Methods for purifying, detecting, and quantifying residual PEI-based transfection reagents

Abstract

The present invention relates to a method for purifying, detecting and quantifying residual PEI-based transfection reagents.The present invention is directed to a method for performing an acidic hydrolysis of a liquid mixture comprising a biological matrix and a polyethylenimine (PEI)-based transfection reagent of general formula (I) as described herein, wherein the biological matrix comprises a recombinant virus or virus-like particle produced using the PEI-based transfection reagent, and wherein said acidic hydrolysis does not degrade the PEI-based transfection reagent.The present invention also relates to a method for purifying, detecting and / or quantifying the PEI-based transfection reagent of general formula (I) as described herein.

Description

[Technical field]

[0001] The present invention relates to a method for purifying, detecting and quantifying residual PEI-based transfection reagents.The present invention is directed to a method for performing an acidic hydrolysis of a liquid mixture comprising a biological matrix and a polyethylenimine (PEI)-based transfection reagent of general formula (I) as described herein, wherein the biological matrix comprises a recombinant virus or virus-like particle produced using the PEI-based transfection reagent, and wherein said acidic hydrolysis does not degrade the PEI-based transfection reagent.The present invention also relates to a method for purifying, detecting and / or quantifying the PEI-based transfection reagent of general formula (I) as described herein. [Background technology]

[0002] Gene therapy aims to provide exogenous genes into cells to correct the expression of defective genes in monogenic diseases or to achieve new beneficial gene expression in cancer, viruses, and immune infections. To achieve this, the exogenous genetic material must be efficiently introduced into the cells to be corrected where gene expression will cause the desired therapeutic effect.

[0003] Exogenous genetic material consists of a gene expression system containing a gene of interest together with sequences that control its expression (promoter, enhancer, exons, etc.) and is present in a nucleic acid construct (plasmid, linear or ligated double-stranded DNA, messenger RNA, etc.).

[0004] Since eukaryotic cells are not permeable to nucleic acid, delivery systems or vectors that allow the introduction of nucleic acid have been developed. Some systems based on viral vectors or non-viral vectors are the dominant delivery systems. Therefore, among them, viral vectors account for the majority of the delivery systems used in clinical applications.

[0005] Viral vectors have taken advantage of the ability of viruses to target and in particular infect cells with great efficiency associated with millions of years of biological evolution. Taking advantage of this efficiency, viral vectors, particularly recombinant viral vectors, have been constructed to utilize viruses as transporters of genomic sequences while suppressing pathogenicity while maintaining cell targeting or infecting capabilities. Among recombinant viral vectors, AAV, retroviruses, lentiviruses, and adenoviruses are the most used in gene cell therapy. This list is not exhaustive (for a review, see "Viral Vectors in Gene Therapy", Kenneth Lundstrom, Diseases 2018, 6, 42) and will change depending on progress and knowledge about other viruses and their potential use as non-pathogenic genome expression vectors.

[0006] Viral vectors, particularly recombinant viral vectors, are produced in cell lines to allow for their replication and propagation while avoiding the production of wild-type virus (for a review, see Forsberg et al., Key Considerations in Gene Therapy Manufacturing for Commercialization, 2018).

[0007] Several cell lines have been used to produce recombinant viral vectors, such as HEK293 cells, HeLa cells, or insect cells. This process consists of introducing into the cells the genetic material of the recombinant virus required for the replication of the recombinant genome, the production of viral capsids and the encapsidation of the recombinant viral genome into the capsids, followed by the recovery of the produced viral vector.

[0008] Several methods are available for introducing the various components required for the production of recombinant viruses, including infection with baculovirus, transfection by chemical methods (calcium phosphate or transfection reagents or formulations), physical methods such as electroporation, or even the generation of stable cell clones (integration of the various viral genome parts required for the production of recombinant viruses into the host genome of the producer cell).

[0009] Transfection by chemical methods is the method mainly used to produce viruses, especially AAV or lentivirus, used in gene cell therapy. The use of calcium phosphate precipitation is unsuitable for large-scale production (mass production) due to the difficulty in controlling and regenerating nucleic acid precipitation, and has been replaced by the use of transfection reagents such as cationic polymers or cationic lipids. Among cationic polymers, PEI polymers are widely used for the production of recombinant viral vectors from HEK293 cells and derivatives.

[0010] By transfection, it is possible to introduce various nucleic acids into cells required for the production of recombinant viruses, usually in the form of plasmids as gene expression vectors. Viral genes are introduced by transfection and can be transiently expressed in virus-producing cells for several days (1-7 days).

[0011] Several methods of detection of polyethyleneimine have been developed using spectrophotometric (Francesca Ungaro et al., J.Pharm.Biomed.Anal.31(2003)143), colorimetric (Qu,Fei Li et al., Langmuir 2013,29,1199-1205) or fluorimetric (Yunyi Zhang et al., Analytical and Bioanalytical Chemistry 2017,409,4771) methods. These methods are based on specific interactions between PEI and metals (copper, silver) (Yu Ling et al., J.Phys.Chem.C 2015,119,27173-27177) and are applicable to solutions that do not contain other nitrogen atoms capable of chelating metals. These tests, which require extraction of PEI from the sample matrix, are not compatible with compositions containing viral vectors, since some residual transfection reagents may be trapped within the virus.

[0012] The cells producing the recombinant viruses are essentially HEK293 cells, their derivatives, subclones or genetically modified cells, or other cells such as HeLa, CHO, etc. These cells can be cultivated in a variety of culture systems, either adherent or non-adherent (in suspension), on plastic or glass supports, or in bioreactor systems.

[0013] The virus-producing cells are cultured in a medium containing or not containing serum, or containing proteins, peptides, vitamins, hormones, amino acids, lipids, salts, or containing various synthetic compounds that are free of animal-derived components, or containing synthetic components such as polymers or substances that allow growth and good viability and avoid aggregation. Culture media are well known, and examples of compositions are described, for example, in the literature of Srivastava A et al. (J Pharm Sci. July 2021; 110(7): 2609-2624).

[0014] The initial success of gene cell therapy has made it possible to extend the use of viral vectors from rare diseases to more common diseases. Regulatory authorities are adapting their manufacturing guidelines to this new endeavor. The methods and processes of manufacturing viral vectors must be governed by regulations to guarantee patient safety. Regulatory authorities have introduced "guidelines" for the manufacture of these new medicines regarding several aspects: safety, identity, quality, purity, impurities and possible substances (WHO good manufacturing practices for biological products Replacement of Annex 1 of WHO Technical Report Series, No. 822). Among these, the manufacturing process of active substances containing or consisting of viral vectors must be controlled in terms of impurities and it must be possible to identify and quantify them if they may represent a safety risk.

[0015] The production of viral vectors by transient transfection in cells such as HEK293 cells or their derivatives consists of two phases. The introduction of nucleic acids containing the virus and optionally additional genes (in the form of a plasmid, 1-4 plasmids or other forms of gene expression vectors) into HEK293 cells cultured under adherent or non-adherent conditions in synthetic or non-synthetic medium containing culture supplements allows the production of recombinant viruses, which may or may not be excreted depending on the type of recombinant virus produced. This recombinant virus production step is called the upstream step. This is followed by harvesting the virus and then purifying it until it may be suitable for use as a drug active substance [drug substance (DS) or drug product (DP)] in the drug composition formulation process, which are defined as downstream steps (J. Fraser Wright, Biotechnology Journal, 2021, 16, 2000022; Martinez-Molina et al., Pharmaceutics 2020, 12(11), 1051; and Christopher Perry and Andrea CMERayat, Viruses 2021, 13, 268).At this final stage of manufacture, additives such as acceptable pharmaceutical excipients or ingredients (US Food and Drug Administration's (FDA's) 21 CFR 210.3(b)(8) guidance, an excipient or inactive ingredient is any component of a drug product other than the active ingredient, and EMEA Guideline on excipients in the dossier for application for marketing authorisation of medicinal product, Doc. Ref. EMEA / CHMP / QWP / 396951 / 2006) can be added to promote good preservation of the virus or to prevent degradation or aggregation of the virus.

[0016] In this viral vector manufacturing process, the transfection reagent is a raw material for the upstream process and may become an impurity in the final product (DS or DP) if not removed during downstream processing. Therefore, the transfection reagent must be identified and quantified throughout the upstream and downstream processes or in the final product (DS or DP). Currently, there are no guidelines regarding the acceptable dose of residual transfection reagent in the viral formulation. However, it seems that the measurement of the residual rate is mandated to provide information to the manufacturing files of the regulatory authorities and to have traceability of residual impurities in viral formulations for therapeutic purposes that may represent a risk of toxicity.

[0017] The growing demand for viral vectors is required to meet the anticipated needs at the commercial level and to meet the needs created by the rapid progress through the different phases of clinical development (Van Der Loo-Human Molecular Genetics_2015). As a result, large-scale production, the development of new producer cell lines, new defined media, or new transfection reagents are being investigated to increase the productivity of viral vectors at the upstream level.

