Methods, systems, kits, and devices comprising peptides

Abstract

Provided herein are compositions, systems, kits, devices, and methods comprising peptides of interest, mixture of individual amino acids, an aqueous solution, and uses thereof. The methods comprising peptides of interest may be characterized as methods of synthesizing a peptide. These peptides of interest may be characterized as hydrophobic or partially hydrophobic. These individual amino acids may be characterized as natural amino acids or unnatural amino acids. The aqueous solution may be characterized as pure or substantially pure water. Various compositions, systems, kits, devices, or methods of the present disclosure may involve synthesizing a peptide of interest from a template peptide.

Description

[0001] METHODS, SYSTEMS, KITS, AND DEVICES COMPRISING PEPTIDES

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] [1] This application claims the benefit of priority to UK Patent Application 2409416.1, filed June 28, 2024, the contents of which are incorporated herein by reference in its entirely.

[0004] BACKGROUND

[0005] [2] A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar-substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. Peptides are also increasingly being used in cosmetic applications for application to skin and hair, for example.

[0006] [3] Peptides are currently generally made by recombinant means using bacterial or eukaryotic cell cultures or solid phase peptide synthesis, using FMOC or FBOC protected amino acids. Both processes are costly and purification of the peptides can be onerous. Moreover, for solid phase synthesis methods copious amounts of toxic organic solvents are required for the chemistry reactions and removal of the resulting waste material is approximately 10% of the overall cost of peptide production.

[0007] [4] Native peptide ligation of peptides has been undertaken to provide proteins from large peptide fragments. Typically, this requires one half of the peptide to have a reactive thioester chemistry to enable the reaction. Further this technique relies on solid phase synthesis of two portions or halves of the desired peptide before these portions or halves are ligated together.

[0008] SUMMARY

[0009] [5] The present disclosure provides compositions, systems, devices, kits, and methods comprising synthesized peptides and uses thereof. Provided herein are compositions, systems, kits, devices, and methods comprising peptides of interest, mixture of individual amino acids, an aqueous solution, and uses thereof. The methods comprising peptides of interest may be characterized as methods of synthesizing a peptide. These peptides of interest may be characterized as hydrophobic or partially hydrophobic. These individual amino acids may be characterized as natural amino acids or unnatural amino acids. The aqueous solution may be characterized as pure or substantially pure water. Various compositions, systems, kits, devices, or methods of the present disclosure may involve synthesizing a peptide of interest from a template peptide.

[0010] [6] The present disclosure relies on studies concerning the synthesis of peptides without the use of complex chemistry, high temperature and / or pressure or DNA templates, or any cellular components, to facilitate peptide synthesis. Unlike solid phase synthesis of two portions or halves of the desired peptide and then ligation of these portions or halves together, the templated method described herein does not rely on having the majority of the peptide presynthesized. The method of synthesizing a peptide from a template peptide described herein mainly requires constituent amino acids.

[0011] [7] Unexpectedly, and as described in the present disclosure, it has been found that a templated synthesis of peptides can occur by providing low amounts of energy to amino acids in solution.

[0012] Certain Embodiments

[0013] [8] The present disclosure provides compositions, systems, devices, kits, and methods comprising synthesized peptides and uses thereof. Provided herein are compositions, systems, kits, devices, and methods comprising a template peptide, a mixture of individual amino acids, an aqueous solution, and optionally a synthesized peptide.

[0014] [9] Provided herein are methods of synthesizing a peptide, the method comprising: (a) providing a template peptide; providing a mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in a template peptide sequence; (b) contacting the template peptide and the mixture of individual amino acids in an aqueous solution, wherein the conditions can comprise: a pH of about 3.0-10.0; a relative humidity of about 10% to about 90%; or a pressure of about 5 mbar to about 10 mbar; (c) providing of energy to the aqueous solution of the template peptide and the mixture of individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the template peptide amino acid sequence; and performing step (d) for a time period sufficient to synthesize the peptide from the mixture of individual amino acids, wherein the amino acid sequence of the peptide is the same as the amino acid sequence of the template peptide. In some embodiments, the template peptide can comprise about 2 amino acids to about 200 amino acids. In some embodiments, the peptide can comprise amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% hydrophobic. In some embodiments, the conditions can further comprise a reaction temperature of about 37°C to about 70°C. In some embodiments, the conditions can further comprise a reaction temperature of about 40°C. In some embodiments, the conditions can further comprise a reaction temperature of about 32°C In some embodiments, the pH is about 6.0. In some embodiments, the pressure is about 8 mbar. In some embodiments, the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the providing of energy to the aqueous solution is by a cyclical provision. In some embodiments, the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light. In some embodiments, periodically exposing the aqueous solution to full spectrum light can comprise a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution. In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the aqueous solution comprises a phosphate buffered saline solution. In some embodiments, the aqueous solution comprises an acid or a base. In some embodiments, the acid or the base is HC1, Formic Acid, or NaOH. In some embodiments, the aqueous solution can further comprise the template peptide and the mixture of individual amino acids after contacting. In some embodiments, the aqueous solution is sterile. In some embodiments, the mixture of individual amino acids comprises only the amino acids that constitute the template peptide. In some embodiments, the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 or 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide. In some embodiments, the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 300 to 1 and 10 to 1, or between 150 to 1 and 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide. In some embodiments, the concentration of the mixture of individual amino acids in the aqueous solution prior of to the providing of energy is from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL. In some embodiments, the time period is from 10 minutes to 5 days. In some embodiments, peptide synthesis is terminated by separation of the peptide from the mixture of individual amino acids in the aqueous solution. In some embodiments, the method is carried out in the presence of oxygen, Nitrogen, hydrogen, or CO2. In some embodiments, the method is performed in the absence of nucleic acids, enzymes, co-enzymes, other cellular material or cells. In some embodiments, the mixture of individual amino acids comprises natural amino acids. In some embodiments, the mixture of individual amino acids comprises unnatural amino acids. In some embodiments, the unnatural amino acids comprise labeled amino acids. In some embodiments, the mixture of individual amino acids comprises: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, or combinations thereof. In some embodiments, synthesizing a peptide is optimized for maximizing peptide yield and minimizing contaminants. In some embodiments, the method further comprises drying the peptide after step (e). In some embodiments, the drying results in 40% to 60% volume reduction. In some embodiments, the drying results in 50% volume reduction.

[0015]

[0010] Also provided herein are devices comprising: a sample interface configured to receive a sample comprising a template peptide; a chamber fluidically connected to the sample interface; and wherein the chamber comprises an aqueous solution, and a mixture of individual amino acids such that a peptide is synthesized with the mixture of individual amino acids in aqueous solution within the chamber. In some embodiments, synthesizing a peptide within the chamber comprises: (a) [1] contacting the template peptide and mixture of individual amino acids in the aqueous solution within the chamber; and (b) providing of energy to the aqueous solution for a time period sufficient to synthesize the peptide with the mixture of individual amino acids, wherein the amino acid sequence of the synthesized peptide is the same as the amino acid sequence of the template peptide. In some embodiments, a peptide is synthesized in conditions comprising: (a) a reaction temperature of about 37°C to about 70°C; (b) a pH of about 3.0-10.0; (c) a relative humidity of about 10% to about 90%; or (d) a pressure of about 5 mbar to about 1000 mbar. In some embodiments, the aqueous solution comprising the peptide is passed through a preparative column to separate the peptide from the aqueous solution after peptide synthesis is terminated. In some embodiments, the preparative column is a Cl 8 column. In some embodiments, the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the providing of energy to the aqueous solution is by cyclical provision. In some embodiments, the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light. In some embodiments, periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 3 cm from the aqueous solution. In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the aqueous solution comprises a phosphate buffered saline solution. In some embodiments, the aqueous solution comprises an acid or a base. In some embodiments, the acid or the base is HC1, Formic Acid, or NaOH. In some embodiments, the device further comprises a pump for vacuum. In some embodiments, the device further comprises a component for reaction temperature regulation. In some embodiments, the device further comprises a component for evaporation and / or condensation regulation. In some embodiments, the device can further comprise a component for addition and removal of reagents. In some embodiments, the device can further comprise a component for purification of synthesized peptide. In some embodiments, the device can further comprise a component for removal of synthesized peptide. In some embodiments, the device can further comprise a membrane to separate the peptide from the aqueous solution after peptide synthesis is terminated. In some embodiments, the membrane is a hydrophilic membrane. In some embodiments, the membrane is a a size-specific membrane. In some embodiments, the device can further comprise tubes with high optical transmission. In some embodiments, the device can further comprise tubes and / or containers in non-reactive or absorptive material such as glass or stainless steel. In some embodiments, the device can further comprise a component for monitoring and documenting the progress of peptide synthesis. In some embodiments, the device can further comprise a component for monitoring and documenting the contamination profile during peptide synthesis and / or after peptide synthesis is terminated. In some embodiments, the device can comprise a component for monitoring the progress of peptide synthesis with visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof. In some embodiments, the device can further comprise a component for sampling the reaction mixture to assess the progress of peptide synthesis. In some embodiments, sampling can comprise inline sampling comprising receiving a sample, pumping the sample down a monolithic column, drying the sample in drying gas, characterizing the sample, or combinations thereof. In some embodiments, sampling the reaction mixture to assess the progress of peptide synthesis indicates whether to add individual amino acids or remove individual amino acids from the aqueous solution. In some embodiments, the device is programmed to adhere to a method of synthesizing a peptide comprising at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof. In some embodiments, the device can comprise multiple detection components. In some embodiments, the device can comprise a detector for measuring the template peptide, aqueous solution, individual amino acids, synthesized peptide, or combinations thereof. In some embodiments, the detector is capable of visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof. In some embodiments, the device can comprise touch screen user interface. In some embodiments, the device is a bench top instrument. In some embodiments, the device is capable of synthesizing 10-100 g of peptide. In some embodiments, the device is a microfluidic device.

[0016]

[0011] Also provided herein are devices for performing the method of synthesizing a peptide as disclosed herein, the device comprising a structural component as well as a composition component, wherein the composition component is a reaction mixture comprising a template peptide, an aqueous solution, a mixture of individual amino acids, and optionally a synthesized peptide.

[0017]

[0012] Also provided herein are systems for synthesizing a peptide from a template peptide, the system comprising: a mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in the template peptide, wherein the mixture is configured to contact the template peptide and the mixture of individual amino acids in an aqueous solution; and an energy generating component that is configured to provide an amount of energy to the aqueous solution of the template peptide and the mixture of individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the template peptide amino acid sequence, wherein the system is configured to synthesize the peptide from the template peptide under conditions that comprise: a pH of about 3.0-10.0; a relative humidity of about 10% to about 90%; or a pressure of about 5 mbar to about 10 mbar. In some embodiments, the template peptide comprises about 2 amino acids to about 200 amino acids. In some embodiments, the peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% hydrophobic. In some embodiments, the conditions further comprise a reaction temperature of about 37°C to about 70°C. In some embodiments, wherein the conditions further comprise a reaction temperature of about 40°C. In some embodiments, the conditions further comprise a reaction temperature of about 32°C. In some embodiments, the pH is about 6.0. In some embodiments, the pressure is about 8 mbar. In some embodiments, the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the providing of energy to the aqueous solution is by a cyclical provision. In some embodiments, the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light. In some embodiments, periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution. In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the aqueous solution comprises a phosphate buffered saline solution. In some embodiments, the aqueous solution comprises an acid or a base. In some embodiments, the acid or the base is HC1, Formic Acid, or NaOH. In some embodiments, the aqueous solution further comprises the template peptide and the mixture of individual amino acids after contacting. In some embodiments, the aqueous solution is sterile. In some embodiments, the mixture of individual amino acids comprises only the amino acids that constitute the template peptide. In some embodiments, the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 and 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide. In some embodiments, the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 300 to 1 and 10 to 1, or between 150 to 1 and 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide. In some embodiments, the concentration of the mixture of individual amino acids in the aqueous solution prior of to the providing of energy is from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL. In some embodiments, the time period is from 10 minutes to 5 days. In some embodiments, peptide synthesis is terminated by separation of the peptide from the mixture of individual amino acids in the aqueous solution. In some embodiments, the method is carried out in the presence of oxygen, Nitrogen, hydrogen, or CO2. In some embodiments, the method is performed in the absence of nucleic acids, enzymes, co-enzymes, other cellular material or cells. In some embodiments, the mixture of individual amino acids comprises natural amino acids. In some embodiments, the mixture of individual amino acids comprises unnatural amino acids. In some embodiments, the unnatural amino acids comprise labeled amino acids. In some embodiments, the mixture of individual amino acids comprises: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, or combinations thereof. In some embodiments, the system is configured to maximize peptide yield and minimize contaminants after peptide synthesis. In some embodiments, the system further comprises a drying component configured to dry peptide after peptide synthesis. In some embodiments, the drying results in 40% to 60% volume reduction.

[0018] In some embodiments, the drying results in 50% volume reduction.

[0019] INCORPORATION BY REFERENCE

[0020]

[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0021] BRIEF DESCRIPTION OF DRAWINGS

[0022]

[0014] FIGURES 1A-1D show the effects of ratio of template to amino acids on peptide WK20 amplification. FIG. 1A shows the calculated change in ratio of template to amino acids (1:N) after each round of peptide amplification. Y axis represents a ratio of template to amino acids (N). X axis represents the number of rounds of amplification. FIG. IB shows the 50% evaporation curve during peptide amplification. Y axes represent that area under the curve (AUC), and the mAU 218 nm. X-axis represents the number of rounds of amplification. FIG. 1C shows the yield of semapeptide with different ratios of sema template to amino acid. Y axes represent peptide amounts (mg), and the mAU 218 nm. X-axis represents ratio of template (1) to amino acids (N). FIG. ID shows the calculated change in ratio of template to amino acids (1:N) after each round of semapeptide amplification. Y axis represents a ration of template to amino acids (N). X axis represents the number of rounds of amplification.

[0023]

[0015] FIGURES 2A-2F show the optimization of the drying step on product recovery. FIG. 2A shows, from left to right, amplification product of WK20 in vials before drying, 3 minutes after drying started (50% volume remaining), 5 minutes after drying started (28% volume remaining), and 7 minutes after drying started (14% volume remaining). FIG. 2B shows the recovery yield for each vial. Y-axis shows the amount of peptide (mg). X-axis shows experimental condition. FIGs. 2C - 2D show chromatograms of sample purification for control (FIG. 2C), 3 minutes (FIG. 2D), 5 minutes(FIG. 2E), and 7 minutes (FIG. 2F). Y-axis represents mAU. X-axis represents retention time.

[0024]

[0016] FIGURES 3A-3B show the mass spectra of peptides amplified according to the methods disclosed herein. FIG. 3A shows the chromatography trace an exemplary peptide verified after amplification. Y-axis represents relative abundance depicted as % intensity. X- axis represents mass-to-charge (m / z) ration. FIG. 3B shows the tandem mass spectra spectra of the same exemplary peptide after amplification. Y-axis represents relative abundance depicted as % intensity. X-axis represents mass-to-charge (m / z) ration.

[0025]

[0017] FIGURE 4 shows the effects of temperature on peptide synthesis. Y axis represents the yield of peptide amplification (in milligrams). X axis represents the temperature.

[0026] DETAILED DESCRIPTION

[0027]

[0018] It is to be understood that both the foregoing general description and the following detailed description are exemplary, and explanatory only, and are not restrictive of the disclosure.

[0028]

[0019] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0029]

[0020] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0030] Definitions

[0031]

[0021] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated or obvious from context, the following terms have the following meanings:

[0032]

[0022] The terms, “a,” “an,” and “the,” as used herein, include plural references unless the context clearly dictates otherwise.

[0033]

[0023] The terms, “or” and “and / or,” as used herein, include any and all combinations of one or more of the associated listed items.

[0034]

[0024] The terms, “including,” “includes,” “included,” and other forms, are not limiting.

