EP4662486A1 - Capillary electrophoresis for biomolecule encapsulation efficiency determination - Google Patents

Abstract

Described and claimed herein are capillary electrophoresis methods and kits for characterizing encapsulation efficiency or characterizing a biomolecule on an encapsulation.

Description

CAPILLARY ELECTROPHORESIS FOR BIOMOLECULE ENCAPSULATION EFFICIENCY DETERMINATION

RELATED APPLICATIONS

[001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63/484,15, filed February 9, 2023 and U.S. Provisional Patent Application Ser. No. 63/583,923, filed September 20, 2023, the content of which is hereby incorporated by reference in its entirety into this disclosure.

BACKGROUND

[002] Biomolecules, such as messenger ribonucleic acid (mRNA), have emerged as promising therapeutic tools in vaccine development and protein replacement therapy. Delivery vehicles, such as lipid nanoparticles (LNPs) and viral vectors, are effective materials for delivering biomolecules. Encapsulation efficiency, the measurement of the percentage of delivery vehicles that successfully encapsulate biomolecules, is a critical quality attribute as it affects the efficacy and safety of the biologies. Encapsulation efficiency has been analyzed using intercalating dye measured by fluorescent detection and light scattering. However, these methods require optimization for different biomolecules, the use of degraded biomolecules, or are biased by detection scatter from the delivery vehicle.

SUMMARY

[003] The inventors have recognized the need for high-precision analysis of encapsulated biomolecules, including encapsulation efficiency. The claimed and described capillary electrophoresis methods offer high resolution and high sensitivity, automated analysis of encapsulated biomolecules, and provide information to determine the encapsulation efficiency.

[004] One aspect of the disclosure is a method for characterizing encapsulation efficiency, the method including loading a first sample comprising an encapsulated biomolecule and a free biomolecule on a capillary electrophoresis (CE) capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix; applying a separation voltage to the CE capillary to separate the free biomolecule; detecting the separated free biomolecule with a detector; producing an electropherogram comprising corrected peak area of the free biomolecule and generating a first corresponding set of values; loading a second sample comprising a total biomolecule, on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix; applying a separation voltage to the CE capillary to separate total biomolecule; detecting the total biomolecule with a detector; producing an electropherogram comprising corrected peak area of the total biomolecule and generating a second corresponding set of values; and wherein encapsulation efficiency is determined using the first and second corresponding set of values.

[005] Another aspect of the disclosure is a method for characterizing a biomolecule on an encapsulation surface, the method including loading a first sample comprising an encapsulated biomolecule and a free biomolecule on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix; applying a separation voltage to the CE capillary to separate the free biomolecule; detecting the separated free biomolecule with a detector; producing an electropherogram comprising corrected peak area of the free biomolecule and generating a first corresponding set of values; and wherein the first corresponding set of values and a measurement value of a free biomolecule and a biomolecule on an encapsulation surface are used to determine an amount of a biomolecule on a surface of an encapsulating material in a sample.

[006] In an aspect, the method further includes obtaining the measurement value of the free biomolecule and the biomolecule on the surface of the encapsulating material in the sample using a known biomolecule quantitation value.

[007] In an aspect, the method further includes loading a second sample comprising a total biomolecule, on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix; applying a separation voltage to the CE capillary to separate total biomolecule; detecting the total biomolecule with a detector; producing an electropherogram comprising corrected peak area of the total biomolecule and generating a second corresponding set of values; and wherein encapsulation efficiency is determined using the first and second corresponding set of values.

[008] In an aspect, the encapsulated biomolecule comprises fully encapsulated and the biomolecule on the surface of the encapsulating material. In an aspect, an amount of the biomolecule on the surface of the encapsulating material is determined using the encapsulation efficiency. In an aspect, an amount of the biomolecule on the surface of the encapsulating material is determined using the encapsulation efficiency and a known biomolecule quantitation value. [009] In an aspect, the total biomolecule comprises free biomolecule and a released biomolecule. In an aspect, the released biomolecule is a biomolecule that has been subjected to a treatment agent, wherein the treatment agent releases the biomolecule from an encapsulating material. In an aspect, the released biomolecule is a biomolecule that was encapsulated inside an encapsulating material and/or on a surface of an encapsulating material that has been subjected to a treatment agent, wherein the treatment agent releases the biomolecule from the encapsulating material.

[010] In an aspect, the treatment agent comprises a detergent, a surfactant, a lysis buffer, an enzyme, endonuclease, a protease, a peptidase, a proteinase, a nuclease, a hypotonic solution, an antibiotic, a chelating agent, a solvent, a chaotropic agent, or a combination thereof.

[Oi l] In an aspect, the detergent comprises Triton or Tween. In an aspect, the lysis buffer comprises urea, thiourea, or a chaotropic agent.

[012] In an aspect, the encapsulating material is a lipid nanoparticle or a viral vector. In an aspect, the encapsulating material is a lipid nanoparticle comprising one or more of an ionizable cationic lipid, a PEGylated lipid, a phospholipid, and/or cholesterol.

[013] In an aspect the method further includes loading a third sample comprising a known amount of a biomolecule on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix, applying a separation voltage to the CE capillary to separate biomolecule; detecting the separated biomolecule with a detector; producing an electropherogram comprising corrected peak area of the separated biomolecule and generating a third corresponding set of values; determining, using the first and third corresponding set of values, a concentration of the free biomolecule in the first sample; determining, using the second and third corresponding set of values, a concentration of the total biomolecule in the treated sample; and using the concentrations to determine the encapsulation efficiency.

[014] In an aspect, the first sample, second sample, and/or third sample are loaded onto separate capillaries or loaded sequentially on the same capillary.

