Method for purification, detection, and quantification of 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 relates to a method for acid hydrolyzing a liquid mixture of a polyethyleneimine (PEI)-based transfection reagent comprising a biological matrix and the 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 the acid hydrolysis does not degrade the PEI-based transfection reagent. The present invention also relates to a method for purifying, detecting and / or quantifying a PEI-based transfection reagent of the general formula (I) as described herein.

Description

[0001] The present invention relates to a method for purifying, detecting and quantifying residual PEI-based transfection reagents. The present invention relates to a method for acid hydrolyzing a liquid mixture comprising a biomatrix and a polyethyleneimine (PEI)-based transfection reagent of general formula (I) as described herein, wherein the biomatrix comprises a recombinant virus or virus-like particle produced using the PEI-based transfection reagent, and wherein the acid hydrolysis does not degrade the PEI-based transfection reagent. The present invention also relates to a method for purifying, detecting and / or quantifying a PEI-based transfection reagent of general formula (I) as described herein.

[0002] Gene therapy aims to deliver exogenous genes into cells to correct the expression of defective genes in monogenic diseases or to provide new, beneficial gene expression in cancer, viral, and immune infections. To do this, the exogenous genetic material must be effectively introduced into the cell to correct the location where the gene expression will trigger the desired therapeutic effect.

[0003] The exogenous genetic material consists of a gene expression system comprising the gene of interest constructed with sequences that control its expression (promoter, enhancer, exons, etc.) and provided on a nucleic acid construct (plasmid, linear DNA or ligated double-stranded DNA, messenger RNA, etc.).

[0004] Since eukaryotic cells are impermeable to nucleic acids, delivery systems or vectors have been developed to allow their introduction. Several systems based on viral or non-viral vectors are effective delivery systems. Therefore, viral vectors represent the majority of delivery systems used in clinical applications.

[0005] Viral vectors have taken advantage of the ability of viruses to target cells, especially infect cells, with a huge efficiency associated with millions of years of biological evolution. Taking advantage of its efficiency, viral vectors, particularly recombinant viral vectors, have been constructed to maintain its ability to target or infect cells, but to suppress its pathogenicity while using it as a genomic sequence transporter. Among recombinant viral vectors, AAV, retroviruses, lentiviruses, and adenoviruses are most commonly used for gene and cell therapy. This list is not exhaustive (see review "Viral Vectors in Gene Therapy", Kenneth Lundstrom, Diseases 2018, 6, 42), and depends on the progress and knowledge of other viruses and their potential use as non-pathogenic genomic expression vectors.

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

[0007] Several cell systems, such as HEK293 cells, HeLa cells, or insect cells, are used to produce recombinant viral vectors. The method involves introducing the genetic material of the recombinant virus required for replication of its recombinant genome, production of viral capsids, and encapsidation of the recombinant viral genome into the cells, followed by recovery of the produced viral vector.

[0008] Several methods can be used to introduce the different components required for the production of the recombinant virus, such as by baculovirus infection, transfection by chemical methods (calcium phosphate or transfection reagents or preparations), 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 the recombinant virus into the host genome of the producer cells).

[0009] Chemical transfection represents the main method used to produce viruses used in gene and cell therapy, particularly AAV or lentiviruses. Calcium phosphate precipitation is not suitable for large-scale production (production volume) due to difficulties in controlling and reproducible nucleic acid precipitation, and its use has been replaced by the use of transfection reagents such as cationic polymers or cationic lipids. Among cationic polymers, PEI polymer is widely used for the production of recombinant viral vectors from HEK293 cells and derivatives.

[0010] Transfection allows the introduction of different nucleic acids, usually in the form of plasmids as gene expression vectors, into the cells required for the production of recombinant viruses. Transfection allows the introduction of viral genes and their transient expression over a period of several days (1 to 7 days) in the cells producing the virus.

[0011] Several polyethyleneimine detection methods have been developed using spectrophotometry (Franceca Ungaro et al., J.Pharm.Biomed.Anal.31 (2003) 143), colorimetry (Qu, Fei Li et al., Langmuir 2013, 29, 1199-1205) or fluorescence (Yunyi Zhang et al., Analytical and Bioanalysis Chemistry 2017, 409, 4771). Based on the specific interaction between PEI and metals (copper, silver) (Yu Ling et al., J.Phys.Chem.C 2015, 119, 27173-27177), these methods are applicable to solutions that do not contain other nitrogen atoms that can chelate metals. These tests that require extraction of PEI from the sample matrix are not suitable for compositions comprising viral vectors because some residual transfection reagents may be trapped inside the virus.

[0012] The cells for producing the recombinant virus are essentially HEK293 cells, their derivatives, subclones or genetically modified or other cells, such as HeLa, CHO, etc. These cells can be cultured in a variable culture system, on plastic or glass carriers or in a bioreactor system in an adherent or non-adherent (suspension) manner.

[0013] The cells producing the virus are cultured in a culture medium containing or without serum, containing proteins, peptides, vitamins, hormones, amino acids, lipids, salts, or containing various synthetic compounds other than those of animal origin, or containing synthetic components such as polymers or substances that allow their proliferation and good vitality and avoid their aggregation. Culture media are well known, and examples of compositions are described in the literature, for example, by Srivastava A et al. (J Pharm Sci. 2021 Jul; 110(7): 2609-2624).

[0014] The initial successes of gene and cell therapies have made it possible to expand the application of viral vectors from rare diseases to more common conditions. Regulatory agencies are adjusting their guidelines for the manufacture of these new products. The methods and processes for producing viral vectors must be regulated to ensure patient safety. Regulatory agencies have already established "Guidance" for the production of these new medicines, covering several aspects of the substance's safety, substance, quality, purity, impurities, and potential (WHO Good Manufacturing Practice for Biological Products, Annex 1 to WHO Technical Report Series No. 822). Among other things, the production process for active substances containing or composed of viral vectors must be controlled for impurities, and if they represent a safety risk, they must be able to be identified and quantified.

[0015] The production of viral vectors in cells such as HEK293 cells or their derivatives by transient transfection methods consists of two stages. A nucleic acid comprising a virus and optional additional genes (in the form of a plasmid, 1 to 4 plasmids or other forms of gene expression vectors) is introduced into HEK293 cells, cultured in a synthetic medium containing or without a culture supplement under adherent or non-adherent conditions to produce recombinant viruses, whether or not to discharge depends on the type of recombinant virus produced. The recombinant virus production step is referred to as an upstream process. This is followed by a step of harvesting the virus and then purifying it until it can be suitable for use as a pharmaceutical active substance [drug substance (DS) or drug product (DP)] in the formulation process of a pharmaceutical composition, which is defined as a downstream process (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). During this final stage of production, additives such as acceptable pharmaceutical excipients or ingredients (U.S. Food and Drug Administration (FDA) 21 CFR 210.3(b)(8) guidance, an excipient or inactive ingredient is any component of a medicinal product other than the active ingredient, and EMEA guidance, Dossiers on Marketing Authorizations Applicable to Medicinal Products, Document No.: EMEA / CHMP / QWP / 396951 / 2006) may be added to promote good preservation of the virus or prevent its degradation or aggregation.

