Systems and methods for making hydroxypropyl-beta-cyclodextrin

Abstract

Provided herein are systems and methods for the manufacture of hydroxypropyl-beta-cyclodextrin.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 351,719, filed on June 13, 2022, entitled “SYSTEMS AND METHODS FOR MANUFACTURING HYDROXYPROPYL-BETA-CYCLODEXTRIN,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to systems and methods for making hydroxypropyl-β-cyclodextrin.Thus, the present disclosure relates generally to the fields of chemistry, pharmacy, and chemical engineering. Background Art

[0004] Hydroxypropyl-β-cyclodextrin (HPBCD) has received increasing attention in the pharmaceutical field due to its potential to treat a variety of disease types. Therefore, new systems and methods are needed to produce HPBCD to meet the growing demand. Summary of the invention

[0005] A reactor system for producing hydroxypropyl-β-cyclodextrin (HPBCD) is provided herein. The system includes propylene oxide feed, β-cyclodextrin (BCD) feed, mass flow meter or mass flow controller and static mixer. In some embodiments, the system also includes a back pressure regulator. In some other embodiments, the system includes a mass flow controller. In further embodiments, the system includes a temperature controller. In some aspects, the static mixer is a spiral static mixer.

[0006] In some embodiments, the propylene oxide feed is pressurized. In other embodiments, the BCD feed is pressurized.

[0007] In some embodiments, the system includes at least two propylene oxide feeds. In some aspects, the at least two propylene oxide feeds are operably connected to a separate mass flow meter or mass flow controller. In some embodiments, the first propylene oxide feed provides a concentration of about 7 to about 15 equivalents of BCD, and the second propylene oxide feed provides a concentration of about 3.5 to about 15 equivalents of BCD.

[0008] In some embodiments, the BCD feed comprises sodium hydroxide (NaOH). In some aspects, the β-cyclodextrin feed comprises NaOH at a concentration of about 5 to about 10 equivalents.

[0009] In some embodiments, the system further comprises a pump.In some aspects, the pump can be a syringe pump operably connected to one or more feeds.

[0010] In some embodiments, the system further comprises a coil. In some embodiments, the system comprises a plug flow reactor. In some aspects, the plug flow reactor comprises at least two coils and a temperature control unit. In some embodiments, a back pressure regulator is operably connected to the plug flow reactor or the coil. In some aspects, the temperature control unit maintains a temperature of about 30°C to about 60°C.

[0011] In some embodiments, propylene oxide is metered in at two locations. In some aspects, propylene oxide is metered before the plug flow reactor. In some additional aspects, at least one dose of propylene oxide is metered before the first coil and at least another dose of propylene oxide is metered before the second coil.

[0012] In some embodiments, the system also includes a holding tank. In some aspects, the holding tank is operably connected to an acid feed. In some further aspects, the acid feed comprises hydrochloric acid, sulfuric acid, lactic acid, acetic acid, formic acid, citric acid, oxalic acid, uric acid, malic acid, fumaric acid, tartaric acid or a combination thereof. In some other aspects, the system provides a total residence time of about 30 minutes to about 70 minutes.

[0013] Also provided herein is a method for making a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, comprising: (a) contacting a hydroxypropyl-β-cyclodextrin (HPBCD) mixture with at least two solvents, the HPBCD mixture comprising a high degree of substitution HPBCD and a low degree of substitution HPBCD; (b) dissolving the high degree of substitution HPBCD in one of the solvents; and (c) removing the low degree of substitution HPBCD by precipitation. In some embodiments, the at least two solvents include ethanol and acetone.

[0014] Also provided herein is a method for making a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, comprising: (a) contacting a hydroxypropyl-β-cyclodextrin (HPBCD) mixture with at least two solvents, the HPBCD mixture comprising a high degree of substitution HPBCD; (b) dissolving the high degree of substitution HPBCD in one of the solvents to form a mother liquor; and (c) filtering out the mother liquor. In some embodiments, the method comprises lyophilizing the mother liquor to produce a solid. In some aspects, the method further comprises analyzing the solid by MALDI-TOF to determine the degree of substitution. In some embodiments, the at least two solvents comprise ethanol and acetone.

[0015] Also provided herein is a composition comprising a mixture of methylated 2-hydroxypropyl-β-cyclodextrin (HPBCD) having a degree of substitution of about 6.5 to about 9.5 and methylated glucose with 0 to 5 2-hydroxypropyl groups. In some examples, the composition may have Fig.25 Mass spectra depicted.

[0016] Also provided herein is a method for oligomeric substitution by methanolysis of a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, the method comprising: (a) mixing HPBCD and methanol; (b) stirring until the HPBCD is dissolved; (c) adding an acid to the mixture; (d) heating the mixture to at least 50°C to about 90°C; (e) stirring the mixture and maintaining heating for at least about 24 hours; (f) neutralizing the mixture with a base; and (g) filtering the mixture.

[0017] Also provided herein is a method for purifying a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, comprising: (a) purifying the HPBCD mixture by nanofiltration; (b) collecting a total of at least 5 diafiltration volumes of nanofiltration permeate; and (c) lyophilizing the resulting retentate to produce solid hydroxypropyl-β-cyclodextrin.

[0018] In some embodiments, purification occurs at a feed pressure of about 200 to about 400 psi (e.g., about 300 psi). In some embodiments, purification by nanofiltration comprises a flat sheet membrane. In some aspects, the flat sheet membrane comprises 0.010 to 0.050 m 2 area.

[0019] In some embodiments, the method comprises collecting a total of at least 7 diafiltration volumes, or more preferably a total of at least 10 diafiltration volumes of nanofiltration permeate.

[0020] Also provided herein is a method for purifying a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, comprising: (a) purifying the HPBCD mixture by nanofiltration; (b) collecting a total of at least 5 diafiltration volumes of nanofiltration permeate; and (c) analyzing the propylene glycol content of the obtained retentate. In some embodiments, the method further comprises freeze-drying the obtained retentate to produce solid hydroxypropyl-β-cyclodextrin.

[0021] In some embodiments, the claimed invention also encompasses compositions, including compositions produced according to any of the methods or systems described herein. For example, the composition may comprise a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 0.3% of unsubstituted β-cyclodextrin ("DS-0") or less than 1% of β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"), wherein the composition is suitable for intrathecal, intravenous, or intraventricular administration to a patient in need thereof. The present invention may also include a composition produced by any of embodiments 26-30 or 33-42 or the method of any of claims 28-32 or 35-45, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and at least 70% of the β-cyclodextrin has a hydroxypropyl group at DS-1; a DS within ±1σ, where σ is the standard deviation. In addition, the present invention may also include a composition produced by any of embodiments 26-30 or 33-42 or any of claims 28-32 or 35-45, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and the mixture comprises 1% to 10% of β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7").

[0022] Alternatively, the composition can be produced by any of the methods described herein (such as the method of any of Embodiments 28-32 or 35-45), and the composition comprises a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and the mixture comprises no more than 25% of β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"). Likewise, the present invention may also include a composition produced by the method of any one of embodiments 26-30 or 33-42 or any one of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and, the mixture comprises no more than 20% of β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"). In another aspect, the composition can be produced by the method of any one of embodiments 26-30 or 33-42 or any one of claims 28-32 or 35-45, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 2.5% of β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"), wherein the composition is suitable for intrathecal, intravenous, or intracerebroventricular administration to a patient in need thereof. The composition can be produced by any of embodiments 26-30 or 33-42 or the method of any of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and, the mixture comprises 5% to 25% of β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6"). Finally, the present invention may also include a composition produced by any of embodiments 26-30 or 33-42 or any of claims 28-32 or 35-45, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and β-cyclodextrin having glucose units having the following structure:

[0023]

[0024] wherein R1, R2 and R3 are independently at each occurrence -H or -HP, wherein HP comprises one or more hydroxypropyl groups, and in the β-cyclodextrin, the total percentage of occurrences in which R1 and R2 combined are HP ranges from 85% to 95%.

[0025] In some embodiments, the present invention simultaneously produces at least two different, at least three different, at least four different, at least five different compositions, wherein each composition comprises a different mixture of β-cyclodextrin molecules. Thus, in some embodiments, the present invention simultaneously produces at least a plurality of different compositions, wherein each composition comprises a different mixture of β-cyclodextrin molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 An example diagram of a system of the present disclosure is shown.

[0027] FIG. 2 shows an exemplary 1 H-NMR spectrum. The spectrum above is that of β-cyclodextrin 1 H-NMR spectrum. The middle spectrum is that of HPBCD with a molecular weight of 1380 Da. 1 H-NMR spectrum. The spectrum below is that of HPBCD with a molecular weight of 1540Da. 1 H-NMR spectrum.

[0028] Figure 3 Shown are the HPLC-ELSD chromatogram (orange line) and the approximate population generated by Monte Carlo rejection sampling, represented as a histogram (blue bars).

[0029] Figure 4 Shown is a plot of predicted DS values ​​versus actual DS values ​​using the fitting parameter equation described in Example 2. The orange points are validation samples that were not used for equation fitting.

[0030] Figure 5 A plot of predicted DS values ​​versus actual DS values ​​using the fitting parameter equation described in Example 2 is shown, showing that the estimated DS for the low DSHPBCD material is negative.

[0031] Figure 6 Shown is a plot of the predicted DS values ​​versus the actual DS values ​​estimated from HPLC-ELSD data using the modified parameter equation (Eq. 3).

[0032] Figure 7 Shown is a plot of the predicted DS values ​​versus the actual DS values ​​estimated from HPLC-ELSD data using the modified parameter equation (Eq. 4).

[0033] Figure 8Shown is a graph of DS values ​​for HPBCD produced using the system described herein.

[0034] Fig. 9 A graph showing actual DS versus calculated DS for HPBCD produced using the system of the present disclosure is shown.

[0035] Fig.10 Shown is a graph of actual HPLC-ELSD peak variance versus calculated HPLC-ELSD peak variance for HPBCD produced using the system of the present disclosure.

[0036] Fig.11 The separation of HPBCD prepared from ethanol / acetone is shown. 1 H-NMR spectrum.

[0037] Fig.12 The HPBCD material remaining in the mother liquor after separation from ethanol / acetone is shown. 1 H-NMR spectrum.

[0038] Fig.13 Shown are overlaid HPLC-ELSD spectra of isolated solid HPBCD, feed material, and mother liquor after separation from ethanol / acetone.

[0039] Fig.14 Shown is the powder x-ray diffraction pattern of isolated solid HPBCD overlaid with the pattern of the starting material.

[0040] Fig.15 The changes in mother liquor concentration and DS with increasing ethanol volume % are shown.

[0041] Fig.16 The changes in the mother liquor concentration and the DS of HPBCD in the mother liquor and solids with increasing vol% of acetone are shown.

[0042] Fig.17 ELSD data of discarded material from fractionation of HPBCD are shown.

[0043] Fig.18 Shown are ELSD data of starting material overlaid with fractionated HPBCD product.

[0044] Fig.19 Shown 1 The MALDI-TOF spectrum of the purified HPBCD showed a DS of 6.9 as determined by H-NMR.

[0045] Fig. 20 Shown 1 MALDI-TOF spectrum of purified HPBCD with a DS of 9.3 as determined by H-NMR.

[0046] Fig.21 Shown are MALDI-TOF data of the crude quench reactor effluent having a DS of 7.7 as determined by HPLC-ELSD analysis.

[0047] Fig. 22 The product distribution of the Cavitron HP7 HPBCD using MALDI-TOF is shown.

[0048] Fig.23 The product distribution of HPBCD prepared using the system of the present disclosure is shown.

[0049] Fig.24 DS and variance of the DoE MALDI-TOF data are shown.

[0050] Fig.25 The mass spectrum of methanolyzed HPBCD is shown.

[0051] Figure 26A-26B An exemplary flow chart of the system of the present disclosure is shown.

[0052] Fig.27A Depicted is a non-limiting example of a single enzyme reaction for converting sucrose to amylose according to embodiments of the present disclosure.

[0053] Fig.27B Depicted is a non-limiting example of a dual enzyme reaction for converting sucrose to amylose according to embodiments of the present disclosure.

[0054] Fig.28 Depicted is a non-limiting example of an enzymatic reaction for converting amylose to α-cyclodextrin according to embodiments of the present disclosure. DETAILED DESCRIPTION

[0055] A reactor system for producing hydroxypropyl-β-cyclodextrin (HPBCD) is provided herein. Figure 1, the reactor system 100 of the present disclosure generally includes a propylene oxide feed 102, a β-cyclodextrin (BCD) feed 104, a mass flow meter 106 or a mass flow controller 108, and a static mixer 110. Propylene oxide from the propylene oxide feed 102 and BCD from the BCD feed 104 are combined and mixed in the static mixer 110. The reactants then pass through a reactor 118 to form a first reactor effluent, after which, optionally, more propylene oxide from the second propylene oxide feed 102 is added. The mixture passes through a second static mixer 110 and then enters a second reactor 118 to form a second reactor effluent. The second reactor effluent 118 is collected in a collection tank 124, where it is quenched with an acid provided by an acid feed 126. The reactor system described herein can be operated to efficiently produce HPBCD with a target degree of substitution.

[0056] The reactor system of the present disclosure can be operated to produce HPBCD according to the following reaction scheme: BCD is reacted with propylene oxide and a base (eg, sodium hydroxide) and then quenched with an acid (eg, hydrochloric acid).

[0057]

[0058] System 100 includes at least one propylene oxide feed 102; however, it is noted that the reactor system may include at least two propylene oxide feeds (i.e., at least a plurality of propylene oxide feeds), at least three propylene oxide feeds, etc. Propylene oxide feed 102 may include a tank with piping and instrumentation operable to deliver propylene oxide to system 100. Propylene oxide may be introduced into the system at one or more locations. Propylene oxide may be introduced at a rate of about 0.1 g / min to about 10 g / min; for example, about 0.1 g / min, 0.2 g / min, 0.3 g / min, 0.4 g / min, 0.5 g / min, 0.6 g / min, 0.7 g / min, 0.8 g / min, 0.9 g / min, 1.0 g / min, 2.0 g / min, 3.0 g / min, 4.0 g / min, 5.0 g / min, 6.0 g / min, 7.0 g / min, 8.0 g / min, 9.0 g / min, or about 10.0 g / min. Propylene oxide may be metered in at one or more locations in system 100. In systems 100 having more than one reactor 118, propylene oxide may be metered in before each reactor. For example, Figure 1As shown in the system 100 of FIG. 1 , propylene oxide may be metered in at two locations. However, generally, at least one dose of propylene oxide is metered in prior to reactor 118. In some embodiments, the propylene oxide feed may comprise a racemic mixture of propylene oxide; in other embodiments, the propylene oxide feed may comprise enantiomerically pure propylene oxide. The propylene oxide may include deuterated propylene oxide.

[0059] The propylene oxide may be metered in at a concentration of about 1 to about 20, about 3.5 to about 20, about 5 to about 20, about 7 to about 20, about 1 to about 15, about 3.5 to about 15, about 5 to about 15, or about 7 to about 15 molar equivalents of BCD. For example, the propylene oxide may be metered in at a concentration of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 molar equivalents of BCD. In embodiments where the propylene oxide is metered in at two locations, the first propylene oxide feed may provide propylene oxide at a concentration of about 7 to about 15 molar equivalents of BCD, and the second propylene oxide feed may provide propylene oxide at a concentration of about 3.5 to about 15 molar equivalents of BCD.

[0060] System 100 includes at least one BCD feed 104. BCD feed 104 may include a tank having piping and instrumentation operable to deliver BCD to system 100. BCD can be introduced at a flow rate of about 0.0 g / min to about 20 g / min, about 0.1 g / min to about 10 g / min, about 0.5 g / min to about 7 g / min, or about 1.0 g / min to about 5 g / min; for example, about 0.1 g / min, about 0.2 g / min, about 0.3 g / min, about 0.4 g / min, about 0.5 g / min, about 0.6 g / min, about 0.7 g / min, about 0.8 g / min, about 0.9 g / min, about 1.0 g / min, about 2.0 g / min, about 3.0 g / min, about 4.0 g / min, about 5.0 g / min, about 6.0 g / min, about 7.0 g / min, about 8.0 g / min, about 9.0 g / min, or about 10.0 g / min. The BCD feed can include deuterated BCD.

[0061] The system may also include a base or sodium hydroxide (NaOH) feed. The base or sodium hydroxide may be provided at a concentration of about 1 to about 10, about 3 to about 10, about 5 to about 10, or about 7 to about 10 molar equivalents of BCD, or more preferably about 5 to about 10 molar equivalents of BCD. In some embodiments, the BCD feed may include a base or sodium hydroxide.

[0062] The propylene oxide feed 102 and / or the BCD feed 104 may be pressurized. When a lower reactant flow rate is desired (e.g., about 1.5 g / min), pressurizing the feed may be helpful. The feed may be pressurized with an inert gas, such as a noble gas (e.g., helium, neon, argon, krypton, or xenon), or another non-reactive gas, such as nitrogen or carbon dioxide. The inert gas may be provided in a pressurized tank 114 operably connected to the feed.

[0063] The propylene oxide feed 102 and / or the BCD feed 104 may be operably connected to a mass flow meter 106. The mass flow meter 106 is operable to determine the mass flow of the propylene oxide or BCD. Mass flow meters and methods of measuring mass flow are well known in the art. Additional mass flow meters may be included at other locations in the system to monitor the mass flow of reactants and / or products.

[0064] The propylene oxide feed 102 and / or the BCD feed 104 may be operably connected to a mass flow controller 108. The mass flow controller may be operable to control the mass flow rate of the propylene oxide or BCD; for example, the mass flow controller may increase, decrease, or maintain a constant mass flow rate of the feed. Mass flow controllers and methods of measuring mass flow are well known in the art.

[0065] The mass flow meter 106 and / or the mass flow controller 108 may be operably connected to the controller. The controller may be operable to communicate electronically or wirelessly with any system component. In general, the controller may include one or more processors and a non-transitory computer-readable storage medium having instructions stored thereon for enabling one or more processors to control any one or more of the various aspects of the system to start, operate or shut down one or more of the various aspects to facilitate safe and efficient operation. For example, if an abnormal condition is detected, the controller may interrupt the power supply to any system component. The controller may also be operable to open or close a valve or adjust other system parameters (such as temperature and pressure) to ensure safe and efficient operation of the system.

[0066] The system 100 may also include at least one static mixer 110. The static mixer is operable to increase turbulence by directing the flow, thereby continuously mixing the fluid flowing through the static mixer without using moving parts. Static mixers are generally well known in the art and may include plates, baffles, spiral elements, or geometric grids. In an exemplary embodiment, the static mixer is a spiral static mixer. The system 100 may include one or more static mixers 110 at various points in the system 100.

[0067] One or more feeds can be operably connected to pump 116. The pump can be any pump known in the art, including a centrifugal pump, a positive displacement pump, a syringe pump, etc. Pump 116 can be operably connected to one or more feeds. In an exemplary embodiment, the system includes a syringe pump operably connected to the BCD feed.

[0068] System 100 may also include reactor 118. Reactor includes a plug flow reactor, which may include at least one coil. In some embodiments, the system may include two or more reactors. In other embodiments, the plug flow reactor may include at least two coils. The reactor may have a volume of about 1 to about 1000 mL, about 1 to about 500 mL, about 1 to about 250 mL, or about 1 to about 100 mL; for example, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, about 100 mL, about 250 mL, about 500 mL, or about 1000 mL. The reactor may also have a volume greater than 100 mL, greater than 250 mL, greater than 500 mL, or greater than 1000 mL.

[0069] The volume of the reactor and the flow of the reactant can be used to determine the residence time of the reactant in the reactor. The residence time of the reactant in the reactor can be from about 1 minute to about 360 minutes, from about 3 minutes to about 180 minutes, from about 5 minutes to about 90 minutes or from about 10 minutes to about 60 minutes; For example, from about 10 minutes, from about 15 minutes, from about 20 minutes, from about 25 minutes, from about 30 minutes, from about 35 minutes, from about 40 minutes, from about 45 minutes, from about 50 minutes, from about 55 minutes or from about 60 minutes. The residence time of the reactant in the reactor can be greater than about 60 minutes, greater than about 90 minutes, greater than about 180 minutes or greater than about 360 minutes.

[0070] The system 100 may also include a temperature control unit 122. The temperature control unit may be operably connected to one or more reactors 118. The temperature control unit 122 may maintain the temperature in the reactor 118 at about 40°C to about 50°C, about 35°C to about 55°C, about 30°C to about 60°C, about 25°C to about 65°C, about 20°C to about 70°C, about 15°C to about 90°C, or about 10°C to about 95°C.

[0071] The system 100 may also include a back pressure regulator 112. The back pressure regulator is operable to maintain a predetermined set pressure upstream of the back pressure regulator. Typically, the back pressure regulator 112 is placed near the end of the system 100; for example, directly before the collection tank 124. Therefore, the back pressure regulator can be operably connected to the collection tank 124. The back pressure regulator can also be operably connected to the reactor. The back pressure regulator can be operable to maintain a back pressure of about 0 psi to about 500 psi, about 1 psi to about 400 psi, about 1 psi to about 300 psi, about 3 psi to about 200 psi, about 5 psi to about 100 psi, about 10 psi to about 50 psi; for example, about 10 psi, about 15 psi, about 20 psi, about 25 psi, about 30 psi, about 35 psi, about 40 psi, about 45 psi or about 50 psi. The back pressure regulator may be operable to maintain a back pressure of greater than 5 psi, greater than 10 psi, greater than 25 psi, greater than 50 psi, greater than 100 psi, greater than 200 psi, greater than 300 psi, greater than 400 psi, or greater than 500 psi.

[0072] The system 100 may also include a collection tank 124. The collection tank may be operable to contain the reaction product and / or any remaining reactants. In addition, the collection tank may be operable to quench the mixture from the reactor 118 with an acid (e.g., hydrochloric acid). The feed amount of the acid may be stoichiometric with the HPBCD produced, or an amount sufficient to reach a predetermined pH value. The contents of the collection tank typically comprise a crude HPBCD mixture. Collection tanks are well known in the art. The collection tank may also include a stirring device to continuously stir the contents and maintain a uniform mixture.

[0073] The system 100 may also include an acid feed 126. The acid feed 126 may be operably connected to the collection tank 124. The acid feed may include hydrochloric acid, sulfuric acid, lactic acid, acetic acid, formic acid, citric acid, oxalic acid, uric acid, malic acid, fumaric acid, tartaric acid, or a combination thereof. Alternatively, the reactor effluent may be reacted with an acidic ion exchange resin (e.g., Amberlyst TM 35 Dry) to quench the reactor effluent.

[0074] The system 100 can provide a total residence time of the components of about 5 minutes to about 360 minutes, 5 minutes to about 180 minutes, 10 minutes to about 100 minutes, or more preferably about 30 minutes to about 70 minutes. For example, the system can provide a total residence time of about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 180 minutes, or about 360 minutes. The system can provide a total residence time of greater than about 90 minutes, greater than about 100 minutes, greater than about 180 minutes, or greater than about 360 minutes.

[0075] In some embodiments, one or more feeds may include a deuterated material (eg, deuterated BCD or deuterated propylene oxide). The use of a deuterated material in one or more feeds may produce a deuterated HPBCD mixture.

[0076] In some embodiments, the crude HPBCD mixture collected in the collection tank 124 may be removed by, for example, Figures 26A-26B Further purification is shown in purification process 200. The purification process can be performed as a batch process or a continuous process.

[0077] Before the purification process 200 is started, the HPBCD produced in the reactor 118 can be monitored at the junction 202 to determine the pH, concentration and / or conductivity of the HPBCD produced and / or other parameters of the HPBCD. If any parameter is determined to be outside a predetermined range, the HPBCD can be recycled back to the reactor 118 before being quenched in the collection tank 124.

[0078] Additionally, the crude HPBCD mixture collected in the collection tank 124 may be monitored at junction 204 to determine the pH, concentration, and / or conductivity, or other parameters of the crude HPBCD mixture, prior to initiation of the purification process 200. If any parameter is determined to be outside of a predetermined range, the HPBCD mixture may be recycled back to the collection tank 124 prior to purification.

[0079] Figures 26A-26B The purification process 200 begins by first subjecting the crude HPBCD mixture collected in the collection tank 124 to liquid filtration using a filter 206. The filter 206 may be a liquid material filter that is capable of removing any large solids and / or biological contaminants from the crude HPBCD mixture.

[0080] Next, the HPBCD mixture can be nanofiltered using membrane filter 208. The membrane can have a pore size of about 10nm to about 1nm, such as about 10nm to about 5nm, or about 5nm to about 1nm. In some aspects, the membrane can have a pore size of about 10nm, about 9nm, about 8nm, about 7nm, about 6nm, about 5nm, about 4nm, about 3nm, about 2nm or about 1nm. The membrane filter 208 can include regenerated cellulose, polyethersulfone, polyvinylidene fluoride, polypropylene, polyamide, polyethyleneimine, polyacrylonitrile, polyethylene, polytetrafluoroethylene, metal organic framework, graphene, ceramics, composite materials or other membrane materials known in the art and combinations thereof. Preferably, the membrane filter 208 includes regenerated cellulose or polyethersulfone.

