Methods and compositions for enhancing the stability and solubility of fractionated inteins

Abstract

Disclosed herein are protein purification systems and methods for making such systems. Specifically, the invention relates to a method for immobilizing an N-terminal intein segment to a solid support, the method comprising the steps of exposing the N-terminal intein segment to a cognate folding partner under conditions that promote association of the N-terminal intein with the cognate folding partner, immobilizing the N-terminal intein to the solid support, subjecting the N-terminal intein to conditions that disrupt association of the N-terminal intein with the cognate folding partner, and washing the solid support to remove unbound material, thereby immobilizing the N-terminal intein segment to the solid support.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63 / 018,084 filed April 30, 2020, which is incorporated herein by reference in its entirety.

[0002] Statements concerning research funded by the federal government This invention was developed with government support under Grant R21GM126543, awarded by the U.S. National Institutes of Health (NIH). The government reserves certain rights to this invention. [Background technology]

[0003] Inteins are naturally occurring self-splicing protein subdomains that can cleave their own protein subdomain from a larger protein structure while simultaneously joining two adjacent peptide regions ("extines") together to form a natural host protein.

[0004] The ability of inteins to rearrange adjacent peptide bonds and retain activity when fused to a protein other than its conventional extein has led to numerous intein-based biotechnological uses. These biotechnological uses include various types such as protein ligation and activation applications, as well as protein labeling and tracking applications. Split inteins have recently attracted attention for affinity chromatography applications, in which an N-intane ligand, which is one different protein from a specific pair, is recombinantly expressed in standard cell culture techniques (usually in microorganisms) and then immobilized on solid chromatography support media (resins, beads, membranes, etc.). The N-intane ligand is the N-terminal intein (INT N ) includes a segment, and the segment can be qualified, and additionally, INT NThe segments may contain functional groups that support the purification, immobilization, or functionalization of the segments. For use in protein purification, the corresponding C-terminal intein segment "tag" is expressed in fusion with a given target protein and then captured by an immobilized N-intane ligand, thereby facilitating the purification of the target protein by acting as a self-cleaving affinity tag (as described, for example, in U.S. Patent No. 10,066,027 B2). However, for the self-cleaving tag to be applicable, the N-intane ligand must be economically manufactured, purified, and immobilized on a solid substrate in a recombinant system.

[0005] In effect, the overall yield in any conventional protein manufacturing process is fundamentally limited by the total amount of protein produced in cell culture and the percentage of that protein that remains soluble when extracted from cells. However, regardless of how efficiently recombinant proteins are produced in cell culture, only soluble proteins can be recovered and purified by conventional chromatographic techniques. This means that any proteins that form insoluble aggregates upstream during any of the steps of expression, harvesting, lysis, clarification, or filtration will be lost and discarded in the manufacturing process. In some cases, proteins expressed as insoluble aggregates can be recovered and refolded in vitro as part of the purification process, but the necessary refolding processes are difficult to develop and are typically inefficient.

[0006] Standard microbial fermentation techniques can overexpress recombinant N-intane ligands at moderately high expression titers; however, due to the inherent structure or lack thereof of the proteins, the resulting proteins are prone to aggregation, degradation, and are often insoluble when extracted from the protein's cell host. This makes it extremely difficult to construct a reliable and economically viable process for producing N-intane ligands. In fact, the majority of the total protein expressed in fermentation, sometimes more than 90%, appears to be insoluble after cell lysis and is lost during production. The net yield of soluble N-intane ligands obtained from standard E. coli expression is at the level of 10–30 mg of protein per liter of expression culture, which is approximately two orders of magnitude lower than most commercially operating recombinant protein production processes. This directly and proportionally increases the commodity and manufacturing costs for split-intane-mediated affinity chromatography platforms, jeopardizing their commercial viability in its existence.

[0007] In general, solubility has been a common problem of heterologous expression that scientists and engineers have grappled with since the early days of protein engineering, and many potential solutions have been employed with varying degrees of success. These most commonly focus on either promoting proper structural assembly in vivo or harsh chemical refolding treatments to resolubilize aggregates ex vivo. Numerous approaches to promoting proper folding of N-intanes have been attempted in vivo, and these approaches have shown moderate but inconsistent improvements in net soluble recovery in manufacturing (as described, for example, in Millipore's U.S. Patent Application WO2016 / 073228 A1 and German Patent Application US 2019 / 0263856 A1). Even when properly folded and expressed solublely in cell cultures, proteins still appear to be highly susceptible to spontaneous idiosyncratic aggregation in inconsistent and unpredictable amounts, even under the same ex vivo handling conditions. This view is based on the wild-type INT published in literature by another research group (Shah, Eryilmaz et al. 2013). N This is reinforced by structural studies of the segments.

[0008] Therefore, what is needed is a method and composition for heterologous protein expression of fragmented inteins that significantly improves the solubility of the expressed product and its stability in downstream manufacturing processes. [Overview of the Initiative] [Means for solving the problem]

[0009] In accordance with the object(s) of the present invention, as embodied and broadly described herein, the present invention relates in one aspect to a method for stabilizing an N-intane ligand during expression and purification, purifying an N-intane ligand, and immobilizing an N-intane ligand onto a solid support. In particular, a method is disclosed comprising the steps of forming a soluble and stable intein complex via the association of an N-intane ligand with a homologue-binding partner (e.g., a corresponding C-terminal intein segment fused with a homologue-binding partner, either alone or cleavable or non-cleavable), purifying the intein complex, and immobilizing the intein complex onto a solid support. The intein complex can then be subjected to conditions that disrupt the association between the N-intane ligand and the homologue-binding partner, wash the solid support to remove the unbound homologue-binding partner, and allow the N-intane ligand to fold into an active state.

[0010] The homologous binding partner is the C-terminal intein (INT), which induces a structured soluble intein complex by binding to an N-intene ligand. C The ) segment may be included. The N-intei ligand and its homologue binding partner can be co-expressed in vivo or trans (expressed in separate cells) in single cells derived from a single plasmid system or a two-plasmid system and mixed before or during the purification process. Such immobilization can be carried out on a solid support such as a chromatography medium, membrane, or magnetic beads. In one example, the chromatography medium may be a solid chromatography resin backbone.

[0011] When an N-intane ligand is stabilized using a homologous binding partner, the N-intane ligand can then be stabilized by any other INT CThe segments cannot be ligated. Therefore, after immobilization, the N-intein ligand must be denatured or otherwise dissociated from the cognate binding partner, thereby enabling removal, washing, or "peeling off" of the cognate binding partner from the N-intein ligand. Once the cognate binding partner is removed, the immobilized N-intein ligand must revert to an active state (able to bind a new partner), thereby forming a functional affinity capture medium.

[0012] A method for producing an affinity medium comprising an N-intein ligand covalently bound to a convenient substrate, and a composition related to the production process are disclosed. The N-intein ligand can include an internal N-terminal intein segment (INT N ) together with a fusion partner that is operably linked. The INT N segment within the N-intein ligand can be obtained from a native intein such as Npu DnaE intein. The INT N segment can further be modified to enhance its utility (e.g., so as not to contain any cysteine residues within the INT N segment, thus facilitating attachment at a single site to the substrate). For example, a tag can be attached to the INT N segment within the region following the C-terminal residue of the INT N segment to assist in the purification, detection, and / or enhancement of the soluble expression of the N-intein ligand. The N-intein ligand can also contain amino acids within the region following the C-terminal residue of the INT N segment, thereby enabling covalent immobilization of the N-intein ligand onto the substrate. The N-intein ligand can further include a sensitivity enhancement motif that renders its cleavage activity sensitive to exogenous conditions. The sensitivity enhancement motif can be fused to the N-terminal of the INT N segment. The exogenous conditions can be pH, temperature, zinc ion concentration, or a combination thereof.

[0013] Also disclosed is a protein purification medium containing an N-intane ligand covalently immobilized on a solid support, wherein more than 90% of the N-intane ligand molecules are associated with a congener-binding partner, and at least 90% of the congener-binding partners are not expressed in a fused state with the desired protein of interest. The congener-binding partner induces a soluble intein complex structured by binding to the N-intane ligand. C It can include segments.

[0014] Furthermore, a protein purification medium is disclosed that contains an N-intane ligand covalently immobilized on a solid support, wherein 0.001% of the N-intane ligand molecule is associated with a congener-binding partner, and at least 90% of the congener-binding partner is not expressed in a fused state with the desired protein of interest. The congener-binding partner is an INT that induces a soluble intein complex structured by binding to the N-intane ligand. C It can include segments.

[0015] Also disclosed are chromatographic resins comprising a base resin having a covalently bonded N-intei ligand, wherein the compressibility difference (ΔC) measured with respect to the resin is less than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% compared to the base resin substrate.

[0016] Also disclosed is a chromatographic resin comprising a base resin having a covalently bonded N-intei ligand, wherein the intrinsic functional compressibility coefficient (IFCF) measured with respect to the resin is 1.10 to 1.25.

[0017] Also disclosed is an expression vector containing an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an N-intane ligand and a homologue-binding partner, the expression vector can encode the N-intane ligand to be expressed with a purified tag, and the homologue-binding partner cannot be encoded for expression fused to the desired protein of interest. Also disclosed is a two-plasmid system in which the N-intane ligand and homologue-binding partner are encoded on two different affinity plasmids housed in a single cell. Also disclosed are cells containing the expression vector(s). The homologue-binding partner can be encoded for expression fused to a protein or peptide other than the desired protein of interest, such as an affinity tag.

[0018] While aspects of the present invention can be described and asserted in a particular legal class, such as a legal class of systems, this is for convenience only, and those skilled in the art will understand that each aspect of the present invention can be described and asserted in any legal class. Unless otherwise specified, none of the methods or aspects disclosed herein are intended to be construed as requiring their steps to be performed in a particular order. Thus, no order is intended to be inferred in any way in the claims or specification, where the claim for a method does not specifically state that the steps are limited to a particular order. This applies to any possible implicit grounds for interpretation, including logical matters relating to the arrangement of steps or operational flows, plain meaning derived from grammatical structure or punctuation, or the number or type of aspects described herein.

[0019] The accompanying drawings, incorporated and constituting parts of this specification, illustrate several embodiments and serve to illustrate the principles of the invention together with this specification. [Brief explanation of the drawing]

[0020] [Figure 1] Figure 1 shows SDS-PAGE analysis comparing cell lysates of N-intei ligands produced by conventional single-product overexpression in E. coli.

[0021] [Figure 2] Figure 2 shows an SDS-PAGE analysis comparing conventional single-product overexpression with co-expression with a congener-binding partner.

[0022] [Figure 3] Figure 3 shows SDS-PAGE analysis demonstrating that homologous binding partners can be modified or expressed by various fusion partners.

[0023] [Figure 4A] Figure 4A shows a comparison of ligand solubility between conventional single-product overexpression and CBP co-expression batches. Each batch was expressed and processed in parallel under identical conditions. Figure 4A shows a comparison of SDS-Pages. [Figure 4B] Figure 4B shows a comparison of ligand solubility for conventional single-product overexpression versus CBP coexpression batches. Each batch was expressed and processed in parallel under identical conditions. Figure 4B shows the retention volume in the conventional method versus ligand and CBP processing. [Figure 4C] Figure 4C shows a comparison of ligand solubility for conventional single-product overexpression versus CBP co-expression batches. Each batch was expressed and processed in parallel under identical conditions. Figure 4C shows the elution peak for normalized yield.

[0024] [Figure 5] Figure 5 shows SDS-PAGE analysis illustrating purification and cleavage kinetic assays for end-use. The resin used in the lower panel was produced using the method disclosed herein.

[0025] [Figure 6A]Figure 6A shows a generalized modular structure of the key components including the disclosed present invention. (Figure 6A) Modular structure of an N-intane ligand comprising a split intein segment and a usably linked fusion partner. The ligand consists of at least an N-terminal intein segment (INTN), but may consist of additional protein / peptide domains / motifs / molets expressed as fusion partners with the INTN segment. These fusion partners may include a sensitivity-enhancing motif (SEM) and various “immobilization” moieties (I), “linker” moieties (L), and / or “tag” moieties (T). [Figure 6B] Figure 6B shows a generalized modular structure of the key components including the disclosed invention. (Figure 6B) A congener-binding partner (CBP) is defined as a peptide / protein capable of binding to an INTN counterpart to induce a folded and stabilized state. The CBP may or may not include an optional tag and linker moiety expressed fused to either end. INTC segments and peptides derived from INTC species constitute a specific subset of CBPs that can be used to induce INTN stabilization. The term “congener-binding partner” is used because the intein complex resulting from the association of an INTN segment and a CBP may not necessarily exhibit cleavage or splicing activity, i.e., a slight but important distinction from a more specific subset of INTCs. [Figure 6C] Figure 6C shows a generalized modular structure of the main components including the disclosed present invention. (Figure 6C) A generalized example of INTN stabilization induced by a binding event between an INTN segment and a congener binding partner.

[0026] [Figure 7] Figure 7 shows a generalized process illustrating various standard heterologous expression techniques that can be used to produce N-intei ligands stabilized by a homologous binding partner for the purpose of manufacturing intein-mediated capture media.

[0027] [Figure 8A] Figures 8A and 8B show a generalized manufacturing process comparing (Figure 8A) a “conventional” bioprocessing step with (Figure 8B) the manufacturing process claimed herein. Both processes produce an affinity capture medium containing an immobilized N-intane ligand of the same sequence composition. The “active” affinity capture medium immediately before final use, as shown in the final “intane-mediated affinity capture” step, is shown in the dotted box in each panel. This illustrates and contrasts the key differences in the manufacturing process required by the introduction of the homologue binding partner. Additional advantages of the present invention are partially revealed in subsequent embodiments for carrying out the invention, partially revealed from embodiments for carrying out the invention, or can be known by carrying out the invention. The advantages of the present invention will be realized and achieved by the elements and combinations particularly pointed out in the appended claims. It should be understood that both the general description above and the subsequent embodiments for carrying out the invention are illustrative and explanatory only and do not limit the invention as claimed. [Figure 8B] Same as above.

