Purification resin and methods for using the same

Abstract

The present invention is generally related to compositions and methods for purification of biologics. More specifically, the present invention relates to a purification resin, and method of using the same.

Description

[0001] PURIFICATION RESIN AND METHODS FOR USING THE SAME

[0002] FIELD OF THE INVENTION

[0003] The present invention is generally related to compositions and methods for purification of biologies. More specifically, the present invention relates to a purification resin, and method of using the same.

[0004] BACKGROUND OF THE INVENTION

[0005] Lentiviruses are enveloped viruses members of the Retroviradae family that have become increasingly relevant for biopharmaceuticals since they lead to stable integration of the transgene(s) to be expressed in both dividing and nondividing cells. Over the past years, the number of gene therapy clinical trials based on lentiviral vectors (LVs) has grown. Their use in gene therapy for several conditions has been reported both ex vivo and in vivo. The success of these clinical trials requires improved methods for purification of LV both quantitively, increasing process capacity and product quality, and qualitatively replacing traditional methods used at small scales that are not economically and / or technically viable for larger scales.

[0006] The current production technologies commonly result in low functional titers, ranging from 106to 108transducing units per mL (TU / mL), depending on the production system, LV pseudotype, harvest conditions, or even the titration technique. Additionally, impurities vary with the production system. When developing a downstream process (DSP), the concentration of LV is pursued along with increasing LV purity, which must consider the impurities profile of the production broth. Just like any other biological product, the degree of concentration and purification needed is fundamentally connected to what the LVs are intended for. Research / small-scale purposes do not generally require a very pure product. However, when the product is intended for clinical use, the safety of the drug is a concern.

[0007] The traditional purification and concentration techniques are mostly used if the needs of LV can be satisfied by small-scale production, for instance, scientific research. The centrifugationbased methods can be used as concentration and / or purification techniques.

[0008] The development and use of DSP with chromatography steps and membrane and monoliths-based separation operations have been described over the past years. Current lentiviral vector (LVV) purification process is based on an anion exchange chromatography (AEX). This allows LVV separation from other contaminants according to their charge. Due to the low selectivity of this purification method, final product contains extracellular vesicles and other negative charged contaminants. In addition, elution using high salt concentration causes loss of infectivity, which results in low recovery yields. Consequently, affinity chromatography appears as an outstanding alternative for LVV purification, due to the high selectivity and greater retention capacity of this type of chromatography columns.

[0009] The patent document US 2022010288A1 discloses a method for purifying a lentivirus particle comprising contacting the lentivirus particle with a fusion protein comprising a peptide derived from LDL-R. The fusion protein described in US 2022 010288A1 comprises a lentivirus domain and a polypeptide with phase behaviour.

[0010] The document Perry Christopher, “Lentiviral vector stability and process purification based on modifications to the viral envelope", 2 november 2022, discloses a method for purification a pseudotyped lentiviral vector comprising a VSV-G protein that natively binds to the LDL-R. The method is based on affinity binding of the viral particles, present in culture medium at pH of at least 7, to the CR2 domain of LDL-R conjugated to magnetic beads. However, this method failed to elute the vector at pH 6.5 and lower continously decrasing pH solution did not indicate transduction ability in the elution supernatants.

[0011] The patent document WO 2006 / 004660A2 discloses an affinity cromatography comprising glutathione-agarose beads binding a GST-LDLR fusion protein.

[0012] There is a need in the art for improved resins and methods for rapidly and cost-effectively purifying viral particles, such as lentiviral particles.

[0013] SUMMARY OF THE INVENTION

[0014] The authors of the present invention have developed an affinity chromatography system based on the interaction between vesicular stomatitis virus G glycoprotein (VSV-G), the envelope protein of the LVV, and the low-density lipoprotein receptor (LDL-R), which is the receptor located on the cell membrane that allows viral infection.

[0015] Thus, in a first aspect, the invention relates to a method for purifying a viral particle or a virus like particle (VLP) comprising an envelope, wherein the envelope comprises the VSVG protein or a functionally equivalent variant or fragment thereof that retains the ability to bind to the low-density lipoprotein receptor (LDL-R), wherein the method comprises: i) contacting a sample comprising the viral particles or VLPs with a purification resin, ii) washing the purification resin with an elution buffer, and iii) collecting the viral particles or VLPs eluted from the purification resin, wherein the purification resin comprises: a) the low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, wherein the fragment or variant retains the ability of LDL-R to bind VSVG, and b) a matrix, wherein the purification resin does not comprise a polypeptide with phase behaviour

[0016] In another aspect, the invention relates to a purification resin, hereinafter the purification resin of the invention, comprising: a) the low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, and b) a matrix, wherein the purification resin does not comprise a polypeptide with phase behaviour, and wherein the purification resin has no magnetic properties.

[0017] In another aspect, the invention relates to the use of the purification resin of the invention for the purification of viral particles or VLPs comprising an envelope comprising the VSVG protein or a functionally equivalent variant or fragment thereof that retains the ability to bind to the LDL-R.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1. Protocol for LVV purification by affinity chromatography using GSTCR2 as affinity ligand.

[0020] Figure 2. Viral particles detected in each step of the purification process determined by ELISA p24.

[0021] Figure 3. Titration results of lentiviral samples before purification (Initial), unbound from the affinity column (FT) and eluted LVV after purification (Elu).

[0022] Figure 4. Determination of host cell proteins amount in the different samples of the purification process. Values are scaled to a fixed viral titer. Determination of residual DNA present in the different samples of the purification process.

[0023] 6. Comparison to commercial affinity purification technology CaptureSelect Lenti

[0024] VSVG (ThermoFisher). Viral particles detected in each step of both purification processes determined by ELISA p24.

[0025] 7. Comparison to commercial affinity purification technology CaptureSelect Lenti

[0026] VSVG (ThermoFisher). Titration results of lentiviral samples before purification (Initial), unbound from the affinity column (FT) and eluted LVV after purification (Elu) for both purification systems.

[0027] 8. Comparison to commercial affinity purification technology CaptureSelect Lenti

[0028] VSVG (ThermoFisher). Determination of host cell proteins amount in the different samples of the purification process. Values are represented per transfection unit.

[0029] Comparison to commercial affinity purification technology CaptureSelect Lenti

[0030] VSVG (ThermoFisher). Determination of residual DNA present in the different samples of the purification process.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] Method of the invention

[0033] The authors of the present invention have developed a novel system for viral particles purification using affinity chromatography. This system is based on the interaction of an envelope protein that comprises the VSVG protein, with specific domains of the LDL-R.

[0034] In a first aspect, the present invention relates to a method, hereinafter the method of the invention, for purifying a viral particle or a virus like particle (VLP) comprising an envelope, wherein the envelope comprises the VSVG protein or a functionally equivalent variant or fragment thereof that retains the ability to bind to the low-density lipoprotein receptor (LDL-R), wherein the method comprises: i) contacting a sample comprising the viral particles or VLPs with a purification resin, ii) washing the purification resin with an elution buffer, and iii) collecting the viral particles or VLPs eluted from the purification resin, wherein the purification resin comprises: a) the low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, wherein the fragment or variant retains the ability of LDL-R to bind VSVG, and b) a matrix, wherein the purification resin does not comprise a polypeptide with phase behaviour.

[0035] A “viral particle” refers to a whole viral particle and not to a protein subunit or peptide. Viral particles consist of two or three parts: the genetic material of the virus made from either DNA or RNA; a protein coat that protects these genes; and, in some cases, an envelope of lipids that surrounds the protein coat when they are outside a cell. The shape of the viral particle ranges from simple helical and icosahedral forms to more complex structures, depending on the virus. A viral envelope is the outermost layer of many types of viruses. It protects the genetic material in their life cycle when traveling between host cells.

[0036] In a particular embodiment, the viral particle comprising an envelope is selected from the group consisting of: vesicular stomatitis virus or retrovirus.

[0037] Thus, in a particular embodiment, the viral particle comprising an envelope is a vesicular stomatitis viral particle.

[0038] Vesicular stomatitis virus (VSV) is an enveloped, negativestrand RNA virus that belongs to the Vesiculovirus genus of the Rhabdovirus family. VSV genome encodes five structural proteins among which a singletransmembrane glycoprotein (G). Glycoprotein G of the vesicular stomatitis virus (VSVG) is involved in receptor recognition at the host cell surface and then, after endocytosis of the virion, triggers membrane fusion via a low pH-induced structural rearrangement. G plays a critical role during the initial steps of virus infection. First, it is responsible for virus attachment to specific receptors. After binding, virions enter the cell by a clathrin-mediated endocytic pathway. In the acidic environment of the endocytic vesicle, G triggers the fusion, between the viral and endosomal membranes, which releases the genome in the cytosol for the subsequent steps of infection. VSVG has been widely used for pseudotyping other viruses and VSV- G-pseudotyped lentiviruses (VSV-G-LVs) exhibit the same broad tropism as VSV.

[0039] The term “pseudotyping” is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped virus particle, also called a pseudovirus. With this method, the foreign viral envelope proteins can be used to alter host tropism or increase or decrease the stability of the virus particles. Pseudotyped particles do not carry the genetic material to produce additional viral envelope proteins, so the phenotypic changes cannot be passed on to progeny viral particles. Pseudotyping allows one to control the expression of envelope proteins. A frequently used protein is the glycoprotein G (VSVG) from the Vesicular stomatitis virus (VS ).

[0040] In another particular embodiment, the viral particle comprising an envelope is a retrovirus particle.

[0041] Retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Retroviruses have many subfamilies in three basic groups.

[0042] Oncoretrovi ruses (cancer-causing retroviruses) include human T-lymphotropic virus (HTLV) causing a type of leukemia in humans, and murine leukemia viruses (MLVs) in mice.

[0043] Lentiviruses (slow viruses) include HIV-1 and HIV-2, the cause of acquired immune deficiency syndrome (AIDS) in humans.

[0044] Spumaviruses (foamy viruses) are benign and not linked to any disease in humans or animals.

[0045] In a particular embodiment, the retrovirus particle is selected from the group consisting of: alpharetrovirus particle, betaretrovirus particle, deltaretrovirus particle, epsilonretrovirus particle, gammaretrovirus particle or lentivirus particle.

[0046] In a more particular embodiment, the viral particle comprising an envelope is a gammaretrovirus particle.

