WO2024194767A1

WO2024194767A1 - Automated drug production system

Info

Publication number: WO2024194767A1
Application number: PCT/IB2024/052540
Authority: WO
Inventors: Manfred Joseph BRUNEN; Anja Kristin HOFFMANN; Rainer KRÖNER
Original assignee: BioNTech SE
Priority date: 2023-03-17
Filing date: 2024-03-15
Publication date: 2024-09-26

Abstract

An automated drug production process includes: performing in vitro DNA transcription via an automated in vitro transcription (IVT) module, thereby producing an RNA solution to be used for producing drug substance; performing at least one tangential flow filtration (TFF) step on the RNA solution; providing a stream of lipid stock; mixing the stream of lipid stock within the RNA solution in an impingement jet mixer, thereby forming an RNA lipid nanoparticle (LNP) preparation; and formulating the RNA-LNP preparation to produce a drug product.

Description

AUTOMATED DRUG PRODUCTION SYSTEM

Cross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/452,903, filed March 17, 2023; and U.S. Provisional Patent Application No. 63/461,769, filed April 25, 2023, the title of each of which is “AUTOMATED DRUG PRODUCTION SYSTEM,” and the content of each of which is incorporated herein by reference in its entirety.

Background

[0002] Nucleic acids represent an important therapeutic modality; lipid nanoparticle technologies have proven to be particularly useful for the delivery of nucleic acid therapeutics, specifically including RNA therapeutics. The ability to deliver lipid nanoparticles, nucleic acids, and/or drug products resulting therefrom, (as well as other drug products) in a time-sensitive manner is often constrained by drug product manufacturing capacity at centralized facilities, the availability of trained manpower and workers, as well as the ability to rapidly adapt drug product manufacturing to current needs.

[0003] There are disadvantages associated with the manual handling of lipids. After opening bulk lipid containers, the vacuum cannot be re-established. Nitrogen, if needed, needs to be added manually. The same applies to transport containers. Lipids that are very susceptible to oxygen risk chemical decomposition. Many lipids are sticky and are not easily transferred to the mixing vessel. Manual weighing and handling of lipids also includes the risk of failures in weighing accuracy, and/or contamination or exposure to unfavourable conditions like temperature, oxygen or contact materials.

Summary

[0004] The present disclosure provides technologies relating to automated and/or modular drug product manufacturing including lipid nanoparticle (LNP) compositions, as well as other drug products. Quickly producing and delivering therapies and/or treatments to patients often involves challenges relating to but not limited to manufacturing capacity, manufacturing flexibility, the overall time required to produce and deliver a drug product to a patient, as well as other considerations. Often, local requirements relating to certification and approval of drugs categorize drug products differently depending on if they are produced within a country or imported from another country. For example, different regulatory approval hurdles may exist for drugs that are produced within a country than those that exist for drugs that are imported. Finding sufficient numbers of trained workers (including operators, scientists, engineers, laboratory technicians, etc.) to operating localized production facilities also poses challenges.

[0005] The present embodiments include a drug production system that may be fully automated and/or modular, and optionally may be shipped anywhere in the world that standard shipping (i.e., “overseas”) containers are able to be shipped. The automated and/or modular drug production system of the present embodiments may be particularly useful for producing RNA-LNP drug products, but may also be useful for any number of other types of drug products. The modular drug production system of the present embodiments enables drugs to be produced within a country and/or localized region in an automated fashion, thereby allowing for drug products that address localized outbreaks and viral strains to be manufactured, in the exact location in which they are needed, without the need for timeconsuming manufacturing processes, or lengthy transport from the manufacturing facility to the outbreak site. In addition, the modular drug production system of the present embodiments allows for an increase in the overall drug manufacturing capacity, and offloads the demand and/or pressure on centralized large scale drug manufacturing facilities to undertake smaller-scale production runs (i.e., to address regional and/or localized viral strains) to the possible detriment of large-scale production (which is primarily focused on helping the greatest number people in the most efficient manner).

[0006] In one aspect, the present disclosed embodiments are directed to an automated drug production process comprising: performing in vitro DNA transcription via an automated in vitro transcription (IVT) module, thereby producing an RNA solution to be used for producing drug substance; performing at least one tangential flow filtration (TFF) step on the RNA solution; providing a stream of lipid stock; mixing the stream of lipid stock within the RNA solution in an impingement jet mixer, thereby forming an RNA lipid nanoparticle (LNP) preparation; formulating the RNA-LNP preparation to produce a drug product.

[0007] In some embodiments, automatically providing a stream of lipid stock includes: using a lipid handling system to automatically puncture at least one lipid cartridge; and aseptically extracting the lipids from the at least one lipid cartridge, thereby providing the stream of lipids.

[0008] In some embodiments, the process includes measuring an RNA concentration within the LNP preparation, and automatically recirculating the LNP preparation within a TFF module in which the second TFF step is being performed until the measured RNA reaches a predetermined threshold.

[0009] In some embodiments, measuring an RNA concentration within the LNP preparation comprises measuring the RNA concentration using a spectrophotometer.

[0010] In some embodiments, the predetermined threshold comprises an RNA concentration in a range from about 1.5 g/L to about 5.0 g/L.

[0011] In some embodiments, the process includes automatically monitoring and controlling multiple IVT process parameters within the IVT module to ensure that an mRNA synthesis subprocess, a DNA hydrolysis subprocess, and a protein hydrolysis subprocess have been completed; and automatically routing RNA solution to the first TFF module when the mRNA synthesis subprocess, DNA hydrolysis subprocess, and protein hydrolysis subprocess have been completed.

[0012] In some embodiments, the multiple IVT process parameters include two or more of: pH, temperature, incubation time, mixing speed, mixing time, level of agitation, and/or hold time.

[0013] In some embodiments, the process includes performing a fill and finish process after the formulating step, to dispose the drug product into containers.

[0014] In some embodiments, the process includes performing a second TFF step after the mixing step.

[0015] In some embodiments, the RNA solution, lipid stock, and RNA-LNP preparation are maintained at a temperature within a range from about 10 degrees C to about 35 degrees C throughout the entire process.

[0016] In some embodiments, the process includes continuously monitoring, via inline instrumentation, at least one operating parameter of each of the in vitro DNA transcription, tangential flow filtration, mixing, and formulating processes. [0017] In some embodiments, the process includes formulating comprises: mixing at least one excipient solution with the RNA-LNP preparation; and mixing at least one buffer with the RNA-LNP preparation.

[0018] In some embodiments, the at least one excipient solution comprises sucrose, and the buffer comprises a TRIS buffer.

[0019] In another aspect, the present embodiments are directed to an automated drug production system comprising: an in vitro transcription (IVT) module for producing an RNA solution; a first tangential flow filtration (TFF) module disposed immediately downstream of the IVT module; a lipid nanoparticle (LNP) formation module disposed immediately downstream of the first TFF module; a second TFF module disposed immediately downstream of the LNP formation module; a formulation module disposed immediately downstream of the second TFF module; and a fill and finish module disposed immediately downstream of the formulation module.

[0020] In some embodiments, a flow existing the LNP formation module is in a range from about 500 ml/min to about 1000 ml/min.

[0021] In some embodiments, the system further includes from about 4 to about 12 standard shipping containers that contain the entire system.

[0022] In some embodiments, the about 2 to about 12 standard shipping containers house the IVT module, the first TFF module, the LNP formation module, the second TFF module, the formulation module, and the fill and finish module.

[0023] In some embodiments, the entire system is located within a single location comprising a footprint within a range from about 100 square meters to about 12,000 square meters.

[0024] In some embodiments, the IVT module comprises at least three (3) bioreactors fluidly connected to each other in series, and fluidly connected to a common supply line in parallel, such that feed materials may be separately and selectively supplied to each of the at least three (3) bioreactors.

[0025] In some embodiments, the system includes at least one bypass line, wherein the at least one bypass line fluidly connects at least one of: an outlet of the first bioreactor to an inlet of the third bioreactor; an outlet of the first bioreactor to an outlet of the third bioreactor; and an outlet of the second bioreactor to an outlet of the third bioreactor. [0026] In some embodiments, each of the first TFF module and the second TFF module comprises: a first TFF cassette (or filter module); a second TFF cassette (or filter module) fluidly coupled downstream of the first TFF cassette; and at least one recirculation line routing flow back to an inlet of the second TFF cassette.

[0027] In some embodiments, the LNP formation module comprises: at least one impingement jet mixing unit; and a lipid handling machine configured to automatically provide a supply of lipids for mixing with an RNA solution in the at least one impingement jet mixing unit.

[0028] In some embodiments, at least one of the first TFF module and the second TFF module comprises: at least one inline probe disposed upstream of the first TFF cassette; one or more dynamic light scattering instruments disposed downstream of the second TFF cassette; and one or more spectroscopy probes disposed downstream of the second TFF cassette.

[0029] In some embodiments, at least one of the first TFF module and the second TFF module comprises a double cross flow configuration comprising: a product flow in a first direction; and an exchange buffer flow in a second direction, the second direction being in an opposite direction to the first direction.

[0030] In some embodiments, the product flow comprises an RNA solution and/or an RNA-LNP preparation.

[0031] In some embodiments, the first TFF module and/or the second TFF module comprises a pressure modulator configured to selectively reverse a transmembrane pressure.

[0032] In some embodiments, the RNA solution, lipid stock, and RNA-LNP preparation are maintained in liquid phase throughout the entire process.

[0033] In some embodiments, the process includes sterilizing at least one component or piece of equipment employed in the process using a vaporized steam comprising hydrogen peroxide.

[0034] In some embodiments, sterilizing at least one component or piece of equipment employed in the process using a solution comprising hydroxyl radicals at a concentration in a range from about 0.005% to about 0.05%.

[0035] In some embodiments, the process is fully automated. [0036] Among other things, the present disclosure identifies the source of a problem that can be encountered with drug production. Without wishing to be bound by any particular theory, the present disclosure proposes automated drug manufacturing systems and methodology that simultaneously address both shortfalls in manpower and worker availability, while also improving quality control of the underlying drug product. Furthermore, the present disclosure provides insights that certain negative impacts may emerge and/or may become particularly impactful when relevant compositions are prepared at large scale.

[0037] The present disclosure provides technologies for improving LNP manufacturing, transport, storage, distribution, and/or automation. Those skilled in the art, reading the present disclosure, will appreciate the significance and applicable breadth of its teachings.

[0038] Among other things, those skilled in the art will appreciate the increasing significance 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.

[0039] Those skilled in the art will further appreciate the importance of delivery technologies, and particularly of LNP delivery technologies, to the success of nucleic acid therapeutics, specifically including therapeutic RNAs e.g., therapeutic mRNAs.

[0040] In some embodiments, provided technologies are useful for manufacturing pharmaceutical-grade RNA therapeutics. In some embodiments, provided technologies may be particularly useful for large scale, medium scale, and small-scale manufacturing of RNA therapeutics, e.g., of pharmaceutical-grade RNA therapeutics. In some embodiments, provided technologies may be particularly useful for the manufacturing of RNA therapeutics and other drugs at distributed locations around the globe close to areas where certain diseases and/or localized strains of diseases are prevalent and/or breaking out.

[0041] Among other things, in some embodiments, the present disclosure identifies the source of one or more challenges that can be associated with manufacturing and/or maintaining certain LNP compositions, specifically including RNA-LNP compositions. Among other things, the present disclosure provides technologies that facilitate consistent manufacturing, for example, satisfying pre-determined in-process controls, and/or lot release specifications (e.g. , high purity, integrity, potency, etc.). In some embodiments, the present disclosure provides robust manufacturing technologies for LNP e.g., RNA-LNP) compositions, including technologies that can be performed at scale, while maintaining particular product attributes, such as high purity, integrity, stability (e.g., to transportation and/or storage) etc. In some embodiments, relevant product attributes may be or include, for example, colloidal stability, particle size (and/or size distribution), LNP topology, amenability to further processing and/or formulation, effectiveness of delivery of encapsulated material from administered compositions, etc.

[0042] Among other things, in some embodiments, the present disclosure provides technologies for automating various stages and/or processes associated with manufacturing LNP compositions. In some embodiments, manufacture described herein can comprise one or more of: lipid stock preparation, preparation of stock of an encapsulated agent (e.g., a nucleic acid agent such as an RNA agent), 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., cryoprotectant), aseptic filling, labelling, storage, and/or characterization of LNP-containing drug product and/or one or more components thereof, any or all of which may best performed under pre-determined conditions and parameters that yield a large-scale, medium-scale, and/or small-scale mass throughput of drug product (e.g., as described herein) while maintaining product attributes (e.g. , as described herein).

[0043] In another aspect, the present embodiments are directed to a lipid handling system comprising: a cartridge handling system; a lipid dissolution system located in the vicinity of the cartridge handling system; a robot configured to take lipid cartridges from the cartridge handling system and place them in the lipid dissolution system; an ethanol supply line configured to supply ethanol to lipid cartridges disposed in the lipid dissolution system; and an outlet line configured to deliver ethanol and lipids from cartridges within the lipid dissolution system to a downstream vessel.

[0044] In some embodiments, each of the ethanol supply line and the outlet line are configured to puncture lipid cartridges disposed within the lipid dissolution system. [0045] In some embodiments, each of the ethanol supply line and the outlet line comprises an angled tip for puncturing the lipid cartridges disposed within the lipid dissolution system.

[0046] In some embodiments, the lipid cartridges comprise at least one of a phospholipid, a PEG-lipid, a sterol, a cationic lipid, and a neutral lipid.

[0047] In some embodiments, the lipid cartridge, once punctured by each of the ethanol supply line and the outlet line, fluidly connects the ethanol supply line to the outlet line.

[0048] In some embodiments, the ethanol supply line is further configured to puncture lipid cartridges disposed in the lipid dissolution system.

[0049] In another aspect, the present embodiments are directed to a lipid handling system comprising: a cartridge handling system; a lipid dissolution system located in the vicinity of the cartridge handling system; a robot configured to take lipid cartridges from the cartridge handling system and place them in the lipid dissolution system; an ethanol supply line configured to puncture lipid cartridges disposed in the lipid dissolution system; and an outlet line configured to puncture cartridges within the lipid dissolution system.

[0050] In some embodiments, the ethanol supply line is configured to supply ethanol to lipid cartridges disposed in the lipid dissolution system; wherein the outlet line is configured to deliver ethanol and lipids from the lipid cartridges within the lipid dissolution system to a downstream vessel.

[0051] In another aspect, the present embodiments are directed to a lipid cartridge containing at least one of a phospholipid, a PEG-lipid, a sterol, a cationic lipid, and a neutral lipid, the cartridge comprising: a generally cylindrical shape; and a septum layer disposed on at least one of a top surface and a bottom surface of the cartridge.

[0052] In some embodiments, the cartridge comprises at least one of aluminum, polyethylene, and polypropylene.

[0053] In some embodiments, the cartridge comprises an aluminum shell coated with at least one of polyethylene and polypropylene.

[0054] In some embodiments, the septum layer comprises silicon.

[0055] In some embodiments, technologies described herein can be useful for manufacturing RNA-LNP compositions for treatment and/or prevention of coronavirus infection, e.g., SARS-CoV-2 infection, as described in Walsh et al. “RNA-based COVID- 19 vaccine BNT162b2 selected for a pivotal efficacy study” medRxiv preprint (2020), which is online accessible at: https://doi.org/10.1101/2020.08.17.20176651; and Milligan et al. “Phase I/II study of COVID-19 RNA vaccine BNT162bl in adults” Nature (2020 August), which is online accessible 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 Drawing

[0056] Figure 1 depicts a schematic of an exemplary drug manufacturing process.

[0057] Figure 2 depicts an overview of an automated drug manufacturing process, according to aspects of the present disclosure.

[0058] Figure 3 depicts an overview of an automated in vitro transcription process, according to aspects of the present disclosure.

[0059] Figure 4 depicts an overview of a first automated tangential flow filtration process, according to aspects of the present disclosure.

[0060] Figure 5 depicts an overview of an automated LNP formation process, according to aspects of the present disclosure.

[0061] Figure 6 depicts an overview of an automated lipid supply machine, according to aspects of the present disclosure.

