Two-step cell lysis for release of viral vectors

Abstract

The present invention provides a method for releasing viral vectors from cell culture. Specifically, a cell culture is contacted with a lysis reagent to produce a lysate composition and incubated, and then the lysate composition is contacted with an acidifying reagent to reduce the pH. The method according to the present invention allows for a high release of viral vectors and reduces the co-release of host cell related impurities. The present invention also provides a method for producing viral vectors. The present invention also provides a kit for releasing viral vectors from cell culture. The present invention also provides a kit for producing viral vectors.

Description

Technical Field

[0001] This invention provides a method for releasing a viral vector from a cell culture. Specifically, the cell culture is contacted with a lysis reagent to generate a lysis composition and incubated, and then the lysis composition is contacted with an acidifying agent to lower the pH. The method according to the invention results in a high viral vector release and reduces co-released host cell-related impurities. This invention also provides a method for producing a viral vector. This invention further provides a kit for releasing a viral vector from a cell culture. This invention also provides a kit for producing a viral vector. Background Technology

[0002] In recent years, viral vectors have been developed as an important means of gene therapy, capable of repairing dysfunctional DNA sequences or introducing compensatory alterations to restore normal cellular physiological function. Well-known examples of viral vectors from which recombinant viral vectors are derived include enveloped viruses, such as retroviruses, lentiviruses, Sendai viruses, and herpesviruses; and non-enveloped viruses, such as adenoviruses and adeno-associated viruses (hereinafter referred to as AAVs). Gene therapy via gamma retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs) is attractive because viruses have the natural ability to enter cells and deliver genetic material. Specifically, vectors based on recombinant AAVs (rAAVs) are suitable for gene therapy applications and AAV vector-mediated gene delivery, and have recently received medical approval for, for example, the treatment of inherited blindness and spinal muscular atrophy, and have shown long-term therapeutic effects for other rare diseases, including hemophilia and Duchenne muscular dystrophy.

[0003] Recombinant viral vectors based on AAV are excellent vectors for in vivo gene therapy because they exhibit broad tropism, are non-pathogenic to humans, and achieve long-term transgene expression stability without requiring genome integration. AAV can infect a variety of cell types, including human cells, and can even infect non-dividing cells that have completed differentiation, including blood cells, muscle cells, and nerve cells. The wild-type AAV genome contains inverted terminal repeats (ITRs) flanked by Rep and Cap genes, which encode genome replication and packaging proteins, as well as capsid proteins. AAV vectors typically have Rep and Cap replaced by the target gene, thus making them replication-deficient and retaining only the ITR for replication and genome packaging. AAV viral particles are also physicochemically stable. For these reasons, AAV has recently gained attention for its practical value as a gene transfer vector used in gene therapy to treat congenital genetic diseases as well as cancer or infections.

[0004] In most cases, viral vectors (such as AAV, adenovirus, and lentivirus-derived vectors) are produced in host cells (e.g., mammalian cell lines). Typically, a method for producing recombinant viral vectors involves introducing genetic elements for forming a viral vector in the form of one or more nucleic acid constructs into a host cell to generate a cell capable of producing a viral vector, and culturing that cell to express the elements used to form the viral vector. Among available cell lines, HEK293 cells or HEK293-derived cells are widely used because they are well-characterized and very easy to transfect. A significant advantage of mammalian cell lines, particularly human-derived cell lines, is their ability to confer specific post-translational modifications (PTMs) onto the vector capsid. These PTMs can influence in vivo stability, infectivity, and immunogenicity, making them critical quality parameters.

[0005] AAV-based drugs are currently the most expensive drugs in the world, with Glybera costing $1.2 million per patient and Zolgensma costing $2 million per patient. The high price of AAV-based drugs is primarily due to their high current manufacturing costs, necessitating process optimization for cost-effective production. In the past, researchers have increasingly proposed various methods for process optimization in viral vector production. In addition to optimizing classic biotechnological parameters in the production process (i.e., upstream processing), transfection processes have been optimized to increase viral vector yield. Furthermore, downstream processes are being continuously improved to separate viral vectors from various impurities generated throughout the upstream production process, aiming to achieve even higher yields.

[0006] Since most of the generated viral vectors remain inside the cell in most cases, it is necessary to break down or disrupt the outer boundary or cell membrane. This release process is called cell lysis. To obtain maximum yield, the cells need to be completely lysed so that essentially intact intracellular DNA, RNA, and proteins can be obtained. Various methods exist in the field of cell lysis, including mechanical methods (such as sonication and high-pressure homogenization), physical methods (such as freeze-thaw cycles), and chemical methods. However, each method has its own advantages and limitations. While mechanical methods of cell lysis are effective at disrupting cells, scaling up can be challenging and may generate heat, leading to denaturation of target molecules or aggregation of viral vectors. Furthermore, these methods often require specialized equipment and can be labor-intensive. Freeze-thaw methods for cell lysis are relatively simple. However, they are not easily scalable and may therefore be unsuitable for large-scale production, as they rely on repeated freeze-thaw cycles, which are time-consuming and impractical for high-volume production. Chemical methods of cell lysis are scalable and versatile because they can be formulated to suit different cell types and target molecules. However, in the field of viral vectors such as AAV, chemical lysis compositions remain relatively inefficient, requiring multiple steps, such as initial cell separation and washing, long incubation times, and sequential nuclease treatment. Furthermore, current lysis compositions and protocols can result in high levels of co-extracted impurities (e.g., host cell nucleic acids), making downstream purification more challenging and reducing yields. Finally, many commonly used cell lysis reagents for AAV vector extraction contain 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (known as "Triton X-100") or their derivatives, which are known to be biohazardous compounds listed in the REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) list and are restricted or prohibited in the EU. In addition to the aforementioned chemical lysis methods, combining chemical lysis with existing mechanical techniques (e.g., microfluidics) or physical methods (e.g., freeze-thaw) has been considered an effective cell lysis technique. However, while mechanical methods of cell lysis effectively destroy cells, they can lead to product loss due to shear stress-induced aggregation. Moreover, these methods typically require specialized equipment. On the other hand, combining chemical methods with freeze-thaw techniques is not an ideal solution either. Although the freeze-thaw method for cell lysis is relatively simple, it is not easy to scale up and may therefore be unsuitable for large-scale production because it relies on repeated freeze-thaw cycles, which is time-consuming and impractical for mass production.

[0007] Therefore, there remains a pressing need to improve viral vector production, particularly by improving the release of viral vectors from cell cultures. Specifically, there is a need to provide simplified cell lysis workflows without sacrificing yield, or even potentially increasing yield. Additionally, there is a need to reduce the time required for cell lysis so that the released viral vectors can be more readily used for downstream purification.

[0008] Therefore, the object of this invention is to improve cell lysis. Specifically, the object of this invention is to increase the amount of virus released, i.e., to increase the vector titer, such as the capsid and / or transducing viral vector titer. Furthermore, the object of this invention may be to reduce impurities released during cell lysis and / or to improve the removal of impurities released along with the viral vector. The object of this invention may also be to shorten the time required for lysis and / or increase the applicability to various sample types, thereby simplifying downstream processing. Summary of the Invention

[0009] This invention addresses the aforementioned needs. It provides a method for releasing viral vectors from cell cultures, wherein a lysis reagent is first provided, which is then contacted with the cell culture to generate a lysis composition and incubated, thereby releasing the viral vector from the cell culture. Subsequently, the lysis composition is contacted with an acidifying agent to lower the pH. The disclosed method advantageously increases the extraction yield of viral vectors by improving the efficiency of cell lysis while simultaneously achieving improved removal of host cell-related impurities. Furthermore, the method is flexibly applicable to a wide range of samples and sample sizes, including cell culture media and even cell pellets, thus possessing broad applicability. Finally, the presence of biohazardous components or process steps is reduced to ensure operator and environmental safety. The cell lysis method and kit according to this disclosure are carefully screened to obtain optimal conditions compared to existing cell lysis reagent formulations and techniques.

[0010] According to a first aspect, a method for releasing a viral vector from a cell culture is provided, the method comprising the following steps: (a) Provide lysis reagents; (b) Contacting cell cultures with a lysis reagent to produce a lysis composition; (c) Incubating the lysis composition to release the viral vector from the cell culture; and (d) Contact the pyrolysis composition with an acidifying agent to lower the pH.

[0011] The method according to the first aspect advantageously enables efficient release of viral vectors while maintaining low levels of host cell-related impurities, such as host cell proteins. Specifically, the lysis method disclosed herein divides the process into two short lysis steps (also referred to herein as "two-step cell lysis" or "two-step lysis") and applies different advantageous conditions in the cell culture medium that cannot be combined in a single step. The inventors have found that a single-step lysis method using an alkaline lysis reagent yields high viral vector yields, but also relatively high levels of host cell-related impurities, while a single-step lysis method using an acidic lysis reagent yields moderate viral vector yields, but with higher purity and, arguably, fewer host cell-related impurities. Surprisingly, by dividing the release of the viral vector into two short lysis steps, the advantageous conditions of the single-step lysis method can be combined. Therefore, high yields of viral vectors can be obtained without compromising purity or requiring more complex or longer downstream purification. Furthermore, the method according to the first aspect of this disclosure has the advantage of being able to be carried out directly in culture vessels such as bioreactors. Therefore, there is no need to transfer or further process (e.g., centrifuge) the cell culture to facilitate the release of the viral vector. Conversely, the lysis reagent can be directly contacted with cell cultures in culture vessels such as bioreactors to generate a lysis composition, followed by steps (c) and (d). Furthermore, the method according to the first aspect can shorten the lysis time, thereby achieving a more efficient lysis process. Finally, advantageously, the two-step method can precipitate impurities, particularly host cell-related impurities such as nucleic acids and / or proteins, which improves downstream purification. In addition, the precipitate enhances filtration capacity.

[0012] According to the second aspect, a method for producing a viral vector is provided, the method comprising the following steps: x.1 Provide cell cultures capable of producing viral vectors; x.2 Cultured cell cultures to produce viral vectors; x.3 According to the method of the first aspect of the invention, a viral vector is released from a cell culture; and x.4 Optionally, purify the released viral vector.

[0013] According to a third aspect, a kit for releasing a viral vector from a cell culture is provided, the kit comprising: (i) lysis reagents; and (ii) Acidifying reagents.

[0014] According to the fourth aspect, a kit for producing viral vectors is provided, the kit comprising: (i) The kit according to the third aspect of the present invention; (ii) Either of the following two: I. Mammalian cells capable of producing viral vectors, which are modified by genome editing of mammalian cells using one or more nucleic acid molecules for viral vector production, thereby enabling stable production of viral vectors; or II. Mammalian cells that can be modified with one or more viral vector production plasmids to produce transient viral transfection, thereby enabling the transient production of viral vectors.

[0015] Other aspects of the invention are disclosed below. Other objects, features, advantages, and aspects of this application will become apparent to those skilled in the art from the following description and appended claims. However, it should be understood that while the following description, appended claims, and specific embodiments illustrate preferred embodiments of the application, they are given by way of illustration only. Attached Figure Description

[0016] Some exemplary embodiments of this disclosure will now be described with reference to the accompanying drawings.

[0017] Figure 1 An exemplary experimental setup is shown for integrating the methods disclosed herein into a biological processing workflow, including downstream processing.

[0018] Figure 2 An image shows the aseptic filtration of crude lysate from HEK293 cells producing the AAV2 vector through a 0.22 µm CA Spin-X filter (Sigma Aldrich). A table shows the variables for the cell lysis conditions (pH, salt molar concentration, incubation time, and detergent addition), where N is an identification number for AAV2 production and cell lysis conditions, with N2, N5, and N6 relating to the acidic lysis reagent according to this disclosure, and N25 relating to the comparison condition at pH 8. The cell lysis assay setup shown was performed without a nuclease digestion step.

[0019] Figure 3 SDS-PAGE analysis of crude lysates from HEK293 cells used to produce the AAV2 vector under reducing and denaturing conditions was performed using sterile filtered gels. Protein bands on the gels were visualized by Coomassie blue staining. Variables of cell lysis conditions (pH, salt molar concentration, incubation time, and detergent addition) are shown in the table above the gel images, where N is an identification number for AAV2 production and cell lysis conditions, and N1, N5, and N9 relate to the acidic lysis reagent according to this disclosure, compared with lysis reagents at pH 8 and pH 9. M in lanes 1 and 13 stands for PageRuler. TM Unstained protein molecular weight standard (Thermo Fisher Scientific).

[0020] Figure 4 It shows the use of Incucyte ® A live-cell analysis system was used to perform transduction assays, determining the functional titer of AAV2 after cell lysis and clarification in the cell culture medium (see Group A, Whole Culture Lysis). The N designations are identification numbers for AAV2 production and cell lysis conditions, where N70, N71, N72, and N73 relate to acidic lysis reagents at pH 2, N74, N75, N76, and N77 relate to acidic lysis reagents at pH 4, and N78, N79, N80, and N81 relate to alkaline lysis reagents at pH 8 according to this disclosure. As a reference lysis reagent, the "Prior Art Cell Lysis Reagent" (N82) and CelLytic were used. TM M(C1), CelLytic TM MT (C2; both are Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. Error bars represent the standard deviation of the mean (n=3).

[0021] Figure 5 The use of Octet is shown ® The AAVX biosensor determines the AAV2 capsid titer after cell lysis and clarification in cell culture medium (see Group A, Whole Culture Lysis). The N designation refers to the identification number of the cell lysis condition, where N70, N71, N72, and N73 relate to an acidic lysis reagent at pH 2, N74, N75, N76, and N77 relate to an acidic lysis reagent at pH 4, and N78, N79, N80, and N81 relate to an alkaline lysis reagent at pH 8 according to this disclosure. As a reference lysis reagent, the "Prior Art Cell Lysis Reagent" (N82) and CelLytic were used. TM M(C1), CelLytic TM MT (C2; both are Sigma-Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. Error bars represent the standard deviation of the mean (n=3).

[0022] Figure 6 It shows the use of Incucyte ®A live-cell analysis system was used to perform transduction assays, determining the functional titer of AAV2 after cell lysis and clarification of the cell pellet (see Group A, Cell Pellet Lysis). The N designation refers to the identification number for AAV2 production and cell lysis conditions, where N86, N87, N88, and N89 relate to an acidic lysis reagent at pH 2 according to this disclosure; N90, N91, N92, and N93 relate to an acidic lysis reagent at pH 4 according to this disclosure; and N94, N95, N96, and N97 relate to an alkaline lysis reagent at pH 8 according to this disclosure. As a reference lysis reagent, the "Prior Art Cell Lysis Reagent" (N98) and CelLytic were used. TM M (C1, Sigma Aldrich), CelLytic TM MT (C2, Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. Error bars represent the standard deviation of the mean (n=3).

[0023] Figure 7 The use of Octet is shown ® The AAVX biosensor determines the AAV2 capsid titer after cell lysis and clarification of a cell pellet (see Group A, Cell Pellet Lysis). The N designation refers to the identification number of the AAV2 production and cell lysis conditions, where N86, N87, N88, and N89 relate to an acidic lysis reagent at pH 2 according to this disclosure, N90, N91, N92, and N93 relate to an acidic lysis reagent at pH 4 according to this disclosure, and N94, N95, N96, and N97 relate to an alkaline lysis reagent at pH 8 according to this disclosure. As a reference lysis reagent, the "Prior Art Cell Lysis Reagent" (N98) and CelLytic were used. TM M (C1, Sigma Aldrich), CelLytic TM MT (C2, Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. Error bars represent the standard deviation of the mean (n=3).

[0024] Figure 8 It shows the use of Incucyte ®A functional titer of AAV2 was determined by transduction assays performed using a live-cell analysis system after cell lysis and clarification in the cell culture medium (see Group B, Whole Culture Lysis). The N designation refers to the identification number of the AAV2 production and cell lysis conditions, where N70A relates to an acidic lysis reagent at pH 2.75, N74A to an acidic lysis reagent at pH 4, and N78A to an alkaline lysis reagent at pH 8. Prior art cell lysis reagents (N82) and lysis reagents containing Triton X100 (TRT) were used as reference lysis reagents. Error bars represent the standard deviation of the mean (n=3).

[0025] Figure 9 The use of Octet is shown ® The AAVX biosensor determines the AAV2 capsid titer after cell lysis and clarification in cell culture medium (see Group B, Whole Culture Lysis). The N designation identifies the AAV2 production and cell lysis conditions, with N70A relating to an acidic lysis reagent at pH 2.75, N74A to an acidic lysis reagent at pH 4, and N78A to an alkaline lysis reagent at pH 8. Prior art cell lysis reagents (N82) and those containing Triton X100 (TRT) were used as reference lysis reagents. Error bars represent the standard deviation of the mean (n=3).

[0026] Figure 10 Phase confluence of adherent HEK293 cells transduced with sterile filtered crude AAV2 samples diluted between 1:10 and 1:320 is shown. Transduction assays were performed using two AAV2 sample types, one lysed with a lysis reagent containing Triton X100 (TRT) and the other with a basic lysis reagent containing Tween 20 (N78, LR2). Incucyte was used for the assay. ® Images captured by a live cell analysis system.

[0027] Figure 11 The results of selective two-step lysis using HEK cell culture to produce AAV8 viral vector as a model are shown. The effects of different lysis reagent solutions and nuclease additions (shown in the table below) on cell viability [A], DNA impurities [B], and host cell protein (HCP) impurities [C] were tested.

[0028] Figure 12 The total cell concentration and viability determined after lysis screening are shown to compare different single-step lysis methods versus two-step lysis methods (S9) using HEK cell cultures as a model for producing adenovirus vectors. Samples S0 and S1 represent unlysolved cell cultures as references.

[0029] Figure 13 Octet with a suitable AV biosensor was demonstrated after cell lysis and centrifugation. ® Determined adenovirus (AV) capsid titer. Samples S0 and S1 represent unlysed cell culture samples used as references. Different single-step lysis methods were compared with a two-step lysis method (S9). The presence of AV capsid aggregates in the samples was compared by measuring the sample supernatant directly after centrifugation (without filtration) and by measuring the sample supernatant after centrifugation and filtration through a 0.2 μm syringe.

[0030] Figure 14 The relative amounts of DNA impurities associated with the amount of adenovirus (AV) capsid determined after cell lysis and centrifugation are shown. Samples S0 and S1 represent unlyzed cell culture samples used as references. Different single-step lysis methods were compared with a two-step lysis method (S9). Sample supernatants were measured directly after centrifugation (without filtration) and after centrifugation followed by filtration through a 0.2 μm syringe (filtered) to compare the presence of impurity aggregates in the samples.

[0031] Figure 15 The relative host cell impurities (HCPs) levels related to the amount of adenovirus (AV) capsid determined after cell lysis and centrifugation are shown. Samples S0 and S1 represent unlysed cell culture samples used as references. Different single-step lysis methods were compared with a two-step lysis method (S9). The sample supernatant was measured directly after centrifugation (without filtration) and after centrifugation followed by filtration through a 0.2 μm syringe (filtered) to compare the presence of impurity aggregates in the samples. Detailed Implementation

[0032] The following description is intended to enhance understanding of this disclosure and should be understood as supplementing the description of exemplary embodiments of this disclosure provided in the foregoing sections of this specification, and should be read in conjunction with the description of exemplary embodiments of this disclosure provided in the foregoing sections of this specification. It should be understood that the invention is not limited to the specific embodiments, methods, schemes, and reagents described herein, as they may vary within the scope set forth in the claims. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the scope of the invention as defined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0033] In the following description, some elements of the invention will be described. These elements may be discussed with particular embodiments; however, it should be understood that they can be combined in any manner and in any number to obtain additional embodiments. The various described embodiments, features, and particular embodiments should not be construed as limiting the invention to the explicitly described embodiments or combinations of explicitly described features. This description should be understood as disclosure and covers embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation and combination of all descriptive elements in this application should be considered as disclosed in this specification.

[0034] As explained in the description of the invention, the various aspects and embodiments of the invention disclosed herein make important contributions to the art by providing improved viral vector release and production.

[0035] Method for releasing viral vectors from cell cultures

[0036] According to a first aspect, a method for releasing a viral vector from a cell culture is provided, the method comprising the following steps: (a) Provide lysis reagents; (b) Contacting cell cultures with a lysis reagent to produce a lysis composition; (c) Incubating the lysis composition to release the viral vector from the cell culture; and (d) Contact the pyrolysis composition with an acidifying agent to lower the pH.

