Systems and methods for cell-free polypeptide synthesis - Patents.com

Abstract

Disclosed herein is a method for cell-free polypeptide synthesis. The method includes the steps of lysing cells to produce a cell lysate, filtering the cell lysate to produce a cell extract, contacting the cell extract with a nucleic acid encoding a polypeptide to produce a reaction mixture, and incubating the reaction mixture to produce the polypeptide. In some embodiments, disclosed herein is a method for cell-free polypeptide synthesis.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 165,335, filed March 24, 2021, which is incorporated by reference in its entirety.

[0002] References 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. Summary of the Invention

[0003] A brief summary In some embodiments, a method for cell-free polypeptide synthesis is disclosed herein. In some embodiments, the method includes lysing cells to produce a cell lysate, passing the cell lysate through a filter to produce a cell extract, contacting the cell extract with a nucleic acid encoding a polypeptide to produce a reaction mixture, and incubating the reaction mixture to produce the polypeptide. In some embodiments, the cell lysate is not subjected to clarification via centrifugation. In some embodiments, the cell extract is not subjected to clarification via centrifugation. In some embodiments, the reaction mixture is not subjected to clarification via centrifugation. In some embodiments, the method includes flocculating the cell lysate prior to passing the filter. In some embodiments, the method includes precipitating the cell lysate prior to passing the filter. In some embodiments, the filter includes a surface filter. In some embodiments, the filter includes a depth filter. In some embodiments, the passing step comprises one or more selected from the group comprising cross-flow filtration, belt filtration, microfiltration, ultrafiltration, microporous filtration, vacuum drum filtration, sieving, membrane filtration, sand filtration, and screen filtration. In some embodiments, the filter comprises pores that retain or exclude particles having a size greater than about 100 microns. In some embodiments, the filter comprises pores that retain or exclude particles having a size greater than about 15 microns. In some embodiments, the filter comprises pores that retain or exclude particles having a size greater than about 5 microns. In some embodiments, the filter comprises pores that retain or exclude particles having a size greater than about 0.4 microns. In some embodiments, the filter comprises pores that retain or exclude particles having a size greater than about 0.2 microns. In some embodiments, the filter comprises pores that retain or exclude particles having a size greater than about 0.1 microns.In some embodiments, the filter includes pores that retain or exclude particles having a size smaller than 100 microns. In some embodiments, the filter includes pores that retain or exclude particles having a size smaller than 15 microns. In some embodiments, the filter includes pores that retain or exclude particles having a size smaller than 5 microns. In some embodiments, the filter includes pores that retain or exclude particles having a size smaller than 0.4 microns. In some embodiments, the filter includes pores that retain or exclude particles having a size smaller than 0.2 microns. In some embodiments, the filter includes pores that retain or exclude particles having a size smaller than 0.1 microns. In some embodiments, the filter includes pores that retain or exclude particles having a size between about 0.4 microns and about 15 microns. In some embodiments, the filter includes pores that retain or exclude particles having a size between about 0.2 microns and about 0.4 microns. In some embodiments, the filter includes pores that retain or exclude particles having a size of about 0.2 microns. In some embodiments, the filter includes at least two filters arranged in series or parallel. In some embodiments, the filter includes at least two filters arranged in series with gradually decreasing pore sizes. In some embodiments, the filter includes a first filter including pores that retain or exclude particles having a size between about 0.5 microns and about 15 microns, a second filter including pores that retain or exclude particles having a size between about 0.2 microns and about 0.5 microns, and a third filter including pores that retain or exclude particles having a size of about 0.2 microns. In some embodiments, the depth filter includes a medium. In some embodiments, the medium forms a flow channel. In some embodiments, the medium includes two fibers bonded or fixed to provide a matrix. In some embodiments, one of the two fibers is a cellulose fiber. In some embodiments, the medium is positively charged.In some embodiments, the medium is neutrally charged. In some embodiments, the medium is negatively charged. In some embodiments, the depth filter comprises a filter aid. In some embodiments, the depth filter comprises pores that retain particles having a size that is either larger or smaller than the size of the pores. In some embodiments, the depth filter comprises a single-use filter. In some embodiments, the depth filter comprises a lenticular filter, a deep bed filter, a pad filter, a panel filter, a cartridge-type depth filter, or a combination thereof. In some embodiments, the filter comprises a first depth filter comprising pores that retain or exclude particles having a size between about 0.5 microns and about 15 microns, a second depth filter comprising pores that retain or exclude particles having a size between about 0.2 microns and about 0.5 microns, and a third filter comprising a sterilizing grade membrane and pores that retain or exclude particles having a size of about 0.2 microns. In some embodiments, the cell lysate is subjected to filtration through the first depth filter, then the second depth filter, and finally the third filter. In some embodiments, the method further comprises obtaining cells from the cell culture. In some embodiments, the obtaining comprises subjecting the cell culture to one or more selected from the group including centrifugation, tangential flow filtration, membrane separation, and combinations thereof. In some embodiments, the membrane separation comprises hollow fiber membrane separation. In some embodiments, the lysing comprises one or more selected from the group including sonication, homogenization, nitrogen cavitation, freeze-thaw, French press lysis, bead crushing, syringing, chemical lysis, enzymatic lysis, osmotic lysis, and combinations thereof. In some embodiments, the method further comprises freezing the cell extract. In some embodiments, the method further comprises lyophilizing the cell extract.In some embodiments, the cell comprises a transgenic cell, a mammalian cell, a bacterial cell, a plant cell, a yeast cell, an insect cell, a fungal cell, an algal cell, or a combination thereof. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the DNA comprises an open reading frame of the polypeptide. In some embodiments, the DNA comprises a promoter of a DNA-dependent RNA polymerase, wherein the open reading frame is operably linked to the promoter. In some embodiments, the DNA comprises a ribosome binding site located upstream of the 5' end of the open reading frame. In some embodiments, the DNA comprises a terminator located downstream of the 3' end of the open reading frame. In some embodiments, the promoter comprises a T7 promoter. In some embodiments, the T7 promoter comprises a sequence having at least 80% sequence identity to 5'-TAATACGACTCACTATAGG-3' (SEQ ID NO:1). In some embodiments, the T7 promoter comprises a sequence having at least 85% sequence identity to SEQ ID NO:1. In some embodiments, the T7 promoter comprises a sequence having at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the T7 promoter comprises a sequence having at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the T7 promoter comprises SEQ ID NO:1. In some embodiments, the terminator comprises a T7 terminator. In some embodiments, the T7 terminator comprises a sequence having at least 80% sequence identity to 5'-CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG-3' (SEQ ID NO:2). In some embodiments, the T7 terminator comprises a sequence having at least 85% sequence identity to SEQ ID NO:2. In some embodiments, the T7 terminator comprises a sequence having at least 90% sequence identity to SEQ ID NO:2.In some embodiments, the T7 terminator comprises a sequence having at least 95% sequence identity to SEQ ID NO:2. In some embodiments, the T7 terminator comprises SEQ ID NO:2. In some embodiments, the reaction mixture comprises a divalent cation. In some embodiments, the passing step is performed at a temperature between about 1° C. and about 40° C. In some embodiments, the reaction mixture comprises amino acids, nucleotides, salts, cofactors, energy source, translation template, or combinations thereof. In some embodiments, the contacting step comprises adding to the cell extract a nucleic acid encoding the polypeptide, and one or more selected from the amino acids, the nucleotides, the salts, the cofactors, the energy source, the translation template, or combinations thereof. In some embodiments, the energy source comprises a phosphate group. In some embodiments, the energy source comprises a non-phosphorylated energy group. In some embodiments, the energy source comprises glutamate and is present at a concentration between about 10 mM and about 400 mM. In some embodiments, the energy source comprises pyruvate and is present at a concentration between about 10 mM and about 400 mM. In some embodiments, the energy source comprises glucose at a concentration between about 10 and about 400 mM. In some embodiments, the salt comprises potassium at a concentration between about 50 mM and about 500 mM. In some embodiments, the salt comprises magnesium at a concentration between about 1 mM and 30 mM. In some embodiments, the salt comprises potassium at a concentration between about 10 mM and about 400 mM and magnesium at a concentration between about 1 mM and 30 mM. In some embodiments, the salt comprises ammonium at a concentration between about 1 mM and about 400 mM. In some embodiments, the method further comprises isolating the polypeptide from the reaction mixture. In some embodiments, the isolating step is performed at a temperature between about 1° C. and 30° C.In some embodiments, the nucleotides comprise a nucleoside monophosphate (NMP), a nucleoside diphosphate (NDP), a nucleoside triphosphate (NTP), or a combination thereof. In some embodiments, the reaction mixture comprises a transfer RNA.

[0004] BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized. The accompanying drawings, in which: [Brief description of the drawings]

[0005] [Figure 1] FIG. 1 compares the current state of the art method for clarification of cell lysates via centrifugation (top) with a method for clarification of cell lysates via filtration (bottom).

[0006] [Diagram 2] FIG. 2 illustrates a multi-step filtration process for clarification of a cell lysate.

[0007] [Diagram 3] FIG. 3 illustrates the state of the art cell-free polypeptide synthesis methods using cell lysate clarified via centrifugation (left) and cell-free polypeptide synthesis methods using cell lysate clarified via filtration (right).

[0008] [Figure 4] FIG. 4 illustrates the yield of superfolder green fluorescent protein (sfGFP) produced from a cell-free polypeptide synthesis method using cell lysate clarified via centrifugation (left) and cell lysate clarified via filtration (right).

[0009] [Diagram 5] FIG. 5 illustrates the effect of different filter pore sizes on the protein yield of CFPS.

[0010] [Figure 6] FIG. 6 illustrates the effect of various filter pore sizes on throughput and average flux achieved during lysate clarification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description Cell-free polypeptide synthesis (CFPS) is a platform technology that uses crude cell extracts to rapidly and reliably synthesize desired polypeptide products. This crude cell extract is the starting point of the cell-free reaction because it utilizes the endogenous machinery that allows polypeptide translation (and, if necessary, mRNA transcription) to generate polypeptide and protein products from nucleic acid templates (e.g., plasmid or linear DNA) and supplemented nucleotides, amino acids, energy sources, and other necessary cofactors. CFPS has many potential advantages over cell expression, including faster expression timelines, the freedom to customize expression conditions, and avoiding the need to generate unique cell lines for each synthesized polypeptide or protein by direct addition of linear or plasmid DNA templates.

[0012] However, cell-free extract preparation methods are time-consuming and laborious. In the CFPS field, much effort has been devoted to streamlining extract preparation, which generally consists of cell growth, cell harvesting, cell lysis, and post-lysis processing. Manufacturing-scale polypeptide or protein synthesis may use alternative methods of post-lysis processing that reduce energy costs and washing while maintaining high protein yields. Post-lysis clarification typically relies on centrifugation. Many reports describe attempts to lower the power demands of the post-lysis centrifugation step (e.g., lower speeds and / or lower spins). For example, operation times for clarifying cell lysates using a centrifuge can range from about 30 minutes to 24 hours. Additional time is required to clean the centrifugation equipment (e.g., up to 48 hours of washing time at large scale). Depending on the scale, centrifugation equipment can cost $10,000 to $1,000,000 to purchase, and annual operating costs can be $50,000 to $200,000. Centrifugation used for high-speed post-lysis processing generally requires expensive and reusable stainless steel equipment with risk of cross-contamination, and requires very time-consuming cleaning validation for use on many products, especially those made under GMP conditions. Thus, the need for centrifugation makes post-lysis processing incompatible with disposable manufacturing methods, which are very versatile, do not have risk of cross-contamination, and are currently standard in the biopharmaceutical industry. Centrifugation can also be difficult to implement in high-throughput formats (e.g., multi-well plates) for high-throughput extract production. Processes that replace the centrifugation step with another clarification method during extract preparation may produce proteins at lower cost and / or in less time per gram, while the titers of proteins produced using the improved extraction method may be comparable. The improved process can be implemented in resource-limited environments, for high-throughput extract production, with less investment, and in existing or new disposable manufacturing environments.

[0013] Thus, there remains a need for scaled-up CFPS systems and methods that can prepare cell extracts efficiently and inexpensively, with reduced time and equipment costs, and that can be implemented in resource-limited or disposable manufacturing environments.

[0014] The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques that are within the skill of the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0015] definition Throughout this disclosure, various embodiments are presented in range format. It should be understood that the description in range format is merely for convenience and simplicity and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual numerical values, within the range to one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual values ​​within that range (e.g., 1.1, 2, 2.3, 5, and 5.9). This is true regardless of the breadth of the range. The upper and lower limits of these intervening ranges may be independently included in the smaller ranges, and are also encompassed within the scope of the disclosure, subject to any specifically excluded boundaries in the specified range. Where the stated range includes one or both of its limits, ranges excluding either or both of those included limits are also encompassed within the disclosure unless the context clearly dictates otherwise.

[0016] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms "a," "an," and "the" are intended to include the plural unless the context clearly dictates otherwise. It is further understood that the terms "comprises" and / or "comprising," as used herein, specify the presence of the specified 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. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0017] Unless specifically specified or clear from the context, as used herein, the term "about" in reference to a numerical value or range of numerical values ​​is understood to mean the specified numerical value and numerical value ±10%, or, with respect to values ​​recited in relation to a range, 10% below the recited lower limit and 10% above the recited upper limit.

[0018] The term "cell lysis" refers to the disruption of cell membranes. Lysis is generally carried out in a buffer configured to limit or prevent the inactivation, denaturation, and degradation of cellular proteins.

[0019] The term "cell lysate" generally includes the contents of lysed cells and components of the lysis buffer. In some cases, the cell lysate includes exogenous materials introduced during cell culture. The terms "cell lysate" and "cell-free lysate" are used interchangeably in this disclosure.

[0020] The terms "clarify", "clarification", and "clarification step" refer to one or more steps used to prepare cell extracts for use with cell-free polypeptide synthesis (CFPS), which are generally performed to remove whole cells, cell debris, and extraneous materials present in the cell lysate that are not required for cell-free polypeptide synthesis.

[0021] The term "depth filter" refers to a filter that traps or absorbs particles both within or on the filter.

[0022] The term "cell debris" refers to cellular proteins, membranes, cell wall components, and / or genomic DNA that are normally present in a cell lysate.

[0023] The term "cell-free extract" refers to the product obtained by clarification of a cell lysate. Cell-free Polypeptide Synthesis

[0024] Cell-free polypeptide synthesis (CFPS) is a platform technology that uses crude cell extracts to rapidly and reliably synthesize desired polypeptide or protein products. CFPS utilizes the cell's endogenous machinery and / or supplemented nucleotides, amino acids, energy sources, and other necessary cofactors to transcribe and translate mRNA from a nucleic acid template (e.g., a plasmid or linear DNA).

[0025] As illustrated in FIG. 1, existing cell-free extract-based CFPS methods (top panel) include the following steps: cell culture or cell growth, cell harvesting by centrifugation, cell lysis by homogenization, cell lysate clarification by centrifugation, and CFPS using cell-free extract clarified via centrifugation. Post-lysis clarification processes usually rely on centrifugation, which can require long operation times and substantial costs for equipment such as centrifuges. The clarification step is intended to remove whole cells, cell debris, and extraneous materials present in the cell lysate that may interfere with the ability of the extract to generate the desired polypeptide or protein product specifically and in high yield and high quality (including post-translational modification, folding, and limited proteolysis), as well as the reproducibility, low bioburden, and material properties of the extract, without requiring CFPS. There is a need for a post-lysis processing method that replaces the centrifugation step with one that can reduce both the time and cost of CFPS and can be performed in a resource-limited or disposable manufacturing environment. Disclosed herein are methods and systems for CFPS using cell extract clarified via filtration.

[0026] Methods and systems for clarification of cell lysates via filtration - Patents.com Method for clarification of cell lysates via filtration As illustrated in FIG. 1, disclosed herein is a method for CFPS that uses filtration instead of centrifugation to clarify cell lysates and obtain cell-free extracts for CFPS (lower panel).

[0027] In some embodiments, methods for cell-free polypeptide synthesis (CFPS) are described herein. In some embodiments, the methods may include obtaining a cell. In some embodiments, the obtaining may include growing the cell. In some embodiments, the obtaining may include culturing the cell. In some embodiments, the cell may include a cell suitable for CFPS. In some embodiments, the cell may include one or more selected from the group including a transgenic cell, a mammalian cell, a bacterial cell, a plant cell, a yeast cell, an insect cell, a fungal cell, and an algal cell. In some embodiments, the cells may comprise one or more selected from the group including Escherichia coli, Spodoptera frugiperda, Saccharomyces cerevisiae, Neurospora crassa, Streptomyces, Vibrio natriegens, Bacillus subtilis, Tobacco, Arabidopsis, Pseudomonas Putida, Bacillus megaterium, Archaea, Leishmania tarentolae, Chai's Bumster ovary cells, rabbit reticulocytes, wheat germ cells, HEK cells, HeLa cells, and genetically modified variants thereof.

[0028] In some embodiments, the method may include harvesting the cells. In some embodiments, the harvesting may include subjecting the cells to one or more selected from the group including centrifugation, tangential flow filtration, and diafiltration centrifugation. In some embodiments, the harvesting may include subjecting the cells in cell culture to one or more selected from the group including centrifugation, tangential flow filtration, and diafiltration centrifugation. In some embodiments, the diafiltration may include tangential flow filtration, normal-flow filtration, or hollow fiber membrane separation. In some embodiments, the harvesting may include resuspending the cells.In some embodiments, the cells are stored in a volume of between about 0.01 ml and 50 ml per gram of wet weight of cells, e.g., about 0.01 ml, 0.02 ml, 0.03 ml, 0.04 ml, 0.05 ml, 0.06 ml, 0.07 ml, 0.08 ml, 0.09 ml, 0.1 ml, 0.11 ml, 0.12 ml, 0.13 ml, 0.14 ml, 0.15 ml, 0.16 ml, 0.17 ml, 0.18 ml, 0.19 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 18 ml, 19 ml, 20 ml, 20 ml, 20 ml, 25 ml, 26 ml, 27 ml, 28 ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38 ml, 39 ml, 40 ml, 41 ml, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml, 49 ml, 50 ml, 51 ml, 52 ml, 53 ml, 54 ml, 55 ml, 56 ml, 57 ml, 58 ml, 59 ml, 60 ml, 61 ml, 62 ml, 63 ml, 64 ml, 65 ml, 66 ml, 67 ml, 68 ml, 69 ml, 70 ml, 71 ml, 72 ml, 73 ml, 74 ml, 75 ml, 76 ml, 77 ml .19ml, 0.2ml, 0.21ml, 0.22ml, 0.23ml, 0.24ml, 0.25ml, 0.26ml, 0.27ml, 0.28ml, 0.29ml, 0.3ml, 0.31ml, 0.3 2ml, 0.33ml, 0.34ml, 0.35ml, 0.36ml, 0.37ml, 0.38ml, 0.39ml, 0.4ml, 0.41ml, 0.42ml, 0.43ml, 0.44ml, 0.45 ml, 0.46ml, 0.47ml, 0.48ml, 0.49ml, 0.5ml, 0.55ml, 0.6ml, 0.65ml, 0.7ml, 0.75ml, 0.8ml, 0.85ml, 0.9ml, 0 .95ml, 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml, 11ml, 12ml, 13ml, 14ml, 15ml, 16ml, 17ml, 18ml, 19ml , 20ml, 21ml, 22ml, 23ml, 24ml, 25ml, 26ml, 27ml, 28ml, 29ml, 30ml, 31ml, 32ml, 33ml, 34ml, 35ml, 36ml, 37ml, 38ml, 39ml, 40ml, 41ml, 42ml, 43ml, 44ml, 45ml, 46ml, 47ml, 48ml, 49ml, or about 50ml or any volume therebetween.

[0029] In some embodiments, the method may include lysing the cells to produce a cell lysate. In some embodiments, the cell lysate is not subjected to clarification via centrifugation. In some embodiments, the lysing may include one or more selected from the group including French press, bead crushing, sonication, homogenization, nitrogen cavitation, freeze-thawing, syringing, chemical lysis, enzymatic lysis, and osmotic lysis.

[0030] In some embodiments, the method may include passing the cell lysate through a filter to produce a cell extract. In some embodiments, the cell lysate is not subjected to clarification via centrifugation. In some embodiments, the cell extract is not subjected to clarification via centrifugation. In some embodiments, the method may include flocculating the cell lysate prior to the filtering step. In some embodiments, the method may include precipitating components within the cell lysate prior to the filtering step.

