Modular manufacturing of pharmaceuticals

Abstract

This specification provides a modular system for producing pharmaceuticals. The modular design of the system enables not only the customization and efficient operation of the system, but also the on-demand supply of pharmaceuticals.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 351,716, filed on June 13, 2022, entitled "MODULAR MANUFACTURING FOR PHARMACEUTICALS", and the entire content thereof is incorporated herein by reference.

[0002] The present disclosure is directed to systems and methods for modular production of pharmaceutical compositions (e.g., liquid pharmaceuticals, solid pharmaceuticals, pharmaceutical formulations, and / or combinations thereof), and thus relates to the fields of pharmacy, medicine, and engineering.

Background Art

[0003] The manufacture of pharmaceuticals often requires special equipment and facilities, and often imposes high economic costs. Such facilities are often built to produce only one product.

Summary of the Invention

[0004] Disclosed herein is a modular system for producing pharmaceutical compositions. The modular system comprises a plurality of modules, the plurality of modules including one or more flow modules, one or more mixing modules, one or more heat exchange modules, and one or more reactor modules. Each of the modules is operably connected to one or more other modules, and at least two of the modules are operably connected to one or more reactors. In some embodiments, one or more of the modules in the system are interchangeable with each other. In some additional embodiments, the modular system further comprises a back - pressure regulator. In still further embodiments, the pharmaceutical composition is a liquid pharmaceutical, a solid pharmaceutical, a pharmaceutical formulation, or a combination thereof.

[0005] In some embodiments, the system further comprises a controller that communicates with at least one or more of a plurality of modules. In some aspects, the controller is electrically or wirelessly connected to at least one or more of the plurality of modules. In some additional aspects, the controller is configured to automatically adjust system parameters selected from the group consisting of temperature, pressure, flow rate, heat transfer rate, solvent content, solvent amount, filtration, or combinations thereof. In still further aspects, the controller is configured to be remotely operated.

[0006] In some embodiments, the plurality of modules are configured to be simultaneously stationary cleaned by a chemical cleaning agent. In some aspects, each module does not need to be cleaned independently.

[0007] In some embodiments, the system includes, as pharmaceutical composition outputs, substituted and / or unsubstituted cyclodextrins, beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and combinations thereof. In other embodiments, the system includes, as a plurality of pharmaceutical composition outputs, substituted and / or unsubstituted cyclodextrins, beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and combinations thereof.

[0008] In some aspects, the system is configured to simultaneously generate a plurality of pharmaceutical composition outputs. In some additional aspects, the system is configured to generate a plurality of pharmaceutical composition outputs that include mixtures of common molecules at different component amounts.

[0009] In some embodiments, the system is configured to maintain the flow rate and heat transfer rate in a plurality of modules at a predetermined level.

[0010] Furthermore, the present specification provides a modular system for producing a pharmaceutical composition, which comprises a plurality of modules. The plurality of modules includes a plurality of flow modules, a plurality of mixing modules, a plurality of heat exchange modules, and a plurality of reactor modules. Each of the modules is operably connected to one or more other modules so as to perform in-line production of the pharmaceutical composition.

[0011] Furthermore, the present specification provides a modular system for producing a pharmaceutical composition, which comprises a plurality of cases. The plurality of cases includes two or more modules selected from one or more flow modules, one or more mixing modules, one or more heat exchange modules, and one or more reactor modules. The two or more modules are stacked vertically within each case.

[0012] Furthermore, the present specification provides a modular system for producing a pharmaceutical composition, which comprises a plurality of modules. Each of the modules is operably connected to one or more other modules, and the modules are stackable.

[0013] Furthermore, the present specification provides a remotely controlled factory comprising one or more of the above-described modular systems.

Brief Description of the Drawings

[0014]

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[0015] The present specification provides a system for modular manufacturing of pharmaceuticals. Specifically, the system provided herein is useful for manufacturing liquid pharmaceuticals, solid pharmaceuticals, pharmaceutical formulations, and / or combinations thereof. The modularity of the system enables reconfiguration and optimization of the system. The system provided herein can be used at geographically remote locations and / or controlled at different physical locations. In one aspect, the invention includes a remotely controlled factory for producing pharmaceutical compositions. In this way, a skilled technician can remotely program, implement, monitor, and modify the in-line manufacture of pharmaceutical compositions without being physically present at the factory or facility. The present invention also results in cost savings due to minimal labor, waste emission limits, and geographical flexibility, reducing the cost of the supply chain. Furthermore, the present invention reduces costs by avoiding long shutdowns or service waiting times by using in-line instruments that can be easily exchanged, replaced, or rerouted. Generally, modularity enables the rapid repair and / or replacement of unit operations when the need arises. For example, if a pump is damaged and needs to be replaced, the pump module is simply replaced with another pump module. Thus, one or more modules can be interchangeable with each other. This minimizes downtime and increases the production rate. There is also the advantage that it becomes possible to produce multiple types of products in the same facility or mass-produce products in high demand. The present applicants also assert that by using the in-line instruments (e.g., one or more flow modules, mixing modules, heat exchange modules, and / or reactor modules) described in the present invention, contamination can be significantly reduced, human error can be minimized, batch variability can be reduced, and the quality of the pharmaceutical compositions produced can be improved.

[0016] Furthermore, the system can be constructed to meet specific regulatory standards and criteria defined by laws. For example, the system and the facility housing the system can be designed and constructed to operate as a cleanroom conforming to ISO-7 / Grade C cleanroom classification. Modules can similarly be constructed to operate within the scope of these standards.

[0017] The system provided herein includes a plurality of modules. Each module can be designed and constructed to perform one or more functions. For example, the module can be a flow module, a mixing module, a heat exchange module, a reactor module, a storage module, or a measurement module (including, for example, an HPLC device, a refractive index device, a pH meter device, an NMR device, an LC-MS-MS device, a MALDI-TOF MS device, a mass spectrometry device, or a combination thereof).

[0018] The system can include a controller that is in electrical or wireless communication with at least one or more of the plurality of modules. Generally, the controller can include one or more processors and a non-transitory computer-readable storage medium having instructions stored thereon that cause one or more of the processors to control one or more of the start-up, operation, or stop of any one or more of the various aspects of the system to facilitate safe and efficient operation. For example, the controller can cut off power to any of the plurality of modules if an abnormal condition is detected. The controller can also be operable to open or close valves or adjust other system parameters to ensure safe and efficient operation of the system.

[0019] The controllers of the plurality of modules may communicate with each other and execute a protocol such that the plurality of modules perform tasks simultaneously to produce a liquid pharmaceutical (note: the term "liquid pharmaceutical" as used herein may alternatively be replaced with a pharmaceutical composition, a solid pharmaceutical, or a pharmaceutical formulation). For example, the controllers of the plurality of modules may communicate with each other and execute a protocol such that a liquid pharmaceutical is sequentially mixed, heated, cooled, pressurized, purified, and / or filtered by the plurality of modules (e.g., each successive purification and / or filtration thereof improves the properties of the liquid pharmaceutical). Without being bound by theory, improvements in the properties of the liquid pharmaceutical produced by the modular manufacturing process / system may be a reduction in metal, a reduction in toxins, a reduction in average particle size, an improvement in water solubility, an improvement in cholesterol solubility, an improvement in bioavailability, an improvement in hydrophobicity, or a combination thereof.

[0020] The pharmaceutical product produced by the present invention can be a homogeneous, dissolved solution that does not contain precipitates or solid particles, and thus can be easily sterilized by filtration and the active pharmaceutical ingredient (API) can be completely or almost completely recovered. The pharmaceutical product produced by the present invention can alternatively be a suspension containing a mixture of both liquid and solid pharmaceuticals. In one aspect, the present invention can include spray drying of a liquid pharmaceutical, which is a process of converting the liquid pharmaceutical into dried solid microparticle form. Optionally, an alternative drying process or a second drying process such as fluid bed drying or vacuum drying may be used to reduce the residual solvent to a pharmaceutically acceptable level. Typically, spray drying involves contacting a highly dispersed liquid suspension or solution with a sufficient amount of hot gas to cause evaporation and drying of the droplets. The preparation to be spray dried can be any solution, coarse suspension, slurry, colloidal dispersion, or paste that can be atomized using a spray drying device. Generally, the pharmaceutical composition or formulation of the present invention can include any dosage form suitable for oral administration, particularly tablets (preferably in a form for swallowing as such (optionally film-coated), or in a form that can rapidly disintegrate (in the mouth after ingestion or with a small amount of liquid before ingestion), including directly compressed tablets and pills), chewable forms, mini tablets, dry powders, granules, capsules or sachets containing these granules, or mini tablets (micro tablets), wafers, lozenges, crystals, microparticles, etc. The form for swallowing as such may be film-coated if desired. The pharmaceutical composition of the present invention also includes powders or granules that may be appropriately compressed or consolidated into tablets.

[0021] Furthermore, the pharmaceutical products produced by the present invention may include the term pharmaceutical formulation (or formulation), which refers to a mixture of an active ingredient (preferably one, two or three) and a pharmaceutically acceptable excipient, and is in a form suitable for the preparation / manufacture of a pharmaceutical product (e.g., a pharmaceutical composition). The choice of fillers and other excipients is determined by the chemical and physical properties of the pharmaceutical, the behavior of the mixture during processing, and the properties of the final pharmaceutical form. In the present invention, the formulation may include powders or granules suitable for making tablets by consolidation or compression or direct compression. In the present invention, the term compression may be directed to any physical consolidation process that results in a solid dosage unit.

[0022] In another aspect, the controllers of the plurality of modules may communicate with each other to execute a protocol such that a liquid pharmaceutical is sequentially mixed, heated, cooled, pressurized, purified, and / or filtered by the plurality of modules arranged in series or in parallel. A single stream may include a plurality of modules arranged in series. The system may also include a plurality of streams arranged in series or in parallel. The system may be configured to produce liquid pharmaceuticals in a continuous process or in batch format. The system may also include one or more recirculation loops.

[0023] In one embodiment, the system includes a plurality of modules arranged in parallel that can communicate with each other to execute a protocol such that a liquid pharmaceutical is sequentially mixed, heated, pressurized, purified, and / or filtered, and the system provides a plurality of liquid pharmaceutical outputs operable to produce a volume of at least about 50 mL, 100 mL, 200 mL, 500 mL, 750 mL, 1 L, 2 L, or 4 L. In such a configuration, if any one of the parallel modules has a mechanical failure or produces an undesirable liquid pharmaceutical output, the problematic module or modules can be isolated, removed, or replaced without endangering the other liquid pharmaceutical outputs in parallel.

[0024] This system generally also includes a controller that can automatically adjust system parameters (e.g., temperature, pressure, flow rate(s), heat transfer rate(s), solvent content and amount(s), residence time, filter selection, number of filtrations performed, and operation and stop connectivity between multiple modules) in response to data input from the instruments included in the system. Each module includes conduits and instruments that are operable to connect to one or more of the other modules and / or the controller. Generally, each system includes at least one reactor module or multiple reactor modules.

[0025] The flow module may include conduits and valves for directing the flow of a substance to a destination (e.g., another module). Any valves and conduits known to those skilled in the art may be used. Further, although it will be understood by those skilled in the art, different valve types and conduit materials may be required based on the process conditions and substances used. The valves can be controlled via the controller. The flow module may also include instruments such as a flow sensor, temperature sensor, backpressure regulator, and pressure sensor that are operably connected to the controller.

[0026] The mixing module includes a mixer operable to mix substances into a homogeneous form. The mixer may include a static mixer (e.g., an in-line helical static mixer) or any other mixer known to those skilled in the art. The mixing module may also include a temperature sensor, flow sensor, pressure sensor, and / or level sensor that is operably connected to the controller. The controller may be operable to adjust parameters such as the mixing speed or temperature in response to data input from the system.

[0027] The heat exchange module includes a heat exchange unit for performing a heat exchange operation. The heat exchanger can be any heat exchanger known in the art, such as a shell and tube heat exchanger, a double tube heat exchanger, a tube-in-tube heat exchanger, or a plate heat exchanger. A heat exchange fluid (e.g., water or steam) may be supplied to the heat exchange module, or the ambient environment may be suitable for heat exchange. Such heat exchange fluids are known to those skilled in the art. The heat exchange module may also include a temperature sensor and / or a flow sensor operably connected to a controller. The controller may be operable to adjust parameters such as flow rate or temperature in response to data input from the system.

[0028] The reactor module includes a reactor for performing a chemical reaction. The reactor can be any reactor known in the art, such as a plug flow reactor, a continuous stirred tank reactor, a batch reactor, a semi-batch reactor, or any other reactor known in the art. In a preferred embodiment, the reactor is a plug flow reactor. The reactor module may include a temperature sensor, a pressure sensor, and / or a flow sensor operably connected to a controller.

[0029] The reactor in each reactor module can include a plug flow reactor that can include at least one coiled tube. In some embodiments, the system can include two or more reactors. In additional embodiments, the plug flow reactor can include at least two coiled tubes. The reactor can 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, such as 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 can also have a volume greater than 100 mL, greater than 250 mL, greater than 500 mL, or greater than 1000 mL.

[0030] Using the volume of the reactor and the flow rate of the reactants in the reactor module, the residence time of the reactants in the reactor can be determined. The reactants can have a residence time in the reactor of 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, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. The reactants can have a residence time in the reactor of greater than about 60 minutes, greater than about 90 minutes, greater than about 180 minutes, or greater than about 360 minutes.

[0031] The storage module includes storage tanks for storing products, reactants, and wastes that are generated during the process or supplied for the process. The storage module can include temperature sensors, flow sensors, pressure sensors, and / or level sensors operably connected to the controller.

[0032] The system of the present disclosure can include additional modules or process units. For example, the system can further include a spray dryer or a spray dryer module. In another example, the system can include a cyclone separator or a cyclone separator module. In yet another example, the system can include an extruder or an extruder module. Spray dryers, cyclone separators, and extruders are generally well known and described in the art. The system can also include and / or be connected to one or more off-the-shelf modules (e.g., Zeton modules) available to those skilled in the art.

[0033] Alternatively, the system may include a storage system separate from the modules of the present disclosure. These storage systems are operably connected to one or more modules of the system of the present disclosure.

[0034] The system may also include one or more filtration modules for purifying the pharmaceutical composition. In particular, the filtration module may include a sterilizing filter (such as a capsule filter), a Nutsche filter, a nanofilter, a tangential flow filtration system, or a combination thereof. Similar to the other components of the system described herein, the filtration module may be operable to connect to one or more of the other modules and / or a controller.

[0035] In embodiments including a nanofilter, the nanofilter may include a flat sheet membrane for achieving nanofiltration. Flat sheet membranes for nanofiltration and methods of making and procuring flat sheet membranes are widely known in the art. The flat sheet membrane may have an area of from about 0.010 m 2 to about 0.500 m 2 , from about 0.050 m 2 to about 0.100 m 2 or from about 0.010 m 2 to about 0.050 m 2 . For example, the flat sheet membrane may have an area of about 0.010 m 2 , about 0.015 m 2 , about 0.020 m 2 , about 0.025 m 2 , about 0.030 m 2 , about 0.035 m 2 , about 0.040 m 2 , about 0.045 m 2 or about 0.050 m 2 . The flat sheet membrane may have an area greater than 0.010 m 2 , greater than about 0.025 m 2 , greater than about 0.050 m 2 , greater than about 0.100 m 2 or greater than about 0.500 m 2 .

[0036] The nanofiltration permeate can be collected for a total of at least one diafiltration volume, at least two diafiltration volumes, at least three diafiltration volumes, at least four diafiltration volumes, or at least five diafiltration volumes. For example, the nanofiltration permeate can be collected for a total of at least five diafiltration volumes, at least six diafiltration volumes, at least seven diafiltration volumes, at least eight diafiltration volumes, at least nine diafiltration volumes, or at least ten diafiltration volumes. In some embodiments, the nanofiltration permeate can be collected for a diafiltration volume of more than ten times.

[0037] The propylene glycol content of the filtration residue may be analyzed. Methods for analyzing the propylene glycol content of a composition are widely known in the art and may include mass spectrometry, high performance liquid chromatography, gas chromatography, and the like.

