period of time is about 60 minutes. In certain embodiments, the period of time is about 2 hours. In certain NAI-1541834789v1 46Attorney Docket No.13371-304-228 embodiments, the period of time is about 5 hours. In certain embodiments, the period of time is about 10 hours. In certain embodiments, the period of time is about 15 hours. In certain embodiments, the period of time is about 20 hours. In certain embodiments, the period of time is about 30 hours. In certain embodiments, the period of time is about 2 days. In certain embodiments, the period of time is about 3 days. In certain embodiments, the period of time is less than 30 minutes. In certain embodiments, the period of time is 30 to 60 minutes. In certain embodiments, the period of time is 1 to 5 hours. In certain embodiments, the period of time is 5 to 10 hours. In certain embodiments, the period of time is 10 to 15 hours. In certain embodiments, the period of time is 15 to 20 hours. In certain embodiments, the period of time is 20 to 30 hours. In certain embodiments, the period of time is 1 to 2 days. In certain embodiments, the period of time is 2 to 3 days. In certain embodiments, the period of time is more than 3 days. In certain embodiments, a person skilled in the art would be able to pick an appropriate period of time for the exposure.

[0109] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 2 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon- centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M as determined using ESR after being exposed to the filter for about 30 minutes. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is NAI-1541834789v1 47Attorney Docket No.13371-304-228 less than the stated amount described in this paragraph per 500 cm² filtration area as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0110] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 2 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon- centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M as determined using ESR after being exposed to the filter for about 15 hours. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than the stated amount described in this paragraph per 500 cm² filtration area as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, no Carbon- centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0111] In another aspect, provided herein is a method of producing a liquid composition NAI-1541834789v1 48Attorney Docket No.13371-304-228 comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein no degradation product of a spin-trap molecule is detectable in an aqueous solution containing the spin-trap molecule following exposure to the filter. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0112] The amount of degradation product of a spin-trap molecule can be determined using any method described herein or known in the art suitable for measuring the amount of degradation product of the spin-trap molecule. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR

[0113] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, wherein the amount of degradation product of a spin- trap molecule in an aqueous solution containing the spin-trap molecule following exposure to the filter is less than the amount of degradation product of the spin-trap molecule in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. The term “substantially identical” is used in this context to describe that the reference aqueous solution is identical or sufficiently similar to the aqueous solution (for example, in terms of their ingredients and the respective concentrations of the ingredients), such that the reference aqueous solution can serve the purpose of being an appropriate control for comparison in the opinion of a person of ordinary skill in the art. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0114] In certain embodiments, the exposure is for about 15 minutes. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 60 minutes. In certain embodiments, the exposure is for about 2 hours. In certain embodiments, the exposure is for about 5 hours. In certain embodiments, the exposure is for about 10 hours. In certain embodiments, the exposure is for about 15 hours. In certain embodiments, the exposure is for about 20 hours. In certain embodiments, the exposure is for NAI-1541834789v1 49Attorney Docket No.13371-304-228 about 30 hours. In certain embodiments, the exposure is for about 2 days. In certain embodiments, the exposure is for about 3 days. In certain embodiments, the exposure is for less than 30 minutes. In certain embodiments, the exposure is for 30 to 60 minutes. In certain embodiments, the exposure is for 1 to 5 hours. In certain embodiments, the exposure is for 5 to 10 hours. In certain embodiments, the exposure is for 10 to 15 hours. In certain embodiments, the exposure is for 15 to 20 hours. In certain embodiments, the exposure is for 20 to 30 hours. In certain embodiments, the exposure is for 1 to 2 days. In certain embodiments, the exposure is for 2 to 3 days. In certain embodiments, the exposure is for more than 3 days. In certain embodiments, a person skilled in the art would be able to pick an appropriate period of time for the exposure.

[0115] The amount of degradation product of a spin-trap molecule can be determined using any method described herein or known in the art suitable for measuring the amount of degradation product of the spin-trap molecule. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) is determined using ESR. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) and the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter are determined using the same method, e.g., ESR.

[0116] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the NAI-1541834789v1 50Attorney Docket No.13371-304-228 amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.5 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than the stated amount per 500 cm² filtration area (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule (e.g., as determined using ESR) after being exposed to the filter for a period of time. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0117] In certain embodiments, the period of time is about 15 minutes. In certain embodiments, the period of time is about 30 minutes. In certain embodiments, the period of time is about 60 minutes. In certain embodiments, the period of time is about 2 hours. In certain embodiments, the period of time is about 5 hours. In certain embodiments, the period of time is about 10 hours. In certain embodiments, the period of time is about 15 hours. In certain embodiments, the period of time is about 20 hours. In certain embodiments, the period of time is about 30 hours. In certain embodiments, the period of time is about 2 days. In certain embodiments, the period of time is about 3 days. In certain embodiments, the period of time is NAI-1541834789v1 51Attorney Docket No.13371-304-228 less than 30 minutes. In certain embodiments, the period of time is 30 to 60 minutes. In certain embodiments, the period of time is 1 to 5 hours. In certain embodiments, the period of time is 5 to 10 hours. In certain embodiments, the period of time is 10 to 15 hours. In certain embodiments, the period of time is 15 to 20 hours. In certain embodiments, the period of time is 20 to 30 hours. In certain embodiments, the period of time is 1 to 2 days. In certain embodiments, the period of time is 2 to 3 days. In certain embodiments, the period of time is more than 3 days. In certain embodiments, a person skilled in the art would be able to pick an appropriate period of time for the exposure.

[0118] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than the stated amount per 500 cm² filtration area (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO. NAI-1541834789v1 52Attorney Docket No.13371-304-228

[0119] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.5 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than the stated amount per 500 cm² filtration area (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO. NAI-1541834789v1 53Attorney Docket No.13371-304-228

[0120] In another aspect, provided herein is a method of producing a liquid composition comprising BoNT / A, said method comprising reconstituting a solid composition (e.g., a powdered pharmaceutical composition) comprising BoNT / A (for example, with a sodium chloride solution).

[0121] In another aspect, provided herein is a method of producing a solid composition (e.g., a powdered pharmaceutical composition) comprising BoNT / A, said method comprising drying (e.g., vacuum-drying, freeze-drying or lyophilizing) a liquid composition described herein or a liquid composition produced using a method described herein.

[0122] In various embodiments and aspects, the filter used to produce a liquid composition described herein is not sterilized using gamma-irradiation. In various embodiments and aspects, the filter used to produce a liquid composition described herein is sterilized by a method other than gamma-irradiation. In various embodiments and aspects, the filter used to produce a liquid composition described herein is sterilized by steaming. In various embodiments and aspects, the filter used to produce a liquid composition described herein is autoclaved.

[0123] In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a hydrophilic membrane.

[0124] In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polyvinylidene fluoride (PVDF) membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polycarbonate membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polyester membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polyethersulfone membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polypropylene membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a cellulose acetate membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a mixed cellulose ester membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a nylon membrane.

[0125] In various embodiments and aspects, the filter used to produce a liquid composition described herein has a pore size of about 0.1 µm to about 0.45 µm (e.g., about 0.1 µm, about 0.2 NAI-1541834789v1 54Attorney Docket No.13371-304-228 µm, about 0.22 µm, or about 0.45 µm). In specific embodiments, the filter used to produce a liquid composition described herein has a pore size of about 0.22 µm.

[0126] In various embodiments and aspects, the filter used to produce a liquid composition described herein has a filtration area of about 50 cm2to about 1000 cm2(e.g., about 50 cm2, about 100 cm2, about 200 cm2, about 300 cm2, about 500 cm2, or about 1000 cm2). In specific embodiments, the filter used to produce a liquid composition described herein has a filtration area of about 100 cm2. In specific embodiments, the filter used to produce a liquid composition described herein has a filtration area of about 500 cm2.

[0127] In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-20 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma- irradiated Millipak®-40 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-60 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-100 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-200 filter.

[0128] In various embodiments and aspects, a gamma-irradiated filter described herein, which is used for purposes of comparison, is substantially identical to the filter used to produce a liquid composition described herein, except that the gamma-irradiated filter is sterilized by gamma-irradiation while the filter used to produce the liquid composition is not. The term “substantially identical” is used in this context to describe that the gamma-irradiated filter is identical or sufficiently similar to the filter used to produce the liquid composition (for example, in terms of their materials and dimensions (e.g., pore sizes and / or filtration areas)), such that the gamma-irradiated filter can serve the purpose of being an appropriate control for comparison in the opinion of a person of ordinary skill in the art.

[0129] In various embodiments and aspects, a gamma-irradiated filter described herein comprises a hydrophilic membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a hydrophilic membrane.

[0130] In various embodiments and aspects, a gamma-irradiated filter described herein comprises a polyvinylidene fluoride (PVDF) membrane. In various embodiments and aspects, a NAI-1541834789v1 55Attorney Docket No.13371-304-228 gamma-irradiated filter described herein comprises a polycarbonate membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a polyester membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a polyethersulfone membrane. In various embodiments and aspects, a gamma- irradiated filter described herein comprises a polypropylene membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a cellulose acetate membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a mixed cellulose ester membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a nylon membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polyvinylidene fluoride (PVDF) membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polycarbonate membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polyester membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polyethersulfone membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polypropylene membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a cellulose acetate membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a mixed cellulose ester membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a nylon membrane.

[0131] In various embodiments and aspects, a gamma-irradiated filter described herein has a pore size of about 0.1 µm to about 0.45 µm (e.g., about 0.1 µm, about 0.2 µm, about 0.22 µm, or about 0.45 µm). In specific embodiments, the gamma-irradiated filter has a pore size of about 0.22 µm. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a pore NAI-1541834789v1 56Attorney Docket No.13371-304-228 size of about 0.1 µm to about 0.45 µm (e.g., about 0.1 µm, about 0.2 µm, about 0.22 µm, or about 0.45 µm). In specific embodiments, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a pore size of about 0.22 µm.

[0132] In various embodiments and aspects, a gamma-irradiated filter described herein has a filtration area of about 50 cm2to about 1000 cm2(e.g., about 50 cm2, about 100 cm2, about 200 cm2, about 300 cm2, about 500 cm2, or about 1000 cm2). In specific embodiments, the gamma- irradiated filter has a filtration area of about 100 cm2. In specific embodiments, the gamma- irradiated filter has a filtration area of about 500 cm2. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a filtration area of about 50 cm2to about 1000 cm2(e.g., about 50 cm2, about 100 cm2, about 200 cm2, about 300 cm2, about 500 cm2, or about 1000 cm2). In specific embodiments, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a filtration area of about 100 cm2. In specific embodiments, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a filtration area of about 500 cm2.

[0133] In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma-irradiated Millipak®-20 filter. In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma-irradiated Millipak®-40 filter. In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma-irradiated Millipak®-60 filter. In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma- irradiated Millipak®-100 filter. In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma-irradiated Millipak®-200 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma- irradiated Millipak®-20 filter, and the gamma-irradiated filter used for comparison is a gamma- irradiated Millipak®-20 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-40 filter, and the gamma-irradiated filter used for comparison is a gamma-irradiated Millipak®-40 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-60 filter, and the gamma-irradiated filter used for comparison is a gamma-irradiated Millipak®-60 filter. In various embodiments and aspects, the NAI-1541834789v1 57Attorney Docket No.13371-304-228 filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-100 filter, and the gamma-irradiated filter used for comparison is a gamma-irradiated Millipak®-100 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-200 filter, and the gamma- irradiated filter used for comparison is a gamma-irradiated Millipak®-200 filter.

[0134] In various embodiments and aspects, a liquid composition described herein is animal product free. In various embodiments and aspects, a solution described herein comprising BoNT / A is animal product free. In various embodiments and aspects, both the liquid composition and the solution comprising BoNT / A are animal product free. In various embodiments and aspects, a solid composition described herein is animal product free. In various embodiments and aspects, a liquid composition described herein does not contain a protease inhibitor. In certain embodiments, a liquid composition described herein does not contain benzamidine hydrocholoride. In various embodiments and aspects, a solution described herein comprising BoNT / A does not contain a protease inhibitor. In certain embodiments, a solution described herein comprising BoNT / A does not contain benzamidine hydrocholoride. In various embodiments and aspects, both the liquid composition and the solution comprising BoNT / A do not contain a protease inhibitor. In certain embodiments, both the liquid composition and the solution comprising BoNT / A do not contain benzamide hydrocholoride. 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 hydrocholoride.

[0135] In various embodiments and aspects, a liquid composition described herein further comprises one or more pharmaceutically acceptable carriers. In various embodiments and aspects, a solution described herein comprising BoNT / A further comprises one or more pharmaceutically acceptable carriers. In various embodiments and aspects, both the liquid composition and the solution comprising BoNT / A further comprise one or more pharmaceutically acceptable carriers. In various embodiments and aspects, a solid composition described herein further comprises one or more pharmaceutically acceptable carriers. In specific embodiments, the one or more pharmaceutically acceptable carriers comprise human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of BoNT / A). In specific embodiments, the one or more pharmaceutically acceptable carriers comprise sodium chloride NAI-1541834789v1 58Attorney Docket No.13371-304-228 (e.g., about 0.9 mg of sodium chloride per 100 units of BoNT / A). In specific embodiments, the one or more pharmaceutically acceptable carriers comprise human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of BoNT / A) and sodium chloride (e.g., about 0.9 mg of sodium chloride per 100 units of BoNT / A). In certain embodiments, the human serum albumin is recombinant human serum albumin. In a specific embodiment, the recombinant human serum albumin is animal product free. In a specific embodiment, the recombinant human serum albumin is not produced from an animal. In a specific embodiment, the recombinant human serum albumin is produced from a microorganism, such as bacteria. In a specific embodiment, the recombinant human serum albumin is produced from a plant-based expression system. In certain embodiments, the human serum albumin is human plasma-derived human serum albumin.

[0136] In various aspects and embodiments, a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) comprises about 50 units of BoNT / A. In various aspects and embodiments, a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) comprises about 100 units of BoNT / A. In various aspects and embodiments, a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) comprises about 200 units of BoNT / A.

[0137] In various embodiments and aspects, a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

[0138] In various aspects and embodiments, the method of producing a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) does not involve using a protease inhibitor. In certain embodiments, the method of producing a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) does not involve using benzamidine hydrocholoride.

[0139] In various aspects and embodiments, the method of producing a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) further comprises producing the BoNT / A drug substance.

[0140] In certain embodiments, the BoNT / A drug substance is produced as described in Example 1, Example 2 or Example 3.

[0141] In certain embodiments, the BoNT / A drug substance is produced using one or more NAI-1541834789v1 59Attorney Docket No.13371-304-228 steps as described in Example 1, Example 2 or Example 3.

[0142] In certain embodiments, the BoNT / A drug substance is produced using a process that does not comprise one or more steps described in Example 1 or Example 2.

[0143] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises one or more steps of column chromatography (e.g., one or more steps of column chromatography performed during purification of the BoNT / A drug substance).

[0144] In preferred embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise hydrophobic interaction chromatography. In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise anion exchange chromatography. In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise cation exchange chromatography.

[0145] In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise hydrophobic interaction chromatography and anion exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, hydrophobic interaction chromatography and anion exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, anion exchange chromatography and hydrophobic interaction chromatography.

[0146] In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise hydrophobic interaction chromatography and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, hydrophobic interaction chromatography and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, cation exchange chromatography and hydrophobic interaction chromatography. NAI-1541834789v1 60Attorney Docket No.13371-304-228

[0147] In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise anion exchange chromatography and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, anion exchange chromatography and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, cation exchange chromatography and anion exchange chromatography.

