Patent Application
20230193239
The contents of the text file named “049386-541C01 US_Sequence Listing.xml” which was created on Nov. 30, 2022 and is 2,803 bytes in size, is hereby incorporated by reference in their entirety.
Messenger RNA, or mRNA, is a key intermediary in the conversion of genetic information into biologically active proteins. Many aspects of biomedical research and drug development depend on the ability to obtain high-quality, purified mRNA. Several properties of mRNA, however, make its purification challenging. Relative to total RNA, mRNA exists in very low copy numbers in cells. Furthermore, mRNA is highly sensitive to degradation by RNase enzymes, further compounding the difficulties of purification.
Current methods of isolating mRNA take advantage of the poly-adenine (poly-A) tail present on mRNA molecules. Oligonucleotides consisting of stretches of the nucleic acid base, deoxythymine (oligo-dT), are used to bind to the complementary poly-A tails of mRNA molecules. Oligo-dT-cellulose affinity chromatography has been used to purify mRNA from total RNA fractions.
Purification of mRNA using traditional chromatographic methods, however, is inefficient. Cellulose and other particle-based chromatography columns contain small pore sizes causing slow diffusion of large biomolecules such as mRNA and large contaminants or other particles. Consequently, mRNA purification over traditional particle-based columns is characterized by low flow rates, poor yields and extensive processing time. This problem is further exacerbated when mRNA, formulated for therapeutic delivery, are to be purified, as such formulated mRNA are even larger than naked mRNA.
There remains a need for improved methods of purifying mRNA molecules and other polynucleotides, both naked and formulated, to high purity with a high yield and lower processing time.
Described herein are compositions and methods for purifying polynucleotides, both naked and formulated, e.g., mRNA formulated in lipid nanoparticles (LNPs). Polynucleotides are purified from contaminants, such as, for example, other biomolecules, such as DNA, ribosomal and transfer RNA, and proteins, using monolithic column chromatography. Where formulated polynucleotides are purified, contaminants also include unformulated polynucleotide (“free” or “naked” polynucleotides).
In one embodiment, the disclosure is directed to a method of separating a formulated polynucleotide from free polynucleotide, the method comprising: a) loading a sample onto a monolith matrix comprising a ligand comprising: i) a reactive moiety coupled to the monolith matrix, and ii) an affinity moiety that binds to the free polynucleotide but not the formulated polynucleotide, wherein the ligand is immobilized to the monolithic matrix via the reactive moiety; and b) collecting the formulated polynucleotide from the column while the free polynucleotide remains immobilized on the monolith matrix. In a particular embodiment, the monolith matrix is contained in a column. In a particular embodiment, the formulated polynucleotide is a formulated mRNA. In a particular embodiment, the mRNA is formulated in a lipid nanoparticle. In a particular embodiment, the ligand is an oligo-dT probe. In a particular embodiment, the ligand is NH2—CX-dTY, where X is a whole number between 1 and 50 and Y is a whole number between 5 and 30. In a particular embodiment, the ligand is NH2—C12-dT18. In a particular embodiment, the ligand further comprises a carbon linker positioned between the reactive moiety and the ligand. In a particular embodiment, the carbon linker is CX, where X is a whole number between about 1 and about 50. In a particular embodiment, the methods described herein further comprise eluting the free polynucleotide from the monolith matrix by reducing the ionic strength of the liquid phase.
In one embodiment, the disclosure is directed to a method of purifying a polynucleotide from a sample, the method comprising: a) loading the sample onto a monolithic matrix comprising a ligand comprising: i) a reactive moiety coupled to the monolithic matrix, and ii) a ligand that binds to the polynucleotide, wherein the ligand is immobilized to the monolithic matrix via the reactive moiety; b) allowing for the polynucleotide to bind to the ligand; and c) eluting the polynucleotide from the monolith matrix after one or more contaminants have been substantially separated from the bound polynucleotide. In a particular embodiment, the reactive moiety is a primary amine. In a particular embodiment, the monolithic matrix is activated with an activating agent selected from carbonyldiimidazole, epoxy, ethylenediamine (EDA), carbodiimide, aldehyde, anhydride, imidoester and NHS ester. In a particular embodiment, the ligand further comprises a carbon linker positioned between the reactive moiety and the ligand. In a particular embodiment, the carbon linker is CX, where X is a whole number between about 1 and about 50. In a particular embodiment, the polynucleotide is mRNA. In a particular embodiment, the ligand is an oligo-dT probe. In a particular embodiment, the ligand is NH2—CX-dTY, where X is a whole number between 1 and 50 and Y is a whole number between 5 and 30. In a particular embodiment, the ligand is NH2—C12-dT18. In a particular embodiment, the methods described herein further comprise washing the column prior to eluting the polynucleotide, e.g., wherein the wash buffer contains a salt concentration of at least 200 mM, and the elution buffer contains a salt concentration of 100 mM or less, wherein the wash buffer comprises one or more salts selected from sodium sulfate, sodium chloride and sodium phosphate. In a particular embodiment, the elution buffer is selected from water and Tris. In a particular embodiment, the flow rate of the column is at least 0.5 mL/min or 0.5 CV/min (e.g., in a 1 mL column). Column volume is abbreviated “CV.”