[0062] Figure 7 depicts an overview of an automated lipid supply machine, according to aspects of the present disclosure.

[0063] Figure 8 depicts an overview of a second automated tangential flow filtration process, according to aspects of the present disclosure.

[0064] Figure 9 depicts an overview of an automated drug formulation process, according to aspects of the present disclosure.

[0065] Figure 10 depicts an overview of an exemplary drug product manufacturing site, according to aspects of the present disclosure.

[0066] Figure 11 depicts an overview of an exemplary manufacturing process for a pharmaceutical-grade composition comprising RNA, according to aspects of the present disclosure. [0067] Figure 12 illustrates an overview of an exemplary DNA template manufacturing process via a PCR-based process, according to aspects of the present disclosure.

[0068] Figure 13 illustrates an exemplary process for manufacturing LNP compositions, according to aspects of the present disclosure.

Certain Definitions

[0069] About or Approximately: The term “about” or “approximately”, when used herein in reference to a value, refers to a value that is similar, in context to a stated reference value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” 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 referred value.

[0070] Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, intradermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may be intramuscular. In some embodiments, administration may involve dosing that is intermittent e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. [0071] Agent'. In general, the term “agent”, as used herein, is used to refer to an entity (e.g., a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc.). In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively, or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and/or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises 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 is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0072] Analog : As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance. [0073] 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 includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc. , as is known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody agent utilized in accordance with the present disclosure is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific 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); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPsTM"); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g., poly-ethylene glycol, etc.]. In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes 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 one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows 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, an included CDR is substantially identical to a reference CDR in that it shows at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

[0074] Antibody agents can be made by the skilled person using methods and commercially available services and kits known in the art. For example, methods of preparation of monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Further 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. Humana Press (September 30, 2013).

[0075] Antibodies may be produced by standard techniques, for example 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 bearing a desired epitope(s), optionally haptenized to another polypeptide. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surfaceactive substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the desired epitope contains antibodies to 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.

[0076] Antigen-. The term “antigen”, as used herein, refers to (i) 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 to an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an antigen elicits a cellular response e.g., involving T-cells whose receptors specifically interact with the antigen). In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may 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 biologic polymer (e.g., other than a nucleic acid or amino acid polymer)) etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some embodiments, antigens utilized in accordance with the present invention are provided in a crude form. In some embodiments, an antigen is a recombinant antigen.

[0077] Binding: It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).

[0078] Bioreactor: The term “bioreactor” as used herein refers to a vessel used for in vitro transcription described herein. A bioreactor can be of any size so long as it is useful for in vitro transcription. For example, in some embodiments, a bioreactor can be at least 0.5 liter, including 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 composed of any material that is suitable for in vitro transcription under the conditions as described herein, including glass, plastic or metal. One of ordinary skill in the art will be aware of and will be able to choose suitable bioreactor volume for use in practicing in vitro transcription.

[0079] Cap : As used herein, the term “cap” refers to a structure comprising or essentially consisting of a nucleoside-5 '-triphosphate that is typically joined to a 5'-end of an uncapped RNA (e.g., an uncapped RNA having a 5'- diphosphate). In some embodiments, a cap is or comprises a guanine nucleotide. In some embodiments, a cap is or comprises a naturally occurring RNA 5’ cap, including, e.g., but not limited to a N7- methylguanosine cap, which has a structure designated as "m7G." In some embodiments, a cap is or comprises a synthetic cap analog that resembles an RNA cap structure and possesses the ability to stabilize RNA if attached thereto, including, e.g., but not limited to anti-reverse cap analogs (ARCAs) known in the art. Those skilled in the art will appreciate that methods for joining a cap to a 5’ end of an RNA are known in the art. For example, in some embodiments, a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, a capped RNA can be obtained by in vitro transcription (IVT) of a DNA template, wherein, in addition to the GTP, an IVT system also contains a cap analog, e.g., as known in the art. Non-limiting examples of a cap analog include a m7GpppG cap analog or an N7-methyl-, 2’-O- methyl -GpppG ARCA cap analog or an N7-methyl-, 3'-O-methyl-GpppG ARCA cap analog, or any commercially available cap analogs, including, e.g., CleanCap (Trilink), EZ Cap, etc. In some embodiments, a cap analog is or comprises a trinucleotide cap analog.

[0080] Comparable : As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0081] Complementary: As used herein, the term “complementary” is used in reference to oligonucleotide hybridization related by base-pairing rules. For example, the sequence “C-A-G-T” is complementary to the sequence “G-T-C-A.” 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 permits oligonucleotide hybridization. Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. Total or complete complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. [0082] Detecting: The term “detecting” is used broadly herein to include appropriate 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, “detecting” may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and/or location of an entity of interest. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest is being detected relative to a control reference, or absolute. As such, the term “quantifying” when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification. Absolute quantification may be accomplished by correlating a detected level of an entity of interest to known control standards (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts 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 to each other.

[0083] Determine: Those of ordinary skill in the art, reading the present specification, will appreciate that a step of “determining” can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, determining involves receiving relevant information and/or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.

[0084] 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 quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0085] Encapsulate: The term “encapsulate” or “encapsulation” is used herein to refer to at least a portion of a component is enclosed or surrounded by another material or another component in a composition. In some embodiments, a component can be fully enclosed or surrounded by another material or another component in a composition.

[0086] Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired property or effect (e.g., desired consistency, delivery, and/or stabilizing effect, etc.). In some embodiments, suitable pharmaceutical excipients to be added to a LNP composition may include, for example, salts, starch, glucose, lactose, sucrose, gelatin, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like.

[0087] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or a single-stranded RNA e.g., an mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target polypeptide agent. In some embodiments, a coding region of a single- stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such a target polypeptide agent, which may be used as a template for transcription of a gene or cDNA.

[0088] 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 an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein. [0089] Fed-batch process: The term “fed-batch process” as used herein refers to a process in which one or more components are introduced into a vessel, e.g., a bioreactor, at some time subsequent to the beginning of a reaction. In some embodiments, one or more components are introduced by a fed-batch process to maintain its concentration low during a reaction. In some embodiments, one or more components are introduced by a fed-batch process to replenish what is depleted during a reaction.

[0090] Five prime untranslated region: As used herein, the terms "five prime untranslated region" or "5' UTR" refer to a 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 an RNA.

[0091] 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 may have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).

[0092] Gene: As used herein, the term “gene” refers to a DNA sequence in a chromosome that codes for a product (e.g. , an RNA product and/or a polypeptide product). In some embodiments, a gene includes coding sequence (i.e., sequence that encodes a particular product); in some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.).

[0093] Gene product or expression product: As used herein, the term “gene product” or “expression product” generally refers to an RNA transcribed from the gene (pre- and/or post-processing) or a polypeptide (pre- and/or post-modification) encoded by an RNA transcribed from the gene.

[0094] Homology: As used herein, the term “homology” or “homolog” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between 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., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as "hydrophobic" or “hydrophilic” amino acids, and/or as having “polar” or “nonpolar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0095] Host cell'. As used herein, refers to a cell into which exogenous material (e.g., DNA such as recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, 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 of the Kingdoms of life that are suitable for expressing an exogenous DNA e.g., a recombinant nucleic acid sequence). Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria 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 such as, for example, hybridomas or quadromas. In some embodiments, a host cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, a host cell is eukaryotic. For example, an eukaryotic host cell may be CHO (e.g., CHO KI, DXB-1 1 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeEa, HepG2, WI38, MRC 5, Colo205, HB 8065, HE-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRE 3 A cell, HT1080 cell, myeloma cell, tumor cell, or a cell line derived from an aforementioned cell.

[0096] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric 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. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence 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 a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the 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, which needs to be introduced for optimal alignment of the two sequences. The 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), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use 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 an NWSgapdna.CMP matrix.

[0097] Improved, increased or reduced: As used herein, these terms, or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be “improved” relative to that obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to 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 that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.

[0098] In vitro: The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel (e.g., a bioreactor), in cell culture, etc., rather than within a multi-cellular organism.

[0099] In vitro transcription-. As used herein, the term "in vitro transcription" or "IVT" refers to the process whereby transcription occurs in vitro in a non-cellular system to produce a synthetic RNA product for use in various applications, including, e.g., production of protein or polypeptides. Such synthetic RNA products can be translated in vitro or introduced directly into cells, where they can be translated. Such synthetic RNA products include, e.g., but not limited to mRNAs, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNAs (e.g., for CRISPR), ribosomal RNAs, small nuclear RNAs, small nucleolar RNAs, and the like. An IVT reaction typically utilizes a DNA template e.g., a linear DNA template) as described and/or utilized herein, ribonucleotides (e.g., non-modified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase.

[0100] In vitro transcription RNA composition -. As used herein, the term “in vitro transcription RNA composition” refers to a composition comprising target RNA synthesized by in vitro transcription. In some embodiments, such a composition can comprise excess in vitro transcription reagents (including, e.g., ribonucleotides and/or capping agents), nucleic acids or fragments thereof such as DNA templates or fragments thereof, polypeptides or fragments thereof such as recombinant enzymes or host cell proteins or fragments thereof, and/or other impurities. In some embodiments, an in vitro transcription RNA composition may have been treated and/or processed prior to one or more purification processes that ultimately produces an RNA transcript preparation comprising RNA transcript at a desired concentration in an appropriate buffer for formulation and/or further manufacturing and/or processing. For example, in some embodiments, an in vitro transcription RNA composition may have been treated to remove or digest DNA template (e.g., using a DNase). In some embodiments, an in vitro transcription RNA composition may have been treated to remove or digest polypeptides (e.g., enzymes such as RNA polymerases, RNase inhibitors, etc.) present in an in vitro transcription reaction (e.g., using a protease).

[0101] In vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal.

[0102] Nanoparticle: As used herein, the term “nanoparticle” refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, a nanoparticle has a diameter of less than 80 nm as defined by the National Institutes of Health. In some embodiments, a nanoparticle comprises one or more enclosed compartments, separated from the bulk solution by a membrane, which surrounds and encloses a space or compartment.

[0103] Nucleic acid/ Polynucleotide: As used herein, the term “nucleic acid” refers to a polymer of at least 2 nucleotides or more, including, e.g., at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxy adenosine, deoxy cytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, 1-methyl-pseudouridine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl- cytidine, 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, a non-natural residue comprises one or more modified sugars (e.g., 2' -fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a 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, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 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 long.

[0104] Pharmaceutical grade: The term “pharmaceutical grade” as used herein refers to standards for chemical and biological drug substances, drug products, dosage forms, compounded preparations, excipients, medical devices, and dietary supplements, established by a recognized national or regional pharmacopeia (e.g., The United States Pharmacopeia and The Formulary (USP-NF)).

[0105] Polypeptide’. The term “polypeptide”, as used herein, typically has its art- recognized meaning of a polymer of at least three amino acids or more. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining at least one activity) of such complete polypeptides. In some embodiments, polypeptides may contain L-amino acids, D-amino acids, or both and/or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics). In some embodiments, a polypeptide may be or comprise an enzyme. In some embodiments, a polypeptide may be or comprise a polypeptide antigen. In some embodiments, a polypeptide may be or comprise an antibody agent. In some embodiments a polypeptide may be or comprise a cytokine.

[0106] 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 to be a pure preparation. In some embodiments, an 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 a preparation.

[0107] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides 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 include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0108] Ribonucleic acid (RN A): As used herein, the term “RNA” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid/ Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments, an RNA is an mRNA. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 3’ end a poly(A) region. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 5’ end, an art- recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, an RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro e.g., by enzymatic synthesis methods and/or by chemical synthesis methods). In some embodiments, an RNA is a single-stranded RNA. In some embodiments, a single-stranded RNA may comprise self-complementary elements and/or may establish a secondary and/or tertiary structure. One of ordinary skill in the art will understand that when a single-stranded RNA is referred to as “encoding,” it can mean that it comprises a nucleic acid sequence that itself encodes or that it comprises a complement of the nucleic acid sequence that encodes. In some embodiments, a singlestranded RNA can be a self-amplifying RNA (also known as self-replicating RNA).

[0109] Recombinant: as used herein, is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that encodes and/or directs 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 is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.).

[0110] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0111] RNA polymerase: As used herein, the term “RNA polymerase” refers to an enzyme that catalyzes polyribonucleotide synthesis by addition of ribonucleotide units to a nucleotide chain using DNA or RNA as a template. The term refers to either a complete enzyme as it occurs in nature, or an isolated, active catalytic or functional domain, or fragment thereof. In some embodiments, an RNA polymerase enzyme initiates synthesis at the 3 '-end of a primer or a nucleic acid strand, or at a promoter sequence, and proceeds in the 5 '-direction along the target nucleic acid to synthesize a strand complementary to the target nucleic acid until synthesis terminates.

[0112] RNA transcript preparation -. The term “RNA transcript preparation” as used herein refers to a preparation comprising RNA transcript that is purified from an in vitro transcription RNA composition described herein. In some embodiments, an RNA transcript preparation is a preparation comprising pharmaceutical-grade RNA transcript. In some embodiments, an RNA transcript preparation is a preparation comprising RNA transcript, in which one or more product quality attributes are characterized and determined to meet a release and/or acceptance criteria (e.g., as 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.

[0113] Room temperature'. As used herein, the term “room temperature” refers to an ambient temperature. In some embodiments, a room temperature is about 18°C-30°C, e.g., about 18°C-25°C, or about 20°C-25°C, or about 20-30°C, or about 23-27°C or about 25°C.

[0114] Sample : As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest, e.g., as described herein. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a mouse). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, 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 appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.

[0115] Stable: The term “stable,” when 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 characteristics (e.g., physical and/or structural characteristics, function, and/or activity) over a period of time under a designated set of conditions (e.g., pH, temperature, light, relative humidity, etc.). In some embodiments, such stability is maintained over a period of time of at least about one hour; in some embodiments, such stability is maintained over a period of time of about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or longer. In some embodiments, such stability is maintained over a period of time within the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc. In some embodiments, such stability is maintained under an ambient condition (e.g., at room temperature and ambient pressure). In some embodiments, such stability is maintained under a physiological condition (e.g., in vivo or at about 37 °C for example in serum or in phosphate buffered saline). In some embodiments, such stability is maintained under cold storage (e.g., at or below about 4 °C, including, e.g., -20 °C, or -70 °C). In some embodiments, such stability is maintained when nucleic acids and/or compositions comprising the same are protected from light (e.g., maintaining in the dark).

[0116] As an example, in some embodiments, the term “stable” is used in reference 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 component(s) thereof maintain one or more aspects of its characteristics (e.g., physical and/or structural characteristics, function(s), and/or activity) over a period of time under a designated set of conditions. For example, in some embodiments, a stable nanoparticle composition (e.g., a lipid nanoparticle composition) is characterized in that average particle size, particle size distribution, and/or polydispersity of nanoparticles is substantially maintained (e.g., within 10% or less, as compared to the initial characteristic(s)) over a period of time (e.g., as described herein) under a designated 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 no detectable amount of degradation products (e.g., associated with hydrolysis and/or enzymatic digestion) is present after it is maintained under a designated set of conditions (e.g., as described herein) over a period of time.

[0117] Synthetic: As used herein, the term “synthetic” refers to an entity that is artificial, or that is made with human intervention, or that results from synthesis rather than naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments by solid-phase synthesis. In some embodiments, the term “synthetic” refers to an entity that is made outside of biological cells. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., an RNA) that is produced by in vitro transcription using a template.

[0118] Tangential flow filtration (TFF)’. As used herein, the term “tangential flow filtration (TFF)” refers to a mode of operation of a filtration system where a fluid passes parallel to a filter membrane (i.e., the flow is tangential to the membrane) to reduce clogging of the filter. In some embodiments, particles larger than the pore size will flow past the membrane as retentate and is recycled back to the feed. In some embodiments, particles smaller than the pore size will pass through and be collected as permeate. [0119] Three prime untranslated region: As used herein, the terms "three prime untranslated region" or "3' UTR" refer 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 stop codon of the coding region of an open reading frame sequence.

