Purified resin and method of use thereof

Abstract

The present invention generally relates to compositions and methods for purifying biological products. More specifically, the present invention relates to purified resins and methods for using the same.

Description

[Technical Field]

[0001] Field of Invention The present invention generally relates to compositions and methods for purifying biological products. More specifically, the present invention relates to purified resins and methods for using the same. [Background technology]

[0002] Background of the Invention Lentiviruses are enveloped retroviral viruses that are gaining increasing importance for biopharmaceuticals because they enable the stable integration of transgenes expressed in both dividing and non-dividing cells. In recent years, the number of clinical trials of gene therapy based on lentiviral vectors (LVs) has increased. Their use in gene therapy for several disease conditions has been reported both ex vivo and in vivo. The success of these clinical trials requires improvements in both quantitative aspects of LV purification methods, such as increasing processing capacity and product quality, and qualitative aspects, such as replacing conventional methods that are used on a small scale but are not economically and / or technically feasible on a large scale.

[0003] Current production techniques generally depend on the production system, false type LV, recovery conditions, or even the titration technique, resulting in a rate of 10 per mL. 6 ~10 8 Only low functional potency in the form of introduction units (TU / mL) can be obtained. Furthermore, impurities vary depending on the production system. In the development of downstream processes (DSP), LV concentration is pursued simultaneously with the improvement of LV purity, but in this case, the impurity profile of the production culture medium must be taken into consideration. As with other biological products, the required degree of concentration and purification is fundamentally related to the application of the LV. For research / small-scale applications, extremely high purity products are usually not required. However, if the product is intended for clinical use, drug safety becomes a concern.

[0004] When the need for LV can be met by small-scale production, such as in scientific research, conventional purification and concentration techniques are mainly used. Methods based on centrifugation can be used as concentration and / or purification techniques.

[0005] In recent years, the development and use of DSPs involving chromatography processes and membrane and monolith-based separation operations have been reported. Current lentiviral vector (LVV) purification processes are based on anion exchange chromatography (AEX). This allows for the separation of LVVs from other contaminants based on their charge. However, the selectivity of this purification method is low, and the final product contains extracellular vesicles and other negatively charged contaminants. Furthermore, elution using high salt concentrations leads to loss of infectivity and a decrease in recovery rate. Therefore, affinity chromatography is emerging as a superior alternative for LVV purification due to the high selectivity and excellent retention capacity of this type of chromatography column.

[0006] Patent document US2022 010288A1 discloses a method for purifying lentiviral particles, comprising contacting the lentiviral particles with a fusion protein containing an LDL-R-derived peptide. The fusion protein described in US2022 010288A1 comprises a lentiviral domain and a polypeptide that exhibits phase behavior.

[0007] In the literature of Perry Christopher on November 2, 2022, “Lentiviral vector stability and process purification based on modifications to the viral envelope”, a purification method for pseudotyped lentiviral vectors containing the VSV-G protein that binds naturally to LDL-R is disclosed. This method is based on affinity binding of virus particles present in a culture medium at least at pH 7 to the CR2 domain of LDL-R bound to magnetic beads. However, in this method, elution of the vector did not succeed at pH 6.5 or lower, and transduction ability was not recognized in the elution supernatant with a solution that continuously decreased the pH.

[0008] Patent document WO2006 / 004660A2 discloses an affinity chromatography containing glutathione-agarose beads that bind to the GST-LDLR fusion protein.

Summary of the Invention

Problems to be Solved by the Invention

[0009] In the art, there is a need for improved resins and methods for rapidly and cost-effectively purifying virus particles such as lentiviral particles.

Means for Solving the Problems

[0010] Summary of the Invention The authors of the present invention developed an affinity chromatography system based on the interaction between the vesicular stomatitis virus G glycoprotein (VSV-G), which is an envelope protein of LVV, and the low-density lipoprotein receptor (LDL-R), which is a receptor present on the cell membrane that enables virus infection.

[0011] Therefore, in a first embodiment, the present invention relates to a method for purifying viral particles or virus-like particles (VLPs), comprising an envelope, the envelope comprising a VSVG protein or a functionally equivalent variant or fragment that retains the ability to bind to a low-density lipoprotein receptor (LDL-R), i) Contacting a sample containing viral particles or VLPs with a purified resin, ii) Washing the purified resin with an elution buffer, and iii) Recovering the virus particles or VLPs eluted from the purified resin. The purified resin includes, a) Low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, wherein the fragment or variant retains the VSVG binding ability of LDL-R. b) Matrix The method includes the above, and the purified resin does not contain polypeptides exhibiting phase behavior.

[0012] In another embodiment, the present invention is a) Low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, b) Matrix A purified resin containing the following, which does not contain polypeptides exhibiting phase behavior and is non-magnetic, is the present invention.

[0013] In another aspect, the present invention relates to the use of the purification resin of the present invention for the purification of viral particles or VLPs comprising an envelope containing a VSVG protein or a functionally equivalent variant or fragment thereof that retains the ability to bind to LDL-R. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1. LVV purification protocol using affinity chromatography with GSTCR2 as the affinity ligand. [Figure 2]Figure 2. Virus particles detected at each stage of the purification process as determined by ELISA p24. [Figure 3] Figure 3. Titer measurement results of the lentivirus sample before purification (initial), the unbound fraction (FT) from the affinity column, and the eluted lentivirus (Elu) after purification. [Figure 4] Figure 4. Determination of host cell protein levels in various samples during the purification process. Values ​​are converted to a predetermined viral titer. [Figure 5] Figure 5. Measurement of residual DNA present in various samples during the purification process. [Figure 6] Figure 6. Comparison with commercially available affinity purification technology CaptureSelect Lenti VSVG (ThermoFisher). Virus particles detected at each stage of both purification processes, as determined by ELISA p24. [Figure 7] Figure 7. Comparison with commercially available affinity purification technology: CaptureSelect Lenti VSVG (ThermoFisher). Titer measurement results for the lentivirus sample before purification (initial), unbound fraction (FT) from the affinity column, and eluted LVV (Elu) after purification for both purification systems. [Figure 8] Figure 8. Comparison with commercially available affinity purification technology: CaptureSelect Lenti VSVG (ThermoFisher). Measurement of host cell proteins in various samples during the purification process. Values ​​are expressed per transfection unit. [Figure 9] Figure 9. Comparison with commercially available affinity purification technology: CaptureSelect Lenti VSVG (ThermoFisher). Measurement of residual DNA present in various samples during the purification process. [Modes for carrying out the invention]

[0015] Detailed description of the invention Method of the present invention The authors of this invention have developed a novel system for purifying viral particles using affinity chromatography. This system is based on the interaction between envelope proteins, including the VSVG protein, and specific domains of LDL-R.

[0016] In a first embodiment, the present invention relates to a method for purifying viral particles or virus-like particles (VLPs), comprising an envelope comprising a VSVG protein or a functionally equivalent variant or fragment that retains the ability to bind to a low-density lipoprotein receptor (LDL-R), i) Contacting a sample containing viral particles or VLPs with a purified resin, ii) Washing the purified resin with an elution buffer, and iii) Recovering the virus particles or VLPs eluted from the purified resin. The purified resin includes, a) Low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, wherein the fragment or variant retains the VSVG binding ability of LDL-R. b) Matrix The present invention relates to a method that includes and, wherein the purified resin does not contain polypeptides exhibiting phase behavior.

[0017] The term "viral particle" refers to the entire viral particle, not a protein subunit or peptide. A viral particle consists of two or three parts: the viral genetic material, which consists of either DNA or RNA; a protein coat that protects these genes; and, if extracellular, a lipid envelope that surrounds the protein coat. The shape of a viral particle varies from simple helices and icosahedrons to more complex structures depending on the virus. The viral envelope is the outermost layer for many virus species. The envelope protects the genetic material during its life cycle as it travels between host cells.

[0018] In certain embodiments, the enveloped viral particles are selected from the group consisting of vesicular stomatitis viruses or retroviruses.

[0019] Therefore, in certain embodiments, the virus particle containing an envelope is a vesicular stomatitis virus particle.

[0020] Vesicular stomatitis virus (VSV) is an enveloped negative-strand RNA virus belonging to the genus Vediclovirus in the family Rhabdoviridae. The VSV genome encodes five structural proteins, including a single-pass transmembrane glycoprotein (G). Glycoprotein G of vesicular stomatitis virus (VSVG) is involved in receptor recognition on the surface of host cells, and subsequently induces membrane fusion via virion endocytosis and structural rearrangement at low pH. G plays a crucial role in the early stages of viral infection. First, it is responsible for the attachment of the virus to specific receptors. After binding, virions enter the cell via the cratrin-dependent endocytosis pathway. Under the acidic environment of the endocytic vesicle, G induces fusion of the viral membrane and the endosomal membrane, thereby releasing the genome into the cytosol and moving to the next stage of infection. VSVG is widely used for pseudotyping other viruses, and VSV-G pseudotyped lentiviruses (VSV-G-LV) exhibit broad tropism similar to that of VSV.

[0021] Pseudotyping is a process of creating a virus or viral vector by combining it with a foreign viral envelope protein. This results in pseudotyped viral particles (also called pseudoviruses). This method allows for altering host tropism or increasing / decreasing the stability of viral particles using foreign viral envelope proteins. Since pseudotyped particles do not possess the genetic material necessary to generate further viral envelope proteins, phenotypic changes are not passed on to subsequent viral particles. Pseudotyping allows for the control of envelope protein expression. A commonly used protein is glycoprotein G (VSVG) derived from vesicular stomatitis virus (VSV).

[0022] In another specific embodiment, the virus particle containing an envelope is a retrovirus particle.

[0023] Retroviruses are a type of virus that alters the genome of an invading host cell by inserting a copy of its own RNA genome into the cell's DNA. Retroviruses belong to three basic groups and have many subfamilies. - Oncoretroviruses (carcinogenic retroviruses) include human T-lymphotropic virus (HTLV), which causes a type of leukemia in humans, and mouse leukemia virus (MLV) in mice. - Lentiviruses (slow-onset viruses) include HIV-1 and HIV-2, which cause acquired immunodeficiency syndrome (AIDS) in humans. - Spumaviruses (foam viruses) are benign and not associated with diseases in humans or animals.

[0024] In a particular embodiment, the retrovirus particle is selected from the group consisting of alpha retrovirus particles, beta retrovirus particles, delta retrovirus particles, epsilon retrovirus particles, gamma retrovirus particles, or lentivirus particles.

[0025] In a more detailed embodiment, the virus particle containing an envelope is a gamma retrovirus particle.

[0026] Gamma retroviruses are a genus of the family Retroviridae. Exemplary species include mouse leukemia virus and feline leukemia virus.

[0027] In another specific embodiment, the virus particle containing an envelope is a lentiviral particle.