In one embodiment, the disclosure is directed to a column for purifying a polynucleotide from a sample, said column comprising: a) a monolithic matrix; and b) a ligand comprising a reactive moiety coupled to the monolithic matrix, and a ligand that binds to the polynucleotide, wherein the ligand is immobilized to the monolithic matrix via the reactive moiety. In a particular embodiment, the reactive moiety is a primary amine. In a particular embodiment, the monolithic matrix is activated with an activating agent selected from carbonyldiimidazole, epoxy, ethylenediamine (EDA), carbodiimide, aldehyde, anhydride, imidoester and NHS ester. In a particular embodiment, the ligand is an oligo-dT probe. In a particular embodiment, the ligand further comprises a carbon linker positioned between the reactive moiety and the oligo-dT probe. In a particular embodiment, the carbon linker is CX, where X is a whole number between 1 and 50. In a particular embodiment, the ligand is NH2—CX-dTY, where X is a whole number between 1 and 50 and Y is a whole number between 5 and 30. In a particular embodiment, the ligand is NH2—C12-dT18.
In one embodiment, the disclosure is directed to a method of preparing a column described herein by a method comprising: a) treating the monolithic matrix with an activating agent to produce an activated monolithic matrix; and b) incubating the activated monolithic matrix in the presence of a ligand comprising a reactive moiety. In a particular embodiment, the activating agent is selected from carbonyldiimidazole, epoxy, ethylenediamine (EDA), carbodiimide, aldehyde, anhydride, imidoester and NHS ester.
Described herein are compositions and methods of purifying polynucleotides and formulated polynucleotides, e.g., DNA, or RNA, e.g., mRNA, oligonucleotides, e.g., probes, primers and siRNA, or artificial or synthetic polynucleotides, from contaminants. Contaminants include, for example, other biomolecules, such as DNA, ribosomal and transfer RNA and proteins. In the case of formulated nucleotides, e.g., polynucleotides enveloped within a lipid nanoparticle (LNP), contaminants also included unformulated nucleotides (“free” polynucleotides). The materials and methods described herein comprise using monolithic column chromatography. The materials and methods described herein relate to unexpected findings that immobilization of polynucleotide ligands, e.g., oligo-deoxythymine (oligo-dT) and sequence-specific or non-specific oligonucleotides or affinity moieties, on monolithic chromatography columns allows for improved purification of polynucleotides, e.g., polynucleotides comprising poly-A. As described herein, any affinity moiety, e.g., a sequence-specific polynucleotide, can be used in conjunction with monolith columns to achieve polynucleotide purification, e.g., separation of formulated polynucleotides from free polynucleotides. The methods described herein are applicable to immobilizing a ligand via an active moiety to an activated monolith matrix, wherein the ligand specifically binds to the polynucleotide to be purified, e.g., through sequence-specific binding, through hybridization or other base-pairing interactions, or through chemical and non-chemical interactions.
The present disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments can be made that still fall within the scope of the appended Claims. The terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. The singular forms “a,” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.
Described herein is a solid support medium comprising attached polynucleotides or affinity ligands for the purification of biomolecules that specifically bind to the attached polynucleotides or affinity ligands. The solid support medium, for example, can be a column used to purify mRNA from a sample, said column comprising a monolithic matrix coupled to, for example, a ligand comprising an oligo-dT probe. The material of interest to be purified, for example, can be the material that binds to the ligand. Alternatively, the material of interest to be purified can be material that does not bind to the ligand, with a primary contaminant being bound to the ligands instead.
