WO2025190867A1

WO2025190867A1 - Two-step intensified cell lysis to release a viral vector

Info

Publication number: WO2025190867A1
Application number: PCT/EP2025/056465
Authority: WO
Inventors: Martin Saballus; Nazguel WAGNER; Markus Kampmann
Original assignee: Sartorius Stedim Biotech GmbH
Current assignee: Sartorius Stedim Biotech GmbH
Priority date: 2024-03-11
Filing date: 2025-03-10
Publication date: 2025-09-18
Anticipated expiration: 2026-09-11

Abstract

The present invention provides methods 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, whereupon the lysis composition is contacted with an acidifying reagent to lower the pH. The method according to the present invention results in high viral vector release and reduction in co-released host cell related impurities. The present invention further provides a method for producing a viral vector. The present invention further provides a kit for releasing a viral vector from a cell culture. The present invention further provides a kit for producing a viral vector.

Description

TWO-STEP INTENSIFIED CELL LYSIS TO RELEASE A VIRAL VECTOR

TECHNICAL FIELD OF THE INVENTION

BACKGROUND OF THE I NVENTION

Viral vectors have been developed over the last years as an important modality for gene therapy, which allows for repairing malfunctioning DNA sequences or to introduce a compensatory change that will restore the normal physiological functions of the cell. Well-known examples of viral vectors from which recombinant viral vectors are derived from include viruses with envelopes, for example retrovirus, lentivirus, Sendai virus, and herpes virus, and viruses without envelopes, for example adenovirus, and adeno-associated virus (hereinafter, referred to as AAV). Gene therapy via gammaretroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAV) is attractive because of the natural ability of viruses to enter and deliver genetic material to cells. Specifically, recombinant AAV (rAAV)-based vectors are ideal for gene therapy applications and AAV-vector- mediated gene delivery which recently led to medical approva ls e.g. for the treatment of inherited blindness and spinal muscular atrophy, and long-term therapeutic effects have been achieved for other rare diseases, including hemophilia and Duchenne muscular dystrophy.

Recombinantly produced viral vectors based on AAV are excellent vectors for in vivo gene therapy due to their wide tropism, absence of pathogenicity in humans, and long-term transgene expression stability without the need for genome integration. AAV can infect a wide variety of cells including human cells, and AAV infects even non-dividing cells whose differentiation has concluded, including blood cells, muscle cells, and nerve cells. The wild-type AAV genome comprises inverted terminal repeat (ITR) flanked Rep and Cap genes that encode genome replication and packaging proteins as well as capsid proteins. AAV vectors typically have Rep and Cap replaced by a gene of interest making them replication defective with only ITRs remaining to ena ble replication and genome packaging. The viral particle of AAV is also physicochemically stable. For these reasons, AAV has recently attracted attention for its utility value as a vector for gene transfer used in gene therapy for the treatment of congenital genetic disease as well as the treatment of cancer or infection.

In most instances, viral vectors such as AAV-, Adenovirus, and lentivirus-derived vectors are produced in host cells, such as mammalian cell lines. Typically, a method for producing a recombinant viral vector comprises introducing genetic elements for formation of a viral vector in the form of one or more nucleic acid constructs into a host cell to generate a cell having the ability to produce a viral vector and culturing the cell to express the elements for formation of the viral vector. Out of the available cell lines HEK293 cells or a derivative of a HEK293 are widely used as these are well- characterized and very susceptible to transfection. A considerable advantage that especially the mammalian cell lines, e.g. such as those of human origin, share is their ability to confer certain post- translational modification (PTM) to the vector capsid. These PTM can affect the stability, infectivity and immunogenicity in vivo, thereby making them a crucial quality parameter.

AAV- based drugs are currently the most expensive drugs in the world, Glybera costing US$1.2 million per patient and Zolgensma US$2 million. The main contributor to the high price point of AAV-based drugs is their current high manufacturing costs requiring process optimization that enables costefficient production. In the past, a growing number of authors presented different approaches towards process optimization in viral vector production. Apart from optimizing the classic biotechnological parameters during production (i.e. upstream processing), the process of transfection was optimized to increase the viral vector yield. In addition, the downstream processing which aims as separating the viral vectors from the various impurities which are generated throughout the upstream production of the viral vectors is continuously improved to obtain higher yields.

Since in most cases the majority of the generated viral vectors remains within the cells, the outer boundary or cell membrane needs to be broken down or destroyed. Such release processes are referred to as cell lysis. In order to obtain maximal yield, the cells need to be completely lysed, such that essentially the complete intracellular DNA, RNA and protein is available. In the field of cell lysis, there exist various methods including mechanical approaches such as sonication and high-pressure homogenization, physical methods like freeze-thaw, and chemical methods. However, each method has its own advantages and limitations. Mechanical cell lysis methods, although effective in cell disruption, can be challenging to scale up and can generate heat that may result in the denaturation of target molecules or aggregation of viral vectors. Additionally, these methods often require specialized equipment and can be labor-intensive. The freeze-thaw method of cell lysis is relatively simple. However, it is not easily scalable and may not be suitable for large-scale production due to its reliance on repeated cycles of freezing and thawing, which can be time-consuming and impractical for large volumes. Chemical cell lysis methods offer scalability and versatility as 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 are still relatively inefficient, requiring multiple steps, such as upfront cell separation and washing steps, long incubation times, and sequential nuclease treatments. Moreover, present lysis compositions and protocols can result in high contents of coextracted impurities, such as host cell nucleic acids, which make the downstream purification more challenging and reduce 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 its derivatives, known as biohazardous compounds listed on the REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals) list, which restrict or prohibit their use in the European Union. Furthermore, in addition to the aforementioned chemical lysis methods, the combination of chemical lysis with established mechanical techniques such as microfluidization or physical approaches like freeze-thaw has been recognized as effective cell lysis techniques. However, mechanical cell lysis methods, while effective in cell disruption, can lead to product loss due to the shear-stress induced aggregation. Additionally, these methods often require specialized equipment. On the other hand, combining the chemical method with freeze-thaw technique is also not an ideal solution. While the freeze-thaw method of cell lysis is relatively simple, it is not easily scalable and may not be suitable for large-scale production due to its reliance on repeated cycles of freezing and thawing, which can be time-consuming and impractical for large volumes.

Consequently, there is still a great need to improve the production of a viral vector, particularly by improving the release of the viral vector from a cell culture. Specifically, there is a need to provide simplified cell lysis workflows without compromising yield or possibly even improving yield. Additionally, there is a need to reduce the time required for cell lysis to render the released viral vectors being more quickly applicable for downstream purification.

Hence, it is an object of the present invention to improve cell lysis. Specifically, it is an object to increase the amount of released viral, i.e. increase the vector titer, e.g. the capsid and/or transducing viral vector titer. Furthermore, it may be an object to reduce impurities released during cell lysis and/or to improve removal of impurities which are released together with the viral vector. It may also be an object to reduce time required for lysis and/or increase applicability for various sample types simplifying downstream processing.

