In vitro dissolution

Abstract

The present disclosure provides an in vitro method for dissolving a solute comprising using a movable paddle to agitate a solution contained in a container, the solution comprising a solute, a medium, and a plurality of beads, and the paddle is submerged in the solution. In some aspects, the plurality of beads are disposed between the solute and the movable paddle. In some aspects, the solute is sandwiched between the plurality of beads, the solute being disposed both above and below the plurality of beads.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Application No. US63 / 184,629, filed May 5, 2021, which is incorporated by reference in its entirety.

[0002] The present disclosure provides an in vitro method for the dissolution of a solute, such as a long-acting injectable drug. [Background technology]

[0003] Long-acting injectables (LAIs) are formulations that exhibit a sustained release profile over days or weeks. LAIs represent an important and often practical approach to unlock the therapeutic value of many development candidates to reduce dosing frequency and improve patient compliance. During the development of LAI formulations and during batch-to-batch quality evaluation of drug products, in vitro drug release and dissolution testing play a key role, with increasing interest and expectations from both regulatory authorities and industry. Summary of the Invention [Problem to be solved by the invention]

[0004] Currently, the most used methods for in vitro elution of LAI are (1) sample separation, (2) dialysis, and (3) flow-through. Although these methods have been generally well applied to small molecule LAIs, they have had limited success when applied to larger molecules such as peptides. Thus, there remains a need in the art for elution methods that exhibit robustness and discrimination with respect to key quality attributes and are predictive of in vivo performance. [Means for solving the problem]

[0005] Some aspects of the present disclosure relate to an in vitro dissolution method that includes agitating a solution contained in a container using a movable paddle, the solution including a solute, a medium, and a plurality of beads, the plurality of beads being disposed between the solute and the movable paddle, and the movable paddle being submerged in the solution.

[0006] In some embodiments, the method includes, prior to stirring, (i) adding a solute to the bottom of the container, (ii) adding a plurality of beads to the bottom of the container, where the plurality of beads are added above the solute, and adding a volume of medium to the container, where the medium is added above the solute and the plurality of beads, thereby placing the solution in the container.

[0007] In some embodiments, the medium is added without agitating the solute or the plurality of beads, hi some embodiments, the movable paddle does not contact the plurality of beads or solute while the movable paddle is in a stationary position.

[0008] In some embodiments, the solute comprises a long-acting injectable agent. In some embodiments, the solute comprises a long-acting injectable microsphere.

[0009] In some embodiments, the plurality of beads comprises one or more glass beads, one or more plastic beads, one or more silicate beads, one or more metal beads, or any combination thereof. In some embodiments, the plurality of beads comprises one or more beads having a diameter of at least about 0.01 mm, at least about 0.05 mm, at least about 0.1 mm, at least about 0.5 mm, at least about 1.0 mm, at least about 1.5 mm, or at least about 2.0 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 1.0 mm.

[0010] In some embodiments, the volume of solution in the container is sufficient to submerge the movable paddle, the plurality of beads, and the solute, hi some embodiments, the volume of solution in the container is at least about 50 mL, at least about 60 mL, at least about 70 mL, at least about 75 mL, at least about 80 mL, at least about 90 mL, or at least about 100 mL.

[0011] In some embodiments, the plurality of beads comprises at least about 3 g of beads per about 100 mL of solution, at least about 4 g of beads per about 100 mL of solution, at least about 5 g of beads per about 100 mL of solution, at least about 6 g of beads per about 100 mL of solution, or at least about 7 g of beads per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 4 g of beads per 100 mL of solution. In some embodiments, the plurality of beads comprises less than about 6 g of beads per 100 mL of solution.

[0012] In some embodiments, the solute comprises a sustained release injectable microsphere loaded with a biologically active moiety.

[0013] In some embodiments, the method further comprises covering the container during lysis, hi some embodiments, the cover reduces or eliminates the loss of evaporated solution during lysis.

[0014] In some embodiments, the pH of the solution is adjusted.

[0015] In some embodiments, an additional volume of a plurality of beads is added below the solute.

[0016] In some embodiments, the method includes (i) adding a solute to the bottom of the container, where the solute comprises sustained injection microspheres; (ii) adding a plurality of beads on top of the solute, where the plurality of beads comprises one or more glass beads; (iii) adding a medium to the solute and the volume of beads, where the medium is added without agitating the solute or the volume of beads, and where the medium is added in a volume sufficient to submerge the movable paddle; and (iv) applying a force to rotate and / or vibrate the movable paddle to agitate the solution.

[0017] In some embodiments, the method includes (i) adding a first plurality of beads to the bottom of the container, the plurality of beads comprising one or more glass beads; (ii) adding a solute on top of the plurality of beads, the solute comprising a long-acting injectable microsphere; (iii) adding a second plurality of beads on top of the solute, the plurality of beads comprising one or more glass beads; (iv) adding a medium to the solute and the volume of beads, the medium being added without agitating the solute or the volume of beads, the medium being added in a volume sufficient to submerge the movable paddle; and (v) applying a force to rotate and / or vibrate the movable paddle to agitate the solution.

[0018] In some embodiments, the method further comprises adding a volume of a media solution to the solute and / or plurality of beads, the volume of media solution being sufficient to wet but not completely submerge the solute and / or plurality of beads.

[0019] In some embodiments, the method further comprises detecting the presence of dissolved solutes in the medium. In some embodiments, the detecting comprises placing a probe in the solution, obtaining a sample of the solution, or a combination thereof. In some embodiments, the presence of dissolved solutes in the medium is detected by probing the solution above the movable paddle with a probe and / or obtaining a sample from a portion thereof.

[0020] In some embodiments, the solute comprises a long-acting injectable agent comprising a biologically active moiety. In some embodiments, the biologically active moiety comprises a small molecule or a polypeptide. In some embodiments, the molecular weight of the polypeptide is less than about 10 kDa, less than about 9 kDa, less than about 8 kDa, less than about 7 kDa, less than about 6 kDa, less than about 5 kDa, or less than about 4 kDa. In some embodiments, the molecular weight of the polypeptide is about 4 kDa.