[0037] The method according to the first aspect enables efficient release of the viral vector while maintaining low levels of host cell-related impurities, such as, in particular, host cell proteins. Specifically, the lysis method disclosed herein divides the process into two short lysis steps (also referred to herein as “two-step cell lysis” or “two-step lysis”), and applies different advantageous conditions in the cell culture medium that cannot be combined in a single step (see Exemplary). Figure 1 The inventors discovered that the single-step lysis method using an alkaline lysis reagent yielded high viral vector yields, but also relatively high levels of host cell-related impurities. In contrast, the single-step lysis method using an acidic lysis reagent yielded moderate viral vector yields, but with higher purity and, in other words, fewer host cell-related impurities (see [link to original text]). Figures 2 to 9 Surprisingly, by dividing the release of the viral vector into two short lysis steps, the advantages of the single-step lysis method can be combined (see...). Figures 11 to 15 Therefore, high yields of viral vectors can be obtained without compromising purity or eliminating the need for more complex or lengthy downstream purification. Simultaneously, biohazardous components or process steps are excluded to ensure operator and environmental safety (see [link]). Figures 8 to 10Furthermore, the advantage of the method according to the first aspect is that it can be carried out directly in culture dishes such as bioreactors, since the lysis reagent and acidifying reagent can both contact the cell culture and the lysis composition separately in the culture dish (see Example 8). This significantly simplifies the release of viral vectors, as there is no need to transfer the cell culture, and therefore no need for any processing steps such as sedimentation or centrifugation. Thus, the method according to the first aspect provides flexibility and simplification in the process of releasing viral vectors (such as AAV or adenovirus) from cell cultures. In addition, the method according to the first aspect can shorten the lysis time, thereby achieving a more efficient lysis process, for example, less than 2 hours, such as 70 minutes (see Example 4). Finally, advantageously, the two-step method can precipitate impurities, for example, particularly host cell-related impurities such as nucleic acids and / or proteins, which improves downstream purification. Furthermore, the precipitate improves filtration capacity.

[0038] The steps and preferred implementation schemes of the method according to the first aspect will now be described in detail.

[0039] Viral vector

[0040] The viral vectors according to this disclosure are any viral material suitable for delivering genetic material into cells (e.g., used alone or in combination with other viruses or biochemical signals). In particular, as used herein, the terms "virus (vector)," "viral vector," and "gene delivery vector" refer to viral particles that act as nucleic acid delivery vectors, containing nucleic acid molecules packaged within the viral particles. Viral vectors are suitable for gene therapy applications, i.e., the ability to use nucleic acids to repair dysfunctional DNA sequences or introduce compensatory alterations, thereby restoring normal physiological function of cells. This gene delivery is also known as transduction. Typically, viral vectors used for gene therapy are recombinant and therefore contain one or more target genes, also known as "transgenic" genes. Thus, according to a preferred embodiment, the viral vector is a recombinant viral vector. The target gene can be transduced into cells, such as patient cells, autologous cells, or allogeneic cells, which can occur in vivo, in vitro, or ex vivo. One or more target genes can then be used directly in cells as non-integrating vectors for protein expression, which may degrade naturally over time or integrate into the cell's nuclear DNA. As used herein, a “transgenic” or “target gene” is a nucleic acid introduced into the genome, including but not limited to: genes or nucleic acids whose sequences are not normally present in viral vector genes (e.g., AAV or adenovirus); genes or nucleic acids whose sequences are present in viral vector genomes (e.g., AAV or adenovirus genomes) but are not normally transcribed and translated (“expressed”) in viral vector genomes (e.g., AAV or adenovirus genomes); or any other gene or nucleic acid that is desired to be located between the ITR sequences of a viral vector repetitive sequence such as AAV. Transgenics may include one or more transcriptional regulatory sequences and any other nucleic acids, such as introns, which may be necessary for optimal expression of the selected nucleic acid. The length of the transgene can be only a few nucleotides, but a preferred length is at least about 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 21. Transgenic sequences can contain 0, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, or 4600 nucleotides (nt). Transgenic sequences can contain either coding or non-coding sequences.

[0041] Importantly, as shown in the following examples, the method for releasing viral vectors according to this disclosure is applicable to a variety of viral vectors and is not limited to a specific type. Specifically, the method according to the first aspect of this disclosure has been used to release different AAV serotypes and adenoviruses, demonstrating broad applicability.

[0042] "Releasing a viral vector" refers to a process in which a viral vector produced from cells of a cell culture can be used in further process steps, such as downstream purification and / or analysis. Typically, viral vectors are primarily located within the cells that produce them, and therefore must be released to make them usable in any subsequent process steps. For this purpose, the cell membrane is usually broken down, i.e., the cells undergo lysis. Therefore, methods for releasing viral vectors from cell cultures include lysing the cells of the cell culture; for example, this method can also refer to methods for lysing the cells of a cell culture to release the viral vector.

[0043] Many viruses are well-suited as viral vectors for gene therapy, as is known to those skilled in the art; see, for example, Roldao et al., 2017, Comprehensive Biotechnology, vol. 1, pp. 633-656. The viral vector can be selected from retroviruses, such as lentiviruses, adenoviruses, herpes simplex virus, vaccinia virus, and adeno-associated virus (AAV). According to a preferred embodiment, the viral vector is non-enveloped, including, for example, adeno-associated virus (AAV) or adenovirus (Ad).

[0044] According to a preferred embodiment, the viral vector is selected from adeno-associated virus (AAV) or adenovirus (Ad), with AAV being the most preferred viral vector. As shown in the following examples, the method according to this disclosure improves the release of AAV and adenovirus, particularly by increasing genomic titer and transduction titer, and reducing the level of host cell-related impurities (e.g., host cell proteins) (see [link to documentation]). Figures 1 to 15 ).

[0045] According to a preferred embodiment, the viral vector is primarily present within the cell. However, a portion of the viral vector may also be present outside the cell, resulting from, for example, cellular degradation or lysis during the entire production process of the viral vector. The application of the method according to this disclosure to release the viral vector to be present outside the cell is also within the scope of this invention, wherein only a portion of the viral vector is present within the cell. For example, some serotypes, such as AAV8, are known to be efficiently released from cells into the surrounding culture medium, thus a portion of the produced viral vector is not present within the cell.

[0046] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, type 1 AAV (e.g., AAV with serotype 1, also known as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), type 11 AAV (e.g., AAV11), and type 12 AAV. Type AAV (e.g., AAV12), Type 13 AAV (e.g., AAV13), Type rh32.33 AAV (e.g., AAVrh32.33), Type rh8 AAV (e.g., AAVrh8), Type rhlO AAV (e.g., AAVrhlO), Type rh74 AAV (e.g., AAVrh74), Type hu.68 AAV (e.g., AAVhu.68), Bird AAV (e.g., AAAV), Cattle AAV (e.g., BAAV), Dog AAV, Horse AAV, Sheep AAV, Snake AAV, Bearded Dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV now known or to be discovered in the future.

[0047] cell culture

[0048] According to the disclosed method for releasing viral vectors, viral vectors are released from cell cultures. According to a preferred embodiment, in step (b), it is not necessary to separate the cell culture from the surrounding liquid before contacting the cell culture with the lysis reagent. Since it is not necessary to separate the cells from the surrounding liquid before contacting the cell culture with the lysis reagent in step (b), no further processing steps are required. In other words, the cells do not need to undergo any type of separation step, such as centrifugation or sedimentation, before contact with the lysis reagent. Therefore, the method of this disclosure simplifies cell lysis and expands its applicability, for example, by directly performing cell lysis in a bioreactor.

[0049] The “cell culture” according to this disclosure includes cells and surrounding fluid, as well as any products generated, such as viral vectors like AAV or adenovirus. The generated products may be present in the cells (intracellular) and / or in the surrounding fluid (extracellular). Typically, a cell culture is a cell suspension containing cells (preferably HEK293 cells or derived cells thereof), surrounding fluid (e.g., cell culture medium), and products generated by the cells (here, viral vectors). Suitable cell culture media are known in the art and are not limited thereto by this disclosure. Examples of suitable cell culture media for culturing HEK293 cells are shown in the following embodiments. Cell culture media may be HEK ViP NB and HEK TF (Sartorius Xell GmbH), and DMEM (Pan-Biotech). In addition to cells and surrounding fluid, other compounds may be present in the cell culture, such as residues of transfection reagents, compounds that enhance viral vector production, etc.

[0050] According to a preferred embodiment, the cell culture is a cell culture medium or cell pellet, preferably a cell culture medium. The cell culture medium contains cells producing a viral vector and surrounding culture medium, and if a viral vector has already been produced, it contains the viral vector. The cell culture may be referred to as a "whole (cell) culture," "cell culture medium," or "cell culture suspension." As shown in the examples, the method according to this disclosure is applicable to cell culture media and other crude samples (see...). Figure 4 and Figure 5 ), and purified or processed samples, such as cell pellets (see Figure 6 and Figure 7 This demonstrates the broad and advantageous applicability of the method according to this disclosure.

[0051] According to the preferred embodiment, cell cultures are not subjected to sonication, high-pressure homogenization, or freeze-thaw cycles. This embodiment is advantageous because sonication, high-pressure homogenization, or freeze-thaw cycles are difficult to scale up and can generate heat that can cause denaturation of target molecules or aggregation of viral vectors. Furthermore, these methods typically require specialized equipment, such as sonication and high-pressure homogenization, and can be labor-intensive, potentially leading to viral contamination. Additionally, freeze-thaw methods are not easily scalable and therefore unsuitable for large-scale production because they rely on repeated freeze-thaw cycles, which are time-consuming and impractical for mass production. On the other hand, the method disclosed herein offers scalability and versatility because the lysis reagent can be formulated according to different cell types and viral vectors.

[0052] According to the preferred embodiment, prior to steps (a), (b), and (c), the cell cultures are not centrifuged, cell precipitated, or have their surrounding fluid exchanged. This avoids centrifugation and sedimentation, simplifying the process and eliminating, for example, labor-intensive and potentially contaminating steps in removing the supernatant.

[0053] According to a preferred embodiment, the cell culture comprises mammalian cells. Mammalian cells are cells derived from mammals. However, the cells do not need to be identical to cells obtained from mammals and can be modified, engineered, or naturally / artificially altered. For example, mammalian cells can be cells of mammalian origin, but may contain one or more genetic modifications to allow for repeated proliferation and potentially unlimited proliferation. The term "one mammalian cell" may be used interchangeably with "multiple mammalian cells" and should encompass both singular and plural forms. The mammalian cells are preferably mammalian cell lines. According to a preferred embodiment, the mammalian cells are human cell lines that advantageously possess the ability to provide suitable post-translational modifications for human treatment. This means that the produced viral vectors have similar post-translational modifications, just as natural viruses infect human cells leading to viral production.

[0054] Mammalian cells can be selected from the following groups: HeLa, human embryonic kidney 293 (HEK293), BSC-1, SW480, juvenile hamster kidney (BHK), BHK-21, Vero E6, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, Chinese hamster ovary (CHO), MCF-7, Y79, SO-Rb50, Hep G2, DUKX-X11, J558L, HuH-7, MDCK, or HepG2 cells, or their derivative cells, such as subcell lines, for example, subcell lines of HEK293 HEK293, HEK293T, HEK293T / 17, ANJOU. 65. HEK293H, HEK293E, HEK293-6E, HEKEBNA1-6E, HEK293F, HEK293FT, HEK293Flp-INT-REx, HEK293FTM, HEK293S, HEK293SG, HEK293SGGD, HEK293MSR, HEK293A, or any modified variant thereof. According to a preferred embodiment, the mammalian cells are selected from the group consisting of HEK293, A549, BSC-1, SW480, juvenile hamster kidney (BHK), Vero E6, and MDCK cells, or derived cells thereof. According to a more preferred embodiment, the mammalian cells are selected from HEK293 cells or derived cells of HEK293 cells. HEK293 is particularly advantageous because it is well-characterized, widely used, and very easy to transfect. HEK293 cells are established by transforming human embryonic kidney cells with spliced ​​adenovirus type 5 DNA. Therefore, mammalian cells can be HEK293 cells or HEK293-derived cells, such as those selected from HEK293, HEK293T, HEK293T / 17, ANJOU 65, HEK293H, HEK293E, HEK293-6E, HEKEBNA1-6E, HEK293F, HEK293FT, HEK293Flp-IN T-REx, HEK293FTM, HEK293S, HEK293SG, HEK293SGGD, HEK293MSR, HEK293A, or any modified variants thereof. HEK293-derived cells may also include HEK293 cells adapted for suspension culture and / or adapted for specific types of cell culture media.

[0055] According to a preferred embodiment, the cell culture includes cells capable of suspension culture. For example, HEK293 cells or HEK293-derived cells have been adapted for suspension culture. This is particularly advantageous because cells capable of suspension culture can yield a greater number of cells compared to adherent cells. Alternatively, native adherent cells (e.g., adherent HEK293 cells) may be used, which have subsequently been adapted for suspension culture and modified to be configured to produce viral vectors (e.g., through transient transfection or genome editing).

[0056] According to a preferred embodiment, mammalian cells are modified to be configured to produce viral vectors, preferably AAV or adenovirus. According to one embodiment, mammalian cells are modified (preferably prior to step (a)) to be configured to produce viral vectors. In other embodiments, mammalian cells capable of producing the desired viral vector can be purchased directly from mammalian cells without any activity modification steps. These mammalian cells are, for example, so-called packaging cells or stable cells containing all the genetic elements required to express the viral vector and typically do not contain the target gene. In some embodiments, the mammalian cells used may themselves express one or more genes required to produce the viral vector from the outset; for example, HEK293 cells are known to express E1A and E1B required for AAV production.

[0057] According to a preferred embodiment, mammalian cells are modified by transiently transfecting them with one or more viral vector production plasmids, thereby enabling transient production of viral vectors. Transiently transfected mammalian cells essentially allow for one viral vector manufacturing run, thus requiring transfection for each manufacturing run. According to an alternative preferred embodiment, mammalian cells are modified by genome editing with one or more viral vector production nucleic acid molecules, thereby enabling stable viral vector production. Stable integration allows for the repeated use of a stockpile of modified mammalian cells (such as a cell bank or master cell bank) without requiring prior genome modification of the mammalian cells before each manufacturing run (compared to transient transfection). The advantage of this is that the established process can be repeated multiple times, thus simplifying the process and improving efficiency without prior transient transfection. On the other hand, transient transfection is more flexible because a different set of genetic elements can be transfected into the cells for each run without prior complex genome editing of the mammalian cells. Both methods of modifying mammalian cells to produce viral vectors are applicable within the framework of the methods according to this disclosure and should not be limited in any way. In fact, combining transient transfection and stable integration is also within the scope of this disclosure, for example, providing some stably integrated genetic elements and other genetic elements via transient transfection. For example, a transgenic cassette can be provided via transient transfection, while the remaining genetic elements can be stably integrated.

[0058] According to a preferred embodiment, one or more plasmids or nucleic acid molecules used for viral vector production encode at least a portion of an adenovirus (Ad) or at least a portion of an adeno-associated virus (AAV). In some cases, not all naturally occurring viral genetic elements need to be present in the one or more plasmids or nucleic acid molecules used for viral vector production; for example, when some genetic elements are already present in mammalian cells, their presence in the plasmid or nucleic acid molecule is unnecessary. For example, HEK293 cells, commonly used for viral vector production, were initially established by transfecting primary human embryonic kidney cells with spliced ​​type 5 adenovirus DNA, and it has been shown that HEK293 cells stably express adenovirus E1A and E1B-55k proteins due to the integration of a 4 kbp adenovirus DNA fragment into chromosome 19. Therefore, E1A and E1B do not need to (but may) be included in the genetic design used to modify such mammalian cells. Additionally, for gene therapy, the produced viral vectors typically contain one or more genes to be delivered to a patient or another cell. Such one or more genes can be referred to as "target genes." These are then used to replace the viral vectors packaged into plasmids to produce relevant elements, since it is generally undesirable to replicate the viral vector in vivo (i.e., in a potential patient). Therefore, instead of delivering the native and complete viral vector genetic elements, only those genetic elements that produce the viral vector, including the target gene, may be delivered. In some embodiments, the target gene is a therapeutic nucleic acid, such as therapeutic DNA or RNA. In other embodiments, the target gene includes a reporter gene. In a preferred embodiment, the reporter gene can be detected by antibody-based analysis. In a further preferred embodiment, the reporter gene is a fluorescent molecule. Exemplary fluorescent molecules suitable as reporter genes are GFP, eGFP, mGFP, eYFP, yellow fluorescent protein variants (citrine), eCFP, mCFP, sky blue fluorescent protein (Cerulean), tandem dimer red fluorescent protein (dtTomato), and any variants thereof. In some embodiments, the reporter gene is β-galactosidase, luciferase, or glutathione S-transferase, or any variant thereof. In certain implementations, the target gene is suitable for screening analyses or markers, such as fluorescent proteins, like green fluorescent protein (GFP) or derivatives thereof, which are used to visualize viral vector transduction.

[0059] According to a preferred embodiment for producing AAV, one or more plasmids or nucleic acid molecules used to produce the viral vector encode one or more of the following genes, preferably all of them: Rep78, Rep68, Rep52, Rep40, VP1, VP2, VP3, ITR, AAP, MAAP, X gene, VA RNA, E4orf6, and E2A, for the production of AAV. According to another embodiment, one or more plasmids or nucleic acid molecules used to produce the viral vector encode one or more of the following genes, preferably all of them, for the production of AAV. In any of these embodiments, the mammalian cell may already encode one or more genetic elements for the production of AAV. For example, E1A and E1B may already be produced by mammalian cells such as HEK293, where the plasmid or nucleic acid molecule can support cellular expression through additional copies of E1A or E1B, or cellular expression may be considered sufficient. The gene encoding can be provided on a single or multiple plasmids or single or multiple nucleic acid molecules. According to one embodiment, more than one plasmid is provided for the production of AAV. For example, two, three, four, five, six, seven, eight, nine, or ten plasmids may be provided.

[0060] According to a preferred embodiment, the viral vector is primarily located within the cell and is preferably selected from adeno-associated virus (AAV) or adenovirus (Ad). Although extracellular viral vectors can also be modified using the methods according to this disclosure (see Example 6), Figure 11 However, this method is most effective when some viral vectors (e.g., at least 10%, at least 20%, at least 30%, or at least 40%) are present inside the cell.

[0061] Step (a)

[0062] Step (a) of the method according to the first aspect defines the provision of the lysis reagent. This disclosure provides various lysis reagents, as shown in the Examples section. Therefore, different lysis reagents may be suitable for provision in step (a).

[0063] Preferably, the lysis reagent is provided in liquid form, such as as a solution or suspension. This is generally easier to handle and for the contact step. Furthermore, this can be more accurate than adding the reagent in solid form. According to another embodiment, the lysis reagent is provided in solid form, such as as a powder. This solid lysis reagent can be present in a container in which the cell culture is added in contact step (b) according to this disclosure. The specific form of the lysis reagent should not be limited to the scope of this disclosure.

[0064] In this document, the term "lysis reagent" may be used interchangeably with the term "cell lysis reagent." As stated above, the advantage of providing lysis reagents for chemical lysis is that it eliminates the need for specialized equipment, such as that required for mechanical lysis using physical lysis methods. Furthermore, chemical lysis methods are suitable for direct use with cell cultures in culture vessels, such as the bioreactors disclosed herein. In addition, while physical methods such as freeze-thaw cycles for cell lysis are relatively simple, their reliance on repeated freeze-thaw cycles makes them time-consuming and impractical for large-scale production, thus hindering scalability and potentially making them unsuitable for mass production.

[0065] As will be readily understood by those skilled in the art, the lysis reagents disclosed herein refer to lysis reagents that at least partially lyse cells, commonly referred to as chemical lysis. Therefore, complex and / or time-consuming procedures are not required. According to a preferred embodiment, the method according to this disclosure does not include a lysis step based on mechanical or physical lysis. According to a preferred embodiment, the method according to this disclosure does not include a step of repeated freeze-thaw cycles for cell lysis or a microfluidization step for cell lysis.

[0066] lysis reagent

[0067] According to a preferred embodiment, the lysis reagent is an alkaline lysis reagent, preferably with a pH greater than 7.0, more preferably greater than 7.5, and most preferably greater than 7.7, such as 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or higher than 8.5. According to a particularly preferred embodiment, the alkaline lysis reagent has a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, such as 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or higher than 8.5. It has been found that this alkaline pH is beneficial for maximizing yield in the method according to the invention. Specifically, the inventors have found that the alkaline lysis reagent enhances the extractability of intracellular AAV carriers (see Examples). Figures 4 to 9 , Figure 12 and Figure 13 By improving AAV vector extraction efficiency through the use of a developed cell lysis reagent, the manufacturing cost of AAV-based drugs can be significantly reduced. AAV2 transduction assays showed that the improved extraction efficiency resulted in a higher yield of active AAV vectors. Higher AAV vector yield means more drug product can be obtained from the same production process, thereby increasing overall productivity and reducing unit costs. Furthermore, the alkaline lysis reagent disclosed herein helps to shorten cell lysis and extraction times. This time saving significantly reduces costs by shortening the total time required for the production cycle.