[0016] In this viral vector production process, the transfection reagent is a raw material in the upstream process and becomes a potential impurity in the final product (DS or DP) if it is not eliminated in the downstream process. Therefore, the transfection reagent must be identified and quantified throughout the upstream and downstream processes or in the final product (DS or DP). To date, there are no guidelines for the acceptable amount of residual transfection reagent in viral preparations. However, the determination of the residual rate seems to be mandatory, requiring the notification of regulatory production documents and the traceability of residual impurities in viral preparations for therapeutic purposes, which may represent a risk of toxicity.

[0017] The increasing demand for viral vectors is needed to meet the expected demand at the commercial level and the demand generated by the rapid progress through the various stages of clinical development (Van Der Loo-Human Molecular Genetics_2015). Therefore, large-scale production, new production cell lines, new synthetic culture media or new transfection reagents are being studied 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 production achieved with PEI, which is considered the gold standard transfection reagent (WO2021 / 023796; WO2021 / 023798). However, determination of the residual content of these new transfection reagents in the produced viral vectors or during their production process (sometimes referred to as "residual determination" or "residual testing") has not been achieved.

[0019] Therefore, the object of the present invention is to develop a specific method for determining the amount of substance in a complex liquid mixture. The substance is present in a liquid mixture also comprising a biomatrix in a very low amount. The biomatrix includes recombinant viruses and / or virus-like particles, and can be cell culture medium, buffer or any solution used during upstream (UP) and downstream processes, and finally produces final medicine. The material defined as a transfection reagent is used to produce viral vectors (AAV, LV, adenovirus, oncolytic virus, baculovirus) in mammalian cells. After producing viral vectors (upstream processes), several steps are needed to eliminate all impurities produced during UP, thereby ultimately obtaining a pure pharmaceutical active ingredient that can be applied to a patient.

[0020] Several residue tests must be performed to ensure patient safety. One of these involves transfection reagents. Transfection reagents typically contain cationic polymers or lipids. Due to their cationic nature, these reagents have the ability to interact with nucleic acids and many other chemicals used throughout the viral vector production process (polymers, salts, small molecules, etc.).

[0021] To ensure that the levels of these transfection reagents are limited to safe amounts, regulatory agencies in the field need to develop analytical methods that can detect and quantify such residues.

[0022] Inventors must consider several criteria to develop accurate residue tests:

[0023] Depending on the biosafety level of the virus, a preliminary viral inactivation step may be required to protect the operator. The inactivation step can be thermal (heat), chemical (detergents, acids, etc.), or physical (UV) and may produce byproducts that can react with the transfection reagent. The inactivation option needs to be ineffective against the transfection reagent, i.e., the transfection reagent needs to be preserved in the test mixture that will ultimately be analyzed.

[0024] • In addition, since the transfection reagent may adhere to or be encapsulated in the virus, in the determination of the residual content of the transfection reagent, the virus should be completely degraded to obtain this potential encapsulated reagent.

[0025] At every step of the viral vector manufacturing process, the transfection reagent is present at very low levels compared to the other components (virus, cell culture medium, detergents, salts, etc.). The difficulty here is detecting 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 large molecule, like DNA, viral proteins, and detergents used during manufacturing or in the final formulation, purification by size exclusion will not be accurate enough to allow meeting LOD (limit of detection) and LOQ (limit of quantification).

[0026] The sensitivity of the analytical method requires a wide range of detection capabilities, from 1 ppm (LOD) to 1000 ppm, which can correspond to the amount of transfection reagent in the concentration stage of the downstream process.

[0027] Because each viral vector manufacturing process is unique, it is difficult to predict the impact of viral vector composition on analytical testing. In order to simplify and develop residual tests that can be generalized to viral vector manufacturing, the inventors decided to focus their attention on the development of orthogonal degradation methods that would not affect the transfection reagents of general formula (I) as described herein ( Figure 1 ), the transfection efficiency of the transfection reagent is well known (Thomas Lorson et al., Biomaterials 178 (2018) 204e280; Ts. Ivanva, E et al., PHARMACIA, 2016 vol. 63, 3; Nico Adams et al., Reviews 59 (2007) 1504-1520).

[0028] PEI derivatives represent a large family of polymers for nucleic acid delivery (DNA, siRNA, mRNA, miRNA, etc.). In particular, linear PEI (or IPEI) 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-oxazoline to produce polyalkyl-(2-oxazoline). Then, polyalkyl-(2-oxazoline) can be fully or partially hydrolyzed to produce IPEI or a combination of IPEI and polyalkyl-(2-oxazoline) (Ryuichi Tanaka et al., Macromolecules 1983, 16, 6, 849-853) ( Figure 2 ).

[0029] Typically, hydrolysis, which involves cleavage of the carbon-nitrogen bond of the amide functional group, is carried out under strongly acidic conditions (Emi Haladjova et al., Polymers 2020, 12, 2609; Alexander B. Cook et al., Polym. Chem., 2019, 10, 1202-1212; Emi Haladjova et al., Macromol. Biosci. 2018, 1700349). Rangelov et al. performed partial degradation of poly(2-methyl-2-oxazoline) by acid hydrolysis in an aqueous solution containing 17.5% HCl at 100°C (Haladjova E et al., J Appl Polym Sci. 2020; e49400; R. Shah et al., J. Mater. Sci. Mater. Med. 2015, 26, 157). The heating time is directly related to the degree of hydrolysis. The degree of hydrolysis (DH) increased from 2-6% to 60% from 15 to 180 minutes at 100°C (Table 1).

[0030] Table 1. Effect of heating time.

[0031]

[0032] Park et al. conducted a 6-hour partial degradation of poly(2-ethyl-2-oxazoline) using various concentrations of HCl at 100°C (Ji Hoon Jeong et al., Journal of Controlled Release 73 (2001) 391-399; R. Tanaka et al., Macromolecules 16 (1983) 849-853). In this study, hydrolysis was performed using different concentrations of HCl at 5.0, 7.5, and 10.0% (v / v). As the HCl concentration increased, the extent of hydrolysis increased, as shown in Table 2.

[0033] Table 2. Effect of HCl concentration.

[0034]

[0035] a Using 400MHz 1 H NMR spectroscopy.

[0036] Knowing that similar acid conditions can be applied to the degradation of nucleic acids or proteins or major components of viral vectors, the inventors have studied the degradation conditions of the acid hydrolysis of viral vectors contained in biological matrices composed of various components present during the manufacture, purification and storage of viral vectors. Several parameters have been studied, such as the source and concentration of the acid, temperature, heating time, in order to design a method capable of specifically degrading viral vectors or in addition to the transfection reagents based on general formula (I) ( Figure 1 ) is a general method for any amide functional group other than those in ).