[0038] In certain embodiments, the purification system may include an activated carbon purification module. The activated carbon purification module includes a container containing activated carbon. In some embodiments, the activated carbon can be prepared by first washing the activated carbon with purified water to remove salts. The pharmaceutical composition produced by the system may be introduced into the activated carbon purification module, and by rocking the activated carbon and the pharmaceutical composition, it may be ensured that the pharmaceutical composition is sufficiently contacted with the activated carbon so that impurities are removed from the pharmaceutical composition.

[0039] The system also includes a plurality of cases. A case as defined herein is a structure that is capable of housing one or more modules and the electronics or conduits necessary to operably connect the one or more modules to other parts of the system, such as a controller or another case. Each case includes one or more of the plurality of modules, and for example, each case may include one module, two modules, three modules, four modules, etc. Further, a case may be designed and constructed to house three modules, but at a given point in time may house only one or two modules depending on the requirements of the system. In a preferred embodiment, the cases are structured such that the modules are stacked vertically within each case.

[0040] Referring now to FIG. 3, an exemplary system of the present disclosure is operable to produce hydroxypropyl beta-cyclodextrin (HPBCD). System 100 includes at least one propylene oxide feed 102. However, it should be noted that the system may include at least two propylene oxide feeds (i.e., multiple propylene oxide feeds), at least three propylene oxide feeds, and so on. Propylene oxide feed 102 may include a tank having conduits and apparatus operable to deliver propylene oxide to system 100. Although not shown in FIG. 3, propylene oxide may be introduced into the system at one or more locations. Propylene oxide may be introduced at a flow rate of from 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 provided at one or more locations within system 100. Generally, at least one dose of propylene oxide is provided prior to reactor 118. In some embodiments, the propylene oxide feed may include a racemic mixture of propylene oxide, and in other embodiments, the propylene oxide feed may include enantiopure propylene oxide. Propylene oxide may include deuterated propylene oxide.

[0041] Propylene oxide can be provided 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 beta - cyclodextrin (BCD). For example, propylene oxide can be provided 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 propylene oxide is provided at two locations, the first propylene oxide feed may supply propylene oxide at a concentration of about 7 to about 15 molar equivalents of BCD, and the second propylene oxide feed may supply propylene oxide at a concentration of about 3.5 to about 15 molar equivalents of BCD.

[0042] System 100 includes at least one BCD feed 104. The BCD feed 104 can include a tank having conduits and apparatus operable to deliver BCD to the system 100. The BCD can be introduced at a flow rate of greater than 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.

[0043] The system may further comprise a base or sodium hydroxide (NaOH) feed 130. The base or sodium hydroxide can be supplied 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 at a concentration of about 5 to about 10 molar equivalents of BCD. In some embodiments, the BCD feed can include the base or sodium hydroxide.

[0044] The propylene oxide feed(s) 102 and / or the BCD feed(s) 104 may be pressurized. Pressurizing the feed can be beneficial when low flow rates (e.g., about 1.5 g / min) of reactants are required. 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 supplied in a pressurized tank operably connected to the feed.

[0045] The propylene oxide feed(s) 102, the BCD feed(s) 104, and / or the base feed or sodium hydroxide feed 130 may be operably connected to mass flow controllers (106a, 106b, 106c, 106d). The mass flow controllers are operable to determine and adjust the mass flow rate of propylene oxide or BCD. Mass flow controllers as well as methods for measuring and controlling mass flow rate are well known in the art. Additional mass flow controllers may be included at other locations within the system to monitor the mass flow rate of reactants and / or products.

[0046] The propylene oxide feed(s) 102, the BCD feed(s) 104, and / or the base feed or sodium hydroxide feed 130 may be operably connected to a mass flow meter. The mass flow meter is operable to determine the mass flow rate of propylene oxide or BCD. Mass flow meters and methods for measuring mass flow rate are well known in the art. Additional mass flow meters may be included at other locations within the system to monitor the mass flow rate of reactants and / or products.

[0047] One or more mass flow meters and / or mass flow controllers (106a, 106b, 106c, 106d) may be operably connected to the controller. The controller may be operable to communicate electronically or wirelessly with any of the system components. Generally, the controller may include one or more processors and a non-transitory computer-readable storage medium storing instructions for causing the one or more processors to control one or more of the start-up, operation, or stop of any one or more of various aspects of the system to facilitate safe and efficient operation. For example, the controller may cut off power to any of the system components if an abnormal condition is detected. The controller may also be operable to open or close a valve or adjust other system parameters (e.g., temperature and pressure) to ensure safe and efficient operation of the system.

[0048] System 100 may further include at least one static mixer (110a, 110b, 110c, 110d). The static mixer is operable to continuously mix the fluid flowing through the static mixer without using moving parts by inducing the flow to increase turbulence. Static mixers are well known in the art and may include plates, baffles, helical elements, or geometric grids. In an exemplary embodiment, the static mixer is a helical static mixer. System 100 may include one or more static mixers at various locations within System 100.

[0049] One or more of the feeds may be operably connected to a pump (116a, 116b, 116c, 116d, 116e). The pump may be any pump known in the art, including centrifugal pumps, positive displacement pumps, syringe pumps, etc. The pump may be operably connected to one or more of the feeds. In an exemplary embodiment, the system includes a syringe pump operably connected to a BCD feed.

[0050] In step 1 of FIG. 3, sodium hydroxide (NaOH) and beta-cyclodextrin (BCD) are pumped and mixed into static mixer 110a and then delivered to heat exchanger 132a. Mass flow controllers 106c and 106d control the flow rates of the respective components to static mixer 110a.

[0051] In step 2, propylene oxide is heated within heat exchange module 132b and split into two streams. The BCD mixture from step 1 and the propylene oxide from step 2 are mixed within static mixer 110b. In step 5 of FIG. 3, this mixture then enters plug flow reactor 118a where the BCD reacts with the propylene oxide to form hydroxypropyl beta-cyclodextrin (HPBCD).

[0052] In step 3, more propylene oxide is added to the mixture obtained from step 5. This mixture is then reacted in a second plug flow reactor 118b in step 6. Next, an acid (such as hydrochloric acid) is added to the mixture to lower the pH of the mixture. The acid can be from acid feed 126. Acid feed 126 may be operably connected to static mixer 110d. Acid feed 126 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 combinations thereof.

[0053] System 100 may further include a backpressure regulator 112. The backpressure regulator is operable to maintain a predetermined set pressure upstream of the backpressure regulator. Generally, the backpressure regulator 112 is disposed near an end of the system 100. Thus, the backpressure regulator may be operably connected to the collection tank 124. The backpressure regulator may also be operably connected to the reactor. The backpressure regulator may be operable to maintain a backpressure 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, or 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 backpressure regulator may be operable to maintain a backpressure 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.

[0054] Next, in step 7, the mixture is pumped to one of two filtration modules (136a, 136b), such as two nanofiltration units. The filtration modules (136a, 136b) may be connected in parallel as shown so that one can be used while the other is offline. Alternatively, in step 7, when the system is being purged or cleaned, the contents of the conduit may be pumped to the waste treatment system 140. Once the mixture has been filtered, the mixture is spray dried in an atomizer 142 under a dry nitrogen atmosphere provided as appropriate by a nitrogen feed 144. The spray dried HPBCD is delivered to a cyclone separator 146 operably connected as appropriate to a vacuum pump 148 and subsequently to an extruder 150. Optionally, at various times, such as in step 1, 4, or 7, the HPBCD may be recycled to the system.

[0055] As will be understood by those skilled in the art, the various pressure transmitters (PTs), temperature transmitters (TTs), valves, and other equipment shown in FIG. 3 can be omitted and removed or configured differently without affecting the overall functionality of the system.

[0056] Referring now to FIG. 4, a facility incorporating the system of the present disclosure can be constructed. The facility can be specially constructed to house the system and to perform additional unit operations. The facility can be prefabricated and deployable anywhere. Such deployability is important for meeting high or rapidly increasing demand in remote locations. Also, such deployability can be important because the system of the present disclosure can be staffed by the general public, unskilled workers, and / or individuals without specialized training (e.g., engineering, chemistry, chemical biology, pharmacology, or pharmacy). The system of the present disclosure is configured to produce liquid pharmaceuticals such that quality and / or system parameters can be controlled remotely and / or at a physical location different from the facility (e.g., more than about 5, 10, 25, 50, 100, 200, 300, 500, 1000, or 1500 miles from the facility).

[0057] The facility can be designed and manufactured to meet cleanroom standards, e.g., to conform to an ISO-7 / Class C cleanroom classification. The facility can have FDA approval and / or GMP certification. The facility may be manufactured to include an HVAC system, an electrical system, and a plumbing system that are operable for use with the system of the present disclosure. In some examples, the facility can have passages and compartments specialized for such operation and kept separate from the system of the present disclosure. The facility can also include amenities for the personnel operating the facility.

[0058] Liquid pharmaceuticals can include, but are not limited to, substituted and / or unsubstituted cyclodextrins, beta-cyclodextrin, mixtures of hydroxypropyl beta-cyclodextrin, and combinations thereof. The systems provided herein are useful for manufacturing high-purity (e.g., 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 99.5% or greater pure) liquid pharmaceuticals, including their isomers, positional isomers, and / or stereoisomers. It is understood that the liquid pharmaceuticals described herein are pharmaceutically acceptable and suitable for administration (e.g., oral, intravenous, intrathecal, topical, subcutaneous, enteral, or parenteral) to a human subject in need thereof.

[0059] In another aspect, the system comprises a feed tank that includes a motor for dissolving reagents. The reactants may be added individually or together. The reactants can be added, for example, to a mixer module or a reactor module as part of a one-pot process. In another aspect, the system further comprises a plurality of modules configured to be simultaneously sanitized in place by a chemically sanitizing agent pumped through module connections. The chemically sanitizing agent can be heated and / or can include an antibacterial agent (e.g., bleach). The in-place sanitization functionality can enable the entire system to be sanitized and prepared for the next liquid pharmaceutical production run without the need to sanitize and certify each individual module.

[0060] Further provided herein is a composition produced using one or more of the systems and / or methods provided herein, the composition comprising a mixture of beta-cyclodextrin molecules, the mixture of beta-cyclodextrin molecules comprising beta-cyclodextrin substituted with 0 hydroxypropyl groups (“DS-0”, also referred to as “unsubstituted”), beta-cyclodextrin substituted with 1 hydroxypropyl group (“DS-1”), beta-cyclodextrin substituted with 2 hydroxypropyl groups (“DS-2”), beta-cyclodextrin substituted with 3 hydroxypropyl groups (“DS-3”), beta-cyclodextrin substituted with 4 hydroxypropyl groups (“DS-4”), beta-cyclodextrin substituted with 5 hydroxypropyl groups (“DS-5”), beta-cyclodextrin substituted with 6 hydroxypropyl groups (“DS-6”), beta-cyclodextrin substituted with 7 hydroxypropyl groups (“DS-7”), beta-cyclodextrin substituted with 8 hydroxypropyl groups (“DS-8”), beta-cyclodextrin substituted with 9 hydroxypropyl groups (“DS-9”), beta-cyclodextrin substituted with 10 hydroxypropyl groups (“DS-10”), beta-cyclodextrin substituted with 11 hydroxypropyl groups (“DS-11”), beta-cyclodextrin substituted with 12 hydroxypropyl groups (“DS-12”), beta-cyclodextrin substituted with 13 hydroxypropyl groups (“DS-13”), and beta-cyclodextrin substituted with 14 hydroxypropyl groups (“DS-14”). The degree of substitution of the mixture of beta-cyclodextrin molecules can be determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).As far as this book is concerned, the number of hydroxypropyl groups per anhydroglucose unit in a mixture of beta - cyclodextrin is the "molar substitution" or "MS", and is determined according to the procedure described in the USP monograph on Hydroxypropyl Betadex (USP NF 2015) (the "USP Hydroxypropyl Betadex monograph"), which is incorporated herein by reference in its entirety. In the present disclosure, the term "average molar substitution" or "MS. a " is used synonymously with the term "MS" used in the USP monograph on Hydroxypropyl Betadex, and the term "glucose unit" is used as a synonym for the term "anhydroglucose unit" used in the USP monograph on Hydroxypropyl Betadex. Further, as far as this book is concerned, the "average number of hydroxypropyl groups per beta - cyclodextrin" is known as the "degree of average substitution", "average DS", or "DS a ", and refers to the total number of hydroxypropyl groups in a population of beta - cyclodextrin divided by the number of beta - cyclodextrin molecules. In an illustrative example, an equal - amount mixture of beta - cyclodextrin containing glucose units each substituted with 1 hydroxypropyl group and beta - cyclodextrin containing glucose units each substituted with 2 hydroxypropyl groups has a DS a = 10.5 (average of equal amounts of beta - cyclodextrin with DS = 7 and DS = 14). In another illustrative example, a mixture of 33.3% beta - cyclodextrin in which only 1 out of 7 glucose units is substituted with a hydroxypropyl group (i.e., DS = 1) and 66.7% beta - cyclodextrin containing glucose units each substituted with 1 hydroxypropyl group (i.e., DS = 7) has a DS a = 5.0. The DS ais determined by multiplying MS by 7. Further, as used in this book, "degree of substitution" or "DS" refers to the total number of hydroxypropyl groups directly or indirectly substituted on the beta-cyclodextrin molecule. For example, a beta-cyclodextrin molecule containing glucose units each substituted with one hydroxypropyl group has DS = 7. In another example, a beta-cyclodextrin molecule in which only one of the seven glucose units is substituted with a hydroxypropyl group and that hydroxypropyl group itself is substituted with another hydroxypropyl group (e.g., the occurrence of HP containing two hydroxypropyl groups is one beta-cyclodextrin) has DS = 2. As used herein, DS a is used synonymously with the term "degree of substitution" as defined in the USP monograph for hydroxypropyl beta dextrin.

[0061] In certain embodiments, the pharmaceutical composition of the present disclosure comprises, as a pharmaceutically active ingredient, a mixture of an unsubstituted beta-cyclodextrin molecule and a beta-cyclodextrin molecule substituted with hydroxypropyl groups at one or more hydroxyl positions, and the mixture has an average number of hydroxypropyl groups per beta-cyclodextrin molecule (DS a ) of about 3 to about 7.

[0062] In some embodiments, DS a is from about 3 to about 5, such as 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 is 3.2 ± 0.2, 3.3 ± 0.2, 3.4 ± 0.2, 3.5 ± 0.2, 3.6 ± 0.2, 3.7 ± 0.2, or 3.8 ± 0.2. In other embodiments, DS a is 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.

[0063] In some embodiments, DS a is from about 3.5 to about 5.5, such as from about 3.5 to about 4.5. In some embodiments, DS a is 3.8 ± 0.3, 4.0 ± 0.3, or 4.2 ± 0.3. In other embodiments, DS a is 3.7 ± 0.2, 3.8 ± 0.2, 3.9 ± 0.2, 4.0 ± 0.2, 4.1 ± 0.2, 4.2 ± 0.2, or 4.3 ± 0.2. In other embodiments, DS a is 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.

[0064] In some embodiments, DS a is from about 4 to about 6, such as 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 is 4.2 ± 0.2, 4.3 ± 0.2, 4.4 ± 0.2, 4.5 ± 0.2, 4.6 ± 0.2, 4.7 ± 0.2, or 4.8 ± 0.2. In other embodiments, DS a is 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.

[0065] In some embodiments, DS a is from about 4.5 to about 6.5, such as from about 4.5 to about 5.5. In some embodiments, DS a is 4.8 ± 0.3, 5.0 ± 0.3, or 5.2 ± 0.3. In other embodiments, DS a is 4.7 ± 0.2, 4.8 ± 0.2, 4.9 ± 0.2, 5.0 ± 0.2, 5.1 ± 0.2, 5.2 ± 0.2, or 5.3 ± 0.2. In other embodiments, DS a is 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.

[0066] In some embodiments, DS a is from about 5 to about 7, such as 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 is 5.2 ± 0.2, 5.3 ± 0.2, 5.4 ± 0.2, 5.5 ± 0.2, 5.6 ± 0.2, 5.7 ± 0.2, or 5.8 ± 0.2. In other embodiments, DS a is 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.

[0067] In some embodiments, DS a is from about 5.5 to about 6.5. In some embodiments, DS a is 5.8 ± 0.3, 6.0 ± 0.3, or 6.2 ± 0.3. In other embodiments, DS a is 5.7 ± 0.2, 5.8 ± 0.2, 5.9 ± 0.2, 6.0 ± 0.2, 6.1 ± 0.2, 6.2 ± 0.2, or 6.3 ± 0.2. In other embodiments, DS a is 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.