[0021] In some embodiments, the solution further comprises a surfactant. In some embodiments, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, TRITON X100, PLURONIC F-68, and any combination thereof. In some embodiments, the solution further comprises an antibacterial agent.

[0022] Some aspects of the present disclosure relate to kits that include a container; a plurality of beads; a medium; a movable paddle, the movable paddle capable of being positioned within the container; an optional cover; and instructions for eluting a solute according to any of the methods disclosed herein. [Brief description of the drawings]

[0023] [Figure 1] Figure 1A is a schematic diagram of the in vitro dissolution method disclosed herein, in which the LAI sample is sandwiched between glass beads at the bottom of a vessel and a movable paddle is positioned above the glass beads. Previous methods are illustrated in Figure 1B ("USP IV") and Figure 1C ("USP II").

[0024] [Diagram 2] FIG. 2 is a graphical representation of drug elution (y-axis) from the LAI versus time (x-axis), where the LAI is placed under 3 g (squares), 4 g (triangles), 6 g (crosses) or 9 g (stars) of glass beads (1 mm diameter).

[0025] [Diagram 3]FIG. 3 is a graphical representation of the elution of drug from the LAI (y-axis) versus time (x-axis), where the LAI is placed under 4 g of glass beads (1 mm diameter) for six runs in six vessels.

[0026] [Figure 4] FIG. 4 is a graphical representation of the elution of the drug exenatide (y-axis) from LAI microspheres versus time (x-axis), where the LAI was placed under 4 g of glass beads (1 mm diameter) at 37° C. and the pH was held at pH 7.8 (diamonds), pH 9.4 (squares), pH 11 (triangles), or pH 12 (crosses).

[0027] [Diagram 5] Figure 5A is a graphical representation of the elution (y-axis) of the drug exenatide from LAI microspheres versus time (x-axis), where the LAI is placed under 4 g of glass beads (1 mm diameter) at 37°C, and the buffer contains 33 mM PO4 and 0.1% TWEEN20 (diamonds) or 0.3% TWEEN20 (squares). Figure 5B is a graphical representation of the elution (y-axis) of the drug exenatide from LAI microspheres versus time (x-axis), where the LAI is placed under 4 g of glass beads (1 mm diameter) at 37°C, and the buffer contains 33 mM PO4 and 0.1% TWEEN20 (diamonds), 0.1% TritanX100 (squares), or 0.1% Pluoronic F-68 (triangles).

[0028] [Figure 6] FIG. 6 is a graphical representation of the elution of the pharmaceutical agent exenatide (y-axis) from LAI microspheres versus time (x-axis), where the LAI was placed under 4 g of glass beads (1 mm diameter) at 37° C. and buffer containing 90 mM PO4 and 0.2% TWEEN 20 (crosses), 20 mM PO4 (squares), 33 mM PO4 (stars), 40 mM PO4 (circles), 50 mM PO4 (diamonds), 60 mM PO4 (solid squares), or 70 mM PO4 (triangles).

[0029] [Figure 7]FIG. 7 is a graphical representation of the elution of the drug exenatide (y-axis) from LAI microspheres versus time (x-axis), where the LAI was placed under 4 g of glass beads (1 mm diameter) at 37° C. and the buffer contained 0.2% sodium azide (diamonds) or no sodium azide (squares).

[0030] [Figure 8] FIG. 8 is a graphical representation of the dissolution of the pharmaceutical agent exenatide (y-axis) from LAI microspheres at pH 12, where the LAI is placed under 4 g of glass beads (1 mm diameter) at (i) 37° C. in 33 mM PO4 buffer (diameter diamonds), (ii) 37° C. in 60 mM PO4 buffer (diameter squares), (iii) 45° C. in 33 mM PO4 buffer (diameter triangles), or (iv) 45° C. in 60 mM PO4 buffer (diameter crosses).

[0031] [Figure 9A] Figures 9A-9C are graphical representations of dissolution of fatty acid-relaxin (FA-RLX) pharmaceuticals at particle size (PS) of less than 20 μm (Figures 9A and 9C), less than 50 μm (Figures 9B-9C), and less than 100 μm (Figures 9A and 9C), where the FA-RLX is placed under 0.5 mm glass beads and subjected to stirring at 35 rpm using a paddle. Figure 9C shows an overlay of the data shown in Figures 9A and 9B. [Figure 9B] Figures 9A-9C are graphical representations of dissolution of fatty acid-relaxin (FA-RLX) pharmaceuticals at particle size (PS) of less than 20 μm (Figures 9A and 9C), less than 50 μm (Figures 9B-9C), and less than 100 μm (Figures 9A and 9C), where the FA-RLX is placed under 0.5 mm glass beads and subjected to stirring at 35 rpm using a paddle. Figure 9C shows an overlay of the data shown in Figures 9A and 9B. [Figure 9C]Figures 9A-9C are graphical representations of dissolution of fatty acid-relaxin (FA-RLX) pharmaceuticals at particle size (PS) of less than 20 μm (Figures 9A and 9C), less than 50 μm (Figures 9B-9C), and less than 100 μm (Figures 9A and 9C), where the FA-RLX is placed under 0.5 mm glass beads and subjected to stirring at 35 rpm using a paddle. Figure 9C shows an overlay of the data shown in Figures 9A and 9B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure provides an in vitro dissolution method comprising agitating a solution contained in a container using a movable paddle, the solution comprising a solute, a medium, and a plurality of beads. In some embodiments, the plurality of beads is disposed between the solute and the movable paddle, and the movable paddle is submerged in the solution. In some embodiments, the solute is disposed at the bottom of the container. In some embodiments, a first portion of the plurality of beads is disposed at the bottom of the container, the solute is disposed on the first portion of the plurality of beads, and a second portion of the plurality of beads is disposed on the solute. The method disclosed herein can be used for dissolution of any solute, including, but not limited to, a long acting injection (LAI), e.g., a pharmaceutical-loaded LAI microsphere.

[0033] I. Terminology So that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly defined otherwise herein, each of the following terms shall have the meaning set forth below. Further definitions are set forth throughout this application.

