Microcarriers with improved rehydration

Abstract

Dried microcarriers comprising polygalacturonic acid (PGA), or partly esterified pectic acid (PE PGA) with a degree of esterification of 40 mol% or less, or salts of the foregoing, are provided. The dried microcarriers are formed with drying solutions comprising a calcium chelator or a positively charged molecule and require less rehydration solution to rehydrate the microcarriers as compared to currently available pectin-based dried microcarriers. The dried microcarriers of the present disclosure may further comprise a filler and / or a lubricant. The process for making the dried microcarriers requires less volume of alcohol and filler when calcium chelators or positively charged molecules that other existing drying processes.

Description

Attorney Docket No. SP24-284MICROCARRIERS WITH IMPROVED REHYDRATION CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S.Provisional Application Serial No. 63 / 719,935 filed on November 13, 2024, the content of which is relied upon and incorporated herein by reference in its entirety.FIELD

[0002] The present disclosure relates to the field of cell culture using microcarriers as substrate for cell attachment and expansion.BACKGROUND

[0003] Cell culture can be performed a variety of ways, including through suspension cultures, 2D cell culture where adherent cells attach and expand on a surface, and 3D cell culture where cells attach to each other to create structures such as spheroids and organoids. 2D cell culture techniques vary but include using cell culture dishes (such as a petri dish) or plates with individual wells, higher volume devices such as roller bottles, and / or microcarriers. Microcarriers can be dissolvable or non-dissolvable, and the microcarriers act as substrate for adherent cells to attach to and expand on, typically cultured in a non-static cell culture medium. Existing hydrogel-based microcarriers are inefficient to make and rehydrate, making them suitable for only smaller-scale productions. A need exists for microcarriers that are more efficient to make and rehydrate for microcarriers to be useful for larger-scale productions.SUMMARY

[0004] Aspect 1. A microcarrier, comprising a pectin-based polymer in an amount between 20 wt.% and 80 wt.% of the microcarrier, calcium, and a calcium chelator. The microcarrier is a dried microcarrier. The pectin-based polymer is polygalacturonic acid (PGA), partly esterified polygalacturonic acid (PE PGA) having a degree of esterification of 40 mol% or less, a salt of any of the foregoing, or a combination of the foregoing. The calcium is present in the microcarrier at an amount between 3 wt.% and 10 wt.% of the microcarrier. The calcium chelator is present in the microcarrier at an amount greater than 0 wt.% and less than 5 wt.% of the microcarrier. Further,the dried microcarrier in its rehydrated form has the pectin-based polymer present at 1.0 w / v% and 3.0 w / v%.

[0005] Aspect 2. The microcarrier of aspect 1, wherein the calcium chelator comprises sodium citrate, ethylenediaminetetraacetic acid (EDTA), aspartic acid, (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N' -tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTP A), N(hydroxyl)-ethylenediaminetetraacetic acid (HETDA), a salt of any of the foregoing, or combinations of any of the foregoing.

[0006] Aspect 3. The microcarrier of any one of aspects 1-2, wherein the microcarrier further comprises a filler and the filler comprises a low molecular weight sugar, a low molecular weight polyethylene glycol (PEG), a low molecular weight positively charged compound, or a combination thereof.

[0007] Aspect 4. The microcarrier of aspect 3, wherein the filler comprises glucose, glucosamine, a low molecular weight polyethylene glycol (PEG), or a combination thereof.

[0008] Aspect 5. The microcarrier of any one of aspects 1-4, wherein the microcarrier further comprises a lubricant.

[0009] Aspect 6. The microcarrier of aspect 5, wherein the lubricant comprises a low molecular weight polyethylene glycol (PEG), dimethyl sulfoxide (DMSO), or a combination of the foregoing.

[0010] Aspect 7. The microcarrier of any one of aspects 1-6, wherein the microcarrier further comprises a positively charged molecule.

[0011] Aspect 8. The microcarrier of aspect 7, wherein the positively charged molecule comprises a weak acid with a net positive charge, a primary amine, a secondary amine, a tertiary amine, or a combination thereof.

[0012] Aspect 9. The microcarrier of any one of aspects 1-8, wherein the microcarrier is a sterilized microcarrier.

[0013] Aspect 10. The microcarrier of any one of aspects 1-9, wherein a diameter of a rehydrated form of the dried microcarrier is at least 80% of a diameter the dried microcarrier’s initial form.

[0014] Aspect 11. The microcarrier of any one of aspects 1-10, wherein the microcarrier, in its rehydrated form, has a diameter of between 10 pm and 500 pm.

[0015] Aspect 12. A microcarrier, comprising a pectin-based polymer in an amount between 20 wt.% and 80 wt.% of the microcarrier, calcium, and a positively charged molecule. The microcarrier is a dried microcarrier. The pectin-based polymer is polygalacturonic acid (PGA), partly esterified polygalacturonic acid (PE PGA) having a degree of esterification of 40 mol% or less, a salt of any of the foregoing, or a combination of any of the foregoing. The calcium is present in the microcarrier in an amount between 3 wt.% and 10 wt.% of the microcarrier. The positively charged molecule is present in the microcarrier in an amount greater than 0 wt.% and less than 5 wt.% of the microcarrier. The dried microcarrier, in its rehydrated form, has the pectin-based polymer present at 1.0 w / v% and 3.0 w / v%.

[0016] Aspect 13. The microcarrier of aspect 12, wherein the positively charged molecule comprises a weak acid with a net positive charge, a primary amine, a secondary amine, a tertiary amine, or a combination thereof.

[0017] Aspect 14. The microcarrier of any one of aspects 12-13, wherein the microcarrier further comprises a filler that comprises a low molecular weight sugar, a low molecular weight polyethylene glycol (PEG), a low molecular weight positively charged compound, or a combination thereof.

[0018] Aspect 15. The microcarrier of aspect 14, wherein the filler comprises glucose, glucosamine, polyethylene glycol, or a combination thereof.

[0019] Aspect 16. The microcarrier of any one of aspects 12-15, wherein the microcarrier further comprises a lubricant.

[0020] Aspect 17. The microcarrier of aspect 16, wherein the lubricant comprises a low molecular weight polyethylene glycol (PEG), dimethyl sulfoxide (DMSO), or a combination of the foregoing.

[0021] Aspect 18. The microcarrier of any one of aspects 12-17, wherein the microcarrier is a sterilized microcarrier.

[0022] Aspect 19. The microcarrier of any one of aspects 12-18, wherein a diameter of a rehydrated form of the dried microcarrier is at least 80% of a diameter the dried microcarrier’s hydrated, initial form.

[0023] Aspect 20. The microcarrier of any one of aspects 12-18, wherein the microcarrier, in its rehydrated form, has a diameter of between 10 pm and 500 pm.

[0024] Aspect 21. A method of drying microcarriers, comprising the steps of: (a) providing an initial form of a microcarrier, (b) providing a drying solution comprising a calcium chelator, a positively charged molecule, or a combination thereof, (c) exposing the initial form of the microcarrier to the drying solution, and (d) drying the microcarrier from step (c). The microcarrier comprises calcium and polygalacturonic acid (PGA), partly esterified polygalacturonic acid (PE PGA) having a degree of esterification of 40 mol% or less, salts of the foregoing, or a combination of the foregoing.

[0025] Aspect 22. The method of aspect 21, wherein the calcium chelator is selected from the group consisting of sodium citrate, ethylenediaminetetraacetic acid (EDTA), aspartic acid, (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N(hydroxyl)-ethylenediaminetetraacetic acid (HETDA), a salt of any of the foregoing, and combinations of any of the foregoing.

[0026] Aspect 23. The method of any one of aspects 21-22, wherein the positively charged molecule comprises a weak acid with a net positive charge, a primary amine, a secondary amine, a tertiary amine, or a combination thereof.

[0027] Aspect 24. The method of any one of aspects 21-23, wherein the drying solution further comprises a filler, a lubricant, or a combination thereof.

[0028] Aspect 25. The method of aspect 24, wherein the filler is a low molecular weight sugar, a low molecular weight polyethylene glycol (PEG), a low molecular weight positively charged compound, or a combination thereof.

[0029] Aspect 26. The method of any one of aspects 24-25, wherein the lubricant comprises a low molecular weight polyethylene glycol (PEG), dimethyl sulfoxide (DMSO), or a combination of the foregoing.

[0030] Aspect 27. The method of any one of aspects 21-26, wherein the drying solution comprises an alcohol.

[0031] Aspect 28. The method of any one of aspects 21-27, wherein the drying is by rotary evaporator, freeze drying, lyophilization, or a combination thereof.

[0032] Aspect 29. The method of any one of aspects 25-28, further comprising the steps of providing a drying solution comprising an alcohol and exposing the initially formed microcarrier to the drying solution comprising an alcohol, wherein both steps are performed before step (b).

[0033] Aspect 30. The method of any one of aspects 21-23, further comprising a step of exposing the initially formed microcarrier to a drying solution comprising a filler, a lubricant, or a combination thereof, wherein the step is performed after step (c) and before step (d).

[0034] Aspect 31. The method of aspect 30, wherein the calcium chelator is selected from the group consisting of sodium citrate, ethylenediaminetetraacetic acid (EDTA), aspartic acid, (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N(hydroxyl)-ethylenediaminetetraacetic acid (HETDA), a salt of any of the foregoing, and combinations of any of the foregoing; and wherein the positively charged molecule is selected from the group consisting of triethanolamine chloride and lysine chloride, glucosamine, or a combination thereof.

[0035] Aspect 32. The method of any one of aspects 21-31, further comprising a step of sterilizing the microcarrier after step (d).

[0036] Aspect 33. The method of aspect 32, wherein the sterilization is performed using high-energy irradiation.

[0037] Aspect 34. The method of any one of aspects 21-33, wherein a diameter of a rehydrated form of the dried microcarrier is at least 80% of a diameter the dried microcarrier’s initial form.

[0038] Aspect 35. A method of rehydrating a dried microcarrier, comprising the steps of: (a) providing the dried microcarrier of any one of aspects 1-20, (b) providing an aqueous solution to the dried microcarrier, (c) combining the dried microcarrier and the aqueous solution to achieve a concentration of dried microcarrier in aqueous solution of greater than 2 mg / mL, and (d) rehydrating the dried microcarrier in the aqueous solution to generate a rehydrated microcarrier. Under a phase contract optical microscope in brightfield mode, the rehydrated microcarrier has a light perimeter with an interior that is, on average, darker than the light perimeter.

[0039] Aspect 36. The method of aspect 35, wherein the optical phase microscope in brightfield mode is at 4x magnification.

[0040] Aspect 37. The method of any one of aspects 35-36, wherein the concentration of dried microcarriers to aqueous solution between 20 mg / mL and 100 mg / mL.

[0041] Aspect 38. The method of any one of aspects 35-37, wherein the concentration of dried microcarriers to aqueous solution is between 40 mg / mL and 100 mg / mL.