[0018] Recently, new transfection reagents based on heterocyclic compounds grafted to cationic polymers have shown improved production yields of viral vectors, including AAV and lentivirus, compared to the production achieved with PEI, which is considered the reference standard for transfection reagents (WO2021 / 023796; WO2021 / 023798). However, assays for the residual content of these new transfection reagents in the generated viral vectors or during their manufacturing process (sometimes called "residual assays" or "residual tests") are not yet available. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] WO2021 / 023796 [Patent Document 2] WO2021 / 023798 [Non-patent literature]

[0020] [Non-Patent Document 1] "Viral Vectors in Gene Therapy", Kenneth Lundstrom, Diseases2018, 6, 42 [Non-Patent Document 2] Forsberg et al., Key Considerations in Gene Therapy Manufacturing for Commercialization, 2018 [Non-Patent Document 3] Francesca Ungaro et al., J. Pharm. Biomed. Anal. 31 (2003) 143 [Non-Patent Document 4] Qu, Fei Li et al., Langmuir 2013, 29, 1199 - 1205 [Non-Patent Document 5] Yunyi Zhang et al., Analytical and Bioanalytical Chemistry 2017, 409, 4771 [Non-Patent Document 6] Yu Ling et al., J. Phys. Chem. C 2015, 119, 27173 - 27177 [Non-Patent Document 7] Srivastava A et al. (J Pharm Sci. July 2021; 110(7): 2609 - 2624) [Non-Patent Document 8] WHO good manufacturing practices for biological products Replacement of Annex 1 of WHO Technical Report Series, No. 822 [Non-Patent Document 9] J. Fraser Wright, Biotechnology Journal, 2021, 16, 2000022 [Non-Patent Document 10] Martinez-Molina et al., Pharmaceutics 2020, 12(11), 1051 [Non-Patent Document 11] Christopher Perry and Andrea C.M. Rayat, Viruses 2021, 13, 268 [Non-Patent Document 12] US Food and Drug Administration's (FDA's) 21 CFR 210.3(b)(8) guidance, an excipient or inactive ingredient is any component of a drug product other than the active ingredient [Non-Patent Document 13] EMEA Guideline on excipients in the dossier for application for marketing authorisation of medicinal product, Doc. Ref. EMEA / CHMP / QWP / 396951 / 2006 [Non-Patent Document 14] Van Der Loo-Human Molecular Genetics_2015 [Non-Patent Document 15] Thomas Lorson et al., Biomaterials 178(2018)204e280 [Non-Patent Document 16] Ts.Ivanova, E. et al., PHARMACIA, 2016, Vol. 63, No. 3 [Non-Patent Document 17] Nico Adams et al., Reviews 59 (2007) pp. 1504-1520 [Non-Patent Document 18] Ryuichi Tanaka et al., Macromolecules 1983, 16, 6, pp. 849-853 [Non-Patent Document 19] Emi Haladjova et al., Polymers 2020, 12, 2609 [Non-Patent Document 20] Alexander B. Cook et al., Polym. Chem., 2019, 10, pp. 1202-1212 [Non-Patent Document 21] Emi Haladjova et al., Macromol.Biosci.2018, 1700349 [Non-Patent Document 22] Haladjova E et al., J Appl Polym Sci.2020;e49400 [Non-Patent Document 23] R. Shah et al., J. Mater. Sci. Mater. Med. 2015, 26, 157 [Non-Patent Document 24] Ji Hoon Jeong et al., Journal of Controlled Release 73 (2001) pp. 391-399 [Non-Patent Document 25] R. Tanaka et al., Macromolecules 16 (1983) pp. 849-853 [Non-Patent Document 26] Sommer JM et al., Molecular therapy: the journal of the American Society of Gene Therapy 2003, 7(1), pp. 122-128 [Non-Patent Document 27] ;Gargi Maheshwari et al., Journal of Virological Methods 2004, 118(2), pp. 141-146 [Non-Patent Document 28] Fabian Krieselet et al., Journal of Virological Methods 2020,276,113768 [Non-Patent Document 29] Araujo, Journal of Chromatography B, 2009, 877(23), pp. 2224-2234. Summary of the Invention [Problem to be solved by the invention]

[0021] The object of the present invention is therefore to develop a specific method that allows to determine the amount of a substance in a complex liquid mixture. This substance is present in very small amounts in a liquid mixture that further comprises a biological matrix. The biological matrix may be a cell culture medium, a buffer or any solution that contains recombinant viruses and / or virus-like particles and is used both in the upstream (UP) process and in the downstream process that finally produces the final drug substance. This substance is obviously a transfection reagent, used to produce viral vectors (AAV, LV, adenovirus, oncolytic virus, baculovirus) in mammalian cells. Following the production of the viral vector (upstream process), several steps are necessary to remove all the impurities produced during the UP and to finally obtain a pure active drug ingredient that can be administered to the patient.

[0022] To ensure patient safety, several residual tests must be performed. One of them concerns transfection reagents. Transfection reagents usually contain cationic polymers or lipids. Due to their cationic properties, these reagents have the ability to interact not only with nucleic acids, but also with many other chemicals (polymers, salts, small molecules, etc.) used throughout the viral vector manufacturing process.

[0023] To ensure that the levels of these transfection agents are kept within safe limits, regulatory authorities in this field require the development of analytical methods that can detect and quantify such residues.

[0024] The inventors had to consider several criteria in order to develop an accurate residue test. Depending on the biosafety level of the virus, a preliminary virus inactivation step may be necessary to protect the manipulator. The inactivation step may be thermal (heat), chemical (detergents, acids, etc.) or physical (UV) and may generate by-products that may react with the transfection reagent. The inactivation choice must be ineffective against the transfection reagent, i.e., preserve the transfection reagent in the test mixture that is finally analyzed. Furthermore, since transfection reagents may attach to or be encapsulated in the virus, assays for residual transfection reagent content require complete degradation of the virus to allow access to this potentially encapsulated reagent. At each step of the viral vector manufacturing process, the transfection reagent is present at very low levels compared to other components (virus, cell culture medium, detergents, salts, etc.). The challenge now is to detect this molecule in a complex liquid mixture. Specific purification steps are required to detect and quantify residual transfection reagent. Considering that this reagent is a macromolecule such as DNA, viral proteins, detergents used during production or in the final formulation, purification by size exclusion is not precise enough to be able to meet LOD (limit of detection) and LOQ (limit of quantification). The sensitivity of the assay should have a wide detection range from 1 ppm (LOD) to 1000 ppm, which can correspond to the amount of transfection reagent in the downstream concentration phase.

[0025] Since the manufacturing process of each viral vector is unique, it is difficult to predict the impact of the composition of the viral vector on the analytical test. In order to simplify and develop a residue test that is generalized to viral vector manufacturing, the inventors decided to focus on developing an orthogonal degradation that does not affect the transfection reagent of general formula (I) described herein (Figure 1), whose transfection efficiency is well known (Thomas Lorson et al., Biomaterials 178 (2018) 204e280; Ts. Ivanova, E et al., PHARMACIA, 2016 Vol. 63, 3; Nico Adams et al., Reviews 59 (2007) pp. 1504-1520).

[0026] PEI derivatives are a large family of polymers used for the delivery of nucleic acids (DNA, siRNA, mRNA, miRNA, etc.). In particular, linear PEI (or lPEI) has shown strong transfection efficiency in both in vitro and in vivo applications. The synthesis of linear PEI is based on the cationic ring-opening polymerization (or CROP) of 2-alkyl-2-oxazolines to generate polyalkyl-(2-oxazolines). The polyalkyl-(2-oxazolines) can then be fully or partially hydrolyzed to generate lPEI or a combination of lPEI and polyalkyl-(2-oxazolines) (Ryuichi Tanaka et al., Macromolecules 1983, 16, 6, 849-853) (Figure 2).

[0027] Typically, hydrolysis, consisting of carbon-nitrogen bond cleavage of amide functional groups, is carried out under strongly acidic conditions (Emi Haladjova et al., Polymers 2020, 12, 2609; Alexander B. Cook et al., Polym. Chem., 2019, 10, pp. 1202-1212; Emi Haladjova et al., Macromol. Biosci. 2018, 1700349). Rangelov et al. carried out partial degradation of poly(2-methyl-2-oxazoline) by acidic hydrolysis at 100 °C in an aqueous solution containing 17.5% HCl (Haladjova E et al., J Appl Polym Sci. 2020; e49400; R. Shah et al., J. Mater. Sci. Mater. Med. 2015, 26, 157). Heating time and degree of hydrolysis were directly correlated. At 100 °C, between 15 and 180 min, the degree of hydrolysis (DH) increased from 2–6% to 60% (Table 1).

[0028] [Table 1]

[0029] Park et al. carried out partial hydrolysis of poly(2-ethyl-2-oxazoline) with various concentrations of HCl at 100°C for 6 hours (Ji Hoon Jeong et al., Journal of Controlled Release 73 (2001) pp. 391-399; R. Tanaka et al., Macromolecules 16 (1983) pp. 849-853). In this study, various concentrations of HCl, 5.0, 7.5, and 10.0% (v / v), were used for hydrolysis. As the concentration of HCl increased, the degree of hydrolysis increased as listed in Table 2.

[0030] [Table 2]

[0031] Knowing that similar acid conditions can be applied to the degradation of nucleic acids or proteins or the main components of viral vectors, the present inventors investigated the conditions for degradation by acid hydrolysis of viral vectors contained in biological matrices consisting of various components present during the production, purification and storage of viral vectors. Several parameters, such as the source and concentration of acid, temperature, heating time, etc., were investigated to design a general method that can specifically degrade viral vectors or any amide functional group except those contained in transfection reagents based on general formula (I) (Figure 1). [Means for solving the problem]

[0032] The inventors provide a test capable of detecting and quantifying a PEI-based transfection reagent in a liquid mixture containing a biological matrix containing a recombinant virus or virus-like particle, the test comprising the following steps: In some cases, inactivation of viral vectors, specific acid hydrolysis which degrades any amide functional group or viral vector other than the general formula PEI-based transfection reagent, i.e. said specific acid hydrolysis does not affect the PEI-based transfection reagent; A purification step capable of separating the PEI-based transfection reagent from by-products generated by acid hydrolysis; Qualitative and quantitative analysis of residual PEI-based transfection reagents.