SUMMARY OF THE INVENTION

The present invention addresses the above-described needs. The present invention provides a method for releasing a viral vector from a cell culture, wherein first a lysis reagent is provided, contacted with the cell culture to generate a lysis composition, and incubated such that a viral vector is released from the cell culture. Subsequently, the lysis composition is contacted with an acidifying reagent to lower the pH. The disclosed method advantageously increases extraction of a viral vector, by enhancing the efficiency of the cell lysis while achieving improved removal of host cell-related impurities. Furthermore, the method can be flexibly applied to a wide range of samples, which include cell broth down cell pellets, and sample sizes, allowing for broad applicability. Finally, the presence of biohazardous components or process steps are mitigated to ensure the safety of operators and the environment. Compared to the existing cell lysis reagent formulations and techniques, the cell lysis methods and kits according to the present disclosure are carefully screened for optimal conditions.

According to a first aspect, a method for releasing a viral vector from a cell culture is provided, the method comprising following steps:

(a) providing a lysis reagent;

(b) contacting the cell culture with the lysis reagent to generate a lysis composition;

(c) incubating the lysis composition such that a viral vector is released from the cell culture; and

(d) contacting the lysis composition with an acidifying reagent to lower the pH.

The method according to the first aspect advantageously allows for releasing the viral vector with high efficiency but keeping the host cell related impurities, such as in particularthe host cell proteins, at a low level. In particular, the herein disclosed lysis method divides the process into two short lysis steps (herein also referred to as "two-step cell lysis" or "two-step lysis"), applying different beneficial conditions in the cell broth that cannot be combined in a single step. The inventors found that a single-step lysis approach with an alkaline lysis reagent achieves high viral vector yields but also relatively high impurity levels of host cell related impurities, whereas single-step lysis approach with an acidic lysis achieves moderate viral vector yields but better purity in sense of less host cell related impurities. Surprisingly, by dividing the release of the viral vector into two short lysis steps, the beneficial conditions of single-step lysis approaches can be combined. Therefore, high yields of viral vector can be obtained without compromising purity or requiring more complex or longer downstream purification. Furthermore, the method according to the first aspect of the present disclosure has the advantage that it can be directly performed in a culture vessel, e.g. a bioreactor. Therefore, the cell culture does not need to be transferred or further processed (e.g. centrifuged) in order to render the cell culture susceptible to release of the viral vector. Rather, the lysis reagent can be directly contacted with the cell culture in the culture vessel, e.g. bioreactor, in order to generate the lysis composition and also subsequent steps (c) and (d). In addition, the method according to the first aspect allows for reducing the lysis time, enabling a more efficient lysis process. Finally, the two- step process advantageously can lead to precipitation of impurities, such as in particular host cell related impurities, e.g. nucleic acid and/or protein, which improves downstream purification. Also, the precipitation increases the filtering capacity.

According to a second aspect, a method for producing a viral vector is provided, the method comprising following steps: x.l providing a cell culture capable of producing a viral vector; x.2 culturing the cell culture to produce a viral vector; x.3 releasing the viral vector from the cell culture according to the method of the first aspect of the invention; and x.4 optionally, purifying the released viral vector.

According to a third aspect, a kit for releasing a viral vector from a cell culture is provided, the kit comprising:

(i) a lysis reagent; and

(ii) an acidifying reagent.

According to a fourth aspect, a kit for producing a viral vector is provided, the kit comprising:

(i) the kit according to the third aspect of the invention;

(ii) either one of

I. a mammalian cell capable of producing the viral vector by being modified by genome editing the mammalian cell with one or more nucleic acid molecules for viral vector production allowing for stable viral vector production; or

II. a mammalian cell, which can be modified to produce the viral transient transfection with one or more plasmids for viral vector production allowing for transient viral vector production. Further aspects of the invention are disclosed below. Other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only.

BRIEF DESCRIPTION OF THE FIGURES

Some exemplary embodiments of the present disclosure will now be described with reference to the accompanying drawings.

Fig. 1 shows an exemplified setup for integrating the herein disclosed methods into a bioprocessing workflow, including downstream processing.

Fig. 2 shows pictures of sterile filtration of crude lysates of HEK293 cells producing AAV2 vectors through 0.22 pm CA Spin-X filters (Sigma Aldrich). Variables of cell lysis conditions (pH, salt molarity, incubation time and the addition of a detergent) are shown in the table, wherein the N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N2, N5, N6, relate to acidic lysis reagents according to the present disclosure and N25 relates to a comparison condition having pH 8. Shown cell lysis setups are conducted without a nuclease digestion step.

Fig. 3 SDS-PAGE analysis of sterile filtered crude lysates of HEK293 cells producing AAV2 vectors under reducing and denaturing conditions. The protein bands on the gel were visualized by Coomassie blue stain. Variables of cell lysis conditions (pH, salt molarity, incubation time and the addition of a detergent) are shown in the table above the gel image, wherein the N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N l, N5, N9, relate to acidic lysis reagents according to the present disclosure, which were compared to pH 8 and pH 9 lysis reagents. M in lanes 1 and 13 stands for PageRuler™ unstained Protein ladder (Thermo Fisher Scientific).

Fig. 4 shows AAV2 functional titers (see Set A, whole culture lysis) determined after cell lysis of a cell broth and clarification by transduction assay using Incucyte® Live-Cell Analysis system. The N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N70, N71, N 72 and N73 relate to pH 2 acidic lysis reagents, N74, N75, N76 and N77 relate to pH 4 acidic lysis reagents, and N78, N79, N80 and N81 relate to pH 8 alkaline lysis reagents according to the present disclosure. As reference lysis reagents, the "prior art cell lysis reagent" (N82) was applied, as well as the CelLytic™ M (Cl), CelLytic™ MT (C2; both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. Error bars represent standard deviation from the mean (n = 3).

Fig. 5 shows AAV2 capsid titers (see Set A, whole culture lysis) determined after cell lysis of a cell broth and clarification using the Octet® AAVX Biosensors. The N-number refers to the identification number of cell lysis conditions, wherein N70, N71, N72 and N73 relate to pH 2 acidic lysis reagents, N74, N75, N76 and N77 relate to pH 4 acidic lysis reagents, and N78, N79, N80 and N81 relate to pH 8 alkaline lysis reagents according to the present disclosure. As reference lysis reagents, the "prior art cell lysis reagent" (N82) was applied, as well as the CelLytic™ M (Cl), CelLytic™ MT (C2; both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. Error bars represent standard deviation from the mean (n = 3).

Fig. 6 shows AAV2 functional titers (see Set A, cell pellet lysis) determined after cel I lysis of a cell pellet and clarification by transduction assay using Incucyte® Live-Cell Analysis system. The N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N86, N87, N88 and N89 relate to pH 2 acidic lysis reagents, N90, N91, N92 and N93 relate to pH 4 acidic lysis reagents, and N94, N95, N96 and N97 relate to pH 8 alkaline lysis reagents according to the present disclosure. As reference lysis reagents, the "prior art cell lysis reagent" (N98) was applied, as well as the CelLytic™ M (Cl), CelLytic™ MT (C2, both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. Error bars represent standard deviation from the mean (n = 3).