[0068] In some embodiments, DS a is 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 is 6.2 ± 0.2, 6.3 ± 0.2, 6.4 ± 0.2, 6.5 ± 0.2, 6.6 ± 0.2, 6.7 ± 0.2, or 6.8 ± 0.2. In other embodiments, DS a is 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.

[0069] In some embodiments, DS a is about 4.1 ± 15%, about 4.2 ± 15%, about 4.3 ± 15%, about 4.4 ± 15%, or about 4.5 ± 15%, for example about 4.1 ± 10%, about 4.2 ± 10%, about 4.3 ± 10%, about 4.4 ± 10%, or about 4.5 ± 10%, for example 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 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%, for example 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%. In certain embodiments, DS a is about 4.34 ± 10%, for example about 4.34 ± 5%.

[0070] In some embodiments, DS a is about 4.3 ± 15%, about 4.4 ± 15%, about 4.5 ± 15%, about 4.6 ± 15%, or about 4.7 ± 15%, for example about 4.3 ± 10%, about 4.4 ± 10%, about 4.5 ± 10%, about 4.6 ± 10%, or about 4.7 ± 10%, for example 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 is about 4.47 ± 10%, about 4.48 ± 10%, about 4.49 ± 10%, about 4.50 ± 10%, about 4.51 ± 10%, about 4.52 ± 10%, or about 4.53 ± 10%, for example about 4.47 ± 5%, about 4.48 ± 5%, about 4.49 ± 5%, about 4.50 ± 5%, about 4.51 ± 5%, about 4.52 ± 5%, or about 4.53 ± 5%. In certain embodiments, DS a is about 4.50 ± 10%, for example about 4.50 ± 5%.

[0071] In some embodiments, DS ais about 6.1±15%, about 6.2±15%, about 6.3±15%, about 6.4±15%, or about 6.5±15%, for example about 6.1±10%, about 6.2±10%, about 6.3±10%, about 6.4±10%, or about 6.5±10%, for example 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 is about 6.34±10%, about 6.35±10%, about 6.36±10%, about 6.37±10%, about 6.38±10%, about 6.39±10%, or about 6.40±10%, for example about 6.34±5%, about 6.35±5%, about 6.36±5%, about 6.37±5%, about 6.38±5%, about 6.39±5%, or about 6.40±5%. In certain embodiments, DS a is about 6.37±10%, for example about 6.37±5%.

[0072] In some embodiments, DS a is about 6.3±15%, about 6.4±15%, about 6.5±15%, about 6.6±15%, or about 6.7±15%, for example about 6.3±10%, about 6.4±10%, about 6.5±10%, about 6.6±10%, or about 6.7±10%, for example about 6.3±5%, about 6.4±5%, about 6.5±5%, about 6.6±5%, or about 6.7±5%. For example, in certain embodiments, DS a 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%, for example 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%. In certain embodiments, DS a is about 6.53±10%, for example about 6.53±5%.

[0073] In a mixture of unsubstituted beta-cyclodextrin molecules and beta-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions, the degree of substitution distribution can vary. For example, an equimolar mixture of beta-cyclodextrin containing glucose units each substituted with one hydroxypropyl group and beta-cyclodextrin containing glucose units each substituted with two hydroxypropyl groups has a DS a = 10.5 (average of equimolar amounts of beta-cyclodextrin with DS = 7 and DS = 14). The DS a = 10.5, but in this example, beta-cyclodextrin having DS = 10 or DS = 11 is not present in the mixture. In other cases, most of the beta-cyclodextrin in the mixture of beta-cyclodextrin has a DS a close to this value.

[0074] In some embodiments of the present disclosure, 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 beta-cyclodextrin in the mixture has a DS a within DS ± Xσ, where σ is the standard deviation and X is 1, 2, or 3. For example, 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 beta-cyclodextrin in the mixture has a DS a within DS ± 1σ. In some embodiments, at least about 70% of the beta-cyclodextrin has a DS a within DS ± 1σ. In some embodiments, at least about 90% of the beta-cyclodextrin has a DS a within DS ± 1σ. In some embodiments, at least about 95% of the beta-cyclodextrin has a DS a within DS ± 1σ.

[0075] In some embodiments, at least about 50% of the beta-cyclodextrin in the mixture, 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% has a DS a within ±2σ of the DS. In some embodiments, at least about 70% of the beta-cyclodextrin has a DS a within ±2σ of the DS. In some embodiments, at least about 90% of the beta-cyclodextrin has a DS a within ±2σ of the DS. In some embodiments, at least about 95% of the beta-cyclodextrin has a DS a within ±2σ of the DS.

[0076] In some embodiments, at least about 50% of the beta-cyclodextrin in the mixture, 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% has a DS a within ±3σ of the DS. In some embodiments, at least about 70% of the beta-cyclodextrin has a DS within ±3σ of the DSa. In some embodiments, at least about 90% of the beta-cyclodextrin has a DS a within ±3σ of the DS. In some embodiments, at least about 95% of the beta-cyclodextrin has a DS a within ±3σ of the DS.

[0077] In some embodiments, at least about 50% of the beta-cyclodextrin, 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% has a DS a within ±1 of the DS. In some embodiments, at least about 70% of the beta-cyclodextrin has a DS a within ±1 of the DS. In some embodiments, at least about 90% of the beta-cyclodextrin has a DS aIt has a DS within ±1. In some embodiments, at least about 95% of beta-cyclodextrin has a DS a It has a DS within ±1.

[0078] In some embodiments, at least about 50% of beta-cyclodextrin, 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% has a DS a It has a DS within ±0.8. In some embodiments, at least about 70% of beta-cyclodextrin has a DS a It has a DS within ±0.8. In some embodiments, at least about 90% of beta-cyclodextrin has a DS a It has a DS within ±0.8. In some embodiments, at least about 95% of beta-cyclodextrin has a DS a It has a DS within ±0.8.

[0079] In some embodiments, at least about 50% of beta-cyclodextrin, 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% has a DS a It has a DS within ±0.6. In some embodiments, at least about 70% of beta-cyclodextrin has a DS a It has a DS within ±0.6. In some embodiments, at least about 90% of beta-cyclodextrin has a DS a It has a DS within ±0.6. In some embodiments, at least about 95% of beta-cyclodextrin has a DS a It has a DS within ±0.6.

[0080] In some embodiments, at least about 50% of beta-cyclodextrin, 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% has a DS aIt has a DS within ±0.5. In some embodiments, at least about 70% of beta-cyclodextrin is the DS a It has a DS within ±0.5. In some embodiments, at least about 90% of beta-cyclodextrin is the DS a It has a DS within ±0.5. In some embodiments, at least about 95% of beta-cyclodextrin is the DS a It has a DS within ±0.5.

[0081] In some embodiments, at least about 50% of beta-cyclodextrin, 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% is the DS a It has a DS within ±0.4. In some embodiments, at least about 70% of beta-cyclodextrin is the DS a It has a DS within ±0.4. In some embodiments, at least about 90% of beta-cyclodextrin is the DS a It has a DS within ±0.4. In some embodiments, at least about 95% of beta-cyclodextrin is the DS a It has a DS within ±0.4.

[0082] In some embodiments, at least about 50% of beta-cyclodextrin, 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% is the DS a It has a DS within ±0.3. In some embodiments, at least about 70% of beta-cyclodextrin is the DS a It has a DS within ±0.3. In some embodiments, at least about 90% of beta-cyclodextrin is the DS a It has a DS within ±0.3. In some embodiments, at least about 95% of beta-cyclodextrin is the DS a It has a DS within ±0.3.

[0083] In some embodiments, at least about 50% of beta-cyclodextrin, 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% has a DS a within ±0.2 of the DS. In some embodiments, at least about 70% of beta-cyclodextrin has a DS a within ±0.2 of the DS. In some embodiments, at least about 90% of beta-cyclodextrin has a DS a within ±0.2 of the DS. In some embodiments, at least about 95% of beta-cyclodextrin has a DS a within ±0.2 of the DS.

[0084] In some embodiments, at least about 50% of beta-cyclodextrin, 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% has a DS a within ±0.1 of the DS. In some embodiments, at least about 70% of beta-cyclodextrin has a DS a within ±0.1 of the DS. In some embodiments, at least about 90% of beta-cyclodextrin has a DS a within ±0.1 of the DS. In some embodiments, at least about 95% of beta-cyclodextrin has a DS a within ±0.1 of the DS.

[0085] In some embodiments, the MS ranges from 0.40 to 0.80, such as 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.

[0086] In certain embodiments, the 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.

[0087] In certain embodiments, the MS is from about 0.571 to about 0.686 (DS a from about 4.0 to about 4.8). In some of these embodiments, the MS is in the range of about 0.58 to about 0.68. In presently preferred embodiments, the MS is in the range of 0.58 to 0.68.

[0088] 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 about 0.70 or less. In specific embodiments, the MS is about 0.69, about 0.68, about 0.67, about 0.66, or about 0.65 or less.

[0089] Further provided herein is a composition produced using one or more of the systems and / or methods provided herein, the composition comprising a mixture of beta-cyclodextrin molecules, the mixture of beta-cyclodextrin molecules comprising beta-cyclodextrin substituted with 4 hydroxypropyl groups (“DS-4”), beta-cyclodextrin substituted with 5 hydroxypropyl groups (“DS-5”), beta-cyclodextrin substituted with 6 hydroxypropyl groups (“DS-6”), beta-cyclodextrin substituted with 7 hydroxypropyl groups (“DS-7”), beta-cyclodextrin substituted with 8 hydroxypropyl groups (“DS-8”), beta-cyclodextrin substituted with 9 hydroxypropyl groups (“DS-9”), beta-cyclodextrin substituted with 10 hydroxypropyl groups (“DS-10”), beta-cyclodextrin substituted with 11 hydroxypropyl groups (“DS-11”), beta-cyclodextrin substituted with 12 hydroxypropyl groups (“DS-12”), beta-cyclodextrin substituted with 13 hydroxypropyl groups (“DS-13”), and beta-cyclodextrin substituted with 14 hydroxypropyl groups (“DS-14”). The degree of substitution of the mixture of beta-cyclodextrin molecules can be determined by MALDI-TOF-MS.

[0090] In some embodiments, the composition can have an average degree of substitution of from about 7 to 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 beta-cyclodextrin molecules is about 7.7.

[0091] In some embodiments, the mixture of beta-cyclodextrin molecules may contain less than 1% of DS-4. For example, the mixture of beta-cyclodextrin molecules may contain about 0.9% of DS-4, about 0.8% of DS-4, about 0.7% of DS-4, about 0.6% of DS-4, about 0.5% of DS-4, about 0.4% of DS-4, about 0.3% of DS-4, about 0.2% of DS-4, or about 0.1% of DS-4. In some aspects, the mixture of beta-cyclodextrin molecules may contain from less than 1% to about 0.9% of DS-4, about 0.9% to about 0.8% of DS-4, about 0.8% to about 0.7% of DS-4, about 0.7% to about 0.6% of DS-4, about 0.7% to about 0.6% of DS-4, about 0.6% to about 0.5% of DS-4, about 0.5% to about 0.4% of DS-4, about 0.4% to about 0.3% of DS-4, about 0.3% to about 0.2% of DS-4, about 0.2% to about 0.1% of DS-4, or less than 0.1% of DS-4. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from less than 1% to about 0.8% of DS-4, from less than 1% to about 0.7% of DS-4, from less than 1% to about 0.6% of DS-4, from less than 1% to about 0.5% of DS-4, from less than 1% to about 0.4% of DS-4, from less than 1% to about 0.3% of DS-4, from less than 1% to about 0.2% of DS-4, from less than 1% to about 0.1% of DS-4, from about 0.9% to about 0.1% of DS-4, from about 0.8% to about 0.1% of DS-4, from about 0.7% to about 0.1% of DS-4, from about 0.6% to about 0.1% of DS-4, from about 0.5% to about 0.1% of DS-4, from about 0.4% to about 0.1% of DS-4, or from about 0.3% to about 0.1% of DS-4. In still further aspects, the mixture of beta-cyclodextrin may contain less than 1% of DS-4, less than 0.9% of DS-4, less than 0.8% of DS-4, less than 0.7% of DS-4, less than 0.6% of DS-4, less than 0.5% of DS-4, less than 0.4% of DS-4, less than 0.3% of DS-4, less than 0.2% of DS-4, or less than 0.1% of DS-4. In still further aspects, the mixture of beta-cyclodextrin molecules may 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% of DS-4.In some embodiments, the amount of DS-4 in the mixture of beta-cyclodextrin molecules 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%.

[0092] In some embodiments, the mixture of beta-cyclodextrin molecules may contain from about 2% to about 5% of DS-5. In some aspects, the mixture of beta-cyclodextrin molecules may contain from about 2% to about 2.5% of DS-5, from about 2.5% to about 3% of DS-5, from about 3% to about 3.5% of DS-5, from about 3.5% to about 4% of DS-5, from about 4% to about 4.5% of DS-5, or from about 4.5% to about 5% of DS-5. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from about 2% to about 3% of DS-5, from about 2% to about 3.5% of DS-5, from about 2% to about 4% of DS-5, from about 2% to about 4.5% of DS-5, from about 2.5% to about 5% of DS-5, from about 3% to about 5% of DS-5, from about 3.5% to about 5% of DS-5, from about 4% to about 5% of DS-5, or from about 3% to about 4% of DS-5. In still further aspects, the mixture of beta-cyclodextrin molecules may 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% of DS-5. In some embodiments, the amount of DS-5 in the mixture of beta-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%.

[0093] In some embodiments, the mixture of beta-cyclodextrin molecules may contain from about 7% to about 13% of DS-6. In some aspects, the mixture of beta-cyclodextrin molecules may contain from about 7% to about 7.5% of DS-6, from about 7.5% to about 8% of DS-6, from about 8% to about 8.5% of DS-6, from about 8.5% to about 9% of DS-6, from about 9% to about 9.5% of DS-6, from about 9.5% to about 10% of DS-6, from about 10% to about 10.5% of DS-6, from about 10.5% to about 11% of DS-6, from about 11% to about 11.5% of DS-6, from about 11.5% to about 12% of DS-6, from about 12% to about 12.5% of DS-6, or from about 12.5% to about 13% of DS-6. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from about 7% to about 8% of DS-6, from about 7% to about 8.5% of DS-6, from about 7% to about 9% of DS-6, from about 7% to about 9.5% of DS-6, from about 7% to about 10% of DS-6, from about 7% to about 10.5% of DS-6, from about 7% to about 11% of DS-6, from about 7% to about 11.5% of DS-6, from about 7% to about 12% of DS-6, from about 7% to about 12.5% of DS-6, from about 7.5% to about 13% of DS-6, from about 8% to about 13% of DS-6, from about 8.5% to about 13% of DS-6, from about 9% to about 13% of DS-6, from about 9.5% to about 13% of DS-6, from about 10% to about 13% of DS-6, from about 10.5% to about 13% of DS-6, from about 11% to about 13% of DS-6, from about 11.5% to about 13% of DS-6, from about 12% to about 13% of DS-6, from about 8% to about 12% of DS-6, or from about 9% to about 11% of DS-6.In still further embodiments, the mixture of beta-cyclodextrin molecules can contain 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% of DS-6. In some embodiments, the amount of DS-6 in the mixture of beta-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%.

[0094] In some embodiments, the mixture of beta-cyclodextrin molecules may contain from about 21% to about 27% of DS-7. In some aspects, the mixture of beta-cyclodextrin molecules may contain from about 21% to about 21.5% of DS-7, from about 21.5% to about 22% of DS-7, from about 22% to about 22.5% of DS-7, from about 22.5% to about 23% of DS-7, from about 23% to about 23.5% of DS-7, from about 23.5% of DS-7 to about 24% of DS-7, from about 24% to about 24.5% of DS-7, from about 24.5% to about 25% of DS-7, from about 25% to about 25.5% of DS-7, from about 25.5% to about 26% of DS-7, from about 26% to about 26.5% of DS-7, or from about 26.5% to about 27% of DS-7. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from about 21% to about 22% of DS-7, from about 21% to about 22.5% of DS-7, from about 21% to about 23% of DS-7, from about 21% to about 23.5% of DS-7, from about 21% to about 24% of DS-7, from about 21% to about 24.5% of DS-7, from about 21% to about 25% of DS-7, from about 21% to about 25.5% of DS-7, from about 21% to about 26% of DS-7, from about 21% to about 26.5% of DS-7, from about 21.5% to about 27% of DS-7, from about 22% to about 27% of DS-7, from 22.5% to about 27% of DS-7, from about 23% to about 27% of DS-7, from about 23.5% to about 27% of DS-7, from about 24% to about 27% of DS-7, from about 24.5% to about 27% of DS-7, from about 25% to about 27% of DS-7, from about 25.5% to about 27% of DS-7, from about 26% to about 27% of DS-7, from about 22% to about 26% of DS-7, or from about 23% to about 25% of DS-7.In still further embodiments, the mixture of beta-cyclodextrin molecules can contain 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%, 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%.