[0148] In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise hydrophobic interaction chromatography, anion exchange chromatography, and cation exchange chromatography. In a preferred embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, hydrophobic interaction chromatography, anion exchange chromatography, and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, hydrophobic interaction chromatography, cation exchange chromatography, and anion exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, anion exchange chromatography, cation exchange chromatography, and hydrophobic interaction chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, anion exchange chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, cation exchange chromatography, hydrophobic interaction chromatography, and anion exchange chromatography.

[0149] In certain embodiments, the BoNT / A drug substance is produced by a process that NAI-1541834789v1 61Attorney Docket No.13371-304-228 does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate (e.g., does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with hydrochloric acid (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with ammonia sulfate (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol (e.g., during purification of the BoNT / A drug substance) and does not comprise a step of precipitation with hydrochloric acid (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol (e.g., during purification of the BoNT / A drug substance) and does not comprise a step of precipitation with ammonia sulfate (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A is produced by a process that does not comprise a step of precipitation with hydrochloric acid (e.g., during purification of the BoNT / A drug substance) and does not comprise a step of precipitation with ammonia sulfate (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol (e.g., during purification of the BoNT / A drug substance), does not comprise a step of precipitation with hydrochloric acid (e.g., during purification of the BoNT / A drug substance) and does not comprise a step of precipitation with ammonia sulfate (e.g., during purification of the BoNT / A drug substance).

[0150] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, and filtering at 0.2 μm with added ammonium sulfate. NAI-1541834789v1 62Attorney Docket No.13371-304-228

[0151] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps and in the following order: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, and filtering at 0.2 μm with added ammonium sulfate.

[0152] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, and subjecting the filtrate to hydrophobic interaction chromatography (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0153] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises one or more of the following steps: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, and subjecting the filtrate to hydrophobic interaction chromatography (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0154] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps and in the following order: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, and subjecting the filtrate to hydrophobic interaction NAI-1541834789v1 63Attorney Docket No.13371-304-228 chromatography (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0155] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, loading the filtrate onto a hydrophobic interaction column, eluting with a descending gradient of ammonium sulfate, and isolating the product peak (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0156] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps and in the following order: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, loading the filtrate onto a hydrophobic interaction column, eluting with a descending gradient of ammonium sulfate, and isolating the product peak (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0157] In certain embodiments, the BoNT / A drug substance is produced by a process that does not involve using a protease inhibitor. In certain embodiments, the BoNT / A drug substance is produced by a process that does not involve using benzamidine hydrocholoride.

[0158] In one aspect, provided herein is a method of producing a powdered pharmaceutical composition comprising a 900 kDa BoNT / A complex (e.g., a powdered pharmaceutical composition comprising about 50 units, about 100 units or about 200 units of a 900 kDa BoNT / A complex), human serum albumin, and sodium chloride, said method comprising filtering a solution comprising the 900 kDa BoNT / A complex with a filter that is not gamma-irradiated to produce a liquid composition comprising the 900 kDa BoNT / A complex, and drying (e.g., vacuum-drying, freeze-drying or lyophilizing) the liquid composition to produce the powdered pharmaceutical composition. In certain embodiments, the method comprises producing the 900 NAI-1541834789v1 64Attorney Docket No.13371-304-228 kDa BoNT / A complex by a process that comprises one or more steps of column chromatography. In specific embodiments, the one or more steps of column chromatography are performed during purification of the 900 kDa BoNT / A complex. In specific embodiments, the one or more steps of column chromatography comprise hydrophobic interaction chromatography. In specific embodiments, the one or more steps of column chromatography comprise anion exchange chromatography. In specific embodiments, the one or more steps of column chromatography comprise cation exchange chromatography. In certain embodiments, the method comprises producing the 900 kDa BoNT / A complex by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate (e.g., during purification of the 900 kDa BoNT / A complex). In specific embodiments, the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum (e.g., the Type A Hall strain of Clostridium botulinum). In specific embodiments, the 900 kDa BoNT / A complex is onabotulinumtoxin A. In specific embodiments, the powdered pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers. In a specific embodiment, the one or more pharmaceutically acceptable carriers comprise human serum albumin, such as recombinant human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of the 900 kDa BoNT / A complex) and sodium chloride (e.g., about 0.9 mg of sodium chloride per 100 units of the 900 kDa BoNT / A complex). In specific embodiments, the powdered pharmaceutical composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg. In specific embodiments, the method does not involve using a protease inhibitor. In a specific embodiment, the method does not involve using benzamidine hydrocholoride. 4.4. BoNT / A Compositions

[0159] In one aspect, provided herein is a liquid composition comprising Clostridium botulinum neurotoxin serotype A (BoNT / A), wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter that is not gamma-irradiated.

[0160] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, said filtration is performed with a filter, and wherein no free radicals are detectable on the filter.

[0161] The amount of free radicals on a filter can be determined using any method described NAI-1541834789v1 65Attorney Docket No.13371-304-228 herein or known in the art suitable for measuring the amount of free radicals. In certain embodiments, the amount of free radicals on the filter is determined using Electron Spin Resonance (ESR).

[0162] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, said filtration is performed with a filter, and wherein the amount of free radicals on the filter is less than that on a gamma-irradiated filter of the same type.

[0163] The amount of free radicals on a filter can be determined using any method described herein or known in the art suitable for measuring the amount of free radicals. In certain embodiments, the amount of free radicals on the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) is determined using ESR. In certain embodiments, the amount of free radicals on the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of free radicals on the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) and the amount of free radicals on the gamma-irradiated filter are determined using the same method, e.g., ESR.

[0164] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, said filtration is performed with a filter, and wherein the amount of free radicals on the filter is less than 250 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 200 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 150 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 100 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 50 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 10 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 1010spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 109spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 108spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 107spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the NAI-1541834789v1 66Attorney Docket No.13371-304-228 filter is less than 1 x 106spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 105spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 104spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 103spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 1 x 102spins / mg as determined using ESR. In specific embodiments, the amount of free radicals on the filter is less than 10 spins / mg as determined using ESR. In certain embodiments, the amount of free radicals on the filter is less than the stated amount described in this paragraph per 500 cm² filtration area as determined using ESR. In specific embodiments, no free radicals are detectable on the filter as determined using ESR.

[0165] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, said filtration is performed with a filter, and wherein no Carbon-centered radicals are detectable in an aqueous solution containing a spin-trap molecule following exposure to the filter. In certain embodiments, the spin-trap molecule is N-tert-butyl-α-phenylnitrone (PBN). In certain embodiments, the spin-trap molecule is 5,5-dimethyl-pyrroline N-oxide (DMPO). In certain embodiments, the spin-trap molecule is 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO).

[0166] The amount of Carbon-centered radicals can be determined using any method described herein or known in the art suitable for measuring the amount of Carbon-centered radicals. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR.

[0167] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, said filtration is performed with a filter, wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule following exposure to the filter is less than the amount of Carbon-centered radicals in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. The term “substantially identical” is used in this context to describe that the reference aqueous solution is identical or sufficiently similar to the aqueous solution (for example, in terms of their ingredients NAI-1541834789v1 67Attorney Docket No.13371-304-228 and the respective concentrations of the ingredients), such that the reference aqueous solution can serve the purpose of being an appropriate control for comparison in the opinion of a person of ordinary skill in the art. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0168] In certain embodiments, the exposure is for about 15 minutes. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 60 minutes. In certain embodiments, the exposure is for about 2 hours. In certain embodiments, the exposure is for about 5 hours. In certain embodiments, the exposure is for about 10 hours. In certain embodiments, the exposure is for about 15 hours. In certain embodiments, the exposure is for about 20 hours. In certain embodiments, the exposure is for about 30 hours. In certain embodiments, the exposure is for about 2 days. In certain embodiments, the exposure is for about 3 days. In certain embodiments, the exposure is for less than 30 minutes. In certain embodiments, the exposure is for 30 to 60 minutes. In certain embodiments, the exposure is for 1 to 5 hours. In certain embodiments, the exposure is for 5 to 10 hours. In certain embodiments, the exposure is for 10 to 15 hours. In certain embodiments, the exposure is for 15 to 20 hours. In certain embodiments, the exposure is for 20 to 30 hours. In certain embodiments, the exposure is for 1 to 2 days. In certain embodiments, the exposure is for 2 to 3 days. In certain embodiments, the exposure is for more than 3 days. In certain embodiments, a person skilled in the art would be able to pick an appropriate period of time for the exposure.

[0169] The amount of Carbon-centered radicals can be determined using any method described herein or known in the art suitable for measuring the amount of Carbon-centered radicals. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) is determined using ESR. In certain embodiments, the amount of Carbon-centered radicals in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) and the amount of Carbon-centered NAI-1541834789v1 68Attorney Docket No.13371-304-228 radicals in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter are determined using the same method, e.g., ESR.

[0170] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 2 x 10-7M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon- centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M as determined using ESR after being exposed to the filter for a period of time. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than the stated amount described in this paragraph per 500 cm² filtration area as determined using ESR after being exposed to the filter for a period of time. In specific embodiments, no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for a period of time. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0171] In certain embodiments, the period of time is about 15 minutes. In certain embodiments, the period of time is about 30 minutes. In certain embodiments, the period of time NAI-1541834789v1 69Attorney Docket No.13371-304-228 is about 60 minutes. In certain embodiments, the period of time is about 2 hours. In certain embodiments, the period of time is about 5 hours. In certain embodiments, the period of time is about 10 hours. In certain embodiments, the period of time is about 15 hours. In certain embodiments, the period of time is about 20 hours. In certain embodiments, the period of time is about 30 hours. In certain embodiments, the period of time is about 2 days. In certain embodiments, the period of time is about 3 days. In certain embodiments, the period of time is less than 30 minutes. In certain embodiments, the period of time is 30 to 60 minutes. In certain embodiments, the period of time is 1 to 5 hours. In certain embodiments, the period of time is 5 to 10 hours. In certain embodiments, the period of time is 10 to 15 hours. In certain embodiments, the period of time is 15 to 20 hours. In certain embodiments, the period of time is 20 to 30 hours. In certain embodiments, the period of time is 1 to 2 days. In certain embodiments, the period of time is 2 to 3 days. In certain embodiments, the period of time is more than 3 days. In certain embodiments, a person skilled in the art would be able to pick an appropriate period of time for the exposure.

[0172] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 2 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon- centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M as determined using ESR after being exposed to the filter for about 30 minutes. In certain embodiments, the NAI-1541834789v1 70Attorney Docket No.13371-304-228 amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than the stated amount described in this paragraph per 500 cm² filtration area as determined using ESR after being exposed to the filter for about 30 minutes. In specific embodiments, no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0173] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 2 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon- centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M as determined using ESR after being exposed to the filter for about 15 hours. In certain embodiments, the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule is less than the stated amount described in this paragraph per 500 cm² filtration area as determined using ESR after being exposed to the filter for about 15 hours. In specific embodiments, no Carbon- centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO. NAI-1541834789v1 71Attorney Docket No.13371-304-228

[0174] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein no degradation product of a spin-trap molecule is detectable in an aqueous solution containing the spin-trap molecule following exposure to the filter. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin- trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0175] The amount of degradation product of a spin-trap molecule can be determined using any method described herein or known in the art suitable for measuring the amount of degradation product of the spin-trap molecule. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR

[0176] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule following exposure to the filter is less than the amount of degradation product of the spin-trap molecule in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution. The term “substantially identical” is used in this context to describe that the reference aqueous solution is identical or sufficiently similar to the aqueous solution (for example, in terms of their ingredients and the respective concentrations of the ingredients), such that the reference aqueous solution can serve the purpose of being an appropriate control for comparison in the opinion of a person of ordinary skill in the art. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0177] In certain embodiments, the exposure is for about 15 minutes. In certain embodiments, the exposure is for about 30 minutes. In certain embodiments, the exposure is for about 60 minutes. In certain embodiments, the exposure is for about 2 hours. In certain embodiments, the exposure is for about 5 hours. In certain embodiments, the exposure is for about 10 hours. In certain embodiments, the exposure is for about 15 hours. In certain embodiments, the exposure is for about 20 hours. In certain embodiments, the exposure is for NAI-1541834789v1 72Attorney Docket No.13371-304-228 about 30 hours. In certain embodiments, the exposure is for about 2 days. In certain embodiments, the exposure is for about 3 days. In certain embodiments, the exposure is for less than 30 minutes. In certain embodiments, the exposure is for 30 to 60 minutes. In certain embodiments, the exposure is for 1 to 5 hours. In certain embodiments, the exposure is for 5 to 10 hours. In certain embodiments, the exposure is for 10 to 15 hours. In certain embodiments, the exposure is for 15 to 20 hours. In certain embodiments, the exposure is for 20 to 30 hours. In certain embodiments, the exposure is for 1 to 2 days. In certain embodiments, the exposure is for 2 to 3 days. In certain embodiments, the exposure is for more than 3 days. In certain embodiments, a person skilled in the art would be able to pick an appropriate period of time for the exposure.

[0178] The amount of degradation product of a spin-trap molecule can be determined using any method described herein or known in the art suitable for measuring the amount of degradation product of the spin-trap molecule. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) is determined using ESR. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter (i.e., the filter used to produce the liquid composition comprising BoNT / A) and the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter are determined using the same method, e.g., ESR.

[0179] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of NAI-1541834789v1 73Attorney Docket No.13371-304-228 degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.5 x 10-7M (e.g., as determined using ESR)after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin- trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin- trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M (e.g., as determined using ESR) after being exposed to the filter for a period of time. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than the stated amount per 500 cm² filtration area (e.g., as determined using ESR) after being exposed to the filter for a period of time. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule (e.g., as determined using ESR) after being exposed to the filter for a period of time. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO.

[0180] In certain embodiments, the period of time is about 15 minutes. In certain embodiments, the period of time is about 30 minutes. In certain embodiments, the period of time is about 60 minutes. In certain embodiments, the period of time is about 2 hours. In certain embodiments, the period of time is about 5 hours. In certain embodiments, the period of time is about 10 hours. In certain embodiments, the period of time is about 15 hours. In certain embodiments, the period of time is about 20 hours. In certain embodiments, the period of time is about 30 hours. In certain embodiments, the period of time is about 2 days. In certain embodiments, the period of time is about 3 days. In certain embodiments, the period of time is NAI-1541834789v1 74Attorney Docket No.13371-304-228 less than 30 minutes. In certain embodiments, the period of time is 30 to 60 minutes. In certain embodiments, the period of time is 1 to 5 hours. In certain embodiments, the period of time is 5 to 10 hours. In certain embodiments, the period of time is 10 to 15 hours. In certain embodiments, the period of time is 15 to 20 hours. In certain embodiments, the period of time is 20 to 30 hours. In certain embodiments, the period of time is 1 to 2 days. In certain embodiments, the period of time is 2 to 3 days. In certain embodiments, the period of time is more than 3 days. In certain embodiments, a person skilled in the art would be able to pick an appropriate period of time for the exposure.

