Plasmid purification methods

Abstract

The presently claimed and described technology provides chromatography processes for purifying plasmid DNA (pDNA) using elution buffers containing at least one basic amino acid and / or at least one denaturant.

Description

Cytiva ref. 2024-24061-P-WOPLASMID PURIFICATION METHODSBACKGROUND

[0001] The global demand for biopharmaceuticals continues to increase and high-quality plasmid DNA (pDNA) is key modality involved the production of mRNA and viral vectors. Chromatographic purification of plasmids by anion exchange chromatography (AEX) is common, however elution yield from AEX can be modest and go below 60%.

[0002] A need exists for plasmid purification methods that can result in increased elution yields.BRIEF SUMMARY

[0003] One aspect of the disclosure is a process for purifying plasmid DNA (pDNA) comprising passing a sample comprising pDNA through a chromatography device, wherein at least a portion of the pDNA binds to the chromatography device; eluting the pDNA from the chromatography device with an elution buffer, wherein the elution buffer comprises at least about 0.05M of at least one basic amino acid or at least about 0.05M of at least one denaturant, and wherein recovery of the purified pDNA is at least about 60%.

[0004] One aspect of the disclosure is a process for purifying plasmid DNA (pDNA) comprising passing a sample comprising pDNA through a chromatography device, wherein at least a portion of the pDNA binds to the chromatography device; eluting the pDNA from the chromatography device with an elution buffer, wherein the elution buffer comprises at least about 0.05M of at least one basic amino acid and at least about 0.0.09M of at least one denaturant, and wherein recovery of the purified pDNA is at least about 60%.

[0005] In an aspect, the elution buffer comprises between about 0.05M and about IM of the at least one basic amino acid, alternatively about 0.075M of at least one basic amino acid, alternatively about 0.1M, alternatively about 0.125M, alternatively about 0.15M, alternatively about 0.2M, alternatively about 0.25M, alternatively about 0.3M, alternatively about 0.35M, alternatively about 0.4M, alternatively about 0.45M, alternatively about 0.5M, alternatively about 0.55M, alternatively about 0.6M, alternatively about 0.65M, alternatively about 0.7M, alternatively about 0.75M, alternatively about 0.8M, alternatively about 0.85M, alternatively about 0.9M, or alternatively about 0.95M.Cytiva ref. 2024-24061-P-WO

[0006] In an aspect, the elution buffer comprises between about 0.05M and about IM of the at least one denaturant, alternatively about 0.075M of at least one denaturant, alternatively about 0.1M, alternatively about 0.125M, alternatively about 0.15M, alternatively about 0.2M, alternatively about 0.25M, alternatively about 0.3M, alternatively about 0.35M, alternatively about 0.4M, alternatively about 0.45M, alternatively about 0.5M, alternatively about 0.55M, alternatively about 0.6M, alternatively about 0.65M, alternatively about 0.7M, alternatively about 0.75M, alternatively about 0.8M, alternatively about 0.85M, alternatively about 0.9M, or alternatively about 0.95M.

[0007] In an aspect, the at least one basic amino acid is selected from the group consisting of arginine, histidine, and lysine.

[0008] In an aspect, the denaturant is urea or guanidine.

[0009]

[0010] In an aspect, the elution buffer comprises about 0. IM arginine and 0. IM urea, alternatively about 0.2M arginine and about 0.2M urea, alternatively about 0.25M arginine and about 0.5M urea, or alternatively about 0.5M arginine and about IM urea.

[0011] In an aspect, the elution buffer comprises about 0.1M arginine and 0.1M guanidine, alternatively about 0.25M arginine and about 0.25M guanidine, or alternatively about 0.25M arginine and about 0.5M guanidine.

[0012] In an aspect, the elution buffer comprises about 0. IM histidine and 0. IM urea, alternatively about 0.125M histidine and about 0.25M urea, alternatively about 0.25M histidine and about 0.5M urea, or alternatively about 0.5M histidine and about IM urea.

