APPARATUS, SYSTEM AND METHODS FOR PROCESSING - Patent application

Abstract

The present invention relates generally to devices, systems, and methods for preparing and processing circular polyribonucleotides, including the use of a pressurized device comprising a reservoir comprising (a) a filtrate chamber and (b) a waste collection chamber separated by (c) a filter; the reservoir further comprising (d) a removable cap in contact with a pressure inlet port; and the device in fluid communication with a source of positive pressure.

Description

[Background technology]

[0001] There is a need for methods for making and processing circular polyribonucleotides. Summary of the Invention [Problem to be solved by the invention]

[0002] The present invention relates generally to devices, systems, and methods for the preparation and processing of circular polyribonucleotides. [Means for solving the problem]

[0003] In one aspect, the invention features a method for processing cyclic polyribonucleotides, the method including the steps of: (a) providing an apparatus, the apparatus comprising: (i) a reservoir having a filter and (ii) a filtrate chamber and (iii) a waste collection chamber disposed therebetween, the reservoir further comprising a removable cap having a pressure inlet port; the apparatus being in fluid communication with a positive pressure source through the pressure inlet port; (b) providing cyclic polyribonucleotides in a first fluid to the filtrate chamber; (c) pressurizing the reservoir with the positive pressure source, such that the first fluid passes through the filter and the cyclic polyribonucleotide does not pass through the filter; (d) applying a second fluid to the reservoir; and (e) pressurizing the reservoir with the positive pressure source, such that the second buffer passes through the filter and the cyclic polyribonucleotide does not pass through the filter.

[0004] In some embodiments, the method further comprises repeating steps (d) and (e) 1-5 times. In some embodiments, the method further comprises (f) aspirating the circular polynucleotide from the filter after step (e). In some embodiments, 1 mg to 1000 mg of circular polyribonucleotide is aspirated from the filter. In some embodiments, 50 mg to 150 mg of circular polyribonucleotide is aspirated from the filter. In some embodiments, about 100 mg of circular polyribonucleotide is aspirated from the filter.

[0005] In some embodiments, the second fluid is applied through a fluid inlet port in the cap. In some embodiments, the fluid inlet port is in fluid communication with a fluid source. In some embodiments, the first and / or second fluid is a liquid. In some embodiments, the liquid is a solution. In some embodiments, the solution is a buffer. In some embodiments, the buffer is a formulation buffer, a storage buffer, or a purification buffer.

[0006] In some embodiments, the pressurizing steps (c) and (d) include pressurizing the reservoir at between 10 PSI and 100 PSI. In some embodiments, the applying step (d) includes applying between 5 mL and 15 mL of fluid to the reservoir.

[0007] In another aspect, the invention features an apparatus that includes a reservoir having a filtrate chamber and a waste collection chamber separated by a filter and a removable cap in contact with a pressure inlet port; the apparatus is in fluid communication with a positive pressure source.

[0008] In some embodiments, the reservoir is a tube. In some embodiments, the tube is a centrifuge tube. In some embodiments, the filtrate chamber is funnel-shaped. In some embodiments, the cap further comprises a fluid inlet port. In some embodiments, the reservoir has a volume between 5 mL and 100 mL. In some embodiments, the filtrate chamber has a volume between 5 mL and 15 mL. In some embodiments, the filter is between 2 kDa and 200 kDa. In some embodiments, the filtrate chamber and filter are stacked on top of, screwed into, or nested within the reservoir. In some embodiments, the pressure inlet port is sealed.

[0009] In some embodiments, the device further comprises a sensor (e.g., a liquid detection sensor). In some embodiments, the liquid detection sensor is selected from the list consisting of an optical sensor, a vibration sensor, an ultrasonic sensor, a buoyancy sensor, a capacitance sensor, a radar sensor, a conductivity sensor, or a resistance sensor. In some embodiments, the sensor is an optical sensor.

[0010] In another aspect, the invention features a system that includes (i) a filter tray having a sample reservoir with a filtrate chamber having a filter, (ii) a lid having a pressure inlet port disposing the filter tray therebetween, and (iii) a waste reservoir; the pressure inlet port is in fluid communication with (iv) a positive pressure source.