[0028] Lentiviruses are enveloped retroviral viruses that are gaining increasing importance for biopharmaceuticals because they enable the stable integration of transgenes, which are expressed in both dividing and non-dividing cells.

[0029] "Virus-like particles" (also called VLPs) are particles that resemble viruses but do not contain viral genetic material. VLPs are formed by the expression and self-assembly of viral structural proteins such as capsid proteins.

[0030] VLPs may be derived from hepatitis B virus (HBV) or may consist of a small HBV-derived surface antigen (HBsAg). VLPs are made from components of various virological families, including parvoviridae (e.g., adeno-associated viruses), retroviridae (e.g., HIV), flaviviridae (e.g., hepatitis C virus), paramyxoviridae (e.g., Nipah), and bacteriophages (e.g., Qβ, AP205). Thus, in certain embodiments, VLPs are derived from HBV, HBsAg, adeno-associated viruses, HIV, hepatitis C virus, Nipah, or bacteriophages.

[0031] The term "envelope" refers to the outermost layer of many virus species. The envelope protects the genetic material during its life cycle as it travels between host cells. The envelope is generally derived from parts of the host cell membrane (phospholipids and proteins), but also includes some viral glycoproteins.

[0032] As described above, the envelope of a viral particle or VLP contains a VSVG protein or a functionally equivalent variant or fragment that retains the ability to bind to the low-density lipoprotein receptor (LDL-R). Therefore, in certain embodiments, the envelope of a viral particle or VLP contains a VSVG protein. The VSVG protein can independently bind to different CR domains of the low-density lipoprotein receptor (LDL-R).

[0033] In certain embodiments, the envelope of a viral particle or VLP contains an extracellular fragment of the VSVG protein. In more detailed embodiments, the VSVG fragment contains or consists of a sequence of extracellular fragments of VSV-G. [ka]

[0034] The low-density lipoprotein receptor (LDL-R) is a mosaic protein consisting of 839 amino acids (after removal of a 21-amino acid signal peptide) that mediates the endocytosis of cholesterol-rich low-density lipoprotein (LDL). LDL-R is a cell surface receptor that recognizes apolipoprotein B100 (ApoB100), embedded in the outer phospholipid layer of very low-density lipoprotein (VLDL), its remnants (i.e., intermediate-density lipoprotein (IDL)), and LDL particles. This receptor also recognizes apolipoprotein E (ApoE), found in chylomicron remnants and IDL. The LDL receptor mediates the endocytosis of cholesterol-rich LDL and thus maintains LDL levels in plasma.

[0035] The LDL-R ectodomain consists of a ligand-binding domain, an epidermal growth factor (EGF) precursor homology domain, and a high-density C-terminal domain rich in O-linked oligosaccharides. The ligand-binding domain consists of seven cysteine-rich repeats (CR1-CR7). Each repeat consists of approximately 40 amino acids, with six cysteine ​​residues linked by three disulfide crosslinks and Ca 2+ It contains acidic residue clusters that coordinate ions. Intracellular release of the cargo is driven by a conformational change of LDL-R from open to closed due to low pH.

[0036] In certain embodiments, LDL-R is a human protein identified by Uniprot accession number P01130 (entry version 261, sequence version 1, June 28, 2023).

[0037] The LDL-R gene family consists of transmembrane receptors located on the cell surface, involved in the uptake of lipoproteins via endocytosis, and involves Ca2+ binding to ligands. 2+ These receptors all share a common intracellular domain containing multiple CR repeats (up to several dozen), an EGF precursor-like repeat, a transmembrane region, and at least one endogenous signaling sequence.

[0038] In another specific embodiment, the envelope of the viral particle or VLP contains a functionally equivalent variant of the VSVG protein that retains its ability to bind to the low-density lipoprotein receptor (LDL-R).

[0039] In the context of the present invention, a “functionally equivalent variant” of the VSVG protein is understood to be any sequence having additions, substitutions, deletions or combinations thereof to its amino acid sequence, and / or being chemically modified to substantially maintain the function of the protein, particularly its ability to bind to LDL-R. Preferably, a functionally equivalent variant of VSVG exhibits at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, and advantageously 99% of the above ability.

[0040] Suitable assays for determining whether VSVG binds to LDL-R include, but are not limited to, co-immunoprecipitation, far-western blotting, protein ligation assays, affinity electrophoresis, and isothermal titration calorimetry.

[0041] Functionally equivalent variants of VSVG proteins preferably have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with these proteins. The degree of identity between the variant and the native protein is determined using computer algorithms and methods widely known to those skilled in the art. For example, identity between two amino acid sequences is determined using the BLASTP algorithm (BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 21 5: 403-410 (1990)), but other similar algorithms can also be used.

[0042] In certain embodiments, functionally equivalent variants of the VSVG protein are preferably sequence-based: [ka] and have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0043] In certain embodiments, functionally equivalent variants of the VSVG protein have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence of VSVG, and retain at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, and advantageously 99% of the LDL-R binding ability of VSVG.

[0044] In the first step of the method of the present invention, a sample containing virus particles or VLPs is brought into contact with a purification resin.

[0045] The term "sample" refers to any sample containing viral particles or VLPs.

[0046] In certain embodiments, virus particles or VLPs are brought into contact with a purified resin at a pH of at least 7.

[0047] In a more detailed embodiment, virus particles or VLPs are brought into contact with a purified resin at a pH of 7-9. In an even more detailed embodiment, virus particles or VLPs are brought into contact with a purified resin at a pH of 7, 7.5, 8, or 8.5.

[0048] In certain embodiments, virus particles or VLPs are brought into contact with a purified resin at a temperature of 2°C to 28°C, preferably 2°C to 10°C, and more preferably 4°C.

[0049] In another specific embodiment, virus particles or VLPs are brought into contact with a purified resin for 10 minutes to 8 hours, preferably 30 minutes to 2 hours, more preferably 1 hour.

[0050] In a more detailed embodiment, virus particles or VLPs are contacted with a purified resin at a temperature of 4°C for 1 hour at pH 7, 7.5, or 8.5.

[0051] In another specific embodiment, virus particles or VLPs are brought into contact with a purified resin for the time and temperature described, and then the sample is centrifuged. After the centrifugation step, bound and unbound fractions are collected.

[0052] In certain embodiments, the first step of the method of the present invention further includes obtaining an unbound fraction of the sample after contacting the sample with a purified resin.

[0053] In certain embodiments, the unbound fraction (flow-through (FT) sample) is obtained by centrifugation of the sample at a rate of 300xg to 1000xg, preferably 500xg. In certain embodiments, the unbound fraction (flow-through (FT) sample) is obtained by centrifugation of the sample for at least 2 minutes, at least 3 minutes, at least 4 minutes, preferably 5 minutes. In other specific embodiments, these rates and times are combined. Those skilled in the art know how to combine or vary the centrifugation rate and centrifugation time conditions by compensatory changes.

[0054] In a more detailed embodiment, the unbound fraction is obtained by centrifuging the sample at a rate of 300 to 1000xg, preferably 300 to 800xg, for example, 500xg, for 2 to 10 minutes, preferably 5 minutes.

[0055] In one embodiment, after obtaining the unbound fraction, the resin is resuspended in a washing buffer and incubated at a temperature of 2°C to 28°C, preferably 2°C to 10°C, more preferably 4°C, for 10 minutes to 8 hours, preferably 30 minutes to 2 hours, more preferably 30 minutes.

[0056] In another embodiment, after incubation of the resin with a washing buffer, the resin fraction is obtained by centrifugation of the sample at a rate of 300xg to 1000xg, preferably 500xg. In one embodiment, the resin fraction is obtained by centrifugation of the sample for at least 2 minutes, at least 3 minutes, at least 4 minutes, preferably 5 minutes. In another specific embodiment, these rates and times are combined. Those skilled in the art know how to combine or vary the conditions of centrifugation rate and centrifugation time by compensatory changes.

[0057] In a more detailed embodiment, the resin fraction is obtained by centrifuging the sample at a rate of 300 to 1000 x g, preferably 300 to 800 x g, for example, 500 x g, for 2 to 10 minutes, preferably 5 minutes.

[0058] In the second step of the method of the present invention, the purified resin is washed with an elution buffer.

[0059] As used herein, "elution buffer" refers to the primary solvent of affinity chromatography. In this invention, the elution buffer is added to cleave the binding interaction and release the target viral particle or VLP, which is then recovered in a purified state. The elution conditions may be specific, such as a competing ligand, or nonspecific, such as a change in pH, ionic strength, or polarity. The elution buffer dissociates the binding partner by extreme pH (low or high), high salt concentration (ionic strength), use of a surfactant or chaotropic agent that denatures one or both molecules, removal of the binding factor, or competition with a counterligand.

[0060] In certain embodiments, the elution buffer has a pH of 5.5 to 6.5. In more detailed embodiments, the elution buffer has a pH of 6.

[0061] In another specific embodiment, the elution buffer is Ca 2+It contains a chelating agent. A chelating agent is an organic compound used to capture metal ions within a cyclic structure (chelating ring) by means of multiple coordination bonds. Most of them contain oxygen, nitrogen, and / or sulfur and are based on ethylenediamine, acetylacetone, and oxine.

[0062] In a more specific embodiment, the elution buffer contains EDTA and / or EGTA.

[0063] EDTA is a common chelating agent for divalent ions and is widely used in biochemistry, molecular biology, and cell biology. EDTA is an abbreviation for ethylenediaminetetraacetic acid (and many other related molecules). EDTA is widely used to sequester divalent and trivalent metal ions (e.g., Ca 2+ and Mg 2+ ). EDTA binds to metals via four carboxyl groups and two amine groups.

[0064] EGTA (ethylene glycol bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid) is a chelating agent with a much higher affinity for Ca 2+ ions than for Ca 2+ .

[0065] In a specific embodiment, the purification resin is incubated with the elution buffer at a temperature of 2°C to 28°C, preferably 2°C to 10°C, more preferably 4°C.

[0066] In another specific embodiment, the purification resin is incubated with the elution buffer for 10 minutes to 8 hours, preferably 30 minutes to 2 hours, more preferably 1 hour.

[0067] In a more specific embodiment, the purification resin is incubated with the elution buffer for 1 hour at a temperature of 4°C.

[0068] In the third step of the method of the present invention, the virus particles or VLPs eluted from the purification resin are recovered.

[0069] In one embodiment, the centrifugation step is performed before the recovery of the virus particles or VLPs. Centrifugation allows for the separation of the eluted fraction from the sample that remains bound to the resin after elution.

[0070] In one embodiment, centrifugation is performed at a speed of 300xg to 1000xg, preferably 500xg. In one embodiment, purified virus particles are obtained after centrifugation for at least 2 minutes, at least 3 minutes, at least 4 minutes, preferably 5 minutes. In another specific embodiment, these speeds and times are combined. Those skilled in the art know how to combine or vary the conditions of centrifugation speed and centrifugation time by compensatory changes.