[0081] In some embodiments, filter 208 may include a flat membrane to achieve nanofiltration. Flat membranes and methods of making and obtaining flat membranes for nanofiltration are well known in the art. Flat membranes may have a diameter of about 0.010 m 2 About 0.500m 2 , about 0.050m 2 About 0.100m 2 or about 0.010m 2 About 0.050m 2 For example, a flat sheet membrane may have an area of ​​about 0.010 m 2 、About 0.015m 2 , about 0.020m 2 , about 0.025m 2 , about 0.030m 2 , about 0.035m 2 , about 0.040m 2 、About 0.045m 2 or about 0.050m 2 The flat membrane may have an area greater than 0.010 m 2 , greater than about 0.025m 2 , greater than about 0.050m 2 , greater than about 0.100m 2 , or greater than about 0.500m 2 area.

[0082] Nanofiltration can be accomplished at a temperature of about 40° C. to about 50° C., for example, about 40° C. to about 45° C., or about 45° C. to about 50° C. In some aspects, nanofiltration can be accomplished at a temperature of about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C.

[0083] Nanofiltration can be completed at a pressure of about 1.5 to about 2.0 MPa, such as about 1.5 MPa to about 1.75 MPa, or about 1.75 MPa to about 2.0 MPa. In some aspects, nanofiltration can be completed at a pressure of about 1.5 MPa, about 1.55 MPa, about 1.6 MPa, about 1.65 MPa, about 1.7 MPa, about 1.75 MPa, about 1.8 MPa, about 1.85 MPa, about 1.9 MPa, about 1.95 MPa, or about 2.0 MPa.

[0084] Alternatively, nanofiltration can be accomplished at a pressure of about 0 psi to about 600 psi, about 50 psi to about 600 psi, about 100 psi to about 500 psi, about 200 psi to about 400 psi, or about 250 psi to about 350 psi. For example, purification can occur at a feed pressure of about 25 psi, about 50 psi, about 75 psi, about 100 psi, about 125 psi, about 150 psi, about 175 psi, about 200 psi, about 225 psi, about 250 psi, about 275 psi, about 300 psi, about 325 psi, about 350 psi, about 375 psi, about 400 psi, about 425 psi, about 450 psi, about 475 psi, or about 500 psi.

[0085] The nanofiltered HPBCD mixture may have a conductivity of about 50 μS / cm or less, such as about 45 μS / cm or less, about 40 μS / cm or less, about 35 μS / cm or less, about 30 μS / cm or less, about 25 μS / cm or less, about 20 μS / cm or less, about 15 μS / cm or less, about 10 μS / cm or less, or about 5 μS / cm or less. Alternatively, the nanofiltered HPBCD mixture may have a conductivity of about 0 μS / cm to about 50 μS / cm. For example, the nanofiltered HPBCD mixture can have a conductivity of about 0 μS / cm to about 10 μS / cm, about 0 μS / cm to about 20 μS / cm, about 0 μS / cm to about 30 μS / cm, about 0 μS / cm to about 40 μS / cm, about 0 μS / cm to about 50 μS / cm, about 10 μS / cm to about 50 μS / cm, about 20 μS / cm to about 50 μS / cm, about 30 μS / cm to about 50 μS / cm, or about 40 μS / cm. In some aspects, the nanofiltered HPBCD mixture can have a conductivity of about 5 μS / cm, about 10 μS / cm, about 15 μS / cm, about 20 μS / cm, about 25 μS / cm, about 30 μS / cm, about 35 μS / cm, about 40 μS / cm, about 45 μS / cm, or about 50 μS / cm.

[0086] The nanofiltered HPBCD mixture may have an impurity concentration of about 0.10 wt % or less. Impurities may include propylene glycol, propylene oxide, endotoxins, etc. For example, the nanofiltered HPBCD mixture may have an impurity concentration of about 0.10 wt % or less, about 0.09 wt % or less, about 0.08 wt % or less, about 0.07 wt % or less, about 0.06 wt % or less, about 0.05 wt % or less, about 0.04 wt % or less, about 0.03 wt % or less, about 0.02 wt % or less, or about 0.01 wt % or less.

[0087] Before continuing, the nanofiltered HPBCD mixture may be monitored at junction 210 to determine the purity and conductivity of the nanofiltered HPBCD mixture. If the purity and / or conductivity of the nanofiltered HPBCD mixture is outside a predetermined range, the HPBCD mixture may be recycled to filter 208 for further nanofiltration at junction 210. Purified water may be added to the HPBCD mixture during recycling to aid in subsequent nanofiltration.

[0088] After the HPBCD mixture has been nanofiltered, the HPBCD mixture can be contacted with activated carbon in container 214. The activated carbon may help remove other impurities, such as propylene oxide. The activated carbon can be prepared by first washing the activated carbon in container 212 with purified water to remove any salts. The activated carbon can be washed with purified water until the conductivity of the wash water is less than 10 μS / cm. The activated carbon can then be placed in container 214 along with the nanofiltered HPBCD mixture and stirred to ensure adequate contact with the HPBCD mixture.

[0089] The contact may occur for 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, or 10 hours or more.

[0090] Contacting can occur at a temperature of about 15° C. to about 30° C. For example, contacting can occur at a temperature of about 15° C. to about 20° C., about 15° C. to about 25° C., about 15° C. to about 30° C., about 20° C. to about 30° C., or about 25° C. to about 30° C. In some examples, contacting can occur at a temperature of about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C.

[0091] After contacting, the HPBCD mixture can then be filtered in filter 216 to remove the activated carbon from the mixture. Any filter capable of removing solid activated carbon from a liquid mixture can be used. Preferably, filter 216 comprises a Nutsche filter.

[0092] Once the activated carbon is filtered from the HPBCD mixture, the propylene oxide concentration in the HPBCD mixture may be less than 0.3 ppm. For example, the HPBCD mixture may have a propylene oxide concentration of about 0.2 ppm or less, about 0.1 ppm or less, about 0.09 ppm or less, about 0.08 ppm or less, about 0.07 ppm or less, about 0.06 ppm or less, about 0.05 ppm or less, about 0.04 ppm or less, about 0.03 ppm or less, about 0.02 ppm or less, or about 0.01 ppm or less. If the HPBCD mixture has a propylene oxide concentration of 0.3 ppm or greater, the step of contacting the HPBCD mixture with the activated carbon may be repeated until the propylene oxide concentration is less than 0.3 ppm.

[0093] Once the activated carbon mixture is filtered, the conductivity of the HPBCD mixture may be less than 90 μS / cm. For example, the conductivity of the HPBCD mixture may be about 80 μS / cm or less, about 70 μS / cm or less, about 60 μS / cm or less, about 50 μS / cm or less, about 40 μS / cm or less, about 30 μS / cm or less, about 20 μS / cm or less, or about 10 μS / cm or less. If the conductivity of the HPBCD mixture is 90 μS / cm or greater, the step of nanofiltering the HPBCD mixture may be repeated until the conductivity of the HPBCD mixture is less than 90 μS / cm.

[0094] Before continuing, the HPBCD mixture may be monitored at junction 218 to determine the propylene oxide concentration and / or the conductivity of the HPBCD mixture. If the purity of the HPBCD mixture is outside a predetermined range, the HPBCD mixture may be recycled to filtration vessel 214 for further purification at junction 218. If the conductivity of the HPBCD mixture is outside a predetermined range, the HPBCD mixture may be recycled to filter 208 at junction 218 for further nanofiltration.

[0095] Once the activated carbon is filtered and the HPBCD mixture has the desired purity and conductivity, the HPBCD mixture can be sterile filtered in filter 220. Sterile filtration reduces the presence of bacteria and other microorganisms in the HPBCD mixture. Sterile filtration systems and methods are generally known to those of ordinary skill in the art. The pore size of the sterile filter is preferably 0.22 μm or less. In some embodiments, the sterile filter can be a capsule filter. The sterile filter membrane can comprise polytetrafluoroethylene, polyethersulfone, polyvinylidene fluoride, nylon, polycarbonate, cellulose acetate, or other materials and combinations thereof known in the art for sterile filtration. The sterile filter preferably comprises a polytetrafluoroethylene membrane.

[0096] The HPBCD mixture can then be filtered in a tangential flow filtration system 222. Tangential flow filtration systems and methods are generally known to those of ordinary skill in the art. In some embodiments, the tangential flow filtration system 222 may include a membrane comprising polyethersulfone, polypropylene, polyurethane, regenerated cellulose, polyvinylidene fluoride, or other materials and combinations thereof known in the art for tangential filtration membranes. Preferably, the membrane comprises polyethersulfone.

[0097] After the HPBCD mixture is filtered in the tangential flow filtration system 222, the HPBCD mixture may be dried in a dryer 224. Preferably, the HPBCD mixture is spray dried.

[0098] In embodiments where the HPBCD mixture is spray dried, the input temperature to the spray dryer can be from about 180°C to about 220°C; for example, the input temperature to the spray dryer can be from about 180°C to about 190°C, from about 180°C to about 200°C, from about 180°C to about 210°C, from about 180°C to about 220°C, from about 190°C to about 200°C, from about 190°C to about 210°C, from about 190°C to about 220°C, from about 200°C to about 210°C, from about 200°C to about 220°C, or from about 210°C to about 220°C. The output temperature of the spray dryer can be from about 100°C to about 120°C; for example, the output temperature of the spray dryer can be from about 100°C to about 105°C, from about 100°C to about 110°C, from about 100°C to about 115°C, from about 100°C to about 120°C, from about 105°C to about 110°C, from about 105°C to about 115°C, from about 105°C to about 120°C, from about 110°C to about 115°C, from about 110°C to about 120°C, or from about 115°C to about 120°C.

[0099] Also provided herein is a method for making a HPBCD mixture. The method can be implemented using any of the systems described above. The method includes: (a) contacting the HPBCD mixture with at least two solvents, the HPBCD mixture comprising a high degree of substitution HPBCD and a low degree of substitution HPBCD; (b) dissolving the high degree of substitution HPBCD in one of the solvents; and (c) removing the low degree of substitution HPBCD by precipitation. The at least two solvents can include ethanol and acetone. The high degree of substitution HPBCD can have an average degree of substitution of about 6.0 or greater, about 6.5 or greater, about 7.0 or greater, about 7.5 or greater, about 8.0 or greater, about 8.5 or greater, about 9.0 or greater, about 9.5 or greater. The low degree of substitution can have an average degree of substitution of less than about 7.5, less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5 or less than about 5.0.

[0100] Alternatively, the method may include: (a) contacting a HPBCD mixture with at least two solvents, the HPBCD mixture comprising a high degree of substitution HPBCD; (b) dissolving the high degree of substitution HPBCD in one of the solvents to form a mother liquor; and (c) filtering the mother liquor. The method may also include crystallizing or lyophilizing the mother liquor to produce a solid. The degree of substitution may be determined by analyzing the solid by MALDI-TOF.

[0101] In some embodiments, a deuterated reactant (eg, deuterated BCD or deuterated propylene oxide) can be used to provide a deuterated product, such as deuterated HPBCD.

[0102] System 100 may also include a purification system to purify HPBCD. The purification system may include absorption chromatography alumina, solvent precipitation, or a combination thereof.

[0103] Also provided herein is a method for oligomeric substitution by methanolysis of a HPBCD mixture. The method generally comprises mixing HPBCD and methanol, stirring until the HPBCD is dissolved, adding an acid to the mixture, heating the mixture to at least about 50°C to about 90°C, stirring the mixture and maintaining heating for at least about 24 hours, neutralizing the mixture with a base, and filtering the mixture. In some embodiments, the HPBCD can be a racemic mixture of HPBCD; in other embodiments, the HPBCD can be enantiomerically pure HPBCD.

[0104] Methanol can be added in an amount of about 100 to about 300 molar equivalents of HPBCD; for example, methanol can be added in an amount of about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 350, or about 400 molar equivalents of HPBCD. In some embodiments, methanol can include deuterated methanol.

[0105] The acid added to the mixture may include hydrochloric acid, sulfuric acid, lactic acid, acetic acid, formic acid, citric acid, oxalic acid, uric acid, malic acid, fumaric acid, tartaric acid, or a combination thereof. In an exemplary embodiment, the acid includes sulfuric acid.

[0106] Heating of the mixture can be maintained for at least 24 hours; for example, heating can be maintained for 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, or more than 48 hours.

[0107] The base used to neutralize the mixture can include sodium hydroxide, potassium hydroxide, lithium hydroxide, magnesium hydroxide, calcium hydroxide, or a combination thereof. In an exemplary embodiment, the base is sodium hydroxide.

[0108] The filtration of the mixture can be carried out by a filtration method known in the art. In a preferred embodiment, the filtration of the mixture is carried out by the following nanofiltration method.

[0109] Also provided herein is a method for purifying a HPBCD mixture, comprising: purifying the HPBCD mixture by nanofiltration, collecting a total of at least 5 diafiltration volumes of nanofiltration permeate, and lyophilizing the resulting retentate to produce solid hydroxypropyl-β-cyclodextrin. In some aspects, different numbers of diafiltration volumes may produce different HPBCD mixtures.

[0110] The HPBCD mixture is purified by nanofiltration. Nanofiltration purification may include a flat sheet membrane to perform the nanofiltration. Flat sheet membranes and methods of making and obtaining flat sheet membranes for nanofiltration are well known in the art. Flat sheet membranes may have a diameter of about 0.010 m 2 About 0.500m 2 , about 0.050m 2 About 0.100m 2 or about 0.010m 2 About 0.050m 2 For example, a flat sheet membrane may have an area of ​​about 0.010 m 2 、About 0.015m 2 , about 0.020m 2 , about 0.025m 2 , about 0.030m 2 , about 0.035m 2 , about 0.040m 2 、About 0.045m 2 or about 0.050m 2 The flat membrane may have an area greater than 0.010 m 2 , greater than about 0.025m 2 , greater than about 0.050m 2 , greater than about 0.100m 2, or greater than about 0.500m 2 area.

[0111] Purification and / or feeding can occur at a feed pressure of about 0 psi to about 600 psi, about 50 psi to about 600 psi, about 100 psi to about 500 psi, about 200 psi to about 400 psi, about 250 psi to about 350 psi. For example, purification can occur at a feed pressure of about 25 psi, about 50 psi, about 75 psi, about 100 psi, about 125 psi, about 150 psi, about 175 psi, about 200 psi, about 225 psi, about 250 psi, about 275 psi, about 300 psi, about 325 psi, about 350 psi, about 375 psi, about 400 psi, about 425 psi, about 450 psi, about 475 psi, or about 500 psi.

[0112] At least 1 diafiltration volume, at least 2 diafiltration volumes, at least 3 diafiltration volumes, at least 4 diafiltration volumes, or at least 5 diafiltration volumes of nanofiltration permeate may be collected. For example, at least 5 diafiltration volumes, at least 6 diafiltration volumes, at least 7 diafiltration volumes, at least 8 diafiltration volumes, at least 9 diafiltration volumes, or at least 10 diafiltration volumes of nanofiltration permeate may be collected. In some embodiments, greater than 10 diafiltration volumes of nanofiltration permeate may be collected.

[0113] The method may further include analyzing the propylene glycol content of the resulting retentate. Methods for analyzing the propylene glycol content in the composition are well known in the art and may include mass spectrometry, high pressure liquid chromatography, gas chromatography, and the like.

[0114] Also provided herein is a method for purifying a HPBCD mixture, comprising: purifying the HPBCD mixture by nanofiltration, collecting a total of at least 5 diafiltration volumes of nanofiltration permeate, and analyzing the resulting retentate for propylene glycol content.

[0115] Also provided herein is a composition comprising a methylated 2-hydroxypropyl-β-cyclodextrin mixture having an average degree of substitution of about 6.5 to about 9.5 and methylated glucose having 0 to about 5 2-hydroxypropyl groups; for example, the average degree of substitution of the methylated 2-hydroxypropyl-β-cyclodextrin mixture can be about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0 or about 9.5. The methylated 2-hydroxypropyl-β-cyclodextrin mixture can have an average degree of substitution of about 6.5 to about 9.5, about 6.5 to about 9.0, about 6.8 to about 9.5, about 6.8 to about 9.0, about 7.0 to about 9.5, about 7.0 to about 9.0, about 7.2 to about 9.5, about 7.2 to about 9.0, about 7.5 to about 9.5, about 7.5 to about 9.0, about 7.8 to about 9.5, about 7.8 to about 9.0, about 8.0 to about 9.5, about 8.0 to about 9.0, about 8.2 to about 9.5, about 8.2 to about 9.0, about 8.5 to about 9.5, about 8.5 to about 9.0, about 8.8 to about 9.5, or about 8.8 to about 9.0. In an exemplary embodiment, the composition has an average degree of substitution of about 6.5 to about 9.5, about 7.0 to about 9.0, about 7.2 to about 9.5, about 7.2 to about 9.0, about 7.5 to about 9.5, about 7.5 to about 9.0, about 7.8 to about 9.5, about 7.8 to about 9.0, about 8.0 to about 9.5, about 8.0 to about 9.0, about 8.2 to about 9.5, about 8.2 to about 9.0, about 8.5 to about 9.5, about 8.5 to about 9.0, about 8.8 to about 9.5, or about 8.8 to about 9.0. Fig.25 Mass spectra depicted.

[0116] Also provided herein are compositions produced using one or more systems and / or methods provided herein, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 0.3% unsubstituted β-cyclodextrin ("DS-0") or less than 1% β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"), wherein the composition is suitable for intrathecal, intravenous or intraventricular administration to a patient in need thereof. The mixture may comprise less than 0.1% DS-0 and less than 0.1% DS-1 in total. For example, the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-0; and / or the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-1. The amount of DS-0 or DS-1 can be determined by the peak height of the electrospray MS spectrum.

[0117] The mixture may have an average molar substitution in the range of about 0.40 to about 0.80; for example, the mixture may have an average molar substitution of about 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or about 0.80. The mixture may have an average degree of substitution ("DSS") of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. a”). For example, the mixture can have an average degree of substitution of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or about 7.

[0118] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC, gas chromatography, or the PG / EG ratio of propylene glycol to ethylene glycol.

[0119] The composition may contain no more than 1 ppm of propylene oxide, no more than 0.9 ppm of propylene oxide, no more than 0.8 ppm of propylene oxide, no more than 0.7 ppm of propylene oxide, no more than 0.6 ppm of propylene oxide, no more than 0.5 ppm of propylene oxide, no more than 0.4 ppm of propylene oxide, no more than 0.3 ppm of propylene oxide, no more than 0.2 ppm of propylene oxide, or no more than 0.1 ppm of propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0120] The total amount of other unspecified impurities in the composition can be less than or equal to 0.05%; for example, the total amount of unspecified impurities in the composition can be 0.05%, less than 0.05%, less than or equal to 0.04%, less than or equal to 0.03%, less than or equal to 0.02%, or less than or equal to 0.01%. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0121] The composition may be suitable for intrathecal, intravenous or intraventricular administration to a patient in need thereof. The patient may be an adult patient or a pediatric patient. The composition may further comprise a pharmaceutically acceptable diluent.

[0122] The composition may dissolve the lipid in an aqueous medium. The lipid may include unesterified or esterified cholesterol. The composition may be provided as a solution wherein a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxy positions is present at a concentration of 20% w / v in solution. The composition may have an affinity for unesterified cholesterol. Solubility may be determined by UV spectroscopy or HPLC.

[0123] In some embodiments, about 200 mg of the composition dissolves at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or at least about 10 mg of unesterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution is capable of dissolving about 2 mg of unesterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0124] The concentration of the composition in the solution may be from about 10 mg / mL to about 200 mg / mL. For example, the concentration of the composition in the solution may be from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 70 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 90 mg / mL, from about 10 mg / mL to about 100 mg / mL. mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 130 mg / mL, about 10 mg / mL to about 140 mg / mL, about 10 mg / mL to about 150 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 170 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 190 mg / mL, about 2 0 mg / mL to about 200 mg / mL, about 30 mg / mL to about 200 mg / mL, about 40 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 60 mg / mL to about 200 mg / mL, about 70 mg / mL to about 200 mg / mL, about 80 mg / mL to about 200 mg / mL, about 90 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 110 mg / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, about 190 mg / mL to about 200 mg / mL, about 200 mg / mL to about g / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 130 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, or about 190 mg / mL to about 200 mg / mL.

[0125] Also provided herein is a composition produced using one or more of the systems and / or methods provided herein, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 2.5% of β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"), wherein the composition is suitable for intrathecal, intravenous, or intracerebroventricular administration to a patient in need thereof. The mixture may contain less than 2.5%, less than 2.4%, less than 2.3%, less than 2.2%, less than 2.1%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-1. The amount of DS-1 can be determined by the peak height of the electrospray MS spectrum.

[0126] The composition may contain no more than 1% unsubstituted β-cyclodextrin ("DS-0"). For example, the composition may contain no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, no more than 0.1%, no more than 0.09%, no more than 0.08%, no more than 0.07%, no more than 0.06%, no more than 0.05%, no more than 0.04%, no more than 0.03%, no more than 0.02%, or no more than 0.01% DS-0. The amount of DS-0 can be determined by the peak height of the electrospray MS spectrum.

[0127] The mixture may have an average molar substitution in the range of about 0.40 to about 0.80; for example, the mixture may have an average molar substitution of about 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or about 0.80. The mixture may have an average degree of substitution ("DSS") of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. a ”). For example, the mixture can have an average degree of substitution of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or about 7.

[0128] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC, gas chromatography, or the PG / EG ratio of propylene glycol to ethylene glycol.

[0129] The composition may contain no more than 1 ppm of propylene oxide, no more than 0.9 ppm of propylene oxide, no more than 0.8 ppm of propylene oxide, no more than 0.7 ppm of propylene oxide, no more than 0.6 ppm of propylene oxide, no more than 0.5 ppm of propylene oxide, no more than 0.4 ppm of propylene oxide, no more than 0.3 ppm of propylene oxide, no more than 0.2 ppm of propylene oxide, or no more than 0.1 ppm of propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0130] The total amount of other unspecified impurities in the composition can be less than or equal to 0.05%; for example, the total amount of unspecified impurities in the composition can be 0.05%, less than 0.05%, less than or equal to 0.04%, less than or equal to 0.03%, less than or equal to 0.02%, or less than or equal to 0.01%. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0131] The composition may be suitable for intrathecal, intravenous or intraventricular administration to a patient in need thereof. The patient may be an adult patient or a pediatric patient. The composition may further comprise a pharmaceutically acceptable diluent.

[0132] The composition may dissolve the lipid in an aqueous medium. The lipid may include unesterified or esterified cholesterol. The composition may be provided as a solution, wherein the concentration of the composition in the solution is 20% w / v. The composition may have an affinity for unesterified cholesterol. Solubility may be determined by UV spectroscopy or HPLC.

[0133] In some embodiments, about 200 mg of the composition dissolves at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or at least about 10 mg of unesterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution is capable of dissolving about 2 mg of unesterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0134] The concentration of the composition in the solution may be from about 10 mg / mL to about 200 mg / mL. For example, the concentration of the composition in the solution may be from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 70 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 90 mg / mL, from about 10 mg / mL to about 100 mg / mL. mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 130 mg / mL, about 10 mg / mL to about 140 mg / mL, about 10 mg / mL to about 150 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 170 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 190 mg / mL, about 2 0 mg / mL to about 200 mg / mL, about 30 mg / mL to about 200 mg / mL, about 40 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 60 mg / mL to about 200 mg / mL, about 70 mg / mL to about 200 mg / mL, about 80 mg / mL to about 200 mg / mL, about 90 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 110 mg / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, about 190 mg / mL to about 200 mg / mL, about 200 mg / mL to about g / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 130 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, or about 190 mg / mL to about 200 mg / mL.

[0135] Also provided herein is a composition produced using one or more of the systems and / or methods provided herein, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and, the mixture comprises 5% to 25% of β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6").

[0136] The mixture may contain less than 0.1% DS-0 and less than 0.1% DS-1 in total. For example, the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-0; and / or the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-1.

[0137] The mixture may contain at least 8% hexahydroxypropyl-substituted β-cyclodextrin ("DS-6"). The mixture may contain at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% DS-6. Alternatively, the mixture may contain from about 8% to about 9%, from about 8% to about 10%, from about 8% to about 11%, from about 8% to about 12%, from about 8% to about 13%, from about 8% to about 14%, from about 8% to about 15%, from about 8% to about 16%, from about 8% to about 17%, from about 8% to about 18%, from about 8% to about 19%, from about 8% to about 20%, from about 8% to about 21%, from about 8% to about 22%, from about 8% to about 23%, from about 8% to about 24%, or from about 8% to about 25%. Alternatively, the mixture may contain no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, or no more than 8% DS-6.

[0138] The amount of DS-0, DS-1 or DS-6 can be determined by the peak height of the electrospray MS spectrum.