[0011] In some embodiments, the filtrate chamber is funnel-shaped. In some embodiments, the volume of the filtrate chamber is between 5 mL and 15 mL. In some embodiments, the waste reservoir comprises a gravity or vacuum drain. In some embodiments, the system further comprises a liquid detection sensor. In some embodiments, the system further comprises a vent. In some embodiments, the filter tray, lid, and waste reservoir are sealably connected together. In some embodiments, the filtrate chamber is stacked on top of, threaded into, or nested within the sample reservoir. In some embodiments, the lid further comprises a fluid inlet port. In some embodiments, the filter tray comprises multiple sample reservoirs.

[0012] definition The present invention will be described with respect to detailed embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Terms as set forth below shall generally be understood in their ordinary meaning unless otherwise indicated.

[0013] As used herein, the terms "cyclic polyribonucleotide" or "circRNA" or "circular RNA" are used interchangeably and refer to a polyribonucleotide molecule having a structure with no free ends (i.e., no free 3' and / or 5' ends), e.g., a polyribonucleotide molecule that forms a circular or endless structure through covalent or non-covalent bonds. In some embodiments, a cyclic polyribonucleotide is a "covalently closed polyribonucleotide" that forms a circular or endless structure through covalent bonds.

[0014] As used herein, the terms "circular polyribonucleotide sample", "circRNA sample", and "circular RNA sample" are used interchangeably and refer to a composition comprising a circRNA molecule and a solution.

[0015] As used herein, the term "expression sequence" refers to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide, or a regulatory nucleic acid.

[0016] "Polypeptide" and "protein" are used interchangeably and refer to a polymer in which two or more amino acids are joined together by covalent bonds (e.g., amide bonds). Polypeptides as described herein can include full-length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally occurring proteins or synthetic polypeptide fragments). Polypeptides can include naturally occurring amino acids (e.g., one of the 20 amino acids known by the one-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V that are commonly found in peptides synthesized in nature) and non-naturally occurring amino acids (e.g., amino acids that are not one of the 20 amino acids that are commonly found in peptides synthesized in nature, including synthetic amino acids, amino acid analogs, and amino acid mimetics).

[0017] As used herein, the term "aptamer sequence" or "aptamer" refers to a non-naturally occurring or synthetic oligonucleotide that specifically binds to a target molecule. Typically, aptamers are 20-250 nucleotides. Typically, aptamers bind to their targets through secondary structure, rather than sequence homology.

[0018] As used herein, the term "binding site" refers to a region in a circular polyribonucleotide that interacts with another entity, e.g., a chemical compound, a protein, a nucleic acid, etc. A binding site can include an aptamer sequence.

[0019] As used herein, a "buffer" is a solution that can withstand a change in pH upon addition of an acidic or basic component. Examples of "buffers" include "formulation buffers" for use in formulating a composition, "storage buffers" for use in storing a composition, "purification buffers" for use in purification, etc.

[0020] The term "buffer exchange" as used herein refers to a process for removing a molecule or particle, such as a polyribonucleotide, from a first buffer and placing it in a second buffer.

[0021] The term "fluid communication," as used herein, refers to a connection between at least two device elements, e.g., reservoirs, pressure sources, etc., that allows fluid to move between such device elements, with or without passing through one or more intervening device elements.

[0022] As used herein, a "solution" is a homogeneous mixture of two or more substances, including a solvent and a solute.

[0023] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0024] Circular polyribonucleotides are described, for example, in U.S. Patent Application Publication No. 2020 / 0306286, and WO 2020 / 181013 and WO 2020 / 023655, which are incorporated by reference herein.