[0031] In some embodiments, various filters may be used to generate the cell extract from the cell lysate through filtration or clarification. In some embodiments, the filter may include one filter. In some embodiments, the filter may include at least two filters, such as two filters, three filters, four filters, five filters, six filters, seven filters, eight filters, nine filters, ten filters, or more than ten filters. In some embodiments, the filter may include a filter disclosed herein, or a combination of two or more of the filters disclosed herein. When the filter includes at least two filters, one of the at least two filters may include a filter disclosed herein. When the filter includes at least two filters, each of the at least two filters may include a filter disclosed herein. In some embodiments, the at least two filters may be arranged in series. In some embodiments, the at least two filters may be arranged in series with gradually decreasing pore sizes. In some embodiments, the passing step of the method may include passing the cell lysate through at least two filters arranged in series to produce the cell extract. In some embodiments, the passing step may include passing the cell lysate through the at least two filters arranged in series such that the cell lysate is filtered through a series of filters with gradually decreasing pore sizes to produce the cell extract. In some embodiments, the at least two filters may be arranged in parallel. In some embodiments, the passing step of the method may include passing the cell lysate through at least two filters arranged in parallel to produce the cell extract. In some embodiments, some but not all of the at least two filters may be arranged in series.In some embodiments, some but not all of the at least two filters may be arranged in parallel, hi some embodiments, some of the at least two filters may be arranged in series and some of the at least two filters may be arranged in parallel.

[0032] In some embodiments, the filter is sized between about 100 microns and about 1000 microns, e.g., about 100 microns, 110 microns, 120 microns, 130 microns, 140 microns, 150 microns, 160 microns, 170 microns, 180 microns, 190 microns, 200 microns, 210 microns, 220 microns, 230 microns, 240 microns, 250 microns, 260 microns, 270 microns, 280 microns, 290 microns, 300 microns, 310 microns, 320 microns, 330 microns, 340 microns, 350 microns, 360 microns, 370 microns, 380 microns, 390 microns, 400 microns, 410 microns, 420 microns, 430 microns, 440 microns, 450 microns, 460 microns, 470 microns, 480 microns, 490 microns, 500 microns, 510 microns, 520 microns, 530 microns, 540 microns , 550 microns, 560 microns, 570 microns, 580 microns, 590 microns, 600 microns, 610 microns, 620 microns, 630 microns, 640 microns, 650 microns, 660 microns, 670 microns, 680 microns, 690 microns, 700 microns, 710 microns, 720 microns, 730 microns, 740 microns, 750 microns, 760 microns, 770 microns, 780 microns, 790 microns, 800 microns, 810 microns, 820 microns, 830 microns, 840 microns, 850 microns, 860 microns, 870 microns, 880 microns, 890 microns, 900 microns, 910 microns, 920 microns, 930 microns, 940 microns, 950 microns, 960 microns, 970 microns, 980 microns, 990 microns, or about 1000 microns, or any size therebetween.

[0033] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 100 microns and about 1000 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 100 microns and about 900 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 100 microns and about 800 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 100 microns and about 700 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 100 microns and about 600 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 100 microns and about 500 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 100 microns and about 400 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 100 microns and about 300 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 100 microns and about 200 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 200 microns and about 1000 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 200 microns and about 900 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 200 microns and about 800 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 200 microns and about 700 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 200 microns and about 600 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 200 microns and about 500 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 200 microns and about 400 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 200 microns and about 300 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 300 microns and about 1000 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 300 microns and about 900 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 300 microns and about 800 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 300 microns and about 700 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 300 microns and about 600 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 300 microns and about 500 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 300 microns and about 400 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 400 microns and about 1000 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 400 microns and about 900 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 400 microns and about 800 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 400 microns and about 700 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 400 microns and about 600 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 400 microns and about 500 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 500 microns and about 1000 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 500 microns and about 900 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 500 microns and about 800 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 500 microns and about 700 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 500 microns and about 600 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 600 microns and about 1000 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 600 microns and about 900 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 600 microns and about 800 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 600 microns and about 700 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 700 microns and about 1000 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 700 microns and about 900 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 700 microns and about 800 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 800 microns and about 1000 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 800 microns and about 900 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 900 microns and about 1000 microns.

[0034] In some embodiments, the filters are sized between about 1 micron and about 100 microns, e.g., about 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, 10 microns, 11 microns, 12 microns, 13 microns, 14 microns, 15 microns, 16 microns, 17 microns, 18 microns, 19 microns, 20 microns, 21 microns, 22 microns, 23 microns, 24 microns, 25 microns, 26 microns, 27 microns, 28 microns, 29 microns, 30 microns, 31 microns, 32 microns, 33 microns, 34 microns, 35 microns, 36 microns, 37 microns, 38 microns, 39 microns, 40 microns, 41 microns, 42 microns, 43 microns, 44 microns, 45 microns, 46 microns, 47 microns, 48 ​​microns, 49 microns, 50 microns, The porous porous material may include pores that retain or remove particles having a size of 51 microns, 52 microns, 53 microns, 54 microns, 55 microns, 56 microns, 57 microns, 58 microns, 59 microns, 60 microns, 61 microns, 62 microns, 63 microns, 64 microns, 65 microns, 66 microns, 67 microns, 68 microns, 69 microns, 70 microns, 71 microns, 72 microns, 73 microns, 74 microns, 75 microns, 76 microns, 77 microns, 78 microns, 79 microns, 80 microns, 81 microns, 82 microns, 83 microns, 84 microns, 85 microns, 86 microns, 87 microns, 88 microns, 89 microns, 90 microns, 91 microns, 92 microns, 93 microns, 94 microns, 95 microns, 96 microns, 97 microns, 98 microns, 99 microns, or about 100 microns, or any size therebetween.

[0035] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 10 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 10 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 10 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 10 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 10 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 10 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 10 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 10 microns and about 30 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 10 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 20 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 20 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 20 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 20 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 20 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 20 microns and about 50 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 20 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 20 microns and about 30 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 30 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 30 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 30 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 30 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 30 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 30 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 30 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 40 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 40 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 40 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 40 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 40 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 40 microns and about 50 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 50 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 50 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 50 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 50 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 50 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 60 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 60 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 60 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 60 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 60 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 70 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 70 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 70 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 80 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 80 microns and about 90 microns.In some embodiments, the filter may include pores that retain or remove particles having a size between about 90 microns and about 100 microns.

[0036] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 1 micron and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 1 micron and about 9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 1 micron and about 8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 1 micron and about 7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 1 micron and about 6 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 1 micron and about 5 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 1 micron and about 4 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 1 micron and about 3 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 1 micron and about 2 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 2 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 2 microns and about 9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 2 microns and about 8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 2 microns and about 7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 2 microns and about 6 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 2 microns and about 5 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 2 microns and about 4 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 2 microns and about 3 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 3 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 3 microns and about 9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 3 microns and about 8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 3 microns and about 7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 3 microns and about 6 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 3 microns and about 5 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 3 microns and about 4 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 4 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 4 microns and about 9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 4 microns and about 8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 4 microns and about 7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 4 microns and about 6 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 4 microns and about 5 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 5 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 5 microns and about 9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 5 microns and about 8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 5 microns and about 7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 5 microns and about 6 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 6 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 6 microns and about 9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 6 microns and about 8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 6 microns and about 7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 6 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 7 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 7 microns and about 9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 7 microns and about 8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 8 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 8 microns and about 9 microns.In some embodiments, the filter may include pores that retain or remove particles having a size between about 9 microns and about 10 microns.

[0037] In some embodiments, the filter is between about 0.001 microns and about 1.0 microns in size, e.g., about 0.001 microns, 0.002 microns, 0.003 microns, 0.004 microns, 0.005 microns, 0.006 microns, 0.007 microns, 0.008 microns, 0.009 microns, 0.01 microns, 0.02 microns, 0.03 microns, 0.04 microns, 0.05 microns, 0.06 microns, 0.07 microns, 0.08 microns, 0.09 microns, 0.1 microns, 0.11 microns, 0.12 microns. , 0.13 microns, 0.14 microns, 0.15 microns, 0.16 microns, 0.17 microns, 0.18 microns, 0.19 microns, 0.2 microns, 0.21 microns, 0.22 microns, 0.23 microns, 0.24 microns, 0.25 microns, 0.26 microns, 0.27 microns, 0.28 microns, 0.29 microns, 0.3 microns, 0.31 microns, 0.32 microns, 0.33 microns, 0.34 microns, 0.35 microns, 0.36 microns, 0.37 microns, 0.38 microns, 0.39 microns, 0.4 microns, 0.41 microns, 0.42 microns, 0.43 microns, 0.44 microns, 0.45 microns, 0.46 microns, 0.47 microns, 0.48 microns, 0.49 microns, 0.5 microns, 0.51 microns, 0.52 microns, 0.53 microns, 0.54 microns, 0.55 microns, 0.56 microns, 0.57 microns, 0.58 microns, 0.59 microns, 0.6 microns, 0.61 microns, 0.62 microns, 0.63 microns, 0.64 microns, 0.65 microns, 0.66 microns, 0.67 microns, 0.68 microns, 0.69 microns, 0.7 microns, 0.71 microns, 0.72 microns, 0.73 microns, 0.74 microns, 0.75 microns, 0.76 microns, 0.77 microns, 0.78 microns, 0.79 microns, 0.8 microns, 0.81 microns, 0.82 microns, 0.83 microns, 0.84 microns, 0.85 microns, 0.86 microns, 0.87 microns, 0.88 microns, 0.89 microns, 0.9 microns, 0.91 microns, 0.92 microns, 0.93 microns, 0.94 microns, 0.95 microns, 0.96 microns, 0.The porous membrane may contain pores that retain or exclude particles having sizes of 97 microns, 0.98 microns, 0.99 microns, or about 1.0 microns, or any size therebetween.

[0038] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 1.0 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 0.9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 0.8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 0.7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 0.6 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 0.5 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 0.4 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 0.3 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 0.2 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 1.0 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 0.9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 0.8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 0.7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 0.6 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 0.5 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 0.4 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 0.3 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 1.0 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 0.9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 0.8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 0.7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 0.6 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 0.5 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 0.4 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 1.0 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 0.9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 0.8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 0.7 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 0.6 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 0.5 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 1.0 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 0.9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 0.8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 0.7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 0.6 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 1.0 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 0.9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 0.8 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 0.7 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 1.0 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 0.9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 0.8 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 1.0 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 0.9 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 1.0 microns.

[0039] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.01 microns and about 0.1 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.01 microns and about 0.09 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.01 microns and about 0.08 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.01 microns and about 0.07 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.01 microns and about 0.06 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.01 microns and about 0.05 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.01 microns and about 0.04 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.01 microns and about 0.03 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.01 microns and about 0.02 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.02 microns and about 0.1 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.02 microns and about 0.09 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.02 microns and about 0.08 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.02 microns and about 0.07 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.02 microns and about 0.06 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.02 microns and about 0.05 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.02 microns and about 0.04 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.02 microns and about 0.03 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.03 microns and about 0.1 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.03 microns and about 0.09 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.03 microns and about 0.08 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.03 microns and about 0.07 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.03 microns and about 0.06 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.03 microns and about 0.05 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.03 microns and about 0.04 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.04 microns and about 0.1 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.04 microns and about 0.09 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.04 microns and about 0.08 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.04 microns and about 0.07 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.04 microns and about 0.06 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.04 microns and about 0.05 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.05 microns and about 0.1 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.05 microns and about 0.09 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.05 microns and about 0.08 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.05 microns and about 0.07 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.05 microns and about 0.06 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.06 microns and about 0.1 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.06 microns and about 0.09 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.06 microns and about 0.08 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.06 microns and about 0.07 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.07 microns and about 0.1 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.07 microns and about 0.09 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.07 microns and about 0.08 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.08 microns and about 0.1 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.08 microns and about 0.09 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.09 microns and about 0.1 microns.

[0040] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.001 microns and about 0.01 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.001 microns and about 0.009 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.001 microns and about 0.008 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.001 microns and about 0.007 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.001 microns and about 0.006 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.001 microns and about 0.005 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.001 microns and about 0.004 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.001 microns and about 0.003 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.001 microns and about 0.002 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.002 microns and about 0.01 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.002 microns and about 0.009 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.002 microns and about 0.008 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.002 microns and about 0.007 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.002 microns and about 0.006 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.002 microns and about 0.005 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.002 microns and about 0.004 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.002 microns and about 0.003 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.003 microns and about 0.01 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.003 microns and about 0.009 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.003 microns and about 0.008 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.003 microns and about 0.007 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.003 microns and about 0.006 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.003 microns and about 0.005 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.003 microns and about 0.004 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.004 microns and about 0.01 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.004 microns and about 0.009 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.004 microns and about 0.008 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.004 microns and about 0.007 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.004 microns and about 0.006 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.004 microns and about 0.005 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.005 microns and about 0.01 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.005 microns and about 0.009 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.005 microns and about 0.008 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.005 microns and about 0.007 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.005 microns and about 0.006 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.006 microns and about 0.01 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.006 microns and about 0.009 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.006 microns and about 0.008 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.006 microns and about 0.007 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.007 microns and about 0.01 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.007 microns and about 0.009 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.007 microns and about 0.008 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.008 microns and about 0.01 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.008 microns and about 0.009 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.009 microns and about 0.01 microns.

[0041] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 95 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 85 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 75 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 65 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 55 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 45 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 35 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 30 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 25 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 15 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.1 microns and about 5 microns.

[0042] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 95 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 85 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 75 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 65 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 55 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 45 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 35 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 30 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 25 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 15 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 5 microns.

[0043] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 95 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 85 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 75 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 65 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 55 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 45 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 35 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 30 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 25 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 15 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.3 microns and about 5 microns.

[0044] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 95 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 85 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 75 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 65 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 55 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 45 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 35 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 30 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 25 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 15 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.4 microns and about 5 microns.

[0045] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 95 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 85 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 75 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 65 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 55 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 45 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 35 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 30 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 25 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 15 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 5 microns.

[0046] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 95 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 85 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 75 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 65 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 55 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 45 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 35 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.16 microns and about 30 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 25 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 15 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.6 microns and about 5 microns.

[0047] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 95 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 85 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 75 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 65 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 55 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 45 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 35 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 30 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 25 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 15 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.7 microns and about 5 microns.

[0048] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 95 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 85 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 75 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 65 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 55 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 45 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 35 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 30 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 25 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 15 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.8 microns and about 5 microns.

[0049] In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 100 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 95 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 90 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 85 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 80 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 75 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 70 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 65 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 60 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 55 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 50 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 45 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 40 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 35 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 30 microns.In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 25 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 20 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 15 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 10 microns. In some embodiments, the filter may include pores that retain or exclude particles having a size between about 0.9 microns and about 5 microns.

[0050] In some embodiments, the filter may include a surface filter. In some embodiments, the filter may include a depth filter. In some embodiments, the filter may include a filter including a sterilizing grade membrane. In some embodiments, the filter including the sterilizing grade membrane may retain particles having a size greater than about 0.1 microns. In some embodiments, the filter including the sterilizing grade membrane may retain particles having a size greater than about 0.2 microns. In some embodiments, the filter including the sterilizing grade membrane may retain particles having a size greater than about 0.4 microns. In some embodiments, the passing step of the CFPS method disclosed herein may be performed using one or more selected from the group including: cross-flow filtration, belt filtration, microfiltration, ultrafiltration, microporous filtration, vacuum drum filtration, sieving, membrane filtration, sand filtration, and screen filtration. In some embodiments, the depth filter may include a medium. In some embodiments, the medium of the depth filter may form a flow channel. In some embodiments, the medium of the depth filter may include a complex, tortuous flow channel. In some embodiments, the depth filter medium may include two fibers. In some embodiments, the depth filter medium may be positively charged. In some embodiments, the depth filter medium may be neutrally charged. In some embodiments, the depth filter medium may be negatively charged. In some embodiments, the two fibers may be bonded or fixed to provide a matrix. In some embodiments, the two fibers may be bonded or fixed to provide a random matrix. In some embodiments, the two fibers may be bonded by a binder. In some embodiments, the binder may include a charged or uncharged resin. In some embodiments, the two fibers may absorb or capture particles in the cell lysate.In some embodiments, one of the two fibers may comprise cellulose fibers. In some embodiments, particles in the cell lysate may be separated via capture by the medium. In some embodiments, particles in the cell lysate may be separated via adsorption to the medium. In some embodiments, particles in the cell lysate may be retained in the filter via size exclusion. In some embodiments, particles in the cell lysate may be retained in the filter by adsorption to the filter medium via hydrophobic, ionic, or other types of interactions. In some embodiments, the depth filter may comprise a filter aid. In some embodiments, the filter aid may comprise diatomaceous earth. In some embodiments, the depth filter may comprise pores that retain particles having a size larger than the size of the pores. In some embodiments, the depth filter may comprise pores that retain particles having a size smaller than the size of the pores. In some embodiments, the depth filter may comprise a single-use filter. In some embodiments, the depth filter may include one or more selected from the group including a lenticular filter, a deep bed filter, a pad filter, a panel filter, and a cartridge type depth filter.

[0051] In some embodiments, the filter may include three filters, a first filter, a second filter, and a third filter. In some embodiments, the first filter may include a filter disclosed herein. In some embodiments, the first filter may include a filter including pores that retain or exclude particles having a size between about 0.5 microns and about 15 microns. In some embodiments, the second filter may include a filter disclosed herein. In some embodiments, the second filter may include a filter including pores that retain or exclude particles having a size between about 0.2 microns and about 0.5 microns. In some embodiments, the third filter may include a filter disclosed herein. In some embodiments, the third filter may include a filter including pores that retain or exclude particles having a size of about 0.2 microns. In some embodiments, at least two of the first filter, second filter, and third filter may be arranged in series. In some embodiments, all three of the first filter, second filter, and third filter may be arranged in series. In some embodiments, at least two of the first, second and third filters may be arranged in parallel, hi some embodiments, all three of the first, second and third filters may be arranged in parallel.

[0052] In some embodiments, the filter may include three filters, a first depth filter, a second depth filter, and a third filter comprising a sterilizing grade membrane. In some embodiments, the first depth filter may include pores that retain or exclude particles having a size between about 0.5 microns and about 15 microns. In some embodiments, the second depth filter may include pores that retain or exclude particles having a size between about 0.2 microns and about 0.5 microns. In some embodiments, the third filter comprising a sterilizing grade membrane may include pores that retain or exclude particles having a size of about 0.2 microns. In some embodiments, at least two of the first depth filter, the second depth filter, and the third filter comprising a sterilizing grade membrane may be arranged in series. In some embodiments, all three of the first depth filter, the second depth filter, and the third filter comprising a sterilizing grade membrane may be arranged in series. In some embodiments, at least two of the first depth filter, the second depth filter, and the third filter comprising sterilizing grade membrane can be arranged in parallel.In some embodiments, all three of the first depth filter, the second depth filter, and the third filter comprising sterilizing grade membrane can be arranged in parallel.When all three of the first depth filter, the second depth filter, and the third filter comprising sterilizing grade membrane are arranged in series, the passing step of the method disclosed herein can include passing the cell lysate through the first depth filter to produce a first cell extract, then through the second depth filter to produce a second cell extract, and finally through the third filter comprising sterilizing grade membrane to produce a third cell extract.

[0053] In some embodiments, based on the solid content in the cell lysate, the passing step can be appropriately configured to optimize extraction. In some embodiments, the passing step can include staged filtration. In some embodiments, the cell lysate can be filtered through at least two filters arranged in series and with decreasing pore size. In some embodiments, the composition of the filter medium can be configured for better flow, particle capture, and cell extract quality. For example, the filter can include two filters, a first filter and a second filter. In some embodiments, the first filter can be used to produce a first clarified cell lysate, which is then passed through the second filter to produce a second clarified cell lysate or clarified cell extract. In some embodiments, the first filter can include pores that exclude or retain particles larger than about 100 microns, about 50 microns, about 30 microns, or about 15 microns. In some embodiments, the second filter may include pores that exclude or retain particles larger than about 5 microns, about 2 microns, about 1 micron, about 500 nm, or about 200 nm. In some embodiments, the cell lysate may be filtered through at least two filters arranged in parallel to make the filtration process less prone to clogging and increase filter surface area. In some embodiments, each of the at least two filters arranged in parallel may include an identical filter. In some embodiments, the at least two filters including an identical filter may be contacted with the cell lysate simultaneously so that the filtration process can continue even if one of the at least two filters is clogged.

[0054] In some embodiments, the method may include a post-filtration step of the cell extract, depending on the characteristics of the cell lysate. In some embodiments, the post-filtration step may include desalting the cell extract. In some embodiments, the post-filtration step may include subjecting the cell extract to buffer exchange. In some embodiments, the post-filtration step may include dialyzing the cell extract. In some embodiments, the post-filtration step may include subjecting the cell extract to diafiltration. In some embodiments, the post-filtration step may include freezing the cell extract. In some embodiments, the post-filtration step may include lyophilizing the cell extract.