The terms “monolith,” “monolithic matrix” and “monolithic column” are used interchangeably herein to refer to a chromatography column composed of a continuous stationary phase made of a polymer matrix. In contrast to particle-based chromatography columns, monolithic columns are made of a porous polymer material with highly interconnected channels and large pore size. While particle-based columns rely on diffusion through pores, separation by monolithic columns occurs primarily by convective flow through relatively large channels (about 1 micron or more). Monolithic columns are commercially available and have been used to purify large biomolecules such as viruses, plasmid DNA, and proteins (Rajamanickam, V. et al., Chromatography, 2:195-212, 2015).
The monolithic matrix may be derived from a variety of materials, such as but not limited to, polymethacrylate, polyacrylamide, polystyrene, silica and cryogels. The monolithic matrix may be activated to promote coupling to a reactive moiety. Coupling to the activated monolithic matrix may occur, for example, through the formation of a covalent bond between the activated monolithic matrix and the reactive moiety. In some embodiments, the monolithic matrix is activated to couple to a primary amine group. Activation of the monolithic matrix can be accomplished through any appropriate methods known in the art (see, e.g., Pfaunmiller, E. et al., Anal. Bioanal. Chem., 405:2133-45, 2013; Hermanson, G., Bioconiugate Techniques, 3rd Ed., 2013). Non-limiting examples of activation agents include carbonyldiimidazole (CDI), epoxy ethylenediamine (EDA), carbodiimide, aldehyde, anhydride, imidoester and NHS ester.
As used herein, the term “ligand” refers to a molecule that preferentially binds, covalently or non-covalently, to a molecule or material of interest. The ligands described herein can further comprise a reactive moiety capable of coupling to a monolithic matrix. An “oligo-dT ligand” is an oligo-dT probe. A “probe” refers to a ligand that selectively interacts, e.g., binds to or hybridizes with, a desired interaction partner, e.g., a specific polynucleotide sequence. A ligand can itself be a polynucleotide, e.g., an oligo-dT probe or an oligonucleotide, that, for example, specifically hybridizes to a sequence of interest, e.g., a poly-A tail or a sequence specific to the polynucleotide to be purified.
An oligo-dT probe consists of a chain of thymine bases or uracil bases or chemically modified bases of any length appropriate to specifically bind to the poly-A tail of mRNA. Non-limiting examples of oligo-dT probes include oligomers of the formula dTY, wherein Y is a whole number between 5 and 30. In specific embodiments, the oligo-dT probe is dT15, dT18, dT20, dT25 or dT30.
The ligands described herein are coupled or attached to the solid support monolith matrix via a reactive moiety. The monolith can be activated, thereby allowing for coupling to the ligand via the active moiety of the ligand. In a particular embodiment, the monolithic matrix is activated with an activation agent to allow coupling to amine groups, and the reactive moiety of the ligand is a primary amine. In one embodiment the activation agent is carbonyldiimidazole.
In some embodiments, the ligand further comprises a carbon linker positioned between the reactive moiety and the oligo-dT probe. Selection of the length of the carbon linker is within capabilities of the skilled person. Non-limiting examples of carbon linkers include linkers of the formula CX, wherein X is any whole number between 5 and 50. In specific embodiments, the carbon linker is C6, C7, C8, C9, C10, C11, C12, C13, C14 or C15.
In some embodiments, the ligand is NH2—CX-dTY, wherein CX is a carbon chain of length X, and X is a whole number between 5 and 50; and dTY is an oligo-dT probe of length Y, and Y is a whole number between 1 and about 100, about 5 and about 50, about 10 and about 30, about 7 and about 26, about 18 and about 24, or between about 5 and about 25. In a particular embodiment the ligand is NH2—C12-dT18.
Also described herein is a method of preparing a column for purifying mRNA from a sample, the method comprising treating a monolithic matrix with an activating agent to produce an activated monolithic matrix; and incubating the activated monolithic matrix in the presence of a ligand comprising a reactive moiety and a polynucleotide, e.g., an oligonucleotide, e.g., an oligo-dT probe. In some embodiments the ligand further comprises a carbon linker positioned between the reactive moiety and the polynucleotide probe. In some embodiments, the reactive moiety is a primary amine. In some embodiments, the activating agent is carbonyldiimidazole.