[0095] In some embodiments, the mixture of beta-cyclodextrin molecules may contain from about 23% to about 29% DS-8. In some aspects, the mixture of beta-cyclodextrin molecules may contain from about 23% to about 23.5% DS-8, from about 23.5% to about 24% DS-8, from about 24% to about 24.5% DS-8, from about 24.5% to about 25% DS-8, from about 25% to about 25.5% DS-8, from about 25.5% to about 26% DS-8, from about 26% to about 26.5% DS-8, from about 26.5% to about 27% DS-8, from about 27% to about 27.5% DS-8, from about 27.5% to about 28% DS-8, from about 28% to about 28.5% DS-8, or from about 28.5% to about 29% DS-8. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from about 23% to about 24% DS-8, from about 23% to about 24.5% DS-8, from about 23% to about 25% DS-8, from about 23% to about 25.5% DS-8, from about 23% to about 26% DS-8, from about 23% to about 26.5% DS-8, from about 23% to about 27% DS-8, from about 23% to about 27.5% DS-8, from about 23% to about 28% DS-8, from about 23% to about 28.5% DS-8, from about 23.5% to about 29% DS-8, from about 24% to about 29% DS-8, from about 24.5% to about 29% DS-8, from about 25% to about 29% DS-8, from about 25.5% to about 29% DS-8, from about 26% to about 29% DS-8, from about 26.5% to about 29% DS-8, from about 27% to about 29% DS-8, from about 27.5% to about 29% DS-8, from about 28% to about 29% DS-8, from about 24% to about 28% DS-8, or from about 25% to about 27% DS-8.In yet a further aspect, 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%.

[0096] In some embodiments, the mixture of beta-cyclodextrin molecules may contain from about 15% to about 21% DS-9. In some aspects, the mixture of beta-cyclodextrin molecules may contain from about 15% to about 15.5% DS-9, from about 15.5% to about 16% DS-9, from about 16% to about 16.5% DS-9, from about 16.5% to about 17% DS-9, from about 17% to about 17.5% DS-9, from about 17.5% to about 18% DS-9, from about 18% to about 18.5% DS-9, from about 18.5% to about 19% DS-9, from about 19% to about 19.5% DS-9, from about 19.5% to about 20% DS-9, from about 20% to about 20.5% DS-9, or from about 20.5% to about 21% DS-9. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from about 15% to about 16% DS-9, from about 15% to about 16.5% DS-9, from about 15% to about 17% DS-9, from about 15% to about 17.5% DS-9, from about 15% to about 18% DS-9, from about 15% to about 18.5% DS-9, from about 15% to about 19% DS-9, from about 15% to about 19.5% DS-9, from about 15% to about 20% DS-9, from about 15% to about 20.5% DS-9, from about 15.5% to about 21% DS-9, from about 16% to about 21% DS-9, from about 16.5% to about 21% DS-9, from about 17% to about 21% DS-9, from about 17.5% to about 21% DS-9, from about 18% to about 21% DS-9, from about 18.5% to about 21% DS-9, from about 19% to about 21% DS-9, from about 19.5% to about 21% DS-9, from about 20% to about 21% DS-9, from about 16% to about 20% DS-9, or from about 17% to about 19% DS-9.In yet a further aspect, the mixture of beta-cyclodextrin molecules can contain 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 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% of 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%.

[0097] In some embodiments, the mixture of beta-cyclodextrin molecules may contain from about 6% to about 12% DS-10. In some aspects, the mixture of beta-cyclodextrin molecules may contain from about 6% to about 6.5% DS-10, from about 6.5% to about 7% DS-10, from about 7% to about 7.5% DS-10, from about 7.5% to about 8% DS-10, from about 8% to about 8.5% DS-10, from about 8.5% to about 9% DS-10, from about 9% to about 9.5% DS-10, from about 9.5% to about 10% DS-10, from about 10% to about 10.5% DS-10, from about 10.5% to about 11% DS-10, from about 11% to about 11.5% DS-10, or from about 11.5% to about 12% DS-10. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from about 6% to about 7% DS-10, from about 6% to about 7.5% DS-10, from about 6% to about 8% DS-10, from about 6% to about 8.5% DS-10, from about 6% to about 9% DS-10, from about 6% to about 9.5% DS-10, from about 6% to about 10% DS-10, from about 6% to about 10.5% DS-10, from about 6% to about 11% DS-10, from about 6% to about 11.5% DS-10, from about 6.5% to about 12% DS-10, from about 7% to about 12% DS-10, from about 7.5% to about 12% DS-10, from about 8% to about 12% DS-10, from about 8.5% to about 12% DS-10, from about 9% to about 12% DS-10, from about 9.5% to about 12% DS-10, from about 10% to about 12% DS-10, from about 10.5% to about 12% DS-10, from about 11% to about 12% DS-10, from about 7% to about 11% DS-10, or from about 8% to about 10% DS-10.In yet a further aspect, the mixture of beta-cyclodextrin molecules can contain 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% of DS-10. In some embodiments, the amount of DS-10 in the mixture of beta-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%.

[0098] In some embodiments, the mixture of beta-cyclodextrin molecules may contain from about 2% to about 6% of DS-11. In some aspects, the mixture of beta-cyclodextrin molecules may contain from about 2% to about 2.5% of DS-11, from about 2.5% to about 3% of DS-11, from about 3% to about 3.5% of DS-11, from about 3.5% to about 4% of DS-11, from about 4% to about 4.5% of DS-11, from about 4.5% to about 5% of DS-11, from about 5% to about 5.5% of DS-11, or from about 5.5% to about 6% of DS-11. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from about 2% to about 3% of DS-11, from about 2% to about 3.5% of DS-11, from about 2% to about 4% of DS-11, from about 2% to about 4.5% of DS-11, from about 2% to about 5% of DS-11, from about 2% to about 5.5% of DS-11, from about 2.5% to about 6% of DS-11, from about 3% to about 6% of DS-11, from about 3.5% to about 6% of DS-11, from about 4% to about 6% of DS-11, from about 4.5% to about 6% of DS-11, from about 5% to about 6% of DS-11, or from about 3% to about 5% of DS-11. In still further aspects, the mixture of beta-cyclodextrin molecules may 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%, about 5.0%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, or about 6.0% of DS-11. In some embodiments, the amount of DS-11 in the mixture of beta-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%.

[0099] In some embodiments, the mixture of beta-cyclodextrin molecules may contain from about 0.5% to about 4% DS-12. In some aspects, the mixture of beta-cyclodextrin molecules may contain from about 0.5% to about 1% DS-12, from about 1% to about 1.5% DS-12, from about 1.5% to about 2% DS-12, from about 2% to about 2.5% DS-12, from about 2.5% to about 3% DS-12, from about 3% to about 3.5% DS-12, or from about 3.5% to about 4% DS-12. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from about 0.5% to about 1.5% DS-12, from about 0.5% to about 2% DS-12, from about 0.5% to about 2.5% DS-12, from about 0.5% to about 3% DS-12, from about 0.5% to about 3.5% DS-12, from about 1% to about 4% DS-12, from about 1.5% to about 4% DS-12, from about 2% to about 4% DS-12, from about 2.5% to about 4% DS-12, from about 3% to about 4% DS-12, or from about 1% to about 3% DS-12. In still further aspects, the mixture of beta-cyclodextrin molecules may contain 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 beta-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%.

[0100] In some embodiments, the mixture of beta-cyclodextrin molecules may contain less than 1% of DS-13. For example, the mixture of beta-cyclodextrin molecules may contain about 0.9% of DS-13, about 0.8% of DS-13, about 0.7% of DS-13, about 0.6% of DS-13, about 0.5% of DS-13, about 0.4% of DS-13, about 0.3% of DS-13, about 0.2% of DS-13, or about 0.1% of DS-13. In some aspects, the mixture of beta-cyclodextrin molecules may contain from less than 1% to about 0.9% of DS-13, from about 0.9% to about 0.8% of DS-13, from about 0.8% to about 0.7% of DS-13, from about 0.7% to about 0.6% of DS-13, from about 0.7% to about 0.6% of DS-13, from about 0.6% to about 0.5% of DS-13, from about 0.5% to about 0.4% of DS-13, from about 0.4% to about 0.3% of DS-13, from about 0.3% to about 0.2% of DS-13, from about 0.2% to about 0.1% of DS-13, or less than 0.1% of DS-13. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from less than 1% to about 0.8% of DS-13, from less than 1% to about 0.7% of DS-13, from less than 1% to about 0.6% of DS-13, from less than 1% to about 0.5% of DS-13, from less than 1% to about 0.4% of DS-13, from less than 1% to about 0.3% of DS-13, from less than 1% to about 0.2% of DS-13, from less than 1% to about 0.1% of DS-13, from about 0.9% to about 0.1% of DS-13, from about 0.8% to about 0.1% of DS-13, from about 0.7% to about 0.1% of DS-13, from about 0.6% to about 0.1% of DS-13, from about 0.5% to about 0.1% of DS-13, from about 0.4% to about 0.1% of DS-13, or from about 0.3% to about 0.1% of DS-13. In still further aspects, the mixture of beta-cyclodextrin may contain less than 1% of DS-13, less than 0.9% of DS-13, less than 0.8% of DS-13, less than 0.7% of DS-13, less than 0.6% of DS-13, less than 0.5% of DS-13, less than 0.4% of DS-13, less than 0.3% of DS-13, less than 0.2% of DS-13, or less than 0.1% of DS-13. In some embodiments, the amount of DS-13 in the mixture of beta-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%.

[0101] In some embodiments, the composition may contain less than 1% of DS-14. For example, a mixture of beta-cyclodextrin molecules may contain about 0.9% of DS-14, about 0.8% of DS-14, about 0.7% of DS-14, about 0.6% of DS-14, about 0.5% of DS-14, about 0.4% of DS-14, about 0.3% of DS-14, about 0.2% of DS-14, or about 0.1% of DS-14. In some aspects, the mixture of beta-cyclodextrin molecules may contain from less than 1% to about 0.9% of DS-14, from about 0.9% to about 0.8% of DS-14, from about 0.8% to about 0.7% of DS-14, from about 0.7% to about 0.6% of DS-14, from about 0.7% to about 0.6% of DS-14, from about 0.6% to about 0.5% of DS-14, from about 0.5% to about 0.4% of DS-14, from about 0.4% to about 0.3% of DS-14, from about 0.3% to about 0.2% of DS-14, from about 0.2% to about 0.1% of DS-14, or less than 0.1% of DS-14. In some additional aspects, the mixture of beta-cyclodextrin molecules may contain from less than 1% to about 0.8% of DS-14, from less than 1% to about 0.7% of DS-14, from less than 1% to about 0.6% of DS-14, from less than 1% to about 0.5% of DS-14, from less than 1% to about 0.4% of DS-14, from less than 1% to about 0.3% of DS-14, from less than 1% to about 0.2% of DS-14, from less than 1% to about 0.1% of DS-14, from about 0.9% to about 0.1% of DS-14, from about 0.8% to about 0.1% of DS-14, from about 0.7% to about 0.1% of DS-14, from about 0.6% to about 0.1% of DS-14, from about 0.5% to about 0.1% of DS-14, from about 0.4% to about 0.1% of DS-14, or from about 0.3% to about 0.1% of DS-14. In still further aspects, the mixture of beta-cyclodextrin may optionally contain less than 1% of DS-14, less than 0.9% of DS-14, less than 0.8% of DS-14, less than 0.7% of DS-14, less than 0.6% of DS-14, less than 0.5% of DS-14, less than 0.4% of DS-14, less than 0.3% of DS-14, less than 0.2% of DS-14, or less than 0.1% of DS-4 as appropriate.In yet a further aspect, the mixture of beta-cyclodextrin molecules can 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% of DS-14. In some embodiments, the amount of DS-14 in the mixture of beta-cyclodextrin molecules can be determined by MALDI-TOF-MS. In some embodiments, DS-14 is not present in the composition.

[0102] In an exemplary embodiment, the composition comprises a mixture of beta-cyclodextrin molecules, the mixture of beta-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 the mixture of beta-cyclodextrin molecules comprises less than 1% of DS-1, DS-2, DS-3, and DS-4.

[0103] Further provided herein is a composition comprising a mixture of beta-cyclodextrin molecules produced using one or more of the systems and / or methods provided herein, wherein the beta-cyclodextrin molecules are substituted with hydroxypropyl groups at one or more hydroxyl positions, the mixture either does not contain unsubstituted beta-cyclodextrin ("DS-0") and beta-cyclodextrin substituted with one hydroxypropyl group ("DS-1") or contains less than 1% thereof, and the mixture contains 5% to 25% of beta-cyclodextrin substituted with six hydroxypropyl groups ("DS-6").

[0104] The mixture may contain less than 0.1% of DS-0 and less than 0.1% of DS-1 combined. For example, the mixture may contain no DS-0, or 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% of DS-0, and / or the mixture may contain no DS-1, or 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% of DS-1. The mixture may not contain DS-0 and / or DS-1.

[0105] The mixture may contain at least 8% of beta-cyclodextrin substituted with 6 hydroxypropyl groups (「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% of 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% of DS-6. Alternatively, the mixture may contain 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, or 8% or less of DS-6.

[0106] The amount of DS-0, DS-1, or DS-6 can be determined by the peak height of the electrospray MS spectrum.

[0107] The mixture can have an average molar substitution in the range of about 0.40 to about 0.80. For example, the mixture can have an average molar substitution of about 0.40, about 0.45, about 0.50, about 0.55, about 0.60, about 0.65, about 0.70, about 0.75, or about 0.80. The mixture can have an average degree of substitution (「DS a 」) in the range of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. For example, the mixture can have an average degree of substitution of about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, or about 7.

[0108] The composition can contain 0.01% or less of propylene glycol. For example, the composition can contain 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% or less of propylene glycol. The composition may not contain propylene glycol. The amount of propylene glycol can be measured by HPLC or gas chromatography.

[0109] The composition can contain 0.01% or less of propylene glycol. For example, the composition can contain 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% or less of 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.

[0110] The composition may contain propylene oxide at 1 ppm or less, 0.9 ppm or less, 0.8 ppm or less, 0.7 ppm or less, 0.6 ppm or less, 0.5 ppm or less, 0.4 ppm or less, 0.3 ppm or less, 0.2 ppm or less, or 0.1 ppm or less. The composition may contain propylene oxide at 0.09 ppm or less, 0.08 ppm or less, 0.07 ppm or less, 0.06 ppm or less, 0.05 ppm or less, 0.04 ppm or less, 0.03 ppm or less, 0.02 ppm or less, or 0.01 ppm or less. The composition may contain propylene oxide at 0.009 ppm or less, 0.008 ppm or less, 0.007 ppm or less, 0.006 ppm or less, 0.005 ppm or less, 0.004 ppm or less, 0.003 ppm or less, 0.002 ppm or less, or 0.001 ppm or less. The composition may not contain propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0111] The total amount of other unspecified impurities in the composition may be 0.05% or less. For example, the total amount of unspecified impurities in the composition may be 0.05%, less than 0.05%, 0.04% or less, 0.03% or less, 0.02% or less, or 0.01% or less. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0112] 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 contain a pharmaceutically acceptable diluent.

[0113] The composition can solubilize lipids in an aqueous medium. The lipids can include non-esterified or esterified cholesterol. The composition may be provided as a solution having a concentration in the solution of the composition of 20 w / v%. The composition can have an affinity for non-esterified cholesterol. The solubilization can be determined by UV spectroscopy or HPLC.

