Continuous spin freeze drying of nucleic acid-containing compositions

Abstract

The present invention relates to the field of freeze-dried compositions, particularly nucleic acid-containing compositions. More specifically, the present invention relates to freeze-dried nucleic acid-containing compositions using the method of continuous spin-freezing and continuous spin-drying (sublimation and desorption) of the composition. The present invention further relates to nucleic acid-containing compositions produced by using the method of the present invention.

Description

[Technical field]

[0001] The present invention relates to the field of freeze-dried compositions, particularly nucleic acid-containing compositions. More specifically, the present invention relates to freeze-dried nucleic acid-containing compositions using the method of continuous spin-freezing and continuous drying (sublimation and desorption) of the composition. The present invention further relates to nucleic acid-containing compositions obtained by the method of the present invention. [Background technology]

[0002] Most biopharmaceutical products have limited stability in aqueous solutions, which can reduce the efficacy of the molecules or cause toxicity. In particular, nucleic acids such as DNA and RNA are highly unstable in aqueous solutions at room temperature for extended periods due to degradation by contaminating nucleases and inherent chemical instability. This instability and loss of integrity are a bottleneck in the application of nucleic acid-based products in the pharmaceutical and biotechnology industries. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2011069528 [Non-patent literature]

[0004] [Non-Patent Document 1] Leys et al., 2020 - European Journal ofPharmaceutics and Biopharmaceutics - A primary drying model-based comparison of conventional batch freeze-drying to continuous spin-freeze-drying for unitdoses Summary of the Invention [Problem to be solved by the invention]

[0005] Freeze drying (also known as lyophilisation) is applied to maintain the shelf life of products and make storage, distribution and transport more convenient. The freeze drying process usually consists of three successive steps: i) a freezing stage where water crystallizes into ice, ii) a primary drying step where the ice crystals are removed by sublimation under vacuum, and iii) a secondary drying step where most of the unfrozen water is removed by diffusion and desorption. Traditional "batch" freeze drying can process many vials in one step (e.g. freezing), but the method is time and energy consuming. The main drawbacks are both uncontrolled freezing and uneven heat transfer, which ultimately result in different process conditions for each individual vial in the batch, leading to uncontrolled vial-to-vial and batch-to-batch variations. [Means for solving the problem]

[0006] To overcome the drawbacks associated with traditional batch freeze-drying, the continuous freeze-drying technology was developed, where all freeze-drying steps i) to iii) are integrated into a single production line, where selected vials filled with the corresponding liquid formulations can be placed in a continuous mode. One important difference is that the vials filled with the liquid formulations are rapidly rotated along their longitudinal axis and cooled simultaneously (i.e., spin-frozen). As a result, the resulting frozen product spreads with a uniform thickness over the entire vial surface (i.e., a large surface area and a thin product layer). With a suitable load-lock system, the frozen vials can be rapidly transferred between the continuous freezing and drying units without affecting the specific conditions of pressure and temperature. In the drying chamber, an endless belt system allows the vials to be transported in front of individually controlled radiators, which transfer the heat required for sublimation and desorption to the vials, so that the temperature can be regulated for each individual vial, which is an advantage over existing freeze-drying technologies. A condenser system is used that can continuously remove condensed water.

[0007] Overall, this continuous integration approach significantly reduces the variability of critical quality attributes and consistently ensures the predefined quality of the spin-lyophilized product. The spin-freeze and dry cycles reduce the chance of exposure to temperatures that may destabilize the product. This applies to both formulated and naked forms of nucleic acid (i.e., unprotected nucleic acid generated in the laboratory). Thus, we have found that spin-lyophilization of nucleic acid-containing compositions improves product stability due to shorter processing times and increased processing uniformity.

[0008] Typically, classical freeze-drying, using a fairly low concentration of cryoprotectant, is used for freeze-drying nucleic acid-containing compositions (Patent Document 1). Furthermore, continuous spin freeze-drying of unit doses has already been disclosed in Non-Patent Document 1, but here the nucleic acid-containing composition is not freeze-dried and no high concentration of cryoprotectant is used. Since the concentration of the cryoprotectant can have a significant effect on the quality and properties of the freeze-dried nucleic acid-containing cake, the inventors aimed to provide a freeze-drying method that allows the most flexibility in changing the concentration of the cryoprotectant. Furthermore, it was found that the spin freeze-drying method of the present invention is much more flexible in terms of the concentration of the cryoprotectant compared to the conventional freeze-drying method. In particular, a concentration of 20% w / v of the cryoprotectant adversely affected the cake quality in the conventional freeze-drying method, while the quality of the cake was well maintained at a similar concentration of the cryoprotectant in the spin freeze-drying method of the present invention.

[0009] Thus, spin-freezing and drying cycles have unexpectedly been found to be beneficial for nucleic acids and nucleic acid-containing compositions, especially when using cryoprotectants at various concentrations. In particular, spin-freezing creates a homogenous thin layer on the inner wall of the vial, which prevents / prevents structural rearrangements at the nucleic acid level or nucleic acid-containing composition near the glass transition temperature (Tg'), and maintains the original configuration of naked and formulated nucleic acids as in the liquid state. As a result, the product is not destabilized and important quality attributes and biological activity are maintained.

[0010] In a first aspect, the present invention provides a method for lyophilizing a nucleic acid-containing composition, the method comprising the steps of: a) filling a container having an inner wall and an outer wall with a nucleic acid-containing liquid formulation, in particular a nucleic acid-containing liquid formulation containing more than 15% (w / v) cryoprotectant; b) rotating (spinning) the container of step a) along an axis, thereby allowing the formulation to form a layer on the circumferential inner wall of the container; c) cooling the container while continuing the rotation of the container of step b) to freeze-dry the formulation on the inner peripheral wall, thereby obtaining a spin-freezing container; d) drying the spin-freeze vessel of step c) by subjecting it to a heat source that provides uniform heat transfer to the inner wall of the vessel to form a dried nucleic acid-containing composition.