[0139] The mixture may have an average molar substitution in the range of about 0.40 to about 0.80; for example, the mixture may have an average molar substitution of about 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or about 0.80. The mixture may have an average degree of substitution ("DSS") of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. a ”). For example, the mixture can have an average degree of substitution of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or about 7.

[0140] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002% or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC or gas chromatography.

[0141] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC, gas chromatography, or the PG / EG ratio of propylene glycol to ethylene glycol.

[0142] The composition may contain no more than 1 ppm of propylene oxide, no more than 0.9 ppm of propylene oxide, no more than 0.8 ppm of propylene oxide, no more than 0.7 ppm of propylene oxide, no more than 0.6 ppm of propylene oxide, no more than 0.5 ppm of propylene oxide, no more than 0.4 ppm of propylene oxide, no more than 0.3 ppm of propylene oxide, no more than 0.2 ppm of propylene oxide, or no more than 0.1 ppm of propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0143] The total amount of other unspecified impurities in the composition can be less than or equal to 0.05%; for example, the total amount of unspecified impurities in the composition can be 0.05%, less than 0.05%, less than or equal to 0.04%, less than or equal to 0.03%, less than or equal to 0.02%, or less than or equal to 0.01%. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0144] The composition may be suitable for intrathecal, intravenous or intraventricular administration to a patient in need thereof. The patient may be an adult patient or a pediatric patient. The composition may further comprise a pharmaceutically acceptable diluent.

[0145] The composition may dissolve the lipid in an aqueous medium. The lipid may include unesterified or esterified cholesterol. The composition may be provided as a solution, wherein the concentration of the composition in the solution is 20% w / v. The composition may have an affinity for unesterified cholesterol. Solubility may be determined by UV spectroscopy or HPLC.

[0146] In some embodiments, about 200 mg of the composition dissolves at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or at least about 10 mg of unesterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution is capable of dissolving about 2 mg of unesterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0147] The concentration of the composition in the solution may be from about 10 mg / mL to about 200 mg / mL. For example, the concentration of the composition in the solution may be from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 70 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 90 mg / mL, from about 10 mg / mL to about 100 mg / mL. mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 130 mg / mL, about 10 mg / mL to about 140 mg / mL, about 10 mg / mL to about 150 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 170 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 190 mg / mL, about 2 0 mg / mL to about 200 mg / mL, about 30 mg / mL to about 200 mg / mL, about 40 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 60 mg / mL to about 200 mg / mL, about 70 mg / mL to about 200 mg / mL, about 80 mg / mL to about 200 mg / mL, about 90 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 110 mg / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, about 190 mg / mL to about 200 mg / mL, about 200 mg / mL to about g / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 130 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, or about 190 mg / mL to about 200 mg / mL.

[0148] Also provided herein is a composition produced using one or more of the systems and / or methods provided herein, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and, the mixture comprises 1% to 10% of β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7").

[0149] The mixture may contain less than 0.1% DS-0 and less than 0.1% DS-1 in total. For example, the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-0; and / or the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-1.

[0150] The mixture may contain from about 1% to about 10% DS-7; for example, the mixture may contain from about 1% to about 2%, from about 1% to about 3%, from about 1% to about 4%, from about 1% to about 5%, from about 1% to about 6%, from about 1% to about 7%, from about 1% to about 8%, from about 1% to about 9%, from about 2% to about 10%, from about 3% to about 10%, from about 4% to about 10%, from about 5% to about 10%, from about 6% to about 10%, from about 7% to about 10%, from about 8% to about 10%, from about 9% to about 10%, from about 2% to about 9%, from about 3% to about 8%, from about 4% to about 7%, or from about 5% to about 6% DS-7. The mixture may contain about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or about 10% DS-7. Alternatively, the composition may have less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% DS-7.

[0151] The amount of DS-0, DS-1 or DS-7 can be determined by the peak height of the electrospray MS spectrum.

[0152] The mixture may have an average molar substitution in the range of about 0.40 to about 0.80; for example, the mixture may have an average molar substitution of about 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or about 0.80. The mixture may have an average degree of substitution ("DSS") of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. a ”). For example, the mixture can have an average degree of substitution of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or about 7.

[0153] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002% or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC or gas chromatography.

[0154] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC, gas chromatography, or the PG / EG ratio of propylene glycol to ethylene glycol.

[0155] The composition may contain no more than 1 ppm of propylene oxide, no more than 0.9 ppm of propylene oxide, no more than 0.8 ppm of propylene oxide, no more than 0.7 ppm of propylene oxide, no more than 0.6 ppm of propylene oxide, no more than 0.5 ppm of propylene oxide, no more than 0.4 ppm of propylene oxide, no more than 0.3 ppm of propylene oxide, no more than 0.2 ppm of propylene oxide, or no more than 0.1 ppm of propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0156] The total amount of other unspecified impurities in the composition can be less than or equal to 0.05%; for example, the total amount of unspecified impurities in the composition can be 0.05%, less than 0.05%, less than or equal to 0.04%, less than or equal to 0.03%, less than or equal to 0.02%, or less than or equal to 0.01%. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0157] The composition may be suitable for intrathecal, intravenous or intraventricular administration to a patient in need thereof. The patient may be an adult patient or a pediatric patient. The composition may further comprise a pharmaceutically acceptable diluent.

[0158] The composition may dissolve the lipid in an aqueous medium. The lipid may include unesterified or esterified cholesterol. The composition may be provided as a solution, wherein the concentration of the composition in the solution is 20% w / v. The composition may have an affinity for unesterified cholesterol. Solubility may be determined by UV spectroscopy or HPLC.

[0159] In some embodiments, about 200 mg of the composition dissolves at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or at least about 10 mg of unesterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution is capable of dissolving about 2 mg of unesterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0160] The concentration of the composition in the solution may be from about 10 mg / mL to about 200 mg / mL. For example, the concentration of the composition in the solution may be from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 70 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 90 mg / mL, from about 10 mg / mL to about 100 mg / mL. mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 130 mg / mL, about 10 mg / mL to about 140 mg / mL, about 10 mg / mL to about 150 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 170 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 190 mg / mL, about 2 0 mg / mL to about 200 mg / mL, about 30 mg / mL to about 200 mg / mL, about 40 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 60 mg / mL to about 200 mg / mL, about 70 mg / mL to about 200 mg / mL, about 80 mg / mL to about 200 mg / mL, about 90 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 110 mg / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, about 190 mg / mL to about 200 mg / mL, about 200 mg / mL to about g / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 130 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, or about 190 mg / mL to about 200 mg / mL.

[0161] Also provided herein is a composition produced using one or more systems and / or methods provided herein, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and, the mixture comprises no more than 50% of β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4").

[0162] The mixture may contain less than 0.1% DS-0 and less than 0.1% DS-1 in total. For example, the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-0; and / or the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-1.

[0163] The mixture may contain no more than 25% of tetrahydroxypropyl-substituted β-cyclodextrin ("DS-4"). The mixture may contain at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% of DS-4. Alternatively, the mixture may contain no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, or no more than 50% of DS-4. The mixture may contain about 5% to about 50%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 10% to about 40%, or about 20% to about 30% DS-4.

[0164] The amount of DS-0, DS-1 or DS-4 can be determined by the peak height of the electrospray MS spectrum.

[0165] The mixture may have an average molar substitution in the range of about 0.40 to about 0.80; for example, the mixture may have an average molar substitution of about 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or about 0.80. The mixture may have an average degree of substitution ("DSS") of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. a ”). For example, the mixture can have an average degree of substitution of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or about 7.

[0166] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002% or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC or gas chromatography.

[0167] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC, gas chromatography, or the PG / EG ratio of propylene glycol to ethylene glycol.

[0168] The composition may contain no more than 1 ppm of propylene oxide, no more than 0.9 ppm of propylene oxide, no more than 0.8 ppm of propylene oxide, no more than 0.7 ppm of propylene oxide, no more than 0.6 ppm of propylene oxide, no more than 0.5 ppm of propylene oxide, no more than 0.4 ppm of propylene oxide, no more than 0.3 ppm of propylene oxide, no more than 0.2 ppm of propylene oxide, or no more than 0.1 ppm of propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0169] The total amount of other unspecified impurities in the composition can be less than or equal to 0.05%; for example, the total amount of unspecified impurities in the composition can be 0.05%, less than 0.05%, less than or equal to 0.04%, less than or equal to 0.03%, less than or equal to 0.02%, or less than or equal to 0.01%. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0170] The composition may be suitable for intrathecal, intravenous or intraventricular administration to a patient in need thereof. The patient may be an adult patient or a pediatric patient. The composition may further comprise a pharmaceutically acceptable diluent.

[0171] The composition may dissolve the lipid in an aqueous medium. The lipid may include unesterified or esterified cholesterol. The composition may be provided as a solution, wherein the concentration of the composition in the solution is 20% w / v. The composition may have an affinity for unesterified cholesterol. Solubility may be determined by UV spectroscopy or HPLC.

[0172] In some embodiments, about 200 mg of the composition dissolves at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or at least about 10 mg of unesterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution is capable of dissolving about 2 mg of unesterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0173] The concentration of the composition in the solution may be from about 10 mg / mL to about 200 mg / mL. For example, the concentration of the composition in the solution may be from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 70 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 90 mg / mL, from about 10 mg / mL to about 100 mg / mL. mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 130 mg / mL, about 10 mg / mL to about 140 mg / mL, about 10 mg / mL to about 150 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 170 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 190 mg / mL, about 2 0 mg / mL to about 200 mg / mL, about 30 mg / mL to about 200 mg / mL, about 40 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 60 mg / mL to about 200 mg / mL, about 70 mg / mL to about 200 mg / mL, about 80 mg / mL to about 200 mg / mL, about 90 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 110 mg / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, about 190 mg / mL to about 200 mg / mL, about 200 mg / mL to about g / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 130 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, or about 190 mg / mL to about 200 mg / mL.

[0174] Also provided herein is a composition produced using one or more of the systems and / or methods provided herein, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and, the mixture comprises no more than 50% of β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5").

[0175] The mixture may contain less than 0.1% DS-0 and less than 0.1% DS-1 in total. For example, the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-0; and / or the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-1.

[0176] The mixture may contain no more than 25% of pentahydroxypropyl-substituted β-cyclodextrin ("DS-5"). The mixture may contain at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% of DS-5. Alternatively, the mixture may contain no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, or no more than 50% of DS-5. The mixture may contain from about 5% to about 50%, from about 5% to about 10%, from about 5% to about 20%, from about 5% to about 30%, from about 5% to about 40%, from about 10% to about 50%, from about 20% to about 50%, from about 30% to about 50%, from about 40% to about 50%, from about 10% to about 40%, or from about 20% to about 30% DS-5.

[0177] The amount of DS-0, DS-1 or DS-5 can be determined by the peak height of the electrospray MS spectrum.

[0178] The mixture may have an average molar substitution in the range of about 0.40 to about 0.80; for example, the mixture may have an average molar substitution of about 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or about 0.80. The mixture may have an average degree of substitution ("DSS") of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. a ”). For example, the mixture can have an average degree of substitution of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or about 7.

[0179] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002% or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC or gas chromatography.

[0180] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC, gas chromatography, or the PG / EG ratio of propylene glycol to ethylene glycol.

[0181] The composition may contain no more than 1 ppm of propylene oxide, no more than 0.9 ppm of propylene oxide, no more than 0.8 ppm of propylene oxide, no more than 0.7 ppm of propylene oxide, no more than 0.6 ppm of propylene oxide, no more than 0.5 ppm of propylene oxide, no more than 0.4 ppm of propylene oxide, no more than 0.3 ppm of propylene oxide, no more than 0.2 ppm of propylene oxide, or no more than 0.1 ppm of propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0182] The total amount of other unspecified impurities in the composition can be less than or equal to 0.05%; for example, the total amount of unspecified impurities in the composition can be 0.05%, less than 0.05%, less than or equal to 0.04%, less than or equal to 0.03%, less than or equal to 0.02%, or less than or equal to 0.01%. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0183] The composition may be suitable for intrathecal, intravenous or intraventricular administration to a patient in need thereof. The patient may be an adult patient or a pediatric patient. The composition may further comprise a pharmaceutically acceptable diluent.

[0184] The composition may dissolve the lipid in an aqueous medium. The lipid may include unesterified or esterified cholesterol. The composition may be provided as a solution, wherein the concentration of the composition in the solution is 20% w / v. The composition may have an affinity for unesterified cholesterol. Solubility may be determined by UV spectroscopy or HPLC.

[0185] In some embodiments, about 200 mg of the composition dissolves at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or at least about 10 mg of unesterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution is capable of dissolving about 2 mg of unesterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0186] The concentration of the composition in the solution may be from about 10 mg / mL to about 200 mg / mL. For example, the concentration of the composition in the solution may be from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 70 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 90 mg / mL, from about 10 mg / mL to about 100 mg / mL. mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 130 mg / mL, about 10 mg / mL to about 140 mg / mL, about 10 mg / mL to about 150 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 170 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 190 mg / mL, about 2 0 mg / mL to about 200 mg / mL, about 30 mg / mL to about 200 mg / mL, about 40 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 60 mg / mL to about 200 mg / mL, about 70 mg / mL to about 200 mg / mL, about 80 mg / mL to about 200 mg / mL, about 90 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 110 mg / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, about 190 mg / mL to about 200 mg / mL, about 200 mg / mL to about g / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 130 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, or about 190 mg / mL to about 200 mg / mL.

[0187] Also provided herein is a composition produced using one or more systems and / or methods provided herein, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and at least 70% of the β-cyclodextrin has a hydroxypropyl group at DS-1. a DS within ±1σ, where σ is the standard deviation.

[0188] At least 70% of β-cyclodextrin has a DS aIn some embodiments, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the β-cyclodextrins have a DS within ±1σ, where σ is the standard deviation. a DS within ±1σ.

[0189] The mixture may contain less than 0.1% DS-0 and less than 0.1% DS-1 in total. For example, the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-0; and / or the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-1.

[0190] The amount of DS-0 or DS-1 can be determined by the peak height of the electrospray MS spectrum.

[0191] The mixture may have an average molar substitution in the range of about 0.40 to about 0.80; for example, the mixture may have an average molar substitution of about 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or about 0.80. The mixture may have an average degree of substitution ("DSS") of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. a ”). For example, the mixture can have an average degree of substitution of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or about 7.

[0192] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002% or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC or gas chromatography.

[0193] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC, gas chromatography, or the PG / EG ratio of propylene glycol to ethylene glycol.

[0194] The composition may contain no more than 1 ppm of propylene oxide, no more than 0.9 ppm of propylene oxide, no more than 0.8 ppm of propylene oxide, no more than 0.7 ppm of propylene oxide, no more than 0.6 ppm of propylene oxide, no more than 0.5 ppm of propylene oxide, no more than 0.4 ppm of propylene oxide, no more than 0.3 ppm of propylene oxide, no more than 0.2 ppm of propylene oxide, or no more than 0.1 ppm of propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0195] The total amount of other unspecified impurities in the composition can be less than or equal to 0.05%; for example, the total amount of unspecified impurities in the composition can be 0.05%, less than 0.05%, less than or equal to 0.04%, less than or equal to 0.03%, less than or equal to 0.02%, or less than or equal to 0.01%. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0196] The composition may be suitable for intrathecal, intravenous or intraventricular administration to a patient in need thereof. The patient may be an adult patient or a pediatric patient. The composition may further comprise a pharmaceutically acceptable diluent.

[0197] The composition may dissolve the lipid in an aqueous medium. The lipid may include unesterified or esterified cholesterol. The composition may be provided as a solution, wherein the concentration of the composition in the solution is 20% w / v. The composition may have an affinity for unesterified cholesterol. Solubility may be determined by UV spectroscopy or HPLC.

[0198] In some embodiments, about 200 mg of the composition dissolves at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or at least about 10 mg of unesterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution is capable of dissolving about 2 mg of unesterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0199] The concentration of the composition in the solution may be from about 10 mg / mL to about 200 mg / mL. For example, the concentration of the composition in the solution may be from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 70 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 90 mg / mL, from about 10 mg / mL to about 100 mg / mL. mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 130 mg / mL, about 10 mg / mL to about 140 mg / mL, about 10 mg / mL to about 150 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 170 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 190 mg / mL, about 2 0 mg / mL to about 200 mg / mL, about 30 mg / mL to about 200 mg / mL, about 40 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 60 mg / mL to about 200 mg / mL, about 70 mg / mL to about 200 mg / mL, about 80 mg / mL to about 200 mg / mL, about 90 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 110 mg / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, about 190 mg / mL to about 200 mg / mL, about 200 mg / mL to about g / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 130 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, or about 190 mg / mL to about 200 mg / mL.

[0200] Also provided herein are compositions produced using one or more of the systems and / or methods provided herein, the composition comprising a mixture of β-cyclodextrin molecules, wherein the mixture of β-cyclodextrin molecules can comprise β-cyclodextrin substituted with zero hydroxypropyl groups ("DS-0", also referred to as "unsubstituted"), β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"), β-cyclodextrin substituted with two hydroxypropyl groups ("DS-2"), β-cyclodextrin substituted with three hydroxypropyl groups ("DS-3"), β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"), β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"), β-cyclodextrin substituted with Six hydroxypropyl substituted β-cyclodextrin ("DS-6"), seven hydroxypropyl substituted β-cyclodextrin ("DS-7"), eight hydroxypropyl substituted β-cyclodextrin ("DS-8"), nine hydroxypropyl substituted β-cyclodextrin ("DS-9"), ten hydroxypropyl substituted β-cyclodextrin ("DS-10"), eleven hydroxypropyl substituted β-cyclodextrin ("DS-11"), twelve hydroxypropyl substituted β-cyclodextrin ("DS-12"), thirteen hydroxypropyl substituted β-cyclodextrin ("DS-13"), and fourteen hydroxypropyl substituted β-cyclodextrin ("DS-14"). The degree of substitution of a mixture of β-cyclodextrin molecules can be determined by MALDI-TOF-MS. In this regard, the number of hydroxypropyl groups per anhydroglucose unit in a β-cyclodextrin mixture is the "molar substitution" or "MS" and is determined according to the procedure described in the USP monograph on hydroxypropyl β-cyclodextrin (USP NF 2015) ("USP Hydroxypropyl β-cyclodextrin Monograph"), which is incorporated herein by reference in its entirety. In this disclosure, the term "average molar substitution" or "MS" is used to refer to the molar substitution of hydroxypropyl β-cyclodextrin. a " is synonymous with the term "MS" as used in the USP hydroxypropyl β-cyclodextrin monograph, and the term "glucose unit" is synonymous with the term "anhydroglucose unit" as used in the USP hydroxypropyl β-cyclodextrin monograph. In further connection herewith, the "average number of hydroxypropyl groups per β-cyclodextrin" is also referred to as the "average degree of substitution", "average DS" or "DS a ” refers to the total number of hydroxypropyl groups in a β-cyclodextrin population divided by the number of β-cyclodextrin molecules. In an illustrative example, the DS of a mixture of equal parts of β-cyclodextrins containing glucose units each substituted with one hydroxypropyl group and β-cyclodextrins containing glucose units each substituted with two hydroxypropyl groups is a =10.5 (the average of equal parts of β-cyclodextrin with DS=7 and DS=14). In another illustrative example, the DS of a mixture of 33.3% β-cyclodextrin in which only one of the seven glucose units is substituted with a hydroxypropyl group (i.e., DS=1) and 66.7% β-cyclodextrin containing glucose units each substituted with a hydroxypropyl group (i.e., DS=7) is a=5.0. DS a Determined by multiplying the MS by 7. In further relation, the "degree of substitution" or "DS" refers to the total number of hydroxypropyl groups substituted directly or indirectly on a β-cyclodextrin molecule. For example, a β-cyclodextrin molecule containing glucose units, each of which is substituted with one hydroxypropyl group, has a DS=7. In another example, a β-cyclodextrin molecule in which only one of the seven glucose units is substituted with a hydroxypropyl group, and the hydroxypropyl group itself is substituted with another hydroxypropyl group (e.g., a β-cyclodextrin with a single HP contains two hydroxypropyl groups), has a DS=2. As used herein, DS a Used synonymously with "degree of substitution," which term is defined in the USP Hydroxypropyl Beta-cyclodextrin monograph.

[0201] In certain embodiments, the pharmaceutical composition of the present disclosure comprises a mixture of unsubstituted β-cyclodextrin molecules and β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions as pharmaceutically active ingredients, wherein the average number of hydroxypropyl groups (DS) per β-cyclodextrin molecule in the mixture is a ) is from about 3 to about 7.

[0202] In some embodiments, DS a is from about 3 to about 5, for example from about 3 to about 4. In some embodiments, DS a is 3.3±0.3, 3.5±0.3 or 3.7±0.3. In other embodiments, DS a In other embodiments, DS a It was 3.1±0.1, 3.2±0.1, 3.3±0.1, 3.4±0.1, ±0.1, 3.6±0.1, 3.7±0.1, 3.8±0.1 or 3.9±0.1.

[0203] In some embodiments, DS a In some embodiments, DS a is 3.8±0.3, 4.0±0.3 or 4.2±0.3. In other embodiments, DS a In other embodiments, DS a It was 3.6±0.1, 3.7±0.1, 3.8±0.1, 3.9±0.1, 4.0±0.1, 4.1±0.1, 4.2±0.1, 4.3±0.1 or 4.4±0.1.

[0204] In some embodiments, DS a From about 4 to about 6, for example, from about 4 to about 5. In some embodiments, DS a is 4.3±0.3, 4.5±0.3 or 4.7±0.3. In other embodiments, DS a In other embodiments, DS a It was 4.1±0.1, 4.2±0.1, 4.3±0.1, 4.4±0.1, 4.5±0.1, 4.6±0.1, 4.7±0.1, 4.8±0.1 or 4.9±0.1.

[0205] In some embodiments, DS a In some embodiments, DS a is 4.8±0.3, 5.0±0.3 or 5.2±0.3. In other embodiments, DS a In other embodiments, DS a It was 4.6±0.1, 4.7±0.1, 4.8±0.1, 4.9±0.1, 5.0±0.1, 5.1±0.1, 5.2±0.1, 5.3±0.1 or 5.4±0.1.

[0206] In some embodiments, DS a From about 5 to about 7, for example, from about 5 to about 6. In some embodiments, DS a is 5.3±0.3, 5.5±0.3 or 5.7±0.3. In other embodiments, DS a In other embodiments, DS a It was 5.1±0.1, 5.2±0.1, 5.3±0.1, 5.4±0.1, 5.5±0.1, 5.6±0.1, 5.7±0.1, 5.8±0.1 or 5.9±0.1.

[0207] In some embodiments, DS a In some embodiments, DS a is 5.8±0.3, 6.0±0.3 or 6.2±0.3. In other embodiments, DS aIn other embodiments, DS a It was 5.6±0.1, 5.7±0.1, 5.8±0.1, 5.9±0.1, 6.0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1 or 6.4±0.1.

[0208] In some embodiments, DS a From about 6 to about 7. In some embodiments, DS a is 6.3±0.3, 6.5±0.3 or 6.7±0.3. In other embodiments, DS a In other embodiments, DS a It was 6.1±0.1, 6.2±0.1, 6.3±0.1, 6.4±0.1, 6.5±0.1, 6.6±0.1, 6.7±0.1, 6.8±0.1 or 6.9±0.1.

[0209] In some embodiments, DS a About 4.1±15%, about 4.2±15%, about 4.3±15%, about 4.4±15%, or about 4.5±15%, such as about 4.1±10%, about 4.2±10%, about 4.3±10%, about 4.4±10%, or about 4.5±10%, such as about 4.1±5%, about 4.2±5%, about 4.3±5%, about 4.4±5%, or about 4.5±5%. For example, in certain embodiments, DS a In certain embodiments, DS is about 4.31 ± 10%, about 4.32 ± 10%, about 4.33 ± 10%, about 4.34 ± 10%, about 4.35 ± 10%, about 4.36 ± 10%, or about 4.37 ± 10%, such as about 4.31 ± 5%, about 4.32 ± 5%, about 4.33 ± 5%, about 4.34 ± 5%, about 4.35 ± 5%, about 4.36 ± 5%, or about 4.37 ± 5%. a It is about 4.34±10%, for example, about 4.34±5%.

[0210] In some embodiments, DS aAbout 4.3±15%, about 4.4±15%, about 4.5±15%, about 4.6±15%, or about 4.7±15%, such as about 4.3±10%, about 4.4±10%, about 4.5±10%, about 4.6±10%, or about 4.7±10%, such as about 4.3±5%, about 4.4±5%, about 4.5±5%, about 4.6±5%, or about 4.7±5%. For example, in certain embodiments, DS a In certain embodiments, DS a It is about 4.50±10%, for example, about 4.50±5%.

[0211] In some embodiments, DS a About 6.1±15%, about 6.2±15%, about 6.3±15%, about 6.4±15%, or about 6.5±15%, such as about 6.1±10%, about 6.2±10%, about 6.3±10%, about 6.4±10%, or about 6.5±10%, such as about 6.1±5%, about 6.2±5%, about 6.3±5%, about 6.4±5%, or about 6.5±5%. For example, in certain embodiments, DS a In certain embodiments, DS a It is about 6.37±10%, for example, about 6.37±5%.