[0028] [Figure 9A] Figure 9A shows the standard calculation principles for efficiency metrics of a column with reduced compressibility, peak asymmetry, and plate height. (Figure 9A) Diagram of the measurement of the bed compressibility during the column packing procedure. [Figure 9B] Figure 9B shows the standard calculation principles for efficiency metrics of columns with reduced compressibility, peak asymmetry, and plate height. (Figure 9B) A generalized example of a tracer pulse injection test chromatogram. Tracer concentration in the column eluent (monitored by A280) is plotted as a function of retention volume. Annotations are added to show and define the parameters used to evaluate column efficiency. [Figure 9C]Figure 9C shows the standard calculation principles for efficiency metrics of columns with reduced compressibility, peak asymmetry, and plate height. (Figure 9C) A list of relevant parameters and relational notations that define the terms used in evaluating column packing and calculating column efficiency metrics. [Figure 9D] Figure 9D shows the standard calculation principle for efficiency metrics of a column with reduced compressibility, peak asymmetry, and plate height. (Figure 9D) Definitions and expressions used to calculate the efficiency metrics of a column.

[0029] [Figure 10A] Figure 10A shows column efficiency data from tracer pulse injection tests performed for two resin batches (+CBP and -CBP, respectively) with and without the support of a homologous binding partner, as described in Example 5. (Figure 10A) Overlay chromatograms from each batch, plotting UV absorbance (A280) in the column eluent against retention time. [Figure 10B] Figure 10B shows column efficiency data from tracer pulse injection tests performed for two resin batches (+CBP and -CBP, respectively) with and without the support of a congenerate binding partner, as described in Example 5. (Figure 10B) A bar graph comparing the column efficiency metrics for each batch, calculated from the chromatogram data shown in Figure 10A. To illustrate the effect of the congenerate binding partner on column packing, Figure 10B summarizes the key column efficiency metrics reported for each batch, namely Cf, As, and h. Figure 10B also shows the extreme and acceptable values / ranges for each metric (indicated by dotted lines and green shaded areas, respectively), provided for comparison with the values ​​calculated from the experimental results for each batch. [Modes for carrying out the invention]

[0030] The present invention can be more readily understood by referring to the embodiments for carrying out the invention described below and the examples contained herein.

[0031] Before the compounds, compositions, articles, systems, apparatus, and / or methods of the present invention are disclosed and described, it should be understood that, unless otherwise specified, they are not limited to specific synthesis methods or specific reagents, and are naturally subject to change as such. It should also be understood that the terms used herein are for the purpose of describing only specific embodiments and are not intended to be limiting. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, but only exemplary methods and materials are described herein.

[0032] All publications referenced herein are incorporated herein by reference to disclose and explain the methods and / or materials to which such publications are related. Publications discussed herein are provided solely for the purpose of disclosing such publications prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention has no prior rights to such publications by prior invention. Furthermore, publication dates provided herein may differ from actual publication dates and may require independent verification.

[0033] A.Definition As used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. Thus, for example, references to "functional group," "alkyl," or "residue" include two or more such functional groups, alkyls, or mixtures of residues.

[0034] A range can be expressed herein as "about" one particular value to and / or "about" another particular value. When such a range is expressed, further aspects include "about" one particular value to and / or other particular values. Similarly, when a value is expressed as an approximation by the use of the antecedent "about", it will be understood that the particular value forms further aspects. It will also be understood that each endpoint of a range is significant with respect to the other endpoints, or independently of the other endpoints. It will also be understood that there are several values ​​disclosed herein, and each value is also disclosed herein not only as the value itself but also "about" its particular value. For example, if the value "10" is disclosed, "about 10" is also disclosed. It will also be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0035] The weight percentage (W%) of a component is based on the total weight of the formulation or composition containing that component, unless otherwise specified.

[0036] As used herein, the terms “optional” or “optional” mean that the events or circumstances described below may or may not occur, and that such descriptions include both cases in which such events or circumstances occur and cases in which they do not occur.

[0037] As used herein, the term “contact” refers to bringing together two biological entities in such a manner that a compound may directly affect the activity of the target, either directly, i.e., by interacting with the target itself, or indirectly, i.e., by interacting with another molecule, cofactor, factor, or protein on which the activity of the target depends. “Contact” can also mean facilitating the interaction between two biological entities, such as peptides, to bind them together, either covalently or otherwise.

[0038] As used herein, “kit” means a collection of at least two components that make up a kit. Together, the components constitute a functional unit for a given purpose. Individual component parts may be physically packaged together or separately. For example, a kit that includes instructions for using the kit may or may not physically include such instructions together with the other individual component parts. Alternatively, such instructions may be supplied as a separate component part, either in paper or electronic form, or as a recorded presentation, which may be supplied on a computer-readable storage device or downloaded from an internet website.

[0039] As used herein, “Instructions” means a document describing the associated materials or methodology associated with the kit. These materials may include any combination of the following: background information, a list of components and their availability information (such as purchasing information), a simple or detailed protocol for using the kit, troubleshooting, references, technical support, and any other relevant documents. Instructions may be supplied with the kit or as a separate component, either in paper or electronic form, either on a computer-readable storage device or downloadable from an internet website, or as a recorded presentation. Instructions may include one or more documents and include future updates.

[0040] As used herein, the terms “target protein,” “protein of interest,” and “therapeutic agent” include any synthetic or naturally occurring protein or peptide. In the context of this invention, “protein of interest” means a protein that will be purified by an end-user in a laboratory or manufacturing environment using fragmented intein purification techniques, as opposed to any context relating to the manufacture of the purification medium itself. This definition will apply to any protein or peptide that requires purification for research or other research applications. In addition, the term encompasses drugs, vaccines, and compounds conventionally considered biologics, including molecules such as proteins and peptides. Examples of therapeutic agents are described in well-known references such as the Merck Index (14th edition), Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (1st edition), and such therapeutic agents include, but are not limited to, pharmaceuticals; substances used to treat, prevent, diagnose, cure or alleviate a disease or illness; substances that affect the structure or function of a body; or prodrugs that become biologically active or more active after being placed in a physiological environment.

[0041] As used herein, “mutant” refers to a molecule that retains the same or substantially similar functional activity as the original sequence. A mutant may originate from the same or different species, or it may be a synthetic sequence based on a natural or conventional molecule. Furthermore, as used herein, “mutant” refers to a molecule having a structure derived from the structure of a parent molecule (e.g., a protein or peptide disclosed herein), and whose structure or sequence is sufficiently similar to those disclosed herein that, based on their similarity, it would be expected by those skilled in the art to exhibit the same or similar activity and utility compared to the parent molecule. For example, a mutant peptide having similar activity to the parent can be obtained by substituting specific amino acids in a given peptide.

[0042] As used herein, the term “amino acid sequence” refers to a list of abbreviations, notations, letters or words representing amino acid residues. The amino acid abbreviations used herein are the conventional single-letter notations for amino acids, and are represented as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.

[0043] As used herein, “peptide” refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A peptide is composed of a sequence of amino acids. The term “peptide” includes naturally occurring or synthetic molecules.

[0044] In addition, as used herein, the term “peptide” refers to amino acids linked to one another by peptide bonds or modified peptide bonds, such as peptide isosteres, and may include modified amino acids other than the 20 amino acids encoded by genes. Peptides can be modified by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Modifications can occur at any site on the peptide, including the peptide backbone, amino acid side chains, and amino or carboxyl terminus. The same type of modification may be present at several sites in a given polypeptide to the same or varying degrees. Furthermore, a given peptide may have many types of modifications. Modifications include, but are not limited to, transfer RNA-mediated addition of amino acids to proteins, such as linking of different domains or motifs, acetylation, acylation, ADP-ribosylation, amidation, covalent crosslinking or covalent cyclization, covalent attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine ​​or pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfated phosphate, and arginylation. (See Proteins-Structure and Molecular Properties 2nd Ed., TECreighton, WH Freeman and Company, New York (1993) and Posttranslational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).)

[0045] As used herein, “isolated peptide” or “purified peptide” means a peptide (or fragment thereof) that substantially does not contain the material to which the peptide normally associates in nature, or the material to which the peptide associates in an artificial expression system or artificial production system, including but not limited to the material to which the peptide normally associates in nature, or the material to which the peptide associates in an artificial expression system or artificial production system, including but not limited to the material to which the peptide associates in an artificial expression system or artificial production system, including expression host cell lysates, growth medium components, buffer components, cell culture supernatants, or components of a synthetic in vitro translation system. The peptides or fragments thereof disclosed herein can be obtained, for example, by extraction from a natural source (e.g., mammalian cells), by expression of recombinant nucleic acids encoding the peptide (e.g., intracellular or in a cell-free translation system), or by chemical synthesis of the peptide. In addition, peptide fragments can be obtained by any of these methods, or by cleaving full-length proteins and / or peptides.

[0046] As used herein, the terms "or" (or "or") mean any one element of a particular list, and also include any combination of the components of that list.

[0047] As used herein, the term “nucleic acid” refers to naturally occurring or synthetic oligonucleotides or polynucleotides, whether single-stranded or double-stranded, sense or antisense, that can be hybridized into complementary nucleic acids by Watson-Crick base pairing, such as DNA or RNA or DNA-RNA hybrids. The nucleic acids of the present invention may also include nucleotide analogs (e.g., BrdU) and non-phosphodiester nucleoside bonds (e.g., peptide nucleic acids (PNA) or thiodiester bonds). In particular, nucleic acids may include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0048] As used herein, “isolated nucleic acid” or “purified nucleic acid” means DNA in which the gene does not contain adjacent genes in the naturally occurring genome of the organism from which the DNA of the present invention originates. Therefore, the term includes recombinant DNA (e.g., a transgene) incorporated into a vector such as an autonomous replicating plasmid or virus, or incorporated into the genomic DNA of a prokaryote or eukaryote, or recombinant DNA existing as a separate molecule (e.g., cDNA or genomic fragment or cDNA fragment produced by PCR, restriction endonuclease digestion, or chemosynthesis or in vitro synthesis). The term also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence. The term “isolated nucleic acid” also refers to mRNA molecules, for example, encoded by an isolated DNA molecule, or chemically synthesized, or isolated from or substantially free from at least some cellular components, such as other types of RNA molecules or peptide molecules.

[0049] "Intein" refers to an in-frame intervening sequence in a protein, as described by Perler (Perler, Davis et al. 1994). Inteins can catalyze the cleavage of the intein itself from the protein after the posttranslational protein splicing process, yielding free intein and mature protein. Inteins can also catalyze the cleavage of the intein-extine bond at either the intein N-terminus, the intein C-terminus, or both ends of the intein-extine. As used herein, "intine" includes mini-intines, modified or mutant inteins, and split inteins.

[0050] The term "split intein" refers to "N-terminal intein segment" (INT N ) and the corresponding "C-terminal intein segment" (INT C) refers to a pair of two distinct and independently translated protein segments, including a binding partner, characterized by at least one of the following properties: (1) INT N Segments and INT C The segments exhibit an intrinsic innate affinity for their individual corresponding proteins, causing the pairs to spontaneously associate, fold, and "bind" to each other via non-covalent bonds, forming an "intene complex." (2) During the association, the intein complex may become “splicing-active” or “cleavage-active,” in which case the complex catalyzes a cleavage or splicing event between the complex and its extein fusion partner. This activity is generally considered to depend on the formation of the intein complex, i.e., INT N INT C However, it does not independently possess this activity in the absence of those binding partners. (3) INT N Segments and INT C The segments contain peptides, protein domains, or amino acid sequences that are identical, similar to, or derived from naturally occurring or artificially fragmented inteins, such as those listed in the so-called "InBase, or intein database," established by Perler (Perler 1999, Perler 2002). Examples of intein species are also listed in Table 2. (4) However, the formation of complexes exhibiting cleavage activity and / or splicing activity is "divided intein" and / or INT N Segment and / or INT C It should be noted that this is not strictly required to satisfy the definition of a segment. In other words, for example, if the “split intein” is modified so that it no longer exhibits splicing activity and / or cleavage activity, this is still covered by the present invention.

[0051] The term “homogeneic binding partner” or “homogene” refers to any “binding-active” INT that comes into contact with each other. N This refers to any peptide or protein segment that can spontaneously associate with its counterpart via a non-covalent bond. The homologous binding partner is an INT that is usably linked to the linker and tag portion of the tag shown in Figure 6(b) and described below. C INT containing peptides C This includes, but is not limited to, a subset of peptide and protein segments, including species defined as peptides. For example, INT C A segment can be an example of a homogeneous bonding partner, but a homogeneous bonding partner is, by definition, INT C It is not strictly necessary for it to be a specific species.

[0052] Furthermore, in this specification, INT C More specifically, INT N It is distinguished from the congener superfamily in that it is a binding partner that associates with other molecules to form an active intein complex.

[0053] INT C is, INT N When it associates with and folds into an intein complex, it is considered a homolog; however, the resulting complex is an inactive intein complex (it does not exhibit splicing or cleavage activity).

[0054] As used herein, the term "extrain" means INT N N-terminus of the segment, INT C This refers to any peptide, protein, domain, or amino acid expressed in a state of covalent fusion to one of the C-terminuses of a segment. Ectein is further characterized as a portion of the intein-fusion polypeptide that can be cleaved or spliced ​​during excision of the intein or intein complex.

[0055] N-terminal extensions (N-EXTs) are specifically INT NIt is an extain that is expressed fused to the N-terminus of the segment. N-EXT is INT N When expressed in a fused state with a segment, it is simply classified as such, but INT N The segment is INT N The existence of N-EXT is not strictly required to satisfy the definition of a segment.

[0056] The C-terminal protein (C-EXT) is specifically INT C C-EXT is a protein that is expressed fused to the C-terminus of a segment or homologous binding partner. C When expressed in a fused state with a segment or homologous binding partner, it is simply classified as such, but INT C Segments and congeneral bonding partners do not strictly require the presence of C-EXT to satisfy their individual definitions.

[0057] Furthermore, the N-EXT domain and the C-EXT domain are their individual INT N Fusion Partners and INT C Despite being cleaved from a fusion partner, it may continue to be identified as such after a cleavage or splicing event has occurred.