[0068] According to a preferred embodiment, the pH range of the alkaline lysis reagent is selected from 7.2 to 10, preferably 7.5 to 9.5, more preferably 8 to 9 or 8.2 to 8. Such a range is beneficial for achieving efficient release of the viral vector.

[0069] According to a preferred embodiment, the alkaline lysis reagent comprises a buffer suitable for buffering the lysis reagent at an alkaline pH, preferably capable of buffering at a pH range selected from 7.2 to 9.5. Buffers that effectively buffer the alkaline lysis reagent (and can later buffer the lysis composition) at a specified acidic pH are particularly suitable for the methods of this disclosure because these buffers maintain a relatively stable pH at alkaline pH.

[0070] According to a preferred embodiment, the lysis reagent comprises a buffer selected from the following: tris(hydroxymethyl)aminomethane (TRIS) buffer, 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, phosphate buffer, glycine-sodium hydroxide buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer, TES buffer, DIPSO buffer, TAPSO buffer, triethanolamine buffer, HEPPSO buffer, POPSO buffer, TRICINE buffer, HEPPS buffer, EPPS buffer, BICIN buffer, TAPS buffer, AMPSO buffer, taurine buffer, CHES buffer, AMP buffer, CAPSO buffer, or combinations thereof, preferably TRIS buffer or BTP buffer. These buffer compounds have been found suitable for alkaline lysis reagents because these buffers effectively buffer the lysis reagent and possible lysis compositions at alkaline pH.

[0071] In one embodiment, the pH of the alkaline lysis reagent is selected from the range of 7.5 to 9.5, wherein the following buffer solutions may be selected: i) TRIS buffer; pH range 7.5 to 9.0, ii) BTP buffer, pH range 6.3 to 9.5. iii) Phosphate buffer; pH range 5.8 to 8.0, or iv) Glycine-sodium hydroxide, pH 8.6 to 10.6.

[0072] According to a preferred embodiment, the lysis reagent comprises a buffer solution with a concentration of 0.01 M to 3 M, preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.05 M to 1 M; ii) For 5x lysis reagent, the concentration is 0.3 M to 1.5 M; or iii) Preferably, for 10x lysis reagent, the concentration is 1 M to 3 M.

[0073] This concentration of buffer solution has been found to be particularly advantageous for releasing viral vectors. For cell culture media, high concentrations of lysis reagent, such as 5x or 10x, preferably 10x, are particularly advantageous because they reduce the volume of lysis reagent that needs to be added to the cell culture. Therefore, when the culture vessel (e.g., a bioreactor) has sufficient open space for filling with lysis reagent, the lysis reagent can be added directly to the culture vessel (e.g., the bioreactor). As illustrated in the examples, by combining the concentrations of buffer and salt, appropriate concentrations can be carefully selected to maximize the release of viral vectors while maintaining a low level of viral vector aggregation, thereby improving viral vector release.

[0074] Throughout this disclosure, the concentration of the compound refers to x parts lysis reagent. This specifically refers to the ratio of cell culture to lysis reagent. For example, 1x lysis reagent is typically added to cell cultures that contain essentially no or very little surrounding fluid, such as cell cultures present in cell pellets or sediments without supernatant. 5x lysis reagent typically involves mixing 4 parts cell culture with 1 part lysis reagent, for example, 4 mL of cell culture with 1 mL of lysis reagent. 10x lysis reagent typically involves mixing 9 parts cell culture with 1 part lysis reagent, for example, 9 mL of cell culture with 1 mL of lysis reagent.

[0075] According to a preferred embodiment, the lysis reagent comprises a salt preferably selected from potassium chloride or sodium chloride. As shown in the examples, these salts are particularly useful for modulating ionic strength, thereby enabling the release of large quantities of viral vectors. Furthermore, these are commonly used buffer salts that are harmless and non-toxic.

[0076] According to a preferred embodiment, the lysis reagent comprises a salt with a concentration of 0.01 M to 5 M, preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.05 M to 1 M; ii) For 5x lysis reagent, the concentration is 0.3 M to 1.5 M; or iii) Preferably, for 10x lysis reagent, the concentration is 0.8 M to 5 M or 1 M to 3 M.

[0077] This concentration has been found to be particularly advantageous for releasing viral vectors. For cell culture media, high concentrations of lysis reagent, such as 5x or 10x, preferably 10x, are particularly advantageous because they reduce the volume of lysis reagent that needs to be added to the cell culture. Therefore, when the culture vessel (e.g., a bioreactor) has sufficient open space for filling with the lysis reagent, it can be added directly to the culture vessel (e.g., the bioreactor).

[0078] According to a preferred embodiment, the lysis reagent comprises a detergent, preferably a nonionic detergent, and more preferably a nonionic detergent not classified as toxic and / or harmful to the environment. In particular, lysis reagents containing detergents, as disclosed herein, have been found to effectively lyse cells, thereby releasing viral vectors from cell cultures. Therefore, according to a specific preferred embodiment, the lysis reagent also comprises a detergent.

[0079] Preferably, the detergents according to this disclosure are beneficial to human and environmental safety, eliminating potential biohazard risks associated with their use (e.g., the use of Triton-X100 or similar detergents). It is advantageous that the lysis reagent may also include such detergents when necessary. According to a preferred embodiment, the detergent is selected from one or more of Tween, Triton, Nonidet, Igepal, or Tergitol. Although any nonionic detergent can be used, examples of nonionic detergents include those from the following: Tween class (Tween-20, Tween-40, Tween-60, Tween-80, etc.), Triton class (X-100, X-114, XL-80N, etc.), Tergitol (XD, TMN-6, etc.), and Nonidet or Igepal (NP-40, etc.). Nonionic surfactants include, but are not limited to, alkyl glucosides, particularly polysorbates such as polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), and polysorbate 80 (Tween 80), as well as polyoxyethylene alkyl ethers such as Triton X-100, Nonidet P40, NP-40, and various nonionic detergents from the Brij class. Other detergents that can be used for pyrolysis and degradation of samples are also well known in the art and therefore need not be described in detail herein. Furthermore, mixtures of detergents may also be used.

[0080] According to a preferred embodiment, the detergent is an alkyl glucoside, preferably a polysorbate, such as polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), and polysorbate 80 (Tween 80). As shown in the examples, such detergents are non-toxic and harmless, especially compared to Triton-X-based detergents (see...). Figure 10 Polysorbates, such as polysorbate 20 (also known as Tween 20), have been found to be particularly useful as cell-dissolving detergents. Polysorbates are biohazard-free detergents, and in addition to their cell-dissolving properties, they can also act as protectants at the gas-liquid interface, thereby preventing the aggregation of target molecules.

[0081] According to one implementation, the detergent is not Triton, for example, not Triton X-100.

[0082] According to another embodiment, the detergent is a polyoxyethylene alkyl ether, such as Triton X-100, Nonidet P40, NP-40, and various nonionic detergents from the Brij class.

[0083] According to a preferred embodiment, the concentration of the detergent is from 0.1% (v / v) to 20% (v / v), preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.1% (v / v) to 2% (v / v). ii) For 5x lysis reagent, 1% (v / v) to 5% (v / v); or iii) For 10x lysis reagent, the concentration is 4% (v / v) to 10% (v / v).

[0084] It has been found that this concentration of detergent is beneficial for the release of viral vectors.

[0085] According to a preferred embodiment, the cleavage reagent comprises a divalent cation, preferably Mg. 2+ Ca 2+ Ba 2+ Cu 2+ Fe 2+ Zn 2 + Mn 2+ Ni 2+ Or combinations thereof, preferably Mg 2+ Cu 2+ Zn 2+ Mn 2+ Ni 2+ Or a combination thereof, Mg is the most preferred. 2+ Divalent cations are particularly advantageous for cell lysis involving nucleases because they can enable or improve nuclease activity, for example, by acting as cofactors for such nucleases. Furthermore, divalent cations (e.g., Mg) are believed to have beneficial effects. 2+ The presence of divalent cations can stabilize viral vectors such as AAV. In some cases, divalent cations may not be necessary, such as when nucleases are not used.

[0086] According to a preferred embodiment, the concentration of the divalent cation in the cleavage reagent is from 0.1 mM to 1000 mM, preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.5 mM to 5 mM; ii) For 5x lysis reagent, the concentration is 5 mM to 15 mM; or iii) Preferably, for 10x lysis reagent, the concentration is 15 mM to 50 mM.

[0087] It has been found that this concentration of divalent cations is conducive to the release of viral vectors.

[0088] According to a preferred embodiment, the lysis reagent comprises a cryoprotectant, preferably a sugar, more preferably sucrose, trehalose, or mannitol, and most preferably sucrose. The capsid and functional viral vector titers, i.e., the release of the viral vector, are not affected by the presence of the cryoprotectant, and therefore this compound is not essential for release (see also Example 5). However, the advantage of the cryoprotectant is that, when optionally added to the lysis reagent according to this disclosure, it improves the storability and freezeability of the composition containing the viral vector after lysis and / or purification. However, the cryoprotectant is not required during cell lysis but may be added subsequently, for example, after step (c) or step (d) of the method according to this disclosure. Including the cryoprotectant in the lysis reagent to avoid a subsequent addition step can be advantageous, i.e., adding the cryoprotectant simplifies the lysis process.

[0089] According to a preferred embodiment, the concentration of the cryoprotectant in the lysis reagent is from 1% (w / v) to 70% (w / v), with the preferred concentration selected from the following range: i) For 1x lysis reagent, 1% (w / v) to 10% (w / v); ii) For 5x lysis reagent, 10% (v / v) to 35% (w / v); or iii) For 10x lysis reagent, the concentration is 20% (w / v) to 60% (w / v).

[0090] It has been found that this concentration of cryoprotectant is beneficial to the stability of viral vectors during cryopreservation.

[0091] Exemplary pyrolysis reagents containing detergents are shown in Tables 1, 2, 4, 5, 9, and 11.

[0092] Other exemplary lysis reagents

[0093] According to a specific embodiment of this method, the lysis reagent is an alkaline lysis reagent with a pH greater than 7.0, preferably greater than 7.5, most preferably greater than 7.7, such as 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or higher, and contains a detergent, preferably an alkyl glucoside, more preferably a polysorbate, such as polysorbate 20 (Tween 20), polysorbate 40 (Tween 40) and polysorbate 80 (Tween 80). In such an embodiment, the concentration of the detergent is preferably from 0.1% (v / v) to 20% (v / v), more preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.1% (v / v) to 2% (v / v). ii) For 5x lysis reagent, 1% (v / v) to 5% (v / v); or iii) For 10x lysis reagent, the concentration is 4% (v / v) to 10% (v / v).

[0094] In such an implementation, the viral vector may be AAV or adenovirus, and the cells of the cell culture are mammalian cells, preferably selected from HEK293 cells or HEK293-derived cells, such as subcell lines of HEK293 including HEK293, HEK293T, HEK293T / 17, ANJOU 65, HEK293H, HEK293E, HEK293-6E, HEKEBNA1-6E, HEK293F, HEK293FT, HEK293Flp-IN T-REx, HEK293FTM, HEK293S, HEK293SG, HEK293SGGD, HEK293MSR, HEK293A, or any modified variant thereof. The HEK293-derived cells may also comprise HEK293 cells adapted for suspension culture and / or adapted for specific types of cell culture media.

[0095] According to the preferred embodiment, the pyrolysis reagent is an alkaline pyrolysis reagent containing a detergent, preferably selected from: i.1x alkaline lysis reagent, pH greater than 7.0, preferably greater than 7.5, most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v); ii.5x alkaline lysis reagent, with a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.3 M to 1.5 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.3 M to 1.5 M; - Descaling agent, preferably polysorbate, with a concentration selected from 1% (v / v) to 5% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 5 mM to 15 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 10% (v / v) to 35% (w / v); or iii. Preferably, the 10x alkaline lysis reagent has a pH greater than 7.0, more preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 1 M to 3 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.8 M to 5 M or 1 M to 3 M; - Descaling agent, preferably polysorbate, at a concentration selected from 4% (v / v) to 10% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 15 mM to 50 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 20% (w / v) to 60% (w / v).

[0096] Step (b)

[0097] Step (b) of the method according to the first aspect defines contacting the cell culture with a lysis reagent to produce a lysis composition. Contact may include adding the lysis reagent to the cell culture, adding the cell culture to the lysed culture, or both simultaneously. According to one embodiment, it is advantageous to add the lysis reagent to the cell culture, for example, when the cell culture is present in a culture dish (e.g., a bioreactor) or when the cells are present in a container as a cell pellet. On the other hand, specific contact methods should not be limited to the scope of this disclosure.

[0098] Preferably, the lysis reagent is provided in liquid form and added to the cell culture in contact step (b). This allows the cell culture to be held in a culture vessel (e.g., a bioreactor or shake flask) and to which the lysis reagent is added, for example, manually or automatically. In addition to the lysis reagent, the cell culture may also be contacted with other compounds. These compounds are used for the culture of mammalian cells, such as cell culture media, or may be necessary for the contact of mammalian cells with the lysis reagent. Such cell culture media are well known to those skilled in the art and should not limit the scope of this disclosure. Specific examples include the cell culture media used in the following embodiments.

[0099] According to a preferred embodiment, in step (b), the pH of the lysis composition is greater than 7.2, preferably greater than 7.5, and most preferably greater than 7.8 or 8.0. According to a preferred embodiment, in step (b), the pH of the lysis composition is selected from the range of 7.2 to 10.0, preferably 7.5 to 9.5. Lysis compositions having specified pH values ​​(see Exemplary Lysis Compositions Tables 5, 9, 11) have been found to be effective for releasing viral vectors (see...). Figures 4 to 9 , Figure 12 and Figure 13 ).

[0100] According to a preferred embodiment, in step (b), the lysis composition comprises a compound at a substantially 1x lysis reagent concentration, which includes a 1x alkaline lysis reagent containing a detergent. Since a 1x lysis reagent is preferably provided to release viral vectors from concentrated cell samples (e.g., cell pellets), the 1x lysis reagent is substantially undiluted or only slightly diluted, for example, less than 20%, preferably less than 10%. Therefore, the concentration defined herein for a 1x lysis reagent substantially corresponds to the concentration obtained in the lysis composition. In an exemplary embodiment, 10x lysis reagent may be mixed with the sample such that the final concentration in the lysis composition corresponds to one-tenth of the 10x lysis reagent, i.e., substantially equivalent to a 1x lysis reagent. Therefore, according to a preferred embodiment, the lysis composition comprises a compound at a substantially 1x lysis reagent concentration as disclosed herein. In this respect, "substantially" means the specified concentration of a 1x lysis reagent, but can be slightly diluted, for example, less than 20%, preferably less than 10%.

[0101] According to a preferred embodiment, in step (b), the pyrolysis composition comprises a compound at a concentration substantially 1x that of the pyrolysis reagent, preferably substantially the concentration disclosed herein. According to a preferred embodiment, the pyrolysis composition has the following characteristics: I. The pyrolysis composition has a pH greater than 7.2, preferably greater than 7.5, most preferably greater than 7.8 or 8.0, and comprises: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v).

[0102] nuclease

[0103] In one embodiment, the nuclease is added to the lysis composition after step (b) (see also the examples); however, the nuclease may also be added to the lysis reagent before or during step (b). Within the scope of this disclosure, the addition of nuclease is generally used to achieve three objectives: (i) reducing nucleic acid contamination, (ii) reducing the viscosity of cell lysates, and (iii) mitigating AAV carrier aggregation caused by the binding of nucleic acids to the surface of AAV carrier particles. According to a preferred embodiment, the nuclease is salt-tolerant. Salt-tolerant nucleases can be used in compositions with higher ionic strengths, such as those present in the lysis compositions and / or lysis reagents according to this disclosure. Examples of nucleases or salt-tolerant nucleases are well known to those skilled in the art, and the specific enzyme used should not limit the scope of this disclosure. Examples of suitable salt-tolerant nucleases are M-SAN HQ and SAN-HQ (ArcticZymes). Typical suitable conditions for nucleases such as “San HQ 2.0” (ArcticZymes) are: i) Temperature: 7°C to 38°C, overnight at 4°C, optimal temperature: 30°C to 38°C ii) Salt concentration (NaCl / KCl): 100 mM to 900 mM, optimal concentration: 400 mM to 650 mM. iii) Mg 2+ >1 mM is beneficial for activity, with an optimal range of 5 mM to 50 mM. iv) pH: 7.3 to 9.2, with an optimal range of 8.2 to 8.8.

[0104] According to a specific embodiment of this method, the lysis reagent is an alkaline lysis reagent with a pH greater than 7.0, preferably greater than 7.5, most preferably greater than 7.7, such as 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or higher, and contains a detergent, preferably an alkyl glucoside, more preferably a polysorbate, such as polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), and polysorbate 80 (Tween 80). In such an embodiment, the concentration of the detergent is preferably from 0.1% (v / v) to 20% (v / v), more preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.1% (v / v) to 2% (v / v). ii) For 5x lysis reagent, 1% (v / v) to 5% (v / v); or iii) For 10x lysis reagent, the concentration is 4% (v / v) to 10% (v / v).

[0105] In such embodiments, the nuclease is added to the lysis composition after step (b), or to the cell culture during or before step (b). Furthermore, in such embodiments, the viral vector may be AAV or adenovirus, and the cells of the cell culture are mammalian cells, preferably selected from HEK293 cells or HEK293-derived cells, such as subcell lines of HEK293: HEK293, HEK293T, HEK293T / 17, ANJOU 65, HEK293H, HEK293E, HEK293-6E, HEKEBNA1-6E, HEK293F, HEK293FT, HEK293Flp-IN T-REx, HEK293FTM, HEK293S, HEK293SG, HEK293SGGD, HEK293MSR, HEK293A, or any modified variants thereof. HEK293 cell-derived cells may also comprise HEK293 cells adapted for suspension culture and / or adapted for a specific type of cell culture medium. Furthermore, in such embodiments, the lysis composition may also comprise the following substances: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v).

[0106] Step (c)

[0107] Step (c) of the method according to the first aspect defines the incubation of the lysis composition, thereby releasing the viral vector from the cell culture. Within the framework of this disclosure, different incubation conditions are suitable. Exemplary conditions include adjusting the temperature of the lysis composition, ranging from 5°C to 50°C, preferably from 25°C to 45°C, more preferably from 30°C to 42°C, such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, and 42°C. According to a particular embodiment, the temperature is 37°C. Exemplary conditions also include shaking or stirring the lysis composition. For example, the lysis composition may be shaken at 10 rpm to 1000 rpm, preferably from 20 rpm to 500 rpm, more preferably from 50 rpm to 250 rpm. In a particular embodiment, the lysis composition is shaken at 120 rpm. According to a particular embodiment, the lysis composition is incubated at 37°C and shaken at 120 rpm. However, as technicians can easily understand, different pyrolysis conditions are suitable and can be flexibly adjusted according to their respective requirements.

[0108] According to a preferred embodiment, in step (c), the cell culture is partially lysed. While partial lysis is generally not preferred, the method of this disclosure is capable of partial lysis because the subsequent step (d), which contacts the lysis composition with an acidifying agent to lower the pH, completes cell lysis. Therefore, partial lysis is acceptable because complete lysis can be achieved in a second lysis step. Consequently, less incubation time is required in the first step, resulting in faster release of the viral vector and thus greater overall efficiency.

[0109] According to the preferred embodiment, the incubation step (c) includes: - Incubation time is less than 6 hours, preferably less than 5 hours or less than 4 hours, more preferably less than 3 hours, such as 2 hours, 1 hour or 0.5 hours; and / or - The incubation time ranges from 15 minutes to 300 minutes, preferably 20 minutes to 250 minutes, 25 minutes to 200 minutes, or more preferably 30 minutes to 150 minutes or 30 minutes to 100 minutes, and most preferably 30 minutes to 90 minutes or 45 minutes to 75 minutes.