[0120] Threshold level (e.g., acceptance criteria) : As used herein, the term “threshold level” refers to a level that are used as a reference to attain information on and/or classify the results of a measurement, for example, the results of a measurement attained in an assay. For example, in some embodiments, a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g., a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria). Thus, a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population. A threshold level can be determined based on one or more control samples or across a population of control samples. A threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold level can be a range of values.

[0121] Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may 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 be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors.”

[0122] 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 foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose.

Detailed Description of Certain Embodiments

[0123] Nucleic acid therapeutics, and particularly RNA therapeutics represent a particularly promising class of therapies for treatment and prevention of various diseases such as cancer, infectious diseases, and/or diseases or disorders associated with overabundance or deficiency in certain proteins.

[0124] RNA therapeutics in particular provide remarkably effective as vaccines to address the COVID 19 pandemic. Particularly given the promise of this technology, and its adaptability to a wide variety of clinical contexts, including massively large scale (e.g., vaccination and/or treatment on a global scale such as is under development for SARS-CoV- 2), improvements to manufacturing technologies, especially those applicable to large-scale production, are especially valuable.

[0125] Development of effective delivery technologies has been central to the success of nucleic acid therapeutics, and lipid nanoparticle technologies have proven to be particularly effective reviewed in, for example, Cullis et al. Molecular Therapy 25:1467, July 5, 2017; See also, US Patent 8058069), specifically including for RNA therapeutics (reviewed in, for example, Hou et al., Nat. Rev. Mater doi.org/10.1038/s41578-021-00358-0, August 10, 2021).

[0126] Technologies provided herein are useful, among other things, to achieve particularly effective and/or efficient production, e.g., on commercial scale and/or under commercial conditions, of pharmaceutical grade LNP preparations and/or compositions (e.g., nucleic acid-LNP preparations, and specifically RNA-LNP preparations). For example, in various embodiments, provided technologies permit and/or facilitate achievement of requirements unique to pharmaceutical-grade (and/or scale) production such as, for example, batch size and/or rate of production, pre-determined in-process controls and/or lot release specifications (e.g., high purity, integrity, potency, and/or stability, etc.), etc.

[0127] The present disclosure provides technologies for manufacturing LNP compositions (e.g., including RNA, e.g., therapeutic RNA such as therapeutic mRNA). In some embodiments, provided technologies are useful for manufacturing pharmaceuticalgrade RNA-LNP therapeutics.

[0128] In some embodiments, provided technologies are 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, technologies provided herein can be used to produce a pharmaceutical-grade batch throughput 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, technologies provided herein can be used to produce a pharmaceutical-grade batch throughput of at least 50L of LNP e.g., nucleic acid-LNP, e.g., RNA-LNP) therapeutics (including e.g., at least 50L, at least 60L, at least 70L, at least 80L, at least 100L, at least 110L, at least 120L, at least 130L, at least 140L, at least 150L or more. In some embodiments, each vial can comprise an RNA drug product in an amount of 0.01 mg to 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).

[0129] Technologies described herein can be useful for manufacturing LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions for treatment and/or prevention of a disease, disorder, or condition (e.g., cancer, infectious diseases, diseases associated with protein deficiency, etc.). In some embodiments, technologies described herein can be useful for manufacturing LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions that comprise or deliver (e.g., by comprising and/or delivering a nucleic acid, such as an RNA, that encodes it) a polypeptide.

[0130] In some particular embodiments, technologies described herein can be useful for manufacturing LNP (e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions for inducing an immune response to an antigen. In some embodiments, technologies described herein can be useful for manufacturing LNP(e.g., nucleic acid-LNP, e.g., RNA-LNP) compositions for treatment and/or prevention of coronavirus infection, e.g., SARS-CoV-2 infection, as described in Walsh et al. “RNA-based CO VID-19 vaccine BNT162b2 selected for a pivotal efficacy study” medRxiv preprint (2020), which is online accessible at: https://doi.org/10.1101/2020.08.17.20176651; and Milligan et al. “Phase I/II study of COVID-19 RNA vaccine BNT162bl in adults” Nature (2020 August), which is online accessible at: https://doi.org/10.1038/s41586-020-2639-4, the contents of each of which are incorporated by reference in their entirety.

Lipid Nanoparticles

[0131] Those skilled in the art are aware that lipid nanoparticles have achieved successful clinical delivery of a wide range of therapeutic agents including, for example, small molecules, and various nucleic acids - e.g., oligonucleotides, siRNAs, and mRNAs (reviewed, for example, in Hu et al., Nat. Rev. Mater, https://doi.org/10.1038/s41578-021- 00358-0, August 10, 2021).

[0132] Various routes of administration for lipid nanoparticle compositions have been proposed and/or tested; those skilled in the art will be aware of appropriate routes for particular compositions (e.g., depending on agent being delivered). To give but a few examples, in some embodiments, LNPs are parenterally administered; most clinical studies have utilized parenteral administration, and particularly intravenous, subcutaneous, intradermal, intravitreal, intratumoral, or intramuscular injection. Intrautero injection has also been described. In some embodiments, topical administration is utilized. In some embodiments, intranasal administration is utilized.

[0133] In some embodiments, administered LNPs are delivered to or accumulate in the liver. Given that the liver is naturally effective at producing and secreting proteins, liver delivery can prove useful for achieving delivery of an LNP-encapsulated agent (and/or, in the case of a nucleic acid agent such as an RNA agent, a polypeptide encoded thereby) into the bloodstream. Such liver delivery has been proposed to be particularly useful, for example, for expression of proteins that are missing in certain metabolic or hematological disorders, or that are effective in provoking immune responses (e.g., particularly antibody responses), for example against infectious agents or cancer cells.

[0134] In some embodiments, administered LNPs are delivered to and/or taken up by antigen-presenting cells (e.g., as may be present in skin, muscle, mucosal tissues, etc.); such administration may be particularly useful or effective for induction of T cell immunity (e.g., for treatment of infectious diseases and/or cancers).

[0135] In various embodiments, lipid nanoparticles can have an average size (e.g., mean 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 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 in accordance with the present disclosure can have an average size e.g., mean diameter) of about 50 nm to about 100 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean 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 in accordance with the present disclosure can have an average size (e.g., mean 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.

[0136] In some embodiments, lipids that form lipid nanoparticles described herein comprise: a polymer-conjugated lipid; a cationic lipid; and a helper neutral lipid. In some such embodiments, total polymer-conjugated lipid may be present in 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, total polymer-conjugated lipid may be present in about 1- 2.5 mol% of the total lipids. In some embodiments, the molar ratio of total cationic lipid to total polymer-conjugated lipid (e.g., PEG-conjugated lipid) 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 lipid to total polymer-conjugated lipid may be about 35:1 to about 25:1.

[0137] In some embodiments involving a polymer-conjugated lipid, a cationic lipid, and a helper neutral lipid in lipid nanoparticles described herein, total cationic lipid is present in 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 lipids. In certain embodiments, total cationic lipid is present in 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 lipids. [0138] In some embodiments involving a polymer-conjugated lipid, a cationic lipid, and a helper neutral lipid in lipid nanoparticles described herein, total neutral lipid is present in about 35-65 mol%, about 40-60 mol%, about 45-55 mol%, or about 47-52 mol% of the total lipids. In some embodiments, total neutral lipid is present in 35-65 mol% of the total lipids. In some embodiments, total non-steroid neutral lipid (e.g., DPSC) is present in about 5-15 mol%, about 7-13 mol%, or 9-11 mol% of the total lipids. In some embodiments, total non-steroid neutral lipid is present in about 9.5, 10 or 10.5 mol% of the total lipids. In some embodiments, the molar ratio of the total cationic lipid to the non-steroid neutral lipid ranges from about 4.1: 1.0 to about 4.9: 1.0, from about 4.5: 1.0 to about 4.8: 1.0, or from about 4.7: 1.0 to 4.8: 1.0. In some embodiments, total steroid neutral lipid (e.g., cholesterol) is present in about 35- 50 mol%, about 39-49 mol%, about 39-46 mol%, about 39- 44 mol%, or about 39-42 mol% of the total lipids. In certain embodiments, total steroid neutral lipid (e.g., cholesterol) is present in about 39, 40, 41, 42, 43, 44, 45, or 46 mol% of the total lipids. 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.

[0139] In some embodiments, a lipid composition comprising a cationic lipid, a polymer-conjugated lipid, and a neutral lipid can have individual lipids present in certain molar percents of the total lipids, or in certain molar ratios (relative to each other) as described in WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0140] In some embodiments, lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid (e.g., PEG-conjugated lipid); a cationic lipid; and a neutral lipid, wherein the polymer-conjugated lipid is present in about 1-2.5 mol% of the total lipids; the cationic lipid is present in 35-65 mol% of the total lipids; and the neutral lipid is present in 35-65 mol% of the total lipids. In some embodiments, lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid (e.g., PEG-conjugated lipid); a cationic lipid; and a neutral lipid, wherein the polymer-conjugated lipid is present in about 1-2 mol% of the total lipids; the cationic lipid is present in 45-48.5 mol% of the total lipids; and the neutral lipid is present in 45-55 mol% of the total lipids. In some embodiments, lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid (e.g., PEG-conjugated lipid); a cationic lipid; and a neutral lipid comprising a non-steroid neutral lipid and a steroid neutral lipid, wherein the polymer-conjugated lipid is present in about 1-2 mol% of the total lipids; the cationic lipid is present in 45-48.5 mol% of the total lipids; the non-steroid neutral lipid is present in 9-11 mol% of the total lipids; and the steroid neutral lipid is present in about 36- 44 mol% of the total lipids. In many of such embodiments, a PEG-conjugated lipid is or

comprises a structure as described in WO

2017/075531 (also described above), or a derivative thereof. In some embodiments, a PEG- conjugated lipid is or comprises 2- [(polyethylene glycol )-2000|-/V,/V-ditctradccylacctamidc. In many of such embodiments, a cationic lipid is or comprises a chemical structure selected from 1-1 to I- 10 of Table 1 herein or a derivative thereof. In some embodiments, a cationic lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate). In many of such embodiments, a neutral lipid comprises DSPC and cholesterol, wherein DSPC is a non-steroid neutral lipid and cholesterol is a steroid neutral lipid.

[0141] In some embodiments, lipid nanoparticles include one or more cationic lipids (e.g., ones described herein). In some embodiments, cationic lipid nanoparticles may comprise at least one cationic lipid, at least one polymer-conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid).

[0142] Figure 1 depicts a schematic of an exemplary drug manufacturing process 10. The process 10 may include the production of drug substance 12 followed by the production of drug product 14. Production of drug substance 12 may include in vitro transcription 16 followed by tangential flow filtration 18, followed by 0.2 micron filtration 20, followed by storage 22 at one or more temperatures in a range from about -20 degrees C to about 8 degrees C. Storage 22 may occur in a different location than the production of drug substance 12. After storage 22, drug subtance 12 may need to be transported to another location for production of drug product 14, which may include lipid nanoparticle (LNP) formation 24, followed by a second tangential flow filtration 26 process, followed by formulaion 28 and 0.2 micron filiation, folllowed by a second storage 30 process at one or more temperatures in a range from about -20 degrees C to about 8 degrees C. Following the second storage 30, the formulation may be transported to a different location for fill and finish steps including filling 32, visual insepction 34, labelling 36, and packaging 38. [0143] Figures 11-13 illustrate an exemplary LNP manufacturing process (for example, an RNA-LNP manufacturing process).

[0144] Figure 11 illustrates an overview of exemplary manufacturing process 520 for a pharmaceutical-grade composition comprising RNA, according to aspects of the present disclosure. The process 520 may include the DNA transcription module 128, the first purification module 130, and the first bioburden reduction (or filtration) module 132, as previously described herein. In the embodiment of Fig. 11, the process 520 includes an exemplary manufacturing process for pharmaceutical-grade RNA comprising an in vitro RNA transcription followed by removal of components utilized or formed in the course of production by a purification process, and filtration to reduce bioburden (e.g., as illustrated in Figure 11). Optional in-process controls may also be completed depending on whether a hold step is performed.

[0145] Figure 12 illustrates an overview of exemplary DNA template manufacturing process 530 via a PCR-based process, according to aspects of the present disclosure. In the embodiment of Fig. 12, the process 530 includes an exemplary manufacturing process of a DNA template via a PCR-based process including the DNA transcription module 128, the first purification module 130, and the first bioburden reduction (or filtration) module 132, as described herein. Initially, a master mix preparation is made. Subsequently, forward primer and vector are added. The PCR-mix is transferred into a reagent reservoir and a PCR plate was filled. A PCR is completed comprising an initial denaturation, a denaturation step, an annealing step, a final extension step for 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. Subsequently, the purified, pooled PCR product may be filtered and quality control tested. Fig. 12 illustrates which portions and/or steps of the process 530 are contained within each module (i.e., 128, 130, 132) of the process flow shown in Fig. 11.

[0146] Figure 13 illustrates an exemplary process 800 for manufacturing LNP compositions. Generally, steps 806, 808, and 810 (as well as equipment associated with those steps, as described herein) will occur and/or be located in the LNP formation module 70, shown in Fig. 5. Generally, steps 812 and 814 (as well as equipment associated with those steps, as described herein) will occur and/or be located in the second purification module (i.e., the second tangential flow filtration module) 80, shown in Fig. 8. Generally, step 816 (as well as equipment associated with step 816, as described herein) will occur and/or be located in the second bioburden reduction module (i.e., formulation) 90 shown in Fig. 9. Steps following 816 may occur in other modules and/or may occur at other facilities (or not at all). For example, as explained herein, freezing and warehousing may not be required in all embodiments.

[0147] Referring still to Fig. 13, as can be seen, the produced compositions are prepared by combining lipids 810 with an aqueous preparation which carries 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 depicted in Fig. 13, the nucleic acid is an RNA (e.g., a therapeutic RNA); in many embodiments of this depicted process, a utilized RNA includes at least one open reading frame (ORF) which may, for example, encode a vaccine antigen, a replacement protein, an antibody agent, a cytokine, etc.). In some embodiments a vaccine antigen may be a cancer vaccine antigen or an infectious disease (e.g., viral) antigen. In some embodiments, an RNA encodes a polypeptide that is or comprises a viral antigen such as a coronaviral antigen, such as a spike protein or portion thereof, or relevant variant of the foregoing (e.g., a SARS-CoV-2 spike protein or receptor binding domain thereof, for example, a prefusion stabilized variant thereof), e.g., as is utilized in one or more of mRNA- BNT162al, mRNA-BNT162bl, mRNA-BNT162b2, mRNA-BNT-162cl, mRNA-1273, CVnCov, CVnCoV2, etc.). In certain embodiments exemplified herein, utilized was an RNA of BNT162b2.

[0148] In some embodiments of the process depicted in Fig. 13, the RNA is prepared by in vitro transcription (e.g., of a DNA template which may, for example be a linear template such as a linearized plasmid or an amplicon).

[0149] Referring to Fig. 13 and the exemplary process that it depicts, at step 808, the process 800 may include LNP formation by adding lipids 810 to an RNA solution 806, as well as high impact mixing (for example, via impingement jet mixing), and stabilization. Typically, the RNA solution is an aqueous solution.

[0150] In many embodiments, the lipids 810 may include one or more of a cationically ionizable (sometimes referred to as “cationic” for simplicity) lipid, a phospholipid, a PEG-lipid, a sterol e.g., a cholesterol) and an appropriate solvent (e.g., ethanol).

[0151] In some embodiments, LNP formation may be performed in presence of a buffer (e.g., a citrate buffer) 812. In some embodiments, the buffer (e.g., a citrate buffer) 812 may be present in the RNA solution 806 prior to mixing with the lipids 810 (for example, via in-line dilution of the water-diluted RNA with the buffer (e.g., citrate buffer) 812 to form the aqueous solution of RNA 806). Stated otherwise, 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 resulting from combining the lipid solution with the aqueous solution 806 (which, as depicted in Fig. 13, is an RNA solution but could, in some embodiments, carry a different agent). In some embodiments, the buffer e.g., citrate buffer) 812 may include citric acid (monohydrate sodium citrate) and/or sodium hydroxide.