[0042] Aspect 39. The method of any one of aspects 35-38, wherein the minimum time required to rehydrate the dried microcarrier is between 20% and 90% less than a minimum time required to rehydrate a dried microcarrier without a calcium chelator and / or a positively charged molecule.

[0043] Aspect 40. The method of any one of aspects 35-39, wherein the minimum volume of aqueous solution required for rehydration is between 20%-90% less than the minimum volume required to rehydrate for a dried microcarrier without a calcium chelator and / or a positively charged molecule.

[0044] Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0045] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide an overview or framework to understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s), and together with the description serve to explain principles and operation of the various embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The following is a description of the figures in the accompanying drawings, given purely by way of non-limiting example. The figures are not necessarily to scale, and certain features and certain views of the figures may be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

[0047] FIG. 1 A is an image of microcarriers formed with a drying solution comprising a calcium chelator (sodium citrate) that were dried with a rotary evaporator and then rehydrated with the volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0048] FIG. IB is an image of microcarriers formed with a drying solution comprising a calcium chelator (sodium citrate) that were dried with a rotary evaporator and then rehydrated with one-fourth of the volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0049] FIG. 1C is an image of microcarriers formed without any drying solution taken before drying, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0050] FIG. ID is an image of microcarriers formed without a drying solution comprising a calcium chelator (sodium citrate) that were dried with a rotary evaporator and then rehydrated with the same volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0051] FIG. 2A is an image of microcarriers formed with a drying solution comprising a calcium chelator (sodium citrate) that were dried by freeze-drying and then rehydrated with the volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0052] FIG. 2B is an image of microcarriers formed with a drying solution comprising a calcium chelator (sodium citrate) that were dried by freeze-drying and then rehydrated with one-fourth of the volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0053] FIG. 2C is an image of microcarriers formed without any drying solution taken before drying, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0054] FIG. 2D is an image of microcarriers formed without a drying solution comprising a calcium chelator (sodium citrate) that were dried by freeze-drying and then rehydrated with the same volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0055] FIG. 3A is an image of microcarriers formed without a drying solution comprising a positively charged molecule that were dried by freeze-drying and then rehydrated with the same volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0056] FIG. 3B is an image of microcarriers formed with a drying solution comprising a neutrally charged molecule (glucose) that were dried by freeze-drying and then rehydrated with the same volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0057] FIG. 3 C is an image of microcarriers formed with a drying solution comprising a positively charged molecule (triethanolamine chloride) that were dried by freeze-drying and then rehydrated with one-third of the volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0058] FIG. 3D is an image of microcarriers formed with a drying solution comprising a positively charged molecule (lysine chloride) that were dried by freeze-drying and then rehydrated with one-third of the volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0059] FIG. 3E is an image of microcarriers formed with a drying solution comprising a positively charged molecule (glucosamine) that were dried by freeze-drying and then rehydrated with one-third of the volume ratio of rehydration solution to dried microcarriers that has previously been required for rehydration, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.

[0060] FIG. 3F is an image of microcarriers formed without drying solution, taken prior to drying, according to aspects of the present disclosure. The image is from a phase contrast optical microscope at 4x magnification.DETAILED DESCRIPTION

[0061] The various aspects and embodiments will now be fully described herein. These aspects and embodiments may, however, be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided so the disclosure will be thorough and complete, and will fully convey the scope of the present subject matter to those skilled in theart. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0062] Modifications of the disclosure will occur to those skilled in the art and to those who make or use the disclosure. Therefore, it is understood that the embodiments shown in the drawings and described above are merely for illustrative purposes and not intended to limit the scope of the disclosure, which is defined by the following claims, as interpreted according to the principles of patent law, including the doctrine of equivalents.A. Definitions

[0063] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, particular methods and materials are now described.

[0064] As used herein the terms “the,” “a,” or “an,” mean “at least one,” and should not be limited to “only one” unless explicitly indicated to the contrary. Thus, for example, reference to “a component” includes embodiments having two or more such components unless the context clearly indicates otherwise.

[0065] Unless otherwise stated, the use of individual numerical values is stated as approximations as though the values were preceded by the word “about” or “approximately.” Similarly, the numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about” or “approximately.” In this manner, variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. As used herein, the terms “about” and “approximately” when referring to a numerical value shall have their plain and ordinary meanings to a person of ordinary skill in the art to which the disclosed subject matter is most closely related or the art relevant to the range or element at issue. The amount of broadening from the strict numerical boundary depends upon many factors. For example, some of the factors which may be considered includethe criticality of the element and / or the effect a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those of skill in the art. As used herein, the use of differing amounts of significant digits for different numerical values is not meant to limit how the use of the words “about” or “approximately” will serve to broaden a particular numerical value or range. Thus, as a general matter, “about” or “approximately” broaden the numerical value. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values plus the broadening of the range afforded by the use of the term “about” or “approximately.” Consequently, recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein.

[0066] As used herein, the term “and / or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing components A, B, and / or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0067] As used herein, “have,” “having,” “include,” “including,” “comprise,” “comprising” or the like are used in their open ended sense, and generally mean “including, but not limited to.”

[0068] “Optional” or “optionally” means that the subsequently described element, component or circumstance may or may not occur, so that the description includes instances where the element, component, or circumstance occurs and instances where it does not.

[0069] In this document, relational terms, such as first and second, top and bottom, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0070] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.B. Introduction

[0071] Microcarriers are tiny substrates that can be used to bind and grow adherent cells during culturing of those cells. For adherent cell cultures to proliferate (expand), adherent cells must attach to a substrate and be provided enough substrate surface area where growth can occur. Unlike traditional foot-print limited culturing methods for adherent cells such as petri dishes, well plates, and roller bottles, the foot-print for an adherent cell culture can be easily scaled up with microcarriers.

[0072] However, despite the ease of foot-print increase for adherent cell cultures with microcarriers, practical limitations in microcarrier manufacturing and use have limited microcarriers to smaller scale applications. Traditional microcarriers such as glass or resin beads must be removed from cell cultures and harsh enzymes such as proteases or alternatively mechanical removal is required to harvest the cells, which can be damaged by the proteases and / or mechanical harvesting. Hydrogel microcarriers offer a less harsh solution by optionally being dissolvable with non-protease enzymes, which not only offers a higher recovery of undamaged cells, but it also simplifies the harvesting step for isolating cultured cells. Yet even hydrogel microcarriers have drawbacks that make them cost prohibitive for large scale usage. These drawbacks include the manufacturing costs due to consumption of large quantities and volumes of raw materials used to make them. Further, the fragility of hydrogel microcarriers requires that they be shipped in a dehydrated form, which also includes the consumption of large quantities and volumes of materials used in the dehydration process in addition to the excess time it requires to perform the dehydration. On the user end, there are likewise inefficiencies in time, material volumes, and associated costs that are required for rehydration of the microcarriers.

[0073] There therefore exists a need to make the manufacturing and use of hydrogel microcarriers more time, resource, and cost efficient. Not only for laboratory-scale cell cultures, but for hydrogel microcarriers to be a viable option for large-scale cultures. The present disclosureprovides improved hydrogel microcarriers that help cut the manufacturing time and cost to make the microcarriers, as well decreasing the associated costs and time for rehydration.

[0074] Additional features and advantages will be set forth in the detailed description which follows and will be apparent to those skilled in the art from the description or recognized by practicing the embodiments as described in the following description, together with the claims and appended drawings.

[0075] Reference will now be made in greater detail to various embodiments of the subject matter of the present disclosure, some embodiments of which are illustrated in the accompanying drawings. The same reference numerals will be used throughout the drawings to refer to the same or similar parts.C. Microcarrier Compositions

[0076] Ionically cross-linked polysaccharide hydrogel microcarriers can be used as a substrate for attaching and growing adherent cells during a cell culture. Methods for forming hydrogel microcarriers are disclosed in commonly-assigned International Application No. PCT / US2014 / 043624, and in U.S. Patent Application No. PCT / US2016 / 036371, the contents of which are incorporated by reference herein in their entirety. Methods of drying, rehydrating, and sterilizing microcarriers using a saccharide and / or a monovalent cation in a drying formulation are disclosed in commonly-assigned U.S. Patent No. 10,900,021, the contents of which are incorporated by reference herein in their entirety.

[0077] Briefly, carboxyl-containing polysaccharides, such as polygalacturonic acid and alginic acid, can be crosslinked with divalent ions such as calcium cations to form stable hydrogels. The hydrogels can be created in the form of microcarriers, which are micron-sized substrates (such as a bead) that can be used for cell culture or for encapsulating living cells, either individually or with three-dimensional cell culture forms such as cysts, spheroids, organoids, etc.

[0078] Once cell cultures are grown or otherwise ready for use, crosslinked hydrogels (including microcarriers) can be digested (at least partially dissolved) by removing the calcium from the hydrogels using chelators such as ethylenediaminetetraacetic acid (EDTA). Thisfacilitates cell release without using proteinases, which can have an adverse effect on cell physiology.

[0079] Sterilization by high-energy irradiation (gamma, x-ray, e-beam), due to its process efficiency and depth of penetration, may be used to sterilize the hydrogels (including microcarriers). However, the presence of water or humidity during high-energy irradiation creates undesired free radicals, which can damage cells. Thus, for hydrogel microcarriers to be sterilized by high-energy irradiation, they must be dried prior to sterilization and then rehydrated after sterilization. Further, compared to hydrated microcarriers, dried microcarriers are easier and more economical to package, ship, and store.

[0080] The drying of polysaccharide hydrogels (crosslinked by divalent cations such as calcium) enables their sterilization for biotechnology applications such as large scale cell culture. Following sterilization, the hydrogels (including microcarriers) are ideally rehydrated and returned to their original (initial) geometry and properties. For hydrogels formed from polysaccharides like PGA or alginic acid, crosslinking occurs between the saccharide repeat units and the divalent cations. This crosslinking is typically reversible by removing the divalent cations from the hydrogel.

[0081] However, when polysaccharide hydrogels proceed through drying process, the crosslinking density increases as carboxyl groups in the polysaccharides become closer together as the drying process progresses. The increased crosslinking prevents the polymer from rehydrating back to its original hydrogel state, resulting in stunted rehydration and deformation of the microcarrier. It has been found that in large-scale processes for making and using dried microcarriers, large quantities of rinse agents are required to remove excess drying formulation, and large quantities of rehydration solution are required to accommodate the short rehydration times in large-scale processes. This is problematic both for the size of vessels required and for the increased costs incurred when manufacturing large scale quantities of dried microcarriers (e.g., the amounts needed for 30 L or 50 L manufacturing bioreactors) and when rehydrating large scale quantities of dried microcarriers. It has also been found that the larger volumes of liquid (volumes over 1 L) needed for rehydrating larger amounts of dried microcarriers is problematic because therehydration times are too short to get uniform rehydration in across the microcarriers in a single batch when larger quantities of dried microcarriers are used.

[0082] It would be advantageous to provide an even lower cost effective dried microcarrier and more efficient methods to dry, sterilize, and then fully rehydrate hydrogel microcarriers where the rehydrated microcarriers exhibit their initial geometry and mechanical properties.

