WO2024184827A1 - Capillary electrophoresis methods for characterizing encapsulated biomolecules - Google Patents

Abstract

The disclosed technology provides methods and kits for analyzing and characterizing biomolecules in communication with a delivery vehicle.

Description

CAPILLARY ELECTROPHORESIS METHODS FOR CHARACTERIZING ENCAPSULATED BIOMOLECULES

RELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application No. 63/489,049, filed March 8, 2023, the content of which is hereby incorporated by reference in its entirety into this disclosure.

BACKGROUND

[0002] Advancements in pharmaceutical development have led to more specialized therapeutics and novel ways in which they are delivered. Emerging drug delivery mechanisms are liposomes that can be utilized as therapeutic transport systems. The emergence of more specialized lipid nanoparticles (LNPs) has further expanded this area.

[0003] The use of LNPs to deliver nucleic acids, such as mRNA, or other therapeutic compounds has increased significantly. In addition to vaccine development, the application of mRNA-LNPs has expanded widely into gene editing and the treatment of aggressive malignancies. As a result, the demand has increased for high-throughput and robust analytical techniques to ensure that mRNA-LNPs are properly synthesized, loaded with the intended nucleic acid product, and thoroughly characterized.

[0004] For example, the ability to determine the presence of empty LNP particles and characterize the full% of mRNA loaded LNP is critical to ensure the safety and efficacy of these molecules.

SUMMARY

[0005] Capillary electrophoresis (CE) has been demonstrated to be a powerful technique for the analysis of a wide variety of molecules and is used for the separation of biological materials such as proteins, peptides, amino acids, and nucleic acids. The claimed and described CE methods and kits offer a simple way for the characterization of a biomolecule in communication with a delivery vehicle.

[0006] One aspect of the disclosure is a method of characterizing a biomolecule in communication with a delivery vehicle, the method including: treating a first sample and a second sample with a nuclease and/or a treatment agent to obtain a treated first sample and a treated second sample, wherein the first sample includes a biomolecule with a known full% and known delivery vehicle concentration wherein the second sample includes a known delivery vehicle concentration and a biomolecule with an unknown full% in communication with a delivery vehicle; loading the treated first sample 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 free nucleic acid; detecting the separated free nucleic acid with a detector; producing an electropherogram including corrected peak area of the free nucleic acid and generating a first corresponding set of values; loading the treated second sample 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 free nucleic acid; detecting the separated free nucleic acid with a detector; producing an electropherogram comprising corrected peak area of the free nucleic acid and generating a second corresponding set of values; wherein the first and second corresponding set of values are used to characterize the biomolecule.

[0007] In an aspect, characterizing the biomolecule includes determining the full% of the biomolecule in communication with a delivery vehicle, the purity of the biomolecule, the integrity of the biomolecule, and/or the size of the biomolecule. In another aspect, the full% of the biomolecule in communication with a delivery vehicle is determined by using the first and second corresponding set of values.

[0008] In an aspect, the biomolecule of the first sample is in communication with a delivery vehicle. In another aspect, the biomolecule is at least partially or fully encapsulated with the delivery vehicle.

[0009] In an aspect, the delivery vehicle is a lipid nanoparticle. In another aspect, the lipid nanoparticle includes one or more of an ionizable cationic lipid, a PEGylated lipid, a phospholipid, and/or cholesterol.

[0010] In an aspect, the nuclease degrades free nucleic acid outside the delivery vehicle. In another aspect, the nuclease is an endonuclease or an exonuclease.

[0011] In an aspect, the treatment agent releases the biomolecule from within the delivery vehicle. In another aspect, the treatment agent includes a detergent, a surfactant, a lysis buffer, or a chaotropic agent. In an aspect, the detergent includes Triton or Tween.

[0012] In an aspect, the biomolecule is a protein, a peptide, a polynucleotide, or a nucleic acid. In another aspect, the biomolecule is a polynucleotide or a nucleic acid selected from the group including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), mixed RNA/DNAs, singlestranded (ss)DNA, 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), and RNA and/or DNA fragments, impurities, or degraded molecules.

[0013] In an aspect, the biomolecule in the first sample has a similar molecular weight, biomolecule amount per delivery vehicle particle and/or length as the biomolecule in the second sample. In another aspect, the biomolecule in the first sample and the biomolecule in the second sample is mRNA.

[0014] In an aspect, the first sample and second sample are loaded onto separate capillaries or loaded sequentially on the same capillary.

[0015] In an aspect, the first sample and/or the second sample is purified, enriched, and/or diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary prior to loading onto the CE capillary. In another aspect, the first sample and/or the second sample is purified or enriched using spin columns, spin tubes, and/or magnetic beads.