[0011] In certain embodiments of the invention, the nucleic acid-containing composition further comprises one or more lipids, liposomes, lipid nanoparticles, polymers, polymer-based nanoparticles, injection buffer / vehicle water, Tris-buffered saline (TBS), and / or phosphate-buffered saline (PBS).

[0012] In another particular embodiment, said nucleic acid-containing liquid formulation further comprises a cryoprotectant. In particular, the cryoprotectant of the present invention may be selected from the list comprising sodium citrate, sodium chloride, sorbitol, polysorbate, trehalose, mannose, mannitol, maltose, sucrose, glucose, fructose, lactose, histidine, arginine, lysine, dextran, maltodextrin, cyclodextrin, polyvinylpyrrolidone (PVP), glycine, glycerol, polyethylene glycol (PEG), propylene glycol, and / or mixtures thereof, and in particular may be sucrose.

[0013] In a further embodiment of the invention, the nucleic acid-containing liquid formulation comprises a concentration of the cryoprotectant in the range of 0.01% to 25% (w / v) or 0.01% to 25% (w / v), more particularly 16% to 25% (w / v).

[0014] In a further embodiment of the method of the invention, in steps b) and c), the vessel is rotated along its longitudinal axis at about 1000-5000 rpm.

[0015] In a further embodiment of the method of the invention, step c) comprises the use of an inert cryogenic gas flux.

[0016] In a further embodiment, the method of the invention is carried out in a two-chamber system comprising a freezing chamber and a drying chamber.

[0017] In another particular embodiment, the spin-freezing container of step c) is rapidly transferred from the freezing chamber to the drying chamber via an intermediate load-lock compartment, thereby enabling a system for a continuous freeze-drying process. In particular, the load-lock compartment is capable of maintaining a pressure of about 5 Pa to 35 Pa and a temperature of about -5°C to -30°C in the freezing chamber and drying chamber, respectively.

[0018] In a particular embodiment of the invention, the drying chamber is an endless belt system that transports the spin-freezing vessel in front of at least one individually controlled non-contact heat source, in particular the individually controlled non-contact heat source may be selected from infrared radiators and / or electric heating pads.

[0019] In a further embodiment, the spin-freezing vessel is slowly rotated along its longitudinal axis at about 5-12 rpm for about 1 minute to 120 minutes in front of at least one individually controlled heating source.

[0020] In a further embodiment of the method of the invention, step d) comprises a primary drying step in which the ice crystals are removed by sublimation under vacuum, and a secondary drying step in which most of the remaining unfrozen liquid is removed by diffusion and desorption, in particular said sublimated ice crystals and desorbed liquid are successively removed using a cryogenic ice condenser.

[0021] In a further aspect, the present invention provides a lyophilized nucleic acid-containing composition obtainable by the methods disclosed herein.

[0022] In particular, the present invention provides a container containing a nucleic acid-containing composition in powder form, said powder being present in a thin layer on the circumferential inner surface of said container. [Brief description of the drawings]

[0023] Now, with particular reference to the figures, it is emphasized that the details shown are exemplary and are for the sole purpose of illustratively illustrating various embodiments of the present invention. They are presented to provide what is believed to be the most useful and easiest way to explain the principles and conceptual aspects of the present invention. In this regard, no attempt has been made to show structural details of the invention beyond the scope necessary for a fundamental understanding of the invention. When described in conjunction with the figures, it will become apparent to those skilled in the art how some forms of the present invention can be actually implemented.

[0024] [Figure 1] Illustrated spin-freezing process: A vessel filled with a nucleic acid-containing liquid formulation is spun at high speed along its longitudinal axis, forming a thin layer of product that spreads across the interior wall of the vial (i.e., spin-freezing). Once a homogenous product layer is obtained, a flow of cold, inert, sterile gas (flux) cools and freezes the solution, solidifying the nucleic acid-containing formulation across the interior wall of the vessel to form a thin product layer of uniform thickness. [Diagram 2] Continuous freezing system connected to a continuous drying system The continuously spin frozen containers are transferred to the continuous drying chamber via a load lock system, which connects the freezing and drying chambers while maintaining specific pressure and temperature conditions. [Diagram 3]Diagram of infrared assisted continuous primary drying of spin frozen vials rotating along their vertical axis in front of individual infrared heaters. In the drying room, an endless belt system transports the spin frozen containers in front of individually controlled radiators, ensuring even and adequate heat transfer across the entire surface of the container for an efficient and homogenous drying action. Each container rotates very slowly in front of a single radiator, allowing individual temperature regulation and optimal drying trajectory for each spin frozen container. [Figure 4] Product temperature profile during the freezing process Freezing is the first step of the freeze-drying process that solidifies the material. The figure shows the temperature change (°C, y-axis) at a cooling rate of 15°C / min as a function of time (min, x-axis) of the freezing step. The thin lines represent the temperature set points of the freezing cycle and the thick lines represent the actual product temperature. The arrow indicates the moment (t=7 min) when the product temperature is fully stabilized at the set temperature. The freezing rate is important for the formation and size of the ice crystals: a faster freezing rate results in the formation of smaller ice crystals and vice versa. [Diagram 5] Product temperature profile during the drying process The drying process of the freeze-drying process consists of two stages: the primary and the secondary stage. The figure shows the evolution of temperature (°C, y-axis) as a function of time (min, x-axis) during the drying process. The arrow (t=0-40 min) indicates the temperature that separates the primary drying stage (sublimation) from the secondary drying stage (desorption). [Figure 6] Appearance of the product before and after reconstitution of the cake with water for injection The resulting dried product forms a thin layer, evenly distributed on the inner wall of the vial. Addition of 500 μL of water for injection to reconstitute the powder resulted in a white to off-white translucent liquid comparable to the appearance of the liquid before lyophilization. The 10-fold diluted product resulted in a clear solution. There were no visible particles in both the reconstituted and 10-fold diluted liquids. Although the particle size increased slightly upon reconstitution of the dried product, the reconstituted sample showed high particle size uniformity (i.e., narrow size distribution), which is reflected in the low polydispersity index value. [Figure 7]Appearance of FD (freeze-dried) mass by conventional (left panel) and spin freeze-drying (right panel) methods using 15% w / v sucrose (A) or 20% w / v sucrose (B). With conventional freeze-drying (left panel), 15% w / v sucrose (A) results in a visually perfect, homogenous, crack-free FD mass. On the other hand, 20% w / v sucrose (B) produces poor quality FD mass with visible shrinkage, brittleness, cracking, and crystallization at the bottom of the vial. With spin freeze-drying (right panel), both 15% w / v (A) and 20% w / v (B) produce perfect, homogenous FD mass without cracks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention is further described below. In the following text, the various aspects of the present invention are defined in more detail. Each aspect thus defined can be combined with other aspects, unless expressly indicated to the contrary. In particular, features indicated as preferred or advantageous can be combined with other features indicated as preferred or advantageous.