[0181] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than the stated amount per 500 cm² filtration area (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule (e.g., as determined using ESR) after being exposed to the filter for about 30 minutes. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO. NAI-1541834789v1 75Attorney Docket No.13371-304-228

[0182] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.5 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 0.2 x 10-7M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-8M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-9M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than 1 x 10-10M (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In certain embodiments, the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule is less than the stated amount per 500 cm² filtration area (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In specific embodiments, no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule (e.g., as determined using ESR) after being exposed to the filter for about 15 hours. In certain embodiments, the spin-trap molecule is PBN. In certain embodiments, the spin-trap molecule is DMPO. In certain embodiments, the spin-trap molecule is TEMPO. NAI-1541834789v1 76Attorney Docket No.13371-304-228

[0183] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein potency of the liquid composition does not decrease after 3 months of storage or decreases less than 15% after 3 months of storage.

[0184] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein potency of the liquid composition does not decrease after 18 months of storage or decreases less than 35% after 18 months of storage.

[0185] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is produced by a method described herein of producing a liquid composition comprising BoNT / A, such as a method described in Section 4.3.

[0186] In another aspect, provided herein is a liquid composition comprising BoNT / A, wherein the liquid composition is produced by reconstitution of a solid composition (e.g., a powdered pharmaceutical composition) comprising BoNT / A (for example, with a sodium chloride solution).

[0187] In another aspect, provided herein is a solid composition, which is a dried product of a liquid composition described herein.

[0188] In another aspect, provided herein is a solid composition (e.g., a powdered pharmaceutical composition) comprising BoNT / A, wherein the solid composition is produced by a process of drying (e.g., vacuum-drying, freeze-drying or lyophilizing) a liquid composition described herein or a liquid composition produced using a method described herein.

[0189] In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 3 months of storage or decreases less than 15% after 3 months of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 3 months of storage or decreases less than 10% after 3 months of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 3 months of storage or decreases less than 5% after 3 months of storage.

[0190] In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 18 months of storage or decreases less than 35% after 18 months of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 18 months of storage or decreases less than 30% after 18 months NAI-1541834789v1 77Attorney Docket No.13371-304-228 of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 18 months of storage or decreases less than 25% after 18 months of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 18 months of storage or decreases less than 20% after 18 months of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 18 months of storage or decreases less than 15% after 18 months of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 18 months of storage or decreases less than 10% after 18 months of storage. In various embodiments and aspects, potency of a liquid composition described herein does not decrease after 18 months of storage or decreases less than 5% after 18 months of storage.

[0191] In various embodiments and aspects, potency of a liquid composition described herein is determined using a cell-based potency assay. In various embodiments and aspects, potency of a liquid composition described herein is determined using a mouse 50% lethal dose (MLD50) assay.

[0192] In various embodiments and aspects, the filter used to produce a liquid composition described herein is not sterilized using gamma-irradiation. In various embodiments and aspects, the filter used to produce a liquid composition described herein is sterilized by a method other than gamma-irradiation. In various embodiments and aspects, the filter used to produce a liquid composition described herein is sterilized by steaming. In various embodiments and aspects, the filter used to produce a liquid composition described herein is autoclaved.

[0193] In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a hydrophilic membrane.

[0194] In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polyvinylidene fluoride (PVDF) membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polycarbonate membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polyester membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polyethersulfone membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a polypropylene membrane. In various NAI-1541834789v1 78Attorney Docket No.13371-304-228 embodiments and aspects, the filter used to produce a liquid composition described herein comprises a cellulose acetate membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a mixed cellulose ester membrane. In various embodiments and aspects, the filter used to produce a liquid composition described herein comprises a nylon membrane.

[0195] In various embodiments and aspects, the filter used to produce a liquid composition described herein has a pore size of about 0.1 µm to about 0.45 µm (e.g., about 0.1 µm, about 0.2 µm, about 0.22 µm, or about 0.45 µm). In specific embodiments, the filter used to produce a liquid composition described herein has a pore size of about 0.22 µm.

[0196] In various embodiments and aspects, the filter used to produce a liquid composition described herein has a filtration area of about 50 cm2to about 1000 cm2(e.g., about 50 cm2, about 100 cm2, about 200 cm2, about 300 cm2, about 500 cm2, or about 1000 cm2). In specific embodiments, the filter used to produce a liquid composition described herein has a filtration area of about 100 cm2. In specific embodiments, the filter used to produce a liquid composition described herein has a filtration area of about 500 cm2.

[0197] In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-20 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma- irradiated Millipak®-40 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-60 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-100 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-200 filter.

[0198] In various embodiments and aspects, a gamma-irradiated filter described herein, which is used for purposes of comparison, is substantially identical to the filter used to produce a liquid composition described herein, except that the gamma-irradiated filter is sterilized by gamma-irradiation while the filter used to produce the liquid composition is not. The term “substantially identical” is used in this context to describe that the gamma-irradiated filter is identical or sufficiently similar to the filter used to produce the liquid composition (for example, in terms of their materials and dimensions (e.g., pore sizes and / or filtration areas)), such that the gamma-irradiated filter can serve the purpose of being an appropriate control for comparison in NAI-1541834789v1 79Attorney Docket No.13371-304-228 the opinion of a person of ordinary skill in the art.

[0199] In various embodiments and aspects, a gamma-irradiated filter described herein comprises a hydrophilic membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a hydrophilic membrane.

[0200] In various embodiments and aspects, a gamma-irradiated filter described herein comprises a polyvinylidene fluoride (PVDF) membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a polycarbonate membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a polyester membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a polyethersulfone membrane. In various embodiments and aspects, a gamma- irradiated filter described herein comprises a polypropylene membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a cellulose acetate membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a mixed cellulose ester membrane. In various embodiments and aspects, a gamma-irradiated filter described herein comprises a nylon membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polyvinylidene fluoride (PVDF) membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polycarbonate membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polyester membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polyethersulfone membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a polypropylene membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a cellulose acetate membrane. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a mixed cellulose ester membrane. In various embodiments and aspects, both the filter used to NAI-1541834789v1 80Attorney Docket No.13371-304-228 produce a liquid composition described herein and the gamma-irradiated filter used for comparison comprise a nylon membrane.

[0201] In various embodiments and aspects, a gamma-irradiated filter described herein has a pore size of about 0.1 µm to about 0.45 µm (e.g., about 0.1 µm, about 0.2 µm, about 0.22 µm, or about 0.45 µm). In specific embodiments, the gamma-irradiated filter has a pore size of about 0.22 µm. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a pore size of about 0.1 µm to about 0.45 µm (e.g., about 0.1 µm, about 0.2 µm, about 0.22 µm, or about 0.45 µm). In specific embodiments, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a pore size of about 0.22 µm.

[0202] In various embodiments and aspects, a gamma-irradiated filter described herein has a filtration area of about 50 cm2to about 1000 cm2(e.g., about 50 cm2, about 100 cm2, about 200 cm2, about 300 cm2, about 500 cm2, or about 1000 cm2). In specific embodiments, the gamma- irradiated filter has a filtration area of about 100 cm2. In specific embodiments, the gamma- irradiated filter has a filtration area of about 500 cm2. In various embodiments and aspects, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a filtration area of about 50 cm2to about 1000 cm2(e.g., about 50 cm2, about 100 cm2, about 200 cm2, about 300 cm2, about 500 cm2, or about 1000 cm2). In specific embodiments, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a filtration area of about 100 cm2. In specific embodiments, both the filter used to produce a liquid composition described herein and the gamma-irradiated filter used for comparison have a filtration area of about 500 cm2.

[0203] In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma-irradiated Millipak®-20 filter. In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma-irradiated Millipak®-40 filter. In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma-irradiated Millipak®-60 filter. In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma- irradiated Millipak®-100 filter. In various embodiments and aspects, a gamma-irradiated filter described herein is a gamma-irradiated Millipak®-200 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma- NAI-1541834789v1 81Attorney Docket No.13371-304-228 irradiated Millipak®-20 filter, and the gamma-irradiated filter used for comparison is a gamma- irradiated Millipak®-20 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-40 filter, and the gamma-irradiated filter used for comparison is a gamma-irradiated Millipak®-40 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-60 filter, and the gamma-irradiated filter used for comparison is a gamma-irradiated Millipak®-60 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-100 filter, and the gamma-irradiated filter used for comparison is a gamma-irradiated Millipak®-100 filter. In various embodiments and aspects, the filter used to produce a liquid composition described herein is a non-gamma-irradiated Millipak®-200 filter, and the gamma- irradiated filter used for comparison is a gamma-irradiated Millipak®-200 filter.

[0204] In various embodiments and aspects, a liquid composition described herein is animal product free. In various embodiments and aspects, a solution described herein comprising BoNT / A is animal product free. In various embodiments and aspects, both the liquid composition and the solution comprising BoNT / A are animal product free. In various embodiments and aspects, a solid composition described herein is animal product free. In various embodiments and aspects, a liquid composition described herein does not contain a protease inhibitor. In certain embodiments, a liquid composition described herein does not contain benzamidine hydrocholoride. In various embodiments and aspects, a solution described herein comprising BoNT / A does not contain a protease inhibitor. In certain embodiments, a solution described herein comprising BoNT / A does not contain benzamidine hydrocholoride. In various embodiments and aspects, both the liquid composition and the solution comprising BoNT / A do not contain a protease inhibitor. In certain embodiments, both the liquid composition and the solution comprising BoNT / A do not contain benzamide hydrocholoride. 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 hydrocholoride.

[0205] In various embodiments and aspects, a liquid composition described herein further comprises one or more pharmaceutically acceptable carriers. In various embodiments and aspects, a solution described herein comprising BoNT / A further comprises one or more NAI-1541834789v1 82Attorney Docket No.13371-304-228 pharmaceutically acceptable carriers. In various embodiments and aspects, both the liquid composition and the solution comprising BoNT / A further comprise one or more pharmaceutically acceptable carriers. In various embodiments and aspects, a solid composition described herein further comprises one or more pharmaceutically acceptable carriers. In specific embodiments, the one or more pharmaceutically acceptable carriers comprise human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of BoNT / A). In specific embodiments, the one or more pharmaceutically acceptable carriers comprise sodium chloride (e.g., about 0.9 mg of sodium chloride per 100 units of BoNT / A). In specific embodiments, the one or more pharmaceutically acceptable carriers comprise human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of BoNT / A) and sodium chloride (e.g., about 0.9 mg of sodium chloride per 100 units of BoNT / A). In certain embodiments, the human serum albumin is recombinant human serum albumin. In a specific embodiment, the recombinant human serum albumin is animal product free. In a specific embodiment, the recombinant human serum albumin is not produced from an animal. In a specific embodiment, the recombinant human serum albumin is produced from a microorganism, such as bacteria. In a specific embodiment, the recombinant human serum albumin is produced from a plant-based expression system. In certain embodiments, the human serum albumin is human plasma-derived human serum albumin.

[0206] In various aspects and embodiments, a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) comprises about 50 units of BoNT / A. In various aspects and embodiments, a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) comprises about 100 units of BoNT / A. In various aspects and embodiments, a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) comprises about 200 units of BoNT / A.

[0207] In various embodiments and aspects, a BoNT / A composition described herein (e.g., a liquid composition or solid composition described herein) has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

[0208] In certain embodiments, the BoNT / A drug substance is produced as described in Example 1, Example 2 or Example 3.

[0209] In certain embodiments, the BoNT / A drug substance is produced using one or more steps as described in Example 1, Example 2 or Example 3. NAI-1541834789v1 83Attorney Docket No.13371-304-228

[0210] In certain embodiments, the BoNT / A drug substance is produced using a process that does not comprise one or more steps described in Example 1 or Example 2.

[0211] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises one or more steps of column chromatography (e.g., one or more steps of column chromatography performed during purification of the BoNT / A).

[0212] In preferred embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise hydrophobic interaction chromatography. In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise anion exchange chromatography. In specific embodiments, the one or more steps of column chromatography (e.g., during purification of the BoNT / A drug substance) comprise cation exchange chromatography.

[0213] In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise hydrophobic interaction chromatography and anion exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, hydrophobic interaction chromatography and anion exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, anion exchange chromatography and hydrophobic interaction chromatography.

[0214] In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise hydrophobic interaction chromatography and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, hydrophobic interaction chromatography and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, cation exchange chromatography and hydrophobic interaction chromatography.

[0215] In specific embodiments, the one or more steps of column chromatography (e.g., NAI-1541834789v1 84Attorney Docket No.13371-304-228 performed during purification of the BoNT / A drug substance) comprise anion exchange chromatography and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, anion exchange chromatography and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, cation exchange chromatography and anion exchange chromatography.

[0216] In specific embodiments, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise hydrophobic interaction chromatography, anion exchange chromatography, and cation exchange chromatography. In a preferred embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, hydrophobic interaction chromatography, anion exchange chromatography, and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, hydrophobic interaction chromatography, cation exchange chromatography, and anion exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, anion exchange chromatography, cation exchange chromatography, and hydrophobic interaction chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, anion exchange chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction chromatography. In a specific embodiment, the one or more steps of column chromatography (e.g., performed during purification of the BoNT / A drug substance) comprise, in the following order, cation exchange chromatography, hydrophobic interaction chromatography, and anion exchange chromatography.

[0217] In certain embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia NAI-1541834789v1 85Attorney Docket No.13371-304-228 sulfate (e.g., does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with cold ethanol (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with hydrochloric acid (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with ammonia sulfate (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with cold ethanol (e.g., during purification of the BoNT / A drug substance) and does not comprise a step of precipitation with hydrochloric acid (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with cold ethanol (e.g., during purification of the BoNT / A drug substance) and does not comprise a step of precipitation with ammonia sulfate (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with hydrochloric acid (e.g., during purification of the BoNT / A drug substance) and does not comprise a step of precipitation with ammonia sulfate (e.g., during purification of the BoNT / A drug substance). In specific embodiments, the BoNT / A drug substance is produced by a process that does not comprise a step of precipitation with cold ethanol (e.g., during purification of the BoNT / A drug substance), does not comprise a step of precipitation with hydrochloric acid (e.g., during purification of the BoNT / A drug substance) and does not comprise a step of precipitation with ammonia sulfate (e.g., during purification of the BoNT / A drug substance).

[0218] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, and filtering at 0.2 μm with added ammonium sulfate. NAI-1541834789v1 86Attorney Docket No.13371-304-228

[0219] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps and in the following order: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, and filtering at 0.2 μm with added ammonium sulfate.

[0220] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, and subjecting the filtrate to hydrophobic interaction chromatography (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0221] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises one or more of the following steps: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, and subjecting the filtrate to hydrophobic interaction chromatography (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0222] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps and in the following order: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, and subjecting the filtrate to hydrophobic interaction NAI-1541834789v1 87Attorney Docket No.13371-304-228 chromatography (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0223] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, loading the filtrate onto a hydrophobic interaction column, eluting with a descending gradient of ammonium sulfate, and isolating the product peak (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0224] In certain embodiments, the BoNT / A drug substance is produced by a process that comprises the following steps and in the following order: subjecting fermentation culture to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 at a temperature below 25° C, subjecting the acid precipitate to tangential flow filtration (e.g., 0.1 μm tangential flow filtration) to concentrate cell mass, adjusting pH to about 6.0, adding one or more nucleases to reduce host cell nucleic acid content, clarifying by centrifugation to remove cell debris, filtering at 0.2 μm with added ammonium sulfate, loading the filtrate onto a hydrophobic interaction column, eluting with a descending gradient of ammonium sulfate, and isolating the product peak (optionally followed by anion exchange chromatography and cation exchange chromatography).

[0225] In certain embodiments, the BoNT / A drug substance is produced by a process that does not involve using a protease inhibitor. In certain embodiments, the BoNT / A drug substance is produced by a process that does not involve using benzamidine hydrocholoride.