[0212] In some embodiments, DS a For example, in certain embodiments, DS aIn certain embodiments, DS is about 6.50±10%, about 6.51±10%, about 6.52±10%, about 6.53±10%, about 6.54±10%, about 6.55±10%, or about 6.56±10%, such as about 6.50±5%, about 6.51±5%, about 6.52±5%, about 6.53±5%, about 6.54±5%, about 6.55±5%, or about 6.56±5%. a It is about 6.53±10%, for example, about 6.53±5%.

[0213] In a mixture of unsubstituted β-cyclodextrin molecules and β-cyclodextrin molecules substituted at one or more hydroxyl positions with hydroxypropyl groups, the distribution of the degree of substitution can vary. For example, the DS of a mixture of equal parts of β-cyclodextrin containing glucose units each substituted with one hydroxypropyl group and β-cyclodextrin containing glucose units each substituted with two hydroxypropyl groups is a = 10.5 (average of equal parts of β-cyclodextrin with DS = 7 and DS = 14). a = 10.5, but in this example, there is no β-cyclodextrin with DS = 10 or DS = 11 in the mixture. In other cases, the DS of most β-cyclodextrins in the β-cyclodextrin mixture is close to DS a .

[0214] In some embodiments of the present disclosure, at least about 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%) of the β-cyclodextrin in the mixture has a DS a DS within ±Xσ, where σ is the standard deviation and X is 1, 2, or 3. For example, in some embodiments, at least about 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%) of the β-cyclodextrin in the mixture has a DS within ±Xσ, where σ is the standard deviation and X is 1, 2, or 3. a In some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±1σ. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±1σ. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±1σ. a DS within ±1σ.

[0215] In some embodiments, at least about 50%, such as at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% of the β-cyclodextrin in the mixture has a DS aIn some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±2σ. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±2σ. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±2σ. a DS within ±2σ.

[0216] In some embodiments, at least about 50%, such as at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% of the β-cyclodextrin in the mixture has a DS a In some embodiments, at least about 70% of the β-cyclodextrin has a DS within ±3σ of DSa. In some embodiments, at least about 90% of the β-cyclodextrin has a DS within ±3σ of DSa. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±3σ. a DS within ±3σ.

[0217] In some embodiments, at least about 50%, such as at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% of the β-cyclodextrin has a DS a In some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±1. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±1. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±1. a DS within ±1.

[0218] In some embodiments, at least about 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%) of the β-cyclodextrin has a DS a In some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±0.8. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±0.8. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±0.8. a DS within ±0.8.

[0219] In some embodiments, at least about 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%) of the β-cyclodextrin has a DS a In some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±0.6. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±0.6. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±0.6. a DS within ±0.6.

[0220] In some embodiments, at least about 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%) of the β-cyclodextrin has a DS a In some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±0.5. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±0.5. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±0.5. a DS within ±0.5.

[0221] In some embodiments, at least about 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%) of the β-cyclodextrin has a DS a In some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±0.4. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±0.4. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±0.4. a DS within ±0.4.

[0222] In some embodiments, at least about 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%) of the β-cyclodextrin has a DS a In some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±0.3. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±0.3.a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±0.3. a DS within ±0.3.

[0223] In some embodiments, at least about 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%) of the β-cyclodextrin has a DS a In some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±0.2. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±0.2. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±0.2. a DS within ±0.2.

[0224] In some embodiments, at least about 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%) of the β-cyclodextrin has a DS a In some embodiments, at least about 70% of the β-cyclodextrins have a DS within ±0.1. a In some embodiments, at least about 90% of the β-cyclodextrins have a DS within ±0.1. a In some embodiments, at least about 95% of the β-cyclodextrins have a DS within ±0.1. a DS within ±0.1.

[0225] In some embodiments, the MS ranges from 0.40 to 0.80, e.g., 0.41 to 0.79, 0.42 to 0.78, 0.43 to 0.77, 0.44 to 0.76, 0.45 to 0.75, 0.46 to 0.74, 0.47 to 0.73, 0.48 to 0.72, 0.49 to 0.71, 0.50 to 0.70, 0.51 to 0.69, 0.52 to 0.68, 0.53 to 0.67, 0.54 to 0.66, 0.55 to 0.65, 0.56 to 0.64, 0.57 to 0.63, 0.58 to 0.62, or 0.59 to 0.61.

[0226] In certain embodiments, MS is about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 0.50, about 0.51, about 0.52, about 0.53, about 0.54, about 0.55, about 0.56, about 0.57, about 0.58, about 0. 59, about 0.60, about 0.61, about 0.62, about 0.63, about 0.64, about 0.65, about 0.66, about 0.67, about 0.68, about 0.69, about 0.70, about 0.71, about 0.72, about 0.73, about 0.74, about 0.75, about 0.76, about 0.77, about 0.78, about 0.79 or about 0.80.

[0227] In certain embodiments, the MS is about 0.571-0.686 (DS a In some of these embodiments, MS is in the range of about 0.58 to about 0.68. In currently preferred embodiments, MS is in the range of 0.58-0.68.

[0228] In various embodiments, the MS is at least about 0.55. In certain embodiments, the MS is at least about 0.56, about 0.57, about 0.58, about 0.59, or about 0.60. In certain embodiments, the MS is no more than about 0.70. In specific embodiments, the MS is no more than about 0.69, about 0.68, about 0.67, about 0.66, or about 0.65.

[0229] Also provided herein are compositions produced using one or more of the systems and / or methods provided herein, the composition comprising a mixture of β-cyclodextrin molecules, wherein the mixture of β-cyclodextrin molecules can comprise β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"), β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"), β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6"), β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-9"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-10"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-11"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-12"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-13"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-14"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-15"), β-cyclodextrin substituted with six hydroxypropyl groups ("DS-16"), β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-17"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-18"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-19"), β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-20"), β-cyclodextrin substituted with Substituted β-cyclodextrin ("DS-8"), β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"), β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10"), β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"), β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"), β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"), and β-cyclodextrin substituted with fourteen hydroxypropyl groups ("DS-14"). The degree of substitution of a mixture of β-cyclodextrin molecules can be determined by MALDI-TOF-MS.

[0230] In some embodiments, the composition can have an average degree of substitution between about 7 and about 9; for example, the average degree of substitution can be about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In an exemplary embodiment, the average degree of substitution of the mixture of β-cyclodextrin molecules is about 7.7.

[0231] In some embodiments, the mixture of β-cyclodextrin molecules may contain less than 1% DS-4; for example, the mixture of β-cyclodextrin molecules may contain about 0.9% DS-4, about 0.8% DS-4, about 0.7% DS-4, about 0.6% DS-4, about 0.5% DS-4, about 0.4% DS-4, about 0.3% DS-4, about 0.2% DS-4, or about 0.1% DS-4. In some aspects, the mixture of β-cyclodextrin molecules may contain less than 1% to about 0.9% DS-4, about 0.9% to about 0.8% DS-4, about 0.8% to about 0.7% DS-4, about 0.7% to about 0.6% DS-4, about 0.7% to about 0.6% DS-4, about 0.7% to about 0.6% DS-4, about 0.6% to about 0.5% DS-4, about 0.5% to about 0.4% DS-4, about 0.4% to about 0.3% DS-4, about 0.3% to about 0.2% DS-4, about 0.2% to about 0.1% DS-4, or less than 0.1% DS-4. In some additional aspects, the mixture of β-cyclodextrin molecules may contain less than 1% to about 0.8% DS-4, less than 1% to about 0.7% DS-4, less than 1% to about 0.6% DS-4, less than 1% to about 0.5% DS-4, less than 1% to about 0.4% DS-4, less than 1% to about 0.3% DS-4, less than 1% to about 0.2% DS-4, less than 1% to about 0.1% DS-4, about 0.9% to about 0.1% DS-4, about 0.8% to about 0.1% DS-4, about 0.7% to about 0.1% DS-4, about 0.6% to about 0.1% DS-4, about 0.5% to about 0.1% DS-4, about 0.4% to about 0.1% DS-4, or about 0.3% to about 0.1% DS-4. In yet further aspects, the β-cyclodextrin mixture may contain less than 1% DS-4, less than 0.9% DS-4, less than 0.8% DS-4, less than 0.7% DS-4, less than 0.6% DS-4, less than 0.5% DS-4, less than 0.4% DS-4, less than 0.3% DS-4, less than 0.2% DS-4, or less than 0.1% DS-4. In yet further aspects, the β-cyclodextrin molecule mixture may contain about 0.001%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or about 1% DS-4. In some embodiments, the amount of DS-4 in the β-cyclodextrin molecule mixture can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-4 in the MALDI-TOF-MS spectrum is 0.73%.

[0232] In some embodiments, the mixture of β-cyclodextrin molecules may include about 2% to about 5% DS-5. In some aspects, the mixture of β-cyclodextrin molecules may include about 2% to about 2.5% DS-5, about 2.5% to about 3% DS-5, about 3% to about 3.5% DS-5, about 3.5% to about 4% DS-5, about 4% to about 4.5% DS-5, or about 4.5% to about 5% DS-5. In some other aspects, the mixture of β-cyclodextrin molecules may include about 2% to about 3% DS-5, about 2% to about 3.5% DS-5, about 2% to about 4% DS-5, about 2% to about 4.5% DS-5, about 2.5% to about 5% DS-5, about 3% to about 5% DS-5, about 3.5% to about 5% DS-5, about 4% DS-5 to about 5% DS-5, or about 3% to about 4% DS-5. In yet further aspects, the mixture of β-cyclodextrin molecules can contain about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, or about 5.0% DS-5. In some embodiments, the amount of DS-5 in the mixture of β-cyclodextrin molecules can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-5 in the MALDI-TOF-MS spectrum is 3.49%.

[0233] In some embodiments, the mixture of β-cyclodextrin molecules may include about 7% to about 13% DS-6. In some aspects, the mixture of β-cyclodextrin molecules may include about 7% to about 7.5% DS-6, about 7.5% to about 8% DS-6, about 8% to about 8.5% DS-6, about 8.5% to about 9% DS-6, about 9% to about 9.5% DS-6, about 9.5% to about 10% DS-6, about 10% to about 10.5% DS-6, about 10.5% to about 11% DS-6, about 11% to about 11.5% DS-6, about 11.5% to about 12% DS-6, about 12% to about 12.5% ​​DS-6, or about 12.5% ​​to about 13% DS-6. In some additional aspects, the mixture of β-cyclodextrin molecules can include about 7% to about 8% DS-6, about 7% to about 8.5% DS-6, about 7% to about 9% DS-6, about 7% to about 9.5% DS-6, about 7% to about 10% DS-6, about 7% to about 10.5% DS-6, about 7% to about 11% DS-6, about 7% to about 11.5% DS-6, about 7% to about 12% DS-6, about 7% to about 12.5% ​​DS-6, about 7.5% to about 1 3% DS-6, about 8% to about 13% DS-6, about 8.5% to about 13% DS-6, about 9% to about 13% DS-6, about 9.5% to about 13% DS-6, about 10% to about 13% DS-6, about 10.5% to about 13% DS-6, about 11% to about 13% DS-6, about 11.5% to about 13% DS-6, about 12% to about 13% DS-6, about 8% to about 12% DS-6, or about 9% to about 11% DS-6.In yet further aspects, the mixture of beta-cyclodextrin molecules can comprise about 7.0%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8.0%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9.0%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, about 10.0%, about 10. .1%, about 10.2%, about 10.3%, about 10.4%, about 10.5%, about 10.6%, about 10.7%, about 10.8%, about 10.9%, about 11.0%, about 11.1%, about 11.2%, about 11.3%, about 11.4%, about 11.5%, about 11.6%, about 11.7%, about 11.8%, about 11.9%, about 12.0%, about 12.1%, about 12.2%, about 12.3%, about 12.4%, about 12.5%, about 12.6%, about 12.7%, about 12.8%, about 12.9%, or about 13.0% DS-6. In some embodiments, the amount of DS-6 in the mixture of β-cyclodextrin molecules can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-6 in the MALDI-TOF-MS spectrum is 10.66%.

[0234] In some embodiments, the mixture of β-cyclodextrin molecules may include about 21% to about 27% DS-7. In some aspects, the mixture of β-cyclodextrin molecules may include about 21% to about 21.5% DS-7, about 21.5% to about 22% DS-7, about 22% to about 22.5% DS-7, about 22.5% to about 23% DS-7, about 23% to about 23.5% DS-7, about 23.5% to about 24% DS-7, about 24% to about 24.5% DS-7, about 24.5% to about 25% DS-7, about 25% to about 25.5% DS-7, about 25.5% to about 26% DS-7, about 26% to about 26.5% DS-7, or about 26.5% to about 27% DS-7. In some additional aspects, the mixture of β-cyclodextrin molecules can include about 21% to about 22% DS-7, about 21% to about 22.5% DS-7, about 21% to about 23% DS-7, about 21% to about 23.5% DS-7, about 21% to about 24% DS-7, about 21% to about 24.5% DS-7, about 21% to about 25% DS-7, about 21% to about 25.5% DS-7, about 21% to about 26% DS-7, about 21% to about 26.5% DS-7, about 21. 5% to about 27% DS-7, about 22% to about 27% DS-7, 22.5% to about 27% DS-7, about 23% to about 27% DS-7, about 23.5% to about 27% DS-7, about 24% to about 27% DS-7, about 24.5% to about 27% DS-7, about 25% to about 27% DS-7, about 25.5% to about 27% DS-7, about 26% to about 27% DS-7, about 22% to about 26% DS-7, or about 23% to about 25% DS-7.In yet further aspects, the mixture of beta-cyclodextrin molecules can comprise about 21.0%, about 21.1%, about 21.2%, about 21.3%, about 21.4%, about 21.5%, about 21.6%, about 21.7%, about 21.8%, about 21.9%, about 22.0%, about 22.1%, about 22.2%, about 22.3%, about 22.4%, about 22.5%, about 22.6%, about 22.7%, about 22.8%, about 22.9%, about 23.0%, about 23.1%, about 23.2%, about 23.3%, about 23.4%, about 23.5%, about 23.6%, about 23.7%, about 23.8%, about 23.9%, about 24.0%, about 24.1%, about 24.2%, about 24.3%, about 24.4%, about 24.5%, about 24.6%, about 24.7%, about 24.8%, about 24.9%, about 25.0%, about 25.1%, about 25.2%, about 25.3%, about 25.4%, about 25.5%, about 25.6%, about 25.7%, about 25.8%, about 25.9%, about 26.0%, about 26.1%, about 26.2%, about 26.3%, about 26.4%, about 26.5%, about 26.6%, about 26.7%, about 26.8%, about 26.9%, about About 25.0%, about 25.1%, about 25.2%, about 25.3%, about 25.4%, about 25.5%, about 25.6%, about 25.7%, about 25.8%, about 25.9%, about 26.0%, about 26.1%, about 26.2%, about 26.3%, about 26.4%, about 26.5%, about 26.6%, about 26.7%, about 26.8%, about 26.9% or about 27.0% of DS-7. In some embodiments, the amount of DS-7 can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-7 in the MALDI-TOF-MS spectrum is 24.10%.

[0235] In some embodiments, the mixture of β-cyclodextrin molecules may include about 23% to about 29% DS-8. In some aspects, the mixture of β-cyclodextrin molecules may include about 23% to about 23.5% DS-8, about 23.5% to about 24% DS-8, about 24% to about 24.5% DS-8, about 24.5% to about 25% DS-8, about 25% to about 25.5% DS-8, about 25.5% to about 26% DS-8, about 26% to about 26.5% DS-8, about 26.5% to about 27% DS-8, about 27% to about 27.5% DS-8, about 27.5% to about 28% DS-8, about 28% to about 28.5% DS-8, or about 28.5% to about 29% DS-8. In some additional aspects, the mixture of β-cyclodextrin molecules can include about 23% to about 24% DS-8, about 23% to about 24.5% DS-8, about 23% to about 25% DS-8, about 23% to about 25.5% DS-8, about 23% to about 26% DS-8, about 23% to about 26.5% DS-8, about 23% to about 27% DS-8, about 23% to about 27.5% DS-8, about 23% to about 28% DS-8, about 23% to about 28.5% DS-8, about 23. 5% to about 29% DS-8, about 24% to about 29% DS-8, about 24.5% to about 29% DS-8, about 25% to about 29% DS-8, about 25.5% to about 29% DS-8, about 26% to about 29% DS-8, about 26.5% to about 29% DS-8, about 27% to about 29% DS-8, about 27.5% to about 29% DS-8, about 28% to about 29% DS-8, about 24% to about 28% DS-8, or about 25% to about 27% DS-8.In yet further aspects, the mixture of beta-cyclodextrin molecules can comprise about 23.0%, about 23.1%, about 23.2%, about 23.3%, about 23.4%, about 23.5%, about 23.6%, about 23.7%, about 23.8%, about 23.9%, about 24.0%, about 24.1%, about 24.2%, about 24.3%, about 24.4%, about 24.5%, about 24.6%, about 24.7%, about 24.8%, about 24.9%, about 25.0%, about 25.1%, about 25.2%, about 25.3%, about 25.4%, about 25.5%, about 25.6%, about 25.7%, about 25.8% , about 25.9%, about 26.0%, about 26.1%, about 26.2%, about 26.3%, about 26.4%, about 26.5%, about 26.6%, about 26.7%, about 26.8%, about 26.9%, about 27.0%, about 27.1%, about 27.2%, about 27.3%, about 27.4%, about 27.5%, about 27.6%, about 27.7%, about 27.8%, about 27.9%, about 28.0%, about 28.1%, about 28.2%, about 28.3%, about 28.4%, about 28.5%, about 28.6%, about 28.7%, about 28.8%, about 28.9% or about 29.0%. In some embodiments, the amount of DS-8 in the composition can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-8 in the MALDI-TOF-MS spectrum is 26.43%.

[0236] In some embodiments, the mixture of β-cyclodextrin molecules may include about 15% to about 21% DS-9. In some aspects, the mixture of β-cyclodextrin molecules may include about 15% to about 15.5% DS-9, about 15.5% to about 16% DS-9, about 16% to about 16.5% DS-9, about 16.5% to about 17% DS-9, about 17% to about 17.5% DS-9, about 17.5% to about 18% DS-9, about 18% to about 18.5% DS-9, about 18.5% to about 19% DS-9, about 19% to about 19.5% DS-9, about 19.5% to about 20% DS-9, about 20% to about 20.5% DS-9, or about 20.5% to about 21% DS-9. In some additional aspects, the mixture of β-cyclodextrin molecules can include about 15% to about 16% DS-9, about 15% to about 16.5% DS-9, about 15% to about 17% DS-9, about 15% to about 17.5% DS-9, about 15% to about 18% DS-9, about 15% to about 18.5% DS-9, about 15% to about 19% DS-9, about 15% to about 19.5% DS-9, about 15% to about 20% DS-9, about 15% to about 20.5% DS-9, about 15% to about 21.5% DS-9. 5% to about 21% DS-9, about 16% to about 21% DS-9, about 16.5% to about 21% DS-9, about 17% to about 21% DS-9, about 17.5% to about 21% DS-9, about 18% to about 21% DS-9, about 18.5% to about 21% DS-9, about 19% to about 21% DS-9, about 19.5% to about 21% DS-9, about 20% to about 21% DS-9, about 16% to about 20% DS-9, or about 17% to about 19% DS-9.In yet further aspects, the mixture of beta-cyclodextrin molecules can comprise about 15.0%, about 15.1%, about 15.2%, about 15.3%, about 15.4%, about 15.5%, about 15.6%, about 15.7%, about 15.8%, about 15.9%, about 16.0%, about 16.1%, about 16.2%, about 16.3%, about 16.4%, about 16.5%, about 16.6%, about 16.7%, about 16.8%, about 16.9%, about 17.0%, about 17.1%, about 17.2%, about 17.3%, about 17.4%, about 17.5%, about 17.6%, about 17.7%, about 17.8%, about 17.9%, about 18.0%, about 18.1%, about 18.2%, about 18.3%, about 18.4%, about 18.5%, about 18.6%, about 18.7%, about 18.8%, about 18.9%, about 19.0%, about 19.1%, about 19.2%, about 19.3%, about 19.4%, about 19.5%, about 19.6%, about 19.7%, about 19.8%, about 19.9%, about 1 About 7.9%, about 18.0%, about 18.1%, about 18.2%, about 18.3%, about 18.4%, about 18.5%, about 18.6%, about 18.7%, about 18.8%, about 18.9%, about 19.0%, about 19.1%, about 19.2%, about 19.3%, about 19.4%, about 19.5%, about 19.6%, about 19.7%, about 19.8%, about 19.9%, about 20.0%, about 20.1%, about 20.2%, about 20.3%, about 20.4%, about 20.5%, about 20.6%, about 20.7%, about 20.8%, about 20.9% or about 21.0% DS-9. In some embodiments, the amount of DS-9 in the composition can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-9 in the MALDI-TOF-MS spectrum is 18.09%.

[0237] In some embodiments, the mixture of β-cyclodextrin molecules may include about 6% to about 12% DS-10. In some aspects, the mixture of β-cyclodextrin molecules may include about 6% to about 6.5% DS-10, about 6.5% to about 7% DS-10, about 7% to about 7.5% DS-10, about 7.5% to about 8% DS-10, about 8% to about 8.5% DS-10, about 8.5% to about 9% DS-10, about 9% to about 9.5% DS-10, about 9.5% to about 10% DS-10, about 10% to about 10.5% DS-10, about 10.5% to about 11% DS-10, about 11% to about 11.5% DS-10, or about 11.5% to about 12% DS-10. In some additional aspects, the mixture of β-cyclodextrin molecules can include about 6% to about 7% DS-10, about 6% to about 7.5% DS-10, about 6% to about 8% DS-10, about 6% to about 8.5% DS-10, about 6% to about 9% DS-10, about 6% to about 9.5% DS-10, about 6% to about 10% DS-10, about 6% to about 10.5% DS-10, about 6% to about 11% DS-10, about 6% to about 11.5% DS-10, about 6.5% to about 12.5% ​​DS-10. % DS-10, about 7% to about 12% DS-10, about 7.5% to about 12% DS-10, about 8% to about 12% DS-10, about 8.5% to about 12% DS-10, about 9% to about 12% DS-10, about 9.5% to about 12% DS-10, about 10% to about 12% DS-10, about 10.5% to about 12% DS-10, about 11% to about 12% DS-10, about 7% to about 11% DS-10, or about 8% to about 10% DS-10.In yet further aspects, the mixture of beta-cyclodextrin molecules can comprise about 6.0%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7.0%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8.0%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9.0% %, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, about 10.0%, about 10.1%, about 10.2%, about 10.3%, about 10.4%, about 10.5%, about 10.6%, about 10.7%, about 10.8%, about 10.9%, about 11.0%, about 11.1%, about 11.2%, about 11.3%, about 11.4%, about 11.5%, about 11.6%, about 11.7%, about 11.8%, about 11.9%, or about 12.0% DS-10. In some embodiments, the amount of DS-10 in the mixture of β-cyclodextrin molecules can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-10 in the MALDI-TOF-MS spectrum is 9.39%.

[0238] In some embodiments, the mixture of β-cyclodextrin molecules may include about 2% to about 6% DS-11. In some aspects, the mixture of β-cyclodextrin molecules may include about 2% to about 2.5% DS-11, about 2.5% to about 3% DS-11, about 3% to about 3.5% DS-11, about 3.5% to about 4% DS-11, about 4% to about 4.5% DS-11, about 4.5% to about 5% DS-11, about 5% to about 5.5% DS-11, or about 5.5% to about 6% DS-11. In some additional aspects, the mixture of β-cyclodextrin molecules may contain about 2% to about 3% DS-11, about 2% to about 3.5% DS-11, about 2% to about 4% DS-11, about 2% to about 4.5% DS-11, about 2% to about 5% DS-11, about 2% to about 5.5% DS-11, about 2.5% to about 6% DS-11, about 3% to about 6% DS-11, about 3.5% to about 6% DS-11, about 4% to about 6% DS-11, about 4.5% to about 6% DS-11, about 5% to about 6% DS-11, or about 3% to about 5% DS-11. In some additional aspects, the mixture of beta-cyclodextrin molecules can comprise about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about In some embodiments, the amount of DS-11 in the mixture of β-cyclodextrin molecules can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-11 in the MALDI-TOF-MS spectrum is 4.58%.