[0055] In some embodiments, the method may include contacting the cell extract with a nucleic acid encoding a polypeptide to produce a reaction mixture. In some embodiments, the cell extract is not subjected to clarification via centrifugation prior to the contacting step. In some embodiments, the reaction mixture is not subjected to clarification via centrifugation. In some embodiments, the nucleic acid may include an RNA translation template. In some embodiments, the nucleic acid may include an exogenous RNA translation template. In some embodiments, the reaction mixture may include a DNA expression template. In some embodiments, the DNA expression template may encode an open reading frame for the polypeptide. In some embodiments, the open reading frame may be operably linked to a promoter of a DNA-dependent RNA polymerase. In some embodiments, the DNA-dependent RNA polymerase may direct transcription of the DNA expression template.

[0056] In some embodiments, the contacting step may include adding a nucleic acid encoding the polypeptide to the cell extract. In some embodiments, the reaction mixture may include amino acids, nucleotides, salts, cofactors, energy source, translation template, or a combination thereof. In some embodiments, the reaction mixture may include divalent cations. In some embodiments, the reaction mixture may include any of the components disclosed herein. In some embodiments, the contacting step may include adding one or more of the group including amino acids, nucleotides, salts, cofactors, energy source, and translation template to the cell extract. In some embodiments, the energy source may include a phosphate group. In some embodiments, the energy source may include a non-phosphorylated energy group. The energy source may include one or more of the group including phosphoenolpyruvate, glutamate, glycerol, pyruvate, glucose, and creatine phosphate. In some embodiments, the nucleotides in the reaction mixture may include nucleoside monophosphates (NMPs), nucleoside diphosphates (NDPs), nucleoside triphosphates (NTPs), or combinations thereof. In some embodiments, the reaction mixture may include transfer RNA (tRNA). In some embodiments, the reaction mixture may include enzyme cofactors. In some embodiments, the reaction mixture may include a metabolite. In some embodiments, the metabolite may include one or more selected from the group including folinic acid, nicotinamide adenine dinucleotide, coenzyme A, and sodium oxalate.

[0057] In some embodiments, the reaction mixture may include an organic anion. In some embodiments, the organic anion may include glutamate, acetate, or a combination thereof. In some embodiments, the organic anion may be at a concentration between about 0 mM and about 300 mM, for example, about 0.001 mM, 0.002 mM, 0.003 mM, 0.004 mM, 0.005 mM, 0.006 mM, 0.007 mM, 0.008 mM, 0.009 mM, 0.01 mM, 0.011 mM, 0.012 mM, 0.013 mM, 0.014 mM, 0.015 mM, 0.016 mM, 0.017 mM, 0.018 mM, 0.019 mM, 0.02 mM, 0.021 mM, 0.022 mM, 0.023 mM , 0.024mM, 0.025mM, 0.026mM, 0.027mM, 0.028mM, 0.029mM, 0.03mM, 0.031mM, 0.032mM, 0.033mM, 0.034mM, 0.035mM, 0.036mM, 0.037mM, 0. 038mM, 0.039mM, 0.04mM, 0.041mM, 0.042mM, 0.043mM, 0.044mM, 0.045mM, 0.046mM, 0.047mM, 0.048mM, 0.049mM, 0.05mM, 0.055mM, 0.06mM , 0.065mM, 0.07mM, 0.075mM, 0.08mM, 0.085mM, 0.09mM, 0.095mM, 0.1mM, 0.11mM, 0.12mM, 0.13mM, 0.14mM, 0.15mM, 0.16mM, 0.17mM, 0.18m M, 0.19mM, 0.2mM, 0.21mM, 0.22mM, 0.23mM, 0.24mM, 0.25mM, 0.26mM, 0.27mM, 0.28mM, 0.29mM, 0.3mM, 0.31mM, 0.32mM, 0.33mM, 0.34mM, 0. 35mM, 0.36mM, 0.37mM, 0.38mM, 0.39mM, 0.4mM, 0.41mM, 0.42mM, 0.43mM, 0.44mM, 0.45mM, 0.46mM, 0.47mM, 0.48mM, 0.49mM, 0.5mM, 0.51mM ,0.52mM,0.53mM,0.54mM,0.55mM,0.56mM,0.57mM,0.58mM,0.59mM,0.6mM,0.61mM,0.62mM,0.63mM,0.64mM,0.65mM,0.66mM,0.67mM,0.68mM、0.69mM、0.7mM、0.71mM、0.72mM、0.73mM、0.74mM、0.75mM、0.76mM、0.77mM、0.78mM、0.79mM、0.8mM、0.81mM、0.82mM、0.83mM、0.84mM、0.85mM、0.86mM、0.87mM、0.88mM、0.89mM、0.9mM、0.91mM、0.92mM、0.93mM、0.94mM、0.95mM、0.96mM、0.97mM、0.98mM、0.99mM、1mM、1.1mM、1.2mM、1.3mM、1.4mM、1.5mM、1.6mM、1.7mM、1.8mM、1.9mM、2mM、2.1mM、2.2mM、2.3mM、2.4mM、2.5mM、2.6mM、2.7mM、2.8mM、2.9mM、3mM、3.1mM、3.2mM、3.3mM、3.4mM、3.5mM、3.6mM、3.7mM、3.8mM、3.9mM、4mM、4.1mM、4.2mM、4.3mM、4.4mM、4.5mM、4.6mM、4.7mM、4.8mM、4.9mM、5mM、5.1mM、5.2mM、5.3mM、5.4mM、5.5mM、5.6mM、5.7mM、5.8mM、5.9mM、6mM、6.1mM、6.2mM、6.3mM、6.4mM、6.5mM、6.6mM、6.7mM、6.8mM、6.9mM、7mM、7.1mM、7.2mM、7.3mM、7.4mM、7.5mM、7.6mM、7.7mM、7.8mM、7.9mM、8mM、8.1mM、8.2mM、8.3mM、8.4mM、8.5mM、8.6mM、8.7mM、8.8mM、8.9mM、9mM、9.1mM、9.2mM、9.3mM、9.4mM、9.5mM、9.6mM、9.7mM、9.8mM、9.9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 2 7mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45 mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63m M, 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, 71mM, 72mM, 73mM, 74mM, 75mM, 76mM, 77mM, 78mM, 79mM, 80mM, 81mM , 82mM, 83mM, 84mM, 85mM, 86mM, 87mM, 88mM, 89mM, 90mM, 91mM, 92mM, 93mM, 94mM, 95mM, 96mM, 97mM, 98mM, 99mM , 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, It may be present at a concentration of 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, 280 mM, 285 mM, 290 mM, 295 mM, or about 300 mM, or any concentration therebetween.

[0058] In some embodiments, the reaction mixture may include a halide anion. In some embodiments, the halide anion may be present at a concentration of about 0 mM to about 300 mM, for example, about 0.001 mM, 0.002 mM, 0.003 mM, 0.004 mM, 0.005 mM, 0.006 mM, 0.007 mM, 0.008 mM, 0.009 mM, 0.01 mM, 0.011 mM, 0.012 mM, 0.013 mM, 0.014 mM, 0.015 mM, 0.016 mM, 0.017 mM, 0.018 mM, 0.019 mM, 0.02 mM, 0.021 mM, 0.022 mM, 0.023 mM, 0.024 mM, 0.0 25mM, 0.026mM, 0.027mM, 0.028mM, 0.029mM, 0.03mM, 0.031mM, 0.032mM, 0.033mM, 0.034mM, 0.035mM, 0.036mM, 0.037mM, 0.038mM, 0.039mM, 0.04 mM, 0.041mM, 0.042mM, 0.043mM, 0.044mM, 0.045mM, 0.046mM, 0.047mM, 0 .048mM, 0.049mM, 0.05mM, 0.055mM, 0.06mM, 0.065mM, 0.07mM, 0.075mM, 0.08mM, 0.085mM, 0.09mM, 0.095mM, 0.1mM, 0.11mM, 0.12mM, 0.13mM, 0.1 4mM, 0.15mM, 0.16mM, 0.17mM, 0.18mM, 0.19mM, 0.2mM, 0.21mM, 0.22mM, 0 .23mM, 0.24mM, 0.25mM, 0.26mM, 0.27mM, 0.28mM, 0.29mM, 0.3mM, 0.31mM , 0.32mM, 0.33mM, 0.34mM, 0.35mM, 0.36mM, 0.37mM, 0.38mM, 0.39mM, 0.4 mM, 0.41mM, 0.42mM, 0.43mM, 0.44mM, 0.45mM, 0.46mM, 0.47mM, 0.48mM, 0 .49mM, 0.5mM, 0.51mM, 0.52mM, 0.53mM, 0.54mM, 0.55mM, 0.56mM, 0.57mM , 0.58mM, 0.59mM, 0.6mM, 0.61mM, 0.62mM, 0.63mM, 0.64mM, 0.65mM, 0.66 mM, 0.67mM, 0.68mM, 0.69mM, 0.7mM, 0.71mM, 0.72mM, 0.73mM, 0.74mM, 0.75mM、0.76mM、0.77mM、0.78mM、0.79mM、0.8mM、0.81mM、0.82mM、0.83mM、0.84mM、0.85mM、0.86mM、0.87mM、0.88mM、0.89mM、0.9mM、0.91mM、0.92mM、0.93mM、0.94mM、0.95mM、0.96mM、0.97mM、0.98mM、0.99mM、1mM、1.1mM、1.2mM、1.3mM、1.4mM、1.5mM、1.6mM、1.7mM、1.8mM、1.9mM、2mM、2.1mM、2.2mM、2.3mM、2.4mM、2.5mM、2.6mM、2.7mM、2.8mM、2.9mM、3mM、3.1mM、3.2mM、3.3mM、3.4mM、3.5mM、3.6mM、3.7mM、3.8mM、3.9mM、4mM、4.1mM、4.2mM、4.3mM、4.4mM、4.5mM、4.6mM、4.7mM、4.8mM、4.9mM、5mM、5.1mM、5.2mM、5.3mM、5.4mM、5.5mM、5.6mM、5.7mM、5.8mM、5.9mM、6mM、6.1mM、6.2mM、6.3mM、6.4mM、6.5mM、6.6mM、6.7mM、6.8mM、6.9mM、7mM、7.1mM、7.2mM、7.3mM、7.4mM、7.5mM、7.6mM、7.7mM、7.8mM、7.9mM、8mM、8.1mM、8.2mM、8.3mM、8.4mM、8.5mM、8.6mM、8.7mM、8.8mM、8.9mM、9mM、9.1mM、9.2mM、9.3mM、9.4mM、9.5mM、9.6mM、9.7mM、9.8mM、9.9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27 mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45m M, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM , 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, 71mM, 72mM, 73mM, 74mM, 75mM, 76mM, 77mM, 78mM, 79mM, 80mM, 81mM, 82mM, 83mM, 84mM, 85mM, 86mM, 87mM, 88mM, 89mM, 90mM, 91mM, 92mM, 93mM, 94mM, 95mM, 96mM, 97mM, 98mM, 99mM, 1 00mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 17 It may be present at a concentration of 5 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, 280 mM, 285 mM, 290 mM, 295 mM, or about 300 mM, or any concentration therebetween.

[0059] In some embodiments, the reaction mixture may include an organic cation. In some embodiments, the organic cation may include a polyamine. In some embodiments, the polyamine may include spermidine, putrescine, or a combination thereof. In some embodiments, the organic cation may be at a concentration between about 0 mM and about 3 mM, for example, about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74 n 59 nM, 60nM, 61nM, 62nM, 63nM, 64nM, 65nM, 66nM, 67nM, 68nM, 69nM, 70nM, 71nM, 72nM, 73nM, 74nM, 75nM, 76nM, 77nM, 78nM, 79nM, 80nM, 8 1nM, 82nM, 83nM, 84nM, 85nM, 86nM, 87nM, 88nM, 89nM, 90nM, 91nM, 92nM, 93nM, 94nM, 95nM, 96nM, 97nM, 98nM, 99nM, 100nM, 110nM, 120 nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, 200nM, 210nM, 220nM, 230nM, 240nM, 250nM, 260nM, 270nM, 280nM, 290nM, 300n M, 310nM, 320nM, 330nM, 340nM, 350nM, 360nM, 370nM, 380nM, 390nM, 400nM, 410nM, 420nM, 430nM, 440nM, 450nM, 460nM, 470nM, 480nM , 490nM, 500nM, 510nM, 520nM, 530nM, 540nM, 550nM, 560nM, 570nM, 580nM, 590nM, 600nM, 610nM, 620nM, 630nM, 640nM, 650nM, 660nM,670nM、680nM、690nM、700nM、710nM、720nM、730nM、740nM、750nM、760nM、77 0nM、780nM、790nM、800nM、810nM、820nM、830nM、840nM、850nM、860nM、870nM 、880nM、890nM、900nM、910nM、920nM、930nM、940nM、950nM、960nM、970nM、9 80nM、990nM、1μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、 1.9μM, 2μM, 2.1μM, 2.2μM, 2.3μM, 2.4μM, 2.5μM, 2.6μM, 2.7μM, 2.8μM, 2.9μM, 3μM, 3.1μM, 3.2μM, 3.3μM, 3.4μM, 3.5μM, 3.6μM, 3.7μM, 3.8μM, 3.9μM, 4μM ,4.1μM,4.2μM,4.3μM,4.4μM,4.5μM,4.6μM,4.7μM,4.8μM,4.9μM,5μM,5.1μM,5.2μM,5.3μM,5.4μM,5.5μM,5.6μM,5.7μM,5.8μM,5.9μM,6μM,6.1μM,6. 2μM, 6.3μM, 6.4μM, 6.5μM, 6.6μM, 6.7μM, 6.8μM, 6.9μM, 7μM, 7.1μM, 7.2μM, 7.3μM, 7.4μM, 7.5μM, 7.6μM, 7.7μM, 7.8μM, 7.9μM, 8μM, 8.1μM, 8.2μM, 8.3μM ,8.4μM,8.5μM,8.6μM,8.7μM,8.8μM,8.9μM,9μM,9.1μM,9.2μM,9.3μM,9.4μM,9.5μM,9.6μM,9.7μM,9.8μM,9.9μM,10μM,11μM,12μM,13μM,14μM,15μM, 16μM, 17μM, 18μM, 19μM, 20μM, 21μM, 22μM, 23μM, 24μM, 25μM, 26μM, 27μM, 28μM, 29μM, 30μM, 31μM, 32μM, 33μM, 34μM, 35μM, 36μM, 37μM, 38μM, 39μM, 40μM, 41μM, 42μM, 43μM, 44μM, 45μM, 46μM, 47μM, 48μM, 49μM, 50μM, 51μM, 52μM, 53μM, 54μM, 55μM, 56μM, 57μM, 58μM, 59μM, 60μM, 61μM, 62μM, 63μM, 64μM, 65μM,66 μM, 67 μM, 68 μM, 69 μM, 70 μM, 71 μM, 72 μM, 73 μM, 74 μM, 75 μM, 76 μM, 77 μM, 78 μM, 79 μM, 80 μM, 81 μM, 82 μM, 83 μM, 84 μM, 85 μM, 86 μM, 87 μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93 μM, 94 μM, 95 μM, 96 μM, 97 μM, 98 μM, 99 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 210 μM, 220 μM, 230 μM, 240 μM, 250 μM, 260 μM, 270 μM, 280 μM, 290 μM, 300 μM, 310 μM, 320 μM, 330 μM, 340 μM, 350 μM, 360 μM, 370 μM, 380 μM, 390 μM, 400 μM, 410 μM, 420 μM, 430 μM, 440 μM, 450 μM, 460 μM, 470 μM, 480 μM, 490 μM, 500 μM, 510 μM, 520 μM, 530 μM, 540 μM, 550 μM, 560 μM, 570 μM, 580 μM, 590 μM, 600 μM, 610 μM, 620 μM, 630 μM, 640 μM, 650 μM, 660 μM, 670 μM, 680 μM, 690 μM, 700 μM, 710 μM, 720 μM, 730 μM, 740 μM, 750 μM, 760 μM, 770 μM, 780 μM, 790 μM, 800 μM, 810 μM, 820 μM, 830 μM, 840 μM, 850 μM, 860 μM, 870 μM, 880 μM, 890 μM, 900 μM, 910 μM, 920 μM, 930 μM, 940 μM, 950 μM, 960 μM, 970 μM, 980 μM, 990 μM, 1000 μM, 1.05 mM, 1.1 mM, 1.15 mM, 1.2 mM, 1.25 mM, 1.3 mM, 1.35 mM, 1.4 mM, 1.45 mM, 1.5 mM, 1.55 mM, 1.6 mM, 1.65 mM, 1.7 mM, 1.75 mM, 1.8 mM, 1.85 mM, 1.9 mM, 1.95 mM, 2 mM, 2.05 mM, 2.1 mM, 2.15 mM, 2.2 mM, 2.25 mM, 2.3 mM, 2.35 mM, 2.4 mM, 2.45 mM, 2.5 mM, 2.55 mM, 2.6 mM, 2.65 mM, 2.7 mM, 2.75 mM, 2.8 mM, 2.85 mM, 2.9 mM, 2.95 mM, or about 3 mM, or may be present at any concentration between these.