[0013] In an aspect, the elution buffer comprises at least one salt in a concentration of between about 25mM and about lOOOmM.

[0014] In an aspect, the salt is selected from the group consisting of ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, and combinations thereof.

[0015] In an aspect, the elution buffer comprises at least one buffering agent and / or a chelating agent.

[0016] In an aspect, the buffering agent and / or chelating agent is selected from the group consisting of Tri s(hydroxymethyl)aminom ethane (TRIS), 2-(N-Morpholino)ethanesulfonic acid (MES), 4-(2-Hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES), 3-(N-Cytiva ref. 2024-24061-P-WOMorpholino)propanesulfonic acid (MOPS), N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid (TAPSO), N-Bis(2 -hydroxy ethyl)-2-aminoethanesulfonic acid (BES), Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), Ethylenediaminetetraacetic acid (EDTA), and combinations thereof.

[0017] In an aspect, the recovery of the purified pDNA is between about 60% and about 100%, alternatively about 61%, alternatively about 62%, alternatively about 63%, alternatively about 64%, alternatively about 65%, alternatively about 66%, alternatively about 67%, alternatively about 68%, alternatively about 69%, alternatively about 70%, alternatively about 71%, alternatively about 72%, alternatively about 73%, alternatively about 74%, alternatively about 75%, alternatively at about 76%, alternatively at about 77%, alternatively at about 78%, alternatively at about 79%, alternatively at about 80%, alternatively at about 81%, alternatively at about 82%, alternatively at about 83%, alternatively at about 84%, alternatively at about 85%, alternatively at about 86%, alternatively at about 87%, alternatively at about 88%, alternatively at about 89%, alternatively at about 90%, alternatively at about 91%, alternatively at about 92%, alternatively at about 93%, alternatively at about 94%, alternatively at about 95%, alternatively at about 96%, alternatively at about 97%, alternatively at about 98%, or alternatively at about 99%.

[0018] In an aspect, the chromatography device comprises an ion exchange chromatography layer, resin or membrane, wherein the portion of the pDNA binds to the ion exchange layer, resin, or membrane.

[0019] In an aspect, the ion exchange chromatography layer, resin, or membrane is an anion exchange chromatography layer, resin, or membrane.

[0020] In an aspect, the sample comprising pDNA is processed prior to passing it through the chromatography device.

[0021] In an aspect, the sample comprising pDNA is derived from bacterial culture, cells, cell culture, cell lysate, recombinant plasmids, genetically modified plasmids, or synthetic plasmids.

[0022] In an aspect, the process is used for large scale purification of pDNA.

[0023] One aspect of the disclosure is a kit for purifying plasmid DNA (pDNA) comprising a chromatography device, an elution buffer, wherein the elution buffer comprises at least about 0.05M of at least one basic amino acid and / or at least about 0.05M of at least one denaturant, and at least one salt in a concentration of between about 25mM and about 1000 mM, and instructions for use.Cytiva ref. 2024-24061-P-WO

[0024] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Various aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0026] FIG 1 shows a process overview for pDNA.

[0027] FIG 2 is a ~6.3 kbp GFP2 plasmid for mRNA IVT.

[0028] FIG 3 is a chromatogram (UV A280) of purification process using standard conditions (blue) and optimized conditions (yellow) with additives according to an aspect of this disclosure.

[0029] FIG. 4 is a design space for pDNA purification with inclusion different concentrations of arginine / urea additives according to an aspect of this disclosure.DETAILED DESCRIPTIONI. Introduction

[0030] Plasmid DNA (pDNA) is commonly used to express recombinant proteins in various host organisms during molecular biology and protein studies. Advances in cloning technology have increased the demand for numerous small-scale pDNA samples. Preparing pDNA on a small scale often involves parallel purifications using semi -automated or fully automated systems. However, extracting pDNA from prokaryotic cultures can be challenging. The process involves a complex lysis and purification procedure to separate pDNA from genomic DNA. The first step in plasmid purification is lysing the cultured cells to release the DNA. After extraction, the DNA must be clarified to remove any cellular debris. FIG. 1 is an exemplary process for pDNA purification.