[0239] In some embodiments, the mixture of β-cyclodextrin molecules may include about 0.5% to about 4% DS- 12. In some aspects, the mixture of β-cyclodextrin molecules may include about 0.5% to about 1% DS-12, about 1% to about 1.5% DS-12, about 1.5% to about 2% DS-12, about 2% to about 2.5% DS-12, about 2.5% to about 3% DS-12, about 3% to about 3.5% DS-12, or about 3.5% to about 4% DS-12. In some additional aspects, the mixture of β-cyclodextrin molecules may contain about 0.5% to about 1.5% DS-12, about 0.5% to about 2% DS-12, about 0.5% to about 2.5% DS-12, about 0.5% to about 3% DS-12, about 0.5% to about 3.5% DS-12, about 1% to about 4% DS-12, about 1.5% to about 4% DS-12, about 2% to about 4% DS-12, about 2.5% to about 4% DS-12, about 3% to about 4% DS-12, or about 1% to about 3% DS-12. In yet further aspects, the mixture of β-cyclodextrin molecules can comprise about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, or about 4.0%. In some embodiments, the amount of DS-12 in the mixture of β-cyclodextrin molecules can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-12 in the MALDI-TOF-MS spectrum is 1.84%.

[0240] In some embodiments, the mixture of β-cyclodextrin molecules may contain less than 1% DS-13; for example, the mixture of β-cyclodextrin molecules may contain about 0.9% DS-13, about 0.8% DS-13, about 0.7% DS-13, about 0.6% DS-13, about 0.5% DS-13, about 0.4% DS-13, about 0.3% DS-13, about 0.2% DS-13, or about 0.1% DS-13. In some aspects, the mixture of β-cyclodextrin molecules may contain less than 1% to about 0.9% DS-13, about 0.9% to about 0.8% DS-13, about 0.8% to about 0.7% DS-13, about 0.7% to about 0.6% DS-13, about 0.7% to about 0.6% DS-13, about 0.7% to about 0.6% DS-13, about 0.6% to about 0.5% DS-13, about 0.5% to about 0.4% DS-13, about 0.4% to about 0.3% DS-13, about 0.3% to about 0.2% DS-13, about 0.2% to about 0.1% DS-13, or less than 0.1% DS-13. In some additional aspects, the mixture of β-cyclodextrin molecules may contain less than 1% to about 0.8% DS-13, less than 1% to about 0.7% DS-13, less than 1% to about 0.6% DS-13, less than 1% to about 0.5% DS-13, less than 1% to about 0.4% DS-13, less than 1% to about 0.3% DS-13, less than 1% to about 0.2% DS-13, less than 1% to about 0.1% DS-13, about 0.9% to about 0.1% DS-13, about 0.8% to about 0.1% DS-13, about 0.7% to about 0.1% DS-13, about 0.6% to about 0.1% DS-13, about 0.5% to about 0.1% DS-13, about 0.4% to about 0.1% DS-13, or about 0.3% to about 0.1% DS-13. In yet further aspects, the β-cyclodextrin mixture may contain less than 1% DS-13, less than 0.9% DS-13, less than 0.8% DS-13, less than 0.7% DS-13, less than 0.6% DS-13, less than 0.5% DS-13, less than 0.4% DS-13, less than 0.3% DS-13, less than 0.2% DS-13, or less than 0.1% DS-13. In some embodiments, the amount of DS-13 in the mixture of β-cyclodextrin molecules can be determined by MALDI-TOF-MS. In an exemplary embodiment, the area of ​​DS-13 in the MALDI-TOF-MS spectrum is 0.70%.

[0241] In some embodiments, the composition may contain less than 1% DS-14; for example, the mixture of β-cyclodextrin molecules may contain about 0.9% DS-14, about 0.8% DS-14, about 0.7% DS-14, about 0.6% DS-14, about 0.5% DS-14, about 0.4% DS-14, about 0.3% DS-14, about 0.2% DS-14, or about 0.1% DS-14. In some aspects, the mixture of β-cyclodextrin molecules may contain less than 1% to about 0.9% DS-14, about 0.9% to about 0.8% DS-14, about 0.8% to about 0.7% DS-14, about 0.7% to about 0.6% DS-14, about 0.7% to about 0.6% DS-14, about 0.7% to about 0.6% DS-14, about 0.6% to about 0.5% DS-14, about 0.5% to about 0.4% DS-14, about 0.4% to about 0.3% DS-14, about 0.3% to about 0.2% DS-14, about 0.2% to about 0.1% DS-14, or less than 0.1% DS-14. In some additional aspects, the mixture of β-cyclodextrin molecules may contain less than 1% to about 0.8% DS-14, less than 1% to about 0.7% DS-14, less than 1% to about 0.6% DS-14, less than 1% to about 0.5% DS-14, less than 1% to about 0.4% DS-14, less than 1% to about 0.3% DS-14, less than 1% to about 0.2% DS-14, less than 1% to about 0.1% DS-14, about 0.9% to about 0.1% DS-14, about 0.8% to about 0.1% DS-14, about 0.7% to about 0.1% DS-14, about 0.6% to about 0.1% DS-14, about 0.5% to about 0.1% DS-14, about 0.4% to about 0.1% DS-14, or about 0.3% to about 0.1% DS-14. In yet further aspects, the β-cyclodextrin mixture may optionally contain less than 1% DS-14, less than 0.9% DS-14, less than 0.8% DS-14, less than 0.7% DS-14, less than 0.6% DS-14, less than 0.5% DS-14, less than 0.4% DS-14, less than 0.3% DS-14, less than 0.2% DS-14, or less than 0.1% DS-14. In yet further aspects, the β-cyclodextrin molecule mixture may optionally contain about 0.001%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% DS-14. In some embodiments, the amount of DS-14 in the mixture of β-cyclodextrin molecules can be determined by MALDI-TOF-MS. In some embodiments, DS-14 is not present in the composition.

[0242] In an exemplary embodiment, the composition comprises a mixture of β-cyclodextrin molecules, wherein the mixture of β-cyclodextrin molecules comprises DS-4, DS-5, DS-6, DS-7, DS-8, DS-9, DS-10, DS-11, DS-12, DS-13 and DS-14, and wherein the mixture of β-cyclodextrin molecules comprises less than 1% DS-1, DS-2, DS-3 and DS-4.

[0243] Also provided herein are compositions produced using one or more systems and / or methods provided herein, the compositions comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and β-cyclodextrin having glucose units of the following structure:

[0244]

[0245] Where R 1 , R 2 and R 3 Each occurrence is independently -H or -HP, wherein HP contains one or more hydroxypropyl groups, and in β-cyclodextrin, the combined R 1 and R 2 The total percentage of occurrence of HP is in the range of 85% to 95%, or more preferably 90% to 95%.

[0246] In some embodiments, HP comprises one hydroxypropyl group. In some embodiments, HP consists essentially of one hydroxypropyl group. In some embodiments, HP consists of one hydroxypropyl group.

[0247] In some embodiments, the combined R 1 and R 2 No more than about 95%, e.g., no more than about 90%, no more than about 85%, no more than about 80%, no more than about 75%, no more than about 70%, no more than about 65%, no more than about 60%, no more than about 55%, or no more than about 50%, of the total occurrences are HP.

[0248] R 3 At least about 5% of the total occurrences of may be HP; for example, R 3 At least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% of the total occurrences of may be HPs.

[0249] In some embodiments, the combined R 1 and R 2The percentage range for HP is from about 5% to about 95%, for example, from about 10% to about 95%, from about 15% to about 95%, from about 20% to about 95%, from about 25% to about 95%, from about 30% to about 95%, from about 35% to about 95%, from about 40% to about 95%, from about 45% to about 95%, from about 50% to about 95%, from about 55% to about 95%, from about 60% to about 95%, from about 65% to about 95%, from about 70% to about 95%, from about 75% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%; for example, from about 5% to about 90%, from about 10% to about 90%, from about 15% to about 90%, from about 20% to about 90%, from about 25% to about 95% to about 90%, about 30% to about 90%, about 35% to about 90%, about 40% to about 90%, about 45% to about 90%, about 50% to about 90%, about 55% to about 90%, about 60% to about 90%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%; for example, about 5% to about 85%, about 10% to about 85%, about 15% to about 85%, about 20% to about 85%, about 25% to about 85%, about 30% to about 85%, about 35% to about 85%, about 40% to about 85%, about 45% to about 85%, about 50% to about 85%, about 55% to about 85%, about 60% to about 85%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%; for example, about 5% to about 80%, about 10% to about 80%, about 15% to about 80%, about 20% to about 80%, about 25% to about 80%, about 30% to about 80%, about 35% to about 80%, about 40% to about 80%, about 45% to about 80%, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%; for example, about 5% to about 75%, about 10% to about 75%, about 15% to about 75%, about 20% to about 80%, about to about 75%, about 25% to about 75%, about 30% to about 75%, about 35% to about 75%, about 40% to about 75%, about 45% to about 75%, about 50% to about 75%, about 55% to about 75%, about 60% to about 75%, about 65% to about 75%, about 70% to about 75%; for example, about 5% to about 70%, about 10% to about 70%, about 15% to about 70%, about 20% to about 70%, about 25% to about 70%, about 30% to about 70%, about 35% to about 70%, about 40% to about 70%, about 45% to about 70%, about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, about 65% to about 70%;For example, from about 5% to about 65%, from about 10% to about 65%, from about 15% to about 65%, from about 20% to about 65%, from about 25% to about 65%, from about 30% to about 65%, from about 35% to about 65%, from about 40% to about 65%, from about 45% to about 65%, from about 50% to about 65%, from about 55% to about 65%, from about 60% to about 65%; for example, from about 5% to about 60%, from about 10% to about 60%, from about 15% to about 60%, from about 20% to about 60%, from about 25% to about 60%, from about 30% to about 60%, from about 35% to about 65%, from about 40% to about 65%, from about 45% to about 65%, about 60%, about 45% to about 60%, about 50% to about 60%, about 55% to about 60%; for example, about 5% to about 55%, about 10% to about 55%, about 15% to about 55%, about 20% to about 55%, about 25% to about 55%, about 30% to about 55%, about 35% to about 55%, about 40% to about 55%, about 45% to about 55%, about 50% to about 55%; for example, about 5% to about 50%, about 10% to about 50%, about 15% to about 50%, about 20% to about 50%, about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, about 40% to about 50%, about 45% to about 50%; for example, about 5% to about 45%, about 10% to about 45%, about 15% to about 45%, about 20% to about 45%, about 25% to about 45%, about 30% to about 45%, about 35% to about 45%, about 40% to about 45%; for example, about 5% to about 40%, about 10% to about 40%, about 15% to about 40%, about 20% to about 40%, about 25% to about 40%, about 30% to about 40%, about 35% to about 40%; for example, about 5% to about 35%, about 10% to about 35%, about 15% to about 35%, about 20% to about 35%, about 25% to about 35%, about 30% to about 35%; for example, about 5% to about 30%, about 10% to about 30%, about 15% to about 30%, about 20% to about 30%, about 25% to about 30%; for example, about 5% to about 25%, about 10% to about 25%, about 15% to about 25%, about 20% to about 25%; for example, about 5% to about 20%, about 10% to about 20%, about 15% to about 20%; for example, about 5% to about 15%, about 10% to about 15%; or about 5% to about 10%. ;

[0250] The mixture may contain less than 0.1% DS-0 and less than 0.1% DS-1 in total. For example, the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-0; and / or the mixture may contain less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% DS-1.

[0251] The amount of DS-0 or DS-1 can be determined by the peak height of the electrospray MS spectrum.

[0252] The mixture may have an average molar substitution in the range of about 0.40 to about 0.80; for example, the mixture may have an average molar substitution of about 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or about 0.80. The mixture may have an average degree of substitution ("DSS") of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. a ”). For example, the mixture can have an average degree of substitution of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or about 7. In other words, the average number of occurrences of HP per β-cyclodextrin can be 3 to 4, 3 to 5, 3 to 6, 3 to 7, 4 to 5, 4 to 6, 4 to 7, 5 to 6, 5 to 7, or 6 to 7.

[0253] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002% or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC or gas chromatography.

[0254] The composition may contain no more than 0.01% propylene glycol; for example, the composition may contain no more than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% propylene glycol. The amount of propylene glycol can be measured by HPLC, gas chromatography, or the PG / EG ratio of propylene glycol to ethylene glycol.

[0255] The composition may contain no more than 1 ppm of propylene oxide, no more than 0.9 ppm of propylene oxide, no more than 0.8 ppm of propylene oxide, no more than 0.7 ppm of propylene oxide, no more than 0.6 ppm of propylene oxide, no more than 0.5 ppm of propylene oxide, no more than 0.4 ppm of propylene oxide, no more than 0.3 ppm of propylene oxide, no more than 0.2 ppm of propylene oxide, or no more than 0.1 ppm of propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0256] The total amount of other unspecified impurities in the composition can be less than or equal to 0.05%; for example, the total amount of unspecified impurities in the composition can be 0.05%, less than 0.05%, less than or equal to 0.04%, less than or equal to 0.03%, less than or equal to 0.02%, or less than or equal to 0.01%. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0257] The composition may be suitable for intrathecal, intravenous or intraventricular administration to a patient in need thereof. The patient may be an adult patient or a pediatric patient. The composition may further comprise a pharmaceutically acceptable diluent.

[0258] The composition may dissolve the lipid in an aqueous medium. The lipid may include unesterified or esterified cholesterol. The composition may be provided as a solution wherein a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxy positions is present at a concentration of 20% w / v in solution. The composition may have an affinity for unesterified cholesterol. Solubility may be determined by UV spectroscopy or HPLC.

[0259] In some embodiments, about 200 mg of the composition dissolves at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or at least about 10 mg of unesterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution is capable of dissolving about 2 mg of unesterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0260] The concentration of the composition in the solution may be from about 10 mg / mL to about 200 mg / mL. For example, the concentration of the composition in the solution may be from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 70 mg / mL, from about 10 mg / mL to about 80 mg / mL, from about 10 mg / mL to about 90 mg / mL, from about 10 mg / mL to about 100 mg / mL. mL, about 10 mg / mL to about 110 mg / mL, about 10 mg / mL to about 120 mg / mL, about 10 mg / mL to about 130 mg / mL, about 10 mg / mL to about 140 mg / mL, about 10 mg / mL to about 150 mg / mL, about 10 mg / mL to about 160 mg / mL, about 10 mg / mL to about 170 mg / mL, about 10 mg / mL to about 180 mg / mL, about 10 mg / mL to about 190 mg / mL, about 2 0 mg / mL to about 200 mg / mL, about 30 mg / mL to about 200 mg / mL, about 40 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 60 mg / mL to about 200 mg / mL, about 70 mg / mL to about 200 mg / mL, about 80 mg / mL to about 200 mg / mL, about 90 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 110 mg / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, about 190 mg / mL to about 200 mg / mL, about 200 mg / mL to about g / mL to about 200 mg / mL, about 120 mg / mL to about 200 mg / mL, about 130 mg / mL to about 200 mg / mL, about 140 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 160 mg / mL to about 200 mg / mL, about 170 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, or about 190 mg / mL to about 200 mg / mL.

[0261] Also provided herein is a composition produced by any of the systems and / or methods described herein, the composition comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: the mixture comprises less than 0.05% of unsubstituted β-cyclodextrin ("DS-0") and less than 0.05% of β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"), the composition comprising an average degree of substitution of 6.02-7.98, wherein the composition is suitable for intrathecal, intravenous, oral, or intraventricular administration to a patient in need thereof. In some embodiments, the pH of the composition is between 6.0 and 7.9. In some embodiments, the true density of the composition is about 1.096-1.098 g / cm 3 . In some embodiments, the osmotic pressure of the composition is about 635-695mOs / kg. In some embodiments, the composition further comprises a container and invisible particulate matter, and the number of invisible particulate matter with a particle size ≥25 microns is ≤600 / container. In some embodiments, the composition comprises no more than 10ppb of propylene glycol as measured by HPLC. In some embodiments, the composition comprises no more than 10ppb of propylene glycol as measured by gas chromatography. In some embodiments, the composition comprises no more than 10ppb of propylene glycol as measured by the PG / EG ratio of propylene glycol to ethylene glycol. In some embodiments, the composition comprises no more than 1ppm of propylene oxide. In some embodiments, the total amount of other unspecified impurities is less than or equal to 0.05% as measured by HPLC. In some embodiments, the composition has a concentration of about 10mg / mL to about 200mg / mL. In some embodiments, the composition exhibits a ratio Cyclo lower toxicity. In some embodiments, the conductivity of the composition is about ≤200 μS / cm. In some embodiments, the composition is stable for at least 6 months. In some embodiments, the composition further comprises at least one of a pharmaceutical excipient, a carrier, a pharmaceutically acceptable diluent, a pH adjuster, and a buffer. In some aspects, the pH adjuster is sodium hydroxide. In some aspects, the buffer comprises sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0262] Also provided herein are compositions produced by any of the systems and / or methods provided herein, the composition comprising a mixture of β-cyclodextrin molecules, wherein the mixture of β-cyclodextrin molecules comprises β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"); β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"); β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6"); β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"); β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"); ; β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"); β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10"); β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"); β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"); β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"); and β-cyclodextrin substituted with fourteen hydroxypropyl groups ("DS-14"); and wherein the mixture of β-cyclodextrin molecules comprises less than 1% DS-4. In some embodiments, the mixture of β-cyclodextrin molecules comprises about 0.5% w / w to about 1% w / w of DS-4. In some embodiments, the mixture of β-cyclodextrin molecules comprises about 2% w / w to about 5% w / w of DS-5. In some embodiments, the mixture of β-cyclodextrin molecules comprises about 7% w / w to about 13% w / w of DS-6. In some embodiments, the mixture of β-cyclodextrin molecules comprises about 21% w / w to about 27% w / w of DS-7. In some embodiments, the mixture of β-cyclodextrin molecules comprises about 23% w / w to about 29% w / w of DS-8. In some embodiments, the mixture of β-cyclodextrin molecules comprises about 15% w / w to about 21% w / w of DS-9. In some embodiments, the mixture of β-cyclodextrin molecules comprises about 6% w / w to about 12% w / w of DS-10. In some embodiments, the mixture of β-cyclodextrin molecules comprises about 2% w / w to about 6% w / w of DS-11. In some embodiments, the mixture of β-cyclodextrin molecules comprises about 0.5% w / w to about 4% w / w of DS-12. In some embodiments, the mixture of β-cyclodextrin molecules comprises less than about 1% w / w of DS-13. In some embodiments, the mixture of β-cyclodextrin molecules is suitable for intravenous, intrathecal or intraventricular administration. In some embodiments, the amounts of DS-1, DS-2, DS-3, DS-4, DS-5, DS-6, DS-7, DS-8, DS-9, DS-10, DS-11, DS-12, and DS-13 in a mixture of β-cyclodextrin molecules are determined by MALDI-TOF-MS.In some embodiments, DS-8 has the highest concentration in the mixture of β-cyclodextrin molecules compared to the concentrations of DS-1, DS-2, DS-3, DS-4, DS-5, DS-6, DS-7, DS-9, DS-10, DS-11, DS-12, and DS-13. In some embodiments, the substitution rate of the β-cyclodextrin molecules at the 2-O position is 35-55%, the substitution rate at the 3-O position is 45-65%, and the substitution rate at the 6-O position is 0-20%. In some embodiments, the substitution rates at the 2-O position, the 3-O position, and the 6-O position are determined by DEPT-ed HSQC. In some embodiments, the composition has an average degree of substitution of about 7 to about 9. In an exemplary embodiment, the composition has an average degree of substitution of about 7.7. In some embodiments, the composition has about 1.095 g / cm. 3 To about 1.100g / cm 3 In some embodiments, the composition has an osmotic pressure of about 600mOs / kg to about 750mOs / kg. In some embodiments, the composition is a transparent colorless solution. In some embodiments, the pH of the composition is about 4.0 to about 6.0. In some embodiments, the viscosity of the composition at 20°C is 1.5cP to about 3.0cP. In some embodiments, the composition contains less than or equal to about 0.05% impurities. In some embodiments, the composition contains less than 600 particles with a diameter greater than or equal to 25 microns per container. In some embodiments, the composition contains less than 6000 particles with a diameter greater than or equal to 10 microns per container.

[0263] Also provided herein are compositions produced by any of the systems and / or methods provided herein, the compositions comprising a mixture of β-cyclodextrin molecules, the compositions having 1 The H-NMR spectrum contains at least one peak at about 5.0-5.4 ppm corresponding to the anomeric protons of the β-cyclodextrin molecule; at least one peak at about 3.2-4.2 ppm corresponding to the protons in the core region of the β-cyclodextrin molecule; and at least one peak at about 1.0-1.2 ppm corresponding to the methyl protons of the side chains of the β-cyclodextrin molecule.

[0264] Also provided herein is a composition produced by any of the systems and / or methods provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl-β-cyclodextrin molecules, the mixture comprising less than 1% of β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"). In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on the area percentage of the MALDI-TOF-MS spectrum. In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on weight percentage. In some embodiments, the composition comprises less than 1% of β-cyclodextrin substituted with three hydroxypropyl groups ("DS-3"), β-cyclodextrin substituted with two hydroxypropyl groups ("DS-2"), and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"). In some embodiments, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules comprises about 1% to about 5% of β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"). In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 7% to about 13% of β-cyclodextrins substituted with six hydroxypropyl groups ("DS-6"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 8% to about 12% of DS-6. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 16% to about 22% of β-cyclodextrins substituted with seven hydroxypropyl groups ("DS-7"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 17% to about 21% of DS-7. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 26% to about 32% of β-cyclodextrins substituted with eight hydroxypropyl groups ("DS-8"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 27% to about 31% of DS-8. In some embodiments, a mixture of isomerically purified hydroxypropyl β-cyclodextrins comprises about 22% to about 28% of β-cyclodextrins substituted with nine hydroxypropyl groups ("DS-9"). In some aspects, a mixture of isomerically purified hydroxypropyl β-cyclodextrins comprises about 23% to about 27% of DS-9. In some embodiments, a mixture of isomerically purified hydroxypropyl β-cyclodextrins comprises about 11% to about 17% of β-cyclodextrins substituted with ten hydroxypropyl groups ("DS-10"). In some aspects, a mixture of isomerically purified hydroxypropyl β-cyclodextrins comprises about 12% to about 16% of DS-10. In some embodiments, a mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises less than 1% of β-cyclodextrins substituted with eleven hydroxypropyl groups ("DS-11"). In some embodiments, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules comprises less than 1% of β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"), β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"), and β-cyclodextrin substituted with fourteen hydroxypropyl groups ("DS-14").In some embodiments, the average degree of substitution of the isomeric purified hydroxypropyl β-cyclodextrin mixture is about 6.4 to about 7.0. In an exemplary embodiment, the average degree of substitution is about 6.69. In some embodiments, about 52% to about 58% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecule are located at the 3-O position. In some aspects, about 55% to about 56% of the hydroxypropyl substitutions in the β-cyclodextrin molecule are located at the 3-O position. In some embodiments, about 41% to about 47% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecule are located at the 2-O position. In some aspects, about 43% to about 45% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecule are located at the 2-O position. In some embodiments, the concentration of the composition does not substantially change the time required for nanofiltration. In some aspects, the length of time for nanofiltration of the composition ranges from 1.04 to 1.20 hours per diafiltration volume (kg solution / m2-hr / L solution). In some embodiments, the conductivity of the composition is from 0 to 8.0 μS / cm, from 0 to 4.5 μS / cm, from 0 to 3 μS / cm, or from 0 to 1.5 μS / cm.

[0265] Also provided herein is a composition produced by any of the systems and / or methods provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules, the mixture comprising: β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"); β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6"); β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"); β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"); β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"); and β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10"), wherein the composition comprises less than 1% of β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4") and less than 1% of β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"). In some embodiments, the composition comprises 0.0-1.0% of a β-cyclodextrin substituted with three hydroxypropyl groups ("DS-3"), 0.0-1.0% of a β-cyclodextrin substituted with two hydroxypropyl groups ("DS-2"), and 0.0-1.0% of a β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"). In some embodiments, the composition comprises less than 1% of a β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"), a β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"), and a β-cyclodextrin substituted with fourteen hydroxypropyl groups ("DS-14"). In some embodiments, DS-8 has the highest concentration in the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules compared to DS-5, DS-6, DS-7, DS-9, and DS-10. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 1% to about 5% DS-5. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 7% to about 13% DS-6. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 16% to about 22% DS-7. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 26% to about 32% DS-8. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 22% to about 28% DS-9. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrins contains about 11% to about 17% DS-10. In some embodiments, the average degree of substitution of the isomerically purified hydroxypropyl β-cyclodextrin mixture is about 6.4 to about 7.0. In an exemplary embodiment, the average degree of substitution is about 6.69. In some embodiments, about 52% to about 58% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 3-O position. In some embodiments, about 41% to about 47% of the hydroxypropyl substitution in the hydroxypropyl β-cyclodextrin molecule is at the 2-0 position.In some embodiments, the composition has a peak at about 653m / z, about 682m / z, about 711m / z, about 741m / z, about 769m / z, about 799m / z, about 828m / z and about 857m / z -ESI-MS spectrum and a peak at about 686m / z, about 715m / z, about 744m / z, about 773m / z, about 802m / z, about 832m / z, about 861m / z and about 890m / z +ESI-MS spectrum. In some embodiments, the composition has a peak at about 1436m / z, about 1495m / z, about 1555m / z, about 1614m / z, about 1674m / z and about 1733m / z MALDI-TOF spectrum. In some embodiments, the osmotic pressure of the composition is about 635-695mOs / kg. In some embodiments, the composition has a true density of about 1.096-1.098 g / cm. 3 . In some embodiments, the composition comprises no more than 10 ppb of propylene glycol as measured by HPLC. In some embodiments, the composition comprises no more than 1 ppm of propylene oxide. In some embodiments, the total amount of other unspecified impurities is less than or equal to 0.05% as measured by HPLC. In some embodiments, the composition further comprises 0 to 10 ppm of chloride. In some embodiments, the composition is nanofiltered. In some embodiments, no significant difference is observed in HPLC-ELSD after nanofiltration of the composition filtered by nanofiltration compared to before nanofiltration. In some embodiments, no significant difference is observed in NMR after nanofiltration of the composition filtered by nanofiltration compared to before nanofiltration.