[0025] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments, which are given by way of example herein. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0026] [Figure 1]A pressurizing device is shown that includes a filter disposed between a filtrate chamber and a waste collection chamber, and a reservoir having a cap with a pressure inlet port, where the pressurizing device is under positive pressure and pressure is exerted through the positive pressure port. [Diagram 2] Shown is a pressurizing device comprising a filter disposed between a filtrate chamber and a waste collection chamber, and a reservoir having a cap having both a pressure inlet port and a fluid inlet port, where the pressurizing device is under positive pressure and pressure is exerted through the pressure inlet port; and where fresh fluid may be applied to the filtrate chamber through the fluid inlet port. [Figure 3A] 1 shows a pressurized system or pressurized robot assembly that includes a lid having at least one port, a filter tray having multiple sample reservoirs with filters, and a waste reservoir. [Figure 3B] 1 shows a pressurized system or pressurized robot assembly that includes a lid having at least one port, a filter tray having multiple sample reservoirs with filters, and a waste reservoir. [Figure 3C] 1 shows a pressurized system or pressurized robot assembly that includes a lid having at least one port, a filter tray having multiple sample reservoirs with filters, and a waste reservoir. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention generally relates to devices, systems and methods for processing circular polyribonucleotide samples, for example for buffer exchange of such samples. The inventors have found that pressurizing a fluid, such as a solution (e.g., a buffer) containing circular polynucleotides, leads to simple and rapid liquid exchange. One embodiment of the present invention is a device configured to transfer circular polyribonucleotides from a first fluid to a second fluid using pressure, for example a combination of pressure and filtration.

[0028] In some embodiments, the cyclic polyribonucleotide is a polyribonucleotide that is cyclic, i.e., does not have free ends.Cyclic polyribonucleotides are described, for example, in US Patent Publication No. 2020 / 0306286, and WO 2020 / 181013 and WO 2020 / 023655, which are incorporated herein by reference. Cyclic polyribonucleotides are also described, for example, in WO 2015 / 034925, WO 2016 / 011222, US 10407683, WO 2017 / 222911, WO 2021 / 041541, WO 2019 / 236673, WO 2020 / 237227, WO 2016 / 197121, WO 2018 / 191722, and WO 2020 / 023595, which are incorporated herein by reference. In some embodiments, cyclic polyribonucleotides exhibit improved stability, increased half-life, reduced immunogenicity, and / or improved functionality (e.g., functionality for the functions described herein) compared to corresponding linear polyribonucleotides.

[0029] In some embodiments, the cyclic polyribonucleotide is at least about 20 base pairs, at least about 30 base pairs, at least about 40 base pairs, at least about 50 base pairs, at least about 75 base pairs, at least about 100 base pairs, at least about 200 base pairs, at least about 300 base pairs, at least about 400 base pairs, at least about 500 base pairs, or at least about 1,000 base pairs.

[0030] In some embodiments, circular polyribonucleotides can be useful that are at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 1,000 nucleotides, at least 2,000 nucleotides, at least 3,000 nucleotides, at least 4,000 nucleotides, at least 5,000 nucleotides, at least 7,500 nucleotides, at least 10,000 nucleotides, at least 15,000 nucleotides, at least 20,000 nucleotides in length.

[0031] In some embodiments, the circular polyribonucleotide comprises at least one expressed sequence, e.g., an expressed sequence that encodes a polypeptide. In some embodiments, the expressed sequence encodes a peptide or a polynucleotide. In some embodiments, the circular polyribonucleotide comprises multiple expressed sequences, which may be the same or different. In some embodiments, the expressed sequence is less than 5000 bp in length (e.g., less than about 5000 bp, 4000 bp, 3000 bp, 2000 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, or less). In some embodiments, the expression sequences can independently or additionally be greater than 10 bp in length (e.g., at least about 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2.0 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3.9 kb, 3.1 kb, 3.2 ...3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 k .8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, 4.9kb, 5kb or more).

[0032] In some embodiments, the circular polyribonucleotide comprises one or more elements, such as an expression sequence, and the one or more elements may be separated from each other by a spacer sequence or linker.In some embodiments, the elements may be separated from each other by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, or about 1000 nucleotides.In some embodiments, the one or more elements are adjacent to each other, for example, lacking a spacer element. In one embodiment, the medicament is about 0.05:1, about 0.06:1, about 0.07:1, about 0.08:1, about 0.09:1, about 0.1:1, about 0.12:1, about 0.125:1, about 0.15:1, about 0.175:1, about 0.2:1, about 0.225:1, about 0.25:1, about 0.3:1, about 0.35:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 0.98 1, about 1:1, about 1.02:1, about 1.05:1, about 1.1:1, about 1.15:1, about 1.2:1, about 1.25:1, about 1.3:1, about 1.35:1, about 1.4:1, about 1.45:1, about 1.5:1, about 1.55:1, about 1.6:1, about 1.65:1, about 1.7:1, about 1.75:1, about 1.8:1, about 1.85:1, about 1.9:1, about 1.95:1, about 1.975:1, about 1.98:1, or about 2:1. In one embodiment, the spacer sequence is configured to provide conformational flexibility between the elements of the circular polyribonucleotide on either side of the spacer sequence.