[0047] Gammaretroviruses are a genus in the Retroviridae family. Example species are the murine leukemia virus and the feline leukemia virus.

[0048] In another particular embodiment, the viral particle comprising an envelope is a lentivirus particle.

[0049] Lentiviruses are enveloped viruses members of the Retroviradae family that have become increasingly relevant for biopharmaceuticals since they lead to stable integration of the transgene(s) to be expressed in both dividing and nondividing cells.

[0050] The term “virus-like particle”, also referred to as “VLP”, relates to particles resembling viruses that do not contain any viral genetic material. VLPs are the result of the expression of viral structural proteins, such as capsid proteins, and their self-assembly.

[0051] VLPs can be derived from the Hepatitis B virus (HBV) or composed of the small HBV derived surface antigen (HBsAg). VLPs have been produced from components of a wide variety of virus families including Parvoviridae (e.g. adeno-associated virus), Retroviridae (e.g. HIV), Flaviviridae (e.g. Hepatitis C virus), Paramyxoviridae (e.g. Nipah) and bacteriophages (e.g. Qp, AP205). Thus, in a particular embodiment, the VLP is derived from HBV, HBsAg, adeno-associated virus, HIV, hepatitic C virus, Nipah or bacteriophages.

[0052] The term “envelope” refers to the outermost layer of many types of viruses. It protects the genetic material in their life cycle when traveling between host cells. The envelopes are typically derived from portions of the host cell membranes (phospholipids and proteins), but include some viral glycoproteins.

[0053] As mentioned above, the envelope of the viral particles or VLPs comprises the VSVG protein or a functionally equivalent variant or fragment thereof that retains the ability to bind to the low-density lipoprotein receptor (LDL-R). Thus, in a particular embodiment, the envelope of the viral particles or VLPs comprises the VSVG protein. The VSVG protein is able to independently bind distinct CR domains of the low-density lipoprotein receptor (LDL-R).

[0054] In a particular embodiment, the envelope of the viral particles or VLPs comprises the extracelular fragment of the VSVG protein. In a more particular embodiment, the VSVG fragment comprises or consists of the sequence of the extracellular fragment of the VSV- G:

[0055] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADG WMCHA SKWVTTCDFR WYGPKYITHSIRSFTPS VEQCKESIEQ TKQG TWLNPGFPPQ SCGYA TVTDAEA VIVQ VTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHS DYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKH WGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLC QETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVG MISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKA QVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSS (SEQ ID NO: 1).

[0056] The low-density lipoprotein receptor (LDL-R) is a mosaic protein of 839 amino acids (after removal of 21 -amino acid signal peptide) that mediates the endocytosis of cholesterol-rich low-density lipoprotein (LDL). It is a cell-surface receptor that recognizes apolipoprotein B100 (ApoB100), which is embedded in the outer phospholipid layer of very low-density lipoprotein (VLDL), their remnants — i.e. intermediate-density lipoprotein (IDL), and LDL particles. The receptor also recognizes apolipoprotein E (ApoE) which is found in chylomicron remnants and IDL. LDL receptor mediates the endocytosis of cholesterol-rich LDL and thus maintains the plasma level of LDL. LDL-R ectodomain is composed of a ligand-binding domain, an epidermal growth factor (EGF) precursor homology domain and a C-terminal domain enriched in O-linked oligosaccharides. The ligand binding domain is made of 7 cysteine-rich repeats (CR1 to CR7). Each repeat is made of approximately 40 amino acids and contains 6 cysteine residues, engaged in 3 disulfide bridges, and an acidic residues cluster that coordinates a Ca2+ion. The intracellular release of the cargo is driven by a low-pH-induced conformational change of LDL-R from an open to a closed conformation.

[0057] In a particular embodiment, the LDL-R is the human protein identified by the Uniprot accesion number P01130 (Entry version 261 , sequence version 1 , 28 June 2023).

[0058] The LDL-R gene family consists of trans-membrane receptors that reside on the cellsurface, are involved in endocytic uptake of lipoproteins, and require Ca2+for ligand binding. All these receptors have in common several CR repeats (up to several tens), EGF precursor-like repeats, a membrane-spanning region and an intracellular domain containing at least one internalization signal sequence.

[0059] In another particular embodiment, the envelope of the viral particles or VLPs comprises a functionally equivalent variant of the VSVG protein that retains the ability to bind to the low-density lipoprotein receptor (LDL-R).

[0060] In the context of the invention, “functionally equivalent variant” of the VSVG protein is understood as any sequence having additions, substitutions, deletions or combinations thereof in its amino acid sequence and / or which has been chemically modified with respect to said sequence and which substantially maintains the function of said protein, particularly the ability to bind to the LDL-R. Preferably, the functional equivalent variants of the VSVG show the aforementioned ability at least by 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.

[0061] Suitable assays for determining whether VSVG binds to LDL-R include, but are not limited to: co-immunoprecipitation, far westernblot, protein ligation assay, affinity electrophoresis and isothermal titration calorimetry.

[0062] The “functionally equivalent variant” of the VSVG protein preferably have a sequence identity with these proteins of 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%. The degree of identity between the variants and the natural proteins is determined by using computer algorithms and methods that are widely known for the persons skilled in the art. For example, the identity between two amino acid sequences is determined by 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), though other similar algorithms can also be used.

[0063] In a particular embodiment, the functionally equivalent variant of the VSVG protein preferably have a sequence identity of 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% with the sequence:

[0064] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADG WMCHA SKWVTTCDFR WYGPKYITHSIRSFTPS VEQCKESIEQ TKQG TWLNPGFPPQ SCGYA TVTDAEA VIVQ VTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHS DYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKH WGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLC QETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVG MISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKA QVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSS (SEQ ID NO: 1)

[0065] In a particular embodiment, the funcionally equivalent variant of the VSVG protein 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% with the sequence of VSVG and maintains at least 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99% of the ability of VSVG to bind to the LDL-R.

[0066] In a first step of the method of the invention, a sample comprising the viral particles or VLPs is contacted with a purification resin.

[0067] The term “sample” relates to any sample which contains viral particles or VLPs.

[0068] In a particular embodiment, the viral particles or VLPs are contacted with the purification resin at a pH of at least 7.

[0069] In a more particular embodiment, the viral particles or VLPs are contacted with the purification resin at a pH between 7 and 9. In a still more particular embodiment, the viral particles or VLPs are contacted with the purification resin at a pH of 7, 7.5, 8 or 8.5.

[0070] In a particular embodiment, the viral particles or VLPs are contacted with the purification resin at a temperature between 2°C and 28°C, preferably at a temperature between 2°C and 10°C, more preferably at a temperature of 4°C. In another particular embodiment, the viral particles or VLPs are contacted with the purification resin for between 10 minutes and 8 hours, preferably between 30 minutes and 2 hours, more preferably for 1 hour.

[0071] In a more particular embodiment, the viral particles or VLPs are contacted with the purification resin at a temperature of 4°C for 1 hour at a pH of 7, 7.5 or 8.5.

[0072] In another particular embodiment, after the viral particles or VLPs are contacted with the purification resin for the time and at the temperature described, the sample is centrifuged. After the centrifugation step the bound and unbound fractions were taken.

[0073] In a particular embodiment, the first step of the method of the invetion further comprises obtaining an unbound fraction of the sample after contacting the sample with the purification resin.

[0074] In a particular embodiment, the unbound fraction (the flowthrough (FT) sample) is obtained by centrifuging the sample at a speed of between 300 xg and 1000 xg, preferably at 500 xg. In a particular embodiment, the unbound fraction (the flowthrough (FT) sample) is obtained by centrifuging the sample for at least 2 minutes, at least 3 minutes, at least 4 minutes, preferably for 5 minutes. In another particular embodiment, these speeds and times are combined. A person skilled in the art will know how to combine and vary the conditions of centrifugation speed and centrifugation time by modifying them in a compensatory manner.

[0075] In a more particular embodiment, the unbound fraction is obtained by centrifuging a sample at a speed of between 300 to 1000 xg; preferably of between 300 to 800 xg, such as at a speed of 500 xg; and for 2-10 minutes, preferably for 5 minutes.

[0076] In an embodiment, after the unbound fraction is taken, the resin is resuspended in wash buffer and is incubated at a temperature between 2°C and 28°C, preferably betwwen 2°C and 10°C, more preferably at a temperature of 4°C and for between 10 minutes and 8 hours, preferably between 30 minutes and 2 hours, more preferably for 30 minutes.

[0077] In another embodiment, after the incubation of the resin with the wash buffer, the resin fraction is obtained by centrifuging the sample at a speed of between 300 xg and 1000 xg, preferably at 500 xg. In an embodiment, the resin fraction is obtained by centrifuging the sample for at least 2 minutes, at least 3 minutes, at least 4 minutes, preferably for 5 minutes. In another particular embodiment, these speeds and times are combined. A person skilled in the art will know how to combine and vary the conditions of centrifugation speed and centrifugation tima by modifying them in a compensatory manner. In a more particular embodiment, the resin fraction is obtained by centrifuging a sample at a speed of between 300 to 1000 xg; preferably of between 300 to 800 xg, such as at a speed of 500 xg; and for 2-10 minutes, preferably for 5 minutes.

[0078] In a second step of the method of the invention, the purification resin is washed with an elution buffer.

[0079] “Elution buffer” as used herein refers to a major solvent in affinity chromatography. In the present invention, the elution buffer is added to break the binding interaction and release the target viral particles or VLPs, which are then collected in their purified form. Elution conditions may be specific, such as a competitive ligand, or nonspecific, such as changing pH, ionic strength, or polarity. Elution buffers dissociate binding partners by extremes of pH (low or high), high salt (ionic strength), the use of detergents or chaotropic agents that denature one or both of the molecules, removal of a binding factor or competition with a counter ligand.

[0080] In a particular embodiment, the elution buffer has a pH between 5.5 and 6.5. In a more particular embodiment the elution buffer has a pH of 6.

[0081] In another particular embodiment, the elution buffer comprises a Ca2+chelating agent. Chelating agents are organic compounds that are used to trap metal ion in circular structures (chelate circles) by several coordinations bounds. Most include oxygen, nitrogen and (or) sulfur, and are base on ethylediamine, acetylacetone, and oxine.

[0082] In more particular embodiment, the elution buffer comprises EDTA and / or EGTA.