[0266] Also provided herein is a composition produced by any system and / or method provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules, the mixture comprising less than 1% of hydroxypropyl β-cyclodextrin ("DS-5") having five hydroxypropyl groups. In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on the area percentage of the MALDI-TOF-MS spectrum. In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on weight percentage. In some embodiments, the composition comprises less than 1% of β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"), β-cyclodextrin substituted with three hydroxypropyl groups ("DS-3"), β-cyclodextrin substituted with two hydroxypropyl groups ("DS-2"), and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"). In some embodiments, the mixture of isomeric purified hydroxypropyl β-cyclodextrin comprises about 0% to about 6% of hydroxypropyl β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 1% to about 5% DS-6. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 8% to about 14% hydroxypropyl β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 9% to about 13% DS-7. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 19% to about 25% hydroxypropyl β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 20% to about 24% DS-8. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 23% to about 29% hydroxypropyl β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 24% to about 28% DS-9. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 17% to about 23% hydroxypropyl β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10". In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 18% to about 22% DS-10. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 9% to about 15% hydroxypropyl β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"). In some aspects, the mixture of isomerically purified β-cyclodextrin molecules comprises about 10% to about 14% DS-11. In some embodiments, the mixture of isomerically purified β-cyclodextrin molecules comprises about 2% to about 8% hydroxypropyl β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12").In some aspects, the mixture of isomeric purified β-cyclodextrin molecules contains about 3% to about 7% DS-12. In some embodiments, the isomeric purified β-cyclodextrin molecule mixture has an average degree of substitution of about 7 to about 8. In an exemplary embodiment, the average degree of substitution is about 7.42. In some embodiments, about 36% to about 42% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 3-O position. In some aspects, about 37% to about 41% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 3-O position. In some embodiments, about 58% to about 64% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 2-O position. In some aspects, about 59% to about 63% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 2-O position. In some embodiments, the concentration of the composition does not substantially change the time required for nanofiltration. In some aspects, the time length range for nanofiltration of the composition is 1.04 to 1.20 hours per diafiltration volume (kg solution / m2-hr / L solution). In some embodiments, the composition is not observed to be significantly different in HPLC-ELSD after nanofiltration compared to before nanofiltration. In some embodiments, wherein the composition is not observed to be significantly different in NMR after nanofiltration compared to before nanofiltration. In some embodiments, the conductivity of the composition is 0 to 8.0 μS / cm, 0 to 4.5 μS / cm, 0 to 3 μS / cm, or 0 to 1.5 μS / cm.

[0267] Also provided herein is a composition produced by any of the systems and / or methods provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules, the mixture comprising: β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6"); β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"); β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"); β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"); β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10"); β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"); and β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"), wherein the composition comprises less than 1% of β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5") and the composition comprises less than 1% of β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"). In some embodiments, the composition comprises less than 1% of four hydroxypropyl substituted β-cyclodextrin ("DS-4"), three hydroxypropyl substituted β-cyclodextrin ("DS-3"), two hydroxypropyl substituted β-cyclodextrin ("DS-2"), and one hydroxypropyl substituted β-cyclodextrin ("DS-1"). In some embodiments, the composition comprises less than 1% of thirteen hydroxypropyl substituted β-cyclodextrin ("DS-13") and fourteen hydroxypropyl substituted hydroxypropyl β-cyclodextrin ("DS-14"). In some embodiments, DS-9 has the highest concentration in the composition compared to DS-6, DS-7, DS-8, DS-10, DS-11, and DS-12. In some embodiments, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules comprises about 0% to about 6% DS-6. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 8% to about 14% DS-7. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 19% to about 25% DS-8. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 23% to about 29% DS-9. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 17% to about 23% DS-10. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 9% to about 15% DS-11. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 2% to about 8% DS-12. In some embodiments, the average degree of substitution of the isomerically purified hydroxypropyl β-cyclodextrin molecules is about 7 to about 8. In some embodiments, about 36% to about 42% of the hydroxypropyl substitution in the hydroxypropyl β-cyclodextrin molecule is at the 3-0 position. In some embodiments, about 58% to about 64% of the hydroxypropyl substitution in the hydroxypropyl β-cyclodextrin molecule is at the 2-0 position.In some embodiments, the composition has a peak at about 682m / z, about 712m / z, about 740m / z, about 770m / z, about 798m / z, about 828m / z, about 856m / z and about 886m / z -ESI-MS spectrum and a peak at about 744m / z, about 773m / z, about 803m / z, about 832m / z, about 860m / z, about 889m / z and about 919m / z +ESI-MS spectrum. In some embodiments, the composition has a peak at about 1497m / z, about 1557m / z, about 1616m / z, about 1675m / z, about 1734m / z, about 1794m / z and about 1914m / z MALDI-TOF-MS spectrum. In some embodiments, the composition has an osmotic pressure of about 635-695 mOs / kg. In some embodiments, the composition has a true density of about 1.096-1.098 g / cm. 3 . In some embodiments, the composition comprises no more than 10ppb of propylene glycol as measured by HPLC. In some embodiments, the composition comprises no more than 1ppm of propylene oxide. In some embodiments, the total amount of other unspecified impurities is less than or equal to 0.05% as measured by HPLC. In some embodiments, the composition comprises 0 to 10ppm of chloride. In some embodiments, the conductivity of the composition is between 0 and 8μS / cm. In some embodiments, the composition is nanofiltered. In some embodiments, no significant difference is observed in HPLC-ELSD after nanofiltration compared to before nanofiltration in the composition filtered through nanofiltration. In some embodiments, no significant difference is observed in NMR after nanofiltration compared to before nanofiltration in the composition filtered through nanofiltration.

[0268] Also provided herein is a composition produced by any of the methods and / or systems provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules, the mixture comprising less than 1% of hydroxypropyl β-cyclodextrin with six hydroxypropyl groups ("DS-6") and less than 1% of β-cyclodextrin substituted with fourteen hydroxypropyl groups ("DS-14"). In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on the area percentage of the MALDI-TOF-MS spectrum. In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on weight percentage. In some embodiments, the composition comprises less than 1% of β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"), β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"), β-cyclodextrin substituted with three hydroxypropyl groups ("DS-3"), β-cyclodextrin substituted with two hydroxypropyl groups ("DS-2"), and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"). In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 1% to about 7% of β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 2% to about 6% of DS-7. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 16% to about 22% of β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 17% to about 21% of DS-8. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 22% to about 28% of β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 23% to about 27% of DS-9. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 19% to about 25% β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10". In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 20% to about 24% DS-10. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 14% to about 20% β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 15% to about 19% DS-11. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 5% to about 11% β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 6% to about 10% DS-12.In some embodiments, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules contains about 1% to about 7% of β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"). In some aspects, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules contains about 2% to about 6% DS-13. In some embodiments, the average degree of substitution of the isomeric purified hydroxypropyl β-cyclodextrin mixture is about 8 to about 9. In an exemplary embodiment, the average degree of substitution of the isomeric purified hydroxypropyl β-cyclodextrin mixture is about 8.53. In some embodiments, about 26% to about 32% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 3-O position. In some aspects, about 27% to about 31% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 3-O position. In some embodiments, about 68% to about 74% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 2-O position. In some aspects, about 69% to about 73% of the hydroxypropyl substitution in the hydroxypropyl beta-cyclodextrin molecule is located at the 2-0 position. In some embodiments, the concentration of the composition does not substantially change the time required for nanofiltration. In some aspects, the length of time for nanofiltration of the composition ranges from 1.04 to 1.20 hours per diafiltration volume (kg solution / m. 2 -hr / L solution). In some embodiments, no significant difference is observed in HPLC-ELSD of the composition after nanofiltration compared to before nanofiltration. In some embodiments, no significant difference is observed in NMR of the composition after nanofiltration compared to before nanofiltration. In some embodiments, the conductivity of the composition is 0 to 8.0 μS / cm, 0 to 4.5 μS / cm, 0 to 3 μS / cm, or 0 to 1.5 μS / cm.

[0269] Also provided herein is a composition produced by any of the systems and / or methods provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules, the mixture comprising: β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"); β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"); β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"); β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10"); β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"); β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"); and β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"), wherein the composition comprises less than 1% of β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6") and less than 1% of β-cyclodextrin substituted with fourteen hydroxypropyl groups ("DS-14"). In some embodiments, the composition comprises less than 1% of five hydroxypropyl substituted β-cyclodextrins ("DS-5"), four hydroxypropyl substituted β-cyclodextrins ("DS-4"), three hydroxypropyl substituted β-cyclodextrins ("DS-3"), two hydroxypropyl substituted β-cyclodextrins ("DS-2"), and one hydroxypropyl substituted β-cyclodextrin ("DS-1"). In some embodiments, DS-9 has the highest concentration in the isomerically purified hydroxypropyl β-cyclodextrin molecule mixture compared to DS-6, DS-7, DS-8, DS-10, DS-11, DS-12, and DS-13. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 16% to about 22% DS-8. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 22% to about 28% DS-9. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 19% to about 25% DS-10. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 14% to about 20% DS-11. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 5% to about 11% DS-12. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 1% to about 7% DS-13. In some embodiments, the average degree of substitution of the isomerically purified hydroxypropyl β-cyclodextrin mixture is about 8 to about 9. In an exemplary embodiment, the average degree of substitution of the isomerically purified hydroxypropyl β-cyclodextrin mixture is about 8.53. In some embodiments, about 26% to about 32% of the hydroxypropyl substitution in the β-cyclodextrin molecules is located at the 3-O position. In some embodiments, about 68% to about 74% of the hydroxypropyl substitution in the β-cyclodextrin molecules is located at the 2-O position. In an exemplary embodiment, the composition has an HPLC-CAD average retention time of about 13.5 minutes.In some embodiments, the composition has a peak at about 741m / z, about 769m / z, about 799m / z, about 828m / z, about 856m / z, about 886m / z-ESI-MS spectrum and a peak at about 773m / z, about 803m / z, about 833m / z, about 860m / z, about 889m / z and about 920m / z +ESI-MS spectrum. In some embodiments, the composition has a peak at about 1557m / z, about 1617m / z, about 1676m / z, about 1736m / z, about 1795m / z, about 1855m / z and about 1915m / z MALDI-TOF spectrum. In some embodiments, the osmotic pressure of the composition is about 635-695mOs / kg. In some embodiments, the composition has a true density of about 1.096-1.098 g / cm. 3 . In some embodiments, the composition comprises no more than 10ppb of propylene glycol as measured by HPLC. In some embodiments, the composition comprises no more than 1ppm of propylene oxide. In some embodiments, the total amount of other unspecified impurities is less than or equal to 0.05% as measured by HPLC. In some embodiments, the composition comprises 0 to 10ppm of chloride. In some embodiments, the composition comprises 0 to 1ppm of chloride. In some embodiments, the conductivity of the composition is between 0 and 8μS / cm. In some embodiments, the composition is nanofiltered. In some embodiments, no significant difference is observed in HPLC-ELSD after nanofiltration compared to before nanofiltration in the composition filtered through nanofiltration. In some embodiments, no significant difference is observed in NMR after nanofiltration compared to before nanofiltration in the composition filtered through nanofiltration.

[0270] Also provided herein is a composition produced by any system and / or method provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules, the mixture comprising less than 1% hydroxypropyl β-cyclodextrin ("DS-6") having six hydroxypropyl groups. In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on the area percentage of the MALDI-TOF-MS spectrum. In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on weight percentage. In some embodiments, the composition comprises less than 1% of β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"), β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"), β-cyclodextrin substituted with three hydroxypropyl groups ("DS-3"), β-cyclodextrin substituted with two hydroxypropyl groups ("DS-2"), and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"). In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 0% to about 6% of β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 1% to about 5% of DS-7. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 13% to about 19% of β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"). In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 14% to about 18% of DS-8. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 22% to about 28% of β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 23% to about 27% of DS-9. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 23% to about 29% β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10". In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 24% to about 28% DS-10. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 12% to about 18% β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 12% to about 18% β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"). In some embodiments, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules comprises about 13% to about 17% DS-11. In some embodiments, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules comprises about 7% to about 13% β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"). In some aspects, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules comprises about 8% to about 12% DS-12. In some embodiments, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules comprises about 2% to about 8% β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13").In some aspects, the mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules contains about 3% to about 7% DS-13. In some embodiments, the average degree of substitution of the isomeric purified hydroxypropyl β-cyclodextrin mixture is about 7.5 to about 8.5. In an exemplary embodiment, the average degree of substitution of the isomeric purified hydroxypropyl β-cyclodextrin mixture is about 8.08. In some embodiments, about 22% to about 28% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 3-O position. In some aspects, about 23% to about 27% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 3-O position. In some embodiments, about 72% to about 78% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 2-O position. In some aspects, about 73% to about 77% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 2-O position. In some embodiments, the concentration of the composition does not substantially change the time required for nanofiltration. In some aspects, the composition is nanofiltered for a period of time ranging from 1.04 to 1.20 hours per diafiltration volume (kg solution / m. 2 -hr / L solution). In some embodiments, no significant difference is observed in HPLC-ELSD of the nanofiltered composition after nanofiltration compared to before nanofiltration. In some embodiments, no significant difference is observed in NMR of the nanofiltered composition after nanofiltration compared to before nanofiltration. In some embodiments, the conductivity of the composition is 0 to 8.0 μS / cm, 0 to 4.5 μS / cm, 0 to 3 μS / cm, or 0 to 1.5 μS / cm.

[0271] Also provided herein is a composition produced by any of the systems and / or methods provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules, the mixture comprising: β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"); β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"); β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"); β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10"); β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"); β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"); β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"); and β-cyclodextrin substituted with fourteen hydroxypropyl groups ("DS-14"), wherein the composition comprises less than 1% of β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6"). In some embodiments, the composition comprises less than 1% of five hydroxypropyl substituted β-cyclodextrins ("DS-5"), four hydroxypropyl substituted β-cyclodextrins ("DS-4"), three hydroxypropyl substituted β-cyclodextrins ("DS-3"), two hydroxypropyl substituted β-cyclodextrins ("DS-2"), and one hydroxypropyl substituted β-cyclodextrin ("DS-1"). In some embodiments, DS-9 has the highest concentration in the isomerically purified mixture of hydroxypropyl β-cyclodextrin molecules compared to DS-7, DS-8, DS-10, DS-11, DS-12, DS-13, and DS-14. In some embodiments, DS-10 has the highest concentration in the isomerically purified mixture of hydroxypropyl β-cyclodextrin molecules compared to DS-7, DS-8, DS-10, DS-11, DS-12, DS-13, and DS-14. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 0% to about 6% DS-7. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 13% to about 19% DS-8. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 22% to about 28% DS-9. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 23% to about 29% DS-10. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 12% to about 18% DS-11. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 7% to about 13% DS-12. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 2% to about 8% DS-13. In some embodiments, the mixture of isomerically purified hydroxypropyl beta-cyclodextrin molecules comprises from about 0% to about 6% DS- 14. In some embodiments, the average degree of substitution of the isomerically purified hydroxypropyl beta-cyclodextrin mixture is from about 7.5 to about 8.5.In some embodiments, about 22% to about 28% of the hydroxypropyl substitution in the hydroxypropyl β-cyclodextrin molecule is located at the 3-O position. In some embodiments, about 72% to about 78% of the hydroxypropyl substitution in the β-cyclodextrin molecule is located at the 2-O position. In some embodiments, the composition has a peak at about 740m / z, about 770m / z, about 798m / z, about 828m / z and about 857m / z-ESI-MS spectrum and a peak at about 803m / z, about 831m / z, about 861m / z, about 889m / z and about 919m / z +ESI-MS spectrum. In some embodiments, the composition has a peak at about 1559m / z, about 1618m / z, about 1678m / z, about 1737m / z, about 1796m / z, about 1857m / z and about 1916m / z MALDI-TOF spectrum. In some embodiments, the composition has an osmotic pressure of about 635-695 mOs / kg. In some embodiments, the composition has a true density of about 1.096-1.098 g / cm. 3 . In some embodiments, the composition comprises no more than 10ppb of propylene glycol as measured by HPLC. In some embodiments, the composition comprises no more than 1ppm of propylene oxide. In some embodiments, the total amount of other unspecified impurities is less than or equal to 0.05% as measured by HPLC. In some embodiments, the composition comprises 0 to 10ppm of chloride. In some embodiments, the conductivity of the composition is between 0 and 8μS / cm. In some embodiments, the composition is nanofiltered. In some embodiments, no significant difference is observed in HPLC-ELSD after nanofiltration compared to before nanofiltration in the composition filtered through nanofiltration. In some embodiments, no significant difference is observed in NMR after nanofiltration compared to before nanofiltration in the composition filtered through nanofiltration.

[0272] Also provided herein is a composition produced by any system and / or method provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules, the mixture comprising less than 1% hydroxypropyl β-cyclodextrin ("DS-7") having seven hydroxypropyl groups. In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on the area percentage of the MALDI-TOF-MS spectrum. In some embodiments, the percentage of hydroxypropyl β-cyclodextrin is based on weight percentage. In some embodiments, the composition comprises less than 1% of β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6"), β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"), β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"), β-cyclodextrin substituted with three hydroxypropyl groups ("DS-3"), β-cyclodextrin substituted with two hydroxypropyl groups ("DS-2"), and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"). In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 6% to about 12% of β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 7% to about 11% DS-8. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 18% to about 24% of β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 19% to about 23% DS-9. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises from about 24% to about 30% β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10". In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises from about 25% to about 29% DS-10. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises from about 18% to about 24% β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises from about 19% to about 23% DS-11. . In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 10% to about 16% of β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 11% to about 15% DS-12. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 4% to about 10% of β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 5% to about 9% DS-13.In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 0% to about 6% of β-cyclodextrin substituted with fourteen hydroxypropyl groups ("DS-14"). In some aspects, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules contains about 1% to about 5% DS-14. In some embodiments, the average degree of substitution of the isomerically purified hydroxypropyl β-cyclodextrin mixture is about 9 to about 10. In an exemplary embodiment, the average degree of substitution of the isomerically purified hydroxypropyl β-cyclodextrin mixture is about 9.65. In some embodiments, about 15% to about 21% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 3-O position. In some aspects, about 16% to about 20% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 3-O position. In some embodiments, about 79% to about 85% of the hydroxypropyl substitutions in the hydroxypropyl β-cyclodextrin molecules are located at the 2-O position. In some aspects, about 80% to about 84% of the hydroxypropyl substitution in the hydroxypropyl β-cyclodextrin molecule is located at the 2-0 position. In some embodiments, the concentration of the composition does not substantially change the time required for nanofiltration. In some embodiments, the length of time for nanofiltration of the composition ranges from 1.04 to 1.20 hours per diafiltration volume (kg solution / m. 2 -hr / L solution). In some embodiments, no significant difference is observed in HPLC-ELSD of the composition after nanofiltration compared to before nanofiltration. In some embodiments, no significant difference is observed in NMR of the composition after nanofiltration compared to before nanofiltration. In some embodiments, the conductivity of the composition is 0 to 8.0 μS / cm, 0 to 4.5 μS / cm, 0 to 3 μS / cm, or 0 to 1.5 μS / cm.

[0273] Also provided herein is a composition produced by any of the systems and / or methods provided herein, the composition comprising a mixture of isomeric purified hydroxypropyl β-cyclodextrin molecules, the mixture comprising: β-cyclodextrin substituted with eight hydroxypropyl groups ("DS-8"); β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9"); β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10"); β-cyclodextrin substituted with eleven hydroxypropyl groups ("DS-11"); β-cyclodextrin substituted with twelve hydroxypropyl groups ("DS-12"); β-cyclodextrin substituted with thirteen hydroxypropyl groups ("DS-13"); and β-cyclodextrin substituted with fourteen hydroxypropyl groups ("DS-14"), wherein the composition comprises less than 1% of β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7"). In some embodiments, the composition comprises less than 1% of six hydroxypropyl substituted β-cyclodextrin ("DS-6"), 1% of five hydroxypropyl substituted β-cyclodextrin ("DS-5"), four hydroxypropyl substituted β-cyclodextrin ("DS-4"), three hydroxypropyl substituted β-cyclodextrin ("DS-3"), two hydroxypropyl substituted β-cyclodextrin ("DS-2"), and one hydroxypropyl substituted β-cyclodextrin ("DS-1"). In some embodiments, DS-10 has the highest concentration in the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules compared to DS-8, DS-9, DS-11, DS-12, DS-13, and DS-14. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 6% to about 12% DS-8. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 18% to about 24% DS-9. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 24% to about 30% DS-10. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 18% to about 24% DS-11. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 10% to about 16% DS-12. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 4% to about 10% DS-13. In some embodiments, the mixture of isomerically purified hydroxypropyl β-cyclodextrin molecules comprises about 0% to about 6% DS-14. In some embodiments, the average degree of substitution of the isomerically purified hydroxypropyl β-cyclodextrin mixture is about 9 to about 10. In some embodiments, about 15% to about 21% of the hydroxypropyl substitution in the hydroxypropyl β-cyclodextrin molecule is at the 3-0 position. In some embodiments, about 79% to about 85% of the hydroxypropyl substitution in the hydroxypropyl β-cyclodextrin molecule is at the 2-0 position.In some embodiments, the composition has a peak at about 770m / z, about 798m / z, about 828m / z, about 857m / z, about 885m / z-ESI-MS spectrum and a peak at about 803m / z, about 831m / z, about 861m / z, about 889m / z and about 919m / z +ESI-MS spectrum. In some embodiments, the composition has a peak at about 1614m / z, about 1673m / z, about 1733m / z, about 1792m / z, about 1852m / z, about 1912m / z and about 1971m / z MALDI-TOF spectrum. In some embodiments, the osmotic pressure of the composition is about 635-695mOs / kg. In some embodiments, the true density of the composition is about 1.096-1.098g / cm. 3 . In some embodiments, the composition comprises no more than 10ppb of propylene glycol as measured by HPLC. In some embodiments, the composition comprises no more than 1ppm of propylene oxide. In some embodiments, the total amount of other unspecified impurities is less than or equal to 0.05% as measured by HPLC. In some embodiments, the composition comprises 0 to 10ppm of chloride. In some embodiments, the conductivity of the composition is between 0 and 8μS / cm. In some embodiments, the composition is nanofiltered. In some embodiments, no significant difference is observed in HPLC-ELSD after nanofiltration compared to before nanofiltration in the composition filtered through nanofiltration. In some embodiments, no significant difference is observed in NMR after nanofiltration compared to before nanofiltration in the composition filtered through nanofiltration.

[0274] It is contemplated that the systems and methods provided herein can be used to produce compositions described in, for example, U.S. Pat. No. 10,933,083, filed on March 2, 2021, and its related applications (e.g., U.S. Pat. No. 9,675,634, filed on June 13, 2017, U.S. Pat. No. 10,258,641, filed on April 16, 2019, and U.S. Pat. No. 10,300,086, filed on May 28, 2019), and U.S. Provisional Application No. 63 / 311,661, filed on February 18, 2022, entitled "COMPOSITIONS OF HYDROXYPROPYL-BETA-CYCLODEXTRIN AND METHODS OF PURIFYING THE SAME". Each of these patents, patent applications, and provisional applications is incorporated herein by reference in its entirety.

[0275] Method for preparing beta-cyclodextrin

[0276] Beta cyclodextrin (BCD) used in the systems and methods described herein can be produced by an enzymatic synthesis process. Suitable enzymatic synthesis processes are disclosed, for example, in PCT / IB2023 / 055977, the disclosure of which is incorporated herein by reference.

[0277] In some cases, the method of producing BCD or producing a composition comprising cyclodextrins comprises (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose into amylose under conditions that allow sucrose to be converted into amylose, thereby producing amylose. In some cases, the method also comprises (b) contacting amylose with an enzyme capable of converting amylose into cyclodextrin under conditions that allow amylose to be converted into cyclodextrin, thereby producing a composition comprising cyclodextrins. In some cases, the enzyme capable of converting amylose into cyclodextrins is a variant enzyme, and relative to the wild-type enzyme capable of converting amylose into cyclodextrins, the variant enzyme is capable of producing a greater amount and / or concentration (e.g., wt%, mol% or w / v) of β-cyclodextrin than α-cyclodextrin, γ-cyclodextrin or both. In some cases, the composition comprising cyclodextrins comprises β-cyclodextrin, and may optionally also comprise α-cyclodextrin, γ-cyclodextrin, or any combination thereof. In some cases, the composition comprising cyclodextrin comprises an amount and / or concentration (e.g., wt%, mol%, or w / v) of β-cyclodextrin greater than α-cyclodextrin, γ-cyclodextrin, or both. In some cases, the amount and / or concentration of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin is measured by high performance liquid chromatography (HPLC).