[0031] While there are purification methods for preparing pDNA, these methods have low recovery yields, are not automatable, or cannot be scaled up. A need exists for a chromatography process that meets large-scale regulatory manufacturing requirements and is scalable. In some instances, the processes disclosed herein comprise passing a sample comprising pDNA through a chromatography device, wherein at least a portion of the pDNA binds to the chromatography device; eluting the pDNA from the chromatography device with an elution buffer, wherein theCytiva ref. 2024-24061-P-WO elution buffer comprises at least about 0.05M of at least one basic amino acid or at least about 0.05M of at least one denaturant, and wherein recovery of the purified pDNA is at least about 60%. In some instances, the processes disclosed herein comprise passing a sample comprising pDNA through a chromatography device, wherein at least a portion of the pDNA binds to the chromatography device; eluting the pDNA from the chromatography device with an elution buffer, wherein the elution buffer comprises at least about 0.05M of at least one basic amino acid and at least about 0.05M of at least one denaturant, and wherein recovery of the purified pDNA is at least about 60%. As shown in FIG. 1, the process disclosed herein may be substituted for the AIEX step.II. Definitions

[0032] 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 to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0033] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0034] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0035] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments or aspects does not imply that other embodiments or aspects are not useful and is not intended to exclude other embodiments or aspects from the scope of the invention.

[0036] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.Cytiva ref. 2024-24061-P-WO

[0037] By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. "Consisting essentially of' means including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0038] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0039] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0040] Reference throughout this specification to "one aspect," "an aspect," "certain aspects," or "some aspects," "one embodiment," "an embodiment," "certain embodiment," or "some embodiment," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features,Cytiva ref. 2024-24061-P-WO configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0041] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0042] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0043] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0044] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.Cytiva ref. 2024-24061-P-WO

[0045] The invention is defined in the claims. However, below is a non-exhaustive listing of nonlimiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. Plasmid DNA (pDNA) purification processes

[0046] Plasmid DNA or pDNA refers to small, circular, double-stranded DNA molecules that are separate from the chromosomal DNA found in the cells of bacteria, yeast, and some other organisms. Unlike chromosomal DNA, plasmids are not essential for the survival of the host organism, but they often carry genes that can provide advantageous traits, such as antibiotic resistance or the ability to metabolize unusual substances.

[0047] Plasmids can replicate independently of the host's chromosomal DNA, making them valuable tools in genetic engineering and molecular biology for cloning, gene expression, and the production of recombinant proteins.

[0048] Chromatography methods, such as those which employ membranes, resins, or layers, are an effective alternative to conventional beaded sorbents for the purification of pDNA. However, while these chromatography methods have much higher binding capacities for large molecules than beaded sorbents, the elution processes still generate pDNA yields of 40-60%, lower than what is required for biopharmaceutical applications.

[0049] An embodiment of this disclosure includes a process for purifying pDNA comprising passing a sample comprising pDNA through a chromatography device, wherein at least a portion of the pDNA binds to the chromatography device and eluting the pDNA from the chromatography device with an elution buffer. A novel aspect of the disclosure is the use of at least one basic amino acid and / or at least one denaturant in the elution buffer. The inventors have surprisingly and unexpectantly found that the addition of at least 0.05M of a basic amino acid or at least 0.05M of a denaturant in the elution buffer allows for at least a 60% recovery of purified pDNA. In some aspects, the addition of at least 0.05M of a basic amino acid and at least 0.05M of a denaturant in the elution buffer allows for at least a 60% recovery of purified pDNA. As used herein “% recovery” refers to the percentage of the initial amount of pDNA that is successfully retrieved after a purification or processing step. A high % recovery indicates an efficient process with minimal loss of the pDNA, while low % recovery suggests significant losses and potential areas for process improvement. In some aspects, % recovery may be referred to as % yield.Cytiva ref. 2024-24061-P-WO

