Lentiviral detection and purification

Abstract

A synthetic polymer which binds vesicular stomatitis virus G (VSV-G) protein wherein the synthetic polymer does not contain any proteinogenic amino acids. A method of preparing a synthetic polymer which binds VSV-G protein comprises providing a polymerisable composition, wherein the polymerisable composition does not contain any proteinogenic amino acids, and effecting polymerisation of the polymerisable composition to produce the synthetic polymer. Compositions comprising the synthetic polymer are also provided. The synthetic polymer and compositions comprising the same can be used in the detection of VSV-G as well as in purification methods.

Description

[0001] Lentiviral Detection and Purification

[0002] Introduction

[0003] The present invention relates to compounds for the detection of vesicular stomatitis virus (VSV) and VSV glycoprotein (VSV-G) pseudotyped lentiviral vectors as well as the purification of the same. It further relates to methods of producing the compounds and to compositions comprising the compounds. More particularly, the invention relates to use of the compounds or compositions in the detection and purification of VSV and VSV-G pseudotyped lentiviral vectors.

[0004] Background to the Invention

[0005] Lentiviral vectors have become a fundamental tool for gene and cell therapies owing to their effectiveness in transferring genetic material and their ability to transduce both dividing and nondividing, or slowly dividing, cells.

[0006] Although the use of lentiviral vectors was initially confined to academic investigations, they have now been used to effectively transduce multiple cell types for the treatment of several different diseases. Indeed, the number of gene therapy clinical trials worldwide using lentiviral vectors for gene transfer has soared in recent years, increasing from 89 in 2014 to 364 as of May 20231.

[0007] Lentiviruses belong to a genus of the Retroviridae family. They contain an RNA genome which is reverse transcribed into DNA in a transduced cell by a viral ly encoded reverse transcriptase leading to integration of the genetic material into the host genome.

[0008] In order for a lentiviral vector to transport its payload into a cell, it must interact with a cellular receptor in order to trigger the fusion of the viral envelope with the cell membrane. Such interaction is facilitated by glycoproteins within the viral envelope. HIV-1 is the most well-studied and used lentivirus in research. However, the wild type HIV glycoprotein, gp120, has very specific tropism for human CD4+ T cells and monocytes. Pseudotyping, a process whereby the wild-type envelope glycoprotein is exchanged for heterologous glycoproteins, means that lentiviral vectors can be endowed with the tropism of the virus from which the heterologous glycoprotein is derived.

[0009] One of the first, and most widely used, viral envelopes for pseudotyping belongs to vesicular stomatitis virus (VSV) owing to its broad tropism, thermal, and physical stability. Indeed, VSV glycoprotein (VSV-G) pseudotyped lentiviral vectors have been shown to be somewhat resistant to both freeze-thaw cycles and ultracentrifugation2’3and are widely regarded as the ‘gold-standard’ in gene therapy applications6.

[0010] Isolates of VSV are enveloped, nonsegmented, negative-strand RNA viruses that belong to the genus Vesiculovirus in the Rhabdoviridae family.

[0011] There are two major serotypes of VSV, Indiana (VSIV) and New Jersey (VSNJV). There is only 50% identity at the amino acid level between the G proteins of VSIV and VSNJV4The VSIV G protein has 511 amino acids, is glycosylated at positions 178 and 335, and contains covalently linked fatty acid in the cytoplasmic domain5. The VSNJV glycoprotein contains 517 amino acids, and the glycosylation sites are identical to those of VSIV G, but it is not acylated5. The standard envelope used for pseudotyping is from VSIV and it recognizes the low-density lipoprotein receptor (LDL- R) which governs attachment and entry into the cell.9

[0012] Though lentiviral vector production and purification processes have evolved significantly over recent years in line with the expansion of the use of such vectors into the industrial sector, the ever-increasing demand for higher quantities with more restrictive purity requirements means that novel approaches focussed on increasing productivity and purity in a cost-effective manner are needed. Means for analysing lentiviral vectors throughout the manufacturing process are also needed.

[0013] The production of lentiviral vectors centres around the use of a packaging cell line such as the human embryonic kidney (HEK) 293 or 293T cell lines to produce the viral vector particles. Large-scale manufacturing of lentiviral vectors generally begins with the expansion of such packaging cells prior to their transient transfection with plasmid DNA encoding the necessary proteins for lentiviral vector production. Over several days the lentiviral vector particles are produced and can be harvested from the culture medium e.g. after 48-72 hours. The quality of the particles harvested is highly dependent on cell viability which impacts the amount of host cell derived impurities. Thus, the choice of the time to harvest must take into account not just the quantity of particles but the quality of the supernatant in which they exist.7 8

[0014] After flow filtration which is intended to remove cells and cell debris from the lentiviral vector containing media, the clarified vector is treated to remove contaminating DNA products, and viral products are purified using various methods such as gradient purification and chromatography. Following purification, the eluted fractions undergo a series of polishing, formulation and sterilisation steps. The polishing and formulation steps are often completed using either chromatographical methods (based on size exclusion) or by tangential flow filtration (TFF). The sterilisation step is usually completed by filtration through a 0.22 pm filter.7

[0015] The degree of purification required is tied to the downstream use of the lentiviral vectors. Research use generally does not require a very pure product; however, if the lentiviral vector is intended for clinical use, especially for in vivo clinical use, purity is of the utmost importance to reduce the chance of an immunogenic response.7

[0016] For research use, traditional purification and concentration techniques are commonly used such as density gradient centrifugation or ultracentrifugation using sucrose or iodixanol. An alternative approach uses polyethylene glycol (PEG) which sterically excludes viral particles from the solution leading to their precipitation. Viral particles can then be recovered by centrifugation.7

[0017] Limitations of such traditional methods include scalability and the concentration of impurities in the product. Although a high degree of purification can be obtained with ultracentrifugation, the stress that the lentiviral vectors are subjected to can reduce the infectiveness of the particles.7

[0018] Scalable purification steps for lentiviral vectors generally use chromatography and / or TFF. With respect to chromatography, anion-exchange and affinity chromatography have both been used. In anion-exchange chromatography, the lentiviral vector particles are purified in a bind-elute mode whereby the lentiviral vector particles, which at working pH have a negative surface charge, are first adsorbed by a positively charged matrix. An example of such a matrix is HiTrap Q HP (Cytiva). They are then eluted with buffer ionic strengths between 0.5-1 M NaCI.

[0019] Membranes and monoliths stationary phases, as with conventional chromatography resins, can be functionalised with strong or weak ion ligands.7

[0020] One downside with anion exchange chromatography is that because binding is driven by electrostatic interactions, the anion exchange matrices are not selective for lentiviral vector particles and negatively charged nucleic acids and proteins can bind. This is compounded by the high salt elution step which also releases bound impurities.7Accordingly, purification by anion exchange chromatography suffers from low recovery rates. Systems are also often single use, leading to high production costs.

[0021] In affinity chromatography, the lentiviral vector particles are purified based on a highly specific interaction between them and a specific immobilised ligand. Thus, one of the advantages of affinity chromatography over anion exchange chromatography is the increased selectivity and thereby increased purity. That said, there are disadvantages with known techniques. For example, immobilised metal affinity chromatography (IMAC) is centred around the binding between an immobilised metal ion and a molecule capable of sharing electrons with it. For lentiviral vector purification, the envelope protein is modified with a histidine tag however such a tag can cause a reduction in both expression and protein function resulting in a reduction in infectivity. The elution step also makes use of imidazole which can inactivate the lentiviral vector particles. An alternative technique makes use of heparin but a disadvantage therewith is the low selectivity. A further alternative is centred around labelling the lentiviral particles with desthiobiotin or biotin mimics that bind to a streptavidin column but any technique that relies on labelling runs the risk of compromising the biological activity of the lentiviral vector particle.7

[0022] There is currently no established affinity reagent available for the purification of VSV- G pseudotyped lentiviral vectors. Throughout the manufacturing process, lentiviral vectors may need to be analysed e.g. to establish viral titre before transduction. Common methods used to detect lentiviral vectors include p24 ELISA, PCR and VSV-G antibodies (where lentiviral vectors are pseudotyped with VSV-G).

[0023] The p24 ELISA technology uses a standard ELISA to quickly determine the titre of any HIV-1 based lentiviral supernatant. In more detail, the wells of the microtitre plate are coated with anti-p24 capture antibody, which quantitatively binds the p24 capsid protein in the sample. Bound p24 is then detected in a typical sandwich ELISA format using biotinylated anti-p24 secondary antibody, a streptavidin-HRP conjugate and a colour-producing substrate. A p24 standard curve quantifies p24 content, which allows the lentivirus titre to be calculated. However, a disadvantage of this approach is that it requires the lentivirus to first be lysed in order for the p24 capsid protein to be accessible. This then allows the lentivirus-associated p24 to mix with free p24, generated by the packaging cells during transient transfection, in the sample. As free p24 can account for a substantial portion of total p24 in a supernatant sample, this can cause a large overestimation in the quantity of lentivirus present.

[0024] The qRT-PCR procedure entails subjecting serial dilutions of a lentiviral vector supernatant and a control RNA of known copy number to one-step qRT-PCR. The lentiviral copy number contained in the supernatant can then be determined by comparing its Ct value to a standard curve. A disadvantage with this approach is that it again requires lentivirus to be lysed thus it provides no information regarding the integrity of the viral particles.

[0025] VSV-G antibodies have also been used however many such antibodies target the transmembrane domain meaning they cannot detect intact lentiviral particles.

[0026] Molecular imprinting methods have been applied for the synthesis of polymeric materials.

[0027] Li et al in “Size matters: Challenges in imprinting macromolecules” reviews developments in the molecularly imprinted polymer area, with a focus on highlighting issues involved in the imprinting of macromolecules and the similarities and differences between imprinting small molecules and imprinting macromolecules.

[0028] WO 2006 / 004536 A1 discloses a method for producing molecular imprinted polymers which can be applied as a thin film to the surface of a support material in which a suspension of at least one functional monomer, one template and one initiator is used for the polymerisation, and with which RAFT agents are employed. The template can be ions, antibodies, antigens, amino acids, peptides, proteins, DNA bases, carbohydrates, drugs, pesticides, nucleic acids, viruses, bacteria or cells.

[0029] Batista et al in “Plastic Antibodies Mimicking the ACE2 Receptor for Selective Binding of SARS-CoV-2 Spike” describes the development of a silane / silica-based core / shell MIP that mimics ACE2 receptor and binds to SARS-CoV-2 spike RBD protein. Ability of such a MIP to bind to pseudoviruses containing SARS-CoV-2 spike proteins, and VSV-G glycoprotein particles was also assessed.

[0030] Accordingly, one aim of the present invention is to provide alternative means for detecting and purifying VSV-G pseudotyped lentiviral vectors. An aim of specific embodiments of the invention includes providing improved detection and purification means.

[0031] Summary of the Invention

[0032] Accordingly, the invention provides a synthetic polymer which binds vesicular stomatitis virus G (VSV-G) protein, wherein the synthetic polymer does not contain any proteinogenic amino acids.

[0033] The invention also provides a method of preparing a synthetic polymer which binds VSV-G protein comprising the steps of:

[0034] (a) providing a polymerisable composition wherein the polymerisable composition does not contain any proteinogenic amino acids, and

[0035] (b) effecting polymerisation of the polymerisable composition to produce the synthetic polymer.

[0036] Further provided are compositions comprising the synthetic polymer. Each of the synthetic polymers of the invention and the compositions of the invention can be used in the detection and purification of VSV-G and lentiviral vectors pseudotyped with VSV-G.

[0037] Details of the Invention

[0038] The invention provides a synthetic polymer which binds vesicular stomatitis virus G (VSV-G) protein, wherein the synthetic polymer does not contain any proteinogenic amino acids.

[0039] Proteinogenic amino acids in this context are amino acids capable of being incorporated biosynthetically into proteins. Such amino acids include glycine, proline, cysteine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid and glutamic acid. Derivatives of such amino acids do not constitute proteinogenic amino acids.

[0040] In some embodiments, the synthetic polymer does not contain any of the nucleotides adenine, thymine, guanine, cytosine and uracil.

[0041] In some embodiments of the invention, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to VSV-G protein.

[0042] In molecular imprinting, functional, and sometimes cross-linking, monomers are polymerised in an appropriate solvent in the presence of the compound to be imprinted. The compound to be imprinted is known as the template. During the polymerisation step, the monomers interact with the template through electrostatic, hydrophobic or other interactions leading to the formation of binding sites in the polymer, known as recognition sites, that are complementary to the template molecule. After removal of the template, the polymer matrix retains the recognition sites that are complementary to the template. Complementary in this sense means that the interacting groups in the polymer are placed such that they are in a suitable position to interact with the interacting groups in the template molecule and other molecules that contain the template molecule as a substructure i.e. in this invention VSV-G protein.

[0043] There are two major serotypes of vesicular stomatitis virus (VSV), Indiana (VSIV) and New Jersey (VSNJV). The standard envelope used for pseudotyping lentiviral vectors is from VSIV10. The amino acid sequences of the VSIV glycoproteins from several strains of virus are known. SED ID NO 1 -7 show the amino acid sequences from the strains of virus known as ‘strain 94GUB Central America’, ‘strain 85CLB South America’, ‘strain 98COE North America’, ‘Glasgow’, ‘Orsay’, ‘Mudd-Summers’ and ‘San Juan’.

[0044] VSV-G is a type III viral fusion protein and exists as a trimer of monomeric subunits, each of approximately 500 amino acids. Each monomer subunit can be classified by four structural domains: a [3-sheet-rich lateral domain (domain I); a central domain that results in the trimerisation of the molecule (domain II); a pleckstrin homology domain (domain III); and a fusion domain (domain IV). VSV-G attaches to viral membranes (e.g. lentivirus) via a ~20 amino acid sequence near the carboxy-terminus. VSV-G undergoes post-translational modification via glycosylation of two asparagine residues (ASN-178 and ASN-335).

[0045] In preferred embodiments of the invention, the ‘at least one recognition site’ is complementary to a template molecule comprising an amino acid sequence corresponding to a subsequence of a VSV-G polypeptide. Preferably the VSV-G polypeptide is from the Indiana serotype of VSV.

[0046] Although the amino acid sequences of the VSIV glycoproteins from several strains of virus are known, and any sequence therein can be used as a template, for some downstream applications it is beneficial to select, as a template, a subsequence that falls outside of the region responsible for attaching to viral membranes. Advantageously, this means that the synthetic polymer can interact with VSV-G when the VSV-G is affixed to a viral membrane. Accordingly, in preferred embodiments of the invention, the subsequence falls outside of the region responsible for attaching to viral membranes. For some downstream applications, it is also beneficial to select, as a template, sequences which are conserved between strains. This ensures that the synthetic polymer can interact with VSV-G from several different strains.

[0047] For some downstream applications, it is also beneficial to select, as a template, sequences which do not include known glycosylation sites. Advantageously, this increases consistency of binding.

[0048] It is also beneficial to select, as a template, a sequence that is accessible and comprises amino acids with side chains containing ring structures or charged groups.

[0049] With the above in mind, in preferred embodiments of the invention, the ‘at least one recognition site’ is complementary to a template molecule comprising an amino acid sequence selected from the group consisting of (i) DYKVKGLCDSNL, (ii) ADKDLFAAARFPE, (iii) PEGSSISAP, (iv) EDGELSSLGKEGT, (v) PKSHKAI, (vi) ADKDLFAAAR, and (vii) CPEGSSISAP.

[0050] Further preferred is that the ‘at least one recognition site’ is complementary to a template molecule comprising the amino acid sequence DYKVKGLCDSNL.

[0051] Further preferred is that the ‘at least one recognition site’ is complementary to a template molecule comprising the amino acid sequence ADKDLFAAARFPE.

[0052] Further preferred is that the ‘at least one recognition site’ is complementary to a template molecule comprising the amino acid sequence PEGSSISAP.

[0053] Further preferred is that the ‘at least one recognition site’ is complementary to a template molecule comprising the amino acid sequence EDGELSSLGKEGT.

[0054] Further preferred is that the ‘at least one recognition site’ is complementary to a template molecule comprising the amino acid sequence PKSHKAI.

[0055] Further preferred is that the ‘at least one recognition site’ is complementary to a template molecule comprising the amino acid sequence ADKDLFAAAR. Further preferred is that the ‘at least one recognition site’ is complementary to a template molecule comprising the amino acid sequence CPEGSSISAP.

