Modular industrial units for pharmaceutical production

Abstract

A portable system for producing a formulation including RNA encapsulated in lipid nanoparticles (LNPs) comprises a first subsystem having a plurality of drug substance formulation modules, the first subsystem comprising a transcription module for forming an RNA solution by in vitro transcription, and a second subsystem operable downstream of the first subsystem having a plurality of drug product formation modules, the second subsystem comprising an LNP formulation module for producing a first RNA-LNP preparation from the RNA solution, each of the transcription module and the LNP formulation module being housed within a separate standard shipping container.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 293,480, filed December 23, 2021, U.S. Provisional Patent Application No. 63 / 301,834, filed January 21, 2022, U.S. Provisional Patent Application No. 63 / 419,690, filed October 26, 2022, and U.S. Provisional Patent Application No. 63 / 420,508, filed October 28, 2022, each entitled "Modular Drug Production System," the contents of each of which are incorporated by reference in their entirety herein. [Background technology]

[0002] Nucleic acids represent an important therapeutic modality. Lipid nanoparticle technology has proven particularly useful for the delivery of nucleic acid therapeutics, including specifically RNA therapeutics. The ability to supply lipid nanoparticles, nucleic acids, and / or drug products derived therefrom (and other drug products) in a time-limited manner is often constrained by the manufacturing capacity of the drug product, as well as the ability to rapidly adapt the manufacturing of the drug product to current needs. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides techniques related to modular drug product manufacturing, including lipid nanoparticle (LNP) compositions, and other drug products. Rapidly producing and providing treatments and / or procedures to patients often involves challenges. The challenges relate to, but are not limited to, manufacturing capacity, manufacturing flexibility, the overall time required to produce and deliver a drug product to a patient, and other considerations. Often, local requirements related to drug certification and approval categorize drug products differently depending on whether the drug product is produced domestically or imported from another country. For example, drugs produced domestically may have different regulatory approval hurdles than drugs that are imported.

[0004] The present embodiment includes a modular drug production system that can be shipped anywhere in the world where standard shipping (i.e., "overseas") containers can be shipped. The modular drug production system of the present embodiment is particularly useful for the production of RNA-LNP drug products, but also for many other types of drug products. The modular drug production system of the present embodiment allows for drug production within a country and / or localized region, thereby allowing drug products to be manufactured exactly where they are needed to address localized outbreaks and virus strains. Additionally, the modular drug production system of the present embodiment allows for an increase in overall drug manufacturing capacity, reducing the demand and / or pressure on centralized large-scale drug manufacturing facilities to take on small-scale production operations (i.e., to address regional and / or localized virus strains), which may be adversely affected for large-scale production (which is primarily focused on serving the greatest number of people in the most efficient manner).

[0005] In one aspect, an embodiment of the present disclosure is directed to a biopharmaceutical manufacturing facility comprising at least one biopharmaceutical production unit comprising two or more modularly arranged containment units housing one or more apparatuses adaptable for: (1) receiving and processing an active ingredient or active ingredient precursor, and a pharmaceutically acceptable carrier or excipient, and producing the resultant biopharmaceutical product; (2) quality control of the biopharmaceutical product; and (3) filling and finishing of the biopharmaceutical product, wherein the two or more modularly arranged containment units are arranged and optionally interconnected for inter-unit transfer of unfinished biopharmaceutical product and supply of finished biopharmaceutical product.

[0006] In some embodiments, the facility includes 1-10 production units (i.e., containers or modules), each of which comprises: (a) a first storage unit housing one or more devices adaptable for receiving and processing an active ingredient or active ingredient precursor, and a pharma- ceutically acceptable carrier or excipient, and producing a resultant biopharmaceutical product; (b) a second storage unit in communication with the first storage unit for the receipt of a biopharmaceutical product, housing one or more devices adaptable for quality control of the biopharmaceutical product; and (3) a third storage unit in communication with the second storage unit for the receipt of a quality control assessed biopharmaceutical product, housing one or more devices adaptable for filling and finishing the quality control assessed biopharmaceutical product, and delivering a finished biopharmaceutical product.

[0007] In some embodiments, at least two of said modularly arranged containment units are arranged horizontally.

[0008] In some embodiments, the manufacturing facility is at least partially enclosed in a housing. In some embodiments, both the active ingredient and the active ingredient precursor are each a nucleotide, and the processing comprises encapsulating the processed nucleotide within a lipid nanoparticle, and the resulting biopharmaceutical product is a nucleotide vaccine. In some embodiments, the active ingredient is mRNA.

[0009] In some embodiments, at least one of the containment units is an ISO container. In some embodiments, the one or more devices adaptable for receiving and processing an active ingredient or an active ingredient precursor and a pharma- ceutically acceptable carrier or excipient, and producing a resultant biopharmaceutical product, comprise a bioreactor adaptable for receiving DNA and in vitro transcription of said DNA into mRNA. In some embodiments, the one or more devices adaptable for receiving and processing an active ingredient or an active ingredient precursor and a pharma- ceutically acceptable carrier or excipient, and producing a resultant biopharmaceutical product, comprise a purification device configured to remove DNA impurities from said mRNA.

[0010] In some embodiments, the biopharmaceutical manufacturing facility produces about 1 million to about 50 million doses of the finished mRNA vaccine per year, or about 3,000 to about 170,000 doses of the finished mRNA vaccine per day. In some embodiments, the equipment adaptable for (1) receiving and processing an active ingredient or active ingredient precursor and a pharma- ceutical acceptable carrier or excipient, and producing the resulting biopharmaceutical product, (2) quality control of the biopharmaceutical product, and (3) filling and finishing the quality control assessed biopharmaceutical product, is in electronic communication with a process control system for remote operation. In some embodiments, the electronic communication is wireless, the process control is at least partially automated, the process control system is controlled by one or more computers, and / or the one or more computers are remote from the facility.

[0011] In another aspect, embodiments of the present disclosure are directed to a portable system for producing a formulation comprising RNA encapsulated in lipid nanoparticles (LNPs), comprising: a first subsystem comprising a plurality of drug substance formulation modules, the first subsystem comprising a transcription module for forming an RNA solution by in vitro transcription (e.g., of a DNA template); and a second subsystem operative downstream of the first subsystem comprising a plurality of drug product formation modules, the second subsystem comprising an LNP formulation module for producing a first RNA-LNP preparation from the RNA solution, wherein each of the transcription module and the LNP formulation module are contained (or transported to a facility) within separate standard shipping containers.

[0012] In some embodiments, each of the separate standard shipping containers comprises a width of about 8 feet (2.43 m), a height of about 8.5 feet (2.59 m), and a length of about 20 feet (6.06 m) to about 40 feet (12.12 m).

[0013] In some embodiments, the LNP formulation module comprises at least one impinging jet mixing unit. In some embodiments, the second subsystem further comprises a purification module operatively disposed downstream of the LNP formulation module, the purification module comprising at least one tangential flow filtration (TFF) unit, the purification modules being disposed within separate standard shipping containers. In some embodiments, the first subsystem and the second subsystem each comprise a bioburden reduction module disposed within separate standard shipping containers, each bioburden reduction module comprising a filtration unit comprising at least one filter having a pore size of about 0.05 μm to about 0.35 μm.

[0014] In another aspect, embodiments of the present disclosure are directed to a portable system for producing a formulation comprising RNA encapsulated in lipid nanoparticles (LNPs), the system comprising: a first module comprising a transcription module for forming an RNA solution by in vitro transcription; a second module comprising a first purification module comprising a first tangential flow filtration (TFF) unit, the second module being operatively downstream of the first module to receive the RNA solution therefrom; a third module comprising a first bioburden reduction module, the third module being operatively downstream of the second module to generate a first RNA-LNP preparation from the RNA solution; a fourth module comprising an LNP formulation module for producing the first RNA-LNP preparation, the fourth module operatively downstream of the third module; a fifth module comprising a second purification module comprising a second tangential flow filtration (TFF) unit, the fifth module operatively downstream of the fourth module and receiving the first RNA-LNP preparation therefrom; and a sixth module comprising a second bioburden reduction module, the sixth module operatively downstream of the fifth module, wherein each of the first through sixth modules are placed (and / or transported to a production facility) within a separate standard shipping container.

[0015] In another aspect, an embodiment of the present disclosure is directed to a drug production system, the system comprising: a drug substance module for producing at least one drug substance; and a drug product module for producing a drug product at least in part from the at least one drug substance, each of the drug substance module and the drug product module being entirely disposed within one or more portable shipping containers, and the drug product is used for treating and / or vaccinating a patient within 1, 2, 4, 8, 12, 24, 48, and / or 72 hours after production.

[0016] In another aspect, embodiments of the present disclosure are directed to a portable LNP formulation system for producing a first RNA-LNP preparation, the system comprising an impinging jet mixing unit, a first fluid conduit for supplying an RNA solution to the impinging jet mixing unit, the first fluid conduit fluidly connecting the impinging jet mixing unit to a source of RNA solution external to the system, a second fluid conduit for supplying a lipid solution to the impinging jet mixing unit, the second fluid conduit fluidly connecting the impinging jet mixing unit to a source of lipid solution external to the system, and a third fluid conduit for supplying the first RNA-LNP preparation to a downstream module external to the system, the system being located (and / or transported to a production facility) within a single standard shipping container.

[0017] In another aspect, embodiments of the present disclosure are directed to a portable LNP formulation system for producing a first RNA-LNP preparation, the system comprising an impinging jet mixing unit and a tangential flow filtration (TFF) unit fluidly coupled downstream of the impinging jet mixing unit, the TFF unit for performing at least one diafiltration step and at least one ultrafiltration step, the system being placed (and / or transported to a production facility) within a single standard shipping container.

[0018] In some embodiments, the system comprises a bioburden reduction unit fluidly coupled downstream of the TFF unit and housed within the standard shipping container.

[0019] In some embodiments, the system comprises a first fluid conduit for supplying an RNA solution to the impinging jet mixing unit, the first fluid conduit fluidly connecting the impinging jet mixing unit to a source of RNA solution external to the system, a second fluid conduit for supplying a lipid solution to the impinging jet mixing unit, the second fluid conduit fluidly connecting the impinging jet mixing unit to a source of lipid solution external to the system, and a third fluid conduit for supplying a first RNA-LNP preparation to the TFF unit.

[0020] In another aspect, embodiments of the present disclosure are directed to a drug production system comprising: a drug substance module for producing at least one drug substance; and a drug product module for producing a drug product including the at least one drug substance, each of the drug substance module and the drug product module being entirely disposed within one or more portable shipping containers.

[0021] In some embodiments, each of the drug substance module and the drug product module are disposed within at least three portable shipping containers. In some embodiments, the drug product includes at least one lipid nanoparticle (LNP). In some embodiments, the system comprises at least one fill and finish module for disposing the drug product in at least one container.

[0022] In some embodiments, the combined power requirements of the drug substance module and the drug product module range from about 200 kW to about 400 kW, and the uninterruptible power requirements range from about 50 kW to about 100 kW.

[0023] In some embodiments, the combined footprint of the drug substance module and the drug product module encompasses an area of ​​about 500 square meters to about 1000 square meters.

[0024] In some embodiments, each of the drug substance module and the drug product module includes at least one airlock through which materials and / or personnel must pass to enter operational areas within the drug substance module and the drug product module, respectively.

[0025] In some embodiments, the system comprises a quality control module comprising a PCR lab, an RNA / DNA lab, an environmental monitoring console, an HPLC lab, a cell culture lab, a general procedure lab, a freezer monitoring device, a bioburden lab, a quality control storage area, a cleaning area, an endotoxin lab, and / or a gowning area.

[0026] In particular, the present disclosure identifies the causes of problems that may be encountered with lipid nanoparticle compositions. Without wishing to be bound by any particular theory, the present disclosure proposes that failure to avoid, detect, or remove air from an LNP composition (e.g., a nucleic acid-LNP composition, specifically an RNA-LNP composition) may result in one or more deleterious effects, such as, for example, loss of colloidal stability, impeded ability to pass through filters, etc. Furthermore, the present disclosure provides insight that certain adverse effects may occur and / or may be particularly significant when the relevant compositions are prepared on a large scale.

[0027] The present disclosure provides techniques for improving the production, transport, and / or storage of LNPs. Those skilled in the art will appreciate the importance and breadth of applicability of its teachings upon reading this disclosure.

[0028] In particular, those skilled in the art will appreciate the growing importance of nucleic acid therapeutics (e.g., oligonucleotide therapeutics, as well as longer DNA and / or RNA therapeutics), including the transformative impact of RNA vaccines during the COVID19 pandemic.

[0029] Those skilled in the art will further appreciate the importance of delivery technologies, particularly LNP delivery technologies, to the success of nucleic acid therapeutics (specifically including therapeutic RNA, such as therapeutic mRNA).

[0030] In some embodiments, the provided technology is useful for the manufacture of pharmaceutical grade RNA therapeutics. In some embodiments, the provided technology can be particularly useful for large-scale manufacture of RNA therapeutics, such as pharmaceutical grade RNA therapeutics.

[0031] Among other things, in some embodiments, the present disclosure identifies the source of one or more challenges that may be associated with the manufacture and / or maintenance of certain LNP compositions, including specifically RNA-LNP compositions. Among other things, the present disclosure provides techniques that facilitate consistent manufacture, for example, meeting predetermined in-process control and / or lot release specifications (e.g., high purity, integrity, potency, etc.). In some embodiments, the present disclosure provides robust manufacturing techniques for LNP (e.g., RNA-LNP) compositions, including techniques that can be performed at large scale while maintaining certain product attributes, such as high purity, integrity, stability (e.g., stability to transport and / or storage). In some embodiments, the relevant product attributes may be or may include, for example, colloidal stability, particle size (and / or size distribution), LNP topology, suitability for further processing and / or formulation, efficacy of delivery of encapsulated materials from an administered composition, etc.

[0032] Among other things, in some embodiments, the present disclosure provides techniques for avoiding the introduction of air in various stages and / or processes associated with the manufacture of LNP compositions. In some embodiments, the manufacture described herein can include one or more of lipid stock preparation, preparation of stocks of encapsulated agents (e.g., nucleic acid agents, such as RNA agents), agent (e.g., nucleic acid, e.g., RNA)-LNP formulation, stabilization (e.g., by dilution), concentration, purification or separation (e.g., buffer exchange and / or filtration), concentration adjustment, addition of one or more excipients (e.g., cryoprotective agents), aseptic filling, labeling, storage, and / or characterization of the LNP-containing drug product and / or one or more components thereof, any or all of which may be performed under predetermined conditions and parameters that provide for large-scale bulk throughput of the drug product (e.g., as described herein) while maintaining product attributes (e.g., as described herein).

[0033] In some embodiments, the techniques described herein can be utilized at various manufacturing scales, hi some embodiments, the techniques described herein can be utilized in parallel to further increase throughput capacity.

[0034] In some embodiments, the technology provided herein can utilize nucleic acids (e.g., RNA) produced in batch sizes within the range of about 0.01 g to about 500 g, about 0.01 g to about 10 g, about 1 g to about 10 g, about 10 g to about 500 g, about 10 g to about 300 g, about 10 g to about 200 g, or about 30 g to about 60 g.

[0035] Among other things, in some embodiments, the present disclosure provides methods for characterizing manufactured LNP products (e.g., nucleic acid-LNP products, such as RNA-LNP products) suitable for use in pharmaceutical products.

[0036] In some embodiments, the present disclosure provides, inter alia, techniques (e.g., systems and / or methods) for reducing the introduction of air into drug product manufacturing processes, thereby reducing the likelihood of destroying and / or otherwise affecting LNP properties (examples of which include, for example, the encapsulation (e.g., of nucleic acids, particularly RNA) within lipid nanoparticles and / or the stability (e.g., colloidal stability) of the nanoparticle preparation), particularly at large scale.