[0083] Disclosed are dried microcarrier compositions, methods of making dried microcarriers, and methods of rehydrating microcarriers that are improved over currently available dried microcarriers and methods of making and rehydrating them.

[0084] The present disclosure provides microcarriers dried with either a calcium chelator or a positively charged molecule, or dried with both a calcium chelator and a positively charged molecule. These microcarriers result in a significant decrease in the amounts of rinse agents required during the drying process and in the amounts of rehydration solution required during the rehydration process while still yielding rehydrated microcarriers that retain at least 80% of their initial diameter upon rehydration.Dried Microcarriers

[0085] In their dried form, the microcarriers of the present disclosure comprise a pectin-based polysaccharide polymer, calcium, and either a calcium chelator or a positively charged molecule (or a combination thereof). Dried microcarriers are free of water, i.e., they are dry to a water content of no more than 10 wt.% (e.g., no more than 1, 2, 4, 5, or 10 wt.%, including any value or range between 0 wt.% and 10 wt.%). The pectin-based polysaccharide polymer may be polygalacturonic acid (PGA), or partly esterified polygalacturonic acid (PE PGA) with a degree of esterification of 40 mol% or less, salts of the foregoing, or a combination of the foregoing. In one embodiment, the microcarrier comprises PGA, or salts thereof. In another embodiment, the microcarrier comprises PE PGA (1-40 mol% esterification), or a salt thereof.

[0086] The composition of the dried microcarrier may comprise the pectin-based polysaccharide polymer in an amount between 20 wt.% and 80 wt.% of the total weight of the dried microcarrier, or at any value or in any range therebetween. For example, the dried microcarrier may comprise the pectin-based polysaccharide polymer in an amount between 25wt.% and 75 wt.%, between 35 wt.% and 75 wt.%, between 40 wt.% and 75 wt.%, between 45 wt.% and 70 wt.%, between 50 wt.% and 70 wt.%, between 55 wt.% and 70 wt.%, between 60 wt.% and 70 wt.%, between 20 wt.% and 65 wt.%, between 20 wt.% and 60 wt.%, between 20 wt.% and 55 wt. %, between 20 wt.% and 50 wt.%, between 20 wt.% and 45 wt.%, between 20 wt.% and 40 wt.%, between 40 wt.% and 60 wt.%, between 45 wt.% and 65 wt.%, or between 50 wt.% and 60 wt.% of the total weight of the dried microcarrier, or at any value or in any other range between the foregoing. In one specific embodiment, the dried microcarrier comprises the pectin-based polysaccharide polymer in an amount between 40 wt.% and 60 wt.% of the total weight of the dried microcarrier.

[0087] The calcium in the microcarrier acts as a crosslinker that crosslinks the pectin-based polysaccharide polymer when it is exposed to a water-soluble calcium salt (e.g., CaCE), or to an insoluble calcium salt (e.g., calcium carbonate) and an acid (e.g., acetic acid). In the case of an insoluble calcium salt and an acid, the acid dissociates the calcium from the insoluble calcium salt, leaving the calcium free to bind to the pectin-based polysaccharide polymer and cause ionotropic gelation. In some embodiments, the calcium in the microcarrier is from a water-soluble calcium salt, an insoluble calcium salt, or a combination thereof. Examples of water-soluble calcium salts include calcium chloride, calcium acetate, calcium gluconate, calcium lactate, and calcium bicarbonate, among others known to those of ordinary skill in the art. Example of insoluble calcium salts include calcium carbonate and hydroxyapatite, among others known to those of ordinary skill in the art. In some embodiments, the calcium in the microcarrier is from calcium chloride, calcium acetate, calcium gluconate, calcium lactate, calcium citrate, calcium bicarbonate, calcium carbonate, hydroxyapatite, or a combination thereof.

[0088] The composition of the dried microcarrier may comprise calcium in an amount between 3 wt.% and 10 wt.% of the total weight of the dried microcarrier, or at any value or in any range therebetween. In some embodiments, the dried microcarrier may comprise calcium in an amount between 3 wt.% and 9 wt.%, between 3 wt.% and 8 wt.%, between 3 wt.% and 7 wt.%, between 3 wt.% and 6 wt.%, between 3 wt.% and 5 wt.%, between 4 wt.% and 10 wt.%, between 5 wt.% and 10 wt. %, between 6 wt.% and 10 wt.%, between 7 wt.% and 10 wt.%, between 8 wt.% and 10 wt.%, between 4 wt.% and 7% of the total weight of the dried microcarrier, or at any value or in any other range between 3 wt.% and 10 wt.% of the total weight of the dried microcarrier. Inone specific embodiment, the dried microcarrier comprises calcium in an amount between 4 wt.% and 7 wt.%.

[0089] When the dried microcarrier comprises a calcium chelator, the calcium chelator may be any chelator of calcium, scavenger of calcium, or replacement ion (e.g., sodium) known to those of ordinary skill in the art. For example, the calcium chelator may be sodium citrate, ethylenediaminetetraacetic acid (EDTA), aspartic acid, (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N' -tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTP A), N(hydroxyl)-ethylenediaminetetraacetic acid (HETDA), salts of any of the foregoing, or combinations of any of the foregoing, among others known to those of ordinary skill in the art. In one specific embodiment, the calcium chelator may be sodium citrate, EDTA, or a combination thereof.

[0090] The composition of the dried microcarrier may comprise a calcium chelator in an amount greater than 0 wt.% and less than or equal to 10 wt.% of the total weight of the dried microcarrier, or at any value or in any range therebetween. In some embodiments, the dried microcarrier may comprise a calcium chelator in an amount greater than 0 wt.% and less than or equal to 9 wt.%, greater than 0 wt.% and less than or equal to 7 wt.%, greater than 0 wt.% and less than or equal to 6 wt.%, greater than 0 wt.% and less than or equal to 5 wt.%, greater than 0 wt.% and less than or equal to 4 wt.%, greater than 0 wt.% and less than or equal to 3 wt.%, greater than 0 wt.% and less than or equal to 2 wt.%, greater than 0 wt.% and less than or equal to 1 wt.%, greater than 0 wt.% and less than or equal to 0.75 wt.%, greater than 0 wt.% and less than or equal to 0.5 wt.%, greater than 0 wt.% and less than or equal to 0.25 wt.%, or greater than 0 wt.% and less than or equal to 0.1 wt.% of the total weight of the dried microcarrier. In one specific embodiment, the dried microcarrier comprises the calcium chelator in an amount greater than 0 wt.% and less than or equal to 5 wt.% of the total weight of the dried microcarrier.

[0091] As used herein, “a positively charged molecule” is a weak acid with a net positive charge (or water-soluble salts thereof), or a primary, secondary, or tertiary amine (or water-soluble salts thereof). In some embodiments, the positively charged molecule is a weak acid with a net positive charge, or water-soluble salts thereof. In some embodiments, the positively charged molecule is a primary, secondary, or tertiary amine, or water-soluble salts thereof. In some embodiments, the positively charged molecule is either a weak acid with a net positive charge, aprimary, secondary, or tertiary amine, or a water-soluble salt of the foregoing. In some embodiments, the positively charged molecule may be triethanolamine chloride, lysine chloride, glucosamine, or a combination thereof. In one specific embodiment, the positively charged molecule may be triethanolamine chloride. In another specific embodiment, the positively charged molecule may be lysine chloride. In yet another specific embodiment, the positively charged molecule may be glucosamine.

[0092] The composition of the dried microcarrier may comprise a positively charged molecule in an amount greater than 0 wt.% and less than or equal to 10 wt.% of the total weight of the dried microcarrier, or at any value or in any range therebetween. For example, the dried microcarrier may comprise a positively charged molecule in an amount greater than 0 wt.% and less than or equal to 9 wt.%, greater than 0 wt.% and less than or equal to 7 wt.%, greater than 0 wt.% and less than or equal to 6 wt.%, greater than 0 wt.% and less than or equal to 5 wt.%, greater than 0 wt.% and less than or equal to 4 wt.%, greater than 0 wt.% and less than or equal to 3 wt.%, greater than 0 wt.% and less than or equal to 2 wt.%, greater than 0 wt.% and less than or equal to 1 wt.%, greater than 0 wt.% and less than or equal to 0.75 wt.%, greater than 0 wt.% and less than or equal to 0.5 wt.%, greater than 0 wt.% and less than or equal to 0.25 wt.%, or greater than 0 wt.% and less than or equal to 0.1 wt.% of the total weight of the dried microcarrier. In one specific embodiment, the dried microcarrier comprises a positively charged molecule in an amount greater than 0 wt.% and less than or equal to 5 wt.% of the total weight of the dried microcarrier.

[0093] The dried microcarrier may comprise a filler. A “filler”, as used herein, is a molecule that acts to physically push apart parts of the polymer (e.g., the PGA or other pectin-based polymer), which prevents at least some crosslinking. Fillers include low molecular weight sugars, low molecular weight positively charged compound, and low molecular weight polyethylene glycols (PEG). As used herein, “low molecular weight PEG” refers to PEGs that have a melting point of less than about 25 °C (room temperature). As used herein a “low molecular weight sugar” is a monosaccharide or oligosaccharide with less than or equal to 10 sugar units. As used herein, “low molecular weight positively charged compound” means mono- or oligomolecules having 5 repeating units or less and no more than a net positive charge of +3. Examples of low molecular weight positively charged compounds include lysine, polylysine, and glucosamine, among others known to those of ordinary skill in the art. In some embodiments, thefiller may be glucose, glucosamine, low molecular weight polyethylene glycol (PEG), or combinations thereof. In some embodiments, the filler is a low molecular weight sugar, a low molecular weight positively charged compound, a low molecular weight PEG, or a combination thereof. In some embodiments, the filler may be a low molecular weight sugar, a low molecular weight positively charged compound, or a low molecular weight PEG.

[0094] The composition of the dried microcarrier may comprise filler in an amount between 20 wt.% and 80 wt.% of the total weight of the dried microcarrier, or at any value or in any range therebetween. For example, the dried microcarrier may comprise a filler in an amount between 20 wt.% and 70 wt.%, between 20 wt.% and 60 wt.%, between 20 wt. % and 50 wt.%, between 20 wt.% and 40 wt.%, between 20 wt.% and 30 wt.%, between 30 wt.% and 80 wt.%, between 40 wt.% and 80 wt.%, between 50 wt.% and 80 wt.%, between 60 wt.% and 80 wt.%, between 70 wt.% and 80 wt.%, between 30 wt.% and 75 wt.%, or between 40 wt.% and 70 wt.% of the total weight of the dried microcarrier. In one specific embodiment, the dried microcarriers comprises a filler in an amount between 30 wt.% and 60 wt.% of total wt.% of the dried microcarrier.