[0056] The size of the template may be in the range up to 50 amino acids; the range may be 5 to 50 amino acids. That said, suitably the template will be more than 5 amino acids and less than 50 amino acids, more suitably the template will be more than 5 amino acids and less than 30 amino acids; most preferred is that the template is more than 5 amino acids and less than 20 amino acids, for example 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, or 19 amino acids.

[0057] Imprinting against peptide templates of the length specified above has several advantages. Peptide templates of the length specified above cost less to produce than whole proteins or the like, synthesis of peptide templates of the length specified above can reach high yields, with fewer variations, than e.g. whole proteins or the like and gives rise to a more consistent imprinting process. Imprinting against peptide templates of the length specified above also has significant safety benefits.

[0058] In some embodiments of the invention, the template comprises only the amino acid sequence to be imprinted. However, in other embodiments, the template may comprise both the amino acid sequence to be imprinted as well as additional amino acids, often added to assist in the method of manufacture. In the Examples described herein, on the basis that a portion of molecularly imprinted polymers were made via solid-phase template imprinting, which requires immobilisation of the peptide templates onto a solid support, the templates comprised both the amino acid sequence to be imprinted as well as additional amino acids added to assist in immobilisation of the template onto the solid support. Additional amino acids may also be included in the template to influence the structure of the template. In the Examples described herein, one specific template was modified with a cysteine residue to cyclise the template. Additional amino acids may also be included as spacers.

[0059] In some embodiments of the invention, the synthetic polymer is a molecularly imprinted polymer comprising more than one recognition site which binds to VSV-G protein for example 2, 3, 4, 5, 6, 7, 8, 9, or 10 recognition sites. The recognition sites may be complementary to the same template molecule or different template molecules. Preferred is that the respective recognition sites are each complementary to a template molecule comprising an amino acid sequence selected from the group consisting of (i) DYKVKGLCDSNL, (ii) ADKDLFAAARFPE, (iii) PEGSSISAP, (iv) EDGELSSLGKEGT, (v) PKSHKAI, (vi) ADKDLFAAAR, and (vii) CPEGSSISAP. In one example, the synthetic polymer is a molecularly imprinted polymer comprising at least two recognition sites which bind to VSV-G protein, the first recognition site being complementary to a template molecule comprising the amino acid sequence DYKVKGLCDSNL and the second recognition site being complementary to a template molecule comprising the amino acid sequence CPEGSSISAP. An advantage of having more than one recognition site which binds VSV-G is that the molecularly imprinted polymer can be made with multiple specificities. For example, when the molecularly imprinted polymer has at least two recognition sites which are complementary to different template molecules, the molecularly imprinted polymer is dual-specific.

[0060] Monomers which can be used in the preparation of the synthetic polymers of the present invention include, but are not limited to: vinyl monomers, allyl monomers, acetylenes, acrylates, methacrylates, acrylamides, methacrylamides, chloroacrylates, itaconates, trifluoromethylacrylates, derivates of amino acids, nucleosides, nucleotides, and carbohydrates.

[0061] Specific examples of monomers suitable for use in the preparation of synthetic polymers of the present invention include, but are not limited to: acrylamide, tert-butyl acrylate, tert-butyl acrylamide, N-(3-aminopropyl) methacrylamide hydrochloride, 2- carboxyethyl acrylate, mono-2-(methacryloyloxy)ethyl maleate, 3-(perfluoro-5- methylhexyl)-2-hydroxypropyl methacrylate, 2-(methacryloxy)ethyl phosphate, (3- acrylamidopropyl)trimethylammonium chloride solution, 3-sulfopropyl acrylate, methacrylamide, 2-acrylamido-2-methylpropane sulfonic acid, (3- acrylamidopropyl)trimethylammonium chloride solution, 3-sulfopropyl methacrylate potassium salt, glycosyloxyethyl methacrylate solution, 2-(dimethylamine)ethyl methacrylate, 3-(acrylamido) phenylboronic acid, 3-vinylbenzoic acid, 2- (trifluoromethyl)acrylic acid, 2-hydroxy-5-N-methacrylamidobenzoic acid, acrylic acid, 1 H,1 H,5H-octafluoropentyl methacrylate, 2-(diethylamino)ethyl acrylate, vinylphosphonic acid, ethyl methacrylate, 2-N-morpholinoethyl methacrylate, 1 H 1 H 2H 2H-perfluorododecyl acrylate, 2,2,2-trifluoroethyl acrylate, acrylonitrile, n-propyl acrylate, 2-(dimethylamino)ethyl methacrylate, 2,2,2-trifluoroethyl methacrylate and cysteamine.

[0062] In some embodiments of the invention, the synthetic polymer is a cross-linked molecularly imprinted polymer. Advantageously, cross-linking can help to stabilise molecularly imprinted polymers. Alternatively, the synthetic polymer may not comprise a cross-linking monomer. Where no cross-linking monomer is present, the synthetic polymer will be a linear polymer (e.g. a linear molecularly imprinted polymer or a linear non-imprinted polymer). Advantageously, linear polymers are flexible which can assist in target binding in that they can access binding sites which are less accessible to larger and more rigid binding molecules.

[0063] Cross-linking monomers which can be used to produce cross-linked molecularly imprinted polymers include, but are not limited to: ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, divinylbenzene, methylene bisacrylamide, ethylene bisacrylamide, N,N’-bisacryloylpiperazine, and N,N'- methylenebis(acrylamide) (BIS).

[0064] In some embodiments of the present invention, the cross-linked molecularly imprinted polymers are nano-molecularly imprinted polymers.

[0065] The size of the nano-molecularly imprinted polymers of the present invention will be to a large extent dictated by the intended use of the nano-molecular imprinted polymer. That said, suitably the molecular imprinted polymer will be less than 500 nm, more suitably less than 250 nm.

[0066] In embodiments where the synthetic polymer is linear, suitably the linear polymer is 1 kDa - 10OkDa, preferably 1 kDa - 75 kDa, more preferably 1 kDa - 50kDa, and most preferably 3.5kDa - 35kDa.

[0067] Also provided by the invention is a composition comprising the synthetic polymer. In embodiments wherein the synthetic polymer is a molecularly imprinted polymer and more specifically a nano-molecularly imprinted polymer, suitably the nano-molecular imprinted polymers in such compositions will have an average size that is less than 500 nm, more suitably less than 250 nm. In embodiments where the synthetic polymer is linear, suitability the linear polymers in such compositions will on average be 1 kDa - 100kDa, preferably 1 kDa - 75 kDa, more preferably 1 kDa - 50kDa, and most preferably 3.5kDa - 35kDa.

[0068] Suitable methods for determining the size of molecularly imprinted polymers include dynamic light scattering e.g. using a Zetasizer Ultra (Malvern Panalytical), disc centrifugation, nanoparticle tracking analysis e.g. using a NanoSight NS300 (Malvern Panalytical), tunable resistive pulse sensing, atomic force microscopy and electron microscopy. Nanoparticle tracking analysis, tunable resistive pulse sensing, atomic force microscopy, and electron microscopy are particularly suitable for determining the size of single molecularly imprinted polymers. In the Examples provided herein, the size of the nano-molecularly imprinted polymers was determined using dynamic light scattering (DLS).

[0069] Size in the context of the nano-molecularly imprinted polymers refers to the diameter of the nano-molecularly imprinted polymers. Indeed, the techniques used to measure the size of molecularly imprinted polymers assume a spherical particle.

[0070] Suitable methods for determining the size of linear polymers include size exclusion chromatography (SEC) methods, such as gel permeation chromatography (GPC), diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY), mass spectrometry methods such as liquid chromatography mass spectrometry (LC-MS) 20 and matrix assisted laser desorption ionisation (MALDI) spectrometry.

[0071] In preferred embodiments of the invention the synthetic polymer comprises at least one monomer listed in Tables 4 - 10.

[0072] In alternative preferred embodiments of the invention, the synthetic polymer comprises at least two monomers listed in Tables 4-10. In still further preferred embodiments of the invention, the synthetic polymer comprises at least three monomers listed in Tables 4-10.

[0073] In a particularly preferred embodiment, the synthetic polymer comprises the monomers (i) N,N'-Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3-Aminopropyl) methacrylamide hydrochloride.

[0074] In a particularly preferred embodiment, the synthetic polymer comprises 2- carboxyethyl acrylate (CEA), optionally in combination with (i) N,N'- Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3- Aminopropyl) methacrylamide hydrochloride.

[0075] In a particularly preferred embodiment, the synthetic polymer comprises 3-sulfopropyl acrylate (SPA), optionally in combination with (i) N,N'-Methylenebis(acrylamide),

[0076] (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3-Aminopropyl) methacrylamide hydrochloride.

[0077] In a particularly preferred embodiment, the synthetic polymer comprises N-(3- aminopropyl) methacrylamide hydrochloride (APMA), optionally in combination with (i) N,N'-Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N- (3-Aminopropyl) methacrylamide hydrochloride.

[0078] In a particularly preferred embodiment, the synthetic polymer comprises mono-2- (methacryloyloxy)ethyl maleate (MAM EE), optionally in combination with (i) N,N'- Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3- Aminopropyl) methacrylamide hydrochloride.

[0079] In a particularly preferred embodiment, the synthetic polymer comprises 2-acrylamido- 2-methylpropane sulfonic acid (AMPSA), optionally in combination with (i) N,N'- Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3- Aminopropyl) methacrylamide hydrochloride.

[0080] In a particularly preferred embodiment, the synthetic polymer comprises 2- (methacryloxy)ethyl phosphate (MAOEP) and 3-(perfluoro-5-methylhexyl)-2- hydroxypropyl methacrylate (PMHM), optionally in combination with (i) N,N'- Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3- Aminopropyl) methacrylamide hydrochloride.

[0081] In a particularly preferred embodiment, the synthetic polymer comprises 3-sulfopropyl methacrylate (SPMA) and 3-vinylbenzoic acid (3VB), optionally in combination with (i) N,N'-Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N- (3-Aminopropyl) methacrylamide hydrochloride.

[0082] In a particularly preferred embodiment, the synthetic polymer comprises 3-sulfopropyl methacrylate (SPMA), optionally in combination with (i) N,N'-

[0083] Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3- Aminopropyl) methacrylamide hydrochloride.

[0084] In a particularly preferred embodiment, the synthetic polymer comprises vinylphosphonic acid (VPA), optionally in combination with (i) N,N'- Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3- Aminopropyl) methacrylamide hydrochloride.

[0085] In a particularly preferred embodiment, the synthetic polymer comprises methacrylamide (MAAm), optionally in combination with (i) N,N'-

[0086] Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3- Aminopropyl) methacrylamide hydrochloride.

[0087] In a particularly preferred embodiment, the synthetic polymer comprises ethyl methacrylate (EMA), optionally in combination with (i) N,N'-Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3-Aminopropyl) methacrylamide hydrochloride.

[0088] In a particularly preferred embodiment, the synthetic polymer comprises 2- (diethylamino)ethyl acrylate (DEAEM) and vinylphosphonic acid (VPA), optionally in combination with (i) N,N'-Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3-Aminopropyl) methacrylamide hydrochloride. In a particularly preferred embodiment, the synthetic polymer comprises 2,2,2- trifluoroethyl acrylate (TFEA), optionally in combination with (i) N,N'- Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3- Aminopropyl) methacrylamide hydrochloride.

[0089] In a particularly preferred embodiment, the synthetic polymer comprises N-(3- aminopropyl) methacrylamide hydrochloride (APMA) and 2-carboxyethyl acrylate (CEA), optionally in combination with (i) N,N'-Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3-Aminopropyl) methacrylamide hydrochloride.

[0090] In a particularly preferred embodiment, the synthetic polymer comprises 2- (methacryloxy)ethyl phosphate (MAOEP) and methacrylic acid (MAAc), optionally in combination with (i) N,N'-Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3-Aminopropyl) methacrylamide hydrochloride.

[0091] In a particularly preferred embodiment, the synthetic polymer comprises 2- carboxyethyl acrylate (CEA) and mono-2-(methacryloyloxy)ethyl maleate (MAM EE), optionally in combination with (i) N,N'-Methylenebis(acrylamide), (ii) Acrylamide, (iii) tert-Butyl acrylate, and (iv) N-(3-Aminopropyl) methacrylamide hydrochloride.

[0092] In a particularly preferred embodiment, the synthetic polymer comprises the monomers (i)N-fluoresceinylacrylam ide, (ii) 3-Sulfopropyl acrylate, (iii) Mono-2 - (methacryloyloxy)ethyl maleate, (iv) 2-Carboxyethyl acrylate, (v) Cysteamine, and (vi) Tert-butyl acrylate.

[0093] In a particularly preferred embodiment, the synthetic polymer comprises the monomers 3-Sulfopropyl acrylate, mono-2-(methacryloyloxy)ethyl maleate and 2- Carboxyethyl acrylate.

[0094] In a particularly preferred embodiment, the synthetic polymer comprises N- isopropylacrylamide (NIPAM), optionally in combination with the monomers:

[0095] (a) 3-Sulfopropyl acrylate, mono-2-(methacryloyloxy)ethyl maleate and 2-

[0096] Carboxyethyl acrylate or (b) N-fluoresceinylacrylamide, 3-Sulfopropyl acrylate, Mono-2- (methacryloyloxy)ethyl maleate, 2-Carboxyethyl acrylate, Cysteamine, and Tert-butyl acrylate.

[0097] In a particularly preferred embodiment, the synthetic polymer comprises acrylamide, optionally in combination with the monomers:

[0098] (a) 3-Sulfopropyl acrylate, mono-2-(methacryloyloxy)ethyl maleate and 2- Carboxyethyl acrylate or

[0099] (b) N-fluoresceinylacrylamide, 3-Sulfopropyl acrylate, Mono-2- (methacryloyloxy)ethyl maleate, 2-Carboxyethyl acrylate, Cysteamine, and Tert-butyl acrylate.

[0100] In a particularly preferred embodiment, the synthetic polymer comprises N-(3- aminopropyl) methacrylamide hydrochloride (APMA) and acrylamide, optionally in combination with the monomers:

[0101] (a) 3-Sulfopropyl acrylate, mono-2-(methacryloyloxy)ethyl maleate and 2- Carboxyethyl acrylate or

[0102] (b) N-fluoresceinylacrylamide, 3-Sulfopropyl acrylate, Mono-2- (methacryloyloxy)ethyl maleate, 2-Carboxyethyl acrylate, Cysteamine, and Tert-butyl acrylate.

[0103] In a particularly preferred embodiment, the synthetic polymer comprises 2,2,2- Trifluoroethyl acrylate, acrylonitrile and ethyl methacrylate, optionally in combination with (i) acrylamide, (ii) N-Fluoresceinylacrylamide, (iii) cysteamine and (iv) tert-butyl acrylate.

[0104] In a particularly preferred embodiment, the synthetic polymer comprises mono-2- (methacryloyloxy)ethyl maleate or (i) 3-Sulfopropyl acrylate, (ii) mono-2- (methacryloyloxy)ethyl maleate and (iii) 2-Carboxyethyl acrylate, optionally in combination with acrylamide and one of (i) tert-butyl acrylate or (ii) tert-butyl acrylamide.

[0105] In a particularly preferred embodiment, the synthetic polymer comprises mono-2- (methacryloyloxy)ethyl maleate or (i) 3-Sulfopropyl acrylate, (ii) mono-2- (methacryloyloxy)ethyl maleate and (iii) 2-Carboxyethyl acrylate, optionally in combination with acrylamide and cysteamine and one of (i) tert-butyl acrylate or (ii) tert-butyl acrylamide.

[0106] In a particularly preferred embodiment, the synthetic polymer comprises the monomers of, or is, LV-F1 -001 , LV-F1 -002, LV-F1 -004, LV-F1-007, LV-F1 -010, LV- F1 -011 , LV-F1 -012, LV-F2-015, LV-F2-016, LV-F3-018, LV-F4-001 , LV-F6-002, LV- F6-003, LV-F4-004, LV-F8-008, LV-F8-003, linear non-imprinted polymer 2, linear non-imprinted polymer 6, linear non-imprinted polymer 4, or linear molecularly imprinted polymer 3, all disclosed below in Example 1 or linear non-imprinted polymer A3 disclosed below in Example 11 . Even further preferred is that the synthetic polymer comprises the monomers of, or is, LV-F1 -011 , LV-F1 -012, LV-F2-016 or linear nonimprinted polymer A3.

