Compositions for encapsulating lyophilised microspheres

Abstract

This disclosure relates to a composition comprising a shell surrounding a core, wherein the core comprises one or more lyophilized microspheres. A method is also described herein comprising providing one or more lyophilized microspheres and coating one or more lyophilized microspheres with the shell under conditions of effective encapsulation of the one or more lyophilized microspheres. This disclosure further relates to a system comprising one or more compositions as described herein and one or more lyophilized cakes, wherein the one or more compositions and one or more lyophilized cakes are combined under conditions of effective formation of a rehydration system. A method for controlling the release of one or more encapsulated microspheres is also described herein, comprising providing a composition as described herein and mixing the composition with a rehydration solution under a first condition of effective control of the release of one or more lyophilized microspheres from the composition.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 174,325, filed on April 13, 2021, the entire contents of which are incorporated herein by reference.

[0002] This disclosure generally relates to components, systems, and methods for manufacturing and using encapsulated freeze-dried microspheres. Prior Technology

[0003] Many current sequencing platforms use sequencing by synthesis (SBS) technology and fluorescence-based detection methods. More cost-effective, rapid, and convenient alternative sequencing and nucleic acid detection methods, along with improved sample preparation procedures, are expected to complement SBS.

[0004] The current standard practice for SBS technology is to use a sample preparation procedure that converts DNA or RNA into fragmented, sequenceable template libraries. Sample preparation methods typically involve multiple steps, material transfer, and expensive equipment to achieve fragmentation, and are therefore usually difficult, cumbersome, costly, and inefficient.

[0005] Libraries of polynucleotides are typically prepared in any suitable manner to attach oligonucleotide transposons to target polynucleotides. Sequencing can lead to the determination of the sequence of the entire or a portion of the target polynucleotide. By using translocase-mediated fragmentation and labeling, the number of steps involved in converting nucleic acids into templates in solution prepared for clustering and sequencing of transposons can be reduced, or in some cases even minimized. This procedure, called "tagmentation," involves modifying nucleic acids with transposome complexes containing transloses that misalign with transposons containing transposon terminal sequences, as described, for example, in WO 2016 / 130704. Methods for immobilization and amplification prior to sequencing are described, for example, in U.S. Patent Nos. 8,053,192, WO 2016 / 130704, 8,895,249, and 9,309,502. Template libraries can be used to prepare cluster arrays of nucleic acid strains by solid-phase amplification and, more specifically, solid-phase isothermal amplification, as described in U.S. Patent Publication No. 2005 / 0100900, U.S. Patent No. 7,115,400, WO 00 / 18957, and WO 98 / 44151.

[0006] Sequencing can be performed using any suitable sequencing technique, and methods for determining the sequence of the immobilized and amplified transposon-target-transposon molecule (including strand recombination) are known in this art and described, for example, in U.S. Patent Nos. 8,053,192, WO2016 / 130704, 8,895,249, and 9,309,502. SBS technology generally involves the enzymatic extension of nascent nucleic acid strands via the repeated addition of nucleotides to a template strand. In conventional SBS methods, a single nucleotide monomer can be delivered to the target nucleotide in the presence of a polymerase at each delivery point. Illustrative SBS systems and methods are described in U.S. Patent Publications 2007 / 0166705, 2006 / 0188901, 2006 / 0240439, 2006 / 0281109, 2012 / 0270305, and 2013 / 0260372, U.S. Patent No. 7,057,026, WO 05 / 065814, U.S. Patent Publications 2005 / 0100900, WO 06 / 064199, and WO 07 / 010,251, and U.S. Patent Publication 2013 / 0079232.

[0007] The stability of reagents used in sample preparation (including, for example, PCR) varies depending on a range of factors. Historically, reagents were wet and therefore often involved freezing for transport and storage. While dried reagents allow for more haphazard transport and storage, they can be more sensitive to environmental conditions than wet reagents. Exposure to undesirable environmental conditions during manufacturing, transport, storage, or library preparation can affect the quality and efficiency of the resulting library. Similarly, the pH of reagents such as SBS buffers can change during sequencing, necessitating improved stabilization of these buffers to enhance SBS performance. Reagents used in sample preparation can be highly sensitive to changes in humidity, light, and moisture, making them difficult to stabilize.

[0008] Furthermore, lyophilized microspheres used in sample preparation often degrade when exposed to mechanical stress during transport and storage, and may adversely detach their outer coating. The resulting powder can cause clogging of membranes used in sample preparation and can lead to changes in the desired final concentration after rehydration. Electrostatic charge also poses a risk to the application and dry mixing of the microspheres.

[0009] Triboelectric charging is achieved through (frictional) contact via particle-particle and / or particle-wall interactions. During contact, charge transfer occurs, and upon separation, two objects with opposite charges are obtained. Static electricity is achieved through the ability of a material (particle or wall) to dissipate electrostatic charge, which is related to the material's conductivity.

[0010] Lyophilized microspheres are typically made from non-conductive materials (e.g., trehalose). The necessity of handling and storing lyophilized microspheres in a dry environment stems from their limited tolerance to ambient humidity. Therefore, electrostatic behavior and triboelectric charging are expected in lyophilized microspheres in a dry environment, manifested by adhesion to the container walls. The associated risk of electrostatics is the difficulty in handling microspheres used for dry filling into containers. During dry storage, microspheres tend to adhere to the container walls, leading to cross-contamination between pores and inaccuracies during rehydration.

[0011] Therefore, improved sample preparation components and procedures are needed. Specifically, sequencing reagents and associated methods with improved stability are required, demonstrating improved workflow and efficiency in generating labeled libraries, thereby increasing the read enrichment of the resulting libraries. Components and methods that improve the read enrichment of the resulting libraries and simplify the workflow are also needed.

[0012] This disclosure relates to overcoming these and other defects in this technology. Summary of the Invention

[0013] The first state relates to a composition comprising a shell surrounding a core, wherein the core comprises one or more freeze-dried microspheres.

[0014] In one embodiment, the shell comprises carrageenan, shellac, trehalose, paraffin, gelatin, hydroxypropyl methylcellulose (HPMC), fullalin, oxygen absorber, alginate, chitosan, slurry, and benzo[a] Borazole-poly(vinyl alcohol) (benzo[] Borazole-PVA (benzoxaborole-poly(vinyl alcohol)), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0015] In one embodiment, the shell comprises a shell additive. In one embodiment, the shell additive comprises an antistatic material, a moisture-proof material, or a combination thereof. In one embodiment, the shell additive is an antistatic material present at a concentration not exceeding 40% w / w of the shell. In one embodiment, the shell additive is a moisture-proof material present at a concentration not exceeding 90% w / w of the shell. In one embodiment, the shell additive is present at a concentration of at least 10% w / w of the shell. In one embodiment, the amount of the shell additive is between about 10% w / w and about 90% w / w of the shell. In one embodiment, the shell additive is a water-insoluble additive, a water-soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, copolymer, block copolymer, second polyvinyl alcohol (PVA), ammonium salt, conductivity promoter, stearate derivative, oleate derivative, laurate derivative, polyether compound, amino acid, tocopheryl acetate, piperidinium sebate, sodium salt, buffer, chelating agent, imidazolium salt, polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearate derivative or oleate derivative is selected from magnesium stearate, triglyceride monostearate, Span® 60, Tween® 60, trioleyl ester, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer solution is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, trialkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methylimidazolium salt or polytetramethylammonium salt or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or a combination thereof. In one embodiment, the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol-grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

[0016] In one embodiment, the core comprises one or more reagents selected from one or more enzymes, salts, surfactants, buffers, enzyme inhibitors, primers, nucleotides, organic osmolite, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof. In one embodiment, the reagent is a polymerase. In one embodiment, the volume of the reagent in the core is between about 0.1 µL and about 50 µL.

[0017] In one embodiment, the shell contains a reagent.

[0018] In one embodiment, the core further comprises one or more additional agents, wherein the additional agents are selected from one or more sugars, one or more amino acids, one or more polymers, one or more mesoporous silicas, one or more quaternary amines, and any combination thereof. In one embodiment, when the additional agent comprises one or more sugars, the sugar is selected from trehalose, mannitol, cyclodextrin, polydextrose, sucrose, and any combination thereof. In another embodiment, the additional agent comprises one or more amino acids having hydrophobic side chains. In yet another embodiment, when the additional agent comprises one or more polymers, the polymer is selected from polyvinylpyrrolidone, polyvinyl alcohol, and combinations thereof.

[0019] In one embodiment, the core comprises a core additive. In one embodiment, the core additive comprises an antistatic material. In one embodiment, the core additive is an antistatic material present at a concentration not exceeding 25% w / w of the core. In one embodiment, the core additive is present at a concentration of at least 0.5% w / w of the shell. In one embodiment, the amount of the core additive is between about 2% w / w and about 10% w / w of the core. In one embodiment, the core additive is a water-insoluble additive, a water-soluble additive, an enteric additive, or any combination thereof. In one embodiment, the core additive comprises one or more of a polymer, copolymer, block copolymer, second polyvinyl alcohol (PVA), conductivity promoter, ammonium salt, imidazolium salt, polyether compound, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the buffer solution is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt or polytetramethylammonium salt or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or a combination thereof. In one embodiment, the composition is used to perform multiple co-assay reactions. In one embodiment, the shell comprises more than one lyophilized microsphere, wherein the reagents in the more than one lyophilized microsphere are different.

[0020] The second aspect relates to a method. The method includes providing one or more lyophilized microspheres; and, under conditions of effectively encapsulating the one or more lyophilized microspheres, coating the one or more lyophilized microspheres with a shell.

[0021] In one embodiment, the method further includes covering the shell with the outer layer while the outer layer effectively surrounds the encapsulated microspheres. In one embodiment, the covering is performed for a period of time sufficient to provide a defined thickness to the outer layer.

[0022] In one embodiment, the shell, the outer layer, or both the shell and the outer layer comprise carrageenan, shellac, trehalose, paraffin, gelatin, hydroxypropyl methylcellulose (HPMC), fullalin, oxygen absorber, alginate, chitosan, slurry, and benzo[a]benzene. Borazole-poly(vinyl alcohol) (benzo[] Borazole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0023] In one embodiment, the shell comprises a shell additive. In one embodiment, the shell additive comprises an antistatic material, a moisture-proof material, or a combination thereof. In one embodiment, the shell additive is an antistatic material present at a concentration not exceeding 40% w / w of the shell. In one embodiment, the shell additive is a moisture-proof material present at a concentration not exceeding 90% w / w of the shell. In one embodiment, the shell additive is present at a concentration of at least 10% w / w of the shell. In one embodiment, the amount of the shell additive is between about 10% w / w and about 90% w / w of the shell. In one embodiment, the shell additive is a water-insoluble additive, a water-soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, copolymer, block copolymer, second polyvinyl alcohol (PVA), ammonium salt, conductivity promoter, stearate derivative, oleate derivative, laurate derivative, polyether compound, amino acid, tocopheryl acetate, piperidinium sebate, sodium salt, buffer, chelating agent, imidazolium salt, polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearate derivative or oleate derivative is selected from magnesium stearate, triglyceride monostearate, Span® 60, Tween® 60, trioleyl ester, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer solution is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, trialkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methylimidazolium salt or polytetramethylammonium salt or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or a combination thereof. In one embodiment, the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol-grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

[0024] In one embodiment, the shell surrounds a core containing one or more reagents selected from one or more enzymes, salts, surfactants, buffers, enzyme inhibitors, primers, nucleotides, organic osmolite, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof. In one embodiment, the reagent is a polymerase. In one embodiment, the volume of the reagent in the core is between about 0.1 µL and about 50 µL.

[0025] In one embodiment, the shell contains a reagent.

[0026] In one embodiment, the encapsulated microspheres have a diameter between approximately 100 µm and 1000 µm.

[0027] In one embodiment, the core further comprises one or more additional agents, wherein the additional agents comprise one or more sugars, one or more amino acids, one or more polymers, one or more mesoporous silicas, one or more quaternary amines, or any combination thereof. In one embodiment, when the additional agent comprises one or more sugars, the sugar is selected from trehalose, mannitol, cyclodextrin, polydextrose, sucrose, or any combination thereof. In another embodiment, the additional agent comprises one or more amino acids having hydrophobic side chains. In yet another embodiment, when the additional agent comprises a polymer, the polymer is selected from polyvinylpyrrolidone, polyvinyl alcohol, or combinations thereof.

[0028] In one embodiment, the core comprises a core additive. In one embodiment, the core additive comprises an antistatic material. In one embodiment, the core additive is an antistatic material present at a concentration not exceeding 25% w / w of the core. In one embodiment, the core additive is present at a concentration of at least 0.5% w / w of the shell. In one embodiment, the amount of the core additive is between about 2% w / w and about 10% w / w of the core. In one embodiment, the core additive is a water-insoluble additive, a water-soluble additive, an enteric additive, or any combination thereof. In one embodiment, the core additive comprises one or more of a polymer, copolymer, block copolymer, second polyvinyl alcohol (PVA), conductivity promoter, ammonium salt, imidazolium salt, polyether compound, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the buffer solution is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt or polytetramethylammonium salt or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or a combination thereof.

[0029] In one embodiment, the method further includes contacting the reagents with a sample to perform multiple co-assay reactions. In one embodiment, the shell comprises more than one lyophilized microsphere, and the reagents in the more than one lyophilized microsphere are different.

[0030] The third type relates to a system. This system includes one or more components as described herein and one or more freeze-dried cakes, wherein the one or more components and the one or more freeze-dried cakes are combined under conditions that effectively form a rehydration system.

[0031] In one embodiment, the system further includes one or more shells positioned between the one or more encapsulated microspheres and the one or more freeze-dried cakes. In another embodiment, the shells comprise a mixture selected from carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), fullalin, oxygen absorbers, alginate, chitosan, a film, and benzo[a]pyrene. Borazole-poly(vinyl alcohol) (benzo[] Materials containing boronazole-PVA, pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0032] In one embodiment, the shell comprises a shell additive. In one embodiment, the shell additive comprises an antistatic material, a moisture-proof material, or a combination thereof. In one embodiment, the shell additive is an antistatic material present at a concentration not exceeding 40% w / w of the shell. In one embodiment, the shell additive is a moisture-proof material present at a concentration not exceeding 90% w / w of the shell. In one embodiment, the shell additive is present at a concentration of at least 10% w / w of the shell. In one embodiment, the amount of the shell additive is between about 10% w / w and about 90% w / w of the shell. In one embodiment, the shell additive is a water-insoluble additive, a water-soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, copolymer, block copolymer, second polyvinyl alcohol (PVA), ammonium salt, conductivity promoter, stearate derivative, oleate derivative, laurate derivative, polyether compound, amino acid, tocopheryl acetate, piperidinium sebate, sodium salt, buffer, chelating agent, imidazolium salt, polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearate derivative or oleate derivative is selected from magnesium stearate, triglyceride monostearate, Span® 60, Tween® 60, trioleyl ester, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer solution is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, trialkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methylimidazolium salt or polytetramethylammonium salt or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or a combination thereof. In one embodiment, the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol-grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

[0033] The fourth aspect relates to a method for controlling the release of one or more encapsulated microspheres. The method includes providing a composition as described herein, and mixing the composition with a rehydration solution under a first condition that effectively controls the release of one or more lyophilized microspheres from the composition.

[0034] In one embodiment, the method further includes modifying the first condition to a second condition. In one embodiment, modifying the first condition includes modifying one or more of the following: temperature, exposure time, pH of the rehydration solution, or the position of the encapsulated microspheres in the rehydration solution. In another embodiment, the temperature in the first and / or second condition is between about 10°C and about 90°C. In yet another embodiment, the pH of the rehydration solution is between about 6.0 and about 10.0.

[0035] In one embodiment, the first condition effectively releases the first lyophilized microspheres. In another embodiment, the second condition effectively releases the second lyophilized microspheres, wherein the content of the second lyophilized microspheres differs from the content of the first lyophilized microspheres. In yet another embodiment, modifying the first condition allows for the sequential release of one or more lyophilized microspheres.

[0036] In one embodiment, the shell comprises carrageenan, shellac, trehalose, paraffin, gelatin, hydroxypropyl methylcellulose (HPMC), fullalin, oxygen absorber, alginate, chitosan, slurry, and benzo[a] Borazole-poly(vinyl alcohol) (benzo[] Borazole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0037] In one embodiment, the shell comprises a shell additive. In one embodiment, the shell additive comprises an antistatic material. In one embodiment, the shell additive is an antistatic material present in an amount not exceeding 40% w / w of the shell. In one embodiment, the shell additive is present in an amount of at least 10% w / w of the shell. In one embodiment, the amount of the shell additive is between about 10% w / w and about 90% w / w of the shell. In one embodiment, the shell additive is a water-insoluble additive, a water-soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, copolymer, block copolymer, second polyvinyl alcohol (PVA), ammonium salt, conductivity promoter, stearate derivative, oleate derivative, laurate derivative, polyether compound, amino acid, tocopheryl acetate, piperidinium sebate, sodium salt, buffer, chelating agent, imidazolium salt, polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearate derivative or oleate derivative is selected from magnesium stearate, triglyceride monostearate, Span® 60, Tween® 60, trioleyl ester, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer solution is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, trialkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt, polyquaternary ammonium salt, or Luviquat® (a copolymer of vinylpyrrolidone and quaternized vinylimidazolium), or combinations thereof. In one embodiment, the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol-grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

[0038] In one embodiment, the core comprises one or more reagents selected from one or more enzymes, salts, surfactants, buffers, enzyme inhibitors, primers, nucleotides, organic osmolite, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof. In one embodiment, the reagent is a polymerase.

[0039] In one embodiment, the first condition is modified to effectively release two or more lyophilized microspheres, wherein the two or more lyophilized microspheres contain different reagents. In one embodiment, the volume of the reagent in the core is between about 0.1 µL and about 50 µL.

[0040] In one embodiment, the shell further contains a reagent.

[0041] In one embodiment, the core and / or the rehydration solution further comprises one or more additional agents, wherein the additional agents are selected from one or more sugars, one or more amino acids, one or more polymers, one or more mesoporous silicas, one or more quaternary amines, or any combination thereof. In one embodiment, when the additional agent comprises a sugar, the sugar is selected from trehalose, mannitol, cyclodextrin, polydextrose, sucrose, or any combination thereof. In another embodiment, the additional agent comprises one or more amino acids having hydrophobic side chains. In yet another embodiment, when the additional agent comprises a polymer, the polymer is selected from polyvinylpyrrolidone, polyvinyl alcohol, or combinations thereof.

[0042] In one embodiment, the core comprises a core additive. In one embodiment, the core additive comprises an antistatic material. In one embodiment, the core additive is an antistatic material present at a concentration not exceeding 25% w / w of the core. In one embodiment, the core additive is present at a concentration of at least 0.5% w / w of the shell. In one embodiment, the amount of the core additive is between about 2% w / w and about 10% w / w of the core. In one embodiment, the core additive is a water-insoluble additive, a water-soluble additive, an enteric additive, or any combination thereof. In one embodiment, the core additive comprises one or more of a polymer, copolymer, block copolymer, second polyvinyl alcohol (PVA), conductivity promoter, ammonium salt, imidazolium salt, polyether compound, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the buffer solution is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt or polytetramethylammonium salt or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or a combination thereof.

[0043] In one embodiment, the method further includes providing an additional component as described herein, and mixing the additional component under a third condition of effectively controlling the release of one or more lyophilized microspheres from the additional component.

[0044] In one embodiment, the method further includes contacting the reagents with a sample to perform multiple co-assay reactions. In one embodiment, the shell comprises more than one lyophilized microsphere, wherein the reagents in the more than one lyophilized microsphere are different. In one embodiment, the method further includes providing one or more lyophilized cakes and rehydrating the one or more lyophilized cakes.

[0045] According to this disclosure, the components, systems, and methods described herein have numerous benefits, including, for example, increased stability of microspheres, macro-encapsulation for multiple operating tubes, and micro-encapsulation for simplified workflows and reduced reagent well numbers.

[0046] Encapsulation of lyophilized microspheres has become a significant and unmet need for increasing the stability of sequencing reagents and simplifying workflows. This disclosure describes components, systems, and methods related to encapsulated lyophilized reagents for sequential release. One method for achieving sequential release of lyophilized reagents is temperature-triggered release, for example, by immersing gelatin capsules filled with microspheres in paraffin wax. This method can release microspheres at different temperatures, for example, between about 30°C and about 50°C for natural gelatin capsules, and between about 50°C and about 90°C for coated capsules. Similarly, this method achieves time-triggered release by adding additives (e.g., amino acids) to the rehydration solution that can delay the rehydration rate of the lyophilized reagent.