[0226] In one aspect, provided herein is a powdered pharmaceutical composition comprising a 900 kDa BoNT / A complex (e.g., a powdered pharmaceutical composition comprising about 50 units, about 100 units or about 200 units of a 900 kDa BoNT / A complex), human serum albumin, and sodium chloride, wherein the powdered pharmaceutical composition is a dried (e.g., a vacuum-dried, freeze-dried or lyophilized) product of a liquid composition comprising the 900 kDa BoNT / A, wherein the liquid composition is a filtration product of a solution comprising the 900 kDa BoNT / A complex, and said filtration is performed with a filter that is not gamma-irradiated. In certain embodiments, the 900 kDa BoNT / A complex is produce by a NAI-1541834789v1 88Attorney Docket No.13371-304-228 process that comprises one or more steps of column chromatography. In specific embodiments, the one or more steps of column chromatography are performed during purification of the 900 kDa BoNT / A complex. In specific embodiments, the one or more steps of column chromatography comprise hydrophobic interaction chromatography. In specific embodiments, the one or more steps of column chromatography comprise anion exchange chromatography. In specific embodiments, the one or more steps of column chromatography comprise cation exchange chromatography. In certain embodiments, the 900 kDa BoNT / A complex is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate (e.g., during purification of the 900 kDa BoNT / A complex). In specific embodiments, the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum (e.g., the Type A Hall strain of Clostridium botulinum). In specific embodiments, the 900 kDa BoNT / A complex is onabotulinumtoxin A. In specific embodiments, the powdered pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers. In a specific embodiment, the one or more pharmaceutically acceptable carriers comprise human serum albumin, such as recombinant human serum albumin (e.g., about 0.5 mg of human serum albumin per 100 units of the 900 kDa BoNT / A complex) and sodium chloride (e.g., about 0.9 mg of sodium chloride per 100 units of the 900 kDa BoNT / A complex). In specific embodiments, the powdered pharmaceutical composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg. In specific embodiments, the powdered pharmaceutical composition does not contain a protease inhibitor. In a specific embodiment, the powdered pharmaceutical composition does not contain benzamidine hydrochloride. 4.4.1. Characterization of BoNT / A Compositions

[0227] In one embodiment, BoNT / A compositions described herein have a potency of at least about 2.0×107units / mg, e.g., about 2.0×107to about 6.0×107units / mg, about 2.4×107to about 6.0×107units / mg, about 2.4×107to about 5.9×107units / mg, about 2.4×107to about 5.8×107units / mg, about 2.4×107to about 5.7×107units / mg, about 2.4×107to about 5.6×107units / mg, about 2.4×107to about 5.5×107units / mg, about 2.4×107to about 5.4×107units / mg, about 2.5×107to about 6.0×107units / mg, about 2.6×107to about 6.0×107units / mg, about 2.7×107to about 6.0×107units / mg, about 2.8×107to about 6.0×107units / mg, about 2.9×107to about 6.0×107units / mg, about 3.0×107to about NAI-1541834789v1 89Attorney Docket No.13371-304-228 6.0×107units / mg, or any numbers between such ranges.

[0228] In one embodiment, BoNT / A compositions described herein have a potency of about 2.4 x 107units / mg to about 5.4 x 107units / mg. In preferred embodiments, the term “unit” used herein refers to the LD50 dose.

[0229] The potency of the BoNT / A compositions described herein can be determined with methods known in the art, including but not limited to, e.g., Light-Chain Activity High- Performance Liquid Chromatography (LCA-HPLC) assay, mouse 50% lethal dose (MLD50) assay, Mouse Digit Abduction Score (DAS) assay, SNAP-25 assay, cell-based potency assay (CBPA), etc.

[0230] The LCA-HPLC assay measures SNAP-25 cleavage specificity. Samples are reacted with a commercially available BoNT / A fluorescent substrate derived from the SNAP-25 sequence. The fluorescently-labeled cleavage products are separated and detected via a reverse- phase (RP)-HPLC method. Further description of LCA-HPLC assay can be found in publications 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.

[0231] In one embodiment, the potency is determined using a mouse 50% lethal dose (MLD50) assay. The mouse 50% lethal dose (MLD50) assay has been 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. Mouse 50% lethal dose (MLD50) assay is a method for measuring the potency of a botulinum toxin by intraperitoneal injection of the botulinum toxin into female mice (about four weeks old) weighing 17–22 grams each at the start of the assay. Each mouse is held in a supine position with its head tilted down and is injected intraperitoneally into the lower right abdomen at an angle of about 30 degrees using a 25 to 27 gauge 3 / 8” to 5 / 8” needle with one of several serial dilutions of the botulinum toxin in saline. The death rates over the ensuing 72 hours for each dilution are recorded. The dilutions are prepared so that the most concentrated dilution produces a death rate of at least 80% of the mice injected, and the least concentration dilution produces a death rate of no greater than 20% of the mice injected. There must be a minimum of four dilutions that fall within the monotone decreasing range of the death rates. The monotone decreasing range commences with a death rate of no less than 80%. Within the four or more monotone decreasing rates, the two NAI-1541834789v1 90Attorney Docket No.13371-304-228 largest and the two smallest rates must be decreasing (i.e., not equivalent). The dilution at which 50% of the mice die within the three day post injection observation period is defined as a dilution which comprises one unit (1 U) of the botulinum toxin.

[0232] Mouse Digit Abduction Score (DAS) assay is an in vivo assessment of toxin-induced muscle paralysis following injection of BoNT / A toxin into the hind limb 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 action. Detailed protocols of DAS assay have been 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) Toxins 12(6):393, each of which is incorporated herein by reference in its entirety. For example, the DAS assay can be performed by injection of a BoNT / A composition described herein into the mouse gastrocnemius / soleus complex, followed by assessment of Digital Abduction Score using the method of Aoki (2001) Toxicon 39: 1815- 1820. In the DAS assay, mice are suspended briefly by the tail in order to elicit a characteristic startle response in which the mouse extends its hind limbs and abducts its hind digits. Following the BoNT / A composition injection, the varying degrees of digit abduction are scored on a five- point scale (0=normal to 4=maximal reduction in digit abduction and leg extension). Safety Ratio, the ratio between the amount of a toxin required for a 10% drop in a bodyweight (measured at peak effect within the first seven days after dosing in a mouse) and the amount of toxin required for a DAS score of 2, can also be determined to assess the therapeutic index of the BoNT / A composition described herein, as described in U.S. Patent. No.9,920,310, which is incorporated by reference herein in its entirety. High Safety Ratio scores are therefore desired, and indicate a toxin that is able to effectively paralyze a target muscle with little undesired off- target effects.

[0233] SNAP-25 assay is an ELISA based method to measure SNAP-25 proteolytic activity of the botulinum toxin. The assay uses a truncated SNAP-25 protein (the 206 amino acid residue peptide) bound to polystyrene 96 well microtiter plates and a monoclonal antibody that recognizes the cleaved product (a 197 amino acid residue peptide) which is made by enzymatic hydrolysis between amino acids 197 and 198 of the 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). Exemplary SNAP-25 methods NAI-1541834789v1 91Attorney Docket No.13371-304-228 are described in Ekong et al. (1997) Microbiology 143:3337-3347, and U.S. patent 7,160,699, each of which is incorporated herein by reference.

[0234] Cell-based potency assay (CBPA) has been described in, e.g., Fernández-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-25197SiMa H1 electrochemiluminescent (ECL) CBPA is used to determine the potency of the BoNT / A compositions described herein. The SNAP-25197SiMa H1 electrochemiluminescent (ECL) CBPA is an in vitro cell-based 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 direct comparison of BoNT / A product biological activity in vitro (Fernández-Salas et al. (2012) PLOS ONE 7(11):e49516; Rupp et al. (2020) Toxins 12(6):393). In brief, human neuroblastoma SiMa H1 cells are plated onto poly-D-lysine (PDL) 96-well plates in serum-free media (SFM) with 25 µg / mL of GT1b for three days and treated with toxin samples for 24 hours. After treatment, toxins are removed, cells are lysed and lysates are transferred to MSD High Bind plates coated with anti-SNAP-25197monoclonal antibody (mAb) 2E2A6. Plates are then washed and incubated with SULFO-TAG NHS-Ester labeled anti-SNAP-25 polyclonal antibody (pAb) for detection. Captured, BoNT / A toxin-cleaved SNAP-25 is then quantitated on a MSD plate reader. 5. EXAMPLE

[0235] Certain embodiments provided herein are illustrated by the following non-limiting examples, which describe different methods for obtaining BoNT / A and demonstrate a correlation between the use of a gamma-irradiated filter in the filtration process for manufacturing a liquid BoNT / A composition and its loss of potency during storage, and which also show that free radicals on a gamma-irradiated filter may be introduced into a liquid BoNT / A composition during the filtration process and thereby causing its loss of potency over time. 5.1. Example 1 – Non-APF (Schantz) Process for Obtaining a Botulinum Toxin

[0236] This example sets forth the prior art Schantz process for obtaining botulinum neurotoxin. The process is a non-APF process using animal derived media and reagents (i.e. beef blood agar plates for culturing, casein in the fermentation medium and use of Rnase and NAI-1541834789v1 92Attorney Docket No.13371-304-228 Dnase enzymes for botulinum neurotoxin purification). The Schantz process has about 16 to 20 major steps, for production scale work uses a 115 L fermentor and takes about 3 weeks to complete. The Schantz process is commenced by thawing a non-APF Clostridium botulinum master cell bank (MCB) vial to room temperature followed by four cultivation steps. First to select colonies with a suitable morphology, aliquots from the thawed MCB vial are streaked on pre-reduced Columbia blood agar (CBA) plates and anaerobically incubated for 30-48 hours at 34 °C ± 1 °C. Second, selected colonies are inoculated into 9 mL test tubes containing a casein growth medium for 6-12 hours at 34 °C. The contents of the 9 mL tube with the most rapid growth and highest density (growth selection step) are then further cultivated through two step- up anaerobic incubations (the third and fourth cultivation steps), being a 12-30 hour incubation at 34 °C in a 600 mL to 1 L seed cultivation bottle, followed by a cultivation in a 15 L to 25 L seed fermentor containing a casein growth medium for 6-16 hours at 35 °C. These two step-up cultivations are carried out in a nutritive media containing 2% casein hydrolysate (a casein [milk protein] digest), 1% yeast extract and 1% glucose (dextrose) in water at pH 7.3.

[0237] The step-up cultivations are followed by a further incubation for 60-96 hours at 35 °C in a commercial scale (i.e.115 L) production fermentor in a casein containing medium under a controlled anaerobic atmosphere. Growth of the bacterium is usually complete after 24 to 36 hours, and during the fermentation step carried out for about 65 to about 72 hours where most of the cells undergo lysis and release botulinum neurotoxin. It is believed that toxin is liberated by cell lysis and activated by proteases present in the media. A filtrate of the culture medium can be prepared using a single layer depth filter to remove gross impurities (i.e. whole and ruptured cells) thereby obtaining a clear solution referred to as a clarified culture. Collection of botulinum neurotoxin from clarified culture is accomplished by lowering the pH of the clarified culture to pH 3.5 with 3M sulfuric acid to precipitate the raw toxin at 20 °C (acidification precipitation). The raw botulinum neurotoxin is then concentrated (to achieve a volume reduction) by ultramicrofiltration (microfiltration) (referred to as MF or UF) followed by diafiltration (DF). A 0.1 µm filter is used for the microfiltration step.

[0238] The harvested crude or raw toxin is then transferred to a digestion vessel and stabilized by addition of the protease inhibitor benzamidine hydrochloride. Dnase and Rnase are added to digest (hydrolyze) nucleic acids. The toxin is then extracted with pH 6.0 phosphate buffer and cell debris removed by clarification. Hydrolyzed nucleic acids and low molecular NAI-1541834789v1 93Attorney Docket No.13371-304-228 weight impurities are then removed by further UF and DF steps. Next three sequential precipitation steps (cold ethanol, hydrochloric acid and ammonia sulfate precipitations) are carried out. The purified botulinum neurotoxin complex (bulk toxin) is stored as a suspension in a sodium phosphate / ammonium sulfate buffer at 2 °C to 8 °C.

[0239] Completion of this Example 1 Schantz (non-APF) process, including the harvesting and purification steps, takes about two to three weeks. The resulting bulk botulinum neurotoxin is a high quality suspension of 900 kDa botulinum toxin type A complex made from the Hall A strain of Clostridium botulinum with a specific potency of ≥2 X 107U / mg, an A260 / A278 of less than 0.6 and a distinct pattern of banding on gel electrophoresis, and suitable for use for the compounding of a botulinum toxin pharmaceutical composition.

[0240] Botulinum neurotoxin can also be obtained from an APF, non-chromatographic process, as set forth in Example 7 of U.S. patent 7,452,697, the complete APF, non- chromatographic process (from beginning of culturing to end of all purification and processing steps) taking about two to three weeks to complete. Alternately, botulinum neurotoxin can also be obtained from an APF, chromatographic process, as set forth in Example 16 of U.S. patent 7,452,697, the APF, chromatographic process (from beginning of culturing to end of all purification and processing steps) taking a week or longer to complete. 5.2. Example 2 – APF, Column Chromatographic Systems and Processes for Obtaining a Botulinum Neurotoxin

[0241] Rapid APF, anion-cation chromatographic based systems and processes are developed for obtaining high yield, high purity botulinum neurotoxin. The process of this Example 2 for production purposes (that is to obtain gram quantities of the final botulinum neurotoxin) uses a 20 L fermentation vessel and takes only 4-7 days, preferably about 4 to about 6 days, to complete all step of the process from initiation of culturing to completion of final purification and toxin storage. Apparatus utilized in the systems herein disclosed are discussed below. Chromatographic media processes are developed and are set forth herein. These chromatographic media processes use one, two, or three of the following: hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography. Specifically, the chromatographic media processes that are developed include: (1) a one media process that uses HIC; (2) a two media process that uses HIC followed by anion exchange chromatography; (3) a two media process that uses HIC followed by cation NAI-1541834789v1 94Attorney Docket No.13371-304-228 exchange chromatography; (4) a three media process that uses HIC followed by anion exchange chromatography followed by cation exchange chromatography; (5) a three media process that uses HIC followed by cation exchange chromatography followed by anion exchange chromatography; (6) a one media process that uses anion exchange chromatography; (7) a two media process that uses anion exchange chromatography followed by HIC; (8) a two media process that uses anion exchange chromatography followed by cation exchange chromatography; (9) a three media process that uses anion exchange chromatography followed by HIC followed by cation exchange chromatography; (10) a three media process that uses anion exchange chromatography followed by cation exchange chromatography followed by HIC; (11) a one media process that uses cation exchange chromatography; (12) a two media process that uses cation exchange chromatography followed by HIC; (13) a two media process that uses cation exchange chromatography followed by anion exchange chromatography; (14) a three media process that uses cation exchange chromatography followed by HIC followed by anion exchange chromatography; and (15) a three media process that uses cation exchange chromatography followed by anion exchange chromatography followed by HIC. The HIC removes impurities such as a 49 kDa impurity (which turns out to be a host cell glucose phosphate isomerase, as discussed below). 5.2.1. Preparation of Working Cell Bank

[0242] A new Clostridium botulinum cell bank is developed (for use to initiate the culturing step) without use of Columbia blood agar plates, and which removes the need for colony selection prior to cultivation and also eliminates the need to carry out the Shantz process step up tube cultivation and multiple seed (cultivation) steps.