[015] In an aspect, the biomolecule is a protein, a polynucleotide, or nucleic acid. In an aspect, the biomolecule is a nucleic acid selected from the group consisting of DNA, single-stranded (ss)DNA, RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small RNA (sRNA), microRNAs (miRNA), piwi-interacting RNA (piRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), small-subunit ribosomal RNA (srRNA), tRNA-derived fragments (tRF), and yRNA-derived fragments (ysRNA).

[016] In an aspect, the biomolecule in the third sample is a nucleic acid with a known biomolecule quantitation value, mRNA with a known biomolecule quantitation value, RNA with a known biomolecule quantitation value, ssDNA with a known biomolecule quantitation value, a protein with a known biomolecule quantitation value, a peptide with a known biomolecule quantitation value, and/or an amino acid with a known biomolecule quantitation value.

[017] In an aspect, the biomolecule is mRNA, the third sample comprises at least a first mRNA with a known concentration and a second mRNA with a known concentration.

[018] In an aspect, the measurement value of the free biomolecule and the biomolecule on the surface of the encapsulating material and/or the known biomolecule quantitation value is generated from a fluorescence -based biomolecule-quantitation assay.

[019] In an aspect, the third sample comprises at least a first known amount of a biomolecule and at least a second known amount of a biomolecule. In an aspect, the third sample comprises a biomolecule that has similar molecular weight and/or length as the biomolecule in the first sample and/or second sample.

[020] In an aspect, the first sample, the second sample, and/or the third sample is purified and/or enriched prior to loading onto the CE capillary. In an aspect, the first sample, the second sample, and/or the third sample is purified or enriched using spin columns, spin tubes, and/or magnetic beads. In an aspect, the first sample, the second sample, and/or the third sample, is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary. In an aspect, the sample solution comprises nuclease-free water, tris(hydroxymethyl)aminomethane (Tris) - ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer), phosphate buffered saline (PBS), PBS with sucrose, formamide, and combinations thereof.

[021] In an aspect, the biomolecule in the first sample, second sample, and/or third sample is fluorescently labeled with a fluorescent dye prior to CE separation. In an aspect, the biomolecule in the first sample, second sample, and/or third sample is fluorescently labeled with a fluorescent dye during the CE separation. In an aspect, the fluorescent dye is added to the buffer. In an aspect, the fluorescent dye is a cyanine-based dye, a prylium-based dye, an FQ dye, or a Tamara dye. In an aspect, the fluorescent dye is selected from the group including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, SYBR Green I, SYBR GOLD, SYBR Green II, PicoGreen, Thiazole orange, Oxazole yellow, Rhodmine, Fluorescein, and Fluorescent Chromeo Py-Dyes.

[022] In an aspect, the detector is a UV detector or fluorescence detector. In an aspect, the detector is a laser-induced fluorescence (LIF) detector, a lamp-based fluorescence detector, or a native fluorescence detector.

[023] One aspect of the disclosure is a kit for characterizing encapsulation efficiency, the kit including a fluorescent dye; a buffer comprising a polymer matrix; a treatment agent; at least one sample comprising a known amount of a biomolecule; and instructions for use. In an aspect, the treatment agent comprises a detergent or a lysis buffer. In an aspect, the biomolecule standard is mRNA with a known concentration.

[024] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will be more fully understood from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

[025] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[026] FIG. 1 shows parameter settings for a conditioning method according to an aspect of the disclosure.

[027] FIG. 2 shows parameter settings for a separation method according to an aspect of the disclosure.

[028] FIG. 3 shows electropherogram results for a 1.9kb firefly luciferase (Flue) mRNA standard that was serially diluted to different concentrations and treated according to an aspect of the disclosure.

[029] FIG. 4 shows a calibration standard curve made with a Flue mRNA standard by plotting the corrected peak area of the Flue mRNA peak against Flue mRNA concentration.

[030] FIG. 5 shows electropherogram results for free mRNA outside of an untreated mRNA-lipid nanoparticle. [031] FIG. 6 shows electropherogram results for the total mRNA of a treated mRNA-lipid nanoparticle, wherein the treatment releases the mRNA from the encapsulation.

[032] FIG. 7 shows parameter settings for a shutdown method according to an aspect of the disclosure.

[033] FIG. 8 shows a workflow according to an aspect of this disclosure.

DETAILED DESCRIPTION

[034] Aspects of the present disclosure include methods for characterizing encapsulation efficiency, in particular, encapsulation efficiency of biomolecules. Aspects of the present disclosure also include characterizing a biomolecule on an encapsulation surface.

[035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.) refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[036] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[037] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[038] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[039] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. [040] By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[041] The singular form "a", "an" and "the" include plural reference unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and/or" unless the content clearly dictates otherwise. The term "and/or" means any one or more of the items in the list joined by "and/or". As an example, "x and/or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and/or y" means "one or both of x and y". As another example, "x, y, and/or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and/or z" means "one or more of x, y and z".

[042] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[043] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[044] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is +/-10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[045] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[046] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[047] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest. [048] The invention is defined in the claims. However, below is a non-exhaustive listing of nonlimiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[049] Biomolecules are organic compounds and can include, for example, proteins, peptides, nucleic acids, and polynucleotides. Non-limiting examples of nucleic acids include DNA, singlestranded (ss)DNA, RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small RNA (sRNA), microRNAs (miRNA), piwi- interacting RNA (piRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), smallsubunit ribosomal RNA (srRNA), tRNA-derived fragments (tRF), and yRNA-derived fragments (ysRNA).

[050] In a non-limiting example, a first sample containing an encapsulated biomolecule and a free biomolecule is loaded on a capillary electrophoresis (CE) capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix. A separation voltage is applied to the CE capillary to separate the free biomolecule, which is then detected with a detector. An electropherogram including the corrected peak area of the free biomolecule is produced, and a first set of values is generated.