[0107] In still further preferred embodiments, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence DYKVKGLCDSNL and wherein the synthetic polymer comprises at least one monomer listed in Table 4.

[0108] In a particularly preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence DYKVKGLCDSNL and wherein the synthetic polymer comprises at least one of (i) N-(3-aminopropyl) methacrylamide hydrochloride, (ii) mono-2-(methacryloyloxy)ethyl maleate, (iii) 3- sulfopropyl acrylate, or (iv) 2-acrylamido-2-methylpropane sulfonic acid.

[0109] In a further preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence ADKDLFAAARFPE and wherein the synthetic polymer comprises at least one monomer listed in Table 5. In another particularly preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence ADKDLFAAARFPE and wherein the synthetic polymer comprises at least one of (i) 2-(methacryloxy)ethyl phosphate, (ii) 3-(perfluoro-5-methylhexyl)-2-hydroxypropyl methacrylate, and (iii) methacrylic Acid (MAAc).

[0110] In a further preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence PEGSSISAP and wherein the synthetic polymer comprises at least one monomer listed in Table 6.

[0111] In another particularly preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence PEGSSISAP and wherein the synthetic polymer comprises (i) 3-sulfopropyl methacrylate and (ii) 3-vinylbenzoic acid.

[0112] In a further preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence EDGELSSLGKEGT and wherein the synthetic polymer comprises at least one monomer listed in Table 7.

[0113] In another particularly preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence EDGELSSLGKEGT and wherein the synthetic polymer comprises 3-sulfopropyl methacrylate.

[0114] In a further preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence ADKDLFAAAR and wherein the synthetic polymer comprises at least one monomer listed in Table 9.

[0115] In another particularly preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence ADKDLFAAAR and wherein the synthetic polymer comprises (i) 2-(diethylamino)ethyl acrylate (DEAEM) and (ii) vinylphosphonic acid (VPA).

[0116] In a further preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence CPEGSSISAP and wherein the synthetic polymer comprises at least one monomer listed in Table 10.

[0117] In another particularly preferred embodiment, the synthetic polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein, wherein the recognition site is complementary to a template molecule comprising the amino acid sequence CPEGSSISAP and wherein the synthetic polymer comprises 2,2,2-trifluoroethyl acrylate (TFEA).

[0118] In another particularly preferred embodiment, the synthetic polymer comprises (i) 3- sulfopropyl acrylate, (ii) mono-2-(methacryloyloxy)ethyl maleate, (iii) 2-carboxyethyl acrylate, and (iv) N-isopropylacrylamide.

[0119] The synthetic polymer of the invention, which binds vesicular stomatitis virus G (VSV- G) protein, and does not contain any proteinogenic amino acids, can be made by a process comprising the steps of:

[0120] (a) providing a polymerisable composition in contact with a template molecule; and (b) effecting polymerisation of the polymerisable composition in contact with the template molecule to produce a synthetic polymer wherein the synthetic polymer is a molecularly imprinted polymer.

[0121] In preferred embodiments, the template molecule comprises an amino acid sequence corresponding to a subsequence of a VSV-G polypeptide. For the reasons recited above in relation to the synthetic polymer, in preferred embodiments the subsequence falls outside of the transmembrane domain. For reasons again recited above in relation to the synthetic polymer, further preferred is that the amino acid sequence is selected from the group consisting of: (i) DYKVKGLCDSNL, (ii) ADKDLFAAARFPE, (iii) PEGSSISAP, (iv) EDGELSSLGKEGT, (v) PKSHKAI, (vi) ADKDLFAAAR, and (vii) CPEGSSISAP. In a particularly preferred embodiment, the amino acid sequence is DYKVKGLCDSNL. In a further particularly preferred embodiment, the amino acid sequence is ADKDLFAAARFPE. In a further particularly preferred embodiment, the amino acid sequence is PEGSSISAP. In a further particularly preferred embodiment, the amino acid sequence is EDGELSSLGKEGT. In a further particularly preferred embodiment, the amino acid sequence is PKSHKAI. In a further particularly preferred embodiment, the amino acid sequence is ADKDLFAAAR. In a further particularly preferred embodiment, the amino acid sequence is CPEGSSISAP.

[0122] In embodiments, the template molecule is immobilised on a carrier substance. Suitable carrier substances include polymer resins, polysaccharides, glass, or metal surfaces.

[0123] The carrier substance may be in the form of beads, fibres, membranes, or capillaries. In preferred embodiments, the carrier substance is glass. Further preferred is that the carrier substance is in the form of glass beads.

[0124] The method by which the template molecule is immobilised on the carrier substance is not critical. However, for some methods, the presence of a cysteine or lysine residue at a termini of the template molecule is advantageous. As such, for embodiments of the invention where the template does not naturally comprise a terminal cysteine or lysine residue, it is preferred that the template is modified to comprise such a residue. Additional residues, such as glycine, functioning as spacers, may be present between the added cysteine or lysine residue and the remainder of the template. Additional residues may also be added to the template to influence template structure.

[0125] In some embodiments of the invention, the template molecule is directly attached to the carrier substance whilst in other embodiments one or more linkers are included between the carrier substance and the template molecule. Suitable linkers for use in the invention are well known to those of skill in the art. In the Examples described herein, the carrier substance used was glass beads and these were silanised using (3-aminopropyl)trimethoxysilane (APTMS) prior to immobilisation of the template molecule. The APTMS acts as a linker between the surface of the glass bead and the template molecule. In the Examples provided herein, the APTMS was modified with either n-succinimidyl iodoacetate (SIA) or glutaraldehyde prior to template immobilisation.

[0126] Linkers and spacers function to prevent adverse effects brought about by steric hindrance.

[0127] In alternative embodiments, the template molecule is in solution.

[0128] In some embodiments, the polymerisable composition is in contact with more than one template molecule, for example 2, 3, 4, 5, 6, 7, 8, 9, or 10 template molecules. When more than one template molecule is present, each of the template molecules can be the same or they can be different. In preferred embodiments, each of the template molecules comprises an amino acid sequence corresponding to a subsequence of a VSV-G polypeptide. For the reasons recited above in relation to the synthetic polymer, in preferred embodiments the subsequence falls outside of the transmembrane domain. For reasons again recited above in relation to the synthetic polymer, further preferred is that the amino acid sequence is selected from the group consisting of: (i) DYKVKGLCDSNL, (ii) ADKDLFAAARFPE, (iii) PEGSSISAP, (iv) EDGELSSLGKEGT, (v) PKSHKAI, (vi) ADKDLFAAAR, and (vii) CPEGSSISAP. In one example, the polymerisable composition is in contact with two template molecules, the first comprising the amino acid sequence DYKVKGLCDSNL and the second comprising the amino acid sequence CPEGSSISAP. When the polymerisable composition is in contact with more than one template molecule, each of the template molecules can either be immobilised on a carrier substance or in solution. Preferences with respect to the carrier substance are set out elsewhere herein. An advantage of having more than one template is that the resulting molecularly imprinted polymer can have multiple specificities. For example, when the polymerisable composition is in contact with two template molecules which are different, the resulting molecularly imprinted polymer will be dual-specific.

[0129] The polymerisable composition must contain the monomers required for the polymerisation. Suitable monomers, and cross-linking monomers where applicable, are discussed elsewhere herein.

[0130] In preferred embodiments of the invention, the polymerisable composition comprises 3-sulfopropyl acrylate.

[0131] In preferred embodiments of the invention, the polymerisable composition comprises 2-carboxyethyl acrylate and mono-2-(methacryloyloxy)ethyl maleate.

[0132] In preferred embodiments of the invention, the polymerisable composition comprises N-fluoresceinylacrylamide, cysteamine, and tert-butyl acrylate.

[0133] In preferred embodiments of the invention, the polymerisable composition comprises N-isopropylacrylam ide.

[0134] In particularly preferred embodiments, the polymerisable composition comprises 3- sulfopropyl acrylate, 2-carboxyethyl acrylate, and mono-2-(methacryloyloxy)ethyl maleate.

[0135] Still further preferred is that the polymerisable composition comprises 3-sulfopropyl acrylate, 2-carboxyethyl acrylate, mono-2-(methacryloyloxy)ethyl maleate, N- fluoresceinylacrylamide, cysteamine, and tert-butyl acrylate. Still further preferred is that the polymerisable composition comprises 3-sulfopropyl acrylate, 2-carboxyethyl acrylate, mono-2-(methacryloyloxy)ethyl maleate, N- fluoresceinylacrylamide, cysteamine, tert-butyl acrylate, and N-isopropylacrylamide.

[0136] Also preferred is that the polymerisable composition comprises 3-sulfopropyl acrylate, 2-carboxyethyl acrylate, mono-2-(methacryloyloxy)ethyl maleate, N- fluoresceinylacrylamide, cysteamine, tert-butyl acrylate, and N-isopropylacrylamide and the template comprises the amino acid sequence DYKVKGLCDSNL.

[0137] Step (b) involves effecting polymerisation of the polymerisable composition. Various techniques for effecting polymerisation are known to those of skill in the art.

[0138] Examples include radical polymerisation such as controlled living radical polymerisation (LRP), living anionic polymerisation, living cationic polymerisation, and controlled polycondensation.

[0139] Polymerisation can be initiated by e.g. heating, applying current (electropolymerisation), by addition of redox catalyst(s), persulfate or peroxides, by irradiation, including gamma radiation or by microwave radiation, or by irradiation with UV or visible light and normally takes minutes to hours. In the Examples described herein, polymerisation was initiated using persulfate.

[0140] Once the molecularly imprinted polymers are synthesised they must be separated from the template molecule and collected. Thus, in preferred embodiments, the process comprises the additional step of separating the synthetic polymer from the template molecule. The separation of molecularly imprinted polymers from the template molecule can be achieved by heating, which disrupts complex formation, by changing solution pH, changing ionic strength, or through the additional of urea, guanidine or a substance which interacts with the template more strongly than the molecularly imprinted polymer. The separation of molecularly imprinted polymers from the template molecule can also be achieved by dialysis. The method chosen for separation of the molecularly imprinted polymer from the template may be influenced by the desired affinity of the molecularly imprinted polymers for the template. If high affinity molecularly imprinted polymers are desired e.g. for downstream uses that involve detection, high temperatures (in the Examples described herein, high affinity molecularly imprinted polymers were separated using ~65 °C ethanol) are commonly used; however, if lower affinity molecularly imprinted polymers are desired e.g. for downstream uses that are focussed on purification, higher temperatures may not be required.

[0141] In embodiments wherein the template is immobilised on a carrier substance, prior to the separation step, weakly bound material is removed by washing. Preferably, weakly bound material is removed by washing at cold or ambient temperatures.

[0142] Once collected, the molecularly imprinted polymers may be purified further e.g. by chromatography, filtration, electrophoresis or dialysis.

[0143] The synthetic polymer of the invention, which binds vesicular stomatitis virus G (VSV- G) protein, and does not contain any proteinogenic amino acids, can also be made by a process comprising the steps of:

[0144] (a) providing a polymerisable composition;

[0145] (b) effecting polymerisation of the polymerisable composition to produce a polymer.

[0146] The polymerisable composition must contain the monomers required for the polymerisation. Suitable monomers are discussed elsewhere herein.

[0147] In preferred embodiments of the invention, the polymerisable composition comprises 3-sulfopropyl acrylate.

[0148] In preferred embodiments of the invention, the polymerisable composition comprises 2-carboxyethyl acrylate and mono-2-(methacryloyloxy)ethyl maleate.

[0149] In preferred embodiments of the invention, the polymerisable composition comprises N-fluoresceinylacrylamide, cysteamine, and tert-butyl acrylate.

[0150] In preferred embodiments of the invention, the polymerisable composition comprises acrylamide. In preferred embodiments of the invention, the polymerisable composition comprises N-isopropylacrylam ide.

[0151] In particularly preferred embodiments, the polymerisable composition comprises 3- sulfopropyl acrylate, 2-carboxyethyl acrylate, and mono-2-(methacryloyloxy)ethyl maleate.

[0152] Still further preferred is that the polymerisable composition comprises 3-sulfopropyl acrylate, 2-carboxyethyl acrylate, mono-2-(methacryloyloxy)ethyl maleate, N- fluoresceinylacrylamide, cysteamine, and tert-butyl acrylate.

[0153] Still further preferred is that the polymerisable composition comprises 3-sulfopropyl acrylate, 2-carboxyethyl acrylate, mono-2-(methacryloyloxy)ethyl maleate, N- fluoresceinylacrylamide, cysteamine, tert-butyl acrylate, and N-isopropylacrylam ide.

[0154] Step (b) involves effecting polymerisation of the polymerisable composition. Various techniques for effecting polymerisation are known to those of skill in the art and discussed elsewhere herein.

[0155] The synthetic polymers and compositions of the present invention may be used in a variety of applications.

[0156] It is specifically envisaged that the synthetic polymers and compositions of the present invention be used in the detection of VSV-G.

[0157] In embodiments, the VSV-G is present on the surface of a lentiviral vector.

[0158] It is also specifically envisaged that the synthetic polymers and compositions of the present invention be used in purification methods, particularly those aimed at purifying VSV-G pseudotyped lentiviral vector.

[0159] For certain downstream applications, it is advantageous for the synthetic polymer to be conjugated to a secondary entity. - l-

[0160] In some embodiments of the invention, the synthetic polymer is conjugated to a sensor element. Preferably, in the conjugated state the sensor element is capable of giving a specific quantitative or semi-quantitative measurement reading. Suitable sensors include electrochemical sensors, bio-layer interferometry sensors, optical sensors, and thermal sensors. In some embodiments of the invention, the synthetic polymer is directly attached to the sensor element whilst in other embodiments one or more linkers are included between the synthetic polymer and the sensor element. Suitable linkers are well known to those of skill in the art and include 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC, sometimes in conjunction with N- hydroxysuccinimide (NHS) or sulfo-N-hydroxysuccinimide (sNHS)), glutaraldehyde, succinimidyl iodoacetate, mercaptoundecanoic acid, lipoic acid, mercaptoundecanol, and aminopropyl triethoxy silane. In preferred embodiments, the synthetic polymer conjugated to a sensor is a molecularly imprinted polymer. Further preferred is that the molecularly imprinted polymer is cross-linked. Such conjugates (cross-linked molecularly imprinted polymer conjugated to a sensor) are preferably used in the detection of VSV-G present on the surface of a lentiviral vector.

[0161] In alternative embodiments, the synthetic polymer is conjugated to a solid support. Suitable solid supports include membranes, resins, magnetic beads, microplates, cell culture plates, porous monoliths, and open cell porous supports. In preferred embodiments, the solid support is a resin. Suitable resins include cross-linked agarose or dextran resins and N-isopropylacrylamide (NIPAM) based resins. In some embodiments of the invention, the synthetic polymer is directly attached to the solid support whilst in other embodiments one or more linkers are included between the synthetic polymer and the solid support. Suitable linkers are well known to those of skill in the art and include glutaraldehyde and other di-aldehyde linkers, heterogeneous linkers such as amino-PEG4-acid, EDC / NHS mediated attachments and click-chemistry-based linkers. In some embodiments, the solid support is suitably modified to enable covalent attachment of the synthetic polymer. Such modification can be accomplished with agents such as sodium periodate or other oxidising agents. In a particularly preferred embodiment of the invention, the synthetic polymer is a non- cross-linked synthetic polymer and is covalently conjugated to Sepharose CL-6B (Sigma Aldrich, CL6B200). Such a conjugate is preferably used in the purification of VSV-G pseudotyped lentiviral vectors.

[0162] In still further embodiments, the synthetic polymer is conjugated to a moiety such as a fluorescent tag, biotin, or horseradish peroxidase (HRP).

[0163] It is also specifically envisaged that the synthetic polymers and compositions of the present invention be used in quantification methods, particularly those aimed at quantifying a lentiviral vector pseudotyped with VSV-G.

[0164] Also provided by the invention is a method of preparing a synthetic polymer which binds VSV-G protein comprising the steps of:

[0165] (a) providing a polymerisable composition wherein the polymerisable composition does not contain any proteinogenic amino acids, and

[0166] (b) effecting polymerisation of the polymerisable composition to produce the synthetic polymer.

[0167] The polymerisable composition must contain the monomers required for the polymerisation. Suitable monomers are discussed elsewhere herein.