[0114] In some embodiments, about 200 mg of the composition solubilizes at least about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or at least about 10 mg of non-esterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution can solubilize about 2 mg of non-esterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0115] A mixture of beta-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions can have a solution concentration of from about 10 mg / mL to about 200 mg / mL. For example, a mixture of beta-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions can have a solution concentration of about 10 mg / mL to about 20 mg / mL, about 10 mg / mL to about 30 mg / mL, about 10 mg / mL to about 40 mg / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 60 mg / mL, about 10 mg / mL to about 70 mg / mL, about 10 mg / mL to about 80 mg / mL, about 10 mg / mL to about 90 mg / mL, about 10 mg / mL to about 100 mg / 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 20 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 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.

[0116] Further provided herein is a composition comprising a mixture of beta-cyclodextrin molecules produced using one or more of the systems and / or methods provided herein and substituted with hydroxypropyl groups at one or more hydroxyl positions, wherein the mixture does not contain or contains less than 1% of unsubstituted beta-cyclodextrin ("DS-0") and beta-cyclodextrin substituted with one hydroxypropyl group ("DS-1"), and the mixture contains 1% to 10% of beta-cyclodextrin substituted with seven hydroxypropyl groups ("DS-7").

[0117] The mixture may contain less than 0.1% of DS-0 and less than 0.1% of DS-1 combined. For example, the mixture may not contain DS-0 or 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% of DS-0, and / or the mixture may not contain DS-1 or 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% of DS-1. The mixture may not contain DS-0 and / or DS-1.

[0118] The mixture may contain from about 1% to about 10% of 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% of DS-7. The mixture may contain about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% of DS-7. Alternatively, the composition may have from about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less of DS-7.

[0119] The amount of DS-0, DS-1, or DS-7 can be determined by the peak height of the electrospray MS spectrum.

[0120] 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, about 0.45, about 0.50, about 0.55, about 0.60, about 0.65, about 0.70, about 0.75, or about 0.80. The mixture may have an average degree of substitution (''DS a '') in the range of about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. For example, the mixture may have an average degree of substitution of about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, or about 7.

[0121] The composition may contain 0.01% or less of propylene glycol. For example, the composition may contain 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% or less of propylene glycol. The composition may not contain propylene glycol. The amount of propylene glycol can be measured by HPLC or gas chromatography.

[0122] The composition may contain 0.01% or less of propylene glycol. For example, the composition may contain 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or about 0.001% or less of 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.

[0123] The composition may contain 1 ppm or less of propylene oxide, 0.9 ppm or less of propylene oxide, 0.8 ppm or less of propylene oxide, 0.7 ppm or less of propylene oxide, 0.6 ppm or less of propylene oxide, 0.5 ppm or less of propylene oxide, 0.4 ppm or less of propylene oxide, 0.3 ppm or less of propylene oxide, 0.2 ppm or less of propylene oxide, or 0.1 ppm or less of propylene oxide. The composition may not contain propylene oxide. The amount of propylene oxide can be measured by HPLC or gas chromatography.

[0124] The total amount of other unspecified impurities in the composition may be 0.05% or less. For example, the total amount of unspecified impurities in the composition may be 0.05%, less than 0.05%, 0.04% or less, 0.03% or less, 0.02% or less, or 0.01% or less. The amount of unspecified impurities can be measured by HPLC or gas chromatography.

[0125] 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.

[0126] The composition may solubilize lipids in an aqueous medium. The lipids may include non-esterified or esterified cholesterol. The composition may be provided as a solution having a concentration of 20 w / v% in the solution of the composition. The composition may have an affinity for non-esterified cholesterol. The solubilization may be determined by UV spectroscopy or HPLC.

[0127] In some embodiments, about 200 mg of the composition solubilizes at least about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or at least about 10 mg of non-esterified cholesterol in distilled water at room temperature. In one example, 1 mL of the solution can solubilize about 2 mg of non-esterified cholesterol at room temperature as measured by UV spectroscopy after about 24 hours.

[0128] A mixture of beta-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions can have a solution concentration of about 10 mg / mL to about 200 mg / mL. For example, a mixture of beta-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions can have a solution concentration of about 10 mg / mL to about 20 mg / mL, about 10 mg / mL to about 30 mg / mL, about 10 mg / mL to about 40 mg / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 60 mg / mL, about 10 mg / mL to about 70 mg / mL, about 10 mg / mL to about 80 mg / mL, about 10 mg / mL to about 90 mg / mL, about 10 mg / mL to about 100 mg / 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 20 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 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.

[0129] Method for producing beta-cyclodextrin The 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.

[0130] In some cases, a method for producing BCD, or a method for producing a composition comprising cyclodextrin, can include (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that allow for the conversion of sucrose to amylose, thereby producing amylose. In some cases, the method further includes (b) contacting the amylose with an enzyme capable of converting amylose to cyclodextrin under conditions that allow for the conversion of amylose to cyclodextrin, thereby producing a composition comprising cyclodextrin. In some cases, the enzyme capable of converting amylose to cyclodextrin is a variant enzyme that can produce a higher amount and / or concentration (e.g., weight %, mol %, or w / v) of beta-cyclodextrin compared to a wild-type enzyme capable of converting amylose to cyclodextrin, alpha-cyclodextrin, gamma-cyclodextrin, or both. In some cases, the composition comprising cyclodextrin comprises beta-cyclodextrin and can further optionally comprise alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof. In some cases, the composition comprising cyclodextrin comprises a higher amount and / or concentration (e.g., weight %, mol %, or w / v) of beta-cyclodextrin compared to alpha-cyclodextrin, gamma-cyclodextrin, or both. In some cases, the amounts and / or concentrations of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin are measured by high performance liquid chromatography (HPLC).

[0131] Method step (a) for the enzymatic conversion of sucrose to amylose The methods provided herein may include the enzymatic conversion of sucrose to amylose. In some cases, the amylose is alpha-amylose. In some embodiments, the method comprises contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that allow for the conversion of sucrose to amylose, thereby producing amylose. In one aspect, the method comprises the use of a single enzyme for converting sucrose to amylose. In an alternative aspect, the method comprises the use of an enzyme mixture (e.g., two enzymes) that collectively or in combination convert sucrose to amylose. In some cases, the sucrose is deuterated sucrose (e.g., one or more hydrogens have been replaced with deuterium). In some cases, the sucrose and / or any one or more reagents used in the synthesis reaction are deuterated.

[0132] One-Enzyme Method for Producing Amylose from Sucrose In some aspects, the enzyme is amylosucrase. FIG. 5A shows a schematic diagram of a one-enzyme method for producing amylose from sucrose. In this example, sucrose is contacted with amylosucrase, which converts sucrose to amylose. In some cases, the amylosucrase is wild-type amylosucrase. For example, the wild-type amylosucrase can be Cellulomonas carboniz T26 amylosucrase (NCBI accession number N868_11335). In some cases, the wild-type Cellulomonas carboniz T26 amylosucrase can have the amino acid sequence of SEQ ID NO: 1. In some cases, the wild-type amylosucrase can be Neisseria polysaccharea amylosucrase (NCBI accession number AJ011781). In some cases, the wild-type Neisseria polysaccharea amylosucrase can have the amino acid sequence of SEQ ID NO: 2. Table 1 below shows non-limiting examples of wild-type amylosucrase enzymes (and their amino acid sequences) that can be used in accordance with the methods provided herein.

Table 1

[0133] In some embodiments, the amylosucrase is a variant amylosucrase comprising at least one amino acid variant relative to wild-type amylosucrase. The variant amylosucrase can include one or more amino acid substitutions, deletions, insertions, and / or modifications relative to wild-type amylosucrase. Optionally, the variant amylosucrase can produce a higher amount and / or concentration of amylose from sucrose relative to wild-type amylosucrase.

[0134] In some cases, the variant amylosucrase comprises at least one amino acid variant relative to wild-type Cellulomonas carboniz T26 amylosucrase (SEQ ID NO: 1). In some cases, the variant amylosucrase comprises at least one amino acid variant relative to wild-type Neisseria polysaccharea 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 wild-type Cellulomonas carboniz 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) with the amino acid sequence of SEQ ID NO: 1, preferably at least about 90% sequence identity.In some cases, 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 wild-type Neisseria polysaccharea amylosucrase, preferably at least about 90% sequence identity. In some cases, 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 SEQ ID NO:2, preferably at least about 90% sequence identity.

[0135] In some cases, at least one amino acid variant contains at least one amino acid substitution relative to the wild-type amylosucrase. In some cases, at least one amino acid variant contains at least one amino acid substitution relative to the wild-type Cellulomonas carboniz T26 amylosucrase. In some cases, at least one amino acid variant contains at least one amino acid substitution relative to the wild-type Neisseria polysaccharea amylosucrase. In some cases, at least one amino acid substitution includes or consists of an amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2. In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L, and R234H. In a preferred embodiment, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, and R234K. In this regard, it will be understood that R234Q indicates that the arginine (R) at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is substituted with glutamine (Q), and so on. In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is R234Q (e.g., SEQ ID NO: 3 in Table 2). In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is R234G (e.g., SEQ ID NO: 4 in Table 2). In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is R234A (e.g., SEQ ID NO: 5 in Table 2). In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is R234S (e.g., SEQ ID NO: 6 in Table 2). In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is R234M (e.g., SEQ ID NO: 7 in Table 2).In some cases, the amino acid substitution at amino acid position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234C (e.g., SEQ ID NO: 8 in Table 2). In some cases, the amino acid substitution at amino acid position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234K (e.g., SEQ ID NO: 9 in Table 2). In some cases, the amino acid substitution at amino acid position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234I (e.g., SEQ ID NO: 10 in Table 2). In some cases, the amino acid substitution at amino acid position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234D (e.g., SEQ ID NO: 11 in Table 2). In some cases, the amino acid substitution at amino acid position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234Y (e.g., SEQ ID NO: 12 in Table 2). In some cases, the amino acid substitution at amino acid position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234W (e.g., SEQ ID NO: 13 in Table 2). In some cases, the amino acid substitution at amino acid position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234E (e.g., SEQ ID NO: 14 in Table 2). In some cases, the amino acid substitution at amino acid position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234L (e.g., SEQ ID NO: 15 in Table 2). In some cases, the amino acid substitution at amino acid position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234H (e.g., SEQ ID NO: 16 in Table 2). In some embodiments, the variant amylosucrase 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 with the amino acid sequence according to any one of SEQ ID NOs: 3 - 16 or 48 shown in Table 2, or with the amino acid sequence according to any one of SEQ ID NOs: 3 - 16 or 48 shown in Table 2.In a preferred embodiment, the variant amylase comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 3 to 9 or 48 shown in Table 2.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

[0136] In some embodiments, 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 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. In this regard, as used throughout this disclosure, the recited sequence identity is calculated based on the entire amino acid sequence of the variant enzyme including amino acid substitutions (i.e., the sequence identity is calculated based on the entire amino acid sequence of the variant enzyme that includes amino acid substitutions). Optionally, 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 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, R234K, R234I, R234D, R234Y, R234W, R234E, R234L, and R234H.In a preferred embodiment, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 234 of SEQ ID NO:2 selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, and R234K. Optionally, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and an amino acid substitution R234Q of SEQ ID NO:2. Optionally, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and an amino acid substitution R234G of SEQ ID NO:2.In some cases, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234A relative to SEQ ID NO:2. In some cases, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234S relative to SEQ ID NO:2. In some cases, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234M relative to SEQ ID NO:2.In some cases, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234C relative to SEQ ID NO:2. In some cases, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234K relative to SEQ ID NO:2. In some cases, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234I relative to SEQ ID NO:2.In some cases, 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 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, 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 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, 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 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, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234E relative to SEQ ID NO:2. In some cases, 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 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, 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 SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234H relative to SEQ ID NO:2.

[0137] In some embodiments, the amylosucrase is derived from microbial cells. Optionally, the amylosucrase is isolated and / or purified from microbial cells. Optionally, the microbial cells are bacterial cells. Optionally, the bacterial cells are Escherichia coli. In some embodiments, the amylosucrase is derived from Neisseria polysaccharea. In some embodiments, the amylosucrase is derived from Cellulomonas carboniz T26. In some embodiments, the amylosucrase can be produced within microbial cells. In some embodiments, the amylosucrase is expressed within recombinant host cells (e.g., from a recombinant polynucleotide). Optionally, the amylosucrase is produced recombinantly. Optionally, the amylosucrase is produced in yeast cells (e.g., produced recombinantly). Optionally, the yeast cells are Pichia yeast cells such as Pichia pastoris cells.

[0138] Two-enzyme method for producing amylose from sucrose In some embodiments, the method comprises contacting sucrose with an enzyme mixture capable of converting sucrose to amylose under conditions that allow the conversion of sucrose to amylose, thereby producing amylose. Optionally, the method comprises contacting sucrose with an enzyme mixture comprising at least two enzymes capable of collectively or combinatorially converting sucrose to amylose. For example, the enzyme mixture can comprise at least sucrose phosphorylase and alpha-glucan phosphorylase. The method can comprise contacting sucrose with at least two enzymes simultaneously or substantially simultaneously. Alternatively, the method can comprise contacting sucrose with at least two enzymes sequentially. Figure 5B shows a schematic of a two-enzyme process for producing amylose from sucrose. In this example, sucrose is contacted with sucrose phosphorylase to convert sucrose to glucose-1-phosphate. Next, glucose-1-phosphate is contacted with alpha-glucan phosphorylase to convert glucose-1-phosphate to amylose. Optionally, 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 sequentially (e.g., sucrose phosphorylase is first contacted with sucrose to produce glucose-1-phosphate, and then alpha-glucan phosphorylase is added to produce amylose). Optionally, glucose-1-phosphate produced from the reaction with sucrose phosphorylase is isolated and / or purified before contacting with alpha-glucan phosphorylase. In other cases, glucose-1-phosphate produced from the reaction with sucrose phosphorylase is not isolated and / or purified before contacting with alpha-glucan phosphorylase. As used in the context of adding two or more components to the reaction mixture described herein, the term "substantially simultaneously" means that two or more components are added to the reaction mixture within 10 seconds of each other.

[0139] In some cases, the sucrose phosphorylase is a wild-type sucrose phosphorylase. For example, the wild-type sucrose phosphorylase can be Bifidobacterium longum sucrose phosphorylase (e.g., NCBI accession number AAO84039). In some cases, the wild-type Bifidobacterium longum sucrose phosphorylase can have the amino acid sequence according to SEQ ID NO: 17. In some cases, the wild-type sucrose phosphorylase can be Leuconostoc mesenteroide sucrose phosphorylase (e.g., NCBI accession number D90314.1). In some cases, the wild-type Leuconostoc mesenteroide sucrose phosphorylase can have the amino acid sequence according to SEQ ID NO: 18. In some cases, the wild-type sucrose phosphorylase can be Streptococcus mutans sucrose phosphorylase (e.g., NCBI accession number NZ_CP013237.1). In some cases, the wild-type Streptococcus mutans sucrose phosphorylase can have the amino acid sequence according to SEQ ID NO: 19 (e.g., NCBI accession number P10249). In some cases, the sucrose phosphorylase enzyme is a variant sucrose phosphorylase enzyme. In some cases, the variant sucrose phosphorylase has one or more amino acid substitutions relative to the wild-type sucrose phosphorylase. In some cases, the variant sucrose phosphorylase has amino acid substitutions at one or more or all of amino acid residues T47, S62, Y77, V128, K140, Q144, N155, and D249 relative to SEQ ID NO: 19. In some cases, the amino acid substitution at amino acid position 47 relative to SEQ ID NO: 19 is T47S. In some cases, the amino acid substitution at amino acid position 62 relative to SEQ ID NO: 19 is S62P. In some cases, the amino acid substitution at amino acid position 77 relative to SEQ ID NO: 19 is Y77H. In some cases, the amino acid substitution at amino acid position 128 relative to SEQ ID NO: 19 is V128L. In some cases, the amino acid substitution at amino acid position 140 relative to SEQ ID NO: 19 is K140M.In some cases, the amino acid substitution at position 144 for SEQ ID NO: 19 is Q144R. In some cases, the amino acid substitution at position 155 for SEQ ID NO: 19 is N155S. In some cases, the amino acid substitution at position 249 for SEQ ID NO: 19 is D249G. In some cases, the variant sucrose phosphorylase has, relative to SEQ ID NO: 19, the amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G. In some cases, the variant sucrose phosphorylase comprises or consists of the amino acid sequence according to SEQ ID NO: 20. Table 3 below shows non-limiting examples of sucrose phosphorylase enzymes (and their amino acid sequences) that can be used according to the methods provided herein.