[0033] In some embodiments, the cyclic polyribonucleotide may contain one or more repeating elements. In some embodiments, the cyclic polyribonucleotide contains one or more modifications. In some embodiments, the cyclic polyribonucleotide contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% modified nucleotides.

[0034] The cyclic polyribonucleotide may include at least one binding site for a target, for example, for a binding moiety of a target. The cyclic polyribonucleotide may include at least one aptamer sequence that binds to a target. In some embodiments, the cyclic polyribonucleotide includes one or more binding sites for one or more targets. Targets include, but are not limited to, nucleic acids (e.g., RNA, DNA, RNA-DNA hybrids), small molecules (e.g., drugs, fluorophores, metabolites), aptamers, polypeptides, proteins, lipids, carbohydrates, antibodies, viruses, virus particles, membranes, multicomponent complexes, organelles, cells, other cellular components, any fragments thereof, and any combinations thereof. In some embodiments, the cyclic polyribonucleotide includes binding sites for a single target or multiple (e.g., two or more) targets. In one embodiment, a single cyclic polyribonucleotide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different binding sites for a single target. In one embodiment, a single cyclic polyribonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the same binding sites for a single target. In one embodiment, a single cyclic polyribonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different binding sites for one or more different targets. In one embodiment, there are two or more targets in a sample, such as a mixture or library of targets, and the sample comprises a cyclic polyribonucleotide comprising two or more binding sites that bind to the two or more targets.

[0035] In processing a cyclic ribonucleotide sample, it may be necessary to transfer the cyclic ribonucleotide from a first fluid to a second fluid, where the second fluid is more suitable for further processing, formulation, storage, purification, etc. The fluid exchange of the present invention may be performed without emptying the reservoir, including the filter with the cyclic polyribonucleotide, providing a less labor-intensive and robust methodology. In one embodiment, this process may be further enhanced with a fluid detection sensor, providing a controlled process that prevents the filter itself from drying out too much. The cyclic polyribonucleotide sample may be dispensed with a fluid. In one embodiment, the fluid is a liquid or gas. In one embodiment, the fluid is a solution. In one embodiment, the solution is a buffer. Buffers to be used in the present invention include any aqueous solution that maintains the cyclic polyribonucleotide at a stable pH and / or stable structure. Buffers may include formulation buffers for use in formulating the composition, storage buffers for use in storing the composition, and purification buffers for use in purification. Buffers can include sodium citrate buffer, phosphate buffered saline (PBS), Tris-based buffers, sodium phosphate, water, etc. The present invention can be used with multiple fluids in series (e.g., one after the other) or in parallel (e.g., together).

[0036] In some embodiments, commercially available filters may be used in the devices and systems, including Amicon® Ultra filters, Pierce™ Protein Concentrator PES filters, and the like. Various filter pore sizes are contemplated, including but not limited to, less than 2 kDa, 3 kDa, 20 kDa, 30 kDa, 50 kDa, 100 kDa, 200 kDa, and the like. Filters with various molecular weight cut-offs (MWCOs) are contemplated, including but not limited to, 10 MWCO, 30 MWCO, and 100 MWCO.

[0037] Various cyclic polyribonucleotide concentrations are contemplated, including but not limited to 0.5 to 2.0 mg / mL.

[0038] Various weights of the resulting cyclic polyribonucleotide for incorporation into a second fluid, such as a buffer, are contemplated, including 1 to 200 mg, e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, or 200 mg.

[0039] 1 shows a pressurizing device (101) comprising a filter (102) disposed between a filtrate chamber (104) and a waste collection chamber (105), as well as a reservoir (103) having a cap (106) with a pressure inlet port (108), where the pressurizing device is under positive pressure and pressure is exerted through the pressure inlet port (108). The pressurizing device (101) may further comprise a fluid connection component (107) by which the reservoir may be placed in fluid communication with a pressure source.