[0083] EDTA is a popular chelating agent for divalent ions, that is widely used in biochemistry, molecular biology and cell biology. EDTA is an abbreviation for EthyleneDiamineTetraAcetic ac (and many other related molecules). EDTA is widely used to sequester di- and trivalent metal ions (Ca2+and Mg2+for example). EDTA binds to metals via four carboxylate and two amine groups.

[0084] EGTA (ethyleneglycol bis(2-aminoethyl ether)-N,N,N',N' tetraacetic acid) is a chelating agent with a much higher affinity for Ca2+than for Ca2+ions.

[0085] In a particular embodiment, the purification resin is incubated with the elution buffer at a temperature between 2°C and 28°C, preferably at a temperature between 2°C and 10°C, more preferaby at a temperature of 4°C.

[0086] In another particular embodiment, the purification resin is incubated with the elution buffer for between 10 minutes and 8 hours, preferably between 30 minutes and 2 hours, more preferably for 1 hour. In a more particular embodiment, the purification resin is incubated with the elution buffer at a temperature of 4°C for 1 hour.

[0087] In a third step of the method of the invention, the viral particles or VLPs that eluted from the purification resin are collected.

[0088] In an embodiment, a centrifugation step is performed prior to the collection of the viral particles or VLPs. Centrifugation allows the separation of the eluted fraction and the sample that remained bound to the resin after the elution.

[0089] In an embodiment, the centrifugation is at a speed of between 300 xg and 1000 xg, preferably at 500 xg. In an embodiment, the purified viral particles are obtained after a centrifugation for at least 2 minutes, at least 3 minutes, at least 4 minutes, preferably for 5 minutes. In another particular embodiment, these speeds and times are combined. A person skilled in the art will know how to combine and vary the conditions of centrifugation speed and centrifugation tima by modifying them in a compensatory manner.

[0090] In a more particular embodiment, the purified viral particles are obtained after a centrifugation step at a speed of between 300 to 1000 xg; preferably of between 300 to 800 xg, such as at a speed of 500 xg; and for 2-10 minutes, preferably for 5 minutes.

[0091] The purification method can be through a chromatography column or by batch purification.

[0092] In a particular embodiment, the purification is through a cromatography column.

[0093] The cromatography column can be prepared by packing a solid adsorbent into a cylindrical glass or plastic tube. The size will depend on the amount of compound being isolated. The base of the tube contains a filter, either a cotton or glass wool plug, or glass frit to hold the solid phase in place. A solvent reservoir may be attached at the top of the column.

[0094] Two methods are generally used to prepare a column: the dry method and the wet method. For the dry method, the column is first filled with dry stationary phase powder, followed by the addition of mobile phase, which is flushed through the column until it is completely wet, and from this point is never allowed to run dry. For the wet method, a slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. The top of the silica should be flat, and the top of the silica can be protected by a layer of sand. Eluent is slowly passed through the column to advance the organic material. In another particular embodiment, the purification is by a batch purification.

[0095] Batch purification involves the binding of the protein fraction to some type of chromatographic matrix that is not packaged within a column or physical support that allows flow-through. Batch method purification can be performed at any scale. In batch method purification, wash and elution fractions are separated from the resin after centrifuging to pellet the resin beads. The liquid cannot be removed completely because some of it is contained within the volume of porous bead pellet.

[0096] In a particular embodiment, 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 viral particle is purified to 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% purity.

[0097] In a particular embodiment, the purified viral particles retain their biological activity and / or structure. In a more particular embodiment, the purified viral particles retain its infectivity.

[0098] As an expert in the field may know, in a purification process, different resins can be used. For example, affinity cromatography is a separation technique that is highly selective for the protein of interest based upon molecular conformation, which frequently utilizes application specific resins. These resins have ligands attached to their surfaces which are specific for the compounds to be separated.

[0099] In the present invention, the purification resin used in the method of the invention comprises the LDL-R or a functionally equivalent fragment or variant of the LDL-R.

[0100] Thus, in a particular embodiment, the purification resin used in the method of the invention comprises the LDL-R. In a more particular embodiment, the LDL-R is the human protein identified under Uniprot accesion number P01130 (entry version 261 , sequence version 1 , 28 June 2023). In another particular embodiment, the purification resin used in the method of the invention comprises a functionally equivalent fragment or variant of the LDL-R.

[0101] In the context of the invention, “functionally equivalent variant” of the LDL-R is understood as any sequence having additions, substitutions, deletions or combinations thereof in its amino acid sequence and / or which has been chemically modified with respect to said sequence and which substantially maintains the function of said receptor, particularly the ability to bind the VSVG . The term variant refers to both full-length protein variants and protein fragment variants. Suitable assays for determining whether VSVG binds to LDL-R include, but are not limited to: co-immunoprecipitation, far werternblot, protein ligation assay, affinity electrophoresis and isothermal titration calorimetry.

[0102] Preferably, the functional equivalent variants of the LDL-R show the aforementioned function at least by 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.

[0103] The “functionally equivalent variant” of the LDL-R preferably have a sequence identity with these proteins of 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%. The degree of identity between the variants and the natural proteins is determined by using computer algorithms and methods that are widely known for the persons skilled in the art. For example, the identity between two amino acid sequences is determined by 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), though other similar algorithms can also be used.

[0104] In a particular embodiment, the functionally equivalent variant of the LDL-R preferably have a sequence identity of 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% with the LDL-R human protein identified under Uniprot accesion number P01130 (Entry version 261 , sequence version 1 , 28 June 2023).

[0105] In a particular embodiment, the funcionally equivalent variant of the 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% with the sequence of LDL-R and maintains at least 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99% of the ability of LDL-R to bind to VSVG.

[0106] In another particular embodiment, the variant of the LDL-R comprises a sequence with at least 75% identity to the CR2 domain (SEQ ID NO: 2) or to the CR3 domain (SEQ ID NO: 3). In a more particular embodiment, the variant of the LDL-R comprising a sequence with at least 75% identity to the CR2 domain of SEQ ID NO: 2 or to the CR3 domain of SEQ ID NO: 3 preserve the ability of these domain to bind VSVG. In a more particular embodiment, the variant of the LDL-R comprises a sequence with 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 to the CR2 domain.

[0107] In another more particular embodiment, the variant of the LDL-R comprises a sequence with 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 to the CR3 domain.

[0108] The term “fragment” as used herein refers to a protein or polypeptide includes a truncated form of the protein or polypeptide. For example, a fragment of LDL-R may include 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.

[0109] In a particular embodiment, the fragment of the LDL-R comprises or consists of the CR2 domain (SEQ ID NO: 2).

[0110] In another particular embodiment, the fragment of the LDL-R comprises or consists of the CR3 domain (SEQ ID NO: 3).

[0111] In another particular embodiment, the purification resin comprises the CR2 and the CR3 domains of LDL-R.

[0112] CR2 and CR3 domains are cysteine-rich domains, whose binding sites on glycoprotein G are identical.

[0113] In the present invention, the purification resin used in the method of the invention comprises a matrix.

[0114] The term “matrix” refers to devices and other substances which allow for cellular retention or cellular traversal, are biocompatible, and are capable of allowing traversal of macromolecules and particles either directly through the substance such that the substance itself is a semi-permeable membrane or used in conjunction with a particular semi-permeable substance. Any suitable matrix for viral particle purification can be used, including natural polymers, such as dextran, starch or cellulose, synthetic polymers, such as polyacrylamide, polymethacrylamide or polyvinyl ether polymers, etc. In a particular embodiment, the matrix is not a protein or peptide matrix. The matrix can optionally be in the form of a resin, including beaded resins, a membrane, monolith (hollow cylinders formed from a single block of polymer with a network of interconnected channels), or nanofibers.

[0115] In a particular embodiment, the matrix is agarose, cross-linked poly(styrene- divinylbenzene), cross-linked agarose, epoxi-activated agarose, polypropylene (PP), polyester, celullose, polyethersulfone (PES), poly(glycidyl methacrylate -co- ethylene dimethacrylate) or a non-cellulose synthetic polymer. In a preferred embodiment, the matrix is agarose.

[0116] In a particular embodiment, the matrix 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).

[0117] The LDL-R or the functionally equivalent variant or fragment thereof is linked to the matrix by any suitable means known, by the skilled person, such as covalentely linkage or affinitty binding.

[0118] In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof and the matrix are affinity or covalently bounded.

[0119] In some embodiments, the LDL-R or the functionally equivalent variant or fragment thereof and the matrix are affinity bounded.

[0120] In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof can be fused to a first member of an affinity binding pair that has the capacity to bind with a high affinity to a second member of a binding pair.

[0121] Thus, in a particular embodiment, i) the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of an affinity binding pair that has the capacity to bind with a high affinity to a second member of a binding pair and ii) the matrix is bound to a second member of the affinity binding pair.

[0122] The term affinity-binding pair refers to any peptide / ligand pair wherein the peptide has the capacity to specifically bind to the ligand. As used herein, the term "specific binding" refers to binding of the first member of the binding pair to the second member of the binding pair with greater affinity and specificity than to other molecules. Examples of affinity binding pairs are: a biotin or a biotin acceptor peptide (BAP) and a biotin-binding region (avidin or steptavidin), glutathione S-transferases (GST) and gluthathione (GSH), histidine tag and Ni or Co ions, and an antibody or antibody derivative and its specific antigen.

[0123] The first member of the binding pair does not involve any particular size or any other structural technical feature different from that of being capable of binding to the second member of the binding pair. By way of non-limiting illustration, said peptide can be a member of a specific binding pair, for example, an amino acid tag (e.g., a histidine tag (his-tag), an arginine tag (arg-tag), etc.), a peptide epitope which can be recognized by an antibody (e.g., c-myc-tag, etc.), a biotin acceptor peptide (BAP), a linear domain of interaction with another protein or proteins (e.g. SH3 proteins, signal transduction proteins, etc.), a post-translational modification sequence (e.g. myristoylation, methylation, phosphorylation, etc.), a sequence of transport to a cell compartment, etc.

[0124] In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of a binding pair, particularly a first member of a binding pair selected from the group comprising: a biotin, a biotin acceptor peptide (BAP), a peptide comprising the tripeptide Arg-Gly-Asp, GST, histidine tag and an antibody derivative.