[0035]

[0025] The terms, “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0036]

[0026] The term, “about,” as used herein in reference to a number or range of numbers, is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0027] The term “actuator,” as used herein in reference to a microfluidic device, refers to a component that causes a machine or other device to operate. An actuator may be a component of a machine that is responsible for moving and controlling a mechanism or system, such as, for example, controlling the opening or closing of a valve.

[0037]

[0028] The terms, “amplification,” “amplifying,” and grammatical equivalents thereof, as used herein, refer to a process by which a peptide copied to generate a plurality of peptides containing the same sequence as the original peptide or a distinguishable portion thereof.

[0038]

[0029] The terms, “bind,” “binding,” “contacting,” “interact” and “interacting,” as used herein, refer to a non-covalent interaction between macromolecules (e.g., between two peptides, between a peptide and a nucleic acid; and the like). While in a state of noncovalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Non-limiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions. Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific.

[0039]

[0030] The term “cancer,” as used herein, can refer to a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication. The term cancer may be used interchangeably with the terms “carcino-,” “onco-,” and “tumor.”

[0040]

[0031] The term, “chamber,” and “channel,” when used interchangeably herein with reference to a device, such as a microfluidic device, refers to a compartment, which is at least partially enclosed, in the device, such as a separate section, area, or passageway, in which a composition, system, sample, fluid, gas, or loose material may be contained in isolation and / or in which an activity, such as a reaction, can occur. A chamber or channel is generally connected or communicating with another component of the device. A chamber or channel may contain or have the ability to contain matter, such as reagents. Contained materials, such as a composition, system, sample, fluid, gas, or loose material, may be obstructed or allowed movement through a structural component of the device in a controlled manner. Contained materials may be allowed movement from one structural component of the device to another. Alternatively or in addition, a chamber or channel can also direct or vent air or gases. By way of non-limiting example, a chamber or channels may comprise one or more hydrogels, a well, a flow strip, a heating element, or combinations thereof. Also, by way of non-limiting example, one or more chambers or channels may be in fluid communication, optical communication, or thermal communication. As another non-limiting example, the chambers or channels may be arranged in a sequence, in parallel, or both.

[0041]

[0032] The term, “detection event,” as used herein in reference to a microfluidic device, generally refers to a moment in which compositions within the detection region of a microfluidic device bind to a synthesized peptide or detect peptide bond formation, in accordance to the assay(s) being performed. A detection event may produce a detectable product or a detectable signal.

[0042]

[0033] The term, “detectable product,” as used herein, refers to a unit produced after peptide bond formation that is capable of being discovered, identified, perceived or noticed. A detectable product can comprise a detectable label and / or moiety that emits a detectable signal. A detectable product may include other components that are not capable of being readily discovered, identified, perceived or noticed at the same time as the detectable signal. For example, a detectable product may comprise remnants of a reporter.

[0043]

[0034] The term, “detectable signal,” as used herein, refers to an act, event, physical quantity or impulse that can be detected, discovered, identified, perceived or noticed using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art.

[0044]

[0035] The term, “detection region,” as used herein in reference to a microfluidic device, generally refers to a structural component which may comprise detection reagents that are immobilized, dried, or otherwise deposited thereto. A detection region may comprise one or more dried and / or immobilized amplification reagents. Accordingly, in some instances, a detection region may comprise a plurality of microwells, detection chambers or channels, in fluid communication with amplification region(s). By way of a non-limiting example, a detection region may comprise parallel detection chambers, each coupled to a single amplification region. One of ordinary skills in the art will recognize that the relative numbers of and relationships between amplification region(s) and detection region(s) may be varied depending on the assay(s) being performed. Also, by way of a non-limiting example, compositions within the detection region of a microfluidic device may be agitated (e.g., via a spring-loaded valve piston).

[0045]

[0036] The terms “heater”, “heating unit”, “heating element”, “heat source”, and the like, as used herein in reference to a device, generally refers to an element that is configured to produce heat and is in thermal communication with a portion of a device.

[0037] The term, “zw vitro ” as used herein, refers to describing something outside an organism. An in vitro system, composition or method may take place in a container for holding laboratory reagents such that it is separated from the biological source from which a material in the container is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. The term “in vivo” is used to describe an event that takes place within an organism. The term “ex vivo” is used to describe an event that takes place in a cell that has been obtained from an organism. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.

[0046]

[0038] The terms, “length” and “linked” as used herein, refer to a nucleic acid (polynucleotide) or polypeptide, may be expressed as “kilobases” (kb) or “base pairs (bp),”. Thus, a length of 1 kb refers to a length of 1000 linked nucleotides, and a length of 500 bp refers to a length of 500 linked nucleotides. Similarly, a protein having a length of 500 linked amino acids may also be simply described as having a length of 500 amino acids.

[0047]

[0039] The terms, “non-naturally occurring” and “engineered,” as used herein, refer to indicate involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a molecule, such as but not limited to, a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid refers to a modification of that molecule (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the natural molecule. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a nonlimiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man.

[0048]

[0040] The terms, “peptide,” “polypeptide” and “protein,” as used herein, refer to a polymeric form of amino acids. A polypeptide may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, polypeptides as described herein may comprise one or more mutations, one or more engineered modifications, or both. It is understood that when describing coding sequences of polypeptides described herein, said coding sequences do not necessarily require a codon encoding an N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein. One skilled in the art would understand that a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell. In some instances, when a heterologous peptide, such as a fusion partner protein, protein tag or NLS, is located at the N terminus of the effector protein, a start codon for the heterologous peptide serves as a start codon for the effector protein as well. Thus, the natural start codon encoding an amino acid residue sufficient for initiating translation (e.g., Methionine (M) or a Valine (V)) of the effector protein may be removed or absent.

[0049]

[0041] The term “reagent mix”, “reagent master mix”, “reagents”, and the like, as used herein, generally refers to a formulation comprising one or more chemicals that partake in a reaction that the formulation is intended for.

[0050]

[0042] [3] The term, “sample,” as used herein, refers to something comprising a peptide of interest. In some instances, the sample is a biological sample, such as a biological fluid or tissue sample. In some instances, the sample is an environmental sample. The sample may be a biological sample or environmental sample that is modified or manipulated. By way of nonlimiting example, samples may be modified or manipulated with purification techniques, digestion techniques, heat, nucleic acid amplification, salts and buffers.

[0051]

[0043] [4] The terms “sample interface” and “sample input,” as used herein in reference to a microfluidic device, generally refer to a structural component capable of receiving a composition comprising a peptide of interest as disclosed herein (e.g., a sample). The composition comprising a peptide of interest may be a sample as defined above, which may be collected with a sample collector (e.g., swab, tube, etc.) before being received in a sample interface. By way of a non-limiting example, the sample may be directly collected at the sample interface (e.g., without the use of a separate sample collector). In some instances, a sample interface may be in fluid communication with a plurality of chambers, channels, or reservoirs of a microfluidic device. In some instances, the sample interface is fluidically connected to the plurality of chambers via lysis, preparation, amplification, or detection regions. The term, “subject,” as used herein, refers to an animal. The subject may be a mammal. The subject may be a human. The subject may be diagnosed or at risk for a disease.

[0052]

[0044] The term, “syndrome,” as used herein, refers to a group of symptoms which, taken together, characterize a condition.

[0045] The terms, “thermostable” and “thermostability” refer to the stability of a composition disclosed herein at one or more temperatures, such as an elevated operating temperature for a given reaction. Stability may be assessed by the ability of the composition to perform an activity, e.g., cleaving a target nucleic acid or reporter. Improving thermostability means improving the quantity or quality of the activity at one or more temperatures.

[0053]

[0046] The term, “template peptide,” as used herein, refers to a peptide that is (designed or intended to be) used as a reference in the synthesis of a peptide, such that the synthesized peptide has the same amino acid sequence as the template peptide.

[0054]

[0047] The terms, “treatment” and “treating,” as used herein, refer to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0055]

[0048] The term, “valve,” as used herein, refers to a mechanism or device for directing, regulating, controlling, or obstructing the passage of fluid, gas, or loose materials through an opening or passageway. A valve may regulate the movement of fluid through an opening in one direction only. A valve may operate automatically, pneumatically, hydraulically, mechanically, electrically, chemically or combinations thereof.

[0056]

[0049] The term, “variant,” as used herein, refers to a form or version of a peptide that differs from the wild-type peptide. A variant may have a different function or activity relative to the wild-type peptide. Introduction

[0057]

[0050] Disclosed herein are compositions, systems, devices, kits, and methods comprising at least one of: a) A template peptide; b) A mixture of amino acids; and c) An aqueous solution.

[0058]

[0051] Also provided herein are compositions, systems, kits, devices, and methods comprising peptides of interest, mixture of individual amino acids, an aqueous solution, and uses thereof. Various compositions, systems, kits, devices, or methods of the present disclosure may involve synthesizing a peptide from a template peptide. The methods comprising peptides may be characterized as methods of synthesizing a peptide. In some embodiments, a template peptide comprises the same amino acid sequence as a peptide of interest. In some embodiments, a synthesized peptide is referred to as a peptide or peptide product. In some embodiments, the template peptide or peptide are characterized as hydrophobic or partially hydrophobic. In some embodiments, the individual amino acids are characterized as natural amino acids or unnatural amino acids. In some embodiments, the aqueous solution is characterized as pure or substantially pure water.

[0059]

[0052] In general, compositions, methods and systems described herein are not found in nature or not naturally occurring. In some embodiments, compositions, systems, devices, kits, and methods described herein comprise at least one non-naturally occurring component. In some embodiments, compositions, systems, devices, kits, and methods comprise at least two components that do not naturally occur together. In some embodiments, compositions, systems, devices, kits, and methods described herein comprise a peptide that is similar to a naturally occurring peptide. In some embodiments, the peptide lacks a portion of the naturally occurring peptide. In some embodiments, the peptide comprises a mutation relative to the naturally occurring peptide, wherein the mutation is not found in nature. In some embodiments, the peptide also comprises at least one additional amino acid relative to the naturally-occurring peptide. Conversely, and for clarity, a peptide that is “natural,” “naturally occurring,” or “found in nature” includes peptides from cells or organisms that have not been genetically modified by a human or machine. Peptide

[0060]

[0053] Provided herein are compositions, systems and methods comprising a peptide or peptide of interest. A peptide of interest as described herein can also be referred to as a template peptide in the present disclosure.

[0061]

[0054] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide. In some embodiments, the methods described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide. In some embodiments, the template peptide is a peptide from a biological sample.

[0062]

[0055] In some embodiments, the peptide comprises a length of at least 2 amino acids to at least 200 amino acids. In some embodiments, the peptide comprises at least 2 amino acids to at least 200 amino acids. In some embodiments, the peptide comprises at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, at least 180 amino acids, at least 190 amino acids, at least 200 amino acids, at least 210 amino acids, at least 220 amino acids.

[0063]

[0056] In some embodiments, the peptide comprises a length of about 2 amino acids to about 200 amino acids. In some embodiments, the peptide comprises about 2 amino acids to about 200 amino acids. In some embodiments, the peptide comprises about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100 amino acids, about 110 amino acids, about 120 amino acids, about 130 amino acids, about 140 amino acids, about 150 amino acids, about 160 amino acids, about 170 amino acids, about 180 amino acids, about 190 amino acids, about 200 amino acids, about 210 amino acids, about 220 amino acids.

[0064]

[0057] In some embodiments, the peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 25% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 30% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 35% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 40% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 45% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 50% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 55% hydrophobic. In some embodiments, the peptide comprises amino acids that are z bout 60% hydrophobic.

[0065]

[0058] In some embodiments, the template peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 25% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 30% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 35% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 40% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 45% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 50% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 55% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 60% hydrophobic.

[0066]

[0059] In some embodiments, the peptide comprises amino acids that are about 25%, 30%,

[0067] 35%, 40%, 45%, 50%, 55%, or 60% hydrophilic. In some embodiments, the peptide comprises amino acids that are about 25% hydrophilic. In some embodiments, the peptide comprises amino acids that are about 30% hydrophilic. In some embodiments, the peptide comprises amino acids that are about 35% hydrophilic. In some embodiments, the peptide comprises amino acids that are about 40% hydrophilic. In some embodiments, the peptide comprises amino acids that are about 45% hydrophilic. In some embodiments, the peptide comprises amino acids that are about 50% hydrophilic. In some embodiments, the peptide comprises amino acids that are about 55% hydrophilic. In some embodiments, the peptide comprises amino acids that are about 60% hydrophilic.

[0068]

[0060] In some embodiments, the template peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% hydrophilic. In some embodiments, the template peptide comprises amino acids that are about 25% hydrophilic. In some embodiments, the template peptide comprises amino acids that are about 30% hydrophilic. In some embodiments, the template peptide comprises amino acids that are about 35% hydrophilic. In some embodiments, the template peptide comprises amino acids that are about 40% hydrophilic. In some embodiments, the template peptide comprises amino acids that are about 45% hydrophilic. In some embodiments, the template peptide comprises amino acids that are about 50% hydrophilic. In some embodiments, the template peptide comprises amino acids that are about 55% hydrophilic. In some embodiments, the template peptide comprises amino acids that are about 60% hydrophilic.

[0069]

[0061] In some embodiments, the peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% positively charged. In some embodiments, the peptide comprises amino acids that are about 25% positively charged. In some embodiments, the peptide comprises amino acids that are about 30% positively charged. In some embodiments, the peptide comprises amino acids that are about 35% positively charged. In some embodiments, the peptide comprises amino acids that are about 40% positively charged. In some embodiments, the peptide comprises amino acids that are about 45% positively charged. In some embodiments, the peptide comprises amino acids that are about 50% positively charged. In some embodiments, the peptide comprises amino acids that are about 55% positively charged. In some embodiments, the peptide comprises amino acids that are about 60% positively charged.

[0070]

[0062] In some embodiments, the template peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% positively charged. In some embodiments, the template peptide comprises amino acids that are about 25% positively charged. In some embodiments, the template peptide comprises amino acids that are about 30% positively charged. In some embodiments, the template peptide comprises amino acids that are about 35% positively charged. In some embodiments, the template peptide comprises amino acids that are about 40% positively charged. In some embodiments, the template peptide comprises amino acids that are about 45% positively charged. In some embodiments, the template peptide comprises amino acids that are about 50% positively charged. In some embodiments, the template peptide comprises amino acids that are about 55% positively charged. In some embodiments, the template peptide comprises amino acids that are about 60% positively charged.

[0071]

[0063] In some embodiments, the peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% negatively charged. In some embodiments, the peptide comprises amino acids that are about 25% negatively charged. In some embodiments, the peptide comprises amino acids that are about 30% negatively charged. In some embodiments, the peptide comprises amino acids that are about 35% negatively charged. In some embodiments, the peptide comprises amino acids that are about 40% negatively charged. In some embodiments, the peptide comprises amino acids that are about 45% negatively charged. In some embodiments, the peptide comprises amino acids that are about 50% negatively charged. In some embodiments, the peptide comprises amino acids that are about 55% negatively charged. In some embodiments, the peptide comprises amino acids that are about 60% negatively charged.

[0072]

[0064] In some embodiments, the template peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% negatively charged. In some embodiments, the template peptide comprises amino acids that are about 25% negatively charged. In some embodiments, the template peptide comprises amino acids that are about 30% negatively charged. In some embodiments, the template peptide comprises amino acids that are about 35% negatively charged. In some embodiments, the template peptide comprises amino acids that are about 40% negatively charged. In some embodiments, the template peptide comprises amino acids that are about 45% negatively charged. In some embodiments, the template peptide comprises amino acids that are about 50% negatively charged. In some embodiments, the template peptide comprises amino acids that are about 55% negatively charged. In some embodiments, the template peptide comprises amino acids that are about 60% negatively charged.