[0037] While not wishing to be bound by any particular theory, the present disclosure proposes that, in some embodiments, the air-liquid interface causes disruption of one or more features of the LNP structure (e.g., separation of the PEG lipids from other lipid components and / or other change(s) that may cause collapse or instability of the LNP colloid and / or the LNPs therein).

[0038] In some embodiments, the probability of such adverse effect(s) is reduced by the use of various systems and / or methodologies to prevent the introduction of air into the manufacturing process at one or more stages. Without wishing to be bound by a particular theory, in some embodiments, additional systems and / or process steps may be utilized in connection with at least three manufacturing steps to avoid the introduction of air, examples of which include 1) impinging jet mixing, 2) tangential flow filtration (TFF), and 3) product transportation (i.e., intermediate drug products, e.g., not necessarily final products, being transported from one facility to another). In some embodiments, additional systems and / or process steps may be utilized in connection with a fourth step, including final filling and finishing, to avoid the introduction of air. In some embodiments, an aqueous solution of RNA and a lipid solution are mixed under operating conditions sufficient to form a first LNP preparation such that substantially no air is introduced. In some embodiments, air bubbles are manually removed from the transport bag by a syringe. In some embodiments, one or more LNP preparations may include a buffer exchange configured to substantially prevent the introduction of air. In some embodiments, TFF may include the use of one or more jejunostomy tubes and / or one or more dip tubes configured to avoid the introduction of air into the one or more LNP preparations.

[0039] In some embodiments, the disclosure provides techniques (i.e., systems and methods) for producing RNA encapsulated in lipid nanoparticles (LNPs), examples of which include flowing an aqueous solution of RNA and a lipid solution independently into a mixing unit, and mixing the aqueous solution of RNA and the lipid solution in the mixing unit under operating conditions sufficient to form a first RNA-LNP preparation comprising LNP-encapsulated RNA and to introduce substantially no air into the first RNA-LNP preparation. In some embodiments, the disclosure provides techniques (i.e., systems and methods) for producing formulations comprising RNA encapsulated in lipid nanoparticles (LNPs), examples of which include an impinging jet mixing unit configured to mix an aqueous solution comprising RNA and a lipid solution to produce a first RNA-LNP preparation comprising LNP-encapsulated RNA. The system may include one or more tangential flow filtration units configured to exchange solvent and / or concentrate the first RNA-LNP preparation to produce a second RNA-LNP preparation, and one or more jejunostomy tubes in fluid communication with the impinging jet mixing unit and / or the one or more tangential flow filtration units configured to transport liquid within the system without substantially introducing air into the liquid.

[0040] The techniques described herein can be useful for producing LNP compositions (e.g., LNP compositions that incorporate nucleic acids, particularly RNA such as mRNA). In some embodiments, the techniques described herein can be useful for producing LNP compositions (e.g., nucleic acid LNPs, e.g., RNA-LNPs) for the treatment and / or prevention of a disease, disorder, or condition (e.g., cancer, infectious disease, disease associated with protein deficiency, etc.).

[0041] In some embodiments, the techniques described herein can be useful for producing LNP compositions that contain or deliver a nucleic acid encoding a polypeptide, hi some embodiments, the techniques described herein can be useful for producing LNPs to induce an immune response against an antigen (e.g., an antigen encoded by a nucleic acid contained in or delivered by the LNP composition).

[0042] In some embodiments, the present disclosure is directed to a system comprising at least one DNA sequencer for sequencing at least one local strain of a disease.

[0043] In some embodiments, the system comprises at least one DNA synthesizer for creating at least one custom DNA molecule.

[0044] In some embodiments, the system comprises at least one computing system for performing at least one of the following tasks: uploading sequence information describing at least one local strain of a disease to a public database; downloading sequence information describing the at least one local strain of a disease from the public database; downloading DNA synthesis data from the public database for use in creating a vaccine targeting the at least one local strain of a disease; and calculating a target strain on which the DNA synthesis data is based based on the sequence information describing the at least one local strain of a disease.

[0045] In another aspect, the present disclosure is directed to a method of producing a vaccine for treating a local strain of a disease, the method comprising: filtering genomic data of the disease near at least one location thereby generating local data; determining a target strain from the local data; transmitting DNA synthesis instructions to a facility within or near the at least one location; and producing the vaccine for treating the local strain based on the DNA synthesis instructions within or near the at least one location.

[0046] In some embodiments, the disease is SARS-CoV-2.

[0047] In some embodiments, the method includes administering the vaccine and distributing the vaccine in or near the at least one location.

[0048] In some embodiments, the method includes accessing a publicly available database that stores the genomic data to be filtered.

[0049] In some embodiments, the method includes sequencing a sample of the local strain of the disease within or near the at least one location, thereby generating local strain sequence data, and uploading the local strain sequence data to a publicly available database that stores the filtered genomic data.

[0050] In some embodiments, the method includes filtering the genomic data based on at least one of a date range and a look-back period, wherein the date range and / or the look-back period correspond to a period during which a localized outbreak of the disease occurred.

[0051] In some embodiments, the method comprises assessing the deviation between the local data and a baseline variant of the disease.

[0052] In some embodiments, the method includes comparing the variance of one or more subsets within the local data.

[0053] In some embodiments, the method includes assessing a level of commonality of the variations of the one or more subsets within the local data.

[0054] In some embodiments, determining a target strain from the local data comprises determining a target strain based at least in part on the deviation between the local data and the baseline variant of the disease.

[0055] In some embodiments, the technology described herein may be useful for the manufacture of RNA-LNP compositions for the treatment and / or prevention of coronavirus infections, such as SARS-CoV-2 infections, as described in Walsh et al. "RNA-based COVID-19 vaccine BNT162b2 selected for a pivotal efficacy study" medRxiv preprint (2020), available online at https: / / doi.org / 10.1101 / 2020.08.17.20176651; and Milligan et al. "Phase I / II study of COVID-19 RNA vaccine BNT162b1 in adults" Nature (2020 August), available online at https: / / doi.org / 10.1038 / s41586-020-2639-4, the contents of each of which are incorporated by reference in their entirety. [Brief description of the drawings]

[0056] [Figure 1] 1 illustrates an exemplary modular drug production system according to an aspect of the present disclosure. [Diagram 2]1 illustrates an overview of an exemplary drug product manufacturing entity and / or process according to an embodiment of the present disclosure. [Diagram 3] 1 illustrates an overview of an exemplary drug product manufacturing facility and / or process according to an embodiment of the present disclosure. [Figure 4] 1 illustrates an overview of an exemplary pharmaceutical product manufacturing facility according to an aspect of the present disclosure. [Diagram 5] 1 illustrates an overview of an exemplary pharmaceutical product manufacturing facility and / or modules according to an embodiment of the present disclosure. [Figure 6] 1 illustrates an overview of an exemplary pharmaceutical product manufacturing facility and / or modules according to an embodiment of the present disclosure. [Figure 7] 1 illustrates an overview of an exemplary drug product quality control facility and / or module according to an embodiment of the present disclosure. [Figure 8] 1 illustrates an overview of an exemplary drug product warehouse facility and / or modules according to an embodiment of the present disclosure. [Figure 9] 1 illustrates an overview of an exemplary pharmaceutical product manufacturing facility according to an aspect of the present disclosure. [Figure 10] 1 illustrates an overview of an exemplary pharmaceutical product manufacturing facility according to an aspect of the present disclosure. [Figure 11] 1 shows an overview of an exemplary manufacturing process for a pharmaceutical grade composition comprising RNA, according to an embodiment of the present disclosure. [Figure 12] 1 shows an overview of an exemplary DNA template production process by a PCR-based process, according to an embodiment of the present disclosure. [Figure 13] 1 illustrates an exemplary process for producing an LNP composition according to an embodiment of the present disclosure. [Figure 14] 1 illustrates an overview of an exemplary pharmaceutical product manufacturing facility and / or modules according to an embodiment of the present disclosure. [Figure 15] 1 illustrates an overview of an exemplary pharmaceutical product manufacturing facility and / or modules according to an embodiment of the present disclosure. [Figure 16] 1 illustrates an overview of an exemplary pharmaceutical product manufacturing facility and / or modules according to an embodiment of the present disclosure. [Figure 17] 1 illustrates a process for making a vaccine according to an embodiment of the present disclosure. [Figure 18]1 illustrates a process for making a vaccine according to an embodiment of the present disclosure. [Figure 19] 1 illustrates an overview of an exemplary drug product manufacturing entity and / or process according to an embodiment of the present disclosure. [Figure 20] 1 illustrates an overview of an exemplary drug product manufacturing entity and / or process according to an embodiment of the present disclosure. [Figure 21] 1 illustrates an overview of an exemplary drug product manufacturing entity and / or process according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] definition About or Approximately: The term "about" or "approximately" used herein in reference to a value refers to a value that is similar in context to the stated reference value. Generally, a person skilled in the art who is familiar with the context will understand the relevant degree of variation that "about" or "approximately" encompasses in that context. For example, in some embodiments, the term "about" or "approximately" may encompass a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the referenced value.

[0058] Administration: As used herein, the term "administration" generally refers to the administration of a composition to a subject or system. Those skilled in the art are aware of the various routes that may be utilized for administration to a subject, e.g., a human, in the appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (e.g., one or more of or including topical to the dermis, intradermal, intradermal, transdermal, etc.), intestinal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, subarachnoid, intravenous, intraventricular, in a particular organ (e.g., in the liver), mucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may be intramuscular. In some embodiments, administration may involve intermittent administration (e.g., multiple administrations separated in time) and / or periodic administration (e.g., individual administrations separated by a common period of time). In some embodiments, administration may involve continuous administration (e.g., perfusion) for at least a selected period of time.

[0059] Agent: Generally, the term "agent" as used herein is used to refer to an entity (e.g., lipids, metals, nucleic acids, polypeptides, polysaccharides, small molecules, etc., or complexes, combinations, mixtures, or systems thereof (e.g., cells, tissues, organisms)) or a phenomenon (e.g., heat, electric current or electric field, magnetic force or magnetic field, etc.). In appropriate circumstances, as will be clear to one of skill in the art from the context, the term may be utilized to refer to an entity that is or includes a cell or organism, or a fragment, extract, or component thereof. Alternatively or additionally, as will be clear from the context, the term may be utilized to refer to a natural product found in and / or obtained from nature. In some cases, also as will be clear from the context, the term may be utilized to refer to one or more entities that have been created by man (in that they have been designed, engineered, and / or produced by the action of the human hand, and / or that are not found in nature). In some embodiments, the agent may be utilized in an isolated or pure form, and in some embodiments, the agent may be utilized in a crude form. In some embodiments, potential agents may be provided as a collection or library that can be screened, for example, to identify or characterize active agents within. In some cases, the term "agent" may refer to a compound or entity that is or includes a polymer, and in some cases, the term may refer to a compound or entity that does not include one or more polymeric moieties. In some embodiments, the term "agent" may refer to a compound or entity that is not a polymer and / or does not include substantially any polymer and / or does not include one or more specific polymeric moieties. In some embodiments, the term may refer to a compound or entity that is also devoid of or substantially free of polymeric moieties.

[0060] Analog: As used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity with a reference substance, e.g., sharing a core structure or consensus structure, but differs in a specific individual manner. In some embodiments, an analog is a substance that can be generated from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated by performing a synthetic process that is substantially similar (e.g., sharing multiple steps) to the one that generates the reference substance. In some embodiments, an analog is generated or can be generated by performing a synthetic process that is different from the one used to generate the reference substance.

[0061] Antibody Agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations.For example, in embodiments, antibody agents utilized in accordance with the present disclosure include intact IgA, IgG, IgE or IgM antibodies, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs) or sets thereof, single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies® minibodies, BiTE®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it has when naturally produced. In some embodiments, antibodies may include covalent modifications (e.g., attachment of glycans), payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.). In many embodiments, antibody agents are or include polypeptides having an amino acid sequence that includes one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs).In some embodiments, the antibody agent is or comprises a polypeptide having an amino acid sequence that comprises at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are either identical in sequence or contain 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDRs has been deleted, added, or substituted compared to the reference CDR, but the included CDRs otherwise have the same amino acid sequence as the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids in the included CDRs have been deleted, added, or substituted compared to the reference CDR, but the included CDRs otherwise have the same amino acid sequence as the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDRs has been substituted compared to the reference CDR, but the included CDRs otherwise have the same amino acid sequence as the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids in the included CDRs have been deleted, added, or substituted compared to the reference CDR, but the included CDRs otherwise have the same amino acid sequence as the reference CDR.In some embodiments, an antibody agent is or comprises a polypeptide having an amino acid sequence that includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain, hi some embodiments, an antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0062] Antibody agents can be produced by those skilled in the art using methods known in the art, commercially available services and kits. For example, methods for preparing monoclonal antibodies are well known in the art and include hybridoma and phage display techniques. Additional antibodies suitable for use in the present disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (September 30, 2013);Making and Using Antibodies: A Practical Handbook, Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (July 29, 2013);Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology). Patrick Chames. Humana Press (August 21, 2012); Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Eds. Vincent Ossipow and Nicolas Fischer. Humana Press (February 12, 2014); and Human Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Michael Steinitz. 2013)).

[0063] Antibodies may be produced by standard techniques, such as by immunization with the appropriate polypeptide or portion(s) thereof, or by using a phage display library. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide carrying the desired epitope(s), optionally haptenized to another polypeptide. Depending on the host species, various adjuvants may be used to enhance the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and surface active substances such as dinitrophenol. Serum from the immunized animals is collected and treated according to known procedures. If the serum containing polyclonal antibodies against the desired epitope contains antibodies against other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography or any other method known in the art. Techniques for producing and processing polyclonal antisera are well known in the art.

[0064] Antigen: As used herein, the term "antigen" refers to an agent that elicits an immune response and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including the production of antigen-specific antibodies), and in some embodiments, an antigen elicits a cellular response (e.g., involving T cells that have receptors that specifically interact with the antigen). In some embodiments, an antigen binds to an antibody and may or may not elicit a specific physiological response in an organism. Generally, an antigen can be or include any chemical entity, such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments, other than a biological polymer (e.g., other than a nucleic acid or amino acid polymer)), etc. In some embodiments, an antigen is or includes a polypeptide. In some embodiments, an antigen is or includes a glycan. Those skilled in the art will appreciate that, generally, antigens may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other substances, e.g., extracts such as cell extracts or other relatively crude preparations of antigen-containing sources). In some embodiments, antigens utilized in accordance with the present invention are provided in crude form. In some embodiments, the antigen is a recombinant antigen.

[0065] Binding: As used herein, the term "binding" will be understood to generally refer to a non-covalent association between two or more entities. "Direct" binding involves physical contact between the entities or moieties. Indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in any of a variety of contexts. Examples include when interacting entities or moieties are studied in isolation or in the context of a more complex system (e.g., when covalently or otherwise associated with a carrier entity and / or within a biological system or cell).

[0066] Bioreactor: As used herein, the term "bioreactor" refers to a vessel used for in vitro transcription as described herein. The bioreactor can be of any size as long as it is useful for in vitro transcription. For example, in some embodiments, the bioreactor can be at least 0.5 liters, examples of which include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 liters or more, or any volume in between. The internal conditions of the bioreactor, including but not limited to pH and temperature, are typically controlled during in vitro transcription. The bioreactor can be constructed of any material suitable for in vitro transcription under the conditions described herein, including glass, plastic, or metal. One of skill in the art can recognize and select a suitable volume of the bioreactor to use in performing in vitro transcription.