Fig. 7 shows AAV2 capsid titers (see Set A, cell pellet lysis) determined after cell lysis of a cell pellet and clarification using the Octet® AAVX Biosensors. The N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N86, N87, N88 and N89 relate to pH 2 acidic lysis reagents, N90, N91, N92 and N93 relate to pH 4 acidic lysis reagents, and N94, N95, N96 and N97 relate to pH 8 alkaline lysis reagents according to the present disclosure. As reference lysis reagents, the "prior art cell lysis reagent" (N98) was applied, as well as the CelLytic™ M (Cl), CelLytic™ MT (C2, both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. Error bars represent standard deviation from the mean (n=3). Fig. 8 shows AAV2 functional titers (see Set B, whole culture lysis) determined after cell lysis of a cell broth and clarification by transduction assay using Incucyte® Live-Cell Analysis system. The N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N70A relate to pH 2.75 acidic lysis reagent, N74A relate to pH 4 acidic lysis reagent, and N78A relate to pH 8 alkaline lysis reagent. As reference lysis reagents, the "prior art cell lysis reagent" (N82) was applied, as well as the Triton XIOO-containing (TRT) lysis reagent. Error bars represent standard deviation from the mean (n = 3).

Fig. 9 shows AAV2 capsid titers (see Set B, whole culture lysis) determined after cell lysis of a cell broth and clarification using the Octet® AAVX Biosensors. The N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N70A relate to pH 2.75 acidic lysis reagent, N74A relate to pH 4 acidic lysis reagent, and N78A relate to pH 8 alkaline lysis reagent. As reference lysis reagents, the "prior art cell lysis reagent" (N82) was applied, as well as the Triton XIOO- containing (TRT) lysis reagent. Error bars represent standard deviation from the mean (n = 3).

Fig. 10 shows Phase contrast confluence of adherent HEK293 cells transduced with sterile filtered crude AAV2 samples, which were diluted between 1:10 and 1:320. The transduction assay has been conducted using two AAV2 sample types, one lysed with Triton XIOO-containing (TRT) lysis reagent and the second one Tween 20- containing alkaline lysis reagent (N78, LR2). The images are taken with Incucyte® Live-Cell Analysis system.

Fig. 11 shows selective two-step lysis results of a screening using HEK cell culture for production of an AAV8 viral vector as a model. The impact of different lysis reagent solutions and nuclease additions (described in the table below) on the cell viability [A], DNA impurities [B], and host cell protein (HCP) impurities [C] was tested.

Fig. 12 shows total cell concentrations and viability rates determined after lysis screening to compare different single step lysis approaches with the two-step lysis approach (S9) using a HEK cell culture for production of an adenoviral vector as a model. The samples SO and SI representing the cell culture without lysis as a reference.

Fig. 13 shows Adenovirus (AV) capsid titers using Octet® with appropriates AV biosensors determined after cell lysis and centrifugation. The samples SO and SI representing cell culture samples without lysis as a reference. Different single step lysis approaches were compared with the two-step lysis approach (S9). The sample supernatants were measured directly after centrifugation (w/o filtration) as well as measured after 0.2 m syringe filtration (w/ filtration) to compare whether AV capsid aggregates are contained in the samples.

Fig. 14 shows relative DNA impurity amounts in correlation to the adenovirus (AV) capsid amounts determined after cell lysis and centrifugation. The samples SO and SI representing cell culture samples without lysis as a reference. Different single step lysis approaches were compared with the two-step lysis approach (S9). The sample supernatants were measured directly after centrifugation (w/o filtration) as well as measured after 0.2 pm syringe filtration (w/ filtration) to compare whether impurity aggregates are contained in the samples.

Fig. 15 shows relative host cell impurity (HCP) impurity amounts in correlation to the adenovirus (AV) capsid amounts determined after cell lysis and centrifugation. The samples SO and SI representing cell culture samples without lysis as a reference. Different single step lysis approaches were compared with the two-step lysis approach (S9). The sample supernatants were measured directly after centrifugation (w/o filtration) as well as measured after 0.2 pm syringe filtration (w/ filtration) to compare whether impurity aggregates are contained in the samples.

DETAILED DESCRIPTION

The following description serves to deepen the understanding of the present disclosure and shall be understood to complement and be read together with the description of exemplary embodiments of the present disclosure as provided in the above section of this description. It is to be understood that this invention is not limited to the particular embodiments, methodologies, protocols and reagents described herein as these may vary within the scope set by the claims. It is also to be understood that terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which is defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following description, certain elements of the present invention will be described. These elements may be discussed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples, features and particular embodiments should not be construed to limit the present invention to only the explicitly described embodiments or to the explicitly described combination of features. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise.

As explained in the summary of the invention, the different aspects and embodiments of the invention disclosed herein make important contributions to the art by providing improved viral vector release and production.

Method for releasing a viral vector from a cell culture

(a) providing a lysis reagent;

The method according to the first aspect advantageously allows for releasing the viral vector with high efficiency but keeping the host cell related impurities, such as in particularthe host cell proteins, at a low level. In particular, the herein disclosed lysis method divides the process into two short lysis steps (herein also referred to as "two-step cell lysis" or "two-step lysis"), applying different beneficial conditions in the cell broth that cannot be combined in a single step (see exemplary Fig. 1). The inventors found that a single-step lysis approach with an alkaline lysis reagent achieves high viral vector yields but also relatively high impurity levels of host cell related impurities, whereas single-step lysis approach with an acidic lysis achieves moderate viral vector yields but better purity in sense of less host cell related impurities (see Figs.2 to 9). Surprisingly, by dividing the release of the viral vector into two short lysis steps, the beneficial conditions of single-step lysis approaches can be combined (see Figs. 11 to 15). Therefore, high yields of viral vector can be obtained without compromising purity or requiring more complex or longer downstream purification. At the same time, no biohazardous components or method steps are comprised which ensures safety for the operator and environment (see Figs. 8 to 10). Moreover, the method according to the first aspect can be advantageously performed directly in the culture vessel, e.g. bioreactor, as the lysis reagent but also the acidifying reagent can be contacted in the culture vessel to the cell culture and lysis composition, respectively (see Example 8). This significantly simplifies the release of the viral vector, as no transfer of the cell culture, let alone any processing steps such as sedimentation or centrifugation need to be performed. Hence, the method according to the first aspect provides flexibility and simplification of the process of releasing a viral vector, such as AAV or Adenovirus, from a cell culture. In addition, the method according to the first aspect allows for reducing the lysis time, enabling a more efficient lysis process, e.g. to less than 2 hours, e.g. 70 min (see Example 4). Finally, the two-step process advantageously can lead to precipitation of impurities, such as in particular host cell related impurities, e.g. nucleic acid and/or protein, which improves downstream purification. Also, the precipitation increases the filtering capacity.

The individual steps and preferred embodiments of the method according to the first aspect will now be described in detail.