[0058] The term “N-intane ligand” refers to a protein that is immobilized (or will be immobilized) onto a solid surface, substrate, or chromatography medium in order to function as an affinity ligand. As defined herein, an N-intane ligand is at least INT N It consists of segments, but INT NThe fusion partner with the segment may consist of additional proteins, peptides, functional domains, amino acid motifs, and / or chemical moieties that are expressible and ligated (Figure 6). Fusion partners containing the N-intei ligand may include, but are not limited to, sensitivity-enhancing motifs (SEMs), as well as various "immobilization moieties," "linker moieties," and / or "tag moieties," collectively referred to as the "ILT moiety."

[0059] The term "sensitivity-enhancing motif" (SEM) is used in INT N SEM refers to an amino acid sequence of three or more residues that is expressed fused to the N-terminus of a segment, thereby making the splicing or cleavage activity of the intein complex highly sensitive to exogenous conditions, as already described in U.S. Patent No. 10,066,027. SEM is a component of the N-intei ligand, but INT N This is distinct from the segment and other fusion partners that may contain the N-intei ligand.

[0060] The "ILT portion" contains an N-intane ligand, N It is a collective term for one or more amino acids expressed as fusion partners with INT. The ILT portion can be further subdivided into constituent groups that include at least one of the following classifications: “Immobilization” (I), “Linker” (L), and / or “Tag” (T). The individual portions are linked together for use and with respect to each other, and INT N These elements may be easily repeated, combined, or rearranged (see, for example, Figure 6).

[0061] The term "immobilization portion" refers to the INT that enables the covalent immobilization of the N-intei ligand (and its fusion partner through extension). N This refers to one or more amino acid residues (e.g., Cys) that are expressed in a fused state with [another molecule].

[0062] The classification "linker part" or "linker" is INTN , providing structure, spacing, or flexibility between the fixed portion and / or other fusion partners, INT N This refers to one or more amino acid residues that are expressed in a fused state with a linker. A common example of a linker is the glycine-serine repeat ((Gly n1 Ser n2 ) n3 ), polyproline dyad ((XaaPro) n ), and α-helix (A(EAAAK) n A) There are linker motifs, but they are not limited to these.

[0063] A “tag portion” or “tag” refers to a peptide, domain, or specific amino acid motif that, when fused to a protein, is expressed to aid in purification, detection, and / or enhance the soluble expression of its fusion partner. Common examples of “tag portions” include, but are not limited to, purification tags (e.g., poly-His, poly-Arg, GST, CBD, MBP, CBP, Strep-tag, FLAG-tag, etc.), detection tags (e.g., GFP, luciferase, epitope tags (i.e., FLAG, HA, c-myc), HRP, etc.), and expression / solubility enhancement tags (e.g., T7-tag, NusA, TrxA, DsbA, DsbC, GST, MBP, etc.).

[0064] INT N , INT C Alternatively, a homologous binding partner domain is considered "binding active" if the segment exhibits affinity for its corresponding binding partner and can participate in the binding event that forms a new intein complex. The terms "binding active" and "non-binding active" refer to individual functionalized inteins. N , INT C and / or the homologous segment (a) already has a partner bound to form an intein complex, or (b) the individual functional INT N , INT CIt is used to distinguish the segment from other segments of identical composition that have misfolded in a way that suppresses the affinity of the segment to potential binding partners of the congener segment and / or other related segments. Importantly, when an intein complex is present, the component INT N , INT C and / or related segments can bind to one another in such a way that they cannot further associate with any additional other compatible binding partners that may be encountered while the intein complex is present. For example, given INT N and INT C These can associate and combine to form an intein complex, but during the formation of the complex, INT N and INT C It may be considered "non-binding activity" in terms of functionality, and neither segment, while containing the intein complex, can be involved in any further binding event. However, when the intein complex is dissolved, INT N and INT C If the segments dissociate and then refold to restore their affinity, the individual segments can become "binding active" again.

[0065] Intein complexes can be further functionally classified as either "non-binding" or "binding" with respect to intein splicing and / or cleavage activity. Non-binding intein complexes exhibit less than 10% cleavage or splicing behavior by their extein fusion partner. Conversely, binding intein complexes catalyze cleavage or splicing events that alter at least one peptide bond of their extein fusion partner.

[0066] Active intein complexes can be further classified by the specific type of canonical intein that catalyzes C-terminal cleavage, N-terminal cleavage, double cleavage, or splicing.

[0067] When an "active intein complex" catalyzes a cleavage or splicing event, the resulting intein complex may not further affect the peptide bond of its fusion partner (splicing and cleavage reactions are irreversible). Therefore, the resulting intein complex can generally be considered an "inactive intein complex" after catalyzing either a cleavage or splicing event. "No further effect" means an effect of less than 10%.

[0068] As used herein, the terms “splice” or “splices” mean the removal of a central portion of a polypeptide to form two or more smaller polypeptide molecules. In some cases, splicing also includes the step of fusing two or more smaller polypeptides together to form a new polypeptide. Splicing can also refer to the linking of two polypeptides encoded in two separate gene products via the action of a splitting intein.

[0069] As used herein, the terms “cleave,” “cleaves,” “cleavage,” and “cleavage event” refer to a chemical reaction in which peptide bonds within a polypeptide are broken, thereby splitting a single polypeptide into two or more smaller polypeptide molecules. In some cases, cleavage is mediated by the addition of an exogenous endopeptidase, which is often called “proteolytic cleavage.” In other cases, cleavage can be mediated by the intrinsic activity of one or both of the cleaved peptide sequences, which is often called “autocleavage.” Cleavage can be controlled by exogenous conditions (such as the pH of a buffer), as in the action of the splitting intein systems described herein.

[0070] The term "fusion" or "in a fused state" means covalently bonding to another. For example, a first peptide is fused to a second peptide when the two peptides are covalently bonded to each other (e.g., via a peptide bond). Peptides and / or protein domains linked by a peptide bond may also be called "fusion partners."

[0071] As used herein, “isolated” or “substantially pure” means a substance that has been separated from its naturally associated components. Typically, a polypeptide is substantially pure when it does not contain at least 50% by weight (e.g., 60%, 70%, 80%, 90%, 95%, and 99% by weight) of other proteins and other naturally occurring organic molecules that it naturally associates with.

[0072] In this specification, “bind,” “binds,” “being bound,” or “binding event” means that one molecule recognizes another molecule in a sample and attaches to it, but does not substantially recognize or attach to any other molecule in the sample. The terms “bind,” “binds,” “being bound,” and “binding event” also mean that the interaction between the two molecules is non-covalent and reversible. 5 ~10 6 When a molecule has a binding affinity greater than liters / moles, it "specifically binds" to that other molecule. These terms are interchangeable with "associate with," "associates with," or "is associated with."

[0073] The nucleic acids, nucleotide sequences, proteins, or amino acid sequences referred to herein can be isolated, purified, chemically synthesized, or produced via recombinant DNA technology. All of these methods are well known in the art.

[0074] As used herein, the terms “modified” or “mutated,” such as “modified intein” or “mutated intein,” refer to one or more modifications to any of the nucleic acids or amino acid sequences mentioned, including intein, compared to the original or naturally occurring structure. Such modifications may be substitutions, additions, or deletions. The modifications may occur at one or more amino acid residues or one or more nucleotides in the structure mentioned, including intein.

[0075] As used herein, “usably ligated” refers to the association of two or more biomolecules in a configuration relative to each other that enables them to perform their normal function. With respect to nucleotide sequences, “usably ligated” refers to the association of two or more nucleic acid sequences in a configuration relative to each other that enables them to perform their normal function, by enzymatic ligation or other means. For example, a nucleotide sequence encoding a presequence or secretion leader is usably ligated to the nucleotide sequence for the polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is usably ligated to the coding sequence if it affects the transcription of the coding sequence; and a ribosome binding site is usably ligated to the coding sequence if it is positioned to facilitate the translation of the sequence.

[0076] "Sequence homology" can refer to a situation where the sequences of nucleic acids or proteins are similar for the reason that they share a common evolutionary origin. "Sequence homology" can indicate that the sequences are very similar. Sequence similarity is observable, and homology can be based on that observation. "Very similar" can mean at least 70% identity, homology, or similarity, at least 75% identity, homology, or similarity, at least 80% identity, homology, or similarity, at least 85% identity, homology, or similarity, at least 90% identity, homology, or similarity, for example, at least 93% or at least 95% or even at least 97% identity, homology, or similarity. The similarity, homology, or identity of nucleotide sequences can be determined using the "alignment" program in Myers et al. (1988) CABIOS 4:11-17, which is available at NCBI. Additionally or alternatively, the similarity, identity, or homology of amino acid sequences can be determined using the BlastP program (Altschul et al. Nucl. Acids Res. 25:3389-3402), which is available at NCBI. Alternatively or additionally, for example with respect to nucleotide sequences, the terms “similarity,” “identity,” or “homology” are intended to indicate a quantitative measure of homology between two sequences.

[0077] Alternatively or additionally, “similarity” of sequences refers to the number of positions with identical nucleotides obtained by dividing by the number of nucleotides in the shorter of the two sequences, and the alignment of the two sequences can be determined according to the Wilbur and Lipman algorithm (1983) Proc. Natl. Acad. Sci. USA 80:726. For example, computer-aided analysis and interpretation of sequence data, including alignment, using a 20-nucleotide window size, a 4-nucleotide word length, and a 4-gap penalty, can be easily performed using commercially available programs (e.g., Intelligenetics® Suite, Intelligenetics Inc. CA). When an RNA sequence is said to be similar to a DNA sequence, or to have a degree of sequence identity, thymidine (T) in the DNA sequence is considered equivalent to uracil (U) in the RNA sequence. The following references also provide algorithms for comparing the relative identity, homology, or similarity of amino acid residues of two proteins, and, additionally or alternatively to the foregoing, the references can be used to determine the percentage of homology, identity, or similarity. Needleman et al. (1970) J.Mol.Biol.48:444-453, Smith et al. (1983) Advances App.Math.2:482-489, Smith et al. (1981) Nuc. Acids Res.11:2205-2220, Feng et al. (1987) J. Molec. Evol. 25:351-360, Higgins et al. (1989) CABIOS 5:151-153, Thompson et al. (1994) Nuc. Acids Res. 22:4673-480, and Devereux et al. (1984) 12:387-395.The term "strict hybridization conditions" is well known in the art; see, for example, Sambrook, "Molecular Cloning, A Laboratory Manual," second ed., CSH Press, Cold Spring Harbor, 1989, and "Nucleic Acid Hybridization, A Practical Approach," Hames and Higgins eds., IRL Press, Oxford, 1985, as well as Figure 2, which includes a sequence comparison, and its explanation herein.

[0078] The terms "plasmid," "vector," and "cassette" refer to extrachromosomal elements that carry genes, typically in the form of circular double-stranded DNA molecules, and are not part of the cell's central metabolism. Such elements may be single-stranded or double-stranded DNA or RNA autonomous replication sequences, genomic integration sequences, linear or circular phage sequences, or nucleotide sequences derived from any source, where some nucleotide sequences are bound or recombined into their own constructs that allow for the introduction of promoter fragment DNA sequences for selected gene products into the cell, along with appropriate 3' untranslated sequences. Typically, a "vector" is a modified plasmid containing multiple additional insertion sites for cloning and an "expression cassette" containing the DNA sequence for a selected gene product (i.e., the transgene) for expression in the host cell. This "expression cassette" typically includes a 5' promoter region, the transgene ORF, and a 3' terminator region, along with all the necessary regulatory sequences required for the transcription and translation of the ORF. Thus, integration of the expression cassette into the host enables the expression of the transgene ORF within the cassette.

[0079] The term "buffer solution" refers to a solution that resists changes in pH due to the action of its conjugate acid-base range.

[0080] The term "loading buffer" or "binding buffer" refers to a buffer containing one or more salts that is mixed with a protein preparation to load the protein preparation onto a column. This buffer is also used to equilibrate the column before loading and to wash the column after loading the protein.

[0081] The term “wash buffer” is used herein to refer to a buffer that passes through a column (e.g.,) after loading the target protein (e.g., one bound to a C-terminal intein fragment) and before elution of the target protein. The wash buffer may function to remove one or more contaminants without substantial elution of the desired protein.

[0082] The term "elution buffer" refers to a buffer used to elute a desired protein from a column. As used herein, the term "solution" refers to either a buffered solution or a non-buffered solution, including water.

[0083] The term "washing" means passing an appropriate buffer solution through or on a solid support, such as a chromatography resin.

[0084] The term "eluting" a molecule (e.g., a desired protein or contaminant) from a solid support means removing that molecule from such material.

[0085] The term “contaminant” or “impurity” refers to any foreign or undesirable molecule, particularly biological macromolecules, present in the sample of protein being purified, such as DNA, RNA, or proteins other than the protein being purified. Contaminants include, for example, other proteins derived from cells that express and / or secrete the protein being purified.

[0086] The terms “separate” or “isolate,” as used in relation to protein purification, refer to separating a desired protein from a mixture of a second protein or other contaminants or impurities in a mixture containing both the desired protein and the second protein or other contaminants or impurities, such that at least a majority of the molecules of the desired protein are removed from the portion of the mixture containing at least a majority of the molecules of the second protein or other contaminants or impurities.

[0087] The term “purify” or “to purify” means to increase the purity of the desired protein in a composition or solution by removing (completely or partially) at least one contaminant from the composition or solution.

[0088] The term "chromatography media" or "chromatographic medium" refers to any type of stationary phase substrate (solid support), scaffold, or matrix used in chromatography or purification, on which N-intei ligands are immobilized, attached, bonded, or graft-polymerized (covalently or otherwise) for the purpose of separating, concentrating, or purifying a secondary molecule of interest. Common examples of chromatography media include, but are not limited to, chromatography resins (e.g., cross-linked agarose, polymers, or silica-based particles / porous beads), functionalized membranes, micro- and nano-scale magnetic particles, and structured pore / structured channel media (e.g., monoliths and monolithic columns).