[0067] Cap: As used herein, the term "cap" refers to a structure that includes or consists essentially of a nucleoside-5'-triphosphate, typically attached to the 5' end of an uncapped RNA (e.g., an uncapped RNA with a 5'-diphosphate). In some embodiments, the cap is or includes a guanine nucleotide. In some embodiments, the cap is or includes a naturally occurring RNA 5' cap. Examples include, but are not limited to, an N7-methylguanosine cap, which has a structure designated as "m7G". In some embodiments, the cap is or includes a synthetic cap analog that resembles an RNA cap structure and has the ability to stabilize the RNA when attached to the RNA. Examples include, but are not limited to, anti-reverse cap analogs (ARCAs), which are known in the art. One of skill in the art will appreciate that methods of attaching a cap to the 5' end of an RNA are known in the art. For example, in some embodiments, capped RNA may be obtained by in vitro capping of RNA having a 5' triphosphate group or RNA having a 5' diphosphate with a capping enzyme system (examples of which include, but are not limited to, the Vaccinia capping enzyme system or the Saccharomyces cerevisiae capping enzyme system). Alternatively, capped RNA may be obtained by in vitro transcription (IVT) of a DNA template, where in addition to GTP, the IVT system also includes, for example, a cap analog, as known in the art. Non-limiting examples of cap analogs include m7GpppG cap analog, or N7-methyl-,2'-O-methyl-GpppG ARCA cap analog, or N7-methyl-,3'-O-methyl-GpppG ARCA cap analog, or any commercially available cap analog, including, for example, CleanCap (Trilink), EZ Cap, and the like. In some embodiments, the cap analog is or includes a trinucleotide cap analog.

[0068] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, states of affairs, sets of conditions, etc. that may not be identical to one another, but are similar enough to allow comparison between them, so that one of skill in the art will understand that one may reasonably draw conclusions based on observed differences or similarities. In some embodiments, comparable sets of conditions, states of affairs, individuals, or populations are characterized by a number of substantially identical characteristics and one or a few different characteristics. One of skill in the art will understand from the context what degree of identity is necessary for two or more such agents, entities, states of affairs, sets of conditions, etc. to be considered comparable in any given situation. For example, one of skill in the art will understand that sets of conditions, individuals, or populations are comparable to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of conditions, individuals, or populations are caused by or represent changes in those characteristics that have been altered.

[0069] Complementary: As used herein, the term "complementary" is used in reference to hybridization of oligonucleotides related by base pairing rules. For example, the sequence "CAGT" is complementary to the sequence "GTCA". Complementarity can be partial or total. Thus, any degree of partial complementarity is intended to be included within the scope of the term "complementary", provided that the partial complementarity allows hybridization of the oligonucleotides. Partial complementarity is a state in which one or more nucleobases are not matched according to the base pairing rules. Total or complete complementarity between nucleic acids is that each and every nucleobase matches another base under the base pairing rules.

[0070] Detection: The term "detection" is used broadly herein to include any suitable means of determining the presence or absence of an entity of interest, or any form of measurement of an entity of interest in a sample. Thus, "detection" may include determining, measuring, evaluating, or analyzing the presence or absence, level, amount, and / or location of an entity of interest. Quantitative and qualitative determinations, measurements, or evaluations, including semi-quantitative ones, are included. Such determinations, measurements, or evaluations may be relative, for example, when the entity of interest is detected compared to a control reference, or may be absolute. Thus, when used in the context of quantifying an entity of interest, the term "quantification" can refer to absolute or relative quantification. Absolute quantification may be achieved by correlating the detected level of the entity of interest with a known control standard (e.g., through the construction of a standard curve). Alternatively, relative quantification can be achieved by comparing the levels or amounts detected between two or more different entities of interest to provide a relative quantification of each of the two or more different entities of interest, i.e., relative quantification to each other.

[0071] Determining: Those of skill in the art reading this specification will understand that the step of "determining" may utilize or be accomplished by the use of any of a variety of techniques available to those of skill in the art, examples of which include, for example, the specific techniques explicitly mentioned herein. In some embodiments, determining involves the manipulation of the physical sample. In some embodiments, determining involves the consideration and / or manipulation of data or information, for example, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, determining involves receiving relevant information and / or material from a source. In some embodiments, determining involves comparing one or more characteristics of the sample or entity to a comparable reference.

[0072] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term "dosage form" may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, such amount is a unit dosage (or a whole fraction thereof) suitable for administration according to a dosing regimen (i.e., a therapeutic dosing regimen) determined to correlate with a desired or beneficial outcome when administered to a relevant population. Those skilled in the art will appreciate that the overall amount of therapeutic composition or agent to be administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0073] Encapsulated: As used herein, the term "enclose" or "enclosed" refers to at least a portion of a component being enclosed or surrounded by another material or component within the composition. In some embodiments, a component may be completely enclosed or surrounded by another material or component within the composition.

[0074] Excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition to, for example, provide or contribute to a desired property or effect (e.g., a desired consistency, delivery, and / or stabilizing effect, etc.). In some embodiments, pharmaceutical excipients suitable for addition to the LNP compositions may include, for example, salts, starch, glucose, lactose, sucrose, gelatin, sodium chloride, glycerol, propylene, glycol, water, ethanol, and the like.

[0075] Encode: As used herein, the term "encode" or "encoding" refers to sequence information of a first molecule that leads to the production of a second molecule having a defined nucleotide sequence (e.g., mRNA) or a defined amino acid sequence. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process involving a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, cDNA, or single-stranded RNA (e.g., mRNA) encodes a polypeptide if the polypeptide is produced in a cell or other biological system by transcription and translation of an mRNA corresponding to that gene. In some embodiments, the coding region of a single-stranded RNA encoding a target polypeptide agent refers to the coding strand whose nucleotide sequence is identical to the mRNA sequence of such target polypeptide agent. In some embodiments, the coding region of a single-stranded RNA encoding a target polypeptide agent refers to the non-coding strand of such target polypeptide agent that may be used as a template for transcription of a gene or cDNA.

[0076] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0077] Fed-batch processing: As used herein, the term "fed-batch processing" refers to a process in which one or more components are introduced into a vessel (e.g., a bioreactor) at some point after the start of a reaction. In some embodiments, one or more components are introduced by fed-batch processing to keep their concentration low during the reaction. In some embodiments, one or more components are introduced by fed-batch processing to replenish those that are depleted during the reaction.

[0078] 5 prime untranslated region: As used herein, the term "5 prime untranslated region" or "5'UTR" refers to the sequence of an mRNA molecule that begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region of the RNA.

[0079] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized. In some embodiments, a biological molecule can have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).

[0080] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes coding sequences (i.e., sequences that encode a particular product), and in some embodiments, a gene includes non-coding sequences. In some particular embodiments, a gene may include both coding sequences (e.g., exons) and non-coding sequences (e.g., introns). In some embodiments, a gene may include one or more regulatory elements that can, for example, control or affect one or more aspects of gene expression (e.g., cell type specific expression, inducible expression, etc.).

[0081] Gene Product or Expression Product: As used herein, the term "gene product" or "expression product" generally refers to the RNA transcribed from a gene (before and / or after processing) or the polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0082] Homologous: As used herein, the term "homologous" or "homologs" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., contain residues with related chemical properties at corresponding positions). For example, as known to those of skill in the art, certain amino acids are typically classified as being similar to one another as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid with another amino acid of the same type can often be considered a "homologous" substitution.

[0083] Host cell: As used herein, the term "host cell" refers to a cell into which exogenous material (e.g., DNA, recombinant or otherwise) has been introduced. A skilled artisan will understand upon reading this disclosure that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Since certain changes may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any kingdom of life suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions, e.g., hybridomas or quadromas. In some embodiments, the host cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the host cell is a eukaryotic cell. For example, the eukaryotic host cell may be CHO (e.g., CHO Kl, DXB-1 1 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL3A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived from the aforementioned cells.

[0084] Identity: The term "identity" as used herein refers to the overall relatedness between polymer molecules, such as between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second sequences, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding positions are then compared. If a position of the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position of the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17) incorporated in the ALIGN program (version 2.0). In some exemplary embodiments, comparison of nucleic acid sequences with the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0085] Improved, increased or reduced: As used herein, these terms, or grammatically equivalent comparative terms, refer to a relative value relative to a comparable reference measurement. For example, in some embodiments, a metric achieved with an agent of interest may be "improved" compared to a metric obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, a metric achieved in a subject or system of interest may be "improved" compared to a metric obtained in the same subject or system under different conditions (e.g., before or after an event such as administration of an agent of interest), or in a different comparable subject (e.g., in a comparable subject or system different from the subject or system of interest in the presence of one or more indicators of a particular disease, disorder, or condition of interest, or in the presence of a previous exposure to a condition, agent, etc.). In some embodiments, the comparative term refers to a statistically relevant difference (e.g., a difference in the number of patients and / or a magnitude sufficient to achieve statistical relevance). One skilled in the art will recognize or be able to readily determine the degree of difference and / or the number of patients necessary or sufficient to achieve such statistical significance in a given context.

[0086] In vitro: As used herein, the term "in vitro" refers to events that occur not within a multicellular organism but in an artificial environment, such as, for example, in a test tube or reaction vessel (e.g., a bioreactor), in cell culture, etc.

[0087] In vitro transcription: As used herein, the term "in vitro transcription" or "IVT" refers to a process in which transcription is performed outside of a cell in a non-cellular system to produce synthetic RNA products for use in a variety of applications, including, for example, the production of proteins or polypeptides. Such synthetic RNA products can be translated outside of a cell or directly introduced into a cell for translation. Such synthetic RNA products include, but are not limited to, for example, mRNA, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNAs (e.g., for CRISPR), ribosomal RNA, small nuclear RNA, small nucleolar RNA, and the like. IVT reactions typically utilize a DNA template (e.g., a linear DNA template), ribonucleotides (e.g., unmodified or modified ribonucleotide triphosphates), and a suitable RNA polymerase as described and / or utilized herein.

[0088] In vitro transcribed RNA composition: As used herein, the term "in vitro transcribed RNA composition" refers to a composition comprising target RNA synthesized by in vitro transcription. In some embodiments, such compositions may contain excess in vitro transcription reagents (e.g., including ribonucleotides and / or capping agents), nucleic acids or fragments thereof (e.g., DNA template or fragments thereof), polypeptides or fragments thereof (e.g., recombinant enzymes or host cell proteins or fragments thereof), and / or other impurities. In some embodiments, the in vitro transcribed RNA composition may have been treated and / or processed prior to formulation and / or purification steps that ultimately produce an RNA transcript preparation comprising a desired concentration of RNA transcripts in an appropriate buffer for further manufacture and / or processing. For example, in some embodiments, the in vitro transcribed RNA composition may have been treated to remove or digest the DNA template (e.g., using DNase). In some embodiments, the in vitro transcribed RNA composition may have been treated to remove or digest polypeptides (e.g., enzymes such as RNA polymerase, RNase inhibitors, etc.) present during the in vitro transcription reaction (e.g., using proteases).

[0089] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals.

[0090] Nanoparticles: As used herein, the term "nanoparticles" refers to particles having a diameter of less than 1000 nanometers (nm). In some embodiments, nanoparticles have a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, nanoparticles have a diameter of less than 100 nm, as defined by the National Institutes of Health. In some embodiments, nanoparticles have a diameter of less than 80 nm, as defined by the National Institutes of Health. In some embodiments, nanoparticles comprise one or more encapsulated compartments separated from the bulk solution by a membrane that surrounds and encapsulates the space or compartment.

[0091] Nucleic acid / polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least two or more nucleotides, for example, at least three nucleotides, at least four nucleotides, at least five nucleotides, at least six nucleotides, at least seven nucleotides, at least eight nucleotides, at least nine nucleotides, at least ten nucleotides, or more. In some embodiments, the nucleic acid is or comprises DNA. In some embodiments, the nucleic acid is or comprises RNA. In some embodiments, the nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, the nucleic acid is or comprises single-stranded nucleic acid. In some embodiments, the nucleic acid is or comprises double-stranded nucleic acid. In some embodiments, the nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, the nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, the nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, the nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, such as "peptide nucleic acids". In some embodiments, a nucleic acid comprises one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-naturally occurring residues.In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, 1-methyl-pseudouridine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template, e.g., in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis.In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 40 In some embodiments, the nucleic acid sequence is 00, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length.

[0092] Pharmaceutical Grade: As used herein, the term "pharmaceutical grade" refers to the standards established by a recognized national or regional pharmacopoeia (e.g., the United States Pharmacopeia and National Formulary (USP-NF)) for chemical and biological drug substances, drug products, dosage forms, compounded preparations, excipients, medical devices, and dietary supplements.

[0093] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three or more amino acids. Those skilled in the art will appreciate that the term "polypeptide" is intended to encompass not only polypeptides having the complete sequence described herein, but also polypeptides that represent functional, biologically active, or characteristic fragments, portions, or domains of such complete polypeptides (e.g., fragments, portions, or domains that retain at least one activity). In some embodiments, a polypeptide may include L-amino acids, D-amino acids, or both, and / or may include any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, a polypeptide may include (e.g., be or include) natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, a polypeptide may be or include an enzyme. In some embodiments, a polypeptide may be or include a polypeptide antigen. In some embodiments, a polypeptide may be or include an antibody agent. In some embodiments, a polypeptide may be or include a cytokine.

[0094] Pure or purified: As used herein, an agent or entity is "pure" or "purified" if it is substantially free of other components. For example, a preparation that contains more than about 90% of a particular agent or entity is typically considered a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure in the preparation.

[0095] Ribonucleotide: As used herein, the term "ribonucleotide" encompasses unmodified and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may contain one or more modifications, examples of which include, but are not limited to, for example, (a) terminal modifications, such as 5'-terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse conjugation, etc.), 3'-terminal modifications (e.g., conjugation, reverse conjugation, etc.), (b) base modifications, such as substitutions with modified bases, stabilized bases, destabilized bases, or bases that base-pair with a broad repertoire of partners, or conjugate bases, (c) sugar modifications (e.g., at the 2' or 4' positions) or sugar substitutions, and (d) internucleoside bond modifications, including modifications or substitutions of phosphodiester bonds. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

[0096] Ribonucleic acid (RNA): As used herein, the term "RNA" refers to a polymer of ribonucleotides. In some embodiments, the RNA is single-stranded. In some embodiments, the RNA is double-stranded. In some embodiments, the RNA includes both single-stranded and double-stranded portions. In some embodiments, the RNA can include a backbone structure as described above in the definition of "nucleic acid / polynucleotide." The RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA). In some embodiments, the RNA is an mRNA. In some embodiments where the RNA is an mRNA, the RNA typically includes a poly(A) region at its 3' end. In some embodiments where the RNA is an mRNA, the RNA typically includes a cap structure at its 5' end, e.g., as recognized in the art, for recognition and binding of the mRNA to ribosomes to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by enzymatic and / or chemical synthesis methods). In some embodiments, the RNA is single-stranded RNA. In some embodiments, the single stranded RNA may contain self-complementary elements and / or may establish secondary and / or tertiary structures. One skilled in the art will understand that when a single stranded RNA is referred to as "encoding", it means that it either contains the encoding nucleic acid sequence itself or contains the complement of the encoding nucleic acid sequence. In some embodiments, the single stranded RNA may be a self-amplifying RNA (also known as a self-replicating RNA).

[0097] Recombinant: As used herein, the term "recombinant" is intended to refer to a polypeptide that is designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is genetically transgenic or otherwise engineered to express a gene(s) or genetic components that encode and / or direct the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or a polypeptide that is prepared, expressed, created, or isolated by any other means, including splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements result from mutagenesis (e.g., in vivo or ex vivo) of known sequence elements, such as from natural or synthetic sources (e.g., germline of a source organism of interest (e.g., human, mouse, etc.)).

[0098] Reference: As used herein, the term "reference" refers to a standard or control against which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or historical control, optionally embodied in a tangible medium. Typically, as will be understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those under evaluation. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0099] RNA polymerase: As used herein, the term "RNA polymerase" refers to an enzyme that catalyzes polyribonucleotide synthesis by adding ribonucleotide units to a nucleotide chain using DNA or RNA as a template. The term refers to either the complete enzyme found in nature, or an isolated active catalytic or functional domain, or fragment thereof. In some embodiments, the RNA polymerase enzyme initiates synthesis at the 3' end of a primer or nucleic acid strand, or at a promoter sequence, and proceeds in the 5' direction along the target nucleic acid, synthesizing a strand complementary to the target nucleic acid, until synthesis terminates.