[0050] In some embodiments, the recovery of the purified pDNA is between about 60% and about 100%, alternatively about 61%, alternatively about 62%, alternatively about 63%, alternatively about 64%, alternatively about 65%, alternatively about 66%, alternatively about 67%, alternatively about 68%, alternatively about 69%, alternatively about 70%, alternatively about 71%, alternatively about 72%, alternatively about 73%, alternatively about 74%, alternatively about 75%, alternatively at about 76%, alternatively at about 77%, alternatively at about 78%, alternatively at about 79%, alternatively at about 80%, alternatively at about 81%, alternatively at about 82%, alternatively at about 83%, alternatively at about 84%, alternatively at about 85%, alternatively at about 86%, alternatively at about 87%, alternatively at about 88%, alternatively at about 89%, alternatively at about 90%, alternatively at about 91%, alternatively at about 92%, alternatively at about 93%, alternatively at about 94%, alternatively at about 95%, alternatively at about 96%, alternatively at about 97%, alternatively at about 98%, or alternatively at about 99%.

[0051] As used herein, “chromatography device” refers to an apparatus used in the process of chromatography. Various chromatography devices are commercially available including the Mustang™ E, Q and S chromatography membranes (Cytiva). The chromatography device may be part of a chromatography system. Various chromatography systems are commercially available including the AKTA avant chromatography system (Cytiva).

[0052] In some aspects, the chromatography device may be an ion exchange chromatography device. Ion exchange chromatography operates on the principle that different ions or charged molecules in a mixture will interact differently with a charged stationary phase (the resin) in the device. The stationary phase consists of ion exchange resins that can either be cationic (negatively charged to attract and bind positively charged ions) or anionic (positively charged to attract and bind negatively charged ions).

[0053] In some embodiments, the chromatography device may include an ion exchange chromatography layer, resin or membrane, wherein at least a portion of the pDNA binds to the ion exchange chromatography layer, resin, or membrane. In another aspect, the ion exchange chromatography layer, resin, or membrane is an anion exchange chromatography layer, resin, or membrane. In this aspect, the negatively charged pDNA molecules bind to the positively charged functional groups on the anion exchange chromatography layer, resin, or membrane.Cytiva ref. 2024-24061-P-WOChromatography devices useful in the disclosed processes are described in U.S. Pub. No. 2023 / 0331773 and U.S. Pat. No. 7094347 each of which is incorporated by reference in its entirety.

[0054] In an aspect, the sample comprising pDNA may be derived from a bacterial culture, cells, cell culture, cell lysate, recombinant plasmids, genetically modified plasmids, and synthetic plasmids. In other aspects the sample may be an environmental sample, clinical sample or food sample. The sample may be a fluid or liquid sample. In some aspects, the sample may be resuspended or reconstituted prior to passing through the chromatography device.

[0055] In some aspects the pDNA is processed prior to purification. Exemplary processing steps are illustrated in FIG. 1 and include, but are not limited to, fermentation, cell concentration, lysis and flocculation lift, depth filtration, tangential flow filtration (TFF) conditioning, and normal flow filtration (NFF). However, these steps are not all required. For example, the inventors surprisingly and unexpectedly discovered that the TFF conditioning (i.e., sample concentration and buffer exchange) can be removed from the pDNA purification workflow while still obtaining high % recovery.

[0056] The bound pDNA is eluted from the chromatography device by using an elution buffer. The elution buffer contains components that compete with the pDNA for binding sites on the chromatography layer, resin, or membrane, for example, causing the pDNA to elute from the chromatography device.

[0057] In an embodiment, the elution buffer comprises between about 0.09M and about IM of at least one basic amino acid. In some embodiments, the elution buffer comprises more than IM of at least one basic amino acid. A basic amino acid is an amino acid that has a side chain (R group) that is positively charged at physiological pH (around 7.4). This positive charge is due to the presence of an extra amino group ( — NEE), which tends to accept a proton (H+). Non-limiting examples of basic amino acids include arginine, histidine, and lysine.