[0033] The method developed by the inventors shows several advantages. The method includes purification of the transfection reagent, meaning that the analysis is specific, sensitive, robust and independent of the sample composition. The method is applicable to different virus types or subtypes, at different concentrations and completely independent of the viral biosafety level. The method is not modified by the nature of the biological matrix (buffers, detergents, pH, etc.). The method is easy to handle and may be used for R&D, clinical or commercial samples. The method can be certified as GMP grade.

[0034] It is therefore an object of the present invention to provide a method for carrying out acidic hydrolysis of a liquid mixture comprising a biological matrix and a polyethylenimine (PEI)-based transfection reagent, comprising: The biological matrix comprises a recombinant virus or virus-like particle produced using a PEI-based transfection reagent; The method comprises the steps of incubating a liquid mixture comprising a biological matrix in an aqueous solution containing 0.1% to 10% (v / v) hydrochloric acid (HCl) at a temperature ranging from 60° C. to 110° C. for 2 hours to 24 hours, preferably in an aqueous solution containing 0.1% (v / v) HCl at a temperature of 110° C. for 2 hours or in an aqueous solution containing 1% (v / v) HCl at a temperature ranging from 60° C. to 80° C. for 2 hours; The acid hydrolysis does not degrade the PEI-based transfection reagent, PEI-based transfection reagents are reagents of general formula (I), or an acceptable salt thereof:

[0035] [ka]

[0036] [In the formula, m represents an integer between 27 and 1200, preferably between 200 and 600, and n represents an integer between 3 and 600, preferably between 20 and 300, with the proviso that n is smaller than m and the sum m+n ranges from 30 to 1200; -X is H or the following formula:

[0037] [ka]

[0038] (wherein q represents an integer between 10 and 800, preferably between 20 and 400). represents a group of - p is an integer between 1 and 4, Z has the formula:

[0039] [ka]

[0040] (Wherein, - Y0, Y1, Y2, Y3 and Y4 may be the same or different and represent C or N, provided that at least two and not more than three of Y0, Y1, Y2, Y3 and Y4 are N; W1, W2, W3 and W4 may be the same or different and are each H, linear or branched, saturated or unsaturated C1-C 18 Alkyl, C6-C 18 Aryl, linear or branched, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, C5-C 10 Heteroaryl, linear or branched, saturated or unsaturated C2-C 18 Heteroalkyl, amine, linear or branched, saturated or unsaturated C1-C 18 Alkylamines, C1-C 12or (i) W1 and W2 or (ii) W2 and W3 or (iii) W3 and W4 together form a fused, optionally substituted, 6-membered aryl ring; or form a fused, optionally substituted, 6-membered heteroaryl ring containing one or less N atoms, with the proviso that at least one, but not more than two, of W1, W2, W3 and W4 are absent. represents the group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] As defined herein, the term “C1-C 18 "Alkyl" refers to any monovalent radical of a linear or branched hydrocarbon chain containing 1 to 18 carbon atoms. 18 Examples of alkyl groups include, but are not limited to, C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl or t-butyl, C6-C8 alkyl groups, such as n-hexyl, n-heptyl or n-octyl, and n-pentyl, 2-ethylhexyl, 3,5,5-trimethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl or n-octadecyl.

[0042] As defined herein, the term “C6-C 18 "Aryl" refers to any monovalent radical of an aromatic hydrocarbon containing 6 to 18 carbon atoms. 18 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, or phenanthrenyl.

[0043] As defined herein, the term “C6-C 18 Aryl-C1~C 18 "Alkyl" refers to an aryl group, as defined herein, combined with an alkyl group, as defined herein. 18 Aryl-C1~C 18Examples of alkyl groups include, but are not limited to, benzyl, phenylethyl (or phenethyl), phenylpropyl, phenylbutyl, phenylpentyl, phenylhexyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylpentyl, naphthylhexyl, anthracenylmethyl, anthracenylethyl, anthracenylpropyl, anthracenylbutyl, anthracenylpentyl, anthracenylhexyl, phenanthrenylmethyl, phenanthrenylethyl, phenanthrenylpropyl, phenanthrenylbutyl, phenanthrenylpentyl, or phenanthrenylhexyl.

[0044] As defined herein, the term “C5-C 10 "Heteroaryl" refers to any monovalent group of monocyclic or bicyclic 5- to 10-membered aromatic groups containing 1 to 3 heteroatoms independently selected from oxygen, nitrogen and sulfur. 10 Examples of heteroaryl groups include, but are not limited to, furyl, thienyl, pyrrolyl, pyrazoyl, imidazolyl, isoxazolyl, isothiazolyl, thiazolyl, oxazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1-benzofuryl, 1-benzothienyl, indolyl, benzimidazolyl, indazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzothiazolyl, benzoxazolyl, benzotriazolyl, pyridyl, pyridinium, quinolinyl, quinolinium, isoquinolinyl, isoquinolinium, pyridazinyl, cinnolinyl, phthalazinyl, pyrimidinyl, quinazolinyl, pyrazinyl, or quinoxalinyl.

[0045] As defined herein, the term “C2-C 18 "Heteroalkyl" refers to an alkyl group as defined herein substituted by one or more heteroatoms such as O, N, or S.

[0046] As defined herein, the term “C1-C 18"Alkylamine" refers to any monovalent group of linear or branched hydrocarbon chains containing 1 to 18 carbon atoms, in which one of the hydrogen atoms bonded to the carbon atom is replaced by an amino group. 18 Examples of alkylamines include, but are not limited to, -(CH2) n -NH2 (n represents an integer between 1 and 18), -CH2NHCH3, -CH2CH(CH3)-NH2, or -(CH2) n N(CH3)2 (n represents an integer between 1 and 6).

[0047] As defined herein, the term “C1-C 12 "Alkoxy" refers to a group of formula -OR', where R' is a C1-C 12 Alkyl. Preferred C1-C 12 Examples of alkoxy groups include, but are not limited to, C1 to C6 alkoxy groups, such as methoxy (-OCH3), ethoxy (-OCH2CH3), t-butoxy (-OC(CH3)3), or -O(CH2)5CH3.

[0048] Unless otherwise stated, the groups and radicals defined herein above may be unsubstituted or substituted with one or more substituents such as halogen, alkyl, alkoxy, aryl, heteroaryl, haloalkyl, haloalkoxy, alkoxycarbonyl, alkanoyl, aroyl, formyl, nitrile, nitro, amido, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, arylsulfinyl, arylsulfonyl, amino, alkylamino, arylamino, dialkylamino, diarylamino, and the like.

[0049] The term "halogen" as defined herein represents an atom of F, Cl, Br or I.

[0050] In a particular embodiment of the invention, the PEI-based transfection reagent of general formula (I) as defined herein is one in which W1, W2, W3 and W4, which may be the same or different, represent H, methyl, cyclopropyl, isopropyl, tert-butyl, phenyl, benzyl, 2-pyridine, 3-pyridine or 4-hydroxyphenethyl, or (i) W1 and W2 or (ii) W2 and W3 or (iii) W3 and W4 together form a fused phenyl, a methyl group, in particular a fused phenyl substituted with two methyl groups, a methoxy group, a carboxyphenyl or Cl, a fused naphthalene, a fused 2-pyridine or a fused 3-pyridine.

[0051] In a particular embodiment of the invention, the PEI-based transfection reagent of general formula (I) as defined herein is one in which X represents a branched PEI, which has the formula:

[0052] [ka]

[0053] (wherein q represents an integer between 10 and 800, preferably between 20 and 400). is the basis.

[0054] Examples of branched PEI are well known to those skilled in the art.

[0055] In a preferred embodiment of the present invention, the PEI-based transfection reagent of general formula (I) defined herein is one in which X represents H, i.e. X is linear PEI. Examples of linear PEI (lPEI) are well known to those skilled in the art.

[0056] In a particular embodiment of the present invention, the PEI-based transfection reagent of general formula (I) defined herein has a grafting ratio defined as (n / (m+n))×100, the grafting ratio being in the range of 1 to 50%, preferably 5 to 30%, more preferably 20%.

[0057] As defined herein, the term "graft ratio" refers to the number of monomer units grafted to primary or secondary amino groups at the side chains divided by the total number of monomer units present in the original cationic polymer backbone (i.e., the PEI polymer backbone). The graft ratio depends on the molecular weight of the cationic polymer, the chemical reactivity of the side chains grafted to the polymer, or the biological effect obtained. The graft ratio can be determined by measurement methods well known in the art, for example by NMR.

[0058] The PEI polymer backbone has the general formula (II):

[0059] [ka]

[0060] (In the formula, m , n and X is as defined herein. It is of the following.

[0061] In certain embodiments of the invention, the PEI polymer backbone has an average molecular weight (Mw) in the range of 1 kDa to 50 kDa, preferably in the range of 5 kDa to 30 kDa or 10 kDa to 25 kDa, more preferably the PEI polymer backbone has an average molecular weight (Mw) of 8, 10, 15, 22, 25 or 30 kDa, preferably 22 kDa.