[0060] In some embodiments, the reaction mixture may include an inorganic cation. In some embodiments, the inorganic cation may include a monovalent cation. In some embodiments, the monovalent cation may include sodium, potassium, lithium, or a combination thereof. In some embodiments, the inorganic cation may include a divalent cation. In some embodiments, the divalent cation may include magnesium, calcium, manganese, or a combination thereof. In some embodiments, the divalent cation includes magnesium. In some embodiments, the magnesium is at a concentration between about 1 mM and about 50 mM, e.g., about 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 ... .4mM, 3.5mM, 3.6mM, 3.7mM, 3.8mM, 3.9mM, 4mM, 4.1mM, 4.2mM, 4.3mM, 4.4mM, 4.5mM, 4.6mM, 4.7mM, 4.8mM, 4.9mM , 5mM, 5.1mM, 5.2mM, 5.3mM, 5.4mM, 5.5mM, 5.6mM, 5.7mM, 5.8mM, 5.9mM, 6mM, 6.1mM, 6.2mM, 6.3mM, 6.4mM, 6.5mM, 6.6mM, 6.7mM, 6.8mM, 6.9mM, 7mM, 7.1mM, 7.2mM, 7.3mM, 7.4mM, 7.5mM, 7.6mM, 7.7mM, 7.8mM, 7.9mM, 8mM, 8.1mM, 8.2mM, 8.3mM, 8.4mM, 8.5mM, 8.6mM, 8.7mM, 8.8mM, 8.9mM, 9mM, 9.1mM, 9.2mM, 9.3mM, 9.4mM, 9.5mM, 9.6mM, 9.7mM , 9.8mM, 9.9mM, 10mM, 10.1mM, 10.2mM, 10.3mM, 10.4mM, 10.5mM, 10.6mM, 10.7mM, 10.8mM, 10.9mM, 11mM, 11.1mM , 11.2mM, 11.3mM, 11.4mM, 11.5mM, 11.6mM, 11.7mM, 11.8mM, 11.9mM, 12mM, 12.1mM, 12.2mM, 12.3mM, 12.4mM, 12.5mM、12.6mM、12.7mM、12.8mM、12.9mM、13mM、13.1mM、13.2mM、13.3mM、13.4mM、13.5mM、13.6mM、13.7mM、13.8mM、13.9mM、14mM、14.1mM、14.2mM、14.3mM、14.4mM、14.5mM、14.6mM、14.7mM、14.8mM、14.9mM、15mM、15.1mM、15.2mM、15.3mM、15.4mM、15.5mM、15.6mM、15.7mM、15.8mM、15.9mM、16mM、16.1mM、16.2mM、16.3mM、16.4mM、16.5mM、16.6mM、16.7mM、16.8mM、16.9mM、17mM、17.1mM、17.2mM、17.3mM、17.4mM、17.5mM、17.6mM、17.7mM、17.8mM、17.9mM、18mM、18.1mM、18.2mM、18.3mM、18.4mM、18.5mM、18.6mM、18.7mM、18.8mM、18.9mM、19mM、19.1mM、19.2mM、19.3mM、19.4mM、19.5mM、19.6mM、19.7mM、19.8mM、19.9mM、20mM、20.1mM、20.2mM、20.3mM、20.4mM、20.5mM、20.6mM、20.7mM、20.8mM、20.9mM、21mM、21.1mM、21.2mM、21.3mM、21.4mM、21.5mM、21.6mM、21.7mM、21.8mM、21.9mM、22mM、22.1mM、22.2mM、22.3mM、22.4mM、22.5mM、22.6mM、22.7mM、22.8mM、22.9mM、23mM、23.1mM、23.2mM、23.3mM、23.4mM、23.5mM、23.6mM、23.7mM、23.8mM、23.9mM、24mM、24.1mM、24.2mM、24.3mM、24.4mM、24.5mM、24.6mM、24.7mM、24.8mM、24.9mM、25mM、25.1mM、25.2mM、25.3mM、25.4mM、25.5mM、25.6mM、25.7mM、25.8mM、25.9mM、26mM、26.1mM、26.2mM、26.3mM、26.4mM、26.5mM、26.6mM、26.7mM、26.8mM、26.9mM、27mM、27.1mM、27.2mM、27.3mM、27.4mM、27.5mM、27.6mM、27.7mM、27.8mM、27.9mM、28mM、28.1mM、28.2mM、28.3mM、28.4mM、28.5mM、28.6mM、28.7mM、28.8mM、28.9mM、29mM、29.1mM、29.2mM、29.3mM、29.4mM、29.5mM、29.6mM、29.7mM、29.8mM、29.9mM、30mM、30.1mM、30.2mM、30.3mM、30.4mM、30.5mM、30.6mM、30.7mM、30.8mM、30.9mM、31mM、31.1mM、31.2mM、31.3mM、31.4mM、31.5mM、31.6mM、31.7mM、31.8mM、31.9mM、32mM、32.1mM、32.2mM、32.3mM、32.4mM、32.5mM、32.6mM、32.7mM、32.8mM、32.9mM、33mM、33.1mM、33.2mM、33.3mM、33.4mM、33.5mM、33.6mM、33.7mM、33.8mM、33.9mM、34mM、34.1mM、34.2mM、34.3mM、34.4mM、34.5mM、34.6mM、34.7mM、34.8mM、34.9mM、35mM、35.1mM、35.2mM、35.3mM、35.4mM、35.5mM、35.6mM、35.7mM、35.8mM、35.9mM、36mM、36.1mM、36.2mM、36.3mM、36.4mM、36.5mM、36.6mM、36.7mM、36.8mM、36.9mM、37mM、37.1mM、37.2mM、37.3mM、37.4mM、37.5mM、37.6mM、37.7mM、37.8mM、37.9mM、38mM、38.1mM、38.2mM、38.3mM、38.4mM、38.5mM、38.6mM、38.7mM、38.8mM、38.9mM、39mM、39.1mM、39.2mM、39.3mM、39.4mM、39.5mM、39.6mM、39.7mM、39.8mM、39.9mM、40mM、40.1mM、40.2mM、40.3mM、40.4mM、40.5mM、40.6mM、40.7mM、40.8mM、40.9mM、41mM、41.1mM、41.2mM、41.3mM、41.4mM、41.5mM、41.6mM、41.7mM、41.8mM、41.9mM, 42mM, 42.1mM, 42.2mM, 42.3mM, 42.4mM, 42.5mM, 42.6mM, 42.7mM, 42.8mM, 42.9mM, 43mM, 43.1mM, 43.2mM, 43.3mM, 43.4mM, 43.5mM, 43.6mM, 43.7mM, 43.8mM, 43.9mM, 44mM , 44.1mM, 44.2mM, 44.3mM, 44.4mM, 44.5mM, 44.6mM, 44.7mM, 44.8mM, 44.9mM, 45mM, 45. 1mM, 45.2mM, 45.3mM, 45.4mM, 45.5mM, 45.6mM, 45.7mM, 45.8mM, 45.9mM, 46mM, 46.1mM, 4 6.2mM, 46.3mM, 46.4mM, 46.5mM, 46.6mM, 46.7mM, 46.8mM, 46.9mM, 47mM, 47.1mM, 47.2m M, 47.3mM, 47.4mM, 47.5mM, 47.6mM, 47.7mM, 47.8mM, 47.9mM, 48mM, 48.1mM, 48.2mM, 48. It may be present at about 3 mM, 48.4 mM, 48.5 mM, 48.6 mM, 48.7 mM, 48.8 mM, 48.9 mM, 49 mM, 49.1 mM, 49.2 mM, 49.3 mM, 49.4 mM, 49.5 mM, 49.6 mM, 49.7 mM, 49.8 mM, 49.9 mM, or about 50 mM, or any concentration therebetween.

[0061] In some embodiments, the reaction mixture may include endogenous nucleotides, i.e., nucleotides present in the cell extract and obtained from the cell, such as nucleoside triphosphates (NTPs). In some embodiments, the reaction mixture may include exogenous nucleotides, i.e., nucleotides added to the cell extract, such as nucleoside triphosphates (NTPs), nucleoside diphosphates (NDPs), or nucleoside monophosphates (NMPs). In some embodiments, the nucleotides may include NTPs, e.g., ATP, GTP, CTP, UTP, or combinations thereof. In some embodiments, the nucleotides may include NMPs, e.g., AMP, GMP, CMP, UMP, or combinations thereof. In some embodiments, the reaction mixture may include a combination of NTPs, NDPs, and NMPs. For example, the reaction mixture may include a mixture of NTPs and NDPs, a mixture of NTPs and NMPs, a mixture of NDPs and NMPs, or a mixture of NTPs, NDPs, and NMPs.

[0062] In some embodiments, each NTP, e.g., ATP, GTP, CTP, or UTP, is present at a concentration between about 0.1 mM and about 5 mM, e.g., about 0.1 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.17 mM, 0.18 mM, 0.19 mM, 0.2 mM, 0.21 mM, 0.22 mM, 0.23 mM, 0.24 mM, 0.25 mM, 0.26 mM, 0.27 mM, 0.28 mM, 0.29 mM, 0.3 mM, 0.31 mM, 0.32 mM, 0.33 mM, 0.34 mM, 0.35 mM, 0.36 mM, 0.38 mM, 0.39 mM, 0.40 mM, 0.41 mM, 0.42 mM, 0.43 mM, 0.44 mM, 0.45 mM, 0.46 mM, 0.47 mM, 0.48 mM, 0.49 mM, 0.50 mM, 0.51 mM, 0.52 mM, 0.53 mM, 0.54 mM, 0.55 mM, 0.56 mM, 0.57 mM, 0.58 mM, 0.59 mM, 0.60 mM, 0.61 mM, 0.62 mM, 0.63 mM, 0.64 mM, 0.65 mM, 0.66 mM, 0.67 mM, 0.68 mM, 0.69 mM, 0.70 mM, 0.71 mM, 0.72 mM, 0.73 mM, 0.74 mM, 0.75 mM, 0.76 mM, 0 M, 0.37mM, 0.38mM, 0.39mM, 0.4mM, 0.41mM, 0.42mM, 0.43mM, 0.44mM, 0.45m M, 0.46mM, 0.47mM, 0.48mM, 0.49mM, 0.5mM, 0.51mM, 0.52mM, 0.53mM, 0.54m M, 0.55mM, 0.56mM, 0.57mM, 0.58mM, 0.59mM, 0.6mM, 0.61mM, 0.62mM, 0.63m M, 0.64mM, 0.65mM, 0.66mM, 0.67mM, 0.68mM, 0.69mM, 0.7mM, 0.71mM, 0.72mM , 0.73mM, 0.74mM, 0.75mM, 0.76mM, 0.77mM, 0.78mM, 0.79mM, 0.8mM, 0.81mM , 0.82mM, 0.83mM, 0.84mM, 0.85mM, 0.86mM, 0.87mM, 0.88mM, 0.89mM, 0.9mM , 0.91mM, 0.92mM, 0.93mM, 0.94mM, 0.95mM, 0.96mM, 0.97mM, 0.98mM, 0.99m M, 1mM, 1.01mM, 1.02mM, 1.03mM, 1.04mM, 1.05mM, 1.06mM, 1.07mM, 1.08mM, 1 .09mM, 1.1mM, 1.11mM, 1.12mM, 1.13mM, 1.14mM, 1.15mM, 1.16mM, 1.17mM, 1 .18mM, 1.19mM, 1.2mM, 1.21mM, 1.22mM, 1.23mM, 1.24mM, 1.25mM, 1.26mM, 1 .27mM, 1.28mM, 1.29mM, 1.3mM, 1.31mM, 1.32mM, 1.33mM, 1.34mM, 1.35mM, 1 .36mM, 1.37mM, 1.38mM, 1.39mM, 1.4mM, 1.41mM, 1.42mM, 1.43mM, 1.44mM, 1.45mM、1.46mM、1.47mM、1.48mM、1.49mM、1.5mM、1.51mM、1.52mM、1.53mM、1.54mM、1.55mM、1.56mM、1.57mM、1.58mM、1.59mM、1.6mM、1.61mM、1.62mM、1.63mM、1.64mM、1.65mM、1.66mM、1.67mM、1.68mM、1.69mM、1.7mM、1.71mM、1.72mM、1.73mM、1.74mM、1.75mM、1.76mM、1.77mM、1.78mM、1.79mM、1.8mM、1.81mM、1.82mM、1.83mM、1.84mM、1.85mM、1.86mM、1.87mM、1.88mM、1.89mM、1.9mM、1.91mM、1.92mM、1.93mM、1.94mM、1.95mM、1.96mM、1.97mM、1.98mM、1.99mM、2mM、2.01mM、2.02mM、2.03mM、2.04mM、2.05mM、2.06mM、2.07mM、2.08mM、2.09mM、2.1mM、2.11mM、2.12mM、2.13mM、2.14mM、2.15mM、2.16mM、2.17mM、2.18mM、2.19mM、2.2mM、2.21mM、2.22mM、2.23mM、2.24mM、2.25mM、2.26mM、2.27mM、2.28mM、2.29mM、2.3mM、2.31mM、2.32mM、2.33mM、2.34mM、2.35mM、2.36mM、2.37mM、2.38mM、2.39mM、2.4mM、2.41mM、2.42mM、2.43mM、2.44mM、2.45mM、2.46mM、2.47mM、2.48mM、2.49mM、2.5mM、2.51mM、2.52mM、2.53mM、2.54mM、2.55mM、2.56mM、2.57mM、2.58mM、2.59mM、2.6mM、2.61mM、2.62mM、2.63mM、2.64mM、2.65mM、2.66mM、2.67mM、2.68mM、2.69mM、2.7mM、2.71mM、2.72mM、2.73mM、2.74mM、2.75mM、2.76mM、2.77mM、2.78mM、2.79mM、2.8mM、2.81mM、2.82mM、2.83mM、2.84mM、2.85mM、2.86mM、2.87mM、2.88mM、2.89mM、2.9mM、2.91mM、2.92mM、2.93mM、2.94mM、2.95mM、2.96mM、2.97mM、2.98mM、2.99mM、3mM、3.01mM、3.02mM、3.03mM、3.04mM、3.05mM、3.06mM、3.07mM、3.08mM、3.09mM、3.1mM、3.11mM、3.12mM、3.13mM、3.14mM、3.15mM、3.16mM、3.17mM、3.18mM、3.19mM、3.2mM、3.21mM、3.22mM、3.23mM、3.24mM、3.25mM、3.26mM、3.27mM、3.28mM、3.29mM、3.3mM、3.31mM、3.32mM、3.33mM、3.34mM、3.35mM、3.36mM、3.37mM、3.38mM、3.39mM、3.4mM、3.41mM、3.42mM、3.43mM、3.44mM、3.45mM、3.46mM、3.47mM、3.48mM、3.49mM、3.5mM、3.51mM、3.52mM、3.53mM、3.54mM、3.55mM、3.56mM、3.57mM、3.58mM、3.59mM、3.6mM、3.61mM、3.62mM、3.63mM、3.64mM、3.65mM、3.66mM、3.67mM、3.68mM、3.69mM、3.7mM、3.71mM、3.72mM、3.73mM、3.74mM、3.75mM、3.76mM、3.77mM、3.78mM、3.79mM、3.8mM、3.81mM、3.82mM、3.83mM、3.84mM、3.85mM、3.86mM、3.87mM、3.88mM、3.89mM、3.9mM、3.91mM、3.92mM、3.93mM、3.94mM、3.95mM、3.96mM、3.97mM、3.98mM、3.99mM、4mM、4.01mM、4.02mM、4.03mM、4.04mM、4.05mM、4.06mM、4.07mM、4.08mM、4.09mM、4.1mM、4.11mM、4.12mM、4.13mM、4.14mM、4.15mM、4.16mM、4.17mM、4.18mM、4.19mM、4.2mM、4.21mM、4.22mM、4.23mM、4.24mM、4.25mM、4.26mM、4.27mM、4.28mM、4.29mM、4.3mM、4.31mM、4.32mM、4.33mM、4.34mM、4.35mM, 4.36mM, 4.37mM, 4.38mM, 4.39mM, 4.4mM, 4.41mM, 4.42mM, 4.43mM, 4.44mM, 4.45mM, 4.46mM, 4.47mM, 4.48mM, 4.49mM, 4.5mM, 4.51mM, 4.52mM , 4.53mM, 4.54mM, 4.55mM, 4.56mM, 4.57mM, 4.58mM, 4.59mM, 4.6mM, 4.61 mM, 4.62mM, 4.63mM, 4.64mM, 4.65mM, 4.66mM, 4.67mM, 4.68mM, 4.69mM, 4. It may be present in the reaction mixture at about 7 mM, 4.71 mM, 4.72 mM, 4.73 mM, 4.74 mM, 4.75 mM, 4.76 mM, 4.77 mM, 4.78 mM, 4.79 mM, 4.8 mM, 4.81 mM, 4.82 mM, 4.83 mM, 4.84 mM, 4.85 mM, 4.86 mM, 4.87 mM, 4.88 mM, 4.89 mM, 4.9 mM, 4.91 mM, 4.92 mM, 4.93 mM, 4.94 mM, 4.95 mM, 4.96 mM, 4.97 mM, 4.98 mM, 4.99 mM, or about 5 mM or any concentration therebetween.

[0063] In some embodiments, each NMP, e.g., AMP, GMP, CMP, UMP, is at a concentration between about 0.1 mM and about 5 mM, e.g., about 0.1 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.17 mM, 0.18 mM, 0.19 mM, 0.2 mM, 0.21 mM, 0.22 mM, 0.23 mM, 0.24 mM, 0.25 mM, 0.26 mM, 0.27 mM, 0.28 mM, 0.29 mM, 0.3 mM, 0.31 mM, 0.32 mM, 0.33 mM, 0.34 mM, 0.35 mM, 0.36 mM, 0. 37mM, 0.38mM, 0.39mM, 0.4mM, 0.41mM, 0.42mM, 0.43mM, 0.44mM, 0.45mM, 0. 46mM, 0.47mM, 0.48mM, 0.49mM, 0.5mM, 0.51mM, 0.52mM, 0.53mM, 0.54mM, 0.5 5mM, 0.56mM, 0.57mM, 0.58mM, 0.59mM, 0.6mM, 0.61mM, 0.62mM, 0.63mM, 0.6 4mM, 0.65mM, 0.66mM, 0.67mM, 0.68mM, 0.69mM, 0.7mM, 0.71mM, 0.72mM, 0.73 mM, 0.74mM, 0.75mM, 0.76mM, 0.77mM, 0.78mM, 0.79mM, 0.8mM, 0.81mM, 0.82 mM, 0.83mM, 0.84mM, 0.85mM, 0.86mM, 0.87mM, 0.88mM, 0.89mM, 0.9mM, 0.91m M, 0.92mM, 0.93mM, 0.94mM, 0.95mM, 0.96mM, 0.97mM, 0.98mM, 0.99mM, 1mM, 1.01mM, 1.02mM, 1.03mM, 1.04mM, 1.05mM, 1.06mM, 1.07mM, 1.08mM, 1.09mM, 1.1mM, 1.11mM, 1.12mM, 1.13mM, 1.14mM, 1.15mM, 1.16mM, 1.17mM, 1.18mM, 1.19mM, 1.2mM, 1.21mM, 1.22mM, 1.23mM, 1.24mM, 1.25mM, 1.26mM, 1.27mM, 1 .28mM, 1.29mM, 1.3mM, 1.31mM, 1.32mM, 1.33mM, 1.34mM, 1.35mM, 1.36mM, 1 .37mM, 1.38mM, 1.39mM, 1.4mM, 1.41mM, 1.42mM, 1.43mM, 1.44mM, 1.45mM, 1.46mM、1.47mM、1.48mM、1.49mM、1.5mM、1.51mM、1.52mM、1.53mM、1.54mM、1.55mM、1.56mM、1.57mM、1.58mM、1.59mM、1.6mM、1.61mM、1.62mM、1.63mM、1.64mM、1.65mM、1.66mM、1.67mM、1.68mM、1.69mM、1.7mM、1.71mM、1.72mM、1.73mM、1.74mM、1.75mM、1.76mM、1.77mM、1.78mM、1.79mM、1.8mM、1.81mM、1.82mM、1.83mM、1.84mM、1.85mM、1.86mM、1.87mM、1.88mM、1.89mM、1.9mM、1.91mM、1.92mM、1.93mM、1.94mM、1.95mM、1.96mM、1.97mM、1.98mM、1.99mM、2mM、2.01mM、2.02mM、2.03mM、2.04mM、2.05mM、2.06mM、2.07mM、2.08mM、2.09mM、2.1mM、2.11mM、2.12mM、2.13mM、2.14mM、2.15mM、2.16mM、2.17mM、2.18mM、2.19mM、2.2mM、2.21mM、2.22mM、2.23mM、2.24mM、2.25mM、2.26mM、2.27mM、2.28mM、2.29mM、2.3mM、2.31mM、2.32mM、2.33mM、2.34mM、2.35mM、2.36mM、2.37mM、2.38mM、2.39mM、2.4mM、2.41mM、2.42mM、2.43mM、2.44mM、2.45mM、2.46mM、2.47mM、2.48mM、2.49mM、2.5mM、2.51mM、2.52mM、2.53mM、2.54mM、2.55mM、2.56mM、2.57mM、2.58mM、2.59mM、2.6mM、2.61mM、2.62mM、2.63mM、2.64mM、2.65mM、2.66mM、2.67mM、2.68mM、2.69mM、2.7mM、2.71mM、2.72mM、2.73mM、2.74mM、2.75mM、2.76mM、2.77mM、2.78mM、2.79mM、2.8mM、2.81mM、2.82mM、2.83mM、2.84mM、2.85mM、2.86mM、2.87mM、2.88mM、2.89mM、2.9mM、2.91mM、2.92mM、2.93mM、2.94mM、2.95mM、2.96mM、2.97mM、2.98mM、2.99mM、3mM、3.01mM、3.02mM、3.03mM、3.04mM、3.05mM、3.06mM、3.07mM、3.08mM、3.09mM、3.1mM、3.11mM、3.12mM、3.13mM、3.14mM、3.15mM、3.16mM、3.17mM、3.18mM、3.19mM、3.2mM、3.21mM、3.22mM、3.23mM、3.24mM、3.25mM、3.26mM、3.27mM、3.28mM、3.29mM、3.3mM、3.31mM、3.32mM、3.33mM、3.34mM、3.35mM、3.36mM、3.37mM、3.38mM、3.39mM、3.4mM、3.41mM、3.42mM、3.43mM、3.44mM、3.45mM、3.46mM、3.47mM、3.48mM、3.49mM、3.5mM、3.51mM、3.52mM、3.53mM、3.54mM、3.55mM、3.56mM、3.57mM、3.58mM、3.59mM、3.6mM、3.61mM、3.62mM、3.63mM、3.64mM、3.65mM、3.66mM、3.67mM、3.68mM、3.69mM、3.7mM、3.71mM、3.72mM、3.73mM、3.74mM、3.75mM、3.76mM、3.77mM、3.78mM、3.79mM、3.8mM、3.81mM、3.82mM、3.83mM、3.84mM、3.85mM、3.86mM、3.87mM、3.88mM、3.89mM、3.9mM、3.91mM、3.92mM、3.93mM、3.94mM、3.95mM、3.96mM、3.97mM、3.98mM、3.99mM、4mM、4.01mM、4.02mM、4.03mM、4.04mM、4.05mM、4.06mM、4.07mM、4.08mM、4.09mM、4.1mM、4.11mM、4.12mM、4.13mM、4.14mM、4.15mM、4.16mM、4.17mM、4.18mM、4.19mM、4.2mM、4.21mM、4.22mM、4.23mM、4.24mM、4.25mM、4.26mM、4.27mM、4.28mM、4.29mM、4.3mM、4.31mM、4.32mM、4.33mM、4.34mM、4.35mM、4.36mM, 4.37mM, 4.38mM, 4.39mM, 4.4mM, 4.41mM, 4.42mM, 4.43mM, 4.44mM , 4.45mM, 4.46mM, 4.47mM, 4.48mM, 4.49mM, 4.5mM, 4.51mM, 4.52mM, 4.53 mM, 4.54mM, 4.55mM, 4.56mM, 4.57mM, 4.58mM, 4.59mM, 4.6mM, 4.61mM, 4. 62mM, 4.63mM, 4.64mM, 4.65mM, 4.66mM, 4.67mM, 4.68mM, 4.69mM, 4.7mM, The phospholipid may be present in the reaction mixture at about 4.71 mM, 4.72 mM, 4.73 mM, 4.74 mM, 4.75 mM, 4.76 mM, 4.77 mM, 4.78 mM, 4.79 mM, 4.8 mM, 4.81 mM, 4.82 mM, 4.83 mM, 4.84 mM, 4.85 mM, 4.86 mM, 4.87 mM, 4.88 mM, 4.89 mM, 4.9 mM, 4.91 mM, 4.92 mM, 4.93 mM, 4.94 mM, 4.95 mM, 4.96 mM, 4.97 mM, 4.98 mM, 4.99 mM, or about 5 mM, or any concentration therebetween.