[0016] In an aspect, the biomolecule in the first sample and/or second sample is fluorescently labeled with a fluorescent dye prior to CE separation.

[0017] One aspect of the disclosure is a kit for characterizing a biomolecule in communication with a delivery vehicle, the kit including: a fluorescent dye; a buffer comprising a polymer matrix; a nuclease; a treatment agent; and instructions for use.

[0018] In an aspect, the treatment agent is a detergent.

[0019] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the disclosure in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

[0020] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0021] FIGs. 1A and IB depict a workflow of a method according to an aspect of this disclosure.

DETAILED DESCRIPTION

[0022] It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure or the appended claims.

[0023] 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.

[0024] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). 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".

[0025] 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%.

[0026] Where ranges are given, endpoints are included. 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. [0027] 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.

[0028] The disclosure generally relates to capillary electrophoresis methods and kits for characterizing a biomolecule in communication with a delivery vehicle. In non-limiting examples, the biomolecule may be a protein, a peptide, a polynucleotide, or nucleic acid.

[0029] "Polypeptide", "protein", and "peptide" may be used interchangeably and refer to polymers of amino acids of any length.

[0030] "Polynucleotide(s)", "nucleic acid", "nucleotide sequence", and "nucleotide(s)" may be used interchangeably and refer to a continuous sequence of any type of nucleic acid molecule(s). Non-limiting examples of nucleic acids include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), mixed RNA/DNAs, single-stranded (ss)DNA, 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), and RNA and/or DNA fragments, impurities, or degraded molecules.

[0031] Referring now to FIGs. 1A and IB, one method of the disclosure 100 includes treating at least two samples (a first sample and a second sample) with a nuclease and/or a treatment agent to obtain a treated first sample and a treated second sample 102.

[0032] A "nuclease" refers to any enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. In an aspect, the nuclease is used to degrade free nucleic acid outside the delivery vehicle. The nuclease may be an endonuclease or an exonuclease or a combination of both. A "treatment agent" refers to any component, compound, or composition that may be used to release the biomolecule from within the delivery vehicle. In some aspects, the treatment agent may include a detergent, a surfactant, a lysis buffer, or chaotropic agents. Non-limiting examples of detergents include Triton or Tween. Triton, commonly described as alkylaryl polyether alcohols, includes a group of nonionic surfactants are prepared by the reaction of octylphenol with ethylene oxide. Non-limiting examples include Triton X-114, Triton X-100, and Triton X-405. Tween is a polysorbate often used in protein extraction. Non-limiting examples include Tween 20 and Tween 80.

[0033] In this aspect, the first sample may include a biomolecule with a known full% and known delivery vehicle concentration. The second sample may include a known delivery vehicle concentration and a biomolecule with an unknown full% in communication with a delivery vehicle. In some non-limiting examples, the biomolecule of the first sample and/or the second sample is at least partially or fully encapsulated with the delivery vehicle. The "full%" is the percentage of the delivery vehicles that are in communication with a biomolecule. This includes, but is not limited to, the percentage of the delivery vehicles that have an encapsulated biomolecule and/or the percentage of the lipid nanoparticles with biomolecule encapsulated inside.

[0034] A "delivery vehicle" refers to a vehicle (e.g., particles, viral vectors, etc.) that facilitates, at least in part, the in vivo, in vitro, or ex vivo delivery of a biomolecule (e.g., such as a therapeutic drug substance). Delivery vehicles include, but are not limited to, viral vectors and particles such as lipid nanoparticles, lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, retrovirus. In a non-limiting example, the delivery vehicle is a lipid nanoparticle and may include one or more of an ionizable cationic lipid, a PEGylated lipid, a phospholipid, and/or cholesterol.

[0035] In some aspects, biomolecule in the first sample has a similar molecular weight, biomolecule amount per delivery vehicle particle and/or length as the biomolecule in the second sample. In a non-limiting example, the biomolecule in the first sample and the biomolecule in the second sample are both mRNA.

[0036] In some aspects, the method further includes loading the treated first sample on a capillary electrophoresis (CE) capillary 104. The CE capillary is filled with a buffer comprising a polymer matrix 104.

[0037] As used herein, "capillary" refers to a channel, tube, or other structure capable of supporting a volume of separation medium for performing electrophoresis. Capillary geometry can vary and includes structures having circular, rectangular, or square cross-sections, channels, grooves, plates, and the like that can be fabricated by technologies known in the art. Capillaries of the present disclosure can be made of materials such as, but not limited to, silica, fused silica, quartz, silicate-based glass such as borosilicate glass, phosphate glass, alumina-containing glass, and other silica-like materials. In some aspects, the methods can be adapted and used in any generally known electrophoresis platform, such as, for example, electrophoresis devices comprising single or multiple microfluidic channels, etched microfluidic capillaries, as well as slab gel and thin-plate gel electrophoresis.