[0037] The inventors provide a test method capable of detecting and quantifying a PEI-based transfection reagent in a liquid mixture, wherein the liquid mixture contains a biological matrix containing a recombinant virus or a virus-like particle, the method comprising the following steps:

[0038] Optional inactivation of viral vectors,

[0039] a specific acid hydrolysis that degrades the viral vector or any amide functional groups other than the amide groups of the general PEI-based transfection reagent, i.e., the specific acid hydrolysis does not affect the PEI-based transfection reagent,

[0040] A purification step to separate PEI-based transfection reagents from byproducts generated by acid hydrolysis,

[0041] Qualitative and quantitative analysis of residual PEI-based transfection reagents.

[0042] The method that the inventor developed shows some benefits.The method includes the purification of transfection reagent, which means that analysis is specific, highly sensitive, stable and does not rely on sample composition.The method is applicable to different virus types or subtypes, different concentrations, and is completely independent of the biosafety level of the virus.The method is not modified by the properties (buffer, detergent, pH etc.) of the biological matrix.The method is easy to operate and can be used for research and development, clinical or commercial samples.The method can be to meet GMP (good manufacturing practice) level requirements.

[0043] Therefore, the object of the present invention is to provide a method for acid hydrolysis of a liquid mixture comprising a biological matrix and a transfection reagent based on polyethyleneimine (PEI),

[0044] wherein the biological matrix comprises recombinant viruses or virus-like particles produced using the PEI-based transfection reagent,

[0045] The method comprises the step of incubating the liquid mixture comprising the biological matrix under the following conditions: incubating in an aqueous solution comprising 0.1% to 10% (v / v) hydrochloric acid (HCl) at a temperature of 60° C. to 110° C. for a period of 2 hours to 24 hours, preferably incubating in an aqueous solution comprising 0.1% (v / v) HCl at a temperature of 110° C. for 2 hours, or incubating in an aqueous solution comprising 1% (v / v) HCl at a temperature of 60° C. to 80° C. for 2 hours,

[0046] wherein the acid hydrolysis does not degrade the PEI-based transfection reagent, and

[0047] The PEI-based transfection reagent has the general formula (I) or an acceptable salt thereof:

[0048]

[0049] in:

[0050] - m represents an integer between 27 and 1200, preferably an integer between 200 and 600, and n represents an integer between 3 and 600, preferably an integer between 20 and 300, with the proviso that n is lower than m and the sum of m+n is in the range of 30 to 1200,

[0051] -X represents H or a group of the formula:

[0052]

[0053] wherein q represents an integer between 10 and 800, preferably an integer between 20 and 400,

[0054] -p represents an integer between 1 and 4,

[0055] -Z represents a group of the formula:

[0056]

[0057] - Y0, Y1, Y2, Y3 and Y4 may be the same or different and represent C or N, provided that at least two, but not more than three, of Y0, Y1, Y2, Y3 and Y4 are N,

[0058] -W1, W2, W3 and W4 can be the same or different and represent 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 Alkylamine, C1-C 12 alkoxy; or (i) W1 and W2 or (ii) W2 and W3 or (iii) W3 and W4 together form a fused, optionally substituted six-membered aryl group; or a fused, optionally substituted six-membered heteroaryl group containing not more than 1 N atom,

[0059] The condition is that at least one, but no more than two, of W1, W2, W3 and W4 do not exist.

[0060] As defined herein, the term "C1-C 18"Alkyl" means any monovalent group of a straight or branched hydrocarbon chain containing 1 to 18 carbon atoms. Suitable C1-C 18 Examples of alkyl groups include, but are not limited to, C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-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.

[0061] As defined herein, the term "C6-C 18 "Aryl" means any monovalent group of an aromatic hydrocarbon containing 6 to 18 carbon atoms. Suitable C6-C 18 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, or phenanthrenyl.

[0062] As defined herein, the term "C6-C 18 Aryl-C1-C 18 "Alkyl" means an aryl group as defined herein in combination with an alkyl group as defined herein. Suitable C6-C 18 Aryl-C1-C 18 Examples 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, phenanthracenylmethyl, phenanthracenylethyl, phenanthracenylpropyl, phenanthracenylbutyl, phenanthracenylpentyl, or phenanthracenylhexyl.

[0063] As defined herein, the term "C5-C 10 "Heteroaryl" means any monovalent group of a monocyclic or bicyclic 5-10 membered aromatic group containing 1-3 heteroatoms independently selected from oxygen, nitrogen and sulfur. Suitable C5-C 10 Examples of heteroaryl groups include, but are not limited to, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, isoxazolyl, isothiazolyl, thiazolyl, oxazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1-benzofuranyl, 1-benzothienyl, indolyl, benzimidazolyl, indazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzothiazolyl, benzoxazolyl, benzotriazolyl, pyridinyl, pyridinium, quinolinyl, quinolinium, isoquinolinyl, isoquinolinium, pyridazinyl, cinnolinyl, phthalazinyl, pyrimidinyl, quinazolinyl, pyrazinyl, or quinoxalinyl.

[0064] As defined herein, the term "C2-C 18 "Heteroalkyl" means an alkyl group as defined herein substituted with one or more heteroatoms such as O, N or S.

[0065] As defined herein, the term “C 1- C 18 "Alkylamine" means any monovalent radical of a linear or branched hydrocarbon chain containing 1 to 18 carbon atoms, in which one of the hydrogen atoms bonded to a carbon atom is replaced by an amino group. Suitable C1-C 18 Examples of alkylamines include, but are not limited to, -(CH2) n -NH2, wherein n represents an integer between 1 and 18, -CH2NHCH3, -CH2CH(CH3)-NH2, or -(CH2) n N(CH3)2, wherein n represents an integer between 1 and 6.

[0066] As defined herein, the term "C1-C 12 "Alkoxy" means a group of formula -OR', wherein R' is C1-C 12 Suitable C1-C 12 Examples of alkoxy groups include, but are not limited to, C1-C6 alkoxy groups, such as methoxy (-OCH3), ethoxy (-OCH2CH3), tert-butoxy (-OC(CH3)3), or -O(CH2)5CH3.

[0067] Unless otherwise indicated, the groups defined 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, acylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, arylsulfinyl, arylsulfonyl, amino, alkylamino, arylamino, dialkylamino and diarylamino.

[0068] As defined herein, the term "halogen" refers to an atom of F, Cl, Br or I.

[0069] In a specific embodiment of the present invention, the PEI-based transfection reagent of general formula (I) defined herein is a transfection reagent wherein W1, W2, W3 and W4 may be the same or different and represent H, methyl, cyclopropyl, isopropyl, tert-butyl, phenyl, benzyl, 2-pyridine, 3-pyridine or 4-hydroxyphenethyl; or wherein (i) W1 and W2 or (ii) W2 and W3 or (iii) W3 and W4 together form a fused phenyl; a fused phenyl substituted by methyl (especially two methyl), methoxy, carboxyphenyl or Cl; a fused naphthalene; a fused 2-pyridine; or a fused 3-pyridine.

[0070] In a particular embodiment of the present invention, the PEI-based transfection reagent of general formula (I) as defined herein is a transfection reagent wherein X represents a branched PEI, which is a group of the formula:

[0071]

[0072] wherein q represents an integer between 10-800, preferably an integer between 20-400.