Table 3-1

Table 3-2

[0140] 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) with the wild-type Bifidobacterium longum sucrose phosphorylase, preferably at least about 90% sequence identity. In some cases, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., 75%, at least about 80%, at least about 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 SEQ ID NO: 17, preferably at least about 90% sequence identity. In some cases, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., 75%, at least about 80%, at least about 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 wild-type Leuconostoc mesenteroides sucrose phosphorylase, preferably at least about 90% sequence identity.In some cases, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., 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 SEQ ID NO: 18, preferably at least about 90% sequence identity. In some cases, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., 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 wild-type Streptococcus mutans sucrose phosphorylase, preferably at least about 90% sequence identity. In some cases, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., 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 SEQ ID NO: 19, preferably at least about 90% sequence identity.In some cases, sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., 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 SEQ ID NO: 20, preferably at least about 90% sequence identity, and comprises the amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G relative to SEQ ID NO: 19.

[0141] In some embodiments, sucrose phosphorylase is derived from a microbial cell. In some cases, sucrose phosphorylase is isolated and / or purified from a microbial cell. In some cases, the microbial cell is a bacterial cell. In some cases, the bacterial cell is 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 within a microbial cell. In some embodiments, sucrose phosphorylase is expressed in a recombinant host cell (e.g., from a recombinant polynucleotide). In some cases, sucrose phosphorylase is produced recombinantly. In some cases, sucrose phosphorylase is produced in a yeast cell (e.g., produced recombinantly). In some cases, the yeast cell is a Pichia yeast cell such as a Pichia pastoris cell.

[0142] In some embodiments, the alpha-glucan phosphorylase is a wild-type alpha-glucan phosphorylase. In some cases, the wild-type alpha-glucan phosphorylase can be a Solanum tuberosum alpha-glucan phosphorylase (e.g., NCBI accession number D00520.1). In some cases, the wild-type Solanum tuberosum alpha-glucan phosphorylase can have the amino acid sequence according to SEQ ID NO: 21. In some cases, the wild-type alpha-glucan phosphorylase can be an S. tokodaii strain 7 alpha-glucan phosphorylase (e.g., NCBI accession number NC_003106.2). In some cases, the wild-type S. tokodaii strain 7 alpha-glucan phosphorylase can have the amino acid sequence according to SEQ ID NO: 22. In some cases, the wild-type alpha-glucan phosphorylase can be a C. callunae DSM 20145 alpha-glucan phosphorylase (e.g., NCBI accession number AY102616.1). In some cases, the wild-type C. callunae DSM 20145 alpha-glucan phosphorylase can have the amino acid sequence according to SEQ ID NO: 23. In some cases, the alpha-glucan phosphorylase enzyme is a variant alpha-glucan phosphorylase enzyme. In some cases, the variant alpha-glucan phosphorylase has one or more amino acid substitutions relative to the wild-type alpha-glucan phosphorylase. In some cases, the variant alpha-glucan phosphorylase has an amino acid substitution in one or all of amino acid residues F39, N135, and T706 relative to SEQ ID NO: 21. In some cases, the amino acid substitution at amino acid position 39 relative to SEQ ID NO: 21 is F39L. In some cases, the amino acid substitution at amino acid position 135 relative to SEQ ID NO: 21 is N135S. In some cases, the amino acid substitution at amino acid position 706 relative to SEQ ID NO: 21 is T706I. In some cases, the variant alpha-glucan phosphorylase has the amino acid substitutions F39L, N135S, and T706I relative to SEQ ID NO: 21.In some cases, the variant alpha-glucan phosphorylase enzyme comprises or consists of the amino acid sequence according to SEQ ID NO: 24. Table 4 below shows non-limiting examples of alpha-glucan phosphorylase enzymes (and their amino acid sequences) that can be used according to the methods provided herein.

Table 4-1

Table 4-2

[0143] In some cases, the alpha - 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) with the wild - type Solanum tuberosum alpha - glucan phosphorylase, preferably at least about 90% sequence identity. In some cases, the alpha - 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) with the amino acid sequence of SEQ ID NO: 21, preferably at least about 90% sequence identity. In some cases, the alpha - 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) with the wild - type S. tokodaii strain 7 alpha - glucan phosphorylase, preferably at least about 90% sequence identity.In some cases, the alpha-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) with the amino acid sequence of SEQ ID NO: 22, preferably at least about 90% sequence identity. In some cases, the alpha-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) with the wild-type C. callunae DSM 20145 alpha-glucan phosphorylase, preferably at least about 90% sequence identity. In some cases, the alpha-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) with the amino acid sequence of SEQ ID NO: 23, preferably at least about 90% sequence identity.In some cases, 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) with the amino acid sequence of SEQ ID NO: 24, preferably at least about 90% sequence identity, and comprises the amino acid substitutions F39L, N135S, and T706I relative to SEQ ID NO: 21.

[0144] In some embodiments, the alpha-glucan phosphorylase is derived from a microbial cell. In some cases, the alpha-glucan phosphorylase is isolated and / or purified from a microbial cell. In some cases, the microbial cell is a bacterial cell. In some cases, the bacterial cell is Escherichia coli. In some embodiments, the alpha-glucan phosphorylase is derived from Solanum tuberosum. In some embodiments, the alpha-glucan phosphorylase is derived from S. tokodaii strain 7. In some embodiments, the alpha-glucan phosphorylase is derived from C. callunae DSM 20145. In some embodiments, the alpha-glucan phosphorylase can be produced within a microbial cell. In some embodiments, the alpha-glucan phosphorylase is expressed within a recombinant host cell (e.g., from a recombinant polynucleotide). In some cases, the alpha-glucan phosphorylase is recombinantly produced. In some cases, the alpha-glucan phosphorylase is produced in a yeast cell (e.g., recombinantly produced). In some cases, the yeast cell is a Pichia yeast cell such as a Pichia pastoris cell.

[0145] Method step (b) for the enzymatic conversion of amylose to beta-cyclodextrin In various embodiments, the method further comprises enzymatically converting amylose (e.g., produced by the methods provided herein (e.g., method step (a))) to cyclodextrin, preferably beta-cyclodextrin. Optionally, the method can include contacting the amylose with an enzyme or enzyme mixture (e.g., two or more enzymes, etc.) that can convert amylose to cyclodextrin under conditions that permit the conversion of amylose to cyclodextrin. Optionally, the enzyme that can convert amylose to cyclodextrin is a variant enzyme that can produce a higher amount and / or concentration of beta-cyclodextrin than an alpha-cyclodextrin, gamma-cyclodextrin, or both, relative to a wild-type enzyme that can convert amylose to cyclodextrin.

[0146] In some embodiments, the enzyme that can convert amylose to cyclodextrin comprises a variant cyclodextrin glucanotransferase. Optionally, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type cyclodextrin glucanotransferase. Figure 6 shows the enzymatic conversion of amylose to beta-cyclodextrin using cyclodextrin glucanotransferase. Preferably, the cyclodextrin glucanotransferase produces beta-cyclodextrin from amylose in a higher amount and / or concentration than the amount and / or concentration of alpha-cyclodextrin and / or gamma-cyclodextrin.

[0147] In some embodiments, the cyclodextrin glucanotransferase is a variant cyclodextrin glucanotransferase comprising at least one amino acid variant relative to the wild-type cyclodextrin glucanotransferase. The variant cyclodextrin glucanotransferase can include one or more amino acid substitutions, deletions, insertions, and / or modifications relative to the wild-type cyclodextrin glucanotransferase. Optionally, the variant cyclodextrin glucanotransferase can produce a higher amount and / or concentration of beta-cyclodextrin from amylose compared to alpha-cyclodextrin and / or gamma-cyclodextrin relative to the wild-type cyclodextrin glucanotransferase.

[0148] In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild-type Bacillus sp. (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 wild-type B. 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 wild-type B. 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) with the amino acid sequence of SEQ ID NO: 25, preferably at least about 90% sequence identity. 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) with the amino acid sequence of SEQ ID NO: 26 or 27, preferably at least about 90% sequence identity.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) with the amino acid sequence of SEQ ID NO: 27, preferably at least about 90% sequence identity.

[0149] In some cases, 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 position 31 relative to the amino acid sequence of SEQ ID NO: 27. In some cases, the amino acid substitution at position 31 relative to the amino acid sequence of SEQ ID NO: 27 is A31R (e.g., SEQ ID NO: 28 in Table 5). In some cases, the amino acid substitution at position 31 relative to the amino acid sequence of SEQ ID NO: 27 is A31P (e.g., SEQ ID NO: 29 in Table 5). In some cases, the amino acid substitution at position 31 relative to the amino acid sequence of SEQ ID NO: 27 is A31T (e.g., SEQ ID NO: 30 in Table 5). In some embodiments, the cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 25 - 30 shown in Table 5.

[0150] In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild-type Paenibacillus macerans cyclodextrin glucanotransferase (e.g., NCBI accession number 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) to the amino acid sequence of wild-type Paenibacillus macerans cyclodextrin glucanotransferase, preferably at least about 90% sequence identity. 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 any one of SEQ ID NOs: 31-34, preferably at least about 90% sequence identity.

[0151] In some cases, at least one amino acid variant contains at least one amino acid substitution relative to wild-type cyclodextrin glucanotransferase. In some cases, at least one amino acid substitution includes an amino acid substitution at position 146 of the amino acid sequence of SEQ ID NO: 34. In some cases, the amino acid substitution at position 146 of the amino acid sequence of SEQ ID NO: 34 is R146A (e.g., SEQ ID NO: 35 in Table 5). In some cases, the amino acid substitution at position 146 of the amino acid sequence of SEQ ID NO: 34 is R146P (e.g., SEQ ID NO: 36 in Table 5). In some cases, at least one amino acid substitution includes an amino acid substitution at position 147 of the amino acid sequence of SEQ ID NO: 34. In some cases, the amino acid substitution at position 147 of the amino acid sequence of SEQ ID NO: 34 is D147A (e.g., SEQ ID NO: 37 in Table 5). In some cases, the amino acid substitution at position 147 of the amino acid sequence of SEQ ID NO: 34 is D147P (e.g., SEQ ID NO: 38 in Table 5). In some cases, at least one amino acid substitution includes amino acid substitutions at positions 146 and 147 of the amino acid sequence of SEQ ID NO: 34. In some cases, the amino acid substitution at position 146 of the amino acid sequence of SEQ ID NO: 34 is R146A and the amino acid substitution at position 147 of 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 position 146 of the amino acid sequence of SEQ ID NO: 34 is R146P and the amino acid substitution at position 147 of 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 position 146 of the amino acid sequence of SEQ ID NO: 34 is R146P and the amino acid substitution at position 147 of the amino acid sequence of SEQ ID NO: 34 is D147P (e.g., SEQ ID NO: 41 in Table 5).

[0152] In some cases, at least one amino acid substitution includes an amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34 is D372K (e.g., SEQ ID NO: 42 (for SEQ ID NO: 32) and SEQ ID NO: 45 (for SEQ ID NO: 34) in Table 5). In some cases, at least one amino acid substitution includes an amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34 is Y89R (e.g., SEQ ID NO: 43 (for SEQ ID NO: 32) and SEQ ID NO: 47 (for SEQ ID NO: 34) in Table 5). In some cases, at least one amino acid substitution includes an amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34 and an amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 32 or 34 is D372K, and the amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 32 or 34 is Y89R (e.g., SEQ ID NO: 44 (for SEQ ID NO: 32) and SEQ ID NO: 47 (for SEQ ID NO: 34) in Table 5).

[0153] In some embodiments, cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 31 to 47 shown in Table 5. In some embodiments, 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, with an amino acid sequence according to any one of SEQ ID NOs: 31 to 47 shown in Table 5.

[0154] In certain embodiments, 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, with the amino acid sequence according to SEQ ID NO: 34.

[0155] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the 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 with the amino acid sequence according to SEQ ID NO: 39, preferably at least about 90% sequence identity.

[0156] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the 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 with the amino acid sequence according to SEQ ID NO: 40, preferably at least about 90% sequence identity.

[0157] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the 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 with the amino acid sequence according to SEQ ID NO: 41, preferably at least about 90% sequence identity.

[0158] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the 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 with the amino acid sequence according to SEQ ID NO: 47, preferably at least about 90% sequence identity.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

[0159] In some embodiments, 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) with the amino acid sequence of SEQ ID NO: 25, preferably at least about 90% sequence identity. In some embodiments, 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) with the amino acid sequence of SEQ ID NO: 26 or 27, preferably at least about 90% sequence identity.

[0160] In some embodiments, 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) with 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) with the amino acid sequence of SEQ ID NO: 27, preferably at least about 90% sequence identity, and the 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) with the amino acid sequence of SEQ ID NO: 27, preferably at least about 90% sequence identity, and the 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) with the amino acid sequence of SEQ ID NO: 27, preferably at least about 90% sequence identity, and an amino acid substitution A31T with respect to SEQ ID NO: 27.

[0161] In some embodiments, 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) with 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. Optionally, 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) with the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, and an amino acid substitution R146A relative to SEQ ID NO: 34. Optionally, 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) with the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, and an amino acid substitution R146P relative to SEQ ID NO: 34.

[0162] In some embodiments, 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) with 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. Optionally, 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) with the amino acid sequence of SEQ ID NO:34, preferably at least about 90% sequence identity, and an amino acid substitution D147P relative to SEQ ID NO:34. Optionally, 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) with the amino acid sequence of SEQ ID NO:34, preferably at least about 90% sequence identity, and an amino acid substitution D147A relative to SEQ ID NO:34.

[0163] In some embodiments, 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) with 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. Optionally, 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) with the amino acid sequence of SEQ ID NO: 34, preferably at least about 90% sequence identity, the amino acid substitution R146A relative to SEQ ID NO: 34, and the 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) with 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) with 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.

[0164] In some embodiments, 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) with 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. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence (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 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.

[0165] In some embodiments, 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) with 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. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence (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 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.

[0166] In some embodiments, 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) with 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. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence (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 SEQ ID NO: 32 or 34, preferably at least about 90% sequence identity, the amino acid substitution D372K relative to SEQ ID NO: 32 or 34, and the amino acid substitution Y89R relative to SEQ ID NO: 32 or 34.

[0167] In some embodiments, cyclodextrin glucanotransferase is derived from microbial cells. In some cases, cyclodextrin glucanotransferase is isolated and / or purified from microbial cells. In some cases, the microbial cells are bacterial cells. In some cases, the bacterial cells are Escherichia coli. In some embodiments, cyclodextrin glucanotransferase is derived from Bacillus sp. (strain number 38-2). In some embodiments, cyclodextrin glucanotransferase is derived from B. circulans strain 251. In some embodiments, cyclodextrin glucanotransferase can be produced within microbial cells. In some embodiments, cyclodextrin glucanotransferase is expressed within recombinant host cells (e.g., from a recombinant polynucleotide). In some cases, cyclodextrin glucanotransferase is produced recombinantly. In some cases, cyclodextrin glucanotransferase is produced in yeast cells (e.g., produced recombinantly). In some cases, the yeast cells are Pichia yeast cells such as Pichia pastoris cells.

[0168] In various embodiments, the methods provided herein produce beta-cyclodextrin at a higher ratio than alpha-cyclodextrin, gamma-cyclodextrin, or both. For example, in some cases, the methods provided herein provide a ratio of beta-cyclodextrin to alpha-cyclodextrin, gamma-cyclodextrin, or both 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, 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. In preferred embodiments, the methods provided herein provide a ratio of beta-cyclodextrin to alpha-cyclodextrin of 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 more. 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 more. In preferred embodiments, the methods provided herein provide a ratio of beta-cyclodextrin to both 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.

[0169] Throughout the present disclosure, methods are outlined for achieving robust enzyme activity at each step to obtain a higher yield of beta-cyclodextrin than the yield currently achievable. In some embodiments, a first enzymatic step of converting sucrose to amylose (e.g., as described herein) is performed over a first period, thereby enabling a catalytic conversion of sucrose to amylose, followed by a second enzymatic step of converting amylose to beta-cyclodextrin (e.g., as described herein) over a second period, thereby enabling a catalytic conversion of amylose to beta-cyclodextrin. In some embodiments, the first enzymatic reaction (e.g., converting sucrose to amylose, as described herein) and the second enzymatic reaction (e.g., converting amylose to beta-cyclodextrin, as described herein) are performed in the same reservoir (e.g., one-pot synthesis method).