[0168] Step (b) involves effecting polymerisation of the polymerisable composition. Various techniques for effecting polymerisation are known to those of skill in the art and described elsewhere herein.

[0169] In some embodiments of the invention, the method comprises the preliminary step of providing a template molecule comprising an amino acid sequence corresponding to a subsequence of a VSV-G polypeptide. For the reasons recited above in relation to the synthetic polymer, in preferred embodiments the subsequence falls outside of the transmembrane domain. For reasons again recited above in relation to the synthetic polymer, further preferred is that the amino acid sequence is selected from the group consisting of: (i) DYKVKGLCDSNL, (ii) ADKDLFAAARFPE, (iii) PEGSSISAP, (iv) EDGELSSLGKEGT, (v) PKSHKAI, (vi) ADKDLFAAAR, and (vii) CPEGSSISAP. In a particularly preferred embodiment, the amino acid sequence is DYKVKGLCDSNL. In a further particularly preferred embodiment, the amino acid sequence is ADKDLFAAARFPE. In a further particularly preferred embodiment, the amino acid sequence is PEGSSISAP. In a further particularly preferred embodiment, the amino acid sequence is EDGELSSLGKEGT. In a further particularly preferred embodiment, the amino acid sequence is PKSHKAI. In a further particularly preferred embodiment, the amino acid sequence is ADKDLFAAAR. In a further particularly preferred embodiment, the amino acid sequence is CPEGSSISAP.

[0170] In embodiments, the template is immobilised on a carrier substance. Suitable, and indeed preferred, carrier substances have been discussed elsewhere herein.

[0171] In other embodiments, the template is in solution.

[0172] In some embodiments, the method comprises the preliminary step of providing more than one template molecule for example 2, 3, 4, 5, 6, 7, 8, 9, or 10 template molecules. When more than one template molecule is present, each of the template molecules can be the same or they can be different. In preferred embodiments, each of the template molecules comprises an amino acid sequence corresponding to a subsequence of a VSV-G polypeptide. For the reasons recited above in relation to the synthetic polymer, in preferred embodiments the subsequence falls outside of the transmembrane domain. For reasons again recited above in relation to the synthetic polymer, further preferred is that the amino acid sequence is selected from the group consisting of: (i) DYKVKGLCDSNL, (ii) ADKDLFAAARFPE, (iii) PEGSSISAP, (iv) EDGELSSLGKEGT, (v) PKSHKAI, (vi) ADKDLFAAAR, and (vii) CPEGSSISAP. In one example, the preliminary step involves providing two template molecules, the first comprising the amino acid sequence DYKVKGLCDSNL and the second comprising the amino acid sequence CPEGSSISAP.

[0173] When more than one template molecule is provided, each of the template molecules can either be immobilised on a carrier substance or in solution. Preferences with respect to the carrier substance are set out elsewhere herein. An advantage of having more than one template is that the resulting molecularly imprinted polymer can have multiple specificities. For example, when two template molecules are provided which are different, the resulting molecularly imprinted polymer will be dual-specific. In embodiments of the invention wherein the method comprises the preliminary step of providing a template molecule comprising an amino acid sequence corresponding to a subsequence of a VSV-G polypeptide, a final step may also be included wherein the polymer is separated from the template. Techniques for separating polymers from templates have been discussed elsewhere herein.

[0174] Once prepared, the synthetic polymers may be subjected to a purification step.

[0175] Advantageously, the synthetic polymers of the present invention are specific, robust, inexpensive to produce, and stable.

[0176] Examples

[0177] The invention is now further described in specific examples with reference to the accompanying drawings in which:

[0178] Figure 1 shows the in silico binding analysis for LV-F1 and the 9 candidate monomers identified in the HPLC screen shown in Table 4. Figure 1a shows the six binding zones for LV-F1 and Figure 1 b shows which amino acid(s) of LV-F1 each of the monomer candidates from the HPLC screen binds to.

[0179] Figure 2 shows the results of molecular dynamic simulations for potential monomer combinations identified with respect to modified peptide template LV-F1.

[0180] Figure 3 shows sensorgrams for the binding of VSV-G (orange) and BSA (green) to surface plasmon resonance (SPR) surfaces functionalised with nanoMIPs. Figure 3a shows the sensorgram for the binding of VSV-G (orange) and BSA (green) to SPR surfaces functionalised with LV-F1 -004 and Figure 3b shows the sensorgram for the binding of VSV-G (orange) and BSA (green) to SPR surfaces functionalised with LV- F 1 -007.

[0181] Figure 4 shows binding of a VSV-G-gold conjugate to membranes functionalised with nanoMIPs imprinted against LV-F1 (Figure 4a), LV-F2 (Figure 4b), LV-F3 (Figure 4c), LV-F4 (Figure 4d), LV-F6 (Figure 4e), and LV-F8 (Figure 4f).

[0182] Figure 5 shows binding of a BSA-gold conjugate to membranes functionalised with nanoMIPs imprinted against LV-F1 , LV-F2, LV-F3, LV-F4, LV-F6, and LV-F8. Figure 6 shows binding of a VSV-G-gold conjugate to Sepharose® functionalised with linear MIPs and linear non-imprinted polymers.

[0183] Figure 7 shows elution of VSV-G-gold conjugate from Sepharose® functionalised with linear MIPs and linear non-imprinted polymers.

[0184] Figure 8 shows binding of a VSV-G-gold conjugate to polymer-functionalised membranes.

[0185] Figure 9 shows a pH scout for nanoMIP (LV-F1 -012) immobilisation on an AR2G biosensor. Figure 9a displays the trace for activation of the AR2G biosensor with EDC / NHS, nanoMIP immobilisation (labelled ‘MIP Loading’), and quenching with ethanolamine. Figure 9b shows the trace for association of the nanoMIP immobilised AR2G biosensor surface with VSV-G and then subsequent dissociation of the VSV-G. Figure 10 shows a nanoMIP loading scout for nanoMIP (LV-F1 -012) immobilisation on an AR2G biosensor. Figure 10a shows the trace for activation of the AR2G biosensor with EDC / NHS, nanoMIP immobilisation (labelled ‘MIP Loading’), and quenching with ethanolamine. Figure 10b shows the trace for association of the nanoMIP immobilised AR2G biosensor surface with VSV-G and then subsequent dissociation of the VSV-G. Figure 11 shows binding of VSV-G to LV-F1-012 nanoMIPs.

[0186] Figure 12 shows binding of lentiviral particles to nanoMIP LV-F1 -012. Figure 12a shows binding of Incucyte® Nuclight Red to nanoMIP LV-F1 -012 and Figure 12b shows binding of Incucyte® Nuclight Green to nanoMIP LV-F1 -012.

[0187] Figure 13 shows binding of lentiviral particles to nanoMIP LV-F2-016.

[0188] Figure 14 shows binding of lentiviral particles to nanoMIP LV-F1 -012 in complex buffer vs standard buffer.

[0189] Figure 15 shows immobilisation of an anti-VSV-G antibody to BLI biosensor tips and subsequent binding to lentiviral particles.

[0190] Figure 16 shows elution of VSV-G from linear non-imprinted polymers.

[0191] Example 1: Synthesis of polymers with the ability to bind VSV-G

[0192] Synthesis of MIPs to VSV-G

[0193] Selection of peptides from VSV-G suitable for imprinting There are two major serotypes of VSV, Indiana (VSIV) and New Jersey (VSNJV). The standard envelope used for pseudotyping lentiviral vectors is from VSIV. The amino acid sequences of the VSIV glycoproteins from several strains of virus known as ‘strain 94GUB Central America’ (UniProt accession number: Q8B0H1 ), ‘strain 85CLB South America’ (UniProt accession number: Q8B0H6), ‘strain 98COE North America’ (UniProt accession number: Q8B0I1 ), ‘strain Glasgow’ (UniProt accession number: P04883), ‘strain Orsay’ (UniProt accession number: P04884), ‘strain Mudd-Summers’ (UniProt accession number: P0C2X0), and ‘strain San Juan’ (UniProt accession number: P03522) are known (see SEQ ID NO.: 1 -7). Using the amino acid sequence of the VSV-G polypeptide from the Mudd-Summers strain, seven peptide sequences (i) DYKVKGLCDSNL (SEQ ID NO. 8), (ii) ADKDLFAAARFPE (SEQ ID No. 9), (iii) PEGSSISAP (SEQ ID NO. 10), (iv) EDGELSSLGKEGT (SEQ ID NO. 11 ), (v) PKSHKAI (SEQ ID NO. 12), (vi) ADKDLFAAAR (SEQ ID NO. 13), and (vii) CPEGSSISAP (SEQ ID NO. 14) were selected by the present inventors as potential candidate templates for imprinting based on several criteria: (i) avoidance of the transmembrane region to increase accessibility, (ii) avoidance of known glycosylation sites to increase consistency in binding, (iii) accessibility, based on the protein crystal structure accessible using UniProt ID: 5I2S, (iv) side chain reactivity (amino acids with side chains containing ring structures or charged groups are preferred), (v) conservation with VSV-G amino acid sequences from alternative strains noted above, and (vii) similarity of conformation between the isolated peptide and the peptide when present in the native protein (assessed using in silico modelling).

[0194] On the basis that a portion of MIPs were made via solid-phase template imprinting, which requires immobilisation of the peptide templates onto a solid support, the peptide sequences listed above were modified at their N-terminus to comprise a terminal cysteine or lysine. One or more glycine residues, functioning as spacers, were also added between the terminal cysteine or lysine residue and the final N-terminal amino acid of the peptide template. One of the peptide templates, CPEGSSISAP, was additionally modified to comprise a C-terminal cysteine residue. This modification renders the peptide template cyclic owing to the disulfide bridge that forms between the cysteine at position 1 and the additional cysteine at the C-terminus. Another of the peptide templates, ADKDLFAAAR, was additionally modified to comprise a C-terminal aspartic acid residue. This modification renders the peptide template helical. The modified templates are shown in Table 1 below.

[0195] Table 1

[0196] Peptide analysis

[0197] Prior to MIP synthesis, the peptide sequences shown in the second column of Table 1 were assessed using common on-line tools for peptides (in this case PEPTIDE 2.0 and PepDraw) to establish theoretical properties which may influence peptide handling, immobilisation, or MIP synthesis (see Table 2).

[0198] Table 2

[0199] Table 2 summarises the properties of the peptides. The properties are based on the native sequences without modifications (i.e. column 2 of Table 1 ). The data in the third column was used to assist solubility studies prior to immobilisation of the templates. Net charge and hydrophobicity were calculated from the sequence ‘within’ protein at pH=7. The ‘within’ protein structure has acetylation on the N-terminus and amidation on the C-terminus. Selection of monomers suitable for imprinting

[0200] In silico screening

[0201] As an initial step, molecular modelling was performed for each of the modified peptide templates, LV-F1 to LV-F8. In more detail, a 3D structure of each of the aforementioned modified peptide templates was prepared.

[0202] The resulting 3D structures of the modified peptide templates were then screened against a database containing over 600 monomers. The estimated binding energies between each of the modified peptide templates and each monomer were then calculated.

[0203] Select monomers with the most negative binding energies (i.e. those with the strongest affinity for a given modified peptide template) were, along with other monomers of lower affinity for comparative purposes, taken forward into high performance liquid chromatography (HPLC) screening as functional monomers i.e. monomers used to provide direct interactions with the template (see below). Table 3 shows a selection of the monomers identified for LV-F1 .

[0204] Table 3

[0205] HPLC screening

[0206] As an initial step, each modified peptide template, LV-F1 to LV-F6 and LV-F8 (ordered from KareBay Biochem, USA, and / or Proteogenix, France), was immobilised onto amine-functionalised silica. Peptides LV-F1 , LV-F2, LV-F4, LV-F5, and LV-F6 were immobilised via succinimidyl iodoacetate (SIA) conjugation chemistry: 5 mL of a 3 mg / mL solution of SIA in acetonitrile was added to 3.2 g of amine-derivatised glass beads. After a 2 hr incubation, the beads were transferred to a solid phase extraction (SPE) cartridge, and were washed with 5 x 10 mL acetonitrile. 5 mL of a 0.4 mg / mL tris(2-carboxyethyl) phosphine hydrochloride (TCEP) solution in PBS, pH 8.0, was added to 10 mg of peptide. The peptide solution was added to the SIA-functionalised glass beads, which were incubated at 4 °C overnight. After incubation, the beads were again transferred into a SPE cartridge, and were washed with 4 x 50 mL ultra-pure water followed by 1 x 10 mL methanol / acetone until dry. The peptide-functionalised glass beads were then packed into a HPLC column.

[0207] Peptides LV-F3 and LV-F8 were immobilised via glutaraldehyde (GA) conjugation chemistry: 2.8 pL of 50% glutaraldehyde solution and 20 mL of PBS, pH 7.4, was added to 3.5 g of amine-derivatised glass beads. After a 2 hr incubation, the beads were transferred to a solid phase extraction (SPE) cartridge, and were washed with 4 x 50 mL ultra-pure water. 20 mL of a 1 .7 mg / mL peptide solution in PBS, pH 7.4, was then added to the GA-functionalised glass beads. After a further 2.5 hrs of incubation, 20 mg of sodium cyanoborohydride was added, and the beads were further incubated for 30 min. The beads were again transferred to a SPE cartridge, and were washed with 4 x 50 mL ultra-pure water followed by 1 x 10 mL ethanol until dry. The peptide- functionalised glass beads were then packed into a HPLC column. A library of 89 linear polymers was then screened against the peptide-functionalised silica stationary phase in a water / acetonitrile (ACN) mobile phase (30:70 ACN:H2O). Each linear polymer contained acrylamide and a variable ‘functional monomer’ in a 94:6 molar ratio. A portion of the functional monomers were identified via the in silico screening described above.

[0208] As a control, the library of 89 linear polymers was also screened against a silica stationary phase comprising silica conjugated to mercaptoethanol. Linear polymers with greater affinity to the peptide-functionalised silica than the control, showed a longer retention time, hence a difference in retention time (At) relative to the control.

[0209] Tables 4-11 show the functional monomers, in descending order, with the largest At for each of LV-F1 to LV-F6 and LV-F8. Table 4 (LV-F1 )

[0210] Table 5 (LV-F2)

[0211] Table 6 (LV-F3)

[0212] Table 7 (LV-F4)

[0213] CAS Number Functional Monomer (FM) At (mins)

[0214] Table 8 (LV-F5)

[0215] Table 9 (LV-F6) Table 10 (LV-F8)

[0216] Those monomers with the largest At were identified as good monomers to use in the generation of MIPs against each peptide.

[0217] Molecular modelling to determine monomer combinations

[0218] In order to identify monomer candidates which may work well cooperatively, further in silico modelling was completed to identify where each of the monomer candidates from the above HLPC screens binds to each of LV-F1 to LV-F8.

[0219] During this screen, binding zones were identified for each of LV-F1 to LV-F8. Each of the monomer candidates was then assigned to a binding zone and the specific amino acid(s) that each monomer bound to therein was identified. Figure 1 a shows the six binding zones for LV-F1 and Figure 1 b shows which amino acid(s) of LV-F1 each of the monomer candidates from the HPLC screen binds to (NB: for in silico modelling the non-salt version of sulfopropyl acrylate and the non-chloride version of (3- acrylamidopropyl)trimethylammonium chloride solution were used).

[0220] The results of this analysis allowed combinations of monomers to be identified which have the potential to be used together to create high affinity MIPs. Indeed, two or more monomers that bind to different regions, or amino acids, of the modified peptide template should be able to be used together to yield high affinity MIPs. The monomer combinations identified for LV-F1 were:

[0221] (i) 2-Carboxyethyl acrylate and mono-2-(methyacryloyloxy)ethyl maleate,

[0222] (ii) N-(3-Aminopropyl) methacrylamide hydrochloride and 2-carboxyethyl acrylate,

[0223] (iii) N-(3-Aminopropyl) methacrylamide hydrochloride and 3-(perfluoro-5- methylhexyl)-2-hydroxypropyl methacrylate, and

[0224] (iv) Trimethyl-[3-(prop-2-enoylamino)propyl]azanium) and mono-2 - (methyacryloyloxy)ethyl maleate.

