System and method for producing pharmaceutical compositions using a peristaltic pump and damping device

Abstract

Provided herein are methods and systems for use with peristaltic pumps. More specifically, provided herein are peristaltic pump systems that include a damping device for reducing pulsations in flow rate from the peristaltic pump system for producing, mixing, transporting, and / or manufacturing pharmaceutical compositions and formulations, including pharmaceutical compositions and formulations that include lipids and RNA.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 075,723, filed on September 8, 2020, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing, and the content of the following submission on an ASCII text file is hereby incorporated by reference in its entirety: the computer - readable format (CRF) of the sequence listing (file name: 146392048140SEQLIST.TXT, recording date: September 3, 2021, size: 9,549 bytes).

[0003] Field The present disclosure relates to methods and systems for producing, mixing, transferring, and / or manufacturing pharmaceutical compositions and formulations. In some aspects, a tubing kit for use with a peristaltic pump for forming a pharmaceutical mixture is provided. More specifically, the present disclosure relates to a peristaltic pump system that includes a damping device for reducing the pulsation of the flow rate from a peristaltic pump system for producing, mixing, transferring, and / or manufacturing pharmaceutical compositions and formulations that include pharmaceutical compositions and formulations containing lipids (e.g., liposomes or lipoplexes) and RNA.

Background Art

[0004] Background A peristaltic pump is a positive displacement pump that can be used to pump various fluids. Typically, a peristaltic pump includes a circulating pump casing with tubes mounted or connected inside the casing and a rotor that compresses the tubes. The rotor includes several rollers mounted on its outer circumference. As the rotor rotates, the compressed portion of the tubes is blocked, thereby forcing the fluid to move through the tubes. Since a constant amount of fluid is pumped out with each rotation, a peristaltic pump can be used to roughly measure the amount of fluid being pumped out.

[0005] One major drawback of peristaltic pumps is that they can provide uneven flow. Because they use rollers to compress the tubing within the pump casing, the flow rate from a peristaltic pump pulsates or oscillates. Therefore, peristaltic pumps are not very suitable when a smooth and consistent flow is required.

[0006] Nucleic acids, such as DNA and RNA, are of increasing interest for a variety of therapeutic applications. Numerous reports describe approaches for the administration of nucleic acids. See, for example, U.S. Patent No. 10485884, incorporated herein by reference for all purposes. One approach is to leverage the ability of cationic liposomes (to induce DNA / RNA enrichment) to facilitate the cellular uptake of DNA or RNA into specific cells. Cationic liposomes typically consist of cationic lipids, such as DOTMA and / or DOTAP, and one or more helper lipids, such as DOPE. So-called "lipoplexes" can be formed from cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids. Lipoplexes can spontaneously form by mixing nucleic acids with liposomes, driven by electrostatic interactions between the positively charged liposomes and the negatively charged nucleic acids. Therefore, methods and systems are needed for producing, mixing, transferring, and / or manufacturing pharmaceutical compositions and formulations containing RNA and lipids (e.g., liposomes). [Overview of the project]

[0007] overview This specification provides peristaltic pump systems that include damping devices for reducing pulsation or oscillation of the flow rate from a peristaltic pump within the system. In some embodiments, these systems are useful for producing, mixing, transferring, and / or manufacturing pharmaceutical compositions and formulations, including, for example, compositions and formulations containing RNA and lipids including lipoplexes or liposomes. As described above, the flow rate from a peristaltic pump may pulsate or oscillate over time due to the nature of peristaltic pumps. Therefore, peristaltic pumps may not be suitable for certain applications where a smooth or consistent flow rate is required. An example of such use may be the use of a peristaltic pump to displace pharmaceutical compositions. These pharmaceutical compositions may contain delicate and expensive components. Furthermore, the amount of a given component in a pharmaceutical composition may be critical to whether the pharmaceutical composition is effective and safe for its intended use. For example, pharmaceutical compositions and formulations containing RNA and lipids (such as lipoplexes or liposomes) are affected by (1) the dynamic ratio of nucleic acids and lipids / liposomes when the pharmaceutical composition is formed during mixing, and (2) the average flow rate used during mixing. If the dynamic flow rate of nucleic acids changes dynamically during the operation, the ratio of nucleic acids to lipids / liposomes changes throughout the mixing operation, resulting in greater heterogeneity of the quality attributes of the resulting lipoplex (size, molecular weight dispersion, surface charge, etc.). A syringe pump can be used to mix nucleic acids and liposomes / lipids (e.g., containing RNA and lipids) to form lipoplexes useful in the manufacture of RNA vaccines (see, for example, Oberli MA et al., Nano Lett. 2017, 17, 1326-1335, or Kauffman, KJ et al. Nano Lett. 2015, 15, 7300-7306; see also International Publication No. 2019077053). Syringe pumps generate a flow with relatively low pulsation, allowing for good control over the mixing ratio of two or more solutions.However, since the inside of the syringe barrel is exposed to the ambient environment before the syringe plunger is pulled to fill the syringe barrel with fluid, a normal syringe does not provide a closed sterile boundary outside of a Grade A cleanroom environment. This is particularly critical for systems where liposomes and / or lipoplexes are too large to pass through sterile-grade filters and cannot ultimately be sterilized without significant degradation. In contrast, peristaltic pumps can be used in completely closed fluid pathways that do not require operation in a Grade A cleanroom environment to maintain sterile processing and sterility assurance. This is a significant advantage, as Grade A cleanroom environments are expensive to maintain and require time-consuming environmental monitoring and control. Accordingly, we have discovered methods and systems using peristaltic pumps, including dampers for dramatically reducing pulsation or vibration from the peristaltic pump, which are useful for producing, mixing, transferring, and / or manufacturing, for example, pharmaceutical compositions and formulations containing RNA and lipids including lipoplexes or liposomes, such as RNA vaccines. Although the damping devices disclosed herein are discussed in combination with peristaltic pumps, the pump system does not necessarily have to be a peristaltic pump system, as the damping devices can be combined with any pump system that generates pulsation as part of its mechanism of action (e.g., including membranes, pistons, etc.). For example, a syringe pump system can use the damping devices disclosed herein. In some embodiments, these methods and systems using a peristaltic pump including a damping device are suitable for ensuring smooth or consistent flow rates for producing, mixing, transferring, and / or manufacturing pharmaceutical compositions containing RNA and lipids (including lipoplexes or liposomes), such as RNA vaccines.

[0008] In some embodiments, a tubing kit for forming a mixture includes: a first portion of tubing configured to be fluidly connected to a container for a first composition; a second portion of tubing configured to be fluidly connected to a container for a second composition; a damping device fluidly connected to the first portion of tubing and to the second portion of tubing; a mixing device for mixing the first composition from the first portion of tubing and the second composition from the second portion of tubing; and a mixture container for collecting the mixed first and second compositions from the mixing device, wherein the first portion of tubing is configured to be connected to at least one peristaltic pump head for pumping the first composition from the container for the first composition to the mixture container, and the second portion of tubing is configured to be connected to at least one peristaltic pump head for pumping the second composition from the container for the second composition to the mixture container. In some embodiments, the damping device contains a sealed volume of fluid. In some embodiments, the fluid is air. In some embodiments, the damping device is a tubing damping device. In some embodiments, the damping device comprises a flexible membrane. In some embodiments, the tubing kit includes a damping device and a first T-connector that fluidly connects a first portion of the tubing and a first mixer input portion of the tubing, the first mixer input portion of the tubing being fluidly connected to the mixer. In some embodiments, the tubing kit includes a damping device and a second T-connector that fluidly connects a second portion of the tubing and a second mixer input portion of the tubing, the second mixer input portion of the tubing being fluidly connected to the mixer. In some embodiments, the first portion of the tubing comprises a first segment of the tubing and a second segment of the tubing, the first segment of the tubing and the second segment of the tubing being fluidly connected in parallel.In some embodiments, a first segment of the tubing is configured to be connected to a first peristaltic pump head, and a second segment of the tubing is configured to be connected to a second peristaltic pump head. In some embodiments, the second portion of the tubing comprises a third segment and a fourth segment, the third and fourth portions of the tubing being fluidically connected in parallel. In some embodiments, the third segment of the tubing is configured to be connected to a third peristaltic pump head, and the fourth segment of the tubing is configured to be connected to a fourth peristaltic pump head. In some embodiments, the mixing device comprises an input section fluidically connected to the first portion of the tubing, an input section fluidically connected to the second portion of the tubing, and an output section fluidly connected to a mixture container. In some embodiments, the mixing device comprises a Y-connector, a helical mixer, or a static mixer. In some embodiments, the tubing kit includes a first damping connector that fluidly connects a first portion of the tubing to the damping and mixing device, and a second damping connector that fluidly connects a second portion of the tubing to the damping and mixing device. In some embodiments, the mixing container is a bag, a container, or a bottle.

[0009] In some embodiments, a system for forming a pharmaceutical composition or a mixture of pharmaceutical compositions includes a first container for containing a first pharmaceutical composition, a second container for containing a second pharmaceutical composition, a first portion of tubing fluidly connected to the first container, a second portion of tubing fluidly connected to the second container, a damping device fluidly connected to the first portion of tubing and fluidly connected to the second portion of tubing, a mixing device for mixing the first pharmaceutical composition from the first portion of tubing and the second pharmaceutical composition from the second portion of tubing, and a mixture container for collecting the mixed first and second pharmaceutical compositions from the mixing device. In some embodiments, the system includes at least one peristaltic pump head connected to the first portion of tubing for pumping the first composition from the container for containing the first composition to the mixture container, and at least one peristaltic pump connected to the second portion of tubing for pumping the second composition from the container for containing the first composition to the mixture container. In some embodiments, the first or second composition comprises nucleic acid, one or more lipids, one or more proteins, or a buffer. In some embodiments, the first composition comprises nucleic acid and the second composition comprises one or more lipids. In some embodiments, the first composition comprises RNA and the second composition comprises one or more lipids. In some embodiments, the RNA comprises one or more polynucleotides encoding 10 to 20 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In some embodiments, the RNA is incorporated into lipoplex nanoparticles or liposomes. In some embodiments, the lipoplex nanoparticles or liposomes comprise one or more lipids forming a multilayer structure that encapsulates the RNA. In some embodiments, the one or more lipids comprise at least one cationic lipid and at least one helper lipid. In some embodiments, one or more lipids include (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propaneaminium chloride (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).In some embodiments, at physiological pH, the total charge ratio of positive to negative charges of liposomes is 1.3:2 (0.65). In some embodiments, the RNA molecule comprises, in the 5'→3' direction, (1) a 5' cap, (2) a 5' untranslated region (UTR), (3) a polynucleotide sequence encoding a secretory signal peptide, (4) a polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen, (5) a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule, (6) a 3'UTR comprising (a) the 3' untranslated region or a fragment thereof of Amino-Terminal Enhancer of Split (AES) mRNA, and (b) a non-coding RNA or a fragment thereof of mitochondrial-encoded 12S RNA, and (7) a poly(A) sequence. In some embodiments, the RNA molecule further comprises a polynucleotide sequence encoding an amino acid linker, wherein the polynucleotide sequence encoding the amino acid linker and one or more neoepitopes forms a first linker-neoepitope module, and the polynucleotide sequence forming the first linker-neoepitope module is located in the 5'→3' direction between a polynucleotide sequence encoding a secretory signal peptide and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule. In some embodiments, the amino acid linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 21). In some embodiments, the polynucleotide sequence encoding the amino acid linker comprises the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 19).In some embodiments, the RNA molecule further comprises at least a second linker-epitope module in the 5'→3' direction, the at least second linker-epitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, the polynucleotide sequence forming the second linker-neoepitope module being located in the 5'→3' direction between a polynucleotide sequence encoding the neoepitope of the first linker-neoepitope module and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule, the neoepitope of the first linker-epitope module being different from the neoepitope of the second linker-epitope module. In some embodiments, the RNA molecule comprises five linker-epitope modules, each of the five linker-epitope modules encoding a different neoepitope. In some embodiments, the RNA molecule comprises 10 linker-epitope modules, each encoding a different neoepitope. In some embodiments, the RNA molecule comprises 20 linker-epitope modules, each encoding a different neoepitope. In some embodiments, the RNA molecule further comprises a second polynucleotide sequence encoding an amino acid linker, the second polynucleotide sequence encoding the amino acid linker being located between a polynucleotide sequence encoding the most distal neoepitope in the 3' direction and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule. In some embodiments, the 5' cap comprises a D1 diastereoisomer of the following structure: JPEG0007872261000001.jpg52170 In some embodiments, the 5'UTR comprises the sequence UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 5). In some embodiments, the 5'UTR comprises the sequence GGCGACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 3). In some embodiments, the secretion signal peptide comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 9). In some embodiments, the polynucleotide sequence encoding the secretion signal peptide comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 7). In some embodiments, at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule contains the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 12). In some embodiments, the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule contains the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC (SEQ ID NO: 10). In some embodiments, the 3' untranslated region of the AES mRNA contains the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO: 15).In some embodiments, the non-coding RNA of 12S RNA encoded by mitochondria includes the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (Sequence ID 17). In some embodiments, the 3'UTR contains the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 13). In some embodiments, the poly(A) sequence contains 120 adenine nucleotides.In some embodiments, the RNA is arranged in the 5'→3' direction, with the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 1); a polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUA GCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCAC GCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 2). In some embodiments, a method for transferring a pharmaceutical composition using a peristaltic pump includes: using at least one peristaltic pump to pump a first composition from a first container through a first portion of tubing; using at least one peristaltic pump to pump a second composition from a second container through a second portion of tubing; and using damping devices fluidly connected to the first portion of tubing and fluidly connected to the second portion of tubing to dampen the pulsations in the fluid flow of the first composition in the first portion of tubing and the pulsations in the fluid flow of the second composition in the second portion of tubing. In some embodiments, the method includes mixing the first composition from the first portion of tubing and the second composition from the second portion of tubing in a mixing device fluidly connected to the first and second portions of tubing. In some embodiments, the method includes depositing the mixture containing the first and second compositions in a mixture container fluidly connected to the mixture. In some embodiments, the first or second composition comprises nucleic acid, one or more lipids, one or more proteins, or a buffer. In some embodiments, the first composition comprises nucleic acid and the second composition comprises one or more lipids. In some embodiments, the first composition comprises RNA and the second composition comprises one or more lipids. In some embodiments, the RNA comprises one or more polynucleotides encoding 10 to 20 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In some embodiments, the RNA is incorporated into lipoplex nanoparticles or liposomes. In some embodiments, the lipoplex nanoparticles or liposomes comprise one or more lipids forming a multilayer structure that encapsulates the RNA. In some embodiments, the one or more lipids comprise at least one cationic lipid and at least one helper lipid. In some embodiments, one or more lipids include (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propaneaminium chloride (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).In some embodiments, at physiological pH, the total charge ratio of positive to negative charges in liposomes is 1.3:2 (0.65). In some embodiments, RNA is oriented in the 5'→3' direction, consisting of (1) a 5' cap and (2) a 5' untranslated region (UTR). (3) A polynucleotide sequence encoding a secretory signal peptide; (4) A polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; (5) A polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; (6) A 3'UTR comprising (a) the 3' untranslated region or a fragment thereof of Amino-Terminal Enhancer of Split (AES) mRNA and (b) a non-coding RNA or a fragment thereof of mitochondrial-encoded 12S RNA; and (7) An RNA molecule comprising a poly(A) sequence. In some embodiments, the RNA molecule further comprises a polynucleotide sequence encoding an amino acid linker, wherein the polynucleotide sequence encoding the amino acid linker and one or more neoepitopes forms a first linker-neoepitope module, and the polynucleotide sequence forming the first linker-neoepitope module is located in the 5'→3' direction between a polynucleotide sequence encoding a secretory signal peptide and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule. In some embodiments, the amino acid linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 21). In some embodiments, the polynucleotide sequence encoding the amino acid linker comprises the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 19).In some embodiments, the RNA molecule further comprises at least a second linker-epitope module in the 5'→3' direction, the at least second linker-epitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, the polynucleotide sequence forming the second linker-neoepitope module being located in the 5'→3' direction between a polynucleotide sequence encoding the neoepitope of the first linker-neoepitope module and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule, the neoepitope of the first linker-epitope module being different from the neoepitope of the second linker-epitope module. In some embodiments, the RNA molecule comprises five linker-epitope modules, each of the five linker-epitope modules encoding a different neoepitope. In some embodiments, the RNA molecule comprises 10 linker-epitope modules, each encoding a different neoepitope. In some embodiments, the RNA molecule comprises 20 linker-epitope modules, each encoding a different neoepitope. In some embodiments, the RNA molecule further comprises a second polynucleotide sequence encoding an amino acid linker, the second polynucleotide sequence encoding the amino acid linker being located between a polynucleotide sequence encoding the most distal neoepitope in the 3' direction and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule. In some embodiments, the 5' cap comprises a D1 diastereoisomer of the following structure: JPEG0007872261000002.jpg53170 In some embodiments, the 5'UTR comprises the sequence UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 5). In some embodiments, the 5'UTR comprises the sequence GGCGACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 3). In some embodiments, the secretory signal peptide comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 9). In some embodiments, the polynucleotide sequence encoding the secretory signal peptide comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 7). In some embodiments, at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule contains the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 12). In some embodiments, the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule contains the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC (SEQ ID NO: 10). In some embodiments, the 3' untranslated region of the AES mRNA contains the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (Sequence ID 15).In some embodiments, the non-coding RNA of 12S RNA encoded by mitochondria includes the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (Sequence ID 17). In some embodiments, the 3'UTR contains the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 13). In some embodiments, the poly(A) sequence contains 120 adenine nucleotides.In some embodiments, the RNA is arranged in the 5'→3' direction, with the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 1); a polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUA GCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCAC GCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 2).

[0010] In some embodiments, a method for transferring a pharmaceutical composition using a peristaltic pump includes: pumping a first composition from a first vessel through a first portion of tubing at a first flow rate using at least one peristaltic pump head; pumping a second composition from a second vessel through a second portion of tubing at a second flow rate using at least one peristaltic pump; and damping the pulsations in the fluid flow of the first composition in the first portion of tubing and the pulsations in the fluid flow of the second composition in the second portion of tubing using a damping device fluidly connected to the first portion of tubing and the second portion of tubing, wherein the flow pulsation level (LoP) of the first flow rate in the first portion of tubing behind the damping device is less than 10, and the flow pulsation level (LoP) of the second flow rate in the second portion of tubing behind the damping device is less than 10.

[0011] In some embodiments, a method for producing a pharmaceutical composition comprising nucleic acids and one or more lipids includes: pumping a first composition comprising nucleic acids from a first vessel through a first portion of tubing using at least one peristaltic pump head at a first flow rate; pumping a second composition comprising one or more lipids from a second vessel through a second portion of tubing using at least one peristaltic pump head at a second flow rate; damping the pulsation of the fluid flow of the first composition in the first portion of tubing and the pulsation of the fluid flow of the second composition in the second portion of tubing using damping devices fluidly connected to the first portion of tubing and the second portion of tubing; mixing the first composition comprising nucleic acids from the first portion of tubing and the second composition comprising one or more lipids from the second portion of tubing in a mixing device fluidly connected to the first and second portions of tubing; and depositing the composition comprising nucleic acids and one or more lipids in a vessel fluidly connected to the mixture.