[0058] In some embodiments, the elution buffer comprises about 0.05M and about IM of the at least one basic amino acid, alternatively about 0.075M of at least one basic amino acid, alternatively about 0.1M of at least one basic amino acid, alternatively about 0.125M of at least one basic amino acid, alternatively about 0.15M of at least one basic amino acid, alternatively about 0.2M of at least one basic amino acid, alternatively about 0.25M of at least one basic amino acid, alternatively about 0.3M of at least one basic amino acid, alternatively about 0.35M of atCytiva ref. 2024-24061-P-WO least one basic amino acid, alternatively about 0.4M of at least one basic amino acid, alternatively about 0.45M of at least one basic amino acid, alternatively about 0.5M of at least one basic amino acid, alternatively about 0.55M of at least one basic amino acid, alternatively about 0.6M of at least one basic amino acid, alternatively about 0.65M of at least one basic amino acid, alternatively about 0.7M of at least one basic amino acid, alternatively about 0.75M of at least one basic amino acid, alternatively about 0.8M of at least one basic amino acid, alternatively about 0.85M of at least one basic amino acid, alternatively about 0.9M of at least one basic amino acid, or alternatively about 0.95M of at least one basic amino acid.

[0059] In some aspects, the elution buffer comprises two or more basic amino acids or three or more basic amino acids.

[0060] In an embodiment, the elution buffer comprises between about 0.05M and about IM of at least one denaturant. In an embodiment, the elution buffer comprises more than about IM of at least one denaturant. A denaturant includes substances, such as, but not limited to urea, guanidine, and detergents (e.g., Sodium dodecyl sulfate), which interfere with non-covalent interactions - like hydrogen bonds, hydrophobic interactions, and ionic bonds.

[0061] In some embodiments, the elution buffer comprises about 0.075M of at least one denaturant, alternatively about 0.1M of at least one denaturant, alternatively about 0.125M of at least one denaturant, alternatively about 0.15M of at least one denaturant, alternatively about 0.2M of at least one denaturant, alternatively about 0.25M of at least one denaturant, alternatively about 0.3M of at least one denaturant, alternatively about 0.35M of at least one denaturant, alternatively about 0.4M of at least one denaturant, alternatively about 0.45M of at least one denaturant, alternatively about 0.5M of at least one denaturant, alternatively about 0.55M of at least one denaturant, alternatively about 0.6M of at least one denaturant, alternatively about 0.65M of at least one denaturant, alternatively about 0.7M of at least one denaturant, alternatively about 0.75M of at least one denaturant, alternatively about 0.8M of at least one denaturant, alternatively about 0.85M of at least one denaturant, alternatively about 0.9M of at least one denaturant, or alternatively about 0.95M of at least one denaturant.

[0062] In some aspects, the elution buffer comprises two or more denaturants or three or more denaturants.Cytiva ref. 2024-24061-P-WO

[0063] In some aspects, the elution buffer comprises at least one basic amino acid and at least one denaturant. For example, in one embodiment, the elution buffer comprises about 0.1M arginine and 0. IM urea, alternatively about 0.2M arginine and about 0.2M urea, alternatively about 0.25M arginine and about 0.5M urea, or alternatively about 0.5M arginine and about IM urea.

[0064] In another embodiment, the elution buffer comprises about 0.1M arginine and 0.1M guanidine, alternatively about 0.25M arginine and about 0.25M guanidine, or alternatively about 0.25M arginine and about 0.5M guanidine.

[0065] In another embodiment, the elution buffer comprises about 0.1M histidine and 0.1M urea, alternatively about 0.125M histidine and about 0.25M urea, alternatively about 0.25M histidine and about 0.5M urea, or alternatively about 0.5M histidine and about IM urea.