[0071] In a more detailed embodiment, the purified virus particles are obtained after a centrifugation step of 2 to 10 minutes, preferably 5 minutes, at a rate of 300 to 1000xg, preferably 300 to 800xg, for example, 500xg.

[0072] Purification methods may include chromatography column chromatography or batch purification.

[0073] In certain embodiments, purification is performed by chromatography column chromatography.

[0074] Chromatography columns can be prepared by packing a solid adsorbent into a cylindrical glass or plastic tube. The size depends on the amount of compound to be isolated. The bottom of the tube contains a filter (cotton or glass wool plug, or glass frit) to hold the solid phase in place. A solvent reservoir can be attached to the top of the column.

[0075] Two methods are generally used to prepare columns: the dry method and the wet method. In the dry method, the column is first packed with dry stationary phase powder, followed by the addition of the mobile phase. The mobile phase is allowed to flow through the column until it is completely wet, and from this point onward, the column must never be allowed to dry out. In the wet method, a slurry of the eluent and stationary phase powder is prepared and then carefully poured into the column. The top of the silica should be flat, and the top of the silica can be protected with a layer of sand. The eluent is slowly passed through the column to advance the organic material.

[0076] In another specific embodiment, purification is performed by batch purification.

[0077] Batch purification involves binding the protein fraction to some type of chromatographic matrix or a physical support that allows flow-through, which is not encapsulated within the column. Batch purification can be performed at any scale. In batch purification, resin beads are centrifuged to form a pellet, and then the wash fraction and elution fraction are separated from the resin. The liquid cannot be completely removed because some of it is contained within the volume of the porous bead pellet.

[0078] In certain embodiments, the purification yield is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In another particular embodiment, the virus particles are purified to a purity of at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0079] In certain embodiments, the purified virus particles retain their biological activity and / or structure. In more detailed embodiments, the purified virus particles retain their infectivity.

[0080] As those skilled in the art would know, various resins can be used in the purification process. For example, affinity chromatography is a separation technique that exhibits high selectivity for target proteins based on the conformation of molecules, and in many cases, resins specific to the application are used. These resins have ligands specific to the compound being separated bound to their surface.

[0081] In the present invention, the purified resin used in the method of the present invention comprises LDL-R or a functionally equivalent fragment or variant of LDL-R.

[0082] Therefore, in certain embodiments, the purified resin used in the method of the present invention comprises LDL-R. In more detailed embodiments, LDL-R is a human protein identified by Uniprot accession number P01130 (entry version 261, sequence version 1, June 28, 2023). In another specific embodiment, the purified resin used in the method of the present invention comprises a functionally equivalent fragment or variant of LDL-R.

[0083] In the context of this invention, a “functionally equivalent variant” of LDL-R is understood to be any sequence having additions, substitutions, deletions, or combinations thereof to its amino acid sequence, and / or chemical modifications to the sequence, which substantially maintains the function of the receptor, particularly its ability to bind to VSVG. The term "variant" refers to both full-length protein variants and protein fragment variants.

[0084] Suitable assays for determining whether VSVG binds to LDL-R include, but are not limited to, co-immunoprecipitation, far-western blotting, protein ligation assays, affinity electrophoresis, and isothermal titration calorimetry.

[0085] Preferably, functionally equivalent variants of LDL-R exhibit at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, and advantageously 99% of the aforementioned functions.

[0086] Functionally equivalent variants of LDL-R preferably have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with these proteins. The degree of identity between the variant and the native protein is determined using computer algorithms and methods widely known to those skilled in the art. For example, identity between two amino acid sequences is determined using the BLASTP algorithm (BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 21 5: 403-410 (1990)), but other similar algorithms can also be used.

[0087] In certain embodiments, functionally equivalent variants of LDL-R preferably have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the human LDL-R protein identified by Uniprot accession number P01130 (entry version 261, sequence version 1, June 28, 2023).

[0088] In certain embodiments, functionally equivalent variants of LDL-R have a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of LDL-R, while maintaining at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, and advantageously 99% of the binding ability of LDL-R to VSVG.

[0089] In another specific embodiment, the variant of LDL-R includes a sequence having at least 75% identity with the CR2 domain (SEQ ID NO: 2) or the CR3 domain (SEQ ID NO: 3). In a more detailed embodiment, the variant of LDL-R including a sequence having at least 75% identity with the CR2 domain of SEQ ID NO: 2 or the CR3 domain of SEQ ID NO: 3 retains the VSVG binding ability of these domains.

[0090] In a more detailed embodiment, the LDL-R variant includes a sequence having at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the CR2 domain.

[0091] In another, more detailed embodiment, the LDL-R variant includes a sequence having at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the CR3 domain.

[0092] As used herein, the term “fragment” refers to a protein or polypeptide, and includes cleaved forms of a protein or polypeptide. For example, a fragment of LDL-R may contain about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 99% of the amino acids of full-length LDL-R.

[0093] In certain embodiments, the LDL-R fragment includes or consists of a CR2 domain (SEQ ID NO: 2).

[0094] In another specific embodiment, the LDL-R fragment includes or consists of a CR3 domain (SEQ ID NO: 3).

[0095] In another specific embodiment, the purified resin contains the CR2 and CR3 domains of LDL-R.

[0096] The CR2 and CR3 domains are cysteine-rich domains, and their binding sites on glycoprotein G are the same.

[0097] In the present invention, the purified resin used in the method of the present invention includes a matrix.

[0098] The term "matrix" refers to a device or other material that allows for the retention or passage of cells, is biocompatible, and allows the passage of polymers and particles either directly by being a semipermeable membrane itself, or in combination with certain semipermeable materials. Any matrix suitable for the purification of viral particles can be used and includes natural polymers such as dextran, starch, or cellulose, and synthetic polymers such as polyacrylamide, polymethacrylamide, or polyvinyl ether polymers. In certain embodiments, the matrix is ​​not a protein or peptide matrix.

[0099] The matrix can optionally take the form of a resin containing bead-like resin, a film, a monolith (a hollow cylinder formed from a single polymer block having a network of interconnected channels), or nanofibers.

[0100] In certain embodiments, the matrix is ​​agarose, crosslinked poly(styrene-divinylbenzene), crosslinked agarose, epoxy-activated agarose, polypropylene (PP), polyester, cellulose, polyethersulfone (PES), poly(glycidyl methacrylate-co-ethylenedimethacrylate), or a non-cellulose synthetic polymer. In preferred embodiments, the matrix is ​​agarose.

[0101] In certain embodiments, the matrix is ​​selected from the group consisting of DEAE Sepharose FF(Trademark) (Cytiva), POROS DM / 50(Trademark) (ThermoFisher), CaptureSelect Lenti VSVG(Trademark) (ThermoFisher), Sartobind Q(Trademark) (Sartorius), Mustang Q(Trademark) (Cytiva), Vivapure IEX(Trademark) (Sartorius), CIM DEAE(Trademark) (Sartorius), and Nereus LentiHERO(Trademark) (Astrea).

[0102] LDL-R or a functionally equivalent variant or fragment is bound to the matrix by any suitable means known to those skilled in the art, such as covalent bonding or affinity bonding.

[0103] In certain embodiments, LDL-R or a functionally equivalent variant or fragment is affinity-bound or covalently bound to the matrix.

[0104] In some embodiments, LDL-R or a functionally equivalent variant or fragment is affinity-bound to the matrix.

[0105] In certain embodiments, LDL-R or a functionally equivalent variant or fragment can be fused with a first member of an affinity binding pair, the first member having the ability to bind to a second member of the binding pair with high affinity.

[0106] Therefore, in certain embodiments, i) LDL-R or a functionally equivalent variant or fragment binds to the first member of the affinity binding pair, and the first member has the ability to bind to the second member of the binding pair with high affinity, and ii) the matrix is ​​bound to the second member of the affinity binding pair.

[0107] The term affinity binding pair refers to any peptide / ligand pair in which a peptide has the ability to specifically bind to a ligand. As used herein, the term "specific binding" refers to the binding of the first member of a binding pair to the second member of a binding pair with higher affinity and specificity than to other molecules. Examples of affinity binding pairs include: - Biotin or biotin acceptor peptide (BAP) and biotin binding domain (avidin or streptavidin), -Glutathione S-transferase (GST) and glutathione (GSH), - Histidine tag and Ni or Co ion, and -Antibodies or antibody derivatives and their specific antigens There is.

[0108] The first member of the binding pair does not have any particular size or other structural or technical features other than being able to bind to the second member of the binding pair. The peptide may be, but is not limited to, a specific member of the binding pair, e.g., an amino acid tag (e.g., a histidine tag (his tag), an arginine tag (arg tag), etc.), an antibody-recognizable peptide epitope (e.g., a c-myc tag), a biotin acceptor peptide (BAP), a linear domain that interacts with another protein or protein (e.g., an SH3 protein, a signaling protein, etc.), a post-translational modification sequence (e.g., myristoylation, methylation, phosphorylation, etc.), a transport sequence to a cellular compartment, etc.

[0109] In certain embodiments, LDL-R or a functionally equivalent variant or fragment is bound to a first member of a binding pair, particularly selected from the group including biotin, biotin acceptor peptide (BAP), peptides containing the tripeptide Arg-Gly-Asp, GST, histidine tags, and antibody derivatives.

[0110] LDL-R, variants, or fragments can be covalently fused to the first member of an affinity-binding pair. In certain embodiments, if the first member of the affinity-binding pair is a protein or peptide, the LDL-R, variant, or fragment can be bound to the first member of the affinity-binding pair by a peptide bond, thereby forming a fusion protein between the LDL-R, variant, or fragment and the first member of the affinity-binding pair.

[0111] The matrix can be covalently fused to the second member of the affinity bond pair.

[0112] In certain embodiments, where LDL-R or a functionally equivalent variant or fragment is bound to the first member of the binding pair, the matrix binds to the second member of the binding pair, and the second member of the binding pair binds to the first member of the binding pair. In certain embodiments, the second member of the binding pair is selected from the group consisting of molecules containing a biotin-binding domain, glutathione, Ni ions, Co ions, and specific antigens.

[0113] Molecules containing the biotin-binding domain according to the present invention include, but are not limited to, avidin, avidin analogs, streptavidin, and streptavidin analogs.