[0026] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "a compound" means one compound or multiple compounds.

[0027] As used herein, the terms "about" or "approximately" are used when referring to a measurable value, such as a parameter, amount, time duration, and the like, and are intended to encompass a variation of no more than + / - 10%, preferably no more than + / - 5%, more preferably no more than + / - 1%, and even more preferably no more than + / - 0.1% from the specified value, to the extent appropriate for practice in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself specifically, and preferably, disclosed.

[0028] Thus, the present invention is particularly directed to a process for lyophilizing nucleic acid-containing compositions utilizing a spin lyophilization process. The inventors of the present invention have discovered that spin lyophilized nucleic acid-containing compositions have superior distribution and storage stability compared to compositions lyophilized using standard lyophilization methods.

[0029] A further advantage of the present invention is that the freeze-drying method according to the present invention is 20-40 times faster than conventional freeze-drying methods, resulting in a significant reduction in the formation of ice crystals.

[0030] Thus, as already detailed above, a first aspect of the present invention provides a method for lyophilizing a nucleic acid-containing composition, said method comprising the steps of: a) filling a container having an inner wall and an outer wall with a nucleic acid-containing liquid formulation, particularly a nucleic acid-containing liquid formulation comprising more than 15% (w / v) cryoprotectant; b) rotating (spinning) the container of step a) along an axis, thereby allowing the formulation to form a layer on the inner peripheral wall of the container; c) cooling the container while continuing the rotation of the container of step b) to freeze-dry the formulation on the inner peripheral wall, thereby obtaining a spin-freeze container; d) drying the spin-freeze vessel of step c) by subjecting it to a heat source that provides uniform heat transfer to the inner wall of the vessel to form a dried nucleic acid-containing composition.

[0031] The term "nucleic acid" as used herein is understood as a biopolymer or large biological molecule composed of nucleotides (monomers made of three components: a five-carbon sugar, a phosphate group, and a nitrogenous base), unless otherwise specified. The term "nucleic acid" is a general term for information-carrying molecules such as RNA (ribonucleic acid, the sugar compound is ribose) and DNA (deoxyribonucleic acid, the sugar compound is deoxyribose). The sequences in question in the present invention may be of natural (human, animal, plant, bacterial, viral) or artificial origin, in particular genomic DNA, cDNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), piwi-interacting RNA (piRNA), small tRNA-derived RNA (tsRNA), small rDNA-derived RNA (srRNA), hybrid sequences, or synthetic or semi-synthetic sequences. The term naked nucleic acid refers to unprotected nucleic acids such as DNA / RNA generated in a laboratory. Furthermore, nucleic acids can vary in size from oligonucleotides to chromosomes and can be single-stranded or double-stranded.

[0032] In the context of the present invention, the term "RNA" refers to a molecule that comprises ribonucleotide residues, preferably a molecule that is completely or substantially composed of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term includes isolated RNA, such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications include the addition of non-nucleotide material to the ends or interior of the RNA, for example, to one or more nucleotides of the RNA. Nucleotides in an RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides, chemically synthesized nucleotides, or deoxynucleotides. These modified RNAs may be referred to as analogs. The nucleic acid may be included in a vector. As used herein, the term "vector" includes any vector known to those of skill in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus and baculovirus, artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes, or analogs of natural RNA.

[0033] According to the present invention, the term "RNA" includes and preferably relates to "mRNA" meaning "messenger RNA", which may be produced using DNA as a template, and relates to "transcripts" that code for peptides or proteins. mRNA usually comprises a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, there are various in vitro transcription kits available commercially. In a particular embodiment of the invention, said mRNA molecule is an mRNA molecule that codes for an immunomodulatory protein.

[0034] In the context of the present invention, the term "mRNA molecule encoding an immune-modulating protein" refers to an mRNA molecule encoding a protein that modifies the function of antigen-presenting cells, particularly dendritic cells. Such molecules can be selected from the list comprising CD40L, CD70, caTLR4, IL-12p70, EL-selectin, CCR7, and / or 4-1 BBL, ICOSL, OX40L, IL-21, and more specifically one or more of CD40L, CD70, and caTLR4. A preferred combination of immune stimulators used in the methods of the present invention is CD40L and caTLR4 (i.e., "DiMix"). In another preferred embodiment, a combination of immune stimulatory molecules of CD40L, CD70, and caTLR4 is used, also referred to herein as "TriMix".