[0089] The disclosure herein relating to the immobilization of N-intane ligands onto "chromatographic media" is presumed to apply in general to any type of "chromatographic media." The basic functional requirement of a "chromatographic media" is to provide a solid support surface for holding N-intane ligands. As such, it is understood that various chromatographic media can be freely and independently substituted for one another with little or no consequence to the function of the immobilized N-intane ligand.

[0090] The term "asymmetry coefficient," indicated by the symbol "As," refers to a column efficiency metric used to evaluate the uniformity of flow through a packed bed chromatography column. The asymmetry coefficient is collected by a standard column efficiency test performed using tracer pulse injection and then determined using data calculated using the formula and definition shown in Figure 9.

[0091] The term "reduced plate height," indicated by the symbol "h," refers to a column efficiency metric based on theoretical plate height, normalized for particle size within a packed bed chromatography column. Reduced plate height is collected by standard column efficiency tests performed using tracer pulse injection and then determined using data calculated with the formulas and definitions shown in Figure 9.

[0092] The term "column efficiency metric" refers collectively to the asymmetry coefficient (As) and reduced plate height (h), which are standard metrics commonly cited to assess the quality of packing and the uniformity of flow through a packed bed chromatography column.

[0093] Symbol "C" f The term “compressibility factor,” as shown by '', refers to the relative change in volume experienced when a compressible chromatography resin is packed into a chromatography column. The common definition used in the industry and by those skilled in the art is that the compressibility factor is usually given by formula (C f =V 膨張 / V 圧縮 ) is calculated by, in the formula, V膨張 V represents the volume of the resin solid when fully expanded or "gravitationally settled". 圧縮 This represents the volume occupied by the same resin solid after compression in the packed resin bed. For columns with a constant cross-sectional area, this formula is C f This can be reduced to =L0 / L, where L0 is the height of the resin floor when fully expanded or "gravity settled," and L is the height of the same resin floor when compressed, as shown in Figure 9(a).

[0094] The term "sufficiently packed" refers to the compressibility factor (C) f This refers to the state of chromatography column packing where the asymmetry coefficient (As) and reduced plate height (h) are all measured within their respective acceptable ranges.

[0095] The column efficiency measurement criteria and the above-mentioned definition of "well packed" are widely recognized in the industry and well established by those skilled in the art.

[0096] The term "Intrinsic Functional Compressibility Factor," also abbreviated as "IFCF," refers to a property of chromatographic resins that indicates the rate of volume change when the resin is packed into a chromatography column under standardized packing conditions. IFCF essentially defines a compression factor (C) that further specifies the measurement method for "standardized criteria." f This is a measured value of ), which is necessary to ensure that the observed floor compression represents only the intrinsic properties of the resin. Where defined herein, IFCF is the calculated compression factor (C) achieved when the resin is packed into a chromatography column in a manner that statistically accounts for all of the following "standardized criteria" conditions. f ) (1) The resin must be suspended as a slurry and packed in a phosphate buffer solution (PBS). (2) The packing resin bed generated during column packing must exhibit an asymmetry coefficient (As) between 0.8 and 1.4. (3) The packing resin bed generated during column packing must have a reduced plate height (h) of less than 5.0. For example, if a resin is suspended as a slurry in PBS, then allowed to settle by gravity in a chromatography column to a bed volume X, and then compressed to produce a packed resin bed volume Y, the packed resin bed will be C f It is said to have a compressibility coefficient of =X / Y. Subsequently, when performing a column efficiency test to confirm that the asymmetry coefficient of the packed resin bed and the reduced plate height satisfy conditions (2) and (3) (for example, an asymmetry coefficient of As=1.0 and a reduced plate height h=3.0), since all the "standard criteria" conditions were met when the resin bed was packed, the intrinsic functional compressibility coefficient of the resin is IFCF=C f It is said that the equation is X / Y.

[0097] In the second example, consider the same gravity-settled resin bed in which the porous semi-elastic particle structure of the resin is broken down, instead being filled with excessive compression, resulting in a smaller packed bed volume Z. This resin bed is produced from the same resin as in the previous example, but C f The calculated compression factor is =X / Z. Comparing these scenarios, the compression factor (C f) is specific to a given packed bed, and volumes Y and Z are partially determined by the intrinsic compressibility of the resin. However, Y differs from Z due to variations in the compression filling force, and it is to be understood that this is exogenous and arbitrary. Therefore, a criterion is specified for normalizing the compression force applied during filling so that further deviations in compression depend only on the intrinsic compressibility of the resin. Conditions (2) and (3) provide this normalized criterion because excessive (or insufficient) compression in the preparation of the packed bed creates irregular hydrodynamics and appears as deviations in the asymmetry coefficient (As) and / or reduced plate height (h). In fact, the asymmetry coefficient (As) and reduced plate height (h) satisfy conditions (2) and (3) only when the degree of compression applied to the bed during filling is functionally appropriate for the mechanical structure of a given resin. In a second example, the resin bed was filled with an inappropriate amount of compression and, therefore, exhibited poor asymmetry coefficient (As) and / or reduced plate height (h) (e.g., As = 0.6 or As = 1.8, and / or h = 6.5), thereby failing to meet the definition of the "normalized criterion". Thus, the measured compression coefficient C f = X / Z should not be regarded as a valid measure of the IFCF of the resin.

[0098] Similarly, resins are often slurried and filled in buffer solutions of various compositions. Considering that alternative buffer compositions have been observed to swell or shrink porous resins to various degrees, measurements of resin compressibility from packed beds prepared with other buffer solutions can lead to different observations of the compression coefficient C f ). Therefore, it is necessary to explain the basis for performing the IFCF measurement in PBS buffer, which ensures that any deviation in the measured compression is due only to differences in resin composition that affect the intrinsic compressibility of the resin.

[0099] It is understood that when the three "standard criteria" of the IFCF are met, the measured compressibility coefficient reflects the intrinsic properties of the resin itself. Therefore, variations in the IFCF can be used as an indirect method for detecting changes in the composition of the resin.

[0100] The term "base resin" refers to a resin-supported substrate to which N-intei ligand or any other ligand is not bound.

[0101] The term "compressibility difference," indicated by the symbol "ΔC," refers to the relative change in the compressibility of a given resin when a ligand binds to it. The compressibility difference is calculated by comparing the functional compressibility coefficient (IFCF) of the bound resin with that of the base resin substrate. 基材 Calculate the percentage difference between (IFCF) and (IFCF). As defined herein, the compression difference is ΔC = |(IFCF) - (IFCF). 基材 )| / (IFCF 基材 It is calculated as ) × 100%. For example, using the data shown in Example 5, the compressibility difference of the "-CBP" resin batch would be calculated as ΔC = |(1.01)-(1.15)| / (1.15) × 100% = 12.2%, which means that the compressibility of the resin changed by more than 12% as a result of binding the N-intane ligand to the resin in the production of the "-CBP" batch. The compressibility difference (ΔC) of a resin may be less than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% relative to its base resin substrate.

[0102] This specification discloses components used to prepare the compositions of the present invention, and the compositions themselves used in the methods disclosed herein. These and other materials are disclosed herein, and when combinations, partial sets, interactions, groups, etc., of these materials are disclosed, specific references to various individual and collective combinations and permutations of these compounds cannot be explicitly disclosed, but it is understood that each is specifically contemplated and described herein. For example, when a particular compound is disclosed and discussed, and several modifications that can be made to several molecules containing the compound are discussed, all possible combinations and permutations of compounds and modifications are specifically contemplated unless the opposite is specifically indicated. Thus, when classes of molecules A, B, and C are disclosed as well as classes of molecules D, E, and F, and an example of combined molecule AD is disclosed, it means that combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered disclosed, even if they are not individually enumerated, each is considered individually and collectively. Similarly, any partial sets or combinations of these are also disclosed. Therefore, for example, the subgroups AE, BF, and CE would be considered disclosed. This concept applies to all embodiments of this application, including but not limited to steps in a method for producing and using the compositions of the present invention. Therefore, where there are various additional steps that can be performed, each of these additional steps can be performed in any specific embodiment or combination of embodiments of the method of the present invention.

[0103] It is understood that the compositions disclosed herein have certain functions. It is understood that certain structural requirements for performing the disclosed functions are disclosed herein, that there are various structures that can perform the same functions as the disclosed structures, and that these structures typically achieve the same results. For example, the compounds used to control pH in the examples shown can be substituted with other buffer compounds to control pH, since pH is a critical variable to be controlled and the specific buffer compound can be changed.

[0104] Method for fixing the BN terminal intein segment Intein-based methods for protein modification and ligation have been developed (U.S. Patents 10,066,027 and 9,796,967, which are incorporated herein by reference in their entirety). Inteins are internal protein sequences that can catalyze protein splicing reactions, which involve cleaving an intein sequence from a precursor protein and attaching adjacent sequences (N- and C-extines) to peptide bonds (Perler et al. (1994)). Hundreds of intein and intein-like sequences have been discovered in various organisms and proteins (Perler et al. (2002), Liu et al. (2003)), which are typically 350–550 amino acids in size and contain homing endonuclease domains, but natural and engineered miniintines having only a splicing domain of about 140 amino acids are sufficient for protein splicing (Liu et al. (2003), Yang et al. (2004), Telenti et al. (1997), Wu et al. (1998), Derbyshire et al. (1997)).

[0105] Both continuous and fragmented inteins are suitable for protein purification applications (US Patent No. 10,066,027 and US Patent No. 9,796,967), in which modified inteins are used to mediate affinity capture of the secondary protein of interest. Fragmented inteins are particularly useful for such applications due to their dimeric structure, binding-dependent cleavage activity, and strong innate affinity between counterpart segments. However, fragmented inteins also suffer from low yield or low solubility when produced using "conventional" bioprocessing techniques (Shah, Dann et al. 2012). Indeed, since protein yields achieved via conventional processing are often very low, the scalable production of fragmented intein-based chromatography media can be prohibitively expensive and therefore not economically viable.

[0106] While the production of any protein-based affinity ligand is indeed a complex, multi-step process involving numerous factors that influence overall yield, obstacles to the production process are typically offset by scaling up the operation of throughput-limiting units. However, this approach appears particularly inefficient with split inteins, as solubility and aggregation are often yield-limiting factors in the production process. Solubility in heterologous protein expression is typically considered a function of cell culture conditions and their effects on protein folding in vivo (e.g., proper formation of secondary and tertiary structures) (Rosano and Ceccarelli 2014) (Dyson and Wright 2005), but split inteins appear to be an exception to this, as illustrated by the example in Figure 1. Therefore, to improve production yields for chromatographic media based on split inteins, we have devised novel processing techniques disclosed herein to mitigate the stability issues inherent in split inteins and their intrinsic structures.

[0107] In the absence of those natural binding partners, INTN Segment and INT C Segments are mainly composed of essentially disordered domains with little or no defined structural conformation (Zheng, Wu et al. 2012, Shah, Eryilmaz et al. 2013, Eryilmaz, Shah et al. 2014). This essential disorder is presumably thought to explain the rapid and long-range high-affinity binding presented between split intein segments (Pontius 1993, Shoemaker, Portman et al. 2000, Wright and Dyson 2009). Essential disorder can endow split inteins with exact properties suitable for affinity capture applications, but this also means that hydrophobic and charged residues within the disordered domain can be accessible or exposed, making split intein segments prone to aggregation and insolubility (Carrio and Villaverde 2002)(Saleh and Perler 2006)(Aranko, Wlodawer et al. 2014). In fact, during basic research on intein folding, the INT N segment from Synechocystis sp. PCC6803 was observed by Zheng et al. (2012) to have low solubility when expressed without its native INT C counterpart, and the authors attributed this to the "disordered" structure of the isolated INT N segment. The authors proposed this view in support of their hypothesis that the intein transitions from a disordered state to a folded state upon complex formation.

[0108] As claimed herein, the N-intein ligand can be stabilized during the manufacturing process by introducing a cognate binding partner to induce a new folded state that improves the stability and solubility of the INT N . This results in a dramatic increase in the yield throughout the manufacturing process, as demonstrated in the example shown in Figure 4.

[0109] However, importantly, the presence of a congeneral bonding partner improves process yield, but it also means that INT N The segment is inactivated as a functional, and the N-intane ligand is used to inactivate any of the INTs it may encounter. C This prevents the fusion protein from binding or associating with it. Given that the fundamental function of affinity capture media is based on their ability to bind to the target protein, introducing excipient proteins that are known to inactivate N-intane ligands during the manufacturing process is, ostensibly, unpredictable.

[0110] Therefore, the feasibility of the disclosed manufacturing process is (1) INT after fixation by covalent bonding. N It is critically dependent on (2) the ability to dissociate the homologous binding partner from the segment, and (3) the ability to return the immobilized N-intei ligand to its binding-active folded state. Neither of these appears to have been previously demonstrated in the literature.

[0111] It is not clear that forced dissociation of split inteins is possible without damaging their structure and / or activity in the process. (Wild-type INT) N Segments and INT CThe binding affinity to segments has been measured in the low nanomolar concentration range (Shi and Muir 2005) (Zettler, Schutz et al. 2009). Since splicing extensions are unnecessary for this application and can therefore be eliminated to reduce steric binding inhibition, this likely underestimates the value of modified fragmented inteins for affinity capture. While it is understood that denaturing agents can destabilize the binding protein complex (O'Brien, Dima et al. 2007), stronger equilibrium binding affinity typically exhibits a significant energy barrier to dissociation (Kastritis and Bonvin 2013). These barriers can be overcome using proportionally harsher denaturing agents, but this often cannot be achieved without irreversible damage to the structure or activity of the protein components. Furthermore, several fragmented inteins have been shown to withstand uniform denaturation conditions while remaining complexed in the presence of denaturing chaotropes such as 6M urea (Southworth, Adam et al. 1998), as well as denaturing detergents and reducing agents such as 2% w / v SDS and 150 mM DTT (Nichols, Benner et al. 2003). Therefore, conventional approaches to detaching protein-based affinity ligands are ineffective against INT. N Segments and INT C It may be logical to conclude that the segment may not be dissociated. This can be overcome by treating the N-intane ligand with increasingly harsh denaturing agents, but this carries the risk of irreversibly damaging the structure and function of the intein.