[0038] In some aspects, the capillary is an uncoated capillary. In some aspects, the capillary is a coated capillary. For example, a capillary can be coated to shield or minimize electrostatic interactions and/or electroosmotic flow. Shielding can comprise non-permanent, replaceable polymeric hydrophilic coatings that adsorb to the capillary surface or permanent hydrophilic coatings including, for example, linear polyacrylamide or polyvinylalcohol that covalently bind the capillary surface.

[0039] The CE capillary may be filled with a buffer comprising a polymer matrix or gel buffer prior to applying a separation voltage and/or loading the samples. In some aspects, the buffer comprising a polymer matrix or gel buffer is placed into a buffer vial(s). These buffer vials may be placed into buffer trays. In some aspects, the buffer comprising a polymer matrix or gel buffer may comprise additional components to facilitate the separation of the biomolecules. Non-limiting examples of a suitable polymer matrix include crosslinked polymer, linear polymers, slightly branched polymers, linear polyacrylamide, polyethylene oxide, polyethylene glycol, and dextran. For example, a gel from a SCIEX RNA 9000 Purity and Integrity Analysis kit may be used for the methods of characterizing a biomolecule described herein.

[0040] In some aspects of the method, a separation voltage may be applied to the CE capillary to separate free nucleic acid 106 and the separated free nucleic acid may be detected with a detector 108. The detector can be, for example, a UV detector.

[0041] As described herein, the biomolecules may be separated using capillary gel electrophoresis or capillary electrochromatography. In an aspect, the separation uses capillary gel electrophoresis (CGE), which separates samples by size and detects the separated nucleic acids using a fluorescent dye that binds to the nucleic acids. In this aspect, the detector may also be a fluorescence detector, 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 an increase in sensitivity, yet it also requires additional sample manipulation.

[0042] If a fluorescence detector is used, the biomolecule in the first sample and/or second sample is fluorescently labeled with a fluorescent dye prior to CE separation. In these aspects, the fluorescent dye may be a cyanine-based dye. Cyanine-based dyes of the disclosure include, not are not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5,5, Cy7, SYBR Green I, SYBR Green II, PicoGreen, Thiazole orange, Oxazole yellow, and p503.

[0043] In some aspects, the method further includes producing an electropherogram comprising the corrected peak area of the free nucleic acid and the generation of a first corresponding set of values 110. An "electropherogram" (or "e-gram") refers to a series of peaks that can be converted to determine the size and/or quantity of a sample. Peaks are integrated for area as a measure of quantity and can be corrected for mobility differences between different sized peaks.

[0044] In some aspects, the method further includes loading the treated second sample on a CE capillary, wherein the CE capillary is filled with a buffer including a polymer matrix 112, applying a separation voltage to the CE capillary to separate free nucleic acid 114, detecting the separated free nucleic acid with a detector 116, and producing an electropherogram comprising corrected peak area of the free nucleic acid and generating a second corresponding set of values 118.

[0045] In some aspects, the first sample and the second sample are loaded onto separate capillaries. In alternative aspects, the first sample and the second sample are loaded sequentially on the same capillary.

[0046] In an aspect, the first sample and/or the second sample is purified, enriched, and/or diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary. The sample solution may be a sample loading solution. The water may be deionized water, CE-grade water, or nuclease-free water. The first sample and/or the second sample may be purified or enriched using spin columns, spin tubes, and/or magnetic beads.

[0047] In an aspect, the first and the second corresponding sets of values may be used to characterize the biomolecule. In non-limiting examples characterizing the biomolecule includes determining the full% of the biomolecule in communication with a delivery vehicle, the purity of the biomolecule, the integrity of the biomolecule, and/or the size of the biomolecule. For example, the full% of the biomolecule in communication with a delivery vehicle may be determined by using the first and second corresponding sets of values.

[0048] One aspect of the disclosure includes a kit for characterizing a biomolecule in communication with a delivery vehicle. The kit may include a fluorescent dye, a buffer comprising a polymer matrix, a nuclease, a treatment agent, and instructions for use. The treatment agent may be a detergent, and the biomolecule standard may be mRNA, a protein, or a peptide with a known concentration.