[0035] In another specific embodiment, said mRNA molecule is an mRNA molecule encoding an antigen and / or a disease specific protein.

[0036] According to the present invention, the term "antigen" includes any molecule, preferably a peptide or protein, that induces an immune response and / or contains at least one epitope against which an immune response is directed. That is, the term antigen is meant to include the minimal epitope derived from the antigen. "Minimal epitope" as defined herein means the minimal structure capable of eliciting an immune response. Preferably, an antigen in the context of the present invention is a molecule that, after optional processing, preferably induces a specific immune response in the antigen or in a cell expressing the antigen. In particular, "antigen" relates to a molecule that, after optional processing, is presented by MHC molecules and reacts specifically with T lymphocytes (T cells).

[0037] In certain embodiments, the antigen is a target-specific antigen, which may be a tumor antigen, a bacterial antigen, a viral antigen, or a fungal antigen, etc. The target-specific antigen may be derived from either total mRNA isolated from a target cell, one or more specific target mRNA molecules, a protein lysate of the target cell, a specific protein from the target cell, or a synthetic target-specific peptide or protein, and synthetic mRNA or DNA encoding the target-specific antigen or its derived peptide.

[0038] For the avoidance of doubt, the LNPs of the present invention may comprise a single mRNA molecule or may comprise multiple mRNA molecules, such as a combination of one or more mRNA molecules encoding an immunomodulatory protein and / or one or more mRNA molecules encoding an antigen and / or disease specific protein.

[0039] In a very specific embodiment, said mRNA molecule encoding an immune-modulating molecule can be combined with one or more mRNA molecules encoding an antigen and / or disease-specific protein. For example, the LNPs of the present invention can include a combination of an mRNA molecule encoding an immune-stimulating molecule CD40L, CD70 and / or caTLR4 (such as Dimix or Trimix) with one or more mRNA molecules encoding an antigen-specific protein and / or disease-specific protein. Thus, in a very specific embodiment, the LNPs of the present invention include an mRNA molecule encoding CD40L, CD70 and / or caTLR4 in combination with one or more mRNA molecules encoding an antigen and / or disease-specific protein.

[0040] In the context of the present invention, the term "composition" refers to the identity of all the different individual substances, compounds, or elements (e.g., drugs, regulators, control agents, etc.) that make up the composition. It can be a solution, suspension, liquid, powder, paste, aqueous or non-aqueous formulation, or a combination thereof. The composition is preferably a pharmaceutical composition that includes one or more pharmaceutical excipients, carriers, diluents.

[0041] In certain embodiments of the invention, the nucleic acid-containing composition further comprises one or more lipids, liposomes, lipid nanoparticles, polymers, polymer-based nanoparticles, injection buffer / media, Tris-buffered saline (TBS), and phosphate-buffered saline (PBS).

[0042] In the context of the present invention, the term "nucleic acid-containing composition" refers to a composition comprising at least one nucleic acid polymer delivered in one or more vehicles, including lipids, liposomes, lipid nanoparticles, polymers, polymer-based nanoparticles, injection buffers / media water, Tris-buffered saline (TBS), and / or phosphate-buffered saline (PBS).

[0043] After lyophilization, the resulting dried nucleic acid may be reconstituted using any suitable medium / buffer, such as, but not limited to, water for injection, Tris-buffered saline (TBS), and / or phosphate-buffered saline (PBS).

[0044] As used herein, lipid refers to any component belonging to the categories of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterols, prenols, glycolipids, polyketides. Furthermore, in the context of the present invention, liposomes are spherical vesicular structures with at least one lipid bilayer forming the shape of a hollow sphere surrounding an aqueous phase. Thus, cargo of interest, such as nucleic acid-containing formulations, pharmaceuticals, proteins / peptides, etc., can be encapsulated within the liposome either in the aqueous compartment (if water-soluble / hydrophilic) or in the lipid bilayer (if lipophilic / lipophilic).

[0045] Further, as used herein, lipid nanoparticles are liposome-like structures specialized for encapsulating various nucleic acids (RNA and DNA) as drugs or vaccines within a non-aqueous core. More specifically, lipid nanoparticles are generally spherical in shape and are composed of a solid lipid core stabilized by surfactants. The core lipids can be fatty acids, acylglycerols, waxes, and mixtures thereof, and biological membrane lipids such as phospholipids, sphingomyelins, bile salts (sodium taurocholate), and sterols (cholesterol) can be utilized as stabilizers. While such LNPs can include many different types of lipids, the LNPs of the present invention are typically composed of a combination of ionized lipids, phospholipids, sterols, and PEG lipids.

[0046] In the context of the present invention, the term "PEG lipid" or "PEGylated lipid" means any suitable lipid modified with a PEG (polyethylene glycol) group.

[0047] In the context of the present invention, the term "ionizable" (or cationic) in the context of a compound or lipid refers to a compound or lipid that contains ions (usually H + "Polycation" refers to the presence of uncharged groups that have the ability to dissociate, generating electrons (ions), thereby becoming positively charged themselves. Alternatively, uncharged groups in the compound or lipid may generate electrons, thereby becoming negatively charged.

[0048] In the context of the present invention, the term "phospholipid" refers to a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two components are most often linked by a glycerol molecule, so that the phospholipids of the present invention are preferably glycerol phospholipids. Furthermore, the phosphate group is often modified with simple organic molecules such as choline (i.e., resulting in phosphocholine) or ethanolamine (i.e., resulting in phosphoethanolamine).