[0125] The LDL-R, variant or fragment can be fused to a first member of the affinity binding pair by a covalent linkage. In a particular embodiment, when the first member of the affinity binding pair is a protein or peptide, the LDL-R, variant or fragment is bound to the first member of the affinity binding pair by peptide linkage, so that the LDL-R, variant or fragment and the first member of the affinity binding pair form a fusion protein.

[0126] The matrix can be fused to the second member of the affinity binding pair by a covalent linkage.

[0127] In a particular embodiment, wherein LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of the binding pair, the matrix is bound to a second member of a binding pair, said second member of the binding pair being bound to the first member of the binding pair. In a particular embodiment, the second member of the binding pair is selected from the group consisting of: a molecule comprising a biotin-binding region, gluthathione, Ni ions, Co ions and an specific antigen.

[0128] Molecules comprising a biotin-binding region according to the invention include, without limitation, avidin, an avidin analog, streptavidin, and streptavidin analog. The term “biotin-binding molecule”, as used herein, relates to a member of a binding pair that binds to biotin. Particularly, the biotin-binding molecule is avidin, streptavidin (SA) or avidin fragments which retain substantial binding activity for biotin, such as at least 50 percent or more of the binding affinity of native SA. As used herein, the term "avidin" refers to a glycoprotein found in egg whites and in tissues of birds, reptiles and amphibian’s protein and which has the capacity to bind to biotin with high affinity as well as any expressed or engineered form of the avidin biotin-binding molecule, such as streptavidin, neutravidin and the like. The term avidin includes both avidin found naturally in the eggs of Gallus gallus (NCBI accession numbers NM_205320.1 I GL45384353en, release as of 14 May 2013) as well as the orthologues of said protein in other species. Streptavidin, corresponding to the protein from Streptomyces avidinii (accession number CAA00084.1 in GenBank, release as of 28 January 1993), as well as the orthologues, homologues and fragments of streptavidin defined in the same manner as avidin. Streptavidin comprises 4 subunits each of which contains a binding site for biotin. Streptavidin (SA) or avidin fragments which retain substantial binding activity for biotin, such as at least 50 percent or more of the binding affinity of native SA or avidin, respectively, may also be used. Preferably, the affinity of the avidin variant for biotin is of at least 1015M'1, 1014M'1, 1013M'1, 1012M'1, 1010M'1or 109M'1.

[0129] Avidin and streptavidin variants suitable for use in the present invention include, without limitation:

[0130] "Core streptavidin" ("CSA"), which is a truncated version of the full-length streptavidin polypeptide which may include streptavidin residues 13-138, 14-138, 13-139 and 14-139. See, e.g., Pahler et al., (J. Biol. Chem. 1987, 262: 13933- 37).

[0131] - Truncated forms of streptavidin and avidin that retain strong binding to biotin (See, e.g. Sano et al., (J Biol Chem., 1995, 270: 28204-09) (describing core streptavidin variants 16-133 and 14-138) (U.S. Pat. No. 6,022,951).

[0132] Mutants of streptavidin and core forms of streptavidin which retain substantial biotin binding activity or increased 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.

[0133] Mutants of avidin and core forms of avidin which retain substantial biotin binding activity or increased biotin binding activity also may be used. See Hiller et al., J. Biochem. (1991) 278: 573-85; Livnah et al. Proc. Natl. Acad. Sci. USA (90: 5076- 80 (1993). - Variants resulting from the chemical modification of avidin such as those resulting from the complete or partial modification of glycosylation and fragments thereof as well as the completely deglycosylated avidin variant known as neutravidin.

[0134] - Avidin mutants as described in W005047317A1

[0135] - Avidin-like proteins as described in W006045891 , Recombinant avidin as described in WO0198349,

[0136] - Avidin variants as described in W00027814,

[0137] Monomeric streptavidin as described in WO06084388,

[0138] Modified streptavidin dimers such as those described in WO06058226,

[0139] - The protein with biotin binding capacity as described in W004018509, Streptavidin having a higher affinity for biotin as described in WO9840396,

[0140] - The modified streptavidin and avidin molecules as described in WO9640761 ,

[0141] - The streptavidin mutants as described in WO9711183,

[0142] - The streptavidin with modified affinity as described in WO9624606.

[0143] For convenience, in the instant description, the terms "avidin" and "streptavidin" as used herein are intended to encompass biotin-binding fragments, mutants and core forms of these binding pair members. Different avidin variants are commercially available, such as Extravidin (Sigma-Aldrich), NeutrAvidin (Thermo Scientific), NeutrAvidin (Invitrogen) and NeutraLite (Belovo). Moreover, the nucleic acid sequences encoding streptavidin and avidin and the streptavidin and avidin amino acid sequences can be found, for example, in GenBank Accession Nos. X65082; X03591 ; NM --205320; X05343; Z21611 ; and Z21554.

[0144] The expression “capable of interacting specifically” or “specific interaction”, as used herein in the context of the first and second members of a binding pair, describes the interaction between a first species and a second species characterized in that the nature of the binding is such that an antibody, a receptor or a nucleic acid binding protein binds to its corresponding binding partner but does not substantially bind to other species. Likewise, as used herein, the term “binding” or “binding to” means the physical association between a first species and a second species.

[0145] In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to GST as the first member of the affinity binding pair and the matrix is bound to gluthathione as the second member of the affinity binding pair.

[0146] Glutathione S-transferases (GSTs), previously known as ligandins, are a family of eukaryotic and prokaryotic phase II metabolic isozymes best known for their ability to catalyze the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification. The GST family consists of three superfamilies: the cytosolic, mitochondrial, and microsomal proteins. The glutathione binding site, or "G-site", is located in the thioredoxin-like domain of both cytosolic and mitochondrial GSTs.

[0147] Gluthathione is an antioxidant in plants, animals, fungi, and some bacteria and archaea. It is a tripeptide with a gamma peptide linkage between the carboxyl group of the glutamate side chain and cysteine. The carboxyl group of the cysteine residue is attached by normal peptide linkage to glycine. Glutathione exists in reduced (GSH) and oxidized (GSSG) states. GST binds specifically to reduced glutathione (GSH) in nearneutral, nondenaturing conditions (e.g., Tris buffer).

[0148] In another particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to biotin as the first member of the affinity binding pair and the matrix is bound to a biotin binding molecule as the second member of the affinity binding pair. In a more particular embodiment, the biotin binding molecule is avidin or streptavidin. In a still more particular embodiment, the biotin binding molecule is avidin.

[0149] In another particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to an His tag as the first member of the affinity binding pair and the matrix in bound to Ni ions or Co ions as the second member of the affinity binding pair.

[0150] In another particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to an antibody derivative and the first member of the affinity binding pair and the matrix is bound to an specific antigen for that antibody as the second member of the affinity binding pair.

[0151] As used herein, the term “antibody” refers to a protein including at least one immunoglobulin variable region, for example, an amino acid sequence providing an immunoglobulin variable domain or a sequence of the immunoglobulin variable domain. An antibody can include, for example, a variable heavy chain (H) region (herein abbreviated as VH) and a variable light chain (L) region (herein abbreviated as VL). Typically, an antibody includes two variable heavy chain regions and two variable light chain regions. The term “antibody” encompasses antigen-binding antibody fragments (for example, single-chain antibodies, Fab fragments, F(ab’)2 fragments, Fd fragments, Fv fragments and dAb fragments) as well as whole antibodies, for example, intact and / or full length immunoglobulins of the IgA, IgG types (for example, lgG1 , lgG2, lgG3, lgG4), IgE, I g D, IgM (as well as subtypes thereof). In a particular embodiment, the matrix is agarose and comprises or consists of the CR2 domain of the LDL-R fused to GST.

[0152] In a particular embodiment, i) the LDL-R or the functionally equivalent variant or fragment thereof and ii) the matrix are affinity bound. In this particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of an affinity binding pair and ii) the matrix is bound to a second member of the affinity binding pair as explained above.

[0153] In another particular embodiment, i) the LDL-R or the functionally equivalent variant or fragment thereof and ii) the matrix are covalently bound.

[0154] Therefore, in some embodiments, the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of a binding pair, particularly GST, without requiring the matrix to be bounded to a second member of a binding pair, particularity GSH.

[0155] In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of a binding pair, particularity GST. In a more particular embodiment, the LDL-R or the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence with at least 75% identity thereto is bound to a first member of a binding pair, particularly GST.

[0156] In another particular embodiment, the first member of the affinity binding pair is selected from the group consisting of: GST, biotin, histidine tag, a biotin acceptor peptide (BAP), an an antibody or an antibody derivative.

[0157] In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof bounded to a first member of a binding pair, particularly GST, and the matrix are covalently bound.

[0158] Therefore, in a particular embodiment, the first member of the affinity binding pair is bound to the matrix by a covalent linkage.

[0159] In a more particular embodiment, the GST is bound to the matrix by a covalent linkage.

[0160] In another particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to the first member of a binding pair, particularly GST, by a covalent linkage. In a more particular embodiment, the LDL-R or the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence with at least 75% identity thereto is bound to the first member of a binding pair, particularly GST, by a covalent linkage. A covalent bond or a covalent linkage is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. Covalent bonding also includes many kinds of interactions, including o-bonding, TT-bonding, metal-to-metal bonding, agostic interactions, bent bonds, three-center two-electron bonds and three-center four-electron bonds.

[0161] In a particular embodiment, the bound between the LDL-R or the functionally equivalent variant or fragment thereof and the matrix is the result of the reaction between two different functional groups found within the LDL-R and the matrix.

[0162] Common functional groups are known to an expert in the field and include: hydrocarbons, groups containing halogen, groups containing oxygen, groups containing nitrogen, groups containing sulfur, groups containing phosphorus, groups containing boron and groups containing metals.

[0163] Some of the most common functional groups are: hydroxyl functional group, aldehyde functional group, ketone functional group, amine functional group, amino functional group, amide functional group, ether functional group, ester functional group, carboxylic acid functional group, thiol functional group and phenyl functional group.

[0164] The hydroxyl functional group [H-OH] is the simplest of all the common organic functional groups. Also known as the alcohol group or hydroxy group, the hydroxyl group is an oxygen atom bonded to a hydrogen atom. Hydroxy groups link biological molecules together via dehydration reactions. Hydroxyls are often written as OH on structures and chemical formulas. While hydroxyl groups are not highly reactive, they do readily form hydrogen bonds and tend to make molecules that contain them soluble in water. Examples of common compounds containing hydroxyl groups are alcohols and carboxylic acids. The hydroxyl functional group consists of a hydrogen atom attached to an oxygen atom.