[0225] Molecular dynamic (MD) simulations were then performed for the selected monomer combinations. Simulations containing monomers (i) N,N'-methylenebis(acrylamide) (BIS), (ii) acrylamide (AA), (iii) tert-butyl acrylate (TBAc) and (iv) N-(3-aminopropyl) methacrylamide hydrochloride (APMA) together with the peptide (LV-F1 to LV-F8) and each functional monomer (FM) combination were generated. The molar ratio of components was BIS(1.5):AA(7.5):TBAc(5.0):APMA(1.0):FM (2.25):FM (2.25).

[0226] The average number of hydrogen bonds between the modified peptide template and each composition of monomers were calculated. A greater number of hydrogen bonds between the mixture of monomers and the modified peptide template indicates a higher affinity composition. Figure 2 shows the results of the molecular dynamic simulations for the monomer combinations identified above for LV-F1. These results suggest that the affinity of the corresponding MIP towards the LV-F1 peptide will be: LV-F1 -013 (N-(3-aminopropyl) methacrylamide hydrochloride and 3-(perfluoro-5- methylhexyl)-2-hydroxypropyl methacrylate) > LV-F1-011 (2-carboxyethyl acrylate and mono-2-(methyacryloyloxy)ethyl maleate) > LV-F1 -012 (N-(3-aminopropyl) methacrylamide hydrochloride and 2-carboxyethyl acrylate) > LV-F1-014 (trimethyl-[3- (prop-2-enoylamino)propyl]azanium) and mono-2-(methyacryloyloxy)ethyl maleate) with a total of 10.08, 7.64, 7.36, and 7.13 monomer-peptide hydrogen bonds respectively.

[0227] Solid-phase synthesis of nanoMIPs to VSV-G

[0228] Modified peptide templates LV-F1 to LV-F6 and LV-F8 were immobilised onto silane- functionalised glass beads prior to nanoMIP synthesis.

[0229] Modified peptide templates comprising terminal cysteines (LV-F1 , LV-F2, LV-F4, LV- F5, and LV-F6) were immobilised using SIA conjugation chemistry, whilst those modified peptide templates comprising terminal lysine residues (LV-F3 and LV-F8) were immobilised using glutaraldehyde (GA) coupling.

[0230] With respect to those modified peptide templates immobilised by SIA conjugation chemistry, peptide immobilisation was carried out as follows: the silanised glass beads were placed in PBS, pH 7.4, with 0.2 mg / mL n-succinimidyl iodoacetate (SIA) for two hr before being washed with acetonitrile. The templates (LV-F1 , LV-F2, LV-F4, LV-F5, or LV-F6) were then immobilised on the surface of the glass beads by incubation in a solution of PBS, pH 7.4, with 0.4 mg / mL tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and 0.1 mg / mL of peptide for a minimum of 4 hr (typically overnight). Excess template was removed by washing with ultra-pure water and ethanol.

[0231] With respect to those modified peptide templates immobilised by glutaraldehyde coupling peptide immobilisation was carried out as follows: the silanised glass beads were placed in PBS, pH 7.4 containing 13% v / v of 50% glutaraldehyde solution for 2 hr before being washed with ultra-pure water. The templates (LV-F3 or LV-F8) were then immobilised on the surface of the glass beads by incubation in a solution of PBS, pH 7.4, and 0.1 mg / mL of peptide for a minimum of 4 hr (typically overnight). Excess template was removed by washing with water and acetonitrile.

[0232] The success of peptide immobilisation via SIA conjugation was quantified by bicinchoninic acid (BCA) assay. Modified peptide templates LV-F1 , LV-F2, and LV-F6 showed particularly good immobilisation densities of 16.65, 21.77, and 7.74 pg of peptide per gram of glass bead respectively. The peptide coated glass beads were used for the synthesis of imprinted nanoMIPs using the following general method. Monomer solutions were prepared in ultra-pure, deionized water (UPW) and were sonicated for 10 min to ensure dissolution. Examples of monomer solutions used with respect to each of LV-F1 , LV-F2, LV-F3, LV-F4, LV- F6, and LV-F8 are shown in Tables 11A-11 F below. Typically 0.345 mmol of total monomers were dissolved in 40 mL of UPW in the ratio BIS:AA:TBAc:APMA: Functional Monomer 1 (functional Monomer 2) ratio of 1 .5:7.5:5.0:1 ,0:2.25(:2.25). Where necessary, monomers were first dissolved in alternative solvents (e.g. ethanol) before addition to the bulk aqueous solution. 40 mL of the monomer solution was then put into a 3-necked round bottom flask containing 30 g of the peptide-functionalised glass beads. The reaction solution was degassed under nitrogen for 15 min. Polymerisation was then initiated by adding 0.25 mL of aqueous initiator solution containing 30 mg of ammonium persulfate (APS) with 15 pL of N,N,N’,N’-tetramethylethylene diamine (TEMED). The polymerisation proceeded at room temperature for 45 min under nitrogen atmosphere. The contents of the flasks were then poured into solid phase extraction (SPE) cartridges fitted with 20 pm porosity frits in order to separate the glass beads with attached nanoparticles from the other components. 10 washing steps, each with approximately 20 mL of room temperature ultra-pure water, were then performed to remove low affinity material. The residue from each of these washing steps was discarded. High affinity nanoMIPs were then detached from the glass beads by performing 6 washing steps, each with 20 mL of ~65 °C ethanol. The residue from each of these washes, containing the nanoMIPs, was collected. The nanoMIPs were then concentrated down to approximately 10 mL and then dialysed against UPW at 40 °C for 24 - 48 hrs.

[0233] Table 11 A-F

[0234] NB: All nanoMIPs listed in Tables 11 A-11 F also contain the following monomers: N,N'- methylenebis(acrylamide) (BIS, Sigma Aldrich, 7279-25G) which functions as a crosslinker, acrylamide (AA, Sigma Aldrich, A8887-100G) which functions as a backbone monomer, tert-Butyl acrylate (TBAc, Sigma Aldrich, 327182) which functions as a backbone monomer and N-(3-aminopropyl) methacrylamide hydrochloride (APMA, Sigma Aldrich, 731099) which functions as an amino monomer. Table 11 A: NanoMIPs imprinted against LV-F1

[0235] Table 11 B: NanoMIPs imprinted against LV-F2

[0236]

[0237] Table 11 C: NanoMIPs imprinted against LV-F3

[0238] Table 11 D: NanoMIPs imprinted against LV-F4

[0239] Table 11 E: NanoMIPs imprinted against LV-F6

[0240]

[0241] Table 11 F: NanoMIPs imprinted against LV-F8 The size of selected nanoMIPs was determined using dynamic light scattering (DLS) on a Zetasizer Ultra (Malvern Panalytical) instrument (Table 11 G). NB: some polymers have two distinct size populations. The numbers in the square brackets indicate the mean diameter of the minor population.

[0242] Table 11 G Once the present inventors had identified monomers likely to give rise to a nanoMIP which binds VSV-G, corresponding linear polymers were also made with and without the use of a template (see above for methodology associated with the synthesis of linear polymers in the presence of a template).

[0243] Solid-phase synthesis of linear polymers (linear MIPs and linear non-imprinted polymers) to VSV-G

[0244] LV-F1 coated glass beads (from above) were used for the synthesis of imprinted linear MIPs using the following general method. The monomers shown in Table 12 were dissolved, in the quantities shown, in 150 mL deionized water to produce a stock monomer solution. 100 mL of this stock monomer solution was then supplemented with 42 mg of acrylamide (CAS: 79-06-1 , Sigma Aldrich, A8887) acting as a backbone monomer to produce the monomer composition for linear MIP 1. The remaining 50 mL of stock monomer solution was supplemented with 33 mg of N-isopropylacrylamide (NIPam (CAS: 2210-25-5, Sigma Aldrich, 415324) acting as a backbone monomer to produce the monomer composition for linear MIP 3.

[0245] 50 mL of the monomer composition for linear MIP 1 and the 50 mL monomer composition for linear MIP 3 were each placed in a 250 mL Duran flask, sonicated for 2 min and nitrogen bubbled for 5 min before 30 g of LV-F1 -functionalised glass beads were added to each. The remaining 50 mL of the monomer composition for linear MIP 1 was added to a 250 mL Duran flask, sonicated for 2 min and nitrogen bubbled for 5 min before 30 g of silanised glass beads (i.e. glass beads without the LV-F1 ) were added (linear non-imprinted polymer 2).

[0246] During nitrogen bubbling, polymerisation was initiated by adding 15 pL of TEMED (CAS: 110-19-9) followed by 30 mg APS (CAS: 7727-54-0), pre-dissolved in 200 uL of distilled water, to each of the solutions. Polymerisation was carried out at room temperature overnight.

[0247] Afterwards, the contents of the Duran flasks were each transferred to individual fritted 60 mL SPE tubes, then washed with 4 x bead bed volumes of water at room temperature, followed by 5 x 20 mL washes with DI water at 70 °C to elute the linear MIPs. The linear MIPs were then evaporated down to approximately 10 mL using a rotary evaporator set to 50 °C, dialysed vs. 2 L of deionized water (3 water changes every 3 hr during daytime) for approx. 2 days with a 3.5 kDa MWCO snakeskin membrane, then transferred to a vial and stored in the fridge.

[0248] Table 12

[0249] * Tert-butyl acrylate acts as a backbone monomer, N-fluoresceinylacrylam ide aids in the visualisation of the polymers, and cysteamine is added to introduce primary amines at the end of the polymer chains. 3-Sulfopropyl acrylate, mono-2- (methacryloyloxy)ethyl maleate, and 2-carboxyethyl acrylate act as functional monomers. Synthesis of N-fluoresceinylacrylam ide The fluorescent monomer N-fluoresceinylacrylamide was prepared by mixing acryloyl chloride (Sigma Aldrich, A24109) with fluoresceinamine (Sigma Aldrich 201626) (molar ratio 1.15 to 1.00) in dry acetone, in the presence of N,N-diisopropylethylamine (Alfa Aesar A11801 ) (1.20 equivalent) (see schematic below). Dry acetone was obtained by means of molecular sieves (3 A), previously activated for 3 hr at 200 °C. The mixture was stirred under nitrogen for about 2 hr. Afterwards, the flask was placed at -18 °C in order to enhance the precipitation of the product. The precipitate obtained was collected by filtration and washed with 15 x 20 mL aliquots of cold acetone (0 °C). The product was dried under vacuum and stored in the dark at 4 °C. All steps were followed by thin layer chromatography (TLC). Yield: 89 % w / w.

[0250] Fluoresceinamine Acryloyl chloride N-fluoresceinylacrylamide

[0251] Synthesis of linear MIPs to VSV-G by solution imprinting

[0252] Owing to the absence of a free thiol group in its structure, LV-F8 was used for the synthesis of linear MIPs by solution imprinting using the following method.

[0253] The monomers shown in Table 13 were dissolved, in the quantities shown, in 50 mL deionized water and placed in a 250 mL Duran flask, sonicated for 2 min and nitrogen bubbled for 5 min before 5 mg of LV-F8 was added.

[0254] During nitrogen bubbling, polymerisation was initiated by adding 15 pL of TEMED (CAS: 110-19-9) followed by 30 mg APS (CAS: 7727-54-0). Polymerisation was carried out at room temperature overnight.

[0255] The linear MIPs (linear MIP 7) were then evaporated down to approximately 10 mL using a rotary evaporator set to 50 °C, dialysed overnight vs. 2 L of deionized water (3 water changes every 3 hr during daytime) for approx. 2 days with a 3.5 kDa MWCO snakeskin membrane, then transferred to a vial and stored in the fridge.

[0256] Table 13

[0257] *Acrylamide and tert-butyl acrylate act as backbone monomers, N- fluoresceinylacrylamide aids in the visualisation of the polymers, 2,2,2-trifluoroethyl acrylate, acrylonitrile, ethyl methacrylate act as functional monomers, cysteamine is added to introduce primary amines at the end of the polymer chains

[0258] Synthesis of linear non-imprinted polymers to VSV-G

[0259] In one example, the monomers shown in Table 12 above were dissolved, in the quantities shown, in 150 mL deionized water to produce a stock monomer solution. 100 mL of this stock monomer solution was then supplemented with 42 mg of acrylamide (CAS: 79-06-1 , Sigma Aldrich, A9099) to produce the monomer composition for linear non-imprinted polymer 4. The remaining 50 mL of stock monomer solution was supplemented with 33 mg of N-isopropylacrylamide (NIPam (CAS: 2210-25-5, Sigma Aldrich, 415324) to produce the monomer composition for linear non-imprinted polymer 6.

[0260] 50 mL of the monomer composition for linear non-imprinted polymer 4 and the 50 mL monomer composition for linear non-imprinted polymer MIP 6 were each placed in a 250 mL Duran flask, sonicated for 2 min and nitrogen bubbled for 5 min. To the remaining 50 mL of the monomer composition for linear non-imprinted polymer 4, 5.5 mg APMA (CAS: 72607-53-5, Sigma Aldrich, 244484) was added (linear nonimprinted polymer 5) and the solution transferred to a 250 mL Duran flask, sonicated for 2 min and nitrogen bubbled for 5 min.

[0261] In another example the monomers shown in Table 13 above were dissolved, in the quantities shown, in 50 mL deionized water (linear non-imprinted polymer 8) and placed in a 250 mL Duran flask, sonicated for 2 min and nitrogen bubbled for 5 min.

[0262] During nitrogen bubbling, polymerisation was initiated by adding 15 pL of TEMED (CAS: 110-19-9) followed by 30 mg APS (CAS: 7727-54-0) to each of the solutions. Polymerisation was carried out at room temperature overnight.

[0263] Polymers were then evaporated down to approximately 10 mL using a rotary evaporator set to 50 °C and then dialysed overnight vs. 2 L of deionized water (3 water changes every 3 hr during daytime) for approx. 2 days with a 3.5 kDa MWCO snakeskin membrane, transferred to a vial and stored in the fridge.

[0264] Example 2: Flow Induced Dispersion Analysis (FIDA) of binding between NanoMIPs and VSV-G peptides

[0265] NanoMIPs were tested for interaction towards the peptide template against which they were imprinted using flow induced dispersion analysis (FIDA) on a 0031.723 model FIDA instrument from Fidabio. Fluorescent tracers were made by conjugating each VSV-G peptide (LV-F1 , LV-F2, LV-F3, LV-F4, LV-F6, and LV-F8) to a fluorescently labelled amino dextran (AD) linker. Each nanoMIP was injected under laminar flow conditions in a solution containing its corresponding peptide tracer (e.g. a nanoMIP imprinted against LV-F1 was injected with an LV-F1 -AD tracer). The dispersion profile was measured for each injection, and was compared to that of the peptide tracer run in isolation.

[0266] If a nanoMIP and a peptide tracer bind in solution, they will diffuse at a slower rate and will generate a broader dispersion profile (compared to that of the peptide tracer in isolation). The FIDA software (V2.34 from Fidabio) extracts the change in size (ARh) due to molecule binding. Hence, a nanoMIP with affinity towards its peptide is indicated by an increase in the measured diameter.

[0267] If a nanoMIP showed binding affinity, it was then tested against a control tracer - a fluorescently tagged amino dextran linker without any peptide attached. A nanoMIP was said to pass the control interaction analysis if it showed no size increase, and therefore no interaction, towards the control tracer.

[0268] Table 14A / B shows the results of FIDA interaction analysis for a selection of nanoMIPs imprinted against LV-F1 , LV-F2, LV-F3, LV-F4, LV-F6, and LV-F8. The analysis was conducted in both phosphate-buffered saline (PBS-P) (prepared in house using 10xPBS, Fisher Bio Reagents, BP399-20, and Tween-20, Fisher Bio Reagents, BP337) (Table 14A) and HEPES-Buffered Saline (HBS-P) (Table 14B) (Cytiva, BR1 00827, diluted to 1 x with ultra-pure water) running buffers. Statistical thresholds were set for both ARh and confidence measurements (SSMD). A nanoMIP passed FIDA interaction analysis if the conditions for both thresholds were met for both the peptide and the control interactions.

[0269] Table 14A

[0270]

[0271] *Pass (Y / N) = Yes if thresholds for both ARh and SSMD are exceeded.

[0272] *Pass (Y / N) = N if thresholds for neither ARh or SSMD are met. *Pass (Y / N) = LC (low confidence) if the threshold for ARh is met, but the threshold for SSMD is not.

[0273] Blank cells indicate unavailable data

[0274] IA: Interaction Analysis

[0275] Table 14B

[0276] Pass (Y / N) = Y if thresholds for both ARh and SSMD are exceeded.