[0243] For this purpose, a previously established Schantz master cell bank (MCB) is used to create an APF research cell bank (RCB) from which a new APF master cell bank (MCB) and a subsequent working cell bank (WCB) are generated. A research cell bank (RCB) is made from a colony from the Schantz (NAPF) MCB. To remove the animal-derived protein from the MCB vial, the cells are washed twice in APF medium containing 2% w / v SPTII (Soy Peptone type II), 1% w / v yeast extract, and 1% w / v glucose. The cells are plated on APF medium under strict anaerobic conditions using a Modular Atmosphere Controlled System (MACS) anaerobic chamber. An isolated colony is further expanded and stored in APF medium containing about 20% NAI-1541834789v1 95Attorney Docket No.13371-304-228 glycerol below −135° C.

[0244] The APF-MCB is made under GMP conditions by expanding the RCB into oxygen- free APF medium (200 mL, reduced for a minimum of 12 hours in an anaerobic chamber) and cultured in a MACS anaerobic chamber at 34.5° C ±1° C (stirred at 60 rpm) until the OD540 of the culture reaches 2.5±1.0 AU. Sterile glycerol is added to the resultant culture to a final concentration of about 20% after which the mixture is transferred into cryovials at 1 mL / vial (APF-MCB vials). The vials are flash frozen in liquid nitrogen, and then stored below -135° C. An APF-WCB is made under GMP conditions by expanding as above. The resultant APF cell banks are characterized for identity, purity, viability and genetic stability. 5.2.2. Upstream Steps (Culturing and Fermentation)

[0245] The Example 2 processes have two general stages: an upstream stage and a downstream stage. The upstream stage includes expansion of a starting cell line (growth and reproduction of Clostridium botulinum bacteria in a substantially APF culture medium), fermentation, harvest (removal of cellular debris) to provide a clarified, harvested culture that is then concentrated and diluted. Thus, in this example the steps of an exemplary three column process can include culturing, fermentation, harvest filtration, concentration, HIC, capture (anion) chromatography, polishing (cation) chromatography, buffer exchange, bioburden reduction and vial fill.

[0246] The upstream stage includes use of a culture medium in a 1 L bottle containing 400 mL of reduced (in an anaerobic chamber) seed APF culture medium (2% w / v SPTII, 1% w / v yeast extract, (adjusted to pH 7.3 with 1 N sodium hydroxide and / or 1 N hydrochloric acid prior to autoclaving)) 1% w / v sterile glucose added post autoclaving of culture media). The culture (seed) medium is inoculated with 400 µL of a thawed Clostridium botulinum WCB. Incubation / culturing occurs at 34.5 °C ± 1.0 °C with 150 rpm agitation in an anaerobic chamber.

[0247] When the optical density of the culture medium at 540 nm is 1.8 ± 1.0 AU, the entire contents of the 1 L bottle (approximately 400 mL) are transferred to a 20 L production fermentor containing APF fermentation medium adjusted with 1 N sodium hydroxide and / or 1 N hydrochloric acid post-steam sterilization to pH 7.3, fermentation medium composed of 3.25% w / v SPTII, 1.2% w / v yeast extract, 1.5% w / v sterile glucose (added post sterilization; sterilization, e.g. at about 122 °C for 0.5 hour). The temperature and agitation are controlled at NAI-1541834789v1 96Attorney Docket No.13371-304-228 35 °C ± 1 °C and 70 rpm, respectively. Nitrogen overlay is set at 12 slpm and headspace pressure set at 5 psig to maintain an anaerobic environment for cell growth. Fermentation pH and cell density are monitored by pH and online turbidity probes, respectively. The three phases for the production fermentation include exponential growth, stationary, and autolysis phases. Cellular autolysis, which releases active BoNT / A complex into the culture medium, is observed to occur consistently between 35 hours and the end of fermentation. At the end of fermentation, the culture is cooled to 25 °C for harvest.

[0248] Once the fermentation medium is cooled to 25 °C, the cell debris is separated from the botulinum neurotoxin type A complex containing lysate by depth filtration, first through a 5 – 0.9 µm nominal retention rating gradient pre-filter to remove cell debris, and then through a positively charged 0.8 – 0.2 µm nominal retention rating gradient to remove DNA (removal of up to about 80%). Both filters are rinsed together with 20 L of water for injection (WFI) before use. A minimum of 15 L of the filtrate is required for further processing, and any excess material is decontaminated after in-process sampling is complete. The filtrate is stored at 4 °C if not immediately processed by ultrafiltration.

[0249] Within a biosafety cabinet (BSC) the filtrate from the harvest step is concentrated from 15 L to 5 ± 0.5 L using a hollow fiber, tangential flow filtration (TFF) membrane from GE Healthcare. The ultrafiltered material is then diluted with 10 mM sodium phosphate pH 6.5 buffer to a final volume of 20 L. This material is purified by use of one chromatography column, two chromatography columns or three chromatography columns (see Section 5.2.3). The diluted, ultrafiltered harvest material is stored at 4 °C if not immediately processed by purification.

[0250] In the Schantz process the culture step is ended and the fermentation step begins based on time and visual observation of culture growth. In contrast, in the Example 2 processes determination of when to end the culturing step is based on analysis of culture fluid optical density, which ensures that the culture is in the logarithmic growth phase at the time of commencement of the fermentation step, and permits reduction of duration of the culturing step to about 8 hours to about 14 hours. The OD parameter terminated culture step maximizes the health of the cultured cells and encourages robust and abundant botulinum toxin resulting from the fermentation step. The average optical density (at 540 nm) of the culture medium at conclusion of culturing is 1.8 AU. The average duration of the fermentation step is 72 hours and the average final turbidity (A890) of the fermentation medium at conclusion of the fermentation NAI-1541834789v1 97Attorney Docket No.13371-304-228 step is 0.15 AU. The average amount of botulinum toxin type A complex present (as determined by ELISA) in the 20 L fermentation medium (whole broth) at the end of the fermentation step for is about 64 μg botulinum toxin type A complex / mL fermentation medium.

[0251] The harvest step uses depth filtration to remove cell debris and nucleic acids, followed by ultrafiltration and dilution to prepare the fermentation medium for the next step in the process. This harvesting / cell debris clearing is fundamentally different from the Schantz harvest process, which uses precipitation by acidification followed by microfiltration and diafiltration to concentrate and exchange buffers in preparation for further processing. 5.2.3. Downstream Steps (Purification)

[0252] Downstream steps include purification by one chromatography column, two chromatography columns, or three chromatography columns (for example, capture of the botulinum neurotoxin on an anion exchange column, elution from the column and further separation from impurities by polishing on a cation exchange column, and preferably (in the three column process), passage of eluent containing desired botulinum neurotoxin through a third column, preferably a hydrophobic interaction column (e.g. chromatography), before (preferably) or after the two-column process), followed by concentration and buffer exchange using tangential flow filtration (TFF), and bioburden reduction (e.g. by further filtration using a 0.2 µm filter) to a final botulinum neurotoxin type A complex optimized for cold storage, preferably freezing, and eventual compounding into a botulinum neurotoxin type A complex pharmaceutical composition. The sequence of the chromatography and filtration stages is intended to remove product and process-related impurities, to remove potential adventitious agents and to control the botulinum neurotoxin type A complex concentration and buffer matrix of the final botulinum neurotoxin type A in order to provide a more stable drug substance.

[0253] Several embodiments of exemplary two-column and three-column downstream processes are described below. Additional embodiments are contemplated in which the order of the columns is changed, and the procedures are adjusted accordingly as deemed appropriate in the opinion and judgment of a person skilled in the art, but which are otherwise similar to or essentially identical to the embodiments described below. Further embodiments of one-column downstream processes are also contemplated, which embodiments involve the use of only one chromatography column but are otherwise similar to or essentially identical to the embodiments NAI-1541834789v1 98Attorney Docket No.13371-304-228 described below. Specifically, this example contemplates a three-column downstream process that involves (1) the use of HIC followed by anion exchange chromatography followed by cation exchange chromatography, (2) the use of HIC followed by cation exchange chromatography followed by anion exchange chromatography, (3) the use of anion exchange chromatography followed by HIC followed by cation exchange chromatography, (4) the use of anion exchange chromatography followed by cation exchange chromatography followed by HIC, (5) the use of cation exchange chromatography followed by HIC followed by anion exchange chromatography, or (6) the use of cation exchange chromatography followed by anion exchange chromatography followed by HIC. This example also contemplates a two-column downstream process that involves (1) the use of HIC followed by anion exchange chromatography, (2) the use of HIC followed by cation exchange chromatography, (3) the use of anion exchange chromatography followed by HIC, (4) the use of anion exchange chromatography followed by cation exchange chromatography, (5) the use of cation exchange chromatography followed by HIC, or (6) the use of cation exchange chromatography followed by anion exchange chromatography. This example also contemplates a one-column downstream process that involves the use of HIC, the use of anion exchange chromatography, or the use of cation exchange chromatography.

[0254] A first detailed embodiment of an exemplary three-column downstream process carried out is as follows. Clarified (diluted) ultrafiltered material (20 L, as disclosed above) is passed through a POROS® 50HQ anion exchange chromatography resin, the captured botulinum neurotoxin is eluted from the anion exchange column and then run through a POROS® 20HS cation exchange chromatography resin, the eluent from which is run through a Phenyl Sepharose HP chromatography resin. Eluent from the HIC column is subjected to 100 kDa tangential flow filtration, followed by 0.2 μm filtration. The resulting botulinum neurotoxin type A complex is frozen for storage.

[0255] In this first embodiment of the exemplary three-column downstream process, the first chromatography step of the downstream process uses a POROS® 50HQ anion exchange chromatography resin packed into a column with an inner diameter of about 8 cm and a column height of about 15 cm. The entire POROS® 50HQ column operation is completed at ambient temperature, and the flow is in the downward direction. The botulinum neurotoxin type A complex is eluted from the anion column using a pH step change where the more negatively charged components such as nucleic acids (e.g. DNAs and RNAs) and other host cell proteins NAI-1541834789v1 99Attorney Docket No.13371-304-228 remain bound to the anion exchange column.

[0256] Particulars of the anion exchange step are: use of the POROS® 50HQ column using 0.1 N sodium hydroxide for a minimum contact time of 30 minutes (at least about 3 column volumes, at 230 cm / hour). The column is then equilibrated with a 50 mM sodium phosphate, pH 6.5 buffer (at least 5 column volumes). Next the clarified ultrafiltered and diluted material (i.e. processed lysate APF fermentation material) is loaded at 230 cm / hour onto the POROS® 50HQ anion exchange column, followed by washing with at least about 20 column volumes of 50 mM sodium phosphate, pH 6.5 at 230 cm / hour until absorbance at 280 nm of column effluent decreases to 0.10 AU, followed by eluting with 50 mM sodium acetate, pH 4.8 at 230 cm / hour. The product pool is collected, when the absorbance at 280 nm (A280) increases to at least about 0.15 AU and through the peak maximum to equal or less than about 0.2 AU on the trailing edge, into a vessel containing 1 column volume of 50 mM sodium acetate, pH 4.8. This elution pool is stored at about 2 °C to about 8 °C for up to 48 hours.

[0257] The second chromatography step in this first embodiment of the exemplary three- column downstream process of this Example 2 uses a POROS® 20HS cation exchange chromatography resin packed into a column with an inner diameter of 8 cm and a column height of 5 cm. The entire POROS® 20HS column operation is completed at ambient temperature, and the flow is in the downward direction. The botulinum neurotoxin type A complex associates with the POROS® 20HS column resin. The botulinum neurotoxin type A complex is then eluted from the column using a salt step change. The product-related impurities are eluted with the wash buffer and decontamination solution.

[0258] Particulars of the cation exchange step are: use of the POROS® 20HS column using 0.1 N sodium hydroxide solution for a minimum contact time of 30 minutes (at least about 3 column volumes, at 230 cm / hour). The column is then equilibrated with a 50 mM sodium acetate, pH 4.8 buffer (at least about 5 column volumes). Next the POROS® 50HQ product pool (collected as described above, fresh or from refrigeration) is loaded onto the POROS® 20HS column. The column is then washed with a 50 mM sodium acetate, pH 4.8 buffer (at least about 3 column volumes) and then washed again with a 50 mM sodium acetate, 150 mM sodium chloride, pH 4.8 buffer. The botulinum neurotoxin type A complex is eluted from the POROS® 20HS column with a 50 mM sodium acetate, 250 mM sodium chloride, pH 4.8 buffer at 200 mL / min, the eluate is diverted into a bioprocess collection bag (containing 1 column volume of NAI-1541834789v1 100Attorney Docket No.13371-304-228 50 mM NaH3C2O2, pH 4.8) when the A280 increases to about ≥ 0.1 AU through peak maximum until the A280 of the trailing edge of the elution peak decreases to a trailing edge value of ≤ 0.1 AU. The POROS® 20HS product pool is stored in the collection bag at ambient temperature for up to about 6 hours.

[0259] In this first embodiment of the exemplary three-column chromatography media process of this Example 2, eluent from the second (cation exchange) column is passed through a HIC column. The HIC column used is a Phenyl Sepharose HP hydrophobic interaction chromatography resin packed into a column with an inner diameter of about 8 cm and a column height of about 5 cm. The entire Phenyl Sepharose HP column operation is completed at ambient temperature, and the flow is in the downward direction. The botulinum neurotoxin type A complex is eluted from the column using a decreasing salt step change. The impurities are eluted during the load and with the wash buffer and decontamination solution.

[0260] Particulars of the hydrophobic interaction chromatography step are: a Phenyl Sepharose HP column is initially sanitized with a 0.1 N sodium hydroxide solution for a minimum contact time of 30 minutes (with at least about 3 column volumes of a 0.1 N sodium hydroxide solution at 200 cm / hour). The column is then equilibrated with at least about 5 column volumes of 50 mM sodium acetate, 0.4 M ammonium sulfate, pH 4.8 buffer. Next the POROS® 20HS (cation exchange column) product pool (from above) is combined 1:1 with a 50 mM sodium acetate, 0.8 M ammonium sulfate, pH 4.8 buffer and loaded onto the Phenyl Sepharose HP column. The column is first washed with at least about 3 column volumes of a 50 mM sodium acetate, 0.4 M ammonium sulfate, pH 4.8 buffer, and then washed with a 50 mM sodium phosphate, 0.4 M ammonium sulfate, pH 6.5 buffer. Botulinum neurotoxin type A complex is eluted from the column with a 10 mM sodium phosphate, 0.14 M ammonium sulfate, pH 6.5 buffer. The eluate is diverted into a bioprocess collection bag when the A280 increases to ≥ 0.05 AU. The eluate is collected until the A280of the trailing edge of the elution peak decreases to a value of ≤ 0.05 AU. The Phenyl Sepharose HP product pool is stored in the collection bag at ambient temperature for up to 6 hours.

[0261] A tangential flow filtration system is used to concentrate and diafilter the Phenyl Sepharose HP chromatography step product pool into the drug substance formulation buffer. Pall® Filtron Minimate cassettes with a 100 kDa molecular weight cut off membrane are used for the concentration and diafiltration steps. The formulated material is then passed through a NAI-1541834789v1 101Attorney Docket No.13371-304-228 Pall Mini Kleenpak ® 0.2 µm filter to reduce the potential bioburden. The UF / DF step concentrates the Phenyl Sepharose HP product pool (eluent of the HIC column) to a BoNT / A complex concentration of 0.7 g / L and diafilters the concentrated material with a 10 mM potassium citrate, pH 6.5 buffer.