[0062] In a particular embodiment of the invention, in the PEI-based transfection reagent of general formula (I), the sum of m+n is in the range of 30 to 1200, preferably 200 to 600, and the average molecular weight (Mw) of the PEI polymer backbone is in the range of 1 kDa to 200 kDa, in particular 1 kDa to 50 kDa, preferably 5 kDa to 30 kDa or 10 kDa to 25 kDa, more preferably 8, 10, 15, 22, 25 or 30 kDa, even more preferably 22 kDa.

[0063] In a particular embodiment of the invention, m represents an integer between 27 and 600 and n represents an integer between 3 and 600. Preferably, m represents an integer between 198 and 300 and n represents an integer between 2 and 300.

[0064] In another particular embodiment of the invention, the sum of m+n ranges from 30 to 1200, preferably from 200 to 600.

[0065] In a preferred embodiment of the invention, the PEI polymer backbone has an average molecular weight (Mw) of 8, 10, 15, 22, 25 or 30 kDa, preferably 22 kDa. When the average molecular weight (Mw) of the PEI polymer backbone is 8, 10, 15, 22, 25 or 30 kDa, the sum of m+n is equal to 180, 220, 340, 500, 570 or 680, respectively.

[0066] In a preferred embodiment of the invention, the PEI-based transfection reagent of general formula (I) is selected from the group consisting of the following compounds:

[0067] [Table 3A]

[0068] [Table 3B]

[0069] In these compounds 1-34, the term "PEI" refers to linear PEI and the term "bPEI" refers to branched PEI.

[0070] Compounds of general formula (I) can be prepared according to various methods well known in the art, for example as disclosed in patent applications WO2021 / 023796 and WO2021 / 023798.

[0071] In certain embodiments of the invention, the biological matrix is ​​selected from the group consisting of cell culture media, in particular media for eukaryotic cells, in particular suspension or adherent cells, buffers, solutions used during the recombinant virus production and purification process, and final compositions comprising purified viruses in final formulations containing pharma- ceutically acceptable buffers and excipients.

[0072] As defined herein, the term "cell culture medium" has its meaning known in the art and refers, for example, to a medium containing at least one of the following components: serum, synthetic medium, animal-free ingredient medium or chemically defined medium, particularly a medium for keeping cells alive or for growing, differentiating or expanding cells, or a medium for enhancing transfection.

[0073] As defined herein, the term "suspension cells" refers to cells that do not require a solid support for growth and are therefore non-anchorage-dependent. Examples of suspension cells include, but are not limited to, NSO cells, U937 cells, Namalwa cells, HL60 cells, WEHI231 cells, Yacl cells, Jurkat cells, THP-1 cells, K562 cells, or U266B1 cells.

[0074] As defined herein, the term "adherent cells" refers to cells that require a solid support for growth and are therefore anchorage-dependent. Examples of adherent cells include, but are not limited to, MRC-5 cells, HeLa cells, Vero cells, NIH-3T3 cells, L293 cells, CHO cells, BHK-21 cells, MCF-7 cells, A549 cells, COS cells, HEK293 cells, HepG2 cells, SNN-BE(2) cells, BAE-1 cells, or SH-SY5Y cells.

[0075] As defined herein, the term "buffer" refers to a buffer solution that contains a buffering agent.

[0076] As defined herein, the term "buffering agent" refers to an agent that adjusts, maintains, or controls the pH of a solution. A buffering agent can be either a weak acid or a weak base that constitutes a buffer solution. Examples of suitable buffering agents include, but are not limited to, sodium carbonate, sodium bicarbonate, sodium hydroxide, calcium bicarbonate, calcium citrate, sodium citrate, magnesium hydroxide, magnesium bicarbonate, potassium acetate, tris acetate, sodium acetate, monopotassium phosphate, potassium carbonate, potassium bicarbonate, potassium citrate, or magnesium oxide.

[0077] As defined herein, the phrase "solutions used during the recombinant virus production and purification process" refers to any solution known in the art that can be used during the recombinant virus production and purification process.

[0078] As defined herein, the term "pharmaceutical acceptable buffers and excipients" refers to a pharmaceutically acceptable solvent, any substance or combination of substances that is physiologically acceptable, i.e., suitable for use in a composition that comes into contact with a host (especially when administered to a host), the host being particularly a human being, and therefore non-toxic. It may refer to a solid, semi-solid or liquid filler, diluent, encapsulating material, or any type of conventional auxiliary formulation. Examples of suitable acceptable excipients include, but are not limited to, glucose, galactose, lactose, dextrose, maltose, mannitol, sucrose, trehalose, polyethylene glycol, or pluronic acid.

[0079] Another object of the present invention is to provide a method for purifying, detecting and / or quantifying a polyethylenimine (PEI)-based transfection reagent of general formula (I) as defined herein, in which the PEI-based transfection reagent of general formula (I) is contained in a liquid mixture comprising a biological matrix, The biological matrix comprises a recombinant virus or virus-like particle produced using a PEI-based transfection reagent of general formula (I), This method is (a) carrying out an acidic hydrolysis of the liquid mixture according to the method of the present invention; (b) purifying the reaction mixture obtained in step (a) to obtain a purified PEI-based transfection reagent of general formula (I); (c) detecting and / or quantifying the purified PEI-based transfection reagent of general formula (I) obtained in step (b); Includes.

[0080] As defined herein, the term "purification" means separating or removing other components of a solution to retain only the transfection reagent solution.

[0081] In a particular embodiment of the invention, prior to carrying out step (a), the method comprises the step of inactivating the recombinant virus or virus-like particle by heating the biological matrix at a temperature in the range of 110°C to 130°C for a period in the range of 30 minutes to 4 hours, preferably at a temperature of 120°C for 30 minutes.

[0082] In certain embodiments of the invention, step (b) is carried out using ultrafiltration or centrifugation.

[0083] In a particular embodiment of the invention, step (c) is carried out using High Performance Liquid Chromatography (HPLC) or Ultra High Performance Liquid Chromatography (UHPLC) analytical techniques, preferably UHPLC.

[0084] In a particular embodiment of the invention, the PEI-based transfection reagent of general formula (I) in step (c) is detected with a limit of detection (LOD) in the range of 1 ppm to 1000 ppm and / or a limit of quantification (LOQ) in the range of 1 ppm to 1000 ppm.

[0085] In a particular embodiment of the present invention, the PEI-based transfection reagent of general formula (I) is detectable in a biological matrix during the recombinant virus production process, said biological matrix being selected from the group consisting of cell culture media, in particular culture media for eukaryotic cells, in particular suspension or adherent cells, buffers, solutions used during the recombinant virus production and purification process, and final compositions comprising the purified virus in a final formulation comprising pharma- ceutically acceptable buffers and excipients.

[0086] In another specific embodiment of the invention, the PEI-based transfection reagent is used during the manufacturing process of an advanced therapeutic medicinal product (ATMP) and is present in residual amounts (1-1000 ppm) relative to the transfection reagent and other components of the liquid mixture provided in the manufacturing process.

[0087] The terms "cell culture medium," "suspension cells," "adherent cells," "buffers," "solutions used during the recombinant virus production and purification process," and "pharmaceutical acceptable buffers and excipients" are defined according to the definitions above and according to the examples provided herein.

[0088] In a particular embodiment of the present invention, the recombinant virus is selected from the group consisting of adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus and baculovirus, preferably adeno-associated virus (AAV) or lentivirus (LV), more preferably adeno-associated virus (AAV).

[0089] In a particular embodiment of the invention, the PEI-based transfection reagent of general formula (I) is a compound selected from the group consisting of compounds 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 30, 31, 32, 33 and 34, and the recombinant virus is an adeno-associated virus (AAV).

[0090] Other features and advantages of the invention will become apparent from the following examples and are illustrated in the drawings. [Brief description of the drawings]

[0091] [Figure 1] FIG. 1 shows a transfection reagent of general formula (I). [Diagram 2] FIG. 1 shows the synthesis of PEI by CROP. [Diagram 3] 1 is a graph showing optimization of heat inactivation of Compound 05. [Figure 4] Figure 4a is a graph showing the hydrolysis of compound 05. Figure 4b is a graph overlay of Figure 4a. [Diagram 5] 1 is a graph showing the measurement of the linearity of hydrolysis of Compound 05. EXAMPLES

[0092] Example 1 General procedure for the preparation of graft polymers

[0093] [ka]

[0094] Step 1: N-alkylation of heterocycles To an oven-dried round-bottom flask under argon was added the corresponding heterocycle (1 equiv.) and DMF (2 ml / 1 mmol of starting material). The solution was cooled to 0°C and sodium hydride (60% dispersed in mineral oil, 1.2 equiv.) was added portionwise. The mixture was allowed to warm slowly to room temperature over 1 h. The corresponding ester was then added dropwise and the reaction was stirred at room temperature for 4-12 h. The mixture was quenched by the addition of water (10 mL / 1 mL DMF) and the aqueous layer was extracted with EtOAc (5 x 2 mL / 1 mL DMF). The combined organic extracts were washed with brine and dried over anhydrous MgSO4. After filtration, the solvent was removed in vacuo and the resulting oil was purified by column chromatography (20-50% EtOAc in heptane).