[0277] Pass (Y / N) = N if thresholds for neither ARh or SSMD are met.

[0278] Pass (Y / N) = LC (low confidence) if the threshold for ARh is met, but the threshold for SSMD is not.

[0279] Blank cells indicate unavailable data

[0280] IA: Interaction Analysis

[0281] Example 3: Surface Plasmon Resonance (SPR) Analysis of binding between NanoMIPs and VSV-G protein

[0282] A Biacore T200 SPR instrument was employed in this study.

[0283] Surface activation and nanoMIP immobilisation

[0284] NanoMIP-SPR sensors were prepared by covalently immobilising nanoMIPs (from Example 1 ) onto 4% mercaptoundecanoic acid (MLIDA) gold chips, utilising the immobilisation wizard on a BiaCore T200 SPR instrument, with a PBS running buffer.

[0285] In more detail, the surface of the chip (which bears carboxyl groups) was activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) I N-hydroxysuccinimide (NHS) (68 pL of 0.4 M EDC (aq), and 68 pL of 0.1 M NHS (aq)). Solutions containing 63 pL of a nanoMIP and 7 pL of acetate buffer (pH 4.0) were then injected over flow cell 2 (FC2), FC3, and FC4 at 5 pL / min for 420 s. Typically, different nanoMIPs were immobilised onto each flow cell. ‘Blank Immobilisation’ was selected for flow cell 1 (FC1 ), such that it acted as a control surface, with no immobilised nanoMIP. All flow cells were then blocked with 130 pL of 0.5% BSA / 0.1 % Pluronic solution. Ideally, immobilisation of nanoMIPs resulted in > 1 ,000 R.U.

[0286] Detection of interaction between nanoMIPs and VSV-G protein VSV-G (recombinant VSV-G protein sourced from ProteoGenix, product code: PX- P2100-100) as a serial dilution in PBS (1 .25 nM, 2.5 nM, 5 nM, 10 nM and 20 nM) was then injected in succession (1 .25 nM to 20 nM) over all flow cells, using a single cycle kinetics method with 5 concentrations per cycle, at a flow rate of 25 pL / min for 310 s in PBS running buffer. After a dissociation period of 1 ,800 s, the binding response to the control surface was subtracted from the binding response to the nanoMIP- functionalised surface yielding 2-1 / 3-1 / 4-1 binding curves.

[0287] The VSV-G binding response to several immobilised nanoMIPs is shown in Table 15. NB: ‘±’ indicates the standard deviation for repeat measurements.

[0288] Table 15

[0289] To ensure the binding response reported above is specific to VSV-G, immobilised nanoMIPs were also tested for affinity towards a control protein, bovine serum albumin (BSA), which was selected as the control protein due to its similar size and isoelectic point (Mr, VSV-G = 52 kDa, Mr, BSA = 66 kDa, pIVSV-G = 6.35, pIBSA = 5.82).

[0290] For each test, nanoMIPs were immobilised onto FC2 and FC3 of an SPR chip, using the immobilisation wizard and the conditions stated above. ‘Blank immobilisation’ was selected for FC1. Using the conditions described above, solutions of VSV-G were injected over FC1 and FC2. Solutions of BSA were prepared to match the concentrations of the VSV-G solutions. The BSA solutions were then injected over FC3.

[0291] The nanoMIPs tested so far showed no significant binding response to BSA. Two representative sensorgrams comparing the binding of VSV-G and BSA to an SPR surface functionalised with (a) nanoMIP LV-F1 -004 and (b) nanoMIP LV-F1 -007 are shown in Figure 3.

[0292] Example 4: Membrane based analysis of binding between NanoMIPs and VSV- G protein

[0293] The ability of nanoMIPs (from Example 1 ) to bind to VSV-G (recombinant VSV-G protein sourced from ProteoGenix, product code: PX-P2100-100) was also tested in a membrane-based assay.

[0294] In more detail, each nanoMIP was immobilised onto two membranes via reaction of the amine surface groups on the nanoMIPs with aldehyde groups on the membrane surface. The membranes (AstreAdept®, Astrea Bioseparations) were first cut into 5 x 5 mm squares, then mounted onto folded 7 mm strips made from lateral flow backing cards (BBI International, SKU GL-28840) with a 4.5 mm hole punched at one end to allow for exposure of the membrane to the test solution. Following mounting on the backing strip, 10 membranes at a time were activated by immersion in 3 mL ultrapure water containing 100 mg sodium periodate for 1 hr, then washed by immersion in 20 mL ultrapure water for 5 min. For nanoMIP immobilisation, 900 uL of each MIP stock (used as prepared) was added to 100 uL of carbonate buffer pH 10 (0.1 M, containing 8.4 g / L of sodium bicarbonate) and two pre-activated membranes were placed into each individual nanoMIP / carbonate buffer solution. Following overnight incubation, membranes with immobilised nanoMIPs were washed with water (as above) then placed for 1 hr in 3 mL 1 x PBS pH 7.4 (Fisher Scientific, SKU 10649743) containing 3 mg / mL sodium cyanoborohydride and 5 mg / mL of ethanolamine hydrochloride, then washed as above and kept in ultrapure water until use. In parallel VSV-G-gold conjugates were prepared through physisorption. In more detail, 4.59 uL of VSV-G at 1 mg / mL (recombinant VSV-G protein sourced from ProteoGenix, product code: PX-P2100-100) were incubated in with 1 mL solution of OD5 colloidal gold nanoparticles (BBI Solutions, product code: SKU HD.GC20.0D5 / 10). The gold surface was incubated with the VSV-G for 30 min at room temperature after which the surface was passivated by addition of 12.5 pL of BSA (200 mg / mL). After centrifugation at 6000 ref for 20 min, the pelleted conjugate product was re-suspended (to a final OD of 45) in phosphate buffer pH 8 (25 mM, 3.4 g / L potassium phosphate, containing 5% sucrose and 10% BSA).

[0295] Detection of interaction between nanoMIPs and VSV-G protein

[0296] Each nanoMIP-functionalised membrane was exposed to the VSV-G-gold conjugate (6 pL of VSV-G-gold conjugate in 200 pL PBS pH 7.4) for 15 min at room temperature. After washing the membranes by incubation in 20 mL of PBS-T (PBS containing 0.005% Tween® 20) for 5 min / wash, VSV-G binding was indicated by the presence of a pink / red spot. The intensity of colour development is indicative of the strength of binding between the VSV-G and nanoMIP. A control membrane, functionalised with only ethanolamine, was also exposed to the VSV-G-gold conjugate under the same conditions.

[0297] As shown in Figures 4A-4F, strong interactions were detected between VSV-G and select nanoMIPs imprinted against all of LV-F1 (e.g. LV-F1-001 ), LV-F2 (e.g. LV-F2- 015), LV-F3 (e.g. LV-F3-018), LV-F4 (e.g. LV-F4-001 ), LV-F6 (e.g. LV-F6-008), and LV-F8 (LV-F8-003) (as indicated by 3 dots). Conversely, no interactions were observed between ethanolamine and VSV-G.

[0298] For nanoMIPs that showed binding to the VSV-G-gold conjugate, a further control was included in which the membrane test was repeated using a BSA-gold conjugate which indicates the specificity of the nanoMIP-functionalised membranes for VSV-G.

[0299] As shown in Figure 5, the strength of binding between the BSA-gold conjugate and the nanoMIP-functionalised membrane was less significant for all nanoMIPs when compared to binding between the VSV-G-gold conjugate and the nanoMIP- functionalised membrane.

[0300] Example 5: Sepharose® based analysis of binding between linear MIPs and linear non-imprinted polymers, and VSV-G protein

[0301] Sepharose® activation and MIP immobilisation

[0302] Aldehyde-activated Sepharose® was prepared as follows:

[0303] 6 mL of Sepharose® slurry was added to a 15 mL SPE tube and washed with 50 mL deionised water to remove storage solution, then excess water removed under vacuum for ca 10 s. This was followed by addition of 6 mL of sodium periodate solution (30 mg / mL in water) to each cartridge, then incubation for 1 hr. The contents of the cartridge were then washed with 50 mL DI water, and excess water was removed during ca. 10 s under vacuum, completing the activation process. The Sepharose® was then transferred to a 18 mL glass vial and suspended in 4 mL deionised water containing 20% ethanol.

[0304] 200 pL of aldehyde-activated Sepharose® slurry was added to 9 SPE tubes (1 mL nominal capacity), each fitted with a 20 pm porosity plastic frit. The aldehyde-activated Sepharose® in the tubes was washed with 1 mL of deionized water and all liquid was drained under vacuum for 10 s. Aldehyde-activated Sepharose® in SPE tubes 1 to 8 was modified with linear MIPs 1 , 3, and 7, and non-imprinted polymers 2, 4, 5, 6, and 8. As a control, aldehyde-activated Sepharose® in SPE tube 9 was modified with ethanolamine. For polymer immobilisation, the polymer stock (used directly as prepared in Example 1 ) was diluted four times in carbonate buffer pH 10 (0.1 M containing 8.4 g / L of sodium bicarbonate, 200 pL total volume) and was then added to the aldehyde-activated Sepharose® in tubes 1 -8 and incubated overnight. SPE tube 9 (control) was incubated overnight with 200 pL of carbonate buffer containing 10 mg / mL ethanolamine. After incubation, the SPE tubes were washed with 1 mL deionized water. Then 200 pL of PBS (pH 7.4) containing 3 mg / mL sodium cyanoborohydride and 10 mg / mL ethanolamine was added to each tube to block residual aldehyde and reduce the Shift bases. After 1 hr incubation followed by four washes with 1 mL PBS, the immobilisation procedure was complete. Sepharose® was stored swollen with 200 L of PBS.

[0305] In parallel to the above, a tenth SPE tube was prepared by adding 200 pL unmodified Sepharose® slurry to a SPE tube fitted with a 20 pm porosity plastic frit, and washed with 50 mL deionised water to remove storage solution, then excess water removed under vacuum for ca 10 s. The unmodified Sepharose® was stored swollen with 200 pL of PBS.

[0306] Detection of interaction between both linear MIPs and linear (non imprinted) polymers, and VSV-G protein

[0307] The PBS present in each SPE tube was drained by gravity before 200 pL of VSV-G- gold solution (prepared by diluting 6 pL of VSV-G-gold stock (from Example 4) in 200 pL PBS) was added to each tube and incubated for 1 hr. The solution was then drained by gravity and the Sepharose® washed with 0.2 mL PBS, which was subsequently drained by gravity.

[0308] Figure 6 shows a photograph of the 10 SPE tubes. As is apparent from the pink colouration, linear non-imprinted polymers 2 and 6, and linear MIP 3, showed the highest binding to VSV-G-gold. Increased binding may be linked to greater affinity of the polymer to the VSV-G or higher capacity (i.e. the polymer binds more copies of the VSV-G).

[0309] For linear non-imprinted polymers 2 and 6, and linear MIP 3, a further control was included in that the test was repeated using a BSA-gold conjugate (from Example 4) in place of the VSV-G-gold conjugate which indicates the specificity of the polymer- immobilised Sepharose® for VSV-G. The BSA-gold conjugate was also added to SPE tube 9 containing ethanolamine-immobilised Sepharose®.

[0310] As shown in Figure 6 no binding between the BSA-gold conjugate and the polymer- immobilised Sepharose® was detected.

[0311] Elution of VSV-G from polymer-immobilised Sepharose® Following the binding test immediately above, 0.5 mL of PBS was added to each of SPE tubes 1 -9 followed by incubation at room temperature for 1 hr. The Sepharose® bed was then gently mixed using a spatula before the PBS was allowed to drain under gravity.

[0312] Figure 7 shows a photograph of the 9 SPE tubes. As is apparent from the reduction in colour, the VSV-G-gold conjugate was successfully eluted from linear non-imprinted polymers 2, 4, 5, 6, and 8, and linear MIP 3. SPE tubes containing Sepharose® functionalised with linear non-imprinted polymers 2 and 6, and linear MIP 3 showed the greatest reduction in colour.

[0313] Table 16 below summarises the binding and elution patterns seen for each of linear MIPs 1 , 3, and 7, as well as linear non-imprinted polymers 2, 4, 5, 6, and 8. Darker colours in the ‘binding performance’ column represent higher binding performance to VSV-G. Darker colours in the ‘release profile’ column represent greater release of VSV-G from the functionalised Sepharose®.

[0314] Table 16

[0315] Example 6: Membrane based analysis of binding between linear MIPs and linear non-imprinted polymers, and VSV-G protein

[0316] Membranes (AstreAdept®, Astrea Bioseparations), were first cut into nine 5 x 5 mm squares, then mounted onto folded 7 mm strips made from lateral flow backing cards (BBI International SKU GL-28840) with a 4.5 mm hole punched at one end to allow for exposure of the membrane to the test solution. Preparation of polymer-functionalised membranes for use in detection analysis Sodium periodate activation was used to create aldehyde groups on the membrane to facilitate polymer coupling.

[0317] In more detail, each of the mounted membranes was incubated at room temperature for 1 hr in a solution containing 3 mL deionized water and 100 mg sodium periodate. Post incubation, the activated mounted membranes were rinsed with deionized water before excess liquid was removed with adsorbent paper.

[0318] Each of activated mounted membranes 1 -8 were placed in a vial containing 400 pL of polymer solution (linear MIPs 1 , 3, and 7, and linear non imprinted polymers 2, 4, 5, 6, and 8) and 400 pL of 0.1 M carbonate buffer, pH 10. As a control, activated mounted membrane 9 was placed in a vial containing 800 pL of 0.1 M carbonate buffer, pH 10 with 10 mg / mL of ethanolamine. All vials were incubated overnight at room temperature.

[0319] Post incubation, all mounted membranes were rinsed with deionized water and placed in a vial containing 3 mL of PBS buffer (pH 7.4), supplemented with 3 mg / mL sodium cyanoborohydride and 10 mg / mL ethanolamine for reduction of Schiff bases and residual aldehyde blocking, respectively. After subsequent rinsing with deionized water, polymer immobilisation was complete.

[0320] Detection of binding between polymers and VSV-G using polymer-functionalised membranes

[0321] Polymer-functionalised membranes were pre-conditioned by incubating in 15 mL PBS- T for 10 seconds, followed by rinsing in a vial containing 20 mL of PBS (pH 7.4). The polymer-functionalised membranes were then transferred to a vial containing 6 pL of VSV-G-gold conjugate (from Example 4) diluted in 200 pL of PBS (pH 7.4) and were incubated for 10 min at room temperature. During incubation, the vials were stirred at 120 rpm using an orbital shaker. Post incubation, the membranes were washed by immersion in a vial containing 20 mL PBS (pH 7.4) and imaged. Figure 8 shows a photograph of the 9 polymer-functionalised membranes post incubation with VSV-G-gold. Linear non-imprinted polymers 2, 4, and 6, and linear MIP 3, exhibited the greatest binding to VSV-G.

[0322] Example 7: Biolayer Interferometry (BLI) Analysis of NanoMIPs to VSV-G

[0323] An Octet® R8 or RED96e instrument (Sartorius) with Octet® Amine Reactive 2ndGeneration (AR2G) Biosensor tips (Sartorius) was employed in this study.

[0324] NanoMIP immobilisation

[0325] NanoMIPs (Example 1 ) were immobilised onto AR2G biosensor tips via 1 -ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) / sulfo-N-hydroxysuccinimide (sNHS) chemistry.

[0326] In order to optimise immobilisation, a pH scout was first performed to identify the most favourable pH for the loading buffer for each nanoMIP. Typically, a series of sodium acetate loading buffers of pH 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 were trialled. For each sample, a 1 :1 mixture of nanoMIPJoading buffer was prepared and loaded onto an activated AR2G biosensor tip. After nanoMIP loading, each tip was blocked with 1 M ethanolamine and then exposed to a 50 nM solution of VSV-G (recombinant VSV-G protein sourced from ProteoGenix, product code: PX-P2100-100) in PBS. The most favourable loading buffer was identified as the one that yeilded the highest MIP- loading response, and the highest binding response to VSV-G. Figure 9 shows that for nanoMIP LV-F1 -012, a loading buffer of pH 3.5 was the most favourable. Parallel experiments showed that for nanoMIP LV-F2-016, a loading buffer of pH 4 was the most favourable.