[0012] In some embodiments, the first and second portions of the tubing are configured to be connected to the pump head of the same peristaltic pump. In some embodiments, the first portion of the tubing, the second portion of the tubing, the damping device, the mixing device, and / or the mixture container are made of disposable material. The tubing kit or system is an aseptic closed tubing kit or system, or the method is carried out within an aseptic closed system.

[0013] In some embodiments, a tubing kit for forming a mixture comprises: a first portion of tubing configured to be fluidly connected to a first container for containing a first composition; a second portion of tubing configured to be fluidly connected to a second container for containing a second composition; a tubing damper containing a sealed volume of fluid, fluidly connected to the first portion of tubing and the second portion of tubing; and a mixing device fluidly connected downstream from the fluid damper to the first portion of tubing and the second portion of tubing, wherein the first composition from the first portion of tubing and the tubing The invention comprises a mixing device configured to mix a second composition from a second portion of the first composition with a mixture container fluidly connected to the mixing device and configured to collect the mixed first and second compositions from the mixing device, wherein the first portion of the tubing is configured to be connected to a first peristaltic pump head upstream of the tubing damper for pumping the first composition from the first container to the mixture container, and the second portion of the tubing is configured to be connected to a second peristaltic pump head upstream of the tubing damper for pumping the second composition from the second container to the mixture container.

[0014] In some embodiments, a system for forming a pharmaceutical composition or a mixture of pharmaceutical compositions comprises a first container for containing a first pharmaceutical composition, a second container for containing a second pharmaceutical composition, a peristaltic pump comprising a first portion of tubing fluidly connected to the first container, a second portion of tubing fluidly connected to the second container, a first peristaltic pump head connected to the first portion of tubing for pumping the first pharmaceutical composition from the first container, and a second peristaltic pump head connected to the second portion of tubing for pumping the second pharmaceutical composition from the second container, and a peristaltic pump The system includes a tubing damper containing a sealed volume of fluid, which is fluidically connected downstream from the tubing to a first portion and a second portion of the tubing; a mixing device, which is fluidically connected downstream from the fluid damper to the first portion and a second portion of the tubing, and configured to mix a first pharmaceutical composition from the first portion of the tubing and a second pharmaceutical composition from the second portion of the tubing; and a mixture container, which is fluidically connected to the mixing device, and configured to collect the mixed first and second pharmaceutical compositions from the mixing device.

[0015] In some embodiments, a method for transferring a pharmaceutical composition using a peristaltic pump includes: using a first peristaltic pump head to pump a first composition from a first container through a first portion of tubing; using a second peristaltic pump head to pump a second composition from a second container through a second portion of tubing; and using a tubing damping device containing a sealed volume of fluid, which is fluidically connected to the first portion of tubing and the second portion of tubing, to transfer the first portion of tubing downstream of the first peristaltic pump head. The method includes attenuating the pulsation of the first composition in the fluid flow at a certain point and attenuating the pulsation of the second composition in the fluid flow at a second portion of the tubing downstream of a second peristaltic pump head; mixing the first composition from the first portion of the tubing and the second composition from the second portion of the tubing in a mixing device fluidly connected to the first and second portions of the tubing downstream from the fluid attenuator; and depositing the mixture containing the first and second compositions in a container fluidly connected to the mixing device.

[0016] In some embodiments, a tubing kit for forming a mixture includes a first portion of tubing configured to be fluidly connected to a container for containing a first composition, a second portion of tubing configured to be fluidly connected to a container for containing a second composition, a first damping device fluidly connected to the first portion of tubing, a second damping device fluidly connected to the second portion of tubing, a mixing device for mixing the first composition from the first portion of tubing and the second composition from the second portion of tubing, and a mixture container for collecting the mixed first and second compositions from the mixing device, wherein the first portion of tubing is configured to be connected to at least one peristaltic pump head for pumping the first composition from the container for containing the first composition to the mixture container, and the second portion of tubing is configured to be connected to at least one peristaltic pump head for pumping the second composition from the container for containing the second composition to the mixture container. In some embodiments, the first and / or second damping devices contain a sealed volume of fluid. In some embodiments, the fluid is air. In some embodiments, the first and / or second damping device is a tubing damping device. In some embodiments, the damping device comprises a flexible membrane. In some embodiments, the tubing kit includes a first damping device and a first T-connector that fluidly connects a first portion of the tubing and a first mixer input portion of the tubing, the first mixer input portion of the tubing being fluidly connected to the mixer. In some embodiments, the tubing kit includes a second damping device and a second T-connector that fluidly connects a second portion of the tubing and a second mixer input portion of the tubing, the second mixer input portion of the tubing being fluidly connected to the mixer. In some embodiments, the first portion of the tubing comprises a first segment of the tubing and a second segment of the tubing, the first segment of the tubing and the second segment of the tubing being fluidly connected in parallel.In some embodiments, a first segment of the tubing is configured to be connected to a first peristaltic pump head, and a second segment of the tubing is configured to be connected to a second peristaltic pump head. In some embodiments, the second portion of the tubing comprises a third segment and a fourth segment, the third and fourth portions of the tubing being fluidically connected in parallel. In some embodiments, the third segment of the tubing is configured to be connected to a third peristaltic pump head, and the fourth segment of the tubing is configured to be connected to a fourth peristaltic pump head. In some embodiments, the mixing device comprises an input section fluidically connected to the first portion of the tubing, an input section fluidically connected to the second portion of the tubing, and an output section fluidly connected to a mixture container. In some embodiments, the mixing device comprises a Y-connector, a helical mixer, or a static mixer. In some embodiments, the tubing kit includes a first damping connector that fluidly connects a first portion of the tubing to a first damping and mixing device, and a second damping connector that fluidly connects a second portion of the tubing to a second damping and mixing device. In some embodiments, the mixing container is a bag, a container, or a bottle.

[0017] In some embodiments, a pulsation damper for a fluid pump includes a bioprocessing bag having a fluid inlet and a fluid outlet, the fluid inlet being configured to be fluidly connected downstream of a fluid pump; a housing configured to receive the bioprocessing bag, the housing comprising a base and a plurality of side walls forming a cavity for the bioprocessing bag, the at least one side wall comprising one or more notches configured to provide access to the fluid inlet and fluid outlet of the bioprocessing bag; and a housing lid attached to the plurality of side walls and configured to close the housing. In some embodiments, the bioprocessing bag has a gas inlet, the gas inlet being configured to be fluidly connected to a gas source. In some embodiments, one or more notches are configured to provide access to the gas inlet of the bioprocessing bag. In some embodiments, the base of the housing comprises a window. In some embodiments, the window comprises an opening in the base of the housing or transparent material in the bottom of the housing. In some embodiments, the housing lid comprises a window. In some embodiments, the window comprises an opening in the housing lid or transparent material in the housing lid. In some embodiments, the damping device includes a front plate configured to be connected to at least one of the side walls and / or lid of the housing, the front plate having at least one opening configured to receive a fluid inlet and a fluid outlet. In some embodiments, the fluid pump is a circulation pump. In some embodiments, the circulation pump is a peristaltic pump. In some embodiments, the fluid outlet is configured to be fluidically connected to a fluid storage container. In some embodiments, the fluid outlet includes a check valve.

[0018] Further advantages will be readily apparent to those skilled in the art from the detailed description below. The examples and descriptions herein should be considered illustrative and not limiting in nature. [Brief explanation of the drawing]

[0019] Exemplary embodiments will be described with reference to the attached drawings.

[0020] [Figure 1] Figure 1 shows an example of an experimental apparatus for measuring the flow rate of a peristaltic pump according to some aspects disclosed in this specification.

[0021] [Figure 2] Figure 2 shows the flow rate of water through the peristaltic pump achieved in Experiment 1.

[0022] [Figure 3] Figure 3 shows an example of an experimental apparatus for measuring the flow rate of a peristaltic pump having a damping device according to some aspects disclosed in this specification.

[0023] [Figure 4] Figure 4 is an image of a syringe damping device having various T-shaped connectors disclosed in this specification.

[0024] [Figure 5] Figure 5 shows the flow rate through the peristaltic pump achieved in Experiment 2.

[0025] [Figure 6] Figure 6 shows the flow rate through the peristaltic pump achieved in Experiment 4.

[0026] [Figure 7] Figure 7 shows the flow rate through the peristaltic pump achieved in Experiment 6.

[0027] [Figure 8] Figure 8 is an image of a membrane damping device having a T-shaped connector disclosed in this specification.

[0028] [Figure 9] Figure 9 shows an example of a T-shaped connector tubing connector according to some aspects disclosed in this specification.

[0029] [Figure 10]Figure 10 shows an example of a cross-shaped tubing connector according to several embodiments disclosed herein.

[0030] [Figure 11] Figure 11 shows an example of an experimental apparatus for measuring the flow rate of a two-source peristaltic pump system with a damping device, according to some embodiments disclosed herein.

[0031] [Figure 12] Figure 12 shows an example of a damping device loop connecting two separate fluid lines.

[0032] [Figure 13] Figure 13 shows the water flow rate through the peristaltic pump system achieved in Experiment 19.

[0033] [Figure 14] Figure 14 shows the water flow rate achieved in Experiment 20 through the peristaltic pump.

[0034] [Figure 15] Figure 15 shows an example of a tubing kit according to several embodiments disclosed herein.

[0035] [Figure 16] Figure 16 shows the general structure of an exemplary RNA molecule (i.e., polyneoepitope RNA). This figure is a schematic diagram of the general structure of an RNA drug substance having a constant 5' cap (β-S-ARCA(D1)), 5' and 3' untranslated regions (hAg-Kozak and FI, respectively), N-terminal and C-terminal fusion tags (sec2.0 and MITD, respectively), and a poly(A) tail (A120), as well as tumor-specific sequences encoding neoepitopes (neo1-10) fused by a GS-rich linker.

[0036] [Figure 17]Figure 17 shows the ribonucleotide sequence (5'→3') of the constant region of an exemplary RNA molecule (SEQ ID NO: 24). The binding between the first two G residues is an abnormal binding (5'→5')-ppsp- as shown in Figure 18 for the 5'-capping structure. The insertion site for the patient's cancer-specific sequence is between the C131 and A132 residues (marked in bold). "N" refers to the position of a polynucleotide sequence encoding one or more (e.g., 1-20) neoepitopes (separated by any linker).

[0037] [Figure 18] Figure 18 shows the 5'-capping structure β-S-ARCA(D1)(m2 7·2'·OGppspG) used at the 5' end of the RNA constant region. The stereogenic P center is in the Rp configuration in the "D1" isomer. Note: Differences between β-S-ARCA(D1) and the basic cap structure m7GpppG; the -OCH3 group at the C2' position of component m7G ​​and the sulfur substitution of the non-crosslinked oxygen in the β-phosphate are shown in red. Due to the presence of the stereogenic P center (labeled with *), the phosphorothioate cap analog β-S-ARCA exists as two diastereomers. Based on the elution order in reverse-phase high-performance liquid chromatography, these were named 01 and 02.

[0038] [Figure 19] Figure 19 is a schematic diagram of the HPPD apparatus used in the experiments described herein.

[0039] [Figure 20] Figure 20 shows the experimental setup using a glass bottle as HPPD.

[0040] [Figure 21] Figure 21 is a graph showing the time required for the glass bottle damping device in Figure 20 to achieve a constant pressure, and the time required for the same HPPD to dissipate this acquired pressure after the pump stops.

[0041] [Figure 22] Figure 22 is a graph showing the displacement of the glass bottle damping device in Figure 20 at various flow rates over a certain period of time.

[0042] [Figure 23] Figure 23 is a graph showing the pressure difference across the glass bottle HPPD device (pressure before HPPD vs. pressure after HPPD) as a function of the increased air pocket size.

[0043] [Figure 24] Figure 24 shows an HPPD based on a laboratory flask (left) and the same device (right) with a removable liner, which theoretically allows the HPPD to be treated as a single-use device.

[0044] [Figure 25] Figure 25 is a graph showing the efficiency of the HPPD attenuation device shown in Figure 24 with and without the liner (i.e., bladder attenuation device).

[0045] [Figure 26] Figure 26 shows a flexible, single-use HPPD design based on a modified bioprocessing bag, which includes a raised inlet tube to prevent backflow washing of the discharged fluid when back pressure is high, and an additional third point that allows for gas insertion to improve the priming efficiency of the device by pre-filling the bag with a gas cushion.

[0046] [Figure 27] Figure 27 shows a bioprocessing bag decay device with a cardboard box cover.

[0047] [Figure 28A] Figure 28A is an exploded view of an HPPD according to several embodiments disclosed herein.

[0048] [Figure 28B]Figure 28B shows an HPPD housing according to several embodiments disclosed herein.

[0049] [Figure 28C] Figure 28C shows HPPD in use according to several embodiments disclosed herein.

[0050] [Figure 29A] Figure 29A shows the peristaltic pump experimental setup and flow profile results.

[0051] [Figure 29B] Figure 29B shows the syringe pump experimental setup and flow profile results.

[0052] [Figure 29C] Figure 29C shows an experimental setup for a peristaltic pump having an HPPD damping device disclosed herein, and the resulting flow profile.

[0053] [Figure 30] Figure 30 shows a commercially available Cole-Parmer HPPD.

[0054] [Figure 31A] Figure 31A shows the flow profile of the Cole-Parmer HPPD device.

[0055] [Figure 31B] Figure 31B shows the flow profile of the HPPD attenuation device disclosed herein.

[0056] [Figure 32] Figure 32 shows the dead volume of the HPPD attenuator disclosed herein and the pressure at the attenuator inlet as the flow rate increases.

[0057] [Figure 33A] Figure 33A shows the effect of a 1.6 mm inner diameter on pulsation, as disclosed herein.

[0058] [Figure 33B] Figure 33B shows the effect of a 3.2 mm inner diameter on pulsation, as disclosed herein.

[0059] [Figure 33C] Figure 33C shows the effect of a 6 mm inner diameter on pulsation, as disclosed herein.

[0060] [Figure 34A] Figure 34A shows the effect of a 1-meter tubing length on pulsation, as disclosed herein.

[0061] [Figure 34B] Figure 34B shows the effect of a 2-meter tubing length on pulsation, as disclosed herein.

[0062] [Figure 34C] Figure 34C shows the effect of a 20-meter tubing length on pulsation, as disclosed herein. [Modes for carrying out the invention]

[0063] Detailed explanation The applicants have discovered methods and systems for using a peristaltic pump that include a damping device for dramatically reducing pulsation or vibration from the peristaltic pump. The methods and systems disclosed herein can minimize the number of parts required to achieve damping. Furthermore, the kits and systems disclosed herein may be single-use, disposable kits and systems and can offer significant advantages for producing, mixing, transferring, and / or manufacturing pharmaceutical compositions and formulations, for example, compositions and formulations comprising RNA and lipids containing lipoplexes or liposomes. In some embodiments disclosed herein, the compositions and formulations comprising RNA and lipids containing lipoplexes or liposomes are RNA vaccines.

[0064] This disclosure also provides peristaltic pumps, dampers, and tubing kit systems suitable for use with two fluid sources, comprising, for example, a first pharmaceutical composition comprising RNA, RNA molecules, or RNA vaccines, and a second pharmaceutical composition comprising one or more lipids, which can be mixed to prepare, transfer, or manufacture the pharmaceutical compositions described herein, particularly the final pharmaceutical compositions comprising RNA-lipoplexes, RNA liposomes, or RNA vaccines. In some embodiments, the methods and systems described herein are useful in GMP manufacturing processes that require a substantial reduction of flow rate pulsation or oscillations typically observed when using peristaltic pumps.

[0065] I. Definition Before describing the present invention in detail, it should be understood that the terms used herein are merely for the purpose of describing specific embodiments and are not intended to limit them.

[0066] Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or expressions used herein are intended to have the same meaning as those generally understood by those skilled in the art in the field to which the claimed subject matter pertains. In some cases, terms having a generally understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from that generally understood in the art.

[0067] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context explicitly indicates otherwise. The term “and / or” as used herein should be understood to refer to and encompass any possible combination of one or more of the enumerated items relating to each other. Where used herein, the terms “includes,” “including,” “comprises,” and / or “comprising” specify the presence of the described features, integers, processes, operations, elements, components, and / or units, but do not exclude the presence or addition of one or more other features, integers, processes, operations, elements, components, units, and / or groups thereof.

[0068] Throughout this disclosure, various aspects are presented in range format. It should be understood that the range format is merely for convenience and conciseness and should not be interpreted as an immutable limitation to this disclosure. Therefore, a range description should be considered to specifically disclose not only all possible sub-ranges but also the individual numerical values ​​within those ranges. For example, where a range of values ​​is provided, it is understood that this disclosure encompasses each intermediate value between the upper and lower limits of that range, as well as any other stated or intermediate values ​​within that stated range. These smaller range upper and lower limits may be independently included within those smaller ranges and are also included in this disclosure, subject to any particularly excluded limits within the stated range. If a stated range includes one or both limits, the range excluding either or both of those included limits is also included in this disclosure. This applies regardless of the width of the range.

[0069] As used herein, the term “about” refers to the normal range of error for each value as is easily understood. References to values ​​or parameters following “about” herein include (and describe) aspects relating to the value or parameter itself. For example, a description referring to “about X” includes a description of “X.” In some embodiments, “about” may mean ±25%, ±20%, ±15%, ±10%, ±5%, or ±1%, as understood by those skilled in the art.

[0070] As used herein, a composition refers to any mixture of one or more products, substances, or compounds, including cells. A composition may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0071] As used herein, “peristallic pump” refers to a type of positive displacement pump that can be used to pump various fluids. Examples of peristaltic pumps include, but are not limited to, the Masterflex Pump (HV-77921-75) and the Watson Marlow Flexicon (PD12I). Typically, peristaltic pumps are used in biopharmaceuticals for two reasons: (1) the system can pump fluid through a closed system (i.e., no exposed pump parts come into contact with the fluid), and (2) the shear stress is low.

[0072] The term "damping device" refers to any component, device, or mechanism that can reduce flow rate pulsation and / or vibration from a pump, including, for example, a peristaltic pump.

[0073] The term "tubing kit" refers to an assembly of tubes / tubing and other components that can interact with them.

[0074] The terms “pharmaceutical composition,” “pharmaceutical preparation,” or “pharmaceutical composition and preparation” refer to a preparation in which the biological activity of the active ingredient contained therein is effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition will be administered. Such preparations are sterile. A “pharmaceutically acceptable” excipient (vehicle, additive) is one that can be administered to a target mammal in a moderate amount to provide an effective dose of the active ingredient to be used. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some embodiments described herein, a pharmaceutical composition comprises nucleic acids (e.g., including RNA, mRNA, or RNA vaccines) and / or one or more lipids (e.g., including cationic lipids and / or neutral “helper” lipids).

[0075] The “individual,” “subject,” or “patient” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain contexts, the individual or subject is human.

[0076] The terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by a sequence of bases, which represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. In this specification, the term nucleic acid molecule includes, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone links or chemically modified residues. “Isolated” nucleic acids refer to nucleic acid molecules separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained within cells containing nucleic acid molecules, but which exist outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0077] The terms “RNA” or “RNA molecule” refer to a molecule containing, and preferably entirely or substantially composed of, ribonucleotide residues. “Ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term includes isolated RNA such as double-stranded RNA, single-stranded RNA, and partially purified RNA; essentially pure RNA; synthetic RNA; recombinantly produced RNA; and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material to one or more nucleotides of RNA, for example, at the ends or within the RNA. Nucleotides in an RNA molecule may also include non-standard nucleotides such as naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be called analogs or analogs of naturally occurring RNA.