[0095] In some embodiments, a dried microcarrier comprises a calcium chelator and a filler. In a further embodiment, the filler is low molecular weight sugar. In some embodiments, a dried microcarrier comprises a positively charged molecule and a filler. In a further embodiment, the filler is a low molecular weight sugar or a low molecular weight positively charged compound. In a different embodiment, a dried microcarrier comprises a positively charged molecule and a filler, and the filler is different from the positively charged molecule. In yet another embodiment, a dried microcarrier comprises both a positively charged molecule and filler, and the filler is the same substance as the positively charged molecule. In this particular embodiment, because the filler and the positively charged molecule are the same, the total amount of the filler and positively charged molecule in the dried microcarrier is greater than or equal to 30 wt.% and less than or equal to 60 wt.%.

[0096] The dried microcarrier may comprise liquid lubricant. As used herein, a “lubricant” is a chemical that is liquid at room temperature, is water soluble, and has a boiling point of at least 120 °C. A lubricant helps protect microcarriers from being damaged when they are dried. Damagein microcarriers may include cracking, flaking, and blemishes that are visually apparent on the surface of microcarriers that have been rehydrated from a dried microcarrier form. Any suitable lubricant known to those of ordinary skill in the art may be used, including low molecular weight PEGs (e.g., PEG-400) and dimethylsulfoxide (DMSO). In some embodiments, the dried microcarrier may comprise a lubricant, and the lubricant may be a low-molecular weight PEG, DMSO, or a combination thereof. In another embodiment, the dried microcarrier may comprise a lubricant, and the lubricant is a low-molecular weight PEG or DMSO.

[0097] The composition of the dried microcarrier may comprise a lubricant in an amount between 0.1 wt.% and 30 wt.% of the total weight of the dried microcarrier, or at any value or in any range therebetween. For example, the dried microcarrier may comprise a lubricant in an amount between 0.1 wt.% and 25 wt.%, between 0.1 wt.% and 20 wt.%, between 0.1 wt. % and 15 wt.%, between 0.1 wt.% and 10 wt.%, between 0.1 wt.% and 5 wt.%, between 5 wt. % and 30 wt.%, between 10 wt.% and 30 wt.%, between 15 wt.% and 30 wt.%, between 20 wt.% and 30 wt.%, between 25 wt.% and 30 wt.%, or between 5 wt.% and 25 wt.% of the total weight of the dried microcarrier. In one specific embodiment, the dried microcarriers comprises a lubricant in an amount between 15 wt.% and 25 wt.% of total wt.% of the dried microcarrier, or at any range or in any range value therebetween.

[0098] It should be understood from the foregoing that fillers, lubricants, or both fillers and lubricants are optional components of the dried microcarriers. Accordingly, a dried microcarrier may not have either a filler or a lubricant, it may have either a filler or a lubricant, or it may have both a filler and a lubricant.

[0099] Dried microcarriers are generated from microcarriers that begin in an initially hydrated form, or as used herein. As used herein, the “initial form” of the microcarrier is the hydrated form of the microcarrier before any drying solutions have been applied. The diameter of an initial form of the microcarrier may be between 50 pm and 500 pm, or at any value or range therebetween. An average diameter of the initial form of the microcarriers may be determined by measuring the diameter (using a phase contrast optical microscope) of each of 90 microcarriers in an image and then calculating the average diameter of those 90 microcarriers. The initial form of the microcarrier may have an average diameter of 50 pm and 500 pm, or at any value or rangetherebetween. In some embodiments, either the diameter of an initial form of a microcarrier or the average diameter of the initial form of the microcarriers may be between 50 m and 450 pm, 50 pm and 400 pm, 50 pm and 350 pm 50 pm, and 300 pm, 50 pm and 250 pm, 50 pm and 200 pm, 50 pm and 150 pm, 50 pm and 100 pm, 100 pm and 500 pm, 150 pm and 500 pm, 200 pm and 500 pm, 250 pm and 500 pm, 300 pm and 500 pm, 350 pm and 500 pm, 400 pm and 500 pm, 450 pm and 500 pm, 100 pm and 400 pm, 100 pm and 300 pm, or 200 pm and 300 pm. In one specific embodiment, the diameter of the initial form of the microcarrier is between 100 pm and 300 pm.

[0100] Dried microcarriers are typically rehydrated before use, resulting in a “rehydrated” microcarrier form. The diameter of a rehydrated microcarrier may be between 50 pm and 500 pm, or at any value or range therebetween. An average diameter of rehydrated microcarriers may be determined using the same method as for the initial form of the microcarriers. In some embodiments the rehydrated form of the microcarrier may have an average diameter of 50 pm and 500 pm, or at any value or range therebetween. In yet other embodiments, either the diameter of a rehydrated form of a microcarrier or the average diameter of the rehydrated form of the microcarriers is between 50 pm and 450 pm, 50 pm and 400 pm, 50 pm and 350 pm 50 pm and 300 pm, 50 pm and 250 pm, 50 pm and 200 pm, 50 pm and 150 pm, 50 pm and 100 pm, 100 pm and 500 pm, 150 pm and 500 pm, 200 pm and 500 pm, 250 pm and 500 pm, 300 pm and 500 pm, 350 pm and 500 pm, 400 pm and 500 pm, 450 pm and 500 pm, 100 pm and 400 pm, 100 pm and 300 pm, or 200 pm and 300 pm. In one specific embodiment, the diameter of the rehydrated form of the microcarrier is between 100 pm and 300 pm.

[0101] The diameter of a rehydrated microcarrier may be characterized as a percentage of the diameter of microcarrier in its hydrated, initial form. In some embodiments, the diameter of a rehydrated form of the dried microcarrier may be the same diameter as the dried microcarrier’s initial form. In other words, the diameter of a rehydrated form of the dried microcarrier may be 100% of the diameter of the dried microcarrier’ s initial form. In some embodiments, the diameter of a rehydrated form of the dried microcarrier may be smaller than or equal to the diameter of the dried microcarrier’s initial form. For example, the diameter of a rehydrated form of the dried microcarrier may be between 50% and 100% of the diameter of the dried microcarrier’s initialform, or at any value or range therebetween. In some embodiments, the diameter of a rehydrated form of the dried microcarrier may be between 55% and 100%, between 60% and 100%, between 65% and 100%, between 70% and 100%, between 75% and 100%, between 80% and 100%, between 85% and 100%, between 90% and 100% or between 95% to 100% of the dried microcarrier’ s initial form. In one specific embodiment, the diameter of a rehydrated form of the dried microcarrier may be between 95% and 100% of the dried microcarrier’s initial form.

[0102] When the dried microcarrier is rehydrated, the rehydrated microcarrier has an amount of the pectin-based polymer per volume of the microcarrier. The amount of pectin-based polymer per volume of rehydrated microcarrier may be between 0.5 w / v% and 3.5 w / v%, or at any value or in any range therebetween. For example, the amount of pectin-based polymer per volume of rehydrated microcarrier is between 0.5 w / v% and 3.0 w / v%, between 0.5 w / v% and 2.5 w / v%, between 0.5 w / v% and 2.0 w / v%, between 0.5 w / v% and 1.5 w / v%, between 1.0 w / v% and 3.5 w / v%, between 1.5 w / v% and 3.5 w / v%, between 2.0 w / v% and 3.5 w / v%, between 2.5 w / v% and 3.5 w / v%, between 1.0 w / v% and 3.0 w / v%, between 1.5 w / v% and 2.5 w / v%, or in any range between these ranges. In one specific embodiment, the amount of pectin-based polymer per volume of rehydrated microcarrier is between 1.0 w / v% and 3.0 w / v%.

[0103] In some aspects of the disclosure, the dried microcarriers described herein are sterilized dried microcarriers. Sterilized dried microcarriers are dried microcarriers that have been irradiated with high-energy irradiation (gamma, x-ray, or e-beam). Sterilization of dried microcarriers may be performed with a dosage of high-energy irradiation that is greater than or equal to 5 kGy and less than or equal to 50 kGy dosage, or at any range or value therebetween. In some embodiments, a dried microcarrier is a sterilized microcarrier, wherein the dosage of high-energy irradiation (gamma, x-ray, or e-beam) is greater than or equal to 15 kGy and less than or equal to 30 kGy, or at any range or value therebetween (e.g., about 20 kGy). In one specific embodiment, a dried microcarrier as described herein is a sterilized dried microcarrier.D. Methods of Making Dried MicrocarriersFormation of Microcarriers1

[0104] Dried microcarriers are generated from hydrated microcarriers that are formed by mixing a precursor hydrogel solution with calcium to form a crosslinked hydrogel microcarrier. The hydrated, cross-linked microcarrier (the hydrated “initial form” of the microcarrier) may be formed by any method known to those of ordinary skill in the art, including those described in commonly-assigned International Application No. PCT / US2014 / 043624, and in commonly-assigned International Application No. PCT / US2016 / 036371, the contents of which are incorporated by reference herein in their entirety.

[0105] As part of the formation of the microcarriers, the microcarriers may be coated or otherwise treated for adherent cell culture, as known to those of ordinary skill in the art. Microcarriers that are formed and coated are still “initial forms” of the microcarrier as that term is used herein.Drying Solutions

[0106] Prior to drying, the initially formed, hydrated microcarriers are exposed to a calcium chelator and / or a positively charged molecule in at least one drying solution. When a calcium chelator is used with the initially formed, hydrated microcarriers prior to and / or during drying of the microcarriers, the calcium chelator may partially remove calcium from the initially formed, hydrated microcarriers. The use of a calcium chelator unexpectedly improves rehydration of the microcarriers as compared to the same microcarriers without the use of the calcium chelator. A dried microcarrier with improved rehydration is a dried microcarrier that requires a decreased volume of a rehydration solution to rehydrate the microcarrier (rehydration solution) and / or has decreased visible damage (cracks, etc.) in its rehydrated form. The improvement is unexpected at least because calcium chelators destabilize ionotropically crosslinked hydrogels, thereby dissolving them.

[0107] When a positively charged molecule is used with the initially formed, hydrated microcarriers prior to and / or during drying of the microcarriers, the positively charged molecule may prevent or displace calcium ions from binding to the polymer prior to and / or during the drying of the microcarriers. The use of a positively charged chelator unexpectedly improves rehydration of the microcarriers. The improvement is unexpected at least because destabilizing ionotropically crosslinked hydrogels removal calcium crosslinkers or preventing calcium ions from crosslinkingdestabilizes the ionotropically crosslinked hydrogels, making it more likely for the hydrogels to dissolve or be damaged during drying and upon rehydration.

[0108] The calcium chelators may be any of those described earlier for the dried microcarriers, including those known to those of ordinary skill in the art. In some embodiments, the drying solution may comprise a calcium chelator with a concentration between 1 mM and 50 mM, or at any value or in any range therebetween. For example, the drying solution may comprise a calcium chelator with a concentration of between 2 mM and 25 mM, or at any value or in any range therebetween. As another example, the drying solution may comprise a calcium chelator with a concentration of between 1 mM and 45 mM, between 1 mM and 40 mM, between 1 mM and 35 mM, between 1 mM and 30 mM, between 1 mM and 25 mM, between 1 mM and 20 mM, between 1 mM and 15 mM, between 1 mM and 10 mM, between 5mM and 50 mM, between 10 mM and 50 mM, between 15 mM and 50 mM, between 20 mM and 50 mM, between 25 mM and 50 mM, between 30 mM and 50 mM, between 35 mM, and 50 mM, between 40 mM and 50 mM, or at any value or in any range between the foregoing. In one specific embodiment, the drying solution may comprise a calcium chelator with a concentration of between 4 mM and 20 mM, or at any value or in any range therebetween.