[0110] According to the preferred embodiment, the time required to perform steps (c) and (d) is as follows: - Less than 360 minutes, preferably less than 300 minutes or less than 240 minutes, more preferably less than 180 minutes or less than 120 minutes, most preferably less than 100 minutes, such as 90 minutes, 80 minutes or 70 minutes or less; and / or - The time range is selected from 15 minutes to 360 minutes, preferably 20 minutes to 300 minutes, 25 minutes to 240 minutes, or more preferably 30 minutes to 180 minutes or 40 minutes to 120 minutes, and most preferably 45 minutes to 90 minutes.

[0111] The method of this disclosure advantageously enables rapid and efficient release of viral vectors from cell cultures. As shown in the examples, the method of this disclosure efficiently releases viral vectors such as AAV within 70 minutes or less. This rapid release of the viral vector improves the overall efficiency of the process.

[0112] Step (d)

[0113] Step (d) of the method according to the first aspect defines contacting the lysis composition with an acidifying agent to lower the pH. Advantageously, the acidification step is capable of releasing any viral vectors not released in steps (b) and / or (c). Therefore, the incubation time in step (c) can be shortened, thereby making the method more efficient. Additionally, as shown in the following examples, by including step (d), the released viral vector has higher purity and contains fewer host cell-related impurities, such as host cell proteins (see...). Figure 3 , Figure 11 and Figure 15 ).

[0114] According to a preferred embodiment, in step (d), the lysis composition is contacted with the acidifying agent to substantially completely lyse the cell culture. In some cases, after step (c), a portion of the cell culture is lysed, allowing some cells to remain viable. For example, less than 90%, preferably less than 50%, more preferably less than 30% of the cells are not lysed. These remaining unlysed cells are then effectively lysed in step (d), causing the acidifying agent to substantially completely lyse the cell culture. Thus, after step (d), more than 90%, preferably more than 95%, more preferably more than 98% or more than 99% of the cells are lysed. In one embodiment, the cells of the cell culture are completely lysed after step (d). As shown in the following examples, viable cells can no longer be detected after step (d) (see examples, such as Table 6).

[0115] According to a preferred embodiment, the contact step (d) includes titrating an acidifying agent into the pyrolysis composition. By titrating the acidifying agent into the pyrolysis composition, the amount of acidifying agent added to the pyrolysis composition can be carefully adjusted. Furthermore, for example, by parallel pH measurements, the pH can be flexibly and precisely adjusted, thereby regulating the conditions in step (d).

[0116] According to a preferred embodiment, step (d) includes one or more steps of pH determination. Advantageously, the pH determination allows for precise pH adjustment during step (d). According to a preferred embodiment, the determination is performed using a pH probe. This pH probe can be supplied separately and contained within a container or dish in which viral vector release can be performed. The pH probe can also be part of a culture dish, such as a bioreactor or other culture dish (e.g., a shake flask), such that the culture dish is equipped with a pH probe for pH determination. Since the pH probe is typically used to monitor pH throughout the culture process, no additional equipment is required, allowing step (d) and the pH determination to be performed directly in the culture dish (e.g., a bioreactor).

[0117] According to a preferred embodiment, the released viral vector is purified. Preferably, purification is performed after step (d) to release the viral vector. According to a preferred embodiment, purification is performed by a single-step or multi-step method, preferably by at least deep filtration, centrifugation, and / or sterile filtration after step (d).

[0118] According to a preferred embodiment, compared to methods omitting step (b) or step (d), the host cell-derived impurities in the purified viral vector are reduced by 10%, preferably by 25% or 30% for host cell proteins. As shown in the examples, host cell-derived impurities (e.g., host cell proteins) can be significantly reduced (see [example]). Figure 11 and Figure 15 ).

[0119] According to a preferred embodiment, the capsid viral vector titer and / or transduced viral vector titer are increased compared to the method without step (d). According to a preferred embodiment, the capsid viral vector titer is increased by at least 10%, preferably at least 25%, compared to the method without step (d). According to a preferred embodiment, the transduced viral vector titer is increased by at least 10%, preferably at least 25%, compared to the method without step (d). According to a preferred embodiment, at least 10% more viral vector is released, preferably at least 25%, compared to the method without step (d).

[0120] Acidifying reagent

[0121] According to a preferred embodiment, in step (d), the acidifying agent lowers the pH to an acidic pH, preferably less than 6.0, more preferably less than 5.0, and most preferably less than 4.5. According to a preferred embodiment, in step (d), the acidifying agent lowers the pH to a pH selected from the range of: 1.5 to 6.0, preferably 2.5 to 5.0, more preferably 3.5 to 4.5, for example 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5. The advantage of lowering the pH is that any remaining cells that have not yet lysed are essentially completely lysed, which also shortens the incubation time and yields a high yield of viral vector. Furthermore, lowering the pH to a specified pH has the advantage of fewer host cell-related impurities, resulting in a higher purity of the released viral vector (see also...). Figure 15 ).

[0122] According to a preferred embodiment, the acidifying agent comprises an acid, preferably an organic acid. As shown in the examples, acids are particularly suitable as acidifying agents to lower the pH. According to a preferred embodiment, the acid is a carboxylic acid. According to a preferred embodiment, the acid is acetic acid or citric acid, preferably acetic acid.

[0123] According to a preferred embodiment, the acidifying agent is provided in liquid or solid form, preferably in liquid form. According to a preferred embodiment, the acidifying agent comprises an acid and a solvent, preferably but not limited to water. In this case, the acid is dissolved in the solvent, allowing the acid to be provided to the pyrolysis composition in liquid form. When the acidifying agent is provided in liquid form, it is generally easier to handle and easier to perform the contact step. Furthermore, this can be more accurate than adding the acidifying agent in solid form. Suitable solvents are readily known to those skilled in the art. According to another embodiment, the acidifying agent is provided in solid form (e.g., powder). The specific form of the acidifying agent should not be limited to the scope of this disclosure.

[0124] The acidifying agent preferably comprises a concentrated acid, such that only a small volume of acid is needed to acidify the pyrolysis composition. Preferably, the acidifying agent comprises a concentrated organic acid, preferably a concentrated carboxylic acid, and most preferably concentrated acetic acid or citric acid. Preferably, the concentration of the concentrated acid (e.g., concentrated acetic acid or concentrated citric acid) is sufficient to lower the pH of the pyrolysis composition to an acidic pH, preferably less than 6.0, more preferably less than 5.0, and most preferably less than 4.5. An example concentration of the acid in the acidifying agent is at least 100 mM, preferably at least 250 mM, at least 500 mM, or at least 750 mM, and most preferably at least 1 M, such as 2 M, 3 M, or 4 M. According to a specific embodiment, the acidifying agent comprises a carboxylic acid, such as acetic acid or citric acid, at a concentration of at least 1 M.

[0125] Other implementation plans

[0126] According to a preferred embodiment, at least one of steps (b), (c), and (d) is carried out in a bioreactor, preferably at least two of these steps are carried out in a bioreactor, and most preferably, all steps (b), (c), and (d) are carried out in a bioreactor. An advantage of carrying one or more steps of the method in a bioreactor is that it eliminates the need to transfer the cell culture to another container or dish, but a bioreactor can be used to release viral vectors from the cell culture, as shown in Example 8 below.

[0127] According to a preferred embodiment, stirring in the bioreactor facilitates mixing in these steps. By stirring the lysis composition in any of steps (b), (c), and / or (d), preferably in all steps (b), (c), and (d), the lysis composition can be thoroughly mixed, resulting in a more homogeneous composition and thus more efficient release of the viral vector.

[0128] For example, this method can be carried out in a tank bioreactor, such as a continuous stirred tank reactor, a rocking bioreactor, a wave bioreactor, a conical flask, a rotating flask, a rotating wall bioreactor, and / or a multi-parallel bioreactor.

[0129] According to a preferred embodiment, the bioreactor is capable of temperature and / or pH control. By including temperature control, the temperature of any of steps (b), (c), and / or (d) can be controlled, thereby providing ideal conditions for the release of the viral vector. It can be advantageous to increase the temperature (e.g., above 30°C) to increase the reaction rate and thus increase the release of the viral vector. On the other hand, excessively high temperatures can be avoided, which would affect the integrity of the viral vector. By controlling the pH, the pH of the lysis composition can be closely monitored and influenced. For example, when insufficient acidifying agent is added in step (d), pH control can facilitate adjustment to an ideal pH state.

[0130] In some implementations, the controller can monitor and / or control one or more parameters related to the process, such as key process parameters (e.g., pH, DO, and temperature). For example, mixing can be performed in stirring, shaking, or track shaking modes.

[0131] According to a preferred embodiment, the bioreactor is capable of pH adjustment, preferably by configuring in step (d) to allow the pyrolysis composition to contact with the acidifying agent.

[0132] In some embodiments, the bioreactor chamber can be a dimensionally stable vessel, such as one made of stainless steel, glass, or plastic. Alternatively, it is conceivable that the bioreactor chamber can be a flexible bag made of at least one suitable polymer, which can be further configured to be placed in a holder to maintain dimensional stability. In the art, the vast majority of flexible bag containers and rigid plastic bioreactors are also referred to as single-use bioreactors (SUBs), and these are all included herein.

[0133] According to one embodiment, in a method for releasing a viral vector from a cell culture, the cell culture is a cell culture medium, and all steps (b), (c), and (d) are carried out in a bioreactor. In such an embodiment, advantageously, the bioreactor (i) is stirred to facilitate mixing in these steps; (ii) is capable of temperature control; and (iii) is capable of pH adjustment, preferably achieved by configuring step (d) to allow contact between the lysis composition and an acidifying agent. Preferably, the viral vector is AAV or adenovirus, and / or the cell culture comprises mammalian cells selected from HEK293 cells or HEK293 cell derivatives.

[0134] According to one embodiment, in a method for releasing a viral vector from a cell culture, the cell culture is a cell culture medium, and all steps (b), (c), and (d) are performed in a bioreactor. Preferably, the viral vector is AAV or adenovirus, and / or the cell culture comprises mammalian cells selected from HEK293 cells or HEK293 cell derivatives. In such an embodiment, the lysis reagent is an alkaline lysis reagent containing a detergent, preferably selected from: 5x alkaline lysis reagent, with a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.3 M to 1.5 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.3 M to 1.5 M; - Descaling agent, preferably polysorbate, with a concentration selected from 1% (v / v) to 5% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 5 mM to 15 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 10% (v / v) to 35% (w / v); or Preferably, the 10x alkaline lysis reagent has a pH greater than 7.0, more preferably greater than 7.5, and most preferably greater than 7.7, and comprises: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 1 M to 3 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.8 M to 5 M or 1 M to 3 M; - Descaling agent, preferably polysorbate, at a concentration selected from 4% (v / v) to 10% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 15 mM to 50 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 20% (w / v) to 60% (w / v).

[0135] In such embodiments, the acidifying agent may comprise a concentrated acid, preferably a concentrated organic acid, with a concentration sufficient to lower the pH of the lysis composition to an acidic pH, preferably less than 6.0, more preferably less than 5.0, most preferably less than 4.5, and optionally, a concentration of at least 1 M. In such embodiments, the method may exclude a lysis step based on mechanical or physical lysis, such as repeated freeze-thaw cycles for cell lysis or a microfluidization step for cell lysis.

[0136] According to a specific embodiment, in step (b), before contacting the cell culture with the lysis reagent, it is not necessary to separate the cell culture used for releasing the viral vector from the cell culture from the surrounding liquid, and the cell culture used for releasing the viral vector from the cell culture comprises mammalian cells selected from HEK293 cells or HEK293 cell derivatives. Preferably, the viral vector is AAV or adenovirus. In such an embodiment, the pH of the lysis composition may be greater than 7.2, preferably greater than 7.5, most preferably greater than 7.8 or 8.0, and may contain: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v).

[0137] In such embodiments, the acidifying agent may comprise a concentrated acid, preferably a concentrated organic acid, with a concentration sufficient to lower the pH of the lysis composition to an acidic pH, preferably less than 6.0, more preferably less than 5.0, most preferably less than 4.5, and optionally, a concentration of at least 1 M. In such embodiments, the method may exclude a lysis step based on mechanical or physical lysis, such as repeated freeze-thaw cycles for cell lysis or a microfluidization step for cell lysis.

[0138] According to a specific embodiment, in a method for releasing a viral vector from a cell culture, in step (c), the cell culture is partially lysed, and in step (d), the lysis composition is contacted with an acidifying agent such that the cell culture is substantially completely lysed. In such an embodiment, the pH of the lysis composition may be greater than 7.2, preferably greater than 7.5, most preferably greater than 7.8 or 8.0, and may contain: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v).

[0139] In such embodiments, the acidifying agent may comprise a concentrated acid, preferably a concentrated organic acid, at a concentration sufficient to lower the pH of the lysis composition to an acidic pH, preferably less than 6.0, more preferably less than 5.0, most preferably less than 4.5, and optionally, at a concentration of at least 1 M. Preferably, the viral vector is AAV or adenovirus, and / or the cell culture comprises mammalian cells selected from HEK293 cells or HEK293 cell derivatives. In such embodiments, the method may exclude a lysis step based on mechanical or physical lysis, such as repeated freeze-thaw cycles or microfluidization steps for cell lysis.

[0140] Methods for producing viral vectors

[0141] According to the second aspect, a method for producing a viral vector is provided, the method comprising the following steps: x.1 Provide cell cultures capable of producing viral vectors; x.2 Cultured cell cultures to produce viral vectors; x.3 According to the method of the first aspect of the invention, a viral vector is released from the cell culture; and x.4 Optionally, purify the released viral vector.

[0142] Advantageously, the method according to the second aspect enables efficient production of viral vectors by utilizing the improved method of releasing viral vectors from cell cultures according to the first aspect. Therefore, the advantages disclosed above can also be found in the method according to the second aspect. Specifically, this method enables the production of viral vectors with improved capsid and functional viral vector yields within a short incubation time. Furthermore, in the method according to the second aspect, releasing viral vectors from cell cultures results in fewer host cell-related impurities, particularly host cell proteins, thereby producing purer viral vectors.

[0143] The steps and preferred embodiments of the method according to the second aspect correspond to the steps and embodiments of the method according to the first aspect. Therefore, referring to the above disclosure, it should also apply to the method according to the second aspect. This specifically includes, but is not limited to, viral vectors, cell cultures, lysis reagents comprising buffers, salts, divalent cations, detergents, cryoprotectants, nucleases, and acidifying agents, as well as steps (a), (b), and (c). Other features will now be described in detail.

[0144] According to a preferred embodiment, step x.3, releasing the viral vector from the cell culture, is performed by contacting the cell culture (which is a cell culture medium) in a culture vessel with a lysis reagent to generate a lysis composition, incubating the lysis composition in the culture vessel to partially lyse the cell culture, and contacting the lysis composition with an acidifying agent to substantially completely lyse the cell culture. According to a preferred embodiment, at least one of steps x.2 and x.3 is performed in a bioreactor, preferably both steps x.2 and x.3 are performed in a bioreactor. These embodiments have the advantage of allowing for well-defined incubation conditions, for example, in a bioreactor, making it easy to adjust temperature and / or mix, stir, or shake. Exemplary conditions include adjusting the temperature of the cell culture and / or lysis composition, ranging from 5°C to 50°C, preferably from 25°C to 45°C, more preferably from 30°C to 42°C, such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, and 42°C. According to a specific embodiment, the temperature is 37°C. Exemplary conditions also include shaking or agitating the cell culture and / or lysis composition. For example, the cell culture and / or lysis composition may be shaken at 10 rpm to 1000 rpm, preferably 20 rpm to 500 rpm, more preferably 50 rpm to 250 rpm. In a specific embodiment, the cell culture and / or lysis composition is shaken at 120 rpm. According to a specific embodiment, the cell culture and / or lysis composition is incubated at 37°C and shaken at 120 rpm. However, as will be readily apparent to those skilled in the art, different conditions are suitable and can be flexibly adjusted according to respective requirements.

[0145] According to an alternative embodiment, step x.3, releasing the viral vector from the cell culture, is performed by contacting the cell culture (which is a cell culture medium) with a lysis reagent outside the culture vessel to generate a lysis composition and incubating the lysis composition outside the culture vessel to release the viral vector from the cell culture. For example, the cell culture can be transferred from the culture vessel (e.g., a bioreactor) via a sterile connector (e.g., a tube), and the lysis reagent can be added online (i.e., by coupling with a sterile connector) or in another vessel in which the lysis composition is incubated.

[0146] According to a preferred embodiment, the method includes step x.4 of purifying the released viral vector, wherein purification may include a single-step or multi-step method, preferably purification by at least deep filtration, centrifugation, and / or sterile filtration. As shown in the examples, downstream purification of the lysis composition according to this disclosure effectively removes host cell-related impurities, such as nucleic acids (e.g., DNA) and proteins. Therefore, when an acidifying agent is used, some impurities can precipitate, thereby allowing these impurities to be removed during downstream purification, for example, by at least deep filtration, centrifugation, and / or sterile filtration.

[0147] According to a preferred embodiment, compared to methods omitting step (b) or step (d), the purified viral vector contains 10% less host cell-derived impurities (e.g., host cell proteins), preferably 25% or 30% less. As illustrated in the examples, this lower content of host cell-derived impurities (e.g., nucleic acids and proteins) is advantageous because less downstream purification is required and a purer viral vector is produced.

[0148] According to a preferred embodiment, the method further includes step x.5 of preparing a viral vector for gene therapy.

[0149] Kits for releasing viral vectors from cell cultures

[0150] According to a third aspect, a kit for releasing a viral vector from a cell culture is provided, the kit comprising: (i) lysis reagents; and (ii) Acidifying reagents.

[0151] Advantageously, the kit according to the third aspect can effectively release viral vectors from cell cultures and can be used within the scope of the methods disclosed herein. Therefore, the advantages disclosed above can also be found for cell lysis according to the third aspect. Specifically, the kit can produce viral vectors with improved capsid and functional viral vector yields within a short incubation time. Furthermore, using the kit according to the third aspect to release viral vectors from cell cultures results in fewer host cell-related impurities, particularly host cell proteins, thereby producing purer viral vectors.

[0152] The various and preferred embodiments of the kit according to the third aspect correspond to the embodiments of the methods according to the first and second aspects. Therefore, referring to the above disclosure, it should also apply to the kit according to the third aspect. This specifically, but not exclusively, includes viral vectors, cell cultures, lysis reagents comprising buffers, salts, divalent cations, detergents, cryoprotectants, nucleases, and acidifying agents. Other features will now be described in detail.

[0153] According to a preferred embodiment, the kit is used for the method for releasing the viral vector according to the first aspect and / or for the method for producing the viral vector according to the second aspect.

[0154] According to the preferred embodiment, the pyrolysis reagent is an alkaline pyrolysis reagent containing a detergent, preferably selected from: i.1x alkaline lysis reagent, pH greater than 7.0, preferably greater than 7.5, most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v); ii.5x alkaline lysis reagent, with a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.3 M to 1.5 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.3 M to 1.5 M; - Descaling agent, preferably polysorbate, with a concentration selected from 1% (v / v) to 5% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 5 mM to 15 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 10% (v / v) to 35% (w / v); or iii. Preferably, the 10x alkaline lysis reagent has a pH greater than 7.0, more preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 1 M to 3 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.8 M to 5 M or 1 M to 3 M; - Descaling agent, preferably polysorbate, at a concentration selected from 4% (v / v) to 10% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 15 mM to 50 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 20% (w / v) to 60% (w / v).

[0155] According to a preferred embodiment, the acidifying agent comprises an acid, preferably an organic acid, more preferably a carboxylic acid, such as acetic acid or citric acid. As shown in the following examples, this acidifying agent composition is particularly suitable for releasing viral vectors.

[0156] According to a preferred embodiment, the acidifying agent is provided in liquid or solid form, preferably in liquid form, wherein the liquid form preferably comprises an acid and a solvent, preferably but not limited to water. Other suitable solvents are readily known to those skilled in the art.

[0157] Reagent kit for producing viral vectors

[0158] According to the fourth aspect, a kit for producing viral vectors is provided, the kit comprising: (i) A kit according to a third aspect of the invention; (ii) One of the following two: I. Mammalian cells capable of producing viral vectors, which are modified by genome editing of mammalian cells using one or more nucleic acid molecules for viral vector production, thereby enabling stable production of viral vectors; or II. Mammalian cells capable of being modified with one or more viral vector production plasmids to produce transient viral transfection, thereby transiently producing viral vectors.

[0159] Advantageously, the kit according to the fourth aspect enables efficient production of viral vectors by utilizing the improved kit for releasing viral vectors from cell cultures according to the third aspect. Therefore, the aforementioned advantages disclosed in the methods of the first and second aspects, as well as the kit according to the third aspect, can also be found in the kit according to the fourth aspect. Specifically, this kit enables the production of viral vectors with improved capsid and functional viral vector yields within a short incubation time. Furthermore, using this kit results in fewer host cell-related impurities, particularly host cell proteins, thereby enabling the production of purer viral vectors.