[0078] The term "RNA" also means "messenger RNA" and includes, preferably, "mRNA" relating to a "transcript" that can be produced using DNA as a template and codes for a peptide or protein. mRNA typically contains a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro. mRNA can be produced by in vitro transcription or chemosynthesis using a DNA template. In vitro transcription methods are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0079] As used herein, “RNA vaccine” refers to an RNA, RNA polynucleotide, or RNA molecule that encodes one or more antigens and induces an immune response (e.g., protective immunity against the antigen) when administered to a subject or individual. Several RNA vaccines have been described. For example, see Pardi et al., “mRNA vaccines—a new era in vaccinology.” Nat Rev Drug Discov 17, 261-279 (2018). https: / / doi.org / 10.1038 / nrd.2017.243.

[0080] The term "lipoplex" or "RNA lipoplex" refers to a complex of lipids and nucleic acids such as RNA. Lipoplexes form spontaneously when cationic lipids and / or liposomes, often including neutral "helper" lipids, are mixed with nucleic acids.

[0081] Where this disclosure refers to charges such as positive, negative, or neutral charges, or cationic compounds, negative compounds, or neutral compounds, this generally refers to the presence of the referred charge at a selected pH, such as physiological pH. For example, the term “cationic lipid” means a lipid that has a net positive charge at a selected pH, such as physiological pH. The term “neutral lipid” means a lipid that has no net positive or negative charge and can exist in the form of an uncharged or neutral amphoteric ion at a selected pH, such as physiological pH. In this specification, “physiological pH” means a pH of about 7.5.

[0082] The lipid carriers described herein for use in the present invention include any substance or vehicle that can associate with RNA, for example, by forming a complex with RNA, or by forming a vesicle in which RNA is enclosed or encapsulated. This may result in increased RNA stability compared to naked RNA. In particular, it may increase RNA stability in the blood.

[0083] Cationic lipids, cationic polymers, and other positively charged substances can form complexes with negatively charged nucleic acids. These cationic molecules can be used to complex nucleic acids, thereby forming, for example, so-called lipoplexes or polyplexes, respectively.

[0084] Liposomes are microscopic lipid vesicles, often having one or more bilayers of vesicle-forming lipids such as phospholipids, that can encapsulate drug or nucleic acid molecules such as RNA. Various types of liposomes, including but not limited to multilayer vesicles (MLVs), small monolayer vesicles (SUVs), large monolayer vesicles (LUVs), sterically stabilized liposomes (SSLs), multilocular vesicles (MVs), and large multilocular vesicles (LMVs), as well as other bilayer forms known in the art, may be used in the context of the present invention. The size and lamination of liposomes depend on the preparation method, and the selection of the type of vesicle to be used depends on the preferred mode of administration. Several other forms of supramolecular structures exist in which lipids can exist in an aqueous medium, including lamellar phases, hexagonal and inverse hexagonal phases, cubic phases, micelles, and inverse micelles composed of monolayers. These phases may also be obtained in combination with DNA or RNA, and interactions with RNA and DNA can substantially influence the phase state.

[0085] Any suitable method for forming liposomes can be used for liposome formation, provided that it provides liposomes suitable for producing the intended RNA lipoplex. Liposomes can be formed using standard methods such as reverse evaporation (REV), ethanol injection, dehydration-rehydration (DRV), sonication, or other suitable methods. After liposome formation, the liposomes can be separated by size to obtain a population of liposomes with a substantially uniform size range.

[0086] Bilayer-forming lipids typically have two hydrocarbon chains, particularly acyl chains, and either polar or nonpolar head groups. Bilayer-forming lipids consist of naturally occurring or synthetically derived lipids, including phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin, where the two hydrocarbon chains are typically about 14 to 22 carbon atoms long and have varying degrees of unsaturation. Other suitable lipids for use in the compositions of the present invention include glycolipids and sterols, such as cholesterol and its various analogues, which can also be used in liposomes.

[0087] Cationic lipids typically have a lipophilic moiety such as a sterol, acyl, or diacyl chain and have an overall net positive charge. The head group of the lipid typically has a positive charge. Cationic lipids preferably have a positive charge of 1 to 10, more preferably 1 to 3, and more preferably 1. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA); dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammoniumpropane; 1,2-dialkyloxy-3-dimethylammoniumpropane; dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium (propanamium) trifluoroacetate (DOSPA). Preferred are DOTMA, DOTAP, DODAC, and DOSPA. The most preferable option is DOTMA.

[0088] II. Overview Peristaltic pump systems including damping devices for reducing flow rate pulsation or oscillation from peristaltic pumps within the system, as well as methods of using these systems for producing, mixing, transferring, and / or manufacturing pharmaceutical compositions and formulations, for example, compositions and formulations comprising RNA and lipids including lipoplexes or liposomes. In some embodiments, the pharmaceutical compositions and formulations comprise RNA, RNA molecules, or RNA vaccines.

[0089] The disclosure also provides peristaltic pumps, damping devices, and tubing kit systems suitable for use with two fluid sources, comprising, for example, a first pharmaceutical composition comprising RNA, RNA molecules, or RNA vaccines, and a second pharmaceutical composition comprising one or more lipids described herein, which can be mixed or transported to produce or manufacture a final pharmaceutical composition comprising an RNA-lipoplex, RNA liposome, or RNA vaccine as described herein.

[0090] III. Peristaltic pumps, damping devices, and tubing kit systems A tubing kit for use with a peristaltic pump system is provided herein. In some embodiments, the peristaltic pump may be a Masterflex pump (HV-77921-75) having four rollers and which can be adapted to have multiple pump heads. In some embodiments, the peristaltic pump may be a Watson Marlow. In some embodiments, the tubing kit can be used to form pharmaceutical compositions, formulations and / or mixtures. In some embodiments, the tubing kit can be used to mix in series to form pharmaceutical compositions, formulations and / or mixtures. In some embodiments, the pharmaceutical compositions, formulations and / or mixtures can be formed by mixing a first pharmaceutical composition with a second pharmaceutical composition. Figure 15 shows an example of tubing kit 1. The tubing kit may include a first portion of tubing configured to be fluidly connected to a container. An example of the first portion of tubing is shown in Figure 15 as part of tubing 2.

[0091] Part of the tubing 2 may include an inlet tubing 2a, pump tubings 2b and 2c, pump outlet tubing 2d, and a rear tubing 2e for the damping device. The inlet tubing 2a may be fluidically connected to a fluid source or container. In some embodiments, a first portion of the tubing may include a connector 4 for fluidly connecting the first portion of the tubing to a fluid source or container. In some embodiments, the connector for fluidly connecting the first portion of the tubing to a fluid source or container may be a sterile connector. In some embodiments, any connector from the container to the tubing may function. In some embodiments, the container may contain a first fluid or composition. In some embodiments, the first composition may be a first pharmaceutical composition containing, for example, a composition for containing lipids or RNA. In some embodiments, the container may be a fluid container such as a bag, bottle, and / or vessel.

[0092] The first portion of the tubing may include a first segment and a second segment. In some embodiments, the first segment and the second segment are fluidly connected in parallel. For example, as shown in Figure 15, the first portion of the tubing includes pump tubing 2b and pump tubing 2c. In some embodiments, the first segment is configured to be connected to or mounted on a first head of a peristaltic pump, and the second segment is configured to be connected to or mounted on a second head of a peristaltic pump.

[0093] In some embodiments, the inlet tubing 2a can be fluidically connected to the pump tubings 2b and 2c via a connector 5. In some embodiments, the connector that fluidly connects the inlet tubing to the pump tubing is a Y-connector or a mixing device (e.g., static, helical, etc.). In some embodiments, the pump tubing does not need to be divided into parallel pump tubes as shown in Figure 15. Therefore, a connector may not be necessary, and the inlet tubing and pump tubing may be the same single unit. In some embodiments, the first portion of the tubing can be configured to connect to or mount to a peristaltic pump. In some embodiments, the peristaltic pump may be a multi-head peristaltic pump, such as a dual-head peristaltic pump. In some embodiments, the pump tubings 2b and 2c are connected to or mounted to the first peristaltic pump. For example, if the peristaltic pump is a dual-head peristaltic pump, the pump tubing 2b may be connected to or mounted to one pump head, and the pump tubing 2c may be connected to or mounted to another pump head. The same logic can be used for pumps with more than two heads. In several embodiments, the first peristaltic pump is a single-head peristaltic pump. Thus, only one of the pump tubings 2b or 2c may be connected to or fitted to the first peristaltic pump, or a single inlet tubing / pump tubing may be connected to or fitted to the first peristaltic pump.

[0094] In some embodiments, the pump tubing or inlet / pump tubing can be fluidly connected to the pump outlet tubing. In some embodiments, the pump tubings 2b and 2c are fluidly connected to the pump outlet tubing 2d via a connector 5 (e.g., a Y-connector). In some embodiments, the pump tubing does not need to be divided into parallel pump tubes. Therefore, a connector after the pump may not be necessary, and the pump tubing and pump outlet tubing may be the same single entity. For example, the tubing may consist of inlet, pump, and pump outlet tubing.

[0095] In some embodiments, a tubing kit may include a damping device fluidly connected to a first portion of the tubing. The first portion of the tubing may be fluidly connected to the damping device via a connector. In some embodiments, such a connector may be a T-connector, a four-way connector, or various other types of connectors. Figure 15 shows a first portion of tubing 2 fluidly connected to a damping device 7 via a connector 6. Specifically, the pump outlet tubing 2d is fluidly connected to the damping device 7 via the connector 6. In some embodiments, the first portion of tubing 2 may be fluidly connected to its own first damping device. In some embodiments, the damping device may also be fluidly connected to the tubing behind the damping device. In some embodiments, the pump outlet tubing is fluidly connected to the tubing behind the damping device via a connector (e.g., connector 5 as shown in Figure 15). In some embodiments, the damping device, pump outlet tubing, connector, and tubing behind the damping device are fluidly connected.

[0096] The tubing kit may also include a second portion of tubing configured to be fluidly connected to a container. An example of a second portion of tubing is shown in Figure 15 as part of tubing 3. In some embodiments, the first and second portions of tubing are fluidly connected in parallel.

[0097] Part of the tubing 3 may include an inlet tubing 3a, pump tubings 3b and 3c, pump outlet tubing 3d, and a tubing 3e behind the damping device. The inlet tubing 3a may be fluidically connected to a fluid source or container. In some embodiments, a second part of the tubing may include a connector 4 for fluidly connecting the second part of the tubing to a fluid source or container. In some embodiments, the connector for fluidly connecting the second part of the tubing to a fluid source or container may be a sterile connector. In some embodiments, any connector from the container to the tubing may function. In some embodiments, the container may contain a second fluid or composition different from the first fluid or composition. In some embodiments, the container may contain the same fluid or composition as the first fluid or composition. In some embodiments, the second composition may be a second pharmaceutical composition containing, for example, a composition containing lipids or RNA. In some embodiments, the container may be a fluid container such as a bag, bottle, and / or vessel.

[0098] The second portion of the tubing may include a third segment and a fourth segment. In some embodiments, the third segment and the fourth segment are fluidly connected in parallel. For example, as shown in Figure 15, the second portion of the tubing includes pump tubing 3b and pump tubing 3c. In some embodiments, the third segment of the tubing is configured to be connected to or mounted on a first head of a peristaltic pump, and the fourth segment of the tubing is configured to be connected to or mounted on a second head of a peristaltic pump. In some embodiments, a peristaltic pump connected to or mounted on the second portion of the tubing is different from a peristaltic pump connected to or mounted on the first portion of the tubing. In some embodiments, the same peristaltic pump is connected to or mounted on both the first and second portions of the tubing. Thus, the first portion of the tubing may be connected to or mounted on one or more first heads of a peristaltic pump, and the second portion of the tubing may be connected to or mounted on one or more second heads of a peristaltic pump.

[0099] In some embodiments, the inlet tubing 3a can be fluidically connected to the pump tubings 3b and 3c via a connector 5. In some embodiments, the connector that fluidly connects the inlet tubing to the pump tubing is a Y-connector or a mixing device (e.g., static, helical, etc.). In some embodiments, the pump tubing does not need to be divided into parallel pump tubes as shown in Figure 15. Therefore, a connector may not be necessary, and the inlet tubing and pump tubing may be the same single unit. In some embodiments, a second portion of the tubing can be configured to connect to or mount a second peristaltic pump. In some embodiments, the peristaltic pump may be a multi-head peristaltic pump, such as a dual-head peristaltic pump. In some embodiments, the pump tubings 3b and 3c are connected to or mounted to a second peristaltic pump. For example, if the peristaltic pump is a dual-head peristaltic pump, the pump tubing 3b may be connected to or mounted to one pump head, and the pump tubing 3c may be connected to or mounted to another pump head. The same logic can be used for pumps with more than two heads. In some embodiments, the second peristaltic pump is a single-head peristaltic pump. Thus, only one of the pump tubings 3b or 3c may be connected to or fitted to the second peristaltic pump, or a single inlet tubing / pump tubing may be connected to or fitted to the second peristaltic pump.

[0100] In some embodiments, the first peristaltic pump is the same pump as the second peristaltic pump, and the various parts of the tubing are configured to be connected to or mounted on separate heads of the pump. Thus, pump tubing 2b can be connected to or mounted on the first pump head, pump tubing 2c can be connected to or mounted on the second pump head, pump tubing 3c can be connected to or mounted on the third pump head, and pump tubing 3b can be connected to or mounted on the fourth pump head. Alternatively, only one of pump tubings 2b or 2c can be connected to or mounted on the first pump head, or the first single inlet tubing / pump tubing can be connected to or mounted on the first pump head, and only one of pump tubings 3b or 3c can be connected to or mounted on the second pump head, or the second single inlet tubing / pump tubing can be connected to or mounted on the second pump head.

[0101] In some embodiments, the pump tubing or inlet / pump tubing can be fluidly connected to the pump outlet tubing. In some embodiments, the pump tubings 3b and 3c are fluidly connected to the pump outlet tubing 3d via a connector 5 (e.g., a Y-connector). In some embodiments, the pump tubing does not need to be divided into parallel pump tubes. Therefore, a connector after the pump may not be necessary, and the pump tubing and pump outlet tubing may be the same single entity. For example, the tubing may consist of inlet, pump, and pump outlet tubing.

[0102] In some embodiments, the tubing kit may include a damping device fluidly connected to a second portion of the tubing. In some embodiments, the damping device is fluidly connected to a first portion of the tubing and also fluidly connected to a second portion of the tubing. In some embodiments, the damping device is fluidly connected to the first portion of the tubing and / or the second portion of the tubing via a connector such as a T-connector or a four-way connector.

[0103] A damping device can be any device in which damping is performed by a sealed volume of fluid. In some embodiments, the volume of fluid in the damping device may depend on the flow rate and / or the exposed surface area. In some embodiments, a damping device dampens pulsations by a sealed volume of air. In some embodiments, a damping device can be a syringe damping device, a membrane damping device (e.g., a flexible membrane damping device), or a tubing damping device. In some embodiments, a tubing damping device can be a dead-end tubing damping device in which one end of the damping device is fluidically connected to a first or second portion of the tubing and the other end of the damping device is closed. In some embodiments, the other end of the damping device can be closed by a clamp, an end cap, and connected to another damping line or another portion in which gas can be sealed from the environment. In some embodiments, a tubing damping device is fabricated from a silicone tube.

[0104] In some embodiments, one end of the tubing damper is fluidically connected to a first portion of the tubing, and the other end of the tubing damper is fluidically connected to a second portion of the tubing, thereby forming a loop tubing damper. In some embodiments, the loop tubing damper is positioned above the first and / or second portions of the tubing so that minimal fluid from the first and / or second portions of the tubing does not enter the damper and air remains within the damper. For example, if the solution is flowing horizontally on a surface, the damper can be vertically deposited at a height exceeding the intended fluid path so that an air pocket remains above the liquid surface during pumping. In some embodiments, the loop tubing damper can be positioned or mounted above the first and second portions of the tubing. In some embodiments, the loop tubing damper can be mounted on a horizontal bar or attached (i.e., taped) above the first and second portions of the tubing.

[0105] A second portion of the tubing can be fluidically connected to a damper via a connector. In some embodiments, such a connector may be a T-connector, a four-way connector, or various other types of connectors. Figure 15 shows a second portion of tubing 3 fluidly connected to a damper 7 via a connector 6. Specifically, the pump outlet tubing 3d is fluidly connected to the damper 7 via connector 6. In some embodiments, a second portion of tubing 3 can be fluidly connected to its own second damper. In some embodiments, the pump outlet tubing 2d is fluidly connected to its own damper, and the pump outlet tubing 3d is connected to its own different damper. In some embodiments, the damper can also be fluidly connected to the tubing behind the damper. In some embodiments, the pump outlet tubing is fluidly connected to the tubing behind the damper via a connector (e.g., connector 5 as shown in Figure 15). In some embodiments, the damper, pump outlet tubing, connector, and tubing behind the damper are fluidly connected.

[0106] In some embodiments, the tubing kit disclosed herein may include a mixing device for mixing a first fluid or composition from a first portion of the tubing with a second fluid or composition from a second portion of the tubing. Thus, the first portion of the tubing and the second portion of the tubing can be fluidically connected to the mixing device. In some embodiments, the first portion of the tubing and the second portion of the tubing are fluidically connected to the mixing device downstream of the damping device. In some embodiments, the tubing behind the damping device of the first portion of the tubing (i.e., the first mixing device input portion of the tubing) and the tubing behind the damping device of the second portion of the tubing (i.e., the second mixing device input portion of the tubing) are fluidically connected to the mixing device. In some embodiments, a connector (e.g., connector 6) fluidly connects the damping device, the first portion of the tubing, and the first mixing device input portion of the tubing. In some embodiments, a connector (e.g., connector 6) fluidly connects the damping device, the second portion of the tubing, and the second mixing device input portion of the tubing. In some embodiments, a first damping device connector fluidically connects a first portion of the tubing to the damping device and mixing device, and a second damping device connector fluidically connects a second portion of the tubing to the damping device and mixing device.

[0107] In some embodiments, the mixing device includes an input section fluidically connected to a first portion of tubing, an input section fluidically connected to a second portion of tubing, and an output section. In some embodiments, the mixing device may be a Y-connector, a helical mixing device, or a static mixing device. In some embodiments, the output section may be fluidically connected to tubing (e.g., output tubing 8). In some embodiments, the mixing device includes an output section fluidically connected to a mixture container (e.g., first and second source mixture containers 9). The mixture container can collect the mixed first fluid or composition and the second fluid or composition from the mixing device. In some embodiments, the mixture container may be a fluid container such as a bag, bottle, and / or container.

[0108] In some embodiments, a first portion of the tubing is configured to be connected to or fitted to a first peristaltic pump or pump head for pumping a first fluid or composition from a container to a mixture container. In some embodiments, a second portion of the tubing is configured to be connected to or fitted to a second peristaltic pump or pump head for pumping a second fluid or composition from a container to a mixture container. In some embodiments, the fluid or composition pumped through the tubing kit is a pharmaceutical composition comprising, for example, a composition containing lipids or RNA. Pharmaceutical compositions disclosed herein may include nucleic acids (e.g., including RNA or mRNA), one or more lipids, proteins, buffers, small molecules, amino acids and / or polypeptides. In some embodiments, the nucleic acids may be RNA (e.g., including mRNA) and / or DNA. In some embodiments, one or more lipids may be in the form of liposomes or lipoplexes. In some embodiments, the pharmaceutical composition may be a component of a personalized cancer vaccine or RNA vaccine comprising nucleic acids and lipids that together form a lipoplex.