[0049] EXAMPLES

[0050] Example 1: CGE-LIF for LNP particle full/emptv determination

[0051] An LNP standard with a known full% (full%(standard)) and an unknown LNP sample are provided. The number of biomolecules can be the same between the LNP standard and the unknown LNP. The free mRNA present in the LNP standard and LNP unknown sample can be cleared by using benzonase nuclease treatment. The encapsulated biomolecule can then be released from the LNP standard and LNP unknown sample using triton (0.2%).

[0052] Using CGE-LIF, the mRNA in the LNP standard and LNP unknown sample can then be measured, and the corresponding values (LNP standard (D); LNP unknown sample (E)) can be used to calculate a ratio = E/D and/or full% of the unknown LNP sample (full%(unknown) =full% (standard)*E/D).

[0053] 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.

[0054] All patents, patent applications, publications, and descriptions mentioned above are herein incorporated by reference in their entirety.

Claims

CLAIMS What is Claimed is:

1. A method of characterizing a biomolecule in communication with a delivery vehicle, the method comprising: treating a first sample and a second sample with a nuclease and/or a treatment agent to obtain a treated first sample and a treated second sample, wherein the first sample comprises a biomolecule with a known full% and known delivery vehicle concentration wherein the second sample comprises a known delivery vehicle concentration and a biomolecule with an unknown full% in communication with a delivery vehicle; loading the treated first sample 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 free nucleic acid; detecting the separated free nucleic acid with a detector; producing an electropherogram comprising corrected peak area of the free nucleic acid and generating a first corresponding set of values; loading the treated second sample 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 free nucleic acid; detecting the separated free nucleic acid with a detector; producing an electropherogram comprising corrected peak area of the free nucleic acid and generating a second corresponding set of values; and wherein the first and second corresponding set of values are used to characterize the biomolecule.

2. The method of claim 1 , wherein characterizing the biomolecule includes determining the full% of the biomolecule in communication with a delivery vehicle, the purity of the biomolecule, the integrity of the biomolecule, and/or the size of the biomolecule.

3. The method of claim 2, wherein the full% of the biomolecule in communication with a delivery vehicle is determined by using the first and second corresponding set of values.

4. The method of any one of the preceding claims, wherein the biomolecule of the first sample is in communication with a delivery vehicle.

5. The method of any one of the preceding claims, wherein the biomolecule is at least partially or fully encapsulated with the delivery vehicle.

6. The method of any one of the preceding claims, wherein the delivery vehicle is a lipid nanoparticle.

7. The method of claim 6, wherein the lipid nanoparticle comprises one or more of an ionizable cationic lipid, a PEGylated lipid, a phospholipid, and/or cholesterol.

8. The method of any one of the preceding claims, wherein the nuclease degrades free nucleic acid outside the delivery vehicle.

9. The method of claim 8, wherein the nuclease is an endonuclease or an exonuclease.

10. The method of any one of the preceding claims, wherein the treatment agent releases the biomolecule from within the delivery vehicle.

11. The method of claim 10, wherein the treatment agent comprises a detergent, a surfactant, a lysis buffer, or a chaotropic agent.

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

13. The method of any one of the preceding claims, wherein the biomolecule is a protein, a peptide, a polynucleotide, or a nucleic acid.

14. The method of any one of the preceding claims, wherein the biomolecule is a polynucleotide or a nucleic acid selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), mixed RNA/DNAs, single-stranded (ss)DNA, 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), and RNA and/or DNA fragments, impurities, and degraded molecules.

15. The method of any one of the preceding claims, wherein the biomolecule in the first sample has a similar molecular weight, biomolecule amount per delivery vehicle particle and/or length as the biomolecule in the second sample.

16. The method of any one of the preceding claims, wherein the biomolecule in the first sample and the biomolecule in the second sample is mRNA.

17. The method of any one of the preceding claims, wherein the first sample and second sample are loaded onto separate capillaries or loaded sequentially on the same capillary.

18. The method of any one of the preceding claims, wherein the first sample and/or the second sample is purified, enriched, and/or diluted with a sample solution, water, or combinations thereof prior to loading on the CE capillary prior to loading onto the CE capillary.

19. The method of claim 18, wherein the first sample and/or the second sample is purified or enriched using spin columns, spin tubes, and/or magnetic beads.

20. The method of any one of the preceding claims, wherein the biomolecule in the first sample and/or second sample is fluorescently labeled with a fluorescent dye prior to CE separation.

21. A kit for characterizing a biomolecule in communication with a delivery vehicle, the kit comprising: a fluorescent dye; a buffer comprising a polymer matrix; a nuclease; a treatment agent; and instructions for use.

22. The kit of claim 21, wherein the treatment agent is a detergent.