[0073]

[0065] In some embodiments, the peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% aromatic. In some embodiments, the peptide comprises amino acids that are about 25% aromatic. In some embodiments, the peptide comprises amino acids that are about 30% aromatic. In some embodiments, the peptide comprises amino acids that are about 35% aromatic. In some embodiments, the peptide comprises amino acids that are about 40% aromatic. In some embodiments, the peptide comprises amino acids that are about 45% aromatic. In some embodiments, the peptide comprises amino acids that are about 50% aromatic. In some embodiments, the peptide comprises amino acids that are about 55% aromatic. In some embodiments, the peptide comprises amino acids that are about 60% aromatic.

[0074]

[0066] In some embodiments, the template peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% aromatic. In some embodiments, the template peptide comprises amino acids that are about 25% aromatic. In some embodiments, the template peptide comprises amino acids that are about 30% aromatic. In some embodiments, the template peptide comprises amino acids that are about 35% aromatic. In some embodiments, the template peptide comprises amino acids that are about 40% aromatic. In some embodiments, the template peptide comprises amino acids that are about 45% aromatic. In some embodiments, the template peptide comprises amino acids that are about 50% aromatic. In some embodiments, the template peptide comprises amino acids that are about 55% aromatic. In some embodiments, the template peptide comprises amino acids that are about 60% aromatic.

[0075]

[0067] In some embodiments, the peptide comprises a secondary structure. In some embodiments, the peptide comprises a helical secondary structure. In some embodiments, the peptide comprises a beta sheet secondary structure. In some embodiments the peptide comprises a random coil secondary structure. In some embodiments, the peptide comprises a secondary structure that is a combination of a helical, random coil and beta sheet. In some embodiments, the peptide comprises a structural conformation determined by one or more disulfide bonds between amino acids in the peptide. In some embodiments, the peptide comprises a structural conformation that requires folding and / or non-linear assembly. In some embodiments, the peptide is any one peptide described in TABLE 1. In some embodiments, the peptide is Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide, Teduglutide, Linaclotide, Pramlintide, Abarelix, Degarelix, Carfilzomib, Mifamurtide, Aviptadil, Atosiban, Carbetocin, Taltirelin, Bremelanotide, Teriparatide, Abaloparatide, Plecanatide, Nesiritide, Angiotensin II, Icatibant, Insulin, Enfuvirtide, Tesamorelin, Ziconotide, Romiplostim, Peginesatide, Lucinactant, Etelcalcetide, Afamelanotide, Pasireotide, Lutetium Lu 177 dotatate, Edotreotide gallium Ga-68, or Setmelanotide. In some embodiments, the peptide is a peptide with a therapeutic target described in TABLE 2. In some embodiments, the peptide is a peptide with a therapeutic target associated with a disease or syndrome described in TABLE 3.

[0076]

[0068] In some embodiments, the peptide is a peptide variant. In some embodiments, the peptide variant is a form or version of a peptide that differs from a naturally occurring peptide or a wild-type peptide. In some embodiments, the peptide variant comprises a different function or activity relative to the naturally occurring peptide or the wild-type peptide. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying the peptide variant, the naturally occurring peptide or the wild-type peptide. In some embodiments, methods for synthesizing and / or amplifying the peptide described herein comprise synthesizing and / or amplifying a peptide comprising the naturally occurring peptide sequence or the wild-type peptide sequence. In some embodiments, methods for synthesizing and / or amplifying the peptide described herein comprise synthesizing and / or amplifying a peptide comprising a peptide variant sequence.

[0077] Aqueous Solution

[0078]

[0069] Provided herein are compositions, systems and methods comprising an aqueous solution. An aqueous solution as described herein can also be referred to as a composition. In some embodiments, the aqueous solution described herein comprises system components or method components.

[0079]

[0070] In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the aqueous solution comprises pure water. For example, the aqueous solution comprises pure or substantially pure water, the template peptide and the individual amino acids capable of forming copies of the template peptide. In some embodiments, the aqueous solution comprises the template peptide and a mixture of individual amino acids.

[0080]

[0071] In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the pure or substantially pure water is MilliQ® water, which is a form of ultrapure water of Type 1 (as defined by ISO 3696 (1987)). In some embodiments, the pure or substantially pure water is provided by capacitive deionization, reverse osmosis, carbon filtering, microfiltration, ultrafiltration, ultraviolet oxidation and the like. In some embodiments, the pure or substantially pure water comprises a low level of solids, low organics and low conductivity. In some embodiments, the pure or substantially pure water comprises less than 5 pg / ml solids, less than 1 pg / ml solids, or even less than 0.1 pg / ml solids. In some embodiments, the pure or substantially pure water comprises organics content of less than 100pg / l, or less than 50pg / l total organic carbon. Additionally or alternatively, pure or substantially pure water may have a conductivity of less than IpS-cm-l, less than 0.1 pS cm- 1, or even less than 0.01 pS.cm-1 at 25°C.

[0081]

[0072] In some embodiments, the aqueous solution is free or substantially free of biological contaminants. In some embodiments, the aqueous solution is free or substantially free of nucleic acids, enzymes, co-enzymes (such as adenosine triphosphate), cells, cellular material and / or organic solvents. For example, the aqueous solution may be free or substantially free from bacteria, viruses, eukaryotic cells and / or components thereof, such as organelles.

[0073] In some embodiments, the aqueous solution is filtered. In some embodiments, the aqueous solution is sterilized. In some embodiments, the aqueous solution is irradiated. In some embodiments, the aqueous solution is irradiated with gamma irradiation. In some embodiments, the aqueous solution is sterile.

[0082]

[0074] In some embodiments, the aqueous solution comprises a buffer. In some embodiments, the buffer comprises phosphate buffers, Tris buffers, HEPES buffers, or combinations thereof. In some embodiments, the buffer comprises a saline solution. In some embodiments, the aqueous solution comprises a phosphate buffered saline solution. In some embodiments, the aqueous solution comprises an acid or a base. In some embodiments, the acid or the base is HC1, Formic Acid, or NaOH. In some embodiments, the aqueous solution comprises the water, the buffer, the saline solution, the acid, the base, or combinations thereof. In some embodiments, the aqueous solution further comprises the template peptide and the individual amino acids capable of forming copies of the template peptide.

[0083]

[0075] In some embodiments, the aqueous solution comprises a source of phosphate. In some embodiments, the aqueous solution comprises phosphate in an amount of at least 50pM, such as lOOpM, or 1 mM. In some embodiments, the aqueous solution comprises water comprising disodium hydrogen phosphate or phosphate buffered saline solution (PBS). In some embodiments, the PBS comprises disodium hydrogen phosphate, sodium chloride. In some embodiments, the PBS comprises potassium chloride and potassium dihydrogen phosphate. In some embodiments, the PBS comprises:

[0084]

[0076] In some embodiments, the aqueous solution comprises a source of phosphate, a peptide, and individual amino acids. In some embodiments, the aqueous solution comprises a source of phosphate, a template peptide, and a mixture of individual amino acids.

[0085]

[0077] In some embodiments, the aqueous solution comprises individual amino acids. In some embodiments, the aqueous solution comprising individual amino acids is contacted with a peptide. In some embodiments, the aqueous solution comprising individual amino acids is contacted with a template peptide. In some embodiments, the aqueous solution comprising individual amino acids is contacted with a peptide of interest from a biological sample.

[0086]

[0078] In some embodiments, the aqueous solution comprises a mixture of individual amino acids. In some embodiments, the aqueous solution comprising a mixture of individual amino acids is contacted with a peptide. In some embodiments, the aqueous solution comprising a mixture of individual amino acids is contacted with a template peptide. In some embodiments, the aqueous solution comprising a mixture of individual amino acids is contacted with a peptide of interest from a biological sample.

[0087]

[0079] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide in an aqueous solution. In some embodiments, the methods described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide in an aqueous solution comprising a mixture of individual amino acids. In some embodiments, the template peptide is a peptide from a biological sample. In some embodiments, the methods described herein comprise an aqueous solution described herein.

[0088] Individual Amino Acids

[0089]

[0080] Provided herein are compositions, systems and methods comprising individual amino acids or a mixture of individual amino acids. In some embodiments, the individual amino acids or the mixture of individual amino acids comprise the amino acids present in a peptide or peptide of interest. In some embodiments, the individual amino acids or the mixture of individual amino acids comprise the amino acids present in a template peptide. In some embodiments, the individual amino acids or the mixture of individual amino acids are present in an aqueous solution as described herein, which can also be referred to as a composition.

[0090]

[0081] In some embodiments, the mixture of individual amino acids comprises natural amino acids. In some embodiments, the mixture of individual amino acids comprises unnatural amino acids. In some embodiments, the unnatural amino acids comprise labeled amino acids. In some embodiments, the mixture of individual amino acids comprises: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, or combinations thereof. In some embodiments the individual amino acids comprise post translationally modified, or chemically modified amino acids which may or may not occur naturally.

[0082] In some embodiments, the mixture of individual amino acids comprises an amount that is at least equal to the stoichiometric amount of each amino acid in a template peptide sequence. In some embodiments, the mixture of individual amino acids comprises only the amino acids that constitute the template peptide. In some embodiments, the mixture of individual amino acids comprises a concentration in the aqueous solution from about 0.001 g / mL to about 5 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL. In some embodiments, the mixture of individual amino acids comprises a concentration in the aqueous solution of about 0.001 g / mL, about 0.002 g / mL, about 0.003 g / mL, about 0.004 g / mL, about 0.005 g / mL, about 0.006 g / mL, about 0.009 g / mL, about 0.010 g / mL, about 0.020 g / mL, about 0.030 g / mL, about 0.040 g / mL, about 0.050 g / mL, about 0.060 g / mL, about 0.070 g / mL, about 0.080 g / mL, about 0.090 g / mL, about 0.1 g / mL, about 0.2 g / mL, about 0.3 g / mL, about 0.4 g / mL, about 0.5 g / mL, about 0.6 g / mL, about 0.7 g / mL, about 0.8 g / mL, about 0.9 g / mL, about 1 g / mL, about 1.1 g / mL, about 1.2 g / mL, about 1.3 g / mL, about 1.4 g / mL, about 1.5 g / mL, about 1.6 g / mL, about 1.7 g / mL, about 1.8 g / mL, about 1.9 g / mL, about 2 g / mL, about 2.1 g / mL, about 2.2 g / mL, about 2.3 g / mL, about 2.4 g / mL, about 2.5 g / mL, about 2.6 g / mL, about 2.7 g / mL, about 2.8 g / mL, about 2.9 g / mL, about 3 g / mL, about 3.1 g / mL, about 3.2 g / mL, about 3.3 g / mL, about 3.4 g / mL, about 3.5 g / mL, about 3.6 g / mL, about 3.7 g / mL, about 3.8 g / mL, about 3.9 g / mL, about 4 g / mL, about 4.1 g / mL, about 4.2 g / mL, about 4.3 g / mL, about 4.4 g / mL, about 4.5 g / mL, about 4.6 g / mL, about 4.7 g / mL, about 4.8 g / mL, about 4.9 g / mL or about 5 g / mL. In some embodiments, the mixture of individual amino acids in the aqueous solution are contacted with the template peptide. In some embodiments, the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 or 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide.

[0091]

[0083] In some embodiments, the total weight of the amino acids and the weight of the template peptide may be provided in a w / w (weight by weight) ratio of anywhere between 20,000 to 1 and 10 to 1. For example, a w / w ratio between 15,000 to 1; 10,000 to 1; 5 000 to

[0092] 1; 1 000 to 1; 100 to 1 and 10 to 1 w / w; By way of further example, the solution may comprise about 30 to 1, about 29.5 to 1, about 29 to 1, about 28.5 to 1, about 28 to 1, about 27.5 to 1 about 27 to 1, about 26.5 to 1, about 26 to 1, about 25.5 to 1, about 25 to 1, about 24.5 to 1 about 24 to 1, about 23.5 to 1, about 23 to 1, about 22.5 to 1, about 22 to 1, about 21.5 to 1 about 21 to 1, about 20.5 to 1, about 20 to 1, about 19.5 to 1, about 19 to 1, about 18.5 to 1 about 18 to 1, about 17.5 to 1, about 17 to 1, about 16.5 to 1, about 16 to 1, about 15.5 to 1 about 15 to 1, about 14.5 to 1, about 14 to 1, about 13.5 to 1, about 13 to 1, about 12.5 to 1, about 12 to 1, about 11.5 to 1, about 11 to 1, about 10.5 to 1, about 10 to 1, about 9.5 to 1, about 9 to 1, about 8.5 to 1, about 8 to 1, about 7.5 to 1, about 7 to 1, about 6.5 to 1, about 6 to 1, about 5.5 to 1, about 5 to 1, about 4.5 to 1, about 4 to 1, about 3.5 to 1, about 3 to 1, about 2.5 to 1, about 2 to 1, about 1.5 to 1, or about 1 to 1 w / w of the amino acids to the template peptide.

[0093]

[0084] In some embodiments, the total weight of the amino acids in a solution result in a concentration between about O.OOlg / mL and 10 g / mL, or between about 0.005 g / mL and 5 g / mL, or between about 0.01 g / mL and 1 g / mL. In some embodiments, the solution comprises a concentration of amino acids about 0.02 g / mL. In some embodiments, the concentration of the solution depends upon the composition of the template peptide. In some embodiments, the template peptide comprises a relatively higher proportion of amino acids having higher water solubility, such that a more concentrated solution may be needed for peptide synthesis.

[0094] Method of Peptide Synthesis

[0095]

[0085] Provided herein are methods comprising a peptide or peptide of interest. Surprisingly, the methods for peptide synthesis described herein can be performed in an aqueous solution as described herein and without the need for chemical solvents or toxic solvents. Thus, the methods for peptide synthesis described herein result in the desired yield of synthesized peptide without the waste of chemical solvents or toxic solvents common in methods of peptide synthesis known in the art.

[0096]

[0086] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide. In some embodiments, the methods described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide. In some embodiments, the template peptide is a peptide from a biological sample (e.g., a blood sample, a tissue sample, a cell sample). In some embodiments, peptide synthesis as described herein comprises a template peptide, mixture of individual amino acids, an aqueous solution, or combinations thereof. In some embodiments, the peptide provided herein is contacted with individual amino acids in an aqueous solution in the presence of a sufficient amount of energy to form a peptide bond. In some embodiments, the peptide of interest as described herein can also be referred to as a template peptide in the present disclosure.

[0097]

[0087] Provided herein is a method of synthesizing a peptide, the method comprising: (a) providing a peptide; (b) providing a mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in a peptide sequence; (c) contacting the peptide and the mixture of individual amino acids in an aqueous solution; (d) providing of energy to the aqueous solution comprising the peptide and the mixture of individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the peptide amino acid sequence; and (e) performing step (d) for a time period sufficient to synthesize a peptide from the mixture of individual amino acids, wherein the amino acid sequence of the synthesized peptide is the same as the amino acid sequence of the original peptide. In some embodiments, the method further comprises drying the synthesized peptide after step (e). In some embodiments, the drying results in 20% to 100% volume reduction. In some embodiments, the drying results in 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% volume reduction. In some embodiments, the drying results in 50% volume reduction.