[0109] The tubing kits, methods, and / or systems disclosed herein include damping devices for reducing flow rate pulsation or vibration from a peristaltic pump from which the fluid comes from one or two sources. In some embodiments, the level of pulsation ("LoP") of the tubing kits, methods, and / or systems disclosed herein, used with a peristaltic pump from which the fluid comes from one or two sources, is less than about 40, less than about 35, less than about 30, less than about 25, less than about 20, less than about 15, less than about 12, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, or less than about 1. In some embodiments, the level of pulsation ("LoP") of the tubing kits, methods, and / or systems disclosed herein, used with a peristaltic pump from which the fluid comes from one or two sources, is about 7 to about 40 or about 10 to about 20. In some embodiments, the level of pulsation ("LoP") of the tubing kit method and / or system disclosed herein, used with a peristaltic pump from which the fluid comes from one or two sources, may be about 7, about 8, about 10, about 15, about 20, or about 25.

[0110] In some embodiments, the level of pulsation reduction ("LoP") of a peristaltic pump from which the fluid comes from one or two sources, using the tubing kits, methods and / or systems disclosed herein, is about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, or about 20% compared to the LoP of a peristaltic pump from which the fluid comes from one or two sources, lacking the damping devices described herein.

[0111] While the majority of the examples and descriptions discuss the use of damping devices for pharmaceutical compositions and formulations, the damping devices and pump systems disclosed herein are not limited to use in pharmaceutical compositions and formulations. For example, the pump systems disclosed herein can be used to fill a work with a peristaltic pump or simply to move a solution between containers, where having a consistent flow rate may be advantageous. The systems disclosed herein can be used with any pump other than peristaltic pumps, including but not limited to pistons, diaphragms, and screws, with any type of damping device and any tubing size and tubing material (e.g., pipe, plastic, stainless steel), at any flow rate. In some embodiments, the tubing system comes into contact with the product, so the tubing system can be sterilized before use. Such sterilization can be performed using an autoclave, gamma ray, etc.

[0112] IV. Fluid pressure type pulsation damping device As explained above, when using circulating pump systems such as peristaltic pumps, pressure and flow rate fluctuations can occur in the fluid path downstream of the pump. This so-called pulsation can be reduced by using various technical solutions, including hydrostatic pressure pulsation dampers (HPPDs), which absorb the pulsation by compressing a confined fluid (e.g., gas) cushion, resulting in reduced pulsation and a stable, smooth fluid flow.

[0113] Early experiments demonstrated that this concept is valid using a glass bottle with an inlet and outlet immersed in a pressurized fluid (e.g., water) to capture a compressible gas (e.g., air) to absorb pulsations. This concept is shown in Figure 19. To simulate a sterile manufacturing scenario, HPDPs prepared from 500 mL laboratory bottles were tested at various flow rates, evaluated over the time required to rise to a certain pressure and fall from that pressure after the pump was stopped (i.e., the time required to normalize the fluid path pressure). Figure 20 shows an early HDDP apparatus consisting of a fluid (water) reservoir, a peristaltic pump, a 500 mL laboratory flask closed with a cap consisting of two ports (one of which is immersed in the pressurized fluid), and a collection container located at the outlet of the fluid path. As shown in Figure 21, at lower flow rates, the time required to increase the pressure increased rapidly up to a flow rate of approximately 150 mL / min, where the rise and fall times remained equivalent at higher flow rates. These measurements took approximately 75 seconds to increase the pressure to a certain level and approximately 40 seconds to release the pressure and return it to zero.

[0114] For this glass bottle damping device, the effect of residual back pressure on the displacement velocity was also measured. Figure 22 confirms that although a considerable amount of time is required to pressurize and depressurize the HPPD system, the actual displacement of the integrated HPPD system is constant and equivalent to a simple setup from pump to outlet.

[0115] The effect of bottle size on the pressure difference was also investigated. Specifically, the effect of air pocket size was investigated by pumping at a constant rate through HPPDs fabricated using experimental bottles of sizes 250 to 2000 mL. Figure 23 demonstrates that the smaller the air volume, the higher the back pressure at the inlet, but the lower the overall pressure loss in the HPPD device compared to directly connecting the pump to the outlet. It was observed that this pressure loss did not affect the smoothness of the flow at the outlet.

[0116] To test the concept of a single-use design for the HPPD, a liner was inserted into the experimental flask, thereby allowing the contact surface and fluid path to be replaced after each use. This is shown in Figure 24. As shown in Figure 25, the addition of this liner was found to significantly reduce the throughput of the HPPD due to the increased flexibility of the flexible liner. The liner damper (i.e., bladder damper) also worked well in terms of normalizing the fluid flow, and the liner damper was advantageous because it prevented the fluid flow from coming into contact with air during the pumping process. However, the significant decrease in pumping efficiency in the form of residual pressure and fluid outflow after the pump was stopped resulted in a significant decrease in the efficiency of the system.

[0117] Therefore, the glass bottle damper was demonstrated to successfully reduce pulsation. Furthermore, the glass bottle damper had the advantage that relatively large, commonly available laboratory bottles could increase the compressibility of the system, resulting in a large dead volume and large air pocket that could reduce the outlet pressure through energy loss. The disadvantage was that, without a liner, the glass bottle was not suitable for single use and required prior assembly. In addition, long priming and outflow times were required to equilibrate and stop the system, resulting in a large dead volume.

[0118] To further reduce dead volume and achieve a robust, single-use Good Manufacturing Practice (GMP) design, the glass vials were replaced with bioprocessing bags, such as the 50 mL FLEXBOY® bioprocessing bag shown in Figure 26. The FLEXBOY® bag can be modified to include a raised inlet tube. The inlet of this inlet tube can be deposited so that it is positioned away from the periphery of the bag and towards the center of the bag, as shown in Figure 26. In contrast, the gas inlet (e.g., sterile air, nitrogen gas) and outlet can be deposited around the periphery of the bag. Depositing the inlet tube to the bioprocessing bag towards the center of the bag may help prevent backflow washing of the pumped fluid at high back pressures. Furthermore, the gas inlet can be modified to allow gas injection to pre-fill the bag with a gas cushion to improve the priming efficiency of the HPPD.

[0119] To add rigidity to the bag, minimize its expansion with increasing pump flow rate and pressure, and thereby improve the flow of the pumped fluid, a prototype casing was also fabricated from a cardboard box, as shown in Figure 27. Specifically, the FLEXBOY® bag was fixed inside the cardboard box casing, as shown in Figure 27. This helped to prevent the bag from expanding further and to create a gas cushion. This was found to help reduce dead volume and result in shorter primer times. However, due to the weakness of the cardboard box material, the HPPD was only stable up to a certain pressure. Therefore, the applicant found that replacing the cardboard box with a more rigid housing was beneficial.

[0120] Specifically, Figure 28A shows an exploded view of the pulsation damper 100 for a fluid pump. In some embodiments, the pulsation damper may include a bioprocessing bag 102. The bioprocessing bag may be a FLEXBOY® bag. For example, the bioprocessing bag may be a 50 mL FLEXBOY® bag, but other sizes of bioprocessing bags (e.g., any of 5 mL to 50 L) may also be used. The bioprocessing bag may include a fluid inlet 105 and a fluid outlet 106. In some embodiments, the fluid inlet and / or fluid outlet may be fluidically connected to tubing. In some embodiments, the fluid inlet and / or fluid outlet may include the tubing itself. In some embodiments, the fluid tubing connected to the fluid inlet and / or fluid outlet or the fluid inlet / outlet may be deposited so as to be away from the periphery of the bag and toward the center of the bag, as shown in Figure 26. In some embodiments, the fluid inlet may be fluidically connected downstream of a fluid pump, such as a circulation pump (e.g., a peristaltic pump). In some embodiments, the fluid outlet can be fluidly connected to a fluid storage container. In some embodiments, the fluid outlet can include a check valve. In some embodiments, the check valve can have a breakthrough resistance of about 0.05–0.5 bar, about 0.05–0.4 bar, about 0.05–0.3 bar, about 0.1–0.2 bar, or about 0.14 bar.

[0121] In some embodiments, the bioprocessing bag may include a gas inlet 107. The gas inlet may be configured to be fluidly connected to a gas source. In some embodiments, the gas source may be air (e.g., sterile air) and / or nitrogen. The gas inlet may supply gas to the bioprocessing bag so that the bag contains a gas cushion for pulsation damping.

[0122] In some embodiments, the pulsation damper 100 may include a housing 101. Figure 28B shows the housing 101 without other components of the pulsation damper. The housing may be configured to receive a bioprocessing bag. In some embodiments, the housing may include a base 111. The base may be a substrate. In some embodiments, the housing may include a plurality of side walls 104. In some embodiments, the plurality of side walls may extend along the periphery of the base and away from the base. In some embodiments, the plurality of side walls may be connected around the base. In some embodiments, the plurality of side walls and the base may form a single integrated component. In some embodiments, the base and the plurality of side walls may form a cavity configured to receive / hold a bioprocessing bag.

[0123] In some embodiments, at least one of the side walls of the housing may have one or more notches 108. As shown in Figure 28B, the housing 101 includes three notches 108 in the side wall 104. One or more notches may be recesses or depressions into at least one side wall of the housing. In some embodiments, one or more notches may be configured to provide access to a fluid inlet, fluid outlet and / or gas inlet of the bioprocessing bag. In some embodiments, one or more notches may be configured to receive a fluid inlet, fluid outlet and / or gas inlet of the bioprocessing bag. As described in more detail below, the housing may be closed via a housing lid. Thus, any fluid or fluid tubing accessing the bioprocessing bag may enter / exit through one or more notches in at least one of the side walls of the housing.

[0124] In some embodiments, the housing may include a window 110. In some embodiments, the base of the housing may include a window. In some embodiments, the window may be an opening in the base of the housing. In some embodiments, the window may be a transparent material (e.g., glass or transparent plastic) in the base of the housing. The window may allow visual inspection of the bioprocessing bag during use.

[0125] In some embodiments, the damping device 100 may include a housing lid 103. The housing may be configured to close so that a bioprocessing bag is enclosed by the housing and the housing lid. In some embodiments, the housing lid may be configured to be attached to multiple side walls of the housing. In some embodiments, the housing lid may be attached to the housing via any mounting mechanism (e.g., adhesive, screws, nails, bolts, Velcro, clips (as shown in Figure 28C), locking mechanisms). In some embodiments, the housing lid may be attached to at least one side wall so that the housing lid acts as a door (i.e., a hinge mechanism) for the housing. In some embodiments, the housing lid may include a window. In some embodiments, the window may be an opening in the housing lid. The window may be a transparent material (e.g., glass or transparent plastic) in the base of the housing, as well as any window in the base of the housing.

[0126] In some embodiments, the damping device 100 may include a front plate 109. In some embodiments, the front plate may be connected to at least one side wall 104 of the housing 101 and / or housing lid 103. The front plate may include at least one opening configured to receive a fluid inlet, fluid outlet and / or gas inlet of a bioprocessing bag. The front plate may be included to ensure that the inlet / outlet of the bioprocessing bag or any of the corresponding tubing is reliably held in place within the housing of the damping device 100. In some embodiments, the front plate may be included to ensure that the inlet / outlet of the bioprocessing bag or the corresponding tubing is reliably under constant and uniform pressure throughout its use.

[0127] In some embodiments, the housing and / or housing lid can be made of a rigid material (e.g., plastic / polymer, metal, ceramic). In some embodiments, the housing and / or housing lid can be 3D printed. In some embodiments, the internal dimensions of the damping device 100 can be 90 × 80 × 10 mm and can be designed to fit a 50 mL FLEXBOY® bioprocessing bag. This damping device can be a single-use assembly for a fluid flow system that can absorb the resulting pulsations using a gaseous cushion to minimize flow rate fluctuations. In some embodiments, the dead volume may depend on the relative position of the bioprocessing bag in the housing. Thus, the height of the damping device can be determined to ensure a constant dead volume and / or optimal damping effect for the requirements of the fluid path (e.g., flow rate and velocity, magnitude of pulsations generated by the pump head, back pressure of the system, etc.).

[0128] The pulsation damping device can contain two fluids: (1) a pulsating fluid to be displaced (e.g., water); and (2) a compressible fluid (e.g., air). When the displaced fluid is pumped through a bioprocessing bag, the captured compressible fluid can absorb the pulsations in the displaced fluid, allowing it to be pumped only in a stable, pulsation-free flow.

[0129] The pulsation damping capabilities of the damping devices disclosed in this section were systematically tested at different flow rates using peristaltic pumps with and without damping devices, as well as a baseline using syringe pumps. As will be described in more detail below, the damping devices were shown to significantly reduce pulsation to levels even lower than those achieved in syringe pump systems assuming no pulsation.

[0130] Figure 29A shows the apparatus of a peristaltic pump using tubing with an inner diameter of 3.6 mm. Pulsation was measured using an ultrasonic flow sensor (Levitronix) with a temporal resolution of 0.1 seconds and a numerical resolution of 0.8 mL / min. Water was pumped into the system. Figure 29A also shows the flow profile of the tested system over a total period of 100 seconds. The system consisted of a peristaltic pump and a flow sensor. The tested flow rates were 50, 60, 70, and 100 mL / min. High pulsation was observed due to the rotation of the peristaltic pump.

[0131] Figure 29B shows a device consisting of a syringe pump using tubing with an inner diameter of 3.6 mm. Pulsation was measured using an ultrasonic flow sensor with a temporal resolution of 0.1 seconds and a numerical resolution of 0.8 mL / min. Water was pumped into the system. Figure 29B also shows the flow profile of the tested system over a total period of 100 seconds. The tested flow rates were 50, 60, 70, and 100 mL / min, and low pulsation was measured.

[0132] Figure 29C shows a device consisting of a peristaltic pump using tubing with an inner diameter of 3.6 mm and an HPPD attenuator disclosed herein connected thereto. The outlet of the HPPD attenuator was connected to a check valve with a breakthrough resistance of 0.14 bar. Pulsation was measured using an ultrasonic flow sensor with a temporal resolution of 0.1 seconds and a numerical resolution of 0.8 mL / min. Water was pumped into the system. Figure 29C also shows the flow profile using the HPPD attenuator. As shown, a significant reduction in pulsation (lower amplitude) was observed. The HPPD effect was successfully observed (numerically). Furthermore, the HPPD device resulted in even lower flow rate fluctuations than the syringe pump.

[0133] To test this robust damping device, a comparability study was conducted between the HPPD damping device disclosed in this section and the Cole-Parmer HPPD, as shown in Figure 30. The damping principle of the Cole-Parmer HPPD is based on the compression of a confined air pocket. Specifically, the internal volume of the Cole-Parmer HPPD is approximately 190 mL. The dead volume during pumping is approximately 40-50 mL. This is a higher dead volume compared to the HPPD damping device disclosed in this section, while using a 50 mL bioprocessing bag. Furthermore, the Cole-Parmer HPPD takes longer to prime and must be positioned in a fixed horizontal position to produce a pulsating damping effect. In contrast, the HPPD device disclosed in this section does not need to be horizontal and can be oriented in any direction during use. As described in more detail below, the HPPD damping device described in this section has a much smaller, more predictable dead volume and reduced priming time compared to the Cole-Parmer HPPD, while exhibiting even higher damping efficiency at a variety of flow rates.

[0134] Figure 31A shows the flow profile using a Cole-Parmer HPPD over a total period of 100 seconds. The system consists of a peristaltic pump, a Cole-Parmer HPPD, and a flow sensor. Water was pumped at test flow rates of 50, 60, 70, and 100 mL / min. The water was pumped using a peristaltic pump through tubing with an inner diameter of 3.6 mm. Pulsation was measured using an ultrasonic flow sensor with a temporal resolution of 0.1 seconds and a numerical resolution of 0.8 mL / min.

[0135] Figure 31B shows the flow profile of the tested system (Figure 29C) over a total period of 100 seconds. Water was tested at flow rates of 50, 60, 70, and 100 mL / min. Water was pumped through tubing with an inner diameter of 3.6 mm using a peristaltic pump. A check valve with a breakthrough resistance of 0.14 bar was connected to the outlet of the HPPD. Again, pulsation was measured using an ultrasonic flow sensor with a resolution of 0.1 seconds and a value resolution of 0.8 mL / min. No significant difference in pulsation attenuation was observed between the Cole-Parmer and the HPPD disclosed in this section.

[0136] Next, when the system reached its operational equilibrium, the packing volume and pressure were recorded as a function of the pumped flow rate after priming. It was shown that the dead volume of the HPPD attenuator disclosed herein can remain constant even after reaching flow rates of approximately 70 mL / min or more. For testing, water was pumped using a peristaltic pump connected to the HPPD attenuator disclosed in this section. Pressure sensors were placed above the inlet of the HPPD attenuator and above the outlet, which has a check valve with a resistance of 0.14 bar. The flow rate was measured using an ultrasonic flow sensor with a time resolution of 0.1 seconds and a value resolution of 0.8 mL / min.

[0137] Figure 32 shows the dead volume and pressure at the damper inlet of the HPPD damper disclosed in this section as the flow rate increases. Specifically, the pressure at the damper inlet increases proportionally to the flow rate. The dead volume stabilizes at a flow rate of 70 mL / min. The dead volume can be reduced by decreasing the total volume of the bioprocessing bag. However, the robust housing played a significant role in achieving a stable damping effect. Additional parameters that may affect the dead volume and pressure may be changes in dynamic pressure, which can be adjusted by the relative positions of the pump, damper, and fluid container. Changes in dynamic pressure can affect the pressure equilibrium within the damper and therefore the dead volume within the damper. This effect may be negligible at higher flow rates (e.g., 200 mL / min). Another parameter may be the volume of empty tubing between the starting fluid container, pump, and damper. The amount of air inside the damper can be determined by the volume of air in the empty tubing that is pressurized into the damper during the initial filling process of the system. In Figure 32, the diamond symbols represent the dead volume values, and the squares represent the measured pressures.

[0138] Next, the effect of tubing diameter and length on pulsation was investigated. The damping effect is obtained by the absorption of pulsation due to the elasticity of the silicone tubing. Three different tubing diameters were investigated at a constant flow rate using a peristaltic pump. It was shown that the measured pulsation decreased significantly as the tubing diameter decreased. To measure, water was pumped using a peristaltic pump (model Watson Marlow 323) connected to tubing of different inner diameters with a constant length of 1 meter. As the inner diameter of the tubing increased, the pumping RPM decreased to achieve a constant flow flux. Pulsation was measured at 20 ms measurement intervals using a MASTERFLEX® ultrasonic flow sensor. For analysis, 300 measurement points were analyzed for each setting, and the arithmetic mean and standard deviation were calculated. A decrease in the flow rate standard deviation indicates improved pulsation damping.

[0139] Figure 33A shows the pulsation study at a pump speed of 285 rpm for tubing with an inner diameter of 1.6 mm and a fixed length of 1 meter. The arithmetic mean was 75.6 mL / min and the standard deviation was 3.4 mL / min. Figure 33B shows the pulsation study at a pump speed of 70 rpm for tubing with an inner diameter of 3.2 mm and a fixed length of 1 meter. The arithmetic mean was 77.3 mL / min and the standard deviation was 6.7 mL / min. Figure 33C shows the pulsation study at a pump speed of 20 rpm for tubing with an inner diameter of 6 mm and a fixed length of 1 meter. The arithmetic mean was 77.3 mL / min and the standard deviation was 6.7 mL / min. Using smaller tubing inner diameters and higher pumping speeds significantly reduced the standard deviation of the flow rate and simultaneously decreased pulsation.