[0034] It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0035] Furthermore, "and / or" as used herein is considered as a specific disclosure of two specified features or components, each of which may or may not have the other feature or component. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0036] Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the technical field to which this disclosure pertains.For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many terms used in this disclosure.

[0038] Units, prefixes, and symbols are written in the format accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range. Nucleotide sequences are written left to right in 5' to 3' orientation unless otherwise indicated. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the disclosure, which may be had by reference to the entire specification. Thus, the terms immediately defined below are more fully defined by reference to the entire specification.

[0039] As used herein, the term "solute" refers to any substance that can be dissolved in a solvent. In some embodiments, the solute is in a solid state, e.g., before or during elution using the methods disclosed herein. In some embodiments, the solute is in a dissolved state, e.g., after elution using the methods disclosed herein. In certain embodiments, the solute is a long acting injection (LAI). The methods disclosed herein can be used to elute any LAI. In some embodiments, the LAI is a microsphere. In some embodiments, the solute comprises an LAI microsphere loaded with a biologically active moiety.

[0040] As used herein, the term "solvent" refers to a liquid substance capable of dissolving a solute. In some embodiments, the solvent comprises a buffer. In some embodiments, the solvent comprises a potassium buffer, such as PBS.

[0041] As used herein, the term "long-acting injectable" or "LAI" refers to a drug formulation that exhibits a sustained release profile, for example, over a period of days or weeks. In some embodiments, the LAI has a release profile of at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, at least about 120 hours, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days, at least about 22 days. In some embodiments, the LAI has a release profile of at least about 1 week, at least about 1.5 weeks, at least about 2 weeks, at least about 2.5 weeks, at least about 3 weeks, at least about 3.5 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, or at least about 8 weeks. In some embodiments, the LAI comprises a bioactive moiety. In some embodiments, the LAI comprises a bioactive moiety and a carrier. In some embodiments, the carrier comprises a soluble microsphere. In some embodiments, the microsphere is a poly-(D,L-lactide-Co-glycolide) ("PLG") microsphere. The formula of PLG is shown in Formula I. [ka] In some embodiments, the LAI comprises a hydrogel. In some embodiments, the LAI comprises a liquid crystal. In some embodiments, the LAI comprises one or more combinations of a microsphere, a hydrogel, and a liquid crystal. In some embodiments, the bioactive moiety is encapsulated within the LAI, for example, within a PLG microsphere.

[0042] As used herein, "biologically active moiety" refers to any substance that can affect any physical or biochemical property of a biological system, pathway, molecule, or interaction involving a living organism. In particular, as used herein, bioactive molecules include, but are not limited to, any substance intended to diagnose, cure, alleviate, treat, or prevent a disease or condition in humans or other animals, such as a disease or condition associated with fibrosis, or any substance intended to otherwise enhance the physical or mental health of humans or animals. Biologically active moieties include polypeptides (e.g., proteins (e.g., enzymes, growth factors, cytokines, chemokines, ligands, receptors, hormones, antibodies, antigens, fragments thereof, or any combination thereof), nucleotides (e.g., DNA (e.g., ssDNA, dsDNA, plasmids, fragments thereof, or any combination thereof), RNA (e.g., mRNA, miRNA, siRNA, dsRNA, fragments thereof, or any combination thereof), antisense oligomers, fragments thereof, or any combination thereof), Cas9 nucleases, TALEN nucleases, zinc finger nucleases, and the like. , a small molecule, or any combination thereof. In some embodiments, the biologically active moiety comprises a small molecule. In some embodiments, the biologically active moiety comprises a polypeptide. In some embodiments, the molecular weight of the polypeptide is less than about 20 kDa, less than about 15 kDa, less than about 14 kDa, less than about 13 kDa, less than about 12 kDa, less than about 11 kDa, less than about 10 kDa, less than about 9 kDa, less than about 8 kDa, less than about 7 kDa, less than about 6 kDa, less than about 5 kDa, or less than about 4 kDa. In some embodiments, the molecular weight of the polypeptide is about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, or about 10 kDa.

[0043] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects in clinical trials, animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays. In some embodiments, the methods described herein are used to measure the solubility of a therapeutically effective amount of a LAI pharmaceutical in vitro.

[0044] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In a preferred embodiment, the subject is a human. As used herein, the terms "subject" and "patient" are used interchangeably.

[0045] The term "about" or "essentially consisting of" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially consisting of" can mean within or more than one standard deviation, according to the practice in the art. Alternatively, "about" or "essentially consisting of" can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude, or up to 5 times, of the value. When a particular value or composition is described in this application and claims, unless otherwise indicated, the meaning of "about" or "essentially consisting of" should be assumed to be within an acceptable error range for that particular value or composition.

[0046] As described herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the stated range, and fractions thereof, where appropriate (e.g., tenths and hundredths of an integer), unless otherwise indicated.

[0047] Various aspects of the disclosure are described in further detail in the following subsections.

[0048] II. Methods of the Disclosure Certain aspects of the present disclosure relate to in vitro dissolution methods that include agitating a solution contained in a container using a movable paddle, the solution comprising a solute, a medium, and a plurality of beads, and the paddle is submerged in the solution. In some aspects, the plurality of beads are disposed between the solute and the movable paddle.

[0049] The methods disclosed herein provide improved dissolution and solution quality over existing methods. The most common methods, especially for in vitro dissolution of LAI, are (1) sample separation, (2) dialysis, and (3) flow-through. See, for example, Seidlitz et al., J. Pharm. Pharmacol. 64:969-85 (2012). Although these methods have been successfully applied to small molecule LAI, they are less suitable for dissolution of formulations containing large molecules such as peptides, causing, for example, peptide aggregation or particle loss. The dialysis method is affected by the molecular weight cut-off value of the dialysis membrane, which may affect peptide diffusion into the bulk after release from the LAI formulation. The flow-through method uses the official USP apparatus 4 ("USP IV"), but has the disadvantage of weak sterilization effect when used for peptide LAI, causing peptide degradation during dissolution testing.