[0152] According to embodiments described herein, step 808 (LNP formation) includes reducing or eliminating the introduction of various impurities into the process and/or various solutions thereof, thereby forming a first RNA-LNP preparation that includes LNP-encapsulated RNA. LNP formation 808 may include the adjusting of one or more process temperatures, process flow rates, and/or ratios of the buffers, solutions and/or suspensions. LNP formation may include independently flowing each of the aqueous solution and lipids 810 (for example, in a lipid solution) into a mixing unit. Each of the aqueous RNA solution 806 and lipid solution 810 may flow into the mixing unit under laminar flow conditions (to avoid the entrapment of gas bubbles (for example, ambient gas bubbles (i.e., primarily comprised of nitrogen)) within the flow).

[0153] Still referring to Fig. 13, at step 814, the process 800 may include buffer exchange and concentration of the first RNA-LNP preparation to form a second RNA-LNP preparation. The buffer exchange and concentration step 814 may be conducted with process parameters including, for example, a feed flow rate, for example within a range of 18 to 50 liter/min (LPM), a trans-membrane pressure (TMP), for example lower than 1200 mbar, a retentate pressure, for example within a range of 130 to 230 mbar, and a permeate pressure, for example within a range of 10 to 70 mbar.

[0154] In some embodiments, buffer exchange 814 of the first RNA-LNP preparation and concentrating 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, the buffer exchange 814 is conducted via diafiltration and the concentration is conducted via ultrafiltration. In some embodiments, the diafiltration and/or the ultrafiltration are conducted via tangential flow filtration (TFF) (for example, in a tangential flow filtration unit and/or TFF skid). In some embodiments, the tangential flow filtration is conducted using one or more jejunostomy tubes and/or one or more dip tubes configured to avoid introducing ambient gas bubbles into the second RNA- LNP preparation. During the tangential flow filtration, a retentate may be recirculated to a feed tank using a dip tube comprising a first end submerged into filtration feed liquid in the feed tank to avoid introducing ambient gas bubbles into the filtration feed liquid. Prior to the buffer exchange and concentration steps, a filtration system for tangential flow filtration may be filled with a buffer to prevent introducing ambient gas bubbles into the second RNA-LNP preparation.

[0155] Referring still to Fig. 13, the buffer exchange and concentration step 814 may include at least two buffer exchanges conducted via diafiltration alternating with at least two concentrations conducted via ultrafiltration. During buffer exchange and concentration 814, process temperatures may be maintained within a desired temperature range (for example, at or below about 25 degrees C, or from about 2 degrees C to about 25 degrees C, or from about 15 degrees C to about 25 degrees C). During buffer exchange and concentration 814, pH may be continuously monitored (and may be maintained in a target range (for example, from about 7.0 to about 7.5, or from about 7.1 to about 7.3)) and shear may be maintained, for example in a range from about 2000 s^A-l to about 6000 s^A-l, or from about 3000 s^A-l to about 5000 s^A-l, or at about 4000 s^A-l (+/- 1%, 5%, and/or 10%).

Following buffer exchange and concentration 814, a recovery flush may be performed, during which time shear may be reduced to under about 2000 s^A-l (for example, under about 1500 s^A-l, or under about 1000 s^A-l). In some embodiments, following buffer exchange 814, the pH may be maintained within a range from about 7.3 to about 7.5, for example following ultrafiltration and/or diafiltration.

[0156] In some embodiments, during buffer exchange and/or concentration 814, the pH of the first RNA-LNP preparation may be maintained at a pH that is higher than that of the cationic lipid (i.e., the cationic lipid in the lipid solution). Without wishing to be bound by any particular theory, it is proposed that doing so may reduce foaming of the liquid nanoparticles.

[0157] In some embodiments, the first and/or second RNA-LNP preparation(s) may be sterilized without introducing any ambient gas into the produced formulation. In some embodiments, a relevant produced formulation may be a product for further manipulation, processing, packaging, and/or shipping. In some embodiments, a produced formulation may be or comprise a drug product formulation, e.g., for administration to humans. [0158] In some embodiments, one or more sterilization steps may be performed by sterile filtration; in some embodiments, sterile (or other) filtration may be conducted at a target pressure with substantially no pressure building up during the filtration process, for example at about 1.03 bar (or from about 1.02 bar to about 1.04 bar, from about 1.01 bar to about 1.05 bar, or from about 1.00 bar to about 1.1 bar).

[0159] In some embodiments, a utilized mixing unit may include one or more impingement jet mixing skids. Prior to mixing, the impingement jet mixing skids may be vented and/or flooded to remove gas bubbles from tubing of the impingement jet mixing skids. Mixing of the aqueous and lipid solutions may be performed within boundaries of the mixing unit and/or one or more impingement jet mixing skids. In some embodiments, prior to mixing, the aqueous solution does not contact the lipid solution. In some embodiments, the flow rate ratio into the mixing unit of the aqueous solution to the lipid solution is about 3:1, or from about 2.75:1 to about 3.25:1, or from about 2.5:1 to about 3.5:1, and/or from about 2.5: to about 3:1. In some embodiments, the mixing speed may be adapted to avoid entrapping ambient gas in the first RNA-LNP preparation. For example, in order to avoid the introduction of ambient gas (and/or other impurities), one or more mixing processes may include increasing the mixing speed gradually until a slight vortex has formed (for example, the mixing speed at or slightly above the point at which a visible vortex has formed), but below the mixing speed at which foam begins to form.

[0160] Still referring to Fig. 13, the system (for example, the impingement jet mixing skids, the TFF system (i.e., the tangential flow filtration unit), and/or components thereof) may be assessed at one or more time points (e.g., monitored over time, e.g., periodically or continuously) for presence of gas bubbles. In the event that gas is detected in the aqueous solution, the lipid solution, the first RNA-LNP preparation, the second RNA- LNP preparation, the mixing unit, and/or 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 gas has been detected somewhere in the system. In some embodiments, gas detection may be performed via one or more flowmeters (for example, via one or more Coriolis flowmeters), and/or by visual assessment e.g., via the human eye and/or various types of cameras), viand/or other detection means.

[0161] In some embodiments, the aqueous solution and/or the lipid solution may be flowed into the mixing unit through one or more inlets disposed at a bottom portion of the mixing unit, and the resulting first RNA-LNP preparation may be released from the mixing unit through one or more outlets disposed at a top portion of the mixing unit. In some embodiments, the mixing may be performed with a submerged mixer. In some embodiments, foam may be generated during and/or after formation of the LNP- encapsulated RNA, and may be subsequently removed from the RNA-LNP preparation (for example, the foam may be removed from the first and/or second RNA-LNP preparation).

[0162] Referring still to Fig. 13, following buffer exchange 814 and concentration, the process 800 may include 0.2 pm filtration and/or the addition of sucrose and PBS for compounding. Following compounding, the process 800 may include bioburden reduction filtration (BBR) 816 following the buffer exchange and concentration 814. Bioburden reduction filtration 816 (formulation) may include filtering with 0.2 pm pore size (or for example, about a 0.22 pm pore size) or smaller filter. Bioburden reduction filtration 816 may also include using other pore sizes (for example, 0.45 pm pore size) as described herein. According to the present embodiments, 0.2 pm pore size filtering may also occur on each of the lipid solution and the aqueous RNA solution prior to mixing, on the first RNA- LNP preparation, and/or on the second RNA-LNP preparation. Bioburden reduction filtration 816 may also include filtering the post TFF-LNP suspension through a particulate reduction filter prior to filtering the suspension through (for example) the 0.2 pm pore size and/or 0.22 pm pore size bioburden reduction filter. In some embodiments, bioburden reduction filtration 816 may also include performing a filter recovery flush.

[0163] Still referring to Fig. 13, following bioburden reduction filtration 816, the process 800 may include filling transport bags (for example, Flexsafe ® bags) with the filtered second RNA-LNP preparation, and performing a visual inspection 818 of the transport bags for gas bubbles. In some embodiments, transport bags may be, for example 12L bags, 50L bags, 100L bags, and/or other suitable bag sizes (e.g., depending on the batch size of the relevant RNA-LNP preparation), including bags that include a volume between 12L and 50L, and/or bags that include a volume between 50L and 100L.

[0164] In some embodiments, filling transport bags may include filling the bags to a volume in a range from about 30% to about 95%, or from about 40% to about 90%, or from about 50% to about 85%, or from about 60% to about 85% or from about 70% to about 85%, and/or other subranges therebetween of the total bag volume.

[0165] Filled bags may be stored and/or shipped at a temperature in a range from about 1 degree C to about 15 degrees C (for example, at about 2 degrees C to about 10 degrees C, or from about 2 degrees C to about 8 degrees C), or alternatively may be frozen to a temperature of about -70 degrees C (for example, in a range from about -60 degrees C to about -80 degrees C). Prior to shipment, the bags may be secured in or on racks and/or within or on any other suitable shelving or storage system so as to minimize movement, rupturing, and/or disruption of the bags during the transport to a fill and finish site. For example, transport bags may be stacked in a specific manner using a stacking system on pallets that include shock absorbers. During transport 820 and/or in preparation for transport 820, as well as following transport, nitrogen with a positive pressure (for example, from about 1-2 bars) may be maintained in and around the environment in which the bags are kept and/or transported, in order to prevent ambient gas from entering the bags. After transport 820, the bags may be assessed for ambient gas content (e.g., visually inspected) 822 a second time. In some embodiments, ambient gas bubbles that are discovered during such second assessment 822 may be removed (e.g., may be manually removed), or alternatively, the bag or bags that include ambient gas bubbles may be selectively discarded (for example, if the volume of ambient gas within a given bag has exceeded a threshold).

[0166] Referring still to Fig. 13, after arriving at a fill and finish site, sterile filtration 824 may be performed (i.e., the second RNA-LNP preparation). In some embodiments, such sterile filtration 824 may be performed after the preparation has been removed from the transport bags, but prior to being disposed within a collection vessel, reservoir, and/or fill tank. In some embodiments, the material (i.e., the filtered preparation) may then be dispersed from the collection vessel, reservoir, and/or fill tank during aseptic fill and finish 826 (for example, to aseptically fill glass vessels with the drug product).

[0167] Visual inspection 828 may be performed on the filled glass vessels. The inspected and filled glass vessels, at step 830 of the process 800, may then be frozen, stored, warehoused and/or distributed, for example, to health care administration sites.

Alternatively, in some embodiments, filled glass vessels may be subjected to lyophilization or other drying process, so that drug product is transported and/or stored in a dry state (e.g., for subsequent resuspension).

[0168] Still referring to Fig. 13, in some embodiments, the fill and finish facility may be located in the same location as the LNP production facility, in which case fill and finish may be performed directly using Point of Fill filtration equipment (in which case the transport 820, bag filling and sealing, and one or more of the visual inspection steps 818, 822, 828 may not be required. In yet other embodiments, the process 800 may include multiple transport steps 820, as well as additional visual inspection steps 818, 822, 828 if the various steps of the process 800 are performed at additional and/or other facilities (or alternatively, if transport is required within a single facility).

[0169] Figure 2 depicts an overview of an automated drug manufacturing process 40, according to aspects of the present disclosure. The process may include in vitro transcription 50, a first tangential flow filtration process 60, LNP formation 70, a second tangential flow filtration 80, formulation 90, and fill and finish 100. The automated drug manufacturing process 40 of the present embodiments (illustrated in Fig. 2) may include a fully automated process occurring all at a single site (for example, at a single location within a single, contiguous footprint (i.e., production facility) comprising a total area of no more than about 8,000-12,000 square meters (for example, within an area comprising from about 100 square meters to about 12,000 square meters, or from about 200 square meters to about 10,000 square meters, or from about 300 square meters to about 8,000 square meters, or from about 200 square meters to about 6,000 square meters, or from about 500 square meters to about 3,000 square meters, or from about 1,000 square meters to about 5,000 square meters, or from about 1,500 square meters to about 4,000 square meters, or from about 750 square meters to about 3,500 square meters, or from about 500 square meters to about 1,750 square meters, and/or other subranges therebetween). As such, several steps of the exemplary process 10 illustrated in Fig. 1 (such as transport between locations, and multiple storage (and thawing) steps) may be able to be eliminated. In addition, because the process 40 is fully automated, less manpower is required to run the process. For example, the process may be monitored remotely, with no human intervention required to run the process once it has been supplied with electricity, a water supply, and/or the necessary input materials.

[0170] Figure 3 depicts an overview of an automated in vitro transcription process flow, method, module, and/or system 50, according to aspects of the present disclosure. The process flow and/or system 50 may include several of the same process steps, equipment, control parameters, and/or other characteristics of the LNP manafucturing processes and systems described above and illustrated in Figs. 11-13. The process flow and/or system 50 may generally include mRNA synthesis through three succesive in vitro transcription steps performed in fluidly connected bioreactors 226, 228, 230 (for example, stirred bioreactors). As such, the process flow and/or system 50 of the present embodiments is semi-continuous meaning that in vitro transcription is occurring in three or more discrete reaction chambers 226, 228, 230 that are connected such that drug substance preparations may automatically move between bioreactors 226, 228, 230 at various stages of in vitro transcription, without requiring human intervention. In addition, as described herein, the bioreactors 226, 228, 230 are connected to temperature -regulated hold tanks 200, 204 of adequate size that are recharged by robot system technology 202 for all raw materials and cleaning agents. Reaction chambers (i.e., bioreactors 226, 228, 230) may be automatically cleaned after use. The sequence of the process flow 50 may include one or more of the following aspects: a robot system 202 transfers components (i.e., drug substance input materials) that may be added to the reaction chambers (226, 228, 230) from the storage location 200 (usually maintained at -20°C, or in a range from -20°C to 8°C ) to a thawing chamber 204 where a controlled thawing of the material may be performed such that the timing of the thawing process is managed to have the material ready for addition exactly when it is needed. Addition of material to the reaction chambers 226, 228, 230 may also be performed by a robot system 202. The robot system 202 may also be cleaned, disinfected, and/or sanitized automatically (for example, using automated mist, spray, vapor, and/or fog disinfecting equipment that intermittently sprays disinfectant (in liquid, gaseous, and/or multi-phase form)) onto external surfaces of the robot system 202.

[0171] Still referring to Fig. 3, the system, method, process flow, and/or module 50 may include a storage chamber 200 where input materials required for in vitro transcription may be stored at a controlled temperature (for example, in a range from about negative 20 degrees C to about 8 degrees C). A robot 202 may be used to automatically move input materials from the storage chamber 200 into the controlled thawing chamber 204. The input materials may be contained within bags, bottles, and/or other suitable containers when they are in the storage chamber 200. The system 206 may include a cleaning agent vessel 206 for containing a cleaning agent, as well as a pump 208 (for example, a centrifugal pump) downstream of the cleaning agent vessel 206 for pumping cleaning agent through a connection line 210 such that the entire system (i.e., the in vitro transcription system 50 of the present embodiments) can be automatically cleaned in between uses. Each of the storage chamber 200, the controlled thawing chamber 204, and the cleaning agent vessel 206 may include an open tank with a lip (to prevent or minimize spillage), as well as other suitable containers. The system 50 may include a pump 212 (for example, a second centrifugal pump) downstream of the connection line 210 which may be used to selectively pump the the input matrials and/or cleaning agent to one or more of the three bioreactors 226, 228, 230.

[0172] Referring still to Fig. 3, the system, method, module, or process flow 50 may include a common line 236 fluidly downstream of the second pump 212 and fluidly connected to first, second, and third branch lines 214, 216, 218 connecting to the first, second, and third bioreactors 226, 228, 230, respectively. Each of the first, second, and third bioreactors 226, 228, 230 may be monitored and controlled via one or more instruments, each communicatively coupled to a control system (not shown). For example, a short interval controller (SIC) 220 may be coupled to each of the first, second, and third bioreactors 226, 228, 230 to provide localized control to each. Each short interval controller (SIC) 220 may be coupled to a plurality of other instruments 224 coupled to each of the first, second, and third bioreactors 226, 228, 230, the plurality of other instruments 224 including a quantity indicator, a weight indicator, and a temperature indicator controller (providing both temperature indication and the ability to control the tempeature of each of the first, second, and third bioreactors 226, 228, 230. The system 50 may include a temperature element 222 including a heat exchanger (with the ability to both heat and cool each bioreactor to a desired temperature) fluidly coupled to each bioreactor 226, 228, 230, and operatively coupled to the respective short interval controllers 220.