[0140] Next, the pulsation damping effect of increasing tubing length was investigated. This demonstrates that, in addition to decreasing tubing diameter, increasing tubing length can further reduce pulsation. However, this results in increased pressure loss. For the test, water was pumped using a peristaltic pump connected to tubing of different lengths and a constant inner diameter of 1.6 mm. Due to high back pressure, the pumping RPM was increased for a tubing length of 20 m to reach a flow rate equivalent to other settings. Pulsation was measured at 20 ms measurement intervals using an ultrasonic flow sensor. For analysis, 300 measurement points were analyzed for each setting, and the arithmetic mean and standard deviation were calculated. A decrease in the flow rate standard deviation indicates improved pulsation damping.

[0141] Figure 34A shows the pulsation study for tubing with a fixed length of 1 meter, an inner diameter of 1.6 mm, and a pump speed of 285 rpm. The arithmetic mean is 75.6 mL / min, and the standard deviation is 3.4 mL / min. Figure 34B shows the pulsation study for tubing with a fixed length of 2 meters, an inner diameter of 1.6 mm, and a pump speed of 285 rpm. The arithmetic mean is 77.7 mL / min, and the standard deviation is 2.0 mL / min. Figure 34C shows the pulsation study for tubing with a fixed length of 20 meters, an inner diameter of 1.6 mm, and a pump speed of 400 rpm. The arithmetic mean is 85.8 mL / min, and the standard deviation is 1.6 mL / min. Using longer tubing and higher pumping speeds can significantly reduce the standard deviation of flow rates and decrease pulsation. Compared to using the HPPD damping device disclosed herein, the dead volume can be further reduced, and if only tubing for pulsation damping is used, a separate priming process can be skipped. However, the pressure loss over the extended length of the tubing was much greater than when using the HPPD damping device.

[0142] The equipment used in the above experiment is shown in the table below: JPEG0007872261000003.jpg74170

[0143] V.RNA vaccine Certain aspects of this disclosure relate to the production, mixing, or manufacture of pharmaceutical compositions including personalized cancer vaccines (PCVs). In some embodiments, the PCV is an RNA vaccine, including, for example, an mRNA vaccine. Exemplary RNA vaccine features are described below. In some embodiments, this disclosure provides an RNA polynucleotide or RNA molecule comprising one or more of the RNA vaccine features / sequences described below. In some embodiments, the RNA polynucleotide or RNA molecule is a single-stranded mRNA polynucleotide. In other embodiments, this disclosure provides a DNA polynucleotide encoding an RNA molecule comprising one or more of the RNA vaccine features / sequences described below.

[0144] Personalized cancer vaccines contain personalized neoantigens (i.e., tumor-associated antigens (TAAs) specifically expressed in the patient's cancer) that have been identified as having potential immunostimulatory activity. In the embodiments described herein, the PCV is a nucleic acid, e.g., messenger RNA. Therefore, although we do not wish to be bound by theory, it is thought that upon administration, the personalized cancer vaccine is taken up, translated, and expressed by antigen-presenting cells (APCs), and the resulting proteins are presented via major histocompatibility complex (MHC) molecules on the surface of the APCs. This results in the induction of both cytotoxic T lymphocyte (CTL) and memory T cell-dependent immune responses against cancer cells expressing the TAA.

[0145] PCV typically consists of multiple neoantigen epitopes ("neoepitopes"), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, or 30 neoepitopes. , or comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, or 30 neoepitopes, optionally having linker sequences between individual neoepitopes. In some embodiments, the neoepitopes used herein refer to novel epitopes that are specific to the patient's cancer but not found in the patient's normal cells. In some embodiments, the neoepitopes are presented to T cells when bound to MHC. In some embodiments, the PCV also comprises a 5' mRNA cap analogue, a 5' UTR, a signal sequence, a domain that promotes antigen expression, a 3' UTR, and / or a poly-A tail. In some embodiments, an RNA vaccine or RNA molecule that can be used with the methods and systems of this disclosure comprises one or more polynucleotides encoding 10 to 20 neoepitopes arising from cancer-specific somatic mutations present in the tumor specimen. In some embodiments, the RNA vaccine or RNA molecule comprises one or more polynucleotides encoding at least 5 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In some embodiments, the RNA vaccine or RNA molecule comprises one or more polynucleotides encoding 5 to 20 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In some embodiments, the RNA vaccine or RNA molecule comprises one or more polynucleotides encoding 5 to 10 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen.

[0146] In some embodiments, the RNA vaccine or RNA molecule that can be used with the methods and systems of this disclosure comprises one or more polynucleotide sequences encoding an amino acid linker. For example, the amino acid linker can be used between two patient-specific neoepitope sequences, between a patient-specific neoepitope sequence and a fusion protein tag (e.g., including a sequence derived from an MHC complex polypeptide), or between a secretory signal peptide and a patient-specific neoepitope sequence. In some embodiments, the RNA vaccine or RNA molecule encodes multiple linkers. In some embodiments, the RNA vaccine or RNA molecule comprises one or more polynucleotides encoding 5 to 20 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen, with each epitope-encoding polynucleotide separated by a polynucleotide encoding a linker sequence. In some embodiments, the RNA vaccine or RNA molecule comprises one or more polynucleotides encoding 5 to 10 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen, with each epitope-encoding polynucleotide separated by a polynucleotide encoding a linker sequence. In some embodiments, the polynucleotide encoding the linker sequence also exists between a polynucleotide encoding an N-terminal fusion tag (e.g., a secretory signal peptide) and a polynucleotide encoding one of the neoepitopes, and / or between a polynucleotide encoding one of the neoepitopes and a polynucleotide encoding a C-terminal fusion tag (e.g., a portion of an MHC polypeptide). In some embodiments, two or more linkers encoded by an RNA vaccine or RNA molecule contain different sequences. In some embodiments, an RNA vaccine or RNA molecule encodes multiple linkers, all of which share the same amino acid sequence.

[0147] Various linker sequences are known in the art. In some embodiments, the linker is a flexible linker. In some embodiments, the linker comprises G, S, A, and / or T residues. In some embodiments, the linker consists of glycine and serine residues. In some embodiments, the linker is about 5 to about 20 amino acid lengths or about 5 to about 12 amino acid lengths, for example, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid lengths. In some embodiments, the linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 21). In some embodiments, the linker of an RNA vaccine or RNA molecule comprises the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 19). In some embodiments, the linker of an RNA vaccine or RNA molecule is encoded by DNA comprising the sequence GGCGGCTCTGGAGGAGGCGGCTCCGGAGGC (SEQ ID NO: 20).

[0148] In some embodiments, RNA vaccines or RNA molecules that can be used with the methods and systems of this disclosure include a 5' cap. The basic mRNA cap structure consists of two nucleosides (e.g., two guanines) and a 7-methyl group on the distal guanine, i.e., m 7 It is known to contain a 5'-5' triphosphate bond between GpppG. Exemplary cap structures can be seen, for example, in U.S. Patent Nos. 8,153,773 and 9,295,717, and in Kuhn, AN et al. (2010) Gene Ther. 17:961-971. In some embodiments, the 5' cap is structure m2 7,2’-O Gpp s It has pG. In some embodiments, the 5' cap is a β-S-ARCA cap. The S-ARCA cap structure is (for example, m 7 The G group includes a 2'-O methyl substitution at the C2' position and an S substitution on one or more phosphate groups. In some embodiments, the 5' cap includes the following structure: JPEG0007872261000004.jpg54170

[0149] In some embodiments, the 5' cap is the D1 diastereoisomer of β-S-ARCA (see, for example, U.S. Patent No. 9,295,717). * in the above structure indicates a stereogenic P center that may be present in the two diastereoisomers (named D1 and D2). β-S-ARCA or the D1 diastereomer of β-S-ARCA (D1) is a diastereomer of β-S-ARCA that elutes first on the HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA(D2)). HPLC is preferably analytical HPLC. In one embodiment, preferably the following format: a 5 μm, 4.6 × 250 mm Supelcosil LC-18-T RP column is used for separation, and a flow rate of 1.3 ml / min can be applied. In one embodiment, a methanol gradient in ammonium acetate within 15 minutes is used, for example, a linear gradient of 0-25% in methanol in 0.05 M ammonium acetate at pH=5.9. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0150] In some embodiments, the RNA vaccine or RNA molecule that can be used with the methods and systems of this disclosure comprises a 5'UTR. Certain untranslated sequences found at 5' relative to the protein-coding sequence in mRNA have been shown to enhance translation efficiency. See, for example, Kozak, M. (1987) J. Mol. Biol. 196:947-950. In some embodiments, the 5'UTR comprises a sequence from human alphaglobin mRNA. In some embodiments, the RNA vaccine or RNA molecule comprises a 5'UTR sequence of UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 5). In some embodiments, the 5'UTR sequence of the RNA vaccine or RNA molecule is encoded by DNA comprising the sequence TTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 6). In some embodiments, the 5'UTR sequence of the RNA vaccine or RNA molecule comprises the sequence GGCGACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 3). In some embodiments, the 5'UTR sequence of an RNA vaccine or RNA molecule is encoded by DNA containing the sequence GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (Sequence ID 4).

[0151] In some embodiments of the methods provided herein, the constant region of the exemplary RNA vaccine comprises the ribonucleotide sequence (5'→3') of SEQ ID NO: 24. The binding between the first two G residues is an abnormal binding (5'→5')-pp, as shown in Table 6 and Figure 18 for the 5' capping structure. s It is p-. "N" refers to the position of a polynucleotide sequence encoding one or more (e.g., 1-20) neoepitopes (separated by any linker). Tumor-specific insertion sites (C131-A132; marked in bold) are shown in bold. See Table 6 for modified bases and uncommon ligations in exemplary RNA sequences. JPEG0007872261000005.jpg38170

[0152] In some embodiments, the RNA vaccine or RNA molecule that can be used with the methods and systems of this disclosure comprises a polynucleotide sequence encoding a secretory signal peptide. As is known in the art, a secretory signal peptide is an amino acid sequence that, at translation, instructs a polypeptide to be transported from the endoplasmic reticulum to the secretory pathway. In some embodiments, the signal peptide is derived from a human polypeptide, such as an MHC polypeptide. See, for example, Kreiter, S. et al. (2008) J. Immunol. 180:309-318, which describes exemplary secretory signal peptides that improve the processing and presentation of MHC class I and II epitopes in human dendritic cells. In some embodiments, at translation, the signal peptide is N-terminal to one or more neoepitope sequences encoded by the RNA vaccine. In some embodiments, the secretory signal peptide comprises the sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 9). In some embodiments, the secretion signal peptide of an RNA vaccine or RNA molecule comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 7). In some embodiments, the secretion signal peptide of an RNA vaccine or RNA molecule is encoded by DNA comprising the sequence ATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC (SEQ ID NO: 8).

[0153] In some embodiments, RNA vaccines or RNA molecules that can be used with the methods and systems of this disclosure include a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains. In some embodiments, the transmembrane and / or cytoplasmic domains are derived from the transmembrane / cytoplasmic domains of the MHC molecule. The term and abbreviation “Major Histocompatibility Complex” “MHC” refers to a complex of genes that occurs in all vertebrates. The function of MHC proteins or molecules in signaling between lymphocytes and antigen-presenting cells in a normal immune response includes their binding to peptides and their presentation for possible recognition by T cell receptors (TCRs). MHC molecules bind to peptides within the intracellular processing compartment and present these peptides on the surface of antigen-presenting cells to T cells. The human MHC region, also called HLA, is located on chromosome 6 and includes a class I region and a class II region. The class I alpha chain is a glycoprotein with a molecular weight of about 44 kDa. This polypeptide chain has a length of somewhat more than 350 amino acid residues. It can be divided into three functional regions: the extracellular, transmembrane, and cytoplasmic regions. The outer region has a length of 283 amino acid residues and is divided into three domains: alpha-1, alpha-2, and alpha-3. These domains and regions are usually encoded by distinct exons of the class I gene. The transmembrane region extends to the lipid bilayer of the plasma membrane. This typically consists of 23 hydrophobic amino acid residues arranged in an alpha helix. The cytoplasmic region, i.e., the portion facing the cytoplasm and connected to the transmembrane region, is typically 32 amino acid residues long and can interact with elements of the cytoskeleton. The alpha chain interacts with beta-2 microglobulin and thus forms an alpha-beta-2 dimer on the cell surface. The terms "MHC class II" or "class II" refer to major histocompatibility complex class II proteins or genes. Within the human MHC class II region are the DP, DQ, and DR subregions of class II α-chain and β-chain genes (i.e., DPα, DPβ, DQα, DQβ, DRα, and DRβ). Class II molecules are heterodimers consisting of alpha and beta chains, respectively.Both chains are glycoproteins with molecular weights of 31–34 kDa(a) or 26–29 kDa(beta). The total length of the alpha chain varies from 229–233 amino acid residues, and the total length of the beta chain varies from 225–238 residues. Both the alpha and beta chains consist of an outer region, a connecting peptide, a transmembrane region, and a cytoplasmic tail. The outer region consists of two domains, alpha-1 and alpha-2, or beta-1 and beta-2. The connecting peptides are beta and 9 residues long, respectively, in the alpha and beta chains. The connecting peptides annex two domains to a 23-amino acid transmembrane region in both the alpha and beta chains. The length of the cytoplasmic region, i.e., the portion facing the cytoplasm and connected to the transmembrane region, varies from 3–16 residues in the alpha chain and from 8–20 residues in the beta chain. Exemplary transmembrane / cytoplasmic domain sequences are described in U.S. Patents 8,178,653 and 8,637,006. In some embodiments, at translation, the transmembrane and / or cytoplasmic domains are located at the C-terminus of one or more neoepitope sequences encoded by the RNA vaccine. In some embodiments, the transmembrane and cytoplasmic domains of the MHC molecule contain the sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 12). In some embodiments, the transmembrane and / or cytoplasmic domains of the MHC molecule contain the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC (SEQ ID NO: 10).In some embodiments, the transmembrane and / or cytoplasmic domains of an MHC molecule are encoded by DNA containing the sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACAGCC (Sequence ID 11).

[0154] In some embodiments, RNA vaccines or RNA molecules that can be used with the methods and systems of this disclosure include both a polynucleotide sequence encoding a secretory signal peptide that is N-terminus to one or more neoepitope sequences and a polynucleotide sequence encoding a transmembrane and / or cytoplasmic domain that is C-terminus to one or more neoepitope sequences. Combining such sequences has been shown to improve the processing and presentation of MHC class I and II epitopes in human dendritic cells. See, for example, Kreiter, S. et al. (2008) J. Immunol. 180:309-318.

[0155] In bone marrow dendritic cells (DCs), RNA is released into the cytosol and translated into poly-neoepitope peptides. These polypeptides contain additional sequences to enhance antigen presentation. In some embodiments, the signal sequence (sec) from the N-terminal MHCI heavy chain of the polypeptide has been used to target the nascent molecule to the endoplasmic reticulum, which has been shown to enhance MHCI presentation efficiency. While we do not wish to be constrained by theory, it is thought that the transmembrane and cytoplasmic domains of the MHCI heavy chain induce polypeptides into the endosomal / lysosomal compartment, which has been shown to improve MHCII presentation.

[0156] In some embodiments, RNA vaccines or RNA molecules that can be used with the methods and systems of this disclosure include a 3'UTR. Certain untranslated sequences found at 3' relative to the protein-coding sequence in mRNA have been shown to improve RNA stability, translation, and protein expression. Polynucleotide sequences suitable for use as 3'UTRs are described, for example, in PG Publication No. 20190071682, U.S. Patent Application Publication No. 20190071682. In some embodiments, the 3'UTR includes the 3' untranslated region of an AES or a fragment thereof and / or the non-coding RNA of 12S RNA encoded in mitochondria. "AES" refers to the amino-terminal enhancer of a split and includes the AES gene (see, for example, NCBI gene ID: 166). The protein encoded by this gene belongs to the protein groucho / TLE family and can function as a homooligomer or as a heterooligomer with other family members to predominantly repress the expression of other family member genes. An exemplary AES mRNA sequence is provided under NCBI reference sequence accession number NM_198969. The term "MT_RNR1" refers to the MT_RNR1 gene (see, for example, NCBI gene ID: 4549) with respect to mitochondrial 12S RNA. This RNA gene belongs to the Mt_rRNA class. Diseases associated with MT-RNR1 include restrictive cardiomyopathy and auditory neuropathy. Among its associated pathways are ribosome biosynthesis and CFTR translational fidelity (class I mutations) in eukaryotes. An exemplary MT_RNR1 RNA sequence is located within the sequence under NCBI reference sequence accession number NC_012920. In some embodiments, the 3'UTR of an RNA vaccine or RNA molecule contains the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC (Sequence ID 15).In some embodiments, the 3'UTR of an RNA vaccine or RNA molecule contains the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (Sequence ID 17). In some embodiments, the 3'UTR of an RNA vaccine or RNA molecule comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCUCUCUGCUAGUUCCAGACACCUCC (Sequence ID 15) and the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (Sequence ID 17). In some embodiments, the 3'UTR of an RNA vaccine or RNA molecule contains the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (Sequence ID 13).In some embodiments, the 3'UTR of an RNA vaccine or RNA molecule is encoded by DNA containing the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC (Sequence ID 16). In some embodiments, the 3'UTR of an RNA vaccine or RNA molecule is encoded by DNA containing the sequence CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG (Sequence ID 18). In some embodiments, the 3'UTR of an RNA vaccine or RNA molecule is encoded by DNA containing the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC (Sequence ID 16) and the sequence CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG (Sequence ID 18).In some embodiments, the 3'UTR of an RNA vaccine or RNA molecule is encoded by DNA containing the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (Sequence ID 14).

[0157] In some embodiments, RNA vaccines or RNA molecules that can be used with the methods and systems of this disclosure include a poly(A) tail at its 3' end. In some embodiments, the poly(A) tail contains more than 50 or more than 100 adenine nucleotides. For example, in some embodiments, the poly(A) tail contains 120 adenine nucleotides. This poly(A) tail has been demonstrated to enhance RNA stability and translation efficiency (Holtkamp, ​​S et al. (2006) Blood 108:4009-4017). In some embodiments, RNA containing a poly(A) tail is produced by transcribing a DNA molecule that contains a polynucleotide sequence encoding at least 50, 100, or 120 consecutive adenine nucleotides and a recognition sequence for IIS-type restriction endonucleases in the 5'→3' direction of transcription. Exemplary poly(A) tails and 3'UTR sequences that improve translation are found, for example, in U.S. Patent No. 9,476,055.

[0158] In some embodiments, an RNA vaccine or RNA molecule that can be used with the methods and systems of the present disclosure comprises the following general structure (in the 5'→3' direction): (1) a 5' cap; (2) a 5' untranslated region (UTR); (3) a polynucleotide sequence encoding a secretory signal peptide; (4) a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; (5) a 3' UTR comprising (a) a 3' untranslated region or fragment thereof of Amino-Terminal Enhancer of Split (AES) mRNA and (b) a non-coding RNA or fragment thereof of mitochondrial-encoded 12S RNA; and (6) a poly(A) sequence.In some embodiments, the RNA vaccine or RNA molecule that can be used with the methods and systems of the present disclosure is a polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 1); and a polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGGAAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGC CCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 2). Advantageously, RNA vaccines comprising this combination and orientation of structure or sequence are characterized by one or more of the following: improved RNA stability, improved translation efficiency, improved antigen presentation and / or processing (e.g., by DCs), and increased protein expression.