[0073] Examples of branched PEIs are well known to those skilled in the art.

[0074] In a preferred embodiment of the present invention, the PEI-based transfection reagent of general formula (I) defined herein is a transfection reagent wherein X represents H, ie X is a linear PEI. Examples of linear PEI (IPEI) are well known to those skilled in the art.

[0075] In a specific 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, wherein the grafting ratio is 1 to 50%, preferably 5 to 30%, more preferably 20%.

[0076] As defined herein, the term "grafting ratio" refers to the number of monomer units grafted onto primary or secondary amino groups via side chains divided by the number of total monomer units present in the original cationic polymer backbone (i.e., PEI polymer backbone). The grafting ratio will depend on the molecular weight of the cationic polymer, the chemical reactivity of the side chains grafted onto the polymer, or the biological effect obtained. The grafting ratio can be determined by measurement methods well known in the art, for example, by NMR.

[0077]

[0078] The PEI polymer backbone has the general formula (II):

[0079] in m 、 n and X are as defined herein.

[0080] In a specific embodiment of the present invention, the average molecular weight (Mw) of the PEI polymer backbone is 1 kDa to 50 kDa, preferably 5 kDa to 30 kDa or 10 kDa to 25 kDa, more preferably the average molecular weight (Mw) of the PEI polymer backbone is 8 kDa, 10 kDa, 15 kDa, 22 kDa, 25 kDa or 30 kDa, preferably 22 kDa.

[0081] In a specific embodiment of the present invention, in the general formula (I) of the PEI-based transfection reagent, the sum of m+n is 30 to 1200, preferably 200 to 600, and the average molecular weight (Mw) of the PEI polymer backbone is 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 kDa, 10 kDa, 15 kDa, 22 kDa, 25 kDa or 30 kDa, even more preferably 22 kDa.

[0082] In a specific embodiment of the present 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.

[0083] In another embodiment of the present invention, the sum of m+n is 30-1200, preferably 200-600.

[0084] In a preferred embodiment of the invention, the mean molecular weight (Mw) of the PEI polymer backbone is 8 kDa, 10 kDa, 15 kDa, 22 kDa, 25 kDa or 30 kDa, preferably 22 kDa. When the mean molecular weight (Mw) of the PEI polymer backbone is 8 kDa, 10 kDa, 15 kDa, 22 kDa, 25 kDa or 30 kDa, the sum of m+n is 180 kDa, 220 kDa, 340 kDa, 500 kDa, 570 kDa or 680 kDa, respectively.

[0085] In a preferred embodiment of the present invention, the PEI-based transfection reagent of general formula (I) is selected from the following compounds:

[0086]

[0087]

[0088]

[0089] In these compounds 1-34, the term "PEI" refers to linear PEI, and the term "bPEI" refers to branched PEI.

[0090] The compounds of general formula (I) can be prepared according to various methods known in the art, such as the methods disclosed in patent applications WO2021 / 023796 and WO2021 / 023798.

[0091] In a specific embodiment of the present invention, the biological matrix is ​​selected from: cell culture medium, in particular culture medium for eukaryotic cells, in particular culture medium for suspension cells or adherent cells, buffers, solutions used in the production and purification of recombinant viruses, and final compositions comprising purified viruses in a final formulation comprising a pharmaceutically acceptable buffer and excipients.

[0092] As defined herein, the term "cell culture medium" has the meaning known in the art and refers, for example, to a culture medium comprising at least one of the following components: serum, a synthetic culture medium, an animal component-free culture medium or a chemically defined culture medium, in particular a culture medium for maintaining cell survival or for growth, for differentiation or for expansion of cells, or for enhancing transfection.

[0093] As defined herein, the term "suspension cells" refers to cells that do not require a solid support for growth and are therefore not anchored. Examples of suspension cells include, but are not limited to, NSO cells, U937 cells, Namalawa cells, HL60 cells, WEHI231 cells, Yac1 cells, Jurkat cells, THP-1 cells, K562 cells, or U266B1 cells.

[0094] As defined herein, the term "adherent cell" refers to a cell that requires a solid support for growth and is 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, Hep G2 cells, SNN-BE(2) cells, BAE-1 cells, or SH-SY5Y cells.

[0095] As defined herein, the term "buffer" refers to a buffered solution comprising a buffering agent. As defined herein, the term "buffer" refers to an agent that regulates, maintains, or controls the pH of a solution. A buffering agent can be a weak acid or a weak base that comprises a buffered 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, potassium dihydrogen phosphate, potassium carbonate, potassium bicarbonate, potassium citrate, or magnesium oxide.

[0096] As defined herein, the expression "a solution used in the production and purification process of a recombinant virus" refers to any solution known in the art that can be used in the production and purification process of a recombinant virus.

[0097] As defined herein, the term "pharmaceutically acceptable buffer and excipient" refers to a pharmaceutically acceptable carrier, which is any substance or combination of substances that is physiologically acceptable and therefore nontoxic, i.e., suitable for use in compositions that are in contact with a host, particularly a human (particularly administered to a host). It can refer to any conventional type of solid, semisolid or liquid filler, diluent, encapsulating material or formulation aid. Examples of suitable acceptable excipients include, but are not limited to, glucose, galactose, lactose, dextrose, maltose, mannitol, sucrose, trehalose, polyethylene glycol or pluronic acid.

[0098] Another object of the present invention is to provide a method for purifying, detecting and / or quantifying a polyethyleneimine (PEI)-based transfection reagent of general formula (I) as defined herein, wherein the PEI-based transfection reagent of general formula (I) is contained in a liquid mixture containing a biological matrix,

[0099] wherein the biological matrix comprises recombinant viruses or virus-like particles produced using a PEI-based transfection reagent of general formula (I),

[0100] The method comprises the following steps:

[0101] (a) performing acid hydrolysis of the liquid mixture according to the method of the present invention,

[0102] (b) purifying the reaction mixture obtained in step (a) to obtain a purified PEI-based transfection reagent of general formula (I),

[0103] (c) detecting and / or quantifying the purified PEI-based transfection reagent of general formula (I) obtained in step (b).

[0104] As defined herein, the term "purified" means that the other components of a solution are separated or removed so that only the transfection reagent remains in the solution.

[0105] In a specific embodiment of the present invention, before performing step (a), the method includes a step of inactivating the recombinant virus or virus-like particle by heating the biological matrix at a temperature of 110°C to 130°C for 30 minutes to 4 hours, preferably at a temperature of 120°C for 30 minutes.

[0106] In a particular embodiment of the invention, step (b) is performed using ultrafiltration or centrifugation.

[0107] In a particular embodiment of the present invention, step (c) is performed using high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC) analytical techniques, preferably UHPLC.

[0108] In a specific embodiment of the present invention, the PEI-based transfection reagent of formula (I) of step (c) is detected with a limit of detection (LOD) of 1 ppm to 1000 ppm and / or a limit of quantification (LOQ) of 1 ppm to 1000 ppm.