[0114] " Biotin-binding moleculeAs used herein, the term "biotin-binding molecule" refers to a member of a binding pair that binds to biotin. In particular, a biotin-binding molecule is an avidin fragment that retains substantial binding activity to avidin, streptavidin (SA), or biotin, for example, at least 50 percent of the binding affinity of natural SA. As used herein, the term "avidin" refers to glycoproteins found in egg white and in the tissues of avian, reptile, and amphibian proteins that have the ability to bind to biotin with high affinity, as well as any expression or modification of avidin-biotin-binding molecules such as streptavidin and neutraavidin. The term avidin includes both avidin found naturally in chicken (Gallus gallus) eggs (NCBI accession number NM_205320.1 / GL45384353en, published May 14, 2013) and homologous molecular species of said protein in other species. Streptavidin corresponds to the Streptomyces avidinii-derived protein (accession number CAA00084.1, GenBank, published January 28, 1993), as well as orthologues, homologs, and fragments of streptavidin, as defined similarly to avidin. Streptavidin contains four subunits, each containing a biotin binding site. Streptoavidin (SA) fragments or avidin fragments retaining substantial biotin binding activity, e.g., at least 50 percent of the binding affinity of natural SA or avidin, respectively, are also usable. Preferably, the affinity of the avidin variant to biotin is at least 10 15 M -1 , 10 14 M -1 , 10 13 M -1 , 10 12 M -1 , 10 10 M -1 or 10 9 M -1 That is the case.

[0115] Avidin and streptavidin variants suitable for use in the present invention include, but are not limited to, the following: - "Core streptavidin" ("CSA") is a cleaved form of the full-length streptavidin polypeptide, potentially containing streptavidin residues 13-138, 14-138, 13-139, and 14-139. See, for example, Pahler et al., (J. Biol. Chem. 1987, 262: 13933-37). -Streptavidin and avidin cleavage forms that retain strong binding to biotin (see, e.g., Sano et al., (J Biol Chem., 1995, 270: 28204-09)) (Core streptavidin variants 16-133 and 14-138 are described) (U.S. Patent No. 6,022,951) - Mutant streptavidin strains and core streptavidin strains that retain substantial biotin-binding activity or have enhanced biotin-binding activity. See Chilcoti et al., Proc Natl. Acad. Sci. USA. Feb. 28, 1995; 92(5):1754-8; Reznik et al., Nat Biotechnol. August 1996; 14(8):1007-1 1. - Mutant avidin strains that retain substantial biotin-binding activity or have enhanced biotin-binding activity, as well as core strains of avidin that retain substantial biotin-binding activity or have enhanced biotin-binding activity, are also available. See Hiller et al., J. Biochem. (1991) 278: 573-85; Livnah et al. Proc. Natl. Acad. Sci. USA (90: 5076-80 (1993)). - Mutants and fragments of avidin resulting from chemical modifications of avidin, such as complete or partial modification of glycosylation, as well as completely deglycosylated avidin mutants known as neutraavidins. - Mutant avidins as described in WO05047317A1 -Avidin-like proteins as described in WO06045891 - Recombinant avidins as described in WO0198349 -Avidin variants such as those described in WO0027814 - Monomeric streptavidins as described in WO06084388 Modified streptavidin dimers, such as those described in -WO06058226 - A protein with biotin-binding ability, as described in WO04018509. -Streptavidins with higher affinity for biotin, as described in WO9840396 Modified streptavidins and avidin molecules as described in WO9640761 - Mutant streptavidins as described in WO9711183 -Streptavidins with modified affinity, as described in WO9624606

[0116] For convenience, the terms “avidin” and “streptavidin” as used herein are intended to encompass biotin-binding fragments, variants, and core types of their binding pair members. For example, various avidin variants are commercially available, such as extraavidin (Sigma-Aldrich), neutraavidin (Thermo Scientific), neutraavidin (Invitrogen), and neutralite (Belovo). Furthermore, the nucleic acid sequences encoding streptavidin and avidin, as well as the amino acid sequences of streptavidin and avidin, can be found, for example, in GenBank accessions X65082;X03591;NM--205320;X05343;Z21611; and Z21554.

[0117] The expressions "specifically interactable" or "specific interaction," as used herein in the context of the first and second members of a binding pair, refer to an interaction between a first and second species characterized by the nature of the binding, in which an antibody, receptor, or nucleic acid-binding protein binds to its corresponding binding partner but substantially does not bind to the other species. Similarly, as used herein, the terms "to bind" or "to bind" mean a physical association between a first and second species.

[0118] In certain embodiments, LDL-R or a functionally equivalent variant or fragment is bound to GST as the first member of the affinity binding pair, and the matrix is ​​bound to glutathione as the second member of the affinity binding pair.

[0119] Glutathione S-transferases (GSTs), formerly known as ligandins, are a family of phase II metabolic isozymes in eukaryotes and prokaryotes best known for their ability to catalyze the conjugation of reduced glutathione (GSH) to xenobiotic substrates for detoxification purposes. The GST family consists of three superfamilies: cytosolic proteins, mitochondrial proteins, and microsomal proteins. The glutathione binding site, or "G-site," is located in the thioredoxin-like domain of both cytosolic and mitochondrial GSTs.

[0120] Glutathione is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is a tripeptide with a γ-peptide bond between the carboxyl group of the glutamic acid side chain and cysteine. The carboxyl group of the cysteine ​​residue is linked to glycine by a normal peptide bond. Glutathione exists in both reduced (GSH) and oxidized (GSSG) forms. GST specifically binds to reduced glutathione (GSH) under nearly neutral, non-denaturing conditions (e.g., Tris-buffer).

[0121] In another specific embodiment, LDL-R or a functionally equivalent variant or fragment is bound to biotin as the first member of an affinity binding pair, and the matrix is ​​bound to a biotin-binding molecule as the second member of an affinity binding pair. In a more detailed embodiment, the biotin-binding molecule is avidin or streptavidin. In an even more detailed embodiment, the biotin-binding molecule is avidin.

[0122] In another specific embodiment, LDL-R or a functionally equivalent variant or fragment is bound to the His tag as the first member of the affinity binding pair, and the matrix is ​​bound to a Ni ion or a Co ion as the second member of the affinity binding pair.

[0123] In another specific embodiment, LDL-R or a functionally equivalent variant or fragment is bound to an antibody derivative and the first member of the affinity binding pair, and the matrix is ​​bound to a specific antigen for that antibody as the second member of the affinity binding pair.

[0124] As used herein, the term “antibody” refers to a protein comprising at least one immunoglobulin variable region, for example, an amino acid sequence or sequence of an immunoglobulin variable domain that provides an immunoglobulin variable domain. An antibody may, for example, comprise a heavy chain variable (H) region (abbreviated herein as VH) and a light chain variable (L) region (abbreviated herein as VL). Generally, an antibody comprises two heavy chain variable regions and two light chain variable regions. The term “antibody” encompasses antigen-binding antibody fragments (e.g., single-chain antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, and dAb fragments) as well as complete antibodies, for example, intact and / or full-length immunoglobulins of type IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgM (and their subtypes).

[0125] In certain embodiments, the matrix is ​​agarose and comprises or consists of the CR2 domain of LDL-R fused with GST.

[0126] In certain embodiments, i) LDL-R or a functionally equivalent variant or fragment and ii) the matrix are affinity-bound. In these particular embodiments, as described above, LDL-R or a functionally equivalent variant or fragment is bound to the first member of the affinity-binding pair, and ii) the matrix is ​​bound to the second member of the affinity-binding pair.

[0127] In another specific embodiment, i) LDL-R or a functionally equivalent variant or fragment and ii) the matrix are covalently bonded.

[0128] Therefore, in some embodiments, LDL-R or its functionally equivalent variant or fragment does not need to bind to the first member of the binding pair, particularly GST, and the matrix does not need to bind to the second member of the binding pair, particularly GSH.

[0129] In certain embodiments, LDL-R or a functionally equivalent variant or fragment binds to the first member of the binding pair, particularly GST. In more detailed embodiments, LDL-R or the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence having at least 75% identity thereto binds to the first member of the binding pair, particularly GST.

[0130] In another specific embodiment, the first member of the affinity-binding pair is selected from the group consisting of GST, biotin, histidine tag, biotin acceptor peptide (BAP), antibody, or antibody derivative.

[0131] In certain embodiments, the matrix is ​​covalently bound to the first member of the binding pair, in particular LDL-R or a functionally equivalent variant or fragment, which is adapted to GST.

[0132] Therefore, in certain embodiments, the first member of the affinity bond pair is covalently bonded to the matrix.

[0133] In a more detailed embodiment, the GST is covalently bonded to the matrix.

[0134] In another specific embodiment, LDL-R or a functionally equivalent variant or fragment is covalently bound to the first member of the binding pair, particularly GST. In a more detailed embodiment, LDL-R or the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence having at least 75% identity thereto is covalently bound to the first member of the binding pair, particularly GST.

[0135] A covalent bond (or a covalent linkage) is a chemical bond in which atoms share electrons to form electron pairs. These electron pairs are known as covalent pairs or bond pairs. Covalent bonds also encompass many types of interactions, including sigma-type, pi-type, metal-metal-type, agostic interactions, bent bonds, three-center two-electron bonds, and three-center four-electron bonds.

[0136] In certain embodiments, the binding between LDL-R or its functionally equivalent variant or fragment and the matrix is ​​a result of a reaction between two different functional groups found in the LDL-R and the matrix.

[0137] Common functional groups are known to those skilled in the art and include hydrocarbons, halogen-containing groups, oxygen-containing groups, nitrogen-containing groups, sulfur-containing groups, phosphorus-containing groups, boron-containing groups, and metal-containing groups.

[0138] Some of the most common functional groups include hydroxyl, aldehyde, ketone, amine, amino, amide, ether, ester, carboxylic acid, thiol, and phenyl functional groups.

[0139] The hydroxyl functional group [H-OH] is the simplest of all common organic functional groups. Also known as the alcohol group or hydroxyl group, the hydroxyl group consists of an oxygen atom bonded to a hydrogen atom. The hydroxyl group links biomolecules through dehydration reactions. In structural and chemical formulas, the hydroxyl group is often represented as OH. Although the hydroxyl group is not highly reactive, it readily forms hydrogen bonds, which tends to make molecules containing it water-soluble. Common examples of compounds containing hydroxyl groups include alcohols and carboxylic acids. The hydroxyl functional group consists of a hydrogen atom bonded to an oxygen atom.

[0140] Aldehyde functional groups consist of a double bond between carbon and oxygen, with hydrogen bonded to carbon. Aldehydes can exist as keto tautomers or enol tautomers. Aldehyde groups are polar. The chemical formula for aldehydes is R-CHO.

[0141] A ketone is a molecule in which a carbon atom and an oxygen atom form a double bond, and it acts as a bridge between the other two parts of a molecule. This group is also known as a carbonyl functional group. Aldehydes are ketones in which one of the R atoms is a hydrogen atom.

[0142] Amine functional groups are derivatives of ammonia (NH3) in which one or more hydrogen atoms are substituted with alkyl or aryl functional groups. Silanation of microchannels using silane coupling agents such as (3-aminopropyl)triethoxysilane (APTES) and (3-aminopropyl)trimethoxysilane (APTMS) is the most common method for generating amine functional groups on the channel surface and promoting the immobilization of biomolecules.