[0109] The amount of calcium chelated by the calcium chelator may be up to about 50% of the calcium in the hydrated, initial form of the microcarrier. In some embodiments, the amount of calcium chelated from the hydrated, initial form microcarriers by the calcium chelator may be between 0.1% and 50% of the calcium in the initial form of the microcarrier, or at any value or in any range therebetween. It should be understood that the amount of calcium chelated is from the microcarrier beads themselves. In other embodiments, the amount of calcium chelated may be between 10% and 50% of the calcium in the initial form of the microcarrier, or at any value or in any range therebetween. In one embodiment, the amount of calcium chelated by the calcium chelator is between 0.5% and 50%, between 5% and 50%, between 15% and 50%, between 20% and 50%, between 25% and 50%, between 30% and 50%, between 35% and 50%, between 40% and 50%, between 45% and 50%, between 0.5% and 45%, between 0.5% and 40%, between 0.5% and 35%, between 0.5% and 30%, between 0.5% and 25%, between 0.5% and 20%, between 0.5% and 15%, between 0.5% and 10%, between 0.5% and 0.5%, between 5% and 45%, between 10% and 45%, between 15% and 45%, or between 20% and 40% of the calcium in the initial form ofthe microcarrier. In a preferred embodiment, the amount of calcium chelated by the calcium chelator is between 25% and 40% of the calcium in the initial form of the microcarrier.

[0110] The amount of calcium in a microcarrier can be measured by inductively coupled plasma (I CP) mass spectroscopy, as is understood by those of ordinary skill in the art. ICP mass spectroscopy is an elemental analysis technique so measures for elements like calcium. One such ICP mass spectrometer (ICP-MS) is the 7700 ICP-MS from Agilent™.

[0111] When a positively charged molecule is used in the drying solution, the positively charged molecule may be any of those described above for the dried microcarriers, including those known to those of ordinary skill in the art. The drying solution may comprise a positively charged molecule with a concentration of between 0.1 w / v% and 10 w / v%, or at any value or in any range therebetween. For example, the drying solution may comprise a positively charged molecule with a concentration of between 0.1 w / v% and 9 w / v%, 0.1 w / v% and 8 w / v%, 0.1 w / v% and 7 w / v%, 0.1 w / v% and 6 w / v%, 0.1 w / v% and 5 w / v%, 2 w / v% and 10 w / v%, 4 w / v% and 10 w / v%, 6 w / v% and 10 w / v%, 8 w / v% and 10 w / v%, or at any value or in any range of the foregoing. As another example, the drying solution may comprise a positively charged molecule with a concentration of between 0.1 w / v% and 4 w / v%, between 0.1 w / v% and 3 w / v%, between 0.1 w / v% and 2 w / v%, between 0.1 w / v% and 1 w / v%, between 0.5 w / v% and 5 w / v%, between 1 w / v% and 5 w / v%, between 2 w / v% and 5 w / v%, between 3 w / v% and 5 w / v%, between 4 w / v% and 5 w / v%, between 0.5 w / v% and 3 w / v%, and between 0.5 w / v% and 2 w / v%, or at any value or in any range between the foregoing. In one specific embodiment, the drying solution may comprise a positively charged molecule with a concentration of between 0.4 w / v% and 3 w / v% or at any value or in any range therebetween.

[0112] In some embodiments, a drying solution may comprise an alcohol. The alcohol may be any type of alcohol that helps prevent the exteriors of the microcarriers from sticking to each other during the drying process, including but not limited to isopropanol and ethanol. In some embodiments, a drying solution comprises isopropanol, ethanol, or a combination thereof.

[0113] In some embodiments, a drying solution may comprise a filler, a lubricant, or a combination thereof. Fillers may assist in the swelling of microcarriers in the rehydration process and / or in small quantities, may help prevent damage to the microcarriers during the drying process.A lubricant may help prevent damage to the microcarriers during the drying process. In some embodiments, more than one filler may be present in the drying solution. In some embodiments, more than one lubricant may be present in the drying solution. In yet some other embodiments, more than one filler and more than one lubricant may be present in drying solution. However, it should be understood that in some embodiments, no filler and / or no lubricant may present in the drying solution.

[0114] Fillers and lubricants for a drying solution may be any of the ones described earlier for dried microcarriers, among others known to those of ordinary skill in the art. When present in a drying solution, a filler may be present in the drying solution in a concentration of between 0.1 w / v% and 10 w / v%, or at any value or in any range therebetween. For example, the drying solution may comprise a filler with a concentration of between 0.1 w / v% and 9 w / v%, between 0.1 w / v% and 8 w / v%, between 0.1 w / v% and 7 w / v%, between 0.1 w / v% and 6 w / v%, between 0.1 w / v% and 5 w / v%, between 0.1 w / v% and 4 w / v%, between 0.1 w / v% and 3 w / v%, between 0.1 w / v% and 2 w / v%, between 1 w / v% and 10 w / v%, between 2 w / v% and 10 w / v%, between 3 w / v% and 10 w / v%, between 4 w / v% and 10 w / v%, between 5 w / v% and 10 w / v%, between 6 w / v% and 10 w / v%, between 7 w / v% and 10 w / v%, between 8 w / v% and 10 w / v%, between 9 w / v% and 10 w / v%, between 2.5 w / v% and 7.5 w / v%, or at any value or in any range between the foregoing. In one specific embodiment, the drying solution may comprise a filler with a concentration of between 0.1 w / v% and 7.5 w / v%, or at any value or in any range therebetween.

[0115] When present in a drying solution, a lubricant may be present in the drying solution in a concentration of between 0.1 w / v% and 10 w / v%, or at any value or in any range therebetween. For example, the drying solution may comprise a lubricant with a concentration of between 0.1 w / v% and 9 w / v%, between 0.1 w / v% and 8 w / v%, between 0.1 w / v% and 7 w / v%, between 0.1 w / v% and 6 w / v%, between 0.1 w / v% and 5 w / v%, between 0.1 w / v% and 4 w / v%, between 0.1 w / v% and 3 w / v%, between 0.1 w / v% and 2 w / v%, between 1 w / v% and 10 w / v%, between 2 w / v% and 10 w / v%, between 3 w / v% and 10 w / v%, between 4 w / v% and 10 w / v%, between 5 w / v% and 10 w / v%, between 6 w / v% and 10 w / v%, between 7 w / v% and 10 w / v%, between 8 w / v% and 10 w / v%, between 9 w / v% and 10 w / v%, between 2.5 w / v% and 7.5 w / v%, or at any value or in any range between the foregoing. In one specific embodiment, the drying solution maycomprise a lubricant with a concentration of between 0.1 w / v% and 7.5 w / v%, or at any value or in any range therebetween.

[0116] After the hydrated, initial form of the microcarriers have been made and before the microcarriers are physically dried (e.g., by freeze drying, lyophilization, rotary evaporation, etc.), the microcarriers are subjected to at least one drying solution that contains at least a calcium chelator or a positively charged molecule. This imparts improved rehydration characteristics when the dried microcarriers are rehydrated. However, it should be understood that more than one drying solution may be used during this stage and each drying solution may have the same composition or may have different compositions, so long as at least one drying solution contains a calcium chelator or a positively charged molecule. For example, a drying solution comprising an alcohol and no calcium chelator, positively charged molecule, filler, or lubricant may be used as a rinse between drying solutions that comprise one ore more of a calcium chelator, a positively charged molecule, a filler, and a lubricant. As another example, a microcarrier may be exposed to a drying solution comprising and alcohol and either a calcium chelator or a positively charged molecule, and then be exposed to a drying solution comprising a filler, a lubricant, or both a filler and a lubricant. Further aspects of the methods of drying (which includes exposures to drying solutions as well as the physically drying step) will now be described.Methods of Drying

[0117] Methods of drying microcarriers are now provided. In one aspect of the disclosure, a method of drying microcarriers include the steps of (a) providing an initial form of a microcarrier comprising calcium and one of polygalacturonic acid, partly esterified polygalacturonic acid having a degree of esterification of 40 mol% or less, a salt of any of the foregoing, or a combination of any of the foregoing; (b) providing a drying solution comprising a calcium chelator, a positively charged molecule, or a combination thereof; (c) exposing the initially formed microcarriers to the drying solution; and then (d) drying the microcarriers from step (c). In some further embodiments, the initially formed microcarrier beads comprising calcium may be drained of any surrounding solution with a sieve or filters (or other like screen) with pores sized to separate the initially formed microcarriers from the solution containing the microcarriers. This can be done with, or between, the above steps. This can also be performed between (or during) exposing the microcarriers torinses and / or to more than one drying solution. The draining of surrounding solution from the microcarriers is not a required step but it reduces the amount fluid used for exchanging out different fluid compositions and any drying solution that is required to dry the microcarriers.

[0118] In some further embodiments, the initially formed microcarriers are exposed to a drying solution of water, an alcohol (e.g., isopropanol, ethanol), or a mixture thereof. This may be done before, with, and / or after exposing the initially formed microcarriers to the drying solution that comprises the calcium chelator, the positively charged molecule, or a combination thereof. For example, in one embodiment, the initially formed microcarriers are exposed to a drying solution of water, which is then removed, and then the microcarriers are exposed to a drying solution comprising alcohol (e.g., isopropanol or ethanol). In another embodiment, the initially formed microcarriers are exposed to a drying solution of an alcohol or a mixture of water and alcohol before the microcarriers are exposed to a drying solution comprising an alcohol and either a calcium chelator or a positively charged molecule.

[0119] In one particular embodiment, the initially formed microcarriers are exposed to a first drying solution comprising a calcium chelator or a positively charged molecule (or both), and then are exposed to a second drying solution comprising a filler, a lubricant, or both a filler and a lubricant. In a further embodiment, the second drying solution further comprises an alcohol.

[0120] In another particular embodiment, the initially formed microcarriers are exposed to a first drying solution comprising a calcium chelator or a positively charged molecule (or both), and then are exposed to a second drying solution comprising a filler, and are then exposed to a third drying solution comprising a lubricant. In a further embodiment, the third drying solution comprises an alcohol.

[0121] The exposing of the initially formed microcarriers to a drying solution may occur for a time between 1 minute and 2 days, or at any amount of time or in any range therebetween. In a preferred embodiment, the exposing of the initially formed microcarriers to a drying solution may occur for a time between 1 minute and 6 hours, or at any amount of time or in any range therebetween. The exposure itself may be by soaking the microcarriers in the drying solution, rinsing the microcarriers with the drying solution, or by any other method of contacting the microcarriers with the drying solution known to those of ordinary skill in the art. The step ofexposing of the initially formed microcarriers in the drying solution may include a further step of stirring the microcarriers in the drying solution.