The viral vector

The viral vector according to the present disclosure is any viral material suitable for delivering genetic material into cells (e.g. alone or in conjunction with further viruses or biochemical cues). In particular, as used herein, the terms "virus (vector)," "viral vector," and "gene delivery vector" refer to a virus particle that functions as a nucleic acid delivery vehicle, and which comprises a nucleic acid molecule packaged within the viral particle. A viral vector may be suitable for application in gene therapy, i.e. allows for using nucleic acids to repair malfunctioning DNA sequences or to introduce a compensatory change that will restore the normal physiological functions of the cell. Such gene delivery is also referred to as transduction. In general, a viral vector for gene therapy is produced recombinantly such that it contains one or more target genes, also referred to as "transgenes". Hence, according to a preferred embodiment, the viral vector is a recombinant viral vector. The target genes can be transduced into a cell, e.g. patient cells, autologous cells, or allogenic cells, which can happen in vivo or in vitro or ex vivo. The one or more target genes are then either directly available for protein expression in the cell as non-integrative vectors which degrade naturally over time or are integrated into the nuclear DNA of the cell. As used herein, a "transgene" or "target gene" is a nucleic acid that is introduced into the genome, including but not limited to genes or nucleic acid having sequences which are not normally present in the viral vector genes, such as an AAV or Adenovirus, which are present but not normally transcribed and translated ("expressed") in the viral vector genome, such as AAV or Adenovirus genome, or any other gene or nucleic acid which one desires to position between the viral vector repeat sequences, such as AAV's ITR sequences. A transgene may include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid. A transgene can be as few as a couple of nucleotides long, but can preferably be at least about 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1.000, 1.100, 1.200, 1.300, 1.400, 1.500, 1.600, 1.700, 1.800, 1.900, 2.000, 2.100, 2.200, 2.300, 2.400, 2.500, 2.600, 2.700, 2.800, 2.900, 3.000, 3.100, 3.00, 3.300, 3.400, 3.500, 3.600, 3.700, 3.800, 3.900, 4.000, 4.100, 4.200, 4.300, 4.400, 4.500, or 4.600 nucleotides (nt) long. A transgene can comprise coding or non-coding sequences.

Importantly, and as demonstrated in the Examples below, the method for releasing a viral vector according to the present disclosure is applicable for a wide range of viral vectors and not limited to a particular type. Specifically, different AAV serotypes and Adenovirus were released by the method according to the first aspect of the present disclosure, demonstrating the broad applicability.

"Releasing a viral vector" refers to a process wherein the viral vector produced by a cell of a cell culture is rendered accessible for further process steps, e.g. downstream purification and/or analysis. Typically, viral vectors are predominantly present intracellularly of the producing cell, such that the viral vector must be released in order to render it accessible for any subsequent process steps. For this purpose, the cell membrane is typically broken down, i.e. the cell undergoes lysis. Hence, the method for releasing a viral vector from a cell culture encompasses lysing a cell of the cell culture, e.g. may equally well refer to a method for lysing a cell of a cell culture to release the viral vector.

A number of viruses have been suitable as viral vectors for gene therapy, which are known by the skilled person, see e.g. Roldao et al., 2017, Comprehensive Biotechnology, vol. 1, pp. 633-656. A viral vector may be selected from retroviruses, such as lentivirus, adenoviruses, herpes simplex, vaccinia, and adeno-associated virus (AAV). According to a preferred embodiment, the viral vector is nonenveloped, including for instance adeno-associated virus (AAV) or adenovirus (Ad).

According to a preferred embodiment, the viral vector is selected from an adeno-associated virus (AAV) or an adenovirus (Ad), most preferably the viral vector is an AAV. As demonstrated in the Examples below, the method according to the present disclosure improves the release of AAV and Adenovirus, particularly by increasing the genomic and transducing titers and reduces the level of host cell related impurities, such as host cell proteins (see Figs. 1 to 15).

According to a preferred embodiment, the viral vector is predominantly present intracellularly. However, a fraction of the viral vector may also be present extracellularly, e.g. due to cell degradation or lysis throughout production of the viral vector. It is also within the scope of the present invention to apply the method according to the present disclosure to release viral vector present extracellularly, wherein only a fraction is present intracellularly. For instance, some serotypes such as AAV8 are known to efficiently be released by the cell into the surrounding culture liquid, such that part of the produced viral vector is not present intracellularly.

As used herein, the term "adeno-associated virus" (AAV), includes but is not limited to, AAV type 1 (e.g., AAV of serotype 1, also referred to as AAV1), AAV type2 (e.g, AAV2), AAV type 3 (e.g, AAV3, including types 3A and 3B, AAV3A and AAV3B), AAV type 4 (e.g, AAV4), AAV type 5 (e.g, AAV5), AAV type 6 (e.g, AAV6), AAV type 7 (e.g, AAV7), AAV type 8 (e.g, AAV8), AAV type 9 (e.g, AAV9), AAV type 10 (e.g, AAV10), AAV type 11 (e.g, AAV11), AAV type 12 (e.g, AAV12), AAV type 13 (e.g, AAV13), AAV type rh32.33 (e.g, AAVrh32.33), AAV type rh8 (e.g, AAVrh8), AAV type rhIO (e.g, AAVrhIO), AAV type rh74 (e.g, AAVrh74), AAV type hu.68 (e.g, AAVhu.68), avian AAV (e.g, AAAV), bovine AAV (e.g, BAAV), canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV now known or later discovered.

The cell culture

According to the disclosed method for releasing a viral vector, the viral vector is released from a cell culture. According to a preferred embodiment, the cell culture is not required to be separated from the surrounding liquid prior to contacting the cell culture with the lysis reagent in step (b). Since the cell does not need to be separated from the surrounding liquid prior to contacting the cell culture with the lysis reagent in step (b), no need to further processing step exists. In other words, the cell does not need to undergo any kind of separation step, e.g. centrifugation or sedimentation, prior to being contacted to the lysis reagent. As a result, the method of the present disclosure allows simplifying cell lysis and widens applicability, e.g. by performing the cell lysis directly in a bioreactor.

A "cell culture" according to the present disclosure comprises a cell and surrounding liquid as well as any produced product, such as the viral vector, e.g. AAV or Adenovirus. The produced product can be present in the cell (intracellular) and/or surrounding liquid (extracellular). Typically, a cell culture is a suspension of cells comprising cells, preferably HEK293 cells or derivative cells thereof, and surrounding liquid, such as cell culture media, and product produced by the cell (here viral vector). Cell culture media suitable for cel I culture are known in the art and shall not be limiting for the present disclosure. Examples of suitable cell culture media for culture of a HEK293 cell can be found in the Examples below. Cell culture media can be H EK ViP NB and HEK TF (Sartorius Xell GmbH), and DMEM (Pan-Biotech). Further compounds may be present apart from the cell and the surrounding liquid in the cell culture, such as remains of a transfection reagent, compounds enhancing viral vector production, etc. According to a preferred embodiment, the cell culture is a cell broth or cell pellet, preferably a cell broth. A cell broth comprises the cells producing the viral vector, as well as the surrounding culture media and, if already produced, the viral vector. It may be referred to as "whole (cell) culture" or "cell culture fluid" or "cell culture suspension". As demonstrated in the examples, the method according to the present disclosure is suitable for cell broth and other crude samples (see Figs. 4 and 5), as well as for purified or processed samples, such as cell pellets (see Figs. 6 and 7). This shows the broad and advantageous applicability of the method according to the present disclosure.