[0040] FIG. 2 shows an alternative embodiment of a pressurizing device (201) comprising a filter (202) disposed between a filtrate chamber (204) and a waste collection chamber (205), and a reservoir (203) having a cap (206) with a pressure inlet port (208) and a fluid inlet port (209), where the pressurizing device (201) is under positive pressure, and pressure is exerted through the pressure inlet port (208); and where fresh fluid may be applied to the filtrate chamber (204) through the fluid inlet port (209). The pressurizing device (201) may further comprise a fluid connection component (207) by which the reservoir may be placed in fluid communication with a pressure source. In some embodiments, the cap comprises a pressure inlet port. The pressure inlet port may be connected to a pressure source by a fluid connection component. In certain devices, methods, and systems described herein, positive pressure may be exerted by a positive pressure source from the pressure inlet port to force fluid through the filter.A range of pressures are contemplated for fluid exchange of the circular polyribonucleotide, including pressures ranging from 10 PSI to 100 PSI, e.g., 10 PSI to 20 PSI, 10 PSI to 25 PSI, 10 PSI to 30 PSI, 10 PSI to 40 PSI, 10 PSI to 50 PSI, 10 PSI to 60 PSI, 10 PSI to 70 PSI, 10 PSI to 80 PSI, 10 PSI to 90 PSI, 20 PSI to 25 PSI, 20 PSI to 30 PSI , 20PSI~40PSI, 20PSI~50PSI, 20PSI~60PSI, 20PSI~70PSI, 20PSI~75PSI, 20PSI~80PSI, 20PSI~90PSI, 20PSI~ 100PSI, 30PSI~40PSI, 30PSI~50PSI, 30PSI~60PSI, 30PSI~70PSI, 30PSI~75PSI, 30PSI~80PSI, 30PSI~90PSI, 30PSI~100PSI, 40PSI~50PSI, 40PSI~60PSI, 40PSI~70PSI, 40PSI~75PSI, 40PSI~80PSI, 40PSI~90PSI, 40PSI~ 100PSI, 50PSI~60PSI, 50PSI~70PSI, 50PSI~75PSI, 50PSI~80PSI, 50PSI~90PSI, 50PSI~100PSI, 60PSI~70PSI , 60-75 PSI, 60-80 PSI, 60-90 PSI, 60-100 PSI, 70-75 PSI, 70-80 PSI, 70-90 PSI, 70-100 PSI, 80-90 PSI, 80-100 PSI, 90-100 PSI, or about 10, 20, 30, 40, 50, 60, 65, 70, 80, 90, or 100 PSI. In one embodiment, the cap having one or more pressure inlet ports and / or one or more fluid inlet ports may be sealed to withstand pressure. In one embodiment, the reservoir, filter, cap, and associated fluid connections are connected together to withstand pressure, e.g., secured by clasps. In one embodiment, metal clasps may be used to secure the device and system together.

[0041] A positive pressure may be maintained across the reservoir for a period of time. The reservoir may be pressurized for one minute to several hours. In one embodiment, the reservoir is pressurized for less than three hours, more preferably less than one hour. In one embodiment, the reservoir is pressurized for less than 10 minutes for the initial fluid wash and for 10 to 55 minutes for the subsequent fluid washes. A range of pressurized filtration times are contemplated, including less than 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 1 hour, 90 minutes, 2 hours, 3 hours, 10 to 40 minutes, 20 to 40 minutes, 30 to 60 minutes, etc.

[0042] In some embodiments, the cap comprises a fluid inlet port in addition to the pressure inlet port. In some embodiments, the cap comprises multiple fluid inlet ports, e.g., 1-3 ports, 1-5 ports, 2-5 ports, or 1, 2, 3, 4, 5, etc. ports. In some embodiments, the fluid inlet port may be in fluid communication with a fluid source. In some embodiments, multiple fluids may be added to the reservoirs through the same fluid inlet port or different fluid inlet ports. In some embodiments, there may be a fluid junction before the fluid inlet port, where each branch of the fluid junction is fluidly connected to a separate fluid source. In some embodiments, the fluid inlet port and the buffer inlet port are the same port. The fluid inlet port may be connected to a fluid source through a connecting component. In some embodiments, the connecting component may be tubing. In some embodiments, the tubing is disposable, allowing for the delivery and elution of fluids to each reservoir in a sterile manner.