[0173] Still referring to Fig. 3, because the common line 236 is connected to each of the branch lines 214, 216, and 218, thawed input materials as well as cleaning agent may be selectively routed to each of the first, second, and third bioreactors 226, 228, and 230. Therefore, the in vitro transcription process occurring in each of the first, second, and third bioreactors 226, 228, 230 may proceed independently and may receive thawed input materials, as needed, via the respective branch lines 214, 216, and 218. The independent in vitro transcription processes may then we synchronized such that there is at least a semi- continuous, if not fully continuous flow of transcripted RNA solution flowing from the outlet line 240 of the in vitro transcription module or system 50 while the system is in operation. In some embodiments, the in vitro transcription module or system 50 may include more than 3 bioreactors (for example, 4, 5, 6, 7, 8, 9, 10, and/or more than 10) in parallel and/or in series configuration, in order to further enhance the continuous processing of transcripted RNA solution. Each of the branch lines 214, 216, and 218 may include a valve 238 to control flow into each bioreactor 226, 228, 230. In addition, a third pump 232 (for example, a centrifugal pump) and valve 238 may be disposed downstream of the first bioreactor 226 and upstream of the second bioreactor 228 such that a first drug substance preparation can be routed from the first bioreactor 226 to the second bioreactor 228. Similarly, a fourth pump 234 (for example, a centrifugal pump) and valve 238 may be disposed downstream of the second bioreactor 228 and upstream of the third bioreactor 230 such that a second drug substance preparation can be routed from the second bioreactor 228 to the third bioreactor 230. The in vitro transcription module or system 50 may include an outlet line 240 that routes flow downstream to the first tangential flow filtration module (or system) 60 (shown in Fig. 4).

[0174] Referring still to Fig. 3, the system, method, module, or process flow 50 may include a first bypass line 237 fluidly connecting a discharge (or outlet) of the first bioreactor 226 to an inlet line of the third bioreactor 230 (i.e., to allow for a flexible workflow in conditions where a batch of solution (i.e., IVT solution) is ready to proceed to the third bioreactor 230 (from the first bioreactor 226) and the second bioreactor 228 is otherwise unavailable (i.e., for example, is processing another batch)). Similarly, in some embodiments, the system 50 may include a second bypass line 239 that fluidly connects a discharge (or outlet) of the first bioreactor 226 to an exit line 240 of the third bioreactor 230 (i.e., to allow discharge from the first bioreactor 226 to bypass both the second and third bioreactors 228, 230, and to proceed to the TFF module 60). Similarly, in some embodiments, the system 50 may include a third bypass line 241 that fluidly connects a discharge (or outlet) of the second bioreactor 228 to an exit line 240 of the third bioreactor 230 (i.e., to allow discharge from the second bioreactor 228 to bypass the third bioreactor 230, and to proceed to the TFF module 60). Each of the first, second and third bypass lines 237, 239, 241 may include at least one valve 238 and at least one pump 232 such that flow through each line may be selectively controlled, as needed, according to various process parameters as described herein. The configuration of the system 50 illustrated in Fig. 3 allows flows in various quantities to be selectively and intermittently delivered into and out of each of the first, second, and third bioreactors 226, 228, 230, independently of the operating status of each other bioreactor, thereby creating a quasi-batch, quasi-continuous in vitro transcription (IVT) system, method, module, or process flow 50. For example, the system 50 may intermittently and selectively deliver batches of solutions into each of the first, second, and third bioreactors 226, 228, 230 (i.e., in more of a batch-type arrangement) while IVT solution exiting the module at exit line 240 is controlled in a continuous or near- continuous manner or arrangement. For example, the system 50 enables the pooling of IVT solution from all three bioreactors 226, 228, 230 at the IVT module exit line 240, from which the IVT solution can be delivered to the TFF module 60 in a more continuous manner. In some embodiments, the system 50 may include a return line (not shown) for example, extending from the second bioreactor 228 back to the first bioreactor 226, and/or from the third bioreactor 230 back to the second and/or first bioreactor 226, 228 in the event that further IVT processing is required on the IVT solution before it is delivered to the first TFF module 60.

[0175] Still referring to Fig. 3, in some embodiments, processing of IVT solution within the system 50 may continue until mRNA synthesis, DNA hydrolysis and protein hydrolysis have occurred. According to aspects of the present embodiments, mRNA synthesis, DNA hydrolysis and protein hydrolysis may each occur entirely within the same bioreactor and/or in different bioreactors (for example, within the first, second, and/or third bioreactors 226, 228, 230, and/or combinations thereof). For example, in some embodiments, a first IVT step (i.e., mRNA synthesis) occurs in the first bioreactor 226, a second IVT step (i.e., DNA hydrolysis) occurs in the second bioreactor 228, and a third IVT step (i.e., protein hydrolysis) occurs in the third bioreactor 230. In some embodiments, more than one of the first, second, and third IVT steps (for example, 2 or all 3) occur in only one of the first, second, and/or third bioreactors 226, 228, 230. According to aspects of the present embodiments, the mRNA synthesis, DNA hydrolysis and protein hydrolysis processes (i.e., subprocesses) may each be controlled automatically by the system 50. For example, controlled mixing of the process materials may be performed by the first, second, and/or third bioreactors 226, 228, 230. Monitoring of pH, temperatures, and other parameters may be performed automatically using sensor data and control systems, as described herein. Temperatures, mixing speed, timing, and agitation can all be monitored and controlled by the bioreactors 226, 228, 230 while pH can be monitored and adjusted through the controlled introduction and mixing of additional buffer, as needed, into bioreactors 226, 228, 230. Process steps (for example, required incubation temperatures, incubation times, hold times, mixing time, mixing speeds, levels of agitation, etc.) may be monitored and carried out by the system 50 and/or components thereof. The system 50 according to the present disclosure is configured to automatically route IVT solution to the TFF module 60 only when each of the various IVT subprocesses have been carried out.

[0176] Figure 4 depicts an overview of a first automated tangential flow filtration method, module, system, and/or process flow 60, according to aspects of the present disclosure. One of ordinary skill in the art will be aware of available membrane filtration modes, for example which can use either microfiltration or ultrafiltration membranes: (1) Direct Flow Filtration (DFF), also known as “dead-end” filtration, applies a feed stream perpendicular to the membrane face and attempts to pass 100% of the fluid through the membrane, and (2) Tangential Flow Filtration (TFF), also known as crossflow filtration, where a feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) while the remainder (retentate) is retained and/or recirculated back to the feed reservoir. In some embodiments, an in vitro transcription RNA composition (e.g., in some embodiments after DNA removal) can be purified by membrane filtration and may be purified by a process comprising direct flow filtration.

[0177] Referring to Fig. 4, and also Fig. 8, in some embodiments the first TFF process flow 60 and second TFF process flow 80 may include an exchange buffer stream flowing in an opposite direction of the product flow (for example, in each of the first and second TFF cassettes 244, 246, for example, flowing from the outlet end 258 of the product stream to the inlet end 260 of the product stream in a double cross flow (that is, cross flow of the product over the membrane and cross flow of the exchange buffer opposite to the product flow) configuration). For example, in some embodiments, the TFF process flow 60 may include exchange buffer flowing across membranes disposed within the TFF cassettes 244, 246 and product flowing in an opposite direction. As discussed herein, in the first TFF process flow 60 shown in Fig. 4, the product flow may include an RNA solution. In the second TFF process flow 80 shown in Fig. 8, the product flow may include an RNA-LNP preparation. In some embodiments, a turbulent exchange buffer flow can be realized by adjusting the dimensions of flow tubes within each of the first and second TFF cassettes 244, 246 to allow for an increased exchange of buffer components. In some instances, the length of each of the first and second TFF cassettes 244, 246 and/or the number of TFF cassettes can be varied (for example, such that each of the first and second TFF cassettes 244, 246 includes multiple cassettes arranged in series) in order to enhance the buffer exchange. In some embodiments the transmembrane pressure can be reversed so that the filtrate flow is pressed into the product flow from the outlet end 258 to the inlet end 260, thereby replacing buffer components by washing out via crossflow. As such, each of the first and second TFF cassettes 244, 246 may include one or more pressure modulators 277 (for example, to be used in connection with pumps, valves, pressure sensors, etc.) to selectively vary the transmembrane pressure. The second TFF process flow 80 (shown in Fig. 8) may also include TFF cassettes with one or more pressure modulators. In some embodiments, the transmembrane pressure can be selectively varied or oscillated between “normal” (i.e., positive transmembrane pressure) and “reversed” (i.e., negative transmembrane pressure) to get a faster exchange of buffer in the product stream.

[0178] In some embodiments, an in vitro transcription RNA composition (e.g., in some embodiments after DNA and/or protein removal and/or digestion) can be purified by a process comprising tangential flow filtration (TFF). In some embodiments, a filtration membrane with an appropriate molecular weight cut-off (MWCO) may be selected for TFF. The MWCO of a TFF membrane determines which solutes can pass through the membrane (i.e., into the filtrate) and which are retained (i.e., in the retentate). The MWCO of a TFF membrane used in accordance with the present disclosure is selected such that substantially all of the solutes of interest (e.g., desired synthesized RNA species) remains in the retentate, whereas undesired components (e.g., excess ribonucleotides, small nucleic acid fragments such as digested or hydrolyzed DNA template, peptide fragments such as digested proteins and/or other impurities) pass into the filtrate. In some embodiments, the retentate comprising desired synthesized RNA species may be re-circulated to a feed reservoir to be re-filtered in additional cycles. In some embodiments, a TFF membrane may have a MWCO of at least 30 kDa (including, e.g., at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, or more). In some embodiments, a TFF membrane may have a MWCO of at least 100 kDa (including, e.g., at least 150 kDa, at least 200 kDa, at least 250 kDa, at least 300 kDa, at least 350 kDa, at least 400 kDa, or more). In some embodiments, a TFF membrane may have a MWCO of about 250-350 kDa. In some embodiments, a TFF membrane (e.g., a cellulose-based membrane) may have a MWCO of about 30-300 kDa; in some embodiments about 50-300 kDa, about 100-300 kDa, or about 200-300 kDa.

[0179] While a skilled artisan may select an appropriate filtration material for a filtration membrane, in some embodiments, a filtration membrane that is particularly useful for TFF purification in accordance with the present disclosure is or comprises a cellulose- based membrane. In some embodiments, a filtration membrane is not a thermoplastic membrane (e.g., poly sulfone or polyethersulfone). In some embodiments, a filtration membrane is a filter cassette.

[0180] In some embodiments, TFF is performed at a transmembrane pressure that is less than, for example, 2 bar (including, e.g., less than 2 bar, less than 1.9 bar, less than 1.8 bar, less than 1.7 bar, less than 1.6 bar, less than 1.5 bar, less than 1.4 bar, less than 1.3 bar, less than 1.2 bar, less than 1.1 bar, less than 1.0 bar, less than 0.9 bar, less than 0.8 bar, less than 0.7 bar, less than 0.6 bar, or lower). In some embodiments, TFF is performed at a transmembrane pressure in a range of about 0.5 bar to 2 bar. In some embodiments, TFF is performed at a transmembrane pressure of about 1 bar. In some embodiments, TFF is performed with a feed flow rate of less than, for example, 400 liters/m²/hour (LMH) (including, e.g., less than 400 LMH, less than 350 LMH, less than 300 LMH, less than 250 LMH, less than 200 LMH, less than 150 LMH, less than 100 LMH, or less). In some embodiments, TFF is performed with a feed flow rate of about 75 LMH to about 500 LMH, or about 50 LMH to about 400 LMH.

[0181] In some embodiments, an in vitro transcription RNA composition following RNA transcription that is subject to TFF purification has not been treated with a protein denaturing agent such as, e.g., urea, guanidinium chloride thiocyanate, salts of alkali metals (e.g., potassium chloride), sodium dodecyl sulfate, sarcosyl, and combinations thereof.

[0182] Referring again to Fig. 4, a purification buffer 266, 268, 270 may be fed into a TFF process in addition to an RNA preparation comprising an RNA transcription mixture. The choice and composition of the purification buffer may influence the efficiency of RNA purification, levels of protein aggregation, RNA-protein separation, and/or RNA stability. Typical buffers may include Tris buffer and citrate buffers. In some embodiments, a purification buffer that may be particularly useful for TFF purification in accordance with the present disclosure may be or comprise HEPES buffer. In some embodiments, a purification buffer e.g., HEPES buffer) may further comprise a chelating agent (e.g., as described herein) and/or a salt(s) (e.g., ammonium acetate, ammonium sulfate, potassium acetate, potassium chloride, potassium sulfate, sodium acetate, sodium chloride, and/or sodium sulfate).

[0183] In some embodiments, TFF purification may be performed without a buffer change. For example, in some embodiments, TFF purification is performed in a buffer that has been utilized for in vitro transcription; in some such embodiments, TFF purification may be performed in a HEPES buffer.

[0184] In some embodiments, a TFF purification process may comprise at least two separate steps of tangential flow filtration (for example, as represented in Fig. 4 by a first TFF cassette 244, and a second TFF cassette 246). For example, in some embodiments, a first step 244 of tangential flow filtration and a second step 246 of tangential flow filtration may utilize different buffers. In some embodiments, a first buffer 268 used in a first step of tangential flow filtration may comprise salt(s) (e.g., ammonium acetate, ammonium sulfate, potassium acetate, potassium chloride, potassium sulfate, sodium acetate, sodium chloride, and/or sodium sulfate), while a second buffer 270 used in a second step of tangential flow filtration may not comprise the same salt(s) as used in the first step (e.g., ammonium acetate, ammonium sulfate, potassium acetate, potassium chloride, potassium sulfate, sodium acetate, sodium chloride, and/or sodium sulfate). In some embodiments, a second buffer 270 used in a second step of tangential flow filtration may not comprise a salt.

[0185] Referring still to Fig. 4, in some embodiments, a first step 244 of tangential flow filtration e.g., for diafiltration) may be performed with a defined number of volume exchanges (e.g., at least one, at least two, at least three, at least four at least five, at least six, at least seven, or more volume exchanges). In some embodiments, a second step 246 of tangential flow filtration (e.g., for diafiltration) may be performed with a defined number of volume exchanges (e.g., at least one, at least two, at least three, at least four at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least or more volume exchanges). In some embodiments, a first step 244 of tangential flow filtration may be performed with a minimum of 5 volume exchanges and a second step 246 of tangential flow filtration may be performed with a minimum of 10 volume exchanges.

[0186] Still referring to Fig. 4, in some embodiments, an in vitro transcription RNA composition described herein (e.g., in some embodiments after DNA and/or protein removal and/or digestion, e.g., flowing into the TFF module 60 from the in vitro transcription module 50 via inlet 240) can be subjected to a suitable purification method known to one of ordinary skill in the art. In some embodiments, an in vitro transcription RNA composition described herein can be subjected to precipitation followed by membrane filtration (e.g., as described in WO2015164773). In some embodiments, an in vitro transcription RNA composition described herein can be subjected to one or more steps of TFF, wherein at least one or more steps of TFF comprises use of a TFF membrane cassette 244, 246 (e.g., as described in WO2016193206). In some embodiments, an in vitro transcription RNA composition described herein can be subjected to a high salt condition chromatography (e.g., by hydrophobic interaction chromatography). In some embodiments, an in vitro transcription RNA composition described herein can be a crude RNA reaction IVT mixture or high- performance liquid chromatography purified RNA which is subsequently subjected to a high salt condition chromatography (e.g., as described in WO2018096179). In some embodiments, an in vitro transcription RNA composition described herein can be subjected to filtering centrifugation. In some embodiments, an RNA is precipitated prior to centrifugation (e.g., as described in WO2018157141). In some embodiments, an in vitro transcription RNA composition described herein can be subjected to a stirred cell or agitated Nutsche filtration device. In some embodiments, a high concentration of salt is added to an RNA composition to denature and solubilize contaminating proteins prior to subjection to a stirred cell or agitated Nutsche filtration device (e.g., as described in WO2018157133). In some embodiments, an in vitro transcription RNA composition described herein can be subjected to standard flow filtration (e.g., a filtration process in which the material to be purified flows in a direction normal, i.e., perpendicular, to the surface of the filter). In some embodiments, RNA is precipitated prior to standard flow filtration e.g., as described in W02020041793). In some embodiments, an in vitro transcription RNA composition described herein can be subjected to precipitation in a buffer comprising high concentration of salts (e.g., guanidinium salts) and a detergent (e.g., as described in W02020097509).