[0160] The various and preferred embodiments of the kit according to the fourth aspect correspond to the methods according to the first and second aspects and the embodiments of the kit according to the third aspect. Therefore, referring to the above disclosure, it should also apply to the kit according to the third aspect. This specifically includes, but is not limited to, viral vectors, cell cultures, lysis reagents containing buffers, salts, divalent cations, detergents, cryoprotectants, nucleases, and acidifying agents. Other features will now be described in detail.

[0161] According to a preferred embodiment, the kit is used in accordance with the method for producing a viral vector according to the second aspect.

[0162] According to a preferred embodiment, the kit includes one or more of the following components: -A cell culture medium suitable for culturing mammalian cells; - Transfection reagent; and / or - A plasmid encoding at least a portion of one or more viral vectors, preferably at least a portion of adeno-associated virus (AAV) or adenovirus (Ad).

[0163] According to the terms of this disclosure

[0164] The following provisions provide for further advantageous implementations of this disclosure: 1. A method for releasing a viral vector from a cell culture, the method comprising the following steps: (a) Provide lysis reagents; (b) Contacting cell cultures with a lysis reagent to produce a lysis composition; (c) Incubating the lysis composition to release the viral vector from the cell culture; and (d) Contact the pyrolysis composition with an acidifying agent to lower the pH.

[0165] 2. The method according to Clause 1, wherein in step (c), the cell culture is partially lysed, and / or wherein in step (d), the lysis composition is contacted with an acidifying agent to cause substantially complete lysis of the cell culture.

[0166] 3. The method according to clause 1 or 2, wherein the method has one or more of the following characteristics: - This method does not include pyrolysis steps based on mechanical or physical pyrolysis, and / or - This method does not include repeated freeze-thaw cycles for cell lysis or microfluidization steps for cell lysis.

[0167] 4. The method according to one or more of clauses 1 to 3, wherein the cell culture is a cell culture medium or a cell precipitate, preferably a cell culture medium.

[0168] 5. The method according to one or more of clauses 1 to 4, wherein in step (b), it is not necessary to separate the cell culture from the surrounding liquid before contacting the cell culture with the lysis reagent.

[0169] 6. The method according to one or more of clauses 1 to 5, wherein at least one of steps (b), (c) and (d) is carried out in a bioreactor, preferably at least two of these steps are carried out in a bioreactor, and most preferably all steps (b), (c) and (d) are carried out in a bioreactor.

[0170] 7. The method according to Clause 6, wherein stirring of the bioreactor facilitates mixing in these steps.

[0171] 8. The method according to clause 6 or 7, wherein the bioreactor is capable of temperature control and / or pH control.

[0172] 9. The method according to one or more of clauses 6 to 8, wherein the bioreactor is capable of pH adjustment, preferably, the pH adjustment is performed by configuring in step (d) to contact the pyrolysis composition with the acidifying agent.

[0173] 10. The method according to one or more of clauses 1 to 9, wherein the contact step (d) comprises titrating the acidifying agent into the pyrolysis composition.

[0174] 11. The method according to one or more of clauses 1 to 10, wherein step (d) includes one or more steps of determining pH, preferably by using a pH probe.

[0175] 12. The method according to one or more of clauses 1 to 11, wherein in step (d): - The acidifying agent lowers the pH to an acidic pH, preferably less than 6.0, more preferably less than 5.0, and most preferably less than 4.5; and / or - The acidifying agent lowers the pH to a range selected from the following: 1.5 to 6.0, preferably 2.5 to 5.0, more preferably 3.5 to 4.5.

[0176] 13. The method according to one or more of clauses 1 to 12, wherein the acidifying agent comprises an acid, preferably an organic acid.

[0177] 14. The method according to Clause 13, wherein the acid is a carboxylic acid.

[0178] 15. The method according to clause 13 or 14, wherein the acid is acetic acid or citric acid, preferably acetic acid.

[0179] 16. The method according to one or more of clauses 13 to 15, wherein the acid is a concentrated acid having a concentration sufficient to reduce the pH of the pyrolysis composition to an acidic pH, preferably less than 6.0, more preferably less than 5.0, most preferably less than 4.5, and optionally, the concentration is at least 1 M.

[0180] 17. The method according to one or more of clauses 1 to 16, wherein the acidifying agent is provided in liquid or solid form, preferably in liquid form.

[0181] 18. The method according to one or more of clauses 1 to 17, wherein the acidifying agent comprises an acid and a solvent, preferably water.

[0182] 19. The method according to one or more of clauses 1 to 18, wherein the lysis reagent is an alkaline lysis reagent, preferably with a pH greater than 7.0, more preferably greater than 7.5, most preferably greater than 7.7, such as 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or higher than 8.5.

[0183] 20. The method according to Clause 19, wherein the pH range of the alkaline lysis reagent is selected from 7.2 to 10, preferably 7.5 to 9.5, more preferably 8 to 9 or 8.2 to 8.

[0184] 21. The method according to clause 19 or 20, wherein the alkaline lysis reagent comprises a buffer suitable for buffering the lysis reagent at an alkaline pH, preferably capable of buffering at a pH range selected from 7.2 to 9.5.

[0185] 22. The method according to one or more of clauses 1 to 21, wherein the lysis reagent comprises a buffer selected from the group consisting of tris(hydroxymethyl)aminomethane (TRIS) buffer, 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, phosphate buffer, glycine-sodium hydroxide buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer, TES buffer, DIPSO buffer, TAPSO buffer, triethanolamine buffer, HEPPSO buffer, POPSO buffer, TRICINE buffer, HEPPS buffer, EPPS buffer, BICIN buffer, TAPS buffer, AMPSO buffer, taurine buffer, CHES buffer, AMP buffer, CAPSO buffer, or combinations thereof, preferably TRIS buffer or BTP buffer.

[0186] 23. The method according to one or more of clauses 1 to 22, wherein the lysis reagent comprises a buffer solution with a concentration of 0.01 M to 3 M, preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.05 M to 1 M; ii) For 5x lysis reagent, the concentration is 0.3 M to 1.5 M; or iii) Preferably, for 10x lysis reagent, the concentration is 1 M to 3 M.

[0187] 24. The method according to one or more of clauses 1 to 23, wherein the cleavage agent comprises a salt, preferably selected from potassium chloride or sodium chloride.

[0188] 25. The method according to Clause 24, wherein the concentration of the salt in the lysis reagent is from 0.01 M to 5 M, preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.05 M to 1 M; ii) For 5x lysis reagent, the concentration is 0.3 M to 1.5 M; or iii) Preferably, for 10x lysis reagent, the concentration is 0.8 M to 5 M or 1 M to 3 M.

[0189] 26. The method according to one or more of clauses 1 to 25, wherein the pyrolysis reagent comprises a detergent, preferably a nonionic detergent, more preferably a nonionic detergent not classified as toxic and / or harmful to the environment.

[0190] 27. The method according to Clause 26, wherein the detergent is selected from one or more of Tween, Triton, Nonidet, Igepal or Tergitol.

[0191] 28. The method according to clause 26 or 27, wherein the detergent is an alkyl glucoside, preferably a polysorbate, such as polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), and polysorbate 80 (Tween 80).

[0192] 29. The method according to one or more of clauses 26 to 28, wherein the detergent is a polyoxyethylene alkyl ether, such as Triton X-100, Nonidet P40, NP-40 and various nonionic detergents from the Brij class.

[0193] 30. The method according to one or more of clauses 26 to 29, wherein the concentration of the detergent is from 0.1% (v / v) to 20% (v / v), preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.1% (v / v) to 2% (v / v). ii) For 5x lysis reagent, 1% (v / v) to 5% (v / v); or iii) For 10x lysis reagent, the concentration is 4% (v / v) to 10% (v / v).

[0194] 31. The method according to one or more of clauses 1 to 30, wherein the cleavage agent comprises a divalent cation, preferably Mg. 2+ Ca 2+ Ba 2+ Cu 2+ Fe 2+ Zn 2+ Mn 2+ Ni 2+ Or combinations thereof, preferably Mg 2+ Cu 2+ Zn 2+ Mn 2+ Ni 2+ Or a combination thereof, Mg is the most preferred. 2+ .

[0195] 32. The method according to clause 31, wherein the concentration of the divalent cation in the cleavage reagent is from 0.1 mM to 1000 mM, preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.5 mM to 5 mM; ii) For 5x lysis reagent, the concentration is 5 mM to 15 mM; or iii) Preferably, for 10x lysis reagent, the concentration is 15 mM to 50 mM.

[0196] 33. The method according to one or more of clauses 1 to 32, wherein the lysis reagent comprises a cryoprotectant, preferably a sugar, more preferably sucrose, trehalose or mannitol, and most preferably sucrose.

[0197] 34. The method according to clause 33, wherein the concentration of the cryoprotectant in the lysis reagent is from 1% (w / v) to 70% (w / v), preferably selected from the following range: i) For 1x lysis reagent, 1% (w / v) to 10% (w / v); ii) For 5x lysis reagent, 10% (v / v) to 35% (w / v); or iii) For 10x lysis reagent, the concentration is 20% (w / v) to 60% (w / v).

[0198] 35. The method according to one or more of clauses 1 to 34, wherein the method further comprises providing a nuclease, wherein the nuclease is added to a lysis reagent, a cell culture and / or a cell lysis composition, preferably to a cell culture and / or a cell lysis composition.

[0199] 36. The method described in Clause 35, wherein the nuclease is salt-tolerant.

[0200] 37. The method according to one or more of clauses 1 to 36, wherein in step (b), the pH of the pyrolysis composition is greater than 7.2, preferably greater than 7.5, most preferably greater than 7.8 or 8.0.

[0201] 38. The method according to one or more of clauses 1 to 37, wherein in step (b), the pH range of the pyrolysis composition is selected from 7.2 to 10.0, preferably 7.5 to 9.5.

[0202] 39. The method according to one or more of clauses 1 to 38, wherein the pyrolysis agent is an alkaline pyrolysis agent containing a detergent, preferably selected from: i.1x alkaline lysis reagent, pH greater than 7.0, preferably greater than 7.5, most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v); ii.5x alkaline lysis reagent, with a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.3 M to 1.5 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.3 M to 1.5 M; - Descaling agent, preferably polysorbate, with a concentration selected from 1% (v / v) to 5% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 5 mM to 15 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 10% (v / v) to 35% (w / v); or iii. Preferably, the 10x alkaline lysis reagent has a pH greater than 7.0, more preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 1 M to 3 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.8 M to 5 M or 1 M to 3 M; - Descaling agent, preferably polysorbate, at a concentration selected from 4% (v / v) to 10% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 15 mM to 50 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 20% (w / v) to 60% (w / v).

[0203] 40. The method according to one or more of clauses 1 to 39, wherein in step (b), the pyrolysis composition comprises a compound at a concentration substantially 1x the pyrolysis reagent, preferably substantially the concentration described in clause 39, and / or the pyrolysis composition has the following characteristics: I. The pyrolysis composition has a pH greater than 7.2, preferably greater than 7.5, most preferably greater than 7.8 or 8.0, and comprises: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v).

[0204] 41. The method according to one or more of clauses 1 to 40, wherein the incubation step (c) comprises: - Incubation time is less than 6 hours, preferably less than 5 hours or less than 4 hours, more preferably less than 3 hours, such as 2 hours, 1 hour or 0.5 hours; and / or - The incubation time ranges from 15 minutes to 300 minutes, preferably 20 minutes to 250 minutes, 25 minutes to 200 minutes, or more preferably 30 minutes to 150 minutes or 30 minutes to 100 minutes, and most preferably 30 minutes to 90 minutes or 45 minutes to 75 minutes.

[0205] 42. The method according to one or more of clauses 1 to 41, wherein the time required to perform steps (c) and (d) is...

[0206] - Less than 360 minutes, preferably less than 300 minutes or less than 240 minutes, more preferably less than 180 minutes or less than 120 minutes, most preferably less than 100 minutes, such as 90 minutes, 80 minutes or 70 minutes or less; and / or

[0207] - The time is selected from the following range: 15 minutes to 360 minutes, preferably 20 minutes to 300 minutes, 25 minutes to 240 minutes, or more preferably 30 minutes to 180 minutes or 40 minutes to 120 minutes, and most preferably 45 minutes to 90 minutes.

[0208] 43. The method according to one or more of clauses 1 to 42, wherein the cell culture is not subjected to sonication, high-pressure homogenization or freeze-thaw.

[0209] 44. The method according to one or more of clauses 1 to 43, wherein prior to steps (a), (b) and (c), the cell culture is not centrifuged, cell precipitated, or the fluid surrounding the cells of the cell culture is exchanged.

[0210] 45. The method according to one or more of clauses 1 to 44, wherein the released viral vector is preferably purified by a single-step or multi-step method after step (d), preferably by at least deep filtration, centrifugation and / or sterile filtration.

[0211] 46. ​​The method according to Clause 45, wherein, compared with a method excluding step (b) or step (d), the host cell-derived impurities (e.g., host cell proteins) in the purified viral vector are reduced by 10%, preferably by 25% or 30%.

[0212] 47. The method according to one or more of clauses 1 to 46, wherein the capsid viral vector titer and / or transduced viral vector titer are increased compared to the method without step (d).

[0213] 48. The method according to Clause 47, wherein the capsid virus vector titer is increased by at least 10%, preferably at least 25%, compared to the method without step (d).

[0214] 49. The method according to clause 47 or 48, wherein the transduced viral vector titer is increased by at least 10%, preferably at least 25%, compared to the method without step (d).

[0215] 50. The method according to one or more of clauses 1 to 49, wherein at least 10% more viral vector is released, preferably at least 25%, compared to a method without step (d).

[0216] 51. A method for producing a viral vector, the method comprising the following steps: x.1 Provide cell cultures capable of producing viral vectors; x.2 Cultured cell cultures to produce viral vectors; x.3 Release of viral vectors from cell cultures as described in one or more of clauses 1 to 50; and x.4 Optionally, purify the released viral vector.

[0217] 52. The method according to Clause 51, wherein step x.3 of releasing the viral vector from the cell culture is performed by: contacting the cell culture (cell culture medium) in the culture vessel with a lysis agent to generate a lysis composition and incubating the lysis composition in the culture vessel to partially lyse the cell culture and contacting the lysis composition with an acidifying agent to substantially completely lyse the cell culture.

[0218] 53. The method according to clause 51 or 52, wherein the method includes purifying the viral vector, wherein purification includes a single-step or multi-step method, preferably purification by at least deep filtration, centrifugation and / or sterile filtration.

[0219] 54. The method according to Clause 53, wherein, compared with the method without step x.3 in step (b) or step (d), the host cell-derived impurities in the purified viral vector released in step x.4 are reduced by 10%, preferably by 25% or 30%.

[0220] 55. The method according to one or more of clauses 51 to 54, wherein the method further comprises the step of preparing a viral vector for gene therapy. x.5.

[0221] 56. The method according to one or more of clauses 51 to 55, wherein at least one of steps x.2 and x.3 is carried out in a bioreactor, preferably both steps x.2 and x.3 are carried out in a bioreactor.

[0222] 57. The method according to one or more of clauses 1 to 56, wherein the viral vector is primarily present within the cell and is preferably selected from adeno-associated virus (AAV) or adenovirus (Ad).

[0223] 58. The method according to one or more of clauses 1 to 57, wherein the cell culture comprises mammalian cells.

[0224] 59. The method according to Clause 58, wherein the mammalian cells are modified to be configured to produce viral vectors, preferably AAV.

[0225] 60. The method according to clause 58 or 59, wherein the mammalian cells are selected from HEK293 cells or cells derived from HEK293 cells.

[0226] 61. The method according to one or more of clauses 1 to 60, wherein the cell culture comprises cells capable of suspension culture.

[0227] 62. A kit for releasing a viral vector from a cell culture, the kit comprising: (i) lysis reagents; and (ii) Acidifying reagents.

[0228] 63. The kit according to clause 62, wherein the lysis reagent is an alkaline lysis reagent containing a detergent, preferably selected from: i.1x alkaline lysis reagent, pH greater than 7.0, preferably greater than 7.5, most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v); ii.5x alkaline lysis reagent, with a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.3 M to 1.5 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.3 M to 1.5 M; - Descaling agent, preferably polysorbate, with a concentration selected from 1% (v / v) to 5% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 5 mM to 15 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 10% (v / v) to 35% (w / v); or iii. Preferably, the 10x alkaline lysis reagent has a pH greater than 7.0, more preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 1 M to 3 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.8 M to 5 M or 1 M to 3 M; - Descaling agent, preferably polysorbate, at a concentration selected from 4% (v / v) to 10% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 15 mM to 50 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 20% (w / v) to 60% (w / v).

[0229] 64. The kit according to clause 62 or 63, wherein the acidifying agent comprises an acid, preferably an organic acid, more preferably a carboxylic acid, such as acetic acid or citric acid.

[0230] 65. A kit according to one or more of clauses 62 to 64, wherein the acidifying agent is provided in liquid or solid form, preferably in liquid form, wherein the liquid form preferably comprises an acid and a solvent, preferably water.

[0231] 66. A kit for producing a viral vector, the kit comprising: (i) A kit according to one or more of clauses 62 to 65; (ii) One of the following two: I. Mammalian cells capable of producing viral vectors, which are modified by genome editing of mammalian cells using one or more nucleic acid molecules for viral vector production, thereby enabling stable production of viral vectors; or II. Mammalian cells capable of being modified with one or more viral vector production plasmids to produce transient viral transfection, thereby transiently producing viral vectors.

[0232] 67. The kit according to Clause 66, wherein the kit comprises one or more of the following components: -A cell culture medium suitable for culturing mammalian cells; - Transfection reagent; and / or - One or more plasmids encoding at least a portion of a viral vector, preferably at least a portion of adeno-associated virus (AAV) or adenovirus (Ad).

[0233] Throughout the specification, when a method, kit, composition, or use is described as having, including, or comprising a particular component or step, it may also be anticipated that the method, kit, composition, or use of the present invention is substantially composed of, or consists of, said component or step.

[0234] In this application, when an element or component is referred to as being included in a list of elements or components and / or selected from the list, it should be understood that the element or component can be any of the elements or components, or the element or component can be selected from a group consisting of two or more of the elements or components.

[0235] Unless otherwise stated herein or explicitly contradicted by the context, the terms “a,” “an,” and “the,” as well as similar references used in the context of describing the invention (particularly in the context of the claims), should be interpreted to cover both the singular and the plural.

[0236] Unless otherwise specifically stated or understood in the context, the use of the terms “include,” “including,” “have,” or “contain,” and their grammatically equivalent expressions, and their use should generally be understood as open-ended and non-restrictive, for example, not excluding other elements or steps not listed.

[0237] When the terms “about” or “approximately” are used before a quantitative value, the invention also includes the specific quantitative value itself, unless otherwise specifically stated. As used herein, unless otherwise stated or inferred, the term “about” means a variation within ±10% of the nominal value.

[0238] Similarly, as used herein, “and / or” means and covers any and all possible combinations of one or more of the relevant listed items, and does not combine when interpreted as an alternative (“or”). The use of an alternative (e.g., “or”) should be understood to refer to any one, both, or any combination of the alternatives.

[0239] All citations are incorporated into this article by reference.

[0240] All individual embodiments and aspects disclosed herein can be combined with each other within the framework and context of this disclosure. It should be understood that the embodiments disclosed herein are merely exemplary, and any feature presented for a particular exemplary embodiment may be used alone in this disclosure, or in combination with any feature presented for the same or other particular exemplary embodiments, and / or in combination with any other feature not mentioned. It will be further understood that any feature presented for exemplary embodiments in a particular category may also be used in a corresponding manner for exemplary embodiments in any other category.

[0241] Example

[0242] It should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the invention in any way. The following examples demonstrate the methods of the present invention for releasing viral vectors (preferably adeno-associated virus (AAV) or adenovirus (Ad)) from cell cultures, methods for producing viral vectors (preferably AAV or adenovirus (Ad)), and the advantages of the kits according to this disclosure. Specifically, the examples show that the two-step lysis method disclosed herein increases the extraction of viral vectors, wherein the two-step lysis method includes the steps of contacting cell cultures with a lysis reagent to generate a lysis composition and incubating the lysis composition, and then contacting the lysis composition with an acidifying agent. This increase in viral vector extraction is achieved by improving the efficiency of cell lysis while simultaneously improving the removal of host cell-related impurities.