[0095] Step 2: Saponification of the acid moiety A solution of the ester in EtOH (2 mL / 1 mmol of ester) was added dropwise with a 5 M solution of NaOH (0.2 mL / 1 mmol of ester) and stirred at room temperature overnight. The solvent was then removed in vacuo and the residue was purified by column chromatography on SiO2 using MeOH 5% in DCM+AcOH 1% or acetonitrile 0-100% in H2O.

[0096] Step 3: Grafting To a round bottom flask was added cationic polymer (1 eq) in water (4 mL / mmol starting material), followed by N-methylmorpholine or NMM (2 eq). Carboxylate (0.3-1 eq) was added, followed by MeOH (16 mL / mmol polymer). After stirring for 10 min, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride or DMTMM (0.6-2 eq) was added and the mixture was stirred at room temperature for 12-24 h. MeOH was then removed in vacuo and water (4 mL / mmol starting material) was added, followed by a solution of 3 M HCl (1 mL / mmol starting material). The residue was purified using a dialysis cassette in a 50 mM HCl bath.

[0097] Step 4: Synthesis of triazoles via "click" chemistry starting from acids.

[0098] [ka]

[0099] Alkyne (1 equiv.), azide (1 equiv.), CuSO4 (0.01 equiv.) and sodium ascorbate (0.03 equiv.) were added to a 2:1 (v / v) solution of nBuOH and water. The reaction was stirred at room temperature for 24 h. NaOH (5 M, 2 equiv.) was then added and the organic solvents were removed in vacuo. The residue was purified by reverse phase flash chromatography using 0-100% CH3CN in water as the eluent.

[0100] Step 5: Synthesis of triazoles via "click" chemistry starting from esters.

[0101] [ka]

[0102] Alkyne (1 equiv.), azide (1 equiv.), CuSO4 (0.01 equiv.) and sodium ascorbate (0.03 equiv.) were added to a 2:1 (v / v) solution of nBuOH and water. The reaction was stirred at room temperature for 24 h. NaOH (5 M, 2 equiv.) was then added and the organic solvents were removed in vacuo. The residue was purified by reverse phase flash chromatography using 0-100% CH3CN in water as the eluent.

[0103] Step 6: Saponification of the ester moiety. A 3M solution of LiOH was added dropwise to the ester in EtOH and the mixture was stirred at room temperature over the weekend. The solvent was then removed in vacuo and the residue was purified by reversed-phase FC on SiO2 using H2O / MeCN as eluent using a Biotage Flash purification system. The resulting acid was lyophilized to give a solid.

[0104] Example 2 Synthesis of PEI-Based Transfection Reagents of the Invention - Synthesis of compound 13 (polymer molecular weight = 22k, heterocyclic graft ratio = 10%)

[0105] [ka]

[0106] Intermediate 13.a was prepared analogously to general procedure, step 1 (example 1). Yield=60%; m=2.30 g; 11H NMR (400 MHz, chloroform-d) δ 7.94 (s, 1H), 7.81 - 7.72 (m, 1H), 7.43 - 7.36 (m, 1H), 7.26 (ddd, J = 13.1, 7.5, 4.9 Hz, 2H), 4.24 (t, J = 7.0 Hz, 2H), 4.09 (q, J = 7.4 Hz, 2H), 2.28 (t, J = 7.0 Hz, 2H), 2.16 (p, J = 7.0 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3H).

[0107]

Chem.

[0108] Intermediate 13.b was prepared in the same manner as in the general procedure, Step 2 (Example 1). Yield = 45%; m = 1.00 g; 1 1H NMR (400 MHz, methanol-d4) δ 8.21 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.33 (dt, J = 17.3, 7.5 Hz, 2H), 4.37 (t, J = 7.4 Hz, 2H), 2.35 (t, J = 7.4 Hz, 2H), 2.18 (p, J = 7.4 Hz, 2H).

[0109]

Chem.

[0110] Compound 13 was prepared in the same manner as in the general procedure, Step 3 (Example 1). Yield = 94%; m = 117 mg; 1 1H NMR (D2O) δ: 1H NMR (400 MHz, heavy water) δ 9.29 - 8.97 (m, 1H), 7.96 - 7.17 (m, 4H), 4.46 (d, J = 42.4 Hz, 2H), 3.45 (s, 39H), 2.35 (dd, J = 135.3, 58.1 Hz, 4H). - Synthesis of compound 14 (polymer molecular weight = 22k, heterocyclic graft ratio = 24%)

[0111] [ka]

[0112] Intermediate 14.a was prepared analogously to general procedure, step 1 (example 1). Yield=63%; m=1.7g; 1 H NMR (400 MHz, chloroform-d) δ 7.63 (d, J = 8.7 Hz, 0H), 7.27 (d, J = 8.8 Hz, 1H), 6.99 - 6.86 (m, 2H), 4.27 - 4.15 (m, 4H), 3.93 (dd, J = 7.3, 1.3 Hz, 3H), 2.65 (dd, J = 3.7, 1.3 Hz, 3H), 2.43 (q, J = 6.8 Hz, 2H), 2.18 (p, J = 7.1 Hz, 2H), 1.33 (td, J = 7.2, 1.3 Hz, 3H).

[0113] [ka]

[0114] Intermediate 14.b was prepared analogously to general procedure, step 2 (example 1). Yield=100%; m=676 mg; 1H NMR (400 MHz, methanol-d4) δ 7.54 (dd, J = 32.8, 8.9 Hz, 1H), 7.18 (dd, J = 33.3, 2.4 Hz, 1H), 7.01 (ddd, J = 21.3, 8.9, 2.3 Hz, 1H), 4.33 (q, J = 6.8, 6.3 Hz, 2H), 3.92 - 3.84 (m, 3H), 2.70 (d, J = 8.0 Hz, 3H), 2.43 (q, J = 6.7 Hz, 2H), 2.12 (p, J = 7.0 Hz, 2H).

[0115] [ka]

[0116] Compound 14 was prepared similarly to general procedure, step 3 (example 1). Yield=100%; m=175 mg; 1 H NMR (400 MHz, heavy water) δ 7.80 - 6.57 (m, 3H), 4.44 - 2.96 (m, 22H), 2.80 - 1.38 (m, 7H). - Synthesis of compound 15 (polymer molecular weight = 22k, heterocyclic graft ratio = 27%)

[0117] [ka]

[0118] Intermediate 15a was prepared analogously to general procedure, step 1 (example 1). Yield=50%; m=820 mg; 11H NMR (400 MHz, chloroform-d) δ 7.94 - 7.21 (m, 8H), 7.05 (d, J = 1.3 Hz, 1H), 5.08 (d, J = 1.3 Hz, 2H), 4.05 (td, J = 8.1, 7.5, 2.9 Hz, 2H), 3.92 (dtd, J = 16.2, 7.8, 6.5 Hz, 2H), 2.50 (dd, J = 12.5, 1.4 Hz, 3H), 2.19 (q, J = 6.3 Hz, 2H), 1.94 (p, J = 7.0 Hz, 2H), 1.04 (ddd, J = 14.3, 7.9, 6.5 Hz, 3H).

[0119]

Chem.

[0120] Intermediate 15.b was prepared in the same manner as in the general procedure, Step 2 (Example 1). Yield = 73%; m = 580 mg; 1 1H NMR (400 MHz, methanol-d4) δ 8.01 (d, J = 8.9 Hz, 1H), 7.83 - 7.48 (m, 7H), 4.34 (tt, J = 7.3, 3.0 Hz, 2H), 2.71 - 2.65 (m, 2H), 2.41 (ddt, J = 10.2, 7.4, 4.2 Hz, 2H), 2.12 (h, J = 7.2 Hz, 2H).

[0121]

Chem.

[0122] Compound 15 was prepared in the same manner as in the general procedure, Step 3 (Example 1). Yield = 84%; m = 171 mg; 1 1H NMR (400 MHz, heavy water) δ 7.92 - 6.49 (m, 8H), 4.50 - 3.10 (m, 17H), 3.03 - 1.78 (m, 7H). - Synthesis of compound 16 (polymer molecular weight = 22k, heterocyclic graft ratio = 30%)

[0123] [ka]

[0124] Intermediate 16a was prepared analogously to general procedure, step 1 (example 1). Yield=91%; m=1.55 g; 1 H NMR (400 MHz, chloroform-d) δ 7.68 - 7.53 (m, 1H), 7.33 - 7.16 (m, 2H), 4.14 (pd, J = 7.4, 1.6 Hz, 4H), 2.61 (d, J = 1.5 Hz, 3H), 2.35 (td, J = 6.9, 3.5 Hz, 2H), 2.09 (h, J = 5.8, 4.7 Hz, 2H), 1.26 (tdd, J = 6.9, 4.5, 1.5 Hz, 3H).

[0125] [ka]

[0126] Intermediate 16b was prepared analogously to general procedure, step 2 (example 1). Yield=60%; m=840 mg; 1 H NMR (400 MHz, methanol-d4) δ 7.85 (s, 1H), 7.75 - 7.60 (m, 1H), 7.45 - 7.38 (m, 1H), 4.39 (q, J = 7.6, 6.5 Hz, 2H), 2.77 (t, J = 2.9 Hz, 3H), 2.49 (t, J = 6.6 Hz, 2H), 2.13 (p, J = 7.3 Hz, 2H).