[0122] It should be understood that either before or after the step of exposing the hydrated, formed microcarriers to the drying solution, the microcarriers can be exposed to one or more different drying solutions. For example, after generating the initially formed microcarriers, the fluid surrounding the microcarriers can be drained and then a first drying solution of water, alcohol, or a mixture of both may be added to the microcarriers, exposing the microcarriers to the first drying solution. After a chosen time, the first drying solution can be discarded, and a second drying solution may be added to the microcarriers that comprises a calcium chelator, a positively charged molecule, or both. The second drying solution may be discarded, and a third drying solution comprising a filler, a lubricant, or both, may be added to the microcarriers. This is merely exemplary, fewer or greater numbers of drying solution may be used and in other orders than the foregoing example.

[0123] It should also be understood that the concentrations for the drying solution described herein are of the concentrations before adding the drying solutions to the microcarriers. Approximately equivalent volumes of drying solution and packed microcarriers are used when treating the microcarriers. The volume of packed microcarriers refers to the volume of microcarriers present after they have settled by gravity.

[0124] The step of drying of the microcarriers that have been exposed to one or more of the drying solutions may occur by rotary evaporation, freeze drying, lyophilization, a combination thereof, or by any other drying technique known to those of ordinary skill in the art. In some embodiments, the drying of the microcarriers exposed to the drying solution may include rotary evaporation, lyophilization, or a combination thereof. In other embodiments, the drying of the microcarriers exposed to the drying solution may include freeze drying, lyophilization or a combination thereof.

[0125] After the step of drying the microcarriers, in some embodiments, the dried microcarriers are sterilized. Sterilization may be performed with a dosage of high-energy irradiation (gamma, x-ray, or e-beam) that is greater than or equal to 5 kGy and less than or equal to 50 kGy dosage, or at any value or in any range therebetween. In one embodiment, sterilizationis performed with a dosage of high-energy irradiation (gamma, x-ray, or e-beam) greater than or equal to 15 kGy and less than or equal to 30 kGy, or at any value or in any range therebetween. In one specific embodiment, the sterilization is performed with a dosage of gamma irradiation of between 10 kGy and 25 kGy.

[0126] An unexpected advantage of the method with the microcarriers of the present disclosure is the significant decrease in volumes of alcohol required throughout the process in embodiments where alcohol is used in drying solutions. This is particularly advantageous for large scale processes where vessel sizes and costs can otherwise be prohibitive to being able to create microcarriers in mass quantities. In one aspect of the disclosure, using a drying solution that comprises a calcium chelator and / or a positively charged molecule decreases the amount of alcohol needed for making the dried microcarriers by 10% to 80% as compared to the same process for making a microcarrier but without the calcium chelator and / or positively charged molecule. For example, using a drying solution that comprises a calcium chelator and / or a positively charged molecule decreases the amount of alcohol needed for making the dried microcarriers by between 10% and 70%, between 10% and 60%, between 10% and 50%, between 10% and 40%, between 10% and 30%, between 10% and 20%, between 20% and 80%, between 30% and 80%, between 40% and 80%, between 50% and 80%, between 60% and 80%, between 70% and 80%, between 30% and 70%, or at any value or in any range between 10% and 80%, as compared to the same process for making a microcarrier but without the calcium chelator and / or positively charged molecule.

[0127] Another unexpected advantage of the method with the microcarriers of the present disclosure is the significant decrease in the amount of filler required throughout the process in embodiments where filler is used in drying solutions. This is particularly advantageous for large scale processes where costs for large amounts of fillers can otherwise be prohibitive to being able to create microcarriers in mass quantities. In one aspect of the disclosure, using a drying solution that comprises a calcium chelator and / or a positively charged molecule decreases the amount of filler needed for making the dried microcarriers by greater than 5% as compared to the same process for making a microcarrier but without the calcium chelator and / or positively charged molecule. For example, using a drying solution that comprises a calcium chelator and / or a positively charged molecule decreases the amount of filler needed for making the driedmicrocarriers by greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%, as compared to the same process for making a microcarrier but without the calcium chelator and / or positively charged molecule.E. Methods of Rehydrating Microcarriers

[0128] Methods of rehydrating dried microcarriers (sterilized or unsterilized) will now be described. The method comprises the steps of (a) providing dried microcarriers, (b) providing an aqueous solution, (c) combining the dried microcarriers with the aqueous solution, and (d) rehydrating the dried microcarriers to create rehydrated microcarriers. For the step of providing a dried microcarrier, any of the dried microcarriers of the present disclosure comprising a calcium chelator and / or a positively charged molecule (or made with a drying solution comprising a calcium chelator and / or a positively charged molecule) may be used. For the step of providing an aqueous solution, the aqueous solution may be water (including but not limited to deionized, purified, and tap water), a buffer (including but not limited to Tris-HCl, phosphate buffered saline (PBS), 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), and 3-(N-Morpholinopropanesulfonic acid) (MOPS)), cell culture medias (including but not limited to Dulbecco's Modified Eagle Medium (DMEM), minimal essential medium (MEM), and Roswell Park Institute’s RPMI 1640 media), and other aqueous solutions suitable for rehydrating the dried microcarriers known to those of ordinary skill in the art.

[0129] Regarding the step of combining the dried microcarriers with the aqueous medium, this may be performed in a vessel where the dried microcarriers are added to an aqueous solution in the vessel or vice-versa. A maximum concentration of microcarriers in the aqueous solution can be determined by dividing the weight of microcarriers added to a vessel by the minimum volume of aqueous solution (e.g., water, buffer, cell culture media) for rehydration that can be added and still achieve rehydrated microcarriers. As used herein, the “rehydration solution” is the aqueous solution used for rehydration of dried microcarriers. If, for example, if the weight of dried microcarriers added to a vessel is 200 mg and the minimum volume that can be added and still achieve rehydrated microcarriers is 2 mL, then the concentration of dried microcarrier in rehydration solution (i.e., the maximum “microcarrier rehydration concentration”) would be 100mg / mL. The microcarrier rehydration concentration can be determined for concentrations other than the maximum microcarrier concentration. For example, if the weight of dried microcarriers added to a vessel is 200 mg and the volume of rehydration solution used to achieve rehydration of the microcarriers is 10 mL, then the microcarrier rehydration concentration would be 20 mg / mL.

[0130] In some embodiments, for the step of combining dried microcarriers with the aqueous medium where the dried microcarriers either comprise or are made with a calcium chelator, the maximum microcarrier rehydration concentration may be greater than or equal to 10 mg / mL. In some embodiments, the maximum microcarrier rehydration concentration for dried microcarriers either comprising or that are made with a calcium chelator may be between 10 mg / mL and 150 mg / mL, or at any value or in any range therebetween. In some embodiments, the maximum microcarrier rehydration concentration for dried microcarriers either comprising or that are made with a calcium chelator may be greater than or equal to 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, or more. In some embodiments, the maximum concentration of dried microcarrier in aqueous solution where the dried microcarriers either comprise or are made with a calcium chelator may be between 10 mg / mL and 120 mg / mL, between 20 mg / mL and 110 mg / mL, between 30 mg / mL and 100 mg / mL, between 15 mg / mL and 100 mg / mL, between 25 mg / mL and 100 mg / mL, between 50 mg / mL and 100 mg / mL, between 65 mg / mL and 90 mg / mL, or at any value or in any range between 10 mg / mL and 120 mg / mL.

[0131] In other embodiments, for the step of combining dried microcarriers with the aqueous medium where the dried microcarriers either comprise or are made with a positively charged molecule, the maximum microcarrier rehydration concentration may be greater than or equal to 3 mg / mL. In some embodiments, the maximum microcarrier rehydration concentration for dried microcarriers either comprising or that are made with a positively charged molecule may be between 3 mg / mL and 100 mg / mL, or at any value or in any range therebetween. In some embodiments, the maximum microcarrier rehydration concentration for dried microcarriers either comprising or that are made with a positively charged molecule may be greater than or equal to 4 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, or more. In some embodiments, the maximum concentration of dried microcarrier in aqueous solution where thedried microcarriers either comprise or are made with a positively charged molecule may be between 3 mg / mL and 75 mg / mL, between 3 mg / mL and 50 mg / mL, between 3 mg / mL and 25 mg / mL, between 5 mg / mL and 50 mg / mL, between 25 mg / mL and 100 mg / mL, between 25 mg / mL and 75 mg / mL, or at any value or in any range therebetween.

[0132] An unexpected advantage of the dried microcarriers comprising a calcium chelator and / or a positively charged molecule (or that are made with a drying solution comprising a calcium chelator and / or a positively charged molecule) of the present disclosure is that significantly less aqueous solution is required to rehydrate dried microcarriers as compared to dried microcarriers without a calcium chelator and / or a positively charged molecule (or that are made without a drying solution comprising a calcium chelator and / or a positively charged molecule). This means that the maximum microcarrier rehydration concentration is higher with the microcarriers of the present disclosure than previous microcarriers. This in turn decreases the operational costs to any users of the dried microcarriers.

[0133] A dried microcarrier is rehydrated when it achieves at least 80% of the diameter of the microcarrier’s hydrated, initial form. A rehydrated microcarrier can be observed with a phase contrast optical microscope in brightfield mode (typically at 4x magnification for a microcarrier having a diameter between 50-500 pm when in either a rehydrated or initial form). Under phase contrast optical microscopy in brightfield mode, a rehydrated microcarrier will have a light perimeter and an interior that is, on average, darker than the perimeter. Examples of rehydrated microcarriers include those in FIGS. 1 A-1B, 2A-2B, and 3C-E. When dried microcarriers are not rehydrated (i.e., no hydration or partial hydration), the microcarriers may have jagged or sharp edges, may have dark perimeters, may have light interiors combined with light perimeters, may have dark perimeters and dark interiors, and combinations thereof. Examples of microcarriers that are not rehydrated or are partially rehydrated are shown in FIGS. ID, 2D, and 3A-3B.

[0134] In some embodiments, the minimum volume of aqueous solution required for rehydrating dried microcarriers comprising a calcium chelator and / or a positively charged molecule may be at least 10% less than the minimum volume of aqueous solution required for rehydrating the same dried microcarriers without a calcium chelator and / or a positively charged molecule. In some embodiments, the minimum volume of aqueous solution for required forrehydrating dried microcarriers comprising a calcium chelator and / or a positively charged molecule may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the minimum volume of aqueous solution required for rehydrating the same dried microcarriers without a calcium chelator and / or a positively charged molecule. In one specific embodiment, the minimum volume of aqueous solution required for rehydrating dried microcarriers comprising a calcium chelator and / or a positively charged molecule may be between 20% and 90% less than the minimum volume of aqueous solution required for rehydrating dried microcarriers without a calcium chelator and / or a positively charged molecule, or at any value or in any range therebetween.