[0047] The compositions, systems, and methods described herein offer numerous advantages. For example, encapsulated lyophilized microspheres provide antistatic protection by neutralizing charge and reducing triboelectric affinity, thereby reducing metering and manufacturing complexity (e.g., mesoporous silica, ionic liquids, quaternary amines). Electrostatic charge has been identified as the highest risk for microsphere formulation and dry mixing because it significantly impacts the metering and mixing of dry microsphere powders during manufacturing. The encapsulated microspheres described in the compositions, systems, and methods herein, by neutralizing the charge of the microspheres, for example, with particles coated with a neutral material having low triboelectric affinity, significantly improve sequencing stability.

[0048] Similarly, the compositions, systems, and methods described herein provide oxygen protection through a low oxygen permeability polymer coating (e.g., polyvinyl alcohol and / or an oxygen absorber in the coating). Likewise, the compositions, systems, and methods described herein provide moisture protection by applying an amphiphilic coating (e.g., amino acids and / or PVP copolymers). The compositions, systems, and methods described herein further provide protection against mechanical stress, for example, by preventing or reducing fragmentation during manufacturing (e.g., by providing a shell with a 40% solute content). This protective coating increases the mechanical robustness of the microspheres and their contents during manufacturing and shipping, and eliminates powder shedding from the microspheres, which could otherwise cause powder blockage of the membrane.

[0049] The compositions, systems, and methods described herein can further provide protection against light exposure, as protecting the reagents from light exposure reduces manufacturing light constraint. Encapsulation of lyophilized microspheres can improve sequencing quality, enable one-pot preparation, and simplify manufacturing. For example, microspheres with a coating or shell may contain dyes or other additives that are opaque or otherwise prevent or reduce the amount of light incident on the microsphere core.

[0050] It is known that the pH of SBS buffers varies during sequential operation. The compositions, systems, and methods described herein can be used to stabilize particles (e.g., buffer solutions) that would otherwise respond to pH changes, thereby increasing SBS performance. The compositions, systems, and methods described herein can further improve the control of the pH of solutions (e.g., incorporation mixtures (“ICMs”)) which may change over time when placed on the instrument. Various solutions can be used throughout the entire length of an SBS cycle that may take several hours, and therefore, reagents present in the solution are readily degraded upon environmental exposure. This is achieved by developing pH-sensitive microspheres that release ions and return the buffer to the desired pH when the buffer drops below a specified pH. Similarly, the compositions, systems, and methods described herein can improve the control of the external charge of microspheres to facilitate dispensing and prevent or reduce stratification in the mixture, and further allow the separation of reagent components from the SBS cleavage mixture to prevent or reduce and / or control unwanted interactions in a single pan or well. For example, lysis mixtures can benefit from reagent separation to reduce the thermosensitivity of the mixed reagents, as achieved in the compositions, systems, and methods described herein. Similarly, the compositions, systems, and methods described herein protect polymerases during polishing of fully functionalized nucleotides ("ffN") and protect photosensitive ffN from photodegradation, especially in cases where the environmental conditions involved in polishing degrade the enzyme.

[0051] The problem of correcting the deblocking of lyophilized ffN within a single well (integrated into the reagent well) when using two incompatible competing polymerases (polishing polymerase and sequencing polymerase) can be solved by spatially and temporally separating the polymerases using the compositions, systems, and methods described herein. Specifically, this problem can be solved by encapsulating one polymerase (the sequencing polymerase, since this polymerase is used after the polishing polymerase) in a water-soluble, slowly dissolving membrane (e.g., polyvinyl alcohol). By tuning the components and their relative amounts in the water-soluble membrane, the timing of the sequencing polymerase's release from its capsule can be synchronized with the completion time of the polishing process. Temperature or photoreactive additives can also be used to achieve a finer level of control.

[0052] Lyophilized ffN achieves increased stability compared to its liquid form, but generates increased 3'OH concentration, increases the pre-phase concentration, and leads to a decrease in operating quality. Laboratory use of the polishing workflow can be complex. The polishing mixture (ffN, polishing polymerase, polishing oligonucleotide, Mg) is prepared and combined separately, incubated at 50°C for up to one hour (to promote the polishing reaction), and then added to the remaining portion of the mixture to incorporate the sequencing polymerase. This level of complexity means that this workflow is undesirable in its current form for both the user and the sequencer, and this would be even more pronounced on a larger scale. As described in the compositions, systems, and methods disclosed herein, solutions with minimal and / or no user touchpoints, which are equally or less complex than current sequencer workflows, represent a feasible improvement to existing workflows.

[0053] In a single reagent well, a loosely packed polishing microsphere (which may include ffN, polishing polymerase, polishing oligonucleotides, and magnesium enzyme cofactor) is applied. Sequencing polymerase microspheres are also placed in this well; however, these are encapsulated in a water-soluble, time-dissolving membrane. This setup achieves several benefits, including, for example, a reduced number of wells. Following current methods of preparing polishing mixtures separately and then mixing them with large ICM mixtures, individual wells may be required for the polishing reagent. The encapsulated components, systems, and methods described herein facilitate multiple sequential reactions within a single well, thereby minimizing the number of wells. This also affects the tube's footprint, creating cascading effects on the environment, including, for example, plastic use and waste incineration. The components, systems, and methods described herein can be easily scaled up while also providing reduced jetting and valveing, thereby reducing sequencer complexity and associated costs. When a rehydration buffer (such as water) is applied to the well, the loosely packed polishing microspheres dissolve rapidly, and the polishing reaction begins to correct any unblocked ffN. This rehydration buffer also begins to dissolve the water-soluble membrane encapsulating the sequencing polymerase.

[0054] In addition to the above, the components, systems, and methods described herein offer several additional benefits. For example, the use of lyophilized materials and isolated lyophilized materials means that additional cofactors such as magnesium can be added to the microspheres themselves rather than having a separate additional rehydration buffer. This allows reagents that require or benefit from varying amounts of cofactors, salts, pH, and more, different concentrations and / or types of enzymes, to be rehydrated using only water or even atmospheric water capture. Furthermore, the reduced weight of concentrated and / or lyophilized reagents contributes to a cascading reduction in the amount of plastic used in sequencing processes and in terms of carbon footprint.

[0055] The encapsulation method described herein can be applied to achieve easily tuned reagent concentrations. For example, smaller capsules can contain less lyophilized reagent than larger capsules, and multiple capsules can be placed in wells as needed by the user. This improves user flexibility in throughput and avoids potential errors in dilution / concentration calculations. A cell-based method, where X capsules = Y runs, achieves this flexibility in a more controlled manner. This method also grants users flexibility in sequencing depth. Applications involving depth sequencing, such as cancer screening, can use many capsules, while surface screening, such as MRSA, can use fewer capsules.

[0056] The compositions, systems, and methods described herein achieve improved concentration control for reagent release (e.g., rehydration of a first reagent followed by delayed rehydration of one or more subsequent reagents after a certain time) as well as mechanical protection, buffer stability, charge control, combination of two or more different reagents in a single microsphere, single well, or single pot, and photoprotection. Specifically, a true one-pot method for library preparation is achieved through time-controlled release of reagents using the encapsulated lyophilized microspheres described herein. By encapsulating PCR reagents and releasing them at a predetermined time, the inhibition of labeling by PCR reagents is addressed.

[0057] To address the issues of static electricity and triboelectric charging, a method is proposed to directly incorporate slurry additives into microspheres (as a freeze-drying matrix) or as a coating for encapsulating microspheres. The basic principles of the additives are, firstly, to prevent or reduce charge accumulation (increasing conductivity), and secondly, to reduce surface charge through diffusion and / or dissipation.

[0058] Preventing or reducing charge buildup can be achieved through higher ion concentrations, crystallinity, or salinity. These ion concentrations, crystallinity, or salinity result in water molecules engaging in competitive shielding, thus reducing their tendency to accept charge or dissipate it more. Water shielding also reduces the internal friction angle and therefore decreases electrostatic charging.

[0059] Examples of such additives include sodium salts (i.e., sodium chloride, sodium bisulfite, sodium citrate), Trizma (Tris.HCl), MOPS, HEPES, ammonium salts (tetraalkylammonium chloride, Efka® IO 6783 or tri(hydroxyethyl)alkylammonium chloride), imidazolium salts (i.e., Efka® IO 6786 or 1-ethyl-3-methylimidazolium salt, polytetramethylammonium or copolymers of vinylpyrrolidone and vinylimidazolium, such as Luviquat® FC550, FC370), polyaniline, and amino acids (isoleucine, leucine, phenylalanine). Simultaneously, by reducing the contact angle with the container wall (i.e., stainless steel) or reducing interparticle friction, the surface charge can also be reduced through the lubrication effect on the microsphere surface.

[0060] Other examples of such additives include stearate derivatives (i.e., magnesium stearate, triglyceride monostearate, Span® 60, Tween® 60), oleate derivatives (i.e., trioleyl ester, Tween® 80), laurate derivatives (i.e., diethanolamine lauryl ether, Tween® 20, sodium lauryl ether sulfate), amino acids, tocopheryl acetate, piperidinyl sebacate, and Makon® 17R4 (polyethylene glycol / polypropylene glycol block copolymer). Simple Explanation of the Diagram

[0061] [Figure 1] illustrates the type of encapsulation.

[0062] Figures 2A through 2E illustrate an overview of the formulations of nuclear reagents used in embodiments of the components, systems, and methods described herein. Figure 2A shows an overview of sample extraction, Figure 2B shows an overview of library preparation, Figure 2C shows an overview of enrichment, Figure 2D shows an overview of clustering, and Figure 2E shows an overview of sequencing.

[0063] [Figure 3] illustrates the various stresses experienced by the microspheres during manufacturing, transportation, storage, and opening.

[0064] Figures 4A to 4B depict one embodiment of this disclosure, wherein the gelatin capsule is filled with microspheres and wherein the gelatin capsule (which is filled with microspheres) can be coated onto an external covering (e.g., paraffin wax). Figure 4A shows an OTS gelatin capsule (102) filled with microspheres and an OTS gelatin capsule (104) filled with microspheres and rapidly immersed in hot wax. Figure 4B shows an OTS gelatin capsule (106) filled with microspheres dissolved at 37°C and an OTS gelatin capsule (108) filled with microspheres dissolved at 58°C and rapidly immersed in hot wax.

[0065] [Figure 5] shows the results of the paraffin coating on the gelatin capsules, and actually the release of freeze-dried microspheres encapsulated at different temperatures.

[0066] [Figure 6] shows the compatibility test results of gelatin during the Nextera Flex labeling process.

[0067] [Figures 7A] and [Figures 7B] illustrate time-controlled release without transfer reaction. Figure 7A shows the percentage of excipients and their respective rehydration times. Figure 7B shows the rehydration of the example compositions described herein.

[0068] Figures 8A through 8C illustrate the one-pot bonding protocol achieved by the encapsulated lyophilized microspheres described in the compositions, systems, and methods of this disclosure. Figure 8A illustrates the steps of the one-pot bonding protocol. Figure 8B illustrates the time-dependent release of the microspheres. Figure 8C shows information on reagent A, reagent B, and additional reagent B'.

[0069] Figures 9A through 9D illustrate one embodiment of encapsulated lyophilized microspheres as described in the compositions, systems, and methods disclosed herein, wherein the integral polishing microspheres may contain ffN, polishing polymerase, and polishing oligonucleotides, while the sequencing polymerase microspheres may contain sequencing polymerase. Figure 9A shows an integral polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligonucleotides. Figure 9B shows a sequencing polymerase microsphere (404) containing sequencing polymerase. Figure 9C shows an encapsulated polymerase microsphere (406) and an integral polishing microsphere (402) in a single well (408). Figure 9D shows an encapsulated polymerase microsphere (406) and an integral polishing microsphere (402) in a single well (408) subjected to high temperature and rehydration with water.

[0070] [Figures 10A] through [Figures 10D] illustrate one embodiment of encapsulated lyophilized microspheres as described in the compositions, systems, and methods disclosed herein, wherein the integral polishing microspheres may contain ffN, polishing polymerase, and polishing oligonucleotides, while the sequencing polymerase microspheres may contain sequencing polymerase, wherein the same unit dose can be repeated across different reagents to achieve a larger dose. Figure 10A shows an integral polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligonucleotides. Figure 10B shows a sequencing polymerase microsphere (404) containing sequencing polymerase. Figure 10C shows encapsulated polymerase microspheres (406) (x3 units) and integral polishing microspheres (402) (x3 units) in a single well (408). Figure 10D shows the same unit dose repeated across different reagents to achieve an overall x3 (triple or triple) dose.

[0071] Figures 11A through 11F illustrate one embodiment of the encapsulated lyophilized microspheres described in the compositions, systems, and methods disclosed herein. Figure 11A shows an integral polishing microsphere (402) that may contain ffN, polishing polymerase, and polishing oligonucleotides. Figure 11B shows a sequencing polymerase microsphere (404) that may contain sequencing polymerase and is encapsulated (406) and located next to the integral polishing microsphere (402) within a single well. Figure 11C shows the encapsulated lyophilized microsphere (406) being rehydrated with water at 50°C. Figure 11D shows the integral microsphere (402) beginning to dissolve and polishing beginning after one hour. Figure 11E shows the integral microsphere (402) dissolving, polishing completed, and the encapsulated polymerase microsphere (406) dissolving after a delay. In Figure 11F, all microspheres are completely dissolved, and the ICM is then ready for use.

[0072] [Figure 12] Provides details of the manufacturing process for encapsulated freeze-dried microspheres as described in the compositions, systems and methods disclosed herein.

[0073] [Figure 13] illustrates the manufacturing and use of the encapsulated freeze-dried microspheres as described in the compositions, systems and methods of this disclosure.

[0074] Figures 14A and 14B depict the manufacturing and use points of the encapsulated freeze-dried microspheres and cakes as described in the compositions, systems, and methods of this disclosure. Figure 14A illustrates the manufacturing and use points of the encapsulated freeze-dried microspheres. Figure 14B shows an embodiment of the first cake, wax, and second cake in a tube.

[0075] Figures 15A through 15C illustrate the application of SBS to the compositions, systems, and methods disclosed herein. Figure 15A shows an AOM SBS pyrolysis mixture having Pd (550) in the core and THP (552) in the shell. Figure 15B shows ffN / Pol beads having Pol (554) in the core and ffN (556) in the shell. Figure 15C shows photoprotection of ffN having ffN (560) in the core and a photoblocking shell (558).

[0076] Figures 16A through 16I illustrate high-throughput sequencing screening of additives. Specific additives, such as Efka® IO 6783, were titrated to identify concentration limits. Figure 16A shows the phase fixation and pre-phase metric for various additives. Figure 16B shows the error rate and Q30 for various additives. Figure 16C shows the intensity of all lanes and channels for the additives in Figures 16A and 16B. Figure 16D shows the phase fixation and pre-phase metric for various additives. Figure 16E shows the error rate and Q30 for various additives. Figure 16F shows the intensity of all lanes and channels for the additives in Figures 16D and 16E. Figure 16G shows the titration of Efka® IO 6783 sequencing, particularly the phase fixation and pre-phase metric. Figure 16H shows the error rate and Q30 for Efka® IO 6783. Figure 16I shows the intensity of all lanes and channels for Figures 16G and 16H.

[0077] [Figures 17A] through [Figure 17F] show the stability of ffC with 1% additive after incubation at 60°C for 1 to 2 days, compared to the control group in liquid form. HPLC analysis results of ffC incubated with the additive described herein and heat-treated at 60°C for 1 and 2 days are shown. The decrease in ffC peak area and the increase in 3'OH, as well as DiP indicating ffN degradation, are shown. The effect of the additive is compared to the control group. Figure 17A shows the peak area of ​​ffN. Figure 17B shows the 3'OH of ffN. Figure 17C shows the diphosphate of ffN. Figure 17D shows the peak area of ​​the second ffN. Figure 17E shows the 3'OH of the second ffN. Figure 17F shows the diphosphate of the second ffN.

[0078] [Figure 18] shows the DNA recombinase activity of the Exclusion Amplification (ExAmp) solution incubated with the additives described herein.

[0079] [Figures 19A] to [Figures 19B] show the clustering performance of the ExAmp peak of the coated material obtained by cBOT first base detection, specifically the average intensity of ExAmp (Figure 19A) and clustering functionality (Figure 19B) of the ExAmp solution incubated with the additives described herein via cBOT first base incorporation kinetics.

[0080] [Figure 20] The charge potential (via Keyence) of powdered freeze-dried cakes containing different concentrations of additives of ExAmp (matrix) is depicted (at 3% RH).

[0081] [Figure 21] shows the charge potential (via Keyence) measurement of powdered freeze-dried cakes containing different concentrations of additives of ExAmp (matrix) at 40% RH.

[0082] [Figure 22] The charge potential (via Keyence) of powdered freeze-dried cakes containing different concentrations of additives of ExAmp (matrix) is depicted (at 3% RH).

[0083] [Figure 23] The charge potential of the powdered freeze-dried cake containing different concentrations of additives (measured via Keyence) at 40% RH.

[0084] [Figure 24] shows the charge potential (via Keyence) measurements of powdered lyophilized cakes of ExAmp (matrix) containing different concentrations of salt / buffer solution (at 3% and 40% RH).

[0085] [Figures 25A] through [Figure 25C] show Atto and FSCN (fluorescein) microspheres containing additives in matrix form. The antistatic properties of the additives were assessed by adhering the microspheres to the container and measuring their charge density using GranuCharge. Low Δq values ​​indicate low triboelectricity. The matrix form of 1% Efka® IO 6783 minimizes triboelectricity. Figure 25A shows the results of the first group of additives tested in terms of both visual results (top) and percentage loss (bottom) (Atto 20%, +1% Efka® IO 6783, +1.5% Tris.HCl, and +1% Tween 20). Figure 25B shows the results of the second group of additives tested in terms of both visual results (top) and percentage loss (bottom) (FSCN 20% control group, +1% Efka® IO 6783, +2% Efka® IO 6783, and +1% Efka® IO 6786). Figure 25C shows that Atto and FSCN (both 20% trehalose) were dry-mixed using the antistatic agent Efka® IO 6783 in matrix form.

[0086] Figures 26A through 26C show ffN microspheres containing additives in matrix form. The antistatic properties of the additives were assessed by adhesion to the container and measured using a GranuCharge. Figure 26A shows the visual results for the first group of additives (ffN + 25%T control group, +1% Efka® IO 6783, +1% Efka® IO 6786, +1.5% Tris.HCl, +2% isoleucine). Figure 26B shows the visual results for the second group of additives (second ffN + 20%T control group, +0.5% LDA, +1% Makon® 17R4, +1.5% Kollidon® VA64, +2% Kollicoat® Protect). Figure 26C shows the charge density of the various additives tested in Figures 26A and 26B.

[0087] [Figures 27A] through [Figures 27E] show FSCN (fluorescein) and MB (methylene blue) microspheres coated with Eudragit® L100 and magnesium stearate. The presence of the coating mitigates the electrostatic and triboelectric behavior of the microspheres, as shown by GranuCharge measurements and dry incorporation experiments. Figure 27A shows an example shell and core according to this disclosure. Figure 27B shows a weight gain of 5% to 7% from the coating. Figure 27C shows the reduction in electrostatics in the coated composition compared to the uncoated composition. Figure 27D shows the increased moisture resistance in the coated composition compared to the uncoated composition. Figure 27E shows the triggered release in the coated composition.

[0088] Figures 28A to 28F show SEM images of fluorescein (FSCN), DNA recombinase / BSA, and ffN microspheres coated with different concentrations of Kollidon® VA64, Efka® IO 6783, and PEG. Figures 28A and 28B show images of multiple (Figure 28A) and a single (Figure 28B) microspheres coated with 20% Wurster-Spray of 800 µm FSCN coated with Kollidon VA64, Efka® IO 6783, and PEG (#6). Figures 28C and 28D show images of multiple (Figure 28C) and a single (Figure 28D) microspheres coated with 15% Rec / BSA coated with Kollidon VA64, Efka® IO 6783, and PEG (#8) via cryo-ion milling SEM. Figures 28E and 28F show images of multiple (Figure 28E) and a single (Figure 28F) microspheres coated with Kollidon VA64, Efka® IO 6783, and PEG (#11) in 10% ffN by cryo-ion milling SEM.

[0089] [Figures 29A] to [Figure 29B] depict ffN microspheres containing additives in coated form. The antistatic properties of the additives were evaluated by adhesion to a container and measured using a GranuCharge. Figure 29A shows a visual representation of various ffN microspheres containing additives in coated form. Figure 29B shows the charge density of the various ffN microspheres in Figure 29A.

[0090] [Figures 30A] and [Figures 30B] show coated DNA recombinase / BSA microspheres containing additives. The antistatic properties of the additives were assessed by adhesion to the container and measured using a GranuCharge. Figure 30A shows a visual representation of various coated DNA recombinase / BSA microspheres containing additives. Figure 30B shows the charge density of the various DNA recombinase / BSA microspheres in Figure 30A.