[0043] The polyribonucleotide may be removed from the filter after pressurization. The polyribonucleotide may be removed by suction, for example, by suction from the filter surface. In some embodiments, 1 mg to 1000 mg of cyclic polyribonucleotide, for example, 1 mg to 5 mg, 1 mg to 10 mg, 1 mg to 25 mg, 1 mg to 50 mg, 1 mg to 75 mg, 1 mg to 100 mg, 1 mg to 125 mg, 1 mg to 150 mg, 1 mg to 200 mg, 5 mg to 25 mg, 5 mg to 50 mg, 10 mg to 50 mg, 25 mg to 50 mg, 25 mg to 50 mg, 25 mg to 75 mg, 25 mg to 100 mg, 25 mg to 125 mg, 25 mg to 150 mg, 50 mg to 100 mg, 50 ... mg to 75 mg, 50 mg to 100 mg, 50 mg to 150 mg, 50 mg to 200 mg, 50 mg to 250 mg, 75 mg to 125 mg, 75 mg to 150 mg, 75 mg to 200 mg, 80 mg to 120 mg, 90 mg to 110 mg, 100 mg to 150 mg, 100 mg to 200 mg, 100 mg to 250 mg, 125 mg to 175 mg, 150 mg to 250 mg, 250 mg to 500 mg, 250 mg to 750 mg, or 500 mg to 1000 mg is aspirated from the filter.

[0044] One embodiment of the present invention is a pressurized system (301) or pressurized robot assembly comprising a lid (302) with at least one port (303), a filter tray (304) with multiple sample reservoirs (305) with filters, and a waste reservoir (306) as seen in Figures 3A-3C. In some embodiments, the pressurized system can process multiple reservoirs in parallel, such as 2 to 24 reservoirs, preferably 8 to 12 reservoirs, e.g., 8 to 10 reservoirs, 10 to 12 reservoirs, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. reservoirs. The pressurized system is configured for a variety of reservoir volumes, e.g., both 50 mL and 15 mL reservoirs. In some embodiments, the pressurization system further comprises a robotic arm.

[0045] In some embodiments, the present devices, systems, and methods may be used to process a plurality of circular polyribonucleotide samples, such as 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more. In some embodiments, multiple pressurized systems are used in conjunction, for example, 10-12 pressurized systems.

[0046] In some embodiments, the device and system includes a sensor that can be used to stop pressurization when the liquid level in the reservoir drops to a certain level. In some embodiments, the liquid detection sensor is selected from the group consisting of an optical sensor, a vibration sensor, an ultrasonic sensor, a buoyancy sensor, a capacitance sensor, a radar sensor, a conductivity sensor, and a resistance sensor. In some embodiments, the sensor is an optical sensor.

[0047] In some embodiments, the devices and systems include at least one safety component or feature to prevent injury to the operator, such as reservoir rupture due to pressurization. For example, high pressure components, such as reservoirs and connections, that can withstand high pressure, or fastening components, such as clasps, may be included. EXAMPLES

[0048] The following examples are provided to illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood that by their illustrative nature, other procedures, methodologies, or techniques known to those skilled in the art may be substituted.

[0049] Example 1 This example demonstrates the use of a pressure device.

[0050] An Amicon® Ultra-15 filter with a filter porosity of 10 kDa, as shown in Figure 2, was adapted for buffer exchange using positive pressure. A cap with a pressure inlet port was made for use with the Amicon® Ultra-15 filter, as shown in Figure 1. The cap included sealing components to help the system withstand higher pressures, e.g., greater than 15 PSI. A system of components for connecting to a positive pressure source was designed for use in pressurizing a buffer exchange device with an Amicon® Ultra-15 filter with a cap attached later.