[0112] In addition to concerns about reversible binding, designing immobilization reactions to selectively immobilize N-intane ligands while simultaneously forming complexes with their homologous binding partners is not straightforward. Complex formation can induce a restricted folding state in the N-intane ligand, which in turn can reduce accessibility to the reactive immobilization site within the ligand. Furthermore, the chemicals used to covalently immobilize proteins to a substrate may be reactive to both the N-intane ligand and its homologous binding partner, potentially leading to graft polymerization of the latter into the substrate.

[0113] Even if a highly selective immobilization reaction could be designed, the homogeneous binding partner would be effectively consumed in the manufacturing process, thus incurring additional manufacturing costs. As shown in Figure 7, the homogeneous binding partner must either be expressed and purified separately and added to the N-intane ligand in trans, or co-expressed with the N-intane ligand in cell culture. The former requires a secondary manufacturing process for the homogeneous binding partner, which would obviously incur additional manufacturing costs, while the latter option, as shown in the example in Figure 2, clearly reduces the expression titer of the N-intane ligand.

[0114] However, it is worth noting that the solubility issue does not completely rule out the production of N-intane ligands using conventional manufacturing processes. In fact, the compositions described in Millipore patent application WO2016 / 073228 A1 and German patent application US2019 / 0263856 A1 mean that N-intane ligands can already be produced without the aid of a stabilizing homologous binding partner. Clearly, acceptable levels of soluble products can be produced by conventional methods, which suggests that improvements in soluble product yield should have only a slight impact on the overall productivity of the manufacturing process. For this reason, it was quite surprising that homologous binding partners enabled an order of magnitude improvement in yield, as shown in Figure 4.

[0115] Considering the additional processing requirements that arise when stabilizing the N-intene ligand with a congener-binding partner—namely, (a) forcing the intein complex to dissociate without damaging the ligand, (b) selectively immobilizing the ligand covalently in the presence of the congener, and (c) increasing the cost and / or reducing the expression titer to produce the ligand—it was unexpected to find that a slight increase in soluble yield could justify the barriers and costs incurred by introducing a congener-binding partner during the production process.

[0116] In this method, the expression of N-intei ligand is INT C This can occur in the presence of a congener-binding partner, such as a segment. As shown in Figure 7, the congener-binding partner and the N-intane ligand can be co-expressed in vivo from a single-plasmid system or a double-plasmid system, or the congener-binding partner can be expressed in separate cells and exposed to the N-intane ligand in trans before downstream processing. Due to the innate affinity between the N-intane ligand and the congener-binding partner, this pair spontaneously associates. This complex induces a “novel” folding state that the N-intane ligand cannot adopt on its own, and the congener-binding partner can shield certain hydrophobic and charged residues in the N-intane ligand that would otherwise lead to nucleation events, aggregation, and insolubility. Through these steps, a functionalized intein capture medium is generated that can capture the C-terminal intein tag for protein purification applications (as described, for example, in U.S. Patent No. 10,066,027 B2).

[0117] The association of intein complexes (defined as N-intanein ligands associated with a congener-binding partner) adopts a spherical structure, which enhances protein stability by limiting the various conformations that N-intanein ligands can adopt. This makes N-intanein ligands more resistant to degradation and / or aggregation during processing. For example, intein complexes may be 10, 20, 30, 40, 50, 60, 70, 80, or 90%, or 1, 2, 3, 4, or many orders of magnitude more soluble and / or resistant to degradation than N-intanein ligands that are not associated with a congener-binding partner. Additionally, the increased structural and chemical stability of N-intanein ligands reduces the formation of product-associated impurities involved in aggregation and degradation processes, thereby conferring greater physical and chemical homogeneity to this protein population than the N-terminal intein segment alone, significantly simplifying downstream separation processes.

[0118] Furthermore, since the solubility of the folded intein complex is significantly greater than that of the N-intein ligand alone, it can be concentrated to significantly higher levels before and during the resin coupling reaction, thereby improving the N-intein ligand density during the immobilization process. For example, the intein complex can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or 1, 2, 3, 4, or many orders of magnitude more soluble than the N-intein ligand alone, thus enabling N-intein ligand densities exceeding 10 mg ligand / mL resin bed volume. For example, the N-intei ligand density may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mg ligand / mL resin bed volume or more.

[0119] Once the intein complex is purified and concentrated, the N-terminal intein segment can be selectively immobilized covalently onto a chromatographic medium using standard bioconjugation techniques. This will be discussed in more detail later. This selectivity is possible through several mutations manipulated into the N-terminal intein segment (also discussed later). After immobilization, the N-terminal intein segment remains inactive to binding due to the induced folding state with its congener folding partner. In this regard, for the resulting intein-capturing resin to become functional, the binding activity to the N-terminal intein segment must be restored. This can be achieved by subjecting the immobilized intein complex to a strong chaotrope, a strong acid, or a strong base (e.g., 6M guanidine hydrochloride, 150mM phosphate, or 0.5M sodium hydroxide, respectively). However, it should be noted that this can perhaps be achieved using any other reagent or condition (e.g., heating) that can effectively denature the N-intane ligand and / or disrupt the association between the N-intane ligand and its homologous binding partner, which can then be washed away or otherwise removed, leaving the immobilized N-intane ligand.

[0120] When referring to “washing away” congener folding partners with chaotropes or acids, it should be noted that while this method removes the majority of congener folding partners, it is possible that less than 1%, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% (or any amount below or between these amounts) of congener binding partners may remain associated with the N-intei ligand. It is important to note that this homologous binding partner is not expressed fused with the desired protein of interest, as discussed herein, but rather is expressed during the remainder of the manufacturing process.

[0121] It should also be noted that disrupting the association between an N-intei ligand and its homologous binding partner must be done in a manner that allows the N-intei ligand to return to an active state, as opposed to permanent inactivation by denaturation conditions. An example is shown in Figure 5 (bottom panel), where the N-intei ligand is destroyed by guanidine hydrochloride, and then the new N-intei ligand of the desired state is reactivated. C Accepts tagged proteins. It should be noted that "interrupting association between" means actively interrupting the association or binding of the N-intene ligand to its homogeneous binding partner. This "detachment" or "interruption" of the homogeneous binding partner can be achieved by subjecting the immobilized intein complex to a chaotrope, a strong acid, or a strong base (e.g., guanidine hydrochloride, phosphoric acid, or sodium hydroxide, respectively), but this can perhaps be achieved using any other reagent or condition (e.g., heating) that can effectively denature the N-intene ligand and / or interrupt the association between the N-intene ligand and its homogeneous binding partner.

[0122] The primary motivation for the methods disclosed herein is to increase the solubility of N-inteinin ligands, but the stabilizing effect of homologous binding partners has been observed to have an unexpectedly beneficial effect on packing intein capture resins into conventional chromatography columns.

[0123] Column packing is an easily overlooked but not obvious aspect of fixed-bed liquid chromatography. Fixed-bed packing quality can significantly impact separation efficiency and is crucial for consistent and reproducible performance. Uniform bed packing is essential for uniform distribution of fluid flow across the column and consistent contact time. Thus, improper packing can lead to channeling, heterogeneous mixing, irregular contact time distribution, and / or underutilized bed (Rathore, Kennedy et al. 2003). These problems can effectively reduce separation efficiency and resolution, decrease product yield and purity, and result in inconsistent performance and poor reproducibility. Unfortunately, when N-intane ligands are conjugated to particle-based chromatography substrates, the bulk fluid behavior of the substrate changes, making it extremely difficult to properly pack the intein-capturing resin.

[0124] Particulate chromatography support substrates (i.e., resins made from cross-linked agarose, cellulose, dextran, polyacrylate, polystyrene, polyacrylamide, polymethacrylamide, or other polymers) are generally porous and compressible when subjected to moderate pressures, such as the differential pressure drop that occurs across the chromatography column during operation. When packed by gravity compression alone, the fixed bed made of these substrates contracts and expands as the flow through the column circulates on and off, respectively. The compression-relaxation cycle can damage the chromatography resin or degrade column performance by destabilizing the integrity of the packing bed, resulting in channeling, void formation, particle abrasion, excessive back pressure, column dead volume, non-uniform flow, and inconsistent residence time distribution. To avoid these problems, it is standard practice in the art to pre-compress the chromatography medium when it is packed into the column and then physically restrain the bed with the compressed volume to limit the potential re-expansion of the medium. This is typically achieved by fluid packing the resin as a slurry (i.e., pumping the slurry into the column at a high flow rate to exceed the normal operating column pressure difference) and / or by applying mechanical compression directly to the resin bed in the axial direction. However, overcompression of the resin can also adversely affect column function, so different chromatographic substrates are usually packed to a precisely defined compression range to ensure acceptable column performance.

[0125] The acceptable range of media compression is typically the compression factor (C). f (C) is specified as a volume ratio, and is obtained by dividing the volume of the fully relaxed / expanded or "gravity-settled" resin by the volume of the (compressed) resin bed in the packed column. f =V 膨張 / V 圧縮 ). C fThe acceptable range of C may vary for different columns depending on the matrix composition of the substrate and the diameter of the column being packed. Generally, substrate manufacturers determine the appropriate C based on an empirical assessment of the substrate matrix and the pressure it is shown to withstand. f Specify. Most soft porous matrices used in preparative bioprocessing are 1.10 in narrow-diameter laboratory-scale columns. <C f <1.15, or 1.15 for large-diameter process-scale columns. <C f Compression in the range of <1.20 is required (Stickel and Fotopoulos 2001).

[0126] If the packed column is not sufficiently compressed to achieve the desired compression coefficient, apply additional mechanical or hydraulic pressure and specify the C f It is obvious that the floor should be further compressed to reach the specified range. However, applying excessive force to the resin floor can cause cracking, fracture, and / or shattering of the substrate particles. Since evidence of over-compression or under-compression can often be detected by evaluating the uniformity of the flow through the packed floor, it is common practice in the art to perform standard column efficiency tests to verify the integrity of the floor after compression packing has been performed, in addition to determining the compression coefficient. Thus, a column is considered "sufficiently packed" only if both the compression coefficient and the column efficiency metrics are within the specified range.

[0127] A common assay used to evaluate column efficiency is the tracer pulse injection test. Numerous variations of this methodology are described in the literature (Rathore, Kennedy et al. 2003, GE-Healthcare 2010, Andres, Broeckhoven et al. 2015), but all methodologies generally follow a consensus procedure that involves operating the column isocratically at a constant flow rate, applying a pulse injection of an inert tracer, monitoring the column effluent as the tracer flows through the packed bed, and then analyzing the tracer distribution to infer the quality and uniformity of the column packing. The concentration of the tracer in the column eluate as a function of time is continuously monitored throughout the test, and the relationships and methodology shown in Figure 9 are used to calculate the standard column efficiency metrics, namely the peak asymmetry factor (As) and the reduced plate height (h). Under optimal packing conditions, the column has an asymmetry factor of As = 1.00 and a reduced plate height of h < 3. In practice, columns exhibiting an asymmetry factor in the range of 0.8 < As < 1.4 and a reduced plate height of h < 5 are generally considered satisfactory with respect to column efficiency metrics. A column asymmetry factor of As < 0.8 is typically an indicator of overpacking or excessive compression, whereas an asymmetry factor of As > 1.4 can indicate loose packing or bed instability.

[0128] For most porous particulate chromatography substrates, regardless of the functionalization of the substrate particles or the composition of the attached ligands, the allowable limits of the column efficiency metrics As and h are also met, along with a specified compression factor C fThe column can be packed up to this point. However, an unexpected finding arising from the development of this research is that once N-intane ligands are conjugated to them, the particulate substrate becomes far less compressible. Given this phenomenon, achieving a sufficiently well-packed resin bed when packing a column with intein-capturing resin has proven to be extremely difficult, if not impossible. Fortunately, the underlying mechanism presumed to be involved in the decrease in resin compressibility is similar to the mechanism thought to drive the aggregation of N-intane ligands, and therefore can be similarly mitigated by including a homologous binding partner during the packing process, as shown in Example 5.

[0129] As mentioned above, one of the defining characteristics of a partitioned intein is that the INT when separated from its respective counterpart is N Domain and INT C The domain has an inherently disordered structure. In this disordered state, the hydrophobic and charged amino acid residues of intein are exposed to the surrounding environment; the association and binding of intein are driven by these exposed residues, which attract and shield complementary residues in their corresponding domains, thereby folding together to form a more stable structured complex (Shah, Eryilmaz et al. 2013). While these exposed residues are essential for the function that makes the isolated intein useful for affinity capture, their inherent instability can also drive self-self interactions when concentrated, potentially leading to undesirable side effects. This is the cause of the ligand solubility problem mentioned above. N In addition to nucleation of domain aggregation, this phenomenon was found to also affect the interactions between resin particles having surface-immobilized N-intei ligands. As shown in Example 5, a naturally compressible agarose-based resin (C f (=1.15) became incompressible when conjugated with an N-intei ligand (C f =1.01). However, this effect is negated when the conjugated ligand is stabilized by the presence of a homologous binding partner, and the resin loses its original pre-conjugation compressibility (Cf It recovered to =1.15). Therefore, the present invention supports column packing, which is important for the usefulness of resin products.

[0130] INT expressed during fusion with the desired protein of the target C The segments are intended by the present invention as part of a protein purification protocol, but it should be noted in this application that they are not used until the N-intane ligand has already covalently bound to the solid support and the homologous binding partner has been removed. In the present invention, similar INT is used in both the production of the intein-capturing resin and the intended end use. C It is important to note that segments are used. Initially, they are used as homologous binding partners to protect the N-intane ligand and to promote its stability during the production of intein capture resins and the packing of intein capture resins into conventional chromatography columns. C The segment may contain a related protein or peptide, but not the desired protein of interest (the target protein, or the protein desired as the final product of this protein purification process). When the N-intei ligand is covalently coupled to a solid support, INT C The segments can be washed away by the methods disclosed herein. The manufacturing process is essentially complete after the N-intei ligand is immobilized and reactivated by washing away the homologous binding partner. In this regard, during the intended final use of the resin, a second INT containing the desired protein of interest is obtained. C The segment can associate with an N-intei ligand during the purification of the desired protein of interest.