[0143] Amino functional groups are basic or alkaline groups. Amino functional groups are widely found in amino acids, proteins, and nitrogen-containing bases used in the construction of DNA and RNA. Although amino groups are NH2, under acidic conditions they incorporate a proton to become NH3. + This is the result.

[0144] An amide functional group is a combination of a carbonyl group and an amine functional group.

[0145] An ether functional group consists of oxygen atoms that form a bridge between two different parts of a molecule. The formula for ether is ROR.

[0146] An ester functional group is another type of bridging group formed by the linkage of a carbonyl group and an ether group. The formula for an ester is RCO2R.

[0147] Carboxylic acid functional groups are also known as carboxyl functional groups. A carboxyl group is an ester in which the substituent R is a hydrogen atom. Carboxyl groups are usually represented as -COOH.

[0148] The thiol functional group is similar to the hydroxyl group except that the oxygen atom in the hydroxyl group is replaced by a sulfur atom in the thiol group. The thiol functional group is also known as the sulfhydryl functional group. The formula for the thiol functional group is -SH.

[0149] The phenyl functional group is a common cyclic group. It is formed by replacing one hydrogen atom of a benzene ring with an R substituent. The formula for the phenyl group is C6H5.

[0150] The purified resin used in the method of the present invention does not contain phase-behavior polypeptides. In certain embodiments, the purified resin used in the method of the present invention does not contain any peptides or proteins. As used herein, the term "polypeptide" refers to a compound composed of amino acid residues covalently linked by peptide bonds.

[0151] As used herein, the term "polypeptide with phase behavior" refers to any polypeptide capable of undergoing a phase transition. Polypeptides undergo phase transitions in response to environmental factors. Exemplary examples of polypeptides with phase behavior include elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs).

[0152] In certain embodiments, the purified resin used in the method of the present invention does not contain elastin-like polypeptides and resin-like polypeptides. In more detailed embodiments, the purified resin used in the method of the present invention has the sequence: (Val-Pro-Gly-Xaa-Gly) n (Sequence ID 4) (In the sequence, X can be any amino acid except proline), (GRGDSPY) n (Sequence ID 5); (GRGDSPH) n (Sequence ID 6); (GRGDSPV) n (Sequence ID 7); (GRGDSPYG) n (Sequence number 8); (RPLGYDS) n (Sequence number 9); (RPAGYDS) n (Sequence ID 10); (GRGDSYP) n (Sequence ID 11); (GRGDSPYQ) n (Sequence ID 12); (GRGNSPYG) n (Sequence ID 13); (GVGVP) n (Sequence ID 14); (GVGVPGLGVPGVGVPGLGVPGVGVP) m (Sequence ID 15); (GVGVPGVGVPGAGVPGVGVPGVGVP) m (Sequence ID 16); (GVGVPGWGVPGVGVPGWGVPGVGVP) m (Sequence number 17); (GVGVPGVGVPGVGVPGVGVPGVGVGVPGEGVPGFGVPGVGVP) m (Sequence number 18); (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP) m (Sequence number 19); and (GAGVPGVGVPGAGVPGVGVPGAGVP) m It does not contain a polypeptide with phase behavior having (Sequence ID 20) (where n is 20 to 360 and m is 4 to 25). In another embodiment, the purified resin used in the method of the present invention has the sequence (GVGVP) m (Sequence code 21); (ZZPXXXXGZ) m ;(ZZPXGZ) m ;(ZZPXXGZ) m ; or (ZZPXXXGZ)m The present invention does not contain polypeptides with phase behavior having the sequence: (wherein m is an integer between 10 and 160 including the endpoint, X is any amino acid except proline or glycine, if present, and Z is any amino acid, if present). In another embodiment, the purified resin used in the method of the present invention has the sequence: (GVGVPGVGVPGAGVPGVGVPGVGVP) m (Sequence ID 16) or (GVGVPGVGVPGLGVPGVGVPGVGVP) m The method does not contain polypeptides with phase behavior having the sequence (Sequence ID 27) (where m is an integer from 2 to 32 including the endpoint). In another embodiment, the purified resin used in the method of the present invention has the sequence: (GVGVPGVGVPGAGVPGVGVPGVGVP) m (Sequence ID 16) (In the array, m is either 8 or 16); (GVGVPGAGVP) m (Sequence ID 29) (In the array, m is an integer between 5 and 80, including the endpoints); or (GXGVP) m (SEQ ID NO: 30) (In the sequence, m is an integer between 10 and 160 including the endpoint, and for each repeat, X is independently selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine) does not contain polypeptides exhibiting phase behavior.

[0153] Resilin-like polypeptides are elastomeric polypeptides that possess desirable mechanical properties such as elasticity, compressive modulus, tensile modulus, shear modulus, elongation at break, maximum tensile strength, hardness, resilience, and compression set.

[0154] Elastin-like polypeptides (ELPs) are biomacromolecules derived from tropoelastin. The ability of ELPs to undergo morphological changes at specific temperatures allows for the separation of specific proteins bound to ELPs from solution using experimental techniques such as centrifugation. The general structure of polymer ELP is (VPGXG)n (SEQ ID NO: 4), where the monomer unit is Val-Pro-Gly-X-Gly, and "X" represents the transition temperature (T t) represents variable amino acids that can affect the general properties of ELPs. Generally, these polymers are T t The lower it is linear, but T t At higher concentrations, they aggregate into spherical clumps. These polymers undergo reversible phase transitions that can be induced by various environmental stimuli such as temperature, pH, or ionic strength. ELP can be used in purification processes. t At lower temperatures, ELP binds linearly to the ligand. t When heated to a temperature exceeding a certain level, ELP forms spherical clumps. After centrifugation, these clumps settle at the bottom of the solution tube, retaining the target protein.

[0155] In certain embodiments, the term “phase-behaving polypeptide” refers to a polypeptide as defined on pages 25, 48, and 49 of the US Patent Application 2022010288A1.

[0156] In another specific embodiment, the purified resin used in the method of the present invention is non-magnetic.

[0157] Therefore, in a particular embodiment, the present invention relates to a method for purifying viral particles or virus-like particles (VLPs) comprising an envelope, the envelope comprising a VSVG protein or a functionally equivalent variant or fragment that retains the ability to bind to a low-density lipoprotein receptor (LDL-R), i) Contacting a sample containing viral particles or VLPs with a purified resin, ii) Washing the purified resin with an elution buffer, and iii) Recovering the virus particles or VLPs eluted from the purified resin. The purified resin includes, a) Low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, wherein the fragment or variant retains the VSVG binding ability of LDL-R. b) Matrix The present invention relates to a method comprising the above, wherein the purified resin does not contain polypeptides with phase behavior, and the purified resin is non-magnetic.

[0158] The term "magnetism" includes ferromagnetism, paramagnetism, diamagnetism, and superparamagnetism.

[0159] Magnetism-based purification refers to a purification method that uses an external magnetic field to separate particles (particle size fractionation) or to separate various compounds with different magnetic susceptibility, such as cations, anions, and those with effective magnetic moments. It can also be used to separate diamagnetic compounds through different strategies such as complex formation with metal ions, reactions with metal complexes, and adhesion to magnetic or magnetized particles.

[0160] In certain embodiments, the purified resin used in the method of the present invention is non-ferromagnetic. Ferromagnetism is the fundamental mechanism by which certain materials (such as iron) form permanent magnets. This means that the compound exhibits permanent magnetism, rather than only exhibiting magnetism in the presence of an external magnetic field. In ferromagnetic elements, the electrons of the atom are grouped into domains, and each domain has the same charge.

[0161] In another specific embodiment, the purified resin used in the method of the present invention is non-paramagnetic. Paramagnetism refers to the magnetic state of an atom having one or more unpaired electrons. Unpaired electrons are attracted to a magnetic field by the magnetic dipole moment of the electron.

[0162] In another specific embodiment, the purified resin used in the method of the present invention does not have diamagnetic properties. Diamagnetic materials are characterized by not having paired electrons, such as unpaired electrons.

[0163] In another specific embodiment, the purified resin used in the method of the present invention is not superparamagnetic. "Superparamagnetic" refers to the form of magnetism exhibited by minute ferromagnetic or antiferromagnetic nanoparticles. At sizes less than 100 nanometers, nanoparticles are single-domain particles, and the magnetization of the nanoparticles can be approximated as one large magnetic moment by summing the individual magnetic moments of each constituent atom.

[0164] In another specific embodiment, the purified resin used in the method of the present invention does not contain complete LDL-R protein. In that specific embodiment, LDL-R protein refers to the human protein identified by Uniprot accession number P01130 (entry version 261, sequence version 1, June 28, 2023). Complete human LDL-R protein contains 860 amino acids.

[0165] The purified resin of the present invention In another embodiment, the present invention is: a) Low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, b) Matrix The present invention relates to a purified resin containing (hereinafter referred to as the purified resin of the present invention), which does not contain polypeptides exhibiting phase behavior and is non-magnetic.

[0166] The terms "LDL-R" and "purified resin" are defined or described in the method of the present invention, and these definitions also apply to the purified resin of the present invention.

[0167] In certain embodiments, the purified resin of the present invention comprises LDL-R. In other specific embodiments, the purified resin of the present invention comprises a functionally equivalent fragment or variant of LDL-R.

[0168] In the context of the purified resin of the present invention, a “functionally equivalent variant” of LDL-R is understood to be any sequence having additions, substitutions, deletions or combinations thereof to its amino acid sequence, and / or chemical modifications to the sequence, which substantially maintains the function of the receptor, particularly its ability to bind to VSVG. The term variant refers to both full-length protein variants and protein fragment variants.

[0169] Suitable assays for determining whether VSVG binds to LDL-R include, but are not limited to, co-immunoprecipitation, far-western blotting, protein ligation assays, affinity electrophoresis, and isothermal titration calorimetry.

[0170] Preferably, functionally equivalent variants of LDL-R exhibit at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, and advantageously 99% of the aforementioned functions.

[0171] Functionally equivalent variants of LDL-R preferably have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with these proteins. The degree of identity between the variant and the native protein is determined using computer algorithms and methods widely known to those skilled in the art. For example, identity between two amino acid sequences is determined using the BLASTP algorithm (BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 21 5: 403-410 (1990)), but other similar algorithms can also be used.

[0172] In certain embodiments, functionally equivalent variants of LDL-R preferably have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the human LDL-R protein identified by Uniprot accession number P01130 (entry version 261, sequence version 1, June 28, 2023).

[0173] In certain embodiments, functionally equivalent variants of LDL-R have a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of LDL-R, while maintaining at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, and advantageously 99% of the binding ability of LDL-R to VSVG.