[0049] In the context of the present invention, the term "sterol", also called steroid alcohols, is a subgroup of steroids that occur naturally in plants, animals and fungi or can be produced by some bacteria. In the context of the present invention, any suitable sterol can be used, suitable sterols being selected from the list including, for example, cholesterol, ergosterol, campesterol, oxysterol, anthrosterol, desmosterol, nicasterol, sitosterol and stigmasterol, preferably cholesterol.

[0050] As used herein, polymer refers to a natural or synthetic substance or material consisting of very large molecules, or macromolecules, composed of many repeating subunits called monomers. The term "polymeric nanoparticles" is understood as spherical particles in the size range of 1-1000 nm that can be loaded with active compounds entrapped within or surface adsorbed onto the polymer core. Furthermore, the term "nanoparticles" is understood as both nanocapsules and nanospheres, where nanocapsules are usually composed of an oily core in which the drug is dissolved and a surrounding polymer shell, whereas nanospheres are based on a continuous polymer network that can hold drugs inside or adsorbed on the surface.

[0051] As used herein, unless otherwise specified, the term "container" is understood as a container or enclosure for storing a product, which protects the product by containing it within its structure. Containers are made of glass, plastic, or other materials and are used to store, package, or transport liquid, powder, tablet, or capsule products, and may be in the shape of, for example, a vial, bottle, container, canister, flask, preferably a cylindrical vial.

[0052] As used herein, unless otherwise specified, the term "liquid formulation" is understood to mean any excipient in liquid or semi-liquid form, comprising at least one component that may or may not be soluble in said liquid or semi-liquid.

[0053] In another specific embodiment, the nucleic acid-containing liquid formulation further comprises a cryoprotectant.In particular, the cryoprotectant of the present invention can be selected from the list comprising sodium citrate, sodium chloride, sorbitol, polysorbate, trehalose, mannose, mannitol, maltose, sucrose, glucose, fructose, lactose, histidine, arginine, lysine, dextran, maltodextrin, cyclodextrin, polyvinylpyrrolidone (PVP), glycine, glycerol, polyethylene glycol (PEG), propylene glycol, and / or mixtures thereof.In a very specific embodiment, the cryoprotectant is sucrose.

[0054] As used herein, unless otherwise specified, the term "cryoprotectant" is understood as a permeable or non-permeable substance used to protect compositions and their related compounds from freezing damage (i.e., damage due to ice formation). Conventional cryoprotectants are generally glycols (alcohols containing at least two hydroxyl groups) and sugars such as ethylene glycol, propylene glycol, glycerol, and trehalose. In particular, trehalose is a non-reducing sugar consisting of two glucose molecules that maintains the structural integrity of cells during freezing and thawing in a non-toxic manner. In previous studies, this disaccharide has been used for the cryopreservation of human cells such as platelets, red blood cells, sperm, oocytes, pancreatic islets, and fetal skin.

[0055] In yet another embodiment of the invention, the nucleic acid-containing liquid formulation comprises said cryoprotectant at a concentration in the range of 0.01% to 25% (w / v) or 0.01% to 25% w / v; in particular 1% to 20% w / v, more particularly greater than 5% w / v, more particularly greater than 10 w / v, more particularly about 15% w / v, more particularly greater than 15% w / v, more particularly about 16% to 25% w / v.

[0056] In some embodiments, the liquid formulation may comprise a minimum amount of sodium citrate of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0057] In some embodiments, the liquid formulation may contain a minimum amount of sodium chloride of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0058] In some embodiments, the liquid formulation may comprise a minimum amount of sorbitol of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0059] In some embodiments, the liquid formulation may comprise a minimum amount of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v) polysorbate.

[0060] In some embodiments, the liquid formulation may comprise a minimum amount of trehalose of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0061] In some embodiments, the liquid formulation may comprise a minimum amount of mannose of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0062] In some embodiments, the liquid formulation may comprise a minimum amount of mannitol of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0063] In some embodiments, the liquid formulation may comprise a minimum amount of maltose of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0064] In some embodiments, the liquid formulation may comprise a minimum amount of sucrose of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0065] In some embodiments, the liquid formulation may comprise a minimum amount of glucose of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0066] In some embodiments, the liquid formulation may contain a minimum amount of fructose of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0067] In some embodiments, the liquid formulation may contain a minimum amount of lactose of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0068] In some embodiments, the liquid formulation may comprise a minimum amount of histidine of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0069] In some embodiments, the liquid formulation may comprise a minimum amount of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v) arginine.

[0070] In some embodiments, the liquid formulation may contain a minimum amount of lysine of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0071] In some embodiments, the liquid formulation may comprise a minimum amount of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v) dextran.

[0072] In some embodiments, the liquid formulation may comprise a minimum amount of maltodextrin of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0073] In some embodiments, the liquid formulation may comprise a minimum amount of cyclodextrin of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0074] In some embodiments, the liquid formulation may contain a minimum amount of polyvinylpyrrolidone (PVP) of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0075] In some embodiments, the liquid formulation may comprise a minimum amount of glycine of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0076] In some embodiments, the liquid formulation may comprise a minimum amount of glycerol of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0077] In some embodiments, the liquid formulation may comprise a minimum amount of polyethylene glycol (PEG) of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v).

[0078] In some embodiments, the liquid formulation may comprise a minimum amount of at least about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% (w / v) propylene glycol.

[0079] In a further embodiment of the method of the present invention, during the rotating and cooling steps, the vessel rotates along its longitudinal axis at about 1000-5000 rpm.