[051 ] In this non-limiting example, a second sample containing a total biomolecule is loaded on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix. In this aspect, the total biomolecule includes free biomolecule and a released biomolecule. A separation voltage is applied to the CE capillary to separate the total biomolecule, which is then detected with a detector. An electropherogram including the corrected peak area of the total biomolecule is produced, and a second set of values is generated. Using the first and second set of values, the encapsulation efficiency may be determined. FIG. 8 depicts an exemplary workflow. In a nonlimiting example, the following equation may be used to calculate encapsulation efficiency:

((second set of values - first set of values)/second set of values) * 100

[052] A released biomolecule is an encapsulated biomolecule that has been subjected to a treatment agent, wherein the treatment agent releases the biomolecule from an encapsulating material. Nonlimiting examples of the treatment agent include a detergent, a surfactant, a lysis buffer, an enzyme, endonuclease, a protease, a peptidase, a proteinase, a nuclease, a hypotonic solution, a chelating agent, a solvent, a chaotropic agent, or a combination thereof. [053] If the treatment agent is a detergent, it may be a nonionic surfactant such as Triton or Tween. Triton is a nonionic surfactant prepared by the reaction of octylphenol with ethylene oxide and are commonly described as alkylaryl polyether alcohols. Non-limiting examples of Triton surfactants include Triton X-100, Triton X-45, Triton X-405, and Triton X-114. Tween is a polysorbate-type nonionic surfactant formed by the ethoxylation of sorbitan monolaurate. Non-limiting examples of Tween include Tween 20 and Tween 80.

[054] If the treatment agent is a lysis buffer, it can be any suitable buffer solution used to disrupt cells or other biological materials. Non-limiting examples of a lysis buffer include urea, thiourea, or a chaotropic agent.

[055] An encapsulated biomolecule is a biomolecule that is in communication with an encapsulating material. In some aspects, the biomolecule is at least partially encapsulated by the encapsulating material. In other aspects, the biomolecule is fully encapsulated by the encapsulating material. The encapsulating material may be, for example, a delivery vehicle. Non-limiting examples include a lipid nanoparticle or a viral vector. In some examples, the lipid nanoparticle includes one or more of an ionizable cationic lipid, a PEGylated lipid, a phospholipid, and/or cholesterol. In a nonlimiting aspect, the biomolecule is mRNA and the encapsulating material is a lipid nanoparticle. The encapsulated mRNA may be referred to as an mRNA-lipid nanoparticle, the first sample may be referred to as an untreated sample, and/or the second sample may be referred to as a treated sample.

[056] In some non-limiting examples, the method further includes loading a third sample comprising a known amount of a biomolecule on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix. A separation voltage is applied to the CE capillary to separate the biomolecule, which is then detected with a detector. An electropherogram including corrected peak area of the separated biomolecule is produced and a third set of values is generated.

[057] In an aspect, the third sample may include at least one biomolecule that is known and/or well- characterized. In another aspect, the third sample includes a known concentration or amount of at least one biomolecule. In a non-limiting example, the third sample may include a known concentration of a known and/or well-characterized biomolecule. The biomolecule in the third sample may include a nucleic acid with a known concentration, mRNA with a known concentration, RNA with a known concentration, ssDNA with a known concentration, a protein with a known concentration, a peptide with a known concentration, and/or an amino acid with a known concentration. In a non-limiting aspect, the third sample is mRNA with a known concentration. The third sample may also be referred to as a standard, standard sample, and/or standard solution.

[058] In some examples, the third sample includes at least one biomolecule, alternatively at least two biomolecules, alternatively at least three biomolecules, alternatively at least four biomolecules, alternatively at least five biomolecules, alternatively at least six biomolecules, alternatively at least seven biomolecules, alternatively at least eight biomolecules, alternatively at least nine biomolecules, or alternatively at least ten biomolecules. In an example, the biomolecule in the third sample is of a similar molecular weight and/or length as the biomolecule in the first sample and/or second sample.

[059] The third corresponding set of values may be used to determine the concentrations of biomolecule in the first and second samples. For example, using the first and third corresponding set of values, a concentration of the free biomolecule in the first sample may be determined. Using the second and third corresponding set of values, a concentration of the total biomolecule in the treated sample may be determined. The resulting concentrations may be used to determine encapsulation efficiency.

[060] In a non-limiting example, the following equation may be used to calculate encapsulation efficiency:

((concentration of total biomolecule - concentration of free biomolecule)/ concentration of total biomolecule) * 100

[061] In a non-limiting example, the first sample and/or the second sample is purified and/or enriched prior to loading onto the CE capillary. The first sample and/or second sample may be purified or enriched using any technique known in the art. In an aspect, the first sample and/or the second sample are purified or enriched using spin columns, spin tubes, and/or magnetic beads. In another non-limiting aspect, the first sample and/or second sample is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary. In some embodiments the sample solution comprises nuclease-free water, tris(hydroxymethyl)aminomethane (Tris) - ethylenediaminetetraacetic acid (EDTA) buffer (i.e., TE buffer), phosphate buffered saline (PBS), PBS with sucrose, a LNP formulation buffer containing salt and stabilizers, and/or formamide. [062] Depending on the type of analysis desired, the first sample, second sample, and/or third sample are loaded onto separate capillaries. Alternatively, the first sample, second sample, and/or third sample and loaded sequentially on the same capillary.