[0091] [Figures 31A] through [Figure 31F] depict the stability of the compositions described herein. Figure 31A shows ffN microspheres. Figure 31B shows 5% Kollidon® VA64 in the dried matrix. Figure 31C shows 5% Makon® 17R4 in the dried matrix. Figure 31D shows 5% Efka® 6783 in the dried matrix. Figure 31E shows 10% Kollicoat® Protect in the dried matrix. Figure 31F shows 7.5% isoleucine in the dried matrix (bottom right) under different humidity and time conditions.

[0092] Figures 32A to 32F show the measurement results of the relative humidity tolerance of microspheres obtained by dynamic vapor adsorption. Isoleucine in the matrix increases the humidity tolerance of ffN microspheres. Figure 32A shows the results for the ffN control group (18% T, 2% HCD). Figure 32B shows the results for the 2% Kollicoat® Protect matrix (10% dry). Figure 32C shows the results for the +1% Efka® IO 6783 matrix (5% dry). Figure 32D shows the results for the 1% Kollidon® VA64 matrix (5% dry). Figure 32E shows the results for the +1.5% Trizma matrix (7.5% dry). Figure 32F shows the results for the +1.5% isoleucine matrix (7.5% dry).

[0093] Figures 33A through 33D depict the results of the Kollicoat® Protect and VA64 coatings, which provide improved moisture protection for Rec / BSA MS. Figure 33A shows the results of the uncoated Rec / BSA (AP1) control group at the 20% RH limit. Figure 33B shows Rec / BSA coated with 15% protect and Efka® IO 6783 at the 30% RH limit. Figure 33C shows Rec / BSA coated with 15% Kollidon® VA64 and Efka® IO 6783 at the 20% to 30% RH limit. Figure 33D shows the images of the coated compositions in Figures 33A through 33C after exposure to humidity.

[0094] [Figures 34A] and [34B] illustrate how the coating minimizes moisture absorption by the Rec / BSA microspheres. Figure 34A shows images of various microspheres under different humidity conditions. Figure 34B shows the effect of %RH on uncoated and coated Rec-BSA microspheres after 24 hours at 35°C with 30 mg of sample placed on an aluminum disc at 30%, 55%, and 80% RH. Specifically, the Kollicoat® Protect coating performs better than Kollidon® VA64 in terms of moisture resistance.

[0095] [Figure 35] illustrates the concept used for solubility screening.

[0096] [Figure 36] shows the results of Kollidon® VA64, Efka®, and Eudragit® being soluble in spray solution (15% water / solvent) and buffer solution.

[0097] Figures 37A to 37C show the phosphodiester concentration of a specific ffC in the presence of a polymer (Figure 37A), the 3'OH and phosphodiester concentration of a specific ffC in the presence of a polymer (Figure 37B), and the phosphodiester concentration of a specific ffC in the presence of a polymer (Figure 37C).

[0098] [Figure 38] shows the fluorescence intensity of a specific ffC in the presence of a polymer.

[0099] [Figure 39] shows the fluorescence intensity of a specific ffC in the presence of a polymer.

[0100] Figures 40A to 40C show the results of the coating material screening based on ordered compatibility. Figure 40A shows the phase sizing and predetermined phase metric of the various coating materials tested. Figure 40B shows the error rate and Q30 of the various coatings tested. Figure 40C shows the strength of all lanes and all channels of the coating materials described in Figures 40A and 40B.

[0101] Figures 41A and 41B show the results of polymer screening for DNA recombinase activity and clustering efficiency (cBOT), as well as the DNA recombinase activity of ExAmp, in the presence of the polymers described herein. Figure 41A shows the DNA recombinase activity of the polymer-coated ExAmp (TCS1 v1.0) solution. Figure 41B shows the DNA recombinase activity after incubation at 40°C for 24 hours.

[0102] Figures 42A and 42B show the results of ExAmp DNA-binding protein activity after phasing in the presence of the polymer described herein, and the results of ExAmp DNA-binding protein activity after phasing in the presence of the polymer described herein. Figure 42A shows the DNA-binding protein 3 activity of the polymer-coated ExAmp (TCX1 v1.0) solution. Figure 42B shows the DNA-binding protein activity after incubation at 40°C for 24 hours.

[0103] Figures 43A through 43D illustrate the improved stability of SBS reagents through microencapsulation. Figure 43A shows the improvement in solubility. Figure 43B shows the improvement in enzyme compatibility. Figure 44C shows the improvement in ffN compatibility. Figure 34D shows the improvement in sequencing.

[0104] [Figure 44] Depicts the physical characteristics of the encapsulated microspheres.

[0105] Figures 45A to 45B show that the quality of the tested ffN was unaffected by the spraying process: the peak area (Figure 45A) and 3'OH concentration (Figure 45B) were comparable to the control group, and no diphosphate was detected.

[0106] [Figures 46A] through [Figures 46C] illustrate how shell encapsulation improves moisture resistance and reduces static electricity. Figure 46A shows natural microspheres after exposure to humid conditions. Figure 46B shows matrix microspheres after exposure to humid conditions. Figure 46C shows core-shell microspheres coated with the components described herein after exposure to humid conditions.

[0107] [Figure 47] illustrates the method as described herein.

[0108] [Figure 48] illustrates the method as described herein.

[0109] Figures 49A to 49C show triple-coated microspheres with sequentially pH-triggered release agents. Figure 49A shows the results of runs 1, 2, and 3 of the composition with coated microspheres having three shells: shell 1: Eudragit S100 (fluorescent), shell 2: Eudragit L100 (pale blue), and shell 3: Eudragit L100-55 (rose red). Figure 49B shows the transfer of the coated microspheres of Figure 49A from pH 5 to pH 6, to pH 7, and to pH 8. Figure 49C shows the release of various coated compositions under different pH conditions.

[0110] It should be understood that the following detailed discussion of all combinations of the foregoing concepts and additional concepts (assuming that such concepts are not contradictory) is intended to be part of the subject matter of the invention disclosed herein and to achieve the benefits and advantages disclosed herein. Implementation

[0111] The first state refers to a composition comprising a shell surrounding a core, wherein the core comprises one or more freeze-dried microspheres.

[0112] It should be understood that certain forms, patterns, embodiments, variations, and features of this disclosure are described below at different levels of detail to provide a substantial understanding of the technical aspects of the invention. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. The use of the terms "comprise(s)" and "comprising" is not restrictive. The use of the terms "having" and other forms is not restrictive. As used in this disclosure, whether in transient phrases or in themselves within the claims, the terms "comprise(s)" and "comprising" are interpreted in an open-ended sense. That is, these terms are interpreted synonymously with the phrases "at least having" or "at least including".

[0113] The terms “substantially,” “approximately,” “about,” “relatively,” or other similar terms that may be used throughout this disclosure (including the claims) are used to describe and take into account small fluctuations in the self-reference or parameters due to processing variations. Such small fluctuations also include zero fluctuations in the self-reference or parameters. For example, fluctuations may refer to less than or equal to ±10%, such as less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0114] To further understand, for clarity, some features described herein in the context of individual embodiments may also be provided in combination with a single embodiment. Conversely, the various features described in the context of a single embodiment may also be provided separately or in any suitable sub-combination for simplicity.

[0115] The terms “connection,” “contact,” and / or “coupled” encompass a variety of configurations and assemblies. Such configurations and techniques include, but are not limited to: (1) directly engaging one component with another component without any intervening component (i.e., the components are in direct physical contact); and (2) engaging one component with another component, where one or more components are present, provided that one component “connected to,” “contacted,” or “coupled to” the other component is in some way (e.g., electrically, fluidly, physically, optically, etc.) operationally connected to the other component (optionally, one or more additional components may be present among them). Components in direct physical contact with each other may or may not be in electrical and / or fluid contact with each other. Furthermore, two components connected, electrically coupled, optically connected, optically coupled, fluidly connected, or fluidly coupled may or may not be in direct physical contact, and one or more other components may be positioned between the two connected components.

[0116] As described herein, the term "array" can include a group of conductive channels or molecules that can be attached to one or more solid substrates such that the conductive channels or molecules can be distinguished from each other based on their location. An array as described herein can include different molecules each located at different identifiable locations on a solid substrate (e.g., at different conductive channels). Alternatively, an array can include individual solid substrates each carrying different molecules, wherein the different probe molecules can be identified based on the location of the solid substrate on the surface to which it is attached or based on the location of the solid substrate in a liquid (such as a fluid flow). Examples of arrays where individual substrates are located on a surface include pores with beads, as described in U.S. Patent No. 6,355,431, U.S. Patent Publication No. 2002 / 0102578, and WO 00 / 63437, all of which are incorporated herein by reference in their entirety. The molecules in an array can be nucleic acid primers, nucleic acid probes, nucleic acid templates, or nucleases, such as polymerases and exonucleases.

[0117] As described herein, the term "attachment" can refer to the joining, fastening, adhesion, connection, or bonding of two objects together. For example, the reaction components of a polymerase can be attached to a solid-phase component, such as a conductive channel, by covalent or non-covalent bonds. As described herein, the phrases "covalent attachment" or "covalent bond" refer to the formation of one or more chemical bonds characterized by the sharing of electron pairs between atoms. Non-covalent bonds do not involve the sharing of electron pairs and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0118] As described herein, the terms "polynucleotide" or "nucleic acid" refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or DNA or RNA analogs made from nucleotide analogs. The terminology used herein also includes cDNA, which is a complementary or duplicate DNA derived from an RNA template, for example, by the action of reverse transcriptase. In one embodiment, the nucleic acid to be analyzed (e.g., by sequencing using the described system) is immobilized on a substrate (e.g., a substrate within a flow-through or one or more beads on a substrate such as a flow-through). Unless otherwise explicitly stated or indicated by context, the term immobilization as used herein is intended to cover direct or indirect, covalent or non-covalent attachment. Under conditions where a support is intended (e.g., in nucleic acid sequencing applications), the analyte (e.g., nucleic acid) may remain immobilized or attached to a support. In one embodiment, the template polynucleotide is one of a plurality of template polynucleotides attached to the substrate. In one embodiment, the plurality of template polynucleotides attached to the substrate includes a duplicate cluster of library polynucleotides as described herein.

[0119] Nucleic acids include naturally occurring nucleic acids or their functional analogs. Particularly useful functional analogs can hybridize with nucleic acids in a sequence-specific manner or can be used as templates for replicating specific nucleotide sequences. Naturally occurring nucleic acids typically have a backbone containing phosphodiester bonds. Analog structures may have alternative backbone bonds, including various others known in this art, such as either peptide nucleic acids (PNAs) or locked nucleic acids (LNAs). Naturally occurring nucleic acids typically contain deoxyribose (e.g., found in deoxyribonucleic acid (DNA)) or ribose (e.g., found in ribonucleic acid (RNA)).

[0120] In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, meaning it lacks the hydroxyl group present in ribose. Nitrogenous heterocyclic bases can be purine or pyrimidine bases. Purine bases include adenine (A) and guanine (G), and their modified derivatives or analogs. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and their modified derivatives or analogs. The C-1 atom of deoxyribose can be bonded to the N-1 of a pyrimidine or the N-9 of a purine.

[0121] Nucleic acids may contain any of the various analogs of such sugar moieties known in this art. Nucleic acids may include natural or non-natural bases. Natural deoxyribonucleic acid may have one or more bases selected from the group consisting of adenine, thymine, cytosine, or guanine, and ribonucleic acid may have one or more bases selected from the group consisting of uracil, adenine, cytosine, or guanine. Non-natural bases that may be included in nucleic acids are known in this art.

[0122] As used herein, nucleotides may include natural nucleotides, their analogues, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, and other molecules known as nucleotides. As described herein, nucleotides may include a nitrogenous heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides may be monomeric units of nucleic acid sequences, such as subunits that recognize DNA or RNA strands. Nucleotides may also include molecules that are not necessarily present in polymers, such as molecules that can be incorporated into polynucleotides in a template-dependent manner by polymerases. Nucleotides may include nucleoside units having, for example, 0, 1, 2, 3, or more phosphates at the 5' carbon. Tetraphosphate nucleotides, pentaphosphate nucleotides, and hexaphosphate nucleotides may be useful, as are nucleotides having more than 6 phosphates at the 5' carbon, such as 7, 8, 9, 10, or more phosphates. Examples of naturally occurring nucleotides include, but are not limited to, ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP.

[0123] Non-natural nucleotides include nucleotide analogs, such as those not present in natural biological systems or substantially not incorporated into polynucleotides by polymerase in their natural environment (e.g., in non-recombinant cells exhibiting polymerase). Non-natural nucleotides include nucleotides incorporated into polynucleotide strands by polymerase at a rate substantially faster or slower than the rate at which another nucleotide (such as a natural nucleotide with the same Warsenberg-Klicke complementary base) is incorporated into that strand by polymerase. For example, the incorporation rate of non-natural nucleotides can be at least 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, or more, compared to the incorporation rate of natural nucleotides. Non-natural nucleotides may be further extended after incorporation into the polynucleotide. Examples include nucleotide analogs having a 3' hydroxyl group or a nucleotide analog having a reversible terminator motif at the 3' position, which can be removed to allow further extension of the polynucleotide into which the nucleotide analog has been incorporated. Examples of reversible terminator motifs are described, for example, in U.S. Patents 7,427,673, 7,414,116, and 7,057,026, and WO 91 / 06678 and WO 07 / 123744, each of which is incorporated herein by reference in its entirety. It should be understood that in some instances, nucleotide analogs having a 3' terminator motif or lacking a 3' hydroxyl group (such as dideoxynucleotide analogs) may be used where the polynucleotide incorporating the nucleotide analog has not been further extended. In some instances, the nucleotide(s) may not include a reversible terminator motif, or the nucleotide(s) may not include an irreversible terminator motif, or the nucleotide(s) may not include a terminator motif at all.

[0124] This disclosure covers nucleotides including fluorescently labeled (or any other detection tags) that can be used on their own in any of the methods disclosed herein or incorporated into or associated with larger molecular structures or conjugates.

[0125] Fluorescent labels may include compounds selected from any known fluorescent substances, such as rose red or cyanin. As disclosed herein, fluorescent labels may be attached to any position on a nucleotide base and may optionally include a linker. The function of the linker is generally to assist the chemical attachment of the fluorescent label to the nucleotide. In certain embodiments, Warsen-Klicki base pairing may still be performed for the resulting analogues. The linker group can be used to covalently attach the dye to the nucleoside or nucleotide. The linker portion may be of sufficient length to link the nucleotide to the compound such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by a nucleic acid replicase. Therefore, the linker may also include a spacer unit. The spacer separates, for example, the nucleotide base from the cleavage site or label.

[0126] As used herein, a "nucleoside" is structurally similar to a nucleotide but lacks the phosphate ester moiety. Examples of nucleoside analogs are those in which the label is linked to a base and lacks a phosphate ester group attached to a sugar molecule. The term "nucleoside" is used herein in its common sense as understood by one of ordinary skill in the art. Examples include, but are not limited to, ribonucleotides including a ribose moiety and deoxyribonucleotides including a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced by a carbon atom and / or a carbon atom has been replaced by a sulfur or oxygen atom. A "nucleoside" is a monomer that may have substituted bases and / or sugar moieties.

[0127] The term "purine base" is used herein in its ordinary sense as understood by one of ordinary skill in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by one of ordinary skill in the art, and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydroxyuracil, and 5-alkylcytosine (e.g., 5-methylcytosine).

[0128] As described herein, the term substrate (or solid support) may include any inert substrate or matrix to which nucleic acids can be attached, such as, for example, glass surfaces, plastic surfaces, latex, polydextrose, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, and silicon wafers. For example, the substrate may be a glass surface (e.g., a flat surface of a flow channel). In one embodiment, the substrate may include an inert substrate or matrix that has been "functionalized," such as by applying a layer or coating of an intermediate material including reactive groups that allow covalent attachment to molecules (such as polynucleotides). The support may include a polyacrylamide hydrogel supported on an inert substrate (such as glass). Molecules (e.g., polynucleotides) may be directly covalently attached to the intermediate material (e.g., the hydrogel). The support may include a plurality of particles or beads, each with a different attached analyte.

[0129] As used herein, “derivative” or “analyte” means a synthetic nucleotide or nucleoside derivative having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed, for example, in Bücher, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlmann et al., “Antisense Oligonucleotides: A New Therapeutic Principle,” Chemical Reviews 90:543-584 (1990), both of which are incorporated herein by reference in their entirety. Nucleotide analogs may also include modified phosphodiester bonds, including thiophosphates, dithiophosphates, alkyl-phosphonates, aniline phosphates, and aminophosphate bonds. As used herein, “derivative,” “analyte,” and “modified” are used interchangeably and are encompassed by the terms “nucleotide” and “nucleoside” as used herein.

[0130] The compositions, systems, and methods described herein include a shell surrounding a core, and the core may include one or more lyophilized microspheres (i.e., the compositions may include encapsulated lyophilized microspheres).

[0131] As described herein, "encapsulate," "encapsulated," and "encapsulation" include encapsulating one or more microspheres as described herein. Microencapsulation as described herein refers to embedding at least one component, such as an active agent, into at least one other material, such as a shell material. Encapsulation according to this disclosure includes, but is not limited to, bulk encapsulation, mega-encapsulation, microencapsulation, nano-encapsulation, single-molecule encapsulation, and ionic encapsulation. According to this disclosure, the compositions, systems, and methods described herein offer numerous benefits, including, for example, increased microsphere stability, the use of mega-encapsulation to achieve multiple operating tubes, and the use of microencapsulation to achieve simplified workflows and reduced reagent well numbers. The compositions, systems, and methods described herein use encapsulation of particles that would otherwise respond to pH changes to stabilize these buffer solutions and increase SBS performance. The compositions, systems, and methods described herein also use encapsulation to reduce the risk of electrostatic charges from microspheres that would otherwise be difficult to dispense and dry mix during manufacturing.

[0132] As used herein, "microsphere" refers to spherical particles comprising a shell and a core, and having a diameter from 0.1 µm to 1,000 µm. For example, microspheres may have diameters of about 0.1 µm, 0.5 µm, 1 µm, 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 60 µm, 70 µm, 80 µm, 90 µm, 100 µm, 150 µm, 200 µm, 300 µm, 400 µm, 500 µm, 600 µm, 700 µm, 800 µm, 900 µm, or 1000 µm, or any diameter between about 0.1 µm and about 1,000 µm. In one embodiment, the encapsulated microsphere has a diameter between about 100 µm and 1,000 µm.

[0133] Microspheres typically comprise a polymer shell, such as a biodegradable polymer. Microspheres according to this disclosure include those prepared using techniques known to those skilled in the art. For example, microspheres can be prepared by freezing a liquid into cryospheres, followed by placing the cryospheres in a dryer, such as a rotary dryer.

[0134] As described herein, a "giant sphere" may include a plurality of microspheres. Giant spheres typically have a diameter larger than that of microspheres, for example, between 0.1 mm and 1,000 mm. The giant spheres described herein may, for example, have diameters of about 0.1 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 50 mm, 100 mm, 200 mm, 300 mm, 400 mm, 500 mm, 600 mm, 700 mm, 800 mm, 900 mm, 1,000 mm, or any diameter between about 0.1 mm and about 1,000 mm. In one embodiment, the giant sphere (e.g., a giant capsule) will have dimensions ranging from about 5 mm x 5 mm x 9 mm to about 7 cm x 7 cm x 2 cm.

[0135] The giant spheres disclosed herein include those prepared using techniques known to those skilled in the art. The compositions, systems, and methods described herein may include a single lyophilized microsphere or a plurality of lyophilized microspheres, thereby forming giant spheres. For example, the compositions described herein may include any number of lyophilized microspheres between 1 and more than 1,000,000. In one embodiment, the composition includes 1 lyophilized microsphere, or less than 100 lyophilized microspheres, or less than 500 lyophilized microspheres, or any number of microspheres between about 1 and about 1,000,000. In one embodiment, when the shell surrounds more than one lyophilized microsphere, the reagents in the core of the lyophilized microsphere are different.

[0136] As described herein, a "shell" includes components surrounding the core. In one embodiment, the shell comprises an outer layer of microspheres and, alternatively, an outer layer of macrospheres. In one embodiment, the shell comprises, for example, a shell material selected from the group consisting of: carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), fullalin, oxygen absorbers, alginate, chitosan, slurry, benzo[a]pyrene. Borazole-poly(vinyl alcohol) (benzo[] The shell material may contain boronazole-PVA, pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof. In one example, the shell may include, but is not limited to, starch, cellulose, hydrocolloid, alginate, collagen, or any combination thereof. The amount of shell material includes, for example, any amount suitable for producing the desired shell result. In one embodiment, the shell material is present in an amount between about 1 wt% and about 100 wt% of the shell. For example, the shell material may be present in an amount of about 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, 100 wt% of the shell, or any amount between these amounts. In one embodiment, the shell material is present in an amount between about 10 wt% and about 90 wt%, or between about 10 wt% and about 80 wt%, or between about 10 wt% and about 70 wt%, or between about 10 wt% and about 60 wt%, or between about 10 wt% and about 50 wt%.