[0187] Referring still to Fig. 4, in some embodiments, an in vitro transcription RNA composition described herein (e.g., in some embodiments after DNA removal and/or digestion, e.g., delivered to the TFF module 60 via inlet 240) can be subjected to a protein digestion or fragmentation process prior to one or more additional purification methods known in the art (including, e.g., precipitation, affinity-based purification, ion exchange chromatography methods, high performance liquid chromatography, hydrophobic interaction chromatography, size exclusion-based methods such as size exclusion chromatography, filtration methods such as, e.g., centrifugal ultrafiltration and/or membrane filtration (e.g., direct flow filtration or tangential flow filtration), etc., or combinations thereof). For example, in some embodiments, an exemplary protein digestion or fragmentation may comprise use of a proteinase (e.g., but not limited to proteinase K).

[0188] Still referring to Fig. 4, in some embodiments, an in vitro transcription RNA composition described herein (e.g., in some embodiments after DNA removal and/or digestion and/or removal of impurities) can be subjected to a method of removing or reducing bioburden (e.g., microbial contamination). In some embodiments, an exemplary method for bioburden removal or reduction may be or comprise filtration (for example, via filter 296). In some embodiments, filtration may be or comprise gravity filtration. In some embodiments, gravity filtration may be performed using a filter with pore size that is small enough to capture bioburden (e.g., a filter with 0.45 pm pore size or smaller, a filter with 0.2 pm pore size or smaller). In some embodiments, filtration may be performed using a 0.45 pm pore filter. In some embodiments, filtration may be performed using a 0.2 pm pore filter. In some embodiments, filtration may be performed first using a 0.45 pm pore filter and subsequently using a 0.2 pm pore filter. In some embodiments, filtration may be performed first using a 0.2 pm pore filter and subsequently using a 0.45 pm pore filter.

[0189] In some embodiments, an in vitro transcription RNA composition described herein (e.g., in some embodiments after DNA removal and/or digestion) can be subjected to at least one or more of purification methods described herein, including, e.g., bind-and elute process (e.g., utilizing solid substrate with high RNA affinity such as magnetic bead-based purification, membrane filtration e.g., tangential flow filtration), and/or filtration (e.g., gravity filtration). In some embodiments, an in vitro transcription RNA composition described herein may be purified by magnetic-bead-based purification (e.g., as described herein) followed by bioburden filtration (e.g., as described herein), to produce an RNA transcript preparation. In some embodiments, an in vitro transcription RNA composition described herein may be purified by a TFF process that may comprise one or a plurality of (e.g., at least two) TFF steps (e.g., as described herein) followed by bioburden filtration (e.g., as described herein), to produce an in vitro transcription RNA composition.

[0190] Referring again to Fig. 4, the first automated tangential flow filtration module, system, and/or process flow 60 may include a small reservoir 241 for receiving drug substance preparations from the in vitro transcription module 50. Because the first automated tangential flow filtration module, system, and/or process flow 60 is fluidly coupled immediately downstream of the in vitro transcription module 50, drug substance preparations (i.e., in vitro transcription RNA composition(s)) from the in vitro transcription module 50 may be routed directly into tangential flow filtration via inlet 240, without needing to be stored, transported, and/or held in a large reservoir for an extended period of time. Drug substance preparations (i.e., in vitro transcription RNA composition(s)) may be routed into the first TFF cassette (for example at a first end 260). The first buffer 268 and/or second buffer 270, as well as dilution 266 may be fed into a second end 258 of the first TFF cassette 244 via a feedline 262, which is fluidly coupled to each of a first buffer line 274, a second buffer line 276, and a dilution feed line 272. Each of the first buffer line 274, the second buffer line 276, and the dilution feed line 272 are joined at a union 280 comprising one or more valves 278 disposed therewithin such that the first and second buffer 268, 270, and the dilution feed may be selectively controlled into the first and/or second cassette 244, 246 as needed. The dilution feed and buffer storage area 286 may be located adjacent to the TFF process, or in an area that is located a short distance away from the TFF (but still connected to the TFF process via feedline 262). The first automated tangential flow filtration module, system, and/or process flow 60 may also include a diaphragm pump 282 (or membrane pump 282) fluidly disposed within the feedline 262 downstream of the union 280, and upstream of each of the first cassette 244 and the second cassette 246. Feedline 262 feeds the respective buffer inlets 254 and 256 of each of the first and second cassettes 244, 246 respectively, while permeate outlets 248 and 250 act to remove permeate from the first and second TFF cassettes 244, 246 respectively. The permeate may subsequently be disposed of. The first automated tangential flow filtration module, system, and/or process flow 60 may also include a connection line 252 connecting an outlet at the second end 258 of the first TFF cassette 244 to an inlet at the first end 260 of the second TFF cassette 246. The first automated tangential flow filtration module, system, and/or process flow 60 may also include a pump 242 (for example, a centrifugal pump 242) disposed in the TFF inlet line 240 upstream of the first TFF cassette 244.

[0191] Still referring to Fig. 4, the first automated tangential flow filtration module, system, and/or process flow 60 may include a rotary mixer 290 disposed fluidly downstream of the second TFF cassette 246, as well as recirculation line 300 coupled to the rotary mixer 290. The recirculation line 300 may recirculate retentate back to the second TFF cassette 246 as needed until desired process parameters are met. For example, the first automated tangential flow filtration module, system, and/or process flow 60 may include a diaphragm pump (or membrane pump) 292 disposed in the recirculation line 300 as well as a concentration control and adjustment instrument 288 disposed in the recirculation line 300 downstream of the diaphragm pump 292, to help monitor and control flow back through the recirculation line 300 as needed. The first automated tangential flow filtration module, system, and/or process flow 60 may also include one or more instruments 294 located in the vicinity of the rotary mixer 290 to be used in connection with recirculation line 300 to achieve the desired process parameters. For example, the one or more instruments 294 may include a pressure indicator controller, a short interval controller, a weight indicator controller, and/or other types of instruments, which may be used in connection with the inline concentration controller 288 to determine if retentate should be recirculated back to the second TFF cassette 246 (i.e., based on whether product concentration adjustments need to be made, etc.). The first automated tangential flow filtration module, system, and/or process flow 60 may also include a filter 296 disposed downstream of the rotary mixer 290, one or more drain or vent lines 298 disposed downstream of the filer 296, as well as a first TFF exit line 302 for delivering drug substance downstream to the LNP formation module 70.

[0192] Referring still to Fig. 4, tangential flow filtration is often a discontinuous process. The TFF process 60 according to the present embodiments may be a continuous process and/or a semi-continuous process. Flows may be routed from one process step to the next in a continuous fashion. The first automated tangential flow filtration module, system, and/or process flow 60 may also be cleaned automatically after use, via cleaning agent which may be automatically routed through the system between uses. Similarly, robotics (for example, robot 264) may be used to automatically fill the first and second buffer reservoirs 268, 270, as well as the dilution feed 266, and to recharge cleaning agents, such that human intervention is not needed to keep the process operating continuously.

[0193] Figure 5 depicts an overview of an automated LNP formation method, system, module and/or process flow 70, according to aspects of the present disclosure. Generally, in the automated LNP formation process, system, and/or workflow 70, an in vitro transcription RNA composition stream 326 (or 324) is mixed with a lipid stream 341 to form RNA LNPs (that is, lipid nanoparticles that encapsulate RNA). The LNP formation process, system, and/or workflow 70 may include a continuous process that is integrated into the continuous product line from a capacity point of view. For example, the in vitro transcription process 50 and first tangential flow filtration process 60 described herein are automated, continuous processes, and the overall process continues with the automated LNP formation process 70 illustrated in Fig. 5, as well as downstream processes (including the second tangential flow filtration process 80 shown in Fig. 8, the formulation process 90 shown in Fig. 10, and fill and finish 100. The capacities of the in vitro transcription process 50 and the first tangential flow filtration process 60 are adapted to the capacity of the LNP formation process, system, and/or workflow 70. Accordingly to aspects of the present embodiments, lipids are provided in prepacked quantities under the conditions needed for lipid storage and/or transport (usually under vacuum or nitrogen) and are opened and solved by an ethanol solvent in time for the LNP formaiton step. A system comparable to “Nespresso” or Keurig (k-cups) in coffee brewing, but adapted to the sterile pharmaconditions may be used to achieve this, as described herein. For example, according to the present embodiments, small vacuum-sealed aluminum packages may be punctured and washed with ethanol, thereby pushing lipids downstream in an asceptic manner.

Robotics (for example, robots 312 and 344) may be used to recharge the lipid supply, the buffer supply, and/or the ethanol supply.

[0194] Referring still to Fig. 5, the automated LNP formation process, system, and/or workflow 70 may include an inlet stream 302 that receives drug substance (for example, an in vitro transcription RNA composition from the first tangential flow module 60). The system 70 may include one or more centrifugal pumps 304 disposed in the inlet stream, as well as a first reservoir 306 fluidly downstream of the centrifugal pump 304 and a first positive displacement pump 308 disposed downstream of the reservoir 306. The system may include a rotary mixer 310 disposed fluidly downstream of the first positive displacement pump 308. The system 70 may also include a buffer stream 303 including a buffer reservoir 314 and a robot 312 for automatically recharging the buffer reservoir 314. The buffer stream 303 may include a pump 316 (for example, a centrifugal pump) disposed downstream of the buffer reservoir 316, as well as a second reservoir 318 downstream of the pump 316. A first buffer line 323 may include a second positive displacement pump 320 for routing flow from the second reservoir 318 to the rotary mixer 310, while a second line 324 may route flow (i.e., a second buffer stream) from the second reservoir 318 downstream to mix with lipids. A third positive displacement pump 322 may be disposed in the second buffer line 324 for facilitating the flow of buffer downstream. Buffer in the first buffer line 323 is mixed with the RNA composition from the inlet stream 302 at the rotary mixer 310 before flowing downstream (via the in vitro transcription RNA composition stream 326) to a first T-mixer 328.

[0195] Still referring to Fig. 5, the RNA stream 326 and the lipid stream 341 are brought together and mixed within the first T-mixer 328, thereby forming RNA lipid nanoparticles. Prior to mixing with the RNA stream, the lipids are processed via an automated lipid handling machine 350, as shown in Figs. 5-7. The automated lipid handling machine 350 may include an ethanol supply 342 fluidly coupled to one or more fluid lines 348 that become fluidly coupled to lipid capsules or cartridges 346 (for example, ready to use lipid capsules 346), as described herein and as illustrated in Figs. 6 and 7. The automated lipid handling machine 350 punctures each lipid capsule 346, and in so doing, fluidly connects it to both the upstream ethanol supply 342, as well as a downstream ethanol-lipid mixing vessel 338 (that is, via outlet line 352). As each lipid capsule 346 is punctured, an ethanol supply line 348 protrudes into the lipid capsule 346 which allows ethanol to flow into the lipid capsule 346 and flush and/or push the lipids out. Concurrent with the ethanol supply line puncturing the lipid capsule 346, an outlet line 352 punctures and protrudes into the lipid capsule 346 to allow the ethanol / lipid mixture to flow downstream to the ethanol-lipid mixing vessel 338. In some embodiments, as shown in Fig. 5, the ethanol supply line 348 and the outlet line 352 puncture the lipid capsule 346 on opposite sides (for example, with the ethanol supply line 348 puncturing the top of the lipid capsule 346 and the outlet line 352 puncturing the bottom of the lipid capsule 346). In some embodiments, as shown in Figs. 6 and 7, the ethanol supply line 348 and the outlet line 352 puncture the lipid capsule 346 on the same side (for example, with both the ethanol supply line 348 and the and the outlet line 352 puncturing the top of the lipid capsule 346). In some embodiments, the lipid handling machine 350 may include multiple ethanol supply lines 348 and outlet lines 352 for puncturing the lipid capsules as well as a manifold 351 fluidly connecting to each of the multiple outlet lines 352, for collecting the lipids and delivering them fluidly downstream to the ethanol-lipid vessel 338. A vent line 340 and a nitrogen supply 354 may be fluidly coupled to both the ethanol-lipid mixing vessel 338, along with several control instruments such as one or more weight indicator controllers, pressure indicator controllers, short interval controllers, and/or temperature indicator controllers for controlling the operation of the ethanol-lipid mixing vessel 338, and the streams fluidly coupled thereto.

[0196] Referring still to Fig. 5, after exiting the ethanol-lipid mixing vessel 338 via outlet line 335, the ethanol-lipid mixture flows downstream to one or more filters 336, then downstream to a fourth positive displacement pump 334, and finally to the T-mixer 328, where it is mixed with the RNA stream. During mixing (for example, impingement jet mixing) within the T-mixer 328, various flow parameters (such as flow rate, mixing rate, Reynolds number, shear, viscosity) and LNP characteristics (for example, particle size, dispersity) are measured directly and/or determined from measured parameters such that the quality and consistency of the mixing process is maintained. The RNA-LNP mixture then exits the T-mixer 328 via a T-mixer exit line 330, and merges with buffer from the second buffer line 324 before exiting the LNP formation module 70 at exit line 358, which delivers the mixture to the second TFF module, system, or process flow 80 (shown in Fig. 8). In some embodiments, the flow exiting the LNP formation module 70 at exit line 358 may be in a range from about 10 to 10,000 ml/min, or from about 100 to 5,000 ml/min, or from about 200 to 2,000 ml/min, or from about 500 to 1,000 ml/min, or from about 600 to 800 ml/min, or from about 700 to 750 ml/min, and or other subranges therebetween. In some embodiments, the size and capacity of the entire process (for example, the size and capacity of the overall process, and all of the modules or sub-processes thereof (that is, as shown in Fig. 2)) are scaled such that the process may be continuous and may match the capacity of the RNA-LNP mixture that exits the LNP formation module 70 at exit line 358. Similarly, in some embodiments, scaling (i.e., increasing or decreasing the capacity of) all of the other modules or sub-processes thereof (that is, as shown in Fig. 2) other than the LNP formation module 70 may be easier than scaling the LNP formation module 70 itself due to the sensitivities of the RNA-LNP mixing process. Accordingly, in some embodiments, the automated RNA-LNP process, systems, and/or workflows 40 of the present disclosure may entail using an LNP formation process that includes flow and/or mixing capacities that must be maintained within certain ranges (for example, about 650 to 800 ml/min, or 700 to 750 ml/min at exit line 358) in order for various performance characteristics to be maintained).

[0197] Figure 6 depicts an overview of an automated lipid supply machine 350, according to aspects of the present disclosure. The automated lipid supply machine 350 may generally include a reservoir of lipid cartridges or capsules 346, a robot 344 for handling the cartridges 346, a lipid dissolution system 364, an ethanol reservoir 342, and an ethanol-lipid mixing vessel 338. Each of the lipid cartridges 362 may initially be store and/or contained within a cartridge handling system 362, which may include columns, stacks, one or more hoppers, racks, trays, shelves, cylinders, reservoirs, and/or other structures for holding and managing lipid cartridges 362 that have yet to be used. The robot 344 may then take each cartridge 346 from the cartridge handling system 362, and may place each cartridge 346 (for example, one at a time) into the lipid dissolution system 364 where the ethanol supply line 348, and the outlet line 352 puncture each cartridge 346 such that ethanol from the ethanol reservoir 342 flows into the cartridge 346 and flushes the lipids out of the cartridge 346 and into the outlet line 352. Although each cartrdige 346 is prepackaged and presumably contains the desired amout of lipid substance, variation may still exist. Accordingly, in some embodiments, the robot 344 may include a built-in scale such that each lipid cartridge 346 may be automatically weighed when it is picked up by the robot to ensure it contains the desired amount of lipid substance. After each cartridge 346 has been flushed out, the robot 344 may then remove each cartridge 346 from the cartridge handling system 362, and dispose of each cartridge 346 for reuse and/or discarding. [0198] Referring still to Fig. 6, in some embodiments, the automated lipid supply machine 350 may include an ethanol reservoir 342 for supplying ethanol to the lipid dissolution system 364. The ethanol reservoir 342 may be coupled to both a nitrogen supply 368 (for example, which may be used for purging the system 350 and/or ethanol reservoir 342 between uses) and an ethanol supply 370. In some embodiments, the automated lipid supply machine 350 may include a heat exchanger 366 disposed in the ethanol supply line 348, downstream of the ethanol reservoir 342 and upstream of the lipid dissolution system 364. The automated lipid supply machine 350 may also include a manifold 351 fluidly downstream of (and coupled to) the outlet line 352, for feeding lipids to the ethanol-lipid mixing vessel 338. The automated lipid supply machine 350 may also include a nitrogen supply 354 fluidly coupled to the ethanol-lipid mixing vessel 338, as well as an outlet line 335, for delivering lipids downstream (for example, to filter 336 shown in Fig. 5).