[0170] In some embodiments, the first 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 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 period is shorter than the second period. In some embodiments, the first period is longer than the second period. In some embodiments, the first period is the same or substantially the same length as the second period. In some embodiments, sucrose is added to the reaction reservoir batch by batch. In some embodiments, the enzyme used in the first enzyme reaction step (e.g., as described herein, e.g., to convert sucrose to amylose) is added once at the start of the reaction period and then readded after a certain period of time has elapsed to promote catalytic activity. In some embodiments, sucrose is added once at the start of the reaction period and then readded after a certain period of time has elapsed to replenish the sucrose. In some embodiments, the enzyme used in the first enzyme reaction step (e.g., as described herein, e.g., to convert sucrose to amylose) is added to the same reaction reservoir at the same time as the enzyme used in the second enzyme reaction step (e.g., to convert amylose to beta-cyclodextrin).In some embodiments, the enzyme used in the first enzyme reaction step (e.g., to convert sucrose to amylose as described herein) is added at a different time (e.g., prior thereto) than the enzyme used in the second enzyme reaction step (e.g., to convert amylose to beta-cyclodextrin).

[0171] In some embodiments, the sucrose concentration is maximized for efficient conversion to amylose. In some embodiments, the starting concentration of sucrose in the reaction is at least about 50 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 100 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 150 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 200 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 250 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 300 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 350 g / L.

[0172] In some embodiments, the reaction time is an important consideration for obtaining the maximum yield of beta-cyclodextrin. In some embodiments, the production of beta-cyclodextrin can involve the decomposition of the product into glucose, maltose, and other sugars. Therefore, it is important to obtain beta-cyclodextrin without decomposition. In some embodiments, the entire reaction (e.g., method step (a) and method step (b)) is carried out over 12 hours or less. In some embodiments, the entire reaction (e.g., method step (a) and method step (b)) is carried out over 8 hours or less. In some embodiments, the entire reaction is carried out over 7 hours or less. In some embodiments, the entire reaction is carried out over 6 hours or less. In some embodiments, the entire reaction is carried out over 5 hours or less. In some embodiments, the entire reaction is carried out over 4 hours or less. In some embodiments, the entire reaction is carried out over 3 hours or less. In some embodiments, the entire reaction is carried out over 2 hours or less. In some embodiments, the entire reaction is carried out over 1 hour or less.

[0173] Temperature is an important consideration for maximizing the yield of beta - cyclodextrin. In some embodiments, one or more of the enzymatic reactions are carried out at about 30°C to about 55°C, such as about 40°C to about 50°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 40°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 41°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 42°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 43°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 44°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 45°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 46°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 47°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 48°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 49°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 50°C. Preferably, one or more of the reactions are carried out at about 45°C.

[0174] In some embodiments, the enzymatic reaction of step (a) is carried out at about 40°C to about 55°C, such as 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) may be carried out at different temperatures, or preferably, step (a) and step (b) are carried out at approximately the same temperature. If 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 also preferably carried out at about 45°C. If step (a) involves the use of at least two enzymes (e.g., sucrose phosphorylase and alpha - 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 also preferably carried out at about 45°C or about 50°C, respectively.

[0175] In one-pot synthesis, even though the optimal temperatures of each enzyme may vary slightly, it is considered that the functionality of the enzyme mixture(s) should be maximized.

[0176] In some embodiments, the reaction is carried out in a reservoir having a reservoir volume of about 1 mL to about 1,000,000 L. For example, the reaction can be carried out in a reservoir having a reservoir volume of about 100 mL to about 10 L, such as a reservoir volume of about 500 mL or about 10 L.

[0177] 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, such as a total reaction volume of about 500 mL or about 5 L. In some embodiments, the total reaction volume is less than the reservoir volume. For example, in a reaction carried out in a reservoir having a reservoir volume of about 10 L, a total reaction volume of about 5 L can be used.

[0178] In some embodiments, the reaction is carried out in a stirred tank reactor (STR), a loop reactor, a plug flow reactor, a single-stage 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, and the reactants are stirred at about 100 to about 200 rpm, such as about 160 rpm.

[0179] The pH of the reaction mixture can be an important consideration for maximizing the yield of beta-cyclodextrin. In some embodiments, one or more of the enzyme reactions are carried out at a pH of about 6 to about 8, for example, the pH can be from about 6.5 to about 7.5. In a preferred embodiment, one or more of the enzyme 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. Steps (a) and (b) may be carried out at different pHs, but preferably, steps (a) and (b) are carried out at the same pH.

[0180] In some embodiments, one or more of the enzymatic reactions are carried out in a reaction mixture containing a buffer. Any suitable buffer known in the art may be used. For example, the buffer may be selected from the group consisting of sodium citrate, disodium hydrogen phosphate, and Tris-HCl. The buffer may be present in the reaction mixture at a concentration of about 50 mM to about 200 mM, such as about 100 mM.

[0181] In some embodiments, one or more of the enzymatic reactions are carried out in a reaction mixture containing an organic solvent, preferably toluene. The reaction mixture preferably also contains water. Without wishing to be bound by any theory described herein, the inventors have confirmed that the addition of the organic solvent surprisingly increases the yield of beta-cyclodextrin obtained from the enzymatic reaction. For example, the addition of the organic solvent can increase the yield of beta-cyclodextrin by at least about 5%, such as at least about 10%, such as at least about 15%, such as at least about 20%, such as at least about 50%, such as at least about 100%, such as at least about 150%, such as at least about 200%, such as at least about 250%, such as at least about 300%, such as at least about 350%, such as at least about 400% compared to the yield obtained from the enzymatic reaction carried out without the organic solvent. It is believed that the addition of the organic solvent decreases the solubility of beta-cyclodextrin in the reaction mixture, causing beta-cyclodextrin to precipitate and reducing the concentration of beta-cyclodextrin in the reaction mixture, thereby increasing the yield of beta-cyclodextrin. This prevents the degradation of beta-cyclodextrin by the enzyme.

[0182] In some embodiments, the amount of the organic solvent (preferably toluene) in the reaction mixture is about 0.1 v / v% to about 40 v / v% of the reaction mixture, such as about 1 v / v% to about 35 v / v%, such as about 5 v / v% to about 25 v / v%.

[0183] In some embodiments, the organic solvent is introduced at the start of or during the enzymatic reaction of step (a). In some preferred embodiments, the organic solvent is introduced at the start of or during the enzymatic reaction of step (b). For example, in embodiments where the entire reaction (e.g., method step (a) and method step (b)) is carried out over 8 hours or less, the organic solvent can be introduced about 1 hour after the start of enzymatic reaction (b).

[0184] In some embodiments, the enzyme used in step (a) is amylosucrase. In some embodiments, the starting 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.

[0185] In some embodiments, the enzyme mixture used in step (a) comprises sucrose phosphorylase and alpha - 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 alpha - 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.

[0186] In some embodiments, the enzyme is provided in a whole cell lysate, and preferably, the ratio of the starting concentration of the enzyme in step (b) (measured as the volume in the whole cell lysate) to the enzyme in step (a) is about 1:1 to about 50:1, such as about 2:1 to about 50:1, such as about 5:1 to about 40:1, such as about 10:1 to about 30:1. In a preferred embodiment, the ratio is about 20:1.

[0187] In certain embodiments, any one of the enzyme reactions provided herein (e.g., the first enzyme reaction that converts sucrose to amylose and / or the second enzyme reaction that converts amylose to beta-cyclodextrin) can occur within a microbial host cell. Optionally, the microbial cell is a bacterial cell. Optionally, the bacterial cell is Escherichia coli. For example, the microbial host cell can contain one or more heterologous nucleic acid molecules encoding one or more of the enzymes provided herein. The microbial host cell can express one or more of the enzymes provided herein. Optionally, sucrose and / or one or more intermediates of the enzyme reaction can be fed to the microbial host cell. For example, sucrose can be fed to the microbial host cell, and the conversion of sucrose to beta-cyclodextrin can occur within the microbial host cell.

[0188] In some embodiments, one or more of the enzymes used in the enzyme reactions provided herein may be immobilized on a resin. For example, the enzyme may be covalently bound to the resin. Alternatively, the enzyme may be non-covalently bound to the resin. For example, the enzyme may be linked to Ni resin via a His tag. For example, the enzyme of (a) can be a variant amylosucrase (e.g., this variant amylosucrase can contain or consist of the amino acid sequence according to SEQ ID NO: 3), and the enzyme may be immobilized on a resin. Alternatively, or additionally, the enzyme of (b) can be a variant cyclodextrin glucanotransferase (e.g., this variant cyclodextrin glucanotransferase can contain or consist of the amino acid sequence according to SEQ ID NO: 28), and the enzyme may be immobilized on a resin. Optionally, the enzyme or enzyme mixture of (a) and the enzyme of (b) are immobilized on the same resin.

[0189] The resin-immobilized enzyme can be reused by the method described in this specification. However, the inventors have found that when the resin-immobilized enzyme is reused, the yield of beta-cyclodextrin tends to decrease. This is presumably due to the fact that the enzyme leaks from the resin during use, resulting in a decrease in the enzyme conversion rate. Therefore, it is desirable to improve the enzyme stability on the resin and thereby prevent enzyme leakage. This is because it enables the resin-immobilized enzyme to be reused more frequently and / or at a higher enzyme conversion rate, thereby increasing the yield of the reaction.

[0190] The inventors have found that enzyme stability can be improved by using freeze-dried enzymes, spray-drying the enzymes, and / or introducing additives.

[0191] In some embodiments, the enzyme is provided in a cell slurry or in a whole cell lysate. For example, a cell slurry containing recombinant cells expressing the enzyme can be suspended, lysed, and centrifuged in a buffer (such as sodium citrate buffer) to provide a whole cell lysate containing the enzyme. Methods of cell lysis are known in the art. For example, cells can be lysed by homogenization, chemical lysis, sonication, freeze / thaw, lytic enzymes, acidic lysis, and / or alkaline lysis. In a preferred embodiment, the cells are lysed by homogenization.

[0192] In some embodiments, the cell slurry or whole cell lysate further comprises an additive. In some embodiments, the additive is selected from the group consisting of 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%, such as about 0.5 w / v% to about 5 w / v% of the cell slurry or whole cell lysate. For example, the additive may be added at 0.5 w / v%, 1.0 w / v%, or 5 w / v% of the cell slurry or whole cell lysate. In a preferred embodiment, the additive is mannitol, sorbitol, sucrose, or a combination thereof.

[0193] In some embodiments, the cell slurry or cell lysate may be lyophilized. For example, the cell slurry or cell lysate may be lyophilized over a period of two days. Methods of lyophilization are known in the art.

[0194] The inventors have found that adding an additive to a cell slurry or whole cell lysate (as described above) increases the enzyme stability compared to a cell slurry or whole cell lysate without the additive, and that lyophilizing the cell slurry or whole cell lysate (as described above) increases the enzyme stability compared to a non-lyophilized cell slurry or whole cell lysate. The cell slurry or cell lysate may be resuspended and shaken to redissolve before use in the methods described herein.

[0195] In some embodiments, the methods described herein produce a composition comprising at least 18 g / L of beta-cyclodextrin. In some embodiments, the method produces a composition comprising at least 25 g / L of beta-cyclodextrin, at least 30 g / L of beta-cyclodextrin, at least 40 g / L of beta-cyclodextrin, at least 50 g / L of beta-cyclodextrin, or at least 60 g / L of beta-cyclodextrin. In a preferred embodiment, the methods described herein produce a composition comprising at least 50 g / L of beta-cyclodextrin.

[0196] In some embodiments, the percent yield of beta-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%, and this percent yield is calculated by dividing the total amount of beta-cyclodextrin produced by the methods described herein by the maximum theoretical amount of beta-cyclodextrin that could be produced from the sucrose reagent of the starting material.

[0197] Also provided herein is a composition comprising cyclodextrin, said cyclodextrin comprising beta-cyclodextrin and optionally further comprising alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof, and said composition comprising cyclodextrin comprises beta-cyclodextrin in an amount and / or concentration higher than alpha-cyclodextrin, gamma-cyclodextrin, or both. Preferably, the composition is obtained from the method provided herein. In some cases, the composition does not comprise alpha-cyclodextrin and / or gamma-cyclodextrin. Preferably, the composition comprises a ratio of beta-cyclodextrin to alpha-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, 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, a ratio of beta-cyclodextrin to gamma-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, 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, or both a ratio of beta-cyclodextrin and a ratio of beta-cyclodextrin to alpha-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, 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. Preferably, the composition comprises a ratio of beta-cyclodextrin to alpha-cyclodextrin of at least 10:1, such as 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, a ratio of beta-cyclodextrin to gamma-cyclodextrin of at least 10:1, such as 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, or both a ratio of beta-cyclodextrin and a ratio of beta-cyclodextrin to alpha-cyclodextrin of at least 10:1, such as 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.

[0198] In a preferred embodiment, the present invention provides a method for producing a composition comprising cyclodextrin, the method comprising: (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that allow the conversion of sucrose to amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with cyclodextrin glucanotransferase, thereby producing a composition comprising cyclodextrin, wherein the cyclodextrin glucanotransferase in (b) is a variant enzyme capable of producing a higher amount and / or concentration of beta-cyclodextrin than the wild-type enzyme capable of converting amylose to cyclodextrin, and the composition comprising cyclodextrin comprises beta-cyclodextrin and may further comprise, optionally, alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof, preferably, the ratio of beta-cyclodextrin in the composition to alpha-cyclodextrin, gamma-cyclodextrin, or both is at least 10:1, steps (a) and (b) are carried out simultaneously, steps (a) and (b) are carried out at about 45 °C to about 55 °C, steps (a) and (b) are carried out at a pH of about 7.0 to about 7.5, steps (a) and (b) are carried out in a reaction mixture comprising water and an organic solvent (preferably toluene), and the entire reaction is carried out over a period of 8 hours or less.

[0199] Also provided herein is beta-cyclodextrin. Preferably, the beta-cyclodextrin is obtained from the method provided herein.

[0200] Also provided herein is the use of sucrose as a starting material for the production of beta-cyclodextrin. Also provided herein is the use of sucrose in a method for producing beta-cyclodextrin, the method not using starch.

[0201] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for converting sucrose to amylose, as described herein.

[0202] Also provided herein is the use of any one of the variant enzymes described herein for converting amylose to cyclodextrin and / or for producing beta-cyclodextrin in an amount and / or concentration higher than that of alpha-cyclodextrin, gamma-cyclodextrin, or both, as described herein.

[0203] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for the production of beta-cyclodextrin, which production does not require starch as a starting material.

[0204] Also provided herein is any one of the enzymes or enzyme mixtures described herein. For example, provided herein are enzymes comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 1-48. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with any one of the amino acid sequences of SEQ ID NOs: 1-48.

[0205] Preferably, the enzyme is a variant amylosucrase enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 3-16 or 48. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with any one of the amino acid sequences of SEQ ID NOs: 3-16 or 48.

[0206] Preferably, this enzyme is a variant sucrose phosphorylase enzyme comprising or consisting of the amino acid sequence of SEQ ID NO: 20. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with the amino acid sequence of SEQ ID NO: 20.

[0207] Preferably, this enzyme is a variant alpha - glucan phosphorylase enzyme comprising or consisting of the amino acid sequence of SEQ ID NO: 24. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with the amino acid sequence of SEQ ID NO: 24.

[0208] Preferably, this enzyme is a variant cyclodextrin glucanotransferase enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 28 - 30 or 35 - 47. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with any one of the amino acid sequences of SEQ ID NOs: 28 - 30 or 35 - 47.

[0209] Also provided herein are enzyme compositions comprising one or more of the enzymes described herein.

[0210] Also provided herein are genes encoding any one of the variant enzymes described herein. Also provided herein are vectors encoding any one of the variant enzymes described herein.

[0211] Also provided herein are recombinant host cells comprising any one of the genes, vectors, or enzymes described herein.

[0212] Also provided herein is the use of an organic solvent, preferably toluene, to increase the yield of beta-cyclodextrin obtained in a method for producing beta-cyclodextrin, such as beta-cyclodextrin obtained from any one of the methods described herein.