[0100] RNA transcript preparation: As used herein, the term "RNA transcript preparation" refers to a preparation comprising RNA transcripts purified from an in vitro transcribed RNA composition described herein. In some embodiments, an RNA transcript preparation is a preparation comprising pharmaceutical grade RNA transcripts. In some embodiments, an RNA transcript preparation is a preparation comprising RNA transcripts whose one or more product quality attributes have been characterized and determined to meet release and / or acceptance criteria (e.g., those described herein). Examples of such product quality attributes include, but are not limited to, appearance, RNA length, identity of drug substance as RNA, RNA integrity, RNA sequence, RNA concentration, pH, osmolality, residual DNA template, residual double-stranded RNA, bacterial endotoxins, bioburden, and combinations thereof.

[0101] Room temperature: As used herein, the term "room temperature" refers to ambient temperature. In some embodiments, room temperature is about 18°C ​​to 30°C, e.g., about 18°C ​​to 25°C, or about 20°C to 25°C, or about 20°C to 30°C, or about 23°C to 27°C, or about 25°C.

[0102] Sample: As used herein, the term "sample" generally refers to an aliquot of material obtained or derived from a source of interest, such as, for example, as described herein. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism, a plant, or an animal (e.g., a mouse). In some embodiments, the source of interest is or includes a biological tissue or biological fluid. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymphatic fluid, and / or cell-passing fluid. In some embodiments, the biological tissue or sample may be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a sample is or comprises cells obtained from a subject. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. In some embodiments, as the context will allow, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). For example, a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by applying one or more techniques to the primary sample, such as amplification or reverse transcription of nucleic acids, isolation and / or purification of specific components, etc.

[0103] Stable: The term "stable" as applied to nucleic acids and / or compositions comprising nucleic acids, e.g., encapsulated in lipid nanoparticles, means that such nucleic acids and / or compositions maintain one or more aspects of their properties (e.g., physical and / or structural properties, function, and / or activity) over a period of time under a specified set of conditions (e.g., pH, temperature, light, relative humidity, etc.). In some embodiments, such stability is maintained for a period of at least about 1 hour. In some embodiments, such stability is maintained for a period of about 5 hours, about 10 hours, about 1 day, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 10 months, about 12 months, about 24 months, about 36 months, or more. In some embodiments, such stability is maintained for a period of time ranging from about 1 day to about 24 months, from about 2 weeks to about 12 months, from about 2 months to about 5 months, etc. In some embodiments, such stability is maintained under ambient conditions (e.g., room temperature and ambient pressure). In some embodiments, such stability is maintained under physiological conditions (e.g., in vivo or at about 37° C., e.g., in serum or phosphate buffered saline). In some embodiments, such stability is maintained under low temperature storage (e.g., at about 4° C. or below, including, e.g., −20° C., or −70° C.). In some embodiments, such stability is maintained when the nucleic acid, and / or compositions comprising the nucleic acid, are protected from light (e.g., kept in the dark).

[0104] As an example, in some embodiments, the term "stable" is used with respect to a nanoparticle composition (e.g., a lipid nanoparticle composition). In such embodiments, a stable nanoparticle composition (e.g., a stable nanoparticle composition) and / or its component(s) maintain one or more aspects of its properties (e.g., physical and / or structural properties, function(s), and / or activity) over a period of time under a specified set of conditions. For example, in some embodiments, a stable nanoparticle composition (e.g., a lipid nanoparticle composition) is characterized in that the average particle size, particle size distribution, and / or polydispersity of the nanoparticles are substantially maintained (e.g., within 10% compared to the initial property(s)) over a period of time (e.g., as described herein) under a specified set of conditions (e.g., as described herein). In some embodiments, a stable nanoparticle composition (e.g., a lipid nanoparticle composition) is characterized in that there is no detectable amount of degradation products (e.g., those associated with hydrolysis and / or enzymatic digestion) after being maintained under a specified set of conditions (e.g., as described herein) over a period of time.

[0105] Synthetic: As used herein, the term "synthetic" refers to an artificial entity, or an entity created by human intervention, or an entity that is not naturally occurring but is produced synthetically. For example, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments, synthesized by solid phase synthesis. In some embodiments, the term "synthetic" refers to an entity that is made outside of a biological cell. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., RNA) that is produced by in vitro transcription using a template.

[0106] 3 prime untranslated region: As used herein, the term "3 prime untranslated region" or "3'UTR" refers to the sequence of an mRNA molecule that begins following the stop codon of the coding region of an open reading frame sequence. In some embodiments, the 3'UTR begins immediately after the stop codon of the coding region of an open reading frame sequence. In other embodiments, the 3'UTR does not begin immediately after the stop codon of the coding region of an open reading frame sequence.

[0107] Threshold level (e.g., acceptance criteria): As used herein, the term "threshold level" refers to a level used as a criterion for obtaining information about, for example, a measurement result obtained in an analysis and / or classifying a measurement result. For example, in some embodiments, the threshold level refers to a value measured in an analysis that defines a boundary between two subsets of a population (e.g., batches that meet quality control criteria vs. batches that do not meet quality control criteria). Thus, values ​​at or above the threshold level define one subset of the population, and values ​​below the threshold level define the other subset of the population. The threshold level can be determined based on one or more control samples, or across the entire population of control samples. The threshold level can be determined before, simultaneously with, or after the measurement of interest is performed. In some embodiments, the threshold level can be a range of values.

[0108] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA to which additional DNA segments can be ligated. Another type of vector is a viral vector, to which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Moreover, certain vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0109] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above may generally be performed according to conventional methods well known in the art and as described generally and more specifically in various references cited and discussed throughout this specification. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), which is incorporated herein by reference for any purpose. [Mode for carrying out the invention]

[0110] Nucleic acid therapeutics, and in particular RNA therapeutics, represent a particularly promising class of therapeutics for the treatment and prevention of a variety of diseases, examples of which include cancer, infectious diseases, and / or diseases or disorders associated with the excess or deficiency of specific proteins.

[0111] In particular, RNA therapeutics could be highly effective as vaccines to combat the COVID19 pandemic. Given the promise of this technology and its applicability to a wide variety of clinical situations, including very large-scale ones (e.g., global vaccinations and / or treatments such as those being developed for SARS-CoV-2), improvements in manufacturing techniques, especially those amenable to large-scale production, would be particularly valuable.

[0112] The development of effective delivery technologies is central to the success of nucleic acid therapeutics, and lipid nanoparticle technology has proven particularly effective (reviewed, e.g., in Cullis et al. Molecular Therapy 25:1467, July 5, 2017; see also US Patent 8058069), especially for RNA therapeutics (reviewed, e.g., in Hou et al., Nat. Rev. Mater doi.org / 10.1038 / s41578-021-00358-0, August 10, 2021).

[0113] The technology provided herein is useful, inter alia, for achieving particularly effective and / or efficient production of pharmaceutical grade LNP preparations and / or compositions (e.g., nucleic acid-LNP preparations, particularly RNA-LNP preparations), e.g., at a commercial scale and / or under commercial conditions. For example, in various embodiments, the technology provided enables and / or facilitates achievement of requirements specific to pharmaceutical grade (and / or scale) production, such as, for example, batch size and / or production rate, predetermined in-process controls and / or lot release specifications (e.g., high purity, integrity, potency, and / or stability, etc.).

[0114] The present disclosure provides techniques for producing LNP compositions (e.g., including RNA, e.g., therapeutic RNA, such as therapeutic mRNA). In some embodiments, the provided techniques are useful for producing pharmaceutical grade RNA-LNP therapeutics.

[0115] In some embodiments, the provided technology is useful for large-scale manufacturing of LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) therapeutics, e.g., pharmaceutical grade therapeutics. For example, in some such embodiments, the technology provided herein can be used to produce pharmaceutical grade batch throughputs of at least 10,000 vials of LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) therapeutics (including, e.g., at least 20,000 vials, at least 30,000 vials, at least 40,000 vials, at least 50,000 vials, at least 60,000 vials, at least 70,000 vials, at least 80,000 vials, at least 90,000 vials, at least 100,000 vials, at least 200,000 vials, at least 300,000 vials, at least 400,000 vials, at least 500,000 vials, or more). For example, in some such embodiments, the technology provided herein can be used to produce pharmaceutical grade batch throughputs of at least 50 L of LNP (e.g., nucleic acid LNPs, e.g., RNA-LNPs) therapeutic (e.g., including at least 50 L, at least 60 L, at least 70 L, at least 80 L, at least 100 L, at least 110 L, at least 120 L, at least 130 L, at least 140 L, at least 150 L, or more). In some embodiments, each vial can contain an amount of RNA drug product between 0.01 mg and 0.5 mg (e.g., 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg).

[0116] The technology described herein can be useful for producing LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions for the treatment and / or prevention of a disease, disorder, or condition (e.g., cancer, infectious disease, disease associated with a protein deficiency, etc.). In some embodiments, the technology described herein can be useful for producing LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions that include or deliver (e.g., by including and / or delivering a nucleic acid, such as an RNA, that encodes) a polypeptide.

[0117] In some specific embodiments, the technology described herein may be useful for producing LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions for inducing an immune response to an antigen. In some embodiments, the technology described herein may be useful for producing RNA-LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions for treating and / or preventing coronavirus infections, e.g., SARS-CoV-2 infections, as described below. Walsh et al. “RNA-based COVID-19 vaccine BNT162b2 selected for a pivotal efficacy study” medRxiv preprint (2020), available online at https: / / doi.org / 10.1101 / 2020.08.17.20176651; and Milligan et al. “Phase I / II study of COVID-19 RNA vaccine BNT162b1 in adults” Nature (2020 August), https: / / doi.org / 10.1038 / s41586-020-2639-4, the contents of each of which are incorporated by reference in their entireties.

[0118] Lipid Nanoparticles Those skilled in the art will recognize that lipid nanoparticles have been successful in the clinical delivery of a wide range of therapeutic agents, including, for example, small molecules and various nucleic acids (e.g., oligonucleotides, siRNA, mRNA) (reviewed, for example, in Hu et al., Nat. Rev. Mater. https: / / doi.org / 10.1038 / s41578-021-00358-0, August 10, 2021).

[0119] Various routes of administration of lipid nanoparticle compositions have been proposed and / or tested, and one of skill in the art will recognize the appropriate route for a particular composition (e.g., depending on the agent being delivered). To give some examples, in some embodiments, LNPs are administered parenterally, and most clinical studies have utilized parenteral administration, particularly intravenous, subcutaneous, intradermal, intravitreal, intratumoral, or intramuscular injection. Intrauterine injection has also been described. In some embodiments, topical administration is utilized. In some embodiments, intranasal administration is utilized.

[0120] In some embodiments, the administered LNPs are delivered to or accumulate in the liver. Given that the liver has a natural ability to produce and secrete proteins, liver delivery may prove useful for delivering LNP-encapsulated agents (and / or, in the case of nucleic acid agents, such as RNA agents, polypeptides encoded thereby) to the bloodstream. Such liver delivery has been proposed to be particularly useful for expressing proteins that are deficient, for example, in certain metabolic or hematological disorders, or proteins that are effective in eliciting an immune response (e.g., particularly an antibody response), for example, against infectious agents or cancer cells.

[0121] In some embodiments, the administered LNPs are delivered to and / or taken up by antigen presenting cells (which may be present, for example, in skin, muscle, mucosal tissue, etc.) Such administration may be particularly useful or effective in inducing T cell immunity (e.g., for treating infectious diseases and / or cancer).

[0122] In various embodiments, the lipid nanoparticles may have an average size (e.g., average diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 50 nm to about 130 nm, about 50 nm to about 110 nm, about 50 nm to about 100 nm, about 50 nm to about 90 nm, or about 60 nm to about 80 nm, or about 60 nm to about 70 nm. In some embodiments, lipid nanoparticles that may be useful according to the present disclosure may have an average size (e.g., average diameter) of about 50 nm to about 100 nm. In some embodiments, the lipid nanoparticles may have an average size (e.g., average diameter) of less than 80 nm, less than 75 nm, less than 70 nm, less than 65 nm, less than 60 nm, less than 55 nm, less than 50 nm, or less than 45 nm. In some embodiments, lipid nanoparticles that may be useful according to the present disclosure may have an average size (e.g., average diameter) of about 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0123] In some embodiments, the lipids forming the lipid nanoparticles described herein include polymer-conjugated lipids, cationic lipids, and helper neutral lipids. In some such embodiments, the total polymer-conjugated lipids may be present at about 0.5-5 mol%, about 0.7-3.5 mol%, about 1-2.5 mol%, about 1.5-2 mol%, or about 1.5-1.8 mol% of the total lipids. In some embodiments, the total polymer-conjugated lipids may be present at about 1-2.5 mol% of the total lipids. In some embodiments, the molar ratio of total cationic lipids to total polymer-conjugated lipids (e.g., PEG-conjugated lipids) may be about 100:1 to about 20:1, or about 50:1 to about 20:1, or about 40:1 to about 20:1, or about 35:1 to about 25:1. In some embodiments, the molar ratio of total cationic lipids to total polymer-conjugated lipids may be about 35:1 to about 25:1.

[0124] In some embodiments involving polymer-conjugated lipids, cationic lipids, and helper neutral lipids in the lipid nanoparticles described herein, the total cationic lipids are present at about 35-65 mol%, about 40-60 mol%, about 41-49 mol%, about 41-48 mol%, about 42-48 mol%, about 43-48 mol%, about 44-48 mol%, about 45-48 mol%, or about 46-49 mol% of the total lipid. In certain embodiments, the total cationic lipids are present at about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol% of the total lipid.

[0125] In some embodiments involving polymer-conjugated lipids, cationic lipids, and helper neutral lipids in the lipid nanoparticles described herein, the total neutral lipids are present at about 35-65 mol%, about 40-60 mol%, about 45-55 mol%, or about 47-52 mol% of the total lipids. In some embodiments, the total neutral lipids are present at 35-65 mol% of the total lipids. In some embodiments, the total non-steroidal neutral lipids (e.g., DPSC) are present at about 5-15 mol%, about 7-13 mol%, or about 9-11 mol% of the total lipids. In some embodiments, the total non-steroidal neutral lipids are present at about 9.5, 10, or 10.5 mol% of the total lipids. In some embodiments, the molar ratio of total cationic lipids to non-steroidal neutral lipids ranges from about 4.1:1.0 to about 4.9:1.0, about 4.5:1.0 to about 4.8:1.0, or about 4.7:1.0 to 4.8:1.0. In some embodiments, the total steroid neutral lipid (e.g., cholesterol) is present at about 35-50 mol%, about 39-49 mol%, about 39-46 mol%, about 39-44 mol%, or about 39-42 mol% of the total lipid. In certain embodiments, the total steroid neutral lipid (e.g., cholesterol) is present at about 39, 40, 41, 42, 43, 44, 45, or 46 mol% of the total lipid. In certain embodiments, the molar ratio of total cationic lipid to total steroid neutral lipid is about 1.5:1 to 1:1.2, or about 1.2:1 to 1:1.2.