[0109] In a specific embodiment of the present invention, the PEI-based transfection reagent of general formula (I) is detectable in a biological matrix during the production process of the recombinant virus, wherein the biological matrix is ​​selected from: cell culture medium, in particular culture medium for eukaryotic cells, in particular culture medium for suspension cells or adherent cells, buffers, solutions used during the production and purification of the recombinant virus, and final compositions comprising the purified virus in a final formulation comprising a pharmaceutically acceptable buffer and excipients.

[0110] In another embodiment of the present invention, the PEI-based transfection reagent is used in the manufacturing process of an advanced therapy medicinal product (ATMP) and is present in a residual amount (1 to 1000 ppm) relative to the transfection reagent and other components of the liquid mixture provided in the manufacturing process.

[0111] The terms "cell culture medium," "suspension cells," "adherent cells," "buffer," "solutions used in recombinant virus production and purification," and "pharmaceutically acceptable buffers and excipients" are defined in accordance with the above definitions and the examples provided herein.

[0112] In a specific embodiment of the present invention, the recombinant virus is selected from 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).

[0113] In a specific embodiment of the present invention, the PEI-based transfection reagent of general formula (I) is a compound selected from compounds 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 30, 31, 32, 33 and 34, and the recombinant virus is adeno-associated virus (AAV).

[0114] Other features and advantages of the present invention will become apparent from the following examples and will be illustrated in the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 : Transfection reagent of general formula (I).

[0116] Figure 2 : Synthesis of PEI via CROP.

[0117] Figure 3: Optimization of thermal inactivation of compound 05.

[0118] Figure 4: Figure 4a It is the hydrolysis of compound 05; Figure 4b yes Figure 4a overlay.

[0119] Figure 5 : Determination of the linearity of the hydrolysis of compound 05. Example

[0120] Example 1. General method for preparing grafted polymers

[0121]

[0122] Step 1: N-alkylation of the heterocycle

[0123] Under argon, in an oven-dried round-bottom flask, the corresponding heterocycle (1 equivalent) and DMF (2 ml / mmol raw material) were added. The solution was cooled to 0°C and sodium hydroxide (60% dispersion in mineral oil, 1.2 equivalents) was added portionwise. The mixture was slowly warmed to room temperature over 1 hour. The corresponding ester was then added dropwise and the reaction was stirred at room temperature for 4-12 hours. The mixture was quenched by adding 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 (EtOAc 20-50% solution in heptane).

[0124] Step 2: Saponification of the acid moiety

[0125] To a solution of the ester in EtOH (2 mL / mmol ester) was added a 5 M NaOH solution (0.2 mL / mmol ester) dropwise and the mixture was stirred at room temperature overnight. The solvent was then removed in vacuo and the residue was purified by column chromatography on SiO using either 5% MeOH in DCM + AcOH 1% or 0-100% acetonitrile in H O.

[0126] Step 3: Grafting

[0127] A solution of the cationic polymer (1 equivalent) in water (4 mL / mmol of starting material) was added to a round-bottom flask, followed by N-methylmorpholine or NMM (2 equivalents). A carboxylate (0.3-1 equivalent) was added, followed by MeOH (16 mL / mmol of polymer). After stirring for 10 minutes, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride or DMTMM (0.6-2 equivalents) was added, and the mixture was stirred at room temperature for 12-24 hours. The MeOH was then removed in vacuo, and water (4 mL / mmol of starting material) was added, followed by a 3M HCl solution (1 mL / mmol of starting material). The residue was purified using a dialysis cassette in a 50 mM HCl bath.

[0128] Step 4: Synthesis of triazoles by "click" chemistry starting from acids.

[0129]

[0130] Alkyne (1 equivalent), azide (1 equivalent), CuSO (0.01 equivalent) and sodium ascorbate (0.03 equivalent) were added to a 2:1 (v / v) solution of n-butanol and water. The reaction was stirred at room temperature for 24 hours. NaOH (5M, 2 equivalents) was then added and the organic solvent was removed in vacuo. The residue was purified by reverse phase flash chromatography using a 0-100% CH CN aqueous solution as eluent.

[0131] Step 5: Synthesis of triazoles via click chemistry starting from esters

[0132]

[0133] Alkyne (1 equivalent), azide (1 equivalent), CuSO (0.01 equivalent) and sodium ascorbate (0.03 equivalent) were added to a 2:1 (v / v) solution of n-butanol and water. The reaction was stirred at room temperature for 24 hours. NaOH (5M, 2 equivalents) was then added and the organic solvent was removed in vacuo. The residue was purified by reverse phase flash chromatography using a 0-100% CH CN aqueous solution as eluent.

[0134] Step 6: Saponification of the ester moiety.

[0135] To the EtOH solution of the ester was added dropwise a 3M solution of LiOH and the mixture was stirred at room temperature over the weekend. The solvent was then removed in vacuo and the residue was purified by reverse phase FC on SiO using a Biotage Flash purification system with H O / MeCN as eluent. The obtained acid was lyophilized to give a solid.

[0136] Example 2. Synthesis of PEI-based transfection reagents of the present invention

[0137] -Synthesis of compound 13 (polymer molecular weight = 22k, heterocyclic grafting = 10%)

[0138]

[0139] Intermediate 13a was prepared analogously to the general procedure, step 1 (Example 1). Yield = 60%; m = 2.30 g; 1 H 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).

[0140]

[0141] Intermediate 13b was prepared analogously to the general procedure, step 2 (Example 1). Yield = 45%; m = 1.00 g; 1 H 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).

[0142]

[0143] Compound 13 was prepared analogously to the general method, step 3 (Example 1). Yield = 94%; m = 117 mg; 1 H NMR (D2O) δ: 1 H 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).

[0144] -Synthesis of compound 14 (polymer molecular weight = 22k, heterocyclic grafting = 24%)

[0145]

[0146] Intermediate 14a was prepared analogously to the general procedure, step 1 (Example 1). Yield = 63%; m = 1.7 g; 1 H NMR (400MHz, chloroform-d) δ7.63(d,J=8.7Hz,0H),7.27(d,J=8.8Hz,1H),6.99–6.86(m,2H),4.27–4.15(m,4H),3.93(dd,J=7 .3, 1.3Hz, 3H), 2.65 (dd, J = 3.7, 1.3Hz, 3H), 2.43 (q, J = 6.8Hz, 2H), 2.18 (p, J = 7.1Hz, 2H), 1.33 (td, J = 7.2, 1.3Hz, 3H).

[0147]

[0148] Intermediate 14b was prepared analogously to General Procedure, Step 2 (Example 1). Yield = 100%; m = 676 mg; 1 H NMR (400MHz, methanol-d4) δ7.54(dd,J=32.8,8.9Hz,1H),7.18(dd,J=33.3,2.4Hz,1H),7.01(ddd,J=21.3,8.9,2.3Hz,1H ),4.33(q,J=6.8,6.3Hz,2H),3.92–3.84(m,3H),2.70(d,J=8.0Hz,3H),2.43(q,J=6.7Hz,2H),2.12(p,J=7.0Hz,2H)

[0149]

[0150] Compound 14 was prepared analogously to the 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).