[0050] The methods disclosed herein overcome the limitations of conventional methods by placing a solute in a container beneath a number of beads, e.g., glass beads, submerging the solute and beads in a medium, and placing a movable paddle submerged in the medium and above the beads such that the movable paddle does not contact the beads when in a stationary position. These methods provide greater consistency, robustness, and solubility in a short period of time than conventional methods.

[0051] In some embodiments, the method includes, prior to stirring, (i) adding a solute to the bottom of the container, (ii) adding a plurality of beads to the bottom of the container, where the plurality of beads are added above the solute, and (iii) adding a volume of medium to the container, where the medium is added above the solute and the plurality of beads, thereby placing the solution in the container.

[0052] Any vessel can be used in the methods disclosed herein. In some embodiments, the vessel is a vial large enough to contain at least about 100 mL of solution and a movable paddle, the movable paddle being submerged in at least about 100 mL of solution. In some embodiments, the vessel is a small volume vessel. In some embodiments, the vessel is a HANSON small volume vessel. In some embodiments, the vessel is placed in a dissolution bath during elution. Any vessel can be used in the methods disclosed herein. In some embodiments, the dissolution bath maintains a temperature of at least about 34°C, at least about 35°C, at least about 36°C, at least about 37°C, at least about 38°C, at least about 39°C, at least about 40°C, at least about 41°C, at least about 42°C, at least about 43°C, at least about 44°C, or at least about 45°C. In some embodiments, the dissolution bath maintains a temperature of about 37°C. In some embodiments, the dissolution bath maintains a temperature of about 38°C. In some embodiments, the dissolution bath maintains a temperature of about 39°C. In some embodiments, the dissolution bath maintains a temperature of about 40° C. In some embodiments, the dissolution bath maintains a temperature of about 41° C. In some embodiments, the dissolution bath maintains a temperature of about 42° C. In some embodiments, the dissolution bath maintains a temperature of about 43° C. In some embodiments, the dissolution bath maintains a temperature of about 44° C. In some embodiments, the dissolution bath maintains a temperature of about 45° C.

[0053] In some embodiments, the solute is added directly to the bottom of the container. In some embodiments, a first plurality of beads is added to the bottom of the container, and then the solute is added on top of the first plurality of beads. Any volume of solute can be applied to the methods disclosed herein. Increasing the amount of solute can be accommodated by increasing the size of the container, the amount of the plurality of beads, and / or the volume of solvent added to the container by one or more times. In some embodiments, the amount of solute corresponds to a single unit dose of a pharmaceutical product, e.g., a therapeutically effective dose. In some embodiments, the amount of solute is intended to match a therapeutically effective dose of a pharmaceutical product, such that the method can be used to accurately predict in vivo dissolution of the pharmaceutical product.

[0054] In some embodiments, the medium solution is added on top of the solute, e.g., to wet the solute. In some embodiments, the medium solution comprises a solute, e.g., a solution used to store a pharmaceutical formulation. In some embodiments, the volume of the medium solution is sufficient to wet the solute and / or the plurality of beads, but not completely submerge them. In some embodiments, less than about 5 mL, less than about 4.5 mL, less than about 4 mL, less than about 3.5 mL, less than about 3 mL, less than about 2.5 mL, less than about 2 mL, less than about 1.5 mL, less than about 1 mL, less than about 0.5 mL of medium solution is added on top of the solute. In some embodiments, about 0.5 mL of medium solution is added to a therapeutically effective amount of LAI.

[0055] In some embodiments, the plurality of beads is added above the solute in the container. Any beads can be used in the methods disclosed herein. In some embodiments, the plurality of beads comprises one or more glass beads, one or more plastic beads, one or more silicate beads, one or more metal beads, or any combination thereof. In some embodiments, the plurality of beads comprises glass beads. In some embodiments, the plurality of beads comprises plastic beads. In some embodiments, the plurality of beads comprises silicate beads. In some embodiments, the plurality of beads comprises metal beads.

[0056] Beads of any size can be used in the methods disclosed herein. In some embodiments, the plurality of beads comprises one or more beads having a diameter of at least about 0.01 mm, at least about 0.02 mm, at least about 0.03 mm, at least about 0.04 mm, at least about 0.05 mm, at least about 0.06 mm, at least about 0.07 mm, at least about 0.08 mm, at least about 0.09 mm, at least about 0.1 mm, at least about 0.2 mm, at least about 0.3 mm, at least about 0.4 mm, at least about 0.5 mm, at least about 0.6 mm, at least about 0.7 mm, at least about 0.8 mm, at least about 0.9 mm, at least about 1.0 mm, at least about 1.1 mm, at least about 1.2 mm, at least about 1.3 mm, at least about 1.4 mm, at least about 1.5 mm, at least about 1.6 mm, at least about 1.7 mm, at least about 1.8 mm, at least about 1.9 mm, or at least about 2.0 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 0.5 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 0.6 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 0.7 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 0.8 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 0.9 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 1.0 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 1.1 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 1.2 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 1.3 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 1.4 mm. In some embodiments, the plurality of beads comprises one or more beads having a diameter of about 1.5 mm.

[0057] In some embodiments, the plurality of beads comprises beads of equal size. In some embodiments, the plurality of beads comprises a mixture of beads of different sizes. In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the beads are about the same size (e.g., within the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 50% of the beads are about the same size (e.g., within the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 60% of the beads are about the same size (e.g., within the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 70% of the beads are about the same size (e.g., within the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 75% of the beads are about the same size (e.g., in the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 80% of the beads are about the same size (e.g., in the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 85% of the beads are about the same size (e.g., in the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 90% of the beads are about the same size (e.g., in the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 95% of the beads are about the same size (e.g., in the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 96% of the beads are about the same size (e.g., in the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 97% of the beads are about the same size (e.g., in the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 98% of the beads are about the same size (e.g., within the range of about 0.8 mm to about 1.2 mm). In some embodiments, at least about 99% of the beads are about the same size (e.g., within the range of about 0.8 mm to about 1.2 mm).