[063] In an aspect, the detector is a UV detector or fluorescence detector. Non-limiting examples of a fluorescence detector include a laser-induced fluorescence (LIF) detector, a lamp-based fluorescence detector, or a native fluorescence detector. The desired quantitation sensitivity will determine the type of detector used. LIF detection offers the benefit of about a 100-fold increase in sensitivity relative to UV detection, yet it also requires additional sample manipulation.

[064] Depending on the type of detector used, the biomolecule in the first sample, second sample, and/or third sample may be fluorescently labeled with a fluorescent dye prior to CE separation. For example, if the biomolecule is a protein or peptide, the biomolecule may be fluorescently labeled prior to separation. In some other aspects, the biomolecule in the first sample, second sample, and/or third sample may be fluorescently labeled with a fluorescent dye during the CE separation. In this aspect, the fluorescent dye is added to the buffer. For example, if the biomolecule is a nucleic acid, the biomolecule may be fluorescently labeled during separation.

[065] Any fluorescent dye known in the art may be used. In some non-limiting aspects, dyes such as Fluorescent Chromeo Py-Dyes may be used if the biomolecule is a protein or peptide. In other non-limiting aspects, dyes such as SYBR Green I, SYBR GOLD, or SYBR Green II may be used if the biomolecule is a nucleic acid.

[066] In a non-limiting example, the fluorescent dye is a cyanine-based dye, a prylium-based dye, an FQ dye, or a Tamara dye. For example, fluorescent dye may be Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, SYBR Green I, SYBR GOLD, SYBR Green II, PicoGreen, Thiazole orange, Oxazole yellow, Rhodmine, Fluorescein, and Fluorescent Chromeo Py-Dyes.

[067] In a non-limiting aspect, the disclosed encapsulation efficiency method and known biomolecule quantitation value can be used to determine the amount of biomolecule on the surface of an encapsulation material. In an aspect, the method includes measuring free-floating biomolecule using capillary electrophoresis and the biomolecule on the surface of an encapsulation material and free-floating biomolecule are detected using, for example, fluorescence-based RNA-quantitation dyes, such as RiboGreen. [068] In another aspect, the method for characterizing the biomolecule on the surface of the encapsulation material includes obtaining a first data set, wherein the first data set comprises an encapsulation efficiency determined using the disclosed method and obtaining a second data set, wherein the second data set includes a known biomolecule quantitation value. The amount of biomolecule on the surface of the encapsulation material is then determined using the first data set and the second data set.

[069] In a non-limiting example, the following equation may be used to determine the amount of biomolecule on the surface of the encapsulation material present: calculated encapsulation efficiency (or first data set) - known biomolecule quantitation value (or second data set)

[070] In some embodiments, a measurement value of a free biomolecule and a biomolecule on the surface of an encapsulation material is the amount of the free biomolecule and the biomolecule on the surface of an encapsulation material in a sample. In some examples, a measurement value is an encapsulation efficiency of a particular sample determine via an analytical method that detects the free biomolecule and the biomolecule on the encapsulation surface at the same time. In some examples, such analytical methods may include RiboGreen dye florescence-based methods, where both free biomolecule and the biomolecule on the encapsulation surface are detected at the same time.

[071] One aspect of the disclosure is a kit for characterizing encapsulation efficiency. The kit may include a fluorescent dye, a CE capillary, a buffer comprising a polymer matrix, a treatment agent, at least one sample comprising a known amount of a biomolecule, and instructions for use. In non-limiting aspects, the treatment agent is a detergent or a lysis buffer. In another aspect, the biomolecule standard is mRNA with a known concentration.

[072] EXAMPLES

[073 ] Instrument and Software

[074] A PA 800 Plus Pharmaceutical Analysis system or a BioPhase 8800 system equipped with LIF detector and solid-state laser with excitation wavelength at 488 nm and a 520 nm band pass emission filter were from SCIEX (Framingham, MA) and a 600 nm band pass emission filter from Edmund Optics (Barrington, NJ). [075 ] Sample Preparation

[076] For building the standard curve: A Flue mRNA sample with a known concentration ( 1 mg/ml) was first diluted with 1 mM sodium citrate to 500 pg/ml, and then further diluted to 20 pg/ml in a diluent buffer containing 6 mM sucrose, 6.85 mM NaCl, 0.54 mM KC1, 0.16 mM Na2HPC>4, 0.4 mM KH2PO4, 0.2 mM sodium citrate, and 0.2% Triton X-100 in nuclease-free water. After that, it was serially diluted with the diluent buffer from 20 pg/ml to 0.39 pg/ml. Samples were incubated at room temperature for 20 minutes, diluted with an equal volume of sample loading solution (SLS), heated at 70°C for 4 minutes, and chilled on ice for at least 5 minutes. Samples were transferred to sample wells on a sample plate for BioPhase 8800 system before CE separation.

[077] Untreated test sample: a sample containing mRNA encapsulated with an LNP and a free mRNA was diluted 5 fold with nuclease-free water, and loaded onto the BioPhase 8800 system for separation. These samples were not heated at 70°C or diluted with SLS to keep the LNP particles intact.

[078] Alternatively, the untreated test sample is purified prior to separation.

[079] Treated test sample: a sample containing mRNA encapsulated with an LNP and a free mRNA was first diluted with lx PBS (137 mM NaCl, 2.7 mM KC1, 8 mM Na₂HPO4, and 2 mM KH2PO4) with 300 mM sucrose to 250 pg/ml, and then further diluted to 10 ug/ml in a diluent buffer containing 6 mM sucrose, 6.85 mM NaCl, 0.54 mM KC1, 0.16 mM Na2HPC>4, 0.4 mM KH2PO4, 0.2 mM sodium citrate, and 0.2% Triton X-100 in nuclease-free water. Samples were incubated at room temperature for 20 minutes. The treatment with Triton X-100 releases the encapsulated mRNA from the LNP. Samples were then diluted with an equal volume of sample loading solution (SLS), heated at 70°C for 4 minutes, and chilled on ice for at least 5 minutes. Samples were transferred to sample wells on a sample plate for BioPhase 8800 system before CE separation. The released and free mRNA may be purified prior to separation.