[0151] -Synthesis of compound 15 (polymer molecular weight = 22k, heterocyclic grafting = 27%)

[0152]

[0153] Intermediate 15a was prepared analogously to the general procedure, step 1 (Example 1). Yield = 50%; m = 820 mg; 1H NMR (400MHz, chloroform-d) δ7.94–7.21(m,8H),7.05(d,J=1.3Hz,1H),5.08(d,J=1.3Hz,2H),4.05(td,J=8.1,7.5,2.9Hz,2H),3.92(dtd,J=1 6.2,7.8,6.5Hz,2H),2.50(dd,J=12.5,1.4Hz,3H),2.19(q,J=6.3Hz,2H),1.94(p,J=7.0Hz,2H),1.04(ddd,J=14.3,7.9,6.5Hz,3H).

[0154]

[0155] Intermediate 15b was prepared analogously to the general procedure, step 2 (Example 1). Yield = 73%; m = 580 mg; 1 H NMR(400MHz, methanol-d4)δ8.01(d,J=8.9Hz,1H),7.83–7.48(m,7H),4.34(tt,J=7.3,3.0H z,2H),2.71–2.65(m,2H),2.41(ddt,J=10.2,7.4,4.2Hz,2H),2.12(h,J=7.2Hz,2H).

[0156]

[0157] Compound 15 was prepared analogously to the general method, step 3 (Example 1). Yield = 84%; m = 171 mg; 1 H NMR (400 MHz, heavy water) δ 7.92–6.49 (m, 8H), 4.50–3.10 (m, 17H), 3.03–1.78 (m, 7H).

[0158] -Synthesis of compound 16 (polymer molecular weight = 22k, heterocyclic grafting = 30%)

[0159]

[0160] Intermediate 16a was prepared analogously to the 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).

[0161]

[0162] Intermediate 16b was prepared analogously to the 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).

[0163]

[0164] Compound 16 was prepared analogously to the 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).

[0165] -Synthesis of compound 17 (polymer molecular weight = 22k, heterocyclic grafting = 26%)

[0166]

[0167] Compound 17 was prepared analogously to the general method, 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).

[0168] -Synthesis of compound 18 (polymer molecular weight = 22k, heterocyclic grafting = 47%)

[0169]

[0170] Intermediate 18a was prepared analogously to the general procedure, step 1 (Example 1). Yield = 15%; m = 211 mg; 1H 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).

[0171]

[0172] Intermediate 18b was prepared analogously to the 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).

[0173]

[0174] Compound 18 was prepared analogously to the 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).

[0175] -Synthesis of compound 19 (polymer molecular weight = 22k, heterocyclic grafting = 25%)

[0176]

[0177] Intermediate 19a was prepared analogously to the general procedure, step 1 (Example 1). Yield = 43%; m = 602 mg; 1 H 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).

[0178]

[0179] Intermediate 19b was prepared analogously to the general procedure, step 2 (Example 1). Yield = 100%; m = 592 mg; 1 H NMR (400 MHz, heavy water) 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).

[0180]

[0181] Compound 19 was prepared analogously to the general procedure, step 3 (Example 1). Yield = 100%; m = 84 mg; 1 H NMR (400 MHz, heavy water) δ 9.67–8.10 (m, 4H), 4.23–3.06 (m, 16H), 2.86–1.90 (m, 4H).

[0182] -Synthesis of compound 20 (polymer molecular weight = 22k, heterocyclic grafting = 22%)

[0183]

[0184] Compound 20 was prepared analogously to the 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).

[0185] -Synthesis of compound 21 (polymer molecular weight = 22k, heterocyclic grafting = 21%)

[0186]

[0187] Compound 21 was prepared analogously to the general method, 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).

[0188] -Synthesis of compound 22 (polymer molecular weight = 22k, heterocyclic grafting = 21%)

[0189]

[0190] Compound 22 was prepared analogously to the 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).

[0191] -Synthesis of compound 23 (polymer molecular weight = 22k, heterocyclic grafting = 26%)

[0192]

[0193] Compound 23 was prepared analogously to the general method, 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).

[0194] -Synthesis of compound 24 (polymer molecular weight = 8k, heterocyclic grafting = 20%)

[0195]

[0196] Compound 24 was prepared analogously to the 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).

[0197] -Synthesis of compound 25 (polymer molecular weight = 10k, heterocyclic grafting = 20%)

[0198]

[0199] Compound 25 was prepared analogously to the 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).

[0200] -Synthesis of compound 26 (polymer molecular weight = 15k, heterocyclic grafting = 17%)

[0201]

[0202] Compound 26 was prepared analogously to the general procedure, step 3 (Example 1). Yield = 84%; m = 25 mg; 1H NMR (400 MHz, heavy water) δ 7.64–6.35 (m, 2H), 4.57–3.13 (m, 25H), 3.13–1.50 (m, 13H).

[0203] -Synthesis of compound 27 (polymer molecular weight = 30k, heterocyclic grafting = 18%)

[0204]

[0205] Compound 27 was prepared analogously to the 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).

[0206] -Synthesis of compound 28 (polymer molecular weight = 25k, heterocyclic grafting = 22%)

[0207] Compound 28 was prepared analogously to General Method 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).

[0208] -Synthesis of compound 29 (polymer molecular weight = 10k, heterocyclic grafting = 29%)

[0209] Compound 29 was prepared analogously to the general method, 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).

[0210] -Synthesis of compound 30 (polymer molecular weight = 22k, heterocyclic grafting = 35%)

[0211]

[0212] Intermediate 30a was prepared analogously to the general procedure, step 1 (Example 1). Yield = 17%; m = 1.00 g; 1H NMR (400MHz, chloroform-d) δ7.95(d,J=8.4Hz,1H),7.45(d,J=8.3Hz,1H),7.38(t,J=7.6Hz,1H),7.30–7.22(m ,1H),4.68–4.57(m,2H),4.01(qd,J=7.1,1.6Hz,2H),2.30–2.16(m,4H),1.13(td,J=7.1,1.6Hz,3H).

[0213]

[0214] Intermediate 30b was prepared analogously to General Procedure Step 2 (Example 1). Yield = 85%; m = 830 mg; 1 H NMR (400MHz, methanol-d4) δ8.03–7.96(m,1H),7.82(d,J=8.5Hz,1H),7.58(t,J=7.3Hz,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).

[0215]

[0216] Compound 30 was prepared analogously to the 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).

[0217] -Synthesis of compound 31 (polymer molecular weight = 22k, heterocyclic grafting = 35%)

[0218]

[0219] Intermediate 31a was prepared analogously to the 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).

[0220]

[0221] 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).

[0222]

[0223] Compound 31 was prepared analogously to the general method, 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).