[0058] The amount of the plurality of beads added to the solute can be adjusted based on the amount of solute, the volume of the container, the shape of the container, the volume of the solution, or a combination thereof. In some embodiments, the amount of beads in the plurality of beads corresponds to the amount required to cover the solute with beads. In some embodiments, the solute is disposed below the beads. In some embodiments, the solute is sandwiched by the beads, e.g., the solute is disposed such that there are beads both above and below the solute. In some embodiments, the plurality of beads comprises a volume equivalent to 2 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. For example, in an embodiment in which the plurality of beads comprises plastic beads, the volume of the plastic beads is less in weight than the volume equivalent to the glass beads, i.e., the volume equivalent to 3 g of glass beads may be 2 g of plastic beads. Conversely, metal beads may have a larger unit mass than glass beads of the same size. Thus, in an embodiment in which the plurality of beads comprises metal beads, the volume equivalent to 3 g of glass beads may be 4 g of metal beads. In some embodiments, the plurality of beads comprises a volume equivalent to 2.5 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 3 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 3.1 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 3.2 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 3.3 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 3.4 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 3.5 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 3.6 g of glass beads, and the average diameter of the glass beads is about 1.0 mm.In some embodiments, the plurality of beads comprises a volume equivalent to 3.7 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 3.8 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 3.9 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.0 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.1 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.2 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.3 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.4 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.5 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.6 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.7 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.8 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 4.9 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 5.0 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 5.5 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 6.0 g of glass beads, and the average diameter of the glass beads is about 1.0 mm.In some embodiments, the plurality of beads comprises a volume equivalent to 6.5 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 7.0 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 7.5 g of glass beads, and the average diameter of the glass beads is about 1.0 mm. In some embodiments, the plurality of beads comprises a volume equivalent to 8.0 g of glass beads, and the average diameter of the glass beads is about 1.0 mm.

[0059] In some embodiments, the plurality of beads comprises at least about 2 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 2.5 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.1 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.2 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.5 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.6 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.7 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.8 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.9 ... At least about 3.3 g of beads (e.g., about 1 mm diameter glass beads) per L of solution, at least about 3.4 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.5 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.6 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.7 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.8 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 3.9 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 4 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 4.1 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 4.2 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 4.3 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, At least about 4.4 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 4.5 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 4.6 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 4.7 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 4.8 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution, at least about 4.In some embodiments, the plurality of beads comprises about 3.5 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 3.6 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 3.7 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 3.8 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 3.9 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 4 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 4.1 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 4.2 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 4.3 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 4.4 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 4.5 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 5 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 5.In some embodiments, the plurality of beads comprises about 6 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 6.5 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 7 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution. In some embodiments, the plurality of beads comprises about 7.5 g of beads (e.g., about 1 mm diameter glass beads) per about 100 mL of solution.

[0060] In some embodiments, after adding the plurality of beads, the container is filled with a medium, such as a solvent and / or a buffer. Any medium can be used in the methods disclosed herein. In some embodiments, the medium is adjusted to the solute. In some embodiments, the medium comprises a phosphate buffer. In some embodiments, the medium comprises phosphate buffered saline (PBS). In some embodiments, the medium comprises at least about 20 mM PO4, at least about 25 mM PO4, at least about 30 mM PO4, at least about 33 mM PO4, at least about 35 mM PO4, at least about 40 mM PO4, at least about 45 mM PO4, at least about 50 mM PO4, at least about 55 mM PO4, at least about 60 mM PO4, at least about 65 mM PO4, at least about 70 mM PO4, at least about 75 mM PO4, at least about 80 mM PO4, at least about 90 mM PO4, at least about 95 mM PO4, or at least about 100 mM PO4. In some embodiments, the medium comprises at least about 33 mM PO4. In some embodiments, the medium comprises at least about 40 mM PO4. In some embodiments, the medium comprises at least about 50 mM PO4. In some embodiments, the medium comprises at least about 60 mM PO4. In some embodiments, the medium comprises at least about 70 mM PO4. In some embodiments, the medium comprises at least about 80 mM PO4. In some embodiments, the medium comprises at least about 90 mM PO4. In some embodiments, the medium comprises at least about 100 mM PO4.

[0061] In some embodiments, the medium further comprises a surfactant. In some embodiments, the surfactant is selected from polysorbate 20 (TWEEN 20), polysorbate 80 (TWEEN 80), TritonX100, pluoronic F-68, and any combination thereof. In some embodiments, the surfactant comprises polysorbate 20 (TWEEN 20). In some embodiments, the surfactant comprises polysorbate 80 (TWEEN 80). In some embodiments, the surfactant comprises TritonX100. In some embodiments, the surfactant comprises pluoronic F-68. In some embodiments, the medium comprises at least about 0.01%, at least about 0.02%, at least about 0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%, at least about 0.07%, at least about 0.08%, at least about 0.09%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1.0% of a surfactant (e.g., polysorbate 20). In some embodiments, the medium comprises about 0.1% of a surfactant (e.g., polysorbate 20). In some embodiments, the medium comprises about 0.2% of a surfactant (e.g., polysorbate 20). In some embodiments, the medium comprises about 0.3% of a surfactant (e.g., polysorbate 20). In some embodiments, the medium comprises about 0.4% surfactant (e.g., polysorbate 20). In some embodiments, the medium comprises about 0.5% surfactant (e.g., polysorbate 20).

[0062] In some embodiments, the medium further comprises an antimicrobial agent. Any antimicrobial agent can be used in the methods disclosed herein. In some embodiments, the microbial agent comprises sodium azide. In some embodiments, the medium comprises at least about 0.01%, at least about 0.02%, at least about 0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%, at least about 0.07%, at least about 0.08%, at least about 0.09%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1.0% antimicrobial agent (e.g., sodium azide). In some embodiments, the medium comprises about 0.1% antimicrobial agent (e.g., sodium azide). In some embodiments, the medium comprises about 0.2% antimicrobial (e.g., sodium azide). In some embodiments, the medium comprises about 0.3% antimicrobial (e.g., sodium azide). In some embodiments, the medium comprises about 0.4% antimicrobial (e.g., sodium azide). In some embodiments, the medium comprises about 0.5% antimicrobial (e.g., sodium azide).