[0165] The aldehyde functional groups are made up of carbon and oxygen double-bonded together and hydrogen bonded to the carbon. An aldehyde may exist as either the keto or enol tautomer. The aldehyde group is polar. Aldehydes have formula R-CHO.

[0166] A ketone is a carbon atom double bonded to an oxygen atom that appears as a bridge between two other parts of a molecule. Another name for this group is the carbonyl functional group. The aldehyde is a ketone where one R is the hydrogen atom.

[0167] The amine functional groups are derivatives of ammonia (NH3) where one or more of the hydrogen atoms are replaced by an alkyl or aryl functional group. Silanization of microchannels with silane coupling agents such as (3-aminopropyl)triethoxysilane (APTES) and (3-aminopropyl)trimethoxysilane (APTMS) is the most frequently used method to generate amine functional groups on the channels' surfaces and enhance the immobilization of biomolecules.

[0168] The amino functional group is a basic or alkaline group. It's commonly seen in amino acids, proteins, and the nitrogenous bases used to build DNA and RNA. The amino group is NH2, but under acidic conditions, it gains a proton and becomes NH3+.

[0169] The amides functional groups are a combination of a carbonyl group and an amine functional group.

[0170] An ether functional group consists of an oxygen atom forming a bridge between two different parts of a molecule. Ethers have formula ROR.

[0171] The ester functional group is another bridge group consisting of a carbonyl group connected to an ether group. Esters have formula RCO2R.

[0172] The carboxylic acid functional group is also known as the carboxyl functional group. The carboxyl group is an ester where one substituent R is a hydrogen atom. The carboxyl group is usually denoted by -COOH.

[0173] The thiol functional group is similar to the hydroxyl group except the oxygen atom in the hydroxyl group is a sulfur atom in the thiol group. Thiol functional group is also known as a sulfhydryl functional group. Thiol functional groups have formula -SH.

[0174] The phenyl functional group is a common ring group. It is a benzene ring where one hydrogen atom is replaced by the R substituent group. Phenyl groups have formula CeHs.

[0175] The purification resin used in the method of the invention does not comprise a phase behaviour polypeptide. In a particular embodiment, the purification resin used in the method of the invention does not comprise any peptide or protein. The term “polypeptide” as used herein refers to a compound comprised of amino acid residues covalently linked by peptide bonds.

[0176] The term “polypeptide with phase behaviour” as used herein refers to any polypeptide that is capable of undergoing a phase transition. The polypeptide undergoes a phase transition due to the application of an environmental factor. Exemplary polypeptides with phase behavior include elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs).

[0177] In a particular embodiment, the purification resin used in the method of the invention does not comprise an elastin-like polypeptide and resin-like polypeptides. In a more particular embodiment, the purification resin used in the method of the invention does not comprise a polypeptide with phase behaviour with the sequence: (Val-Pro-Gly-Xaa- Gly)n(SEQ ID NO: 4), wherein X can be any amino acid except proline, (GRGDSPY)n

[0178] (SEQ ID NO: 5); (GRGDSPH)n(SEQ ID NO: 6); (GRGDSPV)n(SEQ ID NO: 7);

[0179] (GRGDSPYG)n (SEQ ID NO: 8); (RPLGYDS)n(SEQ ID NO: 9); (RPAGYDS)n(SEQ ID

[0180] NO: 10); (GRGDSYP)n(SEQ ID NO: 11); (GRGDSPYQ)n(SEQ ID NO: 12);

[0181] (GRGNSPYG)n (SEQ ID NO: 13); (GVGVP)n(SEQ ID NO: 14);

[0182] (GVGVPGLGVPGVGVPGLGVPGVGVP)m(SEQ ID NO: 15);

[0183] (GVGVPGVGVPGAGVPGVGVPGVGVP)m(SEQ ID NO: 16);

[0184] (GVGVPGWGVPGVGVPGWGVPGVGVP)m(SEQ ID NO: 17);

[0185] (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)m(SEQ ID

[0186] NO: 18); (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)m(SEQ ID NO: 19); and (GAGVPGVGVPGAGVPGVGVPGAGVP)m (SEQ ID NO: 20); wherein: n is 20-360; and m is 4-25. In another embodiment, the purification resin used in the method of the invention does not comprise a polypeptide with phase behaviour with the sequence (GVGVP)m(SEQ ID NO: 21); (ZZPXXXXGZ)m; (ZZPXGZ)m; (ZZPXXGZ)m; or (ZZPXXXGZ)m, wherein m is an integer between 10 and 160, inclusive of endpoints, wherein X if present is any amino acid except proline or glycine, and wherein Z if present is any amino acid. In another embodiment, the purification resin used in the method of the invention does not comprise a polypeptide with phase behaviour with the sequence: (GVGVPGVGVPGAGVPGVGVPGVGVP)m(SEQ ID NO: 16) or (GVGVPGVGVPGLGVPGVGVPGVGVP)m(SEQ ID NO: 27); wherein m is an integer between 2 and 32, inclusive of endpoints. In another embodiment, the the purification resin used in the method of the invention does not comprise a polypeptide with phase behaviour with the sequence: (GVGVPGVGVPGAGVPGVGVPGVGVP)m(SEQ ID NO: 16), wherein m is 8 or 16; (GVGVPGAGVP)m(SEQ ID NO: 29), wherein m is an integer between 5 and 80, inclusive of endpoints; or (GXGVP)m(SEQ ID NO: 30), wherein m is an integer between 10 and 160, inclusive of endpoints, and wherein X for each repeat is independently selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine.

[0187] Resilin-like polypeptides are elastomeric polypeptides with mechanical properties including desirable resilience, compressive elastic modulus, tensile elastic modulus, shear modulus, extension to break, maximum tensile strength, hardness, rebound, and compression set.

[0188] Elastin-like polypeptides (ELPs) are biopolymers derived from tropoelastin. The ability of ELPs to undergo morphological changes at certain temperatures enables specific proteins that are bound to the ELPs to be separated out from the rest of the solution via experimental techniques such as centrifugation. The general structure of polymeric ELPs is (VPGXG)n (SEQ ID NO: 4), where the monomeric unit is Val-Pro-Gly-X-Gly, and the "X" denotes a variable amino acid that can have consequences on the general properties of the ELP, such as the transition temperature (Tt). In general, these polymers are linear below the Tt, but aggregate into spherical clumps above the Tt. These polymers undergo reversible phase transition that can be triggered by various environmental stimuli, such as temperature, pH or ionic strength. ELPs can be used in purification processes. At temperatures below the Tt, the ELP will bind to the ligand in its linear form. Once the solution is heated to a temperature exceeding the Tt, the ELP will form spherical clumps. These clumps will then settle to the bottom of the solution tube following centrifugation, carrying the protein of interest.

[0189] In a particular embodiment, the term “polypeptide with phase behaviour” refers to those polypeptides defined on pages 25, 48 and 49 of the US 2022010288A1 patent application.

[0190] In another particular embodiment, the purification resin used in the method of the invention has no magnetic properties.

[0191] Therefore, in a particular aspect, the present invention relates to a method for purifying a viral particle ora virus like particle (VLP) comprising an envelope, wherein the envelope comprises the VSVG protein or a functionally equivalent variant or fragment thereof that retains the ability to bind to the low-density lipoprotein receptor (LDL-R), wherein the method comprises: i) contacting a sample comprising the viral particles or VLPs with a purification resin, ii) washing the purification resin with an elution buffer, and iii) collecting the viral particles or VLPs eluted from the purification resin, wherein the purification resin comprises: a) the low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, wherein the fragment or variant retains the ability of LDL-R to bind VSVG, and b) a matrix, wherein the purification resin does not comprise a polypeptide with phase behaviour, and wherein the purification resin has no magnetic properties. The term “magnetic properties” include, ferromagnetism properties, paramagnetism properties, diamagnetism properties or superparamagnetism properties.

[0192] The purification based on magnetic properties refers to the purification method which apply an external magnetic field for the separation of particles (size fractionation) or various compounds with different magnetic susceptibilities such as cations, anions, and those with effective magnetic moment. Also, it can be used for the separation of diamagnetic compounds by different strategies such as complexation with metal ions, reacting with metal-complexes, and attachment to magnetic or magnetized particles.

[0193] In a particular embodiment, the purification resin used in the method of the invention has no ferromagnetism properties. Ferromagnetism is the basic mechanism by which certain materials (such as iron) form permanent magnets. This means the compound shows permanent magnetic properties rather than exhibiting them only in the presence of an external magnetic field. In a ferromagnetic element, electrons of atoms are grouped into domains in which each domain has the same charge.

[0194] In another particular embodiment, the purification resin used in the method of the invention has no paramagnetism properties. Paramagnetism refers to the magnetic state of an atom with one or more unpaired electrons. The unpaired electrons are attracted by a magnetic field due to the electrons' magnetic dipole moments.

[0195] In another particular embodiment, the purification resin used in the method of the invention has no diamagnetism properties. Diamagnetic substances are characterized by paired electrons, e.g., no unpaired electrons.

[0196] In another particular embodiment, the purification resin used in the method of the invention has no superparamagnetic properties. “Superparamagnetism” is a form of magnetism exhibited by small ferromagnetic or ferrimagnetic nanoparticles. At sizes of less than a hundred nanometers, the nanoparticles are single-domain particles, allowing the magnetization of the nanoparticles to be approximated as one giant magnetic moment by summing the individual magnetic moments of each constituent atom.

[0197] In another particular embodiment, the purification resin used in the method of the invention does not comprise the complete LDL-R protein. In a particular embodiment, the LDL-R protein refers to the human protein identified under Uniprot accesion number P01130 (Entry version 261 , sequence version 1 , 28 June 2023). The complete human LDL-R protein comprises 860 amino acids.

[0198] Purification resin of the invention In another aspect, the invention relates to a purification resin, hereinafter the purification resin of the invention, comprising: a) the low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, and b) a matrix, wherein the purification resin does not comprise a polypeptide with phase behaviour, and wherein the purification resin has no magnetic properties.

[0199] The terms “LDL-R” and “purification resin” have been defined or explained in the method of the invention and these definitions are applicable to the purification resin of the invention.