[0262] Particulars of the ultrafiltration / diafiltration process used are as follows. The UF / DF unit and Pall 100 kDa polyether sulfone membrane is initially flushed with a minimum of 5 L of water for injection (WFI) to remove the packing solution and sanitized with a minimum of 200 mL of a 1 N sodium hydroxide solution under recirculation conditions for a minimum of 10 minutes, preferably at least 30 minutes, to sanitize the UF / DF unit. Next the membrane and UF / DF system are equilibrated with sufficient volumes of the 10 mM potassium citrate, pH 6.5 formulation buffer until permeate and retentate pH is pH 6.5. After that the Phenyl Sepharose HP product pool is loaded onto the Minimate® tangential flow filtration cassette and the HIC eluate concentrated to 0.7 g / L. Following the concentration step, the retentate pool is diafiltered against a minimum of 5 diafiltration volumes of the drug substance formulation buffer (10 mM potassium citrate, pH 6.5) at a transmembrane pressure of 7.5 psig (pounds per square inch gauge). The permeate outlet is then closed and the UF / DF system run for at least 2 minutes and the system rinsed with 50 mL of 10 mM potassium citrate, pH 6.5 formulation buffer. After the rinse, the concentration of BoNT / A complex in the retentate pool is determined by measuring the offline A278and based on the A278reading, the concentration of the retentate pool is adjusted to 0.5 g / L with 10 mM potassium citrate, pH 6.5 buffer. The concentration-adjusted retentate pool is then filtered through a Pall Mini KleenpakTM0.2 μm filter to reduce potential bioburden. The filtered concentration-adjusted retentate pool is stored in a collection bag at 2 °C – 8 °C for up to 2 days.

[0263] The final purified botulinum neurotoxin type A complex obtained is filled into 1 mL Nunc® cryovials at 700 μL per vial and stored frozen. The filling operation is carried out in a class 100 biosafety cabinet at ambient temperature.

[0264] The downstream process (including use of 1 or 2 or 3 chromatography columns) is completed in only 1 to 3 days and the botulinum neurotoxin type A complex obtained is stored frozen in a potassium citrate, pH 6.5 buffer at a concentration of 0.5 g / L as a solution. In comparison, the Schantz downstream (toxin purification) process uses multiple filtration, precipitation, extraction and centrifugation steps to purify the botulinum neurotoxin type A NAI-1541834789v1 102Attorney Docket No.13371-304-228 complex and requires 1-2 weeks to complete just the downstream steps, and the resultant drug substance (recovered botulinum neurotoxin) is stored refrigerated as an ammonium sulfate suspension at a concentration of approximately 2.7 g / L. The use of chromatography instead of precipitation and the reduced processing time results in a significantly improved, consistent downstream process, as herein disclosed.

[0265] In accordance with one aspect, concentrations of vegetable-based products, such as soy-based products, can be Soy Peptone Type II Hy-Soy® or SE50MK(a Kosher soy peptone) in culture and fermentation media. Hy-Soy® in the seed culture medium can range between 10-200 g / L. Preferably, the concentration of Hy-Soy® in the seed medium ranges between 15-150 g / L. Most preferably, the concentration of Hy-Soy® in the seed medium is approximately between about 20-30 g / L or an amount therebetween. The concentration of glucose in seed medium can range between 0.1 g / L and 20 g / L. Preferably, the concentration of glucose ranges between 0.5- 15 g / L. Most preferably, the concentration of glucose in the culture medium is approximately 10 g / L. Yeast extract amounts can be from about 5-20 g / L, more preferably from about 10-15 g / L or an amount therebetween. For example, the pH of the culture medium prior to growth of Clostridium botulinum can be approximately pH 7.0-7.5, or therebetween, preferably pH 7.3.

[0266] As an example, Hy-Soy® amounts in the production fermentation medium can range between 10-200 g / L. Preferably, the concentration of Hy-Soy® in the fermentation medium ranges between 15-150 g / L. Most preferably, the concentration of Hy-Soy® in the fermentation medium is approximately between about 20-40 g / L or an amount therebetween. The concentration of glucose in fermentation medium can range between 0.1 g / L and 20 g / L. Preferably, the concentration of glucose ranges between 0.5-15 g / L or an amount therebetween. Not necessarily, but as above, the glucose can be sterilized by autoclaving together with the other components of the fermentation medium. The pH level of the fermentation medium prior to growth can be pH 7.0-7.8, preferably about 7.0-7.5 or therebetween, more preferably pH 7.3.

[0267] An embodiment of an exemplary two-column downstream process comprises the following steps: (a) culturing bacteria, such as Clostridium botulinum bacteria from an APF WCB vial, in a seed / culturing bottle, (b) then fermenting Clostridium botulinum bacteria in a fermentor (toxin production fermentor) having APF fermentation medium to expand the cell line, proceeding with fermentation and botulinum toxin production until a desired cell lysis phase is reached. Next, (c) harvesting (e.g. clarifying by filtration,) the APF fermentation medium to NAI-1541834789v1 103Attorney Docket No.13371-304-228 obtain a harvested fermentation medium, (d) proceeding with concentration and dilution resulting in a diluted harvested fermentation medium that is (e) passed through a capture column to remove impurities, (f) contacting eluent from the capture column with a polishing column to further remove impurities, and optionally a second polishing column (g) concentration and buffer exchange of the polishing column eluent, (h) followed by bioburden reduction filtration and the (i) filling of vials.

[0268] In one example, the fermentation volume is 20 L, the total process time for all steps is only 4 to 6 days, and high botulinum neurotoxin yield is obtained.

[0269] The following provides more details of a particular embodiment within the scope of our invention. The fermentation step is carried out in APF medium using a 30 L stainless steel fermentor.

[0270] In this example below, a much-reduced volume of fermentation medium is used while still providing a high yield of high potency botulinum neurotoxin type A complex. By using the following protocol, only 20 L or less, for example, of APF fermentation medium is required, instead of the typically larger, previous volumes (e.g.115 L) of fermentation medium required for producing commercially useful amounts for obtaining a botulinum neurotoxin.

[0271] The MACS anaerobic workstation (Don Whitley) with airlock provides an oxygen- deficient environment in which to manipulate anaerobic organisms. Access to and egress from the chamber is via a porthole system, comprises of inner and outer doors. The unit is temperature controlled to maintain a user setting within the chamber. A humidistat-controlled condensing plate ensures the effective removal of excess moisture in the chamber. The chamber is illuminated for operator use and alarm for: low gas pressure, continuous gas flow, and loss of power conditions. The chamber is equipped with a HEPA filter to reduce viable and non viable particulate levels in the anaerobic chamber. Anaerobic conditions are maintained utilizing the “Anotox” and Palladium Deoxo “D” Catalyst atmospheric scrubbing system. Condensate water from the condensing plate is collected and piped to an external reservoir where it is removed.

[0272] As disclosed above, an APF process is used for preparation of an APF WCB, having cell bank vials stored below -135 °C. An APF WCB cell bank vial is thawed at room temperature for about 15 min before culture medium inoculation, followed by a single cultivation step as disclosed above to establish a “seed” culture. This is carried out in a modular atmospheric controlled system utilizing aseptic techniques throughout, to minimize bioburden. NAI-1541834789v1 104Attorney Docket No.13371-304-228 The modular atmospheric controlled system is cleaned before undertaking inoculation of the completed seed culture vial with APF WCB vial contents. Culture medium is prepared using 1 N hydrochloric acid and 1 N sodium hydroxide (for pH adjustment), D(+) Glucose, Anhydrous (Mallinckrodt Baker, Cat# 7730, 4.00 g), Soy Peptone Type II (SPTII) (Marcor, Cat #1130, 8.00 g), Water for Injection (WFI) 400.0 mL and Yeast Extract (YE) (BD Cat #212730, 4.00 g). The soy peptone Type II and yeast extract solution is made by measuring 300 mL of WFI with a 500 mL graduated cylinder and poured into a seed culture bottle. The seed culture bottle is placed onto a stirrer and the stirrer activated.8.00 g of SPTII and 4.00 g of yeast extract is added to the seed culture bottle and mixed until dissolved. If dissolution is not complete after mixing, the mixture will be heated on low setting. The pH is measured and adjusted to about 7.30 ± 0.05. The medium solution is brought up to about 360 mL with WFI. The seed culture bottle is adequately vented to allow steam and gas transfer. A 10% Glucose solution (w / v) is prepared by measuring about 30 mL of WFI with a 100 mL graduated cylinder and placed into the pre- assembled glucose addition bottle, which is placed onto a stirrer and the stirrer activated. About 4.00 g of glucose is added to the glucose addition bottle and mixed until dissolved (low heat is used if necessary to a dissolution) and qs (quantity sufficient) glucose solution to 40 mL with WFI. The glucose addition is then capped loosely with vent cap. Both the glucose and seed culture bottles are autoclaved at 123 °C for 30 minutes for sterilization. After sterilization, both items are removed from the autoclave and left to cool in a bio-safety cabinet. After cooling aseptically, 10% of the glucose solution is transferred into the seed culture bottle containing the yeast extract and soy peptone II solution and mixed, thereby providing a completed seed culture bottle.

[0273] This completed seed culture bottle is placed into the pre-cleaned MACS (wherein a prepared anaerobic indicator is placed). The cap of the completed seed culture bottle is loosened. The completed seed culture bottle is then placed on a stir plate within the MACS (stir plate activated to about 150 rpm) and the medium in the completed seed culture bottle is reduced for a minimum of 12 hours at about 34.5 °C + / - 1 °C within the MACS, after which a 1 mL medium blank is sampled for optical density measurement (for biomass determination at 540 nm). Afterwards, the completed seed culture bottle, in the MACS (anaerobic) is inoculated. An APF WCB culture vial is obtained from the frozen cell bank and brought into the MACS. The vial is thawed for about 10-15 minutes, after which about 400 μL of the vial contents are placed directly NAI-1541834789v1 105Attorney Docket No.13371-304-228 into the medium in the completed seed culture bottle. The cap on the completed seed culture bottle is loosened completely and the cap is rested on top of the bottle and the stir pace is set to 150 rpm. After at least about 11 hours of incubation in the MACS, fermentation production is undertaken, as described below.

[0274] Probes (e.g. redox probe, pH probe, turbidity probe, e.g. by Broadley James and Optek) and sequence configuration of the fermentor, such as a 30 L stainless steel fermentor, are checked and calibrated, and inserted into their respective fermentor ports and tightened in place. For example, a fermentor can be a ABEC 30 L (VT) Fermentor System consisting of a 30 L volume fermentor vessel, an agitator drive system, piping assembly for utility connections (CIP, clean steam, CDA, Nitrogen, Oxygen, Process Chilled Water, bio-waste, and plant steam), instrumentation (pH, temperature, pressure, ReDox, optical density, and mass flow), and four peristaltic pumps. The bottom mounted agitator speed is controlled using an Allen-Bradley variable frequency drive (VFD). Semi-automatic and automatic control of the system is handled by an Allen-Bradley ControlLogix PLC with programming. The system is designed to provide closed-loop PID (proportional-integral-derivative) control of culture temperature, pressure, pH, and redox during fermentation operations. An Allen-Bradley DeviceNet® (an open device level network) is utilized for control and communication with devices and sensors on the skid.

[0275] For sterile hold, equilibrium, run and harvest modes, agitation, temperature, pressure and Nitrogen overlay are operated with the following set points.

[0276] For sterile hold and equilibrium mode: Controlled Parameter Set Points and RangeControlled Parameter Set Points and RangeNAI-1541834789v1 106Attorney Docket No.13371-304-228 Controlled Parameter Set Points and Range Agitation 150 rpm ± 10 lude D(+) Glucose, AnhydrousSPTII) (Marcor, Cat #1130, 650.0 g), Water for Injection (WFI, 13 L) and Yeast Extract (YE) (BD Cat #212730, 240.0 g), along with standard balances, a carboy (20 L, for example), glass bottle (5 L), graduated cylinders, stir bars and stirrers. About 10 L of WFI is added into the carboy along with a stir bar. The carboy is placed onto a stirrer and the stirrer is activated, after which about 650.0 g of soy peptone type II is added, along with about 240.00 g of YE. The fermentation medium is q.s. (quantity sufficient) to 13 L with WFI, and the carboy is capped. A 10% glucose solution (w / v) is then prepared by adding about 2 L if WFI into a glass 5 L bottle (with stir bar therein). Placed onto a stirrer and with the bar spinning, about 300.00 g of glucose is added into the bottle, and mixed until dissolved. The glucose solution is q.s. to 3 L with WFI and the bottled capped, thus providing a 10% glucose solution.

[0279] The fermentation medium in the carboy is added to the fermentor and pre-steam in place fermentor volume recorded and the fermentation sequence of operation is advanced. At the end of the SIP (steam in place)(122 °C, + / - 1 °C), the post-SIP fermentor volume is noted. A glucose addition assembly, comprising a vessel having tube therefrom with and in-line 0.2 μm filter (PALL Corp.) and peristaltic pump, is connected to the fermentor and the line is subjected to SIP and allowed to cool. An addition valve port is opened and about 3 L of glucose (filter sterilized) is added, and the appropriate amount of WFI (filter sterilized) to q.s. the total fermentor volume to 20 L is added to the glucose addition bottle and pumped into the fermentor through the same glucose filter line. The addition valve port is closed. The production fermentation medium has its pH adjusted thereafter, to about pH 7.3 + / - 0.05, with sterile 1 N sodium hydroxide or 1 N hydrochloric acid, utilizing SIP of addition lines, as required. Afterwards, parameters for sterile hold are set and held for about 12 hours before inoculation. The medium’s starting glucose concentration is measured using a metabolite analyzer and glucose concentration recorded.

[0280] As stated above, at the end of seed culture incubation (about 11 ± 1 hours), 1 mL of NAI-1541834789v1 107Attorney Docket No.13371-304-228 sample is taken for optical density (OD) measurement. OD is measured offline at 540nm using a spectrophotometer and if within the appropriate range the OD value is recorded and culture is used for fermentation. The fermentor turbidity probe is accordingly zeroed. The seed inoculum bottle, from the anaerobic chamber, is brought over to the fermentor and a seed inoculum transfer assembly (a seed vessel with APF culture medium therein, the vessel having a culture inoculum transfer line with a sterile Kleenpak™ Connector assembly available from PALL Corp. or Millipore). The seed inoculum transfer line is then fixed to a peristaltic pump and the inoculum transfer line with sterile Kleenpak connector is connected to the fermentor. The fermentor pressure is lowered to 2 psig and entire volume of the seed inoculum bottle is pumped into the fermentor. At the end of inoculation, the online Absorbance Units (AU) from the fermentor is recorded, fermentor parameters are set to RUN mode and time is recorded.

[0281] Fermentation then proceeds (fermentation runs can be from about 60 hours to about 80 hours, preferably from about 68 hours to about 76 hours, most preferably for about 72 hours) while samples are taken from the fermentor, at 24 and 48 hours, for example, while maintaining aseptic conditions. Tests that are run on at least one sample taken during fermentation can include, but are not limited to, off-line optical density measurements, glucose measurements, ELISA, SDS-PAGE, Western blot, for example. At the end of the fermentation (end of fermentation broth volume is from about 18-19 L, for example), a sample may be taken (for testing by, for example, off-line optical density measurements, glucose measurements, ELISA, SDS-PAGE, western blot and DNA / RNA quantification.