[0327] In order to further optimise immobilisation, a load scout was performed in order to identify the optimal concentration of nanoMIP to load onto the activated AR2G biosensor tips. Typically, a range of MIP concentrations (e.g. 0 pg / mL, 9 pg / mL, 18 pg / mL, 35 pg / mL, 70 pg / mL, 140 pg / mL, 281 pg / mL, and 563 pg / mL for nanoMIP LV- F1 -012) were prepared in the loading buffer determined as the most favourable in the pH scout (described directly above). Each nanoMIP concentration was loaded onto an activated AR2G biosensor tip. After loading, each tip was blocked with 1 M ethanolamine and then exposed to a 50 nM solution of VSV-G (recombinant VSV-G protein sourced from ProteoGenix, product code: PX-P2100-100) in PBS. The most favourable nanoMIP concentration was identified as the one that yeilded the highest nanoMIP-loading response, and the highest binding response to VSV-G, while maintaining ideal association and dissociation behaviours. Figure 10 shows that for nanoMIP LV-F1 -012 the optimal concentration for immobilisation was 70 pg / mL. Parallel experiments showed that for nanoMIP LV-F2-016, a nanoMIP concentration of 71 .5 pg / mL was most favourable.

[0328] Detection of interaction between nanoMIPs and VSV-G protein

[0329] To test the ability of nanoMIPs to recognise and bind VSV-G (recombinant VSV-G protein sourced from ProteoGenix, product code: PX-P2100-100), a set of 8 activated AR2G biosensors were functionalised with nanoMIPs using the optimised conditions noted above (LV-F1 -012 = loading buffer of pH 3.5 and 70 pg / mL nanoMIP).

[0330] As a control, a set of 8 activated AR2G biosensors without nanoMIP served as reference biosensors.

[0331] Each set of biosensors were exposed to VSV-G (recombinant VSV-G protein sourced from ProteoGenix, product code: PX-P2100-100) solution in PBS (0 - 200 nM). The binding response to the reference biosensor set was subtracted from the binding response to the MIP-functionalised biosensor set, yielding the binding curves in Figure 11 and a calculated KD of 0.014 nM.

[0332] Example 8: Biolayer Interferometry Analysis of NanoMIPs to Lentivirus Particles An Octet® R8 or RED96e instrument (Sartorius) with Octet® Amine Reactive 2ndGeneration (AR2G) Biosensor tips (Sartorius) was employed in this study.

[0333] NanoMIP immobilisation

[0334] NanoMIPs (Example 1 ) were immobilised onto AR2G biosensor tips via 1 -ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC)ZN-hydroxysuccinimide (NHS) chemistry.

[0335] In order to optimise immobilisation, a pH scout was first performed to identify the most favourable pH for the loading buffer for each nanoMIP. Typically, a series of sodium acetate loading buffers of pH 3.0, 3.5, 4.0, 4.5, and 5.0 were trialled. For each sample, a 1 :1 mixture of nanoMIPJoading buffer was prepared and loaded onto an activated AR2G biosensor tip. After nanoMIP loading, each tip was blocked with 1 M ethanolamine and then exposed to a 1.72 x 106Tll / mL solution of lentiviral particles (Sartorius, Incucyte® NucLight Red, 4625). The most favourable loading buffer was identified as the one that yeilded the highest nanoMIP-loading response, and the highest binding response to the lentiviral particles. For nanoMIP LV-F1 -012, a loading buffer of pH 3.5 was the most favourable.

[0336] In order to further optimise immobilisation, a load scout was performed in order to identify the optimal concentration of nanoMIP to load onto the activated AR2G biosensor tip. Typically, a range of nanoMIP concentrations (e.g. 0 pg / mL, 9 pg / mL, 18 pg / mL, 35 pg / mL, 70 pg / mL, 140 pg / mL, 281 pg / mL, and 563 pg / mL for nanoMIP LV-F1 -012) were prepared in the loading buffer determined as the most favourable in the pH scout (described directly above). Each nanoMIP concentration was loaded onto an activated AR2G biosensor tip. After loading, each tip was blocked with 1 M ethanolamine and then exposed to a 1.72 x 106Tll / mL solution of lentiviral particles (Sartorius, Incucyte® NucLight Red, 4625). The most favourable nanoMIP concentration was identified as the one that yeilded the highest nanoMIP-loading response, and the highest binding response to lentiviral particles. For nanoMIP LV- F1 -012, the optimal concentration for immobilisation was 281 pg / mL. Parallel experiments showed that for nanoMIPs LV-F1 -011 and LV-F2-016 respectively, nanoMIP concentrations of 131 pg / mL and 76 pg / mL were most favourable.

[0337] Detection of interaction between nanoMIPs and lentivirus

[0338] To test the ability of nanoMIPs to recognise and bind intact lentiviral particles a set of 8 activated AR2G biosensors were functionalised with nanoMIPs (LV-F1 -012) using the optimised conditions noted above (LV-F1-012 = loading buffer of pH 3.5 and 281 pg / mL nanoMIP).

[0339] As a control, a set of 8 activated AR2G biosensors without nanoMIP served as reference biosensors. Each set of biosensors were exposed to Incucyte® Nuclight Red (Sartorius, product code 4625) and Incucyte® Nuclight Green (Sartorius, product code: 4475) at a range of titres. NucLight reagents are 3rdgeneration HIV-based, VSV-G pseudotyped lentiviral particles which carry a fluorescent payload.

[0340] In both cases, a concentration-dependent binding response was observed after reference subtraction (Figure 12 and Table 17), yielding ‘KD’ values of 3.04 x 107Tll / mL (Incucyte® Nuclight Red) and 1 .063 x 105Tll / mL (Incucyte® Nuclight Green).

[0341] Parallel experiments for nanoMIP LV-F2-016 were also performed whereby instead of immobilising nanoMIP LV-F1 -012 on the 8 activated AR2G biosensors, nanoMIP LV- F2-016 was immobilised on to 1 activated AR2G biosensor using the optimised conditions specific for that nanoMIP (i.e. a nanoMIP loading concentration of 76 pg / mL). As a control, 1 activated AR2G biosensor without nanoMIP served as a reference biosensor.

[0342] Both biosensors were exposed to Incucyte® Nuclight Green (Sartorius, product code: 4475) at a titre of 1.72 x 106Tll / mL and a binding response of 0.1984 nm was observed (Figure 13, Table 17).

[0343] Parallel experiments for nanoMIP LV-F1 -011 were also performed whereby the nanoMIP was immobilised onto 4 activated AR2G biosensors, using the optimised conditions specific for that nanoMIP (i.e. a nanoMIP loading concentration of 131 pg / mL). 4 activated AR2G biosensors without nanoMIP immobilised served as reference biosensors.

[0344] Another set of parallel experiments were performed for nanoMIP LV-F2-016, whereby the nanoMIP was immobilised onto 4 activated AR2G biosensors, using the optimised conditions specific for that nanoMIP (i.e. a nanoMIP loading concentration of 76 pg / mL). 4 activated AR2G biosensors without nanoMIP immobilised served as reference biosensors. Both sets of biosensors were exposed to Incucyte® Nuclight Green (Sartorius, EF1a, Bleo, product code 4477) at a range of titres, yielding concentration-dependent responses after reference subtraction (Table 17). Table 17

[0345] Example 9: Further Biolayer Interferometry Analysis of NanoMIPs to Lentivirus

[0346] Particles in complex buffer An Octet® R8 or RED96e instrument (Sartorius) with Octet® Amine Reactive 2nd Generation (AR2G) Biosensor tips (Sartorius) was employed in this study.

[0347] NanoMIP immobilisation

[0348] NanoMIPs were immobilised onto AR2G biosensor tips via 1 -ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC)ZN-hydroxysuccinimide (NHS) chemistry. To test the ability of nanoMIPs to recognise and bind intact lentiviral particles in a complex buffer representative of an elution buffer for lentivirus purification, two sets of 5 activated AR2G biosensors were functionalised with nanoMIPs (Example 1 - LV-F1- 012) using the optimised conditions from Example 8 (loading buffer of pH 3.5 and 281 pg / mL nanoMIP).

[0349] For the purposes of a control and to allow for reference subtraction, two further sets of 5 activated AR2G biosensors without nanoMIP served as reference biosensors.

[0350] The complex elution buffer comprised 80 mM Tris (Fisher Scientific A11379.30), 4 mM MgC (Fisher Scientific 447155000), 2400 mM NaCI (Fisher Scientific 447300010), 2% sucrose (Sigma Aldrich S0389-500G), 2% mannitol (Fisher Scientific A14030.30) and was adjusted to pH 7.2. The buffer for comparison was 1x PBS pH7.4 prepared by diluting 10x PBS (Fisher Scientific 10649743) with deionised water.

[0351] One set of nanoMIP-functionalised biosensors and one set of reference biosensors was exposed to lentivirus particles (in-house prepared) at a range of titres (particles per mL) in the complex buffer solution, and one set of nano-MIP-functionalised biosensors and one set of reference biosensors was exposed to lentivirus particles (in-house prepared) at a range of titres (particles per mL) in 1x PBS. In both cases, a concentration dependent binding response was observed after reference subtraction (Figure 14).

[0352] The differences in responses between the two buffer conditions were to be expected - 2400 mM NaCI is a very high salt concentration compared to normal operation conditions such as PBS which typically has circa. 140 mM NaCI. However, the responses to the lentivirus in the complex buffer indicate that the nanoMIPs could be used to quantify lentivirus at relevant concentrations immediately post-purification, once a calibration curve had been created in the complex elution buffer used.

[0353] Example 10: Biolayer Interferometry Analysis of anti-VSV-G antibody to Lentivirus Particles For the purpose of comparison, binding of an anti-VSV-G antibody (Ab01402-2.0 Anti- VSV-G [1 E9F9] (Absolute Antibody) to lenti viral particles was assessed.

[0354] In more detail, 4 biosensors were functionalised with anti-VSV-G antibody (Ab01402- 2.0 Anti-VSV-G [1 E9F9] prior to exposure to solutions of NucLight LV (Sartorius, Incuctye Green, EF1 a bleomycin, 4477, #50.215260) at titres of approximately 1.16 x 107TU / mL.

[0355] Although the antibody showed successful immobilisation on to the biosensors (immobilisation response of ~2.5nm, Figure 15), no binding response was observed between the lentiviral particles and the anti-VSV-G antibody suggesting that anti-VSV- G antibodies (or at least this clone) are not suitable for lentiviral detection, or quantification, within BLI.

[0356] Example 11: Synthesis of further linear non-imprinted polymers and chromatographic measurement of their binding to VSV-G

[0357] Preparation of linear polymers for purification experiments

[0358] Twelve polymer compositions were prepared in 100 ml Duran bottles, with cysteamine used as a chain-transfer agent to introduce terminal amine groups for subsequent polymer immobilisation onto solid phase supports.

[0359] Monomer stock preparation

[0360] Common backbone monomer stocks were first prepared in deionised water (in a 250 ml Duran bottle) as follows (Table 18):

[0361] Table 18

[0362] Functional monomer stock preparation

[0363] Functional monomer stocks (A-C) were also prepared in water in 18 ml vials as follows (Table 19):

[0364] Table 19 Initiator stock solution preparation

[0365] Initiator stock solution was also prepared in water as follows (Table 20):

[0366] Table 20

[0367] NB: The ammonium persulfate (APS) was first dissolved in 15 ml deionised water, followed by addition of 360 ul of TEMED and this solution was prepared immediately before addition to the polymerisation mixtures.

[0368] Cysteamine stock preparation

[0369] Cysteamine stock solution was prepared as follows (Table 21 ):

[0370] Table 21

[0371] NB: This solution was prepared immediately before addition to the polymerisation mixtures. Compositions of 12 polymer compositions (Table 22)

[0372] Each individual polymer composition was then prepared in 100 ml Duran bottles by dispensing the following volumes of each stock, volumes are in ml: Table 22

[0373] After mixing by hand (swirling the bottles for 5 seconds), 100 pl of cysteamine stock and 1 ml of initiator stock was added to each bottle, then the headspace flushed with nitrogen, swirled by hand again (for 5 seconds) and the bottles closed with a cap. The bottles were then placed in an oven set to 65°C and polymerised for two hours. After polymerisation, polymer solutions were transferred to 28 ml vials and stored in the fridge until required for use. The composition of each polymerisation mixture was therefore as follows (Table 23):

[0374] Table 23

[0375] After an initial screen (not documented here) the polymers that were employed in a series of purification experiments (see below) were: A3, A6, D11 , E3, E7, F3, F7. Chromatography screening of selected linear non-imprinted polymers

[0376] An AKTA Pure 25 chromatography system (Cytiva Life Sciences) was employed in this study.

[0377] Linear polymers A3, A6, D11 , E3, E7, F3 and F7 were each immobilised onto a 13mm disk of electrospun cellulose membrane (Astrea Bioseparations) housed within a

[0378] Swinny Filter Holder (Sigma, XX3001200) by manual injection of immobilisation solutions. Control membranes blocked with ethanolamine (EA) were prepared in the same way. All membranes were then tested for affinity purification of VSV-G-650 in HEK293T spent media. The VSV-G-650 was prepared at a 2 nM concentration in 1 ,5mL of spent media per run. Table 24 summarises the purification method used for the screening. Table 24 All membrane effluents, starting from the sample application, were collected in 96 well plates for measurement at 650nm on a plate reader (CLARIOstar®) for generation of fluorescence chromatograms.

[0379] Figure 16 shows the overlay of the 650nm fluorescence chromatograms. Linear polymers D11 , A3, E3 and E7 eluted the most VSV-G.

[0380] Of note, the fluorescence chromatogram shown in figure 16 displays peak fronting for polymers A3, D11 and A6 due to leakage observed in the load phase of these three runs.

[0381] References

[0382] 1. Jadlowsky J.K. et al. Long-term stability of clinical-grade lentiviral vectors for cell therapy. Molecular Therapy: Methods & Clinical Development Vol. 32 March 2024. 2. Verhoeyen E., Cosset F.L. Surface-engineering of lentiviral vectors. J. Gene Med. 2004;6 doi: 10.1002 / jgm.494

[0383] 3. Ozog S., Chen C.X., Simpson E., Garijo 0., Timberlake N.D., Minder P., Verhoeyen E., Torbett B.E. CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells. Mol. Then Methods Clin. Dev. 2019;13:27-39. doi: 10.1016 / j.omtm.2018.11.006.

[0384] 4. Vesicular Stomatitis Virus Glycoprotein Is a Determinant of Pathogenesis in Swine, a Natural Host

[0385] 5. Martinez I. et al. Vesicular Stomatitis Virus Glycoprotein Is a Determinant of Pathogenesis in Swine, a Natural Host. J Virol. 2003 Jul; 77(14): 8039-8047.

[0386] 6. Munis A.M. et al. A tool with many applications: vesicular stomatitis virus in research and medicine. Expert Opinion on Biological Therapy 2020, Vol. 20, No. 10, 1187-1201.

[0387] 7. Moreira A S. et al. Advances in Lentivirus Purification. Biotechnol. J. 2021 , 16, 2000019.

[0388] 8. Milone M.C. et al. Clinical use of lentiviral vectors. Leukemia (2018) 32:1529- 1541.

[0389] 9. Gutierrez-Guerrero A et al. Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene Therapy. Viruses 2020, 12, 1016; doi:10.3390 / v12091016.

[0390] 10. Duverge A, Negroni M. Pseudotyping Lentiviral Vectors: When the Clothes Make the Virus. Viruses. 2020 Nov 16; 12(11 ): 1311. doi: 10.3390 / v12111311 . PMID: 33207797; PMCID: PMC7697029.

[0391] Sequence Summary

Claims

Claims1 . A synthetic polymer which binds vesicular stomatitis virus G (VSV-G) protein, wherein the synthetic polymer does not contain any proteinogenic amino acids.

2. The synthetic polymer of claim 1 , wherein the polymer is a molecularly imprinted polymer comprising at least one recognition site which binds to vesicular stomatitis virus G (VSV-G) protein.

3. The synthetic polymer of claim 2, wherein at least one recognition site is complementary to a template molecule comprising an amino acid sequence corresponding to a subsequence of a VSV-G polypeptide.

4. The synthetic polymer of claim 3, wherein the amino acid sequence is selected from the group consisting of:(i) DYKVKGLCDSNL,(ii) ADKDLFAAARFPE,(iii) PEGSSISAP,(iv) EDGELSSLGKEGT,(v) PKSHKAI,(vi) ADKDLFAAAR, and(vii) CPEGSSISAP.