[0066] The elution buffer may also comprise other suitable components including salts, buffering agents, chelating agents, such as, Tri s(hydroxymethyl)aminom ethane (TRIS), 2-(N- Morpholino)ethanesulfonic acid (MES), 4-(2-Hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid (TAPSO), N-Bis(2 -hydroxy ethyl)-2-aminoethanesulfonic acid (BES), Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), Ethylenediaminetetraacetic acid (EDTA), ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, or ammonium sulfate.

[0067] In some aspects, the elution buffer may comprise a salt in a concentration of about IM or lower, alternatively in a concentration of about 750 mM or lower, alternatively in a concentration of about 500 mM or lower, alternatively in a concentration of about 250 mM or lower, alternatively in a concentration of about 150 mM or lower, alternatively in a concentration of about 125 mM or lower, alternatively in a concentration of about 100 mM or lower, alternatively in a concentration of about 75 mM or lower, alternatively in a concentration of about 50 mM or lower, or alternatively in a concentration of about 25 mM or lower. In some aspects, the elution buffer may comprise a salt in a concentration of between about 25mM and about lOOOmM. In some aspects, the salt may be ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, or combinations thereof.Cytiva ref. 2024-24061-P-WO

[0068] In some aspects, the process may be used for large scale pDNA purification. In an embodiment, the large scale pDNA purification may include growing large volumes of a sample containing the plasmid of interest, followed by harvesting and lysing the cells to release the DNA. In some embodiments, the sample may be a bacterial cell, such as E. coli. The lysate is then clarified to remove debris, and the plasmid may be purified using the disclosed process. The plasmid may be further purified and / or concentrated. In some aspects, the process may be scaled up using various commercial kits or custom protocols depending on the desired plasmid yield and purity.

[0069] One aspect of the disclosure relates to a kit for purifying plasmid DNA (pDNA) comprising a chromatography device, an elution buffer, wherein the elution buffer comprises at least about 0.09M of at least one basic amino acid and / or at least about 0.09M of at least one denaturant, at least one salt in a concentration of between about 25mM and about 1000 mM, and instructions for use.

[0070] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.

[0071] EXAMPLES

[0072] Exemplary Purification of pDNA Plasmid

[0073] An ~6.3 kbp GFP2 plasmid for mRNA IVT Clone: E. coli DH5a, Plasmid Type: eGFP BBE PNIV201 (Precision Nanosystems Document No.: PPIS010) (FIG. 2) sample was obtained for purification. Purification of the pDNA plasmid was done using control (i.e., conventional buffers) conditions and experimental conditions (i.e., novel elution buffers).

[0074] A chromatography device comprising an anion exchange membrane was primed and cleaned (CIP) using a five (5) membrane volume of IN sodium hydroxide. The device was equilibrated with 10 MV of equilibration buffer (50mM sodium chloride, 25mM Tris pH 8.0, andCytiva ref. 2024-24061-P-WO10 mM EDTA) and was followed by the pDNA load of 10 mL of ~0.2 mg / mL in Tris pH 8.0 buffer at a 1 :2 dilution. The device was then washed with 10 MV equilibration buffer.

[0075] For the control conditions, the device was eluted with 5 MV Control Buffer 1 (50 mM NaCl, 25 mM Tris pH 8.0, 10 mM EDTA) or 5 MV Control Buffer 2 (1 M NaCl, 25 mM Tris pH 8.0, 10 mM EDTA).

[0076] For the experimental conditions, the device was eluted with 5 MV Experimental Buffer containing 50 mM NaCl or 1 M NaCl, 25 mM Tris pH 8.0, 10 mM EDTA and additives listed as listed Table 1.