[0098]

[0088] Also provided herein is a method of synthesizing a peptide, the method comprising: (a) providing a template peptide; (b) providing a mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in a template peptide sequence; (c) contacting the template peptide and the mixture of individual amino acids in an aqueous solution, wherein the conditions comprise: (i) a pH of about 3.0-10.0; or (ii) a pressure of about 5 mbar to about 1000 mbar; (d) providing of energy to the aqueous solution of the template peptide and the mixture of individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the template peptide amino acid sequence; and (e) performing step (d) for a time period sufficient to synthesize the peptide from the mixture of individual amino acids, wherein the amino acid sequence of the peptide is the same as the amino acid sequence of the template peptide. In some embodiments, the template peptide comprises about 2 amino acids to about 200 amino acids. In some embodiments, the peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% hydrophobic. In some embodiments, the conditions further comprise a reaction temperature of about 37°C to about 70°C. In some embodiments, the conditions further comprise a reaction temperature of about 40°C. In some embodiments, the pH is about 6.0. In some embodiments, the pressure is about 8 mbar. In some embodiments, the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the providing of energy to the aqueous solution is by a cyclical provision. In some embodiments, the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light. In some embodiments, periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution. In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the aqueous solution comprises a phosphate buffered saline solution. In some embodiments, the aqueous solution comprises an acid or a base. In some embodiments, the acid or the base is HC1, Formic Acid, or NaOH. In some embodiments, the aqueous solution further comprises the template peptide and the mixture of individual amino acids after contacting. In some embodiments, the aqueous solution is sterile. In some embodiments, the mixture of individual amino acids comprises only the amino acids that constitute the template peptide. In some embodiments, the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 or 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide. In some embodiments, the concentration of the mixture of individual amino acids in the aqueous solution prior of to the providing of energy is from about 0.001 g / mL to about 5 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL. In some embodiments, the concentration of the mixture of individual amino acids in aqueous solution prior to the providing of energy is about 0.001 g / mL, about 0.002 g / mL, about 0.003 g / mL, about 0.004 g / mL, about 0.005 g / mL, about 0.006 g / mL, about 0.009 g / mL, about 0.010 g / mL, about 0.020 g / mL, about 0.030 g / mL, about 0.040 g / mL, about 0.050 g / mL, about 0.060 g / mL, about 0.070 g / mL, about 0.080 g / mL, about 0.090 g / mL, about 0.1 g / mL, about 0.2 g / mL, about 0.3 g / mL, about 0.4 g / mL, about 0.5 g / mL, about 0.6 g / mL, about 0.7 g / mL, about 0.8 g / mL, about 0.9 g / mL, about 1 g / mL, about 1.1 g / mL, about 1.2 g / mL, about 1.3 g / mL, about 1.4 g / mL, about 1.5 g / mL, about 1.6 g / mL, about 1.7 g / mL, about 1.8 g / mL, about 1.9 g / mL, about 2 g / mL, about 2.1 g / mL, about 2.2 g / mL, about 2.3 g / mL, about 2.4 g / mL, about 2.5 g / mL, about 2.6 g / mL, about 2.7 g / mL, about 2.8 g / mL, about 2.9 g / mL, about 3 g / mL, about 3.1 g / mL, about 3.2 g / mL, about 3.3 g / mL, about 3.4 g / mL, about 3.5 g / mL, about 3.6 g / mL, about 3.7 g / mL, about 3.8 g / mL, about 3.9 g / mL, about 4 g / mL, about 4.1 g / mL, about 4.2 g / mL, about 4.3 g / mL, about 4.4 g / mL, about 4.5 g / mL, about 4.6 g / mL, about 4.7 g / mL, about 4.8 g / mL, about 4.9 g / mL or about 5 g / mL. In some embodiments, the time period is from 10 minutes to 5 days. In some embodiments, peptide synthesis is terminated by separation of the peptide from the mixture of individual amino acids in the aqueous solution. In some embodiments, the method is carried out in the presence of oxygen, Nitrogen, hydrogen, or CO2. In some embodiments, the method is performed in the absence of nucleic acids, enzymes, coenzymes, other cellular material or cells. In some embodiments, the mixture of individual amino acids comprises natural amino acids. In some embodiments, the mixture of individual amino acids comprises unnatural amino acids. In some embodiments, the unnatural amino acids comprise labeled amino acids. In some embodiments, the mixture of individual amino acids comprises: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, or combinations thereof. In some embodiments, synthesizing a peptide is optimized for maximizing peptide yield and minimizing contaminants.

[0099]

[0089] In some embodiments, the amino acids and template peptide may be added to the aqueous solution sequentially or simultaneously. In some embodiments, the method facilitates a peptide synthesis, wherein all the reagents required (e.g., the amino acids and template peptide) are added into the solution at the outset of the synthesis. Consequently, the methods described herein may obviate the need to use complex chemistries and / or multistep processes (such as the need to use protecting group strategies and / or solid phase components). By providing a template peptide in the method, it can be possible to obtain copies of the template peptide in high quantity and quality. Typically, at least 80%, 90%, 95%, 99%, 99.5% or even higher amounts of the peptides produced are identical to the template peptide.

[0100]

[0090] In some embodiments, the progress of the peptide synthesis is monitored. For example, the amount of synthesized peptide in the aqueous solution may be monitored using mass- spectrometry (MS) techniques (e.g., matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ion-trap electrospray MS).

[0101]

[0091] In some embodiments, the increase in the amount of synthesized peptide is observed initially, as the amino acids react to form copies of the template peptide. In some embodiments, the amount of synthesized peptide plateaus (e.g., as all the starting amino acids are consumed). In some embodiments, after an initial increase, the amount of synthesized peptide falls (e.g., if additional amino acids start to add to the synthesized peptide, increasing the length of the peptide chain). In some embodiments, peptide synthesis is terminated when an observed amount of synthesized peptide plateaus or decreases.

[0102]

[0092] In some embodiments, the duration of peptide synthesis is dependent upon the length of the template peptide and / or the nature of the amino acids within it. For example, shorter template peptides may be copied more rapidly than longer template peptides. Therefore, monitoring the peptide synthesis can be used to experimentally determine the duration of the peptide synthesis reaction that will provide the desired peptide yield.

[0103]

[0093] In some embodiments, peptide synthesis is terminated by removing the energy provided, cooling the aqueous solution (e.g., to a temperature below 10 °C, below about 5 °C or below about 0 °C), removing the mixture of amino acids from the aqueous solution, or combinations thereof.

[0104] Synthesis of Peptide Fragments

[0105]

[0094] Provided herein are methods for synthesizing a peptide, the methods comprising a template peptide which serves as a reference for the synthesis or amplification of a peptide as described herein. In some embodiments, synthesizing a peptide yields a synthesized peptide comprising an amino acid sequence that is the same amino acid sequence as the original peptide. In some embodiments, synthesizing a peptide yields a synthesized peptide comprising an amino acid sequence that is the same amino acid sequence as the template peptide. In some embodiments, synthesizing a peptide comprises synthesizing a peptide fragment. In some embodiments, the template peptide comprises a template peptide fragment. In some embodiments, the template peptide fragment comprises an amino acid sequence that is the same amino acid sequence as a fragment of the synthesized peptide.

[0106]

[0095] In some embodiments, synthesizing a peptide comprises more than one template peptide. In some embodiments, synthesizing a peptide comprises more than one template peptide fragment. In some embodiments, synthesizing a peptide comprises two or more template peptide fragments. In some embodiments, each of the two or more template peptide fragments comprise a different portion of the amino acid sequence of the original peptide. In some embodiments, peptide synthesis comprising each of the two or more template peptide fragments comprising a different portion of the amino acid sequence yields a synthesized peptide comprising an amino acid sequence that is the same amino acid sequence as the original peptide.

[0107]

[0096] In some embodiments, the method for synthesizing a peptide described herein comprises more than one template peptide fragment. In some embodiments, the method for synthesizing a peptide described herein comprises two or more template peptide fragments. In some embodiments, each template peptide fragment comprises a different portion of the amino acid sequence of the original peptide. In some embodiments, the two or more template peptide fragments comprise a different portion of the amino acid sequence of the original peptide. In some embodiments, the first of the two or more template peptide fragments comprises a first portion of the amino acid sequence of the original peptide. In some embodiments, the second of the two or more template peptide fragments comprises a second portion of the amino acid sequence of the original peptide. In some embodiments, the first of the two or more template peptide fragments is the first template peptide fragment. In some embodiments, the second of the two or more template peptide fragments is the second template peptide fragment. In some embodiments, the first template peptide fragment comprises the first portion of the amino acid sequence of the original peptide. In some embodiments, the second template peptide fragment comprises the second portion of the amino acid sequence of the original peptide. In some embodiments, the first portion of the synthesized peptide is synthesized from the first template peptide fragment. In some embodiments, the second portion of the synthesized peptide is synthesized from the second template peptide fragment. In some embodiments, the first portion of the synthesized peptide is linked / ligated to the second portion of the synthesized peptide, yielding a synthesized peptide comprising an amino acid sequence that is the same amino acid sequence as the original peptide.

[0108]

[0097] In some embodiments, peptide synthesis as described herein comprises a combination of more than one template peptide fragment. In some embodiments, peptide synthesis as described herein comprises a combination of two or more template peptide fragments. In some embodiments, each of the two or more template peptide fragments comprise a different fragment of the amino acid sequence of the original peptide. In some embodiments, a combination of each of the two or more template peptide fragments yields the same synthesized peptide as a template peptide as described herein. In some embodiments, peptide synthesis as described herein comprises a combination of two or more template peptide fragments, a mixture of individual amino acids, an aqueous solution, or combinations thereof. In some embodiments, each template peptide fragment provided herein is contacted with individual amino acids in an aqueous solution in the presence of a sufficient amount of energy to form a peptide bond. In some embodiments, the synthesized peptide fragments are linked / ligated, yielding a synthesized peptide comprising an amino acid sequence that is the same amino acid sequence as the original peptide.

[0109]

[0098] Also provided herein is a method of synthesizing a peptide, the method comprising synthesizing a peptide with two or more template peptide fragments, a mixture of individual amino acids in an aqueous solution, the mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in a template peptide fragment sequence, an aqueous solution, sufficient amount of energy to form a peptide bond, sufficient amount of energy to link / ligate synthesized peptide fragments, or combinations thereof. pH

[0110]

[0099] Provided herein are compositions, systems and methods comprising a peptide or peptide of interest at a controlled pH or a variable pH. In some embodiments, a peptide of interest as described herein can also be referred to as a template peptide. In some embodiments, a peptide of interest as described herein can also be referred to as a peptide of interest from a biological sample.

[0111]

[0100] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a controlled pH. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a variable pH.

[0112]

[0101] In some embodiments, the rate of synthesis and / or amplification changes as a function of pH. In some embodiments, the yield of synthesis and / or amplification changes as a function of pH. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of pH. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of reaction pH. In some embodiments, pH can be adjusted after incorporation of charged amino acids. In some embodiments, pH can be adjusted after incorporation of polar amino acids. In some embodiments, pH can be adjusted after incorporation of hydrophobic amino acids. For example, pH can be adjusted in one direction when incorporating charged or polar amino acids, and in another direction when adding hydrophobic amino acids. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pH of the aqueous solution comprising individual amino acids and a template peptide. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pH of the aqueous solution comprising individual amino acids and a template peptide prior to the provision of energy to the aqueous solution. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pH of the aqueous solution comprising individual amino acids and a template peptide during to the provision of energy to the aqueous solution.

[0113]

[0102] In some embodiments, the provision of energy to the aqueous solution modulates the pH of the aqueous solution. In some embodiments, the provision of energy to the aqueous solution maintains the aqueous solution at a constant pH. In some embodiments, the aqueous solution is maintained at a constant pH while being provided energy. In some embodiments, the aqueous solution is maintained at a constant pH while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a constant pH while being provided energy from full spectrum light.

[0114]

[0103] In some embodiments, the aqueous solution is maintained at a pH of about 6 while being provided energy. In some embodiments, the aqueous solution is maintained at a pH of about 6 while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a pH of about 6 while being provided energy from full spectrum light.

[0115]

[0104] In some embodiments, the pH ranges from about 3.0 to about 10. In some embodiments, the reaction pH ranges from about 3.0 to about 10. In some embodiments, the pH ranges from about 4.0 to about 5.0, from about 4.0 to about 6.0, from about 5.0 to about 7.0, from about 6.0 to about 8.0 from about 7.0 to about 9.0, from about 8.0 to about 10, or from about 5.0 to about 10.

[0116]

[0105] In some embodiments, the pH is about 3.0 to about 10. In some embodiments, the reaction pH is about 3.0 to about 10. In some embodiments, the pH ranges from about 4.0 to about 5.0, from about 4.0 to about 6.0, from about 5.0 to about 7.0, from about 6.0 to about 8.0 from about 7.0 to about 9.0, from about 8.0 to about 10, or from about 5.0 to about 10. In some embodiments, the pH is about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, or about 10. In some embodiments, the pH is about 6.0.

[0117] Pressure

[0118]

[0106] Provided herein are compositions, systems and methods comprising a peptide or peptide of interest at a controlled pressure or a variable pressure. In some embodiments, a peptide of interest as described herein can also be referred to as a template peptide. In some embodiments, a peptide of interest as described herein can also be referred to as a peptide of interest from a biological sample.

[0119]

[0107] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a controlled pressure. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a variable pressure.

[0108] In some embodiments, the rate of synthesis and / or amplification changes as a function of pressure. In some embodiments, the yield of synthesis and / or amplification changes as a function of pressure. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of pressure. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of reaction pressure. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pressure of the aqueous solution comprising individual amino acids and a template peptide. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pressure of the aqueous solution comprising individual amino acids and a template peptide prior to the provision of energy to the aqueous solution. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pressure of the aqueous solution comprising individual amino acids and a template peptide during to the provision of energy to the aqueous solution.

[0120]

[0109] In some embodiments, the aqueous solution is maintained at a variable pressure while being provided energy. In some embodiments, the aqueous solution is maintained at a constant pressure while being provided energy. In some embodiments, the aqueous solution is maintained at a constant pressure while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a constant pressure while being provided energy from full spectrum light.

[0121] [HO] In some embodiments, the aqueous solution is maintained at a pressure of about 8 mbar while being provided energy. In some embodiments, the aqueous solution is maintained at a pressure of about 8 mbar while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a pressure of about 8 mbar while being provided energy from full spectrum light.

[0122] [Hl] In some embodiments, the pressure ranges from about 5 mbar to about 1000 mbar. In some embodiments, the reaction pressure ranges from about 5 mbar to about 20 mbar. In some embodiments, the pressure ranges from about 5 mbar to about 10 mbar, from about 6 mbar to about 9 mbar, from about 7 mbar to about 12 mbar, from about 9 mbar to about 14 mbar, from about 11 mbar to about 16 mbar, from about 13 mbar to about 18 mbar, from about 15 mbar to about 20 mbar, from about 20 mbar to about 50 mbar, from about 50 mbar to about 100 mbar, from about 100 mbar to about 1000 mbar, from about 150 mbar to about 900 mbar, from about 200 mbar to about 800 mbar, from about 250 mbar to about 750 mbar, from about 300 mbar to about 700 mbar, from about 400 mbar to about 600 mbar, from about 500 mbar to about 1000 mbar, or from about 750 mbar to about 1000 mbar..

[0123]

[0112] In some embodiments, the pressure is about 5 mbar to about 1000 mbar. In some embodiments, the reaction pressure is about 5 mbar, about 6 mbar, about 7 mbar, about 8 mbar, about 9 mbar, about 10, mbar, about 11 mbar, about 12 mbar, about 13 mbar, about 14 mbar, about 15 mbar, about 16 mbar, about 17 mbar, about 18 mbar, about 19 mbar, about 20 mbar, about 50 mbar, about 100 mbar, about 150 mbar, about 200 mbar, about 250 mbar, about 300 mbar, about 350 mbar, about 400 mbar, about 450 mbar, about 500 mbar, about 550 mbar, about 600 mbar, about 650 mbar, about 700 mbar, about 750 mbar, about 800 mbar, about 850 mbar, about 900 mbar, about 950 mbar, or about 1000 mbar.

[0124]

[0113] In some embodiments, the pressure is controlled for air or air in the presence of oxygen. In some embodiments, the pressure is controlled in the presence of oxygen, Nitrogen, hydrogen, or CO2.

[0125] Temperature

[0126]

[0114] Provided herein are compositions, systems and methods comprising a peptide or peptide of interest at a controlled temperature or a variable temperature. In some embodiments, a peptide of interest as described herein can also be referred to as a template peptide. In some embodiments, a peptide of interest as described herein can also be referred to as a peptide of interest from a biological sample.