[0137] As described herein, a shell may comprise one layer or multiple layers of different compositions. For example, a shell may comprise one, two, three, four, five, six, seven, eight, nine, ten, or more than ten layers. Each layer may comprise the same or different material as the other layers present in the shell.

[0138] In one embodiment, the shell as described herein may include a shell additive. The shell additive may be present in an amount between about 0.01% w / w and about 99% w / w of the shell. In one embodiment, the shell additive is present in an amount between about 10% w / w and about 90% w / w of the shell. In one embodiment, the shell additive is present in an amount between about 10% w / w and about 40% w / w. In one embodiment, the shell additive is an antistatic material present in an amount not exceeding 40% w / w of the shell. In one embodiment, the shell additive is a moisture-proof material present in an amount not exceeding 90% w / w of the shell. In one embodiment, the shell additive is present in an amount at least 10% w / w of the shell. For example, in one embodiment, the shell additive may be present in an amount between 0.1% w / w and about 15.0% w / w of the shell. For example, the shell additive may be about 0.01% w / w, 0.05% w / w, 0.1% w / w, 0.5% w / w, 1.0% w / w, 1.5% w / w, 2.0% w / w, 2.5% w / w, 3.0% w / w, 3.5% w / w, 4.0% w / w, 4.5% w / w, 5.0% w / w, 5.5% w / w, 6.0% w / w, 6.5% w / w, 7.0% w / w, 7.5% w / w, 8.0% w / w, 8.5% w / w, 9.0% w / w, 9.5% w / w, 10.0% w / w, 10.5% w / w, 11.0% w / w, 11.5% w / w, 12.0% w / w, 12.5% ​​w / w, 13.0% The shell additive is present in amounts of w / w, 13.5% w / w, 14.0% w / w, 14.5% w / w, 15% w / w, or any amounts between thereof. The amount of shell additive can be any suitable amount to reduce triboelectricity of the composition described herein and / or provide suitable moisture resistance. The amount of shell additive can be adjusted to suit specific reagents or reagent combinations, or to suit specific microsphere compositions.

[0139] In one embodiment, the shell additive comprises an antistatic material, a moisture-proof material, or a combination thereof. In one embodiment, the shell additive is an antistatic material present in an amount not exceeding 40% w / w of the shell. In one embodiment, the shell additive is a moisture-proof material present in an amount not exceeding 90% w / w of the shell. In one embodiment, the shell additive is present in an amount at least 10% w / w of the shell. In one embodiment, the amount of the shell additive is between about 10% w / w and about 90% w / w of the shell. In one embodiment, the shell additive is a water-insoluble additive, a water-soluble additive, an enteric additive, or any combination thereof. In one embodiment, the shell additive comprises one or more of a polymer, copolymer, block copolymer, polyvinyl alcohol (PVA), ammonium salt, conductivity promoter, stearate derivative, oleate derivative, laurate derivative, polyether compound, amino acid, tocopheryl acetate, piperidinium sebate, sodium salt, buffer, chelating agent, imidazolium salt, polyaniline, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the stearate derivative or oleate derivative is selected from magnesium stearate, triglyceride monostearate, Span® 60, Tween® 60, trioleyl ester, Tween® 80, or any combination thereof. In one embodiment, the amino acid is selected from leucine, isoleucine, phenylalanine, or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the sodium salt is selected from sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In one embodiment, the buffer solution is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt, polytetraammonium chloride, or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or combinations thereof. In one embodiment, the shell additive comprises an ammonium salt, a copolymer, a polyvinyl alcohol-grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof. The shell may contain a polymer, and the maximum concentration of the polymer in the shell may be about 90% (dry form). One or more antistatic additives may be added to the polymer coating, and the shell additive in the range of about 10% to about 40% may be present in dry form.

[0140] In various embodiments, the shell additive may include a beneficial combination of compounds for improving and preventing unintended compatibility with SBS reagents. For example, the shell additive may include polyether compounds and polymers and / or copolymers, or alternatively, polyether compounds, PVA and / or polymers and / or copolymers. In one embodiment, the shell additive includes polyethylene glycol, Kollidon® VA64, and Efka® IO 6783 or their chemical equivalents. In another embodiment, the shell additive includes polyethylene glycol and Kollidon® VA64 or their chemical equivalents. In yet another embodiment, the shell additive includes polyethylene glycol, Kollicoat® Protect, and Efka® IO 6783® or their chemical equivalents. In still another embodiment, the shell additive includes polyethylene glycol and Kollicoat® Protect or their chemical equivalents. In one embodiment, an ammonium salt acts as a conductivity promoter. In one embodiment, an imidazolium salt acts as a conductivity promoter.

[0141] As described herein, the "core" or "core region" includes any material surrounding the shell. The core according to this disclosure comprises one or more lyophilized microspheres.

[0142] As used herein, the term "compatible" means that they can coexist or occur together without conflict (i.e., without substantially reducing the effectiveness or activity of one or more substances present or occurring together). Similarly, as used herein, the term "incompatible" means that they cannot coexist or occur together without conflict (i.e., without substantially reducing the effectiveness or activity of one or more substances present or occurring together).

[0143] The lyophilization according to this disclosure includes methods known to those skilled in the art. Lyophilization is also referred to herein as freeze-drying. In this disclosure, for example with respect to the compositions, systems, or methods described herein, the terms "lyophilize" or "lyophilizate" will be used as equivalent terms to "lyophilised," "lyophilisate," or "freeze-dried."

[0144] Lyophilizable formulations can be reconstituted into solutions, suspensions, emulsions, or any other form suitable for administration or use. Lyophilizable formulations are generally first prepared as liquids, followed by freezing and lyophilization. The total liquid volume before lyophilization may be less than, equal to, or greater than the final reconstituted volume of the lyophilized formulation. Preferably, the final reconstituted volume of the lyophilized formulation is less than the total liquid volume before lyophilization. The lyophilization process is known to those skilled in the art and generally involves sublimating water from the lyophilized formulation under controlled conditions.

[0145] The freeze-dried formulation can be stored over a wide range of temperatures. It can be stored below 25°C, for example, refrigerated at 2°C to 8°C, or at room temperature (e.g., about 25°C). Preferably, the freeze-dried formulation is stored below about 25°C, more preferably between about 4°C and 20°C; below about 4°C; below about -20°C; about -40°C; about -70°C; or about -80°C. The stability of the freeze-dried formulation can be determined in various ways known in the art, such as by the visual appearance of the microspheres and / or cakes and / or by moisture content. The components disclosed herein can also withstand temperature fluctuations that may occur during transport, such as up to 70°C, for short periods.

[0146] Lyophilized formulations are generally used by rehydration (interchangeably referred to as "resolution") by adding an aqueous solution to dissolve the lyophilized formulation. A wide variety of aqueous solutions can be used to resolution lyophilized formulations, including water, saline, or another electrolyte or non-electrolyte diluent. Preferably, the lyophilized microspheres described herein are resolutioned using water. Lyophilized formulations can be rehydrated using solutions containing water (e.g., USP WFI or water for injection) or antibacterial water (e.g., USP WFI with 0.9% benzyl alcohol). However, solutions containing additives, buffers, excipients, and / or carriers can also be used and described herein.

[0147] Freeze-dried or lyophilized formulations are generally prepared from liquids derived from solutions, suspensions, emulsions, and the like. Therefore, the liquid undergoing freeze-drying or lyophilization preferably contains all the desired components in the final solution formulation. Thus, upon rehydration or reconstitution, the freeze-dried or lyophilized formulation will present the desired liquid formulation. Core and / or shell additives, if present, can be incorporated into the reagent during rehydration of the components described herein.

[0148] In one embodiment, the core includes, but is not limited to, one or more reagents, such as one or more enzymes, salts, surfactants, buffers, enzyme inhibitors, primers, nucleotides, organic osmolite, magnetic beads, molecular probes, crowding agents, small molecules, labeled nucleotides, fluorophores, or any combination thereof. In a preferred embodiment, the core is not an aqueous medium.

[0149] As used herein, the term "reagent" describes a single agent or a mixture of two or more agents used to react with, interact with, dilute, or add to a sample, and may include agents for nucleic acid reactions, including, for example, buffers, chemicals, enzymes, polymerases, primers (including those having a size of less than 50 base pairs), template nucleic acids, nucleotides, labels, dyes, or nucleases. In some embodiments, reagents as described herein may include enzymes such as polymerases, ligases, recombinases, or transposons; binding couplers such as antibodies, epitopes, avidin, avidin, biotin, biotin, lectins, or carbohydrates; or other biochemically active molecules. Other examples of reagents include those for biochemical protocols, such as nucleic acid amplification protocols, affinity-based assay protocols, enzyme-catalyzed assay protocols, sequencing protocols, and / or protocols for analyzing biological fluids. According to some embodiments disclosed herein, depending on a specific workflow and / or downstream application, reagents may include one or more beads, particularly magnetic beads.

[0150] In one embodiment, the reagent according to this disclosure is a polymerase. The polymerase according to this disclosure may include any polymerase that can tolerate phosphate-labeled nucleotides. Examples of polymerases that may be used according to this disclosure include, but are not limited to, phi29 polymerase, Klenow fragment, DNA polymerase I, DNA polymerase GA-1, PZA, phi15, Nf, G1, PZE, PRD1, B103, GA-1, 9oN polymerase, Bst, Bsu, T4, T5, T7, Taq, Vent, RT, pol β, and polγ. Polymerases engineered to possess specific properties may be used. In one example, the nuclear region may include, but is not limited to, polishing microspheres, sequencing microspheres, and any combination thereof. Polishing microspheres as described herein may include, but are not limited to, ffN, polymerases suitable for polishing (“polishing polymerases”), oligonucleotides suitable for polishing (“polishing oligonucleotides”), magnesium enzyme cofactors, and any combination thereof. In another example, sequencing microspheres may include, but are not limited to, polymerases suitable for sequencing (“sequencing polymerases”).

[0151] As disclosed herein, primers comprise nucleic acid molecules capable of hybridizing to the target sequence of interest. In several embodiments, the primer may serve as a substrate to which nucleotides may be polymerized by a polymerase. However, in some instances, the primer may be incorporated into a synthesized nucleic acid strand and provide a site for another primer to hybridize to induce the synthesis of a novel strand complementary to the synthesized nucleic acid molecule. Primers may comprise any combination of nucleotides or their analogues. In some instances, the primer is a single-stranded oligonucleotide or polynucleotide.

[0152] Non-limiting examples of nucleic acid molecules that can be encapsulated within microspheres include those described above, such as DNA, such as genomic DNA or cDNA; RNA, such as mRNA, sRNA, or rRNA; or mixtures of DNA and RNA. The nucleus may further contain labeled nucleotides.

[0153] The term "salt" can include salts prepared from toxic or non-toxic acids or bases, including inorganic acids and bases as well as organic acids and bases. Salts can be prepared from, for example, pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.

[0154] Any surfactant known to a person skilled in the art can be used as a reagent in the core. Surfactants can be nonionic or ionic (particularly cationic or anionic) or amphoteric. Examples of suitable surfactants include, but are not limited to, polyacrylate surfactants, polysiloxane surfactants, and / or other commercially available surfactants or detergents. Examples of cationic surfactants are hexadecyl dimethylammonium acetamide, octadecyl-dimethylammonium acetamide, tetradecyl-dimethylammonium acetamide, dodecyl-dimethylammonium acetamide, hexadecyl trimethylammonium, octadecyl-trimethylammonium, tetradecyl-trimethylammonium, dodecyl-trimethylammonium, dimethyl dioctadecylammonium, dioctadecyl dimethylammonium, and mixtures thereof. Suitable sources of the cations in cationic surfactants include, but are not limited to, alkyltrimethylammonium salts: such as hexadecyltrimethylammonium bromide (CTAB) or hexadecyltrimethylammonium chloride (CTAC); hexadecylpyridinium chloride (CPC); dimethyl dioctadecylammonium chloride; dioctadecyldimethylammonium bromide (DODAB); hexadecyldimethylammonium acetamide bromide; or other similar cationic surfactants, including lipids. Alternatively, the surfactant may be benzylhexadecyldimethylammonium chloride (BHDC). The core may include anionic surfactants having an anionic functional group at one end, such as sulfate, sulfonate, phosphate, and carboxylic acid ester functional groups. An example of anionic surfactant is sodium dodecyl sulfate. The core may contain neutral surfactants, such as polyethylene glycol lauryl ether.

[0155] The nucleus may further or alternatively include enzyme inhibitors, molecular probes, crowding agents, organic osmolite, cyclodextrin, adenosine triphosphate (ATP), ethylenediaminetetraacetic acid (EDTA), creatine kinase, creatine phosphate, palladium, fatty acids, hexaethylene glycol, trihydroxypropanephosphine, sodium ascorbate, or any combination thereof. Enzyme inhibitors as described herein include any molecule that binds to an enzyme and reduces its activity. Molecular probes as described herein include, for example, foxglove ligand, 8-anilinenaphthalene-1-sulfonic acid (“ANS”), porphyrin, BODIPY, anthocyanins, or any combination thereof. Crowding agents as described herein include any crowding agent known to those skilled in the art. Examples include, but are not limited to, polyethylene glycol, sucrose, polydextrose, and serum albumin. Those skilled in the art will understand that additional reagents may be used in the compositions, systems, and methods disclosed herein that are not explicitly described herein.

[0156] In one embodiment, the core as described herein may further comprise one or more additional agents. These additional agents in the core improve the ability to control the release of one or more lyophilized microspheres. In one embodiment, the additional agent is selected from one or more sugars, amino acids, polymers, mesoporous silica, quaternary amines, or any combination thereof. In one embodiment, when the additional agent comprises a sugar, the sugar is selected from trehalose, mannitol, cyclodextrin, polydextrose, sucrose, or any combination thereof. In another embodiment, when the additional agent comprises an amino acid, the amino acid has a hydrophobic side chain. In another embodiment, when the additional agent comprises a polymer, the polymer is selected from polyvinylpyrrolidone, polyvinyl alcohol, or combinations thereof. In some embodiments, the additional agent may be, for example, one or more copolymers, ionic liquids, or any combination thereof. The additional agent may be added in any amount suitable for producing the desired effect, for example, between about 0.1 wt% and about 50 wt% of the core. In one embodiment, the concentration of the additional agent in the core is about 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or any amount between these values.

[0157] In one embodiment, the nucleus further includes a nucleus additive. The nucleus additive may be present in an amount between about 0.01% w / w and about 100% w / w of the nucleus. For example, in one embodiment, the nucleus additive may be present in an amount between 0.1% w / w and about 20.0% w / w of the nucleus. In one embodiment, the nucleus additive may be present in an amount between about 2% w / w and about 10% w / w of the nucleus. For example, the core additive may be about 01% w / w, 0.05% w / w, 0.1% w / w, 0.5% w / w, 1.0% w / w, 1.5% w / w, 2.0% w / w, 2.5% w / w, 3.0% w / w, 3.5% w / w, 4.0% w / w, 4.5% w / w, 5.0% w / w, 5.5% w / w, 6.0% w / w, 6.5% w / w, 7.0% w / w, 7.5% w / w, 8.0% w / w, 8.5% w / w, 9.0% w / w, 9.5% w / w, 10.0% w / w, 10.5% w / w, 11.0% w / w, 11.5% w / w, 12.0% w / w, 12.5% w / w, 13.0% The amount of the core additive, or any amount thereof, is present in w / w, 13.5% w / w, 14.0% w / w, 14.5% w / w, 15.0% w / w, 15.5% w / w, 16.0% w / w, 16.5% w / w, 17.0% w / w, 17.5% w / w, 18.0% w / w, 18.5% w / w, 19.0% w / w, 19.5% w / w, 20.0% w / w, or any amount between these values. The amount of the core additive can be any suitable amount to reduce the triboelectricity of the composition described herein. The amount of the core additive can be adjusted to suit specific reagents or combinations of reagents, or to suit specific microsphere compositions.

[0158] In one embodiment, the core additive comprises an antistatic material. In one embodiment, the core additive is an antistatic material present in an amount not exceeding 25% w / w of the core. In one embodiment, the core additive is present in an amount of at least 0.5% w / w of the shell. In one embodiment, the amount of the core additive is between about 2% w / w and about 10% w / w of the core. In one embodiment, the core additive is a water-insoluble additive, a water-soluble additive, an enteric additive, or any combination thereof. In one embodiment, the core additive comprises one or more of a polymer, copolymer, block copolymer, second polyvinyl alcohol (PVA), conductivity promoter, ammonium salt, imidazolium salt, polyether compound, or any combination thereof. In one embodiment, the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof. In one embodiment, the polymer is neutral, cationic, or anionic. In one embodiment, the buffer solution is Trizma, Tris.HCl, or a combination thereof. In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tri(hydroxyethyl)alkylammonium chloride, or combinations thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt, polytetramethylammonium chloride, or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or combinations thereof. In one embodiment, the composition described herein may be manufactured from about 20% lyophilized formulation (i.e., the formulation contains 20% lyophilized excipients, such as trehalose and other additives). Therefore, additives (static desiccant or moisture resistant agents) may be incorporated into and / or blended into the lyophilized formulation, followed by drying to obtain an appropriate concentration in the dried form. In one embodiment, the ammonium salt acts as a conductivity promoter. In one embodiment, the imidazolium salt acts as a conductivity promoter.

[0159] In one embodiment, the core additive described herein may include water-insoluble additives, water-soluble additives, enteric additives, or any combination thereof. In one embodiment, the core additive may include one or more of a sodium salt, buffer solution, chelating agent, ammonium salt, imidazolium salt, polyaniline, or any combination thereof. In one embodiment, one or more water-soluble core additives are added to the core. In one embodiment, the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof. In another embodiment, the buffer solution is Trizma (Tris.HCl). In one embodiment, the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or any combination thereof. In one embodiment, the imidazolium salt is selected from 1-ethyl-3-methylimidazolium salt or polytetramethylammonium chloride or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or a combination thereof. In another embodiment, one or more water-soluble additives such as Efka® IO 6783, Efka® IO 6786, Tween® 80, Makon® 17R4, diethanolamine laurylate, or any combination thereof may be included in the core additive composition. In another embodiment, one or more water-insoluble additives such as trioleyl ester, polyaniline, piperidinyl sebacate, amino acids, vitamin E (tocopheryl acetate), Span® 60, or any combination thereof may be included in the core additive composition. In one embodiment, Efka® IO 6783 is used as the core additive in an amount suitable for reducing the triboelectric behavior of the composition (e.g., about 5% w / w in the core).

[0160] The composition (i.e., the encapsulated lyophilized microspheres) can be any suitable size or volume suitable for encapsulating one or more reagents and for use in the preparation of a sequencing library. In one embodiment, the composition has a reagent volume in the core region between about 0.1 µL and about 50 µL. For example, the composition (i.e., the encapsulated lyophilized microspheres) can have an active reagent volume of about 0.1 µL, 0.5 µL, 1 µL, 2 µL, 3 µL, 4 µL, 5 µL, 6 µL, 7 µL, 8 µL, 9 µL, 10 µL, 15 µL, 20 µL, 25 µL, 30 µL, 35 µL, 40 µL, 45 µL, or 50 µL, or any volume between about 0.1 µL and about 50 µL. In one embodiment, the active reagent volume is between about 10 µL and about 40 µL. The composition (i.e., the encapsulated lyophilized microspheres) may have a diameter of, for example, from about 2 µm to about 120 µm, such as, for example, a diameter of 2 µm, 3 µm, 4 µm, 5 µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, 15 µm, 20 µm, 25 µm, 30 µm, 35 µm, 40 µm, 45 µm, 50 µm, 60 µm, 70 µm, 80 µm, 90 µm, 100 µm, 110 µm, or 120 µm, or a diameter within the range defined by any two of the foregoing values.

[0161] The components described herein may include additional reagents within the microsphere shell. In one embodiment, the encapsulated microspheres include a reagent or additive within the microsphere shell. The reagent within the shell may include, for example, any of the aforementioned reagents or additives. In one embodiment, the shell does not contain nucleic acid molecules; for example, the shell does not contain DNA. In one embodiment, the shell contains more than one reagent and / or, alternatively, more than one additive.

[0162] Similarly, the components described herein (e.g., encapsulated lyophilized microspheres) may include a low oxygen-permeable polymer coating, such as polyvinyl alcohol and / or an oxygen absorber. Likewise, the components described herein (e.g., encapsulated lyophilized microspheres) may include an amphiphilic coating, such as an amino acid and / or a PVP copolymer. The encapsulated lyophilized microspheres described herein may further provide protection against mechanical stress, for example, by preventing or reducing fragmentation during manufacturing (e.g., by a shell with a 40% solute content). The components described herein (e.g., encapsulated lyophilized microspheres) may further provide protection against light exposure, as the reagents are protected from light exposure, thereby reducing manufacturing light constraint.