[0159] In some embodiments, the RNA vaccine or RNA molecule of the present disclosure contains the sequence of SEQ ID NO: 24 (in the 5'→3' direction). See, for example, Figure 17. In some embodiments, N refers to a polynucleotide sequence encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 different neoepitopes. In some embodiments, N refers to a polynucleotide sequence encoding one or more linker-epitope modules (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 different linker-epitope modules). In some embodiments, N refers to a polynucleotide sequence encoding one or more linker-epitope modules (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 different linker-epitope modules) and an additional amino acid linker at the 3' end.

[0160] In some embodiments, the RNA vaccine or RNA molecule further comprises a polynucleotide sequence encoding at least one neoepitope, the polynucleotide sequence encoding at least one neoepitope located between a polynucleotide sequence encoding a secretory signal peptide and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule, in the 5'→3' direction. In some embodiments, the RNA molecule comprises polynucleotide sequences encoding at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or twenty different neoepitopes.

[0161] In some embodiments, an RNA vaccine or RNA molecule that can be used with the methods and systems of the present disclosure further comprises a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope in the 5'→3' direction. In some embodiments, the polynucleotide sequences encoding the amino acid linker and the neoepitope form a linker-neoepitope module (e.g., a continuous sequence in the 5'→3' direction within the same open reading frame). In some embodiments, the polynucleotide sequences forming the linker-neoepitope module are located in the 5'→3' direction between a polynucleotide sequence encoding a secretory signal peptide and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of an MHC molecule, or between the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 2. In some embodiments, the RNA vaccine or RNA molecule contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, or 30 linker-epitope modules. In some embodiments, each linker-epitope module encodes a different neoepitope. In some embodiments, the RNA vaccine or RNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 linker-epitope modules, and the RNA vaccine or RNA molecule comprises polynucleotides encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 different neoepitopes. In some embodiments, the RNA vaccine or RNA molecule comprises 5, 10, or 20 linker-epitope modules.In some embodiments, each linker-epitope module encodes a different neoepitope. In some embodiments, the linker-epitope modules form a continuous sequence in the 5'→3' direction within the same open reading frame. In some embodiments, the polynucleotide sequence encoding the linker of the first linker-epitope module is the 3' of the polynucleotide sequence encoding the secretory signal peptide. In some embodiments, the polynucleotide sequence encoding the neoepitope of the last linker-epitope module is the 5' of the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

[0162] In some embodiments, the RNA vaccine or RNA molecule that can be used with the methods and systems of this disclosure is at least 800 nucleotides, at least 1000 nucleotides, or at least 1200 nucleotides in length. In some embodiments, the RNA vaccine is less than 2000 nucleotides in length. In some embodiments, the RNA vaccine is at least 800 nucleotides but less than 2000 nucleotides in length, at least 1000 nucleotides but less than 2000 nucleotides, at least 1200 nucleotides but less than 2000 nucleotides, at least 1400 nucleotides but less than 2000 nucleotides, at least 800 nucleotides but less than 1400 nucleotides, or at least 800 nucleotides but less than 2000 nucleotides in length. For example, the constant region of an RNA vaccine containing the above elements is approximately 800 nucleotides in length. In some embodiments, an RNA vaccine containing five patient-specific neoepitopes (e.g., each encoding 27 amino acids) is more than 1300 nucleotides in length. In some embodiments, an RNA vaccine containing 10 patient-specific neoepitopes (e.g., each encoding 27 amino acids) may exceed 1800 nucleotides in length.

[0163] Lipoplex / Liposomes In some embodiments, the RNA vaccine or RNA molecule that can be used with the methods and systems of the present disclosure is formulated into lipoplex nanoparticles or liposomes. In some embodiments, a lipoplex nanoparticle formulation for RNA (RNA-Lipoplex) is used to enable IV delivery of the RNA vaccine of the present disclosure. In some embodiments, a lipoplex nanoparticle formulation for RNA cancer vaccines containing synthetic cationic lipid(R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propaneaminium chloride (DOTMA) and phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is used, for example, to enable IV delivery. The DOTMA / DOPE liposome component is optimized for IV delivery and targeting of antigen-presenting cells in the spleen and other lymphoid organs.

[0164] In some embodiments, RNA molecules that can be used with the methods and systems of this disclosure are mixed with a pharmaceutical composition comprising one or more cationic lipids, for example, (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propaneaminium chloride (DOTMA) and the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the pharmaceutical composition comprises at least one lipid. In one embodiment, the pharmaceutical composition comprises at least one cationic lipid. The cationic lipid may be monocationic or polycationic. Any cationic amphiphilic molecule, for example, a molecule comprising at least one hydrophilic and lipophilic moiety, is a cationic lipid within the scope of the present invention. In one embodiment, the positive charge is occupied by at least one cationic lipid and the negative charge is occupied by RNA. In one embodiment, the pharmaceutical composition comprises at least one helper lipid. The helper lipid may be neutral or anionic. Helper lipids may be natural lipids, such as phospholipids or analogs of natural lipids, or fully synthetic lipids, or lipid-like molecules that are not similar to natural lipids. In one embodiment, cationic lipids and / or helper lipids are bilayer-forming lipids.

[0165] In one embodiment, at least one cationic lipid includes 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) or its analogues or derivatives, and / or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or its analogues or derivatives.

[0166] In one embodiment, at least one helper lipid includes 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or its analogues or derivatives, cholesterol (Chol) or its analogues or derivatives, and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or its analogues or derivatives.

[0167] In one embodiment, the molar ratio of at least one cationic lipid to at least one helper lipid is 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1. In one embodiment, in this ratio, the molar amount of cationic lipid is obtained by multiplying the molar amount of cationic lipid by the number of positive charges in the cationic lipid.

[0168] In one embodiment, the lipid is contained within a vesicle that encapsulates the RNA. The vesicle may be a multilayer vesicle, a simple vesicle, or a mixture thereof. The vesicle may be a liposome.

[0169] The lipoplexes or liposomes described herein can be formed by mixing RNA and cationic lipids, adjusting the positive-to-negative charge ratio according to the (+ / -) charge ratio of cationic lipids to RNA. The + / - charge ratio of cationic lipids to RNA in the pharmaceutical compositions described herein can be calculated by the following formula: (+ / - charge ratio) = [(amount of cationic lipids (mol)) * (total number of positive charges in cationic lipids)] : [(amount of RNA (mol)) * (total number of negative charges in RNA)]. The amounts of RNA and cationic lipids can be easily determined by those skilled in the art by considering the loading during the preparation of nanoparticles. For further descriptions of exemplary pharmaceutical compositions, see, for example, PG Publication No. 20150086612, U.S. Patent Application Publication No.

[0170] In one embodiment, the total charge ratio of positive charge to negative charge in the pharmaceutical composition (e.g., at physiological pH) is 1.4:1 to 1:8, preferably 1.2:1 to 1:4, e.g., 1:1 to 1:3, e.g., 1:1.2 to 1:2, 1:1.2 to 1:1.8, 1:1.3 to 1:1.7, particularly 1:1.4 to 1:1.6, e.g., about 1:1.5. In some embodiments, at physiological pH, the total charge ratio of positive charge to negative charge of nanoparticles is The image is JPEG0007872261000006.jpg9170. In some embodiments, at physiological pH, the total charge ratio of positive charge to negative charge of the pharmaceutical composition is 1.6:2(0.8) to 1:2(0.5) or 1.6:2(0.8) to 1.1:2(0.55). In some embodiments, at physiological pH, the total charge ratio of positive charge to negative charge of the pharmaceutical (pharmacetical) composition is 1.3:2(0.65). In some embodiments, at physiological pH, the total charge ratio of positive charge to negative charge of liposomes is 1.0:2.0 or greater. In some embodiments, at physiological pH, the total charge ratio of positive charge to negative charge of liposomes is 1.9:2.0 or less. In some embodiments, at physiological pH, the total charge ratio of positive charge to negative charge of liposomes is 1.0:2.0 or greater and 1.9:2.0 or less. As will be apparent to those skilled in the art, the pharmaceutical compositions of this disclosure may comprise a first pharmaceutical composition comprising RNA and a second pharmaceutical composition comprising lipids, which, when mixed using the methods and systems of this disclosure, achieve the positive charge-to-negative charge pairing described above.

[0171] In one embodiment, the pharmaceutical composition comprises DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, where the charge ratio of positive charge in DOTMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the pharmaceutical composition comprises DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, where the charge ratio of positive charge in DOTMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the pharmaceutical composition comprises DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, where the charge ratio of positive charge in DOTMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the pharmaceutical composition comprises DOTMA and DOPE in a molar ratio of 2:1 to 1:2, preferably 2:1 to 1:1, where the charge ratio of positive charge in DOTMA to negative charge in RNA is 1.4:1 or less. In one embodiment, the pharmaceutical composition comprises DOTMA and cholesterol in a molar ratio of 2:1 to 1:2, preferably 2:1 to 1:1, where the charge ratio of positive charge in DOTMA to negative charge in RNA is 1.4:1 or less. In one embodiment, the pharmaceutical composition comprises DOTAP and DOPE in a molar ratio of 2:1 to 1:2, preferably 2:1 to 1:1, wherein the charge ratio of positive charge in DOTAP to negative charge in RNA is 1.4:1 or less. As will be apparent to those skilled in the art, the pharmaceutical composition of the present disclosure may comprise a first pharmaceutical composition comprising RNA and a second pharmaceutical composition comprising lipids (e.g., DOTMA, DOPE, DOTAP, and / or cholesterol), and when mixed using the methods and systems of the present disclosure, the above-described positive-to-negative charge ratio and / or molar ratio can be achieved.

[0172] In one embodiment, the zeta potential of a lipoplex or liposome produced or manufactured after combining two or more pharmaceutical compositions described herein according to the methods and systems of this disclosure is -5 or less, -10 or less, -15 or less, -20 or less, or -25 or less. In various embodiments, the zeta potential of the lipoplex or liposome is -35 or more, -30 or more, or -25 or more. In one embodiment, the nanoparticles or liposomes have a zeta potential of 0mV to -50mV, preferably 0mV to -40mV or -10mV to -30mV.

[0173] In some embodiments, the molecular weight dispersibility of lipoplexes or liposomes produced or manufactured after combining two or more pharmaceutical compositions described herein according to the methods and systems of this disclosure is 0.5 or less, 0.4 or less, or 0.3 or less, as measured by dynamic light scattering.

[0174] In some embodiments, nanoparticles or liposomes produced or manufactured after combining two or more pharmaceutical compositions described herein according to the methods and systems of this disclosure have an average diameter in the range of about 50 nm to about 1000 nm, about 100 nm to about 800 nm, about 200 nm to about 600 nm, about 250 nm to about 700 nm, or about 250 nm to about 550 nm, as measured by dynamic light scattering.

[0175] Further provided herein are DNA molecules encoding either RNA vaccines or RNA molecules that can be used in conjunction with the methods and systems of the present disclosure. For example, in some embodiments, the DNA molecules of the present disclosure comprise the following general structures (5'→3' direction): (1) a polynucleotide sequence encoding a 5' untranslated region (UTR); (2) a polynucleotide sequence encoding a secretory signal peptide; (3) a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; (4) a polynucleotide sequence encoding a 3'UTR, wherein the 3'UTR comprises (a) the 3' untranslated region of Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof; and (b) a 3'UTR comprising a non-coding RNA or fragment thereof of mitochondrial-encoded 12S RNA; and (5) a polynucleotide sequence encoding a poly(A) sequence.In some embodiments, the DNA molecule of the present disclosure has the polynucleotide sequence GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC (SEQ ID NO: 22) and the polynucleotide sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGT CACTGACAGCCTAGTAACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAA TGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO: 23).

[0176] In some embodiments, the DNA molecule further comprises a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope in the 5'→3' direction. In some embodiments, the polynucleotide sequences encoding the amino acid linker and the neoepitope form a linker-neoepitope module (e.g., a continuous sequence in the 5'→3' direction within the same open reading frame). In some embodiments, the polynucleotide sequences forming the linker-neoepitope module are located in the 5'→3' direction between a polynucleotide sequence encoding a secretory signal peptide and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule, or between the sequence of SEQ ID NO: 22 and the sequence of SEQ ID NO: 23. In some embodiments, a DNA molecule contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, or 30 linker-epitope modules, each linker-epitope module encoding a different neoepitope. In some embodiments, the DNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 linker-epitope modules, and the DNA molecule comprises polynucleotides encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 different neoepitopes. In some embodiments, the DNA molecule comprises 5, 10, or 20 linker-epitope modules. In some embodiments, each linker-epitope module encodes a different neoepitope. In some embodiments, the linker-epitope modules form a continuous sequence in the 5'→3' direction within the same open reading frame.In some embodiments, the polynucleotide sequence encoding the linker of the first linker-epitope module is the 3' of the polynucleotide sequence encoding the secretory signal peptide. In some embodiments, the polynucleotide sequence encoding the neoepitope of the last linker-epitope module is the 5' of the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

[0177] In some embodiments, the RNA or DNA molecule of the present disclosure allows the RNA to be transcribed under the control of a 5' RNA polymerase promoter and includes an IIS-type restriction cleavage site containing a polyadenylic cassette (poly(A) sequence), where the recognition sequence is located 3' to the poly(A) sequence, but the cleavage site is located upstream and therefore within the poly(A) sequence. Restriction cleavage at the IIS-type restriction cleavage site allows the plasmid to be linearized within the poly(A) sequence, as described in U.S. Patents 9,476,055 and 10,106,800. The linearized plasmid can then be used as a template for in vitro transcription, and the resulting transcript ends with an unmasked poly(A) sequence. Any of the IIS-type restriction cleavage sites described in U.S. Patents 9,476,055 and 10,106,800 may be used.

[0178] In some embodiments of the methods provided herein, the RNA vaccine or RNA molecule comprises one or more polynucleotides encoding 10 to 20 (e.g., any of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In certain embodiments, the RNA vaccine or RNA molecule is incorporated into lipoplex nanoparticles or liposomes. In certain embodiments, the lipoplex nanoparticles or liposomes comprise one or more lipids forming a multilayer structure that encapsulates the RNA of the RNA vaccine. In certain embodiments, the one or more lipids comprise at least one cationic lipid and at least one helper lipid. In certain embodiments, the one or more lipids comprise (R)-N,N,N-trimethyl-2,3-dioleyloxy-1-propaneaminium chloride (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In a particular embodiment, at physiological pH, the total charge ratio of positive to negative charges in liposomes is 1.3:2 (0.65).

[0179] In a particular embodiment, the RNA vaccine comprises, in the 5'→3' direction, (1) a 5' cap; (2) a 5' untranslated region (UTR); (3) a polynucleotide sequence encoding a secretory signal peptide; (4) a polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; (5) a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of a major histocompatibility complex (MHC) molecule; (6) a 3'UTR comprising (a) the 3' untranslated region or a fragment thereof of Amino-Terminal Enhancer of Split (AES) mRNA; and (b) a 3'UTR comprising a non-coding RNA or a fragment thereof of mitochondrial-encoded 12S RNA; and (7) an RNA molecule comprising a poly(A) sequence.

[0180] In certain embodiments, the RNA molecule further comprises a polynucleotide sequence encoding an amino acid linker, wherein the polynucleotide sequence encoding the amino acid linker and one or more neoepitopes forms a first linker-neoepitope module, and the polynucleotide sequence forming the first linker-neoepitope module is located in the 5'→3' direction between a polynucleotide sequence encoding a secretory signal peptide and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule. In certain embodiments, the amino acid linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 21). In certain embodiments, the polynucleotide sequence encoding the amino acid linker comprises the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 19).

[0181] In a particular embodiment, the RNA molecule further comprises at least a second linker-epitope module in the 5'→3' direction, the at least second linker-epitope module comprising a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope, the polynucleotide sequence forming the second linker-neoepitope module being located in the 5'→3' direction between a polynucleotide sequence encoding the neoepitope of the first linker-neoepitope module and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule, the neoepitope of the first linker-epitope module being different from the neoepitope of the second linker-epitope module. In a particular embodiment, the RNA molecule comprises five linker-epitope modules, each of the five linker-epitope modules encoding a different neoepitope. In a particular embodiment, the RNA molecule contains 10 linker-epitope modules, each of which encodes a different neoepitope. In a particular embodiment, the RNA molecule contains 20 linker-epitope modules, each of which encodes a different neoepitope.

[0182] In one embodiment, the RNA molecule further comprises a second polynucleotide sequence encoding an amino acid linker, the second polynucleotide sequence encoding the amino acid linker being located between a polynucleotide sequence encoding the most distal neoepitope in the 3' direction and a polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule.

[0183] In certain embodiments, the 5' cap comprises a D1 diastereoisomer having the following structure: JPEG0007872261000007.jpg49170

[0184] In a particular embodiment, the 5'UTR contains the sequence UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (Sequence ID 5). In a particular embodiment, the 5'UTR contains the sequence GGCGACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (Sequence ID 3).

[0185] In a particular embodiment, the secretory signal peptide comprises the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 9). In a particular embodiment, the polynucleotide sequence encoding the secretory signal peptide comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 7).

[0186] In certain embodiments, at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule contains the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 12). In certain embodiments, the polynucleotide sequence encoding at least a portion of the transmembrane and cytoplasmic domains of the MHC molecule contains the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC (SEQ ID NO: 10).

[0187] In a particular embodiment, the 3' untranslated region of the AES mRNA contains the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCUCUCUGCUAGUUCCAGACACCUCC (Sequence ID 15). In a particular embodiment, the non-coding RNA of the 12S RNA encoded by the mitochondria contains the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (Sequence ID 17). In a particular embodiment, the 3'UTR contains the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (Sequence ID 13).

[0188] In a particular embodiment, the poly(A) sequence contains 120 adenine nucleotides.

[0189] In a particular embodiment, the RNA vaccine has the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 1); a polynucleotide sequence encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAU AGCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCA CGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO: 2). [Examples]

[0190] This disclosure will be better understood by reference to the following embodiments. However, these embodiments should not be construed as limiting the scope of the invention. The embodiments and aspects described herein are for illustrative purposes only, and various modifications or changes in light thereof are proposed to those skilled in the art and should be understood to be included within the spirit and scope of this application and the appended claims.

[0191] Prior to developing the methods and systems disclosed herein, various experiments were conducted to understand, develop, and optimize these methods and systems. Specifically, the experiments were conducted in the following three stages: (1) understanding the pulsation of peristaltic pump flow rate (Example 1); (2) developing and optimizing damping devices and peristaltic pump configurations to maintain a consistent and stable flow rate measured by the level of pulsation ("LoP") (Example 2); and (3) optimizing damping devices and peristaltic pump configurations for flow processes with fluids from two sources (Example 3). These experiments provided an opportunity to evaluate and quantify the effectiveness of numerous parameters affecting LoP.