[0063] In some embodiments, the medium is added above the plurality of beads, and the plurality of beads is disposed above the solute. In some embodiments, the medium is added at a rate that does not cause agitation of the solute and / or the plurality of beads. In some embodiments, the volume of the solution in the container is at least about 50 mL, at least about 60 mL, at least about 70 mL, at least about 75 mL, at least about 80 mL, at least about 90 mL, or at least about 100 mL. In some embodiments, the volume of the solution in the container is about 90 mL. In some embodiments, the volume of the solution in the container is about 100 mL. In some embodiments, the volume of the solution in the container is about 110 mL. In some embodiments, the volume of the solution in the container is about 120 mL. In some embodiments, the volume of the solution in the container is about 130 mL. In some embodiments, the volume of the solution in the container is about 140 mL. In some embodiments, the volume of the solution in the container is about 150 mL. In some embodiments, the volume of the solution in the container is about 160 mL. In some embodiments, the volume of the solution in the container is about 170 mL. In some embodiments, the volume of the solution in the container is about 180 mL. In some embodiments, the volume of the solution in the container is about 190 mL. In some embodiments, the volume of the solution in the container is about 200 mL. In some embodiments, the volume of the solution in the container is about 300 mL. In some embodiments, the volume of the solution in the container is about 400 mL. In some embodiments, the volume of the solution in the container is about 500 mL. The medium is added while the movable paddle is in a stationary position. The total volume of the solution depends on the total amount of solute to be subjected to the method disclosed herein. More solute requires more total volume of the solution. The total volume of the solution must be sufficient to submerge the solute, the plurality of beads, and the movable paddle.

[0064] Any movable paddle can be used in the methods disclosed herein. In some embodiments, the movable paddle is attached to the container such that the movable paddle is positioned within the container below the surface of the solution and above the plurality of beads. In some embodiments, the movable paddle is not attached directly to the container, but is positioned within the container such that the movable paddle is positioned below the surface of the solution and above the plurality of beads. In some embodiments, the size of the paddle is such that the paddle does not contact the wall of the container. The movable paddle of the present disclosure can move, e.g., rotate, to agitate the solution. In some embodiments, after the solute, the plurality of beads, and the medium are added to the container, the method further includes applying a force to the movable paddle to rotate and / or vibrate the movable paddle, thereby agitating the solution. In some embodiments, the movable paddle rotates at a speed of at least about 10 rpm, at least about 15 rpm, at least about 20 rpm, at least about 25 rpm, at least about 30 rpm, at least about 35 rpm, at least about 40 rpm, at least about 45 rpm, at least about 50 rpm, at least about 55 rpm, at least about 60 rpm, at least about 65 rpm, at least about 70 rpm, at least about 75 rpm, at least about 80 rpm, at least about 85 rpm, at least about 90 rpm, at least about 95 rpm, at least about 100 rpm, at least about 105 rpm, at least about 110 rpm, at least about 115 rpm, at least about 120 rpm, at least about 125 rpm, at least about 130 rpm, at least about 135 rpm, at least about 140 rpm, at least about 145 rpm, at least about 150 rpm, at least about 160 rpm, at least about 170 rpm, at least about 180 rpm, at least about 190 rpm, or at least about 200 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 15 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 20 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 25 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 30 rpm.In some embodiments, the movable paddle rotates at a speed of at least about 35 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 40 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 45 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 50 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 55 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 60 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 65 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 70 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 75 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 80 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 85 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 90 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 95 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 100 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 105 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 110 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 115 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 120 rpm. In some embodiments, the movable paddle rotates at a speed of at least about 125 rpm.

[0065] The duration of the elution method disclosed herein may depend on the solute. In some embodiments, the elution method is applied to the solute for a sufficient time for at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the solute to dissolve in the medium. In some embodiments, the elution method is applied to the solute for at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, or at least about 96 hours.

[0066] The elution of the solute can be measured using any method. In some embodiments, a probe is placed in the container below the level of the solution. In some embodiments, the probe is placed above a movable paddle. In some embodiments, the probe includes a UV optical fiber for in situ measurement. In some embodiments, a sample is removed from the solution during the elution process and the concentration of the solute in the sample is measured. In some embodiments, the sample is obtained from the solution above the movable paddle. In some embodiments, after the sample is obtained, a volume of medium is added to the solution corresponding to the volume of the sample removed from the solution.

[0067] In some embodiments, the vessel is covered during the elution process, hi some embodiments, the vessel is covered during the elution process, and the cover is only removed when the sample is taken.

[0068] In some embodiments, the method includes: (i) adding a solute, including sustained release microspheres, to the bottom of the container; (ii) adding a plurality of beads, including one or more glass beads, on top of the solute; (iii) adding a medium to the solute and the volume of beads, where the medium is added without stirring the solute or the volume of beads, the medium is added in a volume sufficient to submerge the movable paddle, and applying a force to rotate and / or vibrate the movable paddle to stir the solution. In some embodiments, the stirring is performed until at least about 80% of the solute is dissolved in the solution. In some embodiments, the stirring is performed until about 100% of the solute is dissolved in the solution.

[0069] In some embodiments, the method includes (i) adding a first plurality of beads, including one or more glass beads, to the bottom of the container; (ii) adding a solute, including sustained injectable microspheres, on top of the plurality of beads; (iii) adding a second plurality of beads, including one or more glass beads, on top of the solute; (iv) adding a medium to the solute and the volume of beads, where the medium is added without stirring the solute or the volume of beads, and the medium is added in a volume sufficient to submerge the movable paddle; and (v) applying a force to rotate and / or vibrate the movable paddle to stir the solution. In some embodiments, the stirring is performed until at least about 80% of the solute is dissolved in the solution. In some embodiments, the stirring is performed until about 100% of the solute is dissolved in the solution.