[0077] Table 1 - Experimental Buffer Compositions

[0078] The inclusion of a basic amino acid or denaturant in the elution buffer resulted in an increase of yield over the standard elution conditions. As shown in FIG. 3, the purification using standard elution conditions (salt gradient) resulted in limited yield (50-70%). The inclusion of 0.5 M Arginine and 1 M Urea in the elution buffer (Experimental Buffer 1) significantly increased the yield (up to 95%) and lowered the conductivity of elution. Tables 2 and 3 and FIG 4 further show that inclusion of a basic amino acid or denaturant in the elution buffer results in an increase in yield over standard conditions. As shown in Table 2, when 0.5 M arginine is added the recovery of the purified pDNA increased from 50% (no basic amino acid or denaturant) to 85% and when 1 M urea is added the recovery of the purified pDNA increased from 50% (no basic amino acid orCytiva ref. 2024-24061-P-WO denaturant) to 80%. The combination of arginine and urea had a synergistic effect resulting in a yield of 93%.

[0079] Table 3 shows that smaller concentrations of basic amino acid or denaturant also have measurable increases. For example, when 0.2 M arginine is added the recovery of the purified pDNA increased from 50% (no basic amino acid or denaturant) to 74% and when 0.2 M urea is added the recovery of the purified pDNA increased from 50% (no basic amino acid or denaturant) to 76%.

[0080] Table 2

[0081] Table 3

[0082] The effect of other basic amino acid (histidine) and denaturants (guanidine) were explored. These also showed a marked improvement in yield of pDNA as compared to the control conditions (Table 4).

[0083] Table 4Cytiva ref. 2024-24061-P-WO

[0084] The results show that pDNA can be eluted from an anion exchange membrane using a basic amino acid or denaturant at high yields even in cases where the concentration of the salt in the elution buffer is low and where the conductivity of the elution buffer is significantly lower than the conductivity of the binding buffer.

[0085] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0086] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Cytiva ref. 2024-24061-P-WOCLAIMSWhat is claimed is:

1. A process for purifying plasmid DNA (pDNA) comprising: passing a sample comprising pDNA through a chromatography device, wherein at least a portion of the pDNA binds to the chromatography device; eluting the pDNA from the chromatography device with an elution buffer, wherein the elution buffer comprises at least about 0.05M of at least one basic amino acid or at least about 0.05M of at least one denaturant, and wherein recovery of the purified pDNA is at least about 60%.

2. The process of claim 1, wherein the elution buffer comprises between about 0.05M and about IM of the at least one basic amino acid, alternatively about 0.075M of at least one basic amino acid, alternatively about 0.1M, alternatively about 0.125M, alternatively about 0.15M, alternatively about 0.2M, alternatively about 0.25M, alternatively about 0.3M, alternatively about 0.35M, alternatively about 0.4M, alternatively about 0.45M, alternatively about 0.5M, alternatively about 0.55M, alternatively about 0.6M, alternatively about 0.65M, alternatively about 0.7M, alternatively about 0.75M, alternatively about 0.8M, alternatively about 0.85M, alternatively about 0.9M, or alternatively about 0.95M.

3. The process of claim 1 or claim 2, wherein the elution buffer comprises between about 0.05M and about IM of the at least one denaturant, alternatively about 0.075M of at least one denaturant, alternatively about 0.1M, alternatively about 0.125M, alternatively about 0.15M, alternatively about 0.2M, alternatively about 0.25M, alternatively about 0.3M, alternatively about 0.35M, alternatively about 0.4M, alternatively about 0.45M, alternatively about 0.5M, alternatively about 0.55M, alternatively about 0.6M, alternatively about 0.65M, alternatively about 0.7M, alternatively about 0.75M, alternatively about 0.8M, alternatively about 0.85M, alternatively about 0.9M, or alternatively about 0.95M.Cytiva ref. 2024-24061-P-WO4. The process of any one of the preceding claims, wherein the at least one basic amino acid is selected from the group consisting of arginine, histidine, and lysine.

5. The process of any one of the preceding claims, wherein the denaturant is urea or guanidine.

6. A process for purifying plasmid DNA (pDNA) comprising: passing a sample comprising pDNA through a chromatography device, wherein at least a portion of the pDNA binds to the chromatography device; eluting the pDNA from the chromatography device with an elution buffer, wherein the elution buffer comprises at least about 0.05M of at least one basic amino acid and at least about 0.0.09M of at least one denaturant, and wherein recovery of the purified pDNA is at least about 60%.