[0127]

[0115] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a controlled temperature. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a variable temperature.

[0128]

[0116] In some embodiments, the rate of synthesis and / or amplification changes as a function of temperature. In some embodiments, the yield of synthesis and / or amplification changes as a function of temperature. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of temperature. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of reaction temperature. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the temperature of the aqueous solution comprising individual amino acids and a template peptide. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the temperature of the aqueous solution comprising individual amino acids and a template peptide prior to the provision of energy to the aqueous solution. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the temperature of the aqueous solution comprising individual amino acids and a template peptide during to the provision of energy to the aqueous solution.

[0129]

[0117] In some embodiments, the provision of energy to the aqueous solution modulates the temperature of the aqueous solution. In some embodiments, the provision of energy to the aqueous solution maintains the aqueous solution at a constant temperature. In some embodiments, the aqueous solution is maintained at a constant temperature while being provided energy. In some embodiments, the aqueous solution is maintained at a constant temperature while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a constant temperature while being provided energy from full spectrum light.

[0130]

[0118] In some embodiments, the aqueous solution is maintained at a temperature of about 40°C while being provided energy. In some embodiments, the aqueous solution is maintained at a temperature of about 40°C while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a temperature of about 40°C while being provided energy from full spectrum light.

[0131]

[0119] In some embodiments, the temperature ranges from about 37°C to about 70°C. In some embodiments, the reaction temperature ranges from about 37°C to about 70°C. In some embodiments, the temperature ranges from about 35°C to about 45°C, from about 35°C to about 40°C, from about 40°C to about 60°C, from about 50°C to about 60°C from about 55°C to about 65°C, from about 60°C to about 70°C, or from about 65°C to about 70°C.

[0132]

[0120] In some embodiments, the temperature is about 37°C to about 70°C. In some embodiments, the reaction temperature is about 37°C to about 70°C. In some embodiments, the temperature is about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51 °C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 69°C, about 60°C, , about 61 °C, about 62°C, about 63 °C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, or about 70°C.

[0133] Humidity

[0134]

[0121] Provided herein are compositions, systems and methods comprising a peptide or peptide of interest at a controlled humidity or a relative humidity. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a controlled humidity. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a relative humidity.

[0135]

[0122] In some embodiments, the rate of synthesis and / or amplification changes as a function of humidity. In some embodiments, the yield of synthesis and / or amplification changes as a function of humidity. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of humidity. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of reaction humidity. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the humidity of the aqueous solution comprising individual amino acids and a template peptide. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the humidity of the aqueous solution comprising individual amino acids and a template peptide prior to the provision of energy to the aqueous solution. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the humidity of the aqueous solution comprising individual amino acids and a template peptide during to the provision of energy to the aqueous solution.

[0136]

[0123] In some embodiments, the provision of energy to the aqueous solution modulates the humidity of the aqueous solution. In some embodiments, the provision of energy to the aqueous solution maintains the aqueous solution at a constant humidity level. In some embodiments, the aqueous solution is maintained at a constant humidity level while being provided energy. In some embodiments, the aqueous solution is maintained at a constant humidity level while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a constant humidity level while being provided energy from full spectrum light.

[0137]

[0124] In some embodiments, the relative humidity is about 20% to about 90%. In some embodiments, the reaction relative humidity is about 10% to about 90%. In some embodiments, the relative humidity is about 5 %, about 10 %, about 15 %, about 20, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, about 50 %, about 55 %, about 60 %, about 65 %, about

[0138] 70 %, about 75 %, about 80 %, about 85 %, about 90 %, about 91 %, about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 %, or about 100%. Energy

[0139]

[0125] Provided herein are compositions, systems and methods comprising a peptide or peptide of interest provided energy. In some embodiments, a peptide of interest as described herein can also be referred to as a template peptide. In some embodiments, a peptide of interest as described herein can also be referred to as a peptide of interest from a biological sample.

[0140]

[0126] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide provided energy. In some embodiments, the provided energy is a variable energy or a controlled energy. In some embodiments, the provided energy is energy from full spectrum light. In some embodiments, the amount of provided energy is related to the concentration of peptide and / or amino acids. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide provided energy. In some embodiments, the amount of provided energy is related to the rate of synthesizing, detecting, and / or amplifying a peptide.

[0141]

[0127] In some embodiments, the rate of synthesis and / or amplification changes as a function of energy. In some embodiments, the yield of synthesis and / or amplification changes as a function of energy. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of provided energy. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of reaction provided energy. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the provided energy of the aqueous solution comprising individual amino acids and a template peptide. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the energy provided to the aqueous solution comprising individual amino acids prior to the providing of a template peptide to the aqueous solution. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the energy provided to the aqueous solution comprising individual amino acids after the provision of the template peptide to the aqueous solution.

[0142]

[0128] In some embodiments, the provision of energy to the aqueous solution modulates the time for synthesis and / or amplification. In some embodiments, the aqueous solution is provided constant energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is provided energy from full spectrum light. In some embodiments, a full spectrum light source may provide light having wavelengths between about 300 nm to about 700 nm. The full spectrum light may be configured to mimic the composition of natural light. In some embodiments, the energy provided is sunlight, full spectrum light, UV light, heat, electromagnetic radiation, or any other form of energy appropriate for peptide bond formation. In some embodiments, the energy provided is any form of energy that allows peptide bond formation, for example light, heat, or other electromagnetic radiation.

[0143]

[0129] In some embodiments, the energy provided is the energy sufficient for peptide bond formation. In some embodiments, the energy provided is the energy sufficient to synthesize the peptide from the mixture of individual amino acids, wherein the amino acid sequence of the peptide is the same as the amino acid sequence of the template peptide. In some embodiments, the energy provided is the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the template peptide amino acid sequence. In some embodiments, the energy provided is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the energy is provided for a time period sufficient to synthesize the peptide from the mixture of individual amino acids, wherein the amino acid sequence of the peptide is the same as the amino acid sequence of the template peptide. In some embodiments, the energy is provided for a time period of at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, or at least 5 hours. In some embodiments, the energy is provided in dark conditions or in the presence of light.

[0144]

[0130] In some embodiments, the energy provided is constant. In some embodiments, the energy provided is a constant source of full spectrum light for the duration of the peptide synthesis.

[0145]

[0131] In some embodiments, the energy provided is cyclical or repetitive. In some embodiments, the energy is provided for a period of time, and then stopped, before providing the energy again for a further period of time.

[0146]

[0132] In some embodiments, the energy provided is by a cyclical provision. In some embodiments, cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light. In some embodiments, periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution. In some embodiments, cyclical provision of energy comprises periodically increasing the heat of the aqueous solution, for example by at least 0.25°C, 0.5°C, 1°C, 5°C, or even 10°C, 15°C, 20°C, or 100 °C. Optionally, the aqueous solution is permitted to cool to ambient temperature, before the application of heat occurs again. In some embodiments, cyclical provision of energy to the aqueous solution corresponds to the rise and fall in ambient temperature during a day / night cycle. This process may be repeated many, hundreds or even thousands of times. In some embodiments, the aqueous solution is exposed to a constant source of light (e.g. full spectrum light) whilst also being subjected to cyclical temperature oscillations as described above. Alternatively, the aqueous solution is exposed to a cyclical provision of light (e.g., full spectrum light) under constant temperature conditions.

[0147]

[0133] In some embodiments, the temperature, energy, pressure, and / or reaction time can be individually or simultaneous adjusted in the reaction as described herein to adjust the rate of evaporation in order to improve efficiency. In some embodiments, the rate of evaporation is from about 0.1% to about 75%, from about 1% to about 50%, or from about 5% to about 20% by weight of the reaction.

[0148] Synthesis, Isolation and Assaying

[0149]

[0134] Peptides of the present disclosure are synthesized, using the method described herein. In some embodiments, the peptides are synthesized in vitro. In some embodiments, the peptides are further processed by isolation and / or purification.

[0150]

[0135] In some embodiments, a peptide provided herein is an isolated peptide. In some embodiments, the peptide is isolated and purified as described herein. In some embodiments, methods described here comprise the step of isolating peptides described herein. Any suitable method to provide isolated peptides described herein is used in the present disclosure, for example, precipitation, filtration, ion-exchange, and / or chromatography. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol. 182, (Academic Press, (1990)). The methods and conditions for biochemical purification of a peptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.

[0151]

[0136] In some embodiments, a peptide provided herein is purified from the aqueous solution. In some embodiments, purification and / or isolation are performed through high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some embodiments, purification comprises binding members which can selectively bind the peptide of interest, for example an antibody or fragment thereof. In some embodiments, the peptide of interest provided by the method is provided with a tag to allow it to be purified. In some embodiments, beads with a binding member specific for the peptide of interest or tag are attached.

[0152]

[0137] In some embodiments, purification is monitored by mass-spectrometry (MS) techniques, including matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ion-trap electrospray MS. Sequencing by Edman degradation sequence analysis, or in tandem with MS, for example, can be used to confirm peptide homogeneity.

[0153]

[0138] In some embodiments, peptides described herein are isolated from a biological sample. In some embodiments, the compositions described herein comprise 20% or more by weight, 75% or more by weight, 95% or more by weight, 98% or more by weight, or 99.5% or more by weight of the peptide, related to the method of preparation of compositions described herein and its purification thereof, wherein percentages refer to total peptide content relative to contaminants. Thus, in some embodiments, the peptide is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-desired peptides or other macromolecules, etc.).

[0154] Detection Reagents / Components and Reporters

[0155]

[0139] In some embodiments, systems disclosed herein comprise detection reagents to facilitate detection of peptides as described herein. Non-limiting examples of detection reagents include reporter nucleic acids, detection moieties, additional polypeptides, or combinations thereof. Upon the occurrence of the detection event, a signal (e.g., a detectable signal or detectable product) can be generated thereby indicating peptide synthesis or detection of a peptide. In some embodiments, any suitable detection reagent may be used. Any reagents suitable with the detection reactions, events, and signals described herein are useful as detection reagents for the systems, compositions, methods, kits, devices, and solutions provided herein, including a buffer, stain reporter, or combinations thereof.

[0156]

[0140] In some embodiments, detection reagents detect a peptide in a sample. In some embodiments, peptide amplification improves at least one of sensitivity, specificity, or accuracy of the detection assay. Accordingly, in some embodiments, detection assay comprises amplification, providing improved sensitive, specific, or rapid detection. In some embodiments, detection reagents comprise peptides, buffers, and / or signal reagents suitable for a detection reaction.

[0141] In some embodiments, detection is performed in a detection region on a support medium, or sample interface. Alternatively, or in combination, the detection is performed in a reagent chamber, and the resulting sample is applied to the support medium, sample interface, or surface within a reagent chamber.

[0157]

[0142] In some embodiments, detection reaction of nucleic acid as described herein is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value 1 to 60 minutes. In some embodiments, the detection reaction is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes. In some embodiments, the detection reaction is performed at a temperature of around 20-80°C. In some embodiments, the detection reaction is performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 53°C, 55°C, 58°C, 60°C, 62°C, 65°C, 68°C, 70°C, 75°C, 80°C or any value 20 °C to 80 °C. In some embodiments, the detection reaction is performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, or 45°C, or any value 20°C to 80°C. In some embodiments, the detection reaction is performed at a temperature of 20°C to 45°C, 35°C to 60°C, 40°C to 70°C, or 50°C to 65°C.

[0158]

[0143] In some embodiments, the reagents or components for detecting a nucleic acid are, for example, consistent for use within various fluidic devices disclosed herein for detection of a peptide within the sample, wherein the fluidic device may comprise multiple pumps, valves, reservoirs, and chambers for sample preparation, amplification, mixing, and detection of a detectable signal arising from peptide synthesis within the fluidic system itself. These reagents are compatible with the samples, solutions, compositions, systems, devices, fluidic devices, methods of detection, and support mediums as described herein. In some embodiments, systems disclosed herein comprise a reporter.

[0159]

[0144] In some embodiments, a reporter comprises a protein that generates a detectable signal or signal. In some embodiments, a reporter is operably linked to the protein that generates a signal. In some embodiments, a signal is a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some embodiments, the reporter comprises a detection moiety. In some embodiments, the reporter is configured to release a detection moiety or generate a signal. In some embodiments, suitable detectable labels and / or moieties provide a signal. In some embodiments, non-limiting example of a suitable detectable label and / or moiety comprises an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent protein; and a quantum dot.

[0145] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, P-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, and glucose oxidase (GO).

[0160]

[0146] In some embodiments, the reporter comprises a detection moiety. In some embodiments, the detection moiety comprises a fluorescent dye. Sometimes the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some embodiments, the detection moiety comprises an infrared (IR) dye. In some embodiments, the detection moiety comprises an ultraviolet (UV) dye. Alternatively, or in combination, the detection moiety comprises a protein. Sometimes the detection moiety comprises an antigen. Sometimes the detection moiety comprises a biotin. Sometimes the detection moiety comprises at least one of avidin or streptavidin. In some embodiments, the detection moiety comprises a polysaccharide, a polymer, or a nanoparticle. In some embodiments, the detection moiety comprises a gold nanoparticle or a latex nanoparticle.

[0161]

[0147] In some embodiments, a detection moiety comprises any moiety that generates a detectable product or detectable signal upon peptide synthesis. In some embodiments, the detectable product comprises a detectable unit generated from the detectable moiety and that emits a detectable signal as described herein. In some embodiments, the detectable product further comprises a detectable label, a fluorophore, a reporter, or a combination thereof. Iln some embodiments, the detectable product is configured to generate a signal indicative of the presence or absence of the synthesized peptide.

[0162]

[0148] In some embodiments, a detection moiety comprises any moiety that generates a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.). or piezo-electric signal. A nucleic acid of a reporter, sometimes, is protein-nucleic acid that generates a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon peptide synthesis. Often a calorimetric signal is heat produced after peptide synthesis. Sometimes, a calorimetric signal is heat absorbed after peptide synthesis. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after peptide synthesis. Sometimes, an optical signal is a change in light absorbance between before and after the peptide synthesis. Often, a piezo-electric signal is a change in mass between before and after the peptide synthesis.

[0163]

[0149] In some embodiments, the detectable signal comprises a colorimetric signal or a signal visible by eye. In some embodiments, the detectable signal may be fluorescent, electrical, chemical, electrochemical, or magnetic. In some embodiments, there is more than one detectable signal. In some embodiments, the detectable signal is generated directly by the peptide bond formation event. Alternatively, or in combination, the detectable signal is generated indirectly by the by the peptide bond formation event. Sometimes the detectable signal is not a fluorescent signal. In some embodiments, the detectable signal comprises a colorimetric or color-based signal. In some embodiments, the detected peptide is identified based on its spatial location on the detection region of the support medium. In some embodiments, the detectable signal is generated after a peptide yield is reached during peptide synthesis reaction.

[0164] Amplification Reagents / Components

[0165]

[0150] In some embodiments, systems described herein comprise a reagent or component for amplifying a peptide. In some embodiments, peptide amplification is isothermal peptide amplification, providing for the use of the system or system in remote regions or low resource settings without specialized equipment for amplification.