[0064] In some embodiments, the reaction mixture may include amino acids. In some embodiments, the amino acids may include endogenous amino acids, i.e., amino acids present in the cell extract and derived from the cell. In some embodiments, the amino acids may include exogenous amino acids, i.e., amino acids added to the cell extract. In some embodiments, the reaction mixture may include one or more standard amino acids selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, each individual amino acid is present at a concentration between about 0.1 mM and about 5 mM, e.g., about 0.1 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.17 mM, 0.18 mM, 0.19 mM, 0.2 mM, 0.21 mM, 0.22 mM, 0.23 mM, 0.24 mM, 0.25 mM, 0.26 mM, 0. 27mM, 0.28mM, 0.29mM, 0.3mM, 0.31mM, 0.32mM, 0.33mM, 0.34mM, 0.35mM, 0.36mM, 0.37mM, 0.38mM, 0. 39mM, 0.4mM, 0.41mM, 0.42mM, 0.43mM, 0.44mM, 0.45mM, 0.46mM, 0.47mM, 0.48mM, 0.49mM, 0.5mM, 0.5 1mM, 0.52mM, 0.53mM, 0.54mM, 0.55mM, 0.56mM, 0.57mM, 0.58mM, 0.59mM, 0.6mM, 0.61mM, 0.62mM, 0. 63mM, 0.64mM, 0.65mM, 0.66mM, 0.67mM, 0.68mM, 0.69mM, 0.7mM, 0.71mM, 0.72mM, 0.73mM, 0.74mM, 0. 75mM, 0.76mM, 0.77mM, 0.78mM, 0.79mM, 0.8mM, 0.81mM, 0.82mM, 0.83mM, 0.84mM, 0.85mM, 0.86mM, 0. 87mM, 0.88mM, 0.89mM, 0.9mM, 0.91mM, 0.92mM, 0.93mM, 0.94mM, 0.95mM, 0.96mM, 0.97mM, 0.98mM, 0.99mM、1mM、1.01mM、1.02mM、1.03mM、1.04mM、1.05mM、1.06mM、1.07mM、1.08mM、1.09mM、1.1mM、1.11mM、1.12mM、1.13mM、1.14mM、1.15mM、1.16mM、1.17mM、1.18mM、1.19mM、1.2mM、1.21mM、1.22mM、1.23mM、1.24mM、1.25mM、1.26mM、1.27mM、1.28mM、1.29mM、1.3mM、1.31mM、1.32mM、1.33mM、1.34mM、1.35mM、1.36mM、1.37mM、1.38mM、1.39mM、1.4mM、1.41mM、1.42mM、1.43mM、1.44mM、1.45mM、1.46mM、1.47mM、1.48mM、1.49mM、1.5mM、1.51mM、1.52mM、1.53mM、1.54mM、1.55mM、1.56mM、1.57mM、1.58mM、1.59mM、1.6mM、1.61mM、1.62mM、1.63mM、1.64mM、1.65mM、1.66mM、1.67mM、1.68mM、1.69mM、1.7mM、1.71mM、1.72mM、1.73mM、1.74mM、1.75mM、1.76mM、1.77mM、1.78mM、1.79mM、1.8mM、1.81mM、1.82mM、1.83mM、1.84mM、1.85mM、1.86mM、1.87mM、1.88mM、1.89mM、1.9mM、1.91mM、1.92mM、1.93mM、1.94mM、1.95mM、1.96mM、1.97mM、1.98mM、1.99mM、2mM、2.01mM、2.02mM、2.03mM、2.04mM、2.05mM、2.06mM、2.07mM、2.08mM、2.09mM、2.1mM、2.11mM、2.12mM、2.13mM、2.14mM、2.15mM、2.16mM、2.17mM、2.18mM、2.19mM、2.2mM、2.21mM、2.22mM、2.23mM、2.24mM、2.25mM、2.26mM、2.27mM、2.28mM、2.29mM、2.3mM、2.31mM、2.32mM、2.33mM、2.34mM、2.35mM、2.36mM、2.37mM、2.38mM、2.39mM、2.4mM、2.41mM、2.42mM、2.43mM、2.44mM、2.45mM、2.46mM、2.47mM、2.48mM、2.49mM、2.5mM、2.51mM、2.52mM、2.53mM、2.54mM、2.55mM、2.56mM、2.57mM、2.58mM、2.59mM、2.6mM、2.61mM、2.62mM、2.63mM、2.64mM、2.65mM、2.66mM、2.67mM、2.68mM、2.69mM、2.7mM、2.71mM、2.72mM、2.73mM、2.74mM、2.75mM、2.76mM、2.77mM、2.78mM、2.79mM、2.8mM、2.81mM、2.82mM、2.83mM、2.84mM、2.85mM、2.86mM、2.87mM、2.88mM、2.89mM、2.9mM、2.91mM、2.92mM、2.93mM、2.94mM、2.95mM、2.96mM、2.97mM、2.98mM、2.99mM、3mM、3.01mM、3.02mM、3.03mM、3.04mM、3.05mM、3.06mM、3.07mM、3.08mM、3.09mM、3.1mM、3.11mM、3.12mM、3.13mM、3.14mM、3.15mM、3.16mM、3.17mM、3.18mM、3.19mM、3.2mM、3.21mM、3.22mM、3.23mM、3.24mM、3.25mM、3.26mM、3.27mM、3.28mM、3.29mM、3.3mM、3.31mM、3.32mM、3.33mM、3.34mM、3.35mM、3.36mM、3.37mM、3.38mM、3.39mM、3.4mM、3.41mM、3.42mM、3.43mM、3.44mM、3.45mM、3.46mM、3.47mM、3.48mM、3.49mM、3.5mM、3.51mM、3.52mM、3.53mM、3.54mM、3.55mM、3.56mM、3.57mM、3.58mM、3.59mM、3.6mM、3.61mM、3.62mM、3.63mM、3.64mM、3.65mM、3.66mM、3.67mM、3.68mM、3.69mM、3.7mM、3.71mM、3.72mM、3.73mM、3.74mM、3.75mM、3.76mM、3.77mM、3.78mM、3.79mM、3.8mM、3.81mM、3.82mM、3.83mM、3.84mM、3.85mM、3.86mM、3.87mM、3.88mM、3.89mM, 3.9mM, 3.91mM, 3.92mM, 3.93mM, 3.94mM, 3.95mM, 3.96mM, 3.97mM, 3.98mM, 3.99mM, 4mM, 4.01mM, 4.02mM, 4.03mM, 4 .04mM, 4.05mM, 4.06mM, 4.07mM, 4.08mM, 4.09mM, 4.1mM, 4.11mM, 4.12mM, 4.13mM, 4.14mM, 4.15mM, 4.16mM, 4.17mM, 4.18 mM, 4.19mM, 4.2mM, 4.21mM, 4.22mM, 4.23mM, 4.24mM, 4.25mM, 4.26mM, 4.27mM, 4.28mM, 4.29mM, 4.3mM, 4.31mM, 4.32mM, 4 .33mM, 4.34mM, 4.35mM, 4.36mM, 4.37mM, 4.38mM, 4.39mM, 4.4mM, 4.41mM, 4.42mM, 4.43mM, 4.44mM, 4.45mM, 4.46mM, 4.47 mM, 4.48mM, 4.49mM, 4.5mM, 4.51mM, 4.52mM, 4.53mM, 4.54mM, 4.55mM, 4.56mM, 4.57mM, 4.58mM, 4.59mM, 4.6mM, 4.61mM, 4 .62mM, 4.63mM, 4.64mM, 4.65mM, 4.66mM, 4.67mM, 4.68mM, 4.69mM, 4.7mM, 4.71mM, 4.72mM, 4.73mM, 4.74mM, 4.75mM, 4.76 4.9mM, 4.77mM, 4.78mM, 4.79mM, 4.8mM, 4.81mM, 4.82mM, 4.83mM, 4.84mM, 4.85mM, 4.86mM, 4.87mM, 4.88mM, 4.89mM, 4.9mM, 4.91mM, 4.92mM, 4.93mM, 4.94mM, 4.95mM, 4.96mM, 4.97mM, 4.98mM, 4.99mM, or about 5mM, or any concentration therebetween.

[0065] In some embodiments, the reaction mixture may include tRNA. In some embodiments, the tRNA may include endogenous tRNA, i.e., tRNA present in the cell extract and derived from the cell. In some embodiments, the tRNA may include exogenous tRNA, i.e., tRNA added to the cell extract. In some embodiments, the tRNA may be present in the cell extract at a concentration between about 10 μg / mL and about 1 mg / mL, for example, about 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL, 26 μg / mL, 27 μg / mL, 28 μg / mL, 29 μg / mL, 30 μg / mL, 31 μg / mL, 32 μg / mL, 33 μg / mL, 34 μg / mL, 35 μg / mL, 36 μg / mL, 37 μg / mL, 38 μg / mL, 39 μg / mL, 40 μg / mL, 41 μg / mL, 42 μg / mL, 43 μg / mL, 44 μg / mL, 45 μg / mL, 46 μg / mL, 47 μg / mL, 48 μg / mL, 49 μg / mL, 50 μg / mL, 51 μg / mL, 52 μg / mL, 53 μg / mL, 54 μg / mL, 55 μg / mL, 56 μg / mL, 57 μg / mL, 58 μg / mL, 0μg / mL, 31μg / mL, 32μg / mL, 33μg / mL, 34μg / mL, 35μg / mL, 36μg / mL, 37μg / mL, 38μg / mL, 39μg / mL, 40μg / mL, 41μg / mL, 42μg / mL, 43 μg / mL, 44μg / mL, 45μg / mL, 46μg / mL, 47μg / mL, 48μg / mL, 49μg / mL, 50μg / mL, 51μg / mL, 52μg / mL, 53μg / mL, 54μg / mL, 55μg / mL, 56μ g / mL, 57μg / mL, 58μg / mL, 59μg / mL, 60μg / mL, 61μg / mL, 62μg / mL, 63μg / mL, 64μg / mL, 65μg / mL, 66μg / mL, 67μg / mL, 68μg / mL, 69μg / mL, 70μg / mL, 71μg / mL, 72μg / mL, 73μg / mL, 74μg / mL, 75μg / mL, 76μg / mL, 77μg / mL, 78μg / mL, 79μg / mL, 80μg / mL, 81μg / mL, 82μg / mL mL, 83μg / mL, 84μg / mL, 85μg / mL, 86μg / mL, 87μg / mL, 88μg / mL, 89μg / mL, 90μg / mL, 91μg / mL, 92μg / mL, 93μg / mL, 94μg / mL, 95μg / m L, 96μg / mL, 97μg / mL, 98μg / mL, 99μg / mL, 100μg / mL, 110μg / mL, 120μg / mL, 130μg / mL, 140μg / mL, 150μg / mL, 160μg / mL, 170μg / mL,180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL, 310 μg / mL, 320 μg / mL, 330 μg / mL, 340 μg / mL, 350 μg / mL, 360 μg / mL, 370 μg / mL, 380 μg / mL, 390 μg / mL, 400 μg / mL, 410 μg / mL, 420 μg / mL, 430 μg / mL, 440 μg / mL, 450 μg / mL, 460 μg / mL, 470 μg / mL, 480 μg / mL, 490 μg / mL, 500 μg / mL, 510 μg / mL, 520 μg / mL, 530 μg / mL, 540 μg / mL, 550 μg / mL, 560 μg / mL, 570 μg / mL, 580 μg / mL, 590 μg / mL, 600 μg / mL, 610 μg / mL, 620 μg / mL, 630 μg / mL, 640 μg / mL, 650 μg / mL, 660 μg / mL, 670 μg / mL, 680 μg / mL, 690 μg / mL, 700 μg / mL, 710 μg / mL, 720 μg / mL, 730 μg / mL, 740 μg / mL, 750 μg / mL, 760 μg / mL, 770 μg / mL, 780 μg / mL, 790 μg / mL, 800 μg / mL, 810 μg / mL, 820 μg / mL, 830 μg / mL, 840 μg / mL, 850 μg / mL, 860 μg / mL, 870 μg / mL, 880 μg / mL, 890 μg / mL, 900 μg / mL, 910 μg / mL, 920 μg / mL, 930 μg / mL, 940 μg / mL, 950 μg / mL, 960 μg / mL, 970 μg / mL, 980 μg / mL, 990 μg / mL, or about 1.0 mg / mL, or may be present in the above reaction mixture at any concentration therebetween.,

[0066] In some embodiments, the reaction mixture may include a chemical that modulates the redox potential of the cell extract. In some embodiments, the chemical may include oxidized glutathione. In some embodiments, the chemical may include reduced glutathione. In some embodiments, the chemical may include a sulfhydryl deactivator as a pretreatment of the extract before initiating the CFPS reaction. In some embodiments, the sulfhydryl deactivator may include iodoacetamide.

[0067] In some embodiments, the chemical is at a concentration between about 0.1 mM and about 30 mM, e.g., about 0.1 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.17 mM, 0.18 mM, 0.19 mM, 0.2 mM, 0.21 mM, 0.22 mM, 0.23 mM, 0.24 mM, 0.25 mM, 0.26 mM, 0.27 mM, 0.28 mM, 0.29 mM, 0.3 mM, 0.31 mM, 0.32 mM, 0.33 mM, 0.34 mM, 0.35 mM, 0.36 mM, 0.37 mM, 0.38 mM, 0.39 mM, 0.40 mM, 0.41 mM, 0.42 mM, 0.43 mM, 0.44 mM, 0.45 mM, 0.46 mM, 0.47 mM, 0.48 mM, 0.49 mM, 0.50 mM, 0.51 mM, 0.52 mM, 0.53 mM, 0.54 mM, 0.55 mM, 0.56 mM, 0.57 mM, 0.58 mM, 0.59 mM, 0.60 mM, 0.61 mM, 0.62 mM, 0.63 mM, 0.64 mM, 0.65 mM, 0.66 mM, 0.67 mM, 0.68 mM, 0.69 mM, 0.70 mM, 0.71 mM, 0.72 mM, 0.73 mM, 0.74 mM, 0.75 mM, 0.76 mM, 0.77 mM, 0.78 mM, mM, 0.4mM, 0.41mM, 0.42mM, 0.43mM, 0.44mM, 0.45mM, 0.46mM, 0.47mM, 0.48 mM, 0.49mM, 0.5mM, 0.51mM, 0.52mM, 0.53mM, 0.54mM, 0.55mM, 0.56mM, 0.57m M, 0.58mM, 0.59mM, 0.6mM, 0.61mM, 0.62mM, 0.63mM, 0.64mM, 0.65mM, 0.66m M, 0.67mM, 0.68mM, 0.69mM, 0.7mM, 0.71mM, 0.72mM, 0.73mM, 0.74mM, 0.75mM , 0.76mM, 0.77mM, 0.78mM, 0.79mM, 0.8mM, 0.81mM, 0.82mM, 0.83mM, 0.84mM , 0.85mM, 0.86mM, 0.87mM, 0.88mM, 0.89mM, 0.9mM, 0.91mM, 0.92mM, 0.93mM , 0.94mM, 0.95mM, 0.96mM, 0.97mM, 0.98mM, 0.99mM, 1mM, 1.1mM, 1.2mM, 1.3 mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM, 2mM, 2.1mM, 2.2mM, 2.3mM, 2. 4mM, 2.5mM, 2.6mM, 2.7mM, 2.8mM, 2.9mM, 3mM, 3.1mM, 3.2mM, 3.3mM, 3.4mM, 3.5mM, 3.6mM, 3.7mM, 3.8mM, 3.9mM, 4mM, 4.1mM, 4.2mM, 4.3mM, 4.4mM, 4.5mM , 4.6mM, 4.7mM, 4.8mM, 4.9mM, 5mM, 5.1mM, 5.2mM, 5.3mM, 5.4mM, 5.5mM, 5.6 mM, 5.7mM, 5.8mM, 5.9mM, 6mM, 6.1mM, 6.2mM, 6.3mM, 6.4mM, 6.5mM, 6.6mM, 6.7mM、6.8mM、6.9mM、7mM、7.1mM、7.2mM、7.3mM、7.4mM、7.5mM、7.6mM、7.7mM、7.8mM、7.9mM、8mM、8.1mM、8.2mM、8.3mM、8.4mM、8.5mM、8.6mM、8.7mM、8.8mM、8.9mM、9mM、9.1mM、9.2mM、9.3mM、9.4mM、9.5mM、9.6mM、9.7mM、9.8mM、9.9mM、10mM、10.1mM、10.2mM、10.3mM、10.4mM、10.5mM、10.6mM、10.7mM、10.8mM、10.9mM、11mM、11.1mM、11.2mM、11.3mM、11.4mM、11.5mM、11.6mM、11.7mM、11.8mM、11.9mM、12mM、12.1mM、12.2mM、12.3mM、12.4mM、12.5mM、12.6mM、12.7mM、12.8mM、12.9mM、13mM、13.1mM、13.2mM、13.3mM、13.4mM、13.5mM、13.6mM、13.7mM、13.8mM、13.9mM、14mM、14.1mM、14.2mM、14.3mM、14.4mM、14.5mM、14.6mM、14.7mM、14.8mM、14.9mM、15mM、15.1mM、15.2mM、15.3mM、15.4mM、15.5mM、15.6mM、15.7mM、15.8mM、15.9mM、16mM、16.1mM、16.2mM、16.3mM、16.4mM、16.5mM、16.6mM、16.7mM、16.8mM、16.9mM、17mM、17.1mM、17.2mM、17.3mM、17.4mM、17.5mM、17.6mM、17.7mM、17.8mM、17.9mM、18mM、18.1mM、18.2mM、18.3mM、18.4mM、18.5mM、18.6mM、18.7mM、18.8mM、18.9mM、19mM、19.1mM、19.2mM、19.3mM、19.4mM、19.5mM、19.6mM、19.7mM、19.8mM、19.9mM、20mM、20.1mM、20.2mM、20.3mM、20.4mM、20.5mM、20.6mM、20.7mM、20.8mM、20.9mM、21mM、21.1mM、21.2mM、21.3mM、21.4mM、21.5mM、21.6mM、21.7mM、21.8mM, 21.9mM, 22mM, 22.1mM, 22.2mM, 22.3mM, 22.4mM, 22.5mM, 22.6mM, 22.7mM, 22.8mM, 22. 9mM, 23mM, 23.1mM, 23.2mM, 23.3mM, 23.4mM, 23.5mM, 23.6mM, 23.7mM, 23.8mM, 23.9mM, 24m M, 24.1mM, 24.2mM, 24.3mM, 24.4mM, 24.5mM, 24.6mM, 24.7mM, 24.8mM, 24.9mM, 25mM, 25.1m M, 25.2mM, 25.3mM, 25.4mM, 25.5mM, 25.6mM, 25.7mM, 25.8mM, 25.9mM, 26mM, 26.1mM, 26.2m M, 26.3mM, 26.4mM, 26.5mM, 26.6mM, 26.7mM, 26.8mM, 26.9mM, 27mM, 27.1mM, 27.2mM, 27.3m M, 27.4mM, 27.5mM, 27.6mM, 27.7mM, 27.8mM, 27.9mM, 28mM, 28.1mM, 28.2mM, 28.3mM, 28.4m The composition may contain oxidized or reduced glutathione at about 28.5 mM, 28.6 mM, 28.7 mM, 28.8 mM, 28.9 mM, 29 mM, 29.1 mM, 29.2 mM, 29.3 mM, 29.4 mM, 29.5 mM, 29.6 mM, 29.7 mM, 29.8 mM, 29.9 mM, or about 30 mM, or any concentration therebetween.