[0163] The components described herein can be used for a variety of sequential co-assays, including sequentially performed dissolution, DNA analysis, RNA analysis, protein analysis, labeling, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, SBS technology, sequencing translocase-accessible colorimetric assay (ATAC-seq), continuous retention translocation (CPT-seq), single-cell combinatorial index sequencing (SCI-seq), or single-cell genome amplification, or any combination thereof. In one embodiment, the components are used to perform multiple co-assay reactions. In one embodiment, the components, systems, and methods described herein (e.g., encapsulating lyophilized microspheres) can improve sequencing quality, enable one-pot library preparation, and simplify manufacturing. As used herein, the term "one-pot reaction" may also be referred to as "transfer-free reaction."

[0164] The compositions, systems, and methods described herein can be prepared for various stages of sequencing, including but not limited to sample extraction, library preparation, enrichment, clustering, and sequencing. In sample extraction compositions, the core may include enzymes, salts, surfactants, buffers, and any combination thereof. Sample extraction can occur at a pH of about 7.5, with a reaction volume between about 1 mL and about 5 mL. In library preparation compositions, the core may include enzyme inhibitors, salts, primers, enzymes, nucleotides, organic osmolite, magnetic beads, and any combination thereof. Library preparation can occur at a pH of about 7, with a reaction volume of about 0.05 mL. In enrichment compositions, the core may include nucleotides, molecular probes, enzymes, magnetic beads, congestants, and any combination thereof. Enrichment can occur at a pH of about 8.5, with a reaction volume between about 0.1 mL and about 0.2 mL. In clustering compositions, the core may include salts, enzymes, one or more nucleotides, small molecules, surfactants, primers, and any combination thereof. Clustering can occur at a pH of about 8.6, with reaction volumes between about 1 mL and about 5 mL. In the sequencing composition, the nucleus may include labeled nucleotides, fluorophores, surfactants, salts, enzymes, small molecules, and any combination thereof. Sequencing can occur at a pH between about 7 and about 10, with reaction volumes between about 30 mL and about 100 mL.

[0165] In one embodiment, the shell can be rehydrated at a pH between 1 and 14. In one embodiment, the shell may comprise one or more shell layers, and each layer may be rehydrated under the same or different conditions. For example, the shell may comprise a plurality of layers rehydrated under different conditions. In one embodiment, the shell may comprise, for example, two or more layers (e.g., three or more layers) that release at different pH concentrations, such as one layer releasing at pH 5, one layer releasing at pH 5.5, one layer releasing at pH 6, one layer releasing at pH 6.5, one layer releasing at pH 7, one layer releasing at pH 7.5, and / or one layer releasing at pH 8.

[0166] The nucleus may include any number of reagents different from those described herein, or any reagent that may be used to facilitate the use of sequencing systems such as SBS technology.

[0167] In one embodiment, the biological sample comes into contact with the composition. Biological samples may include, for example, whole blood, lymphatic fluid, serum, plasma, sweat, tears, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal secretions, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, exudate, exudate, cystic fluid, bile, urine, gastric juice, intestinal juice, fecal samples, fluids containing single or multiple cells, fluids containing organelles, fluidized tissue, fluidized organisms, fluids containing multicellular organisms, biological swabs, and biological washes. Biological samples may include nucleic acids, such as DNA, genomic DNA, RNA, mRNA or analogs thereof; nucleotides, such as deoxyribonucleotides, ribonucleotides or analogs thereof, analogs having a terminator motif, such as those described in the following references: Bentley et al., "Accurate Whole Human Genome Sequencing Using Reversible Terminator Chemistry," Nature 456:53-59 (2008); WO / 2013 / 131962; U.S. Patent No. 7,057,026; WO / 2008 / 042067; WO / 2013 / 117595; U.S. Patent No. 7,329,492; U.S. Patent No. 7,211,414; U.S. Patent No. 7,315,019; U.S. Patent No. 7,405,281; and U.S. Patent Publication No. 20080108082, all of which are incorporated herein by reference in their entirety.

[0168] The second aspect relates to a method. The method includes providing one or more lyophilized microspheres; and, under conditions of effectively encapsulating one or more lyophilized microspheres, coating one or more lyophilized microspheres with a shell.

[0169] This sample is based on the previously described sample, particularly regarding the characteristics of one or more freeze-dried microspheres and their coatings, shells, cores, and encapsulations.

[0170] Any suitable method may be used to form microspheres. Standard microsphere manufacturing techniques are known to those skilled in the art and include the preparation of frozen microspheres and the placement of such microspheres in a dryer as described herein. Various microspheres are conceived based on the compositions, systems, and methods disclosed herein and include, for example, continuous release, immediate pulse, time-pulse release, organic acid Diffucaps® Bead, and alkaline buffer Diffucaps® Bead microspheres. Various types of encapsulations encompassed by the compositions, systems, and methods described herein are also available, including but not limited to bulk encapsulation, micron encapsulation, nano encapsulation, single-molecule encapsulation, and ion encapsulation.

[0171] Modifications to the standard microsphere production process can be made to manufacture the compositions described herein. For example, one or more additional feed buffer tanks and one or more suitable nozzles and / or nozzle plates can be added to the standard microsphere production apparatus. Specifically, other modifications can be made to the coagulation system to produce various types of shells, including compounds described herein, such as hydrocolloids, alginates, and pectins.

[0172] In one embodiment, two liquid solutions are prepared: one for the core and one for the shell. A dual-nozzle system (i.e., a single or multiple-nozzle system with annular gap nozzles) can be installed, allowing production to begin. Additional factors are important and adjustable based on the size and type of the components to be prepared. For example, interfacial tension, core and shell viscosity, the nozzle diameter ratio of the internal to the external nozzles, and the core and shell pressure ratio can all be taken into account and adjusted.

[0173] In one embodiment, an air brush is used to produce the encapsulated lyophilized microspheres. In another embodiment, a filter membrane may be added, which reduces the number of lyophilized microspheres leaving the chamber during air brushing. In yet another embodiment, an atomizer is used.

[0174] According to the composition, system, and method described herein, a liquid is formed, which is then stored under ambient conditions for one to two days. The microspheres are then spray-frozen and can be stored at -80°C. The freeze-dried microspheres can be placed in a tray or rotary dryer and then dry-applied to consumables and / or capsules, and finally heat-sealed with foil on plastic consumables.

[0175] The composition (e.g., encapsulated lyophilized microspheres) may be coated with one or more additional compositions to provide enhanced control over microsphere release. In one embodiment, the method further includes covering the encapsulated microspheres with an outer layer while the outer layer effectively surrounds them.

[0176] The composition can be immersed in a wax coating for a specific period of time to prevent melting and achieve a specific thickness, eliminating the risk of an excessively thick coating. In one embodiment, the coating is performed for a period of time sufficient to provide a defined thickness for the outer layer. In one embodiment, the defined thickness is, for example, about 50 µm. In other embodiments, the defined thickness is less than 50 µm, or alternatively, greater than 50 µm. In one example, the capsule is filled with one or more lyophilized microspheres, and the capsule is immersed in hot wax to provide the outer layer. This process allows for the release of the microspheres at two different temperatures, with the capsule dissolving between about 30°C and about 50°C, and the wax coating dissolving between about 50°C and about 80°C.

[0177] In one embodiment, the outer layer comprises carrageenan, shellac, trehalose, paraffin, gelatin, hydroxypropyl methylcellulose (HPMC), fullalin, oxygen absorber, alginate, chitosan, slurry, and benzo[a]benzene. Borazole-poly(vinyl alcohol) (benzo[] Borazole-PVA), pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0178] This approach can be used to achieve reagent concentration tuning. For example, smaller capsules can contain less lyophilized reagent than larger capsules, and multiple capsules can be placed in the wells as needed by the user. In one embodiment, a cell-based method is applied, where X capsules = Y runs.

[0179] The third type of sample relates to a system. The system includes one or more components as described herein and one or more freeze-dried cakes, wherein the one or more components and one or more freeze-dried cakes are combined under conditions that effectively form a rehydration system.

[0180] This sample is based on the previously described sample, particularly regarding the characteristics of one or more freeze-dried microspheres and their coatings, shells, cores, and encapsulations.

[0181] In one embodiment, the system further includes one or more shells positioned between one or more encapsulated microspheres and one or more freeze-dried cakes. In one embodiment, the shells comprise a material selected from carrageenan, shellac, trehalose, paraffin wax, gelatin, hydroxypropyl methylcellulose (HPMC), fullalin, oxygen absorbers, alginate, chitosan, a film, and benzo[a]benzene. Borazole-poly(vinyl alcohol) (benzo[] Materials including boronazole-PVA, pectin, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or any combination thereof.

[0182] The fourth aspect relates to a method for controlling the release of one or more encapsulated microspheres. The method includes providing a composition as described herein, and mixing the composition with a rehydration solution under a first condition that effectively controls the release of one or more lyophilized microspheres from the composition.

[0183] This sample is based on the previously described sample, particularly regarding the characteristics of one or more freeze-dried microspheres and their coatings, shells, cores, and encapsulations.

[0184] In one embodiment, the method further includes modifying the first condition to a second condition. In one embodiment, modifying the first condition includes modifying one or more of the following: temperature, exposure time, pH of the rehydration solution, or the position of the encapsulated microspheres in the rehydration solution.

[0185] As described herein, in one embodiment, the capsule is filled with one or more lyophilized microspheres, and the capsule is immersed in a coating (e.g., hot wax) to provide an outer layer. This procedure enables controlled release of the microspheres at different temperatures without a transfer reaction. The capsule is capable of releasing the microspheres at a temperature between about 30°C and about 50°C, preferably between about 30°C and about 40°C, while the coating (e.g., hot wax) is capable of releasing the microspheres at a temperature between about 50°C and about 90°C, preferably between about 55°C and about 85°C. The coating may be hot wax, preferably paraffin wax. In one embodiment, the temperature in the first and / or second conditions is between about 10°C and about 90°C; preferably between about 30°C and about 50°C or between about 50°C and about 90°C, depending on the composition of the shell and / or outer coating.

[0186] Similarly, modification of the first condition can be achieved by adding one or more additives to the rehydration solution or the composition described herein using the additives described herein. In one embodiment, and or alternatively, the rehydration solution further comprises one or more additional agents. The additional agents may be any single additional additive described above or any combination of two or more additional agents described above. For example, an amino acid, alone or in combination with another amino acid, may be used to modify the first condition and promote the controlled release of the microspheres. The amounts of the one or more additives will vary and depend on the composition used and the reaction conditions (e.g., time, temperature, and pH). In one embodiment, the one or more additives are between about 0.1 wt% and 40 wt% of the composition and / or the rehydration solution. For example, the concentration of one or more additives may be 0.1 wt%, 0.5 wt%, 1.0 wt%, 5.0 wt%, 10.0 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, or any amount thereof.

[0187] The rehydration (or resolvation) solution used herein may include water, deionized water, saline solution, acidic solution, alkaline solution, detergent solution, and / or buffer solution, and may be based on the rehydration solutions described above. In a preferred embodiment, the rehydration solution is water. In one embodiment, different concentrations, enzyme types, and amounts of cofactors, salts, pH, and other reagents described herein may be used for rehydration with water alone or even by atmospheric water capture. Additional additives as described herein may be provided in the rehydration solution to further improve control over microsphere release.

[0188] In one embodiment, the pH of the hydrated solution is between about 6.0 and about 10.0. The pH of the rehydrated solution can be, for example, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any amount between these values. The rehydration time will vary depending on the composition content and reaction conditions (e.g., reagents, temperature, pH). In one embodiment, the rehydration time can be between 0.1 seconds and 10 hours. For example, the rehydration time can be about 0.1 seconds, 1 second, 10 seconds, 30 seconds, 45 seconds, 60 seconds, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 2 hours, 5 hours, 8 hours, 10 hours, or any time between these values.

[0189] In one embodiment, a first condition effectively releases a first lyophilized microsphere. The first lyophilized microsphere in this embodiment is based on the lyophilized microspheres and composition described herein.

[0190] In one embodiment, a second condition effectively releases a second lyophilized microsphere, wherein the content of the second lyophilized microsphere differs from the content of the first lyophilized microsphere. The second lyophilized microsphere in this embodiment is based on the lyophilized microspheres and composition described herein.

[0191] In one embodiment, modifying the first conditions effectively releases two or more lyophilized microspheres, wherein the two or more lyophilized microspheres contain different reagents. In one embodiment, the contents of the first lyophilized microsphere include a reagent different from that of the second lyophilized microsphere, thereby reducing the heat sensitivity of the mixed reagents.

[0192] In one embodiment, the method further includes providing an additional component according to the composition described herein, and mixing the additional component under a third condition of effectively controlling the release of one or more lyophilized microspheres from the additional component. The lyophilized microspheres in this embodiment are those according to the lyophilized microspheres and composition described herein. In one embodiment, the reagent components in the SBS lysis mixture are separated into at least two different lyophilized microspheres, thereby preventing or reducing and / or controlling undesired interactions.

[0193] "Modification" as described herein includes any variation of one or more of the conditions in the encapsulated microspheres and, alternatively, the rehydration solution. Modifying the conditions in one embodiment allows for the sequential release of one or more lyophilized microspheres. One way to achieve the sequential release of lyophilized reagents is by temperature-triggered release, for example, by immersing a gelatin capsule filled with microspheres in the paraffin described herein. This method can release the microspheres at different temperatures, for example, between about 30°C and about 50°C for natural gelatin capsules, and between about 50°C and about 90°C for coated capsules. Similarly, this method can achieve time-triggered release by adding additives (e.g., amino acids) to the rehydration solution. Other reaction characteristics may be modified, in addition to time or, alternatively, temperature. For example, pH and humidity may be modified to further control the release of one or more encapsulated microspheres and the reagents contained therein.

[0194] In another embodiment, the components, systems, and methods described herein protect a polymerase in a first component (e.g., encapsulated lyophilized microspheres). In one embodiment, the protection of the polymerase in the first component during the polishing of fully functionalized nucleotides ("ffN") protects the photosensitizing ffN from photodegradation.

[0195] The problem of aligning the release time of a reagent (e.g., sequencing polymerase) from its encapsulation with the completion time of a desired reaction (e.g., a polishing process) can be addressed by tuning the various components in the composition and rehydration solution described herein, as well as the relative amounts of those components. Temperature or photoreactive additives can also be used to achieve a finer level of control. For example, the problem of correcting the deblocking of lyophilized ffN in a well (integrated into the reagent well) when using two incompatible competing polymerases (polishing and sequencing) can be solved by spatially and temporally separating the polymerase using the composition, system, and method disclosed herein.

[0196] In one example, two capsules may be in a single tube, and the capsules may be dissolved using different triggering agents. In another example, two or more capsules may be stacked along the y-axis or x-axis in a narrow tube, and the second capsule dissolves upon contact with liquid, releasing the first capsule, followed by the dissolution of a third capsule upon contact with liquid, repeating this process up to the number of capsules present in a given stack. These implementations can also be triggered by temperature modifications (such as heat). In yet another example, the tube comprises a cake formed by freeze-drying, into which wax can be transferred using a pipette, and capsules are added. The user can add liquid that first dissolves the capsules, and then, as the temperature increases, the wax melts and rehydrates the cake. In another example, the tube comprises a first cake formed by freeze-drying, and then wax is transferred into the tube using a pipette. A second liquid is then deposited, freeze-drying is repeated, and then new wax (having, for example, a different melting temperature) is deposited. The user adds liquid to sequentially rehydrate the different cakes as the wax melts.

[0197] In one embodiment, the method further includes providing one or more freeze-dried cakes and rehydrating the one or more freeze-dried cakes.

[0198] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail herein (assuming that such concepts are not inconsistent with each other) are contemplated as part of the subject matter of the invention disclosed herein. Specifically, all combinations of the claimed subjects appearing at the end of this disclosure are contemplated as part of the subject matter of the invention disclosed herein.

[0199] In this disclosure, reference is made to the accompanying drawings, which form a part of the disclosure and illustrate specific embodiments that can be practiced. These embodiments are described in detail to enable those skilled in the art to practice this disclosure, and it should be understood that other embodiments can be utilized and structural, logical, and electrical changes can be made without departing from the scope of this disclosure. Therefore, the following description of exemplary embodiments is not intended to be limiting.

[0200] This disclosure can be further illustrated by referring to the following examples. [Example] []

[0201] The following examples are intended to illustrate, but are in no way intended to limit, the scope of this disclosure as set forth in the appended patent application. [Example] [1-] [Production of encapsulated freeze-dried microspheres.] []

[0202] Here, encapsulated freeze-dried microspheres are produced.

[0203] Microspheres can be produced using standard techniques. First, cryospheres are prepared. Liquid flows into a container with a nozzle diameter at a specific flow rate and vibration frequency. Deflection power (electrostatic ring) is applied at a given tower temperature and height, and cryospheres are produced. Spray towers can be used to prepare microspheres. Trays are fed into a tray dryer, and the microspheres are placed in the dryer (e.g., a rotary dryer) in bulk form. The microsphere production of the encapsulated freeze-dried microspheres described herein can be based on or alternatively modified from standard microsphere production techniques.

[0204] As shown in Figure 1, there are various types of packaging encompassed by the components, systems, and methods described herein, including but not limited to bulk packaging, micron packaging, nano packaging, single-molecule packaging, and ion packaging.

[0205] One embodiment of the encapsulation method disclosed herein includes wax coating of microspheres. An air brush is used to apply the coating to the microspheres contained within a chamber. The air brush can generate airflow within the chamber, causing some microspheres to exit the chamber. Mitigation measures include adding a filter membrane to prevent microspheres from exiting the chamber or to reduce the likelihood of microspheres exiting the chamber. An atomizer can be used instead of an air brush, or an atomizer can be used in addition to an air brush to reduce airflow generation. Atomizers may be more challenging for coating viscous solutions. [Example] [2-] [Encapsulated lyophilized microsphere composition.]

[0206] This document illustrates exemplary formulations of compositions, systems, and methods according to this disclosure. The compositions described herein can be prepared for various stages of sequencing, including but not limited to sample extraction, library preparation, enrichment, clustering, and sequencing, as shown in Figure 2.

[0207] In the sample extraction composition, the core may include enzymes, salts, surfactants, buffers, and any combination thereof. Sample extraction can occur at a pH of approximately 7.5. The reaction volume can be between approximately 1 mL and approximately 5 mL. In the library preparation composition, the core may include enzyme inhibitors, salts, primers, enzymes, nucleotides, organic osmolite, magnetic beads, and any combination thereof. Library preparation can occur at a pH of approximately 7. The reaction volume can be approximately 0.05 mL. In the enrichment composition, the core may include one or more nucleotides, molecular probes, enzymes, magnetic beads, congesting agents, and any combination thereof. Enrichment can occur at a pH of approximately 8.5. The reaction volume can be between approximately 0.1 mL and approximately 0.2 mL. In the clustering composition, the core may include salts, enzymes, nucleotides, small molecules, surfactants, primers, and any combination thereof. Clustering can occur at a pH of approximately 8.6. The reaction volume can be approximately 1 mL. In the sequencing composition, the nucleus may include labeled nucleotides, fluorophores, surfactants, salts, enzymes, small molecules, and any combination thereof. Sequencing can occur at a pH between about 7 and about 10. The reaction volume can be from about 30 mL to about 100 mL.

[0208] In embodiments of the compositions, systems, and methods described herein, various formulations of lyophilization excipients may be added to the core, including, for example, sugars, amino acids, and polymers. Sugars may include trehalose (10% to 25%), mannitol (1% to 10%), cyclodextrin (1% to 10%), polydextrose (1% to 10%), sucrose (1% to 10%), or any combination thereof. Polymers may include polyvinylpyrrolidone (1% to 10%), polyvinyl alcohol (1% to 10%), or combinations thereof. Mesoporous silica and / or quaternary amines may be added to reduce triboelectric affinity.

[0209] In the embodiments of the compositions, systems, and methods described herein, various formulations of shell components may be added to the shell. Examples of shell components include carrageenan, shellac, trehalose (20% to 40%), paraffin wax, gelatin, HPMC, fullalin, oxygen absorbers, alginate, chitosan, gelatin, slurry, and benzo[a]benzyl peroxide. Borazole-PVA, pectin, polyvinylpyrrolidone (1% to 5%), polyvinyl alcohol (1% to 10%), or any combination thereof. For some embodiments, the materials used are free of DNA to avoid contaminating any reactants and to avoid using expensive sequencing real estate.

[0210] As shown in Figure 3, microspheres are subjected to various stresses. During manufacturing, the microspheres are exposed to humidity for a period of time (e.g., 2 to 3 days) before packaging and undergo triboelectric charging during filling and finishing. During transportation and storage, the microspheres are exposed to temperature variations for a period that may last for several months (e.g., 3 to 6 months). Once the consumable is opened (even for less than 10 minutes), various new stresses are present on the microspheres, including, for example, ambient humidity that can affect the microspheres within seconds of exposure. In addition, manufacturing, transportation, and storage subject the microspheres to additional mechanical stresses (such as shock and vibration). The components, systems, and methods described herein (e.g., encapsulating lyophilized microspheres) mitigate the stresses experienced by microspheres that are not lyophilized and are encapsulated.