[080] CE separation of RNA samples with LIE detection: The RNA 9000 Purity & Integrity kit (PN C48231) containing the Nucleic Acid Extended Range Gel, SYBR™ Green II RNA Gel Stain, Acid Wash (Regenerating Solution), CE Grade water and the ssRNA Ladder (0.5-9 kb) was used. The Nucleic Acid Extended Range Gel was warmed to room temperature and then filtered with a 0.2 pm filter from PALL (PN 4612). Then, SYBR Green II RNA Gel Stain was added to the gel at 500- fold dilution in a 50 ml conical tube and mixed well by capping the tube tightly and gently reverting the tube 10 times. The gel was transferred to buffer plates (disposable, from BioPhase sample and reagent plates (4,4,8), PN 5080311) before CE separation with the pre-assembled BioPhase BFS capillary cartridge - 8 x 30 cm (8 capillaries, 30 cm total length, PN 5080121).

[081 ] Generation of Standard Curve

[082] A Flue mRNA standard was generated by serially diluting the Flue mRNA sample to a known concentration. The capillaries are rinsed sequentially with the conditioning method (FIG. 1). Samples were introduced into the inlet of the capillary using pressure at 1 psi for 5s. Separations were be performed as shown in FIG. 2. A EIF detector was configured with a 488-nm laser with an emission filter of 520 nm. SCIEX Analysis software can be used for data processing. FIG. 3 shows electropherogram results for a 1.9kb firefly luciferase (Flue) mRNA standard that was serially diluted to different concentrations and treated under the same conditions as lipid nanoparticle (ENP) samples before capillary electrophoresis.

[083] The corrected peak area was plotted against the Flue mRNA concentration. (FIG. 4). The Flue mRNA calibration standard built with Flue mRNA samples of known concentrations is used to determine the amount of free mRNA present in the untreated and treated samples.

[084] Using CGE-EIF to Determine Encapsulation Efficiency

[085] The untreated sample were introduced into the inlet of the capillary using pressure at 1 psi for 5s. (FIG. 5) Separations were performed as shown in FIG. 2. The EIF detector was configured with a 488-nm laser with an emission filter of 520nm. SCIEX Analysis software can be used for data processing. Using the Flue mRNA standard curve, the concentration of free mRNA present in the untreated sample was calculated at 20.96 pg/ml.

[086] The treated sample were introduced into the inlet of the capillary using pressure at 1 psi for 5s. (FIG. 6) Separations were performed as shown in FIG. 2. The EIF detector was configured with a 488-nm laser with an emission filter of 520nm. SCIEX Analysis software can be used for data processing. Using the Flue mRNA standard curve, the concentration of total mRNA present in the treated sample was calculated at 436.47 pg/ml.

[087] The encapsulation efficiency was calculated by using the following equation: ((concentration of total biomolecule - concentration of free biomolecule)/ concentration of total biomolecule) * 100 ((436.47 - 20.96) / 436.47) *100 = 95.2% [088] The encapsulation efficiency for the same sample determined using the RiboGreen dye was 92%, showing that the disclosed methods are consistent with the RiboGreen dye method.

[0891 Using CGE-LIF to Characterize Biomolecules on a lipid nanoparticle (ENP) surface

[090] The biomolecule may be present inside the LNP, on the outside of the LNP, and as free- floating in the solution. Certain analytical methods could detect both the biomolecule on the LNP surface and free floating biomolecule. As a non-limiting example, some fluorescence -based RNA- quantitation dyes, such as RiboGreen, will bind to both biomolecule on the LNP surface and free floating biomolecule. Using a measurement value generated from such analytical methods, the amount of biomolecule on the surface can be calculated.

[091] In some examples, a sample comprising a free biomolecule and an LNP with encapsulated biomolecule inside and the biomolecule on its surface can be loaded on a capillary electrophoresis (CE) capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix. A separation voltage can then be applied to the CE capillary to separate the free biomolecule; and the separated free biomolecule can be detected with a detector. An electropherogram comprising corrected peak area of the free biomolecule thus can be produced and a first corresponding set of values can be generated (Value FF). A measurement value of the free biomolecule and the biomolecule on the encapsulation surface can also be obtained — e.g., via RiboGreen dye fluoresce assay (Value FFS). Then Value FF and Value FFS can be used to calculate the biomolecule on the encapsulation surface, e.g., but using the following equation:

Amount of the biomolecule on the encapsulation surface = V alue FFS - V alue FF.

[092] In another example, the encapsulation efficiency was calculated by using the following equation (using capillary electrophoresis methods described therein):

((concentration of total biomolecule - concentration of free biomolecule)/ concentration of total biomolecule) * 100 ((436.47 - 20.96) / 436.47) *100 = 95.2% (Value A)

[093] The encapsulation efficiency determined using RiboGreen dye was 92%. (Value B)

[094] The amount of., the biomolecule on the encapsulation surface) was calculated using the following equation:

Value A- Value

[095] This indicated that there is 3% of partially encapsulated biomolecule (e.g., biomolecule on the encapsulation surface) and accounts for the difference between the calculated encapsulated efficiency (using capillary electrophoresis methods described therein) and encapsulation efficiency determined using RiboGreen test.

[096] While the present disclosure has been described with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the present disclosure or appended claims. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present disclosure without departing from its scope. Therefore, it is intended that the present disclosure not be limited to the particular embodiments disclosed, but that the present disclosure will include all aspects falling within the scope of the appended claims.