[0200] In a particular embodiment, the purification resin of the invention comprises the LDL-R. In another particular embodiment, the purification resin of the invention comprises a functionally equivalent fragment or variant of the LDL-R.

[0201] In the context of the purification resin of the invention, “functionally equivalent variant” of the LDL-R is understood as any sequence having additions, substitutions, deletions or combinations thereof in its amino acid sequence and / or which has been chemically modified with respect to said sequence and which substantially maintains the function of said receptor, particularly the ability to bind the VSVG. The term variant refers to both full-length protein variants and protein fragment variants.

[0202] Suitable assays for determining whether VSVG binds to LDL-R include, but are not limited to: co-immunoprecipitation, far werternblot, protein ligation assay, affinity electrophoresis and isothermal titration calorimetry.

[0203] Preferably, the functional equivalent variants of the LDL-R show the aforementioned function at least by 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.

[0204] The “functionally equivalent variant” of the LDL-R preferably have a sequence identity with these proteins of 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%. The degree of identity between the variants and the natural proteins is determined by using computer algorithms and methods that are widely known for the persons skilled in the art. For example, the identity between two amino acid sequences is determined by using the BLASTP algorithm (BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol.

[0205] 21 5: 403-410 (1990), though other similar algorithms can also be used.

[0206] In a particular embodiment, the functionally equivalent variant of the LDL-R preferably have a sequence identity of 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% with the LDL-R human protein identified under Uniprot accesion number P01130 (Entry version 261 , sequence version 1 , 28 June 2023).

[0207] In a particular embodiment, the funcionally equivalent variant of the 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% with the sequence of LDL-R and maintains at least 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99% of the ability of LDL-R to bind to VSVG.

[0208] In another particular embodiment, the variant of the LDL-R comprises a sequence with at least 75% identity to the CR2 domain (SEQ ID NO:2) or to the CR3 domain (SEQ ID NO:3).

[0209] In a more particular embodiment, the variant of the LDL-R comprises a sequence with 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 to the CR2 domain.

[0210] In another more particular embodiment, the variant of the LDL-R comprises a sequence with 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 to the CR3 domain.

[0211] The LDL-R or the functionally equivalent variant or fragment thereof can be fused to a first member of an affinity binding pair that has the capacity to bind with a high affinity to a second member of a binding pair.

[0212] Thus, in a particular embodiment, i) the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of an affinity binding pair that has the capacity to bind with a high affinity to a second member of a binding pair and ii) the matrix is bound to a second member of the affinity binding pair. In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of a binding pair, particularly a first member of a binding pair selected from the group comprising: a biotin, a biotin acceptor peptide (BAP), a peptide comprising the tripeptide Arg-Gly-Asp, GST, histidine tag and an antibody derivative.

[0213] The LDL-R, variant or fragment can be fused to a first member of the affinity binding pair by a covalent linkage. In a particular embodiment, when the first member of the affinity binding pair is a protein or peptide, the LDL-R, variant or fragment is bound to the first member of the affinity binding pair by peptide linkage, so that the LDL-R, variant or fragment and the first member of the affinity binding pair form a fusion protein.

[0214] The matrix can be fused to the second member of the affinity binding pair by a covalent linkage.

[0215] In a particular embodiment, wherein LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of the binding pair, the matrix is bound to a second member of a binding pair, said second member of the binding pair being bound to the first member of the binding pair. In a particular embodiment, the second member of the binding pair is selected from the group consisting of: a molecule comprising a biotin-binding region, gluthathione, Ni ions, Co ions and an specific antigen.

[0216] Molecules comprising a biotin-binding region according to the invention include, without limitation, avidin, an avidin analog, streptavidin, and streptavidin analog.

[0217] The term “biotin-binsing molecule” has been defined or explained in the method of the invention and this definition is applicable to the purification resin of the invention.

[0218] In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to GST as the first member of the affinity binding pair and the matrix is bound to gluthathione as the second member of the affinity binding pair. The terms GST and GSH have been defined or explained above and this definition is applicable to the purification resin of the invention.

[0219] In another particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to biotin as the first member of the affinity binding pair and the matrix is bound to a biotin binding molecule as the second member of the affinity binding pair. In a more particular embodiment, the biotin binding molecule is avidin or streptavidin. In a still more particular embodiment, the biotin binding molecule is avidin. In another particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to an His tag as the first member of the affinity binding pair and the matrix in bound to Ni ions or Co ions as the second member of the affinity binding pair.

[0220] In another particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to an antibody derivative and the first member of the affinity binding pair and the matrix is bound to an specific antigen for that antibody as the second member of the affinity binding pair. The term “antibody” has been defined or explained above and this definitions is applicable to the purification resin of the invention.

[0221] In another particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof and the matrix are affinity bonded. In this particular embodiment, the the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of an affinity binding pair and ii) the matrix is bound to a second member of the affinity binding pair as explained above.

[0222] In another particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof and the matrix are covalently bonded.

[0223] Therefore, in some embodiments, the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of a binding pair, particularlry GST, without requiring the matrix to be bounded to a second member of a binding pair, particularlry GSH.

[0224] In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of a binding pair, particularlry GST. In a more particular embodiment, the LDL-R or the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence with at least 75% identity thereto is bound to a first member of a binding pair, particularly GST.

[0225] In another particular embodiment, the first member of the affinity binding pair is selected from the group consisting of: GST, biotin, histidine tag, a biotin acceptor peptide (BAP), an an antibody or an antibody derivative.

[0226] In a particular embodiment, the LDL-R or the functionally equivalent variant or fragment thereof bounded to a first member of a binding pair, particularly GST, and the matrix are covalently bound.

[0227] Therefore, in a particular embodiment, the first member of the affinity binding pair is bound to the matrix by a covalent linkage. In a more particular embodiment, the GST is bound to the matrix by a covalent linkage.

[0228] Different types of covalent bonds have been explained in the method of the invention and these covalent bonds are applicable to the purification resin of the invention.

[0229] The term “fragment” as used herein refers to a protein or polypeptide includes a truncated form of the protein or polypeptide. For example, a fragment of LDL-R may include 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.

[0230] In a particular embodiment, the fragment of the LDL-R comprises or consists of the CR2 domain (SEQ ID NO: 2).

[0231] In another particular embodiment, the fragment of the LDL-R comprises or consists of the CR3 domain (SEQ ID NO: 3).

[0232] In a particular embodiment, the purification resin comprises a fragment of the low-density lipoprotein receptor (LDL-R) comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant thereof.

[0233] In another particular embodiment, the purification resin of the invention does not comprise the complete LDL-R protein. In a particular embodiment, the LDL-R protein refers to the human protein identified under Uniprot accesion number P01130 (Entry version 261 , sequence version 1 , 28 June 2023). The complete human LDL-R protein comprises 860 amino acids.

[0234] Therefore, in a particular aspect, the invention relates to a purification resin comprising: a) a fragment of the low-density lipoprotein receptor (LDL-R) comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a functionally equivalent variant thereof, and b) a matrix, wherein the purification resin does not comprise a polypeptide with phase behaviour, wherein the purification resin has no magnetic properties, and wherein the purification resin does not comprise the complete LDL-R protein.

[0235] In a particular embodiment, the purification resin of the invention comprises a functionally equivalent variant of the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO:3). Therefore, in another particular embodiment, the purification resin comprises a functionally equivalent variant of SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0236] In the context of the purification resin of the invention, “functionally equivalent variant” of the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) is understood as any sequence having additions, substitutions, deletions or combinations thereof in its amino acid sequence and / or which has been chemically modified with respect to said sequence and which substantially maintains the function of the LDL-R, particularly the ability to bind the VSVG. The term variant refers to protein fragment variants.

[0237] Suitable assays for determining whether VSVG binds to LDL-R include, but are not limited to: co-immunoprecipitation, far werternblot, protein ligation assay, affinity electrophoresis and isothermal titration calorimetry.

[0238] Preferably, the functional equivalent variants of the CR2 (SEQ ID NO: 2) and / or CR3 domain (SEQ ID NO: 3) show the aforementioned function at least by 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.

[0239] The “functionally equivalent variant” of the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) preferably have a sequence identity with these proteins of 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%. The degree of identity between the variants and the natural proteins is determined by using computer algorithms and methods that are widely known for the persons skilled in the art. For example, the identity between two amino acid sequences is determined by 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), though other similar algorithms can also be used.

[0240] In a particular embodiment, the functionally equivalent variant of the CR2 domain of the LDL-R preferably have a sequence identity of 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% with the sequence SEQ ID NO: 2.

[0241] In another particular embodiment, the functionally equivalent variant of the CR3 domain of the LDL-R preferably have a sequence identity of 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% with the sequence SEQ ID NO: 3.

[0242] In a particular embodiment, the funcionally equivalent variant of the CR2 domain of the 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% with the sequence SEQ ID NO: 2 and maintains at least 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99% of the ability of LDL-R to bind to VSVG.

[0243] In another particular embodiment, the functionally equivalent variant of the CR3 domain of the 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% with the sequence SEQ ID NO: 3 and maintains at least 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99% of the ability of LDL-R to bind to VSVG.

[0244] In aparticular embodiment, the variant of the CR2 domain of the LDL-R comprises a sequence with at least 75% identity to the sequence SEQ ID NO:2.

[0245] In another particular embodiment, the variant of the CR3 domain of the LDL-R comprises a sequence with at least 75% identity to the sequence SEQ ID NO:3.

[0246] In a more particular embodiment, the variant of the CR2 domain comprises a sequence with 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 to the sequence SEQ ID NO: 2.

[0247] In another more particular embodiment, the variant of the CR3 domain comprises a sequence with 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 to the sequence SEQ ID NO: 3.

[0248] In a particular embodiment, the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO:2) and / or the CR3 domain (SEQ ID NO: 3) or the functionally equivalent variant thereof and the matrix are affinity or covalently bounded. In some embodiments, the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO:2) and / or the CR3 domain (SEQ ID NO: 3) or the functionally equivalent variant thereof and the matrix are affinity bounded.

[0249] The fragment of the LDL-R comprising the CR2 domain (SEQ ID NO:2) and / or the CR3 domain (SEQ ID NO: 3) or the functionally equivalent variant thereof can be fused to a first member of an affinity binding pair that has the capacity to bind with a high affinity to a second member of a binding pair.