[0211] Furthermore, electrostatic charges can pose a significant risk to the application and dry mixing of microspheres. The encapsulated microspheres described in the compositions, systems, and methods herein significantly reduce this risk and substantially improve the stability of the sequencing.

[0212] Similarly, the compositions, systems, and methods described herein provide improved oxygen protection to encapsulated lyophilized microspheres through a low oxygen permeability polymer coating (e.g., polyvinyl alcohol, oxygen absorbers in the coating). Likewise, the compositions, systems, and methods described herein provide improved moisture protection to encapsulated lyophilized microspheres through the application of an amphiphilic coating (e.g., amino acids and PVP copolymers). The encapsulated lyophilized microspheres described herein can further provide protection against mechanical stress, for example, by preventing or reducing fragmentation during manufacturing (e.g., a shell with 40% solute content). This protective coating increases the mechanical robustness of the microspheres and their contents during manufacturing and shipping, and reduces or even eliminates powder shedding from the microspheres.

[0213] The encapsulated lyophilized microspheres described herein further provide protection against light exposure by protecting reagents from light exposure, thereby reducing manufacturing light constraint. Encapsulation improves sequencing quality, enables one-pot preparation, and simplifies manufacturing. The benefits and applications of encapsulating lyophilized microspheres according to the compositions, systems, and methods described herein are discussed. The application of SBS for encapsulating lyophilized microspheres is illustrated in Figures 15A through 15C.

[0214] AOM SBS lysis mixtures containing Pd (550) in the core and THP (552) in the shell are shown in Figure 15A, where the Pd lysis mixture may require separation of Pd and THP to reduce the thermosensitivity of the mixture. ffN / Pol beads containing Pol (554) in the core and ffN (556) in the shell are shown in Figure 15B; where the polymerase can be protected during ffN polishing if necessary. Photoprotection of ffN containing ffN (560) in the core and a photoprotective shell (558) is shown in Figure 15C, where the photoprotective shell (558) protects the photosensitizing ffN (found in the core (560)) from photodegradation. [Example] [3-] [Sequential release of lyophilized reagents.]

[0215] There has been considerable interest in encapsulating lyophilized microspheres to increase the stability of sequencing reagents and simplify workflows. This disclosure describes compositions, systems, and methods for encapsulating lyophilized reagents in microspheres, enabling the sequential release of lyophilized reagents, as shown, for example, in Figures 47 and 48. In a first method as shown in Figure 47, one or more lyophilized microspheres (602) are provided, and one or more lyophilized microspheres are shell-coated (604) under conditions of effective encapsulation of one or more lyophilized microspheres. In another method as shown in Figure 48, a composition as described herein (702) is provided, and the composition is mixed with a rehydration solution (704) under a first release condition of effective control over the release of one or more lyophilized microspheres from the composition.

[0216] One method to achieve the sequential release of lyophilized reagents is temperature-triggered release, for example, by immersing gelatin capsules filled with microspheres in paraffin wax. This method can release the microspheres at different temperatures, for example, between about 30°C and about 50°C (e.g., 37°C) for natural gelatin capsules, and between about 50°C and about 90°C (e.g., 58°C) for coated capsules. Similarly, this method can achieve time-triggered release by adding additives (e.g., amino acids) to the rehydration solution that can delay the rehydration rate of the lyophilized reagent.

[0217] Here, the encapsulated lyophilized microspheres are used to achieve the sequential release of the lyophilized reagent. As shown in Figures 4A and 4B, in one embodiment of this disclosure, microspheres can be used to fill gelatin capsules, and the microsphere-filled gelatin capsules can be immersed in hot paraffin (or coated with any suitable outer layer) for a period of time. Figure 5 depicts temperature-controlled release for a transfer-free reaction. As shown in Figures 4A, 4B, and 5, the paraffin coating of the gelatin capsules can release the microspheres at different temperatures. Figure 4A shows an OTS gelatin capsule (102) filled with microspheres and an OTS gelatin capsule (104) filled with microspheres and rapidly immersed in hot wax. Figure 4B shows an OTS gelatin capsule (106) filled with microspheres dissolved at 37°C and an OTS gelatin capsule (108) filled with microspheres and rapidly immersed in hot wax dissolved at 58°C. The gelatin capsules (102) and / or (106) are capable of releasing microspheres between about 30°C and about 50°C (e.g., 37°C and / or 50°C), while the paraffin coatings (104) and / or (108) are capable of releasing microspheres between about 50°C and about 90°C (e.g., 58°C and / or 85°C). In this embodiment, the capsule is filled with microspheres and the capsule is rapidly immersed in hot wax, which should be fast enough that the capsule does not begin to melt and limits the thickness of the wax shell.

[0218] Figure 6 illustrates the compatibility test of gelatin during Nextera Flex labeling. Approximately 80 mM of gelatin was tolerated in the labeling reaction without a decrease in library yield. Gelatin (for OTC capsules) was titrated in the Nextera Flex reaction (n=3). Flex was performed according to standard protocols (100 ng input; 5 cycles of amplification). Yields were measured after bifacial SPRI at the qubit. Yields were normalized relative to a gelatin-free control group. Results were compared to a DNA-free / gelatin-free control group.

[0219] Figures 7A and 7B illustrate time-controlled release without transfer reaction. Figure 7A shows the percentage of excipients and their respective rehydration times. Figure 7B shows the rehydration times of the example compositions described herein at drying time (202), after 1 minute (204), after 2 minutes (206), after 5 minutes (208), after 10 minutes (210), and after 12 minutes (212). The addition of amino acid excipients achieves delayed release of the lyophilized material. Different amounts of excipients are added at different rehydration times. In one example, as shown in Figures 7A and 7B, the addition of 1% leucine and 4% trehalose resulted in a rehydration time of 12 minutes (as shown in (202), (204), (206), (208), (210), and (212)). Various other amino acid excipients were tested, including trehalose alone, phenylalanine combined with trehalose, and isoleucine combined with trehalose at different concentrations (resulting in different rehydration times). Therefore, various materials exist that can be added to the encapsulated lyophilized microspheres prior to rehydration, and or alternatively added to the rehydration solution during rehydration, allowing for improved control and stability of the microsphere contents and delayed release. These include amino acids, PVP copolymers, mesoporous silica, ionic liquids, quaternary amines, polyvinyl alcohol, deoxidizers, phenylalanine, leucine, isoleucine, polyvinyl alcohol, alginates, chitosan, carrageenan, gelatin, HPMC, paraffin, film, and benzo[a] Borazole-PVA and pectin. Further examples of additives and measures that can be used to control the release and triggering mechanism of the lyophilized microspheres encapsulated in this disclosure include polyvinyl alcohol, alginate, chitosan, gelatin, carrageenan, POD, hydrogel, film, and benzo[a]. Borazole-PVA, capsules, waxes, pectin, metal-organic frameworks, CNTs, modified RBCs, polymer matrices, and logic gate photocages. The various additives and measures described herein promote the stability of encapsulated lyophilized microspheres, allowing for large-scale encapsulation to achieve multi-operational tubes, and allowing for microencapsulation to achieve simplified workflows, and, for example, reducing the number of reagent wells. [Example] [4-] [Lyophilized microspheres for one-pot bonding protocols and controlled release of the encapsulated lyophilized microspheres.]

[0220] Here, the encapsulated lyophilized microspheres implement a one-pot connection protocol, as shown in Figures 8A to 8C. The steps of the one-pot connection protocol are illustrated in Figure 8A. Figure 8B shows the time-dependent release of microspheres (302) in a single pot containing two different microspheres: (302) corresponding to reagent A in Figure 8A and (304) corresponding to reagent B in Figure 8A. Examples of active reagent volume, number of microspheres, active reagent composition, encapsulation shell composition, and encapsulation release triggers for reagent A, reagent B, and additional reagent B' are shown in Figure 8C.

[0221] The compositions, systems, and methods described herein achieve delayed release of encapsulated lyophilized microspheres (e.g., rehydration of a first reagent in the microsphere, followed by rehydration of one or more subsequent reagents in the same or different microspheres, in the same rehydration solution, after a period of time), as well as mechanical protection, buffer stability, charge control, reagent combination (e.g., combination of two or more different reagents in a single microsphere, a single well, or a single pot), and photoprotection. Specifically, one-pot library preparation is achieved by using encapsulated lyophilized microspheres with time-controlled release of reagents as described herein. By encapsulating PCR reagents and releasing them at a predetermined time, the inhibition of labeling by reagents involved in PCR is solved. Figures 8A to 8C show encapsulated lyophilized microspheres with different nuclear reagents in a single rehydration solution. For example, reagent A can be rehydrated in less than 1 minute, while reagent B in this example is rehydrated after 5 minutes. This avoids any undesirable reaction between reagents A and B, allows reagents A and B to be present in a single pot or well, and permits a transfer-free reaction. This concept can be applied to more than two types of reagents in a single pot or well. For example, as shown in Figures 8A to 8C, reagents A, B, and B', each containing one or more differently packaged lyophilized microspheres with one or more different reagents, can be combined in a single well or pot and rehydrated in a time-dependent manner. Different numbers of active reagents, multiple microspheres, and the composition of the reagents themselves can be used together with different components of the shell at different time periods, all within a single pot or well. This finding allows various steps in PCR for sequencing applications to be performed in the same pot or well and uniquely permits a transfer-free reaction.

[0222] In addition to the above, the components, systems, and methods described herein offer several additional benefits. For example, the use of lyophilized materials and isolated lyophilized materials means that additional cofactors such as magnesium can be added to the microspheres themselves rather than having a separate additional rehydration buffer. This allows reagents that require or benefit from varying amounts of cofactors, salts, pH, and even different concentrations and types of enzymes to be rehydrated using only water or even atmospheric water capture. Furthermore, the reduced weight of concentrated and / or lyophilized reagents contributes to a cascading reduction in the amount of plastic used in sequencing processes and in terms of carbon footprint.

[0223] Encapsulation methods can be applied to achieve easily tuned reagent concentrations. For example, smaller capsules can contain less lyophilized reagent than larger capsules, and multiple capsules can be placed in wells as needed by the user. This increases user flexibility in throughput and avoids potential errors in dilution / concentration calculations. A cell-based method, where X capsules = Y runs, achieves this flexibility in a more controlled manner. Another option includes deeper or longer sequences (2 x 500) using three times the number of capsules ("3X"), while rapid surface screening tests can use fewer times the number of capsules ("X"). [Example] [5-] [Correcting the freeze-drying process by removing the seal in a hole] [ffN] [。]

[0224] The problem of correcting the deblocking of lyophilized ffN within a single well (e.g., incorporated into a mixture reagent well) using two incompatible competing polymerases (polishing polymerase and sequencing polymerase) can be addressed by spatially and temporally separating the polymerases shown in Figures 9A-9D, 10A-10D, and 11A-11F. The temporal and spatial separation of the two polymerases can be solved by encapsulating one polymerase (the sequencing polymerase, as it is used after the polishing polymerase) in a water-soluble, slowly dissolving membrane (e.g., polyvinyl alcohol). By tuning the composition and relative amounts within the water-soluble membrane, the timing of the sequencing polymerase's release from its capsule can be synchronized with the completion time of the polishing process. Temperature- or photoreactive additives can also be used to achieve a finer level of control.

[0225] Lyophilized ffN offers increased stability compared to its liquid form, but it results in elevated 3'OH concentrations, increasing the pre-phase concentration and thus degrading performance. Laboratory-scale polishing workflows can be complex. The polishing mixture (comprising ffN, polishing polymerase, polishing oligonucleotides, and Mg) is prepared and assembled separately, incubated at 50°C for up to one hour (to promote the polishing reaction), and then added to the remainder of the mixture, including the sequencer polymerase. This level of complexity means that this workflow is undesirable in its current form for both the user and the sequencer, and will be even more so on a larger scale. A solution with minimal and / or no user touchpoints (compared to or less complex than current sequencer workflows) is a viable option.

[0226] The compositions, systems, and methods disclosed herein provide a viable alternative. Loose "polishing microspheres" (which may include ffN, polishing polymerase, polishing oligonucleotides, and magnesium enzyme cofactors) can be applied in a single reagent well. Sequencing polymerase microspheres can also be present in this well; however, these can be encapsulated in a water-soluble, time-dissolving membrane, as shown in Figures 19A-9D and 10A-10D. This setup achieves several benefits, including, for example, a reduction in the number of wells. If the current method of preparing polishing mixtures separately and then mixing them with a large ICM mixture is followed, individual wells may be required for the polishing reagent. The encapsulated compositions, systems, and methods described herein facilitate multiple sequential reactions occurring in a single well, thereby minimizing the number of wells. This also affects the tube's footprint, creating cascading effects on the environment, including, for example, plastic use and waste incineration. The compositions, systems, and methods described herein can be easily scaled up while also providing reduced jetting and valveing, thereby reducing sequencer complexity and associated costs. When a rehydration buffer (such as water) is applied to the wells, the loose polished microspheres dissolve rapidly and the polishing reaction begins to correct any unblocked ffN. This rehydration buffer also begins to dissolve the water-soluble membrane encapsulating the sequencing polymerase, see, for example, Figures 10A to 10D and Figures 11A to 11F.

[0227] An integrated polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligonucleotides is shown, for example, in Figure 9A. A sequencing polymerase microsphere (404) containing sequencing polymerase is shown, for example, in Figure 9B. An encapsulated polymerase microsphere (406) and an integrated polishing microsphere (402) in a single well (408) are shown in Figure 9C. The encapsulated polymerase microsphere (406) and the integrated polishing microsphere (402) can be placed in a single well (408) that is heated to a high temperature and rehydrated with water, as shown in Figure 9D.

[0228] An integrated polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligonucleotides is further shown in Figure 10A. A sequencing polymerase microsphere (404) containing sequencing polymerase is shown, for example, in Figure 10B. Encapsulated polymerase microspheres (406) (x3 units) and integrated polishing microspheres (402) (x3 units) in a single well (408) are shown in Figure 10C. The same unit dose can be repeated across different reagents to achieve the total x3 dose shown in Figure 10D.

[0229] An integrated polishing microsphere (402) containing ffN, polishing polymerase, and polishing oligonucleotides is further shown, for example, in Figure 11A. A sequencing polymerase microsphere (404) can be encapsulated (406) and may contain the sequencing polymerase, and can be located next to the integrated polishing microsphere (402) in a single well, as shown in Figure 11C. The encapsulated lyophilized microsphere (406) can be rehydrated with water at 50°C, as shown in Figure 11C. After one hour, the integrated microsphere (402) begins to dissolve, and polishing begins, as shown in Figure 11D. The integrated microsphere (402) dissolves, polishing is complete, and the encapsulated polymerase microsphere (406) dissolves after a delay, as shown in Figure 11E. The microspheres are completely dissolved, and the ICM is then ready for use, as shown in Figure 11F. [Example] [6-] [Discussion and benefits of controlling the release of one or more encapsulated lyophilized microspheres.]

[0230] It is known that the pH of SBS buffer solutions changes during sequencing operations. The components, systems, and methods described herein use encapsulation of particles that would otherwise respond to pH changes to stabilize these buffer solutions and increase SBS performance.

[0231] The compositions, systems, and methods described herein offer numerous advantages. For example, encapsulated lyophilized microspheres provide antistatic protection by neutralizing charge and reducing triboelectric affinity, thereby reducing metering and manufacturing complexity (e.g., using mesoporous silica, ionic liquids, quaternary amines). Electrostatic charge is a significant risk to microspheres because it significantly affects the metering and mixing of dry microsphere powder during manufacturing. Encapsulation can be used to neutralize the charge of microspheres by coating particles with a neutral material having low triboelectric affinity.

[0232] The compositions, systems, and methods described herein further improve the control of solution pH by developing pH-sensitive microspheres, the pH of which may change over time when placed on an instrument (e.g., an ICM). These pH-sensitive microspheres release when the buffer solution drops below a specified pH to release ions and return the buffer solution to the desired pH. Similarly, the compositions, systems, and methods described herein improve the control of the external charge of the microspheres to facilitate dispensing and reduce (if not prevent) stratification in the mix, and further allow the separation of reagent components from the SBS cleavage mixture to reduce the thermosensitivity of the mixed reagents, thereby reducing or preventing and / or controlling undesired interactions. Likewise, the compositions, systems, and methods described herein provide encapsulated lyophilized microspheres that protect the polymerase during ffN polishing and protect the photosensitized ffN from photodegradation, especially in cases where the environmental conditions involved in polishing degrade the enzyme.

[0233] Various applications of the compositions, systems, and methods described herein (e.g., encapsulated lyophilized microspheres). In one example, two or more capsules may be adjacent to each other in a single tube, and these capsules may be dissolved using different triggering agents. In another example, two or more capsules may be stacked along the y-axis or x-axis of a narrow tube, and a second capsule dissolves upon contact with a liquid, releasing the first capsule, followed by the dissolution of a third capsule upon contact with a liquid, repeating this process up to the number of capsules present in a particular stack. These embodiments can also be triggered by temperature modifications (such as heat). In yet another example, the tube may include a cake formed by lyophilization, into which wax may be pipetted, and capsules may be added. A user may add liquid that first dissolves the capsules, and then, as the temperature increases, the wax melts and rehydrates the cake. In another example, the tube may include a first cake formed by lyophilization, into which wax may then be pipetted. A second liquid is deposited, and lyophilization is repeated, followed by the deposition of new wax (having, for example, a different melting temperature). Users add liquid to sequentially rehydrate different cakes as the wax melts.

[0234] The manufacturing process details of one embodiment of the lyophilized microspheres used for the encapsulation described herein are shown in Figure 12. Liquid formation is carried out in large volumes, followed by storage under ambient conditions for a period of time, such as one to two days, after which the microspheres are spray-frozen and stored at -80°C. The lyophilized microspheres are then placed in a tray or rotary dryer and subsequently dry-applied to consumables and / or capsules, and finally heat-sealed with foil onto plastic consumables. Capsules may be placed close to each other in a single tube and dissolved with different triggering agents, or alternatively, they may be stacked in narrow tubes and dissolved upon release of a previous capsule and upon contact with the liquid described herein (this embodiment can also be triggered by heat).

[0235] The manufacturing and point of use of one embodiment of the lyophilized microspheres used for the encapsulation described herein is shown in Figure 13. In one embodiment, during manufacturing, a liquid is applied to a consumable, one or more reagents are freeze-dried, a hot wax is applied, the wax is cooled to solidify the barrier, and a capsule is added to the consumable. A lyophilized cake can be formed by lyophilization, and the wax can then be transferred to a tube using a pipette and can be dripped into the capsule. In one embodiment, at the point of use, the user adds a liquid that dissolves the capsule, rehydrates the lyophilized microspheres from the capsule, raises the temperature to melt the wax, and rehydrates the lyophilized cake.

[0236] The manufacturing and use points of one embodiment of the lyophilized microspheres and cakes used for the encapsulation described herein are shown in Figures 14A and 14B. In one embodiment, during manufacturing, a liquid is applied to a consumable with one or more reagents, the consumable is freeze-dried, a hot wax is applied, and the wax is cooled to solidify the barrier. This process is repeated for additional layers. A lyophilized cake can be formed by lyophilization, and the wax can then be transferred to a tube using a pipette and can be dripped into a capsule. A second liquid is deposited, lyophilization is repeated, followed by the deposition of new wax with different melting temperatures. In one embodiment, at the point of use, the user adds liquid, melts a first wax at a first temperature, rehydrates a first cake, melts a second wax at a second temperature, and rehydrates a second cake. This process is repeated for additional layers. [Example] [7-] [Discussion of the important findings of the constituents described in this article.]

[0237] The key aspects of this disclosure include four main research and development activities: (1) compatibility screening for ffN and sequencing additives; (2) compatibility screening for ExAmp and clustering additives; (3) incorporating additives into lyophilized matrices to reduce static electricity; and (4) incorporating additives into coatings of microspheres to reduce static electricity.

[0238] Although water-soluble additives are focused on here and in Examples 8 to 13 as examples, water-insoluble additives can also be used. Metal nanoparticles, graphene fillers, carbon nanotubes, and mesoporous silica are available and can be incorporated into the lyophilization or drying agents described herein. For example, Lumay et al. reported using mesoporous silica to reduce static electricity and improve powder flow. Lumay et al., "Influence of Mesoporous Silica on Powder Flow and Electrostatic Properties on Short and Long Term," Journal of Drug Delivery Science and Technology 53:101192 (2019), is incorporated herein by reference in its entirety. Fang et al. incorporated polydopamine and tannic acid into uncharged surface coatings. Fang et al., "Universal Nature-Inspired Coatings for Preparing Noncharging Surfaces," ACS Applied Material and Interfaces 9:32220-32226 (2017), is incorporated herein by reference in its entirety. Guo et al. incorporated graphene oxide into photocurable polymers to fabricate electrostatically dissipative nanocomposites. Guo et al., "Highly Flexible, Thermally Stable, and Static Dissipative Nanocomposite With Reduced Functionalized Graphene Oxide Processed Through 3D Printing," Composites Part B: Engineering 208:108598 (2021), is incorporated herein by reference in its entirety. Finally, Baytekin et al. incorporated free radical scavengers into PDMS or polysiloxane to dissipate electrostatic charges. Baytekin et al., "Control of Surface Charges by Radicals as a Principle of Antistatic Polymers Protecting Electronic Circuitry," Science 341:1368-1371 (2013), is incorporated herein by reference in its entirety. As the literature shows, these additives are commonly used in solid plastics or polymers for electronic applications.