[0051] For buffer exchange of the cyclic polyribonucleotide, the cap with the pressure inlet port was first removed, and 5 mL of the cyclic polyribonucleotide in the initial buffer, a solution of 5-10% acetonitrile in 100 mM TEAA, was dispensed onto the filter in the reservoir of the device, which was then recapped. Compressed air was used to apply a pressure of 15 PSI to the reservoir through the pressure inlet port, forcing the first buffer through the filter to a waste collection area, while the cyclic polyribonucleotide remained on the filter. The first pressurization took about 4 minutes. The compressed air was then turned off, and the cap was slowly opened. 5 mL of water was then dispensed onto the filter into the reservoir, recapped, and pressure restored. The pressure was temporarily stopped, the cap was removed, the second buffer was added, the cap was recapped, and the pressure was restored, and this process was repeated five times, resulting in a total of six buffer washes with the second buffer. Subsequent pressurized filtrations took 5-40 minutes. The cap was then opened and 200 μL of circular polyribonucleotide was aspirated from the filter for incorporation into downstream processing.

[0052] Example 2 This example demonstrates the use of a pressure device.

[0053] Amicon® Ultra-30 filter units with a filter porosity of 10 kDa were adapted for buffer exchange using pressure as opposed to centrifugation. A cap with a pressure inlet port was made for use with Amicon® Ultra-30 filter units. The cap included sealing components to help the system withstand higher pressures, e.g., greater than 65 PSI. A system of components for connecting to a positive pressure source was designed for use in pressurizing the device with Amicon® Ultra-30 filter units with a cap attached later.

[0054] For buffer exchange of the cyclic polyribonucleotide, the cap with the pressure inlet port was first removed and 45 mL of 3.1 mg of cyclic polyribonucleotide in a solution of 5-10% acetonitrile in 100 mM TEAA was dispensed onto the filter in the reservoir of the device and then recapped. A pressure of 65 PSI was applied to the reservoir through the pressure inlet port, forcing the first buffer through the filter to a waste collection area, while the cyclic polyribonucleotide remained on the filter. The first pressure filtration took about 4 minutes. The pressure was then turned off and the cap was opened. Next, 10 mL of sodium citrate was added to the reservoir above the filter, recapped, and pressure was restored. The pressure was temporarily stopped, the cap was removed, the second buffer was added, the cap was recapped, and pressure was restored, and the process was repeated five times for a total of six buffer washes with the second buffer. Subsequent pressure filtrations took 35-50 minutes. The cap was then opened and 2 mg (1335 ng / μL) of circular polyribonucleotide was aspirated from the filter for incorporation into downstream processing.

[0055] Example 3 This example demonstrates the use of a pressure device.

[0056] An Amicon® Ultra-15 filter with a filter porosity of 10 kDa was adapted for buffer exchange using pressure as opposed to centrifugation. A cap with a pressure inlet port and a buffer inlet port was fabricated for use with the Amicon® Ultra-15 filter, as shown in FIG. 1. The cap included sealing components to help the system withstand higher pressures, e.g., greater than 65 PSI. A system of components for connecting to a positive pressure source and a buffer supply source was designed for use in pressurizing the buffer exchange device and supplying fresh buffer for the buffer exchange.

[0057] For buffer exchange of the cyclic polyribonucleotide, the device was first uncapped and 4 mg of cyclic polyribonucleotide in 5-10% acetonitrile in 100 mM TEAA was dispensed onto the filter in the device's reservoir and then recapped. 65 PSI pressure was applied to the reservoir through the pressure inlet port, forcing the first buffer through the filter to a waste collection area while the cyclic polyribonucleotide remained on the filter. The first pressurized filtration took approximately 12 minutes. The pressure was then temporarily stopped, after which 15 mL of sodium citrate was added onto the filter via the buffer inlet port into the reservoir and pressure was restored. The process of temporarily stopping the pressure and adding the second buffer through the buffer inlet port to restore pressure was repeated five times, for a total of six buffer washes with the second buffer. Subsequent pressurizations took 18-50 minutes. The cap was then opened and 3.42 mg (1898.1 ng / μL) of cyclic polyribonucleotide was aspirated from the filter for incorporation into downstream processing.

[0058] Example 4 This example demonstrates the use of an automatic pressurization device.

[0059] Amicon® Ultra-15 filters with a filter porosity of 10 kDa can be adapted for automated buffer exchange using pressure as opposed to centrifugation. A cap with a pressure inlet port and a buffer inlet port was made for use with Amicon® Ultra-15 filters as shown in FIG. 1. The cap included sealing components to help the system withstand higher pressures, e.g., greater than 60 PSI. A system of components for connecting to a positive pressure source and a buffer supply source was designed for use in pressurizing the buffer exchange device and supplying fresh buffer for buffer exchange. The device may also be adapted to include a fluid detection sensor.