[097] All patents, patent applications, publications, and descriptions mentioned above are herein incorporated by reference in their entirety.

Y1

Claims

CLAIMS What is Claimed Is:

1. A method for characterizing a biomolecule on an encapsulation surface, the method comprising: loading a first sample comprising an encapsulated biomolecule and a free biomolecule on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix; applying a separation voltage to the CE capillary to separate the free biomolecule; detecting the separated free biomolecule with a detector; producing an electropherogram comprising corrected peak area of the free biomolecule and generating a first corresponding set of values; and wherein the first corresponding set of values and a measurement value of a free biomolecule and a biomolecule on an encapsulation surface are used to determine an amount of a biomolecule on a surface of an encapsulating material in a sample.

2. The method of claim 1 , further comprising obtaining the measurement value of the free biomolecule and the biomolecule on the surface of the encapsulating material in the sample using a known biomolecule quantitation value.

3. The method of claim 1 or claim 2, wherein the method further comprises loading a second sample comprising a total biomolecule, on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix; applying a separation voltage to the CE capillary to separate total biomolecule; detecting the total biomolecule with a detector; producing an electropherogram comprising corrected peak area of the total biomolecule and generating a second corresponding set of values; and wherein encapsulation efficiency is determined using the first and second corresponding set of values.

4. The method of claim 1 or claim 2, wherein the encapsulated biomolecule comprises fully encapsulated and the biomolecule on the surface of the encapsulating material.

5. The method of claim 4, wherein an amount of the biomolecule on the surface of the encapsulating material is determined using the encapsulation efficiency.

6. The method of claim 4, wherein an amount of the biomolecule on the surface of the encapsulating material is determined using the encapsulation efficiency and a known biomolecule quantitation value.

7. The method of claim 3, wherein the total biomolecule comprises free biomolecule and a released biomolecule.

8. The method of claim 7, wherein the released biomolecule is a biomolecule that has been subjected to a treatment agent, wherein the treatment agent releases the biomolecule from an encapsulating material.

9. The method of claim 7, wherein the released biomolecule is a biomolecule that was encapsulated inside an encapsulating material and/or on a surface of an encapsulating material that has been subjected to a treatment agent, wherein the treatment agent releases the biomolecule from the encapsulating material.

10. The method of claim 9, wherein the treatment agent comprises a detergent, a surfactant, a lysis buffer, an enzyme, endonuclease, a protease, a peptidase, a proteinase, a nuclease, a hypotonic solution, an antibiotic, a chelating agent, a solvent, a chaotropic agent, or a combination thereof.

11. The method of claim 10, wherein the detergent comprises Triton or Tween.

12. The method of claim 10, wherein the lysis buffer comprises urea, thiourea, or a chaotropic agent.

13. The method of claim 1 or claim 2, wherein the encapsulating material is a lipid nanoparticle or a viral vector.

14. The method of claim 13, wherein the encapsulating material is a lipid nanoparticle comprising one or more of an ionizable cationic lipid, a PEGylated lipid, a phospholipid, and/or cholesterol.

15. The method of claim 1 or claim 2, further comprising: loading a third sample comprising a known amount of a biomolecule on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix, applying a separation voltage to the CE capillary to separate biomolecule; detecting the separated biomolecule with a detector; producing an electropherogram comprising corrected peak area of the separated biomolecule and generating a third corresponding set of values; determining, using the first and third corresponding set of values, a concentration of the free biomolecule in the first sample; determining, using the second and third corresponding set of values, a concentration of the total biomolecule in the treated sample; and using the concentrations to determine the encapsulation efficiency.

16. The method of claim 1 or claim 2, wherein the first sample is loaded onto separate capillaries or loaded sequentially on the same capillary.

17. The method of claim 3, wherein the second sample is loaded onto separate capillaries or loaded sequentially on the same capillary.

18. The method of claim 15, wherein the third sample are loaded onto separate capillaries or loaded sequentially on the same capillary.

19. The method of claim 1 or claim 2, wherein the biomolecule is a protein, a polynucleotide, or nucleic acid.

20. The method of claim 1 or claim 2, wherein the biomolecule is a nucleic acid selected from the group consisting of DNA, single-stranded (ss)DNA, RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small RNA (sRNA), microRNAs (miRNA), piwi-interacting RNA (piRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), small-subunit ribosomal RNA (srRNA), tRNA-derived fragments (tRF), and yRNA-derived fragments (ysRNA).

21. The method of claim 15, wherein the biomolecule in the third sample is a nucleic acid with a known biomolecule quantitation value, mRNA with a known biomolecule quantitation value, RNA with a known biomolecule quantitation value, ssDNA with a known biomolecule quantitation value, a protein with a known biomolecule quantitation value, a peptide with a known biomolecule quantitation value, and/or an amino acid with a known biomolecule quantitation value.

22. The method of claim 21, wherein the biomolecule is mRNA, the third sample comprises at least a first mRNA with a known concentration and a second mRNA with a known concentration.

23. The method of claim 1 or claim 2, wherein the measurement value of the free biomolecule and the biomolecule on the surface of the encapsulating material and/or the known biomolecule quantitation value is generated from a fluorescence-based biomolecule-quantitation assay.

24. The method of claim 15, wherein the third sample comprises at least a first known amount of a biomolecule and at least a second known amount of a biomolecule.

25. The method of claim 15, wherein the third sample comprises a biomolecule that has similar molecular weight and/or length as the biomolecule in the first sample and/or second sample.

26. The method of claim 1 or claim 2, wherein the first sample is purified and/or enriched prior to loading onto the CE capillary.