[0278] Method step (a): Enzymatic conversion of sucrose into amylose

[0279] The method provided herein may involve the enzymatic conversion of sucrose to amylose. In some cases, amylose is α-amylose. In some embodiments, the method involves contacting sucrose with an enzyme or enzyme mixture that can convert sucrose into amylose under conditions that allow sucrose to be converted into amylose, thereby producing amylose. In one aspect, the method involves using a single enzyme to convert sucrose into amylose. In alternative aspects, the method involves using an enzyme mixture (e.g., two enzymes) that converts sucrose into amylose together or in combination. In some cases, sucrose is deuterated sucrose (e.g., one or more hydrogens have been replaced by deuterium). In some cases, any one or more reagents used in sucrose and / or synthesis reactions are deuterated.

[0280] A single enzyme method for producing amylose from sucrose

[0281] In some aspects, the enzyme is amylosucrase. Fig.27AA schematic diagram of a single enzyme method for producing amylose from sucrose is depicted. In this example, sucrose is contacted with an amylosucrase, which converts sucrose into amylose. In some cases, the amylosucrase is a wild-type amylosucrase. For example, the wild-type amylosucrase may be a carbon source Cellulomonas T26 amylosucrase (NCBI accession number N868_11335). In some cases, the wild-type carbon source Cellulomonas T26 amylosucrase may have the amino acid sequence of SEQ ID NO:1. In some cases, the wild-type amylosucrase may be a polysaccharide Neisseria (Neisseria polysaccharea) amylosucrase (NCBI accession number AJ011781). In some cases, the wild-type polysaccharide Neisseria amylosucrase may have the amino acid sequence of SEQ ID NO:2. Table 1 below depicts non-limiting examples of wild-type amylosucrases (and their amino acid sequences) that can be used according to the method provided herein.

[0282] Table 1. Non-limiting examples of wild-type amylosucrases

[0283]

[0284]

[0285] In some embodiments, amylosucrase is a variant amylosucrase comprising at least one amino acid variant relative to wild-type amylosucrase. Relative to wild-type amylosucrase, the variant amylosucrase may comprise one or more amino acid substitutions, deletions, insertions and / or modifications. In some cases, relative to wild-type amylosucrase, the variant amylosucrase is capable of producing a greater amount and / or concentration of amylose from sucrose.

[0286] In some cases, the variant amylosucrase comprises at least one amino acid variant relative to the wild-type carbon source Cellulomonas T26 amylosucrase (SEQ ID NO: 1). In some cases, the variant amylosucrase comprises at least one amino acid variant relative to the wild-type polysaccharide Neisseria amylosucrase (SEQ ID NO: 2). In some cases, the variant amylosucrase comprises at least one amino acid variant relative to the wild-type carbon source Cellulomonas T26 amylosucrase (SEQ ID NO: 2). In some cases, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) with the amino acid sequence of the wild-type carbon source Cellulomonas T26 amylosucrase, preferably at least about 90% sequence identity. In some cases, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some cases, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of wild-type Neisseria polysaccharida amylosucrase. In some cases, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0287] In some cases, at least one amino acid variant comprises at least one amino acid replacement relative to wild-type amylosucrase. In some cases, at least one amino acid variant comprises at least one amino acid replacement relative to wild-type carbon source Cellulomonas T26 amylosucrase. In some cases, at least one amino acid variant comprises at least one amino acid replacement relative to wild-type polysaccharide Neisseria amylosucrase. In some cases, at least one amino acid replacement comprises relative to SEQ ID NO:2 amino acid sequence, amino acid replacement at amino acid position 234 or consisting of it. In some cases, relative to SEQ ID NO:2 amino acid sequence, amino acid replacement at amino acid position 234 is selected from the group consisting of the following: R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L and R234H. In a preferred embodiment, relative to the amino acid sequence of SEQ ID NO:2, the amino acid substitution at amino acid position 234 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C and R234K. In this regard, it should be understood that R234Q means that relative to the amino acid sequence of SEQ ID NO:2, the arginine (R) at amino acid position 234 is replaced by glutamine (Q), etc. In some cases, relative to the amino acid sequence of SEQ ID NO:2, the amino acid substitution at amino acid position 234 is R234Q (e.g., SEQ ID NO:3 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO:2, the amino acid substitution at amino acid position 234 is R234G (e.g., SEQ ID NO:4 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO:2, the amino acid substitution at amino acid position 234 is R234A (e.g., SEQ ID NO:5 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234S (e.g., SEQ ID NO: 6 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234M (e.g., SEQ ID NO: 7 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234C (e.g., SEQ ID NO: 8 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234K (e.g., SEQ ID NO: 9 in Table 2).In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234I (e.g., SEQ ID NO: 10 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234D (e.g., SEQ ID NO: 11 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234Y (e.g., SEQ ID NO: 12 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234W (e.g., SEQ ID NO: 13 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234E (e.g., SEQ ID NO: 14 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234L (e.g., SEQ ID NO: 15 in Table 2). In some cases, the amino acid substitution at amino acid position 234 is R234H relative to the amino acid sequence of SEQ ID NO: 2 (e.g., SEQ ID NO: 16 in Table 2). In some aspects, the variant amylosucrase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 3-16 or 48 as depicted in Table 2, or an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to any one of SEQ ID NOs: 3-16 or 48 as depicted in Table 2. In a preferred embodiment, the variant amylosucrase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 3-9 or 48 depicted in Table 2.

[0288] Table 2. Non-limiting examples of variant amylosucrases.

[0289]

[0290]

[0291]

[0292]

[0293]

[0294] In some aspects, the variant amylosucrase comprises an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 234 relative to SEQ ID NO: 2, or consists of. In this regard, and as used throughout the disclosure, the sequence identity includes amino acid substitutions (i.e., the sequence identity is calculated based on the entire amino acid sequence of the variant enzyme, including the amino acid substitutions). In some cases, the variant amylosucrase comprises an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3. NO:2 An amino acid substitution at amino acid position 234 selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L and R234H, or consisting thereof. In preferred embodiments, the variant amylosucrase comprises an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 234 relative to SEQ ID NO: 2 selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C and R234K, or consisting thereof.In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234Q relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234G relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution R234A relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234S relative to SEQ ID NO: 2.In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234M relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234C relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234K relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234I relative to SEQ ID NO: 2.In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution R234D relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234Y relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234W relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234E relative to SEQ ID NO: 2.In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and the amino acid substitution R234L relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234H relative to SEQ ID NO: 2.

[0295] In some embodiments, amylosucrase is derived from microbial cells. In some cases, amylosucrase is separated and / or purified from microbial cells. In some cases, microbial cells are bacterial cells. In some cases, bacterial cells are Escherichia coli. In some embodiments, amylosucrase is derived from polysaccharide Neisseria. In some embodiments, amylosucrase is derived from carbon source Cellulomonas T26. In some embodiments, amylosucrase can be produced in microbial cells. In some embodiments, amylosucrase is expressed in recombinant host cells (for example, from recombinant polynucleotides). In some cases, amylosucrase is recombinantly produced. In some cases, amylosucrase is produced in yeast cells (for example, recombinantly produced). In some cases, yeast cells are Pichia cells, such as Pichia pastoris cells.

[0296] A dual-enzyme method for producing amylose from sucrose

[0297] In some aspects, the method involves contacting sucrose with an enzyme mixture capable of converting sucrose into amylose under conditions that allow sucrose to be converted into amylose, thereby producing amylose. In some cases, the method involves contacting sucrose with an enzyme mixture containing at least two enzymes, which together or in combination can convert sucrose into amylose. For example, the enzyme mixture can contain at least sucrose phosphorylase and alpha-glucan phosphorylase. The method can involve contacting sucrose with the at least two enzymes simultaneously or substantially simultaneously. Alternatively, the method can involve contacting sucrose with the at least two enzymes in sequence. Fig.27B Describe the schematic diagram of the double enzyme method of producing straight-chain starch by sucrose.In this example, sucrose is contacted with sucrose phosphorylase, and sucrose is converted into glucose-1-phosphate.Then glucose-1-phosphate is contacted with alpha-glucan phosphorylase, and glucose-1-phosphate is converted into straight-chain starch.In some cases, sucrose phosphorylase and alpha-glucan phosphorylase are contacted with sucrose simultaneously or substantially simultaneously.In other cases, sucrose phosphorylase and alpha-glucan phosphorylase are added successively (for example, sucrose phosphorylase is first contacted with sucrose to generate glucose-1-phosphate, and then alpha-glucan phosphorylase is added to generate straight-chain starch).In some cases, before glucose-1-phosphate is contacted with alpha-glucan phosphorylase, glucose-1-phosphate generated by the reaction of self-utilizing sucrose phosphorylase is separated and / or purified.In other cases, before glucose-1-phosphate is contacted with alpha-glucan phosphorylase, glucose-1-phosphate generated by the reaction of self-utilizing sucrose phosphorylase is not separated and / or purified. When used in the context of adding two or more components to a reaction mixture as described herein, the term "substantially simultaneously" means that the two or more components are added to the reaction mixture within 10 seconds or less of each other.

[0298] In some cases, sucrose phosphorylase is a wild-type sucrose phosphorylase. For example, a wild-type sucrose phosphorylase may be a long bifidobacterium (Bifidobacterium longum) sucrose phosphorylase (e.g., NCBI accession number AAO84039). In some cases, a wild-type long bifidobacterium sucrose phosphorylase may have an amino acid sequence according to SEQ ID NO: 17. In some cases, a wild-type sucrose phosphorylase may be a mesenteroide-like leuconostocmesenteroide sucrose phosphorylase (e.g., NCBI accession number D90314.1). In some cases, a wild-type mesenteroide-like leuconostocmesenteroide sucrose phosphorylase may have an amino acid sequence according to SEQ ID NO: 18. In some cases, a wild-type sucrose phosphorylase may be a mutans streptococcus (Streptococcus mutans) sucrose phosphorylase (e.g., NCBI accession number NZ_CP013237.1). In some cases, wild-type mutans Streptococcus sucrose phosphorylase can have according to SEQ ID NO:19 amino acid sequence (for example, NCBI accession number P10249). In some cases, sucrose phosphorylase is variant sucrose phosphorylase. In some cases, variant sucrose phosphorylase has one or more amino acid substitutions relative to wild-type sucrose phosphorylase. In some cases, variant sucrose phosphorylase has amino acid substitutions relative to SEQ ID NO:19 at one or more or all of amino acid residues T47, S62, Y77, V128, K140, Q144, N155 and D249. In some cases, relative to SEQ ID NO:19, the amino acid substitution at amino acid position 47 is T47S. In some cases, relative to SEQ ID NO:19, the amino acid substitution at amino acid position 62 is S62P. In some cases, relative to SEQ ID NO:19, the amino acid substitution at amino acid position 77 is Y77H. In some cases, the amino acid substitution at amino acid position 128 is V128L relative to SEQ ID NO: 19. In some cases, the amino acid substitution at amino acid position 140 is K140M relative to SEQ ID NO: 19. In some cases, the amino acid substitution at amino acid position 144 is Q144R relative to SEQ ID NO: 19. In some cases, the amino acid substitution at amino acid position 155 is N155S relative to SEQ ID NO: 19. In some cases, the amino acid substitution at amino acid position 249 is D249G relative to SEQ ID NO: 19. In some cases, the variant sucrose phosphorylase has amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G relative to SEQ ID NO: 19.In some cases, the variant sucrose phosphorylase comprises or consists of an amino acid sequence according to SEQ ID NO: 20. Table 3 below depicts non-limiting examples of sucrose phosphorylases (and amino acid sequences thereof) that can be used according to the methods provided herein.

[0299] Table 3. Non-limiting examples of sucrose phosphorylases

[0300]

[0301]

[0302]

[0303] In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type Bifidobacterium longum sucrose phosphorylase. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type Leuconostoc mesenteroides sucrose phosphorylase. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type mutans Streptococcus sucrose phosphorylase.In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:20, and comprises amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G relative to SEQ ID NO:19.

[0304] In some embodiments, sucrose phosphorylase is derived from microbial cells. In some cases, sucrose phosphorylase is isolated and / or purified from microbial cells. In some cases, microbial cells are bacterial cells. In some cases, bacterial cells are Escherichia coli. In some embodiments, sucrose phosphorylase is derived from Bifidobacterium longum. In some embodiments, sucrose phosphorylase is derived from Leuconostoc mesenteroides. In some embodiments, sucrose phosphorylase is derived from Streptococcus mutans. In some embodiments, sucrose phosphorylase can be produced in microbial cells. In some embodiments, sucrose phosphorylase is expressed in recombinant host cells (e.g., from recombinant polynucleotides). In some cases, sucrose phosphorylase is recombinantly produced. In some cases, sucrose phosphorylase is produced in yeast cells (e.g., recombinantly produced). In some cases, yeast cells are Pichia cells, such as Pichia pastoris cells.

[0305] In some aspects, the α-glucan phosphorylase is a wild-type α-glucan phosphorylase. In some cases, the wild-type α-glucan phosphorylase can be a potato (Solanum tuberosum) α-glucan phosphorylase (e.g., NCBI accession number D00520.1). In some cases, the wild-type potato α-glucan phosphorylase can have an amino acid sequence according to SEQ ID NO:21. In some cases, the wild-type α-glucan phosphorylase can be a S. tokodaii strain 7 α-glucan phosphorylase (e.g., NCBI accession number NC_003106.2). In some cases, the wild-type S. tokodaii strain 7 α-glucan phosphorylase can have an amino acid sequence according to SEQ ID NO:22. In some cases, wild-type α-glucan phosphorylase can be Corynebacterium callunae DSM 20145 (C.callunae DSM20145) α-glucan phosphorylase (e.g., NCBI accession number AY102616.1). In some cases, wild-type Corynebacterium callunae DSM 20145 α-glucan phosphorylase can have an amino acid sequence according to SEQ ID NO:23. In some cases, α-glucan phosphorylase is a variant α-glucan phosphorylase. In some cases, variant α-glucan phosphorylase has one or more amino acid substitutions relative to wild-type α-glucan phosphorylase. In some cases, variant α-glucan phosphorylase has amino acid substitutions relative to SEQ ID NO:21 at one or more or all of amino acid residues F39, N135 and T706. In some cases, relative to SEQ ID NO:21, the amino acid substitution at amino acid position 39 is F39L. In some cases, the amino acid substitution at amino acid position 135 is N135S relative to SEQ ID NO: 21. In some cases, the amino acid substitution at amino acid position 706 is T706I relative to SEQ ID NO: 21. In some cases, the variant α-glucan phosphorylase has amino acid substitutions F39L, N135S and T706I relative to SEQ ID NO: 21. In some cases, the variant α-glucan phosphorylase comprises or consists of an amino acid sequence according to SEQ ID NO: 24. Table 4 below depicts non-limiting examples of α-glucan phosphorylases (and amino acid sequences thereof) that can be used according to the methods provided herein.

[0306] Table 4. Non-limiting examples of α-glucan phosphorylases

[0307]

[0308]

[0309]

[0310] In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type potato α-glucan phosphorylase. In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:21. In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type Sulfolobus solanum strain 7 α-glucan phosphorylase. In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:22.In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to wild-type Corynebacterium heatherum DSM 20145 α-glucan phosphorylase. In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:23. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:24, and comprises amino acid substitutions F39L, N135S, and T706I relative to SEQ ID NO:21.

[0311] In some embodiments, α-glucan phosphorylase is derived from microbial cells. In some cases, α-glucan phosphorylase is isolated and / or purified from microbial cells. In some cases, microbial cells are bacterial cells. In some cases, bacterial cells are Escherichia coli. In some embodiments, α-glucan phosphorylase is derived from potato. In some embodiments, α-glucan phosphorylase is derived from Sulfolobus solanum strain 7. In some embodiments, α-glucan phosphorylase is derived from Corynebacterium heatherum DSM 20145. In some embodiments, α-glucan phosphorylase can be produced in microbial cells. In some embodiments, α-glucan phosphorylase is expressed in recombinant host cells (e.g., from recombinant polynucleotides). In some cases, α-glucan phosphorylase is recombinantly produced. In some cases, α-glucan phosphorylase is produced in yeast cells (e.g., recombinantly produced). In some cases, yeast cells are Pichia cells, such as Pichia pastoris cells.

[0312] Method step (b): Enzymatic conversion of amylose into β-cyclodextrin

[0313] In various aspects, the method further comprises enzymatically converting amylose (e.g., produced by the methods provided herein (e.g., method step (a))) into a cyclodextrin, preferably β-cyclodextrin. In some cases, the method comprises contacting amylose with an enzyme or enzyme mixture (e.g., two or more enzymes) capable of converting amylose into cyclodextrin under conditions that allow the conversion of amylose into cyclodextrin. In some cases, the enzyme capable of converting amylose into cyclodextrin is a variant enzyme that is capable of producing a greater amount and / or concentration of β-cyclodextrin than α-cyclodextrin, γ-cyclodextrin, or both, relative to a wild-type enzyme capable of converting amylose into cyclodextrin.

[0314] In some aspects, the enzyme capable of converting amylose to cyclodextrin comprises a variant cyclodextrin glucanotransferase. In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type cyclodextrin glucanotransferase. Fig.28 Depicted is the enzymatic conversion of amylose to β-cyclodextrin using a cyclodextrin glucanotransferase. Preferably, the cyclodextrin glucanotransferase produces β-cyclodextrin from amylose in an amount and / or concentration greater than that of α-cyclodextrin and / or γ-cyclodextrin.

[0315] In some embodiments, cyclodextrin glucanotransferase is a variant cyclodextrin glucanotransferase, which comprises at least one amino acid variant relative to wild-type cyclodextrin glucanotransferase. Relative to wild-type cyclodextrin glucanotransferase, variant cyclodextrin glucanotransferase may comprise one or more amino acid substitutions, deletions, insertions and / or modifications. In some cases, relative to wild-type cyclodextrin glucanotransferase, variant cyclodextrin glucanotransferase is capable of producing greater amounts and / or concentrations of β-cyclodextrin (relative to α-cyclodextrin and / or γ-cyclodextrin) by amylose.

[0316] In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type Bacillus (strain number 38-2) cyclodextrin glucanotransferase (e.g., NCBI accession number M19880.1; SEQ ID NO: 25). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type Bacillus circulans strain 251 cyclodextrin glucanotransferase (e.g., NCBI accession number X78145.1; SEQ ID NO: 26 or 27). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type Bacillus circulans strain 251 cyclodextrin glucanotransferase of SEQ ID NO: 27. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:25. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or 27. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 27.

[0317] In some cases, the at least one amino acid variant comprises at least one amino acid substitution relative to a wild-type cyclodextrin glucanotransferase. In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 31 relative to the amino acid sequence of SEQ ID NO: 27. In some cases, relative to the amino acid sequence of SEQ ID NO: 27, the amino acid substitution at amino acid position 31 is A31R (e.g., SEQ ID NO: 28 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO: 27, the amino acid substitution at amino acid position 31 is A31P (e.g., SEQ ID NO: 29 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO: 27, the amino acid substitution at amino acid position 31 is A31T (e.g., SEQ ID NO: 30 in Table 5). In some aspects, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to any one of SEQ ID NO: 25-30 as depicted in Table 5.

[0318] In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type Paenibacillus macerans cyclodextrin glucanotransferase (e.g., NCBI Accession No. AAA22298.1 or X59045.1; e.g., SEQ ID NOs: 31-34). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to any one of SEQ ID NOs: 31-34. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of wild-type Paenibacillus macerans cyclodextrin glucanotransferase. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31-34.

[0319] In some cases, the at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type cyclodextrin glucanotransferase. In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 34. In some cases, relative to the amino acid sequence of SEQ ID NO: 34, the amino acid substitution at amino acid position 146 is R146A (e.g., SEQ ID NO: 35 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO: 34, the amino acid substitution at amino acid position 146 is R146P (e.g., SEQ ID NO: 36 in Table 5). In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 34. In some cases, relative to the amino acid sequence of SEQ ID NO: 34, the amino acid substitution at amino acid position 147 is D147A (e.g., SEQ ID NO: 37 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO: 34, the amino acid substitution at amino acid position 147 is D147P (e.g., SEQ ID NO: 38 in Table 5). In some cases, at least one amino acid substitution comprises amino acid substitutions at amino acid positions 146 and 147 relative to the amino acid sequence of SEQ ID NO: 34. In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 34 is R146A, and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147P (e.g., SEQ ID NO: 39 in Table 5). In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 34 is R146P, and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147A (e.g., SEQ ID NO: 40 in Table 5). In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 34 is R146P, and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147P (e.g., SEQ ID NO: 41 in Table 5).

[0320] In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34 is D372K (e.g., SEQ ID NO: 42 (relative to SEQ ID NO: 32) and SEQ ID NO: 45 (relative to SEQ ID NO: 34) in Table 5). In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34 is Y89R (e.g., SEQ ID NO: 43 (relative to SEQ ID NO: 32) and SEQ ID NO: 47 (relative to SEQ ID NO: 34) in Table 5). In some cases, the at least one amino acid substitution includes an amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34, and an amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 32 or 34 is D372K, and the amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 32 or 34 is Y89R (e.g., SEQ ID NO: 44 (relative to SEQ ID NO: 32) and SEQ ID NO: 47 (relative to SEQ ID NO: 34) in Table 5).

[0321] In some aspects, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 31-47 as depicted in Table 5. In some aspects, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence of any one of SEQ ID NOs: 31-47 as depicted in Table 5.

[0322] In specific aspects, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 34, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 34.

[0323] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 39, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 39.

[0324] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 40, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 40.

[0325] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 41, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 41.

[0326] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 47, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 47.

[0327] Table 5. Non-limiting examples of cyclodextrin glucanotransferases

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339] In some aspects, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 25, preferably at least about 90% sequence identity. In some aspects, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or 27.

[0340] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 27, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 31 relative to SEQ ID NO: 27. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 27, preferably at least about 90% sequence identity, and an amino acid substitution A31R relative to SEQ ID NO: 27. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 27, preferably at least about 90% sequence identity and an amino acid substitution A31P relative to SEQ ID NO: 27. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 27, preferably at least about 90% sequence identity, and an amino acid substitution A31T relative to SEQ ID NO: 27.

[0341] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 34. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, and the amino acid substitution R146A relative to SEQ ID NO: 34. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, and the amino acid substitution R146P relative to SEQ ID NO: 34.

[0342] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 34. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity and amino acid substitution D147P relative to SEQ ID NO: 34. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, and amino acid substitution D147A relative to SEQ ID NO: 34.

[0343] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 34, and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 34. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, an amino acid substitution R146A relative to SEQ ID NO: 34, and an amino acid substitution D147P relative to SEQ ID NO: 34. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, an amino acid substitution R146P relative to SEQ ID NO: 34, and an amino acid substitution D147A relative to SEQ ID NO: 34.In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, an amino acid substitution R146P relative to SEQ ID NO: 34, and an amino acid substitution D147P relative to SEQ ID NO: 34.

[0344] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 32 or 34, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 32 or 34. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 32 or 34, preferably at least about 90% sequence identity and an amino acid substitution D372K relative to SEQ ID NO: 32 or 34.

[0345] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 32 or 34, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 32 or 34. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 32 or 34, preferably at least about 90% sequence identity and an amino acid substitution Y89R relative to SEQ ID NO: 32 or 34.

[0346] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 32 or 34, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 32 or 34, and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 32 or 34. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 32 or 34, preferably at least about 90% sequence identity, an amino acid substitution D372K relative to SEQ ID NO: 32 or 34, and an amino acid substitution Y89R relative to SEQ ID NO: 32 or 34.

[0347] In some embodiments, cyclodextrin glucanotransferase is derived from microbial cells. In some cases, cyclodextrin glucanotransferase is separated and / or purified from microbial cells. In some cases, microbial cells are bacterial cells. In some cases, bacterial cells are Escherichia coli. In some embodiments, cyclodextrin glucanotransferase is derived from Bacillus (strain number 38-2). In some embodiments, cyclodextrin glucanotransferase is derived from Bacillus circulans strain 251. In some embodiments, cyclodextrin glucanotransferase can be produced in microbial cells. In some embodiments, cyclodextrin glucanotransferase is expressed in recombinant host cells (for example, from recombinant polynucleotides). In some cases, cyclodextrin glucanotransferase is recombinantly produced. In some cases, cyclodextrin glucanotransferase is produced in yeast cells (for example, recombinantly produced). In some cases, yeast cells are Pichia cells, such as Pichia pastoris cells.