[0070] Certain aspects of the present disclosure relate to kits that include (i) a container, (ii) a plurality of beads, (iii) a medium, (iv) a movable paddle, the movable paddle being positionable within the container, and (v) instructions for eluting a solute according to any of the methods disclosed herein. Certain aspects of the present disclosure relate to kits that include (i) a container, (ii) a plurality of beads, (iii) a medium, (iv) a movable paddle, the movable paddle being positionable within the container, (v) a cover for the container, and (vi) instructions for eluting a solute according to any of the methods disclosed herein.

[0071] All references cited above, and all references cited herein, are incorporated herein by reference in their entirety.

[0072] The following examples are illustrative and not limiting. EXAMPLES

[0073] Example 1 A new setting for dissolution of LAI was developed, in which the LAI sample was sandwiched at the bottom of the container with glass beads and a paddle was placed above the glass beads for stirring (Figure 1). In this example, the application of this method to LAI (PLGA microsphere and hydrogel formulations) containing a peptide drug is demonstrated. The molecular weight of the peptide used in this example is about 4 kDa. The drug loading of the microspheres is 5 wt% for 2 mg API / dose and is designed for weekly injection.

[0074] Small volume vessels (Hanson) on a Distek 2500 dissolution bath were used, with 100 mL of media in each vessel and held at 37°C. A mini paddle was used at 100 rpm. A single dose equivalent of the microsphere formulation was first placed in the vessel, followed by the addition of 0.5 mL of media to wet the microspheres. Glass beads (1 mm diameter) were added on top of the sample, followed by the addition of 100 mL of media, carefully avoiding disturbing the glass beads.

[0075] The following method parameters were evaluated: (1) amount of glass beads (3 g, 4 g, 6 g, and 9 g), (2) pH of phosphate buffer (7.4, 9.8, 11, and 12), (3) buffer concentration (20, 33, 40, 50, 60, 70, and 90 mM), (4) surfactant (TWEEN 20, Triton X100, and Pluoronic F-68), (5) surfactant concentration (0.1%, 0.2%, and 0.3%), (6) temperature of the medium (37°C and 45°C), (7) antibiotic (yes and no), and (8) evaporation control (yes and no).

[0076] Drug release was studied by withdrawing 1 mL aliquots from the bulk at pre-determined time points (1, 2, 4, 8, 12, 24, 48 hours etc.) and replenishing with an equal volume of fresh medium and performing size exclusion chromatography (SEC).

[0077] result

[0078] This dissolution method was well tolerated in dissolution testing of peptide-PLGA microsphere LAI formulations. Data showed that 4 g of glass beads most rapidly eluted drug product (Figure 2), with reproducible results across six separate trials (Figure 3). Phosphate buffer at pH 7.4 was biorelevant, but pH 12 was used when accelerated dissolution testing and shorter turnaround times were required during development (Figure 4). However, pH is likely solute dependent. The specific type and amount of surfactant did not significantly affect dissolution (Figures 5A-5B), the buffer concentration did not significantly affect dissolution (Figure 6), and the temperature of the medium did not appear to significantly affect dissolution rate (Figure 8). No degradation of released peptide was observed when 0.02% sodium azide was added to the medium (Figure 7). Evaporation of the medium was measured to be approximately 1 mL / day / 100 mL (data not shown). Based on these results, 33 mM phosphate buffer supplemented with 0.2% TWEEN 20 was selected.

[0079] Example 2 The novel method disclosed herein was compared with the conventional USP2 and USP4 methods. When the USP2 method was used for the dissolution of LAI microsphere formulations, the microspheres were observed floating on the surface of the dissolution medium, despite the use of a wetting agent for sample introduction. The microspheres also tended to aggregate and float around the shaft, affecting sampling and detection. When the USP2 method was applied to hydrogel and liquid crystal formulations, the samples piled up at the bottom of the container wall, slowing down drug release due to reduced exposure to the medium.

[0080] When the same formulation was tested using USP4, microbial growth occurred in the vessels despite the addition of sodium azide to the medium. No suitable method has been found to overcome this growth. The microbial growth led to cleavage of peptides in the LAI formulation during dissolution and drug release testing, resulting in non-specific peaks in the chromatograms.

[0081] Sandwiching microsphere, hydrogel, or liquid crystal LAI samples using glass beads facilitates drug release testing at relatively low agitation speeds using paddles, and allows in situ measurement during dissolution using UV fiber optics without sampling, improving testing simplicity, productivity, and reducing variability.

[0082] Example 3 The dissolution method disclosed herein was used to dissolve a fatty acid-relaxin (FA-RLX) sustained release injectable formulation. A suspension of FA-RLX was designed as a sustained release injectable formulation. FA-RLX particle sizes (PS) of <20 μm, <50 μm, and <100 μm were tested. FA-RLX of the indicated particle sizes were suspended in Miglyol 812N at a concentration of 17.5 mg / mL. 0.5 mL of each suspension was then placed under 0.5 mm glass beads in a 200 mL vessel filled with 50 mL of 4% hydroxypropyl-β-cyclodextrin in Dulbecco's phosphate buffered saline (PBS). A paddle was placed above the glass beads and the dissolution was performed at 34°C with an agitation speed of 35 rpm. The percentage of FA-RLX dissolved was measured by second derivative at 293-323 nm (2 mm probe path length) every 5 min for a total of 288 measurements over 24 h.

[0083] The glass bead under paddle method showed reproducibility for suspensions with PS <20 μm and PS <100 μm FA-RLX formulations (Figure 9A). A faster release can be observed in the dissolution profile of the PS <20 μm suspension. Approximately 90% release was observed after 60 min for the PS <20 μm suspension. A slower release can be observed in the dissolution profile of the PS <100 μm suspension. Approximately 55% release was observed after 60 min for the PS <100 μm suspension. Differences can be observed between the dissolution profile of the PS <20 μm suspension and the other dissolution profiles. However, the dissolution profile of the PS <100 μm suspension was indistinguishable from the PS <50 μm suspension due to the large variability in the dissolution profile of the PS <50 μm suspension (Figure 9C).