27. The method of claim 3, wherein the second sample is purified and/or enriched prior to loading onto the CE capillary.

28. The method of claim 15, wherein the third sample is purified and/or enriched prior to loading onto the CE capillary.

29. The method of claim 1 or claim 2, wherein the first sample is purified or enriched using spin columns, spin tubes, and/or magnetic beads.

30. The method of claim 3, wherein the second sample is purified or enriched using spin columns, spin tubes, and/or magnetic beads.

31. The method of claim 15, wherein the third sample is purified or enriched using spin columns, spin tubes, and/or magnetic beads.

32. The method of claim 1 or claim 2, wherein the first sample is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary.

33. The method of claim 32, wherein the sample solution comprises nuclease-free water, tris(hydroxymethyl)aminomethane (Tris) - ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer), phosphate buffered saline (PBS), PBS with sucrose, formamide, and combinations thereof.

34. The method of claim 3, wherein the second sample is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary.

35. The method of claim 34, wherein the sample solution comprises nuclease-free water, tris(hydroxymethyl)aminomethane (Tris) - ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer), phosphate buffered saline (PBS), PBS with sucrose, formamide, and combinations thereof.

36. The method of claim 15, wherein the third sample is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary.

37. The method of claim 36, wherein the sample solution comprises nuclease-free water, tris(hydroxymethyl)aminomethane (Tris) - ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer), phosphate buffered saline (PBS), PBS with sucrose, formamide, and combinations thereof.

38. The method of claims 1 or 2, wherein the biomolecule in the first sample is fluorescently labeled with a fluorescent dye prior to CE separation.

39. The method of claim 3, wherein the biomolecule in the second sample is fluorescently labeled with a fluorescent dye prior to CE separation.

40. The method of claim 15, wherein the biomolecule in the third sample is fluorescently labeled with a fluorescent dye prior to CE separation.

41. The method of claim 1 or claim 2, wherein the biomolecule in the first sample is fluorescently labeled with a fluorescent dye during the CE separation.

42. The method of claim 41, wherein the fluorescent dye is added to the buffer.

43. The method of claim 41, wherein the fluorescent dye is a cyanine -based dye, a prylium- based dye, an FQ dye, or a Tamara dye.

44. The method of claim 41, wherein the fluorescent dye is selected from the group consisting of Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, SYBR Green I, SYBR GOLD, SYBR Green II, PicoGreen, Thiazole orange, Oxazole yellow, Rhodmine, Fluorescein, and Fluorescent Chromeo Py-Dyes.

45. The method of claim 3, wherein the biomolecule in the second sample is fluorescently labeled with a fluorescent dye during the CE separation.

46. The method of claim 45, wherein the fluorescent dye is added to the buffer.

47. The method of claim 45, wherein the fluorescent dye is a cyanine -based dye, a prylium- based dye, an FQ dye, or a Tamara dye.

48. The method of claim 45, wherein the fluorescent dye is selected from the group consisting of Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, SYBR Green I, SYBR GOLD, SYBR Green II, PicoGreen, Thiazole orange, Oxazole yellow, Rhodmine, Fluorescein, and Fluorescent Chromeo Py-Dyes.

49. The method of claim 1 or claim 2, wherein the detector is a UV detector or fluorescence detector.

50. The method of claim 49, wherein the detector is a laser-induced fluorescence (LIF) detector, a lamp-based fluorescence detector, or a native fluorescence detector.

51. A method for characterizing encapsulation efficiency, the method comprising: loading a first sample comprising an encapsulated biomolecule and a free biomolecule on a capillary electrophoresis (CE) capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix; applying a separation voltage to the CE capillary to separate the free biomolecule ; detecting the separated free biomolecule with a detector; producing an electropherogram comprising corrected peak area of the free biomolecule and generating a first corresponding set of values; loading a second sample comprising a total biomolecule, on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix; applying a separation voltage to the CE capillary to separate total biomolecule; detecting the total biomolecule with a detector; producing an electropherogram comprising corrected peak area of the total biomolecule and generating a second corresponding set of values; and wherein encapsulation efficiency is determined using the first and second corresponding set of values.

52. The method of claim 51 , wherein the encapsulated biomolecule comprises fully encapsulated and the biomolecule on the surface of the encapsulating material.

53. The method of claim 52, wherein an amount of the biomolecule on the surface of the encapsulating material is determined using the encapsulation efficiency.

54. The method of claim 53, wherein an amount of the biomolecule on the surface of the encapsulating material is determined using the encapsulation efficiency and a known biomolecule quantitation value.

55. The method of claim 52, wherein the total biomolecule comprises free biomolecule and a released biomolecule.

56. The method of claim 55, wherein the released biomolecule is a biomolecule that has been subjected to a treatment agent, wherein the treatment agent releases the biomolecule from an encapsulating material.

57. The method of claim 55 or claim 56, wherein the released biomolecule is a biomolecule that was encapsulated inside an encapsulating material and/or on a surface of an encapsulating material that has been subjected to a treatment agent, wherein the treatment agent releases the biomolecule from the encapsulating material.

58. The method of claim 57, wherein the treatment agent comprises a detergent, a surfactant, a lysis buffer, an enzyme, endonuclease, a protease, a peptidase, a proteinase, a nuclease, a hypotonic solution, an antibiotic, a chelating agent, a solvent, a chaotropic agent, or a combination thereof.

59. The method of claim 58, wherein the detergent comprises Triton or Tween.

60. The method of claim 58, wherein the lysis buffer comprises urea, thiourea, or a chaotropic agent.

61. The method of claim 51, wherein the encapsulating material is a lipid nanoparticle or a viral vector.

62. The method of claim 61, wherein the encapsulating material is a lipid nanoparticle comprising one or more of an ionizable cationic lipid, a PEGylated lipid, a phospholipid, and/or cholesterol.