[0348] In various aspects, the methods provided herein produce a higher ratio of beta-cyclodextrin to alpha-cyclodextrin, gamma-cyclodextrin or both. For example, in some cases, the ratio of beta-cyclodextrin to alpha-cyclodextrin, gamma-cyclodextrin or both provided by the methods provided herein is at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 20: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, at least 100: 1 or greater. In a preferred embodiment, the ratio of beta-cyclodextrin to alpha-cyclodextrin provided by the methods provided herein is at least 10: 1. For example, the ratio can be at least 20: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, at least 100: 1 or greater. In preferred embodiments, the methods provided herein provide a ratio of beta-cyclodextrin to gamma-cyclodextrin of at least 5: 1. For example, the ratio can be at least 10: 1, at least 20: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, at least 100: 1 or greater. In preferred embodiments, the methods provided herein provide a ratio of beta-cyclodextrin to alpha-cyclodextrin and gamma-cyclodextrin of at least 3.5: 1. For example, the ratio can be at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, or more.

[0349] This disclosure outlines throughout the text a method for obtaining robust enzyme activity in each step to obtain a higher yield of β-cyclodextrin than is currently possible. In some embodiments, a first enzymatic step of converting sucrose to amylose (e.g., as described herein) is performed for a first time period, thereby enabling catalytic conversion of sucrose to amylose, and then a second enzymatic step of converting amylose to β-cyclodextrin (e.g., as described herein) is performed for a second time period, thereby enabling catalytic conversion of amylose to β-cyclodextrin. In some embodiments, the first enzymatic reaction (e.g., converting sucrose to amylose, e.g., as described herein) and the second enzymatic reaction (e.g., converting amylose to β-cyclodextrin, e.g., as described herein) are performed in the same container (e.g., a one-pot synthesis).

[0350] In some embodiments, the first time period is at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 85 minutes, at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 135 minutes, at least 150 minutes, at least 165 minutes, at least 180 minutes, at least 195 minutes, at least 210 minutes, at least 225 minutes, at least 240 minutes, at least 255 minutes, at least 270 minutes, at least 285 minutes or at least 300 minutes. In some embodiments, the second time period is at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 85 minutes, at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 135 minutes, at least 150 minutes, at least 165 minutes, at least 180 minutes, at least 195 minutes, at least 210 minutes, at least 225 minutes, at least 240 minutes, at least 255 minutes, at least 270 minutes, at least 285 minutes or at least 300 minutes. In some embodiments, the first time period is shorter than the second time period. In some embodiments, the first time period is longer than the second time period. In some embodiments, the length of the first time period is the same or substantially the same as the second time period. In some embodiments, sucrose is added to the reaction vessel in batches. In some embodiments, the enzyme used in the first enzymatic reaction step (e.g., sucrose is converted into amylose, for example, as described herein) is added once at the beginning of the reaction period, and then added again after a period of time has passed to accelerate the catalytic activity. In some embodiments, sucrose is added once at the beginning of the reaction period, and then added again after a period of time has passed to supplement sucrose. In some embodiments, the enzyme participating in the first enzymatic reaction step (e.g., sucrose is converted into amylose, for example, as described herein) and the enzyme participating in the second enzymatic reaction step (e.g., amylose is converted into beta-cyclodextrin) are added simultaneously in the same reaction vessel. In some embodiments, the enzyme participating in the first enzymatic reaction step (e.g., sucrose is converted into amylose, for example, as described herein) and the enzyme participating in the second enzymatic reaction step (e.g., amylose is converted into beta-cyclodextrin) are added at different times (e.g., the former before the latter).

[0351] In some embodiments, the sucrose concentration is maximized to achieve efficient conversion to amylose. In some embodiments, the initial concentration of sucrose in the reaction is at least about 50 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 100 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 150 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 200 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 250 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 300 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 350 g / L.

[0352] In some embodiments, reaction time is an important consideration for obtaining the maximum yield of β-cyclodextrin. In some embodiments, the production of β-cyclodextrin may be accompanied by the decomposition of the product into glucose, maltose and other sugars. Therefore, it is very important to obtain it without allowing β-cyclodextrin to decompose. In some embodiments, the total reaction (e.g., method steps (a) and method steps (b)) is carried out for no more than 12 hours. In some embodiments, the total reaction (e.g., method steps (a) and method steps (b)) is carried out for no more than 8 hours. In some embodiments, the total reaction is carried out for no more than 7 hours. In some embodiments, the total reaction is carried out for no more than 6 hours. In some embodiments, the total reaction is carried out for no more than 5 hours. In some embodiments, the total reaction is carried out for no more than 4 hours. In some embodiments, the total reaction is carried out for no more than 3 hours. In some embodiments, the total reaction is carried out for no more than 2 hours. In some embodiments, the total reaction is carried out for no more than 1 hour.

[0353] Temperature is an important consideration for maximizing the yield of β-cyclodextrin. In some embodiments, one or more enzymatic reactions are carried out at about 30°C to about 55°C, for example, about 40°C to about 50°C. In some embodiments, one or more enzymatic reactions are carried out at about 40°C. In some embodiments, one or more enzymatic reactions are carried out at about 41°C. In some embodiments, one or more enzymatic reactions are carried out at about 42°C. In some embodiments, one or more enzymatic reactions are carried out at about 43°C. In some embodiments, one or more enzymatic reactions are carried out at about 44°C. In some embodiments, one or more enzymatic reactions are carried out at about 45°C. In some embodiments, one or more enzymatic reactions are carried out at about 46°C. In some embodiments, one or more enzymatic reactions are carried out at about 47°C. In some embodiments, one or more enzymatic reactions are carried out at about 48°C. In some embodiments, one or more enzymatic reactions are carried out at about 49°C. In some embodiments, one or more enzymatic reactions are carried out at about 50°C. Preferably, one or more reactions are carried out at about 45°C.

[0354] In some embodiments, the enzymatic reaction of step (a) is carried out at about 40°C to about 55°C, for example, about 45°C to about 50°C. In some embodiments, the enzymatic reaction of step (b) is carried out at about 40°C to about 50°C. Step (a) and step (b) can be carried out at different temperatures, or preferably, step (a) and step (b) are carried out at about the same temperature. When step (a) involves the use of a single enzyme (e.g., amylosucrase), the enzymatic reaction of step (a) is preferably carried out at about 45°C. In this embodiment, the enzymatic reaction of step (b) is preferably also carried out at about 45°C. When step (a) involves the use of at least two enzymes (e.g., sucrose phosphorylase and α-glucan phosphorylase), the enzymatic reaction of step (a) is preferably carried out at about 45°C or about 50°C. In this embodiment, the enzymatic reaction of step (b) is preferably also carried out at about 45°C or about 50°C, respectively.

[0355] In a one-pot synthesis, the enzyme mixture should function maximally, although the optimal temperature for each enzyme may be slightly different.

[0356] In some embodiments, the reaction is carried out in a vessel having a vessel volume of about 1 mL to about 1,000,000 L. For example, the reaction can be carried out in a vessel having a vessel volume of about 100 mL to about 10 L, such as a vessel volume of about 500 mL or about 10 L.

[0357] In some embodiments, the total reaction volume is from about 1 mL to about 1,000,000 L. For example, the total reaction volume can be from about 100 mL to about 10 L, for example, the total reaction volume is about 500 mL or about 5 L. In some embodiments, the total reaction volume is less than the container volume. For example, a reaction carried out in a container having a container volume of about 10 L can use a total reaction volume of about 5 L.

[0358] In some embodiments, the reaction is carried out in a stirred tank reactor (STR), a loop reactor, a plug flow reactor, a single or multi-stage continuous stirred tank reactor, or any other suitable reactor known in the art. In some embodiments, the reaction is carried out in a stirred tank reactor, wherein the reaction is stirred at about 100 to about 200 rpm, such as about 160 rpm.

[0359] The pH of the reaction mixture may be an important consideration for maximizing the yield of β-cyclodextrin. In some embodiments, one or more enzymatic reactions are carried out at a pH of about 6 to about 8, for example, the pH may be about 6.5 to about 7.5. In a preferred embodiment, one or more enzymatic reactions are carried out at a pH of about 7.0 to about 7.5. Preferably, step (a) is carried out at a pH of about 7.0 to about 7.5. Preferably, step (b) is carried out at a pH of about 7.0 to about 7.5. Step (a) and step (b) may be carried out at different pHs, but preferably step (a) and step (b) are carried out at the same pH.

[0360] In some embodiments, one or more enzymatic reactions are carried out in a reaction mixture comprising a buffer. Any suitable buffer known in the art can be used. For example, the buffer can be selected from the group consisting of sodium citrate, disodium hydrogen phosphate and Tris-HCl. The concentration of the buffer in the reaction mixture can be from about 50mM to about 200mM, for example, about 100mM.

[0361] In some embodiments, one or more enzymatic reactions are carried out in a reaction mixture comprising an organic solvent (preferably toluene). The reaction mixture preferably also comprises water. Without wishing to be bound by any theory described herein, the inventors have found that the addition of an organic solvent can unexpectedly increase the yield of β-cyclodextrin obtained by the enzymatic reaction. For example, compared with the yield obtained by the enzymatic reaction without using an organic solvent, the addition of an organic solvent can increase the yield of β-cyclodextrin by at least about 5%, for example, at least about 10%, for example, at least about 15%, for example, at least about 20%, for example, at least about 50%, for example, at least about 100%, for example, at least about 150%, for example, at least about 200%, for example, at least about 250%, for example, at least about 300%, for example, at least about 350%, for example, at least about 400%. It is believed that the addition of an organic solvent can reduce the solubility of β-cyclodextrin in the reaction mixture, thereby causing β-cyclodextrin precipitation, which reduces the concentration of β-cyclodextrin in the reaction mixture, thereby increasing the yield of β-cyclodextrin. This can prevent the enzyme from decomposing β-cyclodextrin.

[0362] In some embodiments, the amount of organic solvent (preferably toluene) in the reaction mixture is about 0.1% to about 40% v / v of the reaction mixture, such as about 1% to about 35% v / v, such as about 5% to about 25% v / v.

[0363] In some embodiments, an organic solvent is introduced at the beginning or during the enzymatic reaction of step (a). In some preferred embodiments, an organic solvent is introduced at the beginning or during the enzymatic reaction of step (b). For example, in embodiments where the total reaction (e.g., method step (a) and method step (b)) is performed for no more than 8 hours, an organic solvent may be introduced about 1 hour after the enzymatic reaction (b) begins.

[0364] In some embodiments, the enzyme used in step (a) is amylosucrase. In some embodiments, the initial concentration of amylosucrase in the reaction mixture is about 1 to about 30 U / mL, such as about 5 to about 25 U / mL, such as about 8 to about 25 U / mL.

[0365] In some embodiments, the enzyme mixture used in step (a) comprises sucrose phosphorylase and α-glucan phosphorylase. In some embodiments, the starting concentration of sucrose phosphorylase in the reaction mixture is about 1 to about 30 U / mL, such as about 5 to about 25 U / mL, such as about 8 to about 25 U / mL. In some embodiments, the starting concentration of α-glucan phosphorylase in the reaction mixture is about 1 to about 30 U / mL, such as about 5 to about 25 U / mL, such as about 8 to about 25 U / mL.

[0366] In some embodiments, the enzyme is provided in the form of a whole cell lysate, preferably wherein the ratio of the starting concentration of the enzyme in step (b) (measured by volume of the whole cell lysate) to the enzyme in step (a) is from about 1:1 to about 50:1, such as from about 2:1 to about 50:1, such as from about 5:1 to about 40:1, such as from about 10:1 to about 30: 1. In a preferred embodiment, the ratio is about 20:1.

[0367] In certain embodiments, any enzymatic reaction provided herein (e.g., a first enzymatic reaction for converting sucrose into amylose and / or a second enzymatic reaction for converting amylose into beta-cyclodextrin) can occur in a microbial host cell. In some cases, the microbial cell is a bacterial cell. In some cases, the bacterial cell is an Escherichia coli. For example, the microbial host cell can include one or more heterologous nucleic acid molecules encoding one or more enzymes provided herein. The microbial host cell can express one or more enzymes provided herein. In some cases, sucrose and / or one or more enzymatic reaction intermediates can be supplied to the microbial host cell. For example, sucrose can be supplied to the microbial host cell, and the conversion of sucrose to beta-cyclodextrin can occur in the microbial host cell.

[0368] In some embodiments, one or more enzymes used in the enzymatic reaction provided herein can be fixed on a resin. For example, the enzyme can be covalently attached to the resin. Or, the enzyme can be non-covalently attached to the resin. For example, the enzyme can be connected to the Ni resin by a His tag. For example, the enzyme of (a) can be a variant amylosucrase (for example, wherein the variant amylosucrase can include the amino acid sequence according to SEQ ID NO:3 or be composed of it) and the enzyme can be fixed on a resin. Or or additionally, the enzyme of (b) can be a variant cyclodextrin glucanotransferase (for example, wherein the variant cyclodextrin glucanotransferase can include the amino acid sequence according to SEQ ID NO:28 or be composed of it) and the enzyme can be fixed on a resin. Optionally, the enzyme of (a) or the enzyme mixture and the enzyme of (b) are fixed on the same resin.

[0369] The immobilized resin enzyme can be reused in the methods described herein. However, the inventors have found that when the immobilized resin enzyme is reused, the yield of β-cyclodextrin tends to decrease, which may be due to the leaching of the enzyme from the resin during use, resulting in a lower enzyme conversion rate. Therefore, it is necessary to improve the stability of the enzyme on the resin to prevent the leaching of the enzyme, because this will allow the immobilized resin enzyme to be reused more frequently and / or have a higher enzyme conversion rate, thereby increasing the yield of the reaction.

[0370] The present inventors have found that the stability of the enzyme can be improved by using freeze-dried enzyme, spray-dried enzyme and / or introducing additives.

[0371] In some embodiments, enzyme is provided in the form of cytoplasm or whole cell lysate. For example, the cytoplasm of the recombinant cell containing the expressed enzyme can be suspended in a buffer (e.g., sodium citrate buffer), cracked and centrifuged to provide a whole cell lysate containing the enzyme. Cell lysis methods are known in the art. For example, cells can be lysed by homogenization, chemical lysis, ultrasonic treatment, freezing / thawing, lyase, acid lysis and / or alkaline lysis. In a preferred embodiment, cells are lysed by homogenization.

[0372] In some embodiments, the cytoplasm or whole cell lysate also comprises an additive. In some embodiments, the additive is selected from the group consisting of the following: PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch and combinations thereof. In some embodiments, the additive is added in an amount of about 0.1% w / v to about 10% w / v of the cytoplasm or whole cell lysate, such as about 0.5% w / v to about 5% w / v. For example, the additive can be added with 0.5% w / v, 1.0% w / v or 5% w / v of the cytoplasm or whole cell lysate. In a preferred embodiment, the additive is mannitol, sorbitol, sucrose or a combination thereof.

[0373] In some embodiments, the cell paste or cell lysate can be freeze-dried. For example, the cell paste or cell lysate can be freeze-dried within two days. Freeze-drying methods are known in the art.

[0374] The inventors have found that adding additives to a cell paste or whole cell lysate (as described above) improves the stability of the enzyme compared to a cell paste or whole cell lysate without the additive, and that freeze drying a cell paste or whole cell lysate (as described above) improves the stability of the enzyme compared to a cell paste or whole cell lysate that has not been freeze dried. Prior to use in the methods described herein, the cell paste or cell lysate may be resuspended and shaken to redissolve.

[0375] In some embodiments, the methods described herein produce compositions comprising at least 18 g / L of β-cyclodextrin. In some embodiments, the methods produce compositions comprising at least 25 g / L of β-cyclodextrin, at least 30 g / L of β-cyclodextrin, at least 40 g / L of β-cyclodextrin, at least 50 g / L of β-cyclodextrin, or at least 60 g / L of β-cyclodextrin. In preferred embodiments, the methods described herein produce compositions comprising at least 50 g / L of β-cyclodextrin.

[0376] In some embodiments, the percent yield of β-cyclodextrin is at least about 10%, such as at least about 20%, such as at least about 30%, such as at least about 40%, or such as at least about 50%, such as at least about 60%, wherein the percent yield is calculated by dividing the total amount of β-cyclodextrin produced in the methods described herein by the maximum theoretical amount of β-cyclodextrin that can be produced from the starting sucrose reagent.

[0377] Also provided herein is a composition comprising cyclodextrin, wherein the cyclodextrin comprises β-cyclodextrin and may optionally further comprise α-cyclodextrin, γ-cyclodextrin or any combination thereof, and wherein the composition comprising cyclodextrin comprises an amount and / or concentration of β-cyclodextrin greater than α-cyclodextrin, γ-cyclodextrin or both. Preferably, the composition is obtained by the method provided herein. In some cases, the composition does not comprise α-cyclodextrin and / or γ-cyclodextrin. Preferably, the composition comprises a ratio of β-cyclodextrin to α-cyclodextrin, a ratio of β-cyclodextrin to γ-cyclodextrin, or a ratio of β-cyclodextrin and β-cyclodextrin to α-cyclodextrin of at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1,...

Claims

1. A hydroxypropyl-β-cyclodextrin (HPBCD) reactor system, comprising: (a) propylene oxide feed; (b) β-cyclodextrin feed; (c) mass flow meters or mass flow controllers; as well as (d) Static mixer.

2. The reactor system of claim 1, wherein the propylene oxide feed is pressurized.

3. The reactor system of claim 1 or claim 2, wherein the β-cyclodextrin feed is pressurized.

4. The reactor system of any one of the preceding claims, comprising at least two propylene oxide feeds.

5. The reactor system of claim 4, wherein the at least two propylene oxide feeds are operably connected to separate mass flow meters or mass flow controllers.

6. The reactor system of any one of the preceding claims, further comprising a back pressure regulator.

7. The reactor system of any one of the preceding claims, further comprising a mass flow controller.

8. The reactor system of any one of the preceding claims, further comprising a temperature controller.

9. The reactor system of any one of the preceding claims, wherein the β-cyclodextrin feed comprises NaOH.

10. The reactor system of any one of the preceding claims, wherein the static mixer is a helical static mixer.

11. The reactor system of any one of the preceding claims, wherein one or more of the feeds are operably connected to a syringe pump.

12. The reactor system of any one of the preceding claims, further comprising a coil.

13. The reactor system of any one of the preceding claims, further comprising a plug flow reactor.

14. The reactor system of claim 13, wherein the plug flow reactor comprises at least two coils and a temperature control unit.

15. The reactor system as claimed in any one of the preceding claims, wherein propylene oxide is metered in at least two locations.

16. The reactor system of claim 13, wherein at least one dose of propylene oxide is metered prior to the plug flow reactor.

17. The reactor system of claim 14, wherein at least one dose of propylene oxide is metered in before the first coil and at least another dose of propylene oxide is metered in before the second coil.

18. The reactor system of claim 6, wherein the back pressure regulator is operably connected to a plug flow reactor or a coil.

19. The reactor system of any one of the preceding claims, further comprising a collection tank.

20. The reactor system of claim 19, wherein the collection tank is operably connected to an acid feed.

21. The reactor system of claim 8, wherein the temperature control unit maintains a temperature of about 30°C to about 60°C.

22. The reactor system of claim 19, wherein the system provides a total residence time of about 30 minutes to about 70 minutes.

23. The reactor system of any of the preceding claims, wherein the first propylene oxide feed provides a concentration of about 7 to about 15 equivalents and the second propylene oxide feed provides a concentration of about 3.5 to about 15 equivalents.

24. The reactor system of claim 9, wherein the β-cyclodextrin feed comprises NaOH at a concentration of about 5 to about 10 equivalents.

25. The reactor system of claim 20, wherein the acid feed comprises hydrochloric acid, sulfuric acid, lactic acid, acetic acid, formic acid, citric acid, oxalic acid, uric acid, malic acid, fumaric acid, tartaric acid, or combinations thereof.

26. The reactor system of any one of the preceding claims, further comprising a purification process comprising a vessel for contacting the crude HPBCD mixture with activated carbon.

27. The reactor system of claim 26, wherein the purification process further comprises a sterile filter.

28. A method for producing a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, comprising: (a) contacting a hydroxypropyl-β-cyclodextrin (HPBCD) mixture with at least two solvents, wherein the HPBCD mixture comprises a high-substituted HPBCD and a low-substituted HPBCD; (b) dissolving the high-substituted HPBCD in one of the solvents; and (c) removing the low-substituted HPBCD by precipitation.

29. A method for producing a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, comprising: (a) contacting a hydroxypropyl-β-cyclodextrin (HPBCD) mixture with at least two solvents, wherein the HPBCD mixture comprises a high degree of substitution HPBCD; (b) dissolving the high-substituted HPBCD in one of the solvents to form a mother solution; (c) filtering off the mother liquor.

30. The method of claim 29, further comprising lyophilizing the mother solution to produce a solid.

31. The method of claim 30, further comprising analyzing the solid by MALDI-TOF to determine the degree of substitution.

32. The method of any one of claims 28-31, wherein the at least two solvents comprise ethanol and acetone.

33. A composition comprising a mixture of methylated 2-hydroxypropyl-β-cyclodextrins (HPBCD) having a degree of substitution of from about 6.5 to about 9.5 and methylated glucose having from 0 to 5 2-hydroxypropyl groups.

34. The composition of claim 33, wherein the composition has a mass spectrum as depicted in Figure 25.

35. A method for oligomeric substitution by methanolysis of a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, the method comprising: (a) Mixing HPBCD and methanol; (b) stirring until the HPBCD is dissolved; (c) adding an acid to the mixture; (d) heating the mixture to a temperature of at least 50° C. to about 90° C.; (e) stirring the mixture and maintaining heating for at least about 24 hours; (f) neutralizing the mixture with a base; and (g) filtering the mixture.

36. A method for purifying a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, comprising: (a) Purification of HPBCD mixture by nanofiltration; (b) collecting a total of at least 5 diafiltration volumes of nanofiltration permeate; as well as (c) lyophilizing the resulting retentate to produce solid hydroxypropyl-β-cyclodextrin.

37. The method of claim 36, wherein the purifying occurs at a feed pressure of about 200 psi to about 400 psi.

38. The method of claim 36 or claim 37, wherein the purification by nanofiltration comprises a flat sheet membrane.

39. The method of claim 38, wherein the flat sheet membrane comprises 0.010 to 0.050 m 2 area.

40. The method of any one of claims 36-39, comprising collecting a total of at least 7 diafiltration volumes of nanofiltration permeate.

41. The method of any one of claims 36-40, comprising collecting a total of at least 10 diafiltration volumes of nanofiltration permeate.

42. A method for purifying a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, comprising: (a) Purification of HPBCD mixture by nanofiltration; (b) collecting a total of at least 5 diafiltration volumes of nanofiltration permeate; as well as (c) The obtained retentate was analyzed for propylene glycol content.

43. The method of claim 42, further comprising lyophilizing the resulting retentate to produce solid hydroxypropyl-β-cyclodextrin.

44. The method of claim 42 or claim 43, further comprising: (d) contacting the HPBCD mixture with activated carbon.

45. A method for producing a hydroxypropyl-β-cyclodextrin (HPBCD) mixture, comprising: (a) contacting a first propylene oxide feed with a β-cyclodextrin feed to form a first reaction effluent, and (b) contacting a second propylene oxide feed with the first reaction effluent to form a second reaction effluent, The second reaction effluent comprises a HPBCD mixture comprising unsubstituted β-cyclodextrin molecules and β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions.

46. ​​A composition produced by the method of any one of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: The mixture comprises 0% to 0.3% of unsubstituted β-cyclodextrin ("DS-0") or 0% to 1% of β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"), wherein the composition is suitable for intrathecal, intravenous, or intracerebroventricular administration to a patient in need thereof.

47. A composition produced by the method of any one of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: The mixture comprises 0% to 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and at least 70% of the β-cyclodextrin has a DS a DS within ±1σ, where σ is the standard deviation.

48. A composition produced by the method of any one of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: The mixture contains 0% to 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and the mixture contains 1% to 10% of β-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7").

49. A composition produced by the method of any one of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: The mixture contains 0% to 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and the mixture contains no more than 25% of β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4").

50. A composition produced by the method of any one of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: The mixture contains 0% to 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and the mixture contains no more than 20% of β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5").

51. A composition produced by the method of any one of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: The mixture comprises 0% to 2.5% of β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"), wherein the composition is suitable for intrathecal, intravenous, or intracerebroventricular administration to a patient in need thereof.

52. A composition produced by the method of any one of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: The mixture contains 0% to 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and the mixture contains 5% to 25% of β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6").

53. A composition produced by the method of any one of claims 28-32 or 35-45, comprising a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein: The mixture comprises 0% to 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); and β-cyclodextrin having a glucose unit of the following structure: wherein R1, R2 and R3 are independently at each occurrence -H or -HP, wherein HP comprises one or more hydroxypropyl groups, and in the β-cyclodextrin, the total percentage of occurrences of R1 and R2 combined being HP is in the range of 85% to 95%.

Images