[0166]

[0151] Often, the peptide amplification is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value 1 to 60 minutes. In some embodiments, the amplification reaction is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes. In some embodiments, the amplification reaction is performed at a temperature of around 20°C to 45°C. In some embodiments, the amplification reaction is performed at a temperature of around 20°C to 70°C. In some embodiments, the amplification reaction is performed at a temperature no greater than 20°C, 25°C, 27°C, 30°C, 35°C, 37°C, 40°C, 45°C, 47°C, 50°C, 55°C, 57°C, 60°C, 65°C, 67°C, 70°C, 75°C, 77°C, 80°C, or any value 20 °C to 80 °C. In some embodiments, the amplification reaction is performed at a temperature of at least 20°C, 25°C, 27°C, 30°C, 35°C, 37°C, 40°C, 45°C, 47°C, 50°C, 55°C, 57°C, 60°C, 65°C, 67°C, 70°C, 75°C, 77°C, 80°C, or any value 20 °C to 80 °C. In some embodiments, the amplification reaction is performed at a temperature of 20°C to 45°C, 25°C to 40°C, 30°C to 40°C, 35°C to 40°C, 40°C to 45°C, 45°C to 50°C, 50°C to 55°C, 55°C to 60°C, 35°C to 40°C, 50°C to 65°C, 65°C to 70°C, 70°C to 80°C, or 75°C to 80°C. In some embodiments, the amplification reaction is performed at a temperature of around 30 °C to 35 °C. In some embodiments, the amplification reaction is performed at a temperature of around

[0167] 32 °C. In some embodiments, the amplification reaction is performed at a temperature of around

[0168] 33 °C. In some embodiments, the amplification reaction is performed at a temperature of around

[0169] 34 °C. In some embodiments, the amplification reaction is performed at a temperature of around

[0170] 35 °C.

[0171] Additional System Components

[0172]

[0152] Provided herein are systems comprising a peptide or peptide of interest. In some embodiments, the systems described herein are systems for synthesizing, detecting, and / or amplifying a peptide. In some embodiments, the systems described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide. In some embodiments, the template peptide is a peptide from a biological sample. In some embodiments, the systems described herein comprise a template peptide, an aqueous solution, a mixture of individual amino acids, and optionally a peptide that is the product of synthesis or amplification. In some embodiments, the systems described herein are used for the methods of synthesizing, detecting, and / or amplifying a peptide described herein.

[0173]

[0153] In some embodiments, systems include a package, carrier, or container that is compartmentalized to receive one or more containers such as vials, or tubes, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, test wells, bottles, vials, syringes, and test tubes. In some embodiments, the containers are formed from a variety of materials such as glass, plastic, or polymers. In some embodiments, the system or systems described herein contain packaging materials. Examples of packaging materials include, but are not limited to, pouches, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for intended mode of use.

[0154] In some embodiments, systems described herein include labels listing contents and / or instructions for use, or package inserts with instructions for use. In some embodiments, the systems include a set of instructions and / or a label is on or associated with the container. In some embodiments, the label is on a container when letters, numbers or other characters forming the label are attached, molded, or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container (e.g., as a package insert). In some embodiments, the label is used to indicate that the contents are to be used for a specific therapeutic application. In some embodiments, the label indicates directions for use of the contents, such as in the methods described herein. In some embodiments, after packaging the formed product and wrapping or boxing to maintain a sterile barrier, the product is terminally sterilized by heat sterilization, gas sterilization, gamma irradiation, or by electron beam sterilization. Alternatively, in some embodiments, the product is prepared and packaged by aseptic processing.

[0174] Devices

[0175]

[0155] Provided herein are devices comprising a peptide or peptide of interest. In some embodiments, the devices described herein are devices for synthesizing, detecting, and / or amplifying a peptide. In some embodiments, the devices described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide. In some embodiments, the template peptide is a peptide from a biological sample. In some embodiments, the devices described herein comprise a template peptide, an aqueous solution, a mixture of individual amino acids, and optionally a peptide that is the product of synthesis or amplification. In some embodiments, the devices described herein are used for the methods of synthesizing, detecting, and / or amplifying a peptide described herein.

[0176]

[0156] In some embodiments, devices comprise components comprising one or more of: compositions described herein; systems described herein; other components or appurtenances as described herein; or combinations thereof. In some embodiments, devices comprise compositions or systems described herein. In some embodiments, devices perform methods described herein.

[0177]

[0157] In some embodiments, a device comprising: a sample interface configured to receive a sample comprising a template peptide; a chamber fluidically connected to the sample interface; wherein the chamber comprises an aqueous solution, and a mixture of individual amino acids such that a peptide is synthesized with the mixture of individual amino acids in aqueous solution within the chamber; and wherein the aqueous solution comprising the peptide is filtered through a membrane to separate the peptide from the aqueous solution after peptide synthesis is terminated. In some embodiments, synthesizing a peptide within the chamber comprises: (a) contacting the template peptide and mixture of individual amino acids in the aqueous solution within the chamber; and (b) providing of energy to the aqueous solution for a time period sufficient to synthesize the peptide with the mixture of individual amino acids, wherein the amino acid sequence of the synthesized peptide is the same as the amino acid sequence of the template peptide. In some embodiments, a peptide is synthesized in conditions comprising: (a) a reaction temperature of about 37°C to about 70°C; (b) a pH of about 3.0- 10.0; or (c) a pressure of about 5 mbar to about 20 mbar. In some embodiments, the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the providing of energy to the aqueous solution is by cyclical provision. In some embodiments, the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light. In some embodiments, periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution. In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the aqueous solution comprises a phosphate buffered saline solution. In some embodiments, the aqueous solution comprises an acid or a base. In some embodiments, the acid or the base is HC1, Formic Acid, or NaOH. In some embodiments, the device further comprises a pump for vacuum.

[0178]

[0158] In some embodiments, a device comprising: a sample interface configured to receive a sample comprising a template peptide; a chamber fluidically connected to the sample interface; wherein the chamber comprises an aqueous solution, and a mixture of individual amino acids such that a peptide is synthesized with the mixture of individual amino acids in aqueous solution within the chamber; and wherein the aqueous solution comprising the peptide is passed through a preparative column to separate the peptide from the aqueous solution after peptide synthesis is terminated. In some embodiments, the preparative column is a solid phase extraction column (e.g., a C8 column, a Cl 8 column, or other suitable column). In some embodiments, the preparative column is a Cl 8 column. In some embodiments, the preparative column is a reversed phase Cl 8 column.

[0159] In some embodiments, the device further comprises a component for reaction temperature regulation. In some embodiments, the device further comprises a component for evaporation and / or condensation regulation. In some embodiments, the temperature, pressure, and / or reaction time can be individually or simultaneous adjusted in the device as described herein to adjust the rate of evaporation in order to improve efficiency. In some embodiments, the device further comprises a component for addition and removal of reagents. In some embodiments, the device further comprises a component for purification of synthesized peptide. In some embodiments, the device further comprises a component for removal of synthesized peptide. In some embodiments, the device further comprises a membrane to separate the peptide from the aqueous solution after peptide synthesis is terminated. In some embodiments, the membrane is a hydrophilic membrane. In some embodiments, the membrane is a size-specific membrane. In some embodiments, the device further comprises tubes with high optical transmission. In some embodiments, the device further comprises tubes and / or containers in non-reactive or absorptive material such as glass or stainless steel.

[0179]

[0160] In some embodiments, a device comprises an in-line sampler that can be used to extract a small amount from the reaction mixture for characterization or other purposes. In some embodiments, characterization comprises an activity assay. In some embodiments, characterization comprises a peptide activity assay. In some embodiments, characterization comprises characterization of pH, temp, amino acid concentration, template peptide, synthesized peptide, waste or contaminants, detection reagents, amplification reagents, or combinations thereof. In some embodiments, sampling comprises in-line sampling comprising receiving a sample, pumping the sample down a monolithic column, drying the sample in drying gas, characterizing the sample, or combinations thereof. In some embodiments, sampling the reaction mixture to assess the progress of peptide synthesis indicates whether to add individual amino acids or remove individual amino acids from the aqueous solution.

[0180]

[0161] In some embodiments, the device further comprises a component for monitoring and documenting the progress of peptide synthesis. In some embodiments, the device further comprises a component for monitoring and documenting the contamination profile during peptide synthesis and / or after peptide synthesis is terminated.

[0181]

[0162] In some embodiments, the device comprises a component for monitoring the progress of peptide synthesis with visual detection, pH detection, mass spectrometer (MS), high- performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof. In some embodiments, the device further comprises a component for sampling the reaction mixture to assess the progress of peptide synthesis. In some embodiments, the device is programmed to adhere to a method of synthesizing a peptide comprising at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof. In some embodiments, the device comprises multiple detection components. In some embodiments, the device comprises a detector for measuring the template peptide, aqueous solution, individual amino acids, synthesized peptide, or combinations thereof. In some embodiments, the detector is capable of visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof. In some embodiments, the device comprises touch screen user interface. In some embodiments, the device is a bench top instrument. In some embodiments, the device is capable of synthesizing 10-100 g of peptide. In some embodiments, the device is capable of synthesizing 10-500 g of peptide. In some embodiments, the device is a microfluidic device.

[0182]

[0163] In some embodiments, a device for performing a method of described herein, the device comprising a structural component as well as a composition component, wherein the composition component is a reaction mixture comprising a template peptide, an aqueous solution, a mixture of individual amino acids, and optionally a synthesized peptide. In some embodiments, the composition component is also referred to as a sample component.

[0183] Device Components

[0184]

[0164] In general, device components comprise a structural component as well as sample components, including compositions, solutions, and systems described herein. Often, a sample component comprises or consists essentially of compositions, or systems described herein. Additional device components may comprise one or more hydrogels or surfaces with immobilized reporters. In some embodiments, a device’s sample component may be contained in at least one structural device component, such as a sample interface, which may be in fluid communication with a chamber. In some embodiments, the sample interface is fluidically connected to a chamber. By way of non-limiting example, a device’s sample component may be simultaneously contained in a sample interface and a chamber. In some embodiments, by being in fluid communication with a chamber, a device’s sample component may flow from the sample interface to the chamber. In some embodiments, a device’s sample component may flow from the sample interface into a chamber by way of the fluid connection. In some embodiments, a reporter is immobilized to a surface or support medium within the chamber, which may be a hydrogel. In some embodiments, a chamber comprises more than one peptide or more than one peptide type. In some embodiments, the devices described herein comprise a plurality of hydrogels each comprising reporter molecules (e.g., in order to facilitate multiplexing and / or improve signal).

[0185]

[0165] Any of the devices described herein comprise one or more compartments, chambers, channels, or locations. In some embodiments, two or more of the compartments or chambers are in fluid communication, optical communication, thermal communication, or any combination thereof with one another. In some embodiments, two or more compartments or chambers are arranged in a sequence. In some embodiments, two or more compartments or chambers are arranged in parallel. In some embodiments, two or more compartments or chambers are arranged in sequence, parallel, or both. In some embodiments, one or more compartments or chambers comprise a well. In some embodiments, one or more compartments or chambers comprise a flow strip. In some embodiments, one or more compartments or chambers comprise a heating element. In some embodiments, one or more compartments or chambers comprise a vacuum element. In some embodiments, one or more compartments or chambers comprise a vacuum pump.

[0186]

[0166] Any of the devices described herein comprise a sample interface, which are in fluid communication with a valve and / or a chamber or comprising configuration to be fluidically connected to a valve and / or a chamber. In some embodiments, a chamber comprises configuration to comprise compositions, systems, one or more reagents for amplification (z.e., amplification reagents), one or more reagents for detection (z.e., detection reagents), or combinations thereof. In some embodiments, a chamber and / or a valve comprises configuration to be thermally connected to a heating element. In some embodiments, each of the valves of the plurality of valves is thermally connected to a heating element. In some embodiments, each of the valves is filled with a material configured to change between liquid and solid phases when heated by a heating element.

[0187]

[0167] In general, the buffers described herein are compatible for use in the devices described herein. In some embodiments, the device is a microfluidic device. In some embodiments, the device is a handheld device. In some embodiments, the device is a point-of-need device. In some embodiments, the device comprises any one of the device configurations described herein. In some embodiments, the device comprises one or more parts of any one of the device configurations described herein.

[0168] Generally, a sample comprises one or more peptides and a chamber (e.g., a reaction chamber) comprises one or more of: aqueous solution, individual amino acids, or combinations thereof. In some embodiments, a sample flows from a sample interface into a chamber by way of the fluid connection wherein the sample interacts with the components of the compositions, systems, and solutions contained therein. In some embodiments, contacting the template peptide with the mixture of amino acids in aqueous solution, provided energy sufficient to form a peptide bond, results in a peptide is synthesized.

[0188]

[0169] Any of the devices described herein are compatible with any of the compositions, systems, kits, or methods disclosed herein, including methods of synthesizing, detecting, and / or amplifying a peptide.

[0189] Kits

[0190]

[0170] In some embodiments, compositions and / or system components are assembled in a kit. Accordingly, disclosed herein are kits for synthesizing a peptide or detecting a peptide. In some embodiments, kits are compatible with any methods disclosed herein, including methods used for synthesizing or detection, which may be related to a treatment, and / or diagnosis of a disease or disorder.

[0191]

[0171] Any of the kits described herein are compatible with any of the compositions, systems, kits, or methods disclosed herein, including methods used for synthesizing or detection, which may be related to a treatment, and / or diagnosis of a disease or disorder. By way of non-limiting example, in some embodiments, the kits described herein are used in synthesis of peptide. In some embodiments, the kits described herein are used in synthesis of peptide described in TABLE 1. By way of non-limiting example, in some embodiments, the kits described herein are used in synthesis of a peptide having a therapeutic target described in TABLE 2. In some embodiments, the kits described herein are used in detection of peptide described in TABLE 1. In some embodiments, the kits described herein are used in detection of peptide associated with any one of the diseases or disorders recited in TABLE 3.

[0192]

[0172] In some embodiments, kits are compatible with methods of detection as disclosed herein, wherein a kit further comprises a detectable label or a nucleic acid encoding a detectable label. In some embodiments, the components of the kit are in same container. In some embodiments, the components of the kit are in separate containers. Kit Components

[0193]

[0173] In general, kit components comprise structural components as well as sample components, including compositions and systems described herein. Often, kits comprise one or more containers compatible for containing the samples, compositions, and systems described herein. In some embodiments, components of the samples, compositions, and systems are contained in the same container or in separate containers. In some embodiments, a container is a syringe, test wells, bottles, chambers, channels, vials, or test tubes. In one embodiment, the containers are formed from a variety of materials such as glass, plastic, or polymers. The system or systems described herein contain packaging materials. Examples of packaging materials include, but are not limited to, pouches, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for intended mode of use.

[0194]

[0174] In some embodiments, a kit comprises components, compositions, systems, and / or reagents for performing any methods disclosed herein. In some embodiments, a kit comprises components, compositions, and / or reagents for performing an assay disclosed herein. In some embodiments, a kit comprises other therapeutic agents, carriers, buffers, containers, and / or devices for administration. In some embodiments, kits described herein comprise a solid support. In some embodiments, a peptide is attached to a solid support. For example, in some embodiments, the solid support is an electrode or a bead. In some embodiments, the bead is a magnetic bead. In some embodiments, the peptide flows through a chamber into a mixture comprising a substrate. When the peptide meets the substrate, a reaction occurs, such as a colorimetric reaction, which is then detected.

[0195]

[0175] In some embodiments, the kit comprises labels and / or instructions for synthesizing or detecting a peptide. In some embodiments, the kit comprises labels and / or instructions for use. In some embodiments, labeling and / or instructions includes, for example, information concerning the amount, frequency and method of introduction and / or administration of the compositions, systems, and / or nucleic acid constructs described herein. In some embodiments, a label is on a container when letters, numbers or other characters forming the label are attached, molded, or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein. After packaging the formed product and wrapping or boxing to maintain a sterile barrier, in some embodiments, the product is terminally sterilized by heat sterilization, gas sterilization, gamma irradiation, or by electron beam sterilization. Alternatively, in some embodiments, the product is prepared and packaged by aseptic processing.

[0196]

[0176] In some embodiments, the instructions for practicing the methods are recorded on a suitable recording medium. In some embodiments, the instructions are printed on a substrate, such as paper or plastic, etc. In some embodiments, the instructions are present in the kits as a package insert, in the labeling of the container of the kit or components thereof ( / .< ., associated with the packaging or subpackaging) etc. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g. via the Internet), are provided. In some embodiments, the kit includes a web address where the instructions are viewed and / or from which the instructions are downloaded.