[0131] INT disclosed herein N Segments and INT C All segments can be obtained, for example, from the Npu DnaE intelligence.

[0132] The N-intene ligands defined herein may be derived from native inteins (e.g., Npu DnaE; SEQ ID NO: 1), but may include additional modifications both inside and outside the standard defined intein sequence. For example, the INT encoded by the Npu DnaE gene N The segment is INT N The molecule (SEQ ID NO: 2) can be modified by conventional targeted mutagenesis to remove cysteine ​​residues from the portion. It may also have additional amino acids (defined as "within the N-terminal or C-terminal region") added to its N-terminus and / or C-terminus to improve cleavage performance and enable covalent fixation to resins. This has been described in detail previously. The generalized structures of the N-intei ligand and its main components are shown in Figure 6(a).

[0133] For example, the N-intane terminal segment can be modified such that at least one internal cysteine ​​residue is mutated to at least one serine residue, a peptide sequence can be added to the C-terminus to allow for easy purification and fixation to resin, and a sensitivity-enhancing peptide sequence can be added to the N-terminus to promote rapid pH-sensitive cleavage (see SEQ ID NO: 5 and the additional examples described below). The fully modified sequence would be referred to as the “N-intane ligand” as described herein (SEQ ID NO: 5) and would contain the Npu-intane sequence as well as the described mutations and attached sequences.

[0134] N-intane ligands may also contain immobilization moieties that enable or enhance covalent immobilization. For example, one or more amino acids within the C-terminal region may be cysteine ​​residues. This is desirable to eliminate side reactions associated with the nonspecific immobilization of N-intane ligands onto solid supports.

[0135] An example of an N-intane ligand with a mutated cysteine ​​residue can be found in SEQ ID NO: 2. The first cysteine ​​residue that is replaced (INT NIt should be noted that the first amino acid of the segment can be replaced with either alanine or glycine to eliminate intein splicing in the constructed intein complex.

[0136] The methods disclosed herein allow for the immobilization of an intein complex stabilized by a homologous binding partner onto a solid support substrate. Various supports can be used. For example, the solid support may be a polymer medium that enables the immobilization of the N-intane ligand, which may occur covalently or via affinity tags, with or without a suitable linker. When a linker is used, the linker may be an additional amino acid residue expressed fused with the N-intane ligand, or it may be another known linker for attaching the peptide to the support.

[0137] The N-intane ligands disclosed herein may include affinity tags as shown in Figure 6(a). These ligands minimize steric interference with the intein cleavage active site while enabling INT N Linker sequences can also be used to create distance between segments and affinity tags. It is generally accepted that linkers contain relatively unstructured amino acid sequences, and that the design and use of linkers are common in the art of designing fusion peptides. Various protein linker databases exist that will be familiar to those skilled in the art. This includes findings from Argos et al. J Mol Biol 1990 Feb 20;211(4)943-58, Crasto et al. Protein Eng 2000 May;13(5)309-12, George et al. Protein Eng 2002 Nov;15(11)871-9, Arai et al. Protein Eng 2001 Aug;14(8)529-32, and Robinson et al. PNAS May 26, 1998 vol.95 no.11 5929-5934, the entirety of which is incorporated herein by reference with respect to the instruction of the linker example.

[0138] Table 1 shows exemplary sequences of the N-terminal and C-terminal intein segments. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0139] In one example, the solid support substrate may be a solid chromatography resin skeleton such as crosslinked agarose. It may also be a membrane, monolith, or magnetic beads. The term “solid support matrix” or “solid matrix” refers to a solid skeletal material of a resin containing reactive functional groups that enable covalent bonding of ligands (such as N-intei ligands) to it. The skeletal material may be inorganic (e.g., silica) or organic. When the skeletal material is organic, it is preferably a solid polymer, and suitable organic polymers are well known in the art. Examples of solid support matrices suitable for use with the resins described herein include cellulose, regenerated cellulose, agarose, silica, coated silica, dextran, polymers (e.g., polyacrylate, polystyrene, polyacrylamide, polymethacrylamide, etc., including commercially available polymers such as fructgel, enzacryl, and azulactone), copolymers (e.g., copolymers of styrene and divinylbenzene), and mixtures thereof. Furthermore, copolymers, terpolymers, and higher-order polymers can be used if at least one monomer contains a reactive functional group in the resulting polymer, or can be derivatized to contain such a group. In additional embodiments, the solid support matrix may contain ionizable functional groups incorporated into its backbone.

[0140] Reactive functional groups of solid support matrix substrates that enable covalent bonding of N-intane ligands are well known in the art. Such functional groups react with certain peptide moieties, including hydroxyl, carboxyl, thiol, and amino groups. Conventional chemistry makes it possible to covalently bond ligands, such as N-intane ligands, using these functional groups. In addition, conventional chemistry makes it possible to incorporate such groups on solid support matrices. For example, carboxyl groups can be directly incorporated by using acrylic acid or its esters in the polymerization process. When acrylic acid is used during polymerization, carboxyl groups are present, or when acrylic acid esters are used, the polymer can be derivatized to contain carboxyl groups.

[0141] Affinity tags can be peptides or protein sequences expressed fused to the N-terminus or C-terminus of a protein, conferring specific chemical or physical properties that can aid in the purification of proteins from cells. Cells expressing peptides containing affinity tags can be pelleted, lysed, and the cell lysates applied to a column, resin, or other solid support that presents the ligand to the affinity tag. The affinity tag and any fusion peptide can also be bound to a solid support and washed several times with buffer to remove unbound (contaminated) proteins. If the target protein is bound to the affinity tag, it can be eluted from the solid support via a buffer that dissociates the affinity tag from the ligand, resulting in a purified protein, or it can be cleaved from the bound affinity tag using a soluble protease.

[0142] Examples of affinity tags can be found in Kimple et al. Curr Protoc Protein Sci 2004 Sep, Arnau et al. Protein Expr Purif 2006 Jul;48(1)1-13, Azarkan et al. J Chromatogr B Analyt Technol Biomed Life Sci 2007 Apr 15;849(1-2)81-90, and Waugh et al. Trends Biotechnol 2005 Jun;23(6)316-20, all of which are incorporated herein by reference in their entirety for teaching examples of affinity tags.

[0143] Affinity tags can also be used to facilitate the purification of a target protein using the disclosed modified peptides through a variety of methods, including but not limited to selective precipitation, ion exchange chromatography, binding to a precipitable ligand, dialysis (by altering the size and / or charge of the target protein), and other highly selective separation methods.

[0144] N-intane ligands may further include sensitivity-enhancing motifs (SEMs) that make the splicing or cleavage activity of the polymerized intein complex more sensitive to exogenous conditions. These sensitivity-enhancing motifs may enhance the cleavage activity of the intein complex (the desired INT). C The N-intei ligand bound to the tagged protein can be made more cleavable under certain conditions. Therefore, the sensitivity-enhancing motif can make the fragmented intein more sensitive to exogenous conditions compared to the native or naturally occurring intein.

[0145] The list of inteins can be found in Table 2 below. All inteins have the potential to become split inteins, although some inteins naturally exist in their split form. All inteins listed in Table 2 either exist as split inteins or have the potential to become split inteins. Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 4-11

[0146] The divided inteins of the disclosed compositions or the divided inteins that can be used in the disclosed manner may be modified or mutated inteins. The modified inteins are INT N Segment, INT C Modifications may include those to the segments, or both. Modifications may include additional amino acids fused to the N-terminal or C-terminal regions of either segment of the split intein, or they may be within either segment of the split intein. Table 3 lists the amino acids, their abbreviations, polarity, and charge. [Table 5-1] [Table 5-2]

[0147] Once obtained, the homologous binding partner and N-intei ligand can be separated and purified by appropriate combinations of known techniques. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation; methods utilizing molecular weight differences such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods utilizing charge differences such as ion exchange column chromatography; methods utilizing specific affinity such as affinity chromatography; methods utilizing hydrophobicity differences such as reverse-phase high-performance liquid chromatography; and methods utilizing isoelectric point differences such as isoelectric focusing electrophoresis. This will be discussed in more detail later.

[0148] C. Compositions and systems for protein purification A protein purification system is also disclosed, comprising an intein complex covalently immobilized on a solid support, wherein 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the N-inteiin ligands comprising the intein complex are associated with a congener-binding partner, and 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the congener-binding partners are not expressed in a fused state with the desired protein of interest.

[0149] N-intane ligands can be folded with congener-binding partners to stabilize them and to increase their soluble recovery rate while they are being processed and covalently immobilized on a solid support substrate. Furthermore, when N-intane ligands and congener-binding partners associate and fold within the intein complex, they have a more uniform size and charge distribution than the N-intane ligand alone, which can reduce the complexity of downstream processing.

[0150] Also disclosed are chromatographic resins comprising a base resin having a covalently bound N-intane ligand, wherein the compressibility difference (ΔC) measured with respect to the resin is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% compared to the base resin substrate. As defined herein, the term “base resin” refers to a resin-supported substrate to which the N-intane ligand or any other ligand is not bound. The definition of “compressibility difference (ΔC)” is provided elsewhere herein.

[0151] Also disclosed is a chromatographic resin comprising a base resin having a covalently bonded N-intei ligand, wherein the intrinsic functional compressibility coefficient (IFCF) measured with respect to the resin is 1.10 to 1.25. The definition of “intrinsic functional compressibility coefficient” (IFCF) is provided elsewhere in this specification.

[0152] It should be noted that the compressibility differences and intrinsic functional compressibility coefficients of the disclosed resin(s) are understood to be unique mechanical properties resulting from the stabilization of the bound N-intane ligand, induced by the presence of a congeneral bonding partner. Therefore, considering a particulate medium containing N-intane ligands covalently bonded to a solid resin, a compressibility difference of ΔC < 10% and / or an intrinsic functional compressibility coefficient (IFCF) between 1.10 and 1.25 can indicate the presence of a congeneral bonding partner.

[0153] As discussed in relation to the previous method, N-intane ligands covalently bonded to a resin can be stabilized by a homologous binding partner. The homologous binding partner is the C-terminal intein segment (INT C ) may include. The N-intane ligand can be stabilized by association with a congeneral binding partner in any processing step preceding the covalent immobilization of the ligand to the resin substrate. The N-intane ligand density on a solid surface can exceed 10 mg of N-intane ligand / mL resin volume. The N-intane ligand can be obtained from a conventional intein such as Npu DnaE intein. The congeneral binding partner can be derived from Npu DnaE intein. The N-intane ligand can be purified and INT N The N-intane ligand may include segments. The N-intane ligand does not need to contain any cysteine ​​residues within the INTN portion of the N-intane ligand. The N-intane ligand may include a naturally occurring INTN segment modified such that at least one internal cysteine ​​residue is mutated to at least one serine residue. The purified tag may contain one or more histidine residues.

[0154] In the filled resin bed described herein, the N-intei ligand may include one or more amino acids constituting the immobilized portion. The amino acids are INT NThe N-intane ligand can be encoded to be expressed by direct fusion to the C-terminus of a segment, or to be ligated to the C-terminus for use. One or more amino acids within the immobilization region may be cysteine ​​residues. The N-intane ligand may further include a sensitivity-enhancing motif to make it highly sensitive to exogenous conditions. The sensitivity-enhancing motif may be located in the N-terminal region of the N-intane ligand. Exogenous conditions may be pH, temperature, zinc ion concentration, or a combination thereof. The N-intane ligand may include SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, or 9. The homologous binding partner may include SEQ ID NOs: 10, 11, 12, 13, 14, 15, or 16.

[0155] Importantly, in this particular example of a protein purification system, the homogeneity-binding partner is not expressed fused with the target protein. This means that the homogeneity-binding partner does not contain, or link, bind to, or associate with, the desired protein or peptide as the final product of the protein purification system itself during the manufacturing process. This is because the N-intane ligand is expressed fused with the desired target protein in the previous protein purification system. C This distinguishes the "secondary" use of this protein purification system from its association (binding) to segments. It is also important to note that the homologous binding partners described herein may be expressed fused to other proteins or peptides, such as the linker or tagging moieties described earlier.

[0156] Also disclosed herein are solid affinity capture media comprising N-intane ligands covalently bound to their surface, and further comprising less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% of the bound N-intane ligands, but less than 0. 0.01, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 5.0, or more than 10% (or any amount above, between, or below these amounts) are associated with a congenerate-binding partner (forming an intein complex), while 50, 60, 70, 80, 90, or 100% (or any amount above, between, or below these amounts) of the congenerate-binding partner are not associated with the desired protein of interest.

[0157] This composition describes the properties of an affinity capture medium after an intein complex is exposed to a solid substrate, the N-intein ligand is immobilized on the substrate surface, the congeneral binding partners are dissociated from the N-intein ligand, and the unbound material, which includes the majority of the congeneral binding partners, is removed. It should be noted that when the resin is exposed to conditions that interfere with association and then washed, the residual amount of N-intein ligand remains associated with its congeneral binding partners. This creates a capture medium with a unique composition that does not exist except when a specific manufacturing method utilizing the congeneral binding partners is carried out, as described herein.

[0158] Kits are also disclosed herein. A kit may, for example, include an intein complex as described herein. Importantly, the intein complex may consist of an N-intane ligand and a homologue-binding partner, where the homologue-binding partner does not contain the desired protein of interest. A kit may include one or more vectors encoding the homologue complex. For example, a kit may include one vector encoding the N-terminal intein and another vector encoding the homologue-binding partner. In another example, they may be encoded by the same vector. A kit may also include instructions for use.

[0159] D. Experiment The following examples are provided to those skilled in the art to fully disclose and illustrate how the compounds, compositions, articles, apparatus, and / or methods claimed herein are made and evaluated, and are intended to be purely illustrative of the invention and not intended to limit the scope of what the inventors consider to be the invention. While efforts have been made to ensure accuracy with respect to numbers (e.g., quantities, temperatures, etc.), some errors and deviations should be taken into consideration.

[0160] Example 1: SDS-PAGE analysis comparing cell lysates of N-intei ligands N-intei ligand (SEQ ID NO: 5) expression was performed in three separate 1.0 L culture batches under identical culture conditions. After each expression culture batch, cells were harvested, aliquoted, and ligand solubility was examined. A fixed volume of the sample was resuspended in lysis buffer of the indicated concentration and lysed under identical conditions.