[0135] In some embodiments, the minimum volume of aqueous solution required for rehydrating dried microcarriers made with a drying solution comprising a calcium chelator and / or a positively charged molecule may be at least 10% less than the minimum volume of aqueous solution required for rehydrating the same dried microcarriers made without a drying solution comprising a calcium chelator and / or a positively charged molecule. In some embodiments, the minimum volume of aqueous solution for required for rehydrating dried microcarriers made with a drying solution comprising a calcium chelator and / or a positively charged molecule may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the minimum volume of aqueous solution required for rehydrating the same dried microcarriers made without a drying solution comprising a calcium chelator and / or a positively charged molecule. In one specific embodiment, the minimum volume of aqueous solution required for rehydrating dried microcarriers made with a drying solution comprising a calcium chelator and / or a positively charged molecule may be between 20% and 90% less than the minimum volume of aqueous solution required for rehydrating dried microcarriers made without a drying solution comprising a calcium chelator and / or a positively charged molecule, or at any value or in any range therebetween.

[0136] A second unexpected advantage of the dried microcarriers comprising (or made with) a calcium chelator and / or a positively charged molecule of the present disclosure is that the amount of time needed to rehydrate the microcarriers is significantly reduced. As used herein, the minimum time required to rehydrate dried microcarriers refers to amount of time required to rehydrate a dried microcarrier to a diameter that is at least 95% of the diameter of the microcarrier’ s initial form. The minimum time required to rehydrate dried microcarriers comprising a calciumchelator and / or a positively charged molecule may be at least 10% less than the minimum time required to rehydrate the same dried microcarriers without a calcium chelator and / or a positively charged molecule. In some embodiments, the minimum time required to rehydrate dried microcarriers comprising a calcium chelator and / or a positively charged molecule may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the minimum time required to rehydrate the same dried microcarriers without a calcium chelator and / or a positively charged molecule. In one specific embodiment, the minimum time required to rehydrate dried microcarriers comprising a calcium chelator and / or a positively charged molecule may be between 20% and 90% less than the minimum time to rehydrate the same dried microcarriers without a calcium chelator and / or a positively charged molecule, or at any value or in any range therebetween.

[0137] In some embodiments, the minimum time required to rehydrate dried microcarriers made with a drying solution comprising a calcium chelator and / or a positively charged molecule may be at least 10% less than the minimum time required to rehydrate the same dried microcarriers made without a drying solution a comprising calcium chelator and / or a positively charged molecule. In some embodiments, the minimum time required to rehydrate dried microcarriers made with a drying solution comprising a calcium chelator and / or a positively charged molecule may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the minimum time required to rehydrate the same dried microcarriers made without a drying solution comprising calcium chelator and / or a positively charged molecule. In one specific embodiment, the minimum time required to rehydrate dried microcarriers made with a drying solution comprising a calcium chelator and / or a positively charged molecule may be between 20% and 90% less than the minimum time to rehydrate the same dried microcarriers made without a drying solution comprising calcium chelator and / or a positively charged molecule, or at any value or in any range therebetween.

[0138] While the present disclosure includes a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the present disclosure.EXAMPLES

[0139] The embodiments described herein will be further clarified by the following examples.Example 1

[0140] Hydrated, formed microcarriers were made by generating drops of 1.75 wt.% polygalacturonic acid sodium salt solution with laminar jet vibration (NISCO™) and submerging them in an aqueous solution that included 4 wt.% CaCh and 1.5 wt.% Tween 80. The hydrated, newly formed microcarriers were photographed with a phase contrast optical microscope in brightfield mode at 4x magnification, as shown in FIGS. 1C, 2C and 3F. In these figures, the perimeter of the hydrated, initial form of the microcarriers are light in color and the interior of the initial form of the microcarriers is, on average, darker in color than the perimeter. The lightness of the perimeter and the determination that the interior is, on average, darker than the perimeter was performed by visual observation of the 4x magnified image. The crosslinked hydrogel microcarriers ranged in diameter from approximately 220 pm to 240 pm with an average diameter of 230 pm as measured by optical microscope in brightfield mode.Example 2

[0141] 100 mL packed volume of hydrated, formed microcarriers from Example 1 were filtered (pore size 100-200 pm) from the aqueous solution. 100 mL of 10 mM sodium citrate (calcium chelator) was then added to the microcarriers, and the slurry was stirred gently with an impeller mixer at 100 rpm for 15 minutes. The citrate solution was then drained from the microcarriers. Next, 100 mL of isopropanol was added to the microcarriers, and the slurry was stirred gently (100 rpm, impeller mixer) for 15 minutes. The isopropanol rinse was then drained from the microcarriers. Then, 100 mL of isopropanol with 5 w / v% PEG400 was added to resuspend the microcarriers, and the slurry was transferred to a 500 mL round bottom flask to dry in rotary evaporator until the microcarriers were a powder. The powder was then placed in a manifold lyophilizer to vacuum dry for 24 hours.Example 3

[0142] The process of Example 2 was repeated, except that no sodium citrate solution was added to the microcarriers, and no sodium citrate solution was filtered from the microcarriers.Example 4

[0143] 100 mL packed volume of hydrated, formed microcarriers from Example 1 were filtered (pore size 100-200 pm) from the aqueous solution. 100 mL of 10 mM sodium citrate was then added to the microcarriers, and the slurry was stirred gently (100 rpm, impeller mixer) for 15 minutes. The citrate solution was then drained from the microcarriers. Next, 100 mL of a 3.5 wt.% glucose solution was added to the drained microcarriers, and the slurry was stirred gently (100 rpm, impeller mixer) for 15 minutes. The glucose solution was then drained from the microcarriers. Next, 100 mL of isopropanol with 2 w / v% PEG400 was added to resuspend the microcarriers, and then the slurry was transferred to a screen with a 45 pm pore size and the isopropanol / PEG400 solution was drained. The screen with the retained microcarriers was frozen at -20 °C overnight. The powder was then placed in a manifold lyophilizer to vacuum dry for 48 hours.Example 5

[0144] The process of Example 4 was repeated, except that no sodium citrate solution was added to the microcarriers, and no sodium citrate solution was filtered from the microcarriers.Example 6

[0145] 100 mL packed volume of hydrated, formed microcarriers from Example 1 were filtered (pore size 100-200 pm) from the aqueous solution. 100 mL of a 1 wt.% glucose solution was added to the drained microcarriers, and the slurry was stirred gently (100 rpm, impeller mixer) for 15 minutes. The glucose solution was then drained from the microcarriers. Next, 100 mL of 50 v / v% isopropanol and 50% v / v% of 2 wt.% PEG 400 was added to resuspend the microcarriers, and then the slurry was transferred to a screen with a 45 pm pore size and the isopropanol / PEG400 solution was drained. The screen with the retained microcarriers was frozen at -20 °C overnight. The powder was then placed in a manifold lyophilizer to vacuum dry for 72 hours.Example 7

[0146] The process of Example 6 was repeated, except 1% triethanolamine chloride solution was used in place of the 1% glucose solution.Example 8

[0147] The process of Example 6 was repeated, except 1% lysine chloride solution was used in place of the 1% glucose solution.Example 9

[0148] The process of Example 6 was repeated, except 1 % glucosamine solution was used in place of the 1% glucose solution.Example 10

[0149] The process of Example 6 was repeated, except the no glucose solution was added to the microcarriers and no glucose solution was filtered from the microcarriers.Example 11

[0150] Rehydration was investigated for 120 mg of the dried microcarriers from Example 2 (a drying solution with sodium citrate (a calcium chelator) and drying by rotary evaporation and a manifold lyophilizer) that were rehydrated in either 2 mL or 8 mL of deionized water. The 8 mL volume corresponds to previously required volumes to rehydrate dried microcarriers. More specifically, 120 mg of dried microcarriers from Example 2 were rehydrated in 8 mL of deionized water for a microcarrier rehydration concentration of 15 mg / mL. The microcarriers rehydrated within 15 minutes and the rehydrated microcarriers were photographed with a phase contrast optical microscope at 4x magnification (FIG. 1 A). Separately, 120 mg of dried microcarriers from Example 2 were rehydrated in 2 mL of deionized water for a microcarrier rehydration concentration of 60 mg / mL. The microcarriers rehydrated within 15 minutes and the rehydrated microcarriers were photographed with a phase contrast optical microscope at 4x magnification (FIG. IB). FIGS. 1A-1B depict approximately spherical, rehydrated microcarriers with each microcarrier having (by visual observation) a light perimeter and an interior, on average, that is darker than the perimeter.Example 12

[0151] Rehydration was investigated for 120 mg dried microcarriers from Example 3 (no sodium citrate and drying by rotary evaporation a manifold lyophilizer) that were rehydrated in 2 mL of deionized water, for a microcarrier rehydration concentration of 60 mg / mL. The microcarriers did not rehydrate with rehydration solution. The microcarriers were then photographed with a phase contrast optical microscope at 4x magnification (FIG. ID). As seen in FIG. ID, the microcarriers did not rehydrate in the absence of the sodium citrate. The microcarriers are significantly smaller, and they do not have light perimeters with darker interiors.Example 13

[0152] Rehydration was investigated for 60 mg of the dried microcarriers from Example 4 (a drying solution with sodium citrate (a calcium chelator) and drying by freeze drying and a manifold lyophilizer) that were rehydrated in either 3 mL or 8 mL of deionized water. The 8 mL volume corresponds to previously required volumes to rehydrate dried microcarriers. More specifically, 60 mg of dried microcarriers from Example 4 were rehydrated in 8 mL of deionized water for a microcarrier rehydration concentration of 7.5 mg / mL. The microcarriers rehydrated within 5 minutes and the rehydrated microcarriers were photographed with a phase contrast optical microscope at 4x magnification (FIG. 2A). Separately, 60 mg of dried microcarriers from Example 4 were rehydrated in 3 mL of deionized water for a microcarrier rehydration concentration of 20 mg / mL. The microcarriers rehydrated within 5 minutes and the rehydrated microcarriers were photographed with a phase contrast optical microscope at 4x magnification (FIG. 2B). FIGS. 2A-2B depict approximately spherical, rehydrated microcarriers with each microcarrier having (by visual observation) a light perimeter and an interior, on average, that is darker than the perimeter.Example 14

[0153] Rehydration was investigated for 60 mg of the dried microcarriers from Example 5 (no sodium citrate and drying by freeze drying and a manifold lyophilizer) that were rehydrated in 3 mL of deionized water, for a microcarrier rehydration concentration of 20 mg / mL. The microcarriers did not rehydrate with rehydration solution. The microcarriers were then photographed with a phase contrast optical microscope at 4x magnification (FIG. 2D). As seen inFIG. 2D, the microcarriers did not rehydrate in the absence of the sodium citrate. The microcarriers are significantly smaller, agglomerated, and they do not have light perimeters with darker interiors.Example 15

[0154] Rehydration was investigated for 60 mg of each of the dried microcarriers from Examples 6-10 that were rehydrated in 9 mL of deionized water, for a microcarrier concentration of 5 mg / mL. More specifically, 60 mg of dried microcarriers from Example 6 (1% glucose) were rehydrated in 9 mL of deionized water for a microcarrier rehydration concentration of 5 mg / mL. The microcarriers only partially rehydrated and the rehydrated microcarriers were photographed with a phase contrast optical microscope at 4x magnification (FIG. 3B). Separately, 60 mg of dried microcarriers from Example 7 (1% triethanolamine chloride) were rehydrated in 9 mL of deionized water for a microcarrier rehydration concentration of 5 mg / mL. The microcarriers rehydrated within 5 minutes and the rehydrated microcarriers were photographed with a phase contrast optical microscope at 4x magnification (FIG. 3C). In another container, 60 mg of dried microcarriers from Example 8 (1% lysine chloride) were rehydrated in 9 mL of deionized water for a microcarrier rehydration concentration of 5 mg / mL. The microcarriers rehydrated within 5 minutes and the rehydrated microcarriers were photographed with a phase contrast optical microscope at 4x magnification (FIG. 3D). In yet another container, 60 mg of dried microcarriers from Example 9 (1% glucosamine) were rehydrated in 9 mL of deionized water for a microcarrier rehydration concentration of 5 mg / mL. The microcarriers rehydrated within 5 minutes and the rehydrated microcarriers were photographed with a phase contrast optical microscope at 4x magnification (FIG. 3E). Finally, 60 mg of dried microcarriers from Example 10 (no positively charged molecule drying solution) were rehydrated in 9 mL of deionized water for a microcarrier rehydration concentration of 5 mg / mL. The microcarriers only partially rehydrated, and partially rehydrated microcarriers were photographed with a phase contrast optical microscope at 4x magnification (FIG. 3A).