[0192] The ultimate goal of these experiments was to develop methods and systems that have an acceptable level of performance (LoP), including similarity to and / or improvement of other alternatives such as syringe pumps, and that are easy to implement in a sterile, closed, Good Manufacturing Practice ("GMP") environment using single-use materials (i.e., study of structural materials and sterilization for use with the pharmaceutical formulations described herein). Structural materials can be important because the product contact surface must not leach into the product (and the product must not extract any species from the material). Therefore, such materials should be suitable for use in manufacturing or transporting pharmaceutical compositions, including the pharmaceutical compositions and formulations described herein, such as platinum-cured silicone tubing.

[0193] Measurements and calculations used in the examples Non-product contact flowmeters (e.g., Keyence input model FD-XA1) were used to monitor flow rate and LoP in the experiments described in the examples. Based on the outer diameter of the target tubing, the sensor head FD-Xs8 was paired with clamp set FD-XCR2 or FD-XCR1. Each Keyence meter was connected to a Keysight high-speed data logger (e.g., U2541A), and data was recorded every 10 ms using the accompanying Keysight software. The flowmeters were used to measure the dynamic flow rate in the system, and the LoP was determined using the dynamic flow rate in the system during steady-state operation.

[0194] To eliminate inlet effects from flow measurements, the flowmeter was positioned at least 10mm in diameter away from any changes in the flow path, such as dampers, connectors, or sharp curves. Before each experiment, each meter was initialized and the flow rate was set to 0 mL / min. A minimum of 7 seconds was recorded at a sample rate of 100 Hz. Since multiple periods (i.e., distances between flow peaks) existed within one second, a representative second, typically the fourth or fifth second, was used for both pulsation calculations and data plots. In some embodiments, data was obtained from a 10-second period once a steady-state flow was established.

[0195] Pulsation Level: The pulsation level ("LoP") was calculated for representative seconds used in the data plot. The calculation is as follows:

[0196] (Maximum flow rate - Minimum flow rate) / Average flow rate x 100.

[0197] Maximum and minimum flow rate percentages: The maximum and minimum flow rate percentages were calculated as follows:

[0198] Maximum flow rate percentage = ((Maximum flow rate - Average flow rate) / Average flow rate × 100)

[0199] Minimum flow rate percentage = ((Average flow rate - Minimum flow rate) / Average flow rate × 100)

[0200] For all experiments, a single peristaltic pump was used. In experiments using two pump heads, two pump heads were attached to a single pump. In experiments with four product lines in contact with the roller (dual-head pumps with two separate flows), a single pump with two heads was used, supplying both lines to a single pump head, as opposed to using two separate pumps with two pump heads. Unless otherwise specified, the average flow rate of the pumps described in the examples was approximately 50 mL / min.

[0201] [Example 1] Understanding flow rate pulsation in peristaltic pumps As described herein, the flow rate from a peristaltic pump may pulsate or oscillate over time due to the nature of peristaltic pumps. See, for example, Experiment 1 (described in Table 1A (Experimental Setup Details) and Table 1B (Results of Experiment 1)) in which the flow rate of water moving through a peristaltic pump was measured over a given period of time. Briefly, Experiment 1 was conducted using a single pump head without a damper and with 30 cm of tubing downstream from the damper. This setup represented an experimental baseline of the expected LoP for a peristaltic pump system without any attempt to reduce flow rate fluctuations. Figure 1 shows the experimental setup for Experiment 1. This setup included a water container, a peristaltic pump, and a flow meter for measuring the flow of water exiting the peristaltic pump. The average flow rate of the pump was approximately 50 mL / min.

[0202] Figure 2 shows the flow rate of water through the peristaltic pump measured over time. As shown in Figure 2, the flow rate fluctuates significantly over time. Tables 1A and 1B provide further details and a summary of the results of Experiment 1. JPEG0007872261000008.jpg79170

[0203] The minimum flow rate observed in Experiment 1 reached a value less than 0. However, this was due to a combination of the following: (1) inherent solution suck-back; and (2) slight sensor measurement error.

[0204] [Example 2] Optimizing the damping device configuration in a peristaltic pump system to induce a consistent steady flow rate. One possible method for reducing pulsation from a peristaltic pump is to use a damper. Therefore, various designs and configurations of dampers after peristaltic pumps were investigated. Because they are highly compressible, air and / or gases can be used as dampers. Thus, the design of a damper can either be open directly to air or have a membrane in contact with the fluid and air. Figure 3 shows an experimental setup for measuring flow rate using one or two peristaltic pump heads and one or more dampers after peristaltic pumps. Dampers can reduce or eliminate pressure and flow fluctuations generated by peristaltic pumps. Specifically, dampers can smooth the flow rate by absorbing excess fluid during peak flow and releasing it during declines. Experiments 1-3 used one pump head. All other experiments described herein used two pump heads.

[0205] The experimental setup in Figure 3 includes a damping device after the peristaltic pump. As shown in Figure 3, the damping device is a syringe damping device with a T-shaped connector. The T-shaped connector may be a T-shaped connector with two outlets at a 90-degree angle to the connection to the main line. The T-shaped connector may be a short pipe piece with a lateral outlet. The relative size / opening of the T-shape may affect the damping efficiency. The damping device and T-shaped connector are fluidically connected to the outlet tubing from the peristaltic pump, as shown in Figure 3. An example of a damping device is a syringe damping device. The tubing after the peristaltic pump can be fluidically connected to the syringe damping device. In some embodiments, the tubing after the peristaltic pump can be fluidically connected to a T-shaped connector that is fluidly connected to the syringe. Examples of images of syringe damping devices with various T-shaped connectors are shown in Figure 4. The syringe plunger can be retracted so that the volume of air in the syringe can be adjusted to suit a given application. The air inside the syringe can act as a space buffer, temporarily storing excess fluid when the peristaltic pump is operating. This temporary storage can increase the air pressure, pushing out the fluid when the flow rate from the peristaltic pump decreases.

[0206] Several experiments (i.e., Experiments 2-16) using water were conducted according to the experimental setup shown in Figure 3 by adjusting the following variables: (1) the number of pump heads used (using N pump heads, the flow rate can be approximately multiplied by N if the pump settings are the same. Most pumps can be adjusted to many flow rates depending on the minimum and maximum RPM of the pump's rollers); (2) syringe dampener or no syringe dampener; (3) the size of the T-connector used (in most cases, the T-connector may not affect the flow rate unless there is a rare scenario in which the T-connector has a small opening and is currently limiting the flow rate); (4) tubing outlet length (the length of the outlet tubing can affect pulsation. The longer the outlet, the lower the LoP can be, as the flexibility of the tubing naturally dampens the system); (5) the volume of air in the syringe. Further details and results of these experiments (Experiments 2-9 and 12-16) are summarized in Table 2A (Experimental Setup Details) and Table 2B (Experimental Results). JPEG0007872261000009.jpg245170JPEG0007872261000010.jpg168170JPEG0007872261000011.jpg161170

[0207] The flow rates over time achieved in experiments 2, 4, and 6 are shown in Figures 5, 6, and 7, respectively. As shown in these figures, the flow rate in experiment 6 (Figure 7), using two pump heads, a 60 mL syringe attenuation device at the maximum air volume (i.e., 60 mL), and a 60 cm tubing outlet length, significantly reduced pulsation throughout the experiment.

[0208] Experiment 4 evaluated the effect of a second pump head (e.g., a single pump fitted with two pump heads driven by the same motor). This was done by opening a valve adjacent to the second pump head. See Figure 3. The LoP decreased to 134, but flow fluctuations still existed as shown in Figure 6. Adding subsequent pump heads may further reduce the LoP. However, this increases the complexity of the product flow path and tubing kit for the system, and many peristaltic pumps only have two fixed heads.

[0209] Experiments 2 and 5 incorporated a syringe attenuation device. This was achieved by opening a valve attached to a T-connector and a 60 mL syringe. See Figure 3. Before opening the valve, the syringe was adjusted to its maximum position (marked 60 mL), connected to a Luer lock T-connector, and the T-connector was connected in series to the outlet tubing, where the solution flowed without sharp bends. In Experiments 2 and 5, the LoP values ​​decreased significantly to 35 and 23, respectively. Thus, the syringe attenuation device proved effective, although slight pulsation was still observed.

[0210] Another possible method to reduce flow rate fluctuations is to increase the length of the outlet tubing. In experiments 3 and 6, the length of the outlet tubing was changed from 30 cm to 60 cm compared to those in experiments 2 and 5. The LoP values ​​decreased from 35 and 23 (see Table 2B, experiments 2 and 5, respectively) to 25 and 21 (see Table 2B, experiments 3 and 6, respectively).

[0211] In experiments 12–16, the volume of air in a 60 mL syringe attenuator was investigated to better understand the minimum volume at which the flow could be effectively attenuated. In these experiments, the plunger position was adjusted to the volume measurement scales of 60, 40, 20, 10, and 5 cc, as reported in Table 2B, and the LoPs were 22, 21, 22, 30, and 47, respectively. Thus, there was no significant difference between the LoPs until the plunger position was adjusted to 10 cc and 5 cc, where the effect of the attenuator was further reduced.

[0212] During the experiment, the effectiveness of the damping device was influenced by the location of the air-liquid interface in the damping device / T-shaped system. Therefore, although Experiment 7 was conducted using the same apparatus as Experiment 6, unlike Experiment 6 where the solution remained in the T-shape during the test, in Experiment 7, the solution rose beyond the T-shape into the valve and remained in the valve throughout the experiment. The LoP increased from 21 to 28 between Experiment 6 and Experiment 7 (see Table 2B), suggesting that the air-liquid interface surface area can affect the effectiveness of the damping device. The inner diameter of the Luer tee was 3.1 mm, and the inner diameter of the Luer valve was 4.1 mm. Since this Luer tee proved insufficient, a larger tee with an inner diameter of 9.5 mm was tested in Experiment 9. The LoP decreased to 18, confirming that the air-liquid interface surface in the damping device / tee system is a parameter that should be controlled.

[0213] The ability to implement a peristaltic pump attenuation device can depend on many variables, including structural materials, sterilization capabilities, and shape (i.e., how quickly air pressure is applied in response to fluid pulses). Experiments 1–16 above demonstrated that flow pulsation can be reduced using attenuation devices, but that syringe attenuation devices may not meet the criteria for use with pharmaceutical compositions described herein, including those containing RNA or lipids, such as RNA vaccines (e.g., LoP and / or sterility robustness for GMP applications, as described below). Experiments 1–16 provided proof of concept that LoP can be reduced and that additional parameters were further optimized to increase the observed reduction in LoP. One such parameter was the development of an attenuation device that could be easily implemented for use with pharmaceutical formulations containing RNA or lipids, such as RNA vaccines.

[0214] Besides syringe attenuation devices, another type of pulsation attenuation device that can be used is a membrane attenuation device. Thus, tubing after a peristaltic pump can be fluidically connected to a membrane attenuation device. In some embodiments, tubing after a peristaltic pump can be fluidly connected to a T-connector fluidically connected to a membrane. An exemplary membrane attenuation device is shown in Figure 8. Membrane attenuation devices are similar to other attenuation devices disclosed herein in that a compressible gas performs an attenuation device in which a membrane acts as a barrier. In some cases, a closed system can be used with a compressible gas attenuation device having a membrane that acts as a barrier between the gas and the solution. In the experiments described herein, the gas was air, so this scenario was not sufficient, but closed systems can be designed using a compressible gas and a flexible membrane. Fine-tuning of the membrane (flexibility, surface area, hardness, etc.) and the compressible gas can help reduce LoP.

[0215] To investigate the potential effects of the membrane attenuation device, an experiment using water was conducted according to the experimental setup shown in Figure 3, but the syringe attenuation device was replaced with a membrane attenuation device. Further details and results of this experiment (Experiment 10) are summarized in Table 3A (Experimental Setup Details) and Table 3B (Experimental Results). JPEG0007872261000012.jpg57170JPEG0007872261000013.jpg34170

[0216] Experiment 10 evaluated a T-shaped attenuation device with a flexible membrane. Theoretically, the membrane, combined with atmospheric pressure, should act as an attenuation device. However, the LoP of this system was 65, suggesting that the membrane is only slightly effective in reducing flow pulsation and is still too high for use with pharmaceutical compositions described herein, including those containing RNA or lipids, such as RNA vaccines.

[0217] In addition to membranes or syringes, the gas-filled tubing itself can potentially be used as a pulsation damper. Thus, tubing after a peristaltic pump can be fluidically connected to a tubing damper. Exemplary configurations of tubing dampers are shown in Figures 9 and 10. As shown in Figure 9, the tubing damper can be a T-connector tubing damper. When such a T-connector tubing damper is used, tubing after a peristaltic pump can be fluidly connected to a T-connector that is fluidly connected to the tubing damper. The tubing of the tubing damper can be made from the same or different material as the tubing after the peristaltic pump. In some embodiments, the tubing of the tubing damper can be silicone. In some embodiments, the tubing damper can include clamps or other objects so that the end of the tubing damper opposite the end fluidly connected to the T-connector is closed. The tubing damper functions similarly to other dampers in that the enclosed gas can perform the damping.

[0218] Figure 10 shows an example of a tubing damper with a cross-shaped or four-way connector. Instead of a T-shaped connector, the four-way connector in Figure 10 (the damping effect may depend on the valve opening dimensions) allows both ends of the tubing damper to be connected to the four-way connector instead of using a clamp or other device to close one end of the tubing damper. When a four-way tubing damper is used, the tubing after the peristaltic pump can be fluidically connected to the four-way connector which is fluidically connected to the tubing damper. Thus, the fluid from the peristaltic pump can enter through one of the openings of the four-way connector and exit through another, while the tubing damper can be connected to the other two unused openings so that both ends of the tubing damper are fluidically connected to the four-way connector. The four-way tubing damper can function like other dampers in that the sealed gas can be damped.

[0219] To investigate the potential effects of T-shaped connector tubing attenuation devices (shown in Figure 9) or four-way tubing attenuation devices (shown in Figure 10), experiments using water were conducted according to the experimental setup shown in Figure 3, but the syringe attenuation device was replaced with either a T-shaped connector tubing attenuation device or a four-way connector tubing attenuation device. Further details and results of these experiments (Experiments 11, 17, and 18) are summarized in Table 4A (Experimental Setup Details) and Table 4B (Experimental Results). JPEG0007872261000014.jpg124170JPEG0007872261000015.jpg56170

[0220] As preparation, Experiment 11 replaced the T-shaped / damping device with a 30 cm thin-walled flexible tubing (outer diameter 7.9 mm, wall thickness 0.8 mm). The idea of ​​using thin-walled flexible tubing as a damping device is based on Experiments 3 and 6 (see Table 2B), where longer tubing outlet lengths reduced the LoP. While not bound by theory, fluid is damped by tubing, and the damping effect increases as the flexibility of the tubing increases (i.e., from rigid tubing to flexible tubing). From this, it can be assumed that shorter lengths of thin-walled flexible tubing can achieve the same LoP as the tubing used in Experiments 3 and 6. To test this hypothesis, Experiment 11 was conducted, and using 30 cm thin-walled tubing increased the LoP from 134 (see Table 2B, Experiment 4, without damping device) to 152 (see Table 3B). An increase in LoP was initially observed by using thin-walled, flexible tubing, but the concept behind using tubing as an attenuator was further explored, as described in Experiments 17 and 18. If a certain volume of air sealed within a piece of tubing can replace the air in the syringe and effectively reduce LoP, many process requirements such as sterilization and solution hold-up can be met.

[0221] Therefore, in Experiment 17, instead of a syringe with a tubing length of 42 cm (approximately 30 cc of air in the tubing), a silicone tubing with a closed end (i.e., clamped or otherwise secured) (i.e., a T-shaped connector tubing damper as shown in Figure 9) was used. Furthermore, the position of the T-shape was changed to facilitate installation, but it was assumed that this would not affect the damper. As reported in Table 3B, the observed LoP was 19, which was comparable to the value obtained using a syringe damper (see, e.g., Table 2B). These results demonstrate that the damping level of the closed-end tubing is comparable to that of the syringe damper. As mentioned above, the suitability of using closed-end tubing instead of a syringe is that such a system can meet GMP process requirements for pharmaceutical compositions and formulations. In some embodiments, the damper may be a tubing damper with an open end (i.e., open to the atmosphere).

[0222] In Experiment 18, the T-shaped connector was replaced with a four-way connector arranged in an "X" or "cross" shape (as shown in Figure 10), with two ports connected to a single silicone tubing forming a loop, and the other two ports used for fluid flow. The ultimate goal of Experiment 18 was to evaluate whether any advantages existed between this damping device and Experiment 17. The LoP in Experiment 18 was identical to that of Experiment 17. See Table 3B.

[0223] The inventors observed a significant reduction in LoP using several damping device and tubing kit configurations, even achieving low LoP values ​​of 18–22. However, the pulsation and oscillation levels were still not optimal for mixing and / or manufacturing the pharmaceutical compositions described herein, such as RNA vaccines or lipid-containing lipoplexes or liposomes, and were generally higher than the LoP values ​​observed when alternative syringe pump configurations were used (see Tables 5A and 5B, Examples 24–26). Therefore, additional parameters were evaluated to further reduce LoP values. Regardless of the inventors' goal of reducing LoP values, those skilled in the art will understand that the LoP reduction observed in Example 2 may be suitable for other applications and pharmaceutical compositions, including, for example, the transfer and / or filling of pharmaceutical compositions into containers such as bags or vials.

[0224] [Example 3] Application of damping device configuration to a flow process having two fluid sources Experiments 1-18 primarily focused on systems with a single fluid source. However, peristaltic pumps can also be used in systems with two or more fluid sources. One such example is the mixing or combination of two pharmaceutical compositions to form a final pharmaceutical composition, including, for example, the combination of a first pharmaceutical composition containing RNA or an RNA vaccine with a second pharmaceutical composition containing one or more lipids to form a final pharmaceutical composition containing an RNA-lipoplex or RNA liposome. Since pharmaceutical compositions may contain delicate and expensive components, the amount of these components used in the final pharmaceutical composition or formulation can be critical to whether the final pharmaceutical composition is effective, safe, and cost-effective. Because the components of the final pharmaceutical composition originate from different sources or containers, it may be important that the flow rate of these components or intermediate pharmaceutical compositions in the peristaltic pump system does not produce pulsations that prevent the effective mixing of these components or intermediate pharmaceutical compositions in the appropriate proportions necessary for the final pharmaceutical composition containing the intermediate pharmaceutical composition to be effective.

[0225] Since the pharmaceutical compositions described herein, including those containing RNA or lipids such as RNA vaccines, are often mixed to produce final pharmaceutical compositions containing RNA-lipoplexes or RNA liposomes, we conducted experiments to evaluate two different fluid sources having two peristaltic pumps. In particular, we conducted experiments to evaluate the type of attenuation device that can achieve a consistent flow rate across both peristaltic pumps. Figure 11 shows an experimental setup for measuring the flow rate of two fluid source systems using one peristaltic pump and one or more attenuation devices after one or more peristaltic pumps. Although only one peristaltic pump is shown in Figure 11, in some embodiments the peristaltic pump may be a dual-head peristaltic pump or a peristaltic pump having two or more heads. Thus, tubing from each fluid source can be mounted to the head of a dual-head peristaltic pump so that only one peristaltic pump is required. In some embodiments, each fluid source may have its own peristaltic pump. However, in experiments 19-20, the inlet from each source was split into two flows so that each pump head of the dual pump head for each peristaltic pump was used. Experiments 19 and 20 used a single peristaltic pump, but the pump had two motor-driven heads. Experiments 21-22 used only one pump head of each dual-head peristaltic pump.