[0060] The buffer exchange may follow the process of Example 1 or Example 2, except that a liquid detection sensor is used to automate the process. The liquid detection sensor detects when a volume of the first or second buffer or any subsequent buffer is forced through the filter to the waste collection area. When the liquid level in the reservoir above the filter area reaches a target level, the pressure will be temporarily stopped and a volume of the second buffer may be added to the reservoir above the filter. The pressure may then be restored. The temporary stop of the pressure due to the detection of the target liquid level, the addition of the second buffer, and the restoration of the pressure may be repeated five times for a total of six buffer washes with the second buffer, after which the cap is removed and the circular polyribonucleotide on the filter is sucked out and taken up in either the second buffer solution or the third buffer solution.

Claims

1. (a) providing an apparatus comprising a reservoir comprising a filter disposed between a filtrate chamber and a waste collection chamber; the reservoir further comprising a removable cap having a pressure inlet port; and the apparatus being in fluid communication with a source of positive pressure through the pressure inlet port; (b) providing cyclic polyribonucleotides in a first fluid to the filtrate chamber; (c) pressurizing the reservoir with the positive pressure source, wherein the first fluid passes through the filter and the cyclic polyribonucleotide does not pass through the filter; (d) applying a second fluid to the reservoir; and (e) pressurizing the reservoir with the positive pressure source, wherein the second fluid passes through the filter and the cyclic polyribonucleotide does not pass through the filter. A method for processing a cyclic polyribonucleotide, comprising:

2. 10. The method of claim 1, further comprising repeating steps (d) and (e) 1 to 5 times.

3. 3. The method of claim 1 or 2, further comprising the step of: (f) aspirating the circular polynucleotide from the filter after step (e).

4. The method of claim 1 or 2, wherein the second fluid is applied through a fluid inlet port in the cap.

5. The method of claim 4 , wherein the fluid inlet port is in fluid communication with a fluid source.

6. 3. The method of claim 1 or 2, wherein pressurizing steps (c) and (e) comprise pressurizing the reservoir at 10 to 100 PSI.

7. 3. The method of claim 1, wherein the applying step (d) comprises applying between 5 mL and 15 mL of fluid to the reservoir.

8. 3. The method of claim 1, wherein the first and / or second fluid is a liquid.

9. 1. A device comprising: a reservoir comprising a filtrate chamber and a waste collection chamber separated by a filter; the reservoir further comprising a removable cap in contact with a pressure inlet port; and the device in fluid communication with a positive pressure source.

10. 10. The device of claim 9, wherein the filtrate chamber is funnel-shaped.

11. 10. The device of claim 9, wherein the reservoir has a volume of 5 to 100 mL and the filtrate chamber is between 5 mL and 15 mL.

12. 12. The device of claim 9 or claim 11, wherein the filter has a molecular weight cutoff of between 2 kDa and 200 kDa.

13. 12. A device according to any one of claims 9 to 11, wherein the filtrate chamber and filter are stacked on, screwed into or nested within the reservoir.

14. An apparatus according to any one of claims 9 to 11, wherein the pressure inlet port is sealed.

15. 1. A system comprising a filter tray having a sample reservoir with a filtrate chamber having a filter, the filter tray disposed between a lid having a pressure inlet port and a waste reservoir; the pressure inlet port being in fluid communication with a positive pressure source.

16. (i) the filtrate chamber is funnel-shaped; and / or (ii) the volume of the filtrate chamber is 5 mL to 15 mL; 16. The system of claim 15.

17. 17. The system of claim 15 or claim 16, further comprising a liquid detection sensor or a vent.

18. 17. The system of claim 15 or 16, wherein the filter tray, lid, and waste reservoir are sealably connected together.

19. 17. The system of claim 15 or 16, wherein the filtrate chamber is stacked on top of, threaded into, or nested within the sample reservoir.

20. (a) the lid further comprises a fluid inlet port; or (b) the filter tray comprises a plurality of sample reservoirs; 17. A system according to claim 15 or 16.

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