[0126] In some embodiments, lipid compositions comprising cationic lipids, polymer-conjugated lipids, and neutral lipids can have the individual lipids present at specific molar percentages of the total lipids or in specific molar ratios (relative to each other), as described in WO2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0127] In some embodiments, the lipids forming the lipid nanoparticles include a polymer-conjugated lipid (e.g., a PEG-conjugated lipid), a cationic lipid, and a neutral lipid, where the polymer-conjugated lipid is present at about 1-2.5 mol% of the total lipid, the cationic lipid is present at 35-65 mol% of the total lipid, and the neutral lipid is present at 35-65 mol% of the total lipid. In some embodiments, the lipids forming the lipid nanoparticles include a polymer-conjugated lipid (e.g., a PEG-conjugated lipid), a cationic lipid, and a neutral lipid, where the polymer-conjugated lipid is present at about 1-2 mol% of the total lipid, the cationic lipid is present at 45-48.5 mol% of the total lipid, and the neutral lipid is present at 45-55 mol% of the total lipid. In some embodiments, the lipids forming the lipid nanoparticles include a polymer-conjugated lipid (e.g., a PEG-conjugated lipid), a cationic lipid, and a neutral lipid, including a non-steroidal neutral lipid and a steroidal neutral lipid, wherein the polymer-conjugated lipid is present at about 1-2 mol % of the total lipid, the cationic lipid is present at 45-48.5 mol % of the total lipid, the non-steroidal neutral lipid is present at 9-11 mol % of the total lipid, and the steroidal neutral lipid is present at about 36-44 mol % of the total lipid. In many such embodiments, the PEG-conjugated lipid has a structure as described in WO2017 / 075531 (see above). [ka] or a derivative thereof. In some embodiments, the PEG-conjugated lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In many such embodiments, the cationic lipid is or comprises a chemical structure selected from I-1 to I-10 in Table 1 herein, or a derivative thereof. In some embodiments, the cationic lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate). In many such embodiments, the neutral lipid comprises DSPC and cholesterol, where DSPC is a non-steroidal neutral lipid and cholesterol is a steroidal neutral lipid.

[0128] In some embodiments, the lipid nanoparticles include one or more cationic lipids (e.g., those described herein). In some embodiments, the cationic lipid nanoparticles can include at least one cationic lipid, at least one polymer-conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid).

[0129] FIG. 1 illustrates an exemplary modular drug production system 100 according to aspects of the present disclosure. In the embodiment of FIG. 1, the system 100 may include three shipping containers 102 at the bottom and another two or three shipping containers 102 stacked on top of the bottom three for a total of five or six shipping containers 102. The system 100 may include various exterior platforms 104 and / or stairs 106 as needed to allow personnel access within the system 100. The system 100 includes various equipment 108 necessary for the production of drug products. The equipment 108 may include tanks, filtration equipment, mixers, valves, sensors, process control and testing equipment (including computers), and other devices. In some embodiments, the system 100 may include HVAC (heating, ventilation, and air conditioning) equipment to maintain the system 100 within a desired temperature range (e.g., about 15° C. to about 25° C., and / or about 10° C. to about 30° C., as well as other suitable temperature ranges). In one or more embodiments, the system may include a first set of three containers 102 stacked on top of a second set of three containers 102, where the first set of three containers 102 houses the API production module and / or drug product production module, and the second set of three containers houses the HVAC equipment. In some embodiments, the HVAC equipment may be housed on the top floor and the API production module and / or drug product production module may be housed on the bottom floor. In some embodiments, the relative positions may be reversed (i.e., the HVAC equipment is at the bottom).

[0130] FIG. 2 illustrates an overview of an exemplary drug product manufacturing entity 110 and / or process according to aspects of the disclosure. In the embodiment of FIG. 2, the entity 110 and / or process 110 may include building a module and outfitting the module (i.e., container 102) at step 112 at a production facility (e.g., BioNTech facility in Germany and / or one or more contractor and / or partner facilities located at various locations). At step 114, the entity 110 and / or process 110 may include transporting the built container 102 and / or equipment to the drug production facility. At step 116, the entity 110 and / or process 110 may include installing the built module (i.e., container 102) and / or equipment at the drug production facility. At step 118, the entity 110 and / or process 110 may include transporting supplies (i.e., drug substance and / or process input materials required for production of the drug product) to the drug production facility. In some embodiments, step 118 may occur simultaneously with step 114 (i.e., process input supplies are transported to the facility with container 102). In step 122, entity 110 and / or process 110 may include beginning production of the drug substance and / or drug product.

[0131] FIG. 3 illustrates an overview of an exemplary drug product manufacturing facility, system, and / or process 120 according to aspects of the disclosure. In the embodiment of FIG. 3, the system 120 may generally comprise eight containers 102. In some embodiments, the system 120 may comprise fewer than eight containers 102 and / or more than eight containers 102. The system 120 may comprise a first group of one or more containers 102 dedicated to drug substance production 124 and a second group of one or more containers 102 dedicated to drug product production 126. Additionally, the system 120 may comprise a quality control module (and / or container) 140 and a fill and finish module (and / or container) 142. The first group of one or more containers 102 dedicated to drug substance production 124 may comprise a DNA transcription module 128 (or container), a first purification module 130 (or container), and a first bioburden reduction module 132 (or container). A second group of one or more containers 102 dedicated to drug product production 126 may include an LNP formation module 134 (or container), a second purification module 136 (or container), and a second bioburden reduction module 138 (or container). Each module or container may receive input from a previous or adjacent module. For example, the LNP formation module 134 may receive the drug substance as input from the first bioburden reduction module 132.

[0132] 4 illustrates an overview of an exemplary drug product manufacturing facility 150 according to aspects of the disclosure. In the embodiment of FIG. 4, the facility 150 may comprise a first group 124 of one or more containers 102 dedicated to drug substance production and a second group 126 of one or more containers 102 dedicated to drug product production. Each of the first group 124 and the second group 126 may comprise six containers 102, with three of the six containers in each group housing HVAC equipment (e.g., on a first level or on a second level), as well as three of the six containers housing processing modules as shown in FIG. 3 (i.e., the first group of three containers 124 housing a DNA transcription module 128, a first purification module 130, and a first bioburden reduction module 132, and the second group of three containers 126 containing a LNP formation module 134, a second purification module 136, and a second bioburden reduction module 138). In some embodiments, facility 150 may include a different number of containers and may include other modules discussed herein (e.g., quality control module 140, fill and finish module 142, and other modules described herein). In some embodiments, first group 124 and second group 126 are approximately 800 m 2 It may encompass the following total area (or footprint (e.g., facility footprint)) and may be capable of producing approximately 50 million doses of vaccine per year:

[0133] 5 and 6 show an overview of an exemplary drug product manufacturing facility and / or module 160 according to aspects of the disclosure. In the embodiment of FIG. 5, module 160 may be or comprise any of the individual modules 128, 130, 132, 124, 126, 128, 140, and / or 142 (including combinations thereof) as shown in FIG. 3 and described herein. With reference to FIGS. 5 and 6, in some embodiments, module 160 may comprise all relevant equipment required for the production of drug substance and / or drug product in a single container (e.g., for use in medium-, small-, and / or micro-scale production applications). For example, module 160 may be located in a hospital or other location where individualized treatment and / or therapy is needed on a relatively small scale in a time-sensitive environment. Module 160 may include equipment 144 for receiving and / or processing drug substance (and / or other process input supplies), tanks 148 and / or mixing equipment 148 (e.g., for LNP (i.e., RNA-LNP production)), filtration equipment 152, process control equipment 154, fill and finish equipment 146, and / or storage or refrigeration equipment 156. In terms of larger scale production, certain equipment such as mixing and filtration equipment are only commercially available in fixed sizes, which in part determines the scale at which drug production may occur. At smaller scales, the availability of incrementally sized equipment may be significantly greater, meaning that the entire drug production (including the manufacture of drug substance and drug product) may conveniently fit within a single shipping container 102. Furthermore, certain process steps or modules such as refrigeration and / or warehousing may not be required if the drug product is supplied (i.e., administered) to the patient as soon as it is ready. In some embodiments, module 160 may be used in connection with the treatment of a single individual with individual needs. In some embodiments, module 160 may be used to manufacture medicinal products for a group of individuals suffering from the same illness (eg, a localized viral outbreak or strain).

[0134] 7 shows an overview of an exemplary drug product quality control facility and / or module 180 according to aspects of the present disclosure. In some embodiments, the quality control assessment involves evaluation of the presence of air and / or one or more indications that air has been present (e.g., loss of polydispersity, disruption of nanoparticle structure and / or colloidal structure of the LNP composition, etc.).

[0135] FIG. 8 illustrates an overview of an exemplary drug product warehouse facility and / or module 190 according to an embodiment of the present disclosure.

[0136] FIG. 9 illustrates an overview of an exemplary pharmaceutical product manufacturing facility 200 according to aspects of the disclosure. In the embodiment of FIG. 9, the facility 200 may include about 15 modules or containers 102. In some embodiments, the facility 200 may include about 10 to about 20 modules or containers 102. The facility 200 may include various numbers of each type of container 102 other than those shown in FIG. 9. The facility 200 may also include other types of modules or containers 102 (e.g., HVAC modules), but in some embodiments may not include each and every type of module or container 102 shown in FIG. 9. The facility 200 may include each of the modules or containers 102 shown in FIG. 3 as well as one or more staging area containers 158 (e.g., for receiving shipments and / or visitors to the facility 200). The facility 200 may also include a clean room module 162 to allow personnel to change into clean work clothes and / or clothing. The facility 200 may include one or more refrigeration modules 164 for freezing the drug products and one or more warehousing modules 166 for storing the drug products. The facility 200 may be contained within a facility boundary 168, which may include, for example, large tents, hangars (e.g., inflatable hangars), and / or other types of temporary or permanent structures, fences, warehouses, and / or buildings.

[0137] FIG. 10 illustrates an overview of an exemplary drug product manufacturing facility 210 according to aspects of the disclosure. In the embodiment of FIG. 10, the facility 210 may include about 40 modules or containers 102. In some embodiments, the facility 210 may include about 30 to about 50 modules or containers 102, or about 10 to about 60 modules, and / or more than 60 modules. The facility 210 may include various numbers of each type of container 102 other than those illustrated in FIG. 10. The facility 210 may also include other types of modules or containers 102 (e.g., HVAC modules), but in some embodiments may not include each and every type of module or container 102 illustrated in FIG. 10. In addition to each module or container 102 illustrated in FIG. 9, the facility 210 may include a first drug production facility 172 and a second drug production facility 174, both located within the facility boundary 168. The first and second drug production facilities 172, 174 may be used to produce the same drug product and / or different drug products, as desired. The facility 210 may include a number of utilities 176, including power generation, water storage, water treatment, and / or wastewater treatment. The facility 210 may also include one or more patient treatment areas 178, office areas 182, and / or warehousing modules 166.

[0138] Figure 11 shows an overview of an exemplary manufacturing process 220 for pharmaceutical grade compositions comprising RNA, according to aspects of the present disclosure. Process 220 may include a DNA transcription module 128, a first purification module 130, and a first bioburden reduction (or filtration) module 132, as previously described herein. In the embodiment of Figure 11, process 220 includes an exemplary manufacturing process for pharmaceutical grade RNA, which includes in vitro RNA transcription, followed by purification steps to remove components utilized or formed during production, and filtration to reduce bioburden (e.g., as shown in Figure 11). Optional in-process controls may also be completed, depending on whether a hold step is performed.

[0139] FIG. 12 shows an overview of an exemplary DNA template manufacturing process 230 by PCR-based processing according to an aspect of the present disclosure. In the embodiment of FIG. 12, the process 230 includes an exemplary DNA template manufacturing process by PCR-based processing as described herein, which includes a DNA transcription module 128, a first purification module 130, and a first bioburden reduction (or filtration) module 132. First, a master mix is ​​prepared. Then, a forward primer and a vector are added. The PCR mix is ​​transferred to a reagent reservoir and loaded into a PCR plate. The PCR is completed, including an initial denaturation, a denaturation step, an annealing step, a final extension step of 20-30 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) cycles, and a hold step. The PCR products can be pooled and purified. The purified and pooled PCR products are then filtered and quality control tested. FIG. 12 illustrates which portions and / or steps of process 230 are contained within each module or container.

[0140] 13 illustrates an exemplary process 800 for producing an LNP composition. Generally, steps 806, 808, and 810 (and the equipment associated with these steps, as described herein) are performed and / or located in the LNP formation module 134. Generally, steps 812 and 814 (and the equipment associated with these steps, as described herein) are performed and / or located in the second purification module 136. Generally, step 816 (and the equipment associated with step 816, as described herein) is performed and / or located in the second bioburden reduction module 138. Steps following 816 may be performed in other modules and / or in other facilities (or may not be performed at all). For example, as described herein, refrigeration and warehousing may not be required in all embodiments.

[0141] 13, as can be seen, the composition produced is prepared by combining lipid 810 with an aqueous preparation carrying an agent of interest (e.g., an active agent). In many embodiments, the agent of interest is a nucleic acid (e.g., a nucleic acid therapeutic). As shown in FIG. 13, the nucleic acid is an RNA (e.g., a therapeutic RNA). In many embodiments of this illustrated process, the RNA utilized includes at least one open reading frame (ORF) that may encode, for example, a vaccine antigen, a replacement protein, an antibody agent, a cytokine, and the like. In some embodiments, the vaccine antigen may be a cancer vaccine antigen or an infectious disease (e.g., viral) antigen. In some embodiments, the RNA encodes a polypeptide that is or includes a viral antigen, such as a coronavirus antigen (e.g., a spike protein or portion thereof, or a related variant thereof as described above (e.g., SARS-CoV-2 spike protein or a receptor binding domain thereof, e.g., a prefusion stabilized variant thereof) (e.g., as utilized in one or more of mRNA-BNT162a1, mRNA-BNT162b1, mRNA-BNT162b2, mRNA-BNT-162c1, mRNA-1273, CVnCov, CVnCoV2, etc.). In certain embodiments exemplified herein, the RNA of BNT162b2 was utilized.

[0142] In some embodiments of the process shown in FIG. 13, RNA is prepared by in vitro transcription (e.g., transcription of a DNA template, which may be a linear template such as a linearized plasmid or amplicon).

[0143] In particular, as described herein, the present disclosure identifies the source of problems associated with certain LNP compositions and / or their preparation, recognizing, for example, that the presence of air may have undesirable effect(s). Without wishing to be bound by any particular theory, it is proposed that excess air, particularly in preparations or systems exposed to transport conditions, may adversely affect the LNP composition, for example, resulting in aggregation or other loss of colloidal stability and / or one or more other adverse effects of polydispersity. In some embodiments, the present disclosure provides insight that such negative effects may be particularly likely and / or particularly detrimental in large-scale preparations. Alternatively or additionally, the present disclosure provides insight that such negative effects may be particularly problematic for preparations intended for filtration before, during, and / or after the fill / finish step, for example as shown in FIG. 13.

[0144] 13 and the exemplary process depicted therein, in step 808, process 800 may include LNP formation by adding lipids 810 to the RNA solution 806, as well as high impact mixing (e.g., by impinging jet mixing) and stabilization. Typically, the RNA solution is an aqueous solution.

[0145] In many embodiments, the lipids 810 may include one or more of a cationic ionizable lipid (sometimes referred to as "cationic" for simplicity), a phospholipid, a PEG lipid, a sterol (e.g., cholesterol), and a suitable solvent (e.g., ethanol).

[0146] In some embodiments, LNP formation may be performed in the presence of a buffer (e.g., citrate buffer) 812. In some embodiments, the buffer (e.g., citrate buffer) 812 may be present in the RNA solution 806 prior to mixing with the lipids 810 (e.g., by in-line dilution of RNA diluted in water with a buffer (e.g., citrate buffer) 812 to form an aqueous solution of RNA 806). In other words, the buffer (e.g., citrate buffer) 812 may be added to the RNA solution prior to mixing with the lipid solution 810. In some embodiments, the buffer (e.g., citrate buffer) 812 may also or alternatively be added to the mixture obtained by combining the lipid solution with the aqueous solution 806 (which is an RNA solution, as shown in FIG. 13, but which in some embodiments may hold different agents). In some embodiments, the buffer (e.g., citrate buffer) 812 may include citric acid (sodium citrate monohydrate) and / or sodium hydroxide.