[0250] Thus, in a particular embodiment, i) the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or the functionally equivalent variant thereof is bound to a first member of an affinity binding pair that has the capacity to bind with a high affinity to a second member of a binding pair and ii) the matrix is bound to a second member of the affinity binding pair.

[0251] The term affinity-binding pair refers to any peptide / ligand pair wherein the peptide has the capacity to bind to the ligand. Examples of affinity binding pairs are: a biotin or a biotin acceptor peptide (BAP) and a biotin-binding region (avidin or steptavidin), glutathione S-transferases (GST) and gluthathione (GSH), histidine tag and Ni or Co ions, and an antibody derivative and its specific antigen.

[0252] In a particular embodiment, the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or the functionally equivalent variant thereof is bound to a first member of a binding pair, particularly a first member of a binding pair selected from the group comprising: a biotin, a biotin acceptor peptide (BAP), a peptide comprising the tripeptide Arg-Gly-Asp, GST, histidine tag and an antibody derivative.

[0253] The fragment of the LDL-R or variant can be fused to a first member of the affinity binding pair by a covalent linkage. In a particular embodiment, when the first member of the affinity binding pair is a protein or peptide, the fragment of the LDL-R or variant is bound to the first member of the affinity binding pair by peptide linkage, so that the fragment of the LDL-R orvariant and the first member of the affinity binding pair form a fusion protein.

[0254] The matrix can be fused to the second member of the affinity binding pair by a covalent linkage. In a particular embodiment, wherein fragment of LDL-R or the functionally equivalent variant thereof is bound to a first member of the binding pair, the matrix is bound to a second member of a binding pair, said second member of the binding pair being bound to the first member of the binding pair. In a particular embodiment, the second member of the binding pair is selected from the group consisting of: a molecule comprising a biotin-binding region, gluthathione, Ni ions, Co ions and an specific antigen.

[0255] Molecules comprising a biotin-binding region according to the invention include, without limitation, avidin, an avidin analog, streptavidin, and streptavidin analog.

[0256] The term “biotin-binding molecule” has been defined or explained in the method of the invention and this definition is applicable to the purification resin of the invention.

[0257] In a particular embodiment, the fragment of the LDL-R or the functionally equivalent variant thereof is bound to GST as the first member of the affinity binding pair and the matrix is bound to gluthathione as the second member of the affinity binding pair. The terms GST and GSH have been defined or explained above and this definition is applicable to the purification resin of the invention.

[0258] In another particular embodiment, the fragment of the LDL-R or the functionally equivalent variant thereof is bound to biotin as the first member of the affinity binding pair and the matrix is bound to a biotin binding molecule as the second member of the affinity binding pair. In a more particular embodiment, the biotin binding molecule is avidin or streptavidin. In a still more particular embodiment, the biotin binding molecule is avidin.

[0259] In another particular embodiment, the fragment of the LDL-R or the functionally equivalent variant thereof is bound to an His tag as the first member of the affinity binding pair and the matrix in bound to Ni ions or Co ions as the second member of the affinity binding pair.

[0260] In another particular embodiment, the fragment of the LDL-R or the functionally equivalent variant thereof is bound to an antibody derivative and the first member of the affinity binding pair and the matrix is bound to an specific antigen for that antibody as the second member of the affinity binding pair. The term “antibody” has been defined or explained above and this definitions is applicable to the purification resin of the invention.

[0261] As explained above, in a particular embodiment, the fragment of the LDL-R or the functionally equivalent variant thereof and the matrix are affinity bonded. In this particular embodiment, the the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of an affinity binding pair and ii) the matrix is bound to a second member of the affinity binding pair as explained above. In another particular embodiment, the fragment of the LDL-R or the functionally equivalent variant thereof and the matrix are covalently bonded.

[0262] Therefore, in some embodiments, the fragment of the LDL-R comprisng the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or the functionally equivalent variant thereof is bound to a first member of a binding pair, particularly GST, without requiring the matrix is bounded to a second member of a binding pair, particularly GSH.

[0263] In a particular embodiment, the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or the functionally equivalent variant thereof is bound to a first member of a binding pair, particularly GST. In a more particular embodiment, the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence with at least 75% identity thereof is bound to a first member of a binding pair, particularly GST.

[0264] In another particular embodiment, the first member of the affinity binding pair is selected from the group consisting of: GST, biotin, histidine tag, a biotin acceptor peptide (BAP), an antibody or an antibody derivative.

[0265] In a particular embodiment, the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or the functionally equivalent variant thereof bounded to a first member of a binding pair, particularly GST, and the matrix are covalently bound.

[0266] Therefore, in a particular embodiment, the first member of the affinity binding pair is bound to the matrix by a covalent linkage.

[0267] In a more particular embodiment, GST is bound to the matrix by a covalent linkage.

[0268] In another particular embodiment, the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or the functionally equivalent variant thereof is bound to a first member of a binding pair, particularly GST, by a covalent linkage. In a still more particular embodiment, the fragment of the LDL-R comprising the CR2 domain (SEQ ID NO: 2) and / or the CR3 domain (SEQ ID NO: 3) or a sequence with at least 75% identity thereof is bound to a first member of a binding pair, particularly GST, by a covalent linkage.

[0269] Different types of covalent bonds have been explained in the method of the invention and these covalent bonds are applicable to the purification resin of the invention.

[0270] The purification resin of the invention does not comprise a phase behaviour polypeptide.

[0271] In a particular embodiment, the purification resin does not comprise any peptide or protein. The terms “polypeptide” and “polypeptide with phase behaviour” have been defined or explained in the method of the invention and these definitions are applicable to the purification resin of the invention.

[0272] The purification resin of the invention has no magnetic properties. The term “magnetic properties” has been defined or explained above and this definition is applicable to the purification resin of the invention.

[0273] The purification based on magnetic properties refers to the purification method which apply an external magnetic field for the separation of particles (size fractionation) or various compounds with different magnetic susceptibilities such as cations, anions, and those with effective magnetic moment. Also, it can be used for the separation of diamagnetic compounds by different strategies such as complexation with metal ions, reacting with metal-complexes, and attachment to magnetic or magnetized particles.

[0274] In a particular embodiment, the purification resin of the invention has no ferromagnetism properties. Ferromagnetism has been defined or explained above and this definition is applicable to the purification resin of the invention.

[0275] In another particular embodiment, the purification resin of the invention has no paramagnetism properties. Paramagnetism has been defined or explained above and this definition is applicable to the purification resin of the invention.

[0276] In another particular embodiment, the purification resin of the invention has no diamagnetism properties. Diamagnetic substances are characterized by paired electrons, e.g., no unpaired electrons.

[0277] In another particular embodiment, the purification resin of the invention has no superparamagnetic properties. “Superparamagnetism” has been defined or explained above and this definition is applicable to the purification resin of the invention.

[0278] In a particular embodiment, the purification resin of the invention comprises agarose, cross-linked poly(styrene-divinylbenzene), cross-linked agarose, epoxi-activated agarose, polypropylene (PP), polyester, celullose, polyethersulfone (PES), ligand Quaternary ammonium (Q), sulfonic acid (S), diethylamine (D), poly(glycidyl methacrylate -co- ethylene dimethacrylate) or non-cellulose synthetic polymers. In a more particular embodiment, the purification resin is an agarose resin.

[0279] In a particular embodiment, the purification resin is an agarose resin and the matrix or consists of the CR2 domain of the LDL-R fused to GST. In another particular embodiment, the purification 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)

[0280] In another particular embodiment, the purification resin forms a chromatography column. “Cromatography colum” has been defined or explained in the method of the invention and this definition is applicable to the purification resin of the invention.

[0281] In another particular embodiment, the purification resin is a batch resin.

[0282] The term “batch” have been defined or explained in the method of the invention and this definition is applicable to the purification resin of the invention.

[0283] In another aspect, the invention relates to the use of the purification resin of the invention for the purification of viral particles or VLPs comprising an envelope comprising the VSVG protein or a functionally equivalent variant or fragment thereof that retains the ability to bind to the LDL-R.

[0284] The terms “viral particles”, “VLPs”, “VSVG protein”, “functionally equivalent variant” and “LDL-R” have been defined or explained in the method of the invention and these definitions are applicable to the use of the invention.

[0285] EXAMPLES

[0286] The following examples illustrate the invention and must not be considered as limiting the scope thereof.

[0287] Material and Methods

[0288] Cloning, expression and purification of GSTCR2

[0289] GSTCR2 was cloned into the pGEX-6P-1 expression vector. This was later transformed into BL21 (DE3) competent cells and expressed overnight at 20°C after induction with IPTG. Cell pellet was resuspended in Lysis buffer (50mM Tris pH 8.0, 300mM NaCI, 2mM CaCh, 1mM DTT) and lysis was performed by sonication. Supernatant was incubated with reduced glutathione resin for 1 h at 4°C and later poured into the column and FT fraction was discarded. A wash step was performed by addition of Lysis buffer. Resin was finally resuspended 5ml of elution buffer (50mM Tris pH 8.0, 150mM NaCI, 30mM reduced glutathione, 2mM CaCh, 1mM DTT), incubated for 30min at 4°C and placed in the column, collating the eluted fraction. Resin functionalization

[0290] 50pL of GSTCR2 at 20pM were incubated with 200|JL of GST Resin equilibrated with binding buffer (50mM PIPES pH 7.0, 150mM NaCI, 2mM CaCh, 1mM DTT). Incubation was performed for 1 h at 4°C.

[0291] LVV purification with GSTCR2 functionalized resin

[0292] Functionalized resin was centrifuged for 5min at 500xg and supernatant was discarded. 500pL of LVV were added to the resin, incubated for 1 h at 4°C and centrifuged for 5min at 500xg, taking the Flowthrough (FT) sample. Resin was resuspended in 1 ml of wash buffer (1mM PIPES pH 7.0, 150mM NaCI, 2mM CaCh, 1mM DTT) and incubated for 30min at 4°C. Wash and Resin fractions were collected after centrifugation. Resin was finally resuspended in 500pL elution buffer (50mM MES pH 6.0, 150mM NaCI, 1 mM DTT), incubated for 1h at 4°C and centrifuged for 5min at 500xg, taking elution (Elu) and resin samples.