[0068] In some embodiments, the chemical is at a concentration between about 1 μM and about 5000 μM, e.g., about 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3 μM, 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, 3.6 μM, 3.7 μM, 3.8 μM, 3.9 μM, 4 μM, 4.1 μM, 4.2 μM, 4.3 μM, 4.4 μM, 4.5 μM, 4.6 μM, 4.7 μM, 4.8 μM, 4.9 μM, 4. 5μM, 4.6μM, 4.7μM, 4.8μM, 4.9μM, 5μM, 5.1μM, 5.2μM, 5.3μM, 5.4μM, 5.5μM, 5.6μM, 5.7μM, 5.8μM, 5.9μM, 6μM, 6.1μM, 6.2μM, 6.3μM, 6.4μM, 6.5μM, 6.6μM M, 6.7μM, 6.8μM, 6.9μM, 7μM, 7.1μM, 7.2μM, 7.3μM, 7.4μM, 7.5μM, 7.6μM, 7. 7μM, 7.8μM, 7.9μM, 8μM, 8.1μM, 8.2μM, 8.3μM, 8.4μM, 8.5μM, 8.6μM, 8.7μM, 8 .8μM, 8.9μM, 9μM, 9.1μM, 9.2μM, 9.3μM, 9.4μM, 9.5μM, 9.6μM, 9.7μM, 9.8μM , 9.9μM, 10μM, 11μM, 12μM, 13μM, 14μM, 15μM, 16μM, 17μM, 18μM, 19μM, 20μM, 21μM, 22μM, 23μM, 24μM, 25μM, 26μM, 27μM, 28μM, 29μM, 30μM, 31μM, 32μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46μM, 47μM, 48μM, 49μM, 50μM, 51μM, 52μM, 53μM, 54μM, 55μM, 56μM, 57μM, 58 μM, 59 μM, 60 μM, 61 μM, 62 μM, 63 μM, 64 μM, 65 μM, 66 μM, 67 μM, 68 μM, 69 μM, 70 μM, 71μM, 72μM, 73μM, 74μM, 75μM, 76μM, 77μM, 78μM, 79μM, 80μM, 81μM, 82μM, 83 μM, 84 μM, 85 μM, 86 μM, 87 μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93 μM, 94 μM, 95 μM,96μM, 97μM, 98μM, 99μM, 100μM, 110μM, 120μM, 130μM, 140μM, 150μM, 160μM, 170μM, 180μM, 190μM, 200μM, 210μM, 220μM, 230μM, 240μM, 250μM, 260μM, 270 μM, 280μM, 290μM, 300μM, 310μM, 320μM, 330μM, 340μM, 350μM, 360μM, 370μM, 380μM, 390μM, 400μM, 410μM, 420μM, 430μM, 440μM, 450μM, 460μM, 470μM, 48 0μM, 490μM, 500μM, 510μM, 520μM, 530μM, 540μM, 550μM, 560μM, 570μM, 580μM, 590μM, 600μM, 610μM, 620μM, 630μM, 640μM, 650μM, 660μM, 670μM, 680μM, 6 90μM, 700μM, 710μM, 720μM, 730μM, 740μM, 750μM, 760μM, 770μM, 780μM, 790μM, 800μM, 810μM, 820μM, 830μM, 840μM, 850μM, 860μM, 870μM, 880μM, 890μM, 900μM, 910μM, 920μM, 930μM, 940μM, 950μM, 960μM, 970μM, 980μM, 990μM, 1000μM, 1020μM, 1040μM, 1060μM, 1080μM, 1100μM, 1120μM, 1140μM, 1160μM, 11 80μM, 1200μM, 1220μM, 1240μM, 1260μM, 1280μM, 1300μM, 1320μM, 1340μM, 1360μM, 1380μM, 1400μM, 1420μM, 1440μM, 1460μM, 1480μM, 1500μM, 1520μM, 1 540μM, 1560μM, 1580μM, 1600μM, 1620μM, 1640μM, 1660μM, 1680μM, 1700μM, 1720μM, 1740μM, 1760μM, 1780μM, 1800μM, 1820μM, 1840μM, 1860μM, 1880μM, 1900μM, 1920μM, 1940μM, 1960μM, 1980μM, 2000μM, 2020μM, 2040μM, 2060μM, 2080μM, 2100μM, 2120μM, 2140μM, 2160μM, 2180μM, 2200μM, 2220μM, 2240μM,2260μM、2280μM、2300μM、2320μM、2340μM、2360μM、2380μM、2400μM、2420 μM、2440μM、2460μM、2480μM、2500μM、2520μM、2540μM、2560μM、2580μM、26 00μM、2620μM、2640μM、2660μM、2680μM、2700μM、2720μM、2740μM、2760μM 、2780μM、2800μM、2820μM、2840μM、2860μM、2880μM、2900μM、2920μM、2940 μM、2960μM、2980μM、3000μM、3020μM、3040μM、3060μM、3080μM、3100μM、3 120μM、3140μM、3160μM、3180μM、3200μM、3220μM、3240μM、3260μM、3280μM 、3300μM、3320μM、3340μM、3360μM、3380μM、3400μM、3420μM、3440μM、3460 μM、3480μM、3500μM、3520μM、3540μM、3560μM、3580μM、3600μM、3620μM、36 40μM、3660μM、3680μM、3700μM、3720μM、3740μM、3760μM、3780μM、3800μM 、3820μM、3840μM、3860μM、3880μM、3900μM、3920μM、3940μM、3960μM、3980 μM、4000μM、4020μM、4040μM、4060μM、4080μM、4100μM、4120μM、4140μM、41 60μM、4180μM、4200μM、4220μM、4240μM、4260μM、4280μM、4300μM、4320μM、 4340μM、4360μM、4380μM、4400μM、4420μM、4440μM、4460μM、4480μM、4500 μM、4520μM、4540μM、4560μM、4580μM、4600μM、4620μM、4640μM、4660μM、46 80μM、4700μM、4720μM、4740μM、4760μM、4780μM、4800μM、4820μM、4840μM、 4860μM、4880μM、4900μM、4920μM、4940μM、4960μM、4980μM、or about 5000μM、or any concentration therebetween.

[0069] In some embodiments, the reaction mixture may include at least one chemical that acts to promote proper folding and / or post-translational modification of the polypeptide or protein to be produced. In some embodiments, the chemical may include a protein. In some embodiments, the chemical may include a chaperone. In some embodiments, the chemical may include an enzyme. In some embodiments, the chemical may include a glycosyltransferase. In some embodiments, the chemical may include a sugar donor. In some embodiments, the polypeptide or protein to be produced may be expressed from a plasmid or the genome of the cell used to produce the cell extract.

[0070] In some embodiments, the reaction mixture may contain an alcohol. In some embodiments, the alcohol may contain a polyol. In some embodiments, the alcohol is at a concentration between about 0% (v / v) and about 25% (v / v), such as about 0.1% (v / v), 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), 0.6% (v / v), 0.7% (v / v), 0.8% (v / v), 0.9% (v / v), 1% (v / v), 1.1% (v / v), 1.2% (v / v), 1.3% (v / v), 1.4% (v / v), 1.5% (v / v), 1.6% (v / v), 1.7% (v / v), 1.8% (v / v), 1.9% (v / v), 2% (v / v), 2.1% (v / v), 2.2% (v / v), 2.3% (v / v), 2.4% (v / v), 2.5% (v / v), 2.6% (v / v), 2.7% (v / v), 2.8% (v / v), 2.9% (v / v), 3% (v / v), 3.1% (v / v), 3.2% (v / v), 3.3% (v / v), 3.4% (v / v), 3.5% (v / v), 3.6% (v / v), 3.7% (v / v), 3.8% (v / v), 3.9% (v / v), 4% (v / v), 4.1% (v / v), 4.2% (v / v), 4.3% (v / v), 4.4% (v / v), 4.5% (v / v), 4.6% (v / v), 4.7% (v / v), 4.8% (v / v), 4.9% (v / v), 5% (v / v), 5.1% (v / v), 5.2% (v / v), 5.3% (v / v), 5.4% (v / v), 5.5% (v / v), 5.6% (v / v), 5.7% (v / v), 5.8% (v / v), 5.9% (v / v), 6% (v / v), 6.1% (v / v), 6.2% (v / v), 6.3% (v / v), 6.4% (v / v), 6.5% (v / v), 6.6% (v / v), 6.7% (v / v), 6.8% (v / v), 6.9% (v / v), 7% (v / v), 7.1% (v / v), 7.2% (v / v), 7.3% (v / v), 7.4% (v / v), 7.5% (v / v), 7.6% (v / v), 7.7% (v / v), 7.8% (v / v), 7.9% (v / v), 8% (v / v), 8.1% (v / v), 8.2% (v / v), 8.3% (v / v), 8.4% (v / v), 8.5% (v / v), 8.6% (v / v), 8.7% (v / v), 8.8% (v / v), 8.9%(v / v)、9%(v / v)、9.1%(v / v)、9.2%(v / v)、9.3%(v / v)、9.4%(v / v)、9.5%(v / v)、9.6%(v / v)、9.7%(v / v)、9.8%(v / v)、9.9%(v / v)、10%(v / v)、10.1%(v / v )、10.2%(v / v)、10.3%(v / v)、10.4%(v / v)、10.5%(v / v)、10.6%(v / v)、10.7%(v / v)、10.8%(v / v)、10.9%(v / v)、11%(v / v)、11.1%(v / v)、11.2%(v / v)、11. 3%(v / v)、11.4%(v / v)、11.5%(v / v)、11.6%(v / v)、11.7%(v / v)、11.8%(v / v)、11.9%(v / v)、12%(v / v)、12.1%(v / v)、12.2%(v / v)、12.3%(v / v)、12.4%(v / v)、12.5%(v / v)、12.6%(v / v)、12.7%(v / v)、12.8%(v / v)、12.9%(v / v)、13%(v / v)、13.1%(v / v)、13.2%(v / v)、13.3%(v / v)、13.4%(v / v)、13.5%(v / v)、13 .6%(v / v)、13.7%(v / v)、13.8%(v / v)、13.9%(v / v)、14%(v / v)、14.1%(v / v)、14.2%(v / v)、14.3%(v / v)、14.4%(v / v)、14.5%(v / v)、14.6%(v / v)、14.7%(v / v)、14.8%(v / v)、14.9%(v / v)、15%(v / v)、15.1%(v / v)、15.2%(v / v)、15.3%(v / v)、15.4%(v / v)、15.5%(v / v)、15.6%(v / v)、15.7%(v / v)、15.8%(v / v)、1 5.9%(v / v)、16%(v / v)、16.1%(v / v)、16.2%(v / v)、16.3%(v / v)、16.4%(v / v)、16.5%(v / v)、16.6%(v / v)、16.7%(v / v)、16.8%(v / v)、16.9%(v / v)、17%(v / v)、17.1%(v / v)、17.2%(v / v)、17.3%(v / v)、17.4%(v / v)、17.5%(v / v)、17.6%(v / v)、17.7%(v / v)、17.8%(v / v)、17.9%(v / v)、18%(v / v)、18.1%(v / v)、18.2%(v / v)、18.3%(v / v)、18.4%(v / v)、18.5%(v / v)、18.6%(v / v)、18.7%(v / v)、18.8%(v / v)、18.9%(v / v)、19%(v / v)、19.1%(v / v)、19.2%(v / v)、19.3%(v / v)、19.4%(v / v)、19.5%(v / v)、19.6%(v / v)、19.7%(v / v)、19.8%(v / v)、19.9%(v / v) 、20%(v / v)、20.1%(v / v)、20.2%(v / v)、20.3%(v / v)、20.4%(v / v)、20.5%(v / v)、20.6%(v / v)、20.7%(v / v)、20.8%(v / v)、20.9%(v / v)、21%(v / v)、21.1%(v / v)、21.2%(v / v)、21.3%(v / v)、21.4%(v / v)、21.5%(v / v)、21.6%(v / v)、21.7%(v / v)、21.8%(v / v)、21.9%(v / v)、22%(v / v)、22.1%(v / v)、22.2%(v / v)、22.3%(v / v)、22.4%(v / v)、22.5%(v / v)、22.6%(v / v)、22.7%(v / v)、22.8%(v / v)、22.9%(v / v)、23%(v / v)、23.1%(v / v)、23.2%(v / v)、23.3%(v / v)、23.4%(v / v)、23.5%( v / v)、23.6%(v / v)、23.7%(v / v)、23.8%(v / v)、23.9%(v / v)、24%(v / v)、24.1%(v / v)、24.2%(v / v)、24.3%(v / v)、24.4% (v / v)、24.5%(v / v)、24.6%(v / v)、24.7%(v / v)、24.8%(v / v ).

[0071] In some embodiments, the energy source in the reaction mixture is at a concentration between about 10 mM and about 400 mM, e.g., about 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61 mM, 62mM, 63mM, 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, 71mM, 72mM, 73mM , 74mM, 75mM, 76mM, 77mM, 78mM, 79mM, 80mM, 81mM, 82mM, 83mM, 84mM, 85mM, 8 6mM, 87mM, 88mM, 89mM, 90mM, 91mM, 92mM, 93mM, 94mM, 95mM, 96mM, 97mM, 98mM , 99mM, 100mM, 101mM, 102mM, 103mM, 104mM, 105mM, 106mM, 107mM, 108mM, 10 9mM, 110mM, 111mM, 112mM, 113mM, 114mM, 115mM, 116mM, 117mM, 118mM, 119m M, 120mM, 121mM, 122mM, 123mM, 124mM, 125mM, 126mM, 127mM, 128mM, 129mM, 130mM, 131mM, 132mM, 133mM, 134mM, 135mM, 136mM, 137mM, 138mM, 139mM, 140 mM, 141mM, 142mM, 143mM, 144mM, 145mM, 146mM, 147mM, 148mM, 149mM, 150mM , 151mM, 152mM, 153mM, 154mM, 155mM, 156mM, 157mM, 158mM, 159mM, 160mM, 1 61mM, 162mM, 163mM, 164mM, 165mM, 166mM, 167mM, 168mM, 169mM, 170mM, 171 mM, 172mM, 173mM, 174mM, 175mM, 176mM, 177mM, 178mM, 179mM, 180mM, 181mM,182mM、183mM、184mM、185mM、186mM、187mM、188mM、189mM、190mM、191mM、192mM、193mM、194mM、195mM、196mM、197mM、198mM、199mM、200mM、201mM、202mM、203mM、204mM、205mM、206mM、207mM、208mM、209mM、210mM、211mM、212mM、213mM、214mM、215mM、216mM、217mM、218mM、219mM、220mM、221mM、222mM、223mM、224mM、225mM、226mM、227mM、228mM、229mM、230mM、231mM、232mM、233mM、234mM、235mM、236mM、237mM、238mM、239mM、240mM、241mM、242mM、243mM、244mM、245mM、246mM、247mM、248mM、249mM、250mM、251mM、252mM、253mM、254mM、255mM、256mM、257mM、258mM、259mM、260mM、261mM、262mM、263mM、264mM、265mM、266mM、267mM、268mM、269mM、270mM、271mM、272mM、273mM、274mM、275mM、276mM、277mM、278mM、279mM、280mM、281mM、282mM、283mM、284mM、285mM、286mM、287mM、288mM、289mM、290mM、291mM、292mM、293mM、294mM、295mM、296mM、297mM、298mM、299mM、300mM、301mM、302mM、303mM、304mM、305mM、306mM、307mM、308mM、309mM、310mM、311mM、312mM、313mM、314mM、315mM、316mM、317mM、318mM、319mM、320mM、321mM、322mM、323mM、324mM、325mM、326mM、327mM、328mM、329mM、330mM、331mM、332mM、333mM、334mM、335mM、336mM、337mM、338mM、339mM、340mM、341mM、342mM、343mM、344mM、345mM、346mM、347mM、348mM, 349mM, 350mM, 351mM, 352mM, 353mM, 354mM, 355mM, 356mM, 357mM, 358mM, 359mM, 360mM, 361mM, 36 2mM, 363mM, 364mM, 365mM, 366mM, 367mM, 368mM, 369mM, 370mM, 371mM, 372mM, 373mM, 374mM, 375mM, 376m The energy source may be present at about 400 mM, 377 mM, 378 mM, 379 mM, 380 mM, 381 mM, 382 mM, 383 mM, 384 mM, 385 mM, 386 mM, 387 mM, 388 mM, 389 mM, 390 mM, 391 mM, 392 mM, 393 mM, 394 mM, 395 mM, 396 mM, 397 mM, 398 mM, 399 mM, or about 400 mM, or any concentration therebetween. In some embodiments, the concentrations recited above may be applicable to any of the energy sources disclosed herein, including, but not limited to, phosphate groups, non-phosphorylated energy groups, or one or more selected from phosphoenolpyruvate, glutamate, glycerol, pyruvate, glucose, and creatine phosphate.

[0072] In some embodiments, the salt in the reaction mixture is at a concentration between about 50 mM and about 500 mM, e.g., about 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, 117 mM, 118 mM, 119 mM, 120 mM, 121 mM, 122 mM, 123 mM, 124 mM, 125 mM, 126 mM, 127 mM, 128 mM, 129 mM, 130 mM, 131 mM, 132 mM, 133 mM, 134 mM, 135 mM, 136 mM, 137 mM 0mM, 91mM, 92mM, 93mM, 94mM, 95mM, 96mM, 97mM, 98mM, 99mM, 100mM, 101mM, 1 02mM, 103mM, 104mM, 105mM, 106mM, 107mM, 108mM, 109mM, 110mM, 111mM, 112m M, 113mM, 114mM, 115mM, 116mM, 117mM, 118mM, 119mM, 120mM, 121mM, 122mM, 123mM, 124mM, 125mM, 126mM, 127mM, 128mM, 129mM, 130mM, 131mM, 132mM, 133 mM, 134mM, 135mM, 136mM, 137mM, 138mM, 139mM, 140mM, 141mM, 142mM, 143mM , 144mM, 145mM, 146mM, 147mM, 148mM, 149mM, 150mM, 151mM, 152mM, 153mM, 15 4mM, 155mM, 156mM, 157mM, 158mM, 159mM, 160mM, 161mM, 162mM, 163mM, 164m M, 165mM, 166mM, 167mM, 168mM, 169mM, 170mM, 171mM, 172mM, 173mM, 174mM, 1 75mM, 176mM, 177mM, 178mM, 179mM, 180mM, 181mM, 182mM, 183mM, 184mM, 185 mM, 186mM, 187mM, 188mM, 189mM, 190mM, 191mM, 192mM, 193mM, 194mM, 195mM, 196mM, 197mM, 198mM, 199mM, 200mM, 201mM, 202mM, 203mM, 204mM, 205mM, 206 mM, 207mM, 208mM, 209mM, 210mM, 211mM, 212mM, 213mM, 214mM, 215mM, 216mM,217mM、218mM、219mM、220mM、221mM、222mM、223mM、224mM、225mM、226mM、227mM、228mM、229mM、230mM、231mM、232mM、233mM、234mM、235mM、236mM、237mM、238mM、239mM、240mM、241mM、242mM、243mM、244mM、245mM、246mM、247mM、248mM、249mM、250mM、251mM、252mM、253mM、254mM、255mM、256mM、257mM、258mM、259mM、260mM、261mM、262mM、263mM、264mM、265mM、266mM、267mM、268mM、269mM、270mM、271mM、272mM、273mM、274mM、275mM、276mM、277mM、278mM、279mM、280mM、281mM、282mM、283mM、284mM、285mM、286mM、287mM、288mM、289mM、290mM、291mM、292mM、293mM、294mM、295mM、296mM、297mM、298mM、299mM、300mM、301mM、302mM、303mM、304mM、305mM、306mM、307mM、308mM、309mM、310mM、311mM、312mM、313mM、314mM、315mM、316mM、317mM、318mM、319mM、320mM、321mM、322mM、323mM、324mM、325mM、326mM、327mM、328mM、329mM、330mM、331mM、332mM、333mM、334mM、335mM、336mM、337mM、338mM、339mM、340mM、341mM、342mM、343mM、344mM、345mM、346mM、347mM、348mM、349mM、350mM、351mM、352mM、353mM、354mM、355mM、356mM、357mM、358mM、359mM、360mM、361mM、362mM、363mM、364mM、365mM、366mM、367mM、368mM、369mM、370mM、371mM、372mM、373mM、374mM、375mM、376mM、377mM、378mM、379mM、380mM、381mM、382mM、383mM, 384mM, 385mM, 386mM, 387mM, 388mM, 389mM, 390mM, 391mM, 392mM, 393mM, 394mM, 395mM, 396mM, 397mM, 39 8mM, 399mM, 400mM, 401mM, 402mM, 403mM, 404mM, 405mM, 406mM, 407mM, 408mM, 409mM, 410mM, 411mM, 412mM, 413m M, 414mM, 415mM, 416mM, 417mM, 418mM, 419mM, 420mM, 421mM, 422mM, 423mM, 424mM, 425mM, 426mM, 427mM, 428mM, 429mM, 430mM, 431mM, 432mM, 433mM, 434mM, 435mM, 436mM, 437mM, 438mM, 439mM, 440mM, 441mM, 442mM, 443mM, 444 mM, 445mM, 446mM, 447mM, 448mM, 449mM, 450mM, 451mM, 452mM, 453mM, 454mM, 455mM, 456mM, 457mM, 458mM, 459mM , 460mM, 461mM, 462mM, 463mM, 464mM, 465mM, 466mM, 467mM, 468mM, 469mM, 470mM, 471mM, 472mM, 473mM, 474mM, 4 The potassium concentration may be 75 mM, 476 mM, 477 mM, 478 mM, 479 mM, 480 mM, 481 mM, 482 mM, 483 mM, 484 mM, 485 mM, 486 mM, 487 mM, 488 mM, 489 mM, 490 mM, 491 mM, 492 mM, 493 mM, 494 mM, 495 mM, 496 mM, 497 mM, 498 mM, 499 mM, or about 500 mM, or any concentration therebetween.