[0197] Detection of a Peptide

[0198]

[0177] Described herein are various methods of sample amplification and detection in a single reaction volume. In some embodiments, any of the devices described herein are configured to perform amplification and detection in a same well, chamber, channel, or volume in the device. In some embodiments, methods include simultaneous amplification and detection in the same volume and / or in the same reaction. In some embodiments, methods include sequential amplification and detection in the same volume. In some embodiments, amplification and detection occur in a single reaction or in a single volume.

[0199]

[0178] In some embodiments, a detection reaction is used for detecting the presence of a peptide in the same. In some embodiments, the detection reaction produces a detectable signal, as described elsewhere herein, in the presence of a peptide. In some embodiments, a detection reaction is used for detecting the concentration of a peptide in the same. In some embodiments, the detection reaction produces a detectable signal, as described elsewhere herein, in the presence of a concentration of a peptide.

[0200]

[0179] In some embodiments, a detection reaction is used for detecting the presence of a template peptide in a reaction volume or a sample. In some embodiments, a detection reaction is used for detecting the presence of a peptide of interest in a biological sample. Disease or Disorder

[0201]

[0180] In some embodiments, the peptide described herein has a therapeutic target associated to a disease or disorder.

[0202]

[0181] In some embodiments, the peptide described herein has a therapeutic target and can be administered as a method of treatment to treat a disease or disorder. In some embodiments, treating, preventing, or inhibiting disease or disorder in a subject comprises contacting a peptide described herein to a therapeutic target associated to a disease or disorder in a patient in need thereof. In some embodiments, the therapeutic target is a therapeutic target described in TABLE 2. In some embodiments, the disease or disorder is a disease or disorder described herein in TABLE 3. In some embodiments, the methods of treating, preventing, or inhibiting a disease or disorder involves administration of a peptide described herein. In some embodiments, a peptide described herein is used for therapy.

[0203]

[0182] In some embodiments, the use of the peptide described herein in the manufacture of a medicament. In some embodiments, the use of the peptide described herein in the manufacture of a medicament for therapeutic and / or prophylactic treatment of a disease or condition described herein. For example, the disease or condition is Cancer, Advanced prostate cancer, Multiple myeloma, High-grade non-metastatic osteosarcoma, Resectable non-metastatic osteosarcoma, Type 2 Diabetes Mellitus, Short bowel syndrome and malabsorption, Irritable bowel syndrome (IBS), Irritable bowel syndrome (IBS) with constipation and chronic idiopathic constipation, Type 1 Diabetes Mellitus, Type 2 Diabetes Mellitus, Type 1 and Type 2 Diabetes Mellitus, Obesity, Chronic weight management, Neurodegenerative disease, Alzheimer’s disease, Parkinson’s disease, Erectile dysfunction, Imminent pre-term birth, Postpartum hemorrhage, Spinocerebellar degeneration, Hypoactive sexual desire disorder, Osteoporosis, Chronic idiopathic constipation, Acute decompensated heart failure, Sepsis, Septic Shock, Hereditary angioedema, HIV-1, HIV lipodystrophy, Severe chronic pain, Chronic immune thrombocytopenic purpura, Anemia, Chronic kidney disease, Respiratory distress syndrome, Secondary hyperparathyroidism, Phototoxicity, Cushing’s disease, Somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors, Somatostatin receptor positive neuroendocrine tumors. In some embodiments, the disease or condition is an infectious disease. Non-limiting examples of infectious disease include: Diphtheria, Pertussis, Urinary Tract Infection, Gastroenteritis, Cellulitis, Epiglottitis, Septic Arthritis, Pneumococcal, Pharyngitis, Gonorrhea, Common Cold, Mumps, Rubella, Chickenpox, Hand-Foot-and-Mouth Disease, Gastroenteritis, Acquired Immunodeficiency Syndrome (AIDS), Human Immunodeficiency Virus (HIV), Influenza, Syphilis, Salmonella, Conjunctivitis, Sinusitis, Tuberculosis, Lymphadenitis, Leprosy, Chlamydia, Typhus, Parasitic Diseases, Bacterial diseases, or viral diseases.

[0204] Cancer

[0205]

[0183] In some embodiments, the disease comprises cancer. Non-limiting examples of cancers include: acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphocytic leukemia; acute myelogenous leukemia; acute myeloid leukemia (adult / childhood); adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytoma; atypical teratoid / rhabdoid tumor; basal-cell carcinoma; bile duct cancer; extrahepatic (cholangiocarcinoma); bladder cancer; bone osteosarcoma / malignant fibrous histiocytoma; brain cancer (adult / childhood); brain tumor; cerebellar astrocytoma (adult / childhood); brain tumor, cerebral astrocytoma / malignant glioma brain tumor; brain tumor, ependymoma; brain tumor, medulloblastoma; brain tumor, supratentorial primitive neuroectodermal tumors; brain tumor, visual pathway and hypothalamic glioma; brainstem glioma; breast cancer; bronchial adenomas / carcinoids; bronchial tumor; Burkitt lymphoma; cancer of childhood; carcinoid gastrointestinal tumor; carcinoid tumor; carcinoma of adult, unknown primary site; carcinoma of unknown primary; central nervous system embryonal tumor; central nervous system lymphoma, primary; cervical cancer; childhood adrenocortical carcinoma; childhood cancers; childhood cerebral astrocytoma; chordoma, childhood; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; desmoplastic small round cell tumor; emphysema; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; Ewing sarcoma in the Ewing family of tumors; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastric carcinoid; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor; germ cell tumor: extracranial, extragonadal, or ovarian gestational trophoblastic tumor; gestational trophoblastic tumor, unknown primary site; glioma; glioma of the brain stem; glioma, childhood visual pathway and hypothalamic; hairy cell leukemia; head and neck cancer; heart cancer; hepatocellular (liver cancer); Hodgkin’s lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell carcinoma (endocrine pancreas); Kaposi Sarcoma; kidney cancer (renal cell cancer); Langerhans cell histiocytosis; laryngeal cancer; lip and oral cavity cancer; liposarcoma; liver cancer (primary); lung cancer, non-small cell; lung cancer, small cell; lymphoma, primary central nervous system; macroglobulinemia, Waldenstrom; male breast cancer; malignant fibrous histiocytoma of bone / osteosarcoma; medulloblastoma; medulloepithelioma; melanoma; melanoma, intraocular (eye); Merkel cell cancer; Merkel cell skin carcinoma; mesothelioma; mesothelioma, adult malignant; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell neoplasm; mycosis fungoides, myelodysplastic syndromes; myelodysplastic / myeloproliferative diseases; myelogenous leukemia, chronic; myeloid leukemia, adult acute; myeloid leukemia, childhood acute; myeloma, multiple (cancer of the bone-marrow); myeloproliferative disorders, chronic; nasal cavity and paranasal sinus cancer; nasopharyngeal carcinoma; neuroblastoma, non-small cell lung cancer; non-Hodgkin’s lymphoma; oligodendroglioma; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial cancer (surface epithelial-stromal tumor); ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; pituitary tumor, islet cell; papillomatosis; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal astrocytoma; pineal germinoma; pineal parenchymal tumors of intermediate differentiation; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary tumor; pituitary adenoma; plasma cell neoplasia / multiple myeloma; pleuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell carcinoma (kidney cancer); renal pelvis and ureter, transitional cell cancer; NUT midline carcinoma; retinoblastoma; rhabdomyosarcoma, childhood; salivary gland cancer; sarcoma, Ewing family of tumors; Sezary syndrome; skin cancer (melanoma); skin cancer (non-melanoma); small cell lung cancer; small intestine cancer soft tissue sarcoma; soft tissue sarcoma; spinal cord tumor; squamous cell carcinoma; squamous neck cancer with occult primary, metastatic; stomach (gastric) cancer; subependymal glioma; supratentorial primitive neuroectodermal tumor; T-cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); testicular cancer; throat cancer; thymoma; thymoma and thymic carcinoma; thyroid cancer; thyroid cancer, childhood; transitional cell cancer of the renal pelvis and ureter; urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; and Wilms Tumor.

[0206] Method of Making Peptides

[0207]

[0184] In some embodiments, the methods as disclosed herein can generate a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a vaccine polypeptide, and a reporter polypeptide. For examples, the peptides developed from the methods disclosed herein can be used for cosmetics, dermatology, nutraceuticals, supplements, vaccines, chemical defense, antivenom, or antitoxin. In some embodiments, the peptides are formulated against nerve agents, anthrax, or toxins. Further, the peptides of the present disclosure can find use as supplement to food and beverages, and as pharmaceutical formulations not associated with food, suitable for consumption by an individual and usually sold in medicinal forms which may be solid formulations such as caplets, tablet, capsules, soft gel capsules, gel caps and the like, or liquid formulations, such as solutions or suspensions. In some embodiments, the peptides developed from the methods disclosed herein are suitable for topical administration to aging skin, damaged skin, skin after cosmetic procedures, skin after dermatological procedures, atrophied skin, injured skin, or skin in contact with mucosal membranes. In some embodiments, peptides can be formulated to improve or maintain the aesthetic appearance of the skin, hair, nails or mucous membranes. In some embodiments, peptides can be formulated to improve or maintain the aesthetic appearance of skin affected by eczema, contact dermatitis, atopic dermatitis, or psoriasis.

[0208] SEQUENCES AND TABLES

[0209]

[0185] TABLE 1 provides exemplary peptides of interest that are useful in the compositions, systems, devices, kits, and methods described herein.

[0210] TABLE 1. EXEMPLARY PEPTIDES

[0211]

[0186] TABLE 2 provides exemplary peptide therapeutic targets that are useful in the compositions, systems, devices, kits, and methods described herein.

[0212] TABLE 2. EXEMPLARY THERAPEUTIC TARGETS

[0213]

[0187] TABLE 3 provides illustrative diseases and syndromes associated with the exemplary peptide therapeutic targets described herein.

[0214] TABLE 3. EXEMPLARY DISEASES AND SYNDROMES

[0215] EXAMPLES

[0216]

[0188] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0217] Example 1: Synthesizing a Peptide using a Template Peptide

[0218]

[0189] An in vitro method is carried out to synthesize a peptide, the method comprising contacting a template peptide (e.g., a peptide comprising about 2 amino acids to about 200 amino acids) with a mixture of individual amino acids in an aqueous solution. Briefly, the template peptide is contacted with the mixture of individual amino acids in the aqueous solution in the presence of variable conditions (e.g., a reaction temperature of about 30°C to about 70°C, a pH of about 3.0- 10.0, a pressure of about 5 mbar to about 1000 mbar, or combinations thereof). The aqueous solution comprising the template peptide and the mixture of individual amino acids was provided sufficient energy (e.g., at least about 1.2 kcal / mol of energy) to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the template peptide amino acid sequence. The mixture of individual amino acids was provided sufficient energy for a time period (e.g., 10 minutes, 20 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 1 / 2 day, 1 day, 2 days, 3 days, 4 days, or 5 days) sufficient to synthesize the peptide from the mixture of individual amino acids, such that the peptide has the same amino acid sequence as the template peptide. Peptide synthesis is optimized as a factor of the reaction conditions (e.g., temperature, pH, pressure controlled by a vacuum) and / or the hydrophobicity of the template peptide. Without being bound by theory, peptide synthesis involving a template peptide with high hydrophobicity (e.g., peptide is about 65%, 70%, 75%, 80%, 85%, 90%, or 100% hydrophobic) results in a higher yield of synthesized peptide (i.e., peptide product).

[0219]

[0190] In some instances, the method of synthesizing a peptide comprises at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof. In brief, the reaction mixture is sampled during the sampling step to assess the progress of synthesizing a peptide. The yield of synthesized peptide may be optimized after sampling by adding or removing individual amino acids from the aqueous solution. After peptide synthesis has resulted in the desired yield of synthesized peptide, the reaction mixture is filtered using a membrane to separate the synthesized peptide from the aqueous solution and contaminants, such that the synthesized peptide (e.g., insulin) is packaged and the aqueous solution is removed as waste.

[0220] Example 2: Synthesizing a Peptide using a Template Peptide in a Device

[0221]

[0191] An in vitro method is carried out to synthesize a peptide, the method comprising contacting a template peptide (e.g., a peptide comprising about 2 amino acids to about 200 amino acids) with a mixture of individual amino acids in an aqueous solution using a microfluidic device. Briefly, the template peptide is contacted with the mixture of individual amino acids in the aqueous solution within a chamber (e.g., a chamber of non-reactive or absorptive material such as stainless steel or glass with high optical transmission) of the microfluidic device in the presence of variable conditions (e.g., a reaction temperature of about 37°C to about 70°C, a pH of about 3.0-10.0, a pressure of about 5 mbar to about 20 mbar, or combinations thereof). The aqueous solution comprising the template peptide and the mixture of individual amino acids was provided sufficient energy (e.g., at least about 1.2 kcal / mol of energy) to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the template peptide amino acid sequence. The mixture of individual amino acids was provided sufficient energy for a time period (e.g., 10 minutes, 20 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 1 / 2 day, 1 day, 2 days, 3 days, 4 days, or 5 days) sufficient to synthesize the peptide from the mixture of individual amino acids, such that the peptide has the same amino acid sequence as the template peptide. Peptide synthesis is optimized as a factor of the variable reaction chamber conditions (e.g., temperature, pH, pressure controlled by a vacuum) and / or the hydrophobicity of the template peptide. Without being bound by theory, peptide synthesis involving a template peptide with high hydrophobicity (e.g., peptide is about 65%, 70%, 75%, 80%, 85%, 90%, or 100% hydrophobic) results in a higher yield of synthesized peptide (i.e., peptide product).

[0222]

[0192] The device is programmed to adhere to a method of synthesizing a peptide comprising at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof. Overall, the device monitors the progress of synthesizing a peptide with detection (e.g., visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof) and documents the progress of synthesizing a peptide.

[0193] The reaction mixture is sampled (e.g., in-line sampling comprising receiving a sample, pumping the sample down a monolithic column, drying the sample in drying gas, characterizing the sample, or combinations thereof) to assess the progress of synthesizing a peptide. The yield of synthesized peptide may be optimized after sampling by adding or removing individual amino acids from the aqueous solution.

[0223]

[0194] After peptide synthesis has resulted in the desired yield of synthesized peptide, the reaction mixture is filtered using a membrane to separate the synthesized peptide from the aqueous solution and contaminants, such that the synthesized peptide (e.g., insulin) is packaged and the aqueous solution is removed as waste.

[0224] Example 3: Effects of Ratios of Template to Amino Acids on Peptide Amplification

[0225]

[0195] To assay the effects of ratios of peptide template to amino acids on peptide amplification, an exemplary peptide WK20 (WLEHNWEGNAVNLMPSK, SEQ ID NO: 1) was synthesized according to the method of Example 1. Briefly, 1 mg of the template WK20 peptide of SEQ ID NO: 1 was mixed with an aqueous solution of 100 mg of heavy labeled amino acids (heavy labeled amino acids Lysine and Arginine were used as they result in a mass shift of 6 daltons) in molar and % ratios, in 10 ml of 0.1% Formic acid in MilliQ water. The samples were evaporated at 8 mbar, 35 °C, and 600 RPM to 5 mL in volume. The sample was reconstituted to 10ml and 30ul was taken for analytical LC. This was repeated lOx with samples taken at every second cycle. The area under the curve and the absorbance at 218 nm was recorded. The change in ratio of template to amino acids after each round of amplification were calculated and plotted as shown in FIG. 1A. Based on the assumption that the same amount of peptide was made during each round, after each amplification round the ratio of template peptide to amino acids increases as the amino acids were incorporated into the newly synthesized peptide. In tandem with the increase of synthesized peptides, an evaporation was performed to determine the rate of synthesis. Thus, the area under the curve (AUC) after 50% evaporation was monitored for 4 rounds of synthesis with a 1 : 100 ratio of template to amino acids. Notably, as shown in FIG. IB, the AUC increased from rounds 0 to round 1, and demonstrated a modest increase from rounds 2 to rounds 4. Collectively, this data demonstrated that the methods disclosed herein resulted in an increase in synthesized peptides, and a decrease in amino acids from round 1 to round 4.