According to a preferred embodiment, the cell culture is not subjected to sonication, high-pressure homogenization, orfreeze-thaw. Such embodiment is advantageous, as it can be challenging to scale up and can generate heat that may result in the denaturation of target molecules or aggregation of viral vectors. Additionally, these methods often require specialized equipment, e.g. for sonication and high-pressure homogenization, can be labor-intensive, and can also cause contaminations of the released virus. Furthermore, the freeze-thaw method is not easily scalable and may not be suitable for large-scale production due to its reliance on repeated cycles of freezing and thawing, which can be time-consuming and impractical for large volumes. On the other side, the method of the present disclosure offers scalability and versatility as the lysis reagents can be formulated to suit different cell types and viral vectors.

According to a preferred embodiment, the cell culture is not subjected to centrifugation, cell pelleting or an exchange of the liquid surrounding the cells of the cell culture prior to steps (a), (b) and (c). Avoiding centrifugation or also sedimentation simplifies the process and avoids a labor intensive and potentially contaminating step, e.g. when removing the supernatant.

According to a preferred embodiment, the cell culture comprises a mammalian cell. A mammalian cell is a cell that is of mammalian origin. However, the cell does not need to be identical to a cell obtained in a mammalian but can be modified, engineered or naturally/artificially changed. For instance, the mammalian cell may be a cell with mammalian origin, however, may contain one or more genetic changes to propagate it repeatedly and possibly also infinitely. The term "mammalian cell" may be interchangeable used with "mammalian cells" and shall cover both the singular and plural form. The mammalian cell is preferably a mammalian cell line. According to a preferred embodiment, the mammalian cell is a human-derived cell line, which advantageously has the ability to provide the suitable post-translational modifications for therapy in humans. This means that the produced viral vector has similar post-translational modification, as if the native virus would have infected a human cell resulting in virus production.

The mammalian cell can be selected from the group consisting of HeLa, Human embryonic kidney 293 (HEK293), BSC-1, SW480, Baby hamster kidney (BHK), BHK-21, Vero E6, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, Chinese hamsterovary (CHO), MCF-7, Y79, SO-Rb50, Hep G2, DUKX-X11, J558L, HuH-7, MDCK, or HepG2 cells or derivatives thereof, e.g. sub-cell lines such as for HEK293 also 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. According to a preferred embodiment, the mammalian cell is selected from the group consisting of HEK293, A549, BSC-1, SW480, Baby hamster kidney (BHK), Vero E6 and MDCK cells or a derivative thereof. According to an even more preferred embodiment, the mammalian cell is selected from a HEK293 cell or a derivative of a HEK293 cell. HEK293 is particularly advantageous as it is well-characterized, widely used and very susceptible to transfection. The HEK293 cell was established by transforming human embryonic kidney cells with sheared adenovirus type 5 DNA. Accordingly, the mammalian cell may be a HEK293 cell or a derivative of a HEK293 cell, e.g. 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. A derivative of a HEK293 cell may also encompass a HEK293 cell that is adapted to suspension culture and/or adapted to a particular type of cell culture medium.

According to a preferred embodiment, the cell culture comprises a cell capable of being cultured in suspension. For instance, a HEK293 cell or the derivative of a HEK293 cell has been adapted for suspension culture. This is particularly advantageous, as it allows for obtaining higher cell numbers compared to adhesive cells. It may also be that originally adhesive cells, e.g. adhesive HEK293 cells, are used which are subsequently suspension adapted and then modified such that it is configured to produce a viral vector (e.g. by transient transfection or genome editing).

According to a preferred embodiment, the mammalian cell is modified to be configured to produce a viral vector, preferably AAV or Adenovirus. According to one embodiment, the mammalian cell is modified (preferably prior to step (a)) to be configured to produce a viral vector. In other embodiments, it may also be started directly with the mammalian cell without any active modification step by purchasing a mammalian cell that is capable of producing the desired viral vector, e.g. a so- called packaging cell or stable cell which contains all genetic elements required for expressing the viral vector, typically without the target gene(s). In some embodiments, the applied mammalian cell may from the beginning on also be itself capable of expressing one or more genes which are required for producing the viral vector, e.g. HEK293 cells are known to express E1A and E1B which are required for producing AAV.

According to a preferred embodiment, the mammalian cell is modified by transiently transfecting the mammalian cell with one or more plasmids for viral vector production allowing for transient viral vector production. Transiently transfected mammalian cells allow essentially for a single run of viral vector manufacturing, such that for each manufacturing run a transfection has to be performed. According to an alternative preferred embodiment, the mammalian cell is modified by genome editing the mammalian cell with one or more nucleic acid molecules for viral vector production allowing for stable viral vector production. The stable integration a I lows for using a stock, e.g. cell bank or master cell bank, of modified mammalian cells multiple times without the need to genetically modifying the mammalian cells in advance of each manufacturing run (compared to the transient transfection). This has the advantage that an established process can be repeated multiple times without any necessity to perform transient transfection in advance, simplifying the process and rendering it more efficient. On the other hand, the transient transfection is more flexible, as for each run a different set of genetic elements can be transfected into the cells without complicated genome editing of the mammalian cells in advance. Both ways of modifying the mammalian cells for producing a viral vector are applicable in frame of the method according to the present disclosure and shall not be limiting in any way. Indeed, it is also in scope of the present disclosure to combine transient transfection and stable integration, e.g. by providing some genetic elements stably integrated and other genetic elements via transient transfection. For instance, the transgene gene cassette may be provided by transient transfection, whereas the remaining genetic elements may be stably integrated.

According to a preferred embodiment, the one or more plasmids or the one or more nucleic acid molecules for viral vector production encode at least part of an Adenovirus (Ad) or at least part of an adeno-associated virus (AAV). In some cases, not all of the virus's genetic elements that are natively present need to be present in the one or more plasmids or the one or more nucleic acid molecules for viral vector production, e.g. if part of the genetic elements is already present in the mammalian cells. For instance, HEK293 cells which are commonly used for producing a viral vector were originally established by transfection of primary human embryonic kidney cells with sheared adenovirus 5 DNA, and it has been shown that HEK293 cells stably express the adenoviral E1A and ElB-55k proteins due to integration of a 4 kbp adenoviral DNA fragment in chromosome 19. Hence, E1A and E1B do not need to (but can) be including in the genetic design for modifying such mammalian cell. In addition, for gene therapy, the produced viral vector generally contains one or more genes to be delivered to a patient or another cell. Such one or more genes may be referred to as "target gene(s)". These then typically replace the viral vector production machinery, which is packaged into the plasmid, as it is typically not desired to replicate the viral vector in vivo, i.e. in a potential patient. Therefore, not the native and full viral vector genetic elements may be delivered but only those genetic elements to produce the viral vector including the target gene(s). In some embodiments, the target genes are therapeutic nucleic acids, such as therapeutic DNA or RNA. In other embodiments, the target genes include a reporter gene. In preferred embodiments, the reporter gene can be detected by antibody-based assays. In further preferred embodiments, the reporter gene is a fluorescent molecule. Exemplary fluorescent molecules suitable as reporter gene are GFP, eGFP, mGFP, eYFP, citrine, eGFP, mCFP, Cerulean, dtTomato, and any variants thereof. In some embodiments, the reporter gene is a beta-galactosidase, luciferase or glutathione S-transferase, or any variant thereof. In particular embodiments, the target gene is suitable for screening assays or markers, e.g. fluorescence proteins, such as green fluorescent protein (GFP) or a derivative thereof, which are used for visualizing transduction of the viral vector.