[0199] In connection with the present disclosed embodiments (for example, in connection with the lipid nanoparticle formation process, system, and/or workflow 70 shown in Fig. 5, and the automated lipid supply machine 350 shown in Fig, 6) the mRNA production process uses lipid-ethanol solutions containing 4 or more different lipids (for example, phospholipid, a PEG-lipid, a sterol, a cationic lipid, and/or a neutral lipid) solved in specific concentrations in an ethanol solution. The ethanol solution may include dilutions of up to 99% ethanol. As disclosed herein, the ethanol solution is used in the mixing step or lipid nanoparticle formation. According to the present embodiments, the automated lipid supply machine 350 may automate and/or streamline the preparation of lipid solution from pre-weighed, aliquoted lipids and ethanol, without the need for manual interference.

[0200] Figure 7 depicts an overview of a lipid dissolution system 364 of the automated lipid supply machine 350, according to aspects of the present disclosure. As disclosed herein, each of the ethanol supply line 348 and the outlet line 352 (through which the ethanol and lipids exit the cartridge 346) are used to puncture the cartridge 346, and in so doing, fluidly connect thereto. In some embodiments, the ethanol supply line 348 may include a smaller inner diameter than that of the outlet line 352 to facilitate maintaining a seal within the cartridge 346, and to reduce the flow resistance in the outlet line 352 in order to ease the flushing of lipids out of the cartridge 346. For example, in some embodiments, the outlet line 352 may have a diameter that is about 1.5 to about 3 (or from about 1.5 to about 2.5, or from about 2 to about 3, or from about 2 to about 2.5) times larger than the ethanol supply line 348. Each of the the ethanol supply line 348 and the outlet line 352 may include an angled tip, sharp edge, needle, or point 372 for puncturing the cartridge 346. For example, in some embodiments, each of the ethanol supply line 348 and the outlet line 352 may include an angled tip 372 that is angled from about 10 to about 45 degrees (or from about 10 to about 35 degrees, or from about 15 to about 35 degrees, or from about 15 to about 30 degrees, or from about 20 to about 30 degrees, or from about 20 to about 35 degrees, or from about 20 to about 45 degrees, or from about 25 to about 45 degrees, and/or other subranges therebetween) from a longitudinal axis of the respective ethanol supply line 348 and outlet line 352. In some embodiments, the angled tip 372 is composed of stainless steel.

[0201] Referring still to Fig. 7, in some embodiments, each of the ethanol supply line 348 and the outlet line 352 may include an internal diaphragm, seal, and/or one-way valve 374 adjacent or proximate to the respecive angled tips 372 such that the lines 348, 352 remain sealed when they are not protruding into the cartridge 346. Pressure within the ethanol supply line 348 is selectively adjusted (for example, in a range from about 0 to 1 bar (guage pressure; for example, from 0 to 0.5 bar, 0.5 to 1.0 bar, 0.25-0.75 bar, 0.4-0.8 bar, and/or other subranges therebetween)) such that once the ethanol supply line 348 and the outlet line 352 have punctured each cartridge 346, the pressure within the ethanol supply line is high enough to push ethanol (and lipids) through the two internal diaphragms, seals, and/or one-way valves 374. The pressure in the ethanol supply line 348 may then be reduced such that the sealing force of the internal diaphragm, seal, and/or one-way valve 374 is able to hold the ethanol within the ethanol supply line 348 when the lines 348, 352 are no longer disposed within the cartridge 346. Each internal diaphragm, seal, and/or one-way valve 374 may be composed of a resilient material that also includes some amount of rigidity and/or flexibility such that it allows flow therethrough when the pressure is high enough, but returns to its original position or shape when the pressure drops. For example, each internal diaphragm, seal, and/or one-way valve 374 may be composed of polymers, thermoplastic, hardened rubber, aluminum, PEEK (poly ether ether ketone), PEKK (polyether ketone kethone), and/or other suitable materials. Each cartridge 346 may include a tight septum 376 (for example, a silicon layer or layer composed of other suitable materials such as aluminum, polypropylene, polyethylene, and/or other suitable materials, i.e., a “septum layer”) that is pliable and resilient enough to allow the angled tips 372 of each of the ethanol supply line 348 and the outlet line 352 to puncture the cartridge, while otherwise maintaining its structure, thereby forming a tight seal around each of the ethanol supply line 348 and the outlet line 352 while they are disposed within the cartridge 346. In some embodiments, the septum layer 376 is composed of a solid material that is dissolvable in ethanol. In some embodiments the septum layer 376 is disposed only on a top surface of each cartridge. In some embodiments (for example, in connection with the system depicted in Fig. 5), each cartridge 346 includes a tight septum/layer 376 on both the top and bottom surfaces, to allow for puncturing and sealing of the cartridges 346 in embodiments when the cartridges are punctured from opposite sides by the ethanol supply line 348 and the outlet line 352. In some embodiments, the septum layer 376 includes one or more puncture regions or locations where it is to be punctured by the ethanol supply line 348 and/or outlet line 352, the one or more puncture regions or locations being composed of a softer material than the rest of the septum 376 to help facilitate puncturing of the septum 376 while allowing proper sealing to be maintained during ethanol flushing. The lipid dissolution system 364 may include structures 378 (for example, robotic arms or clamps, among other suitable structures) for holding the cartridges 346 in place while they are in dissolution position within the lipid dissolution system 364. In some embodiments, the robot 344 holds each lipid cartridge 346 in place while each cartridge 346 is being punctured and flushed with ethanol.

[0202] Referring to Figs. 5-7, in operation, lipids may be provided by the supplier of lipids in vacuum or nitrogen-overlayed closed single use containers, capsules or cartridges 346. In some embodiments, the cartridges are composed of material that is inert with regards to reaction with lipids (for example, aluminium, polyethylene, polypropylene, and/or or polyethylene, polypropylene covered aluminium) which also serves to protect lipids from exposure to light. In some embodiments, the cartridges 346 are inserted into the machine 350 into a reservoir specific to each lipid (for example, the vertically aligned columns in Fig. 6 that are part of the cartridge handling system 362). Different reservoirs, columns, and/or systems may be used for different scales and different lipids. The machine 350 (for example, via robot 344) selects and grabs the desired lipid cartridge 346 automatically depending on the recipe installed on the machine 350. Each cartridge 346 is positioned in the ethanol stream and is opened by the machine 350 via two needles (inlet/outlet, for example, ethanol supply line 348 and outlet line 352) or other automatic opening. Opening of each cartridge 346 may be performed without allowing the contents of the cartridge 346 to come in contact with the environment or air. As such, ensuring a tight seal is necessary, as discussed above in connection with Fig. 7. Via the puncturing action of the machine 350 (specifically, the lipid dissolution system 364), each cartridge 346 temporarily serves as the vessel, connector, or conduit that fluidly connects the ethanol supply line 348 and the outlet line 352.

[0203] Still referring to Figs. 5-7, in some embodiments, ethanol is heated by the machine 350 (for example, via heat exchanger 366) to the desired temperature to dissolve the lipid (for example, a temperature of about 30°C, or from about 25°C to about 35°C), and is flushed through the cartridge 346 until the lipid material is completely flushed out. The lipid-ethanol solution may then be flushed downstream via manifold 351 into a mixing tank 338 (which is a part of the machine 350) where the lipid-ethanol solution is temperature adjusted and mixed. The same procedure may then be performed with the next lipid until the programmed recipe mixture is complete. Final adjustment of the lipid-ethanol solution may be achieved by adding additional ethanol until a final concentration is reached. From the lipid-ethanol tank 338 of the machine 350, the lipid-ethanol solution may be directly provided to the mixing device (for example, T-mixer 328). The areas of the machine 350 that are in contact with ethanol or lipids are flushed under nitrogen (for example, via nitrogen supply 354) any time lipids or ethanol is handled. The machine 350 may be cleaned with ethanol using an empty “cleaning” cartridge 346. The machine 350 (in connection with the lipid dissolution system 364) may also be used to produce other highly pure solvent mixtures.

[0204] Referring still to Figs. 5-7, aseptic extraction of the lipids from the cartridge 346 may be accomplished according to the present embodiments. For example, by providing internal diaphragms, seals, and/or one-way valves 374 into each of the ethanol supply line 348 and outlet line 352, contaminants are prevented from entering each of the ethanol supply line 348 and outlet line 352 when they are exposed to open air. In addition, by providing a septum layer 376 (i.e., a pliable yet resilient septum layer, as described herein) on top of the cartridge 346, an airtight seal may be maintained between the interior of the cartridge 346 and exterior of the cartridge system, even after the cartridge 346 has been punctured by each of the ethanol supply line 348 and outlet line 352. Finally, by providing positive pressure and suction to the ethanol supply line 348 and outlet line 352 respectively, only after the cartridge 346 has been punctured, undesired fluid flow through the internal diaphragms, seals, and/or one-way valves 374 in each of the ethanol supply line 348 and outlet lines 352 can be avoided. Accordingly, the automated lipid supply machine 350 (or system) of the present embodiments enables aseptic extraction of lipids from each cartridge 346 (i.e., the system 350 aseptically and automatically extracts the lipids from each cartridge), thereby enabling the supply of lipids to the outlet lines 352 (i.e., lipid supply line 352) and eventually to the downstream ethanol-lipid mixing vessel 338, while also minimizing the risk of contamination.

[0205] Figure 8 depicts an overview of a second automated tangential flow filtration process flow, system, and/or method 80, according to aspects of the present disclosure. The second automated tangential flow filtration process flow, system, and/or method 80 may generally include a two-stage filtration process (similar to the first TFF process 60 illustrated in Fig. 4) including a first TFF cassette 390 fluidly connected to a downstream second TFF cassette 400. The system 80 may include an inlet line 358 for receiving the RNA-LNP mixture from the lipid formation module 70. The system may include a second T-mixer 380 downstream of the inlet line 358, a heat exchanger 382 downstream of the T-mixer 380, and a reservoir 384 downstream of the heat exchanger 382. The system 80 may include one or more weight indicator controllers 388 and one or more short interval controllers 386 operatively coupled to the reservoir 384 for controlling the volume and quality of the stream flowing into the first TFF cassette 390. The second automated tangential flow filtration process flow, system, and/or method 80 may also include a buffer reservoir 396 that is coupled to a manifold 402 coupled to first and second pumps (for example, centrifugal pumps) 392, 394, which may be used to pump buffer downstream via delivery line 397 to the first and second TFF cassettes 390, 400. In some embodiments, the first and second pumps 392, 394 are configured in a parallel flow configuration with one another where both are in fluid communication with the buffer reservoir via the manifold 402, but are positioned within separate lines. In some embodiments, the manifold 402 may also be fluidly coupled via connection line 398 to the buffer system 286 of the first TFF process 60 such that buffer can be shared among both TFF processes 60, 80. In some embodiments, a robot 404 automatically recharges and/or resupplies the buffer reservoir 396. Downstream of the second TFF cassette 400, the system 80 may include a reservoir 410, a concentration control sensor 408 disposed in the vicinity of the reservoir 410, at least one diaphragm (or membrane) pump 406 disposed downstream of the concentration control sensor 408, at least one pressure indicator controller 412 disposed downstream of the diaphragm pump 406, at least one filter 414 downstream of the pressure indicator controller 412, a vent or drain line 416 downstream of the filter 414, and an outlet line 418 for delivering the RNA-LNP preparation downstream to a formulation process 90 (shown in Fig. 9). [0206] Referring still to Fig. 8, the second TFF module 80 includes in-line monitoring probes 386, 388, 408, 412 for quality and concentration upstream and/or downstream of the first and second TFF cassettes 390, 400. If the monitored parameters are not at the desired levels, the system 80 can keep recirculating the RNA-LNP preparation back to the second TFF cassette 400 via recirculation line 399, until the desired parameters are reached. In some embodiments, the probes 386, 388, 408, 412 may be used to measure mRNA concentration via spectroscopy. In some embodiments, the probes also measure the size and distribution of lipid nanoparticles (LNPs) and/or the concentraction of LNPs using one or more dynamic light scattering instruments and/or one or more spectroscopy probes. Monitoring both upstream and downstream of the two TFF cassettes 390, 400 is important for ensuring proper control and process validation. As described herein, the size of each of the first and second TFF cassettes 390, 400 is adapted to match the volume of the first and/or second T-mixer 328, 380. In some embodiments, the first and/or second T-mixer 328, 380 may be or include an impingement jet mixing unit. In some embodiments, the concentration control sensor 408 uses UV spectroscopy to measure RNA concentration. In some embodiments, the concentration control sensor 408 includes a spectrophotometer configured to measure absorbance (for example, in a range from about 50 nm to about 600 nm, or from about 100 nm to about 500 nm, or from about 150 nm to about 450 nm, or from about 200 nm to about 400 nm, or from about 200 nm to about 350 nm, or from about 250 nm to about 300 nm, and/or from about 260 nm to about 280 nm). In some embodiments, the system 80 continues to recirculate the RNA-LNP preparation back to the second TFF cassette 400 via recirculation line 399 until an RNA concentration within the RNA-LNP preparation reaches a predetermined threshold, as measured by, the concentration control sensor 408. In some embodiments, the predetermined RNA concentration threshold is within a range from about 1.50 g/L mRNA to about 5.00 g/L mRNA, or from about 2.00 g/L mRNA to about 4.50 g/L mRNA, and/or from about 2.50 g/L mRNA to about 4.0 g/L mRNA (for example, corresponding to absorbance units as measured by the concentration control sensor 408 of from about 37 to about 125, or from about 50 to 113, or from about 62 to about 100, respectively).

[0207] Figure 9 depicts an overview of an automated drug formulation process flow, method, system and/or module 90, according to aspects of the present disclosure. The automated drug formulation process flow, method, system and/or module 90 may include an inlet line 418 for receiving RNA-LNP preparation from the second TFF module 80, as well as a small reservoir 419, as needed for temporarily holding materials received from the second TFF module 80. The inlet line 418 may be coupled to both an excipient addition line 430 and a buffer line 432. An excipient reservoir 420 and a buffer reservoir 426 may both be coupled to a common manifold 424 as well as the respective excipient addition line 430 and buffer line 432, for providing excipient and buffer to the RNA-LNP preparation. A robot 422 may be used to automatically recharge and/or resupply excipient and buffer to the respective excipient and buffer reservoirs 420, 426. The excipient may include sucrose while the buffer may include a TRIS buffer, among other types of suitable excipients and buffers. At least one pressure indicator controller 434 may be disposed fluidly downstream of the buffer line 432 while one or more filters 436 may be disposed downstream of the pressure indicator controller 434. The system 90 may also include a vent or drain line 438, a reservoir 440 downstream of the vent or drain line 438, one or more short interval controllers 444 disposed in the vicinity of the reservoir 440, one or more weight indicator controllers 442 disposed in the vicinity of the reservoir 440, and an exit line 446 for delivering the final formulation to a downstream fill and finish process 100.