5. The synthetic polymer of claim 4, wherein the amino acid sequence is DYKVKGLCDSNL.

6. The synthetic polymer of claim 4, wherein the amino acid sequence is ADKDLFAAARFPE.

7. The synthetic polymer of claim 4, wherein the amino acid sequence is PEGSSISAP.

8. The synthetic polymer of claim 4, wherein the amino acid sequence is EDGELSSLGKEGT.

9. The synthetic polymer of claim 4, wherein the amino acid sequence is PKSHKAI.

10. The synthetic polymer of claim 4, wherein the amino acid sequence is ADKDLFAAAR.

11. The synthetic polymer of claim 4, wherein the amino acid sequence is CPEGSSISAP.

12. The synthetic polymer of claim 1 , wherein the polymer is made by a process comprising the steps of:(a) providing a polymerisable composition in contact with a template molecule; and(b) effecting polymerisation of the polymerisable composition in contact with the template molecule to produce a synthetic polymer wherein the synthetic polymer is a molecularly imprinted polymer.

13. The synthetic polymer of claim 12, wherein the process comprises the additional step of separating the synthetic polymer from the template.

14. The synthetic polymer of any one of claims 12-13, wherein the template molecule comprises an amino acid sequence corresponding to a subsequence of a VSV-G polypeptide.

15. The synthetic polymer of claim 14, wherein the amino acid sequence is selected from the group consisting of:(i) DYKVKGLCDSNL,(ii) ADKDLFAAARFPE,(iii) PEGSSISAP,(iv) EDGELSSLGKEGT,(v) PKSHKAI,(vi) ADKDLFAAAR, and(vii) CPEGSSISAP.

16. The synthetic polymer of any one of claims 14-15 wherein the amino acid sequence is DYKVKGLCDSNL.

17. The synthetic polymer of any one of claims 14-15 wherein the amino acid sequence is CPEGSSISAP.

18. The synthetic polymer of any one of claims 12-17 wherein the template is immobilised on a carrier substance.

19. The synthetic polymer of claim 18, wherein the carrier substance is a glass bead.

20. The synthetic polymer of any one of claims 12-17, wherein the template is in solution.

21. The synthetic polymer of any one of claims 12-20, wherein the polymerisable composition comprises 3-sulfopropyl acrylate (SPA).

22. The synthetic polymer of any one of claims 12-21 , wherein the polymerisable composition comprises 2-carboxyethyl acrylate (CEA) and mono-2- (methacryloyloxy)ethyl maleate (MAM EE).

23. The synthetic polymer of any one of claims 12-22, wherein the polymerisable composition comprises N-fluoresceinylacrylamide, cysteamine, and tert-butyl acrylate.

24. The synthetic polymer of any one of claims 12-23, wherein the polymerisable composition comprises N-isopropylacrylamide (NIPam).

25. The synthetic polymer of any one of claims 12-23, wherein the polymerisable composition comprises acrylamide.

26. The synthetic polymer of any one of claims 12-20, wherein the polymerisable composition comprises 2,2,2-Trifluoroethyl acrylate, acrylonitrile and ethyl methacrylate.

27. The synthetic polymer of claim 26, wherein the polymerisable composition comprises acrylamide, N-Fluoresceinylacrylamide, cysteamine and tert-butyl acrylate.

28. The synthetic polymer of claim 1 , wherein the polymer is made by a process comprising the steps of:(a) providing a polymerisable composition;(b) effecting polymerisation of the polymerisable composition to produce a polymer.

29. The synthetic polymer of claim 28, wherein the polymerisable composition comprises 3-sulfopropyl acrylate (SPA).

30. The synthetic polymer of any one of claim 28-29, wherein the polymerisable composition comprises 2-carboxyethyl acrylate (CEA) and mono-2- (methacryloyloxy)ethyl maleate (MAM EE).

31. The synthetic polymer of any one of claims 28-30, wherein the polymerisable composition comprises N-fluoresceinylacrylamide, cysteamine, and tert-butyl acrylate.

32. The synthetic polymer of any one of claims 28-31 , wherein the polymerisable composition comprises acrylamide.

33. The synthetic polymer of any one of claims 28-31 , wherein the polymerisable composition comprises N-isopropylacrylamide (NIPam).

34. The synthetic polymer of claim 32, wherein the polymerisable composition comprises N-(3-Aminopropyl) methacrylamide hydrochloride (APMA).

35. The synthetic polymer of claim 28, wherein the polymerisable composition comprises 2,2,2-Trifluoroethyl acrylate, acrylonitrile and ethyl methacrylate.

36. The synthetic polymer of claim 35, wherein the polymerisable composition comprises acrylamide, N-Fluoresceinylacrylamide, cysteamine and tert-butyl acrylate.

37. The synthetic polymer of claim 28, wherein the polymerisable composition comprises mono-2-(methacryloyloxy)ethyl maleate (MAM EE).

38. The synthetic polymer of any one of claims 28-30 and 37, wherein the polymerisable composition comprises acrylamide and one of (i) tert-butyl acrylate or (ii) tert-butyl acrylamide.

39. The synthetic polymer of any one of claims 28-30 or 37-38, wherein the polymerisable composition comprises cysteamine.

40. The synthetic polymer of any one of claims 3-27, wherein the template is no more than 50 amino acids in length.

41. The synthetic polymer of any one of claims 1 -40, wherein the polymer comprises a cross-linking monomer.

42. The synthetic polymer of any one of claims 1 -40, wherein the polymer does not comprise a cross-linking monomer.

43. The synthetic polymer of any preceding claim, wherein the polymer is conjugated to a sensor element.

44. The synthetic polymer of any one of claims 1 -42, wherein the polymer is conjugated to a solid support.

45. The synthetic polymer of claim 44, wherein the solid support is a resin.

46. A method of preparing a synthetic polymer which binds VSV-G protein comprising the steps of:(a) providing a polymerisable composition wherein the polymerisable composition does not contain any proteinogenic amino acids, and(b) effecting polymerisation of the polymerisable composition to produce the synthetic polymer.

47. The method of claim 46, comprising the preliminary step of providing a template molecule comprising an amino acid sequence corresponding to a subsequence of a VSV-G polypeptide.

48. The method of claim 47, wherein the amino acid sequence is selected from the group consisting of:(i) DYKVKGLCDSNL,(ii) ADKDLFAAARFPE,(iii) PEGSSISAP,(iv) EDGELSSLGKEGT,(v) PKSHKAI,(vi) ADKDLFAAAR, and(vii) CPEGSSISAP.

49. The method of claim 48, wherein the amino acid sequence isDYKVKGLCDSNL.

50. The method of claim 48, wherein the amino acid sequence isADKDLFAAARFPE.

51. The method of claim 48, wherein the amino acid sequence is PEGSSISAP.

52. The method of claim 48, wherein the amino acid sequence EDGELSSLGKEGT.

53. The method of claim 48, wherein the amino acid sequence is PKSHKAI.

54. The method of claim 48, wherein the amino acid sequence ADKDLFAAAR.

55. The method of claim 48, wherein the amino acid sequence CPEGSSISAP.

56. The method of any one of claims 47-55, wherein the method comprises the additional step of separating the synthetic polymer from the template.

57. The method of any one of claims 47-56, wherein the template is immobilised on a carrier substance.

58. The method of claim 57, wherein the carrier substance is a glass bead.

59. The method of any one of claims 47-56, wherein the template is in solution.

60. The method of any one of claims 47-59, wherein the template is no more than 50 amino acids in length.61 . The method of any one of claims 46-60, wherein the polymerisable composition comprises the monomers:(i) N,N'-Methylenebis(acrylamide),(ii) Acrylamide,(iii) tert-Butyl acrylate, and(iv) N-(3-Aminopropyl) methacrylamide hydrochloride.

62. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 2-carboxyethyl acrylate (CEA).

63. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 3-sulfopropyl acrylate (SPA).

64. The method of any one of claims 46-61 , wherein the polymerisable composition comprises N-(3-aminopropyl) methacrylamide hydrochloride (APMA).

65. The method of any one of claims 46-61 , wherein the polymerisable composition comprises mono-2-(methacryloyloxy)ethyl maleate (MAMEE).

66. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 2-acrylamido-2-methylpropane sulfonic acid (AMPSA).

67. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 2-(methacryloxy)ethyl phosphate (MAOEP) and 3-(perfluoro-5- methylhexyl)-2-hydroxypropyl methacrylate (PMHM).

68. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 3-sulfopropyl methacrylate (SPMA) and 3-vinylbenzoic acid (2VB).

69. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 3-sulfopropyl methacrylate (SPMA).

70. The method of any one of claims 46-61 , wherein the polymerisable composition comprises vinylphosphonic acid (VPA).

71. The method of any one of claims 46-61 , wherein the polymerisable composition comprises methacrylamide (MAAm).

72. The method of any one of claims 46-61 , wherein the polymerisable composition comprises ethyl methacrylate (EMA).

73. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 2-(diethylamino)ethyl acrylate (DEAEM) and vinylphosphonic acid (VPA).

74. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 2,2,2-trifluoroethyl acrylate (TFEA).

75. The method of any one of claims 46-61 , wherein the polymerisable composition comprises N-(3-aminopropyl) methacrylamide hydrochloride (APMA) and 2- carboxyethyl acrylate (CEA).

76. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 2-(methacryloxy)ethyl phosphate (MAOEP) and methacrylic Acid (MAAc).

77. The method of any one of claims 46-60, wherein the polymerisable composition comprises the monomers:(i) N-fluoresceinylacrylamide(ii) 3-Sulfopropyl acrylate(iii) Mono-2-(methacryloyloxy)ethyl maleate(iv) 2-Carboxyethyl acrylate(v) Cysteamine, and(vi) Tert-butyl acrylate.

78. The method of any one of claims 46-60, wherein the polymerisable composition comprises the monomers 3-Sulfopropyl acrylate, mono-2- (methacryloyloxy)ethyl maleate and 2-Carboxyethyl acrylate.

79. The method of any one of claims 77-78, wherein the polymerisable composition additionally comprises N-isopropylacrylamide (NIPAM).

80. The method of any one of claims 77-78, wherein the polymerisable composition additionally comprises acrylamide.

81. The method of claim 80, wherein the polymerisable composition additionally comprises N-(3-aminopropyl) methacrylamide hydrochloride (APMA).

82. The method of any one of claims 46-60, wherein the polymerisable composition comprises 2,2,2-Trifluoroethyl acrylate, acrylonitrile and ethyl methacrylate.

83. The method of claim 82, wherein the polymerisable composition comprises acrylamide, N-Fluoresceinylacrylamide, cysteamine and tert-butyl acrylate.

84. The method of any one of claims 65 or 78, wherein the polymerisable composition comprises acrylamide and one of (i) tert-butyl acrylate or (ii) tertbutyl acrylamide.

85. The method of any one of claims 65, 78 or 84, wherein the polymerisable composition comprises cysteamine.

86. Use of the synthetic polymer of any one of claims 1 -45, 90-116 or 118 in the detection of VSV-G.

87. The use of claim 86, wherein the VSV-G is present on the surface of a lentiviral vector.

88. Use of the synthetic polymer of any one of claims 1 -45, 90-116 or 118 in a method of purification.

89. The use of claim 88, wherein the method of purification comprises the step of purifying a lentiviral vector pseudotyped with VSV-G.

90. The synthetic polymer of any of claims 1 -20, 28 or 40-45, wherein the synthetic polymer comprises the monomers:(i) N,N'-Methylenebis(acrylamide),(ii) Acrylamide,(iii) tert-Butyl acrylate, and(iv) N-(3-Aminopropyl) methacrylamide hydrochloride.

91. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises 2-carboxyethyl acrylate (CEA).

92. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises 3-sulfopropyl acrylate (SPA).

93. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises N-(3-aminopropyl) methacrylamide hydrochloride (APMA).

94. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises mono-2-(methacryloyloxy)ethyl maleate (MAMEE).

95. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises 2-acrylamido-2-methylpropane sulfonic acid (AM PSA).

96. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises 2-(methacryloxy)ethyl phosphate (MAOEP) and 3-(perfluoro-5-methylhexyl)-2-hydroxypropyl methacrylate (PMHM).

97. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises 3-sulfopropyl methacrylate (SPMA) and 3- vinylbenzoic acid (3VB).

98. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises 3-sulfopropyl methacrylate (SPMA).

99. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90 claim, wherein the synthetic polymer comprises vinylphosphonic acid (VPA).

100. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises methacrylamide (MAAm).

101. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises ethyl methacrylate (EMA).

102. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises 2-(diethylamino)ethyl acrylate (DEAEM) and vinylphosphonic acid (VPA).

103. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises 2,2,2-trifluoroethyl acrylate (TFEA).

104. The synthetic polymer of any one of claims 1-20, 28, 40-45 or 90, wherein the synthetic polymer comprises N-(3-aminopropyl) methacrylamide hydrochloride (APMA) and 2-carboxyethyl acrylate (CEA).

105. The synthetic polymer of any one of claims 1-20, 28, 40-45 or 90, wherein the synthetic polymer comprises 2-(methacryloxy)ethyl phosphate (MAOEP) and methacrylic acid (MAAc).

106. The synthetic polymer of any of claims 1-20, 28 or 40-45, wherein the synthetic polymer comprises the monomers:(i) N-fluoresceinylacrylamide(ii) 3-Sulfopropyl acrylate(iii) Mono-2-(methacryloyloxy)ethyl maleate(iv) 2-Carboxyethyl acrylate(v) Cysteamine, and(vi) Tert-butyl acrylate.

107. The synthetic polymer of any one of claims 1-20, 28 or 40-45, wherein the synthetic polymer comprises the monomers 3-Sulfopropyl acrylate, mono- 2-(methacryloyloxy)ethyl maleate and 2-Carboxyethyl acrylate.

108. The synthetic polymer of any one of claims 106-107, wherein the synthetic polymer additionally comprises N-isopropylacrylamide (NIPAM).

109. The synthetic polymer of any one of claims 106-107, wherein the synthetic polymer additionally comprises acrylamide.

110. The synthetic polymer of claim 109, wherein the synthetic polymer additionally comprises N-(3-aminopropyl) methacrylamide hydrochloride (APMA).

111. The synthetic polymer of any one of claims 1-20, 28 or 40-45, wherein the synthetic polymer comprises 2,2,2-Trifluoroethyl acrylate, acrylonitrile and ethyl methacrylate.

112. The synthetic polymer of claim 111 , wherein the synthetic polymer comprises acrylamide, N-Fluoresceinylacrylamide, cysteamine and tert-butyl acrylate.

113. The synthetic polymer of any one of claims 94 or 107, wherein the polymerisable composition comprises acrylamide and one of (i) tert-butyl acrylate or (ii) tert-butyl acrylamide.

114. The synthetic polymer of any one of claims 94, 107 or 113, wherein the synthetic polymer comprises cysteamine.

115. The synthetic polymer of claim 1 , wherein the polymer comprises the monomers of, or is, LV-F1 -001 , LV-F1 -002, LV-F1 -004, LV-F1-007, LV-F1-010, LV-F1 -011 , LV-F1 -012, LV-F2-015, LV-F2-016, LV-F3-018, LV-F4-001 , LV-F6- 002, LV-F6-003, LV-F4-004, LV-F8-008, LV-F8-003, linear non-imprinted polymer 2, linear non-imprinted polymer 6, linear non-imprinted polymer 4, or linear molecularly imprinted polymer 3, all disclosed in Example 1 or linear nonimprinted polymer A3 disclosed in Example 11.

116. The synthetic polymer of claim 115, wherein the polymer comprises the monomers of, or is, LV-F1 -011 , LV-F1-012, LV-F2-016 or linear non-imprinted polymer A3.

117. The method of any one of claims 46-61 , wherein the polymerisable composition comprises 2-carboxyethyl acrylate (CEA) and mono-2- (methacryloyloxy)ethyl maleate (MAMEE).

118. The synthetic polymer of any one of claims 1 -20, 28, 40-45 or 90, wherein the synthetic polymer comprises 2-carboxyethyl acrylate (CEA) and mono-2-(methacryloyloxy)ethyl maleate (MAMEE).

119. Use of the synthetic polymer of any one of claims 1 -45 or 90-116 or 118 in a method of quantification.

120. The use of claim 119, wherein the method of quantification comprises a step of quantifying a lentivi ral vector pseudotyped with VSV-G.

Images