[0293] LVV purification analysis by p24 measurement p24 analysis of the samples taken in the purification steps (FT, Res1 , Elu, Res2) were measured using Lenti-X p24 rapid titer kit (Takara Bio), following the protocol indicated in the kit.

[0294] LVV purification analysis by infective titer determination

[0295] FT and Elution fractions were defrosted and diluted with DMEM +1 % Antibiotic + 8pg / ml polybrene in MW24 plate. Serial dilutions were prepared and each dilution series was performed in independent duplicates. Then, 250pl of each dilution were distributed to a new MW24 plate and 250pl of the cell suspension, with a density of 75.000 HEK293T cells per well in DMEM +1 % Antibiotic +8pg / ml polybrene, were added per well. The plates were incubated for 2h at 37°C. Afterwards, 1.5ml of DMEM +1% Antibiotic +15% FBS were added to each well and incubated for 72h at 37°C. Finally, the infective titer of the samples (FT, Elu) was determined by flow cytometry taking the transduced cells.

[0296] HCP content determination

[0297] HCP content of the samples taken in the purification steps (FT, Elu) were measured using HEK 293 HCP Elisa kit, 3G (cygnus technologies), following the protocol indicated in the kit.

[0298] LVV purification with CaptureSelect Lenti VSVG resin (ThermoFisher) 200pL of CaptureSelect Lenti VSVG resin were equilibrated with buffer (50mM HEPES pH 7.5, 150mM NaCI). 500pL of LVV were added to the resin, incubated for 1 h at 4°C and centrifuged for 5min at 500xg, taking the Flowthrough (FT) sample. Resin Wash was performed by adding 1ml of buffer (50mM HEPES pH 7.5, 150mM NaCI) and incubated for 30min at 4°C. Then, Wash and Resin (Res1) fractions were collected after centrifugation for 5min at 500xg. Resin was finally resuspended in 500pL elution buffer (50mM HEPES pH 7.5, 150mM NaCI, 0.8M Arginine), incubated for 1h at 4°C and centrifuged for 5min at 500xg, taking elution (Elu) and resin (Res2) samples.

[0299] Results

[0300] Protocol for LVV purification by affinity chromatography

[0301] LVV purification was performed in batch using GSTCR2 bound to an agarose-based glutathione resin. Lentiviral sample was incubated with the functionalized chromatography resin at pH 7.0 and samples of the bound (Res1) and unbound (FT) fractions were taken after centrifugation. After a wash step, LVV were eluted from the resin by incubation with buffer solution at pH 6.0. Again, centrifugation allowed the separation of the eluted fraction (Elu) and the sample that remained bound to the resin after elution (Res2). A scheme of the described purification protocol is shown in Figure 1.

[0302] In order to evaluate the efficiency of the purification system, the amount of LVV present in each sample was determined by ELISA p24. Results showed that about 70% of the initial sample binds to the column after interacting with GSTCR2, while 30% comes out in the flow through (Figure 2). After pH changing, almost all LVV bound to the resin elute from the column and only a residual amount of p24 can be detected in the column after elution.

[0303] These results showed that purification of lentiviral vectors by affinity chromatography using GSTCR2 appears as an effective purification method, allowing LVV elution under mild conditions.

[0304] Recovery of infective titer after purification by affinity chromatography

[0305] The LVV affinity purification process described involves product elution using low pH, which could result in a decrease of LVV infectivity. In addition, p24 results showed the presence of this protein in the FT, indicating that a fraction of the initial sample did not bind to GTSCR2 in the column. Therefore, a titration was performed in order to determine the infectivity of LVV in both, FT and eluted samples, and the influence of elution at pH 6.0 in LVV infectivity.

[0306] Results shown in Figure 3 expose that recovery of lentiviral infectivity is higher than 70% when compared to the initial sample. This represents a significant increase (more than 20% increase) in the recovery of the purification step in comparison to the currently use anion exchange chromatography. Also, titration results revealed how FT sample did not contain any infective vectors. The amount of p24 observed in this sample (Figure 2) can be explained if having into account free p24 protein present in lentiviral samples as well as p24 that is not associated to the functional vectors and, therefore, cannot bind to GSTCR2 in the resin. This demonstrated the specific enrichment of viral preparation in infective lentiviral vector.

[0307] Contaminants determination in final product after purification by affinity chromatography The final aim of the DSP process in lentiviral vector manufacturing is to obtain a final product with maximum purity and no contaminants. Therefore, the amount of host cell proteins (HCPs) and residual DNA was determined for LVV samples purified using GSTCR2 affinity chromatography. In addition, these contaminants were also measured in LVV samples purified using the anion exchange chromatography currently used in the DSP process, in order to compare the efficiency for contaminants removal of both methods.

[0308] Results showed that HCPs content decreases in more than 95% after purification with affinity chromatography compared to the amount in the initial sample (Figure 4). In accordance to this result, FT fraction is enriched in HCPs, indicating that this purification process is efficient for HCPs removal. Furthermore, the amount of HCPs in the sample of LVV purified by AEX is 1.25x higher than those vectors purified using the resin with GSTCR2. This demonstrate that affinity chromatography is more efficient for HCPs removal than the currently used anion exchange column.

[0309] In addition to HCPs, the amount of residual DNA was also measured in all samples. Here, the decrease in DNA content present in LVV eluted from the GSTCR2 resin is significantly higher than in those purified by anion exchange (Figure 5). Sample purified by affinity chromatography shows a DNA removal higher than 95%, while in the AEX product DNA content decrease does not even reach 60% compared to the initial sample. Furthermore, the amount of DNA detected in the FT sample from affinity purification contains the majority of the residual DNA, in agreement with the nearly complete elimination observed in the purified product.

[0310] Contaminants removal is a critical aspect of the DSP process, being of special importance for lentiviral vectors used in in vivo therapies, which are becoming more frequent in the recent years. This reveals the use of affinity chromatography as an excellent alternative for LVV purification, due to the higher selectivity that allows to obtain a final product with higher purity and homogeneity.

[0311] Comparison to commercial affinity purification technology: CaptureSelect Lenti VSVG (ThermoFisher)

[0312] It has recently been described an affinity chromatography resin for purification of VSV-G pseudotyped LVV. In order to determine the performance of this new technology in comparison to the GTSCR2 affinity chromatography developed by the authors of the present invention, lentiviral samples were purified using both affinity resins in parallel. It was performed using the same temperature, concentrations and incubation times than those used for GSTCR2 resin, taking samples after all purification steps. Elution was carried out by addition of 0.8M arginine. In order to evaluate the efficiency of the purification process, the amount of LVV present in each sample was determined by ELISA p24.

[0313] Results showed in Figure 6 indicates that elution efficiency is very similar for both purification systems, eluting around 85-90% of the LVV that remained bound to the resin after incubation.

[0314] In order to evaluate product recovery using ThermoFisher affinity system and to determine if elution using 0.8M arginine affected LVV infectivity, a titration was performed. Results shown in Figure 7 showed how recovery of infective titer in purified product using CaptureSelect Lenti VSVG resin is less than 10% when compared to the initial sample. This recovery is around 7x lower than the observed in LVV eluted from GSTCR2 affinity resin.

[0315] In addition, contaminants present in the purified product were determined for FT and eluted samples from both affinity purification systems. The ng of HCPs per TU (infective titer) in each case are represented in Figure 8. Results showed that LVV samples purified using CaptureSelect Lenti VSVG resin present 7x more host cell proteins per transfection unit than those purified using GTSCR2 affinity chromatography. Also, the amount of residual DNA that is co-purified with the lentiviral vectors was determined for each sample (Figure 9). Here, LVV purified using the authors developed technology present 18x less DNA that those vectors purified using CaptureSelect Lenti VSVG affinity resin.

[0316] Therefore, and according to the results, it can be concluded that the affinity resin based on the interaction with GTSCR2 shows clear advantages compare to CaptureSelect Lenti VSVG technology, offering a higher recovery and infectivity in purified product as well as a greater contaminants removal.

Claims

CLAIMS1 . A method for purifying a viral particle or a viral like particle (VLP) comprising an envelope, wherein the envelope comprises the VSVG protein or a functionally equivalent variant or fragment thereof that retains the ability to bind to the low- density lipoprotein receptor (LDL-R), wherein the method comprises: i) contacting a sample comprising the viral particles or VLPs with a purification resin, ii) washing the purification resin with an elution buffer, and iii) collecting the viral particles or VLPs eluted from the purification resin, wherein the purification resin comprises: a) the low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, wherein the fragment or variant retains the ability of LDL-R to bind VSVG, and b) a matrix, wherein the purification resin does not comprise a polypeptide with phase behaviour.

2. The method according to claim 1 , wherein the viral particles or VLPs are contacted with the purification 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 between 5.5 and 6.5, preferably has a pH of 6.

4. The method according to any one of claims 1 or 2, wherein the elution buffer comprises a Ca2+chelating agent, preferably comprises EDTA and / or EGTA.

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

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

7. The method according to claim 6, wherein the first member of the affinity binding pair is bound to the matrix by a covalent linkage.

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, a biotin acceptor peptide (BAP), an antibody or an antibody derivative.

9. The method according to any one of claims 1 to 5, wherein i) the LDL-R or the functionally equivalent variant or fragment thereof is bound to a first member of an 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 wherein the first member is biotin and the second member is avidin, or wherein the first member is a histidine tag and the second member is Ni or Co ions.

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

12. The method according to any one of claims 1 to 11 , wherein the purification resin has no magnetic properties.

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 through a chromatography column or by a batch purification.

15. A purification resin comprising: a) the low-density lipoprotein receptor (LDL-R) or a functionally equivalent fragment or variant thereof, and b) a matrix, wherein the purification resin does not comprise a polypeptide with phase behaviour and wherein the purification resin has no magnetic properties.

16. The purification resin according to claim 15, wherein the fragment of the LDL-R or the functionally equivalent variant thereof and the matrix are affinity or covalently bound.

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

18. The purification resin according to claim 17, wherein the first member of an affinity pair is bound to the matrix by a covalent linkage.

19. The purification 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), an antibody or antibody derivative.

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

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

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

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

24. The purification resin according to any one of claims 15 to 23, wherein the purification resin is an agarose resin.

25. The purification resin according to any one of claims 15 to 24, wherein the purification resin 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 comprising the VSVG protein or a functionally equivalent variant or fragment thereof that retains the ability to bind to the LDL-R.

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