[0147] According to embodiments described herein, step 808 (LNP formation) includes substantially no air being introduced into the process and / or into the various solutions, thereby forming a first RNA-LNP preparation that includes RNA encapsulated in LNPs. LNP formation 808 may include adjusting one or more of process temperatures, process flow rates, and / or ratios of buffers, solutions, and / or suspensions. LNP formation may include independently flowing each of the aqueous solution and lipids 810 (e.g., lipid solution) into a mixing unit. The aqueous RNA solution 806 and lipid solution 810 may each flow into the mixing unit under laminar flow conditions (to avoid entrapment of air bubbles in the flow).

[0148] 13, in step 814, process 800 may include buffer exchanging and concentrating the first RNA-LNP preparation to form a second RNA-LNP preparation. Buffer exchange and concentration step 814 may be performed using process parameters including, for example, a feed flow rate in the range of, for example, 18-50 liters per minute (LPM), a transmembrane pressure (TMP) of, for example, less than 1200 mbar, a retentate pressure in the range of, for example, 130-230 mbar, and a permeate pressure in the range of, for example, 10-70 mbar.

[0149] In some embodiments, buffer exchange 814 of the first RNA-LNP preparation and concentration of the first RNA-LNP preparation are performed in alternating steps. In one or more embodiments, a TRIS (i.e., tris(hydroxymethyl)aminomethane) buffer may be used. In some embodiments, buffer exchange 814 is performed by diafiltration and concentration is performed by ultrafiltration. In some embodiments, diafiltration and / or ultrafiltration are performed by tangential flow filtration (TFF) (e.g., in a tangential flow filtration unit and / or TFF skid). In some embodiments, tangential flow filtration is performed using one or more jejunostomy tubes and / or one or more dip tubes configured to avoid the introduction of air into the second RNA-LNP preparation. During tangential flow filtration, retentate may be recirculated to the feed tank using a dip tube with a first end immersed in the filtrate feed in the feed tank to avoid the introduction of air into the filtrate feed. Prior to the buffer exchange and concentration steps, the filtration system for tangential flow filtration may be filled with buffer to prevent the introduction of air into the second RNA-LNP preparation.

[0150] 13, the buffer exchange and concentration step 814 may include at least two alternating buffer exchanges performed via diafiltration and at least two alternating concentrations performed via ultrafiltration. During the buffer exchange and concentration 814, the process temperature may be maintained within a desired temperature range (e.g., about 25°C or less, or about 2°C to about 25°C, or about 15°C to about 25°C). During the buffer exchange and concentration 814, the pH may be continuously monitored (and maintained within a target range (e.g., about 7.0 to about 7.5, or about 7.1 to about 7.3)) and the shear may be maintained, for example, in a range of about 2000s^-1 to about 6000s^-1, or about 3000s^-1 to about 5000s^-1, or about 4000s^-1 (±1%, 5%, and / or 10%). Following buffer exchange and concentration 814, a recovery flush may be performed during which shear may be reduced to less than about 2000 s^-1 (e.g., less than about 1500 s^-1, or less than about 1000 s^-1). In some embodiments, after buffer exchange 814, e.g., after ultrafiltration and / or diafiltration, the pH may be maintained within the range of about 7.3 to about 7.5.

[0151] In some embodiments, the pH of the first RNA-LNP preparation may be maintained at a higher pH than the pH of the cationic lipids (i.e., the cationic lipids in the lipid solution) during buffer exchange and / or concentration 814. Without wishing to be bound by any particular theory, it has been proposed that doing so may reduce foaming of the liquid nanoparticles.

[0152] In some embodiments, the first and / or second RNA-LNP preparation(s) may be sterilized without substantially introducing air into the produced formulation. In some embodiments, the relevant produced formulation may be a product for further manipulation, processing, packaging, and / or shipping. In some embodiments, the produced formulation may be or include a drug product formulation, for example for administration to a human.

[0153] In some embodiments, one or more sterilization steps may be performed by sterile filtration. In some embodiments, the sterile (or other) filtration may be performed at a target pressure, such as about 1.03 bar (or about 1.02 bar to about 1.04 bar, about 1.01 bar to about 1.05 bar, or about 1.00 bar to about 1.1 bar), without a substantial increase in pressure during the filtration process.

[0154] In some embodiments, the mixing unit utilized may comprise one or more impinging jet mixing skids. The impinging jet mixing skids may be aerated and / or flooded to remove air from the tubes of the impinging jet mixing skids prior to mixing. Mixing of the aqueous solution and the lipid solution may be performed within the confines of the mixing unit and / or one or more impinging jet mixing skids. In some embodiments, the aqueous solution does not contact the lipid solution prior to mixing. In some embodiments, the flow rate ratio of the aqueous solution to the lipid solution into the mixing unit is about 3:1. In some embodiments, the mixing speed may be adjusted to avoid introducing air into the first RNA-LNP preparation. For example, to avoid the introduction of air (and / or other impurities), the mixing process or processes may include gradually increasing the mixing speed until a slight vortex is formed (e.g., a mixing speed at or just above the point at which a visible vortex is formed), but below the mixing speed at which bubbles begin to form.

[0155] With further reference to FIG. 13 , the system (e.g., the impinging jet mixing skid, the TFF system (i.e., tangential flow filtration unit), and / or components thereof) may be evaluated for the presence of air bubbles at one or more time points (e.g., monitored over time, e.g., periodically or continuously). If air is detected in the aqueous solution, the lipid solution, the first RNA-LNP preparation, the second RNA-LNP preparation, the mixing unit, and / or the tubing providing the aqueous solution of RNA, the lipid solution, the first RNA-LNP preparation, and / or the second RNA-LNP preparation, an alert or notification may be sent indicating that air has been detected somewhere in the system. In some embodiments, detection of air may be performed by one or more flow meters (e.g., one or more Coriolis flow meters), and / or by visual evaluation (e.g., by the human eye and / or various types of cameras), and / or by other detection means.

[0156] In some embodiments, the aqueous and / or lipid solutions may be flowed into the mixing unit through one or more inlets located in a bottom portion of the mixing unit, and the resulting first RNA-LNP preparation may be discharged from the mixing unit through one or more outlets located in an upper portion of the mixing unit. In some embodiments, mixing may be performed using an immersion mixer. In some embodiments, bubbles may be generated during and / or after formation of the LNP-encapsulated RNA and may then be removed from the RNA-LNP preparation (e.g., bubbles may be removed from the first and / or second RNA-LNP preparations).

[0157] 13, after buffer exchange 814 and concentration, process 800 may include 0.2 μm filtration and / or addition of sucrose and PBS for combination. After combination, process 800 may include bioburden reduction filtration (BBR) 816 following buffer exchange and concentration 814. Bioburden reduction filtration 816 may include filtration through a filter with a pore size of 0.2 μm or less (or, for example, a pore size of about 0.22 μm). Bioburden reduction filtration 816 may also include using other pore sizes (e.g., 0.45 μm pore size) as described herein. According to this embodiment, 0.2 μm pore size filtering may also be performed for each of the lipid solution and the aqueous RNA solution prior to mixing, for the first RNA-LNP preparation, and / or for the second RNA-LNP preparation. Bioburden reduction filtration 816 may also include filtering the post-TFF-LNP suspension through a particle reduction filter prior to filtering the suspension through a 0.2 μm pore size and / or a 0.22 μm pore size bioburden reduction filter (for example). In some embodiments, bioburden reduction filtration 816 may also include performing a filter recovery flush.

[0158] 13, following bioburden reduction filtration 816, process 800 may include loading the filtered second RNA-LNP preparation into a transport bag (e.g., a Flexsafe® bag) and performing a visual inspection 818 of the transport bag for air bubbles. In some embodiments, the transport bag may be, for example, a 12 L bag, a 50 L bag, a 100 L bag, and / or other suitable bag size (e.g., depending on the batch size of the RNA-LNP preparation involved), examples of which include bags with volumes between 12 L and 50 L, and / or bags with volumes between 50 L and 100 L. As noted herein, the present disclosure recognizes that the adverse effect(s) of introduced air may be particularly problematic when LNP compositions (e.g., the RNA-LNP preparations exemplified herein) need to be manufactured and / or transported on a relatively large scale.

[0159] In some embodiments, filling the transport bag may include filling the bag to a volume ranging from about 30% to about 95%, or about 40% to about 90%, or about 50% to about 85%, or about 60% to about 85%, or about 70% to about 85% of the total volume of the bag, and / or other partial ranges therebetween. In some embodiments, prior to filling, the bag may be emptied, unfilled, and / or otherwise unexpanded to avoid the introduction of air bubbles.

[0160] In some embodiments, after the bag is filled to a desired volume, air bubbles may be removed (e.g., manually removed from the bag with a syringe) and the bag may be sealed. In some embodiments, care is taken to ensure that no air bubbles are introduced or allowed to be introduced during sealing. After the bag is filled and sealed, a visual inspection 818 may be performed and may include a visual inspection 818 using a human eye and / or a camera. In some embodiments, if air bubbles are found in the bag, an effort may be made to remove the air bubbles (e.g., manually with a syringe). Alternatively or additionally, in some embodiments, the bag with air bubbles (e.g., a bag with visibly observable air bubbles) may be discarded.

[0161] The filled bags may be stored and / or transported at a temperature ranging from about 1° C. to about 15° C. (e.g., from about 2° C. to about 10° C., or from about 2° C. to about 8° C.), or alternatively, frozen at a temperature of about −70° C. (e.g., in the range of about −60° C. to about −80° C.). Prior to transport (e.g., once any air bubbles have been removed), the bags may be secured in or on racks and / or in or on any other suitable shelving or storage system to minimize movement, bursting, and / or damage of the bags during transport to the filling and finishing facility. Bags for transport may be stacked in a specific manner, for example, using a stacking system on a pallet with shock absorbers. During and / or in preparation for transport 820, as well as after transport, a positive pressure of nitrogen (e.g., about 1-2 bar) may be maintained in and around the environment in which the bags are stored and / or transported to prevent air from entering the bags. After transport 820, the bags may be evaluated a second time 822 for air content (e.g., visually inspected). In some embodiments, any air bubbles found during such second evaluation 822 may be removed (e.g., manually removed), or alternatively, the bag(s) containing air bubbles may be selectively discarded (e.g., if the amount of air in a given bag exceeds a threshold value).

[0162] 13, after arrival at the fill and finish facility (and, in some embodiments, after undergoing a second air evaluation), sterile filtration 824 may be performed (i.e., second RNA-LNP preparation). In some embodiments, such sterile filtration 824 may be performed after the preparation is removed from the shipping bag and prior to placement in a collection vessel, reservoir and / or filling tank. In some embodiments, the material (i.e., the filtered preparation) may then be dispersed from the collection vessel, reservoir and / or filling tank during sterile fill and finish 826 (e.g., for sterile filling of a drug product into glass containers).

[0163] A third air evaluation 828 (e.g., by visual inspection) may be performed on the filled glass containers to again ensure that no air bubbles have been introduced. The inspected and filled glass containers may then be frozen, stored, warehoused, and / or distributed, for example to a health care management facility, at step 830 of process 800. Alternatively, in some embodiments, the filled glass containers may be subjected to lyophilization or other drying process to transport and / or store the drug product in a dry state (e.g., for subsequent resuspension).

[0164] In many embodiments, as shown in FIG. 13, aseptic filling and finishing 826 at the fill and finish facility is performed such that substantially no air is introduced into the product.

[0165] 13, in some embodiments, the fill and finish facility may be co-located with the LNP production facility, in which case fill and finish may be performed directly using a point of fill filtration device (in which case transport 820, bag filling and sealing, and one or more visual inspection steps 818, 822, 828 may not be required). In still other embodiments, process 800 may include multiple transport steps 820, and additional visual inspection steps 818, 822, 828, if various steps of process 800 are performed in additional and / or other facilities (or, alternatively, if transport within a single facility is required).

[0166] FIG. 14 illustrates an overview of an exemplary pharmaceutical product manufacturing facility 900 according to aspects of the disclosure. In the embodiment of FIG. 14, the facility 900 may include a facility boundary 902 that defines various areas located in the facility 900. In some embodiments, filling and finishing operations 904 may be performed at different locations (or may occur at the facility 900). The facility 900 comprises a total of 6,000 m 2 ~8,000m 2 , or approximately 5,000 m 2 ~about 10,000m 2 The secured ground area covers a total of approximately 3,000 m 2 ~6,000m2 , or approximately 4,000 m 2 ~about 5,000m 2 Facility 900 may encompass a total protected area (i.e., total building area) of approximately 800 m. Facility 900 may include a drug substance module 906 and a drug product module 908, both enclosed within a clean area perimeter 910. Each of the drug substance module 906 and drug product module 908 may be arranged in a six container configuration similar to the arrangement shown in FIG. 4 (i.e., similar to drug substance module 124 and drug production module 126). The drug substance module 906 and drug product module 908 may encompass an area of ​​approximately 800 m. 2 , or about 500m 2 ~about 1,000m 2 Adjacent to the drug substance module 906 and drug product module 908, the facility may include a changing area 912 and a preparation area 914, which in some embodiments may encompass a combined area of ​​approximately 300 m 2 A technical area 916 for electronics, utilities, facility controls, and other equipment may be located adjacent to the changing area 912.

[0167] 14, the facility 900 may include a warehouse area 918, a freezer area 920, a buffer preparation area 924, a utility and waste area 922, an office area 926, and a quality control area 928. The warehouse area 918 is approximately 600 m 2 , or about 500m 2 ~about 800m 2The freezer area 920 may encompass a total area of ​​up to 1000 mm and may be configured with inbound and / or outbound logistics to accommodate 1,000 pallet positions (e.g., each having a standard pallet size of about 1000 mm x 1200 mm or about 800 mm x 1200 mm). The freezer area 920 may include at least three environmental zones, with a first zone maintaining a temperature at or about -70°C (e.g., about -60°C to about -80°C), a second zone maintaining a temperature at or about -20°C (e.g., about -10°C to about -30°C), and a third zone maintaining a temperature in the range of about 2°C to about 8°C. In some embodiments, each of the buffer preparation area 924, the utilities and waste area 922, the office area 926, the quality control area 928, and / or the fill and finish area 904 may be provided (and / or initially constructed) by the local country and / or region in which the facility is located, rather than being shipped to the facility in one or more standard shipping containers. In some embodiments, the quality control area 928 is approximately 400 m 2 and the buffer preparation area 924 may encompass an area of ​​approximately 200 m 2 The utility and waste area 922 may encompass an area of ​​approximately 200 m 2 The filling and finishing area 904 may encompass an area of ​​approximately 400 m 2 The office area 926 may encompass an area of ​​approximately 800 m 2 Facility 900 may encompass an area of ​​approximately 300 m. Facility 900 may also include a security gate / guard station and / or other suitable structures. In some embodiments, the facility may include a second drug product module 908 (which may serve to increase production capacity (e.g., to greater than 50 million doses per year, e.g., to about 80 million to 100 million doses per year)), which may add approximately 300 m to the facility's footprint. 2 may be added.

[0168] 14, the drug substance module (or area) 906 and drug production module (or area) 908 may be housed in a building having a ceiling height of at least 8 m, a point load capacity of at least 9,000 kg, and gates and / or doors having a width of at least 4 m and a height of at least 7 m. The warehouse area 918 (as well as other areas such as quality control area 928, buffer preparation area 924, utility and waste area 922, fill and finish area 904, drug substance module 906, drug product module 908, etc.) may be maintained at a controlled humidity and temperature of about 15° C. to about 25° C. The freezer area 920 may include space to accommodate about 10 to 20 freezers and about 5 to about 10 refrigerators. The utilities and waste area 922 may provide a normal power supply in the range of about 2,000 kW to about 4,000 kW (e.g., about 3,000 kW) and may include an uninterruptible power supply of at least 250 kW (e.g., about 200 kW to about 300 kW) to power the entire facility. The drug substance module 906 and drug product module 908 may require a total (i.e., combined) normal power of about 300 kW (e.g., in the range of about 200 kW to about 400 kW) and an uninterruptible power of about 70 kW (e.g., in the range of about 60 kW to about 80 kW, or about 50 kW to about 100 kW). The utilities and waste area 922 may provide a power supply of at least 4 m 3 14. A potable water supply capable of providing 1000 liters / hr of potable water may also be included. A utility and waste area 922 may also include network connections to the facility 900 as well as equipment for providing compressed air, nitrogen, carbon dioxide, and / or oxygen (e.g., provided in bottled containers). The facility 900 may also include various other components, layouts, and arrangements of modules beyond those shown in FIG. 14.