[0080] As used herein, and unless otherwise specified, the term "rotating (spinning)" means to spread the formulation as evenly as possible over the inside surface of the container by rotating the container about its axis, allowing the formulation to form a layer on the inner circumferential wall of the container (FIG. 1). The rotation (or spinning) of the container may be performed continuously during the freeze-drying process, but not necessarily at the same speed (also called revolutions per minute (rpm)).

[0081] As used herein, unless otherwise specified, the term "axis" is understood to be either a vertical axis or a horizontal axis.

[0082] In some embodiments, the term "longitudinal axis" is understood to be the axis running from the top to the bottom of a designated vessel.

[0083] As used herein, unless otherwise specified, the term "lateral axis" is to be understood as the axis passing horizontally through the center of a given vessel.

[0084] In some embodiments of the method of the present invention, the rotation of the container during the cooling step is performed at about 1000-5000 rpm, preferably about 1500-4000 rpm, and more preferably about 2000-3700 rpm.

[0085] As used herein, unless otherwise specified, the term "cooling" is understood as reducing the temperature of a product below 0°C, typically to about -90°C to -20°C, more preferably -70°C, by means of a liquid or solid gas, to convert the liquid state of the product to a solid state and form ice crystals on the inner surface of a specifically designated container.

[0086] The temperature profile during this cooling step depends on the composition being cooled and may vary from linear cooling (e.g., 15°C / min) to more complex dynamic temperature profiles. The cooling step lasts for about 1 to 30 minutes, more preferably 15 minutes.

[0087] As used herein, unless otherwise specified, the term "ice crystals" should be understood as structures formed at low temperatures by condensation of water vapor onto a solid, which may be a particle or other substance.

[0088] In a further embodiment of the method of the present invention, the cooling step comprises the use of an inert cryogenic gas flux.

[0089] As used herein, unless otherwise specified, the inert cryogenic gas flux can be selected from the list including helium, carbon dioxide, and nitrogen, preferably carbon dioxide, and most preferably nitrogen, and can surround at least one container to obtain a thin frozen product layer.

[0090] In some embodiments of the methods of the present invention, the thin frozen product layer has a thickness of about 0.1-10 nm, preferably about 0.5-5 nm, and most preferably about 1-3 nm thick.

[0091] In a further embodiment, the rotating or spinning vessel is continuously cooled for about 1 to 60 minutes, preferably 5 to 30 minutes, more preferably 10 to 20 minutes.

[0092] In yet another embodiment, the method of the present invention is carried out in a two-chamber system comprising a freezing chamber and a drying chamber (FIG. 2).

[0093] As used herein, unless otherwise specified, the term "drying" refers to the process of removing water or other solvents from a solid, semi-solid, or liquid composition. This can be accomplished by exposing the composition to a heat source, a desiccant, placing the composition under vacuum, or a combination thereof.

[0094] As used herein, unless otherwise specified, a "two-chamber system" means two chambers (a freezing chamber and a drying chamber) that are connected to each other.

[0095] In another specific embodiment, the spin-frozen containers are rapidly transferred from the freezing chamber to the drying chamber via an intermediate load lock chamber, allowing the system to perform a continuous freeze-drying process (FIG. 2).

[0096] In a particular embodiment, the load lock chamber can maintain a pressure of about 5 Pa to about 35 Pa. In another particular embodiment, the temperature of the load lock chamber can be about -5°C to about -30°C within the freezing chamber and the drying chamber. In a further embodiment, the load lock chamber can maintain a pressure of about 5 Pa to about 35 Pa and a temperature of about -5°C to about -30°C within the freezing chamber and the drying chamber.

[0097] As used herein, unless otherwise specified, the term "load-lock system" is understood as a system used to rapidly transfer spin-frozen vials between the continuous freezing and drying chambers while ensuring process continuity without disturbing the specific conditions of pressure and temperature within the freezing and drying chambers.

[0098] In some embodiments of the method of the present invention, the freezing chamber, drying chamber, and load lock chamber are a closed loop system, and the load lock chamber and drying chamber are preferably under pressure, i.e., under vacuum. The container is placed under vacuum, whether or not it is exposed to a heat source, and a process occurs in which water vapor is produced, which is removed using a condenser.

[0099] In some embodiments of the method of the present invention, the pressure in the load lock chamber is between 0 Pa and 70 Pa, preferably between 5 Pa and 40 Pa, and most preferably between 5 Pa and 35 Pa, and can be adjusted so during the process.

[0100] In some embodiments of the method of the present invention, the temperature of the load lock chamber is between −5 and −30° C., preferably between −5 and −25° C., and most preferably about −10° C., and can be adjusted thereto during the process.

[0101] In yet another embodiment of the method of the present invention, the drying process comprises a primary drying step in which the ice crystals are removed by sublimation under vacuum, and a secondary drying step in which most of the remaining unfrozen liquid is removed by diffusion and desorption, in particular, said sublimated ice crystals and desorbed liquid are successively removed using a cryogenic ice condenser.

[0102] In some embodiments, the term "drying" refers to the term "sublimation" (also called the "primary drying step"), or the term "diffusion and desorption" (also called the "secondary drying step"), or both.

[0103] As used herein, unless otherwise specified, the term "sublimation" is understood to mean the change of a substance from a solid state directly to a gaseous state under vacuum without passing through a liquid state.

[0104] In some embodiments of the method of the present invention, the pressure during the sublimation step is 0 Pa to 70 Pa, preferably 5 Pa to 40 Pa, most preferably 5 Pa to 35 Pa, and can be appropriately adjusted during the process.

[0105] As used herein, unless otherwise specified, the term "diffusion" is understood as the thermal motion of all particles (liquid or gas) at temperatures above absolute zero under vacuum. Diffusion describes the net flow of molecules from an area of ​​higher concentration to an area of ​​lower concentration.