[0196] The effects of the template versus amino acids ratios were further confirmed with synthesis of a semaglutide peptide. The sequence of the sema peptide is: H-Aib- EGTFTSDVSSYLEGQAAKEFIAWLVRGRG, SEQ ID NO: 2), wherein Aib means a- aminoisobutyric acid (a non-standard amino acid replacing alanine). 0.5 mg of semaglutide was solubilized with 25 mg, 50 mg, 100 mg, and 150 mg of the required amino acids in molar and % ratios, in 10 ml of 0.1% Formic acid in MilliQ water. The samples were evaporated at 8 mbar, 35 °C, and 600 RPM to 5 mL in volume. The sample was reconstituted to 10 mL and the drying cycles repeated lOx. The samples were then purified using Cl 8 preparative chromatography (with the absorbance measured), and the product weighed. Similarly, the change in ratio of template to amino acids after each round of amplification were calculated and plotted as shown in FIG. 1C. The yield of synthesized semaglutide of SEQ ID NO: 2 with different ratios (1 :50, 1 :100, 1 :200, and 1:300) was verified with UV-Vis Spectroscopy (FIG. ID). The results indicated that an increase in the amount of amino acid used in the method resulted in generation of higher yield of semaglutid.

[0226] Example 4: Drying Effects on Peptide Recovery After Amplification

[0227]

[0197] To assay the drying effects on product recovery, synthesized Peptides WK20 were subjected to a drying titration experiment. Briefly, WK20 peptide was amplified according to Example 3, at a template: individual amino acid ratio of 1: 100. Ten rounds of amplification was performed at a pressure of 8 mbar, 6000 rpm, and at a reaction temperature of 35°C according to the methods of Example 1, and generated approximately 10 ml of synthesized peptide. As shown in FIG. 2A, from left to right, amplification product of WK20 in vials before drying (control), 3 minutes after drying started (50% volume remaining), 5 minutes after drying started (28% volume remaining), and 7 minutes after drying started (14% volume remaining). Theoretically, drying to completeness obtains the highest yield. However, as shown in FIG. 2B, there was a negative ramification of drying to completeness in scale production as after 5 minutes of drying there was a decrease in product recovery. This was potentially due to shearing and grinding occurring during the extended drying process, and this was detrimental to product recovery. Notably, as shown in FIGs. 2C - 2F, the 50% volume fraction, which was after 3 minutes of drying, demonstrated an increased yield of synthesized peptide as compared to control and other drying times tested. Collectively, the results indicated that 3 minutes of drying was sufficient to reduce the volume, and increase the concentration of synthesized peptide product.

[0228] Example 5: Mass Spectroscopy Verification of Peptides after Recovery

[0229]

[0198] To confirm the amino acid composition of the newly synthesized peptides tandem mass spectrometry was performed. Briefly, the synthesized peptide WK20 in the aqueous solution synthesized in Example 4 was further verified using tandem mass-spectrometry (FIGS. 3A-3B). The main peaks in the actual spectra (FIG. 3A) generally matched the expected m / z values (FIG. 3B), this indicated that the correct peptide was synthesized. Notably, as shown in FIG. 3B, the ions were observed at a higher intensity in the actual spectra as compared to the predicted spectra.

[0230] Example 6: Effects of Temperature on Peptide Amplification

[0231]

[0199] To confirm the effects of temperature on peptide amplification, peptide WK20 was amplified using the method described in Example 1 at different temperatures. Briefly, 1 mg of Wk20 was solubilized with lOOmg of the required amino acids in molar and % ratios, in 10 mL of 0.1% Formic acid in MilliQ water. The samples were evaporated at 8 mbar, 25 °C, 25 °C(replicate), 30 °C, 32 °C, 34 °C, 35 °C, 45 °C and 55 °C at 600 RPM to 5 mL in volume. The sample was reconstituted to 10 ml. This was repeated lOx with samples taken at every second cycle. The samples were then purified using Cl 8 preparative chromatography (with the absorbance measured), and the product weighed. As shown in FIG. 4, the yield of amplification product increased as the temperature raised from 25 °C to 32 °C, however a decrease in peptide yield was observed at temperatures higher than 32 °C, this data suggested that increase in temperature above 32 °C increased instability for synthesized WK20 peptides.

Claims

CLAIMSWhat is claimed is:

1. A method of synthesizing a peptide, the method comprising:(a) providing a template peptide;(b) providing a mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in a template peptide sequence;(c) contacting the template peptide and the mixture of individual amino acids in an aqueous solution, wherein the conditions comprise: i. a pH of about 3.0-10.0; ii. a relative humidity of about 10% to about 90%; or iii. a pressure of about 5 mbar to about 10 mbar;(d) providing of energy to the aqueous solution of the template peptide and the mixture of individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the template peptide amino acid sequence; and(e) performing step (d) for a time period sufficient to synthesize the peptide from the mixture of individual amino acids, wherein the amino acid sequence of the peptide is the same as the amino acid sequence of the template peptide.

2. The method of claim 1 , wherein the template peptide comprises about 2 amino acids to about 200 amino acids.

3. The method of claim 1, wherein the peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% hydrophobic.

4. The method of claim 1 , wherein the conditions further comprise a reaction temperature of about 37°C to about 70°C.

5. The method of claim 1, wherein the conditions further comprise a reaction temperature of about 40°C.

6. The method of claim 1, wherein the conditions further comprise a reaction temperature of about 32°C.

7. The method of claim 1, wherein the pH is about 6.0.

8. The method of claim 1, wherein the pressure is about 8 mbar.

9. The method of claim 1 , wherein the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol.

10. The method of claim 1 , wherein the providing of energy to the aqueous solution is by a cyclical provision.

11. The method of claim 10, wherein the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light.

12. The method of claim 1, wherein periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution.

13. The method of claim 1, wherein the aqueous solution comprises pure or substantially pure water.

14. The method of claim 1, wherein the aqueous solution comprises a phosphate buffered saline solution.

15. The method of claim 1, wherein the aqueous solution comprises an acid or a base.

16. The method of claim 14, wherein the acid or the base is HC1, Formic Acid, or NaOH.

17. The method of claim 1 , wherein the aqueous solution further comprises the template peptide and the mixture of individual amino acids after contacting.

18. The method of claim 1, wherein the aqueous solution is sterile.

19. The method of claim 1, wherein the mixture of individual amino acids comprises only the amino acids that constitute the template peptide.

20. The method of claim 1, wherein the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 and 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide.

21. The method of claim 1, wherein the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 300 to 1 and 10 to 1, or between 150 to 1 and 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide.

22. The method of claim 1 , wherein the concentration of the mixture of individual amino acids in the aqueous solution prior of to the providing of energy is from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL.

23. The method of claim 1, wherein the time period is from 10 minutes to 5 days.

24. The method of claim 1 , wherein peptide synthesis is terminated by separation of the peptide from the mixture of individual amino acids in the aqueous solution.

25. The method of claim 1, wherein the method is carried out in the presence of oxygen, Nitrogen, hydrogen, or CO2.

26. The method of claim 1 , wherein the method is performed in the absence of nucleic acids, enzymes, co-enzymes, other cellular material or cells.

27. The method of claim 1 , wherein the mixture of individual amino acids comprises natural amino acids.

28. The method of claim 1, wherein the mixture of individual amino acids comprises unnatural amino acids.

29. The method of claim 1, wherein the unnatural amino acids comprise labeled amino acids.

30. The method of claim 1, wherein the mixture of individual amino acids comprises: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, or combinations thereof.

31. The method of claim 1 , wherein synthesizing a peptide is optimized for maximizing peptide yield and minimizing contaminants.

32. The method of claim 1, further comprising drying the peptide after step (d).

33. The method of claim 32, wherein the drying results in 40% to 60% volume reduction.

34. The method of claim 32, wherein the drying results in 50% volume reduction.

35. A device comprising: a sample interface configured to receive a sample comprising a template peptide; a chamber fluidically connected to the sample interface; and wherein the chamber comprises an aqueous solution, and a mixture of individual amino acids such that a peptide is synthesized with the mixture of individual amino acids in aqueous solution within the chamber.

36. The device of claim 35, wherein synthesizing a peptide within the chamber comprises:(a) contacting the template peptide and mixture of individual amino acids in the aqueous solution within the chamber; and(b) providing of energy to the aqueous solution for a time period sufficient to synthesize the peptide with the mixture of individual amino acids, wherein the amino acid sequence of the synthesized peptide is the same as the amino acid sequence of the template peptide.

37. The device of claim 35, wherein a peptide is synthesized in conditions comprising:(a) a reaction temperature of about 37°C to about 70°C;(b) a pH of about 3.0-10.0;(c) a relative humidity of about 10% to about 90%; or(d) a pressure of about 5 mbar to about 1000 mbar.

38. The device of claim 35, wherein the aqueous solution comprising the peptide is passed through a preparative column to separate the peptide from the aqueous solution after peptide synthesis is terminated.

39. The device of claim 35, wherein the preparative column is a Cl 8 column.

40. The device of claim 36, wherein the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol.

41. The device of claim 36, wherein the providing of energy to the aqueous solution is by cyclical provision.

42. The device of claim 40, wherein the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light.

43. The device of claim 41, wherein periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 3 cm from the aqueous solution.

44. The device of claim 35, wherein the aqueous solution comprises pure or substantially pure water.

45. The device of claim 35, wherein the aqueous solution comprises a phosphate buffered saline solution.

46. The device of claim 35, wherein the aqueous solution comprises an acid or a base.

47. The device of claim 44, wherein the acid or the base is HC1, Formic Acid, or NaOH.

48. The device of claim 35, wherein the device further comprises a pump for vacuum.

49. The device of claim 35, wherein the device further comprises a component for reaction temperature regulation.

50. The device of claim 35, wherein the device further comprises a component for evaporation and / or condensation regulation.

51. The device of claim 35, wherein the device further comprises a component for addition and removal of reagents.

52. The device of claim 35, wherein the device further comprises a component for purification of synthesized peptide.

53. The device of claim 35, wherein the device further comprises a component for removal of synthesized peptide.

54. The device of claim 35, wherein the device further comprises a membrane to separate the peptide from the aqueous solution after peptide synthesis is terminated.

55. The device of claim 53, wherein the membrane is a hydrophilic membrane.

56. The device of claim 53, wherein the membrane is a size-specific membrane.

57. The device of claim 35, wherein the device further comprises tubes with high optical transmission.

58. The device of claim 35, wherein the device further comprises tubes and / or containers in non- reactive or absorptive material such as glass or stainless steel.

59. The device of claim 35, wherein the device further comprises a component for monitoring and documenting the progress of peptide synthesis.

60. The device of claim 35, wherein the device further comprises a component for monitoring and documenting the contamination profile during peptide synthesis and / or after peptide synthesis is terminated.

61. The device of claim 35, wherein the device comprises a component for monitoring the progress of peptide synthesis with visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof.

62. The device of claim 35, wherein the device further comprises a component for sampling the reaction mixture to assess the progress of peptide synthesis.

63. The device of claim 61, wherein sampling comprises in-line sampling comprising receiving a sample, pumping the sample down a monolithic column, drying the sample in drying gas, characterizing the sample, or combinations thereof.

64. The device of claim 61, wherein sampling the reaction mixture to assess the progress of peptide synthesis indicates whether to add individual amino acids or remove individual amino acids from the aqueous solution.

65. The device of claim 35, wherein the device is programmed to adhere to a method of synthesizing a peptide comprising at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof.

66. The device of claim 35, wherein the device comprises multiple detection components.

67. The device of claim 35, wherein the device comprises a detector for measuring the template peptide, aqueous solution, individual amino acids, synthesized peptide, or combinations thereof.

68. The device of claim 66, wherein the detector is capable of visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof.

69. The device of claim 35, wherein the device comprises touch screen user interface.

70. The device of claim 35, wherein the device is a bench top instrument.

71. The device of claim 35, wherein the device is capable of synthesizing 10-500 g of peptide.

72. The device of claim 35, wherein the device is a microfluidic device.

73. A device for performing the method of synthesizing a peptide of claim 1-34, the device comprising a structural component as well as a composition component, wherein the composition component is a reaction mixture comprising a template peptide, an aqueous solution, a mixture of individual amino acids, and optionally a synthesized peptide.

74. A system for synthesizing a peptide from a template peptide, the system comprising:(a) a mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in the template peptide, wherein the mixture is configured to contact the template peptide and the mixture of individual amino acids in an aqueous solution; and(b) an energy generating component that is configured to provide an amount of energy to the aqueous solution of the template peptide and the mixture of individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the template peptide amino acid sequence, wherein the system is configured to synthesize the peptide from the template peptide under conditions that comprise: i. a pH of about 3.0-10.0; ii. a relative humidity of about 10% to about 90%; or iii. a pressure of about 5 mbar to about 10 mbar.

75. The system of claim 74, wherein the template peptide comprises about 2 amino acids to about 200 amino acids.

76. The system of claim 74, wherein the peptide comprises amino acids that are about 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% hydrophobic.

77. The system of claim 74, wherein the conditions further comprise a reaction temperature of about 37°C to about 70°C.

78. The system of claim 74, wherein the conditions further comprise a reaction temperature of about 40°C.

79. The system of claim 74, wherein the conditions further comprise a reaction temperature of about 32°C.

80. The system of claim 74, wherein the pH is about 6.0.

81. The system of claim 74, wherein the pressure is about 8 mbar.

82. The system of claim 74, wherein the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol.

83. The system of claim 74, wherein the providing of energy to the aqueous solution is by a cyclical provision.

84. The system of claim 83, wherein the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light.

85. The system of claim 74, wherein periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution.

86. The system of claim 74, wherein the aqueous solution comprises pure or substantially pure water.

87. The system of claim 74, wherein the aqueous solution comprises a phosphate buffered saline solution.

88. The system of claim 74, wherein the aqueous solution comprises an acid or a base.

89. The system of claim 88, wherein the acid or the base is HC1, Formic Acid, or NaOH.

90. The system of claim 74, wherein the aqueous solution further comprises the template peptide and the mixture of individual amino acids after contacting.

91. The system of claim 74, wherein the aqueous solution is sterile.

92. The system of claim 74, wherein the mixture of individual amino acids comprises only the amino acids that constitute the template peptide.

93. The system of claim 74, wherein the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 and 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide.

94. The system of claim 74, wherein the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 300 to 1 and 10 to 1, or between 150 to 1 and 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide.

95. The system of claim 74, wherein the concentration of the mixture of individual amino acids in the aqueous solution prior of to the providing of energy is from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL.

96. The system of claim 74, wherein the time period is from 10 minutes to 5 days.

97. The system of claim 74, wherein peptide synthesis is terminated by separation of the peptide from the mixture of individual amino acids in the aqueous solution.

98. The system of claim 74, wherein the method is carried out in the presence of oxygen, Nitrogen, hydrogen, or CO2.

99. The system of claim 74, wherein the method is performed in the absence of nucleic acids, enzymes, co-enzymes, other cellular material or cells.

100. The system of claim 74, wherein the mixture of individual amino acids comprises natural amino acids.

101. The system of claim 74, wherein the mixture of individual amino acids comprises unnatural amino acids.

102. The system of claim 74, wherein the unnatural amino acids comprise labeled amino acids.

103. The system of claim 74, wherein the mixture of individual amino acids comprises: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, or combinations thereof.

104. The system of claim 74, wherein the system is configured to maximize peptide yield and minimize contaminants after peptide synthesis.

105. The system of claim 74, further comprising a drying component configured to dry peptides after peptide synthesis.

106. The system of claim 105, wherein the drying results in 40% to 60% volume reduction.

107. The system of claim 105, wherein the drying results in 50% volume reduction.

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