[0213] Purification method Also provided herein is a method for purifying beta-cyclodextrin, the method comprising: i. preparing a crude composition containing beta-cyclodextrin; ii. obtaining a first precipitate containing beta-cyclodextrin from the crude composition, for example, by filtering the crude composition, subjecting the crude composition to centrifugation, subjecting the crude composition to a sedimentation operation, and / or washing with water; iii. dissolving the first precipitate, for example, by dissolving the first precipitate in water to obtain a first solution containing beta-cyclodextrin; iv. filtering the first solution to obtain a second solution containing beta-cyclodextrin; and v. crystallizing and / or precipitating the second solution to obtain a purified beta-cyclodextrin composition.

[0214] Step (ii) and / or (iv) The filtration step (iv) can remove insoluble substances.

[0215] In some embodiments, step (ii) and / or (iv) includes washing the material obtained by filtration with, for example, water or alkaline water.

[0216] In some embodiments, step (iv) includes filtration with a filter aid. In some embodiments, the filter aid includes silicon dioxide. An example of a suitable filter aid is 1% Celite® commercially available from Sigma-Aldrich. The use of a filter aid can be advantageous for shortening the overall filtration time of step (iv).

[0217] The filtration step (iv) can be carried out at a temperature of about 4°C to about 25°C.

[0218] Dissolution step (iii) In some embodiments, step (iii) includes dissolving the first precipitate in an alkaline solution. The precipitate may be dissolved in NaOH, for example 1M NaOH, by adding a plurality (e.g., 5) volumes of 1M NaOH.

[0219] In some embodiments, step (iii) may include heating the solution until beta-cyclodextrin dissolves. For example, this may require heating the solution to about 60°C or higher, such as about 65°C or higher, such as about 70°C or higher, such as about 75°C or higher. Next, the temperature of the solution may be lowered, for example by about 5°C or more, before subsequent steps.

[0220] Crystallization step (v) Step (v) may include neutralizing the second solution. Optionally, the neutralization includes the addition of HCl. For example, the neutralization may include the addition of 6M HCl.

[0221] Step (v) may include seeding the second solution with crystalline beta-cyclodextrin.

[0222] In some embodiments, step (v) may further include heating the solution until the beta-cyclodextrin dissolves. For example, this may require heating the solution to about 60 °C or higher, such as about 65 °C or higher, such as about 70 °C or higher, such as about 75 °C or higher. In a preferred embodiment, the solution is heated to about 75 °C. Next, before adding the crystalline beta-cyclodextrin as seed crystals, the temperature of the solution may be lowered, for example, by about 5 °C or more. In a preferred embodiment, the solution is heated to about 75 °C and then lowered to about 70 °C before adding the seed crystals.

[0223] In some embodiments, step (v) may include cooling the solution to below room temperature, such as about 20 °C or lower, about 15 °C or lower, about 10 °C or lower, or about 5 °C or lower, after adding the seed crystals. In a preferred embodiment, the solution is cooled to about 4 °C. In some embodiments, the solution is cooled over about 1 to about 12 hours. In some embodiments, the solution is cooled over about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In a preferred embodiment, the solution is cooled to about 4 °C over about 4 hours.

[0224] The solution after adding the seed crystals may be maintained under conditions suitable for beta-cyclodextrin crystal formation. For example, the solution may be maintained at below room temperature, such as about 20 °C or lower, about 15 °C or lower, about 10 °C or lower, or about 5 °C or so. In a preferred embodiment, the solution is maintained at about 4 °C. In some embodiments, the solution is maintained at below room temperature for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, or at least about 12 hours. Preferably, the solution is maintained at about 4 °C for 12 hours or more.

[0225] The crystallization step (v) may include a filtration step. The filtration step may include vacuum filtration.

[0226] In some embodiments, step (v) further comprises washing the composition with water.

[0227] In some embodiments, step (v) further comprises drying the composition, optionally drying the composition at about 45 °C (e.g., in a vacuum oven).

[0228] Precipitation step (v) Step (v) may include neutralizing the second solution, optionally the neutralization includes the addition of HCl. For example, the neutralization may include the addition of about 6M HCl.

[0229] Step (v) may include the addition of an antisolvent. The antisolvent can increase the yield of purified beta-cyclodextrin in the composition obtained by the purification method. The antisolvent is a solvent in which beta-cyclodextrin is poorly soluble, for example, a solvent in which beta-cyclodextrin does not dissolve at about 50 °C and about 60 °C. The antisolvent can be THF, AcN, EtOH, toluene, acetone, or a mixture of acetone and water (e.g., a 10:90, or 20:80, or 30:70, or 40:60, or 50:50, or 60:40, or 70:30, or 80:20, or 90:10 acetone:water mixture). In some embodiments, when the antisolvent is a mixture of acetone and water, this mixture can be 10-90%, 20-80%, 30-70%, 40-60%, or about 50% acetone. Preferably, the antisolvent used is a mixture of acetone and water, for example, a 50% acetone and 50% water mixture.

[0230] In some embodiments, step (v) may include cooling the solution to a temperature below room temperature, such as below about 20°C, below about 15°C, below about 10°C, or below about 5°C, after the addition of the antisolvent. In a preferred embodiment, the solution is cooled to about 4°C. In some embodiments, the solution is cooled over about 1 to about 12 hours. In some embodiments, the solution is cooled over about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In a preferred embodiment, the solution is cooled to about 4°C over about 4 hours.

[0231] The solution may be maintained under conditions suitable for beta-cyclodextrin precipitate formation. For example, the solution may be maintained at a temperature below room temperature, such as below about 20°C, below about 15°C, below about 10°C, or about 5°C. In a preferred embodiment, the solution is maintained at about 4°C. In some embodiments, the solution is maintained below room temperature for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, or at least about 12 hours. Preferably, the solution is maintained at about 4°C for 12 hours or more.

[0232] In some embodiments, the solution is cooled to about 4°C over about 4 hours and then maintained at about 4°C for about 12 hours.

[0233] The precipitation in step (v) may include a filtration step. The filtration step may include vacuum filtration.

[0234] In some embodiments, step (v) further includes washing the composition with water.

[0235] In some embodiments, step (v) further includes drying the composition, optionally drying the composition at about 45°C (e.g., under vacuum).

[0236] Composition Preferably, the crude composition of step (i) is obtained by any one of the enzymatic methods described and claimed herein.

[0237] In some embodiments, the crude composition is cooled prior to step (ii). For example, the crude composition may be cooled to room temperature for at least about 3 hours and then to about 4 °C for at least about 3 hours.

[0238] Also provided herein is a purified beta-cyclodextrin composition. The purified beta-cyclodextrin composition can be obtained from any one of the purification methods described and claimed herein. The beta-cyclodextrin in the composition can have a purity of 75 wt% or more, such as 80 wt% or more, such as 85 wt% or more, such as 90 wt% or more, or such as 95 wt% or more.

[0239] The purity of beta-cyclodextrin 1 is measurable by 1H-NMR and can provide the anhydrous amount of beta-cyclodextrin.

[0240] Preferably, the purified beta-cyclodextrin composition consists essentially of beta-cyclodextrin and optional water, and preferably consists of beta-cyclodextrin and optional water. The purified beta-cyclodextrin composition may contain up to 2 wt% toluene, for example, it may contain no toluene. The purified beta-cyclodextrin composition may contain up to 1 wt% sucrose, fructose and / or amylose, for example, it may contain no sucrose, fructose and / or amylose. The purified beta-cyclodextrin composition may contain up to 5 wt%, preferably up to 1 wt% alpha and / or gamma-cyclodextrin, for example, it may contain no alpha and / or gamma-cyclodextrin.

[0241] The recovery rate of beta-cyclodextrin by the purification method described in this specification can be at least 50%, at least 60%, at least 70%, or at least 80%. In other words, the amount of beta-cyclodextrin in the purified composition can be at least 50% (or at least 60%, at least 70%, or at least 80%) of the amount of beta-cyclodextrin in the crude composition. The amounts of beta-cyclodextrin and any other components in the composition are 1 measurable by 1H-NMR (in wt%) or by HLPC-ELSD (in g / L).

[0242] Concentrations, amounts, and other numerical data may be expressed or presented in range format herein. Such range format is used merely for convenience and brevity and should be interpreted flexibly so as to include not only the numerical values explicitly listed as the limits of the range, but also all individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly listed. By way of example, a numerical range of "about 2 to about 50" should be interpreted to include not only the explicitly listed numerical values of 2 to 50, but also all individual numerical values and sub-ranges within the indicated range. Thus, included within this numerical range are individual values such as, for example, 2, 2.4, 3, 3.7, 4, 5.5, 10, 10.1, 14, 15, 15.98, 20, 20.13, 23, 25.06, 30, 35.1, 38.0, 40, 44, 44.6, 45, 48, etc., and sub-ranges such as, for example, 1 to 3, 2 to 4, 5 to 10, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 50, 2 to 10, 2 to 20, 2 to 30, 2 to 40, 2 to 50, etc. The same principle applies to ranges that describe only one numerical value as a minimum or maximum value. Further, such interpretation should apply regardless of the breadth or nature of the range described.

[0243] As used herein, the term "about" is used to provide flexibility to the endpoints of a numerical range by providing that a given value may be "slightly above" or "slightly below" the endpoint. For example, the endpoint may be within 10%, 8%, 5%, 3%, 2%, or 1% of the recited value. Further, for purposes of convenience and brevity, the numerical range of "about 50 mg / mL to about 80 mg / mL" should be understood to also support the range of "50 mg / mL to 80 mg / mL".

[0244] As used herein, the term "filtering" or "filtration" explicitly includes nanofiltration and nanofiltering.

[0245] As used herein, the term "liquid pharmaceutical" includes a pharmaceutical active ingredient. During modular manufacturing processes and / or systems, the liquid pharmaceutical may further include one or more solvents, excipients, intermediates, reactants, precursors, catalysts, and / or impurities.

[0246] While the foregoing refers to specific preferred embodiments, it should be understood that the present invention is not limited thereto. Those skilled in the art will find that various modifications can be made to the disclosed embodiments, and such modifications are intended to be within the scope of the present invention. All published documents, patent applications, and patents cited herein are hereby incorporated by reference in their entirety. [Embodiments]

[0247] 1. A modular system for producing a pharmaceutical composition, comprising a plurality of modules, wherein the plurality of modules include one or more flow modules, one or more mixing modules, one or more heat exchange modules, and one or more reactor modules and each of the modules is operably connected to one or more other modules, ​The modular system, wherein at least two modules are operably connected to the one or more reactors.

[0248] 2. The modular system according to Embodiment 1, further comprising a controller that communicates with at least one or more of the plurality of modules.

[0249] 3. The modular system according to Embodiment 2, wherein the controller is electrically or wirelessly connected to at least one or more of the plurality of modules.

[0250] 4. The modular system according to Embodiment 2, wherein the controller is configured to automatically adjust system parameters selected from the group consisting of temperature, pressure, flow rate, heat transfer rate, solvent content, solvent amount, filtration, or combinations thereof.

[0251] 5. The modular system according to Embodiment 2, wherein the controller is configured to be remotely operated.

[0252] 6. The modular system according to Embodiment 1, wherein the one or more modules in the system are interchangeable with each other.

[0253] 7. The modular system according to Embodiment 1, wherein a plurality of modules are configured to be simultaneously stationary cleaned with a chemical cleaning agent.

[0254] 8. The modular system according to Embodiment 7, which does not require each module to be cleaned independently.

[0255] 9. The modular system according to Embodiment 1, further comprising a back pressure regulator.

[0256] 10. The modular system according to embodiment 1, wherein the system comprises, as a pharmaceutical composition output, substituted and / or unsubstituted cyclodextrin, beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and combinations thereof.

[0257] 11. The modular system according to embodiment 1, wherein the system comprises, as a plurality of pharmaceutical composition outputs, substituted and / or unsubstituted cyclodextrin, beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and combinations thereof.

[0258] 12. The modular system according to embodiment 11, wherein the system is configured to simultaneously generate a plurality of pharmaceutical composition outputs.

[0259] 13. The modular system according to embodiment 12, wherein the system is configured to simultaneously generate a plurality of pharmaceutical composition outputs comprising mixtures of a common molecule in different component amounts.

[0260] 14. The modular system according to embodiment 1, wherein the system is configured to maintain the flow rate and heat transfer rate in the plurality of modules at a predetermined level.

[0261] 15. The modular system according to embodiment 1, wherein the pharmaceutical composition is a liquid pharmaceutical, a solid pharmaceutical, a pharmaceutical formulation, or a combination thereof.

[0262] 16. A modular system for producing a pharmaceutical composition, comprising a plurality of modules, wherein the plurality of modules comprise a plurality of flow modules, a plurality of mixing modules, a plurality of heat exchange modules, and A plurality of reactor modules comprising, each of said modules being operably connected to one or more other modules so as to perform in-line production of said pharmaceutical composition, said modular system.

[0263] 17. A modular system for producing a pharmaceutical composition, comprising a plurality of cases, said plurality of cases comprising one or more flow modules, one or more mixing modules, one or more heat exchange modules, and one or more reactor modules selected from two or more modules, said modular system, wherein said two or more modules are stacked vertically within each case.

[0264] 18. A modular system for producing a pharmaceutical composition, comprising a plurality of modules, each of said modules being operably connected to one or more other modules, said modular system, wherein said modules are stackable.

[0265] 19. A remotely controlled factory comprising the modular system according to any one of the preceding embodiments 1 to 18.

Claims

1. A modular system for producing pharmaceutical compositions comprising multiple modules, wherein the multiple modules are One or more flow modules, One or more mixed modules, One or more heat exchange modules, and One or more reactor modules Includes, Each of the aforementioned modules is operably connected to one or more other modules. The modular system comprising at least two modules operably connected to one or more reactors.

2. The system further comprises a controller that communicates with at least one of the plurality of modules, optionally, (a) The controller is electrically or wirelessly connected to at least one of the plurality of modules; (b) The controller is configured to automatically adjust system parameters selected from the group consisting of temperature, pressure, flow rate, heat transfer coefficient, solvent content, solvent volume, filtration, or a combination thereof, and / or (c) The modular system according to claim 1, wherein the controller is configured to be remotely operated.

3. The modular system according to claim 1, wherein one or more modules in the system are interchangeable with one another.

4. The modular system according to claim 1, wherein multiple modules are configured to be simultaneously cleaned in place by a chemical cleaning agent, and it is not necessary for each module to be cleaned independently in an optional manner.

5. The modular system according to claim 1, further comprising a back pressure regulator.

6. The modular system according to claim 1, wherein the system includes substituted and / or unsubstituted cyclodextrins, beta-cyclodextrins, hydroxypropyl beta-cyclodextrins, and combinations thereof as a pharmaceutical composition output.

7. The modular system according to claim 1, wherein the system comprises, as a plurality of pharmaceutical composition outputs, substituted and / or unsubstituted cyclodextrins, beta-cyclodextrins, hydroxypropyl beta-cyclodextrins, and combinations thereof.

8. The modular system according to claim 1, wherein the system is configured to simultaneously produce a plurality of pharmaceutical composition outputs, and optionally, the system is configured to simultaneously produce a plurality of pharmaceutical composition outputs comprising a mixture of common molecules in different component amounts.

9. The modular system according to claim 1, wherein the system is configured to maintain the flow rate and heat transfer coefficient in the plurality of modules at predetermined levels.

10. The modular system according to claim 1, wherein the pharmaceutical composition is a liquid pharmaceutical, a solid pharmaceutical, a pharmaceutical preparation, or a combination thereof.

11. A modular system for producing pharmaceutical compositions comprising multiple modules, wherein the multiple modules are Multiple flow modules, Multiple mixed modules, Multiple heat exchange modules, and Multiple reactor modules Includes, The modular system, wherein each of the modules is operably connected to one or more other modules to perform in-line manufacturing of the pharmaceutical composition.

12. A modular system for producing a pharmaceutical composition comprising multiple cases, wherein the multiple cases are One or more flow modules, One or more mixed modules, One or more heat exchange modules, and One or more reactor modules Includes two or more modules selected from, The modular system wherein two or more of the aforementioned modules are stacked vertically within each case.

13. A modular system for producing pharmaceutical compositions, comprising multiple modules, Each of the aforementioned modules is operably connected to one or more other modules. The modular system wherein the modules are stackable.

14. A remotely controlled factory comprising the modular system according to any one of claims 1 to 13.

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