[0243] Specifically, the lysis method disclosed herein divides the process into two short lysis steps and applies different, advantageous conditions in the cell culture medium that cannot be combined in a single step. In the first step, a concentrated (e.g., 10x) high-salt alkaline lysis buffer containing detergent and salt-tolerant nuclease is added to the bioreactor to initiate cell lysis. The bioreactor's stirring function is used to mix all components and keep the cells in suspension. Furthermore, activation is performed at a controlled temperature of 37°C to activate maximum nuclease activity. The regulated lysis conditions are maintained for approximately 1 hour. After this, most cells are lysed, so only a small fraction (<20%) remain viable. At this point, the pH is abruptly reduced to pH 4 by titration with a concentrated acidifying agent (e.g., an acid, such as 2 M acetic acid). In this second step, the low pH causes loss of nuclease activity but also causes immediate lysis of the remaining cells. Furthermore, the low pH causes impurities, primarily host cell-related impurities such as host cell proteins or nucleic acids and cell debris, to precipitate almost immediately. Therefore, the subsequent clarification process can begin quickly, for example, a few minutes after pH 4. The addition of acidifying agents can also be achieved using the pH controller of the bioreactor system, ensuring that the amount added is sufficient to acidify the lysis composition while monitoring the pH, for example, via a pH probe within the bioreactor. Alternatively, the acid can be added and mixed online when the cell culture medium is transferred to the filtration stage for clarification. Another advantage of lowering the pH to an acidic level (e.g., pH 4) is its applicability to subsequent downstream steps. Conventional capture steps typically require the virus to be in a specific pH environment, usually achieved by adjusting the pH through ultrafiltration and osmosis. Depending on the virus's stability, the pH can be neutralized in a later step. However, this should only be done after clarification, meaning only after the precipitate has separated from the supernatant. Figure 1 Possible setups for integrating a solution based on the method of this disclosure into a biological treatment solution are shown.

[0244] Materials and methods

[0245] Cell culture and viral vector production

[0246] A two-step cell lysis method was developed and applied to suspension-domesticated HEK293 cells (Expi293F) capable of transiently producing viral vectors such as adeno-associated virus (AAV) or adenovirus (Ad). TM Other lysis methods from Thermo Fisher Scientific Inc. were compared. HEK293 cells were cultured in stirred bioreactors of varying batch sizes. HEK293 cells were modified by transient transfection within defined culture times to initiate viral vector production. Multiple viral vectors were produced, and the feasibility of two-step lysis for different products was demonstrated using cell culture media.

[0247] As an AAV model, two different serotypes, AAV2 and AAV8, were produced by transiently transfecting cells with one or more plasmids. As an adenovirus (Ad) vaccine model, the ChAdOx1 nCoV-19 adenovirus vector was used. This Ad vector is based on a modified Y25 chimpanzee adenovirus serotype and was developed by the Jenner Institute at the University of Oxford (see Joeet. al. 2021 (bioRxiv 2021.12.22.473478)). Transient production was initiated by infecting HEK293 cell cultures with purified virus. Following transfection (AAV) or infection (Ad), cells were cultured for a defined timeframe for viral vector production, typically 48 to 72 hours. Cell cultures were then immediately used for lysis experiments.

[0248] lysis reagent

[0249] To regulate lysis conditions in whole-cell culture while minimizing cell dilution caused by the addition of lysis reagent, a concentrated lysis reagent solution was prepared. Preferably, a 10x concentrated lysis reagent solution was used (in some cases, even lower concentrations), but this is limited by the solubility of the components. To initiate the first lysis step, 1 volume portion of the 10x lysis reagent solution was added to 9 volumes of cell culture, resulting in a viral vector dilution of approximately 11%.

[0250] The lysis reagent solution used in the first step of the two-step lysis method may contain buffer, salt, divalent cation, cryoprotectant, and detergent, which will be described in more detail below. It should be noted that a nuclease (e.g., San HQ 2.0, ArcticZymes, Norway) may also be added in the first lysis step, but it is preferred to store and add it separately to avoid loss of enzyme activity due to concentrated lysis reagent.

[0251] The lysis reagent solution contains a detergent, such as polysorbate 20, which effectively dissolves cell membranes and thus promotes lysis. To adjust and stabilize the pH of the lysate, the lysis reagent contains a buffer component, such as TRIS hydrochloride for adjusting the pH to 8.0. However, a small amount of buffer should be added (e.g., 25 mM to 140 mM in the final lysis composition) to avoid high acid consumption due to low pH changes during the second lysis step. Another important component of the lysis reagent solution is a salt, such as potassium chloride (KCl) or sodium chloride (NaCl), designed to facilitate permeation lysis of cells, inhibit viral aggregation, and precipitate impurities. In cases where nucleases are added to the cell culture medium, a suitable divalent cation, such as magnesium chloride (MgCl2), can be included in the lysis reagent solution to enhance nuclease activity. Optionally, a cryoprotectant can be added to the lysis reagent solution to protect the virus from degradation during storage, freezing, and thawing. However, in a preferred embodiment, the cryoprotectant is added before freezing, in a subsequent process step after ultrafiltration and permeation, to reduce fluid viscosity and product dilution during the filtration step.

[0252] To lower the pH (acidification) in the second lysis step, a separate solution is used. This solution contains a concentrated acid, such as 2 M acetic acid. A high concentration is employed to reduce the dilution of the viral product during titration. However, it is important to ensure that the type and concentration of the acid chosen, and the way the system is mixed, do not damage the virus due to localized over-acidification during the acidification process.

[0253] The two-step cleavage method was compared with an internally developed single-step cleavage method and a publicly available single-step cleavage method that serves as a reference standard, which will be described in more detail below.

[0254] As a reference standard, publicly available buffer formulations and corresponding lysis methods were used, based on reference Fedosyuk et al., 2019. This has been disclosed, for example, in reference Joe et. al. 2021 (bioRxiv2021.12.22.473478) and has become a laboratory standard. Another standard lysis reagent used in this study is a lysis reagent (“TRT”) containing 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (which is known as Triton X-100). Triton X-100 and its derivatives have historically been commonly used for AAV extraction, but due to their biohazardousness, they have been included in the REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) list, restricting or prohibiting their use within the European Union.

[0255] Single-step pyrolysis method

[0256] HEK293 cells (Expi293F) were suspension-acclimated using transiently produced viral vectors (e.g., AAV2 and adenovirus). TM Thermo Fisher Scientific Inc. tested different cell lysis reagents. A single-step cell lysis process is as follows: Figure 2 As shown, the process was initiated by adding a cell lysis reagent to HEK293 cells. As a reference for the lysis reagent, a "pre-existing technology cell lysis reagent" was used (e.g., see Fedosyuk et al., 2019, Vaccine, Vol. 37, pp. 6951-6961), requiring the addition of a nuclease for optimal performance. After an incubation period of 0.5 to 3.5 hours, the next step involved purification via sterile filtration using a low-protein-binding filtration unit. The collected filtrate was then analyzed using various quantitative methods.

[0257] Two-step pyrolysis method

[0258] To investigate and develop a two-step lysis method, the following steps were performed in all experiments, regardless of the different viral vectors, reagents, and additives: - The first lysis step is initiated by the following: adding HEK293 suspension cell culture (Expi293F) TM ThermoFisher Scientific Inc. adds concentrated cell lysis reagents and, in some cases, a defined amount of nuclease to produce the target viral vector. With the addition of the components, an incubation phase begins. During the addition and incubation phases, the lysis composition is continuously mixed to avoid concentration gradients and to keep the biomass (cells and cell debris) in suspension. Additionally, the temperature of the lysis composition is controlled within the range of 30°C to 37°C. After a defined incubation period (e.g., 60 minutes), a second lysis step is initiated by acidifying the cell suspension. At this point, most, but not all, of the cells are lysed, and therefore the virus is not fully released at this stage.

[0259] - Manually acidify (lower the pH) by adding concentrated organic acid while continuing to mix the pyrolysis composition. Once the predetermined low pH is reached, begin a second incubation period, which is shorter than the first incubation period (e.g., 5 minutes).

[0260] Finally, the lysis process is completed by sampling and / or clarifying the lysate. Samples for studying cell culture medium properties are analyzed immediately. Additionally, supernatant samples are generated by centrifuging the biomass (5,000 x g, 5 min) for analysis of viral titers and host cell-related impurities. Samples are stored at -80°C prior to analysis.

[0261] Clarification of pyrolysis products

[0262] To investigate the impact of different pyrolysis methods on subsequent clarification operations, a small-scale filter setup was used. The filter arrangement consisted of a two-layer depth filter, with the first layer having an average rejection rate of 8 µm and the second layer having an average rejection rate of 0.8 µm (SartoClear). ® Cap DL60, 25 cm 2 Sartorius Stedim Biotech GmbH), and the sterile filter is 0.2 µm (Sartopore). ® 2 x LG, 17.3 cm 2 SartoScale 47, Sartorius Stedim Biotech GmbH. A peristaltic pump (SciLog) will be equipped with an online SU pressure sensor (17525SP-10, Sartorius Stedim Biotech GmbH) at the front end of each filtration step. ® Parker) is used for process control. Using 150 L / m 2 The volumetric flux per hour, using pure water (50 L / m³), is calculated. 2 Pre-rinse the deep filter at 6.3 mL / min (150 L / m³). 2 Filtration was performed at a constant flow rate ( / h) until the pressure reached 1.3 bar. To improve mAb recovery, a buffer wash (20 L / m) was performed with the appropriate 1x lysis buffer at a constant pressure limited to 1.5 bar. 2 The total filtration flux is calculated by dividing the filtration volume of the harvested liquid by the area of ​​the depth filter.

[0263] Functional titer quantification of AAV2

[0264] The yield of active AAV2 vectors is determined by AAV2 transduction assays (also known as potency, activity, infectivity assays, or assays). This quantitative method is used to determine the potency of AAV2 vectors. Regulatory agencies such as the FDA and the European Pharmacopoeia (Ph. Eur.) require potency testing for cell and gene therapy products, making it an important aspect of the development process.

[0265] The potency assay performed in this study complies with FDA requirements for potency testing of cell and gene therapy products. This in vitro assay was used to evaluate the transfer of genetic material from a viral vector to a susceptible cell line, followed by quantification of transgenic expression. This robust and reliable assay provides valuable information on the efficacy and functionality of the AAV vector being evaluated.

[0266] For AAV2 transduction assays, adherent HEK293 cells were seeded in 24-well or 96-well plates and immediately transduced with serially diluted AAV2 filtrate. After 72 hours of incubation, the transduction was performed using Incucyte. ® Live cell analysis system (SartoriusStedim Biotech GmbH) analyzes GFP expression.

[0267] Quantitative determination of capsid titer

[0268] For capsid titer quantification, Octet is used. ® The biolayer interferometry platform (Sartorius StedimBiotech GmbH) determined the binding rate of serially diluted, clarified cell lysates. AAV samples were subjected to [the following method / process] on a commercially available Octet [system / platform]. ® The AAVX biosensor (Sartorius Stedim Biotech GmbH) was loaded for 1800 seconds. The binding rate was compared with the corresponding commercially available AAV2 and AAV8 standards (Progen).

[0269] Adenovirus (Ad) samples were loaded onto Octet pre-coated with Ad-specific antibodies (Abcam). ® The ProA biosensor (Sartorius Stedim Biotech GmbH) was used. The preparation and titer quantification of the Ad probe were performed according to the protocol published by Kruse et al. in 2023 (https: / / www.sartorius.com / en / pr / octet / adenovirus-quantitation-application-note?utm_source=marketo&utm_medium=email&utm_campaign=octet-bioprocessing&utm_term=adenovirus&utm_content=application-note%C2%A0#id-1487168).

[0270] Analysis of host cell-related impurities (proteins and DNA)

[0271] To quantify DNA, use Quant-iT according to the manufacturer's instructions. TM PicoGreen TMdsDNA assay kit (P7589, ThermoFisher Scientific, USA). Dilute samples with TRIS-EDTA buffer (10 mM TRIS, 1 mM EDTA, 0.1% SDS) to achieve a detection range of 3.13 ng / mL to 200 ng / mL.

[0272] To determine HCP impurities, the HEK 293 HCP 3G ELISA kit (F650S, Cygnus Technologies, USA) was used according to the manufacturer's instructions.

[0273] Both assays used an ELISA reader. ® 200, Tecan, Switzerland). All samples were analyzed in at least duplicate.

[0274] Other quantitative methods

[0275] Viable cell concentration (VCC), total cell concentration (TCC), viability, and mean cell diameter were analyzed using an automated cell counting system based on trypan blue staining (Cedex HiRes Analyzer, Roche, Germany). For turbidity determination, a turbidimeter (TL2350, Hach, Germany) with 10 mL cuvettes was used.

[0276] Example 1: Single-step cleavage reagent for the cleavage production of AAV2 from HEK293

[0277] In Example 1, after culturing and producing AAV2, the single-step lysis reagent according to this disclosure was used to lyse suspension-acclimated HEK293 cells. Cell lysis was performed by centrifugation at 300 g for 5 minutes for collection. 1x cell lysis buffer was added to the cell pellet, followed by an incubation step for 0.5 to 3.5 hours. Finally, a clarification step was performed using a low-protein binding filtration unit. Table 1 lists examples of the cell lysis reagents tested.

[0278] Table 1: Examples of single-step cell lysis reagents

[0279] Filterability of crude pyrolysis products

[0280] To test the filtration capability of crude lysate, crude lysate from HEK293 cells (Expi293, Thermo Fisher Scientific) producing the AAV2 vector was aseptically filtered through a 0.22 µm CA Spin-X filter (SigmaAldrich). Variables of cell lysis conditions (pH, salt molar concentration, incubation time, detergent addition) and photographs of the spin filter are shown below. Figure 2 As shown. The cell lysis assay shown was performed without a nuclease digestion step.

[0281] Figure 2 The photograph highlights how the single-step acidic lysis reagent, when added to cells, forms a lysis composition that is very easily filtered. Specifically, a clear permeate containing AAV2 is formed, while precipitated impurities (such as host cell-associated impurities) remain on the filter. In contrast, the lysis reagent at pH 8 produces lysates with high viscosity, requiring additional nuclease digestion to facilitate the filtration step (see [link to image]). Figure 2 Experimental setup N25 for the nuclease-free digestion step shown.

[0282] Host cell impurity analysis

[0283] To determine host cell-related impurities, SDS-PAGE analysis was performed on sterile filtered crude lysates of HEK293 (Expi293) cells producing the AAV2 vector under reducing and denaturing conditions. Protein bands on the gel were visualized by Coomassie blue staining. Table 1 shows the cell lysis condition variables (pH, salt molar concentration, incubation time, and detergent addition) for the single-step cell lysis reagent. M in lanes 1 and 13 represents PageRuler. TM Unstained protein molecular weight standard (ThermoFisher Scientific).

[0284] Figure 3 The gel bands shown indicate that acidic pH significantly reduced host cell-related impurities compared to alkaline pH lysis reagents. Therefore, the results suggest that lower pH is beneficial for improving the purity of the viral vector (AAV2 in this case), leading to more efficient downstream purification while improving the safety of the viral vector product. Furthermore, the gel bands correlated with observed improved filterability (see [link to documentation]). Figure 2 This is because better filtered samples contain fewer host cell-related impurities. Furthermore, this advantageous property of the acidic lysis reagent according to this disclosure is consistently observed for different salt concentrations ranging from 50 mM to 500 mM, where slightly fewer host cell-related impurities appear to be present under higher salt concentration conditions.

[0285] in conclusion

[0286] Example 1 demonstrates that the single-step lysis reagent solves the following technical problems by implementing several key improvements: i) Facilitates the removal of host cell impurities: Acidic cell lysis reagents, due to their low pH, facilitate the precipitation and effective removal of host cell-related impurities, thereby achieving improved purification and yielding a higher quality final product. The removal of host cell proteins was confirmed by densitometric analysis of SDS-PAGE gels stained with Coomassie blue (see [link to original text]). Figure 3 The experimental setups shown are N1, N5, and N9. Acidic reagents precipitate most protein and nucleic acid impurities during the cell lysis step while maintaining high target molecule yield and functionality. Therefore, a higher purity final product can be obtained with fewer downstream processing steps. This helps reduce product loss and achieve more efficient purification.

[0287] ii) Reduction of AAV vector aggregates: Improved filterability and purity indicate that acidic cell lysis reagents minimize the formation of AAV vector aggregates, thereby simplifying the purification process and potentially achieving higher titers.

[0288] iii) Effective filtration without the need for additional nuclease digestion (see Figure 2 The experimental setups shown are N2, N5, and N6. In contrast, when cell lysis was performed using a reagent at pH 8, the resulting lysates were highly viscous, requiring additional nuclease digestion to facilitate the filtration step (see [link to experiment description]). Figure 2 Experimental setup N25 for the nuclease-free digestion step shown.

[0289] iv) Shorter incubation time: The single-step cell lysis reagent enables efficient cell lysis and AAV vector extraction in a short time. Shortening the incubation time in the production process can reduce overall production costs.

[0290] v) No biohazard: The reagents are formulated using ingredients that are safe for humans and the environment, eliminating the potential biohazard risks associated with the use of the reagents (e.g., Triton-X100).

[0291] Overall, the improved viral vector extraction efficiency provided by the single-step lysis reagent of this disclosure is likely to significantly impact the manufacturing cost of viral vector-based drugs. These technological and economic advances, through increased yield and simplified manufacturing processes, promise to make viral vector-based therapies more accessible and affordable for patients in need.

[0292] Example 2: Analysis of different single-step lysis reagent formulations

[0293] In Example 2, different formulations of single-step buffer systems and lysis reagents were tested. Typically, concentrated lysis reagents, such as 5x or 10x lysis reagents, were used to achieve concentrations equivalent to 1x lysis reagents. As shown below, the lysis reagents disclosed herein address technical problems commonly encountered in cell lysis, including low cell lysis efficiency and / or long lysis duration, high AAV carrier aggregation, and the potential need for physical or mechanical lysis steps (e.g., freeze-thaw cycles).

[0294] In Example 2, a lysis reagent was used to lyse whole-cell cultures and cell pellets. Specifically, according to this disclosure, suspension-acclimated HEK293 cells cultured and used to produce AAV2 were used for this purpose. To lyse whole-cell cultures, a 5x or 10x concentrated lysis reagent is typically used to achieve a concentration comparable to the 1x lysis reagent in the previous examples. To lyse the cell pellet, an equal volume of whole-cell culture was first centrifuged at 300 g for 5 minutes, and the supernatant was removed. The resulting cell pellet was then treated with the 1x lysis reagent so that the concentrations of the components of the cell lysis reagent were comparable between the whole-cell culture and the cell pellet. The single-step cell lysis reagents used are listed in Table 2 below. As shown below, the single-step lysis reagents address common technical problems encountered in cell lysis, including low cell lysis efficiency and / or long lysis duration, high AAV carrier aggregation, and the potential need for physical or mechanical lysis steps (e.g., freeze-thaw cycles). Furthermore, the following demonstrates that the lysis reagents are suitable for various sample types, such as cell cultures and cell pellets, and yield consistent results. This is particularly advantageous because different types of viral vectors can exist primarily intracellularly, extracellularly, or both. By being applicable to different sample types, these lysis reagents are suitable for intracellular and / or extracellular viral vectors.

[0295] Table 2: Formulation of the single-step cell lysis reagent used in Example 2

[0296] AAV2 functional titer in cell culture medium

[0297] After cell lysis and clarification in whole-cell culture medium, the functional titer of AAV2 was determined using the aforementioned Incucyte-based transduction assay. For comparison, "prior art cell lysis reagents" and CelLytic were used. TM M (C1, SigmaAldrich), CelLytic TMMT (C2, Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents were used as reference lysis reagents. Results are as follows: Figure 4 As shown.

[0298] like Figure 4 As shown, CelLytic TM M(C1), CelLytic TM The MT (C2) and M-PER (C3) lysis reagents showed very low functional titers, less than 0.5 × 10⁻⁶. 6 TU / mL. This result was expected, as these lysis reagents were only indicated for use with cell pelleting. On the other hand, "existing technology cell lysis reagents" (see N82) also produced relatively low functional titers, approximately 4 × 10⁻⁶. 6 TU / mL. The single-step cell lysis reagent disclosed herein can achieve nearly 10 TU / mL. 7 Functional titer of TU / mL.