7. The process of claim 6, wherein the elution buffer comprises about 0.1M arginine and 0.1M urea, alternatively about 0.2M arginine and about 0.2M urea, alternatively about 0.25M arginine and about 0.5M urea, or alternatively about 0.5M arginine and about IM urea.

8. The process of claim 6, wherein the elution buffer comprises about 0.1M arginine and 0.1M guanidine, alternatively about 0.25M arginine and about 0.25M guanidine, or alternatively about 0.25M arginine and about 0.5M guanidine.

9. The process of claim 6, wherein the elution buffer comprises about 0.1M histidine and 0.1M urea, alternatively about 0.125M histidine and about 0.25M urea, alternatively about 0.25M histidine and about 0.5M urea, or alternatively about 0.5M histidine and about IM urea.Cytiva ref. 2024-24061-P-WO10. The process of any one of the preceding claims, wherein the elution buffer comprises at least one salt in a concentration of between about 25mM and about lOOOmM.

11. The process of claim 10, wherein the salt is selected from the group consisting of ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, magnesium nitrate, sodium citrate, ammonium sulfate, and combinations thereof.

12. The process of any one of the preceding claims, wherein the elution buffer comprises at least one buffering agent and / or a chelating agent.

13. The process of any claim 12, wherein the buffering agent and / or chelating agent is selected from the group consisting of Tri s(hydroxymethyl)aminom ethane (TRIS), 2-(N- Morpholino)ethanesulfonic acid (MES), 4-(2-Hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-Tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid (TAPSO), N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-Acetamido)-2- aminoethanesulfonic acid (ACES), Ethylenediaminetetraacetic acid (EDTA), and combinations thereof.

14. The process of any one of the preceding claims, wherein the recovery of the purified pDNA is between about 60% and about 100%, alternatively about 61%, alternatively about 62%, alternatively about 63%, alternatively about 64%, alternatively about 65%, alternatively about 66%, alternatively about 67%, alternatively about 68%, alternatively about 69%, alternatively about 70%, alternatively about 71%, alternatively about 72%, alternatively about 73%, alternatively about 74%, alternatively about 75%, alternatively at about 76%, alternatively at about 77%, alternatively at about 78%, alternatively at about 79%, alternatively at about 80%, alternatively at about 81%, alternatively at about 82%, alternatively at about 83%, alternatively at about 84%, alternatively at about 85%, alternatively at about 86%, alternatively at about 87%, alternatively at about 88%, alternatively at about 89%, alternatively at about 90%, alternatively19Cytiva ref. 2024-24061-P-WO at about 91%, alternatively at about 92%, alternatively at about 93%, alternatively at about 94%, alternatively at about 95%, alternatively at about 96%, alternatively at about 97%, alternatively at about 98%, or alternatively at about 99%.

15. The process of any one of the preceding claims, wherein the chromatography device comprises an ion exchange chromatography layer, resin or membrane, wherein the portion of the pDNA binds to the ion exchange layer, resin, or membrane.

16. The process of claim 15, wherein the ion exchange chromatography layer, resin, or membrane is an anion exchange chromatography layer, resin, or membrane.

17. The process of any one of the preceding claims, wherein the sample comprising pDNA is processed prior to passing it through the chromatography device.

18. The process of claim 17, wherein the sample comprising pDNA is derived from bacterial culture, cells, cell culture, cell lysate, recombinant plasmids, genetically modified plasmids, or synthetic plasmids.

19. The process of any one of the preceding claims, wherein the process is used for large scale purification of pDNA.

20. A kit for purifying plasmid DNA (pDNA) comprising a chromatography device, an elution buffer, wherein the elution buffer comprises at least about 0.05M of at least one basic amino acid and / or at least about 0.05M of at least one denaturant, and at least one salt in a concentration of between about 25mM and about 1000 mM, and instructions for use.

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