[0208] Referring still to Fig. 9, the inline process monitoring and control sensors 434, 442, and 444 can be used to determine how much sucrose (excipient) and buffer (for example, TRIS buffer) should be added based on inline concentration of LNPs and mRNA. The automated drug formulation process flow, method, system and/or module 90 may include mixing technology to perform in-line dilution, mixing and concentration adjustments. For example, the respective flow rates of the sucrose (i.e., excipient), buffer, and LNP solution may be accurately adjusted with high precision equipment (for example, pumps and flow rate analytical devices including, but not limited to massflow meters and precision pumps). According to aspects of the present embodiments, fill and finish processes 100 may occur at the site in smaller batches, thereby reducing the need for transport, freezing and/or warehousing of the LNP solution. The number of freeze cycles is thereby reduced as well, leading to enhanced product quality control due to the elimination of a freeze cycle, which can act as a source of product variability. Therefore, the present disclosed embodiments describe a bulk, continuous, production line from in vitro transcription 50 through fill and finish 100.

[0209] Figure 10 depicts an overview of an exemplary drug product manufacturing site 150, according to aspects of the present disclosure. International patent application number PCT/US22/53695 describes using shipping containers to construct modular drug production facilities in any location where shipping containers are able to be shipped. The modular drug production facilities described in international patent application number PCT/US22/53695 may be used in connected with the automated drug production methods, systems, and process flows described in the present embodiments. In the embodiment of Fig. 10, the site 150 may include one or more modules (for example, a first module 124 and a second module 126) each including about 6 containers 102. For example, the first module 124 (and/or the second module 126) may include 4 containers used for housing the in vitro transcription 50, first TFF 60, LNP formation 70, second TFF 80, and formulation 90 in a total of 4 containers, with fill and finish 100 contained in a total of 2 containers resulting in a total of six containers used to house the entire process. In other embodiments, other numbers of containers 120 can be used to house the entire process 40 (shown in Fig. 2).

[0210] According to aspects of the present embodiments, the scale of each successive step in the disclosed overall process is adapted to match the adjacent processes. Generally, each process or module is adapted to match the capacity of the lipid formation process 70. Because the entire process, and each sub process is being operated continuously, only small reservoirs are needed to temporarily contain the various preparations and solutions before they are routed to the subsequent step(s) of the process. In addition, because the overall process is occurring continuously at a single site, many steps such as freezing, intermediate thawing, storage, and transport between sites can be removed.

Throughout the entire process, in addition to the instrumentation explicitly described herein, other parameters such as pressure, temperature, and flow rates are measured via inline instrumentation. Generally, each instrument, sensor, and/or probe included in the present embodiments are capable of being monitored and controlled remotely. As such, each instrument, sensor, and/or probe included in the present embodiments is either individually connected to a network, or is operatively and/or communicatively coupled to a local computer, control system, edge device, and/or base unit that is in turn communicatively and operatively coupled to a computer network. Although not necessarily mentioned, the systems of the present embodiments may include additional valves, pumps, adapters, couplings, piping, nozzles, orifice plates, flow restrictors, and other components. It is also understood that, although not explicitly illustrated, the present embodiments include various environmental systems such as heating, ventilation, and air conditioning (HVAC). The exact configurations of the embodiments disclosed herein illustrate how the systems and processes work. Other configurations are possible, according to aspects of the present embodiments. [0211] The present embodiments provide a continuous or semicontinuous process which improves overall cycle time compared to discontinous processes, which may include at least 4 separate processes and the need for intermediate storage of materials between the separate processes. In addition, transport, freezing, and thawing may be required between the separate processes of the discontinuous processes whereas those steps are not required for the present embodiments, thereby bringing down the overall drug production cycle time. As such, the intermediate drug substance preparations and solutions may be maintained at a temperature within a range from about 10 degrees C to about 40 degrees C (or from about 10 degrees C to about 30 degrees C, or from about 15 degrees C to about 35 degrees C, or from about 20 degrees C to about 35 degrees C, or from about 20 degrees C to about 40 degrees C, or from about 25 degrees C to about 35 degrees C, or from about 25 degrees C to about 40 degrees C, and other subranges therebetween) for the entire duration of the process (that is, from IVT 50 to fill and finish 100). Each stage and module (for example, processes/modules 50, 60, 70, 80, 90, and 100 shown in Fig. 2) of the present disclosed embodiments is directly fluidly connected to adjacent modules (for example, using direct piping connections), thereby allowing materials to flow directly to the next stage for immediate processing. Human handling is not required at any stage during the process. Manual weighing of components, handling of lipids, buffers, ethanol, nitrogen, excipients, and other materials are not required, as the robots of the present embodiments ensure material supplies are continuously accessible, weighed, refreshed and/or recharged.

Operators are not required to enter clean rooms to intervene in the drug manufacturing processes, thereby reducing or eliminating a source of potential contamination. In addition, the systems may be automatically cleaned out and flushed, as described herein.

[0212] According to aspects of the present embodiments, each of the processes/modules 50, 60, 70, 80, 90, and 100 shown in Fig. 2 includes structures and/or systems that enable the automated, continuous process flow described herein. For example, the in vitro transcription module 50 includes feed lines 214, 216, 218 that are separately connected to each of the three (or more) bioreactors 226, 228, 230, thereby allowing materials to be routed into the bioreactors 226, 228, 230 as needed based on the process controllers 220, 224. Materials may then be automatically routed between bioreactors 226, 228, 230 when ready (again based on process controllers 220, 224). In the first TFF module 60, the small reservoir 241 may receive materials and then may selectively route them into the first TFF cassette 244, according to information from the in-process controllers. Similarly, downstream controllers 288, 294 are used to determine when flow should be routed back to the second TFF cassette 246 (via recirculation line 300). In the LNP formation module 70, a reservoir 306 and in-process controllers may be used to determine how and when process flows are routed through the system. In addition, the lipid handling machine 350 automatically feeds the amounts and types of lipids into the system based on the specific recipe being produced, and feedback from the inline controllers. Similar to the first TFF module 60, the second TFF module 80 has a receiving reservoir 380, in process controllers 386, 388, 408, 412, and recirculation line 399, thereby enabling a continuous, automated process. The formulation process 90 similarly uses inline process controllers 434, 442, 444 to determine how much buffer and excipient is required to mix with the LNP solution, as well as a receiving reservoir 419 for selectively controlling flows through the system. Finally, the fill and finish process / module 100 is automated and produces filled drug product containers with drug product received from the formulation module 90.

[0213] According to aspescts of the present embodiments, each of the bioreactors 226, 228, 230 used during in vitro transcription 50 may include volume indicator and controllers, accumulators and timers to track mixing time and mixing rate, temperature probes, flow rate meters, pressure transducers / transceivers, and speed controllers to track and control the rotational speed (for example, in rpm) of one or more impellers disposed within each bioreactor 226, 228, 230. The process controls of the present embodiments may also include inline analysis and control of microorganisms, in which light-scattering and fluorescent light scattering probes are used to analyze air samples by differentiating between dispersed microorganisms and inert particles and pollen.

[0214] In some embodiments, various hardware components as described herein may be composed of stainless steel, thereby facilitating the ease of cleaning (for example, using cleaning agents, hydrogen peroxide, etc.). Sterilization of components described herein may also be performed via vaporized steam (a fog-like dispersion of extremely small H2O2 droplets that permeates all equipment, in connection with the robots, cleaning agents, and dispersion nozzles (not shown) described herein). In some embodiments, sterilization of components described herein may also be performed via a solution containing hydroxyl radicals (for example, at a concentration from about 0.001% to about 0.2%, or at a concentration from about 0.002% to about 0.1%, or at a concentration from about 0.005% to about 0.05%, or at a concentration from about 0.008% to about 0.02%, and/or at a concentration from about 0.01% to about 0.015%). For example, in some embodiments, the systems and methods described herein may include automatically introducing a solution containing hydroxyl radicals into at least one system component following use (for example, following the production of a batch of drug substance and/or drug product) such that the system component(s) is sanitized in preparation for a subsequent production or development run.

[0215] The present embodiments allow for a reduction of personnel resulting in a significant positive impact on the cost of goods. Lower risk of contamination during the production process may be realized, resulting in a lower risk of material loss. Higher reproducibility may also be realized by removing the inherent variability caused by personnel. The present embodiments also support both production and development processes via standardized, ready to use, uncompromised delivery of solutions of lipids in ethanol for all potential process needs (for example, for mRNA-Lipid Nanoparticle formation, among other potential applications).

Equivalents

[0216] 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. Therefore, the scope of the present invention is not intended to be limited to the above Description.

Claims

Claims We claim:

1. An automated drug production process comprising: performing in vitro DNA transcription via an automated in vitro transcription (IVT) module, thereby producing an RNA solution to be used for producing drug substance; performing at least one tangential flow filtration (TFF) step on the RNA solution; automatically providing a stream of lipid stock; and mixing the stream of lipid stock with the RNA solution in an impingement jet mixer, thereby forming an RNA-lipid nanoparticle (LNP) preparation.

2. The process of claim 1, wherein automatically providing a stream of lipid stock comprises: using a lipid handling system to automatically puncture at least one lipid cartridge; and aseptically extracting the lipids from the at least one lipid cartridge, thereby providing the stream of lipids.

3. The process of claim 1, comprising formulating the RNA-LNP preparation to produce a drug product.

4. The process of claim 3, comprising performing a fill and finish process after the formulating step, to dispose the drug product into containers.

5. The process of claim 1, comprising performing a second TFF step after the mixing step.

6. The process of claim 1, comprising automatically measuring an RNA concentration within the LNP preparation, and automatically recirculating the LNP preparation within a TFF module in which the second TFF step is being performed until the measured RNA reaches a predetermined threshold.

7. The process of claim 6, wherein measuring an RNA concentration within the LNP preparation comprises measuring the RNA concentration using a spectrophotometer.

8. The process of claim 6, wherein the predetermined threshold comprises an RNA concentration in a range from about 1.5 g/L to about 5.0 g/L.

9. The process of claim 1, wherein the RNA solution, lipid stock, and RNA-LNP preparation are maintained at a temperature within a range from about 10 degrees C to about 35 degrees C throughout the entire process.

10. The process of claim 1, comprising: automatically monitoring and controlling multiple IVT process parameters within the IVT module to ensure that an mRNA synthesis subprocess, a DNA hydrolysis subprocess, and a protein hydrolysis subprocess have been completed; and automatically routing RNA solution to the first TFF module when the mRNA synthesis subprocess, DNA hydrolysis subprocess, and protein hydrolysis subprocess have been completed.

11. The process of claim 10, wherein the multiple IVT process parameters comprise two or more of: pH, temperature, incubation time, mixing speed, mixing time, level of agitation, and/or hold time.

12. The process of claim 3, comprising continuously monitoring, via inline instrumentation, at least one operating parameter of each of the in vitro DNA transcription, tangential flow filtration, mixing, and formulating processes.

13. The process of claim 3, wherein formulating comprises: mixing at least one excipient solution with the RNA-LNP preparation; and mixing at least one buffer with the RNA-LNP preparation.

14. The process of claim 13, wherein the at least one excipient solution comprises sucrose, and wherein the buffer comprises a TRIS buffer.

15. An automated drug production system comprising: an in vitro transcription (IVT) module for producing an RNA solution; a first tangential flow filtration (TFF) module disposed immediately downstream of the IVT module; a lipid nanoparticle (LNP) formation module disposed immediately downstream of the first TFF module; a second TFF module disposed immediately downstream of the LNP formation module; a formulation module disposed immediately downstream of the second TFF module; and a fill and finish module disposed immediately downstream of the formulation module.

16. The system of claim 15, wherein a flow existing the LNP formation module is in a range from about 500 ml/min to about 1000 ml/min.

17. The system of claim 15, further comprising from about 4 to about 12 standard shipping containers that contain the entire system.

18. The system of claim 17, wherein the about 4 to about 12 standard shipping containers house the IVT module, the first TFF module, the LNP formation module, the second TFF module, the formulation module, and the fill and finish module.

19. The system of claim 15, wherein the entire system is located within a single location comprising a footprint within a range from about 100 square meters to about 12,000 square meters.

20. The system of claim 15, wherein the IVT module comprises at least three (3) bioreactors fluidly connected to each other in series, and fluidly connected to a common supply line in parallel, such that feed materials may be separately and selectively supplied to each of the at least three (3) bioreactors.

21. The system of claim 15, wherein each of the first TFF module and the second TFF module comprises: a first TFF cassette; a second TFF cassette fluidly coupled downstream of the first TFF cassette; and at least one recirculation line routing flow back to an inlet of the second TFF cassette.

22. The system of claim 15, wherein the LNP formation module comprises: at least one impingement jet mixing unit; and a lipid handling machine configured to automatically provide a supply of lipids for mixing with an RNA solution in the at least one impingement jet mixing unit.

23. The system of claim 15, wherein at least one of the first TFF module and the second TFF module comprises: at least one inline probe disposed upstream of the first TFF cassette; one or more dynamic light scattering instruments disposed downstream of the second TFF cassette; and one or more spectroscopy probes disposed downstream of the second TFF cassette.

24. The process of claim 1, wherein the RNA solution, lipid stock, and RNA-LNP preparation are maintained in liquid phase throughout the entire process.

25. The process of claim 1, comprising sterilizing at least one component or piece of equipment employed in the process using a vaporized steam comprising hydrogen peroxide.

26. The process of claim 1, comprising sterilizing at least one component or piece of equipment employed in the process using a solution comprising hydroxyl radicals at a concentration in a range from about 0.005% to about 0.05%.

27. A lipid handling system comprising: a cartridge handling system; a lipid dissolution system located in the vicinity of the cartridge handling system; a robot configured to take lipid cartridges from the cartridge handling system and place them in the lipid dissolution system; an ethanol supply line configured to supply ethanol to lipid cartridges disposed in the lipid dissolution system; and an outlet line configured to deliver ethanol and lipids from cartridges within the lipid dissolution system to a downstream vessel.

28. The system of claim 27, wherein each of the ethanol supply line and the outlet line are configured to puncture lipid cartridges disposed within the lipid dissolution system.

29. The system of claim 28, wherein each of the ethanol supply line and the outlet line comprises an angled tip for puncturing the lipid cartridges disposed within the lipid dissolution system.

30. The system of claim 27, wherein the lipid cartridges comprise at least one of a phospholipid, a PEG-lipid, a sterol, a cationic lipid, and a neutral lipid.

31. The system of claim 28, wherein the lipid cartridge, once punctured by each of the ethanol supply line and the outlet line, fluidly connects the ethanol supply line to the outlet line.

32. The system of claim 27, wherein the ethanol supply line is further configured to puncture lipid cartridges disposed in the lipid dissolution system.

33. The system of claim 32, wherein the ethanol supply line is configured to supply ethanol to lipid cartridges disposed in the lipid dissolution system; and wherein the outlet line is configured to deliver ethanol and lipids from the lipid cartridges within the lipid dissolution system to a downstream vessel.

34. A lipid cartridge containing at least one of a phospholipid, a PEG-lipid, a sterol, a cationic lipid, and a neutral lipid, the cartridge comprising: a generally cylindrical shape; and a septum layer disposed on at least one of a top surface and a bottom surface of the cartridge.

35. The cartridge of claim 34, wherein the cartridge comprises at least one of aluminum, polyethylene, and polypropylene.

36. The cartridge of claim 35, wherein the cartridge comprises an aluminum shell coated with at least one of polyethylene and polypropylene.

37. The cartridge of claim 34, wherein the septum layer comprises silicon.

38. The process of claim 1, wherein the process is fully automated.

39. The system of claim 15, wherein at least one of the first TFF module and the second TFF module comprises a double cross-flow configuration comprising: a product flow in a first direction; and an exchange buffer flow in a second direction, the second direction being in an opposite direction to the first direction.

40. The system of claim 39, wherein the product flow comprises at least one of an RNA solution and an RNA-ENP preparation.

41. The system of claim 39, wherein at least one of the first TFF module and the second TFF module comprises a pressure modulator configured to selectively reverse a transmembrane pressure.

42. The system of claim 20, further comprising at least one bypass line, wherein the at least one bypass line fluidly connects at least one of: an outlet of the first bioreactor to an inlet of the third bioreactor; an outlet of the first bioreactor to an outlet of the third bioreactor; and an outlet of the second bioreactor to an outlet of the third bioreactor.