[0169] With further reference to FIG. 14, the fill and finish module 904 (or area) may comprise the necessary equipment to perform the following process steps: pooling liquid or frozen bulk material (e.g., Grade C conditions); sterile filtration (e.g., by isolator technology and / or RABS) into one or more aseptic filling lines under Grade A conditions; capping and crimping (e.g.) 2R ISO 8362-1 standard vials of (e.g.) up to 2.25 mL; performing manual, semi-automated, and / or fully automated visual inspection of filled vials; serializing and labeling the vials; freezing and storing filled vials; packaging and shipping under dry ice. The fill and finish module 904 (or area) may comprise the necessary equipment to process approximately 10 million vials per year (equivalent to 50 million doses). In a preferred embodiment, the fill and finish module 904 meets one or more standards (e.g., cGMP standards and / or a process control system such as Siemens PCS7). In some embodiments, the filling and finishing area 904 is located on the facility 900, and in some embodiments, the filling and finishing area 904 is located off the facility.

[0170] FIG. 15 shows an overview of an exemplary drug substance module 906 and / or drug product module 908 according to an aspect of the disclosure. The diagram in FIG. 15 shows a possible layout that can be used for both the drug substance module 906 and the drug product module 908. The drug substance module 930 and / or drug product module 930 may include three containers 932, 934, 936 on the first floor (e.g., upper or lower floor) of the production facility. The embodiment of FIG. 15 is similar in layout to the six container drug substance module 124 and / or drug product module 126 shown in FIG. 4. Thus, FIG. 15 may include an additional three containers (e.g., lower or higher than the floor shown in FIG. 15) that house HVAC equipment. The first container 932 may be used primarily for assembly and to ensure that the airlock is maintained, while the second and third containers may be used primarily to house the operation suite (i.e., for the manufacture of drug substances and / or drug products). At a first end, the first container 932 may contain a personnel entry section 940 adjacent to a personnel airlock area 942. At a second end, the first container 932 may contain a material entry area 946 adjacent to a material airlock area 944. Upon entering the first container 932, the personnel and / or materials must pass through the respective personnel airlock section 942 and / or material airlock section 944. The purpose is to flush out the surrounding air and / or environmental air (e.g., via vacuum pumps and / or blowers in conjunction with sealed doors and passageways) to ensure that no impurities enter the operations suite 938 upon entry of the personnel and / or materials. During the course of normal operations, personnel and materials should only enter the operations suite 938 through the respective airlocks. The drug substance module 906 and / or drug product module 908 may include manufacturing and / or production equipment within the operations suite 938 as described in this disclosure. The positions of equipment, walls, doors, and / or other structures may be adjusted from the positions shown in FIG. 15 as needed to optimize a given production run.

[0171] 15, the drug substance module 906 and / or drug product module 908 may comprise an ATEX or hazardous equipment area 954 for housing any materials and / or equipment that are energized, pressurized, heated, and / or otherwise create a risk of fire, combustion, and / or explosion (e.g., equipment within the scope of the ATEX 114 "Equipment" Directive 2014 / 34 / EU and / or the ATEX 137 "Workplace" Directive 1999 / 92 / EC). The hazardous equipment area 954 may also be maintained as a clean room to reduce the possibility of flammable materials and / or debris entering the hazardous equipment area 954. In some embodiments, the hazardous equipment area 954 (or "ATEX room") may be used to house one or more drug product components or sub-components used in lipid preparation, which may require the use of ethanol, which may be stored (along with a sterilant) in one or more safety cabinets within the ATEX room 954. The drug substance module 906 and / or drug product module 908 may also include one or more stairs 950 (e.g., stairs leading up or down to an HVAC level) and one or more ramps 948 to facilitate easy transport of materials (and / or personnel) to one or more platforms 956 leading to the personnel corridors 940 and material corridors 946. The drug substance module 906 and / or drug product module 908 may also include additional external equipment 952, examples of which include transformers, tanks, HVAC equipment, and other necessary components that are located external to the containers 932, 934, 936, but that are in fluid, electrical, thermal, and / or operational communication with components internal to the containers 932, 934, 936.

[0172] FIG. 16 illustrates an overview of an exemplary quality control module 928 according to an embodiment of the present disclosure. The quality control module 928 may include a PCR lab 958, an RNA / DNA lab 962, an environmental monitoring console 964, a high performance liquid chromatography (HPLC) lab 966, a cell culture lab 968, an overall procedure lab 970 (e.g., to monitor parameters such as pH, color, sample logistics, and / or storage metrics), a freezer monitor 972, a bioburden lab 974, a quality control storage area 976, a cleaning area 978, a chemical / endotoxin lab 980, and / or a changing area 982. Quality control processes (e.g., within each process and / or lab illustrated in FIG. 16) may be tightly controlled and / or standardized to reduce the introduction of sources of variability into each lab. The quality control module 928 may be configured to allow staff / personnel access to each lab area only via the changing area to ensure that appropriate disinfection and sanitization measures are taken before entering the lab area within the quality control module 928.

[0173] In association with the modular drug production system 100 of this embodiment, various personnel and staffing needs must be met to ensure consistent and satisfactory operation of the modular drug production system 100. For example, in association with the drug substance module 906 and / or drug product module 908, qualified operators / personnel are needed to operate the bioreactors and ensure laminar flow, monitor purification protocols, monitor formulation and filtration processes, process support protocols, and production and / or automation engineers, scientists and / or technicians. In association with the quality control module 928, scientists and / or technicians with laboratory experience are needed to perform various tests to ensure quality control is maintained. In association with the fill and finish module 904, staff are needed to perform filling operations, optical controls, labeling, and packaging. In association with the warehouse area 918, logistics and warehousing personnel are needed. On the other hand, office area 928 requires personnel with a wide range of skills, examples of which include (but are not limited to) subject matter experts, scientists, engineers, automation experts, quality control / quality assurance experts, procurement / supply chain specialists, finance experts, human resources experts, training managers, IT administrators, and overall operations coordinators.

[0174] The modular drug production system 100 of this embodiment may be transported (and further constructed or developed as necessary) to a facility with robust road access, train and / or ship access to allow transport of containers and other equipment and materials to the facility. In some embodiments, the modular drug production system 100 may be transported (and further constructed at) an existing pharmaceutical facility, thereby utilizing existing laboratory equipment (e.g., used as quality control module 928) and / or fill and finish capabilities. Proximity to a university may also be an advantage, allowing manpower needs (e.g., including employees / personnel with the required technical background) to be more easily met. In preferred embodiments, the facility is located within an hour of an airport, seaport, and / or train station to help facilitate logistics and supply chain operations. In some embodiments, the facility may be located in a more remote location. In this case, additional time for materials and equipment to arrive at the facility must be considered, and careful consideration must be given to the availability of qualified personnel required to support the operation of the modular drug production system 100. Additionally, as discussed in this disclosure, the facility or location in which the modular drug production system 100 is installed must have access to power, potable water, an internet / network connection, and be capable of being secured continuously (i.e., 24 hours a day, 7 days a week).

[0175] The modular drug production system 100 of the present embodiment may be used in connection with production scales of 12.8 grams, 25.6 grams, 38.4 grams, 40 grams, and / or other sizes. For example, the modular drug production system 100 of the present embodiment may be used in connection with drug product output including 25 L bags of 12 gram batches. Various numbers of containers and container types are described in connection with the embodiments of the present disclosure. It should be understood that the modular drug production system 100 of the present embodiment may be used in connection with types and numbers of containers other than those explicitly described herein. Standard shipping containers described herein may include widths of about 8 feet (2.43 m), heights of about 8.5 feet (2.59 m), and lengths of about 20 feet (6.06 m) to about 40 feet (12.12 m). Other standard sizes, half sizes, and / or large or small sizes may also be used. The modular drug production system 100 of the present embodiment may be used in connection with processing of various production scales. Thus, while some of the process parameters may change, much of the overall process flow and equipment remains substantially the same for microscale, small-scale, mid-scale, and large-scale drug production. Although the modular drug production system 100 of the present embodiment is generally described in connection with RNA-LNP drug production, it may also be used in connection with the production of other drugs.

[0176] Early Warning System The present embodiments, in one or more aspects, are directed to a localized early warning system (EWS) for identifying variants of concern (VOC) of a disease. The present embodiments, in one or more aspects, are directed to systems and methods for determining and producing vaccines that target and treat local strains of a disease. In some embodiments, the early warning system is used to identify variants of concern (i.e., in terms of global spread) based on one or more of the transmissibility of the strain, the rate at which the strain grows in the human body, the ability of the strain to evade existing vaccines and boosters, and the severity of the strain to humans. (See Early Computational Detection of Potential High Risk SARS-CoV-2 Variants; Beguir et al, (doi:https: / / doi.org / 10.1101 / 2021.12.24.474095)).

[0177] FIG. 17 illustrates a process (method) 1700 for making a vaccine according to an embodiment of the present disclosure. In step 1702, the method 1700 may include collecting sequence data (e.g., using a local sequencing device based on test samples taken from a local area). In step 1704, the method 1700 may include uploading the local data to a public database, such as GISAID, NCBI, EWS database, and / or other databases. In step 1706, the method 1700 may include analyzing the data (i.e., data local to the area of ​​interest) to determine a target strain. The target strain may be an actual strain prevalent in the area and / or a hybrid that combines different segments of the sequence of interest to target a hybrid strain or a "synthetic" strain representative of one or more local strains. In step 1708, the method 1700 may include sending synthesis instructions from a database and / or a cloud-based computing system to a local computing system, the synthesis instructions including sequencing information so that DNA can be created by DNA synthesis. At step 1710, method 1700 may include locally manufacturing, administering, and / or distributing the vaccine based on the compounding order, as described herein. At step 1712, method 1700 may include repeating steps 1702-1710 as necessary.

[0178] FIG. 18 illustrates a process 1800 for making a vaccine according to aspects of the present disclosure. In step 1802, the method 1800 may include collecting sequence data. In step 1804, the method 1800 may include uploading the local data to a public database, such as GISAID, NCBI, EWS database, and / or other databases. In step 1806, the method 1800 may include running a conventional EWS algorithm on the collected data to identify and evaluate potential variants of concern based on one or more of the strain's transmissibility, the strain's growth rate in the human body, the strain's ability to evade existing vaccines and / or boosters, and the severity of the strain's impact on humans. In some embodiments, the execution of the EWS algorithm includes a one-week lookback period (i.e., evaluation of data collected in the most recent week). At step 1808, method 1800 may include determining an EWS score (i.e., quantifying and assigning a score to each strain evaluated to determine if a variant of concern has been identified for further investigation and possible future actions may be taken based thereon). At step 1810, method 1800 may include determining a region of interest (e.g., the local area in which the facility is located and / or adjacent or proximate areas where particular outbreak activity may be occurring). In some embodiments, the region of interest may include a simple radius around a geographic area (e.g., within 50 miles, 100 miles, 500 miles, 1000 miles, etc. of the facility). In some embodiments, the region of interest may include particular population centers that do not fall neatly within a particular radius (as population densities are rarely uniform and transportation between regions is usually non-uniform (and well-traveled routes can be transmission routes for disease spread)).

[0179] Still referring to FIG. 18, at step 1812, method 1800 may include filtering the data on the public database based on a geographic region of interest (i.e., to include only sequence data from strains within the geographic region of interest). At step 1814, method 1800 may include adjusting the lookback period (e.g., filtering the data based on a particular time period of interest). Adjusting the lookback period may include looking back more than one week (e.g., looking back one month or several weeks, as the case may be). Adjusting the lookback period may include selecting a range of dates that coincide with dates corresponding to a local outbreak, etc. At step 1816, method 1800 may include evaluating the filtered data and deviations from one or more baseline variants. Evaluating the deviations may include creating deviations between each sequence in the filtered dataset and the baseline variants. Evaluating the deviations may also include creating deviations between a particular subset of sequences in the filtered dataset and the baseline variants. For example, sequences in the filtered dataset may be placed into one or more bins or subsets and averaged to determine an average or representative sequence for each subset. The deviation between each representative sequence and the baseline sequence may then be determined. In some embodiments, assessing the deviation may include identifying regions of the representative sequence that deviate from the baseline variant. In some embodiments, assessing the deviation may also include a specific mutation associated with each nucleotide that deviates from the baseline variant. In some embodiments, the baseline variant may include strains that are currently or previously prevalent in the region of interest and have been previously identified. In some embodiments, the baseline variant may include target strains on which existing vaccines are based.

[0180] Still referring to FIG. 18, at step 1818, method 1800 may include comparing deviations from the baseline variant for each subset of data within the radius and lookback period (i.e., within the filtered data set) to determine if there is commonality between the regions of the sequence where the deviations occur and / or between the specific mutations of each deviation. At step 1820, method 1800 may include establishing a locality score for each subset within the filtered data subset, the locality score being based on the commonality each subset shares with other subsets within the filtered data (thereby identifying the most common strains specific to the location and time period of interest). At step 1822, method 1800 may include comparing the locality score and the EWS score to assess and / or inform future direction of variants of concern and / or local vaccine decision making. For example, a low EWS score means that it is highly likely that no variants of concern have been identified from a global outbreak perspective. On the other hand, if the locality score is high, it means that the strain in the region of interest and time period of interest differs significantly from the baseline variant, existing vaccines may not be particularly suitable for treating local strains in the region of interest, and / or that the local strain has already deviated significantly from the baseline variant and is therefore more likely to develop mutations in the future.

[0181] At step 1824, method 1800 may include determining a target strain based on the EWS score, the locality score, and / or a combination thereof. At step 1826, method 1800 may include final approval of one or more vaccines (i.e., a target strain that is a real strain and / or a hybrid or synthetic strain that prioritizes aspects of sequences obtained from both EWS and local strain analysis based on the target strain in a given region of interest). At step 1828, method 1800 may include transmitting synthesis instructions from a database (or network, cloud, and / or command center) to a facility. At step 1830, method 1800 may include performing DNA synthesis based on the synthesis instructions using a DNA synthesizer located in the transcription module (and / or in a different module) according to the present disclosure. According to aspects of an embodiment of the present invention, DNA synthesis may be followed by transcription as described herein. At step 1832, method 1800 may include performing RNA transcription according to Figures 11 and 12. At step 1834, method 1800 may include performing LNP formation according to FIG. 13. At step 1836, method 1800 may include performing finishing steps (i.e., filling and finishing) as described herein. At step 1838, method 1800 may include distributing and / or administering the local vaccine within the region of interest. At step 1840, method 1800 may include evaluating the effectiveness of the local vaccine to treat the local strain of disease. At step 1842, method 1800 may include uploading the effectiveness results to one or more public databases. At step 1844, method 1800 may include repeating any of steps 1802-1842 as necessary.

[0182] FIG. 19 illustrates an overview of an exemplary drug product manufacturing entity and / or process according to aspects of the disclosure. In the embodiment of FIG. 19, certain aspects of the entity may be located or occur at a facility 1922 or alternatively on a cloud (or network) 1912. According to aspects of an embodiment of the present invention, the facility 1922 may comprise one or more sequencers 1902 to generate sequences of local strains. The facility 1922 may also comprise one or more DNA synthesizers 1906 for synthesizing DNA (which may create a vaccine according to the present embodiment). DNA synthesis is performed based on the target strain, as determined from the cloud or network 1912. The cloud or network communicatively coupled to the facility 1922 and capable of both transmitting data to and receiving data from the facility 1922 may comprise several functions and components, examples of which include (but are not limited to) machine learning 1916, public database 1920, data storage 1918, and computing system 1914.