[0174] In another specific embodiment, the LDL-R variant includes a sequence having at least 75% identity with the CR2 domain (SEQ ID NO: 2) or the CR3 domain (SEQ ID NO: 3).

[0175] In a more detailed embodiment, the LDL-R variant includes a sequence having at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the CR2 domain.

[0176] In another, more detailed embodiment, the LDL-R variant includes a sequence having at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the CR3 domain.

[0177] LDL-R or its functionally equivalent variants or fragments can be fused to the first member of an affinity-binding pair that has the ability to bind with high affinity to the second member of the binding pair.

[0178] Therefore, in certain embodiments, i) LDL-R or a functionally equivalent variant or fragment binds to the first member of an affinity binding pair having the ability to bind with high affinity to the second member of the binding pair, and ii) the matrix binds to the second member of the affinity binding pair.

[0179] In certain embodiments, LDL-R or a functionally equivalent variant or fragment is bound to a first member of a binding pair, particularly selected from the group including biotin, biotin acceptor peptide (BAP), peptides containing the tripeptide Arg-Gly-Asp, GST, histidine tags, and antibody derivatives.

[0180] LDL-R, variants, or fragments can be covalently fused to the first member of an affinity-binding pair. In certain embodiments, if the first member of the affinity-binding pair is a protein or peptide, LDL-R, variants, or fragments can be bound to the first member of the affinity-binding pair by a peptide bond, thereby forming a fusion protein between LDL-R, variants, or fragments and the first member of the affinity-binding pair.

[0181] The matrix can be covalently fused to the second member of the affinity bond pair.

[0182] In certain embodiments, LDL-R or a functionally equivalent variant or fragment is bound to the first member of the binding pair, the matrix is ​​bound to the second member of the binding pair, and the second member of the binding pair is bound to the first member of the binding pair. In certain embodiments, the second member of the binding pair is selected from the group consisting of molecules containing a biotin-binding domain, glutathione, Ni ions, Co ions, and specific antigens.

[0183] Molecules containing the biotin-binding domain according to the present invention include, but are not limited to, avidin, avidin analogs, streptavidin, and streptavidin analogs.

[0184] The term "biotin-binding molecule" is defined or described in the methods of the present invention, and these definitions also apply to the purified resins of the present invention.

[0185] In certain embodiments, LDL-R or a functionally equivalent variant or fragment is bound to GST as the first member of the affinity binding pair, and the matrix is ​​bound to glutathione as the second member of the affinity binding pair. The terms GST and GSH are defined or described above, and these definitions also apply to the purified resin of the present invention.

[0186] In another specific embodiment, LDL-R or a functionally equivalent variant or fragment is bound to biotin as the first member of an affinity binding pair, and the matrix is ​​bound to a biotin-binding molecule as the second member of an affinity binding pair. In a more detailed embodiment, the biotin-binding molecule is avidin or streptavidin. In an even more detailed embodiment, the biotin-binding molecule is avidin.

[0187] In another specific embodiment, LDL-R or a functionally equivalent variant or fragment is bound to the His tag as the first member of the affinity binding pair, and the matrix is ​​bound to a Ni ion or a Co ion as the second member of the affinity binding pair.

[0188] In another specific embodiment, LDL-R or a functionally equivalent variant or fragment is bound to an antibody derivative as the first member of an affinity-binding pair, and the matrix is ​​bound to an antigen specific to that antibody as the second member of the affinity-binding pair. The term “antibody” is defined or described above, and these definitions also apply to the purified resin of the present invention.

[0189] In another specific embodiment, LDL-R or a functionally equivalent variant or fragment is affinity-bound to the matrix. In this specific embodiment, as described above, LDL-R or a functionally equivalent variant or fragment is bound to the first member of the affinity-binding pair, and ii) the matrix is ​​bound to the second member of the affinity-binding pair.

[0190] In another specific embodiment, LDL-R or a functionally equivalent variant or fragment is covalently bonded to the matrix.

[0191] Therefore, in some embodiments, LDL-R or its functionally equivalent variant or fragment does not need to bind to the first member of the binding pair, particularly GST, and the matrix does not need to bind to the second member of the binding pair, particularly GSH.

[0192] In certain embodiments, LDL-R or a functionally equivalent variant or fragment binds to the first member of the binding pair, particularly GST. In more detailed embodiments, LDL-R or the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence having at least 75% identity thereto binds to the first member of the binding pair, particularly GST.

[0193] In another specific embodiment, the first member of the affinity-binding pair is selected from the group consisting of GST, biotin, histidine tag, biotin acceptor peptide (BAP), antibody, or antibody derivative.

[0194] In certain embodiments, the matrix is ​​covalently bound to the first member of the binding pair, particularly LDL-R or a functionally equivalent variant or fragment bound to GST.

[0195] Therefore, in certain embodiments, the first member of the affinity bond pair is covalently bonded to the matrix.

[0196] In a more detailed embodiment, the GST is covalently bonded to the matrix.

[0197] Various types of covalent bonds are described in the method of the present invention, and these covalent bonds also apply to the purified resin of the present invention.

[0198] As used herein, the term “fragment” refers to a protein or polypeptide, and includes cleaved forms of a protein or polypeptide. For example, a fragment of LDL-R may contain about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 99% of the amino acids of full-length LDL-R.

[0199] In certain embodiments, the LDL-R fragment includes or consists of a CR2 domain (SEQ ID NO: 2).

[0200] In another specific embodiment, the LDL-R fragment includes or consists of a CR3 domain (SEQ ID NO: 3).

[0201] In certain embodiments, the purified resin contains fragments of a low-density lipoprotein receptor (LDL-R) or functionally equivalent variants, including a CR2 domain (SEQ ID NO: 2) and / or a CR3 domain (SEQ ID NO: 3).

[0202] In another specific embodiment, the purified resin of the present invention does not contain the complete LDL-R protein. In that specific embodiment, the LDL-R protein refers to the human protein identified by Uniprot accession number P01130 (entry version 261, sequence version 1, June 28, 2023). The complete human LDL-R protein contains 860 amino acids.

[0203] Therefore, in a particular embodiment, the present invention is a) A fragment of a low-density lipoprotein receptor (LDL-R) containing a CR2 domain (SEQ ID NO: 2) and / or a CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant, b) Matrix This invention relates to a purified resin containing the following, which does not contain polypeptides exhibiting phase behavior, is non-magnetic, and does not contain complete LDL-R protein.

[0204] In certain embodiments, the purified resin of the present invention comprises functionally equivalent variants of the LDL-R fragment containing a CR2 domain (SEQ ID NO: 2) and / or a CR3 domain (SEQ ID NO: 3). Thus, in another particular embodiment, the purified resin comprises functionally equivalent variants of SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0205] In the context of the purified resin of the present invention, "functionally equivalent variants" of the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) are understood to be any sequence having additions, substitutions, deletions or combinations thereof to its amino acid sequence, and / or being chemically modified to substantially maintain the function of LDL-R, particularly its ability to bind to VSVG. The term "variant" refers to a protein fragment variant.

[0206] Suitable assays for determining whether VSVG binds to LDL-R include, but are not limited to, co-immunoprecipitation, far-western blotting, protein ligation assays, affinity electrophoresis, and isothermal titration calorimetry.

[0207] Preferably, functionally equivalent variants of the CR2 (SEQ ID NO: 2) and / or CR3 domain (SEQ ID NO: 3) exhibit at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, and advantageously 99% of the aforementioned functions.

[0208] Functionally equivalent variants of the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) preferably have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with these proteins. The degree of identity between the variant and the native protein is determined using computer algorithms and methods widely known to those skilled in the art. For example, identity between two amino acid sequences is determined using the BLASTP algorithm (BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 21 5: 403-410 (1990)), but other similar algorithms may also be used.

[0209] In certain embodiments, functionally equivalent variants of the CR2 domain of LDL-R preferably have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 2.

[0210] In another specific embodiment, functionally equivalent variants of the CR3 domain of LDL-R preferably have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 3.

[0211] In certain embodiments, functionally equivalent variants of the CR2 domain of LDL-R have a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 2, while maintaining at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, and advantageously 99% of the binding ability of LDL-R to VSVG.

[0212] In another specific embodiment, a functionally equivalent variant of the CR3 domain of LDL-R has a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 3, and maintains at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, and advantageously 99% of the binding ability of LDL-R to VSVG.

[0213] In certain embodiments, the variant of the CR2 domain of LDL-R includes a sequence having at least 75% identity with the sequence of Sequence ID No. 2.

[0214] In another specific embodiment, the variant of the CR3 domain of LDL-R includes a sequence having at least 75% identity with the sequence of Sequence ID No. 3.

[0215] In a more detailed embodiment, the variant of the CR2 domain includes a sequence that has at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of Sequence ID No. 2.

[0216] In another, more detailed embodiment, the variant of the CR3 domain includes a sequence having at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 3.

[0217] In certain embodiments, fragments of LDL-R containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or functionally equivalent variants are affinity-bound or covalently bound to the matrix.

[0218] In some embodiments, fragments of LDL-R containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or functionally equivalent variants are affinity-bound to the matrix.

[0219] A fragment of LDL-R containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant can be fused to the first member of an affinity-binding pair, which has the ability to bind with high affinity to the second member of the binding pair.

[0220] Therefore, in certain embodiments, i) a fragment of LDL-R containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant binds to the first member of an affinity-binding pair having the ability to bind to the second member of the binding pair with high affinity, and ii) the matrix binds to the second member of the affinity-binding pair.

[0221] The term affinity pair refers to any peptide / ligand pair in which a peptide has the ability to bind to a ligand. Examples of affinity pairs are: - Biotin or biotin acceptor peptide (BAP) and biotin binding domain (avidin or streptavidin), -Glutathione S-transferase (GST) and glutathione (GSH), - Histidine tag and Ni or Co ion, and - Antibody derivatives and their specific antigens There is.

[0222] In certain embodiments, a fragment of LDL-R containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant is bound to a first member of a binding pair, in particular, a first member of a binding pair selected from the group including biotin, biotin acceptor peptide (BAP), peptides containing the tripeptide Arg-Gly-Asp, GST, histidine tags, and antibody derivatives.

[0223] A fragment or variant of LDL-R can be covalently fused to the first member of an affinity-binding pair. In certain embodiments, if the first member of the affinity-binding pair is a protein or peptide, the LDL-R fragment or variant can be bound to the first member of the affinity-binding pair by a peptide bond, thereby forming a fusion protein between the LDL-R fragment or variant and the first member of the affinity-binding pair.

[0224] The matrix can be covalently fused to the second member of the affinity bond pair.

[0225] In certain embodiments, a fragment of LDL-R or a functionally equivalent variant is bound to the first member of the binding pair, the matrix is ​​bound to the second member of the binding pair, and the second member of the binding pair is bound to the first member of the binding pair. In certain embodiments, the second member of the binding pair is selected from the group consisting of molecules containing a biotin-binding domain, glutathione, Ni ions, Co ions, and specific antigens.