[0161] The results can be seen in Figure 1. The lanes are labeled according to the type of sample: whole cell lysate (WCL), clarified lysate (CL), and pellet (P). The WCL lane shows whole cell protein production, the CL lane represents fractional proteins that remain soluble through clarification of the lysate, and the P lane represents the fraction of insoluble proteins lost when the lysate is centrifuged. A rough approximation of the solubility of N-intane ligands can be estimated by visually comparing the size and intensity of the ligand bands (arrows) in each batch. This is done by estimating the amount of soluble ligand appearing in lane CL as part of the total ligand initially present in lane WCL for the same lysis batch.

[0162] Referring again to Figure 1, a comparison of expression batches A and B shows characteristic inter-batch variability in the percentage of all ligands that remain soluble. Normally, protein solubility is determined in vivo and is presumed to be primarily a result of properly formed secondary and tertiary structures. However, analysis of multiple lots obtained from expression batch C demonstrates that post-expression processing can dramatically affect the solubility of N-intei ligands. For example, the lysis of lot B-1 appears to show ligand solubility of over 90%, which means that "proper" in vivo synthesis was achieved in expression batch B. However, when the lysis and centrifugation are replicated with a second fixed amount (lot B-2) from batch B after one day, the apparent solubility drops to less than 10%, despite being supplied from the same expression culture and lysis under the same conditions. Lane P from lot B-2 confirms that almost all ligands initially present in the lysate precipitated during centrifugation. This data indicates that the N-intei ligand is unstable and can form insoluble aggregates regardless of proper in vivo synthesis and folding.

[0163] Example 2: Co-expression with a homologous binding partner Conventional single-product overexpression was compared with co-expression with a congener-binding partner by performing 1.0 L expression batches side-by-side under identical culture conditions. Each batch was inoculated with E. coli (BLR) strains transformed with pET vectors encoding the individual expression constructs to be compared. The control batch (conventional single-product overexpression) was transformed with a vector encoding only the N-intane ligand (SEQ ID NO: 5). The co-expression batch (co-expression of ligand + CBP-GFP fusion) was transformed with bisistronic vectors encoding the N-intane ligand (SEQ ID NO: 5) and the congener-binding partner-GFP tag fusion (SEQ ID NO: 13) separately for simultaneous co-expression. The second co-expression batch (co-expression of ligand + CBP) was transformed with different bisistronic vectors encoding the N-intane ligand (SEQ ID NO: 5) and the congener-binding partner (SEQ ID NO: 14) separately for simultaneous co-expression. All batches were processed side-by-side, and a constant 10 mL of LB growth medium was inoculated from LB-agar plates and grown at 37°C for approximately 16 hours using ampicillin as a selection marker. These seed cultures were then inoculated into flasks containing 1.0 L of concentrated growth medium and ampicillin, and grown in a shaking incubator at 37°C. The cultures reached the logarithmic metaphase (OD). 600 Once the concentration reached approximately 5.0, IPTG was added to a final concentration of 1.0 mM to induce expression, and the incubator temperature was lowered to 20°C to promote proper folding and solubility. The induced cultures were incubated with shaking for a further 16 hours, then collected separately by centrifugation and weighed. Cells collected from each batch were resuspended in lysis buffer proportional to the wet cell weight, effectively normalizing the concentration of each batch relative to its cultured cell density. A fixed volume of each normalized resuspension was mechanically dissolved and sampled, and the lysate was clarified by centrifugation at 20,000 × g for 10 minutes. The clarified lysate was sampled, decanted, and the residual solid was resuspended in an equal volume of buffer, and then sampled again. These samples—whole cell lysate (WCL), clarified lysate (CL), and pellet (P)—were then analyzed via SDS-PAGE to determine ligand solubility in each expression culture.

[0164] The results shown in Figure 2 demonstrate that co-expression of a congener-binding partner (CBP) in vivo increases cellular metabolism. Cellular resources are finite, and therefore, introducing a secondary co-expression product consumes important materials and energy that the cell could otherwise allocate to synthesizing the primary overexpression product.

[0165] Furthermore, the congenerate-binding partner stabilizes the ligand on a 1:1 stoichiometric basis, meaning that the addition of the congenerate-binding partner is structurally beneficial to the ligand only when it is present in an equal or excess molar amount. This means that the useful co-expression of any congenerate-binding partner requires that it be produced in an amount proportional to the ligand, thus consuming a significant portion of the cell's limited resources and effectively reducing the total productivity of the ligand.

[0166] In Figure 2, this effect can be clearly seen by comparing the WCL lanes from each processing method in conventional overexpression of a single ligand product, where the larger size and density of the ligand band indicate a higher level of expression compared to the corresponding WCL lane of the ligand co-expressed with its homologous binding partner.

[0167] Since co-expression of a homologue-binding partner reduces the ligand's titer, it was not expected that introducing a homologue-binding partner would have a positive impact on the net productivity of the manufacturing process. In fact, considering that association with a homologue-binding partner functionally inactivates the ligand, requiring further processing steps to remove the homologue-binding partner and reactivate the ligand, this approach is actually quite unpredictable on the surface.

[0168] However, the increased ligand stability and solubility induced by CBP may have positive effects elsewhere in the manufacturing process that can offset the relatively reduced ligand productivity titer caused by co-expression of cognate binding partners.

[0169] As seen in Figure 3, the presence of cognate binding partners clearly has a dramatic effect on ligand solubility. This effect is observed for both (SEQ ID NO: 13) and (SEQ ID NO: 14), regardless of the different mutations within each INT C -derived domain, as well as the presence (or absence) of GFP and His6 tags expressed in fusion with the cognate binding partner. This supports the idea that the important ability to induce intein complex formation is conserved and that various cognate binding partners can be devised to increase the solubility of N-intein ligands, as long as mutations within the cognate binding partner and / or substitutions with various fusion partners can be readily made. This trend can also be observed with several other cognate binding partners, such as any of those listed in SEQ ID NOs: 10 - 16.

[0170] Example 3: Ligand Solubility Figure 4 shows Coomassie-stained SDS-PAGE analyses for each batch of samples of whole cell lysate (WCL), clarified lysate (CL), and pellet (P). The WCL lane shows the total cellular productivity titer of the ligand, the P lane shows the relative proportion of ligand lost when insoluble debris is centrifuged away and discarded, and the CL lane represents the feedstock containing the fraction of soluble ligand (arrow) available for loading and capture by subsequent IMAC purification.

[0171] Figure 4 also shows chromatograms tracking absorbance at 280 nm (A280) through parallel IMAC purification performed in batches of conventional single-product overexpression (top) and CBP coexpression (bottom). A280 provides a quantitative approximation of the total protein concentration in the mobile phase as it exits each IMAC column. The total amount of ligand recovered in each purification can be estimated by integrating the A280 peaks that occur during the elution phase (normalized retention volume > 21 CV). As shown in the right panel, samples taken from the peaks labeled E1 and E2 were further analyzed by SDS-PAGE to assess purity and confirm accurate A280 quantification.

[0172] Figure 4 shows SDS-PAGE analysis of samples taken from parallel IMAC elution peaks E1 (conventional single-product overexpression) and E2 (CBP co-expression). Each fraction shows highly purified and concentrated ligand products with similar levels of minor contamination from co-purified host cell proteins. The total ligand mass recovered by each IMAC purification was calculated by integrating the A280 signal throughout the entire elution phase. To account for differences in cell density between expression batches, the total mass recovered in each elution is normalized against the total biomass (wet cell weight) lysed to prepare the feedstock for its purification. This normalized yield is reported for each purification under its corresponding elution lane.

[0173] Example 4: Dynamics of purification and cleaning for end use Two batches of intein-capturing resin were prepared using the same immobilized N-intane ligand (SEQ ID NO: 5). The first batch was prepared using conventional single-product overexpression and standard bioprocessing techniques, while the second batch was prepared using the novel manufacturing process claimed herein.

[0174] For a novel manufacturing process, an N-intane ligand (SEQ ID NO: 5) was co-expressed with a congener-binding partner (SEQ ID NO: 13). The co-expressed products bound to each other to form an intein complex, which was then purified, concentrated, buffer-exchanged, and covalently immobilized on a chromatography resin. The resin was then treated with a 6M GdnHCl gradient wash to dissociate the complex and refold the N-intane ligand. Since the immobilization reaction occurs selectively with the N-intane ligand, the ligand is retained by its covalent bond to the resin while the dissociated congener-binding partner is washed away. This "activates" the resin, and as a result, the N-intane ligand is, here, the desired INT C It can freely capture tagged proteins.

[0175] After manufacturing is complete, the resin from each batch is packed into a gravity flow chromatography column, which is then used to obtain the desired INT C Identical parallel purification was performed on tagged proteins (SEQ ID NO: 17). For these purifications, the target INT was used. C A single batch of lysis solution containing the tagged protein was processed from a single expression batch, and then equally divided and applied to each column to ensure comparability when evaluating the performance of each resin batch. These purifications also demonstrate the intended end use of the intein capture medium.

[0176] In Figure 5, the upper panel shows the performance of conventionally manufactured materials, which appears only superficially different from the performance of the lower panel, where the capture medium was manufactured using the method disclosed herein. This comparison demonstrates that a strong chaotrope wash (6M GdnHCl) can effectively dissociate the congener-binding partner from the intein complex and reactivate the immobilized N-intene ligand. Extended, this also demonstrates that the presence of the congener-binding partner during manufacturing does not adversely affect the performance of the final product (intene capture medium).

[0177] Example 5: Column packing of chromatography resin supported by a homologous binding partner Batches of purified N-intane ligand were prepared using the novel homologous binding partner stabilization technique described herein. As shown in Figure 7, E. coli (BLR) was transformed with a single-vector bicistronic plasmid to separately encode the N-intane ligand (SEQ ID NO: 18) and homologous binding partner (SEQ ID NO: 13) for in vivo ligand stabilization. The N-intane ligand and homologous binding partner were coexpressed, recovered, and purified using standard preparative liquid chromatography techniques. The resulting product, i.e., the intein complex formed by the spontaneous association of the N-intane ligand and homologous binding partner, was then dispensed into two reaction batches for covalent immobilization onto chromatography resin.

[0178] The immobilization reaction involves a 6% crosslinked agarose chromatography resin (average particle size d) derivatized with thiol-reactive functional groups. p The procedure was carried out using a cysteine-immobilized portion (90 μm). A fixed amount of purified cysteine ​​was reacted with this resin to selectively conjugate the N-intane ligand via the manipulated cysteine-immobilized portion. Each reaction batch was then passivated with excess thiols to inactivate any remaining immobilized sites on the resin. After the reaction and passivation, the first resin reaction batch (denoted as "-CBP") was subjected to a denaturing low-pH exfoliation treatment in a stirred vessel to dissociate and remove the congeneral binding partner from the resin (shown in Figures 7 and 8(b)). The second resin reaction batch (denoted as "+CBP") was left untreated to allow the congeneral binding partner to remain complexed with the resin-immobilized N-intane ligand. This allowed for direct comparison and evaluation of the resin properties when the N-intane ligand was stabilized by the congeneral binding partner. Next, both batches were treated with a final wash by passing more than 20 volume equivalents of phosphate-buffered salt (PBS) pH 7.4 through each batch to remove any remaining solvent, reactants, unreacted ligands, and / or dissociated homologous binding partners. The resin was drained using a funnel, then resuspended with fresh PBS, transferred to a graduated cylinder, allowed to settle by gravity for at least 12 hours, and then adjusted to a 50% slurry by pipetting.

[0179] Next, these resins were flow-packed side-by-side on the same chromatography column to evaluate the effect of homologous binding partners on column packing and overall flow uniformity of the packed bed. For each resin batch, 4.0 mL of 50% slurry was added to a 6.6 mm diameter chromatography column, and the remaining headspace of each column was packed with additional PBS to replace any air in the column. The column was then sealed at the column inlet with a height-adjustable flow adapter and connected to the FPLC. The flow adapter was first set to the expanded position using the inlet frit approximately 5 cm above the settled resin bed, and then PBS was pumped through the column at a linear column velocity of 50 cm / hour to ensure resin settling. The height of the settled resin bed (L0) was measured and recorded for each column. Next, the column inlet was vented, and the height of the flow adapter was adjusted so that the inlet frit was positioned 0.5 cm above the settled resin bed. The column inlet was then reconnected to the FPLC and constant-pressure flow packing was started. Additional PBS was passed through the column at a PID-controlled flow rate set to maintain a pressure drop across the column of ΔP = 2.0 bar. After the bed compression stabilized, the packing flow was maintained for at least 5 minutes, and then the flow adapter was adjusted further downward until the inlet frit physically contacted the top of the compressed resin bed. The FPLC flow was restarted at a constant flow rate equivalent to 50 cm / hour and pumped for another 5 minutes. The resin bed was visually inspected to confirm that no additional bed compression or void formation had occurred during the final packing step. The height of the settled resin bed (L) was measured and recorded for each column. Using these measurements, Equation C f =L0 / L is used to calculate the volume compressibility of the bed for each resin (C f The result was calculated. The result is shown in Figure 10.

[0180] After column packing was complete, a standard column efficiency test using inert tracer pulse injection was performed on each column to evaluate the flow uniformity of the entire packed bed. Each test was performed using PBS running buffer pumped at a constant linear velocity of 50 cm / hour. After equilibration, 200 μL pulsed tracer solution (PBS pH 7.4 + 1.0 M NaCl + 0.1% (v / v) acetone) was injected into the column. Isocratic elution of the tracer was continuously monitored for an additional 5 CV by inline UV spectroscopy, and the tracer concentration in the column eluate was measured by absorbance (A) at a wavelength of λ = 280 nm. 280 ) was shown. Chromatograms from tracer pulse experiments performed for each resin are shown in Figure 10(a). Applying the methodology commonly practiced by those skilled in the art, as shown in Figure 9, these data were then used to determine the peak asymmetry coefficient (A) for each batch. s The reduced plate height (h) was calculated, and the column packing quality for each resin batch was verified. f , A s Figure 10(b) reports on , and h for each batch to demonstrate the effect of filling with intein-capturing resin with and without the support of a homologous bonding partner.