[0299] AAV2 capsid titer in cell culture medium

[0300] After cell lysis and clarification in whole-cell culture medium, the above-mentioned Octet was used. ® The AAVX biosensor determines AAV2 capsid titer. For comparison, "prior technology cell lysis reagents" and CelLytic were used. TM M (C1, SigmaAldrich), CelLytic TM MT (C2, Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents were used as reference lysis reagents. Results are as follows: Figure 5 As shown.

[0301] like Figure 5 As shown, CelLytic TM M(C1), CelLytic TM The MT (C2) and M-PER (C3) lysis reagents did not produce any measurable capsid titers. This result was expected, as these lysis reagents are only indicated for use with cell pelleting. On the other hand, the "prior art cell lysis reagent" (see N82) also produced relatively low capsid titers, approximately 3 × 10⁻⁶. 11 Capsids / mL. The single-step reagent of this disclosure can yield up to 7 × 10⁻⁶ capsids / mL. 11 Capsule titer per mL.

[0302] Overall, the results indicate that the single-step lysis reagent is not only well-suited for cell culture media, but also yields improved capsid titers, demonstrating its beneficial effects and more effective cell lysis.

[0303] AAV2 functional titer in cell pellet

[0304] Following cell lysis and clarification of the cell pellet, the functional titer of AAV2 was determined using the aforementioned Incucyte-based transduction assay. For comparison, "prior art cell lysis reagents" and CelLytic were used. TM M (C1, Sigma Aldrich), CelLytic TM MT (C2, Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents were used as reference lysis reagents. Results are as follows: Figure 6 As shown.

[0305] like Figure 6 As shown, CelLytic TM The M(C1) and M-PER(C3) lysis reagents, as well as the "Prior Art Cell Lysis Reagents" (see N98), showed very low functional titers, less than 0.5 × 10⁻⁶. 6 TU / mL. Only CelLytic TM MT(C2) was obtained with a measurable functional titer of approximately 4 × 10⁻⁶. 6 TU / mL. The single-step reagent of this disclosure can yield approximately 6 × 10⁻⁶ TU / mL. 6 Functional titer of TU / mL.

[0306] AAV2 capsid titer in cell pellet

[0307] After cell lysis and clarification of the cell pellet, the above-mentioned Octet was used. ® The AAVX biosensor determines AAV2 capsid titer. For comparison, "prior technology cell lysis reagents" and CelLytic were used. TM M (C1, Sigma Aldrich), CelLytic TM MT (C2, Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents were used as reference lysis reagents. Results are as follows: Figure 7 As shown.

[0308] like Figure 7 As shown, CelLytic TMThe M(C1) and M-PER(C3) lysis reagents, as well as the "Prior Art Cell Lysis Reagents" (see N98), did not produce any measurable capsid titers. Only CelLytic... TM MT(C2) yielded a measurable capsid titer of approximately 6 × 10⁻⁶. 11 Capsids / mL. This disclosed single-step cell lysis reagent can achieve up to 7 × 10⁶ capsids / mL. 11 Capsule titer per mL.

[0309] Overall, the results indicate that the single-step lysis reagent according to this disclosure is not only well suited for cell precipitation, but also yields improved capsid titers and functional titers, demonstrating its beneficial effects and more effective cell lysis.

[0310] Beneficial but non-essential ingredients

[0311] also, Figure 4 , Figure 5 , Figure 6 and Figure 7 The data shown indicate that the functional titers and capsid titers obtained with sucrose-free (i.e., cryoprotectant-free) single-step lysis reagents (see N80, N81, N96, and N97) are substantially equivalent to those with sucrose-containing reagents (see N78, N79, N94, and N95). These results highlight that cryoprotectants (e.g., sucrose) are only optionally added to the single-step lysis reagents according to this disclosure. Specifically, these components are added to improve the storability and freezeability of the viral vector-containing composition after lysis and / or purification. However, cryoprotectants are not required for cell lysis. The advantage of these optional compounds as lysis reagents is that the addition of cryoprotectants (e.g., sugars such as sucrose) in subsequent steps (e.g., after cell lysis and / or purification) can be avoided.

[0312] Furthermore, regarding the detergent polysorbate 20, it was observed that, depending on the lysis conditions of full culture and cell precipitation lysis, the inclusion of polysorbate 20 resulted in an increase of up to 30% in both capsid titer and functional titer. Another advantage of polysorbate 20 is its ability to prevent cavitation effects in subsequent downstream treatments (such as filtration), thereby reducing the risk of product loss.

[0313] Based on the filterability and pH of the pyrolysis composition disclosed herein

[0314] Initially, the 10x concentrated reagent was applied to whole-cell cultures to achieve a final concentration of 1x lysis reagent. Subsequently, specific nucleases as shown in Table 2 were added, and the mixture was incubated for 1 hour. After incubation, the filterability and pH of the crude lysis composition were evaluated. The results are shown in Table 3 below: Table 3: Evaluation of the filterability and pH of the crude lysis composition of Example 2. Filterability was qualitatively evaluated and categorized into four levels: A for excellent filterability, B for good filterability, C for acceptable filterability, and D for moderate filterability. "Nuc." indicates nuclease. "na" indicates not applicable because the composition of the commercially available lysis reagent is unknown.

[0315]

[0316] Table 3 shows how the filterability of the crude lysis compositions varies depending on cell lysis conditions. These variations are related to different cell lysis efficiencies and changes in host cell impurities. Acidic lysis reagents N70 to N77 exhibit excellent filterability and are therefore classified as Group A. In contrast, basic lysis reagents exhibit good to moderate filterability. Notably, N78 (…) was identified as the most effective lysis reagent in this disclosure. Figure 4 and Figure 5 It shows moderate filterability.

[0317] in conclusion

[0318] Example 1 demonstrates that the lysis reagent according to this disclosure consistently produces high capsid and functional titers in various types of cell culture samples. Specifically, it shows that the single-step lysis reagent of this disclosure can be used in whole cell culture media and cell pellets. The comparison results of cell lysis efficiency with the best-performing alkaline reagent are as follows: - Alkaline reagents: 100% (cell culture medium) and 100% (cell pellet) - Acidic reagents: ~80% (cell culture medium) and ~45% (cell pellet) - Existing technology reagents: ~45% (cell culture medium) and ~0% (cell pellet) - CelLytic TM MT: ~0% (cell culture medium) and ~80% (cell pellet) Therefore, cells can undergo a separation step from the surrounding liquid (e.g., by centrifugation / sedimentation and removal of supernatant), or the cell culture medium can be used without any treatment. This is particularly advantageous because the cell lysis reagent of this disclosure is flexible and has broad applicability. Furthermore, this application allows for in-situ use of the cell lysis reagent according to this disclosure, i.e., after generating a viral vector (e.g., AAV), the cell lysis reagent can be added directly to the culture vessel (e.g., a bioreactor or shake flask).

[0319] Example 3: The lysis reagent is effective and non-toxic.

[0320] In Example 3, the efficacy of lysis reagents containing Triton X-100 and single-step lysis reagents for whole-cell culture lysis was evaluated. While reagents containing Triton X-100 are renowned for their effectiveness and have historically been widely used for cell lysis and intracellular product extraction, their use is restricted due to their inclusion in the EU's REACH list of Substances of Very High Concern, meaning their use should be avoided to protect human health and the environment from potential risks posed by chemicals. For whole-culture lysis, HEK293 cells cultured and subsequently acclimated to AAV2 production were used for this purpose, according to this disclosure. Typically, 5x or 10x concentrated lysis reagents were used to achieve concentrations equivalent to the 1x lysis reagent in previous examples. The cell lysis reagents used are listed in Table 4 below.

[0321] Table 4: Formulation of the cell lysis reagent used in Example 3

[0322] For different lysis reagents, the functional titer of AAV2 in cell culture medium

[0323] After cell lysis and clarification in whole-cell culture medium, the functional titer of AAV2 was determined using the Incucyte-based transduction assay described above. For comparison, a prior art cell lysis reagent and a lysis reagent containing Triton X-100 were used as reference lysis reagents. Results are as follows: Figure 8 As shown.

[0324] like Figure 8 As shown, the single-step cell lysis reagent can achieve up to 9 × 10⁹ cells / day. 6 TU / ml, while the "Prior Art Cell Lysis Reagent" (see N82) is 4 × 10⁻⁶. 6 TU / ml. Cell lysis reagent containing Triton X-100 (see TNT) up to 7 × 10⁻⁶. 6 The TU / ml figure demonstrates efficiency comparable to the reagent disclosed in this embodiment, but its use is limited due to the cytotoxicity mentioned above and further illustrated below. Therefore, the results indicate that the single-step lysis reagent can efficiently lyse cells and increase the yield of functional titers without any cytotoxic components.

[0325] AAV2 capsid titer in cell culture medium for different lysis reagents

[0326] After cell lysis and clarification in whole-cell culture medium, the above-mentioned Octet was used. ®The AAVX biosensor determined the AAV2 capsid titer. For comparison, a prior art cell lysis reagent, as well as a lysis reagent containing Triton X-100, were used as a reference lysis reagent. Results are as follows: Figure 9 As shown.

[0327] like Figure 9 As shown, the single-step cell lysis reagent can achieve up to 9 × 10⁹ cells / day. 11 One capsid per ml, while "existing technology cell lysis reagents" (see N82) produce almost 4 × 10⁻⁶ capsids per ml. 11 Capsids per ml. A commonly used cell lysis reagent containing Triton X-100 (see TNT) contains approximately 8 × 10⁻⁶ cells / ml. 11 The yield of capsids per ml demonstrates efficiency comparable to the reagent disclosed in this embodiment, but its use is limited due to the cytotoxicity mentioned above and further illustrated below. Therefore, the results indicate that the single-step lysis reagent can efficiently lyse cells and increase capsid titer yield without any cytotoxic components.

[0328] Toxicity testing

[0329] The potential toxicity of Triton X-100 compared to detergents used in the lysis reagents according to this disclosure was investigated. AAV crude cell lysis compositions derived from lysis reagents containing Triton X-100 or polysorbate 20 as shown in Table 4 were initially concentrated at 1x in the final crude lysate after a sterile filtration step. They were then further diluted in the range of 1:10 to 1:320. The resulting diluted samples were applied to adherent HEK293 cells according to the previously described transfection assay protocol. Subsequently, Incucyte was used… ® A live-cell analysis system was used to analyze the phase difference confluence of these cells. The results are as follows: Figure 10 As shown.

[0330] Figure 10 The obtained phase contrast images showed that the presence of Triton X-100 had a significant effect on cells (see TRT), causing a decrease in phase confluence from greater than 90% to 12%. In contrast, regardless of whether the buffer composition was acidic or alkaline (see N70A, N74A, or N78A), even at the highest concentration of Tween 20, cell confluence remained at a high level of 94%, without any decrease. These results indicate that Triton X-100 has a strong cytotoxic effect on cells.

[0331] in conclusion

[0332] Example 3 demonstrates that the single-step lysis reagent consistently achieves high capsid and functional titers across different types of cell culture media, and yields results similar to (or even better than) those obtained with Triton X-100-based lysis reagents. On the other hand, Triton X-100-based lysis reagents are toxic, posing subsequent problems in viral vector purification and waste management. The polysorbate-based lysis reagents used herein do not present this concern, thus offering advantages for safety and waste management reasons.

[0333] Example 4: Comparison of two-step and single-step fragmentation of AAV2

[0334] In Example 4, the method for releasing viral vectors from cell cultures according to this disclosure is compared with single-step lysis, wherein, in particular, the second step of acidifying the lysis composition is not performed. As observed in Examples 1 to 3 above, the alkaline lysis reagent yielded high AAV capsid and functional titers, but simultaneously resulted in an increase in the content of host cell-related impurities. On the other hand, the acidic lysis reagent tested above effectively removed host cell-related impurities and improved filterability in downstream purification, but was not very efficient in releasing the highest viral vector capsid and functional titers. However, in most cases, the acidic lysis reagent was still more effective than the reference lysis reagent tested. However, the inventors decided to adopt a different approach, combining the advantages of the two different single-step lysis methods (Examples 1 and 2) into two independent steps, with the aim of maximizing viral vector yield while keeping host cell-related impurities at a low level. As shown below, this process advantageously maintains high viral vector capsid and functional titers and low levels of host cell-related impurities throughout.

[0335] HEK293 cells from the fed-batch process used to produce AAV2 were used as lysis material, with a viable cell density of 1.7 × 10⁻⁶. 6Cells / mL and a final viability of 59% (after AAV2 production). Lysis reagent was added to approximately 320 mL of fed-batch culture, and the lysis composition was incubated at 30°C to 37°C for 60 minutes. For the two-step method disclosed herein for releasing viral vectors from cell cultures, an acidification step of the lysis composition was subsequently performed by adding 2 M acetate solution to adjust the pH to 4.0. To test the effect of the lysis method on subsequent clarification, the lysate was clarified sequentially using a deep filter and a sterile filter as described above. Subsequently, the capsid titer, functional titer, host cell protein, and DNA content of the sample were quantified as described above. The reagents tested are listed in Table 5. As nucleases, 50 U / mL San HQ 2.0 salt-tolerant nuclease (ArcticZymes, Norway) was used in both the two-step lysis method and the single-step lysis method at pH 8. This nuclease was not added to the single-step lysis method at pH 4 because it is unstable and rapidly denatures at pH 4.

[0336] Table 5: Overview of single-step and two-step lysis methods, including the compositions used and whether nucleases were added. In the two-step method and the single-step method at pH 4, the pH was adjusted to 4 using a 2 M acetate solution after lysis began.

[0337]

[0338] Results of pyrolysis

[0339] After lysis as described above (see Table 5 in particular), the total cell concentration (TCC), cell viability, turbidity, host cell protein (HCP), DNA, AAV capsid titer, and AAV functional titer of the lysed cell culture medium were measured as described above. These results are shown in Table 6 below.

[0340] Table 6: Results of total cell concentration (TCC), cell viability (Viab.), turbidity, host cell protein (HCP), DNA, AAV capsid titer, and AAV functional titer after lysis.

[0341]

[0342] The results in Table 6 indicate that single-step lysis using an alkaline lysis reagent (pH 8) still yielded viable cells (63%), suggesting insufficient cell lysis. This is likely due to the short incubation time of 60 minutes, while longer incubation times (e.g., 120 minutes) would have resulted in complete lysis. On the other hand, the two-step method and single-step lysis at pH 4 yielded 0% viability, indicating essentially complete cell lysis. Cell lysis not only led to the release of AAV but also significantly increased HCP and DNA content. However, changes in pH and conductivity in the lysate may have resulted in the precipitation of at least some impurities, which can be facilitated by subsequent removal (e.g., by centrifugation and filtration). The precipitate showed a high turbidity of 115 NTU, compared to 50 NTU for the two-step method and 39 NTU for the single-step method at pH 8. Finally, the capsid and functional AAV titers measured by single-step lysis at pH 8 were very low compared to those obtained by two-step lysis and single-step lysis at pH 4, likely due to insufficient lysis completed within 60 minutes. On the other hand, compared to single-step lysis at pH 4, the two-step lysis method further enhances the results by achieving higher capsid and functional titers, despite requiring a further 25% dilution due to pH adjustment to 4. Without dilution, the titers are even higher. Therefore, the two-step method enables complete cell lysis with the highest AAV yield (capsid and functional titers), demonstrating its advantage over the single-step method. Furthermore, the extremely short lysis time of approximately 70 minutes (60 minutes for basic cells and 10 minutes for acidified cells) is highly efficient compared to commonly used lysis protocols in the art that typically require several hours (e.g., 4 to 6 hours).

[0343] Clarification Results

[0344] As described above, three different lysates were clarified using an industrially relevant and scalable filtration system. Filtration performance and final filtrate composition were determined and compared. Specifically, for filtration performance, DNA removal, HCP removal, and functional AAV recovery were determined. For the final filtrate composition, HCP content, relative HCP content (related to functional AAV), DNA content, relative DNA content (related to functional AAV), and the total amount of functional AAV were determined. The results are shown in Tables 7 and 8 below.

[0345] Table 7: Filtration performance of lysed cell culture medium.

[0346] The continuous release of viruses throughout the filtration process leads to an artificially high functional recovery rate. No filter clogging was observed during the filtration process, and a low final pressure was measured. This low pressure indicates a depth filtration capacity greater than 150 L / m³. 2 The two-step and single-step methods at pH 4 showed high HCP and DNA removal rates, while the single-step method at pH 8 yielded only low HCP removal. Furthermore, the functional AAV recovery rates for both the two-step and single-step methods at pH 4 were 100%. Note that the functional AAV recovery rate for the single-step method at pH 8 was artificially inflated and should be considered an artifact, as lysis continued throughout the filtration process, releasing more functional AAV. It can be expected that, under the test conditions, the single-step lysis at pH 8 would require a longer time, particularly 2 hours or longer.

[0347] Table 8: Final filtrate composition, including relative values ​​of HCP and DNA in relation to functional AAV concentration.

[0348]

[0349] After clarification, all three lysis methods tested showed low concentrations of residual DNA impurities. The single-step lysis method at pH 4 achieved the highest DNA reduction; however, the values ​​achievable by the two-step method were also low, and therefore may be easily removed by subsequent purification steps.

[0350] Further analysis of the final filtrate composition demonstrated the advantages of the two-step lysis method compared to the single-step method. Specifically, it yielded the highest functional titer of AAV while maintaining low levels of host cell-associated impurities, particularly HCP. The two-step method doubled the functional titer compared to the single-step method at pH 8 and exhibited greater compositional purity in terms of relative HCP content. Therefore, the two-step lysis method achieves rapid (<70 min) and complete (100% recovery) lysis, the highest functional titer, and low impurity levels due to the precipitation of DNA and HCP.

[0351] in conclusion

[0352] Example 4 demonstrates that, compared to the single-step method, the two-step lysis method disclosed herein yields high functional viral vector yields (50% higher than single-step lysis at pH 4) and low levels of host cell-associated impurities, with DNA reduced by over 60% after digestion and subsequent precipitation, and HCP impurity levels reduced by approximately 50% after HCP precipitation compared to single-step lysis at pH 8. Furthermore, the two-step method ensures robust and complete cell lysis within a short time (<70 minutes, compared to >120 minutes for conventional methods). Moreover, impurity precipitation facilitates removal, as evidenced by significantly lower final pressures in the filtration apparatus for the pH 4 lysis method. Therefore, acidification has significant potential to achieve higher filtration capacities compared to high-salt lysis at pH 8 (data not shown). Thus, the two-step lysis method overall achieves highly efficient lysis, yielding more and purer viral vectors.

[0353] Example 5: Two-step pyrolysis without cryoprotectant

[0354] In Example 5, the two-step lysis method of Example 4 was further tested. Specifically, it was tested whether a cryoprotectant was needed in the lysis reagent. The inventors assumed that sucrose was not needed because the compound was added primarily to stabilize the viral vector for freezing. Therefore, sucrose could also be added before freezing, but its effect on cell lysis would likely be minimal. Therefore, the following conditions were tested at pH 8: 0.5% (v / v) Tween 20, 25 mM Tris / HCl, 400 mM KCl, 5 mM MgCl2, and salt-tolerant nuclease (San HQ (50 U / mL)). The lysis reagent, as a 5x concentrate, was added to the cell culture medium (TCC 2.7 × 10⁻⁶). 6 The samples were collected at a concentration of c / mL (78% viability) and, 60 minutes after lysis, the pH was adjusted to 4 using 2 M acetate buffer and held for 5 minutes. Then, filtration was performed.

[0355] A two-step lysis method without cryoprotectant in the lysis reagent achieved nearly complete lysis with a measured viability of 0%. Large flocculent material was obtained during mixing, indicating precipitation of host cell-related impurities (HCP, DNA), which could therefore be easily filtered out. Specifically, the measured filtration efficiency was 10⁶ L / m³. 2 Furthermore, 82% of the DNA and 30% of the HCP were removed. The final concentrate consisted of 5.2E+03 µg / L HCP, 2.3E+02 µg / L DNA, and 1.8E+06 Vp / mL functional AAV. The high functional AAV titer and the low level of impurities detected indicate that lysis does not require sucrose, and that sucrose can be added as a cryoprotectant in a later step before freezing to make the viral vector more stable for freezing.

[0356] Example 6: Comparison of two-step and single-step fragmentation of AAV8

[0357] In Example 6, HEK293 cells producing AAV8 were lysed using a two-step lysis method. For reference, a further single-step protocol was used. This example demonstrates that the two-step lysis method is applicable to another AAV serotype (compared to Examples 1-5 for AAV2 production). The two-step lysis method produces high AAV8 titers while maintaining low levels of host cell-related impurities. Therefore, the two-step cell lysis method is widely applicable and efficient for obtaining large quantities of pure viral vectors.