[0127] [ka]

[0128] Compound 16 was prepared similarly to general procedure, step 3 (example 1). Yield=100%; m=193 mg; 1 H NMR (400 MHz, heavy water) δ 7.83 - 6.65 (m, 3H), 4.52 - 3.09 (m, 15H), 3.04 - 2.32 (m, 5H), 2.30 - 1.72 (m, 2H). - Synthesis of compound 17 (polymer molecular weight = 22k, heterocyclic graft ratio = 26%)

[0129] [ka]

[0130] Compound 17 was prepared similarly to general procedure, step 3 (example 1). Yield=97%; m=198 mg; 1 H NMR (400 MHz, heavy water) δ 8.19 - 6.14 (m, 6H), 4.51 - 0.73 (m, 24H). - Synthesis of compound 18 (polymer molecular weight = 22k, heterocyclic graft ratio = 47%)

[0131] [ka]

[0132] Intermediate 18a was prepared analogously to general procedure, step 1 (example 1). Yield=15%; m=211 mg; 1 H NMR (400 MHz, chloroform-d) δ 8.52 (dd, J = 4.4, 1.3 Hz, 1H), 8.19 (d, J = 1.0 Hz, 1H), 7.77 (dt, J = 8.5, 1.2 Hz, 1H), 7.24 (dd, J = 8.6, 4.4 Hz, 1H), 4.46 - 4.38 (m, 2H), 4.04 (q, J = 7.2 Hz, 2H), 2.28 - 2.13 (m, 4H), 1.17 (t, J = 7.1 Hz, 3H).

[0133] [ka]

[0134] Intermediate 18b was prepared analogously to general procedure, step 2 (example 1). Yield=100%; m=887 mg; 1 H NMR (400 MHz, heavy water) δ 8.24 (dd, J = 4.5, 1.3 Hz, 1H), 7.92 (d, J = 1.0 Hz, 1H), 7.76 (dt, J = 8.7, 1.2 Hz, 1H), 7.20 (dd, J = 8.7, 4.4 Hz, 1H), 4.21 - 4.13 (m, 2H), 2.02 - 1.87 (m, 4H).

[0135] [ka]

[0136] Compound 18 was prepared similarly to general procedure, step 3 (example 1). Yield=97%; m=103 mg; 1 H NMR (400 MHz, heavy water) δ 8.92 - 7.42 (m, 4H), 4.61 - 4.25 (m, 2H), 4.04 - 3.09 (m, 10H), 2.67 - 1.88 (m, 5H). - Synthesis of compound 19 (polymer molecular weight = 22k, heterocyclic graft ratio = 25%)

[0137] [ka]

[0138] Intermediate 19a was prepared analogously to general procedure, step 1 (example 1). Yield=43%; m=602 mg; 11H NMR (400 MHz, chloroform-d) δ 9.00 (s, 1H), 8.29 (d, J = 5.6 Hz, 1H), 8.04 (d, J = 0.8 Hz, 1H), 7.61 (dd, J = 5.6, 1.3 Hz, 1H), 4.61 - 4.52 (m, 2H), 4.09 (q, J = 7.1 Hz, 2H), 2.34 - 2.21 (m, 4H), 1.20 (t, J = 7.1 Hz, 3H).

[0139]

Chem.

[0140] Intermediate 19b was prepared in the same manner as in the general procedure, Step 2 (Example 1). Yield = 100%; m = 592 mg; 1 1H NMR (400 MHz, deuterium oxide) δ 8.77 (s, 1H), 8.04 - 7.93 (m, 2H), 7.57 (dd, J = 5.8, 1.3 Hz, 1H), 4.37 - 4.29 (m, 2H), 2.07 - 1.93 (m, 4H).

[0141]

Chem.

[0142] Compound 19 was prepared in the same manner as in the general procedure, Step 3 (Example 1). Yield = 100%; m = 84 mg; 1 1H NMR (400 MHz, deuterium oxide) δ 9.67 - 8.10 (m, 4H), 4.23 - 3.06 (m, 16H), 2.86 - 1.90 (m, 4H). - Synthesis of Compound 20 (Polymer molecular weight = 22k, Heterocyclic grafting rate = 22%)

[0143]

Chem.

[0144] Compound 20 was prepared similarly to general procedure, step 3 (example 1). Yield=89%; m=68 mg; 1 H NMR (400 MHz, heavy water) δ 8.63 - 6.72 (m, 4H), 4.57 - 2.78 (m, 20H), 2.75 - 1.47 (m, 4H). - Synthesis of compound 21 (polymer molecular weight = 22k, heterocyclic graft ratio = 21%)

[0145] [ka]

[0146] Compound 21 was prepared similarly to general procedure, step 3 (example 1). Yield=97%; m=73 mg; 1 H NMR (400 MHz, heavy water) δ 9.12 - 7.56 (m, 4H), 4.58 - 4.36 (m, 2H), 4.09 - 3.01 (m, 19H), 2.90 - 1.77 (m, 4H). - Synthesis of compound 22 (polymer molecular weight = 22k, heterocyclic graft ratio = 21%)

[0147] [ka]

[0148] Compound 22 was prepared similarly to general procedure, step 3 (example 1). Yield=52%; m=39 mg; 1 H NMR (400 MHz, heavy water) δ 8.21 - 6.38 (m, 5H), 4.47 - 1.37 (m, 25H). - Synthesis of compound 23 (polymer molecular weight = 22k, heterocyclic graft ratio = 26%)

[0149] [ka]

[0150] Compound 23 was prepared analogously to general procedure, step 3 (example 1). Yield=97%; m=169 mg; 1 H NMR (400 MHz, heavy water) δ 7.74 - 7.19 (m, 4H), 4.41 - 3.00 (m, 13H), 2.83 - 2.52 (m, 3H), 2.48 - 1.99 (m, 2H), 1.95 - 1.00 (m, 6H). - Synthesis of compound 24 (polymer molecular weight = 8k, heterocyclic grafting rate = 20%)

[0151] [ka]

[0152] Compound 24 was prepared similarly to general procedure, step 3 (example 1). Yield=70%; m=22 mg; 1 H NMR (400 MHz, heavy water) δ 7.80 - 6.15 (m, 2H), 4.50 - 3.21 (m,22H), 3.17 - 0.97 (m, 13H). - Synthesis of compound 25 (polymer molecular weight = 10k, heterocyclic grafting rate = 20%)

[0153] [ka]

[0154] Compound 25 was prepared similarly to general procedure, step 3 (example 1). Yield=73%; m=23 mg; 1 H NMR (400 MHz, heavy water) δ 7.98 - 6.15 (m, 2H), 4.62 - 3.06 (m, 22H), 2.99 - 1.39 (m, 13H). - Synthesis of compound 26 (polymer molecular weight = 15k, heterocyclic graft ratio = 17%)

[0155] [ka]

[0156] Compound 26 was prepared analogously to general procedure, step 3 (example 1). Yield=84%; m=25 mg; 1 H NMR (400 MHz, heavy water) δ 7.64 - 6.35 (m, 2H), 4.57 - 3.13 (m, 25H), 3.13 - 1.50 (m, 13H). - Synthesis of compound 27 (polymer molecular weight = 30k, heterocyclic graft ratio = 18%)

[0157] [ka]

[0158] Compound 27 was prepared similarly to general procedure, step 3 (example 1). Yield=68%; m=21 mg; 1 H NMR (400 MHz, heavy water) δ 7.74 - 6.35 (m, 2H), 4.50 - 3.01 (m, 24H), 2.97 - 1.41 (m, 13H). - Synthesis of compound 28 (polymer molecular weight = 25k, heterocyclic graft ratio = 22%)

[0159] [ka]

[0160] Compound 28 was prepared similarly to the general procedure, step 3, using branched polyethyleneimine (bPEI, 25K, Sigma-Aldrich). Yield = 94%; m = 282 mg; 1 H NMR (400 MHz, heavy water) δ 7.42 - 6.52 (m, 2H), 4.45 - 1.51 (m, 33H). - Synthesis of compound 29 (polymer molecular weight = 10k, heterocyclic grafting rate = 29%)

[0161] [ka]

[0162] Compound 29 was prepared similarly to general procedure, step 3, using branched polyethyleneimine (bPEI, 10K, Alfa Aesar). Yield=99%; m=351 mg; 1 H NMR (400 MHz, heavy water) δ 7.37 - 6.50 (m, 2H), 4.57 - 1.44 (m, 29H). - Synthesis of compound 30 (polymer molecular weight = 22k, heterocyclic graft ratio = 35%)

[0163] [ka]

[0164] Intermediate 30a was prepared similarly to general procedure, step 1 (example 1). Yield=17%; m=1.00 g; 1 H NMR (400 MHz, chloroform-d) δ 7.95 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.30 - 7.22 (m, 1H), 4.68 - 4.57 (m, 2H), 4.01 (qd, J = 7.1, 1.6 Hz, 2H), 2.30 - 2.16 (m, 4H), 1.13 (td, J = 7.1, 1.6 Hz, 3H).

[0165] [ka]

[0166] Intermediate 30b was prepared analogously to general procedure, step 2 (example 1). Yield=85%; m=830 mg; 1H NMR (400 MHz, methanol-d4) δ 8.03 - 7.96 (m, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.58 (t, J = 7.3 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 4.80 (dt, J = 7.0, 4.3 Hz, 2H), 2.37 (t, J = 7.0 Hz, 2H), 2.30 (q, J = 7.0 Hz, 2H).