According to a preferred embodiment for producing AAV, the one or more plasmids or the one or more nucleic acid molecules for viral vector production encode one or more of the group comprising Rep78, Rep68, Rep52, Rep40, VP1, VP2, VP3, ITR, AAP, MAAP, X Gene, VA RNA, E4orf6, and E2A, preferably all of the aforementioned, for producing an AAV. According to another embodiment, the one or more plasmids or the one or more nucleic acid molecules for viral vector production encode one or more of the group comprising Rep78, Rep68, Rep52, Rep40, VP1, VP2, VP3, ITR, AAP, MAAP, X Gene, VA RNA, E4orf6, E1A, E1B, and E2A, preferably all of the aforementioned, for producing an AAV. In any of these embodiments, the mammalian cell may already encode one or more of the genetic elements for producing the AAV. For instance, E1A and E1B may already be produced by the mammalian cell, e.g. HEK293, wherein a plasmid or nucleic acid molecule can support the cellular expression by additional copies of E1A or E IB or the cellular expression may be considered sufficient. The encoded genes may be provided on a single or multiple plasmids or a single or multiple nucleic acid molecules. According to one embodiment, more than one plasmid is provided for producing the AAV. For instance, two, three, four, five, six, seven, eight, nine or ten plasmids may be provided.

According to a preferred embodiment, the viral vector is predominantly present intracellularly and preferably is selected from an adeno-associated virus (AAV) or an adenovirus (Ad). While extracellular viral vector can also be improved using the method according to the present disclosure (see Example 6, Fig. 11), the method is most effective when some viral vector is present intracellularly, e.g. at least 10%, at least 20%, at least 30% or at least 40%.

Step (a)

Step (a) according to the method of the first aspect defines providing a lysis reagent. The present disclosure provides various lysis reagents as illustrated in the Examples section. Hence, different lysis reagents may be suitable for being provided in step (a).

Preferably, the lysis reagent is provided in liquid form, e.g. as a solution or suspension. This is typically easier to handle and perform the contacting step. Also, this can be more exact than adding the reagent in solid form. According to another embodiment, the lysis reagent is provided in solid form, e.g. as a powder. Such solid lysis reagent may be present in a container, wherein the cell culture is added in contacting step (b) according to the present disclosure. The particular form of lysis reagent shall not be limiting in scope of the present disclosure.

The term "lysis reagent" may herein be interchangeable used with the term "cell lysis reagent". As disclosed above, providing the lysis reagent for chemical lysis has the advantage that no specialized equipment is needed, as e.g. for mechanical of physical lysis approaches. Also, chemical lysis approaches are applicable to be used directly on the cell culture in the cultivation container such as a bioreactor as disclosed herein. Moreover, while physical methods such as the freeze-thaw method of cell lysis is relatively simple, it is not easily scalable and may not be suitable for large-scale production due to its reliance on repeated cycles of freezing and thawing, which can be timeconsuming and impractical for large volumes.

As is readily understood by the person skilled in the art, a lysis reagent as disclosed herein refers to a lysis reagent that at least partially lyses the cells, commonly referred to as chemical lysis. Hence, no complex and/or time-consuming procedure is required. According to a preferred embodiment, the method according to the present disclosure does not comprise a lysis step based on mechanical or physical lysis. According to a preferred embodiment, the method according to the present disclosure does not encompass a step of repeated cycles of freezing and thawing for cell lysis or a step of microfluidization for cell lysis. Lysis reagent

According to a preferred embodiment, the lysis reagent is an alkaline lysis reagent, preferably having a pH of more than 7.0, preferably more than 7.5, most preferably more 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 of more than 7.0, preferably more than 7.5, most preferably more 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. Such alkaline pH values have been found advantageous for the method according to the present invention for maximizing yield. Specifically, the inventors found that the alkaline lysis reagents enhance extractability of intracellular AAV vectors (see Examples, Figs. 4-9, 12 and 13). By improving the AAV-vector extraction efficiency by using the developed cell lysis reagents, it is possible to significantly reduce manufacturing costs for AAV-based drugs. The enhanced extraction efficiency, demonstrated through the AAV2- Transduction assay results, leads to higher yields of active AAV vectors. Higher AAV vector yields mean that more drug product can be obtained from the same production process, thereby increasing the overall productivity, and reducing cost per unit. Also, the alkaline lysis reagent disclosed herein facilitate shorter cell lysis and extraction time. This time saving aspect can lead to significant cost reductions by reducing the overall time required for production cycles.

According to a preferred embodiment, the alkaline lysis reagent has a pH selected from the range of 7.2 to 10, preferably 7.5 to 9.5, more preferably 8 to 9 or 8.2 to 8. Such ranges are advantageous for achieving efficient release of the viral vector.

According to a preferred embodiment, the alkaline lysis reagent comprises a buffer suitable for buffering the lysis reagent at alkaline pH, preferably being capable of buffering at a pH selected from the range of 7.2 to 9.5. Buffers which efficiently buffer the alkaline lysis reagent (and may later buffer the lysis composition) at the indicated acidic pH are particularly suitable for the method of the present disclosure, as these keep the pH relatively stable at the alkaline pH.

According to a preferred embodiment, the lysis reagent comprises a buffer selected from Tris(hydroxymethyl)-aminomethan (TRIS) buffer, l,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, phosphate buffer, glycine-sodium hydroxide buffer, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) buffer, TES buffer, DIPSO buffer, TAPSO buffer, triethanolamine buffer, HEPPSO buffer, POPSO buffer, TRICINE buffer, HEPPS buffer, EPPS buffer, BIGIN 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 the alkaline lysis reagent, as these efficiently buffer the lysis reagent and possibly the lysis composition at the alkaline pH.

In one embodiment, the pH of the alkaline lysis reagent is selected from the range of 7.5 to 9.5 for which following buffers may be chosen from: 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.

According to a preferred embodiment, the lysis reagent comprises a buffer having a concentration of 0.01 to 3 M, preferably a concentration selected from the following ranges: i) 0.05 to 1 M for a lx lysis reagent; ii) 0.3 to 1.5 M for a 5x lysis reagent; or iii) preferably, 1 to 3 M for a lOx lysis reagent.