[0183] Figure 20 illustrates an overview of another exemplary drug product manufacturing entity and / or process 2000 according to aspects of the present disclosure. In the embodiment of Figure 20, sequencing data flow (i.e., to deduplication 2002), socio-economic and medical data flow (i.e., to conversion and filling 2004), external data flow (i.e., to modeling and analysis), and internal data flow 2008 are shown according to aspects of an embodiment of the present invention.

[0184] FIG. 21 illustrates an overview of another exemplary drug product manufacturing entity and / or process (or method) 2100 according to aspects of the disclosure. At step 2102, the method 2100 may include undergoing a test (e.g., at a facility). At step 2104, the method 2100 may include genomic sequencing one or more samples from the test. At step 2106, the method 2100 may include entering the genomic sequencing data into a database. At step 2108, the method 2100 may include matching the entered genomic sequencing data to one or more global databases. In some embodiments, matching the entered genomic sequencing data to one or more global databases may include comparing the entered genomic sequencing data to other local genomic sequencing data and / or genomic sequencing data from a global sequence repository. If the entered genomic sequencing data is not present in the repository, it may be added according to aspects of an embodiment of the present invention. At step 2110, the method 2100 may include performing one or more risk assessments on the genomic sequencing data using machine learning or artificial intelligence. At step 2112, method 2100 may include identifying a list of potential variants of concern based on the one or more AI-based risk assessments. At step 2114, method 2100 may include performing in vitro lab testing to validate and confirm the prediction(s) from the AI-based risk assessment. At step 2124, method 2100 may include providing feedback from the in vitro lab testing to the AI ​​or machine learning model to further refine the AI ​​or machine learning model.

[0185] With further reference to FIG. 21 , at step 2116, method 2100 may include identifying one or more variants of concern (VOCs) following confirmation and / or validation during in vitro lab testing. At step 2118, method 2100 may include creating a new VOC vaccine to treat the one or more variants of concern (VOCs). At step 2120, method 2100 may include identifying one or more local variants following confirmation and / or validation during in vitro lab testing. At step 2122, method 2100 may include creating a new local variant vaccine to treat the one or more local variants. In some embodiments, the bolded steps shown in FIG. 21 (i.e., steps 2102, 2104, and 2122) may be performed at a local facility, and the non-bolded steps (i.e., the remaining steps of FIG. 21) may be performed at the entity level and / or cloud level.

[0186] According to embodiments of the present disclosure, sequencing data of variants and / or local strains from surveillance field testing can be processed in real-time (or near real-time) without the need to transport samples to a centralized testing location (which can add days to the turnaround time). AI and machine learning running on a supercomputing infrastructure (e.g., leveraging cloud-based networks) allows for on-site availability of results and analysis. In some embodiments, the disclosed systems and methods rank all known variants for their infectivity and potential immune escape risk, allowing for live monitoring of global pandemics and local outbreaks. Novel mutant sequences can be scored and ranked in silico (i.e., by computer modeling) in just a few hours (e.g., less than 4 hours, less than 3 hours, less than 2 hours, etc.). The present embodiments allow new variants to be identified more quickly and thoroughly and added to a global pool or repository. Similarly, the present disclosure allows for the detection of the most infectious variants as well as those with the strongest immune escape. The present embodiments also provide a ranked list of variants that need to be monitored and tested in vitro, which helps prioritize how laboratory resources can be most effectively utilized.

[0187] The present embodiments provide the capability to produce mRNA drug products (e.g., vaccines) at locations around the world. By incorporating a distributed network of modular drug production facilities into a worldwide Early Warning System (EWS), the present embodiments provide infrastructure and local capabilities to help combat ongoing and future pandemics. The present disclosure provides systems and methodologies that enable broader access to vaccines and disease treatments regardless of socio-economic status and other factors. Furthermore, localized production facilities are well suited to provide a tailored real-time (or near real-time) response to local outbreaks. The ecosystem provided also improves testing and research capabilities and fosters collaboration. The EWS of the present embodiments enables identification of potential pathogens that pose pandemic threats through AI-enabled risk assessment and confirmatory laboratory testing. Furthermore, having a global network of local facilities may provide increased transparency into pandemic threats by more rapid detection of new variants and / or pathogens. The local modular mRNA vaccine production unit of the present embodiments also reduces state and national reliance on external drug supplies and suppliers, thereby ensuring rapid and local response to possible future pandemic situations.

[0188] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein, and therefore it is not intended that the scope of the invention be limited to the above description.

Claims

1. 1. A biopharmaceutical manufacturing facility comprising at least one biopharmaceutical production unit, the biopharmaceutical production unit comprising two or more modularly arranged containment units housing one or more apparatuses adaptable for: (1) receiving and processing an active ingredient or active ingredient precursor, and a pharmaceutical acceptable carrier or excipient, and producing the resultant biopharmaceutical product; (2) quality control of the biopharmaceutical product; and (3) filling and finishing of the biopharmaceutical product, the two or more modularly arranged containment units being arranged and optionally interconnected for inter-unit transfer of unfinished biopharmaceutical product and supply of finished biopharmaceutical product.

2. The facility includes 1 to 10 production units, each of the production units having: (a) a first containment unit housing one or more devices adapted for receiving and processing an active ingredient or an active ingredient precursor, and a pharma- ceutically acceptable carrier or excipient, and for producing a resulting biopharmaceutical product; (b) a second storage unit in communication with the first storage unit for said receipt of the biopharmaceutical product, the second storage unit housing one or more devices applicable for quality control of the biopharmaceutical product; (3) a third containment unit in communication with the second containment unit for the receipt of a quality control assessed biopharmaceutical product and containing one or more devices adaptable for filling and finishing the quality control assessed biopharmaceutical product, and for supplying a finished biopharmaceutical product.

3. The biopharmaceutical manufacturing facility of claim 1 or 2, wherein at least two of the modularly arranged storage units are arranged horizontally.

4. 10. The biopharmaceutical manufacturing facility of claim 1, wherein the manufacturing facility is at least partially enclosed in a housing.

5. 3. The biopharmaceutical manufacturing facility of claim 2, wherein both the active ingredient and the active ingredient precursor are each a nucleotide, and the processing comprises encapsulation of the processed nucleotide within a lipid nanoparticle, and the resulting biopharmaceutical product is a nucleotide vaccine.

6. The biopharmaceutical manufacturing facility of claim 5 , wherein the active ingredient is mRNA.

7. The biopharmaceutical manufacturing facility of claim 5 or 6, wherein at least one of the containment units is an ISO container.

8. 7. The biopharmaceutical manufacturing facility of claim 6, wherein the one or more devices adaptable for receiving and processing an active ingredient or active ingredient precursor, and a pharma- ceutical acceptable carrier or excipient, and producing a resultant biopharmaceutical product, comprise a bioreactor adaptable for receiving DNA and for the in vitro transcription of said DNA into mRNA.

9. 10. The biopharmaceutical manufacturing facility of claim 8, wherein the one or more equipment adaptable for receiving and processing an active ingredient or active ingredient precursor, and a pharma- ceutical acceptable carrier or excipient, and producing a resultant biopharmaceutical product, comprises a purification device configured to remove DNA impurities from the mRNA.

10. 10. The biopharmaceutical manufacturing facility of claim 9, wherein the biopharmaceutical manufacturing facility produces from about 3,000 to about 170,000 doses of the finished mRNA vaccine per day.

11. 10. The biopharmaceutical manufacturing facility of claim 1, wherein the equipment adaptable for: (1) receiving and processing active ingredients or active ingredient precursors and pharma- ceutical acceptable carriers or excipients, and producing the resulting biopharmaceutical product; (2) quality control of the biopharmaceutical product; and (3) filling and finishing the quality control assessed biopharmaceutical product, is in electronic communication with a process control system for remote operation.

12. said electronic communication being wireless; The process control is at least partially automated; the process control system is controlled by one or more computers; and / or 12. The biopharmaceutical manufacturing facility of claim 11, wherein the one or more computers are remote from the facility.

13. 1. A portable system for producing a formulation comprising RNA encapsulated in a lipid nanoparticle (LNP), the system comprising: A first subsystem comprising a plurality of drug substance formulation modules, the first subsystem comprising: the first subsystem comprising a transcription module for forming an RNA solution by in vitro transcription; a second subsystem operatively downstream of the first subsystem comprising a plurality of drug product formation modules, the second subsystem comprising: the second subsystem comprising an LNP formulation module for producing a first RNA-LNP preparation from the RNA solution; The system, wherein each of the transcription module and the LNP formulation module are housed within separate standard shipping containers.

14. 14. The system of claim 13, wherein each of the separate standard shipping containers comprises a width of about 8 feet (2.43 m), a height of about 8.5 feet (2.59 m), and a length of about 20 feet (6.06 m) to about 40 feet (12.12 m).

15. 14. The system of claim 13, wherein the LNP formulation module comprises at least one impinging jet mixing unit.

16. 14. The system of claim 13, wherein the second subsystem further comprises a purification module operably positioned downstream of the LNP formulation module, the purification module comprising at least one tangential flow filtration (TFF) unit, the purification module being positioned within a separate standard shipping container.

17. 14. The system of claim 13, wherein each of the first and second subsystems comprises a bioburden reduction module disposed within a separate standard shipping container, each bioburden reduction module comprising a filtration unit comprising at least one filter having a pore size of about 0.05 μm to about 0.35 μm.

18. 1. A portable system for producing a formulation comprising RNA encapsulated in a lipid nanoparticle (LNP), the system comprising: a first module including a transcription module for forming an RNA solution by in vitro transcription; a second module comprising a first purification module comprising a first tangential flow filtration (TFF) unit, the second module being operatively downstream of the first module and receiving the RNA solution therefrom; a third module including a first bioburden reduction module, the third module being operatively downstream of the second module; a fourth module comprising an LNP formulation module for producing a first RNA-LNP preparation from an RNA solution, the fourth module being operatively downstream of the third module; a fifth module comprising a second purification module comprising a second tangential flow filtration (TFF) unit, the fifth module operatively downstream from the fourth module and configured to receive the first RNA-LNP preparation therefrom; a sixth module comprising a second bioburden reduction module, the sixth module being operatively downstream of the fifth module; The system, wherein each of the first through sixth modules is disposed within a separate standard shipping container.

19. an API module for producing at least one API; a drug product module for producing a drug product at least in part from said at least one drug substance; each of the drug substance module and the drug product module is disposed entirely within one or more portable shipping containers; A drug production system, wherein the drug product is used for treating and / or vaccinating a patient within 1, 2, 4, 8, 12, 24, 48 and / or 72 hours after production.

20. A portable LNP formulation system for producing a first RNA-LNP preparation, comprising: an impinging jet mixing unit; a first fluid conduit for supplying an RNA solution to the impinging jet mixing unit, the first fluid conduit fluidly connecting the impinging jet mixing unit to a source of RNA solution external to the system; a second fluid conduit for supplying a lipid solution to the impinging jet mixing unit, the second fluid conduit fluidly connecting the impinging jet mixing unit to a source of lipid solution external to the system; a third fluid conduit for supplying the first RNA-LNP preparation to a downstream module external to the system; The system is placed (and / or transported to a production facility) within a single standard shipping container.

21. A portable LNP formulation system for producing a first RNA-LNP preparation, comprising: an impinging jet mixing unit; a tangential flow filtration (TFF) unit fluidly coupled downstream of the impinging jet mixing unit, the TFF unit for performing at least one diafiltration step and at least one ultrafiltration step; The system is placed (and / or transported to a production facility) within a single standard shipping container.

22. 22. The system of claim 21, further comprising a bioburden reduction unit fluidly coupled downstream of the TFF unit and contained within the standard shipping container.

23. a first fluid conduit for supplying an RNA solution to the impinging jet mixing unit, the first fluid conduit fluidly connecting the impinging jet mixing unit to a source of RNA solution external to the system; a second fluid conduit for supplying a lipid solution to the impinging jet mixing unit, the second fluid conduit fluidly connecting the impinging jet mixing unit to a source of lipid solution external to the system; The system of claim 21, further comprising a third fluid conduit for supplying the first RNA-LNP preparation to the TFF unit.

24. an API module for producing at least one API; a drug product module for producing a drug product comprising the at least one drug substance; A drug production system, wherein each of the drug substance module and the drug product module are disposed entirely within one or more portable shipping containers.

25. 25. The system of claim 24, wherein each of the drug substance modules and the drug product modules are disposed within at least three portable shipping containers.

26. 25. The system of claim 24, wherein the drug product comprises at least one lipid nanoparticle (LNP).

27. 25. The system of claim 24, further comprising at least one fill and finish module for disposing the drug product in at least one container.

28. 25. The system of claim 24, wherein the combined power requirements of the drug substance module and the drug product module range from about 200 kW to about 400 kW and the uninterruptible power requirements range from about 50 kW to about 100 kW.

29. 25. The system of claim 24, wherein the combined footprint of the drug substance module and the drug product module encompasses an area of ​​about 500 square meters to about 1000 square meters.

30. 25. The system of claim 24, wherein each of the drug substance module and the drug product module includes at least one airlock through which at least one of materials and personnel must pass to enter an operational area within each of the drug substance module and the drug product module.

31. 25. The system of claim 24, further comprising a quality control module comprising at least one of a PCR lab, an RNA / DNA lab, an environmental monitoring console, an HPLC lab, a cell culture lab, a general procedure lab, a freezer monitoring device, a bioburden lab, a quality control storage area, a cleaning area, an endotoxin lab, and a changing area.

32. 25. The system of claim 24, further comprising at least one DNA sequencer for sequencing at least one local strain of a disease.

33. 25. The system of claim 24, further comprising at least one DNA synthesizer for creating at least one custom DNA molecule.

34. uploading sequence information describing at least one local strain of the disease to a public database; downloading sequence information describing the at least one local strain of a disease from the public database; downloading DNA synthesis data from said public database for use in creating a vaccine targeting said at least one local strain of a disease; calculating a target strain on which DNA synthesis data is based based on said sequence information describing at least one local strain of a disease; 25. The system of claim 24, further comprising at least one computing system for performing at least one of the tasks of.

35. 1. A method for producing a vaccine to treat a local strain of a disease, comprising: filtering the disease genomic data by at least one location, thereby generating localized data; determining a target strain from the localized data; and transmitting DNA synthesis instructions to a facility at or near said at least one location; and producing, in or near said at least one location, said vaccine for treating said local strain based on said DNA synthesis instructions.

36. 36. The method of claim 35, wherein the disease is SARS-CoV-2.

37. 36. The method of claim 35, further comprising at least one of administering the vaccine and distributing the vaccine at or near the at least one location.

38. 36. The method of claim 35, further comprising accessing a publicly available database that stores the filtered genomic data.

39. sequencing samples of the local strain of the disease at or near the at least one location, thereby generating local strain sequence data; 36. The method of claim 35, further comprising uploading the local strain sequence data to a publicly available database that stores the filtered genomic data.

40. filtering the genomic data based on at least one of a date range and a look-back period; 36. The method of claim 35, wherein the date range and / or the look-back period corresponds to a period during which a localized outbreak of the disease occurred.

41. 36. The method of claim 35, further comprising evaluating the deviation between the local data and a baseline variant of the disease.

42. 42. The method of claim 41, further comprising comparing the variance of one or more subsets within the local data.

43. 43. The method of claim 42, further comprising assessing a level of commonality of the variations of the one or more subsets within the local data.

44. 42. The method of claim 41, wherein determining a target strain from the local data comprises determining a target strain based at least in part on the deviation between the local data and the baseline variant of the disease.

Images