[0239] In pharmaceutical or biotechnology applications, the use of such additives is influenced by their compatibility with the active ingredients. The dried reagents in the form of lyophilized microspheres contain enzymes, oligonucleotides, and ffN (nucleotides). Therefore, the selection of additives in reagent microspheres must be driven by the compatibility and function of the additives to mitigate the electrostatic behavior of the dried microspheres. Specifically, the lyophilized microspheres described herein are treated in a dry environment, which facilitates the accumulation of electrostatic charges. Incorporating this additive into the microspheres as a matrix or coating helps to reduce electrostatic and triboelectric charging during dry filling and mixing (blending). After rehydration, the additives in the matrix or coating of the lyophilized microspheres are expected to be soluble in aqueous solutions. However, if an in-situ separation method for the additives is available, i.e., a filtration membrane, it is technically possible to apply the aforementioned water-insoluble additives and the coating for the microspheres.

[0240] In Table 1, additives were screened based on their solubility in aqueous solutions and organic solvents (i.e., isopropanol). [ [surface] [1.]] [] [The solubility of additives was screened to reduce static electricity and triboelectric charging of freeze-dried microspheres. The weight of soluble additives is summarized.] [%] [and its counterpart solution] [pH] [The impact of this (promising additive).] Static reduction additives water IPA MOPS pH 7.5 ICM pH 9.9 Efka® IO 6783 2.0% 7.44 2.0% 2.0% 7.55 2.0% 7.55 Efka® IO 6786 2.0% - 2.0% 2.0% - 2.0% - Larostat 902A 2.0% 6.89 2.0% 2.0% 7.51 2.0% 7.51 Sodium lauryl sulfate 0.5% 8.91 0.1% 0.5% 7.57 0.5% 7.57 Sodium oleate x x x x stearic acid x 0.5% x x magnesium stearate x x x x Trisodium citrate dihydrate 2.0% 8.52 x 2.0% 7.5 2.0% 7.5 Sodium L-ascorbate 2.0% 7.59 x 2.0% 7.55 2.0% 9.95 Span® 60 x x x x Tween® 60 x (with 1% plus 1% DMSO) 1.0% x x Polyethyleneimine (80% ethoxylated) (37 wt% in water) 2.0% 11.19 2.0% 2.0% 8.04 2.0% 10.1 diethanolamine lauryl acid 2.0% 9.82 2.0% x 2.0% 9.99 Luviquat® FC370 (40 wt% in water) 10.0% 7.16 x 10.0% 7.60 10.0% 9.75 Luviquat® FC550 (40 wt% in water) 10.0% 6.75 x 10.0% 7.36 10.0% 9.76 Triglyceride monostearate x x x x Piperidinyl sebacate x (with 0.1% plus 0.1% DMSO) 2.0% x (with 0.1% plus 0.1% DMSO) x (with 0.1% plus 0.1% DMSO) Vitamin E / Tocopheryl Acetate x (with 0.1% plus 0.1% DMSO) 2.0% x (with 1% plus 1% DMSO) x (with 0.1% plus 0.1% DMSO) Trioleic acid glycerides x 2.0% x x polyaniline x (with 0.1% plus 0.1% DMSO) x x (1% plus 0.1% DMSO) x (with 0.1% plus 0.1% DMSO) Coumarin 6 x x x x Tween 80 2.0% 7.45 2.0% 2.0% 7.52 2.0% 9.91 Makon® 17R4 2.0% 7.6 2.0% 2.0% 7.52 2.0% 9.94

[0241] Organic solvent solubility can be beneficial when additives are applied to lyophilized microspheres as a coating. In addition to solubility in aqueous solutions (i.e., MOPS and ICM buffers), it is important to note that the additives do not alter the pH of the buffer solution, which is used for storing or stabilizing the active ingredient.

[0242] In this example, water-soluble additives, such as Efka® IO 6783, 6786, Tween® 80, Makon® 17R4, and diethanolamine laurylate, are beneficial because they can be directly incorporated into the matrix of the lyophilized microspheres. Water-insoluble additives, such as trioleic acid esters, polyaniline, piperidinyl sebacate, vitamin E (tocopherol acetate), and Span® 60, can be incorporated into the microspheres using DMSO. [Example] [8-] [against] [ffN] [And compatibility screening of sequencing additives.]

[0243] To screen compatibility for sequencing performance, additives were incorporated into the incorporation mixture (IMX) containing ffN and polymerase. Adverse effects were monitored by increasing phase fixation, pre-phase measurement, error rate, and decreasing Q30. As shown in Figures 16A to 16I, diethanolamine laurylate, Makon® 17R4, and Efka® IO 6783 were compatible due to their sequencing results being comparable to the control group (non-incorporated IMX).

[0244] Promising materials (such as Efka® IO 6783) were screened at different concentrations of IMX to measure the compatibility limit (i.e., 2% w / v).

[0245] Additives reported as antistatic agents (such as ethoxylated PEI and Luviquat® FC550) may be incompatible with the sequencing. The sequencing results of the additives are summarized in Table 2 below, which helps in the downward selection of antistatic additives for SBS microspheres (ffN and polymerase). [ [surface] [2.]] [] [Enhanced with additives] [IMX] [(] [SBS] [Summary of sequencing performance when incorporated into mixtures] [] Classification of Additives IMX enhanced with additives Phasing% (≤ 0.2) Pre-order percentage (≤ 0.2) Q30 (% ≥ 90) Error rate Buffer / Salt 2% Tris.HCl √ √ 92.50 0.51 0.1% sodium chloride × √ 57.34 43.20 solvent 5% DMSO √ √ 92.27 0.41 Static reduction additives 1% Efka® IO 6783 √ √ 92.78 0.41 0.5% Efka® IO 6786 √ √ 92.99 0.37 0.5% isoleucine √ √ 92.93 0.34 0.5% diethanolamine lauryl laurate √ √ 94.14 0.33 1% Makon® 17R4 √ √ 93.01 0.61 0.5% tocopheryl acetate plus 0.5% DMSO √ √ 96.97 0.17 0.5% piperidinyl sebacate plus 0.5% DMSO √ √ 96.97 0.20 0.5% Triethyl Citrate √ √ 95.96 0.21 0.5% Tween® 80 × √ 98.45 34.51 0.4% Luviquat® FC370 × × 48.60 43.22 0.4% Luviquat® FC550 × √ 61.53 4.52 0.1% sodium citrate × √ 54.88 11.69 0.1% Sodium Lauryl Sulfate × √ 85.54 2.90 0.4% ethoxylated polyethyleneimine × × 59.32 30.50

[0246] In addition to incorporating IMX, the compatibility of additives with ffN was screened (Figures 17A to 17F). Two blue ffCs from different sequencing platforms were selected because they are thermosensitive to deblocking (3'OH generation) and triphosphate hydrolysis (DiP generation). The additives were incubated with aqueous ffN formulations (or rehydrated lyophilized formulations) at 60°C for 1 and 2 days. Compared with the control group, a significant increase in 3'OH and DiP was observed, along with a decrease in peak area. Besides Tris.HCl, promising additives such as Efka® IO 6783 are compatible with ffN. [Example] [9-] [against] [ExAmp] [And compatibility screening of clustered additives.]

[0247] The same additives were also incubated with the aqueous ExAmp formulation (TCX V1.0). The activity of DNA recombinase (the sensitive enzyme component of ExAmp) was evaluated in the presence of 0.1% and 1% w / v of the additives (Figure 18). Except for Efka® IO 6786, Luviquat®, and Eudragit®, the activities of most additives were comparable to those of the control group.

[0248] The clustering function of ExAmp was evaluated using cBOT first-base incorporation kinetics (Figures 19A to 19B). The results confirmed the DNA recombinase activity assay. Incompatible additives (such as Luviquat® and Eudragit®) exhibited low clustering strength. On the other hand, Efka® IO 6783 slightly impaired DNA recombinase activity and clustering function, especially when ExAmp was fractionated at higher temperatures (i.e., 40°C for 1 day). Therefore, the concentration of Efka® IO 6783 can benefit from being maintained at less than approximately 1% w / v in ExAmp solution (or rehydrated microspheres).

[0249] These additives were also incorporated into ExAmp as freeze-dried cakes. To measure the antistatic properties of the additives, the cakes were ground into powder, and their charge potential was measured using Keyence (as a matrix). Static electricity was more pronounced in a dry environment (3% relative humidity) (Figure 20). Surprisingly, Efka® IO 6783 was effective even at 0.1% w / v in a 20% freeze-dried formulation.

[0250] In addition, Kollidon® VA64, Efka® IO 6786, and Luviquat® are effective at 0.1% and 1% w / v. However, Luviquat® and Efka® IO 6786 are not fully compatible with ExAmp.

[0251] At higher relative humidity (40% RH, Figure 21), the charge potential of the powdered freeze-dried cake decreases, indicating small electrostatic and triboelectric behavior.

[0252] Because of interest in a larger library of antistatic additives, water-insoluble additives were explored in this ExAmp compatibility test. DMSO of the same additive concentration was also added to improve the water solubility of these hydrophobic additives. The lyophilized formulation of TCX V1.0 contains 7.5% HPBCD, which contributes to the water solubility of the additives.

[0253] Figures 22 and 23 show the charge potential measurements of ExAmp powder freeze-dried cakes at 3% and 40% relative humidity, respectively. Diethanolamine lauryl ether, coumarin 6, tocopheryl acetate, bis(4-piperidinyl sebacate), and even DMSO were shown to reduce the charge potential of the ExAmp powder.

[0254] The effects of excipients in lyophilization formulations on the charge potential of ExAmp powder pellets were investigated (Figure 24). Many of these excipients are salts and buffers used in lyophilization formulations. HEPES, MOPS, and tetraalkylammonium chloride were found to effectively reduce the charge potential of the ExAmp powder at two concentrations (0.1% and 1% w / v in a 20% lyophilization formulation). This unexpected finding is helpful in designing lyophilization formulations, particularly those using salt / buffers, and may also help mitigate electrostatic and triboelectric charging in lyophilized microsphere form. Specifically, they were shown to be compatible with the DNA recombinase activity in ExAmp. [Example] [10-] [Additives are incorporated into the lyophilized matrix to reduce static electricity.]

[0255] To test the antistatic effectiveness of the additives in the microspheres, these additives were freeze-dried together in matrix form. Figures 25A to 25C show the inclusion of additives in matrix form (both containing 20% ​​trehalose) in Atto microspheres and fluorescein (FSCN) microspheres. The antistatic properties of the additives were assessed by attaching the microspheres to the container and measuring their charge density using GranuCharge. A low Δq value indicates low triboelectricity. The matrix form of 1% Efka® IO 6783 minimizes triboelectricity. Figure 25A shows the results of the first group of additives tested in terms of both visual results (top) and percentage loss (bottom) (Atto 20%, +1% Efka® IO 6783, +1.5% Tris.HCl, and +1% Tween 20). Figure 25B shows the results of the second group of additives tested in terms of both visual results (top) and percentage loss (bottom) (FSCN 20% control group, +1% Efka® IO 6783, +2% Efka® IO 6783, and +1% Efka® IO 6786). Figure 25C shows that Atto and FSCN (both 20% trehalose) were dry-mixed using the matrix-form antistatic agent Efka® IO 6783.

[0256] Microspheres were adhered to the glass and plastic (polypropylene) walls of containers to demonstrate their electrostatic and triboelectric behavior. A lyophilized formulation matrix doped with 1% Efka® IO 6783 exhibited low electrostatic and triboelectric properties. Charge density measurements using GranuCharge confirmed the antistatic and anti-triboelectric behavior (low q0 and Δq values) of Efka® IO 6783. Furthermore, the doping of Atto microspheres and FSCN microspheres with Efka® IO 6783 resulted in more homogeneous blending.

[0257] The electrostatic and triboelectric behavior of ffN microspheres was screened in the presence of additives (as a matrix). Efka® IO 6783, diacetyl laurate, and isoleucine appeared to be effective in glass containers. However, Efka® IO 6783, Efka® IO 6786, and Makon® 17R4 appeared to be effective in plastic containers. GranuCharge measurements provided the charge density of microspheres containing additives in matrix form (Figures 26A to 26C). ffN microspheres containing additives in matrix form are shown in Figures 26A to 26C. The antistatic properties of the additives were assessed by adhesion to the container and measured using GranuCharge. Figure 26A shows the visual results for the first group of additives (ffN + 25% T control group, +1% Efka® IO 6783, +1% Efka® IO 6786, +1.5% Tris.HCl, +2% isoleucine). Figure 26B shows the visual results for the second group of additives (second ffN + 20%T control group, +0.5% LDA, +1% Makon® 17R4, +1.5% Kollidon® VA64, +2% Kollicoat® Protect). Figure 26C shows the charge density of the various additives tested in Figures 26A and 26B.

[0258] The low charge density of Efka® IO 6783 and isoleucine containing microspheres is corroborated by its behavior within the glass container. [Example] [11-] [Additives are incorporated into the coating of the microspheres to reduce static electricity.]

[0259] Water-insoluble additives, such as magnesium stearate, can be applied as a coating to the surface of microspheres, as organic solvents are generally used in microsphere coating processes. Figures 27A and 27E illustrate how Eudragit® L100 and magnesium stearate coatings reduce the charge density of microspheres as measured by GranuCharge. As shown in matrix form, the adhesion of coated microspheres to glass and plastic containers is significantly reduced due to the presence of additives in the coating. Dry blending of microspheres (core: trehalose and dye) can be achieved through coating. However, rehydration of magnesium stearate may be challenging due to its hydrophobicity. If this additive can be separated in situ by centrifugation or filtration membrane, magnesium stearate can be an attractive antistatic additive for microspheres.

[0260] Based on ffN, sequencing, and ExAmp compatibility screening, Efka® IO 6783, Kollidon® VA64, Kollicoat® IR, and PEG were selected as alternative water-soluble coating materials. Figures 28A to 28F show SEM images of fluorescein (FSCN), DNA recombinase / BSA, and ffN microspheres coated with different concentrations of Kollidon® VA64, Efka® IO 6783, and PEG. Coatings were observed, encapsulating the lyophilized microspheres. Images of multiple (Figure 28A) and single (Figure 28B) microspheres coated with 20% 800 µm FSCN Wurster-Spray of Kollidon VA64, Efka® IO 6783, and PEG (#6) are shown in Figures 28A and 28B. Images of multiple (Fig. 28C) and a single (Fig. 28D) microspheres coated with Kollidon VA64, Efka® IO 6783, and PEG (#8) at 15% Rec / BSA via cryo-ion milling are shown in Fig. 28C and Fig. 28D. Images of multiple (Fig. 28E) and a single (Fig. 28F) microspheres coated with Kollidon VA64, Efka® IO 6783, and PEG (#11) at 10% ffN via cryo-ion milling are shown in Fig. 28E and Fig. 28F.

[0261] The electrostatic and triboelectric behavior of the coated microspheres was evaluated compared to the uncoated control microspheres (Figures 29A-29B). While the uncoated control microspheres exhibited high triboelectric charge in container adhesion tests and GranuCharge measurements, the presence of the coating appeared to reduce the charge density of the microspheres. Specifically, the presence of Efka® IO 6783 reduced the adhesion of the coated microspheres to the glass container. However, the coating containing Kollicoat® Protect appeared to exhibit lower triboelectric charge as measured by GranuCharge.

[0262] The same coating formulation (A: Kollidon® VA64 vs. C: Kollicoat® Protect) was applied to DNA recombinase / BSA microspheres. Different coating concentrations were investigated in the coating process for these microspheres (Figures 30A to 30B). A coating concentration of 7.5% (by weight gain) did not improve the adhesion of the microspheres to both glass and plastic containers. However, at a higher coating concentration of 15%, the adhesion of the coated microspheres to plastic containers was significantly reduced. This was evidenced by a lower charge density Δq value measured by GranuPharce, indicating a reduction in triboelectric charging. Notably, the Kollicoat® Protect coating appeared to be more effective on glass containers, while both coating materials (Kollidon® VA64 and Kollicoat® Protect) reduced the adhesion of the coated microspheres to plastic containers when they contained Efka® IO 6783.

[0263] The stability of the components described in this article (including ffN microspheres, 5% Kollidon® VA64 in the dry matrix, 5% Makon® 17R4 in the dry matrix, 5% Efka® 6783 in the dry matrix, 10% Kollicoat® Protect in the dry matrix, and 7.5% isoleucine in the dry matrix) under different moisture and time conditions is shown in Figures 31A to 31F.

[0264] The results of the measurement of relative humidity tolerance of microspheres obtained by dynamic vapor adsorption are shown in Figures 32A to 32F. Isoleucine in the matrix increases the humidity tolerance of ffN microspheres. Figure 32A shows the results of the ffN control group (18% T, 2% HCD), Figure 32B shows the results of the 2% Kollicoat® Protect matrix (10% dry), Figure 32C shows the results of the +1% Efka® IO 6783 matrix (5% dry), Figure 32D shows the results of the 1% Kollidon® VA64 matrix (5% dry), Figure 32E shows the results of the +1.5% Trizma matrix (7.5% dry), and Figure 32F shows the results of the +1.5% isoleucine matrix (7.5% dry).

[0265] Results demonstrating improved moisture resistance of Rec / BSA MS with Kollicoat® Protect and VA64 coatings are shown in Figures 33A-33D. The coatings minimize moisture absorption by the Rec / BSA microspheres (Figures 34A-34B). Rec / BSA microspheres were tested using two different coating materials: Kollicoat® VA64 (#8) and Kollicoat® Protect (#9). Coating thickness was adjusted by coating concentration or the weight of coating applied to the microsphere surface. In this case, coating concentrations of 7.5% and 15% are shown, controlled by the amount of coating material sprayed onto the microspheres. Specifically, the Kollicoat® Protect coating performed better than Kollicoat® VA64 in terms of moisture resistance. This was an unexpected finding. Microspheres with a 15% coating concentration showed improvement compared to the uncoated control group. Specifically, it exhibits higher tolerance to 10% humidity (via dynamic vapor phase adsorption, DVS), slower moisture absorption at different humidity concentrations, and less shrinkage and melting of the coated microspheres. Microspheres containing Kollicoat® Protect at coating concentrations of 7.5% and 15% outperform Kollidon® VA64.

[0266] In discovering this disclosure, several unexpected results were found. For example, additives compatible with ExAmp activity were identified. Second, additives compatible with ffN stability were identified. Third, additives compatible with sequencing were identified. Fourth, additives exhibiting antistatic properties (minimizing triboelectric charging) in the dried form of lyophilized microspheres (matrix form) were identified. Fifth, additives exhibiting antistatic properties (minimizing triboelectric charging) in coated microspheres (excipient blends as coating formulations) were identified. [Example] [12-] [Discussion on shell and core additives.]

[0267] Various shell additives were discovered to improve the stability of both the shell and core of the compositions described herein.

[0268] Water-soluble polymers such as hydroxypropyl methylcellulose (HPMC), polyvinylpyrrolidone, and polyvinyl alcohol have been used in film coatings for pharmaceutical tablets. For example, HPMC is found to be commercially available as Methocel or Pharmacoat, polyvinylpyrrolidone as Kollidon®, and polyvinyl alcohol as Gohsenol or Opadry AMB, as reviewed in Maroni et al., "Film Coating for Oral Pulsatile Release," International Journal of Pharmaceutics 457:362 (2013) and Joshi et al., "Film coatings for taste masking and moisture protection," International Journal of Pharmaceutics, 457:395 (2013), both of which are incorporated herein by reference in their entirety. According to this disclosure, these materials are used as a reference due to their commercial availability.

[0269] Delayed release can be exemplified by HPMC or Methocel coatings, as reported in Zema et al., "Different HPMC Viscosity Grades as Coating Agents for an Oral Time and / or Site-Controlled Delivery System: An Investigation into the Mechanisms Governing Drug Release," International Journal of Pharmaceutical Sciences 96:1527 (2007), and Maroni et al., "Film Coating for Oral Pulsatile Release," International Journal of Pharmaceutics 457:362 (2013), which are incorporated herein by reference in their entirety.