[0226] In addition to the different configuration compared to Figure 3, further modifications were made to the experimental setup in Figure 11. First, the peristaltic pump used was a Watson Marlow Flexicon PD12l, and the tubing outlet dimension was 2.4 mm inner diameter instead of 3.2 mm inner diameter. Experiment 20 established a baseline for the LoP observed in this setup without using damping devices for the two inlet lines. The minimum flow rate was approximately 80 mL / min for each inlet, which differed from the approximately 50 mL / min previously used. As shown in Figure 11, flow meters were placed not only downstream of both inlet pumps but also downstream of the Y-connector.

[0227] Experiment 19 implemented an attenuation device. In some embodiments, the attenuation device may be two separate, end-closed tubing attenuation devices. However, the applicants found that a single tubing attenuation device can be used simultaneously at both inlets when forming an attenuation tubing loop. Figure 12 shows an example of an attenuation device loop. The attenuation loop can be connected to both T-connectors on the pump back inlet line. Furthermore, the attenuation loop was mounted above the flow path to prevent solution from entering the loop.

[0228] Experiments 21 and 22 evaluated the possibility of further simplifying the tubing kit by utilizing a single pump head for each inlet, in contrast to having dual pump heads. This eliminates the need for Y-connectors immediately upstream and downstream of the pump head. However, Y-connectors are still required for mixing the two separate inlets.

[0229] Further details and results of experiments 19-22 using the setup shown in Figure 11 are summarized in Table 5A (Details of the experimental setup) and Table 5B (Results of the experimental setup).

[0230] Experiments 24, 25, and 26 were conducted using two types of syringe pumps with the aim of directly comparing the LoP values ​​with those observed in Experiment 19 (peristaltic pump with loop attenuation device). Further details and results of Experiments 24-26 are summarized in Table 5A (Details of the experimental setup) and Table 5B (Experimental results). JPEG0007872261000016.jpg236170JPEG0007872261000017.jpg250170JPEG0007872261000018.jpg112170JPEG0007872261000019.jpg247170

[0231] The results for experiments 19.1, 20.1, 21.1, and 22.1 are all from flowmeters attached to the first source inlet. The results for experiments 19.2, 20.2, 21.2, and 22.2 are all from flowmeters attached to the second source inlet. The results for experiments 19.3, 20.3, 21.3, and 22.3 are all from flowmeters after the Y-connector or mixer of the combined first and second sources. As reported in Table 5B, the flow rates measured after mixing both inlets were approximately 91 mL / min and 161 mL / min, and in experiments 21 and 22, their corresponding LoPs were approximately 15 (14-16) and approximately 9 (8-10), respectively. The LoP was less than 10 at higher flow rates (experiment 22), but increased in experiment 21, suggesting that a single pump head setup may not be robust across a wide range of flow rates.

[0232] As reported in Table 5B, the LoPs at each inlet in Experiment 20 (no attenuation) were 80 and 87, and the LoP at the outlet was 80. In contrast, the LoPs in Experiment 19 (attenuated) using a loop attenuation device were both 7, and the LoP at the outlet was 8. These results meet the acceptable objective of achieving a LoP of less than 10 (for example, to properly control the flow rates of multiple pumps and / or fluid sources to ensure proper mixing of pharmaceutical compositions, and to achieve LoP values ​​equivalent to or better than those commonly achieved with syringe pumps), for producing, mixing, transferring, and / or manufacturing the pharmaceutical compositions and formulations described herein, such as RNA or lipid-containing pharmaceutical compositions, such as RNA vaccines, and the attenuated loop meets all of the GMP process objectives summarized earlier, while being easy to implement, cost-effective, and suitable for single-use offerings. Figure 13 shows the flow rate of water through the peristaltic pump system by Experiment 19, and Figure 14 shows the flow rate of water through the peristaltic pump by Experiment 20.

[0233] The results of experiments 24.1, 25.1, and 26.1 are all from flow meters attached to the inlet of the first source. The results of experiments 14.2, 25.2, and 26.2 are all from flow meters attached to the inlet of the second source. The results of experiments 24.3, 25.3, and 26.3 are all from flow meters after the Y-connector or mixing device of the combined first and second sources.

[0234] As reported in Table 5B, the flow rates measured after mixing both inlets were approximately 111 mL / min and 57 mL / min, and in experiments 24 and 25, their corresponding LoPs averaged approximately 25.5 (25-26) and approximately 24.5 (21-28), respectively. These experiments used off-the-shelf syringe pumps not specifically designed to reduce pump pulsation, demonstrating that standard units do not meet the acceptable target with respect to achieving LoP values ​​below 10. Experiment 26 also used an off-the-shelf syringe pump. While the pulsation of commercially available syringe pumps can vary, this type of system is not pulsation-free. As reported in Table 5B, the flow rate measured after mixing both inlets was approximately 141 mL / min, and the corresponding LoP averaged approximately 14 (10-18). These results confirmed that Experiment 19 (loop attenuator) achieved better LoP values ​​than both syringe pump systems.

[0235] Figure 15 describes a peristaltic pump, damping device, and tubing kit system that can achieve less than 10 LoP from two fluid sources, such as a pharmaceutical composition containing RNA, RNA molecules, or RNA vaccines, and another pharmaceutical composition containing one or more lipids, which can be mixed to produce, transfer, or manufacture the pharmaceutical compositions described herein, in particular, the final pharmaceutical compositions containing RNA-lipoplexes or RNA liposomes.

[0236] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in whole for the same degree as each individual publication is incorporated by reference individually. If any definition contained herein contradicts or is inconsistent with any definition contained herein in a patent, application, published application, or other publication incorporated by reference, the definition contained herein shall prevail over the definition incorporated by reference.

[0237] The present invention is not intended to be limited to specific disclosed embodiments provided, for example, to illustrate various aspects of the invention. Various modifications to the described apparatus and methods will become apparent from the description and teachings herein. Such modifications can be carried out without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the disclosure. Abbreviated sequence list All polynucleotide sequences are written in the 5'→3 direction. All polypeptide sequences are written in the N-terminus to C-terminus direction. Complete PCV RNA 5' constant sequence (SEQ ID NO: 1) GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC Complete PCV RNA 3' constant sequence (SEQ ID NO: 2) AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU Full-length PCV Kozak RNA (SEQ ID NO: 3) GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC Full-length PCV Kozak DNA (SEQ ID NO: 4) GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC Short Kozak RNA (SEQ ID NO: 5) UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC Short Kozak DNA (SEQ ID NO: 6) TTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC sec RNA (SEQ ID NO: 7) AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCCUGACAGAGACAUGGGCCGGAAGC sec DNA (SEQ ID NO: 8) ATGAGAGTGATGGCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC sec protein (SEQ ID NO: 9) MRVMAPRTLILLLSGALALTETWAGS MITD RNA (Sequence ID 10) AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAGCCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGCAGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACUGACAGCC MITD DNA (Sequence ID 11) ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACAGCC MITD protein (SEQ ID NO: 12) IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA Complete PCV FI RNA (SEQ ID NO: 13) CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU Complete PCV FI DNA (SEQ ID NO: 14) CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT F element RNA (SEQ ID NO: 15) CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC F element DNA (SEQ ID NO: 16) CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC I-element RNA (SEQ ID NO: 17) CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG I-element DNA (SEQ ID NO: 18) CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG Linker RNA (SEQ ID NO: 19) GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC Linker DNA (SEQ ID NO: 20) GGCGGCTCTGGAGGAGGCGGCTCCGGAGGC Linker protein (SEQ ID NO: 21) GGSGGGGSGG Complete PCV DNA 5' constant sequence (SEQ ID NO: 22) GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGC Complete PCV DNA 3' constant sequence (SEQ ID NO: 23) ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACAGCCTAGTAACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT Full-length PCV RNA with a 5’GG from the cap (SEQ ID NO: 24) GGGGCGAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACCAUGAGAG UGAUGGCCCC CAGAACCCUG AUCCUGCUGC UGUCUGGCGC CCUGGCCCUG ACAGAGACAU GGGCCGGAAG CNAUCGUGGGA AUUGUGGCAG GACUGGCAGU GCUGGCCGUG GUGGUGAUCG GAGCCGUGGU GGCUACCGUG AUGUGCAGAC GGAAGUCCAG CGGAGGCAAG GGCGGCAGCU ACAGCCAGGC CGCCAGCUCU GAUAGCGCCC AGGGCAGCGA CGUGUCACUG ACAGCCUAGU AACUCGAGCU GGUACUGCAU GCACGCAAUG CUAGCUGCCC CUUUCCCGUC CUGGGUACCC CGAGUCUCCC CCGACCUCGG GUCCCAGGUA UGCUCCCACC UCCACCUGCC CCACUCACCA CCUCUGCUAG UUCCAGACAC CUCCCAAGCA CGCAGCAAUG CAGCUCAAAA CGCUUAGCCU AGCCACACCC CCACGGGAAA CAGCAGUGAU UAACCUUUAG CAAUAAACGA AAGUUUAACU AAGCUAUACU AACCCCAGGG UUGGUCAAUU UCGUGCCAGC CACACCGAGA CCUGGUCCAG AGUCGCUAGC CGCGUCGCUA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA

Claims

1. A tubing kit for forming a mixture, A first portion of tubing configured to be fluidly connected to a container containing a first composition, A second portion of tubing configured to be fluidly connected to a container for containing a second composition, A single tubing damping device is fluidically connected to a first portion of the tubing and to a second portion of the tubing, and contains a certain volume of sealed fluid. A mixing apparatus for mixing the first composition from the first portion of the tubing with the second composition from the second portion of the tubing, A mixing container for collecting the mixed first composition and second composition from the mixing apparatus, Equipped with, A tubing kit wherein a first portion of the tubing is configured to be connected to a first peristaltic pump head for pumping the first composition from a container containing the first composition to a mixture container, and a second portion of the tubing is connected to a second peristaltic pump head for pumping the second composition from a container containing the second composition to the mixture container, thereby fluidly connecting the single tubing damping device to the first and second peristaltic pump heads.

2. The tubing kit according to claim 1, wherein the fluid is air.

3. The tubing kit according to claim 1 or 2, further comprising the single tubing damping device, a first T-shaped connector that fluidly connects a first portion of the tubing and a first mixing device input portion of the tubing, wherein the first mixing device input portion of the tubing is fluidly connected to the mixing device.

4. The tubing kit according to claim 3, further comprising the single tubing damping device, a second T-shaped connector for fluidly connecting the second portion of the tubing and the second mixing device input portion of the tubing, wherein the second mixing device input portion of the tubing is fluidly connected to the mixing device.

5. The tubing kit according to any one of claims 1 to 4, wherein the first portion of the tubing comprises a first segment of the tubing and a second segment of the tubing, and the first segment of the tubing and the second segment of the tubing are fluidly connected in parallel.

6. The tubing kit according to claim 5, wherein the first segment of the tubing is configured to be connected to the first peristaltic pump head, and the second segment of the tubing is configured to be connected to the second peristaltic pump head.

7. The tubing kit according to any one of claims 1 to 5, wherein the second portion of the tubing comprises a third segment of the tubing and a fourth segment of the tubing, and the third portion of the tubing and the fourth portion of the tubing are fluidly connected in parallel.

8. The tubing kit according to claim 7, wherein the third segment of the tubing is configured to be connected to a third peristaltic pump head, and the fourth segment of the tubing is configured to be connected to a fourth peristaltic pump head.

9. The tubing kit according to any one of claims 1 to 8, wherein the mixing device comprises an input unit fluidly connected to a first portion of the tubing, an input unit fluidly connected to a second portion of the tubing, and an output unit fluidly connected to the mixture container.

10. The tubing kit according to any one of claims 1 to 9, wherein the mixing device comprises a Y-shaped connector, a helical mixing device, or a static mixing device.

11. The tubing kit according to any one of claims 1 to 10, further comprising: a first damping device connector for fluidly connecting a first portion of the tubing to the single tubing damping device and the mixing device; and a second damping device connector for fluidly connecting a second portion of the tubing to the single tubing damping device and the mixing device.

12. The tubing kit according to any one of claims 1 to 11, wherein the mixture container is a bag, a container, or a bottle.

13. A system for forming a pharmaceutical composition or a mixture of pharmaceutical compositions, A first container for containing a first pharmaceutical composition, A second container for containing a second pharmaceutical composition, A first portion of the tubing fluidly connected to the first container, The second portion of the tubing, which is fluidly connected to the second container, A single tubing damping device is fluidically connected to a first portion of the tubing and to a second portion of the tubing, and contains a certain volume of sealed fluid. A mixing device for mixing the first pharmaceutical composition from the first portion of the tubing with the second pharmaceutical composition from the second portion of the tubing, A mixing container for collecting the mixed first pharmaceutical composition and second pharmaceutical composition from the mixing apparatus, A first peristaltic pump head connected to the first portion of the tubing for pressurizing the first pharmaceutical composition from the first container containing the first pharmaceutical composition to the mixture container, A second peristaltic pump head connected to the second portion of the tubing for pressurizing the second pharmaceutical composition from the second container containing the second pharmaceutical composition to the mixture container, and Equipped with, The single tubing damping device is fluidly connected to the first and second peristaltic pump heads. system.

14. A method for transferring a pharmaceutical composition using a peristaltic pump, Using a first peristaltic pump, the first composition is pumped from the first container through the first portion of the tubing. Using a second peristaltic pump, the second composition is pumped from the second container through the second portion of the tubing. A single tubing damping device, which is fluidically connected to the first portion of the tubing and to the second portion of the tubing, and contains a sealed volume of fluid, is used to dampen the pulsating flow in the fluid flow of the first composition in the first portion of the tubing and to dampen the pulsating flow in the fluid flow of the second composition in the second portion of the tubing, Methods that include...

15. A method for transferring a pharmaceutical composition using a peristaltic pump, The first composition is pumped from the first container through the first portion of the tubing at a first flow rate using a first peristaltic pump, The second composition is pumped from the second container through the second portion of the tubing using a second peristaltic pump at a second flow rate, Attenuating the pulsating flow in the fluid flow of the first composition in the first portion of the tubing and the pulsating flow in the fluid flow of the second composition in the second portion of the tubing, using a single tubing damper containing a certain volume of sealed fluid, which is fluidly connected to the first and second peristaltic pumps, the flow rate pulsation level (LoP) of the first flow rate in the first portion of the tubing behind the single tubing damper is less than 10, and the flow rate pulsation level (LoP) of the second flow rate in the second portion of the tubing behind the single tubing damper is less than 10. Methods that include...

16. A method for producing a pharmaceutical composition comprising nucleic acids and one or more lipids, A first composition containing nucleic acid is pumped from a first container through a first portion of tubing using a first peristaltic pump head at a first flow rate, At a second flow rate, using a second peristaltic pump head, a second composition containing one or more lipids is pumped from a second container through a second portion of the tubing. A single tubing damping device, which is fluidically connected to the first portion of the tubing and to the second portion of the tubing, and contains a sealed volume of fluid, is used to dampen the pulsating flow in the fluid flow of the first composition in the first portion of the tubing and to dampen the pulsating flow in the fluid flow of the second composition in the second portion of the tubing, Mixing the first composition containing the nucleic acid from the first portion of the tubing and the second composition containing one or more lipids from the second portion of the tubing in a single mixing device fluidly connected to the first and second portions of the tubing, The composition comprising the nucleic acid and one or more lipids is deposited in a container fluidly connected to the mixing device. Methods that include...

17. A tubing kit for forming a mixture, A first portion of tubing configured to be fluidly connected to a first container containing a first composition, A second portion of tubing configured to be fluidly connected to a second container containing a second composition, A single tubing damping device containing a certain volume of fluid is fluidly connected to the first and second peristaltic pump heads, and is fluidly connected to the first and second portions of the tubing and the second portion of the tubing. A mixing device, which is fluidly connected downstream from the single tubing damping device to a first portion and a second portion of the tubing, and is configured to mix the first composition from the first portion of the tubing with the second composition from the second portion of the tubing, A mixture container fluidly connected to the mixing device, configured to collect the mixed first and second compositions from the mixing device, Equipped with, The first portion of the tubing is configured to be connected to the first peristaltic pump head upstream of the single tubing damper for pumping the first composition from the first container to the mixture container, and the second portion of the tubing is configured to be connected to the second peristaltic pump head upstream of the single tubing damper for pumping the second composition from the second container to the mixture container. Tubing kit.

18. A system for forming a pharmaceutical composition or a mixture of pharmaceutical compositions, A first container for containing a first pharmaceutical composition, A second container for containing a second pharmaceutical composition, A first portion of the tubing fluidly connected to the first container, The second portion of the tubing, which is fluidly connected to the second container, A peristaltic pump comprising: a first peristaltic pump head connected to a first portion of the tubing for pumping the first pharmaceutical composition from the first container; and a second peristaltic pump head connected to a second portion of the tubing for pumping the second pharmaceutical composition from the second container; A single tubing damping device containing a certain volume of fluid is fluidly connected to the first and second peristaltic pump heads, and is fluidly connected to the first and second peristaltic pump heads, and is fluidly connected to the first and second portions of the tubing downstream from the first and second peristaltic pump heads, A mixing device, which is fluidly connected downstream from the single tubing damping device to a first portion and a second portion of the tubing, and is configured to mix the first pharmaceutical composition from the first portion of the tubing with the second pharmaceutical composition from the second portion of the tubing, A mixture container fluidly connected to the mixing device, configured to collect the mixed first pharmaceutical composition and second pharmaceutical composition from the mixing device, A system that includes these features.

19. A method for transferring a pharmaceutical composition using a peristaltic pump, Using a first peristaltic pump head, the first composition is pumped from the first container through the first portion of the tubing. Using a second peristaltic pump head, the second composition is pumped from the second container through the second portion of the tubing. A single tubing damper, which is fluidically connected to the first and second peristaltic pump heads, and which contains a sealed volume of fluid, is used to dampen the pulsations in the fluid flow of the first composition in the first portion of the tubing downstream of the first peristaltic pump head and to dampen the pulsations in the fluid flow of the second composition in the second portion of the tubing downstream of the second peristaltic pump head. Mixing the first composition from the first portion of the tubing and the second composition from the second portion of the tubing in a mixing device that is fluidly connected downstream from the single tubing damping device to the first portion of the tubing and the second portion of the tubing, The composition comprising the mixed first composition and the second composition is deposited in a container fluidly connected to the mixing device, Methods that include...

20. A tubing kit for forming a mixture, A first portion of tubing configured to be fluidly connected to a container containing a first composition, A second portion of tubing configured to be fluidly connected to a container for containing a second composition, A single tubing damping device is fluidically connected to the first and second portions of the tubing and contains a certain volume of fluid sealed inside, A first damping device connector fluidly connects the first portion of the tubing to the single tubing damping device and mixing device, The second portion of the tubing is connected to the single tubing damping device and the mixing device via a second damping device connector, A mixing apparatus for mixing the first composition from the first portion of the tubing with the second composition from the second portion of the tubing, A mixing container for collecting the first composition and the second composition mixed from the mixing device, Equipped with, A tubing kit wherein a first portion of the tubing is configured to be connected to a first peristaltic pump head for pumping the first composition from a container containing the first composition to a mixture container, and a second portion of the tubing is configured to be connected to a second peristaltic pump head for pumping the second composition from a container containing the second composition to the mixture container, and the single tubing damping device is fluidly connected to the first and second peristaltic pump heads.

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