[0073] In some embodiments, the salt in the reaction mixture is at a concentration between about 1 mM and about 30 mM, e.g., about 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, 4.5mM, 4.6mM, 4.7mM, 4.8mM, 4.9mM, 5mM, 5.1mM, 5.2mM, 5.3mM, 5.4mM, 5.5m M, 5.6mM, 5.7mM, 5.8mM, 5.9mM, 6mM, 6.1mM, 6.2mM, 6.3mM, 6.4mM, 6.5mM, 6.6 mM, 6.7mM, 6.8mM, 6.9mM, 7mM, 7.1mM, 7.2mM, 7.3mM, 7.4mM, 7.5mM, 7.6mM, 7 .7mM, 7.8mM, 7.9mM, 8mM, 8.1mM, 8.2mM, 8.3mM, 8.4mM, 8.5mM, 8.6mM, 8.7mM, 8.8mM, 8.9mM, 9mM, 9.1mM, 9.2mM, 9.3mM, 9.4mM, 9.5mM, 9.6mM, 9.7mM, 9.8m M, 9.9mM, 10mM, 10.1mM, 10.2mM, 10.3mM, 10.4mM, 10.5mM, 10.6mM, 10.7mM, 1 0.8mM, 10.9mM, 11mM, 11.1mM, 11.2mM, 11.3mM, 11.4mM, 11.5mM, 11.6mM, 11 .7mM, 11.8mM, 11.9mM, 12mM, 12.1mM, 12.2mM, 12.3mM, 12.4mM, 12.5mM, 12.6 mM, 12.7mM, 12.8mM, 12.9mM, 13mM, 13.1mM, 13.2mM, 13.3mM, 13.4mM, 13.5m M, 13.6mM, 13.7mM, 13.8mM, 13.9mM, 14mM, 14.1mM, 14.2mM, 14.3mM, 14.4mM, 14.5mM, 14.6mM, 14.7mM, 14.8mM, 14.9mM, 15mM, 15.1mM, 15.2mM, 15.3mM, 1 5.4mM, 15.5mM, 15.6mM, 15.7mM, 15.8mM, 15.9mM, 16mM, 16.1mM, 16.2mM, 16.3mM、16.4mM、16.5mM、16.6mM、16.7mM、16.8mM、16.9mM、17mM、17.1mM、17.2mM、17.3mM、17.4mM、17.5mM、17.6mM、17.7mM、17.8mM、17.9mM、18mM、18.1mM、18.2mM、18.3mM、18.4mM、18.5mM、18.6mM、18.7mM、18.8mM、18.9mM、19mM、19.1mM、19.2mM、19.3mM、19.4mM、19.5mM、19.6mM、19.7mM、19.8mM、19.9mM、20mM、20.1mM、20.2mM、20.3mM、20.4mM、20.5mM、20.6mM、20.7mM、20.8mM、20.9mM、21mM、21.1mM、21.2mM、21.3mM、21.4mM、21.5mM、21.6mM、21.7mM、21.8mM、21.9mM、22mM、22.1mM、22.2mM、22.3mM、22.4mM、22.5mM、22.6mM、22.7mM、22.8mM、22.9mM、23mM、23.1mM、23.2mM、23.3mM、23.4mM、23.5mM、23.6mM、23.7mM、23.8mM、23.9mM、24mM、24.1mM、24.2mM、24.3mM、24.4mM、24.5mM、24.6mM、24.7mM、24.8mM、24.9mM、25mM、25.1mM、25.2mM、25.3mM、25.4mM、25.5mM、25.6mM、25.7mM、25.8mM、25.9mM、26mM、26.1mM、26.2mM、26.3mM、26.4mM、26.5mM、26.6mM、26.7mM、26.8mM、26.9mM、27mM、27.1mM、27.2mM、27.3mM、27.4mM、27.5mM、27.6mM、27.7mM、27.8mM、27.9mM、28mM、28.1mM、28.2mM、28.3mM、28.4mM、28.5mM、28.6mM、28.7mM、28.8mM、28.9mM、29mM、29.1mM、29.2mM、29.3mM、29.4mM、29.5mM、29.6mM、29.7mM、29.8mM、29.9 mM, or 30 mM, or any concentration therebetween. In some embodiments, the salts in the reaction mixture may include potassium at the concentrations disclosed above and magnesium at the concentrations disclosed above.

[0074] In some embodiments, the salt in the reaction mixture is at a concentration between about 1 mM and about 400 mM, e.g., about 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, , 4.5mM, 4.6mM, 4.7mM, 4.8mM, 4.9mM, 5mM, 5.1mM, 5.2mM, 5.3mM, 5.4mM, 5.5 mM, 5.6mM, 5.7mM, 5.8mM, 5.9mM, 6mM, 6.1mM, 6.2mM, 6.3mM, 6.4mM, 6.5mM, 6 .6mM, 6.7mM, 6.8mM, 6.9mM, 7mM, 7.1mM, 7.2mM, 7.3mM, 7.4mM, 7.5mM, 7.6mM , 7.7mM, 7.8mM, 7.9mM, 8mM, 8.1mM, 8.2mM, 8.3mM, 8.4mM, 8.5mM, 8.6mM, 8.7m M, 8.8mM, 8.9mM, 9mM, 9.1mM, 9.2mM, 9.3mM, 9.4mM, 9.5mM, 9.6mM, 9.7mM, 9. 8mM, 9.9mM, 10mM, 10.1mM, 10.2mM, 10.3mM, 10.4mM, 10.5mM, 10.6mM, 10.7m M, 10.8mM, 10.9mM, 11mM, 11.1mM, 11.2mM, 11.3mM, 11.4mM, 11.5mM, 11.6mM , 11.7mM, 11.8mM, 11.9mM, 12mM, 12.1mM, 12.2mM, 12.3mM, 12.4mM, 12.5mM, 1 2.6mM, 12.7mM, 12.8mM, 12.9mM, 13mM, 13.1mM, 13.2mM, 13.3mM, 13.4mM, 13 .5mM, 13.6mM, 13.7mM, 13.8mM, 13.9mM, 14mM, 14.1mM, 14.2mM, 14.3mM, 14.4 mM, 14.5mM, 14.6mM, 14.7mM, 14.8mM, 14.9mM, 15mM, 15.1mM, 15.2mM, 15.3m M, 15.4mM, 15.5mM, 15.6mM, 15.7mM, 15.8mM, 15.9mM, 16mM, 16.1mM, 16.2mM,16.3mM、16.4mM、16.5mM、16.6mM、16.7mM、16.8mM、16.9mM、17mM、17.1mM、17.2mM、17.3mM、17.4mM、17.5mM、17.6mM、17.7mM、17.8mM、17.9mM、18mM、18.1mM、18.2mM、18.3mM、18.4mM、18.5mM、18.6mM、18.7mM、18.8mM、18.9mM、19mM、19.1mM、19.2mM、19.3mM、19.4mM、19.5mM、19.6mM、19.7mM、19.8mM、19.9mM、20mM、20.5mM、21mM、21.5mM、22mM、22.5mM、23mM、23.5mM、24mM、24.5mM、25mM、25.5mM、26mM、26.5mM、27mM、27.5mM、28mM、28.5mM、29mM、29.5mM、30mM、30.5mM、31mM、31.5mM、32mM、32.5mM、33mM、33.5mM、34mM、34.5mM、35mM、35.5mM、36mM、36.5mM、37mM、37.5mM、38mM、38.5mM、39mM、39.5mM、40mM、40.5mM、41mM、41.5mM、42mM、42.5mM、43mM、43.5mM、44mM、44.5mM、45mM、45.5mM、46mM、46.5mM、47mM、47.5mM、48mM、48.5mM、49mM、49.5mM、50mM、51mM、52mM、53mM、54mM、55mM、56mM、57mM、58mM、59mM、60mM、61mM、62mM、63mM、64mM、65mM、66mM、67mM、68mM、69mM、70mM、71mM、72mM、73mM、74mM、75mM、76mM、77mM、78mM、79mM、80mM、81mM、82mM、83mM、84mM、85mM、86mM、87mM、88mM、89mM、90mM、91mM、92mM、93mM、94mM、95mM、96mM、97mM、98mM、99mM、100mM、101mM、102mM、103mM、104mM、105mM、106mM、107mM、108mM、109mM、110mM、111mM、112mM、113mM、114mM、115mM、116mM、117mM、118mM、119mM、120mM、121mM、122mM、123mM, 124mM, 125mM, 126mM, 127mM, 128mM, 129mM, 130mM, 131mM, 132mM, 133mM, 134mM, 135mM, 136mM, 137mM, 13 8mM, 139mM, 140mM, 141mM, 142mM, 143mM, 144mM, 145mM, 146mM, 147mM, 148mM, 149mM, 150mM, 151mM, 152mM, 153mM , 154mM, 155mM, 156mM, 157mM, 158mM, 159mM, 160mM, 161mM, 162mM, 163mM, 164mM, 165mM, 166mM, 167mM, 168mM, 1 69mM, 170mM, 171mM, 172mM, 173mM, 174mM, 175mM, 176mM, 177mM, 178mM, 179mM, 180mM, 181mM, 182mM, 183mM, 184m M, 185mM, 186mM, 187mM, 188mM, 189mM, 190mM, 191mM, 192mM, 193mM, 194mM, 195mM, 196mM, 197mM, 198mM, 199mM, 200mM, 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275 The ammonium concentration may be about 400 mM, 280 mM, 285 mM, 290 mM, 295 mM, 300 mM, 305 mM, 310 mM, 315 mM, 320 mM, 325 mM, 330 mM, 335 mM, 340 mM, 345 mM, 350 mM, 355 mM, 360 mM, 365 mM, 370 mM, 375 mM, 380 mM, 385 mM, 390 mM, 395 mM, or about 400 mM, or any concentration therebetween.

[0075] In some embodiments, the reaction mixture may include a nucleic acid transcription template. In some embodiments, the reaction mixture may include a nucleic acid translation template. In some embodiments, the reaction mixture may include both a nucleic acid transcription template and a nucleic acid translation template. In some embodiments, the reaction mixture may include a polymerase. In some embodiments, the polymerase may generate a nucleic acid translation template from a nucleic acid transcription template. In some embodiments, the polymerase may include a DNA-dependent RNA polymerase. In some embodiments, the polymerase may include an endogenous polymerase, i.e., a polymerase present in the cell extract and derived from the cell. In some embodiments, the polymerase may include an exogenous polymerase, i.e., a polymerase added to the cell extract. In some embodiments, the polymerase may include a T7 RNA polymerase. In some embodiments, the polymerase may be expressed from a plasmid present in the cell used to prepare the cell extract. In some embodiments, the polymerase may be expressed from a site in the genome of the cell used to prepare the cell extract.

[0076] In some embodiments, the nucleic acid encoding the polypeptide may comprise DNA. In some embodiments, the DNA may comprise an open reading frame of the polypeptide. The open reading frame is an amino acid coding sequence of the polypeptide. In some embodiments, the DNA may comprise a promoter of a DNA-dependent RNA polymerase. In some embodiments, the open reading frame of the polypeptide may be operably linked to the promoter. In some embodiments, the DNA may comprise a ribosome binding site located at or upstream of the 5' end of the open reading frame. In some embodiments, the DNA may comprise a terminator located at or downstream of the 3' end of the open reading frame. In some embodiments, the promoter may comprise a T7 promoter. In some embodiments, the T7 promoter may comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to 5'-TAATACGACTCACTATAGG-3' (SEQ ID NO: 1). In some embodiments, the T7 promoter may comprise SEQ ID NO: 1. In some embodiments, the terminator may comprise a T7 terminator. In some embodiments, the T7 terminator may comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to 5'-CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG-3' (SEQ ID NO:2). In some embodiments, the T7 terminator may comprise SEQ ID NO:2.

[0077] In some embodiments, the method may include incubating the reaction mixture to produce the polypeptide. In some embodiments, the reaction mixture is not subjected to clarification via centrifugation prior to the incubating step. In some embodiments, the method for CFPS disclosed herein may further include isolating the polypeptide from the reaction mixture.

[0078] In some embodiments, the methods disclosed herein can be performed at temperatures between about 1° C. and about 40° C., e.g., about 1° C., 1.5° C., 2° C., 2.5° C., 3° ​​C., 3.5° C., 4° C., 4.5° C., 5° C., 5.5° C., 6° C., 6.5° C., 7° C., 7.5° C., 8° C., 8.5° C., 9° C., 9.5° C., 10° C., 10.5° C., 11° C., 11.5° C., 12° C., 12.5° C., 13° C., 13.5° C., 14° C., 14.5° C., 15° C., 15.5° C., 16° C., 16.5° C., 17° C., 17.5° C., 18° C., 18.5° C., 19° C., 19.5° C., 20° C., It may be performed at 20.5°C, 21°C, 21.5°C, 22°C, 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C, 28°C, 28.5°C, 29°C, 29.5°C, 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C, or about 40°C, or any temperature in between. In some embodiments, the temperatures disclosed above may be applicable to one or more steps of the methods disclosed herein, including, but not limited to, growing the cells, harvesting the cells, lysing the cells to produce the cell lysate, passing the cell lysate through the filter to produce the cell extract, contacting the cell extract with a nucleic acid encoding the polypeptide to produce the reaction mixture, incubating the reaction mixture to produce the polypeptide, and isolating the polypeptide from the reaction mixture.

[0079] In some embodiments, the step of incubating the reaction mixture to produce the polypeptide may be carried out at a suitable temperature. In some embodiments, the step of incubating the reaction mixture to produce the polypeptide may be carried out at a temperature between about 10°C and about 42°C, for example, about 10°C, 10.5°C, 11°C, 11.5°C, 12°C, 12.5°C, 13°C, 13.5°C, 14°C, 14.5°C, 15°C, 15.5°C, 16°C, 16.5°C, 17°C, 17.5°C, 18°C, 18.5°C, 19°C, 19.5°C, 20°C, 20.5°C, 21°C, 21.5°C, 22°C, 22.5°C, 23°C, 23.5°C, The reaction may be carried out at temperatures of 24°C, 24.5°C, 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C, 28°C, 28.5°C, 29°C, 29.5°C, 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C, 40°C, 40.5°C, 41°C, 41.5°C, or about 42°C, or any temperature therebetween.

[0080] In some embodiments, the step of isolating the polypeptide from the reaction mixture is carried out at a temperature between about 1° C. and about 30° C., e.g., at about 1° C., 1.5° C., 2° C., 2.5° C., 3° ​​C., 3.5° C., 4° C., 4.5° C., 5° C., 5.5° C., 6° C., 6.5° C., 7° C., 7.5° C., 8° C., 8.5° C., 9° C., 9.5° C., 10° C., 10.5° C., 11° C., 11.5° C., 12° C., 12.5° C., 13° C., 13.5° C., 14° C., 14.5° C. , 15°C, 15.5°C, 16°C, 16.5°C, 17°C, 17.5°C, 18°C, 18.5°C, 19°C, 19.5°C, 20°C, 20.5°C, 21°C, 21.5°C, 22°C, 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C, 28°C, 28.5°C, 29°C, 29.5°C, or 30°C, or any temperature therebetween.

[0081] In some embodiments, the method may be performed at a temperature between about 1° C. and about 40° C. In some embodiments, the method may be performed at a temperature between about 2° C. and about 39° C. In some embodiments, the method may be performed at a temperature between about 3° C. and about 38° C. In some embodiments, the method may be performed at a temperature between about 4° C. and about 37° C. In some embodiments, the method may be performed at a temperature between about 5° C. and about 36° C. In some embodiments, the method may be performed at a temperature between about 6° C. and about 35° C. In some embodiments, the method may be performed at a temperature between about 7° C. and about 34° C. In some embodiments, the method may be performed at a temperature between about 8° C. and about 33° C. In some embodiments, the method may be performed at a temperature between about 9° C. and about 32° C. In some embodiments, the method may be performed at a temperature between about 10° C. and about 31° C. In some embodiments, the method may be performed at a temperature between about 11° C. and about 30° C. In some embodiments, the method may be performed at a temperature between about 12° C. and about 29° C. In some embodiments, the method may be performed at a temperature between about 13°C and about 28°C. In some embodiments, the method may be performed at a temperature between about 14°C and about 27°C. In some embodiments, the method may be performed at a temperature between about 15°C and about 26°C. In some embodiments, the method may be performed at a temperature between about 16°C and about 25°C. In some embodiments, the method may be performed at a temperature between about 17°C and about 24°C. In some embodiments, the method may be performed at a temperature between about 18°C ​​and about 23°C. In some embodiments, the method may be performed at a temperature between about 19°C and about 22°C. In some embodiments, the method may be performed at a temperature between about 20°C and about 21°C.In some embodiments, the temperatures disclosed above may be applicable to one or more steps of the methods disclosed herein, including, but not limited to, growing cells, harvesting cells, lysing the cells to produce the cell lysate, passing the cell lysate through the filter to produce the cell extract, contacting the cell extract with a nucleic acid encoding the polypeptide to produce the reaction mixture, and incubating the reaction mixture to produce the polypeptide.

[0082] Characterization of cell extracts clarified via filtration The properties of the clarified cell extracts according to the methods disclosed herein can be characterized. For example, the biological activity of the cell extracts can be characterized by the volumetric yield of a batch reaction that produces a reporter protein, such as green fluorescent protein (GFP) or luciferase.

[0083] In some embodiments, the cell extract clarified according to the method disclosed herein can be part of a CFPS reaction mixture. A reaction mixture is complete when it contains all the reagents necessary to carry out a cell-free polypeptide synthesis reaction. The components of the reaction mixture can be stored separately in separate containers, each containing one or more of the above components. The components of the reaction mixture can be packaged separately for commercialization, and a commercially available kit can contain one or more of the reaction components for the reaction mixture. The cell extract clarified according to the method disclosed herein can be packaged separately as a component of a CFPS reaction mixture kit.

[0084] System for clarification of cell lysates via filtration - Patent application In some embodiments, a system for cell-free polypeptide synthesis (CFPS) is disclosed herein. In some embodiments, the system can be used for the preparation of clarified cell extracts for CFPS. In some embodiments, the system can work in batch, continuous, or semi-continuous mode. In some embodiments, the system can be contained within a closed sterile environment. In some embodiments, the system can perform the entire clarification process under sterile conditions.

[0085] In some embodiments, a system for CFPS is disclosed herein. In some embodiments, the system may include one or more of the devices disclosed herein. In some embodiments, the system may include a filter disclosed herein. In some embodiments, the filter may receive a cell lysate and clarify the cell lysate via filtration to generate a cell extract for CFPS. In some embodiments, the system may include a pump. In some embodiments, the pump may be configured to control the flow and / or pressure of the cell lysate within the system. In some embodiments, the filter may include at least one, two, or more of the filters disclosed herein. For example, in some embodiments, the filter may include a medium including pores configured to remove particles having a size greater than about 0.4 microns, about 0.2 microns, or about 0.1 microns from the cell lysate. In some embodiments, the filter may include a first filter and a second filter. In some embodiments, the first filter may include a depth filter. In some embodiments, the depth filter may include a lenticular filter, a deep bed filter, a pad filter, a panel filter, a cartridge type depth filter, or a combination thereof. In some embodiments, the second filter may include a surface filter. In some embodiments, the second filter may include a depth filter. In some embodiments, the second filter may include a filter that includes a sterilizing grade membrane. In some embodiments, the system may include other devices disclosed herein, including but not limited to a cell culture vessel, a cell harvesting device, a cell lysis device, a device for CFPS, or a polypeptide isolation device.