[0224] -Synthesis of compound 32 (polymer molecular weight = 22k, heterocyclic grafting = 22%)

[0225]

[0226] Intermediate 32a was prepared analogously to the general procedure, step 1 (Example 1). Yield = 23%; m = 1.69 g; 1 H NMR (400MHz, chloroform-d) δ7.78 (dd, J=9.1, 1.9Hz, 1H), 7.34 (dd, J=9.1, 1.9Hz, 0H), 7.26 (d, J=2.3Hz,0H),7.05(dd,J=9.0,2.2Hz,0H),6.89(dd,J=9.1,2.2Hz,1H),6.73(d,J=2.2 Hz, 1H), 4.55 (dtd, J=13.6, 6.7, 1.9Hz, 2H), 4.01 (q, J=7.1Hz, 2H), 3.78 (dd, J=6.6, 1. 8Hz,3H),3.04(s,0H),2.22(ddd,J=19.8,7.7,4.2Hz,4H),1.13(td,J=7.1,1.8Hz,3H)

[0227]

[0228] Intermediate 32b was prepared analogously to General Procedure, Step 2 (Example 1). Yield = 70%; m = 1.16 g; 1H NMR (400MHz, methanol-d4) δ7.81(dd,J=9.1,2.7Hz,1H),7.67(dd,J=8.9,2.7Hz,0H),7.33(s,0H),7.24–7.15(m,1H),7.05(dd,J=9.1,2.7H z, 1H), 4.73 (qd, J = 7.1, 2.3Hz, 2H), 3.91 (dd, J = 13.2, 2.8Hz, 3H), 3.33 (d, J = 3.2Hz, 0H), 2.35 (t, J = 6.7Hz, 2H), 2.26 (t, J = 7.3Hz, 2H).

[0229]

[0230] Compound 32 was prepared analogously to the general method, 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).

[0231] -Synthesis of compound 33 (polymer molecular weight = 22k, heterocyclic grafting = 18%)

[0232]

[0233] Intermediate 33a was prepared analogously to the general procedure, step 1 (Example 1). Yield = 18%; m = 254 mg; 1 H NMR (400MHz, chloroform-d) δ8.53 (dd, J=4.4, 1.5Hz, 1H), 7.82 (dd, J=8.4, 1.5Hz, 1H), 7.24 (dd, J=8.4, 4. 4Hz, 1H), 4.54 (t, J = 6.8Hz, 2H), 3.89 (q, J = 7.1Hz, 2H), 2.20–2.05 (m, 4H), 1.01 (t, J = 7.1Hz, 3H).

[0234]

[0235] Intermediate 33b was prepared analogously to the general procedure, step 2 (Example 1). Yield = 98%; m = 242 mg; 1 H NMR (400 MHz, heavy water) 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).

[0236]

[0237] Compound 33 was prepared analogously to the general method, step 3 (Example 1). Yield = 75%; m = 54 mg; 1 H NMR (400 MHz, heavy water) δ 9.06–6.76 (m, 3H), 4.16–2.96 (m, 20H), 2.93–1.52 (m, 4H).

[0238] -Synthesis of compound 34 (polymer molecular weight = 22k, heterocyclic grafting = 17%)

[0239]

[0240] Intermediate 34a was prepared analogously to the general procedure, step 1 (Example 1). Yield = 52%; m = 600 mg; 1 H 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).

[0241]

[0242] 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).

[0243]

[0244] Compound 34 was prepared analogously to the general method, 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).

[0245] Example 3. Heat inactivation of viral vectors.

[0246] Heat inactivation of viral vectors is typically performed at around 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), 122-128; Gargi Maheshwari, et al., Journal of Virological Methods 2004, 118(2)141-146; Fabian Krieselet, Journal of Virological Methods 2020, 276, 113768). These inactivation methods typically take an hour. However, to avoid the addition of chemicals and reduce heating time, the inventors investigated heat inactivation at 120°C for 30 minutes.

[0247] Therefore, the stability of compound 05 was evaluated at 120°C for 24 hours. The overlay of different chromatograms at different time points is shown in Figure 3 No degradation was observed within 4 hours. After 6 hours, a peak was detected around the compound of interest, 05, indicating slight degradation. Based on this stability study, any residual reagents in the sample were not affected by the autoclave treatment during the 30-minute period.

[0248] Example 4. Optimization of acid hydrolysis.

[0249] The inventors studied three different parameters to define the optimal conditions under which compound 05 would not degrade:

[0250] Type of acid,

[0251] ·temperature,

[0252] Heating time.

[0253] Acid hydrolysis was performed with 20 ppm of compound 05 using HCl, H2SO4, and HNO3, which are three acids known to degrade proteins or nucleic acids.

[0254] To a distilled water solution of compound 05 (20 ppm) were added hydrochloric acid, sulfuric acid or nitric acid. The three mixtures were heated at 100°C overnight. For each acid, the following method was followed:

[0255] - Precision for the reference substance Compound 05 was performed in triplicate. The test consisted of hydrolyzing 20 ppm of the reference substance with different acids and treating on a centrifugal filter. The acid hydrolysis was performed over 46 hours with HCl (15 hours for the other acids).

[0256] - Recovery of viruses spiked with 20 ppm of compound 05. This parameter was determined with three injections.

[0257] The different results are summarized in Table 3, demonstrating that any conditions were able to avoid the degradation of compound 05. However, hydrochloric acid showed the best reproducibility compared to sulfuric acid or nitric acid.

[0258] Table 3. Recovery results of different acids used for acid hydrolysis.

[0259]

[0260] The concentration of HCl and the heating time are critical for acid hydrolysis. Therefore, the inventors evaluated these two criteria:

[0261] HCl concentration: 10%, 1%, 0.1%,

[0262] Heating time at 60°C, 80°C, and 100°C: t0, +1h, +2h, +3h, +4h, +5h, +6, and +24h.

[0263] Finally, the optimized conditions were fixed as acid hydrolysis in 0.1% hydrochloric acid solution at 100 °C for 2 h. Figure 4a 、 Figure 4b and Figure 5 The linearity in ΔΨ shows LOD = 1.97 and LOQ = 6.55.

[0264] Figure 4b Shown Figure 4a Overlay graph. 1 represents compound 5 at 110°C and 5 ppm; 2 represents compound 5 at 110°C and 10 ppm; 3 represents compound 5 at 110°C and 20 ppm; 4 represents compound 5 at 110°C and 25 ppm; 5 represents compound 5 at 110°C and 50 ppm; 6 represents compound 6 at 110°C and 75 ppm; 7 represents compound 5 at 110°C and 100 ppm; 8 represents compound 5 at 110°C and 150 ppm.

[0265] Example 5. Qualitative and quantitative analysis of residual products.

[0266] The HPLC method is described below in Table 4. This analytical method was developed throughout the development of the present invention due to two main issues:

[0267] - the presence of carry-over that may affect the quantitative amount,

[0268] - The symmetry of the different peaks is not optimal considering the potential validation of the method.

[0269] The inventors have carried out different " in-house " tests, in order to solve the carryover problem by changing different parameters such as column type (grafted C4 versus C18, AX versus Peptide), mobile phase composition, gradient slope and flow rate. After these different screenings, a new analytical method was obtained and is described in Table 4 below.

[0270] Table 4. Description of analytical methods used during the development of the residue tests.

[0271]

[0272]

[0273] Example 6. Experimental part.

[0274] The inventors demonstrated the efficiency of the method for a variety of transfection reagents (e.g. described in WO2021 / 023796; WO2021 / 023798), which showed strong activity in transfection, in particular for the production of viral vectors.