Also described herein are methods for purifying polynucleotides, e.g., oligonucleotides, e.g., mRNA or siRNA from a sample. Such methods include, for example, a) loading a sample onto a column comprising: i) a monolithic matrix with an attached ligand comprising: A) a reactive moiety coupled to the monolithic matrix, and B) a polynucleotide, e.g., oligo-dT, probe; b) washing the column; c) eluting the polynucleotide from the column; and d) collecting at least one elution fraction from the column. In one embodiment, step b) comprises washing the column with at least one wash buffer. In another embodiment, step c) comprises eluting the polynucleotides from the column with at least one elution buffer. In another embodiment, the elution fractions of step d) contain mRNA. In some embodiments, the wash buffer contains a salt concentration between about 150 mM to about 1 M. In particular embodiments, the wash buffer contains a salt concentration of at least about 200 mM, at least 400 mM or at least about 750 mM. In some embodiments, the elution buffer contains a salt concentration between 0 and about 100 mM. As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. In particular embodiments the elution buffer has a salt concentration of 100 mM or less. In particular embodiments the wash buffer comprises one or more salts selected from sodium sulfate, sodium chloride and sodium phosphate.
In one embodiment, the elution buffer is water. In another embodiment, the elution buffer comprises Tris. Tris buffer may be used at a concentration from about 1 mM to about 20 mM. In a particular embodiment, the elution buffer comprises 10 mM Tris.
Selection of the flow rate of the column is within capabilities of the skilled person. In some embodiments, the flow rate of the column is from about 1 mL/min to about 5 mL/min. In particular embodiments the flow rate is at least 2 mL/min, at least 3 mL/min or at least 4 mL/min.
The molecule or material of interest is separated from contaminants, and can come off the column in any of the flow-through, wash or elution fraction, depending on the nature of the molecule or material of interest and the major contaminant(s).
The following examples are included for illustrative purposes only and are not intended to limit the scope of the claims.
Polynucleotides can be applied to monolith matrices as described herein for mRNA. The mRNA transcripts used in this example are described in Table 1; additional mRNA transcripts purified by the methods described herein are described in Table 2. Transcripts used were either LiCl precipitated or used straight after the transcription reaction following EDTA treatment.
CDI (carbonyldiimidazole or carboxydiimidazole)-activated monolith disk columns (0.34 mL) were purchased from BIA Separations through High Purity New England (Smithfield, R.I.). Ligands for immobilization on the CDI-monolithic columns were designed and purchased from Integrated DNA Technologies (Coralville, Iowa). Two ligands were used in these studies: an NH2—C12-dT18 ligand, containing a primary amine followed by a 12-carbon linker chain and 18 deoxythymine bases; and an NH2—C6-dT18 ligand, containing a primary amine followed by a 6-carbon linker chain and with 18 deoxythymine bases.
Experiments were run using a GE AKTA Avant 25 preparative chromatography system (GE Healthcare Life Sciences).
A syringe was used to load the oligo-dT ligand onto the monolithic column. All steps were performed at room temperature. The CDI disk column was assembled in the housing according to the manufacturer's instructions. The assembled column was flushed with at least 10 column volumes (CV) of Milli-Q water. The column was then equilibrated with at least 10 CV of suitable buffer (0.5 M Na Phosphate pH 8.0).
The oligo-dT ligand was dissolved in 0.5 M sodium phosphate (pH 8.0) to a final stock concentration of about 100 mg/mL. Then, 1.5-2.0 mL of ligand was diluted to 3 mg/mL with equilibration buffer and was pushed through the column using a syringe to completely fill the monolith channels. The column was then disconnected from the syringe and sealed with blind fittings. The column was stored at room temperature for 20-24 hours.
Following incubation with the oligo-dT ligand, the column was rinsed with at least 10 CV of suitable buffer (0.5 M Na Phosphate pH 8.0), and the column was then flushed with at least 10 CV Milli-Q water. The column was equilibrated with loading buffer (50 mM sodium phosphate, 750 mM sodium sulfate, 10 mM EDTA pH 7.0) for testing with samples of RNA.
Initial testing of mRNA binding to the monolithic column with immobilized oligo-dT ligand was done as described in Table 3, in the order stated.
The starting buffers used for purification testing were: Buffer A: 50 mM sodium phosphate, 1.0 M Na2SO4, 10 mM EDTA pH 7.0; and Buffer B: 50 mM sodium phosphate, 10 mM EDTA pH 7.0. Fractions from the flow-through were desalted as needed and analyzed appropriately.
Initial conditions for testing purification of mRNA on the NH2—C12-dT18 immobilized monolithic column were designed using a high salt binding buffer. The RNA bound to the monolith in 750 mM sodium sulfate, 50 mM phosphate buffer, 10 mM EDTA at pH 7.0 and was eluted with water. Various salt conditions were tested and are listed in Table 4. These experiments were completed using LiCl purified material.