63. The method of claim 51, further comprising: loading a third sample comprising a known amount of a biomolecule on a CE capillary, wherein the CE capillary is filled with a buffer comprising a polymer matrix, applying a separation voltage to the CE capillary to separate biomolecule; detecting the separated biomolecule with a detector; producing an electropherogram comprising corrected peak area of the separated biomolecule and generating a third corresponding set of values; determining, using the first and third corresponding set of values, a concentration of the free biomolecule in the first sample; determining, using the second and third corresponding set of values, a concentration of the total biomolecule in the treated sample; and using the concentrations to determine the encapsulation efficiency.

64. The method of claim 51 , wherein the first sample and/or second sample are loaded onto separate capillaries or loaded sequentially on the same capillary.

65. The method of claim 63, wherein the third sample is loaded onto separate capillaries or loaded sequentially on the same capillary.

66. The method of claim 51, wherein the biomolecule is a protein, a polynucleotide, or nucleic acid.

67. The method of claim 51, wherein the biomolecule is a nucleic acid selected from the group consisting of DNA, single-stranded (ss)DNA, RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small RNA (sRNA), microRNAs (miRNA), piwi-interacting RNA (piRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), small-subunit ribosomal RNA (srRNA), tRNA-derived fragments (tRF), and yRNA-derived fragments (ysRNA).

68. The method of claim 63, wherein the biomolecule in the third sample is a nucleic acid with a known biomolecule quantitation value, mRNA with a known biomolecule quantitation value, RNA with a known biomolecule quantitation value, ssDNA with a known biomolecule quantitation value, a protein with a known biomolecule quantitation value, a peptide with a known biomolecule quantitation value, and/or an amino acid with a known biomolecule quantitation value.

69. The method of claim 68, wherein the biomolecule is mRNA, the third sample comprises at least a first mRNA with a known concentration and a second mRNA with a known concentration.

70. The method of claim 51, wherein the measurement value of the free biomolecule and the biomolecule on the surface of the encapsulating material and/or the known biomolecule quantitation value is generated from a fluorescence-based biomolecule-quantitation assay.

71. The method of claim 63, wherein the third sample comprises at least a first known amount of a biomolecule and at least a second known amount of a biomolecule.

72. The method of claim 63, wherein the third sample comprises a biomolecule that has similar molecular weight and/or length as the biomolecule in the first sample and/or second sample.

73. The method of claim 51, wherein the first sample and/or the second sample is purified and/or enriched prior to loading onto the CE capillary.

74. The method of claim 63, wherein the third sample is purified and/or enriched prior to loading onto the CE capillary.

75. The method of claim 51, wherein the first sample and/or the second sample is purified or enriched using spin columns, spin tubes, and/or magnetic beads.

76. The method of claim 63, wherein the third sample is purified or enriched using spin columns, spin tubes, and/or magnetic beads.

77. The method of claim 51, wherein the first sample and/or the second sample is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary.

78. The method of claim 77, wherein the sample solution comprises nuclease-free water, tris(hydroxymethyl)aminomethane (Tris) - ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer), phosphate buffered saline (PBS), PBS with sucrose, formamide, and combinations thereof.

79. The method of claim 63, wherein the third sample, is diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary.

80. The method of claim 79, wherein the sample solution comprises nuclease-free water, tris(hydroxymethyl)aminomethane (Tris) - ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer), phosphate buffered saline (PBS), PBS with sucrose, formamide, and combinations thereof.

81. The method of claim 51, wherein the biomolecule in the first sample and/or second sample is fluorescently labeled with a fluorescent dye prior to CE separation.

82. The method of claim 63, wherein the biomolecule in the third sample is fluorescently labeled with a fluorescent dye prior to CE separation.

83. The method of claim 51, wherein the biomolecule in the first sample and/or second sample is fluorescently labeled with a fluorescent dye during the CE separation.

84. The method of claim 83, wherein the fluorescent dye is added to the buffer.

85. The method of claim 83, wherein the fluorescent dye is a cyanine -based dye, a prylium- based dye, an FQ dye, or a Tamara dye.

86. The method of claim 83, wherein the fluorescent dye is selected from the group consisting of Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, SYBR Green I, SYBR GOLD, SYBR Green II, PicoGreen, Thiazole orange, Oxazole yellow, Rhodmine, Fluorescein, and Fluorescent Chromeo Py-Dyes.

87. The method of claim 63, wherein the biomolecule in the third sample is fluorescently labeled with a fluorescent dye during the CE separation.

88. The method of claim 87, wherein the fluorescent dye is added to the buffer.

89. The method of claim 87, wherein the fluorescent dye is a cyanine -based dye, a prylium- based dye, an FQ dye, or a Tamara dye.

90. The method of claim 87, wherein the fluorescent dye is selected from the group consisting of Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, SYBR Green I, SYBR GOLD, SYBR Green II, PicoGreen, Thiazole orange, Oxazole yellow, Rhodmine, Fluorescein, and Fluorescent Chromeo Py-Dyes.

91. The method of claim 51 , wherein the detector is a UV detector or fluorescence detector.

92. The method of claim 91 , wherein the detector is a laser-induced fluorescence (LIF) detector, a lamp-based fluorescence detector, or a native fluorescence detector.

93. A kit for characterizing encapsulation efficiency, the kit comprising: a fluorescent dye; a buffer comprising a polymer matrix; a treatment agent; at least one sample comprising a known amount of a biomolecule; and instructions for use.

94. The kit of claim 93, wherein the treatment agent comprises a detergent or a lysis buffer.

95. The kit of claim 93 or claim 94, wherein the biomolecule standard is mRNA with a known concentration.