[0275] The inventors studied the stability of all transfection reagent compounds 01 to 34 under the above acid hydrolysis (Table 5). Most molecules are completely stable under acidic conditions. In addition, based on the limit of detection (LOD) and limit of quantification (LOQ) studies, the analytical method is applicable to a wide variety of PEI-based polymers.

[0276] Table 5. Examples of polymers used in the present invention (nd = not detected, Experiment: Transfection reagent (20 ppm) was heated in HCl (2 mL, 0.1%) solution at 110°C for 2 hours. After cooling, the experiment was directly analyzed by HPLC.)

[0277]

[0278]

[0279]

[0280]

[0281] The inventors then applied the entire residue test described above to a selection of molecules listed in Table 6 below.

[0282] Table 6. Residual test. nd = not detected; Method of incorporation before hydrolysis (addition of known amount of transfection agent; Araujo, Journal of Chromatography B, 2009, 877(23), 2224-2234): HCl solution (0.37%, 108 μL), a certain volume of pure water (92 μL), and transfection agent solution (concentration: 100 ppm, 80 μL) were added to a viral vector (AAV, 200 μL) solution.

[0283]

[0284]

[0285] The tube was sealed and then heated at 100°C for 2 hours. After cooling to room temperature, the mixture was diluted in 3.5 mL of pure water and then filtered using a centrifugal filter with a minimum cutoff of 1 kDa at 5000 g for 30 minutes to obtain 250-300 μL of solution. The solution was diluted to 4 mL and filtered at 5000 g for 60 minutes. The same procedure was repeated two more times. The final retentate was diluted with pure water to obtain 500 μL of solution, which was analyzed by HPLC.

[0286] Post-hydrolysis incorporation method: HCl solution (0.37%, 108 μL), a certain volume of pure water (92 μL), and a transfection reagent solution (concentration: 100 ppm, 80 μL) were added to a viral vector (AAV, 200 μL) solution. The tube was sealed and then heated at 100°C for 2 hours. After cooling, the transfection reagent solution (concentration: 100 ppm, 80 μL) was added before the filtration process.

[0287] Examples of buffers used with Compound 17 are disclosed in Table 7.

[0288] Table 7. Examples of buffers containing compound 17.

[0289]

[0290]

[0291]

Claims

1. A method for acid hydrolysis of a liquid mixture comprising a biological matrix and a polyethyleneimine (PEI)-based transfection reagent, wherein the biological matrix comprises adeno-associated virus (AAV) produced using a PEI-based transfection reagent, The method comprises the steps of incubating the liquid mixture containing the biological matrix under the following conditions: incubating in an aqueous solution containing 0.1% (v / v) HCl at a temperature of 110° C. for 2 hours, wherein the acid hydrolysis does not degrade the PEI-based transfection reagent, and The PEI-based transfection reagent has the general formula (I) or an acceptable salt thereof: in: - m represents an integer between 198 and 300, and n represents an integer between 2 and 300, provided that n is lower than m and the sum of m+n is in the range of 200 to 600, -X means H, -p represents an integer between 1 and 4, -Z represents a group of the formula: - Y0, Y1, Y2, Y3 and Y4 may be the same or different and represent C or N, provided that at least two, but 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 represent H, methyl, cyclopropyl, isopropyl, tert-butyl, phenyl, benzyl, 2-pyridine, 3-pyridine or 4-hydroxyphenethyl; or wherein (i) W1 and W2 or (ii) W2 and W3 or (iii) W3 and W4 together form a fused phenyl; a fused phenyl substituted by methyl, methoxy, carboxyphenyl or Cl; a fused naphthalene; a fused 2-pyridine; or a fused 3-pyridine, The condition is that at least one, but no more than two, of W1, W2, W3 and W4 do not exist.

2. The method according to claim 1, wherein the PEI-based transfection reagent of general formula (I) has a grafting ratio defined as (n / (m+n))*100, and wherein the grafting ratio is 1 to 50%. The method according to claim 2 , wherein the grafting ratio is 5 to 30%. The method according to claim 3 , wherein the grafting ratio is 20%. The method of claim 1 , wherein the average molecular weight (Mw) of the PEI polymer backbone is from 1 kDa to 50 kDa. The method according to claim 5 , wherein the average molecular weight (Mw) of the PEI polymer backbone is from 5 kDa to 30 kDa.

7. The method of claim 6, wherein the average molecular weight (Mw) of the PEI polymer backbone is from 10 kDa to 25 kDa.

8. The method of claim 6, wherein the average molecular weight (Mw) of the PEI polymer backbone is 8 kDa, 10 kDa, 15 kDa, 22 kDa or 30 kDa.

9. The method of claim 8, wherein the average molecular weight (Mw) of the PEI polymer backbone is 22 kDa.

10. The method according to claim 1, wherein the PEI-based transfection reagent of general formula (I) is selected from the following compounds:

11. The method of claim 1 , wherein the biological matrix is ​​selected from the group consisting of: cell culture medium, buffers, solutions used during the manufacture and purification of AAV, and final compositions comprising purified manufactured AAV in a final formulation comprising a pharmaceutically acceptable buffer and excipients. The method according to claim 11 , wherein the cell culture medium is a culture medium for eukaryotic cells.

13. The method of claim 12, wherein the eukaryotic cells are suspension cells or adherent cells.

14. A method for purifying, detecting and / or quantifying a polyethyleneimine (PEI)-based transfection reagent, wherein the polyethyleneimine (PEI)-based transfection reagent is a PEI-based transfection reagent of the general formula (I) as defined in the method of claim 1, wherein the PEI-based transfection reagent of the general formula (I) is contained in a liquid mixture containing a biological matrix, wherein the biological matrix comprises adeno-associated virus (AAV) produced using a PEI-based transfection reagent of general formula (I), The method comprises the following steps: (a) carrying out acid hydrolysis of the liquid mixture according to the method of claim 1, (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).

15. The method of claim 14, wherein step (c) is performed using high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC) analytical techniques.

16. The method of claim 15, wherein step (c) is performed using UHPLC.

17. The method of claim 14, wherein the PEI-based transfection reagent of general formula (I) of step (c) is detected at a limit of detection (LOD) of 1 ppm-1000 ppm and / or a limit of quantification (LOQ) of 1 ppm-1000 ppm.

18. The method of claim 1, wherein the PEI-based transfection reagent of formula (I) is detectable in a biological matrix during the manufacturing process of AAV, wherein the biological matrix is ​​selected from the group consisting of: cell culture medium, buffer, solutions used during the manufacturing and purification of AAV, and a final composition comprising purified AAV in a final formulation comprising a pharmaceutically acceptable buffer and excipients. The method according to claim 18 , wherein the cell culture medium is a culture medium for eukaryotic cells.

20. The method of claim 19, wherein the eukaryotic cells are suspension cells or adherent cells.

21. The method of claim 10, wherein the PEI-based transfection reagent of general formula (I) is a compound selected from compounds 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 30, 31, 32, 33 and 34.

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