[0270] Cationic and anionic charged polymers are used as film coatings for pharmaceutical coatings due to their enteric or enteroprotective properties, as reviewed in Joshi et al., "Film coatings for taste masking and moisture protection", International Journal of Pharmaceutics, 457:395 (2013), which is incorporated herein by reference in its entirety. Examples of cationic charged polymers include aminodimethyl or diethyl methacrylate copolymers, known commercially available as Eudragit® E and Kollicoat® Smartseal 30D. Examples of anionic charged polymers include methacrylic acid copolymers or Eudragit® S / L, where L100-55, L100, and S100 are used to trigger release at pH 5.5, 7.0, and 8.0, respectively. Another type of anionic polymer is cellulose derivatives, such as carboxymethyl cellulose (Akucel), cellulose acetate phthalate (Aquacoat CPD), and cellulose acetate butyrate (Eastman CAB). According to this disclosure, these charged polymer systems are used as a reference due to their commercial availability.

[0271] Water-insoluble polymers are summarized in the review by Joshi et al., "Film coatings for taste masking and moisture protection", International Journal of Pharmaceutics, 457:395 (2013), which is hereby incorporated by reference. Typically, these polymers are designed to achieve sustained or prolonged drug release. As coating materials, these polymers can be blended with water-soluble polymers (such as PEG or PVP), where the latter forms pores (porogens) to allow the drug to diffuse slowly from the core. Examples of water-insoluble polymers include ethyl cellulose (Ethocel), cellulose acetate (Opadry CA), ammonium methacrylate copolymers (Eudragit® RS100 / RL100), and polyvinyl acetate (Kollicoat® SR). According to this disclosure, these polymers are used as a reference due to their commercial availability.

[0272] In summary, materials for controlled release may include, for example, materials for immediate release, such as Opadry AMB (II), Opadry II (TF), AquaPolish, Starch 1500, Methocel E3, E5, Kollidon® VA64, Kollicoat® Protect / IR, and Soluplus. Other materials for controlled release may include sustained-release materials, such as Eudragit® RL PO, Eudragit® RS PO, Methocel E15, K4M, Kollidon® SR (30D), Surelease, Ethocel / Klucel, Methocel E15, K4M, Aquacoat, and Opadry CA. Other materials for controlled release may include delayed-release materials, such as Kollicoat® MAE-100, Eudragit® L100, Eudragit® S100, Sureteric, HPMC acetate succinate, or CMC.

[0273] Generally, polymers used in moisture-proof film coatings exhibit low water permeability, typically through the use of water-insoluble polymers. A review of materials used in moisture-proof film coatings is available in the following references: Joshi et al., "Film coatings for taste masking and moisture protection", International Journal of Pharmaceutics, 457:395 (2013) and Yang et al., "An Update of Moisture Barrier Coating for Drug Delivery", Pharmaceutics, 11:436 (2019), both of which are incorporated herein by reference in their entirety. However, water-soluble polymers, such as Kollidon® VA64, Kollicoat® Protect, and Eudragit® L100 / S100, have also been studied for their moisture absorption, as discussed in the references. High coating thickness / content (≥20%) or the addition of hydrophobic / water-insoluble additives, such as stearic acid, typically helps reduce moisture absorption.

[0274] The materials that can be used further include those described in Table 3 below. These materials are summarized based on their chemical and physical properties. Their potential functions for moisture protection and static electricity reduction are estimated based on commercial product specifications and chemical properties. [ [surface] [3.]] [] [Materials Review and Selection.] [Function] [Water-soluble] [Viscosity] [Moisture-proof] [Antistatic] PVP copolymer That is, BASF Kollidon® VA64 Tablet adhesives, film coatings Yes, 6:4 PVP: PVAc, can combine with sugars. Low: 20 mPa·s (MW 45-75 kDa) in water at 20% concentration. Yes, its hygroscopicity is lower than Kollidon® 30. No, it may contain TiO2. Luviquat® Polyquaternium 16 Conditioner, antistatic (hair care) Yes, PVP and quaternary polyvinyl-imidazolium At K-30, the K value is low: at 10% <10 mPs.s (40-80 kDa). No, it is very hydrophilic. Yes, Luviquat® Excellence and FC 550 (6.0 and 3.3) HPMC (i.e., Dow Methocel K3, E5) Film coating. ShinEtsu Pharmacoat 603-615 Yes, at 0℃-30℃: K3, E3, E5, E6, E15, E50 Low (LV): 2-50 mPa·s (MW 10-25 kDa) at 2% No, it can be mixed with MC A15 or SM-4. No, it may contain TiO2 (in Opadry II). PVA (ie, Colorcon Opadry® AMB II) Film coating. Exists as II, AMB, and AMB II. Yes, AMB containing PEG may be better. Medium: 110 mPa·s at 20%, lower than AMB Yes, it is better than AMB and II. Possibly due to TiO2 PVA (i.e., BASF Kollicoat® IR, Protect) Film coating. As an IR / Protect (+40% PVA) coating. Yes, 3:1 PVA:PEG graft. Protect: +40% PVA Medium: At 20%, IR 115 mPa.s, MW 45 kDa. Protect 240 mPa.s. Yes, Kollicoat® Protect No, it may contain TiO2. It inherently contains 0.2% SiO2. Eudragit® L100 (L12.5) L100-55 Enteric coating. 1:1 PMAA: PMMA 1:1 PMAA: PEA Yes, pH > 10 (1N NaOH) or pH > 7 (1N buffer, i.e., Tris). Medium: L100 MW 125 kDa (50-200 mPa.s) L100-55 MW 320 kDa Yes, the L100-55 is better than the L100. No, it may contain TiO2. [Example] [13-] [nuclear] [-] [Shell form: Excipient to be dissolved in the spray solvent and rehydration buffer.] []

[0275] The concept used for solubility screening is shown in Figure 35.

[0276] The results for Kollidon® VA64, Efka®, and Eudragit®, which are soluble in the spray solution (15% water / solvent) and buffer solution, are shown in Figure 36.

[0277] The polymer solubility and pH screening results are described in Table 4. Polymers such as Kollicoat® IR, Protect, Kollidon® VA64, Methocel, and Metalose are preferred due to their solubility in aqueous solutions. [ [surface] [4.]] [] [Solubility screening of polymers selected to minimize water absorption by freeze-dried microspheres. Weight of soluble polymers] [%] [and its counterpart solution] [pH] [The impact of this (promising additive).] [polymer] (Moisture-proof) [water] [IPA] [MOPS ph 7.5] [IMX pH 9.9] Kollicoat® Protect 10.0% 6.92 x 10.0% 7.71 10.0% 8.83 Kollicoat® IR 10.0% 6.58 x 10.0% 7.56 10.0% 8.93 Kollicoat ®MAE-100P x x 1.0% 6.83 1.0% 6.87 Collidon ®V64 10.0% 5.21 10.0% 10.0% 7.53 10.0% 9.06 Collidon ®30 10.0% 3.84 10.0% 10.0% 7.40 p.m. 10.0% 9.52 Kollicoat ®SR x x x x Methocel LV E3 10.0% 7.51 x 10.0% 7.67 10.0% 9.78 Methocel LV E5 10.0% 7.85 x 10.0% 7.62 10.0% 9.75 Methocel LV E15 10.0% 8.07 x 10.0% 7.57 10.0% 9.76 Opadry II x x x x Opadry AMB II x x x x Opadry TF x x x x Eudragit®L100-55 x 1.0% 1.0% 6.82 1.0% 6.25 Eudragit®L100 x 1.0% 1.0% 6.74 1.0% 6.36 Metolose SM-4 1.0% 8.24 x 1.0% 7.54 1.0% 9.9 Goshenol EG-05PW 1.0% 8.18 x x x Opadry SGR x x x x

[0278] For coating applications, polymers that are insoluble in organic solvents (such as IPA) can be formulated as solid dispersions in water / alcohol mixtures. For example, Kollicoat® IR and Methocel are insoluble in solvents and use water as the flow rate; their parameters are described in Table 5 below.

[0279] Adding IPA to the pre-dissolved Kollicoat® maintains 2.5% polymer (25% water / IPA) in the suspension. Reducing water flow leads to precipitation. Adding IPA to the pre-dissolved HPMC maintains 1.4% HPMC (14% water) in the solution. It was found that replacing IPA with ethanol (and removing acetone) improves the formulation by keeping the polymer in a stable solid dispersion, which is considered sprayable for coating processes. [ [surface] [5.] [] [With water flow] [Kollicoat , ® , IR ] [and] [Methocel] [。] [] polymer 10:7:3 IPA:Acetone:Water (15% water) 10:6:4 IPA:Acetone:Water (20% water) 17:3 Ethanol:Water (15% water) Kollicoat® Protect x x 3% w / v (dispersion) Kollicoat® IR x x 3% w / v (dispersion) Kollidon® V64 10% w / v NA N / A Kollidon® 30 10% w / v NA N / A Methocel LV E3 <1% w / v 1% w / v 5% w / v Methocel LV E5 <1% w / v 1% w / v 5% w / v Methocel LV E15 x x 5% w / v Eudragit® L100-55 10% w / v NA N / A Eudragit® L100 10% w / v NA N / A Eudragit® S100 10 w / v NA N / A

[0280] Apart from Eudragit®, many of the polymers tested did not significantly lower the pH of the IMX at a concentration of 1%. Kollidon® V64 and K30 are soluble in aqueous solutions and 10% w / v IPA, making them suitable for the relevant spraying procedures. Other polymers tested were insoluble in IPA; water / solvent mixtures may be beneficial, in which greater than 15% v / v of water may be used. Most polymers lowered the pH of the IMX, with Eudragit® L100 being more effective and HPMC less effective (it is the better choice) (see Table 6 below). [ [surface] [6.]] [] [Polymer incorporated into buffer solution] [(IMX pH 9.9)] [Solubility of [something] and its effect on solutions] [pH] [The impact of this.] polymer The weight percentage of polymers soluble in IMX (pH 9.9) and their effect on the pH of IMX. Kollicoat® Protect 10% 8.83 5% 9.42 1% 9.78 0.1% 9.88 Kollicoat® IR 10% 8.93 5% 9.42 1% 9.77 0.1% 9.84 Kollidon® V64 10% 9.06 5% 9.43 1% 9.75 0.1% 9.88 Kollidon® 30 10% 9.52 5% 9.72 1% 9.84 0.1% 9.89 Methocel LV E3 10% 9.78 5% 9.80 1% 9.87 0.1% 9.89 Methocel LV E5 10% 9.75 5% 9.81 1% 9.88 0.1% 9.89 Methocel LV E15 10% 9.76 2% 9.83 0.5% 9.88 0.1% 9.88 Eudragit® L100-55 1% 6.25 0.1% 9.79 - - - - Eudragit® L100 1% 6.36 0.1% 9.78 - - - -

[0281] The polymers described herein contain a considerable amount of 3'OH and diphosphates (as shown in Figures 38, 39, 40A to 40C, 41A to 41B, 42A to 42B and 43A to 43D).

[0282] The screening results of coating materials based on order compatibility are shown in Table 7. [ [surface] [7.]] [] [Coating Material Screening Results] [] Classification of Additives IMX enhanced with additives Phasing% (≤ 0.2) Pre-order percentage (≤ 0.2) Q30 (% ≥ 90) Error rate polymer 0.5% Kollidon® VA64 √ √ 90.86 0.82 1% Kollicoat® Protect √ √ 94.15 0.35 1% Kollicoat® IR √ √ 93.22 0.37 1% Methocel E3 √ √ 88.90 0.66 1% Methocel E5 √ √ 86.99 0.88 0.5% PEG4k √ √ 94.22 0.29 0.1% Eudragit® L100 × × 65.15 15.26 0.1% Eudragit® L100-55 × × 58.88 42.17

[0283] The screening results of the polymers described in this article for DNA recombinase activity and clustering efficacy (cBOT) are shown in Figures 45A-45B and 46A-46C. The quality of the tested ffN was substantially unaffected by the spraying process: the peak area (Figure 45A) and 3'OH concentration (Figure 45B) were comparable to the control group, and no diphosphate was detected. Figures 46A-46C show that the shell packaging was shown to improve moisture protection and reduce static electricity (Figures 46A-46C).

[0284] In the presence of the polymer described herein, the corresponding DNA recombinase and DNA-binding protein activities of ExAmp after staging are shown in Figures 41A to 41B and Figures 42A to 42B.

[0285] A summary of polymer coating materials based on their ExAmp and clustering capabilities is shown in Table 8. [ [surface] [8.]] [] [based on] [ExAmp] [A summary of cluster-compatible polymers and static-reducing additives.] [] Liquid ExAmp enhanced with additives Solubility in TCX1 v.1.0 (ExAmp plus 7.5% HPBCD) DNA recombinase activity was comparable to that of the control group. Cbot was comparable to the control group (incubated for 20 min). Cbot was comparable to the control group (60 min incubation). Methocel E5 √ √ √ √ Kollidon® VA64 √ √ √ √ Kollicoat® IR (Protect) √ √ √ √ Eudragit® L100-55 √ x x x Gohsenol EG 05 √ √ x √ Luviquat® FC370 √ x x x Sodium poly(4-styrenesulfonate) √ x x x Ethoxylated PEI √ x x x polyaniline x (Requires 1:1 DMSO) √ √ √ Efka® IO 6783 √ √ √ < 1.0% √ < 1.0% Efka® IO 6786 √ √ < 1.0% x x Tween® 80 √ √ < 1.0% √ < 1.0% √ < 1.0% diethanolamine lauryl acid √ √ < 1.0% √ < 1.0% √ < 1.0% Makon® 17R4 √ √ √ √ α-Tocopherol Acetate x (Requires 1:1 DMSO) √ √ √ Trioleic acid glycerides x (Requires 1:1 DMSO) √ √ √ [Example] [14-] [Improvements through micro-packaging] [SBS] [Reagent stability.]

[0286] The microencapsulation used to improve the stability of SBS reagents is shown in Figures 43A to 43D. The formulations that reduce the static point and increase moisture resistance are selected downwards via a screening pipeline.

[0287] Table 9 shows a summary of the materials of the components described in this article. [ [surface] [9.]] [] [Summary of materials list.] polymer Potential for use as a moisture barrier Antistatic (Matrix) solubility water solubility IPA IMX pH* Viscosity* (mPa.s) ExAmp compatibility ffN compatibility Sequencing Eudragit® L100-55 √ √ pH > 7, 1% w / v 10% w / v 6.25-9.79 1.14-1.85 × √ × Kollidon® VA64 √ × 10% w / v 10% w / v 9.06-9.88 1.01-1.22 √ √(≤0.5%) √(≤0.1%) Kollicoat® IR / Protect √(Protect®) × 10% w / v × 8.83-9.88 1.11-1.33 √ √(≤1%) √(≤1%) Methocel LV E5 × N / A 10% w / v With 15% water 9.78-9.89 1.08-2.27 √ √(≤1%) √(≤1%) Opadry AMB II √ N / A × × N / A N / A N / A N / A N / A Static reduction additives Potential for use as a moisture barrier Antistatic (Matrix) solubility water solubility IPA IMX pH* Viscosity* (mPa.s) ExAmp compatibility ffN compatibility MiniSeq diethanolamine lauryl acid ×(Water-soluble, liquid) √ 2% w / v 2% w / v 9.99 1.04 √(0.1%) √ √(<0.5%) Efka® 6783 (Ammonium) ×(Water-soluble, liquid) √ 2% w / v 2% w / v 9.78-9.92 1.00-1.13 √ √ √(≤1%) Makon® 17R4 (EO / PO segment) ×(Water-soluble, liquid) √ 2% w / v 2% w / v 9.94 1.10 √ ×(<1%) √(≤1%) Efka® 6786 (Imidazolium) ×(Water-soluble, liquid) × 2% w / v 2% w / v 9.78-9.92 1.00-1.13 ×(<1%) √ √(≤0.5%) magnesium stearate √ √ × 2% w / v N / A N / A N / A N / A N / A Luviquat® FC370 × (Water-soluble) × 2% w / v 2% w / v 9.7 1.03-1.49 × × × Tocopheryl acetate √ √ 0.5% plus 0.5% DMSO 1% w / v Add 0.5% The value is 9.9 at 0.5% DMSO. 0.5% plus 1.0 at 0.5% DMSO √ √ √(≤0.5%)

[0288] Using microspheres (~20% w / v), ffN, and both DNA recombinase / BSA, the target coating concentration resulted in a 10% to 15% weight gain. The tested coating formulations were prepared with the following dry contents: Formulation A: PEG (plasticizer), Kollidon® VA64, Efka® IO 6783; Formulation B: PEG, Kollidon® VA64; Formulation C: PEG, Kollicoat® Protect, Efka® IO 6783; and Formulation D: PEG, Kollicoat® Protect. Physical characterization of the encapsulated microspheres was nearly complete, and the staging and activity tests are shown in Figure 44.

[0289] The quality of ffN was substantially unaffected by the spraying process: the peak area and 3'OH concentration were comparable to the control group, and no diphosphate was detected (see Figures 45A to 45B).

[0290] The shell encapsulation improves moisture protection and reduces static electricity (see Figures 46A to 46C). Matrix encapsulation may be limited to static electricity reduction, while the core-shell encapsulation addresses both, as shown and described in Figures 46A to 46C.

[0291] Triple-coated spheres (e.g., Eudragit® S100 (fluorescent) in shell 1, Eudragit® L100 (pale blue) in shell 2, and Eudragit® L100-55 (rose red) in shell 3) provide improved results regarding the sequential release of pH-triggered agents (e.g., pH 5, pH 6, pH 7, pH 8), as shown in Figures 49A to 49C.

[0292] Although preferred embodiments have been described and illustrated in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions and similar modifications may be made without departing from the spirit of the invention, and such modifications, additions, substitutions and similar modifications are therefore considered to be within the scope of the invention as defined in the claims followed.

[0293] none

Claims

1. A composition for encapsulating lyophilized microspheres, comprising: a shell surrounding a core, wherein the shell comprises a shell additive and the shell additive comprises an antistatic material, a moisture-proof material, or a combination thereof, wherein the core comprises one or more lyophilized microspheres, and wherein the core comprises a polymerase.

2. The composition of claim 1, wherein the shell additive is an antistatic material present in an amount not exceeding 40% w / w concentration of the shell.

3. The composition of claim 1, wherein the shell additive is a moisture-proof material present in an amount not exceeding 90% w / w concentration of the shell.

4. The composition of claim 1, wherein the shell additive comprises one or more of a polymer, an ammonium salt, a stearate derivative, an oleate derivative, a laurate derivative, an amino acid, tocopheryl acetate, piperidinium sebate, a sodium salt, a buffer solution, a chelating agent, an imidazolium salt, or any combination thereof.

5. The composition of claim 4, wherein the polymeric copolymer, second polyvinyl alcohol (PVA), polyether compound, or polyaniline.

6. The composition of claim 4, wherein the polyether compound is selected from polyethylene glycol, polypropylene glycol, block copolymers derived from ethylene oxide (EO) and propylene oxide (PO), or any combination thereof.

7. The composition of claim 4, wherein the amino acid is selected from one or more of leucine, isoleucine, phenylalanine, or any combination thereof.

8. The composition of claim 4, wherein the sodium salt is selected from one or more of sodium chloride, sodium bisulfite, sodium citrate, or any combination thereof.

9. The composition of claim 4, wherein the buffer solution is Trizma, Tris.HCl, or a combination thereof.

10. The composition of claim 4, wherein the ammonium salt is selected from tetraalkylammonium chloride, tris(hydroxyethyl)alkylammonium chloride, or a combination thereof.

11. The composition of claim 4, wherein the imidazolium salt is selected from 1-ethyl-3-methyl-imidazolium salt, polytetraammonium salt, or Luviquat® (a copolymer of vinylpyrrolidone and tetramethylvinylimidazolium), or a combination thereof.

12. The composition of claim 1, wherein the shell additive comprises an ammonium salt, a polyvinyl alcohol-grafted polyethylene glycol copolymer, polyvinyl alcohol (PVA), or any combination thereof.

13. The composition of claim 1, wherein the shell additive comprises a copolymer.

14. The composition of claim 1, wherein the core further comprises one or more additional agents, wherein the additional agents are selected from one or more sugars, one or more amino acids, one or more polymers, one or more mesoporous silicas, one or more quaternary amines, and any combination thereof.

15. The composition of claim 14, wherein when the additional agent contains one or more sugars, the sugar is selected from trehalose, cyclodextrin, polydextrose, sucrose, or any combination thereof.

16. The composition of claim 14, wherein the additional agent comprises mannitol.

17. A composition as claimed in claim 14, wherein the additional agent comprises one or more amino acids having hydrophobic side chains.

18. A composition for encapsulating lyophilized microspheres, comprising: a shell surrounding a core, wherein the core comprises a core additive and the core additive comprises an antistatic material; wherein the core comprises a polymerase; and wherein (i) the core additive is an antistatic material present in an amount not exceeding 25% w / w of the core, or (ii) the amount of the core additive is between 2% w / w and 10% w / w of the core.

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