[0086] In some embodiments, the CFPS system may include a device used in the CFPS method disclosed herein. In some embodiments, the system may include a filter disclosed herein for clarification of cell lysate. In some embodiments, the filter may be configured to receive cell lysate. In some embodiments, the filter may include at least two filters disclosed herein. In some embodiments, the at least two filters may be in fluid communication with each other. In some embodiments, the filter may include a depth filter disclosed herein. In some embodiments, the system may include a filter disclosed herein that retains particles having a size greater than about 0.5 microns. In some embodiments, the system may include a first filter disclosed herein that retains particles having a size greater than about 15 microns. In some embodiments, the system may include a first filter disclosed herein that retains particles having a size between about 0.5 microns and about 15 microns. In some embodiments, the system may include a second filter disclosed herein that retains particles having a size greater than about 0.5 microns. In some embodiments, the system may include a second filter as disclosed herein that retains particles having a size between about 0.2 microns and about 0.5 microns. In some embodiments, the system may include a filter with a sterilizing grade membrane as disclosed herein. In some embodiments, the filter with a sterilizing grade membrane may retain particles having a size greater than about 0.1 microns. In some embodiments, the filter with a sterilizing grade membrane may retain particles having a size greater than about 0.2 microns. In some embodiments, the filter with a sterilizing grade membrane may retain particles having a size greater than about 0.4 microns.

[0087] In some embodiments, the system may further include a bioreactor. In some embodiments, the bioreactor may include cells disclosed herein that are used to prepare cell extracts for CFPS. In some embodiments, the cells may include cultured cells disclosed herein. In some embodiments, the cells may include at least two types of cells. In some embodiments, the cells may include cell cultures disclosed herein. In some embodiments, the bioreactor may include a batch bioreactor, a fed-batch bioreactor, a continuous bioreactor, or a combination thereof. In some embodiments, the continuous bioreactor may include a continuous perfusion fermentation bioreactor. In some embodiments, the bioreactor may include any suitable material. In some embodiments, the material may include stainless steel, plastic, or a combination thereof. In some embodiments, the bioreactor may be of any suitable size. In some embodiments, the bioreactor may include a single-use bioreactor. In some embodiments, the single-use bioreactor may be in the form of a flexible, collapsible bag.

[0088] In some embodiments, the system may include a cell lysis apparatus as disclosed herein. In some embodiments, the cell lysis process as disclosed herein may be performed in the bioreactor. In some embodiments, the system may include a shearing or sonication device used to disrupt the cell membranes to generate the cell lysate. In some embodiments, the bioreactor may be in fluid communication with another component of the system. In some embodiments, fluid may be recirculated from the bioreactor to a cell lysis device. In some embodiments, fluid may be recirculated from the bioreactor to a homogenizer. In some embodiments, the bioreactor may be in fluid communication with the filter. In some embodiments, cell lysate may flow from the bioreactor to the filter.

[0089] In some embodiments, the bioreactor may be used only for cell culture and not for the cell lysis process, which is carried out in a separate cell lysis apparatus (e.g., cell lysis vessel). In some embodiments, the cell lysis device disclosed herein may be in fluid communication with a filter disclosed herein. In some embodiments, the cell lysis vessel may include a vessel suitable for the cell lysis process disclosed herein. In some embodiments, the cell lysis vessel may be selected based on the volume of the cell culture, the volume of cells harvested, or the volume of cells resuspended to be used in the CFPS method disclosed herein. In some embodiments, the cell lysis vessel may include a tank, a test tube, a beaker, a flask, a multi-well plate, or a combination thereof. In some embodiments, the cell lysis device may include a homogenizer, a sonicator, a heat exchanger, a nitrogen cavitation device, a freeze-thaw device, a sonic lysis device, a bead-based disruption system, a syringe, or a combination thereof. In some embodiments, the lysis device may be operably linked to the lysis vessel to generate a cell lysate.

[0090] In some embodiments, the system may include a device configured to concentrate the cell culture used for preparation of the CFPS disclosed herein. In some embodiments, the device for concentrating the cell culture may include a centrifuge, a tangential flow filtration device, a hollow fiber filtration apparatus, a membrane separation device, or a combination thereof. In some embodiments, the membrane separation device may include a woven membrane, a nonwoven membrane, an electrospun membrane, a hollow fiber membrane, a rolled sheet membrane, a flat sheet membrane, or a combination thereof.

[0091] In some embodiments, the system may include a reservoir. In some embodiments, the reservoir may include a suspension medium configured to provide the medium to a cell lysis device of the system. In some embodiments, the reservoir may include a suspension medium configured to provide the medium to a device for resuspending cells within a lysis device. In some embodiments, the reservoir may include a suspension medium configured to provide the medium to a cell lysis device disclosed herein. In some embodiments, a device for resuspending cells within the lysis device may include a device for mixing the cells.

[0092] In some embodiments, one component of the system disclosed herein may include an inlet. In some embodiments, one component of the system disclosed herein may include an outlet. In some embodiments, one component of the system may be in fluid communication with another component of the system. In some embodiments, one component of the system may be in fluid communication with at least another component of the system. In some embodiments, two components of the system disclosed herein may both include an inlet and an outlet and may be in fluid communication with each other such that flow can occur from the outlet of one component to the inlet of the other component.

[0093] In some embodiments, the system may include a post-clarification device for processing the clarified cell extract. In some embodiments, the system may include an incubator for performing a run-off reaction. In some embodiments, the system may include an incubator for verifying the sterility of the clarified cell extract. In some embodiments, the system may include a membrane device. In some embodiments, the membrane device may be used for desalting or dialysis. In some embodiments, the membrane device may include a tangential flow filtration (TFF) device, a hollow fiber, a dialysis membrane, or a combination thereof.

[0094] In some embodiments, the systems disclosed herein may be configured to deliver the clarified cell extract to a reactor vessel for CFPS. In some embodiments, the systems may be configured to flash freeze or freeze-dry the clarified cell extract. In some embodiments, the systems may be configured to store the clarified cell extract. In some embodiments, the systems may be configured to store the clarified cell extract in liquid nitrogen. In some embodiments, the system maintains the clarified cell extract at a temperature between about -200°C and 25°C, e.g., about -200°C, -199°C, -198°C, -197°C, -196°C, -195°C, -194°C, -193°C, -192°C, -191°C, -190°C, -189°C, -188°C, -187°C, -186°C, -185°C, -184°C, -183°C, -182°C, -181°C, -180°C, -179°C, - 178℃, -177℃, -176℃, -175℃, -174℃, -173℃, -172℃, -171℃, -170℃, -169℃, -168℃, -167℃, -166℃, -165℃, -164℃, -163℃, -162℃, -161℃, -160℃, -159℃, -158℃, -157℃, -156℃, -155℃, -154℃, -153℃, -152℃, -151℃, -150℃, -149℃, -148℃, -147℃, -146℃, -145℃, -144℃, -143℃, -142℃, -141℃, -140℃, -139℃, -138℃, -137℃, -136℃, -135℃, -134℃, -133℃, -132℃, -131℃, -130℃, -129℃, -128℃, -127℃, -126℃, -125℃, -124℃, -123℃, -122℃, -121℃, -120℃, -119℃, -118℃, -117℃, -116℃, -115℃ ,-114℃,-113℃,-112℃,-111℃,-110℃,-109℃,-108℃,-107℃,-106℃,-105℃,-104℃,-103℃,-102℃,-101℃,-100℃,-99℃,-98℃,-97℃,-96℃,-95℃,-94℃,-93℃,-92℃,-91℃,-90℃,-89℃,-88℃,-87℃,-86℃,-85℃,-84℃,-83℃,-82℃,-81℃,-80℃,-79℃, -78℃, -77℃, -76℃, -75℃, -74℃, -73℃, -72℃, -71℃, -70℃, -69℃, -68℃, -67℃, -66℃, -65℃, -64℃, -63℃, -62℃, -61℃, -60℃, -59℃, -58℃, -57℃, -56℃, -55℃, -54 °C, -53°C, -52°C, -51°C, -50°C, -49°C, -48°C, -47°C, -46°C, -45°C, -44°C, -43°C, -42°C, -41°C, -40°C, -39°C, -38°C, -37°C, -36°C, -35°C, -34°C, -33°C, -32°C, -31°C, -30°C, -29°C, - The liquid may be configured for storage at 28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C, or any temperature therebetween. In some embodiments, the system may include an instrument for isolating or extracting a polypeptide generated via CFPS.

[0095] In some embodiments, the components of the system disclosed herein may utilize a process equipment unit. In some embodiments, the process equipment unit may include pumps, valves, sensors, or device holders for controlling the flow of fluids including cell lysates and cell extracts, or combinations thereof. In some embodiments, the pumps may include vacuum pumps or peristaltic pumps. In some embodiments, the system may be configured for continuous fluid communication between two or more components of the system. In some embodiments, the system may be configured such that fluids may flow directly through each component of the system without interruption. In some embodiments, the system may include one or more selected from the group including valves, sensors, detectors, surge tanks, and equipment for changing any in-line solutions so that the flow of fluids inside the system may be temporarily interrupted to replace or remove certain unit operations.

[0096] In some embodiments, the system may include sensors and / or probes for controlling and / or monitoring process parameters of the system. In some embodiments, the process parameters may include temperature, pressure, pH, conductivity, dissolved oxygen (DO), dissolved carbon dioxide, mixing rate, flow rate, any other product parameter, or combinations thereof. In some embodiments, the sensor may include an optical sensor. In some embodiments, the system may be configured such that process control is achieved in a manner that does not compromise the sterility of the system. In some embodiments, the sensor and / or probe may be connected to a sensor electronics module such that the output of the sensor and / or probe is sent to a terminal board and / or relay box. In some embodiments, the results of the sensing operation may be input into a computer-implemented control system for calculation and control of various parameters, including but not limited to temperature, weight / volume measurements, and purity, as well as for display and user interface. In some embodiments, the control system may include one or more selected from an electronic system, a mechanical system, a pneumatic system to control at least one process parameter. It will be apparent to one of ordinary skill in the art that the control system may perform other functions and that the embodiments disclosed herein do not limit the control system in any way to any particular function or set of functions.

[0097] The following examples are presented to more clearly illustrate to one of ordinary skill in the art the principles and practice of the embodiments disclosed herein, and should not be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are by weight. EXAMPLES

[0098] Working Example The following examples are presented for the purpose of illustrating various embodiments of the present disclosure, and are not meant to limit the present disclosure in any manner. The examples, together with the methods described herein, are representative of currently preferred embodiments, are illustrative, and are not intended as limitations on the scope of the present disclosure. Modifications therein and other uses encompassed within the spirit of the present disclosure, as defined by the scope of the claims, will occur to those skilled in the art.

[0099] Example 1: Cell-free polypeptide synthesis using cell lysate clarified via filtration This example illustrates a method for preparing a crude cell extract clarified via filtration instead of the traditional method of clarification via centrifugation. This example clearly shows that the clarification method via filtration results in substantial yields of protein produced from a cell-free synthesis reaction.

[0100] Cells were cultured, harvested, and lysed according to conventional methods as illustrated in FIG. 1. Cell-free polypeptide synthesis results were compared between cell extracts clarified via centrifugation and clarified via filtration. The cell extracts were clarified via centrifugation to produce E. coli extracts as previously reported in the E. coli extract preparation protocol. The cell extracts clarified via filtration were prepared according to the process illustrated in FIG. 2. Briefly, a peristaltic pump was used to drive the flow of the cell extract through the filtration media. The homogenized cell lysate was filtered through a first depth filter (15 μm to 0.5 μm) to produce a first clarified cell lysate. The first clarified cell lysate was then filtered through a second depth filter (0.5 μm to 0.2 μm) to produce a second clarified cell lysate. Finally, the second clarified cell lysate was filtered through a sterile filter (0.2 μm) to generate a clarified cell extract for the cell-free polypeptide synthesis reaction.

[0101] The cell extracts clarified via centrifugation and via filtration were then used for cell-free polypeptide synthesis based on the process illustrated in Figure 3. A DNA template encoding superfolder green fluorescent protein (sfGFP) was added to both the cell extracts clarified via centrifugation and via filtration. The sfGFP DNA contained a T7 promoter and a strong ribosome binding site at the 5' end of the coding sequence of sfGFP, and a T7 terminator at the 3' end of the coding sequence of sfGFP. The T7 promoter sequence was SEQ ID NO:1, and the T7 terminator sequence was SEQ ID NO:2. These regulatory elements ensured high levels of transcription and translation within the complex extract environment.

[0102] As shown in FIG. 4, protein yields from cell extracts clarified via filtration were comparable to those from cell extracts clarified via centrifugation, as clearly demonstrated by the synthesis of sfGFP.

[0103] Example 2: Effect of filter pore size on protein yield of CFPS This example illustrates the effect of filter pore size on the cell extract clarified via filtration and protein produced from CFPS using the filtered cell extract.

[0104] Filters with a range of pore sizes, 12-10 microns, 5 microns, 1.2 microns, 3 microns / 0.2 microns, and 0.2 microns, as illustrated in FIG. 5 and listed in Table 1, were used to filter the cell lysates and evaluate their effect on the clarified cell extract activity and overall tolerability of CFPS. When compared to the current state of the art method for clarification of cell lysates via centrifugation, filtration caused a decrease in cell-free polypeptide synthesis yield, but was still tolerable. The cell extract activity was related to its host cell protein concentration and optical density. As the filter pore size used in the filtration process decreased, the host cell protein concentration also decreased (lines displayed as % relative [protein] (relative protein percentage)). Table 1 [Table 1] Table 1: Protein concentration and OD under different clarification methods. 600 , and the relative protein percentage (Relative [Protein] (%)) values The results, as listed in Table 1, showed that the filters captured host cell proteins, some of which could be utilized for CFPS. Similarly, the optical density readings (OD 600 ) also decreased with decreasing pore size (OD 600 (line labeled with ). This indicates further and better clarification. These two variables, extract protein concentration and degree of extract clarification, need to be balanced to produce a fully functional cell extract. Comparison of the activity of cell extracts prepared using 3 micron / 0.2 micron filters and 0.2 micron filters showed that depth filtration trains down to 0.2 micron pore size help retain some of those host cell proteins useful for CFPS while achieving similar levels of clarification.

[0105] Example 3: Effect of filter pore size on throughput and mean flow rate for CFPS This example illustrates the effect of filter pore size on the parameters of throughput and average flow rate during the lysate clarification process.

[0106] Throughput (i.e., volume of lysate filtered per filter surface area) and average filtrate flow rate (i.e., volume of lysate filtered per filter surface area per time) are two parameters of the filtration-mediated clarification process of cell lysate that could be used to optimize the filtration process. The following filters were used: 12-10 micron, 1.2 micron, 3 micron / 0.2 micron, 0.2 micron.

[0107] As illustrated in FIG. 6 and listed in Table 2, the throughput and average flow rates were measured in a series of filtration processes using various filter pore sizes. Table 2 [Table 2] Table 2: Throughput and average flow rates for clarification methods using filters of different pore sizes. The driving force used during the filtration process was centrifugal force as opposed to pressure, which was monitored. This may explain the discrepancy between the throughput of the 12-10 micron and 5 micron filter trains, since the 12-10 micron filters would be expected to have a greater or similar throughput to the 5 micron filters given their relatively large pore size. The average flow rate decreased as the pore size of the filters decreased, as would be expected. The throughput of the 3 micron / 0.2 micron depth filter was greater than that of the 1.2 micron filter, despite the fact that the 3 micron / 0.2 micron filter ultimately had the ability to extrude through the smaller pore size and produce a better clarified extract. The 3 micron / 0.2 micron depth filter also had a greater throughput than the 0.2 micron filter alone. This indicated that depth filters or filter trains with successively smaller holes could not only produce a more productive extract with a similar degree of clarification, but also a more efficient process that required less time or filter area, thus lowering operating costs.

[0108] Example 4: Effect of Filtration Train on CFPS This example illustrates the effect of filter type and filter pore size on CFPS.

[0109] To evaluate depth filtration trains, various depth filters, membrane filters, and combinations thereof into a filter train were used for clarification of cell lysates for CFPS to evaluate their efficiency in the extract clarification process. The filters used in this example included the depth filters 0.2-3.0 microns, 0.4-15.0 microns, 6.0-30.0 microns, 0.2-0.8 microns, 0.1-0.3 microns, 0.2-0.5 microns, as well as several membrane filters with PES or PES hybrid media.

[0110] For the purposes of lysate clarification, throughput and average constant filtrate flow rate were considered appropriate metrics. In optimizing these metrics, a train of two 0.4-15 micron depth filters followed by a 0.2-0.5 micron depth filter was found to have the highest throughput and the highest average constant filtrate flow rate. This train was found to be better than a 2× 0.2-0.8 micron train, a 2× 0.2-3.0 micron train to a 0.1-0.3 micron train, and a 6-30 micron to 0.2-3.0 micron train. Various membrane filters were tested along with the selected depth filtration train, and a PES filter was found to have the highest throughput. That throughput metric was rigorously examined for scale-up purposes.

[0111] A modified version of the final filter train was used to confirm the retained extract activity after the lysate clarification process. The modified filter train included one 0.4-15 micron filter, followed by 0.2-0.5 micron and 0.2 micron PES membrane filters. Protein yields of cell-free extracts filtered using the modified filter train, as discussed in Example 1 above, are illustrated in FIG. 4.

[0112] Although preferred embodiments of the present disclosure have been shown and described above, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various modifications to the embodiments of the disclosure described herein can be used in implementing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for cell-free polypeptide synthesis, the method comprising: (a) lysing cells to generate a cell lysate; (b) filtering the cell lysate to produce a cell extract; (c) contacting the cell extract with a nucleic acid encoding a polypeptide to produce a reaction mixture; and (d) incubating the reaction mixture to produce the polypeptide; The method includes:

2. 10. The method of claim 1, wherein any one or more of the cell lysate, the cell extract, or the reaction mixture is not subjected to clarification via centrifugation.

3. 2. The method of claim 1, further comprising flocculating, precipitating, or both flocculating and precipitating the cell lysate prior to the passing through the filter.

4. The method of claim 1 , wherein the filter comprises a surface filter or a depth filter.

5. 2. The method of claim 1, wherein the passing step comprises one or more selected from the group including cross-flow filtration, belt filtration, microfiltration, ultrafiltration, microporous filtration, vacuum drum filtration, sieving, membrane filtration, sand filtration, and screen filtration.

6. The method of claim 1 , wherein the filter comprises pores that exclude particles having a size between 0.1 microns and 100 microns.

7. 10. The method of claim 1, wherein the filter comprises at least two filters arranged in series or in parallel, or the filter comprises at least two filters arranged in series with decreasing pore sizes.

8. The filter comprises: (a) a first filter containing pores that exclude particles having a size between 0.5 microns and 15 microns; (b) a second filter containing pores that exclude particles having a size between 0.2 microns and 0.5 microns; and (c) a third filter containing pores that exclude particles having a size of 0.2 microns; The method of claim 7, comprising:

9. The method of claim 1 further comprising obtaining the cells from a cell culture.

10. 10. The method of claim 9, wherein the obtaining step comprises subjecting the cell culture to one or more steps selected from the group comprising centrifugation, tangential flow filtration, membrane separation, and combinations thereof.

11. 2. The method of claim 1, wherein the lysing step comprises one or more selected from the group comprising sonication, homogenization, nitrogen cavitation, freeze-thaw, syringing, chemical lysis, enzymatic lysis, osmotic lysis, French press lysis, bead crushing, and combinations thereof.

12. The method of claim 1, further comprising freezing or lyophilizing the cell extract.

13. 10. The method of claim 9, wherein the cell comprises a transgenic cell, a mammalian cell, a bacterial cell, a plant cell, a yeast cell, an insect cell, a fungal cell, an algal cell, or a combination thereof.

14. The method of claim 1 , wherein the nucleic acid comprises DNA comprising a promoter and a terminator located downstream of the 3′ end of the open reading frame.

15. The method of claim 14, wherein the promoter comprises a T7 promoter comprising a sequence having at least 80% sequence identity with 5'-TAATACGACTCACTATAGG-3' (SEQ ID NO: 1), and the terminator comprises a T7 terminator comprising a sequence having at least 80% sequence identity with 5'-CTAGCATAACCCCTTGGGGCCTCTAAAACGGGTCTTGAGGGGTTTTTTTG-3' (SEQ ID NO: 2).

16. 10. The method of claim 1, wherein the reaction mixture comprises amino acids, nucleotides, salts, cofactors, an energy source, a translation template, or a combination thereof.

17. 17. The method of claim 16, wherein the energy source comprises one or more of glutamate, pyruvate or glucose and is present at a concentration between 10 mM and 400 mM.

18. 17. The method of claim 16, wherein the salt comprises potassium at a concentration between 50 mM and 500 mM, or magnesium at a concentration between 1 mM and 30 mM, or ammonium at a concentration between 1 mM and 400 mM.

19. The method of claim 1 , further comprising the step of isolating said polypeptide from said reaction mixture.

20. 17. The method of claim 16, wherein the nucleotide comprises a nucleoside monophosphate (NMP), a nucleoside diphosphate (NDP), a nucleoside triphosphate (NTP), or a combination thereof.

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