[0358] In this embodiment, HEK293 cells were transiently transfected with a plasmid to produce AAV8 with the GFP coding sequence as a transgene. After AAV8 production, 9 mL of cell culture medium was mixed with the corresponding reagents described in Table 9. As another reference, the cell pellet was lysed. Since most of the AAV8 was secreted into the culture medium, with only a small amount of viral vector remaining in the cells, this embodiment demonstrates that conventional lysis methods suitable for cell pellets are insufficient to obtain a sufficient quantity of AAV8.

[0359] Table 9: Pyrolysis conditions for obtaining AAV8.

[0360]

[0361] After lysis, the TCC, cell viability, and cell diameter of the samples were analyzed, and the pH was determined. The crude lysed samples were then centrifuged (5000×g, 5 min), and the DNA, HCP, and AAV8 capsid titers were determined. The results are shown in Table 10.

[0362] Table 10: Results of total cell concentration (TCC), cell viability (Viab.), cell diameter, and pH of crude lysed samples, as well as host cell protein (HCP), DNA, and AAV8 capsid titers after lysis and clarification. After incubation, adjust the pH to 4.

[0363]

[0364] The results in Table 10 indicate that most AAV8 was present in the supernatant because the capsid titer in the cell culture medium (a, b) was significantly higher than that in the lysed cell pellet (i). In the unlysed state, the viability of the cell culture medium reference sample remained almost unchanged during the experiments (69.6% initially in a, and 68% after 60 minutes in b). For the single-step lysis method at pH 8 (see d), viable cells were still detected after 60 minutes of incubation (1.2%), indicating incomplete cell lysis. Similarly, using only a detergent-free lysis reagent resulted in insufficient cell lysis (see g), with 13.6% viable cells after lysis. All other conditions resulted in substantially complete lysis. The pH measured in the compositions was approximately pH 8 (d, e, f, d, h) or approximately pH 4 (c, i). For the two-step lysis method (e, f, g, h), acetic acid was added to adjust the pH to approximately 4.

[0365] Because the AAV8 capsid titer is high in the supernatant of undylated cell cultures (see a and b), lysing cells only slightly increases the AAV8 titer. However, the model process can be used to obtain some information about cell lysis efficiency and impurity release or reduction.

[0366] Overall, the highest AAV8 capsid titer was measured for single-step cleavage at pH 8, but it also exhibited the highest impurity level (HCP of 1.14 × 10⁻⁶). 4 µg / L, while DNA was 9.36×10 µg / L. 2 On the other hand, single-step lysis at pH 4 produced lower AAV8 levels, close to the reference level (see c, compared to a and b), but lower levels of DNA and HCP impurities. The two-step lysis method generally produced higher AAV8 capsid titers (range 2.98 × 10⁻⁶) compared to the single-step method at pH 4. 10 Capsids / mL up to 3.13 × 10⁻⁶ 10 (capsids / mL), but the two-step lysis method has lower impurity levels compared to the single-step method at pH 4 (see e, f, g, h).

[0367] To better compare two-stage lysis methods using different lysis reagents in the first lysis step, data from Table 10 (e, f, g, h) are extracted and presented below. Figure 11 Similarly, Figure 11 [A] indicates that a detergent is required for rapid and complete lysis. Furthermore, it shows that while nucleases are not essential for rapid cell lysis, they still play a positive role in reducing DNA impurities even after subsequent precipitation at pH 4. Figure 11 [A, B]). As a negative control, the concentration of DNA without nuclease (f) is approximately twice that of the method with nuclease (e, h). Figure 11[A, B, C] shows another aspect: the lysis reagent (h) without the addition of the cryoprotectant sucrose has no adverse effect on lysis, so it can be added in a later step.

[0368] In this embodiment, the conditions of the two-step cell lysis method were further varied. Specifically, cell culture medium was mixed with a 10x lysis reagent containing buffer, salt, divalent cations, cryoprotectant, and detergent, along with a nuclease (e), and compared with conditions without nuclease (f), without detergent (g), and without cryoprotectant (h). The results showed that, in principle, all conditions produced high capsid titers and low impurity content. Therefore, different lysis compositions are suitable for the initial lysis step. Notably, the presence of detergent appears to be necessary to achieve complete cell lysis and thus obtain higher viral vector yields, especially for viral vectors present within the cells. On the other hand, the addition of nuclease helps reduce DNA content, thereby obtaining viral vectors with lower DNA impurities. The presence or absence of cryoprotectant (sucrose) had no effect on yield or purity, supporting its use as an alternative compound for cell lysis.

[0369] In summary, this embodiment demonstrates the applicability of two-step cell lysis to different AAV serotypes, indicating its applicability to different viral vectors. Two-step cell lysis enables high viral vector yield while maintaining low levels of host cell-related impurities.

[0370] Example 7: Comparison of two-step and one-step lysis of adenovirus

[0371] In Example 7, different lysis protocols were applied to cell cultures for adenovirus (Ad) production, and various parameters were measured. This example demonstrates that the two-step lysis method is suitable for host cells producing Ad, consistently achieving high Ad yields while maintaining low levels of host cell-related impurities.

[0372] In this embodiment, the cell culture medium of HEK293 cells used for adenovirus production had a TCC of 25.6 × 10⁻⁶. 6 Cells / mL, viability 80.4%. For each lysis condition, 27 mL of cell culture medium was lysed in a 50 mL tube with 3 mL of 10x lysis reagent and incubated at 37°C. Mixing was achieved by placing the tube on a swirl plate. After 90 minutes, some lysis conditions required the addition of acidifying reagent or salt. Samples were taken after 120 minutes and different properties were determined. The lysis conditions disclosed in Table 11 were applied.

[0373] Table 11: Lysis conditions for cell culture medium used in adenovirus production.

[0374]

[0375] Changes in the properties of cell culture medium due to lysis

[0376] After lysis as described above, TCC and viability were measured. The results are as follows: Figure 12 As shown. Except for reference samples S0, S1, and S7, viability was generally low, with single-step lysis at pH 4 performed without the addition of nucleases. S5 still showed slight viability, with sample lysis via a single-step method using an alkaline lysis reagent. In contrast, the two-step cell lysis method disclosed herein achieves efficient cell lysis with no detectable residual viability (see S9).

[0377] Effect on capsid titer

[0378] In addition, the Ad capsid titers of the crude pyrolysis sample and the sample filtered through 0.2 µm were determined. Figure 13 As shown, high capsid titers were generally obtained except for S7 (where the sample was not fully lysed). Slightly lower capsid titers were obtained for single-step cell lysis at pH 4 and condition 2 (see S8). For all other lysis methods, capsid titers were measured in the range of 1.5 × 10⁻⁶. 12 One capsid / mL to 2×10 12 Between one capsid per mL. Therefore, the Ad capsid titer obtained by the two-step cell lysis method is comparable to that of conventional methods used in the art (see S2 and S11). It should be noted that the lysis time for all samples was equal, 2 hours, but as shown in Examples 3 and 4, the lysis time of the two-step lysis method (S9) may be shortened.

[0379] Effects on DNA and HCP impurities

[0380] In addition, Ad impurities (HCP and DNA) were determined in both the crude lysed sample and the filtered sample. Figure 14 and Figure 15 In this study, a correlation analysis was performed between impurities and the obtained titers. For example... Figure 14 and Figure 15 As shown, the two-step cleavage method yielded the lowest HCP content (see S9), with only S8 reaching a similar level, but S8 had a lower capsid titer (see [reference missing]). Figure 13Furthermore, the DNA content of the two-step method is relatively low (see S9), similar to S2, S3, S8, and S11. Even lower DNA levels were obtained only with the incorporation of pDADMAC (polydiallyl dimethyl ammonium chloride; a cationic flocculant replacing the nuclease) into the internal lysis reagent (see S4). However, the two-step method has the advantage of avoiding the use of hazardous substances such as Triton X-100 and pDADMAC, which are environmentally harmful and should be avoided in pharmaceutical compositions, such as viral vector formulations intended for gene therapy.

[0381] in conclusion

[0382] Example 7 demonstrates that the two-step lysis method is suitable for lysing cells producing adenovirus, highlighting its broad applicability to different viral vectors. Therefore, the two-step lysis method is highly efficient in cell lysis, and lysis time may be further reduced. Furthermore, the results again highlight that the two-step lysis method provides an optimal balance between achieving high viral vector yields and minimizing host cell-related contaminants. Specifically, high adenovirus capsid titers were achieved while keeping HCP and DNA levels low.

[0383] Example 8: Implementing two-step cell lysis in a bioprocess

[0384] In Example 8, the preferred experimental setup and workflow for the two-stage pyrolysis are described in more detail. The process experimental setup is as follows: Figure 1 As shown, it aims to simplify workflows, improve safety, and increase product output.

[0385] In the first step, a concentrated (e.g., 10x) high-salt alkaline lysis buffer containing detergent and salt-tolerant nuclease is added to the bioreactor to initiate cell lysis. Even if the bioreactor reaches its maximum working volume at the end of the culture, this can be achieved by stopping culture aeration and using the bioreactor headspace (typically an additional 25% of the working volume) to add a 10x concentrated lysis reagent (approximately an additional 11% of the working volume) to the cell culture for this purpose. Furthermore, the bioreactor's stirring function is used to mix all components and keep the cells in suspension. Additionally, a 37°C temperature control is kept active to achieve maximum nuclease activity. The adjusted lysis conditions are maintained for approximately 1 hour. After this, most cells are lysed, leaving only a small fraction (<20%) of cells still viable. At this point, the pH is rapidly reduced to 4 by titration with a concentrated acidifying reagent (e.g., an acid, such as 2 M acetic acid). For this purpose, a pH probe and pH controller already used during the culture process can also be used for titration. Alternatively, the acidifying reagent can be added using the bioreactor's pH dosing pump.

[0386] In this second step, the low pH reduces nuclease activity but also causes residual cells to lyse immediately. Furthermore, the low pH causes impurities, primarily host cell-related impurities such as host cell proteins or nucleic acids and cell debris, to precipitate almost immediately. Therefore, the subsequent clarification process can begin quickly, for example, within minutes at pH 4. Alternatively, acid can be added and mixed online when the cell culture medium is transferred to the filtration stage for clarification. The precipitation of impurities also has the advantage of enabling higher filtration capacity, thus requiring less filter material in the clarification step.

[0387] After lysis and clarification, it is recommended to proceed directly to the next capture step to avoid viral vector degradation. Prior to this, a tangential flow filtration (TFF) step may be necessary to adapt the capture chromatography buffer conditions, remove some soluble impurities, and concentrate the product. In a simplified experimental setup, TFF can be performed in single-pass (SP) mode. The viral vector is stabilized by removing most of the remaining contaminants (such as proteases) in the subsequent capture step. If the product needs to be stored after capture, for example, frozen at -80°C, further adaptation of the buffer conditions is recommended. This includes adjusting / neutralizing the pH and adding a cryoprotectant (such as sucrose). As an alternative to storage or after storage, the viral vector product can be further processed by purification chromatography and transferred to filling and finishing steps.

[0388] in conclusion

[0389] The above embodiments demonstrate that the method of this disclosure is highly effective in lysing cells to obtain increased capsid and functional viral vector titers, as well as reducing host cell-related impurities. Notably, the two-step method yields significantly higher functional viral yields compared to known single-step methods (see, for example, Example 3: a 50% increase compared to high-salt lysis without nucleases at pH 4). Furthermore, the two-step lysis ensures highly efficient and complete cell lysis. DNA is reduced by more than 60% after digestion and subsequent precipitation. Moreover, the lysis time is reduced by nearly 50% (<70 minutes, compared to over 120 minutes for conventional methods), thereby improving efficiency. HCP precipitation reduces HCP impurity levels by approximately 50% compared to high-salt lysis at pH 8. Furthermore, precipitation of impurities through acidification facilitates their removal in the filtration step, resulting in a significantly lower filtration pressure compared to high-salt lysis at pH 8. Simultaneously, by using lysis reagents that avoid toxic compounds (such as Triton X-100), i.e., detergent-containing lysis reagents according to this disclosure containing polysorbate detergents (such as Tween 20), reduced toxicity and improved waste management can be achieved. Finally, the provided embodiments demonstrate that implementing a two-step lysis method in a bioprocess can simplify the release of viral vectors from cell cultures and achieve precise process control.

Claims

1. A method for releasing a viral vector from a cell culture, the method comprising the steps of: (a) Provide lysis reagents; (b) Contacting the cell culture with the lysis reagent to generate a lysis composition; (c) Incubate the lysis composition to release the viral vector from the cell culture; as well as (d) Contact the pyrolysis composition with an acidifying agent to lower the pH.

2. The method according to claim 1, wherein in step (c), the cell culture is partially lysed, and / or in step (d), the lysis composition is contacted with an acidifying agent to cause the cell culture to be substantially completely lysed.

3. The method according to claim 1 or 2, wherein the cell culture is a cell culture medium or cell precipitate, preferably a cell culture medium, and / or wherein in step (b), the cell culture does not need to be separated from the surrounding liquid before contacting the cell culture with the lysis reagent.

4. The method according to one or more of claims 1 to 3, wherein the method has one or more of the following features: - The method does not include pyrolysis steps based on mechanical or physical pyrolysis, and / or The method does not include repeated freeze-thaw cycles for cell lysis or microfluidization steps for cell lysis.

5. The method according to one or more of claims 1 to 4, wherein at least one of steps (b), (c), and (d) is carried out in a bioreactor, preferably at least two of these steps are carried out in a bioreactor, most preferably all steps (b), (c), and (d) are carried out in a bioreactor, optionally wherein said bioreactor has one or more of the following characteristics: - The stirring of the bioreactor facilitates mixing in these steps; -The bioreactor is capable of temperature and / or pH control; and / or - The bioreactor is capable of pH adjustment, preferably by configuring in step (d) to allow the pyrolysis composition to contact the acidifying agent.

6. The method according to one or more of claims 1 to 5, wherein in step (d): - The acidifying agent lowers the pH to an acidic pH, preferably less than 6.0, more preferably less than 5.0, and most preferably less than 4.5; and / or - The acidifying agent lowers the pH to a range selected from the following: 1.5 to 6.0, preferably 2.5 to 5.0, more preferably 3.5 to 4.

5.

7. The method according to one or more of claims 1 to 6, wherein the acidifying agent comprises an acid, preferably an organic acid, more preferably a carboxylic acid, and most preferably, the acid is acetic acid or citric acid.

8. The method according to one or more of claims 1 to 7, wherein the lysis reagent is an alkaline lysis reagent, preferably, the alkaline lysis reagent has: - pH greater than 7.0, preferably greater than 7.5, most preferably greater than 7.7, such as 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or higher than 8.5; and / or - The pH range is selected from 7.2 to 10, preferably 7.5 to 9.5, more preferably 8 to 9 or 8.2 to 8.

9. The method according to one or more of claims 1 to 8, wherein the pyrolysis reagent comprises a detergent, preferably a nonionic detergent, more preferably a nonionic detergent not classified as toxic and / or harmful to the environment.

10. The method of claim 9, wherein the detergent is an alkyl glucoside, preferably a polysorbate, such as polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), and polysorbate 80 (Tween 80); and / or the concentration of the detergent is from 0.1% (v / v) to 20% (v / v), preferably selected from the following range: i) For 1x lysis reagent, the concentration is 0.1% (v / v) to 2% (v / v). ii) For 5x lysis reagent, 1% (v / v) to 5% (v / v); or iii) For 10x lysis reagent, the concentration is 4% (v / v) to 10% (v / v).

11. The method according to one or more of claims 1 to 10, wherein the method further comprises providing a nuclease, wherein the nuclease is added to the lysis reagent, the cell culture and / or the cell lysis composition, preferably to the cell culture and / or the cell lysis composition, preferably wherein the nuclease is salt-tolerant.

12. The method according to one or more of claims 1 to 11, wherein the pyrolysis reagent is an alkaline pyrolysis reagent containing a detergent, preferably selected from: i. 1x alkaline lysis reagent, having a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v); ii. A 5x alkaline lysis reagent, having a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.3 M to 1.5 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.3 M to 1.5 M; - Descaling agent, preferably polysorbate, with a concentration selected from 1% (v / v) to 5% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 5 mM to 15 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 10% (v / v) to 35% (w / v); or iii. Preferably, the 10x alkaline lysis reagent has a pH greater than 7.0, more preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 1 M to 3 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.8 M to 5 M or 1 M to 3 M; - Descaling agent, preferably polysorbate, at a concentration selected from 4% (v / v) to 10% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 15 mM to 50 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 20% (w / v) to 60% (w / v).

13. The method according to one or more of claims 1 to 12, wherein in step (b), the pyrolysis composition has one or more of the following characteristics: - The pH of the pyrolysis composition is greater than 7.2, preferably greater than 7.5, and most preferably greater than 7.8 or 8.0; - The pH of the pyrolysis composition is selected from the range of 7.2 to 10.0, preferably 7.5 to 9.5; and / or - The pyrolysis composition comprises a compound at a concentration substantially equal to 1x the pyrolysis reagent, preferably substantially at the concentration according to claim 11, and optionally, the pyrolysis composition has the following characteristics: I. The pyrolysis composition has a pH greater than 7.2, preferably greater than 7.5, most preferably greater than 7.8 or 8.0, and comprises: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v).

14. The method according to one or more of claims 1 to 13, wherein: -Incubation step (c) includes Incubation time is less than 6 hours, preferably less than 5 hours or less than 4 hours, more preferably less than 3 hours, such as 2 hours, 1 hour or 0.5 hours; and / or The incubation time is selected from 15 minutes to 300 minutes, preferably 20 minutes to 250 minutes, 25 minutes to 200 minutes, or more preferably 30 minutes to 150 minutes or 30 minutes to 100 minutes, most preferably 30 minutes to 90 minutes or 45 minutes to 75 minutes; and / or -in, Time required to perform steps (c) and (d): Less than 360 minutes, preferably less than 300 minutes or less than 240 minutes, more preferably less than 180 minutes or less than 120 minutes, most preferably less than 100 minutes, such as 90 minutes, 80 minutes or 70 minutes or less; and / or The time is selected from the following range: 15 minutes to 360 minutes, preferably 20 minutes to 300 minutes, 25 minutes to 240 minutes, or more preferably 30 minutes to 180 minutes or 40 minutes to 120 minutes, and most preferably 45 minutes to 90 minutes.

15. A method for producing a viral vector, the method comprising the following steps: x.1 Provide cell cultures capable of producing viral vectors; x.2 The cell culture is cultured to produce a viral vector; x.3 The viral vector is released from the cell culture according to one or more of claims 1 to 14; and x.4 Optionally, purify the released viral vector.

16. The method according to one or more of claims 1 to 15, wherein the viral vector is primarily present within cells and is preferably selected from adeno-associated virus (AAV) or adenovirus (Ad); and / or the cell culture comprises mammalian cells, preferably selected from HEK293 cells or cells derived from HEK293 cells.

17. A kit for releasing a viral vector from a cell culture, the kit comprising: (i) lysis reagents; and (ii) An acidifying agent, preferably comprising an acid, more preferably an organic acid, and most preferably a carboxylic acid, such as acetic acid or citric acid; Optionally, the pyrolysis reagent is an alkaline pyrolysis reagent containing a detergent, preferably selected from: i. 1x alkaline lysis reagent, having a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.05 M to 1 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.05 M to 1 M; - Descaling agent, preferably polysorbate, at a concentration selected from 0.1% (v / v) to 2% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 0.5 mM to 5 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 1% (w / v) to 10% (w / v); ii. A 5x alkaline lysis reagent, having a pH greater than 7.0, preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 0.3 M to 1.5 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.3 M to 1.5 M; - Descaling agent, preferably polysorbate, with a concentration selected from 1% (v / v) to 5% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 5 mM to 15 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 10% (v / v) to 35% (w / v); or iii. Preferably, the 10x alkaline lysis reagent has a pH greater than 7.0, more preferably greater than 7.5, and most preferably greater than 7.7, comprising: - Buffer, preferably tris(hydroxymethyl)aminomethane (TRIS) buffer or 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, with a concentration selected from 1 M to 3 M; - Salt, preferably potassium chloride or sodium chloride, with a concentration selected from 0.8 M to 5 M or 1 M to 3 M; - Descaling agent, preferably polysorbate, at a concentration selected from 4% (v / v) to 10% (v / v); -Optional divalent cation, preferably Mg 2+ The concentration is selected from 15 mM to 50 mM; and -Optional cryoprotectant, preferably sugar, with a concentration selected from 20% (w / v) to 60% (w / v).

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