It was found that such concentrations of buffer are particularly advantageous for releasing the viral vector. For applying a cell broth, a high concentration of lysis reagent, such as 5x or lOx, preferably lOx, is particularly advantageous, as it reduces the volume of lysis reagent that needs to be added to the cell culture. As a result, it can be possible to add the lysis reagent directly to the culture vessel, e.g. bioreactor, in case it has enough open space to be filled by the lysis reagent. As demonstrated in the examples, the concentrations of buffer and salt together allow for carefully selecting the appropriate concentration in order to achieve an ionic strength that maximized the amount of released viral vector while keeping aggregation of viral vector low enabling improved release of the viral vector.

Throughout the present disclosure, the concentration of compounds related to the x-fold lysis reagent. This in particular refers to the ratio of cell culture to lysis reagent. For instance, a lx lysis reagent is typically added to a cell culture which essentially comprises no or very few surrounding liquid, e.g. as is present in a cell pellet or sedimented cell culture without the supernatant. A 5x lysis reagent typically involved 4 parts of cell culture mixed with 1 part of lysis reagent, e.g. 4 mL cell culture mixed with 1 mL lysis reagent. A lOx lysis reagent typically involved 9 parts of cell culture mixed with 1 part of lysis reagent, e.g. 9 mL cell culture mixed with 1 mL lysis reagent.

According to a preferred embodiment, the lysis reagent comprises a salt, preferably selected from potassium chloride or sodium chloride. As demonstrated in the Examples, these salts are particularly useful to adjust the ionic strength such that high amounts of viral vector can be released. Moreover, these are commonly used buffer salts, which are nonhazardous and non-toxic.

According to a preferred embodiment, the salt in the lysis reagent has a concentration of 0.01 to 5 M, preferably a concentration selected from the following ranges: i) 0.05 M to 1 M for a lx lysis reagent; ii) 0.3 M to 1.5 M for a 5x lysis reagent; or iii) preferably, 0.8 M to 5 M or 1 M to 3 M for a lOx lysis reagent.

It was found that such concentrations are particularly advantageous for releasing the viral vector. For applying a cell broth, a high concentration of lysis reagent, such as 5x or lOx, preferably lOx, is particularly advantageous, as it reduces the volume of lysis reagent that needs to be added to the cell culture. As a result, it can be possible to add the lysis reagent directly to the culture vessel, e.g. bioreactor, in case it has enough open space to be filled by the lysis reagent.

According to a preferred embodiment, the lysis reagent comprises a detergent, preferably a nonionic detergent, more preferably a non-ionic detergent that is not classified as toxic and/or hazardous to the environment. It has in particular been found that the lysis reagents as disclosed herein containing a detergent effectively lyse the cells and thus release the viral vector from the cell culture. Hence, according to a particular preferred embodiment, the lysis reagent comprises further a detergent.

Preferably, the detergent according to the present disclosure is advantageously safe for humans and the environment, eliminating potential biohazard risk associated with their usage, e.g. such as Triton- X100 or similar detergents. While this is advantageous, the lysis reagents may also include such detergents if desired. According to a preferred embodiment, the detergent is selected from one or more of Tween, Triton, Nonidet, Igepal or Tergitol. Although any nonionic detergent may be used, examples of non-ionic detergents are those from the Tween class (Tween-20, Tween-40, Tween-60, Tween-80, etc.), the Triton class (X-100, X-114, XL-80N, etc), Tergitols (XD, TMN-6, etc.) and Nonidets or Igepal (NP-40, etc.). Non-ionic surfactants include but are not limited to alkyl glucosides, in particular polysorbates such as polysorbate 20 (Tween 20), polysorbate 40 (Tween 40) and polysorbate 80 (Tween 80) and polyoxyethylen alkyl ethers such as Triton X-100, Nonidet P40, NP- 40 and respective non-ionic detergents from the Brij class. Further detergents that are useful for lysis, respectively degradation of a sample are also well-known in the prior art and thus, need no detailed description here. Also, a mixture of detergents can be used. 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 demonstrated in the examples, such detergents are nontoxic and non-hazardous, especially in comparison to Triton-X based detergents (see Fig. 10). As a cell lysis detergent, polysorbates, such as polysorbate 20, also known as Tween 20, has been found particularly useful. Polysorbates are a biohazard-free detergent, which, in addition to its cell lysis properties acts as a protectant at air-liquid interfaces, preventing aggregation of the target molecules.

According to one embodiment, the detergent is not Triton, e.g. not Triton X-100.

According to another embodiment, the detergent is a polyoxyethylen alkyl ethers such as Triton X- 100, Nonidet P40, NP-40 and respective non-ionic detergents from the Brij class.

According to a preferred embodiment, the detergent has a concentration of 0.1% (v/v) to 20% (v/v), preferably selected from the following ranges: i) 0.1% (v/v) to 2% (v/v) for a lx lysis reagent; ii) 1% (v/v) to 5% (v/v) for a 5x lysis reagent; or iii) 4% (v/v) to 10% (v/v) for a lOx lysis reagent.

It was found that such concentrations of detergent are advantageous for releasing the viral vector.

According to a preferred embodiment, the lysis reagent comprises a divalent cation, preferably Mg2+, Ca2+, Ba2+, Cu2+, Fe2+, Zn2+, Mn2+, Ni2+, or a combination thereof, more preferably Mg2+, Cu2+, Zn2+, Mn2+, Ni2+, or a combination thereof, most preferably Mg2+. The divalent cation is particularly advantageous for cell lysis including a nuclease, as this enables or improves the nuclease activity, e.g. as co-factor of such nuclease. Furthermore, it is believed that the presence of the divalent cation, such as Mg2+, may stabilize the viral vector, such as AAV. In some cases, a divalent cation may not be required, e.g. when not applying a nuclease.

According to a preferred embodiment, the divalent cation in the lysis reagent has a concentration of 0.1 to 1000 mM, preferably a concentration selected from the following ranges: i) 0.5 to 5 mM for a lx lysis reagent; ii) 5 to 15 mM for a 5x lysis reagent; or iii) preferably, 15 to 50 mM for a lOx lysis reagent.

It was found that such concentrations of divalent cation are advantageous for releasing the viral vector. According to a preferred embodiment, the lysis reagent comprises a cryo-protectant, preferably a sugar, more preferably sucrose, trehalose or mannitol, 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 cryo-protectant, such that the compound is non-essential for the release (see also Example 5). However, the cryo-protectant has the advantage that when optionally added to the lysis reagents according to the present disclosure it improves the storability and freezing of the viral vector containing compositions after lysis and/or purification. However, the cryo-protectant does not need to be present during cell lysis but may be added subsequently, e.g. after step (c) or after step (d) of the method according to the present disclosure. It can be advantageous to include the cryoprotectant in the lysis reagent to avoid subsequent addition steps, i.e. adding the cryo-protectant simplifies the lysis process.