[0167] [ka]

[0168] Compound 30 was prepared similarly to general procedure, step 3 (example 1). Yield=100%; m=189 mg; 1 H NMR (400 MHz, heavy water) δ 7.41-6.55 (m, 4H), 4.58 - 3.02 (m, 14H), 2.90-1.31 (m, 3H). - Synthesis of compound 31 (polymer molecular weight = 22k, heterocyclic graft ratio = 35%)

[0169] [ka]

[0170] Intermediate 31a was prepared similarly to general procedure, step 1 (example 1). Yield=34%; m=2.00 g; 1 H NMR (400 MHz, chloroform-d) δ 7.73 (ddt, J = 7.4, 4.1, 2.2 Hz, 2H), 7.25 (ddt, J = 9.4, 4.0, 2.2 Hz, 2H), 4.68 (dd, J = 7.3, 5.5 Hz, 2H), 3.99 (ddd, J = 9.1, 7.2, 6.0 Hz, 2H), 2.40 - 2.12 (m, 4H), 1.11 (tt, J = 7.3, 1.3 Hz, 3H).

[0171] [ka]

[0172] Intermediate 31b was prepared analogously to general procedure, step 2 (example 1). Yield=53%; m=1.00 g; 1 H NMR (400 MHz, methanol-d4) δ 7.86 (dd, J = 6.9, 3.4 Hz, 2H), 7.42 (dd, J = 6.9, 3.4 Hz, 2H), 4.83 (d, J = 13.0 Hz, 2H), 2.37 (d, J = 4.3 Hz, 4H).

[0173] [ka]

[0174] Compound 31 was prepared similarly to general procedure, step 3 (example 1). Yield=100%; m=166 mg; 1 H NMR (400 MHz, heavy water) δ 7.83-6.50 (m, 4H), 4.61-3.90 (m, 2H), 3.88-2.51 (m, 11H), 2.49-1.35 (m, 4H). - Synthesis of compound 32 (polymer molecular weight = 22k, heterocyclic graft ratio = 22%)

[0175] [ka]

[0176] Intermediate 32a was prepared analogously to general procedure, step 1 (example 1). Yield=23%; m=1.69 g; 1H NMR (400 MHz, クロロホルム-d) δ 7.78 (dd, J = 9.1, 1.9 Hz, 1H), 7.34 (dd, J = 9.1, 1.9 Hz, 0H), 7.26 (d, J = 2.3 Hz, 0H), 7.05 (dd, J = 9.0, 2.2 Hz, 0H), 6.89 (dd, J = 9.1, 2.2 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 4.55 (dtd, J = 13.6, 6.7, 1.9 Hz, 2H), 4.01 (q, J = 7.1 Hz, 2H), 3.78 (dd, J = 6.6, 1.8 Hz, 3H), 3.04 (s, 0H), 2.22 (ddd, J = 19.8, 7.7, 4.2 Hz, 4H), 1.13 (td, J = 7.1, 1.8 Hz, 3H).

[0177]

change

[0178] Intermediate 32b, general formula, process 2 (Example 1) and the same formula. Yield=70%;m=1.16g; 1 H NMR (400 MHz, メタノール-d4) δ 7.81 (dd, J = 9.1, 2.7 Hz, 1H), 7.67 (dd, J = 8.9, 2.7 Hz, 0H), 7.33 (s, 0H), 7.24 - 7.15 (m, 1H), 7.05 (dd, J = 9.1, 2.7 Hz, 1H), 4.73 (qd, J = 7.1, 2.3 Hz, 2H), 3.91 (dd, J = 13.2, 2.8 Hz, 3H), 3.33 (d, J = 3.2 Hz, 0H), 2.35 (t, J = 6.7 Hz, 2H), 2.26 (t, J = 7.3 Hz, 2H).

[0179]

change

[0180] Compound 32 was prepared similarly to general procedure, step 3 (example 1). Yield=96%; m=153 mg; 1 H NMR (400 MHz, heavy water) δ 7.99-6.22 (m, 3H), 4.49-4.03 (m, 2H), 3.96-2.80 (m, 21H), 2.63-1.55 (m, 4H). - Synthesis of compound 33 (polymer molecular weight = 22k, heterocyclic graft ratio = 18%)

[0181] [ka]

[0182] Intermediate 33a was prepared analogously to general procedure, step 1 (example 1). Yield=18%; m=254 mg; 1 H NMR (400 MHz, chloroform-d) δ 8.53 (dd, J = 4.4, 1.5 Hz, 1H), 7.82 (dd, J = 8.4, 1.5 Hz, 1H), 7.24 (dd, J = 8.4, 4.4 Hz, 1H), 4.54 (t, J = 6.8 Hz, 2H), 3.89 (q, J = 7.1 Hz, 2H), 2.20 - 2.05 (m, 4H), 1.01 (t, J = 7.1 Hz, 3H).

[0183] [ka]

[0184] Intermediate 33b was prepared analogously to general procedure, step 2 (example 1). Yield=98%; m=242 mg; 11H NMR (400 MHz, deuterium oxide) δ 8.59 (dq, J = 4.5, 1.5 Hz, 1H), 8.21 (dt, J = 8.5, 1.6 Hz, 1H), 7.52 (ddt, J = 8.5, 4.5, 1.4 Hz, 1H), 4.70 - 4.62 (m, 2H), 2.21 - 2.04 (m, 4H).

[0185]

Chem.

[0186] Compound 33 was prepared in the same manner as in the general procedure, Step 3 (Example 1). Yield = 75%; m = 54 mg; 1 1H NMR (400 MHz, deuterium oxide) δ 9.06 - 6.76 (m, 3H), 4.16 - 2.96 (m, 20H), 2.93 - 1.52 (m, 4H). - Synthesis of Compound 34 (Polymer molecular weight = 22k, Heterocyclic grafting rate = 17%)

[0187]

Chem.

[0188] Intermediate 34a was prepared in the same manner as in the general procedure, Step 1 (Example 1). Yield = 52%; m = 600 mg; 1 1H NMR (400 MHz, chloroform - d) δ 9.51 - 9.16 (m, 1H), 8.60 - 8.50 (m, 1H), 7.96 - 7.52 (m, 1H), 4.90 - 4.71 (m, 2H), 4.10 (dq, J = 8.6, 7.1 Hz, 2H), 2.43 - 2.30 (m, 4H), 1.23 (t, J = 7.2 Hz, 3H).

[0189]

Chem.

[0190] Intermediate 34b was prepared analogously to general procedure, step 2 (example 1). Yield=97%; m=550 mg; 1 H NMR (400 MHz, heavy water) δ 9.27 - 9.11 (m, 1H), 8.43 - 8.27 (m, 1H), 7.95 - 7.69 (m, 1H), 4.84 - 4.63 (m, 2H), 2.28 - 2.05 (m, 4H).

[0191] [ka]

[0192] Compound 34 was prepared analogously to general procedure, step 3 (example 1). Yield=95%; m=68 mg; 1 H NMR (400 MHz, heavy water) δ 10.08 - 8.17 (m, 3H), 4.21 - 2.84 (m, 25H), 2.83 - 1.64 (m, 4H).

[0193] Example 3 Heat inactivation of viral vectors Thermal inactivation of viral vectors is often performed at about 70°C in the presence of 0.05-0.1% SDS (Sommer JM et al., Molecular therapy: the journal of the American Society of Gene Therapy 2003, 7(1), pp. 122-128; Gargi Maheshwari et al., Journal of Virological Methods 2004, 118(2) pp. 141-146; Fabian Krieselet et al., Journal of Virological Methods 2020, 276, 113-768). Typically, these inactivation protocols take 1 hour to perform. However, to avoid the addition of chemicals and to shorten the heating time, the inventors considered thermal inactivation at 120°C for 30 minutes.

[0194] Therefore, the stability of compound 05 at 120 °C over a period of 24 hours was evaluated. An overlay of the different chromatograms at different time points is shown in Figure 3. No decomposition was observed beyond 4 hours. After 6 hours, peaks were detected around the desired compound 05, signifying slight decomposition. Based on this stability study, any reagents that may remain in the sample are not affected by autoclaving for 30 minutes.

[0195] Example 4 Optimization of acid hydrolysis The inventors considered three different parameters to define the optimal conditions under which compound 05 is not degraded: Type of acid, · Temperature, Heating time.

[0196] Acidic hydrolysis of 20 ppm of compound 05 was carried out with HCl, H2SO4, and HNO3, three acids known to degrade proteins or nucleic acids.

[0197] To a solution of Compound 05 (20 ppm) in distilled water, hydrochloric acid, sulfuric acid, or nitric acid were added, respectively. The three mixtures were heated at 100° C. overnight. The following method was followed for each acid. - Accuracy in triplicate for standard compound 05. The test consisted of hydrolyzing 20 ppm standard with various acids and centrifugal filtering. Acid hydrolysis with HCl was carried out for 46 hours (15 hours for other acids). - Recovery of virus spiked with 20 ppm of compound 05. This parameter was determined in triplicate injections.

[0198] The various results are summarized in Table 3 and show that all conditions are able to avoid the decomposition of compound 05. However, hydrochloric acid shows the best reproducibility compared to sulfuric or nitric acid.

[0199] [Table 4]

[0200] The concentration of HCl and the heating time are important for acid hydrolysis, therefore, we evaluated these two criteria. HCl concentration: 10%, 1%, 0.1%, Heating times at 60°C, 80°C, 100°C: t0, +1h, +2h, +3h, +4h, +5h, +6, +24h.

[0201] Finally, the optimized conditions were determined as acid hydrolysis in 0.1% hydrochloric acid solution for 2 h at 100° C. The linearity in Figures 4a, 4b and 5 shows LOD=1.97 and LOQ=6.55.