[0155] FIGS. 3C-3D (microcarriers with positively charged molecules in the drying solution) depict approximately spherical, rehydrated microcarriers with each microcarrier having (by visual observation) a light perimeter and an interior, on average, that is darker than the perimeter. These rehydrated microcarriers are similar to the hydrated form of the microcarriersbefore drying shown in FIG. 3F. In contrast, the microcarriers dried with glucose (a neutral charge molecule) or without a positively charged molecule drying solution only partially rehydrated as seen in FIGS. 3A-3B. The partially rehydrated microcarriers had varying sizes and had significant light sections in the interior of the microcarrier images as compared to the rehydrated microcarriers shown in FIGS. 3C-3E and the original hydrated form of the microcarrier before drying (FIG. 3F).

Claims

CLAIMSWhat Is Claimed Is:

1. A microcarrier, comprising:a pectin-based polymer in an amount between 20 wt.% and 80 wt.% of the microcarrier, wherein the pectin-based polymer is polygalacturonic acid (PGA), partly esterified polygalacturonic acid (PE PGA) having a degree of esterification of 40 mol% or less, a salt of any of the foregoing, or a combination of any of the foregoing;calcium, having an amount between 3 wt.% and 10 wt.% of the microcarrier; anda calcium chelator, having an amount greater than 0 wt.% and less than 5 wt.% of the microcarrier;wherein the microcarrier is a dried microcarrier; andwherein the dried microcarrier in its rehydrated form has the pectin-based polymer present at 1.0 w / v% and 3.0 w / v%.

2. The microcarrier of claim 1, wherein the calcium chelator comprises sodium citrate, ethylenediaminetetraacetic acid (EDTA), aspartic acid, (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N' -tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTP A), N(hydroxyl)-ethylenediaminetetraacetic acid (HETDA), a salt of any of the foregoing, or combinations of any of the foregoing.

3. The microcarrier of any of claims 1-2, wherein the microcarrier further comprises a filler and the filler comprises a low molecular weight sugar, a low molecular weight polyethylene glycol (PEG), a low molecular weight positively charged compound, or a combination thereof.

4. The microcarrier of claim 3, wherein the filler comprises glucose, glucosamine, a low molecular weight polyethylene glycol (PEG), or a combination thereof.

5. The microcarrier of any of claims 1-4, wherein the microcarrier further comprises a lubricant.

6. The microcarrier of claim 5, wherein the lubricant comprises a low molecular weight polyethylene glycol (PEG), a dimethyl sulfoxide (DMSO), or a combination thereof.

7. The microcarrier of any of claims 1-6, wherein the microcarrier further comprises a positively charged molecule.

8. The microcarrier of claim 7, wherein the positively charged molecule comprises a weak acid with a net positive charge, a primary amine, a secondary amine, a tertiary amine, or a combination thereof.

9. The microcarrier of any of claims 1-8, wherein the microcarrier is a sterilized microcarrier.

10. The microcarrier of any of claims 1-9, wherein a diameter of a rehydrated form of the dried microcarrier is at least 80% of a diameter the dried microcarrier’s initial form.

11. The microcarrier of any of claims 1 -9, wherein the microcarrier, in its rehydrated form, has a diameter of between 10 pm and 500 pm.

12. A microcarrier, comprising:a pectin-based polymer in an amount between 20 wt.% and 80 wt.% of the microcarrier, wherein the pectin-based polymer is polygalacturonic acid (PGA), partly esterified polygalacturonic acid (PE PGA) having a degree of esterification of 40 mol% or less, a salt of any of the foregoing, or a combination of any of the foregoing;calcium, having an amount between 3 wt.% and 10 wt.% of the microcarrier; anda positively charged molecule, having an amount greater than 0 wt.% and less than 5 wt.% of the microcarrier;wherein the microcarrier is a dried microcarrier; andwherein the dried microcarrier in its rehydrated form has the pectin-based polymer present at 1.0 w / v% and 3.0 w / v%.

13. The microcarrier of claim 12, wherein the positively charged molecule comprises a weak acid with a net positive charge, a primary amine, a secondary amine, a tertiary amine, or a combination thereof.

14. The microcarrier of any of claims 12-13, wherein the microcarrier further comprises a filler that comprises a low molecular weight sugar, a low molecular weight polyethylene glycol (PEG), a low molecular weight positively charged compound, or a combination thereof.

15. The microcarrier of claim 14, wherein the filler comprises glucose, glucosamine, polyethylene glycol, or a combination thereof.

16. The microcarrier of any of claims 12-15, wherein the microcarrier further comprises a lubricant.

17. The microcarrier of claim 16, wherein the lubricant comprises a low molecular weight polyethylene glycol (PEG), a dimethyl sulfoxide (DMSO), or a combination thereof.

18. The microcarrier of any of claims 12-17, wherein the microcarrier is a sterilized microcarrier.

19. The microcarrier of any of claims 12-18, wherein a diameter of a rehydrated form of the dried microcarrier is at least 80% of a diameter the dried microcarrier’s hydrated, initial form.

20. The microcarrier of any of claims 12-18, wherein the microcarrier, in its rehydrated form, has a diameter of between 10 pm and 500 pm.

21. A method of drying microcarriers, comprising the steps of:(a) providing an initial form of a microcarrier, the microcarrier comprising:polygalacturonic acid (PGA), partly esterified polygalacturonic acid (PE PGA) having a degree of esterification of 40 mol% or less, a salt of any of the foregoing, or a combination of any of the foregoing; andcalcium;(b) providing a drying solution comprising a calcium chelator, a positively charged molecule, or a combination thereof;(c) exposing the initial form of the microcarrier to the drying solution; and(d) drying the microcarrier from step (c).

22. The method of claim 21, wherein the calcium chelator is selected from the group consisting of sodium citrate, ethylenediaminetetraacetic acid (EDTA), aspartic acid, (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N' -tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTP A), N(hydroxyl)-ethylenediaminetetraacetic acid (HETDA), a salt of any of the foregoing, and combinations of any of the foregoing.

23. The method of any one of claims 21-22, wherein the positively charged molecule comprises a weak acid with a net positive charge, a primary amine, a secondary amine, a tertiary amine, or a combination thereof.

24. The method of any one of claims 21-23, wherein the drying solution further comprises a filler, a lubricant, or a combination thereof.

25. The method of claim 24, wherein the filler is a low molecular weight sugar, a low molecular weight polyethylene glycol (PEG), a low molecular weight positively charged compound, or a combination thereof.

26. The method of any one of claims 24-25, wherein the lubricant comprises a low molecular weight polyethylene glycol (PEG), a dimethyl sulfoxide (DMSO), or a combination thereof.

27. The method of any one of claims 21-26, wherein the drying solution comprises an alcohol.

28. The method of any one of claims 21-27, wherein the drying is by rotary evaporator, freeze drying, lyophilization, or a combination thereof.

29. The method of any one of claims 25-28, further comprising the steps of providing a drying solution comprising an alcohol and exposing the initially formed microcarrier to the drying solution comprising an alcohol, wherein both steps are performed before step (b).

30. The method of any one of claims 21-23, further comprising a step of exposing the initially formed microcarrier to a drying solution comprising a filler, a lubricant, or a combination thereof, wherein the step is performed after step (c) and before step (d).

31. The method of claim 30, wherein the calcium chelator is selected from the group consisting of sodium citrate, ethylenediaminetetraacetic acid (EDTA), aspartic acid, (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N' -tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTP A), N(hydroxyl)-ethylenediaminetetraacetic acid (HETDA), a salt of any of the foregoing, and combinations of any of the foregoing; andwherein the positively charged molecule is selected from the group consisting of triethanolamine chloride and lysine chloride, glucosamine, or a combination thereof.

32. The method of any one of claims 21-31, further comprising a step of sterilizing the microcarrier after step (d).

33. The method of claim 32, wherein the sterilization is performed using high-energy irradiation.

34. The method of any one of claims 21-33, wherein a diameter of a rehydrated form of the dried microcarrier is at least 80% of a diameter the dried microcarrier’s initial form.

35. A method of rehydrating a dried microcarrier, comprising the steps of:(a) providing the dried microcarrier of any one of claims 1-20;(b) providing an aqueous solution to the dried microcarrier;(c) combining the dried microcarrier and the aqueous solution to achieve a concentration of dried microcarrier in aqueous solution of greater than 2 mg / mL; and(d) rehydrating the dried microcarrier in the aqueous solution to generate a rehydrated microcarrier;wherein the rehydrated microcarrier has a light perimeter with an interior that is, on average, darker than the light perimeter, as observed with a phase contrast optical microscope in brightfield mode.

36. The method of claim 35, wherein the optical phase microscope in brightfield mode is at 4x magnification.

37. The method of any one of claims 35-36, wherein the concentration of dried microcarriers to aqueous solution between 20 mg / mL and 100 mg / mL.

38. The method of any one of claims 35-37, wherein the concentration of dried microcarriers to aqueous solution is between 40 mg / mL and 100 mg / mL.

39. The method of any one of claims 35-38, wherein the minimum time required to rehydrate the dried microcarrier is between 20% and 90% less than a minimum time required to rehydrate a dried microcarrier without a calcium chelator and / or a positively charged molecule.

40. The method of any one of claims 35-39, wherein the minimum volume of aqueous solution required for rehydration is between 20%-90% less than the minimum volume required to rehydrate for a dried microcarrier without a calcium chelator and / or a positively charged molecule.

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