[0226] Molecules containing the biotin-binding domain according to the present invention include, but are not limited to, avidin, avidin analogs, streptavidin, and streptavidin analogs.

[0227] The term "biotin-binding molecule" is defined or described in the methods of the present invention, and these definitions also apply to the purified resins of the present invention.

[0228] In certain embodiments, a fragment of LDL-R or a functionally equivalent variant is bound to GST as the first member of the affinity binding pair, and the matrix is ​​bound to glutathione as the second member of the affinity binding pair. The terms GST and GSH are defined or described above, and these definitions also apply to the purified resin of the present invention.

[0229] In another specific embodiment, a fragment of LDL-R or a functionally equivalent variant is bound to biotin as the first member of an affinity binding pair, and the matrix is ​​bound to a biotin-binding molecule as the second member of an affinity binding pair. In a more detailed embodiment, the biotin-binding molecule is avidin or streptavidin. In an even more detailed embodiment, the biotin-binding molecule is avidin.

[0230] In another specific embodiment, a fragment of LDL-R or a functionally equivalent variant is bound to the His tag as the first member of the affinity binding pair, and the matrix is ​​bound to a Ni ion or a Co ion as the second member of the affinity binding pair.

[0231] In another specific embodiment, a fragment of LDL-R or a functionally equivalent variant is bound to the antibody derivative and the first member of the affinity-binding pair, while the matrix is ​​bound to a specific antigen for that antibody as the second member of the affinity-binding pair. The term “antibody” is defined or described above, and these definitions also apply to the purified resin of the present invention.

[0232] As described above, in certain embodiments, the matrix is ​​affinity-bound to a fragment of LDL-R or a functionally equivalent variant. In these particular embodiments, as described above, the LDL-R or a functionally equivalent variant or fragment is bound to the first member of the affinity-binding pair, and the matrix is ​​bound to the second member of the affinity-binding pair.

[0233] In another specific embodiment, the matrix is ​​covalently bound to a fragment of LDL-R or a functionally equivalent variant.

[0234] Therefore, in some embodiments, the LDL-R fragment containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant binds to the first member of the binding pair, particularly GST, and the matrix does not need to bind to the second member of the binding pair, particularly GSH.

[0235] In certain embodiments, a fragment of LDL-R containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant is bound to the first member of the binding pair, particularly GST. In more detailed embodiments, a fragment of LDL-R containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence having at least 75% identity thereto is bound to the first member of the binding pair, particularly GST.

[0236] In another specific embodiment, the first member of the affinity-binding pair is selected from the group consisting of GST, biotin, histidine tag, biotin acceptor peptide (BAP), antibody, or antibody derivative.

[0237] In certain embodiments, the matrix is ​​covalently bound to a fragment of LDL-R containing the first member of the binding pair, particularly the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant bound to GST.

[0238] Therefore, in certain embodiments, the first member of the affinity bond pair is covalently bonded to the matrix.

[0239] In a more detailed embodiment, the GST is covalently bonded to the matrix.

[0240] In another specific embodiment, a fragment of LDL-R containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant is covalently bound to the first member of the binding pair, particularly the GST. In a more detailed embodiment, a fragment of LDL-R containing the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence having at least 75% identity thereto is covalently bound to the first member of the binding pair, particularly the GST.

[0241] In the method of the present invention, various types of covalent bonds are described, and these covalent bonds are also applicable to the purification resin of the present invention.

[0242] The purification resin of the present invention does not contain phase behavior polypeptides. In certain embodiments, the purification resin does not contain any peptides or proteins. The terms "polypeptide" and "phase-behaving polypeptide" are defined or described in the method of the present invention, and these definitions are also applicable to the purification resin of the present invention.

[0243] The purification resin of the present invention is not magnetic. The term "magnetic" is defined or described above, and this definition is also applicable to the purification resin of the present invention.

[0244] Purification based on magnetism refers to a purification method that separates particles (particle size fractionation) by applying an external magnetic field or separates various compounds with different magnetic susceptibilities, such as cations, anions, and those with an effective magnetic moment. It can also be used for the separation of diamagnetic compounds by different strategies such as complex formation with metal ions, reaction with metal complexes, and attachment to magnetic particles or magnetized particles.

[0245] In certain embodiments, the purification resin of the present invention is not ferromagnetic. Ferromagnetism is defined or described above, and this definition is also applicable to the purification resin of the present invention.

[0246] In another certain embodiment, the purification resin of the present invention is not paramagnetic. Paramagnetism is defined or described above, and this definition is also applicable to the purification resin of the present invention.

[0247] In another certain embodiment, the purification resin of the present invention does not have diamagnetic properties. Diamagnetic substances are characterized by having paired electrons, for example, no unpaired electrons.

[0248] In another certain embodiment, the purification resin of the present invention is not superparamagnetic. "Superparamagnetism" is defined or described above, and this definition is also applicable to the purification resin of the present invention.

[0249] In certain embodiments, the purified resin of the present invention includes agarose, crosslinked poly(styrene-divinylbenzene), crosslinked agarose, epoxy-activated agarose, polypropylene (PP), polyester, cellulose, polyethersulfone (PES), quaternary ammonium ligand (Q), sulfonic acid (S), diethylamine (D), poly(glycidyl methacrylate-co-ethylenedimethacrylate), or non-cellulose synthetic polymers. In more detailed embodiments, the purified resin is an agarose resin.

[0250] In certain embodiments, the purified resin is an agarose resin, and the matrix consists of CR2 domains of LDL-R fused to GST.

[0251] In another specific embodiment, the purified resin is selected from the group consisting of DEAE Sepharose FF (Cytiva), POROS DM / 50 (ThermoFisher), CaptureSelect Lenti VSVG (ThermoFisher), Sartobind Q (Sartorius), Mustang Q (Cytiva), Vivapure IEX (Sartorius), CIM DEAE (Sartorius), and Nereus LentiHERO (Astrea).

[0252] In another specific embodiment, the purified resin forms a chromatography column. The term "chromatography column" is defined or described in the method of the present invention, and this definition also applies to the purified resin of the present invention.

[0253] In another specific embodiment, the purified resin is a batch resin.

[0254] The term "batch" is defined or described in the method of the present invention, and this definition also applies to the purified resin of the present invention.

[0255] In another embodiment, the present invention relates to the use of the present invention's purification resin for purifying viral particles or VLPs comprising an envelope containing a VSVG protein or a functionally equivalent variant or fragment that retains the ability to bind to LDL-R.

[0256] The terms “viral particle,” “VLP,” “VSVG protein,” “functionally equivalent variant,” and “LDL-R” are defined or described in the methods of the present invention, and these definitions also apply to the use of the present invention. [Examples]

[0257] The following embodiments illustrate the present invention and should not be considered to limit its scope.

[0258] material and method Cloning, expression, and purification of GSTCR2 GSTCR2 was cloned into a pGEX-6P-1 expression vector. This was then used to transform BL21(DE3) competent cells, which were induced with IPTG and expressed overnight at 20°C. The cell pellet was resuspended in lysis buffer (50mM Tris pH8.0, 300mM NaCl, 2mM CaCl2, 1mM DTT) and lysed by sonication. The supernatant was incubated with reduced glutathione resin at 4°C for 1 hour, then poured onto a column, and the FT fraction was discarded. A washing step was performed by adding lysis buffer. Finally, the resin was resuspended in 5 ml of elution buffer (50mM Tris pH8.0, 150mM NaCl, 30mM reduced glutathione, 2mM CaCl2, 1mM DTT), incubated at 4°C for 30 minutes, loaded onto a column, and the eluted fraction was collected.

[0259] Functionalization of resins 50 μL of 20 μM GSTCR2 was incubated with 200 μL of GST resin and equilibrated with binding buffer (50 mM PIPES pH 7.0, 150 mM NaCl, 2 mM CaCl2, 1 mM DTT). Incubation was performed at 4°C for 1 hour.

[0260] Purification of LVV using GSTCR2 functionalized resin The functionalized resin was centrifuged at 500 x g for 5 minutes, and the supernatant was discarded. 500 μL of LVV was added to the resin, incubated at 4 °C for 1 hour, and centrifuged at 500 x g for 5 minutes to collect the flow-through (FT) sample. The resin was resuspended in 1 ml of wash buffer (1 mM PIPES pH 7.0, 150 mM NaCl, 2 mM CaCl2, 1 mM DTT) and incubated at 4 °C for 30 minutes. After centrifugation, the wash fraction and resin fraction were collected. Finally, the resin was resuspended in 500 μL of elution buffer (50 mM MES pH 6.0, 150 mM NaCl, 1 mM DTT), incubated at 4 °C for 1 hour, and centrifuged at 500 x g for 5 minutes to collect the elution (Elu) sample and resin sample.

[0261] Purification analysis of LVV by p24 measurement P24 analysis of the samples (FT, Res1, Elu, Res2) collected during the purification process was performed using the Lenti-X p24 rapid titer kit (Takara Bio) according to the protocol specified in the kit.

[0262] LVV purification analysis by infectivity titer measurement The FT and elution fractions were thawed and diluted with DMEM + 1% antibiotic + 8 μg / ml polybrene in a MW24 plate. Dilution series were prepared, and each dilution series was performed in 2 independent replicates. Next, 250 μl of each dilution was pipetted into a new MW24 plate, and 250 μl of a cell suspension with a density of 75,000 HEK293T cells per well in DMEM + 1% antibiotic + 8 μg / ml polybrene was added to each well. The plate was incubated at 37 °C for 2 hours. Then, 1.5 ml of DMEM + 1% antibiotic + 15% FBS was added to each well, and the plate was incubated at 37 °C for 72 hours. Finally, the infectivity titer of the samples (FT, Elu) was measured by flow cytometry, and transduced cells were collected.

[0263] Measurement of HCP content The HCP content of samples (FT, Elu) collected during the purification process was measured using the HEK 293 HCP Elisa kit, 3G (cygnus technologies), according to the protocol indicated in the kit.

[0264] Purification of LVV using CaptureSelect Lenti VSVG resin (ThermoFisher) 200 μL of CaptureSelect Lenti VSVG resin was equilibrated in buffer (50 mM HEPES pH 7.5, 150 mM NaCl). 500 μL of LVV was added to the resin, incubated at 4°C for 1 hour, and centrifuged at 500xg for 5 minutes to collect a flow-through (FT) sample. The resin was washed with 1 ml of buffer (50 mM HEPES pH 7.5, 150 mM NaCl) and incubated at 4°C for 30 minutes. Next, the washing fraction and the resin (Res1) fraction were collected after centrifugation at 500xg for 5 minutes. Finally, the resin was resuspended in 500 μL of elution buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.8 M arginine), incubated at 4°C for 1 hour, and centrifuged at 500xg for 5 minutes to collect the eluted (Elu) sample and the resin (Res2) sample.