[0202] Figure 4b shows an overlay of Figure 4a, where 1 represents 5 ppm of compound 5 at 110°C, 2 represents 10 ppm of compound 5 at 110°C, 3 represents 20 ppm of compound 5 at 110°C, 4 represents 25 ppm of compound 5 at 110°C, 5 represents 50 ppm of compound 5 at 110°C, 6 represents 75 ppm of compound 6 at 110°C, 7 represents 100 ppm of compound 5 at 110°C, and 8 represents 150 ppm of compound 5 at 110°C.

[0203] Example 5 Qualitative and quantitative analysis of residual products The HPLC method is described below in Table 4. This analytical method was developed throughout the development of this invention due to two main issues: - the presence of carryover that may affect quantification, - Symmetry of the different peaks, which is not optimal considering the validation possibilities of this method.

[0204] We performed various "in-house" tests to address the carryover issue by varying various parameters such as column type (grafted C4 vs C18, AX vs peptide), mobile phase composition, gradient slope, and flow rate etc. After these various screens, a new analytical method was implemented and is listed in Table 4 below.

[0205] [Table 5]

[0206] Example 6 Experimental Section The inventors have demonstrated the efficiency of this method in various transfection reagents (e.g., those described in WO2021 / 023796; WO2021 / 023798) that showed strong activity in transfection, particularly in the production of viral vectors.

[0207] We investigated the stability of all transfection reagent compounds 01-34 under acidic hydrolysis as described above (Table 5). Most molecules were completely stable under acidic conditions. Furthermore, based on both limit of detection (LOD) and limit of quantification (LOQ) investigations, this analytical method was applicable to a wide variety of PEI-based polymers.

[0208] [Table 6A]

[0209] [Table 6B]

[0210] [Table 6C]

[0211] We then applied the full residue test described above to a selection of molecules listed in Table 6 below.

[0212] [Table 7A]

[0213] [Table 7B]

[0214] The tube was sealed and then heated at 100 °C for 2 h. After cooling to room temperature, the mixture was diluted with 3.5 mL of pure water and then filtered at 5.000 g for 30 min by centrifugation using a centrifugal filter with a minimum cut-off of 1 kDa to obtain a solution between 250 and 300 μL. The solution was worked up to 4 mL and filtered at 5.000 g for 60 min. The same procedure was repeated two more times. The final retentate was diluted with pure water to obtain 500 μL of solution and analyzed by HPLC.

[0215] Post-hydrolysis addition procedure: To the solution of viral vector (AAV, 200 μL), a solution of HCl (0.37%, 108 μL), a given volume of pure water (92 μL), and a solution of transfection reagent (concentration: 100 ppm, 80 μL) were added. The tube was sealed and heated at 100 °C for 2 h. After cooling, the solution of transfection reagent (concentration: 100 ppm, 80 μL) was added before the filtration step.

[0216] Examples of buffers for use with compound 17 are disclosed in Table 7.

[0217] [Table 8A]

[0218] [Table 8B]

Claims

1. 1. A method for performing acidic hydrolysis of a liquid mixture comprising a biological matrix and a polyethylenimine (PEI)-based transfection reagent, comprising the steps of: the biological matrix comprises a recombinant virus or virus-like particle produced using the PEI-based transfection reagent; The method comprises the steps of incubating the liquid mixture comprising the biological matrix in an aqueous solution containing 0.1% to 10% (v / v) hydrochloric acid (HCl) at a temperature ranging from 60° C. to 110° C. for 2 hours to 24 hours, preferably in an aqueous solution containing 0.1% (v / v) HCl at a temperature of 110° C. for 2 hours or in an aqueous solution containing 1% (v / v) HCl at a temperature ranging from 60° C. to 80° C. for 2 hours; the acidic hydrolysis does not degrade the PEI-based transfection reagent; The method, wherein the PEI-based transfection reagent is a reagent of general formula (I), or an acceptable salt thereof: 【Chemistry 1】 [In the formula, m represents an integer between 27 and 1200 and n represents an integer between 3 and 600, with the proviso that n is less than m and the sum m+n ranges from 30 to 1200; X is H or the formula: 【Chemistry 2】 (wherein q represents an integer between 10 and 800). represents a group of - p is an integer between 1 and 4, Z has the formula: 【Chemistry 3】 (In the formula, Y 0 , Y 1 , Y 2 , Y 3 and Y 4 may be the same or different and represent C or N, with the proviso that Y 0 , Y 1 , Y 2 , Y 3 and Y 4 At least two, but not more than three, of the - W 1 , W 2 , W 3 and W 4 may be the same or different, H, linear or branched, saturated or unsaturated C 1 ~C 18 Alkyl, C 6 ~C 18 Aryl, linear or branched, saturated or unsaturated C 6 ~C 18 Aryl-C 1 ~C 18 Alkyl, C 5 ~C 10 Heteroaryl, linear or branched, saturated or unsaturated 2 ~C 18 Heteroalkyl, amine, linear or branched, saturated or unsaturated C 1 ~C 18 Alkylamines, C 1 ~C 12 (i) W 1 and W 2 or (ii) W 2 and W 3 or (iii) W 3 and W 4 together form a fused, optionally substituted, 6-membered aryl ring; or form a fused, optionally substituted, 6-membered heteroaryl ring containing up to one N atom; However, W 1 , W 2 , W 3 and W 4 At least one, but not more than two of the following are present: represents the group.

2. W 1 , W 2 , W 3 and W 4 may be the same or different and represent H, methyl, cyclopropyl, isopropyl, tert-butyl, phenyl, benzyl, 2-pyridine, 3-pyridine, or 4-hydroxyphenethyl; or (i) W 1 and W 2 or (ii) W 2 and W 3 or (iii) W 3 and W 4 2. The method according to claim 1, wherein together they form a fused phenyl, a fused phenyl substituted with a methyl group, in particular two methyl groups, a methoxy group, a carboxyphenyl or Cl, a fused naphthalene, a fused 2-pyridine or a fused 3-pyridine.

3. 3. The method of claim 1 or 2, wherein X represents H.

4. 4. The method according to any one of claims 1 to 3, wherein the PEI-based transfection reagent of general formula (I) has a grafting ratio defined as (n / (m+n)) x 100, the grafting ratio being in the range of 1 to 50%, preferably 5 to 30%, more preferably 20%.

5. 5. The method of any one of claims 1 to 4, wherein the PEI polymer backbone has an average molecular weight (Mw) in the range of 1 kDa to 50 kDa, preferably in the range of 5 kDa to 30 kDa or 10 kDa to 25 kDa, more preferably an average molecular weight (Mw) of 8, 10, 15, 22, 25 or 30 kDa, preferably 22 kDa.

6. 6. The method according to claim 1, wherein the PEI-based transfection reagent of general formula (I) is selected from the group consisting of the following compounds: 【Table 1A】 【Table 1B】

7. 7. The method according to any one of claims 1 to 6, wherein the biological matrix is ​​selected from the group consisting of cell culture media, in particular culture media for eukaryotic cells, in particular suspension or adherent cells, buffers, solutions used during the recombinant virus production and purification process, and final compositions comprising the purified produced recombinant virus in a final formulation comprising pharma- ceutically acceptable buffers and excipients.

8. A method for purifying, detecting and / or quantifying a polyethylenimine (PEI)-based transfection reagent of general formula (I) as defined in any one of claims 1 to 6, comprising comprising said PEI-based transfection reagent of general formula (I) in a liquid mixture comprising a biological matrix, said biological matrix comprises a recombinant virus or virus-like particle produced using a PEI-based transfection reagent of general formula (I), This method, (a) carrying out an acidic hydrolysis of the liquid mixture according to any one of claims 1 to 7; (b) purifying the reaction mixture obtained in step (a) to obtain a purified PEI-based transfection reagent of general formula (I); (c) detecting and / or quantifying the purified PEI-based transfection reagent of general formula (I) obtained in step (b); A method comprising:

9. 9. The method according to claim 8, wherein step (c) is carried out using High Performance Liquid Chromatography (HPLC) or Ultra High Performance Liquid Chromatography (UHPLC) analytical techniques, preferably UHPLC.

10. 10. The method according to claim 8 or 9, wherein the PEI-based transfection reagent of general formula (I) in step (c) is detected with a limit of detection (LOD) in the range of 1 ppm to 1000 ppm and / or a limit of quantification (LOQ) in the range of 1 ppm to 1000 ppm.

11. 11. The method according to any one of claims 1 to 10, wherein the PEI-based transfection reagent of general formula (I) is detectable in a biological matrix during the recombinant virus production process, said biological matrix being selected from the group consisting of cell culture media, in particular culture media of eukaryotic cells, in particular suspension or adherent cells, buffers, solutions used during the recombinant virus production and purification process, and final compositions comprising the purified virus in a final formulation comprising pharmaceutically acceptable buffers and excipients.

12. 12. The method according to any one of claims 1 to 11, wherein the recombinant virus is selected from the group consisting of adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus and baculovirus, preferably adeno-associated virus (AAV) or lentivirus (LV), more preferably adeno-associated virus (AAV).

13. 13. The method according to any one of claims 6 to 12, wherein the PEI-based transfection reagent of general formula (I) is a compound selected from the group consisting of compounds 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 30, 31, 32, 33 and 34, and the recombinant virus is an adeno-associated virus (AAV).

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