A chromatogram of LiCl precipitated RNA001 bound at 200 mM sodium sulfate buffer is shown in
Following these initial experiments, RNA021 (which had no poly-A tail to interact with the oligo-dT ligand) was assessed using the immobilized monolith disk column. RNA021 was tested using the conditions described in Table 5, in the order stated, and compared to the RNA001 that contains a poly-A tail.
The resulting chromatogram is shown in
The NH2—C12-dT18 immobilized monolithic column was evaluated using transcription reactions that had not been purified further following in vitro transcription. Chromatography conditions for these samples are described in Table 6. The RNA loads in Table 6 were transcription reactions treated with EDTA only. There were slight adjustments made to the CV amount for the wash (increased from 6 to 10 CV) and elution (decreased from 25 to 15 CV).
The resulting chromatogram (
The results indicate that a transcription reaction can be applied directly to an immobilized monolithic column and purified to the same degree as applying RNA that has been initially purified by LiCl precipitation and buffer exchange.
To test the influence of flow rate on mRNA purification over the ligand-immobilized monolithic column, flow rates up to 4 mL/minute were tested using the same samples and process conditions. Pressures were below acceptable levels for all flow rates. Overlays of chromatograms at 2, 3 and 4 mL/minute (
Binding of RNA transcripts where the poly-A tail was absent from the RNA001 transcript was accomplished by digesting the DNA template (DNA001,
Table 8 describes the resulting RNA transcripts following digestion and transcription of DNA001.
Following transcription of these templates, the single RNA was loaded on the ligand-immobilized monolithic column using the conditions listed in Table 5. RNA transcripts without poly-A tails flowed through when applied to the column. An equal parts mixture of the three RNA transcripts listed in Table 8 were applied to the monolithic column. The resulting chromatogram is shown in
The shorter transcripts lacking a poly-A tail do not bind to the column and are found in FT fractions (
To evaluate the effect of the ligand linker on purification efficiency of RNA containing a poly-A tail, a shorter linker (C6 vs C12) between the amino group and oligo-dT probe was tested. The ligand was attached to a new monolithic column using the same method as described in EXAMPLE 1. Once the newly immobilized ligand was attached, the column was washed and tested with RNA001 to compare binding to the C12 linker version of the ligand.
To evaluate the effect of different salts on the purification efficiency, sodium sulfate was replaced with sodium chloride in the equilibration/loading and wash buffers, keeping the phosphate buffer, EDTA and pH the same.
Experiments for the binding and elution conditions for the RNA from oligo-dT monolithic columns looked at loading and washing in a high salt (at least 200 mM) followed by removal of the salt component of the buffer system. The remaining phosphate and EDTA did not elute the RNA; however the subsequent ultra-pure water flush eluted the RNA in a single tight peak. The absence of conductivity proved to be a potent elution condition.
Analysis of the chromatograms indicated that the pH of the elution drifted upwards from pH 7 to as high as pH 9. To control the pH during the elution step, a low conductivity buffer (10 mM Tris pH 7.5) was tested on the 1 mL NH2—C12-oligo-dT18 monolith and implemented for elution of the RNA.
Although mRNA has a short half-life in vivo, high doses of free mRNA can transfect cells and tissues. Additionally, unwanted systemic introduction of mRNA can trigger an immune response before degradation and clearance.
Lipid Nanoparticles (LNPs) can be used to encapsulate and deliver, for example, mRNA. LNPs typically have at least 80% encapsulation of mRNA, i.e., mRNA that is located inside of an LNP as opposed to outside (“free” mRNA). This EXAMPLE evaluates the ability of monolith, oligo-dT purification to remove unencapsulated mRNA to produce a purified LNP.
The results (
LNPs were formulated with mRNA with encapsulation efficiency greater than 80%. Chromatograms and gels demonstrated that LNPs eluted in the flow-through fractions (
It is to be understood that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. References cited herein are herein incorporated by reference in their entireties.
This application is a continuation of U.S. application Ser. No. 16/303,207, filed Nov. 20, 2018, which is a U.S. National Stage entry filed under 35 U.S.C. § 371 of International Application No. PCT/2017/34193, filed May 24, 2017, which claims the benefit of and priority to U.S. Provisional Application No. 62/341,140, filed May 25, 2016, the contents of each of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62341140 | May 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16303207 | Nov 2018 | US |
| Child | 18057489 | US |