[0282] At the end of the fermentation, online optical density, EFT (elapsed fermentation time), and fermentation end time is recorded, as well as agitation rpm, temperature in °C, pressure psig and Nitrogen overlay slpm and redox mV. Next, the production fermentation broth is subjected to harvesting, i.e. the production fermentation broth is clarified through filtration whereby, for example, about 15 L of filtrate is collected. The fermentation parameters are set for HARVEST and the filter assembly for clarification is prepared (CUNO, 3M filtration) which includes a pre-filter, depth filter and at least one pressure gauge. The pre-filter and depth filter are flushed with about 20 L of water for injection. After flushing, the filtration assembly is attached to the harvest / drain port of the fermentor. The fermentor temperature is decreased to about 25 °C, after which clarification of the fermentation broth begins (record clarification start time, initial online OD, initial pH, initial temperature and initial volume of fermentor). The NAI-1541834789v1 108Attorney Docket No.13371-304-228 pressure in the fermentor is increased at a rate of about 1 psi (pound per square inch) about every 10 minutes during filtration, until a pressure of about 6 psi is reached, at which the pressure is held until the end of harvesting. This filter removes approximately 80% of the RNA / DNA in the APF fermentation medium (the remainder essentially removed during later chromatography steps, as discussed below), thus doing away with prior reliance / use of RNase and / or DNase to remove such components from the fermentation broth. Process parameters, such as pre-filter inlet pressure, depth filter inlet pressure, fermentor pressure, agitation and filtrate volume are monitored at every 2 L of filtrate collected, at the end of which the clarification end time and volume of filtrate collected is recorded. Following completion of harvest step, the systems are decontaminated and cleaned.

[0283] The filtrate carboy is brought into the BSC for sampling, from which about ≤10 mL of filtrate is sampled for offline OD measurements and other analysis (e.g. ELISA, SDS-PAGE, DNA / RNA and western blot).

[0284] The filtrate is then subjected to ultrafiltration / dilution. A tangential flow filter (TFF) unit assembly is assembled. The TFF unit is rinsed for about 90 minutes with WFI at a preferred rate of about 2 L per minute and then the TFF unit is sanitized by running 0.1 N sodium hydroxide (re-circulated) therethrough for about 60 minutes, after which 1 L of 10 mM sodium phosphate buffer, pH 6.5 is run therethrough, followed by a rinse with WFI for about 30 minutes. The filtrate from the harvest step (about 15 L) is then passed through the TFF (this is carried out in a bio-safety cabinet), concentrating the filtrate down to about 5 L + / - 0.5 L (the concentration step proceeds at about 2 L per minute and at a trans-membrane pressure of about 5 psig). A sample of the permeate can be taken and subjected to ELISA, dsDNA, SDS-PAGE and western blot tests, for example. Once concentrated to about 5 L + / - 0.5 L, the retentate pool is then diluted up to about 20 L with about 15 L of sterile filtered 10 mM sodium phosphate buffer, pH 6.5, through the TFF, at about a rate of 2 L per minute. A sample can be then again be taken and subjected to ELISA, DNA / RNA, SDS-PAGE and western blot tests, for example. The ultrafiltration / dilution material (retentate) is stored at 4 °C.

[0285] Following use all systems are decontaminated using either 1N sodium hydroxide or sterilization (steam) temperatures and cleaned.

[0286] The following materials, equipment and procedures are used to make the solutions, buffers, etc, set forth below for use in an exemplary process, that is in the purification of the NAI-1541834789v1 109Attorney Docket No.13371-304-228 fermentation medium obtained from the Example 2 processes so as to obtain a purified botulinum neurotoxin type A complex. Exemplary buffers utilized (filtered through a 0.2-micron vacuum filter and their conductivity measured in mS / cm, for recordkeeping) include:10 mM sodium phosphate, pH 6.5; 50 mM sodium phosphate, pH 6.5; 50 mM sodium acetate, pH 4.8; 50 mM sodium acetate, 170 mM sodium chloride, pH 4.8; 50 mM sodium acetate, 250 mM sodium chloride, pH 4.8; 50 mM sodium acetate, 1 M sodium chloride, pH 4.8; 50 mM sodium acetate, pH 4.0 and 10 mM citrate, pH 6.5.

[0287] The following is an example of operations for purification and obtaining botulinum neurotoxin type A from the Example 2 processes. All product-contact parts are designed and constructed to ensure that they are non-reactive and non-absorptive. Additionally, all equipments are designed to allow the utilization of single use disposable systems or are designed and constructed to facilitate sanitization, cleaning and decontamination as per documented, validated methods. The systems or skids are designed to be non-product contacting while the flow paths are designed to be single use disposable, including the chromatography columns and the all associated tubing. Chromatography components are obtained from AlphaBio and UF / DF components are obtained from Scilog Inc. The chromatography set ups used include a peristaltic pump for solution delivery with variable speed drive, inlet valve manifold with 5 inlets, a column valve manifold with an array of 3 automated valves, outlet valve manifold with 3 outlets, column effluent monitoring, including pH, conductivity, and UV, peak collection based on UV absorbance, and instrumentation and controls required to complete the purification operations. The control system has both the software and hardware designed to control the purification process. Commands and data are entered via a HMI (Human Machine Interface) terminal. The operator initiates all automated process functions by commands at the HMI and monitors and adjusts process parameters such as feed flow rates, pressure, conductivity, pH, UV absorbance and individual valve positions.

[0288] The UF / DF system includes a recirculation pump, diafiltration pump, 2 balances and a tangential flow filter (TFF) holder. The recirculation pump interfaces with 3 disposable pressure sensors and one of the balances (located under the permeate reservoir) to control the flow rate to maintain a defined transmembrane pressure and stop, based on the weight of the permeate reservoir. The diafiltration pump interfaces with the second balance (located under the retentate reservoir) to start and stop, based on maintaining a constant weight of the retentate NAI-1541834789v1 110Attorney Docket No.13371-304-228 reservoir.

[0289] After concentration and dilution of retentate material from the harvesting step (harvesting the animal protein free fermentation medium), the material is loaded onto an anion exchange column. The following is the procedure used for packing and testing the anion exchange column useful in the exemplary Example 2 two-column process.

[0290] Pre-packed columns are used for all three chromatographic steps. First, feed material (harvested APF media that has been subjected to ultrafiltration / dilution) is passed through the anion exchange column (Poros 50HQ, from ABI as described above). At least 5 column volumes (CVs) of 50mM sodium phosphate, pH 6.5, are utilized to equilibrate the anion exchange column (in this example, a capture column).

[0291] After equilibration, the loading step is performed, where feed material (post harvesting step harvested fermentation broth, of about 20 L, for example)) is loaded onto the anion exchange column at a rate of about 200 cm / hr for example. After 0.5 column volume of loaded material has passed through the anion exchange column, the flow through (FT) pool is collected into a receptacle such as a polyethersulfone vessel, while toxin complex is bound to the anion exchange column material. This is followed by a wash step, where at least about 15 column volumes of the wash buffer (e.g.50 mM sodium phosphate at a pH of 6.5) is passed through the anion exchange column. The wash step is stopped when the UV, measured at the column outlet, in real time, decreases to less than or equal to about 80 mAU. The wash buffer volume and the flow through / wash pool volume are recorded, and a 1 mL sample of the flow through / wash pool is taken and tested, for example, for toxin concentration, nucleic acid content, whole cell proteins, SDS-PAGE, qPCR, 2D LC and ELISA.

[0292] The next step is the elution step, where elution buffer (e.g.50 mM sodium acetate, pH 4.8) is pumped onto the anion exchange column. When the UV reading at the column outlet, in real-time, increases to about 150 mAU or more, collection of eluate in a container pre-filled with 1 CV of elution buffer (50 mM sodium acetate, pH 4.8) is begun. Collection of eluate pool is stopped when the UV reading decreases to less than or equal to about 200 mAU (volume collected at this point is between about 1 to about 2 CVs). The chromatography system is then decontaminated and cleaned using 1 N sodium hydroxide.

[0293] The eluate pool from the anion exchange column is then prepared for addition onto the cation exchange column. The anion exchange eluate volume, pH, conductivity and feed NAI-1541834789v1 111Attorney Docket No.13371-304-228 temperature are recorded and the eluate pool from the anion exchange column is diluted with 1 CV of 50 mM sodium acetate, pH 4.8.

[0294] Following the run-through of the anion exchange column, cation exchange chromatography operation is undertaken. The cation exchange column (e.g. Poros® 20HS) is equilibrated with a minimum of 5 CVs of equilibration buffer (50 mM sodium acetate, pH 4.8). After equilibration, the diluted eluate pool from the anion exchange column is loaded onto the cation exchange column and the total volume loaded is recorded. After 0.5 column volume of loaded diluted eluate pool has passed through the cation exchange column, the flow through (FT) pool is collected. A first wash of the cation exchange column is conducted where about 3-5 CVs of 50mM sodium acetate, pH 4.8, is passed through the cation exchange column (volume of first wash buffer utilized is recorded). A second wash is performed, where about 3 CVs of 170 mM sodium chloride, 50 mM sodium acetate, pH 4.8, is pumped through the column, this eluate being collected in a new container labeled “WASH 2 Peak”. Collection is begun when the UV readings increase to greater than or equal to 50 mAU. 1 CV is collected and the second wash buffer volume utilized is recorded.

[0295] Elution of bulk toxin complex from the cation exchange column is carried out utilizing elution buffer (e.g.250 mM sodium chloride in 50 mM sodium acetate, pH 4.8) which is pumped onto the cation exchange column. When the UV reading of the elution reaches at least about 100 mAU, eluate collection begins into containers pre-filled with dilution buffers (40 mL of 100 mM potassium phosphate, pH 6.8 and 60 mL of 10 mM potassium citrate, pH 6.5). Collection of eluate from the cation exchange column continues until UV readings decreases to about 100 mAU or less. The total volume of elute, after dilution, is recorded. The cation exchange chromatography system is then decontaminated and cleaned.

[0296] Following elution from the cation exchange column, the eluate is subjected to filtration. A tangential flow filtration (TFF) system is utilized, using three 100K MWCO membranes (Sartorius AG, Goettingen, Germany) stacked one atop the other. The cation exchange eluate pool initial volume is noted, as are the diafiltration / equilibration and sanitation solution descriptions. For example, the diafiltration solution can be 10 mM potassium citrate, pH 6.5 and the sanitation solution can be 0.1 N sodium hydroxide. System set up proceeds with connection of one tube from the reservoir containing either eluate from the cation column (IAPF) or HIC column (FAPF) , the eluate containing botulinum toxin, through the ultrafiltration pump NAI-1541834789v1 112Attorney Docket No.13371-304-228 head into the inlet of the tangential flow filtration membrane. A second tube from the permeate outlet of the tangential flow filtration membrane is connected to the ultrafiltration (UF) permeate container. A tube from the retentate outlet of the tangential flow filtration membrane to the retentate reservoir is secured, and a fourth tube from the diafiltration (DF) buffer through the diafiltration pump head and into the retentate reservoir is also secured. The storage buffer of the system is flushed, as is the membrane, by flushing the membrane with at least about 720 mL of water for injection (WFI) with the retentate directed to waste, after which the membrane is further flushed with at least about 4200 mL of water for injection with the retentate recirculating to the reservoir. After this, membrane sanitation (if necessary) is carried out by flushing the membrane with at least about 200 mL of 1N sodium hydroxide with the retentate directed to waste, followed by a flushing of the membrane with at least about 200 mL of 1N NaOH with the retentate recirculating to the reservoir for a minimum of 30 minutes. Equilibration is then performed, by flushing the membrane with equilibration buffer (10 mM potassium citrate at a pH of 6.5), with retentate directed to waste until the retentate and permeate pH is within + / - 0.2 units of the pH of the equilibration buffer (for example, within + / - 0.2 units of pH 6.5).

[0297] The concentration of the material (eluate (product pool) from the cation exchange column) is determined, to see if dilution or concentration (exemplary processing) is appropriate (an example target concentration can be about 0.7 mg / mL). Dilution is accomplished utilizing 10 mM potassium citrate, pH 6.5. A target volume is determined, for example for a 0.7 mg / mL product concentration (target vol= (starting concentration / starting vol) / 0.7 mg / mL).

[0298] The product pool (eluate (accordingly processed or not) from cation exchange column) is loaded onto the membrane and recirculation (with permeate outlet closed) of the system (TFF system) is run for at least 2 minutes with no backpressure, after which the permeate valve is slowly opened while adjusting the retentate back pressure valve to a target of about 7 psig transmembrane pressure. For dilution, 10 mM potassium citrate, pH 6.5 is added to target volume, and moved onto diafiltration without ultrafiltration; for concentration, ultrafiltration is begun. For diafiltration: permeate waste is collected in a new container (target diafiltration volume is 5X diafiltration volume) and diafiltered with at least 5 diafiltration volumes of 10 mM potassium citrate, pH 6.5. Diafiltration process data is collected at a minimum of 10-minute intervals (permeate weight g / vol mL, inlet pressure (psig), retentate pressure (psig), permeate pressure (psig) and transmembrane pressure (psig)). For recirculation / and rinse: with the NAI-1541834789v1 113Attorney Docket No.13371-304-228 permeate outlet filter closed, the system is recirculated / run for at least 2 minutes with no backpressure and the system is rinsed with at least 20 mL of 10 mM potassium citrate, pH 6.5. The product pool includes the retentate and the rinse. A sample can be taken from the product pool and subjected to verification analysis including, for example, UV at 278nm, SDS-Page, LcHPLC, SE-HPLC, qPCR, RP-HPLC, Native-Page, AUC, Limulus amebocyte lysate, Western Blot and ELISA tests. For post-use cleaning, the system is flushed with 1N sodium hydroxide, recirculated for at least 10 minutes, after which the system is flushed and stored with 0.1 N sodium hydroxide therein.

[0299] Sterile filtration and filling are then conducted for storing and dividing the bulk neurotoxin. Concentration adjustment is performed to adjust toxin concentration, using 10 mM potassium citrate, pH 6.5, to about 0.5 mg / mL with the post rinse sample. If toxin concentration is less than about 0.5 mg / mL, then no concentration adjustment is needed.

[0300] Using a sterile pipette, 10mL / 0.75mL aliquots into each of sterile 15 mL / 1.5mL sample tubes are made. The product container is gently stirred by hand and transfer the required amount of solution (containing bulk drug substance, i.e. bulk botulinum toxin) into each vial. The samples are stored a maximum of 5 days at 2 °C – 8 °C refrigerator or 0.75 mL of the filtrate product pool is transferred to cryovials. The cryovials are stored at -70 °C + / - 5 °C. 5.3. Example 3 - A Column Chromatographic Process for Obtaining a Botulinum Neurotoxin

[0301] This example describes a column chromatographic process for obtaining a botulinum neurotoxin. It is contemplated that certain steps described in Example 1, certain steps described in Example 2, and certain steps described in this Example 3 can be combined or switched with each other for obtaining the botulinum neurotoxin as deemed appropriated in the opinion and judgment of a person skilled in the art.

[0302] Clostridium botulinum bacteria are cultured and allowed to grow until fermentation is complete. The fermentation culture is then used in the following purification procedure:

[0303] The fermentation culture is subjected to acid precipitation using 3M sulfuric acid to reduce pH to 3.5 a...

Claims

Attorney Docket No.13371-304-228 What is claimed is:

1. A liquid composition comprising Clostridium botulinum neurotoxin serotype A (BoNT / A), wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, wherein: (A) the filter is not gamma-irradiated; (B) no free radicals are detectable on the filter, optionally wherein the amount of free radicals on the filter is determined using Electron Spin Resonance (ESR); or (C) the amount of free radicals on the filter is less than that on a gamma-irradiated filter of the same type, optionally wherein (1) the gamma-irradiated filter has a pore size of about 0.22 µm, (2) the amount of free radicals on the gamma-irradiated filter is determined using ESR, and / or (3) the amount of free radicals on the filter is determined using ESR.

2. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein the amount of free radicals on the filter is less than 250 x 1010spins / mg as determined using ESR or less than 100 x 1010spins / mg as determined using ESR, or no free radicals are detectable on the filter as determined using ESR.

3. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein: (A) no Carbon-centered radicals are detectable in an aqueous solution containing a spin- trap molecule following exposure to the filter, optionally wherein (1) the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR and / or (2) the exposure is for about 30 minutes or about 15 hours; (B) the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule following exposure to the filter is less than the amount of Carbon-centered radicals in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution, optionally wherein (1) NAI-1541834789v1 119Attorney Docket No.13371-304-228 the gamma-irradiated filter has a pore size of about 0.22 µm, (2) the amount of Carbon- centered radicals in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR, (3) the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR, and / or (4) the exposure is for about 30 minutes or about 15 hours; (C) the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes or less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes, or no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes; (D) the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours or less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours, or no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours; (E) no degradation product of a spin-trap molecule is detectable in an aqueous solution containing the spin-trap molecule following exposure to the filter, optionally wherein: (1) the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR and / or (2) the exposure is for about 30 minutes or about 15 hours; (F) the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule following exposure to the filter is less than the amount of degradation product of the spin-trap molecule in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution, optionally wherein: (1) the gamma-irradiated filter has a pore size of about 0.22 µm, (2) the amount of degradation product of the spin-trap molecule in the reference aqueous solution containing the spin-trap molecule following exposure to the NAI-1541834789v1 120Attorney Docket No.13371-304-228 gamma-irradiated filter is determined using ESR, (3) the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR, and / or (4) the exposure is for about 30 minutes or about 15 hours; (G) the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes or less than 0.2 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes, or no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes; or (H) the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours or less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours, or no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours; optionally wherein the spin-trap molecule is N-tert-butyl-α-phenylnitrone (PBN).

4. The liquid composition of any one of claims 1-3, wherein potency of the liquid composition (1) does not decrease after 3 months of storage, or decreases less than 15% or less than 10% after 3 months of storage, and / or (2) does not decrease after 18 months of storage, or decreases less than 35% or less than 30% after 18 months of storage; optionally wherein potency of the liquid composition is determined using a cell-based potency assay or a mouse 50% lethal dose (MLD50) assay.

5. A liquid composition comprising BoNT / A, wherein the liquid composition is a filtration product of a solution comprising BoNT / A, and said filtration is performed with a filter, and wherein potency of the liquid composition (1) does not decrease after 3 months of storage, or decreases less than 15% or less than 10% after 3 months of storage, and / or (2) does not decrease after 18 months of storage, or decreases less than 35% or less than 30% after 18 months of storage; optionally wherein potency of the liquid composition is determined using a cell-based potency assay or a mouse 50% lethal dose (MLD50) assay. NAI-1541834789v1 121Attorney Docket No.13371-304-228 6. The liquid composition of any one of claims 1-5, wherein the filter has a pore size of about 0.22 µm and / or is autoclaved.

7. The liquid composition of any one of claims 1-6, wherein the BoNT / A is a 900 kDa BoNT / A complex, optionally wherein the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum, further optionally wherein the Type A strain of Clostridium botulinum is the Type A Hall strain of Clostridium botulinum, and still further optionally wherein the BoNT / A is onabotulinumtoxinA.

8. The liquid composition of any one of claims 1-7, which is animal product free.

9. The liquid composition of any one of claims 1-8, further comprising one or more pharmaceutically acceptable carriers, optionally wherein the one or more pharmaceutically acceptable carriers comprise human serum albumin and sodium chloride, and further optionally wherein (1) the one or more pharmaceutically acceptable carriers comprise about 0.5 mg of human serum albumin per 100 units of BoNT / A, (2) the one or more pharmaceutically acceptable carriers comprise about 0.9 mg of sodium chloride per 100 units of BoNT / A, and / or (3) the human serum albumin is recombinant human serum albumin.

10. The liquid composition of any one of claims 1-9, which comprises about 50 units, about 100 units or about 200 units of BoNT / A.

11. The liquid composition of any one of claims 1-10, which has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

12. The liquid composition of any one of claims 1-11, wherein the BoNT / A is produced by a process that comprises one or more steps of column chromatography, optionally wherein the one or more steps of column chromatography are performed during purification of the BoNT / A and / or the one or more steps of column chromatography comprise hydrophobic interaction chromatography, and further optionally wherein the one or more steps of column chromatography further comprise anion exchange chromatography and / or cation exchange chromatography.

13. The liquid composition of any one of claims 1-12, wherein the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate, optionally wherein the BoNT / A is produced by a process that NAI-1541834789v1 122Attorney Docket No.13371-304-228 does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the BoNT / A.

14. The liquid composition of any one of claims 1-13, which does not contain a protease inhibitor, optionally wherein the protease inhibitor is benzamidine hydrochloride.

15. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, wherein: (A) the filter is not gamma-irradiated; (B) no free radicals are detectable on the filter, optionally wherein the amount of free radicals on the filter is determined using ESR; or (C) the amount of free radicals on the filter is less than that on a gamma-irradiated filter of the same type, optionally wherein (1) the gamma-irradiated filter has a pore size of about 0.22 µm, (2) the amount of free radicals on the gamma-irradiated filter is determined using ESR, and / or (3) the amount of free radicals on the filter is determined using ESR.

16. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein the amount of free radicals on the filter is less than 250 x 1010spins / mg as determined using ESR or less than 100 x 1010spins / mg as determined using ESR, or no free radicals are detectable on the filter as determined using ESR.

17. A method of producing a liquid composition comprising BoNT / A, said method comprising filtering a solution comprising BoNT / A with a filter to produce said liquid composition, and wherein: (A) no Carbon-centered radicals are detectable in an aqueous solution containing a spin- trap molecule following exposure to the filter, optionally wherein (1) the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR and / or (2) the exposure is for about 30 minutes or about 15 hours; (B) the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule following exposure to the filter is less than the amount of Carbon-centered radicals in a reference aqueous solution containing the spin-trap molecule following NAI-1541834789v1 123Attorney Docket No.13371-304-228 exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution, optionally wherein (1) the gamma-irradiated filter has a pore size of about 0.22 µm, (2) the amount of Carbon- centered radicals in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR, (3) the amount of Carbon-centered radicals in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR, and / or (4) the exposure is for about 30 minutes or about 15 hours; (C) the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.5 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes or less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes, or no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes; (D) the amount of Carbon-centered radicals in an aqueous solution containing a spin-trap molecule is less than 2.3 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours or less than 1.0 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours, or no Carbon-centered radicals are detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours; (E) no degradation product of a spin-trap molecule is detectable in an aqueous solution containing the spin-trap molecule following exposure to the filter, optionally wherein (1) the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR and / or (2) the exposure is for about 30 minutes or about 15 hours; (F) the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule following exposure to the filter is less than the amount of degradation product of the spin-trap molecule in a reference aqueous solution containing the spin-trap molecule following exposure to a gamma-irradiated filter of the same type, and wherein the reference aqueous solution is substantially identical to the aqueous solution, optionally wherein (1) the gamma-irradiated filter has a pore size of NAI-1541834789v1 124Attorney Docket No.13371-304-228 about 0.22 µm, (2) the amount of degradation product of the spin-trap molecule in the reference aqueous solution containing the spin-trap molecule following exposure to the gamma-irradiated filter is determined using ESR, (3) the amount of degradation product of the spin-trap molecule in the aqueous solution containing the spin-trap molecule following exposure to the filter is determined using ESR, and / or (4) the exposure is for about 30 minutes or about 15 hours; (G) the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 0.3 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes or less than 0.2 x 10-7M as determined using ESR after being exposed to the filter for about 30 minutes, or no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 30 minutes; or (H) the amount of degradation product of a spin-trap molecule in an aqueous solution containing the spin-trap molecule is less than 1.5 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours or less than 1 x 10-7M as determined using ESR after being exposed to the filter for about 15 hours, or no degradation product of the spin-trap molecule is detectable in the aqueous solution containing the spin-trap molecule as determined using ESR after being exposed to the filter for about 15 hours; optionally wherein the spin-trap molecule is PBN.

18. The method of any one of claims 15-17, wherein the filter has a pore size of about 0.22 µm and / or is autoclaved.

19. The method of any one of claims 15-18, wherein the BoNT / A is a 900 kDa BoNT / A complex, optionally wherein the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum, further optionally wherein the Type A strain of Clostridium botulinum is the Type A Hall strain of Clostridium botulinum, and still further optionally wherein the BoNT / A is onabotulinumtoxinA.

20. The method of any one of claims 15-19, wherein the solution comprising BoNT / A and / or the liquid composition is animal product free.

21. The method of any one of claims 15-20, wherein the solution comprising BoNT / A and / or the liquid composition comprises one or more pharmaceutically acceptable carriers, NAI-1541834789v1 125Attorney Docket No.13371-304-228 optionally wherein the one or more pharmaceutically acceptable carriers comprise human serum albumin and sodium chloride, and further optionally wherein (1) the one or more pharmaceutically acceptable carriers comprise about 0.5 mg of human serum albumin per 100 units of BoNT / A, (2) the one or more pharmaceutically acceptable carriers comprise about 0.9 mg of sodium chloride per 100 units of BoNT / A, and / or (3) the human serum albumin is recombinant human serum albumin.

22. The method of any one of claims 15-21, wherein the liquid composition comprises about 50 units, about 100 units or about 200 units of BoNT / A.

23. The method of any one of claims 15-22, wherein the liquid composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

24. The method of any one of claims 15-23, wherein the BoNT / A is produced by a process that comprises one or more steps of column chromatography, optionally wherein the one or more steps of column chromatography are performed during purification of the BoNT / A and / or the one or more steps of column chromatography comprise hydrophobic interaction chromatography, and further optionally wherein the one or more steps of column chromatography further comprise anion exchange chromatography and / or cation exchange chromatography.

25. The method of any one of claims 15-24, wherein the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate, optionally wherein the BoNT / A is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the BoNT / A.

26. The method of any one of claims 15-25, wherein the method does not involve using a protease inhibitor, optionally wherein the protease inhibitor is benzamidine hydrocholoride.

27. A liquid composition comprising BoNT / A, wherein the liquid composition is produced by the method of any one of claims 15-26.

28. A solid composition, which is a dried product of the liquid composition of any one of claims 1-14 and 27, optionally wherein the solid composition is a vacuum-dried, freeze- dried, or lyophilized product of the liquid composition. NAI-1541834789v1 126Attorney Docket No.13371-304-228 29. The solid composition of claim 28, which does not contain a protease inhibitor, optionally wherein the protease inhibitor is benzamidine hydrochloride.

30. The solid composition of claim 28 or 29, which comprises about 50 units, about 100 units or about 200 units of BoNT / A.

31. The solid composition of any one of claims 28-30, which has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

32. A powdered pharmaceutical composition comprising a 900 kDa BoNT / A complex, human serum albumin, and sodium chloride, wherein the powdered pharmaceutical composition is a dried product of a liquid composition comprising the 900 kDa BoNT / A, wherein the liquid composition is a filtration product of a solution comprising the 900 kDa BoNT / A complex, and said filtration is performed with a filter that is not gamma- irradiated, optionally wherein:. (A) the powdered pharmaceutical composition is a vacuum-dried, freeze-dried, or lyophilized product of the liquid composition; (B) the 900 kDa BoNT / A complex is produced by a process that comprises one or more steps of column chromatography, optionally wherein the 900 kDa BoNT / A complex is produced by a process that comprises one or more steps of column chromatography and / or the one or more steps of column chromatography comprise hydrophobic interaction chromatography, and further optionally wherein the one or more steps of column chromatography further comprise anion exchange chromatography and / or cation exchange chromatography; (C) the 900 kDa BoNT / A complex is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate, optionally wherein the 900 kDa BoNT / A complex is produced by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate during purification of the 900 kDa BoNT / A complex; (D) the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum, optionally wherein the Type A strain of Clostridium botulinum is the Type A Hall strain of Clostridium botulinum, and further optionally wherein the 900 kDa BoNT / A complex is onabotulinumtoxin A; NAI-1541834789v1 127Attorney Docket No.13371-304-228 (E) the powdered pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, optionally wherein the one or more pharmaceutically acceptable carriers comprise human serum albumin and sodium chloride, and further optionally wherein (1) the one or more pharmaceutically acceptable carriers comprise about 0.5 mg of human serum albumin per 100 units of BoNT / A, (2) the one or more pharmaceutically acceptable carriers comprise about 0.9 mg of sodium chloride per 100 units of BoNT / A, and / or (3) the human serum albumin is recombinant human serum albumin; (F) the powdered pharmaceutical composition comprises about 50 units, about 100 units or about 200 units of the 900 kDa BoNT / A complex; and / or (G) the powdered pharmaceutical composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg.

33. A method of producing a powdered pharmaceutical composition comprising a 900 kDa BoNT / A complex, human serum albumin, and sodium chloride, said method comprising filtering a solution comprising the 900 kDa BoNT / A complex with a filter that is not gamma-irradiated to produce a liquid composition comprising the 900 kDa BoNT / A complex, and drying the liquid composition to produce the powdered pharmaceutical composition, optionally wherein: (A) the step of drying is vacuum-drying, freeze-drying, or lyophilizing; (B) the method comprises producing the 900 kDa BoNT / A complex by a process that comprises one or more steps of column chromatography, optionally wherein the one or more steps of column chromatography are performed during purification of the 900 kDa BoNT / A complex and / or the one or more steps of column chromatography comprise hydrophobic interaction chromatography, and further optionally wherein the one or more steps of column chromatography further comprise anion exchange chromatography and / or cation exchange chromatography; (C) the method comprises producing the 900 kDa BoNT / A complex by a process that does not comprise a step of precipitation with cold ethanol, hydrochloric acid, or ammonia sulfate, optionally wherein the method comprises producing the 900 kDa BoNT / A complex by a process that does not comprise a step of precipitation with cold NAI-1541834789v1 128Attorney Docket No.13371-304-228 ethanol, hydrochloric acid, or ammonia sulfate during purification of the 900 kDa BoNT / A complex; (D) the 900 kDa BoNT / A complex is produced by a Type A strain of Clostridium botulinum, optionally wherein the Type A strain of Clostridium botulinum is the Type A Hall strain of Clostridium botulinum, and further optionally wherein the 900 kDa BoNT / A complex is onabotulinumtoxin A; (E) the powdered pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, optionally wherein the one or more pharmaceutically acceptable carriers comprise human serum albumin and sodium chloride, and further optionally wherein (1) the one or more pharmaceutically acceptable carriers comprise about 0.5 mg of human serum albumin per 100 units of BoNT / A, (2) the one or more pharmaceutically acceptable carriers comprise about 0.9 mg of sodium chloride per 100 units of BoNT / A, and / or the human serum albumin is recombinant human serum albumin; (F) the method does not involve using a protease inhibitor, optionally wherein the protease inhibitor is benzamidine hydrocholoride; (G) the powdered pharmaceutical composition comprises about 50 units, about 100 units or about 200 units of the 900 kDa BoNT / A complex; and / or (H) the powdered pharmaceutical composition has a potency of about 2.4 x 107units / mg to about 6.0 x 107units / mg. NAI-1541834789v1 129

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