[0084] All publications, patents, and patent applications disclosed in this specification are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An in vitro dissolution method comprising agitating a solution contained in a container using a movable paddle, the solution comprising a solute, a medium, and a plurality of beads, the plurality of beads being disposed between the solute and the movable paddle, and the movable paddle being submerged in the solution.

2. Before mixing, (i) adding a solute to the bottom of a container; (ii) adding a plurality of beads to a bottom of the container, the plurality of beads being added above the solute; (iii) adding a volume of a medium to the container, the medium being added over the solute and the plurality of beads; 2. The in vitro dissolution method of claim 1, comprising placing the solution in a container by:

3. 3. The in vitro dissolution method of claim 2, wherein the medium is added without agitation of the solute or the plurality of beads.

4. 10. The in vitro elution method of claim 1, wherein the movable paddle does not contact the plurality of beads or solutes while the movable paddle is in the stationary position.

5. 2. The in vitro dissolution method of claim 1, wherein the solute comprises a sustained release injectable agent.

6. 2. The in vitro dissolution method of claim 1, wherein the solute comprises a sustained release injectable microsphere.

7. 2. The in vitro elution method of claim 1, wherein the plurality of beads comprises one or more glass beads, one or more plastic beads, one or more silicate beads, one or more metal beads, or any combination thereof.

8. 2. The in vitro dissolution method of claim 1, wherein the plurality of beads comprises one or more beads having a diameter of at least about 0.01 mm, at least about 0.05 mm, at least about 0.1 mm, at least about 0.5 mm, at least about 1.0 mm, at least about 1.5 mm, or at least about 2.0 mm.

9. 2. The in vitro dissolution method of claim 1, wherein the plurality of beads comprises one or more beads having a diameter of about 1.0 mm.

10. 10. The in vitro dissolution method of claim 1, wherein the volume of solution in the container is sufficient to submerge the movable paddle, the plurality of beads, and the solute.

11. 2. The in vitro dissolution method of claim 1, wherein the volume of the solution in the container is at least about 50 mL, at least about 60 mL, at least about 70 mL, at least about 75 mL, at least about 80 mL, at least about 90 mL, or at least about 100 mL.

12. 2. The in vitro dissolution method of claim 1, wherein the plurality of beads comprises at least about 3 g of beads per about 100 mL of solution, at least about 4 g of beads per about 100 mL of solution, at least about 5 g of beads per about 100 mL of solution, at least about 6 g of beads per about 100 mL of solution, or at least about 7 g of beads per about 100 mL of solution.

13. 2. The in vitro dissolution method of claim 1, wherein the plurality of beads comprises about 4 g of beads per 100 mL of solution.

14. 10. The in vitro dissolution method of claim 1, wherein the plurality of beads comprises less than about 6 g of beads per 100 mL of solution.

15. 2. The in vitro dissolution method of claim 1, wherein the solute comprises sustained injectable microspheres carrying a biologically active moiety.

16. 10. The in vitro dissolution method of claim 1, further comprising covering the container during dissolution.

17. 17. The in vitro dissolution method of claim 16, wherein the cover reduces or eliminates the loss of evaporated solution during dissolution.

18. 2. The in vitro dissolution method of claim 1, wherein the pH of the solution is adjusted.

19. 10. The in vitro elution method of claim 1, wherein an additional volume of a plurality of beads is added below the solute.

20. (i) adding a solute, including sustained release injectable microspheres, to the bottom of a container; (ii) adding a plurality of beads, including one or more glass beads, above the solute; (iii) adding a medium to the solute and the volume of beads, the medium being added without stirring the solute or the volume of beads, and the medium being added in a volume sufficient to submerge the movable paddle; (iv) applying a rotating and / or vibrating force to the movable paddle to stir the solution; 2. The in vitro dissolution method of claim 1, comprising:

21. (i) adding a first plurality of beads comprising one or more glass beads to a bottom of a container; (ii) adding a solute comprising sustained release injectable microspheres onto the plurality of beads; (iii) adding a second plurality of beads comprising one or more glass beads above the solute; (iv) adding a medium to the solute and the volume of beads, the medium being added without stirring the solute or the volume of beads, and the medium being added in a volume sufficient to submerge the movable paddle; (v) applying a rotating and / or vibrating force to the movable paddle to stir the solution; 2. The in vitro dissolution method of claim 1, comprising:

22. 10. The in vitro dissolution method of claim 1, further comprising adding a volume of a media solution to the solute and / or plurality of beads, the volume of media solution being sufficient to wet but not completely submerge the solute and / or plurality of beads.

23. 10. The in vitro dissolution method of claim 1, further comprising detecting the presence of the dissolved solute in the medium.

24. 24. The in vitro elution method of claim 23, wherein detecting comprises placing a probe in the solution, obtaining a sample of the solution, or a combination thereof.

25. 25. The in vitro dissolution method of claim 23 or 24, wherein the presence of the dissolved solute in the medium is detected by probing and / or taking a sample from a portion of the solution above the moveable paddle.

26. 2. The in vitro dissolution method of claim 1, wherein the solute comprises a sustained release injectable formulation comprising a biologically active moiety.

27. 27. The in vitro elution method of claim 26, wherein the biologically active moiety comprises a small molecule or a polypeptide.

28. 28. The in vitro elution method of claim 27, wherein the molecular weight of the polypeptide is less than about 10 kDa, less than about 9 kDa, less than about 8 kDa, less than about 7 kDa, less than about 6 kDa, less than about 5 kDa, or less than about 4 kDa.

29. 29. The in vitro elution method of claim 27 or 28, wherein the molecular weight of the polypeptide is about 4 kDa.

30. 10. The in vitro dissolution method of claim 1, wherein the solution further comprises a surfactant.

31. 31. The in vitro dissolution method of claim 30, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, TRITON X100, PLURONIC F-68, and any combination thereof.

32. 10. The in vitro dissolution method of claim 1, wherein the solution further comprises an antimicrobial agent.

33. (i) a container; and (ii) a plurality of beads; (iii) a medium; and (iv) a movable paddle, the movable paddle positionable within the vessel; and (v) a suitable cover; and (vi) instructions for eluting a solute according to the method of claim 1; Kit including:

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