[0265] result Protocol for LVG purification by affinity chromatography Lentivirus (LVV) was purified using GSTCR2 bound to an agarose-based glutathione resin. Lentivirus samples were incubated with functionalized chromatography resin at pH 7.0, and after centrifugation, bound (Res1) and unbound (FT) fractions were collected. After washing, LVV was eluted from the resin by incubation with a pH 6.0 buffer solution. In this case as well, separation of the eluted fraction (Elu) from the sample still bound to the resin after elution (Res2) was possible by centrifugation. The scheme of the purification protocol described is shown in Figure 1.

[0266] To evaluate the effectiveness of the purification system, the amount of LVV present in each sample was measured using ELISA p24. The results showed that approximately 70% of the initial sample bound to the column after interacting with GSTCR2, and 30% flowed out as flow-through (Figure 2). After changing the pH, almost all of the LVV bound to the resin eluted from the column, and the p24 detected in the column after elution was merely residual.

[0267] These results demonstrate that affinity chromatography using GSTCR2 is an effective purification method for lentiviral vectors, enabling LVV elution under mild conditions.

[0268] Recovery rate of infectious titer after purification by affinity chromatography The described LVV affinity purification process involves elution at a low pH, which may lead to a decrease in LVV infectivity. Furthermore, the p24 results indicate the presence of this protein in the FT, suggesting that some of the initial samples did not bind to GTSCR2 in the column. Therefore, titer measurements were performed to determine the infectivity of LVV in both the FT and eluted samples, and to assess the effect of elution at pH 6.0 on LVV infectivity.

[0269] The results shown in Figure 3 clearly demonstrate that the recovery rate of lentiviral infectivity was 70% higher than that of the initial sample. This represents a significant increase in the recovery rate of the purification process (an increase of over 20%) compared to the currently used anion exchange chromatography. Furthermore, titer analysis revealed that the FT sample did not contain the infectious vector. The amount of p24 observed in this sample (Figure 2) can be explained by considering the free p24 protein present in the lentiviral sample and the p24 that does not bind to the functional vector and therefore cannot bind to GSTCR2 in the resin. This demonstrates the specific enrichment of the viral preparation in the infectious lentiviral vector.

[0270] Measurement of impurities in the final product after affinity chromatography purification. The ultimate goal of the DSP method in lentiviral vector production is to obtain a final product of the highest purity and free from contaminants. Therefore, the amount of host cell protein (HCP) and residual DNA in LVV samples purified using GSTCR2 affinity chromatography was measured. Furthermore, these contaminants in LVV samples purified using anion exchange chromatography, currently used in DSP methods, were also measured, and the contaminant removal efficiency of both methods was compared.

[0271] The results showed that after purification by affinity chromatography, the HCP content decreased by more than 95% compared to the amount in the initial sample (Figure 4). Consistent with this result, the FT fraction was rich in HCP, suggesting that this purification method is effective in removing HCP. Furthermore, the amount of HCP in the LVV sample purified by AEX was 1.25 times that of the vector purified with resin using GSTCR2. This indicates that affinity chromatography is more effective in removing HCP than currently used anion exchange columns.

[0272] In addition to HCP, residual DNA levels were also measured in all samples. The reduction in DNA content in LVV eluted from GSTCR2 resin was significantly higher than that in samples purified by anion exchange (Figure 5). While the DNA removal rate exceeded 95% in samples purified by affinity chromatography, the reduction in DNA content in AEX products was less than 60% compared to the initial sample. Furthermore, the amount of DNA detected in FT samples obtained from affinity purification included the majority of residual DNA, which is consistent with the almost complete removal observed in the purified products.

[0273] Removal of impurities is a crucial aspect of DSP (Digital Spectroscopy), and is particularly important in lentiviral vectors used in increasingly frequent in vivo therapies. This demonstrates that affinity chromatography can be used as an excellent alternative to LVV purification because it offers higher selectivity and yields a purer, more homogeneous final product.

[0274] Comparison with commercially available affinity purification technology: CaptureSelect Lenti VSVG (ThermoFisher) Recently, affinity chromatography resins for the purification of VSV-G pseudotype LVV have been described. To evaluate the performance of this new technology in comparison with the GTSCR2 affinity chromatography developed by the present inventors, lentivirus samples were purified using both affinity resins in parallel. Purification was performed using the same temperature, concentration, and incubation time as used with the GTSCR2 resin, and samples were collected after all purification steps. Elution was performed by adding 0.8M arginine. To evaluate the efficiency of the purification method, the amount of LVV present in each sample was measured by ELISA p24.

[0275] The results shown in Figure 6 indicate that the elution efficiency of both purification systems is very similar, showing that approximately 85-90% of the LVV that remains bound to the resin after incubation is eluted.

[0276] Product recovery rates using the ThermoFisher affinity system were evaluated, and titer measurements were performed to determine whether elution with 0.8M arginine affected the infectivity of LVV. The results shown in Figure 7 indicate that the recovery rate of infectivity in the purified product using CaptureSelect Lenti VSVG resin was less than 10% compared to the initial sample. This recovery rate is approximately one-seventh of the recovery rate observed for LVV eluted from GSTCR2 affinity resin.

[0277] Furthermore, the presence of impurities in the purified products was determined for both the FT samples and eluted samples obtained from the two affinity purification systems. Figure 8 shows the number of ng of HCP per TU (infectious titer) in each case. The results showed that the host cell protein per transfection unit in the LVV sample purified using CaptureSelect Lenti VSVG resin was seven times higher than that in the sample purified using GTSCR2 affinity chromatography.

[0278] Furthermore, the amount of residual DNA co-purified with the lentiviral vector was measured for each sample (Figure 9). Here, the amount of DNA in the LVV purified using the technology developed by the authors was 1 / 18th of that in the vector purified using CaptureSelect Lenti VSVG affinity resin.

[0279] Therefore, the results suggest that affinity resins based on interaction with GTSCR2 offer clear advantages compared to CaptureSelect Lenti VSVG technology, resulting in improved recovery and infectivity of purified products, as well as enhanced removal of impurities.

Claims

1. A method for purifying viral particles or virus-like particles (VLPs) comprising an envelope, wherein the envelope comprises a VSVG protein or a functionally equivalent variant or fragment that retains the ability to bind to a low-density lipoprotein receptor (LDL-R), i) Contacting a sample containing virus particles or VLPs with a purified resin, ii) Washing the purified resin with an elution buffer, and iii) Recovering the virus particles or VLPs eluted from the purified resin. The purified resin includes, a) Low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or mutant thereof, wherein the fragment or mutant retains the VSVG binding ability of LDL-R. b) Matrix A method comprising the above, wherein the purified resin does not contain polypeptides exhibiting phase behavior.

2. The method according to claim 1, wherein the virus particles or VLP are brought into contact with the purified resin at a pH of at least 7.

3. The method according to any one of claims 1 or 2, wherein the elution buffer has a pH of 5.5 to 6.5, preferably pH 6.

4. The elution buffer is Ca ２＋ The method according to any one of claims 1 or 2, comprising a chelating agent, preferably comprising EDTA and / or EGTA.

5. The method according to any one of claims 1 to 4, wherein the matrix is ​​affinity-bound or covalently bound to the LDL-R or a functionally equivalent variant or fragment thereof.

6. The method according to any one of claims 1 to 5, wherein the LDL-R or a functionally equivalent variant or fragment is bound to the first member of the binding pair.

7. The method according to claim 6, wherein the first member of the affinity bond pair is covalently bonded to the matrix.

8. The method according to any one of claims 6 or 7, wherein the first member of the affinity binding pair is selected from the group consisting of GST, biotin, histidine tag, biotin acceptor peptide (BAP), antibody, or antibody derivative.

9. i) the LDL-R or a functionally equivalent variant or fragment is bound to a first member of the affinity binding pair, and ii) the matrix is ​​bound to a second member of the affinity binding pair.

10. The method according to claim 9, wherein the first member of the affinity binding pair is GST and the second member is glutathione, or the first member is biotin and the second member is avidin, or the first member is a histidine tag and the second member is a Ni or Co ion.

11. The method according to any one of claims 1 to 10, wherein the functionally equivalent fragment of LDL-R comprises a CR2 domain (SEQ ID NO: 2) and / or a CR3 domain (SEQ ID NO: 3), or a sequence having at least 75% identity thereto.

12. The method according to any one of claims 1 to 11, wherein the purified resin is non-magnetic.

13. The method according to any one of claims 1 to 12, wherein the matrix is ​​agarose.

14. The method according to any one of claims 1 to 13, wherein the purification is performed by chromatography column or batch purification.

15. a) Low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, b) Matrix A purified resin containing the above, which does not contain polypeptides with phase behavior and is non-magnetic.

16. The purified resin according to claim 15, wherein the matrix is ​​affinity-bound or covalently bound to a fragment of LDL-R or a functionally equivalent variant thereof.

17. The purified resin according to any one of claims 15 or 16, wherein a fragment of LDL-R or a functionally equivalent variant is bound to the first member of the affinity binding pair.

18. The purified resin according to claim 17, wherein the first member of the affinity pair is covalently bonded to the matrix.

19. The purified resin according to any one of claims 17 or 18, wherein the first member of the affinity binding pair is selected from the group consisting of GST, biotin, histidine tag, biotin acceptor peptide (BAP), antibody, or antibody derivative.

20. i) the LDL-R or a functionally equivalent variant or fragment is bound to a first member of the affinity binding pair, and ii) the matrix is ​​bound to a second member of the affinity binding pair, according to any one of claims 15 or 16.

21. The purified resin according to claim 20, wherein the first member of the affinity binding pair is GST and the second member is glutathione, or the first member is biotin and the second member is avidin, or the first member is a histidine tag and the second member is Ni or Co ion.

22. The purified resin according to any one of claims 15 to 21, wherein the functionally equivalent fragment of LDL-R comprises a CR2 domain (SEQ ID NO: 2) and / or a CR3 domain (SEQ ID NO: 3) or a sequence having at least 75% identity thereto.

23. The purified resin according to claim 15, comprising a fragment of a low-density lipoprotein receptor (LDL-R) containing a CR2 domain (SEQ ID NO: 2) and / or a CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant, but not containing a complete LDL-R protein.

24. The purified resin according to any one of claims 15 to 23, which is an agarose resin.

25. A purified resin according to any one of claims 15 to 24, which forms a chromatography column or is a batch resin.

26. Use of the purification resin according to any one of claims 15 to 25 for the purification of viral particles or VLPs comprising an envelope containing a VSVG protein or a functionally equivalent variant or fragment that retains the ability to bind to LDL-R.

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