[0181] Interestingly, the agarose resin-based matrix (i.e., the base resin without the immobilized ligand) is C f It can be packed down to a compressibility factor of 1.15, but when N-intei ligand is conjugated (-CBP batch), the resin is no longer compressible when slurry is packed at ΔP = 2.0 bar, C fA compression coefficient of only 1.01 was achieved. When attempting to further compress the resin bed by mechanical compression, asymmetry occurred, the measurement criteria for the plate height test decreased outside the allowable range, and excessive pressure could crack or crush the resin base material, thus indicating that the integrity of the packed bed was impaired. However, under the same conditions in other respects, when filling with a resin batch stabilized by a cognate binding partner (+CBP batch), the compressibility of the resin was restored. As seen in Figure 10(b), +CBP maintains an acceptable asymmetry and reduced test measurement criteria for plate height while f it was possible to slurry-fill up to a compression coefficient of 1.15, reflecting the performance of the unmodified base resin.

[0182] Aspects of the present invention are further described in the following sections: [Section 1] A method for stabilizing an N-intei ligand during expression and purification, The steps include forming an intein complex through the assembly of an aN-intein ligand and a homologous binding partner, b. A step of purifying the intein complex, c. The step of immobilizing the intein composite onto a solid support, The method, including the method described above. [Section 2] moreover, d. The step of subjecting the intein complex to conditions that disrupt the association between the N-intein ligand and the homologous binding partner, e. A step of providing conditions that cause the N-intei ligand to fold into an active state while remaining immobilized, The method described in item 1 above, including the method described in item 1 above. [Section 3] The method according to item 1 or 2, wherein the homologous binding partner includes a C-terminal intein segment. [Section 4] The method according to any one of items 1 to 3 above, wherein in step a), the N-intei ligand and the homologue-binding partner are co-expressed in vivo. [Section 5] The method according to item 4, wherein the N-intei ligand and the homologue-binding partner are expressed in a single cell derived from a single plasmid or a two-plasmid system. [Section 6] The method according to any one of items 1 to 3, wherein in step a), the N-intei ligand is exposed in trans to the homologous binding partner after the expression of the N-intei ligand. [Section 7] The method according to any one of items 1 to 6, wherein in step c), the N-terminal intein segment is covalently immobilized to the solid support. [Section 8] The method according to any one of items 1 to 7, wherein the solid support is a conventional chromatography medium comprising a porous resin, a membrane, a monolith, or magnetic beads. [Section 9] The method according to item 8, wherein the chromatography medium is a solid chromatography resin skeleton. [Section 10] The method according to any one of items 7 to 9 above, wherein the N-intane ligand density on the solid support exceeds 10 mg of N-intane ligand / mL resin volume. [Section 11] The method according to any one of items 1 to 10, wherein conditions can be created to prevent association between the N-intei ligand and the homologous binding partner by using a chaotrope agent or a basic or acidic solution. [Section 12] The method according to item 2, wherein the association between the N-intane ligand and the homologous binding partner is disrupted, followed by a condition that returns the N-intane ligand to an active state in which it can accept a new binding partner. [Section 13] The method according to item 12, wherein the destructive condition includes one of the following: a chaotrope such as guanidine hydrochloride, an acid such as phosphoric acid, or a base such as sodium hydroxide. [Section 14] The method according to any one of items 1 to 13 above, wherein the N-intei ligand is derived from a conventional intein. [Section 15] The method according to item 14 above, wherein the N-intei ligand is derived from Npu DnaE intein. [Section 16] The method according to item 14, wherein the homologous binding partner is derived from Npu DnaE intein. [Section 17] The aforementioned N-intene ligand is a purified tag and INT N A method according to any one of items 1 to 16 above, including a segment. [Section 18] The N-intane ligand is the INT of the N-intane ligand. N The method described in item 17 above, wherein the portion contains no cysteine ​​residues. [Section 19] Naturally occurring INT N The method according to item 17 or 18, wherein the N-intei ligand containing the segment is modified such that at least one internal cysteine ​​residue is mutated to at least one serine residue. [Section 20] The method according to item 17, wherein the purified tag comprises one or more histidine residues. [Section 21] The method according to any one of items 1 to 20, wherein the N-intei ligand comprises one or more amino acids constituting an immobilization portion. [Section 22] The aforementioned amino acid, N The method according to item 21, wherein the segment is encoded to be directly fused to the C-terminus for expression, or to be usably ligated to the C-terminus, thereby enabling covalent fixation of the N-intei ligand. [Section 23] The method according to item 21 or 22, wherein one or more amino acids in the immobilized portion are cysteine ​​residues. [Section 24] The method according to any one of items 1 to 23, wherein the N-intei ligand further comprises a sensitivity-enhancing motif that makes it highly sensitive to exogenous conditions. [Section 25] The method according to item 24, wherein the sensitivity-enhancing motif is located in the N-terminal region of the N-intei ligand. [Section 26] The method according to item 24 or 25, wherein the exogenous conditions are pH, temperature, zinc, or a combination thereof. [Section 27] The method according to any one of items 1 to 26, wherein the N-intei ligand includes SEQ ID NOs. 2, 3, 4, 5, 6, 7, 8, 9, or 18. [Section 28] The method according to any one of items 1 to 26, wherein the homologous bonding partner includes sequence numbers 10, 11, 12, 13, 14, 15, or 16. [Section 29] A protein purification medium comprising an N-intane ligand covalently immobilized on a solid support, wherein 90% or more of the N-intane ligand is associated with a congener-binding partner, and at least 90% of the congener-binding partner is not expressed in a fused state with the desired protein of interest. [Section 30] The aforementioned homologous binding partner is a C-terminal intein (INT C The media described in item 29 above, including the segment. [Section 31] A chromatography resin comprising a base resin having a covalently bound N-intane ligand, wherein more than 0.001% of the N-intane ligand is associated with a congener-binding partner, and furthermore, 90% of the congener-binding partner is not expressed in a fused state with the desired protein of interest. [Section 32] The aforementioned homologous binding partner is a C-terminal intein (INT C ) A chromatography resin as described in item 31 above, including a segment. [Section 33] An expression vector comprising an exogenous nucleic acid, wherein the exogenous nucleic acid encodes an N-intane ligand and a homologue-binding partner, the N-intane ligand is encoded to be expressed together with a purified tag, and the homologue-binding partner is not encoded for expression in a fused state with the desired protein of interest. [Section 34] A cell containing the expression vector described in item 33 above. [Section 35] The vector according to item 34, wherein the homologue-binding partner is encoded to be expressed fused with a protein or peptide that is not the desired protein of interest. [Section 36] The vector according to item 35 above, wherein the protein or peptide is an affinity tag. [Section 37] A chromatography resin comprising a base resin having a covalently bonded N-intei ligand, wherein the measured compressibility difference (ΔC) of the resin is less than 10% compared to that of the base resin substrate. [Section 38] A chromatographic resin comprising a base resin having a covalently bonded N-intei ligand, wherein the intrinsic functional compressibility coefficient (IFCF) measured with respect to the resin is 1.10 to 1.25. [Section 39] The resin according to items 37-38, wherein the N-intei ligand is stabilized by a homologous binding partner. [Section 40] The aforementioned homologous binding partner is the C-terminal intein segment (INT C The resins described in item 39 above, including ). [Section 41] The resin according to any one of items 37 to 40, wherein the N-intane ligand density on the solid surface exceeds 10 mg of N-intane ligand / mL resin volume. [Section 42] The resin according to any one of items 37 to 41, wherein the N-intei ligand is derived from a conventional intein. [Section 43] The resin according to item 42 above, wherein the N-intei ligand is derived from Npu DnaE intein. [Section 44] The resin according to item 39, wherein the aforementioned homologous bonding partner is derived from Npu DnaE intein. [Section 45] The aforementioned N-intene ligand is a purified tag and INT N A resin according to any one of items 37 to 44 above, including a segment. [Section 46] The N-intane ligand is the INT of the N-intane ligand. N The resin described in item 45 above, which does not contain any cysteine ​​residues within a portion. [Section 47] Naturally occurring INT N The resin according to item 45, wherein the N-intei ligand containing the segment is modified such that at least one internal cysteine ​​residue is mutated to at least one serine residue. [Section 48] The resin according to item 45, wherein the purified tag contains one or more histidine residues. [Section 49] The resin according to any one of the above items 37 to 48, wherein the N-intei ligand comprises one or more amino acids constituting the immobilization portion. [Section 50] The aforementioned amino acid, N The resin according to item 49, which is coded to be expressed by direct fusion to the C-terminus of a segment, or to be usably linked to the C-terminus. [Section 51] The resin according to item 49 or 50, wherein one or more amino acids in the immobilized portion are cysteine ​​residues. [Section 52] The resin according to any one of the above items 37 to 48, wherein the N-intei ligand further comprises a sensitivity-enhancing motif that makes it highly sensitive to exogenous conditions. [Section 53] The resin according to item 45, wherein the sensitivity-enhancing motif is located in the N-terminal region of the N-intei ligand. [Section 54] The resin according to item 52 or 53, wherein the exogenous conditions are pH, temperature, zinc, or a combination thereof. [Section 55] The resin according to any one of the above items 37 to 54, wherein the N-intei ligand comprises SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 18. [Section 56] The resin according to any one of the above items 39 to 55, wherein the homologous bonding partner includes sequence numbers 10, 11, 12, 13, 14, 15, or 16. 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Claims

1. A method for stabilizing an N-intei ligand during its expression and purification, a. N-terminal intein (INT Ｎ A step of forming an intein complex via the assembly of an N-intene ligand containing a segment and a homologue-binding partner, wherein the homologue-binding partner is a C-terminal intein (INT Ｃ ) Steps including segments, b. A step of purifying the intein complex, c. The step of immobilizing the intein composite onto a solid support, The method, including the method described above.

2. moreover, d. The step of subjecting the intein complex to conditions that disrupt the association between the N-intein ligand and the homologous binding partner, e. A step of providing conditions that cause the N-intei ligand to fold into an active state while remaining immobilized, The method according to claim 1, including the method described in claim 1.

3. The method according to claim 1 or 2, wherein in step a), the N-intei ligand and the homologue-binding partner are co-expressed in the cell.

4. The method according to claim 3, wherein the N-intane ligand and the homologue-binding partner are expressed from a single plasmid or a two-plasmid system within a single cell, wherein in the two-plasmid system, the N-intane ligand and the homologue-binding partner are encoded on two different coupling plasmids, respectively.

5. The method according to claim 1 or 2, wherein in step a), the N-intane ligand is exposed to the homologue-binding partner expressed in another cell after the expression of the N-intane ligand.

6. The method according to any one of claims 1 to 5, wherein in step c), the N-terminal intein segment is covalently immobilized to the solid support.

7. The method according to any one of claims 1 to 6, wherein the solid support is a chromatography medium comprising a porous resin, a membrane, a monolith, or magnetic beads.

8. The method according to claim 7, wherein the chromatography medium is a solid chromatography resin skeleton.

9. The method according to claim 8, wherein the N-intane ligand density on the solid support exceeds 10 mg of N-intane ligand / mL resin volume.

10. The method according to any one of claims 1 to 9, wherein conditions can be created to prevent association between the N-intei ligand and the homologous binding partner by using a chaotrope agent or a basic or acidic solution.

11. The method according to claim 2, wherein, after conditions are provided for disrupting the association between the N-intane ligand and the homologous binding partner, conditions are provided for restoring the N-intane ligand to an active state in which the N-intane ligand can accept a new homologous binding partner.

12. The method according to claim 11, wherein the destructive conditions include exposure to one of a chaotrope agent, guanidine hydrochloride, acid, phosphoric acid, base, or sodium hydroxide.

13. The method according to any one of claims 1 to 12, wherein the N-intei ligand is derived from a naturally occurring intein.

14. The method according to claim 13, wherein the N-intane ligand is derived from NpuDnaEintane.

15. The method according to claim 13, wherein the homologous bonding partner is derived from NpuDnaEintein.

16. The aforementioned N-intene ligand is a purified tag and INT Ｎ A method according to any one of claims 1 to 15, comprising a segment.

17. The N-intane ligand is the INT of the N-intane ligand Ｎ The method according to claim 16, wherein the portion does not contain any cysteine ​​residues.

18. Naturally occurring INT Ｎ The method according to claim 16 or 17, wherein the N-intei ligand containing the segment is modified such that at least one internal cysteine ​​residue is mutated to at least one serine residue.

19. The method according to claim 16, wherein the purified tag comprises one or more histidine residues.

20. The method according to any one of claims 1 to 19, wherein the N-intei ligand comprises one or more amino acids constituting an immobilization portion.

21. The aforementioned amino acid, Ｎ The method according to claim 20, wherein the segment is encoded to be directly fused to the C-terminus for expression, or to be usably linked to the C-terminus, thereby enabling covalent fixation of the N-intei ligand.

22. The method according to claim 20 or 21, wherein one or more amino acids in the immobilized portion are cysteine ​​residues.

23. The method according to any one of claims 1 to 22, wherein the N-intei ligand further comprises a sensitivity-enhancing motif that makes it highly sensitive to exogenous conditions.

24. The method according to claim 23, wherein the sensitivity-enhancing motif is located in the N-terminal region of the N-intei ligand.

25. The method according to claim 23 or 24, wherein the exogenous conditions are pH, temperature, zinc concentration, or a combination thereof.

26. The method according to any one of claims 1 to 25, wherein the N-intei ligand comprises the sequence shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, or 18.

27. The method according to any one of claims 1 to 25, wherein the homologous bonding partner comprises the sequence shown in SEQ ID NOs: 10, 11, 12, 13, 14, 15, or 16.

28. N-terminal intein (INT) covalently immobilized on a solid support Ｎ A protein purification medium comprising an N-intane ligand containing a segment, wherein more than 90% of the N-intane ligand is associated with a homologous binding partner, and the homologous binding partner is a C-terminal intein (INT Ｃ The protein purification medium includes a segment, wherein at least 90% of the homologue-binding partners are not expressed in a fused state with other proteins.

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