[0106] As used herein, unless otherwise specified, the term "desorption" is understood as the phenomenon in which a substance is released from or through a surface into the gas phase under vacuum. In particular, most of the unfrozen water (i.e., water dissolved in the amorphous phase of the solid) binds to the inner surface of the container by adsorption and absorption and is gradually removed by desorption.

[0107] In some embodiments of the method of the present invention, the pressure during the diffusion and desorption steps is from 0 Pa to 70 Pa, preferably from 5 Pa to 40 Pa, most preferably from 5 Pa to 35 Pa, and can be adjusted so during the process.

[0108] As used herein, unless otherwise specified, the term "cryogenic ice condenser" is understood as a system designed to rapidly absorb steam and reduce the pressure in a containment vessel.

[0109] In a further embodiment, during the drying process, the spin-freeze vessel is slowly rotated along its longitudinal axis at about 5-12 rpm for about 1 to 120 minutes in front of at least one individually controlled heating source.

[0110] In some embodiments of the invention, the continuous rotation (spin) of the vessel is at about 150-600 rpm / s along its longitudinal axis. 2 The rotation speed can be preferably about 3 to 25 rpm, and more preferably about 5 to 12 rpm.

[0111] In some embodiments of the method of the present invention, the primary drying step (also called sublimation) is carried out for about 1-60 minutes, preferably 5-30 minutes, more preferably 10-20 minutes, and most preferably until the residual moisture content is 0-30%, preferably 2-25%, more preferably 3-20%, and most preferably 5-15%.

[0112] In some embodiments of the method of the present invention, the secondary drying step (also called diffusion and desorption) may take about 1-60 minutes, preferably 5-30 minutes, more preferably 10-20 minutes. Alternatively, secondary drying is continued until the composition has an acceptable moisture content for long term storage, most preferably a residual moisture content of about 0-5%, preferably 0.2-4%, most preferably 0.5-3%.

[0113] In another embodiment of the method of the present invention, the temperature during the secondary drying step is between 10°C and 70°C, more preferably between 20°C and 60°C, most preferably between 30°C and 50°C.

[0114] In some embodiments of the method of the present invention, the entire drying step is carried out for a period of at least 60 minutes, preferably no more than 120 minutes.

[0115] In a particular embodiment of the invention, the drying chamber is an endless belt system that transports the spin freezing vessel in front of at least one individually controlled non-contact heat source, in particular, the individually controlled non-contact heat source may be selected from infrared radiators and / or electric heating pads.

[0116] As used herein, unless otherwise specified, the term "endless belt system" is to be understood as a transport system that continuously places containers in front of a heat source.

[0117] As used herein, unless otherwise specified, the term "heat source" is used to refer to a heater that is at least temporarily associated with a nearby container and is preferably driven to provide heat primarily to that container without providing thermal energy to other containers. This device delivers energy uniformly by radiation or conduction for drying (sublimation and desorption) and can be tuned for each unit dose.

[0118] In some embodiments of the method of the present invention, the heater may be an infrared radiator, which allows for non-contact heat transfer only to opaque objects and not to the surrounding air through infrared heating.

[0119] In another embodiment, the infrared radiator reaches a temperature of about 100-450° C. and can be adapted as such during the process.

[0120] As used herein, unless otherwise specified, the term "infrared" is also understood as infrared (IR) thermography, which allows non-contact, real-time and spatial monitoring of the product temperature at the sublimation interface. In a further aspect, the present invention provides a lyophilized nucleic acid-containing composition obtained by the method disclosed herein.

[0121] In particular, the present invention provides a container containing a powdered nucleic acid-containing composition, the powder being in the form of a thin layer on the inner circumferential surface of the container. EXAMPLES

[0122] Example 1

[0123] Materials and Methods

[0124] Lipid nanoparticles containing mRNA were prepared using a mixing device, after which the ethanol was removed, filtered through a 0.22 μm filter, and filled into 2 mL type I clear glass vials (filling volume per vial was 0.5 mL).

[0125] A pharmaceutical formulation containing 500 μL of lipid nanoparticles (containing ionized lipids, phospholipids, sterols, and PEG-lipids) encapsulating mRNA in Tris-buffered saline (TBS) supplemented with 15% trehalose was subjected to continuous lyophilization in a single-vial apparatus.

[0126] During spin freezing (Figure 1), the vial is rotated (spun) around its vertical axis at approximately 2500 revolutions per minute (rpm), which suggests that the product layer will spread evenly and that the maximum thickness difference between the bottom and top of the product layer will be 10%. Process conditions applied: 15°C / min cooling rate to -70°C, conservative drying setting, 150 rpm / s 2Ramp-up during spin-freezing. The drying cycle was performed at a conservative setting with an infrared (IR) heater switched off at approximately 0.6 watts, the energy value required to sublimate 500 mg of ice. The duration of the freeze-drying cycle was 1.5 hours. After reconstitution, particle size, polydispersity index, and appearance were measured.

[0127] result

[0128] The temperature of the product was decreased at a cooling rate of 15°C / min to −70°C, at which point the product was completely stabilized to the set temperature (Figure 4).

[0129] FIG. 5 shows the increase in temperature in the primary and secondary drying stages to the left and right of the arrows.

[0130] The resulting dried product forms a thin layer and is evenly distributed around the inner wall of the vial (Figure 6). Reconstitution of the powder with water results in a white to pale white translucent liquid. The 10-fold diluted product yielded a clear solution. There were no visible particles in both the reconstituted and 10-fold diluted solutions (Table 1). Although the particle size increased slightly upon reconstitution of the dried product, the reconstituted sample showed high particle size uniformity (i.e., narrow size distribution), which is reflected in the low polydispersity index value.

[0131] Table 1. Appearance and particle characteristics of the dried and reconstituted products [Table 1]

[0132] Example 2

[0133] In this example, lyophilized cakes using different concentrations of sucrose were compared using conventional and spin freeze-drying methods.

[0134] LNP generation:

[0135] The mRNA in acidic buffer was mixed with the lipid mixture in ethanol using a T-junction mixer. After mixing, the LNP dispersion was buffer exchanged into a neutral buffer by dialysis and concentrated by centrifugal concentration. Various concentrations of sucrose (5%, 8%, 10%, 15%, 20% w / v) were added to the final LNP dispersion as a cryoprotectant.

[0136] Lyophilization and analysis:

[0137] Conventional freeze-drying was performed using a Martin Christ EPSILON 2-6 LSCplus freeze-dryer. Spin freeze-drying was performed using a RheaVita multi-vial unit according to the methods disclosed herein.

[0138] After lyophilization, samples were reconstituted with WFI (water for injection). The appearance of the samples before and after reconstitution was evaluated.

[0139] result

[0140] The appearance of the lyophilized mass was good at low cryoprotectant (sucrose) content, but above 15% sucrose content, the mass started to crack and shrink, showing signs of dissolution (see Figure 7 for concentrations of 15% and 20% w / v). Thus, the spin freeze-drying technique improved the appearance of the FD mass at high cryoprotectant addition levels, enhancing its visual quality. All standard Critical Quality Attributes (CQAs) were comparable, with little difference in size, PDI, pH, osmolality, mRNA content and encapsulation efficiency.

[0141] Liquid drug formulation Initiate spinning Liquid spreads over entire vial surface Initiate cooling Start cooling Product solidified over entire vial surface Bottom view spin-freezing load-lock system detail of example of load-lock system secondary freezing Continuous drying chamber Primary drying progress Primary drying progress Water vapour flow IR heater IR heater Rotary stage connected to moving belt Frozen product Ice-free product Ice crystals – dried product interface Temperature Setpoint Product temperature Product temperature Liquid products Dried product(s) Reconstituted product(s) Conventional FD sucrose Spin FD

Claims

1. a) filling a container having an inner wall and an outer wall with a nucleic acid-containing liquid formulation comprising greater than 15% (w / v) cryoprotectant; b) rotating the container of step a) along an axis, thereby allowing the formulation to form a layer on the inner circumferential wall of the container; c) cooling the container while continuing the rotation of the container of step b) to freeze-dry the formulation on the inner peripheral wall, thereby obtaining a spin-freezing container; d) drying the spin-freezing vessel of step c) by subjecting it to a heat source that provides uniform heat transfer to the inner wall of the vessel to form a dried nucleic acid-containing composition; A method for lyophilizing a nucleic acid-containing composition, comprising the steps of:

2. 10. The method of claim 1, wherein the nucleic acid-containing composition further comprises one or more lipids, liposomes, lipid nanoparticles, polymers, polymer-based nanoparticles, injection buffer / vehicle water, Tris-buffered saline (TBS), and / or phosphate-buffered saline (PBS).

3. 3. The method according to claim 1 or 2, wherein the cryoprotectant is selected from the list comprising sodium citrate, sodium chloride, sorbitol, polysorbate, trehalose, mannose, mannitol, maltose, sucrose, glucose, fructose, lactose, histidine, dextran, maltodextrin, cyclodextrin, polyvinylpyrrolidone (PVP), glycine, glycerol, polyethylene glycol (PEG), propylene glycol, and / or mixtures thereof, in particular sucrose.

4. The method of claim 3, wherein the nucleic acid-containing liquid formulation comprises a concentration of the cryoprotectant in the range of 16% to 25% (w / v).

5. The method according to any one of claims 1 to 4, wherein in steps b) and c) the vessel is rotated along its longitudinal axis at about 1000 to 5000 rpm.

6. The method according to any one of claims 1 to 5, wherein step c) comprises the use of an inert cryogenic gas flux.

7. The method according to any one of claims 1 to 6, wherein the method is carried out in a two-chamber system comprising a freezing chamber and a drying chamber.

8. 8. The method of claim 7, wherein the spin-freezing vessel of step c) is rapidly transferred from the freezing chamber to the drying chamber via an intermediate load lock chamber, thereby allowing a system for a continuous freeze-drying process.

9. 9. The method of claim 8, wherein the load lock chamber maintains a pressure of about 5 Pa to 35 Pa and a temperature of about -5° C. to -30° C. in each of the freezing and drying chambers.

10. 10. The method according to any one of claims 7 to 9, wherein the drying chamber is an endless belt system which transports the spin freezing vessel in front of at least one individually controlled non-contact heating source, in particular the individually controlled non-contact heating source is selected from infrared radiators and / or electric heating pads.

11. 11. The method of claim 10, wherein the spin-freezing vessel is slowly rotated along its longitudinal axis at about 5-12 rpm for about 1 minute to 120 minutes in front of at least one individually controlled heating source.

12. 12. The method according to any one of claims 1 to 11, wherein step d) comprises a primary drying step in which the ice crystals are removed by sublimation under vacuum, and a secondary drying step in which most of the remaining unfrozen liquid is removed by diffusion and desorption.

13. 13. The method of claim 12, wherein the sublimated ice crystals and desorbed liquid are continuously removed using a cryogenic ice condenser.

14. A lyophilized nucleic acid-containing composition obtainable by the method according to any one of claims 1 to 13.

15. A container containing a nucleic acid-containing composition in powder form, said powder being present in the form of a thin layer on the inner circumferential surface of said container.

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