According to a preferred embodiment, the cryo-protectant in the lysis reagent has a concentration of 1% (w/v) to 70% (w/v), preferably a concentration selected from the following ranges: i) 1% (w/v) to 10% (w/v) for a lx lysis reagent; ii) 10% (v/v) to 35% (w/v) for a 5x lysis reagent; or iii) 20% (w/v) to 60% (w/v) for a lOx lysis reagent.

It was found that such concentrations of cryo-protectant are advantageous for stability of the viral vector during cryo-preservation.

Exemplary lysis reagents comprising the detergent can be found in Tables 1, 2, 4, 5, 9, and 11.

Further exemplary lysis reagents

According to a particular embodiment of the present method, the lysis reagent is an alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, such as 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or higherthan 8.5, and containing 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 embodiment, the detergent preferably has a concentration of 0.1% (v/v) to 20% (v/v), more preferably selected from the following ranges: i) 0.1% (v/v) to 2% (v/v) for a lx lysis reagent; ii) 1% (v/v) to 5% (v/v) for a 5x lysis reagent; or iii) 4% (v/v) to 10% (v/v) for a lOx lysis reagent.

In such an embodiment, the viral vector may be AAV or Adenovirus and the cell of the cell culture is a mammalian cell, preferably, selected from a HEK293 cell or a derivative of a HEK293 cell, e.g. sub- cell lines such as for HEK293 also 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. A derivative of a HEK293 cell may also encompass a HEK293 cell that is adapted to suspension culture and/or adapted to a particular type of cell culture medium.

According to a preferred embodiment, the lysis reagent is a detergent containing alkaline lysis reagent, preferably selected from: i. lx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v); ii. 5x alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.3 M to 1.5 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.3 M to 1.5 M; a detergent, preferably a polysorbate, having a concentration selected from 1% (v/v) to 5% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 5 mM to 15 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 10% (v/v) to 35% (w/v); or iii. preferably, lOx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 1 M to 3 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.8 M to 5 M or 1 M to 3 M; a detergent, preferably a polysorbate, having a concentration selected from 4% (v/v) to 10% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 15 mM to 50 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 20% (w/v) to 60% (w/v).

Step (b)

Step (b) according to the method of the first aspect defines contacting the cell culture with the lysis reagent to generate a lysis composition. Contacting may comprise adding the lysis reagent to the cell culture or adding the cell culture to the lysis culture orjoining both simultaneously. According to one embodiment, the lysis reagent is added to the cell culture, which is for instance advantageous in case the cell culture is present in the culture vessel, e.g. bioreactor, or if the cells are present as a cell pellet in a container. On the other hand, the particular mode of contacting shall not be limiting in scope of the present disclosure.

Preferably, the lysis reagent is provided in liquid form and added to the cell culture in contacting step (b). This allows for keeping the cell culture in the cultivation container, e.g. bioreactor or shake flasks and adding the lysis reagent thereto, e.g. manually or in an automated fashion. Apart from the lysis reagent, the cell culture may also be contacted with further compounds. Such compounds can be useful for the culture of the mammalian cell, e.g. cell culture medium, or may be required for contacting the mammalian cell with the lysis reagent. Such cell culture media are well-known to the skilled person and shall not limit the scope of the present disclosure. Specific examples include the cell culture media used in the Examples below.

According to a preferred embodiment, in step (b) the lysis composition has a pH of more than 7.2, preferably more than 7.5, most preferably more than 7.8 or 8.0. According to a preferred embodiment, in step (b) the lysis composition has a pH selected from the range of 7.2 to 10.0, preferably 7.5 to 9.5. Lysis compositions having the indicated pH (see exemplary lysis compositions

Table 5, 9, 11) were found efficient for releasing the viral vector (see Figs. 4-9, 12, and 13).

According to a preferred embodiment, in step (b) the lysis composition comprises the compounds in essentially the concentrations of a lx lysis reagent, which includes lx detergent containing alkaline lysis reagent. Since, the lx lysis reagent is preferably provided in order to release viral vector from a concentrated cell sample, such as a cell pellet, the lx lysis reagent is essentially not or only insignificantly diluted, e.g. less than 20%, preferably less than 10%. As a result, the concentrations defined herein for the lx lysis reagents essentially correspond to the concentrations obtained in the lysis compositions. In an exemplary embodiment, a lOx lysis reagent may be mixed with the sample such that the final concentration in the lysis composition corresponds to a tenth of the lOx lysis reagent, i.e. essentially a corresponding lx lysis reagent. Hence, according to a preferred embodiment, the lysis composition comprises the compounds in essentially the concentrations of a lx lysis reagent as disclosed herein. "Essentially" in this respect refers to the indicated concentrations for the lx lysis reagent but allowing a minor dilution, e.g. less than 20%, preferably less than 10% dilution.

According to a preferred embodiment, in step (b) the lysis composition comprises the compounds in essentially the concentrations of a lx lysis reagent, preferably essentially the concentrations disclosed herein. According to a preferred embodiment, the lysis composition has the following characteristics:

I. the lysis composition has a pH of more than 7.2, preferably more than 7.5, most preferably more than 7.8 or 8.0, and comprises: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v). Nuclease

In one embodiment, the nuclease is added to the lysis composition after step (b) (see also Examples), however, the nuclease may equally well be added to the lysis reagent before or during step (b). Nuclease addition in scope of the present disclosure is typically employed to achieve three objectives: (i) reduce nucleic acid contaminations, (ii) decrease the viscosity of cell lysate, and (iii) mitigate AAV vector aggregation caused by the association of nucleic acids with the surface of AAV vector particles. According to a preferred embodiment, the nuclease is salt-tolerant. Salt-tolerant nucleases allow for use in a composition of higher ionic strength, as can be present in the lysis compositions and/or lysis reagents according to the present disclosure. Examples of nucleases or salt-tolerant nucleases are well-known by the skilled person and the particular enzyme used shall not be limiting the scope of the present disclosure. Examples of applicable salt-tolerant nucleases are NISAN HQ and SAN -HQ (ArcticZymes). Typical suitable conditions for a nuclease such as„San HQ 2.0" (ArcticZymes) are: i) Temperature: 7 - 38°C, 4°C overnight, optimal: 30 - 38°C, ii) Salt concentration (NaCI / KCI): 100 - 900 mM, optimal: 400 - 650 mM, iii) Mg²⁺: >1 mM is beneficial for activity, optimal 5 - 50 mM, iv) pH: 7.3 - 9.2, optimal 8.2 - 8.8.

In such an embodiment, the nuclease is added to the lysis composition after step (b) or to the cell culture during or before step (b). Furthermore, in such embodiment, the viral vector may be AAV or Adenovirus and the cell of the cell culture is a mammalian cell, preferably, selected from a HEK293 cell or a derivative of a HEK293 cell, e.g. sub-cell lines such as for HEK293 also 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. A derivative of a HEK293 cell may also encompass a HEK293 cell that is adapted to suspension culture and/or adapted to a particular type of cell culture medium. Furthermore, in such an embodiment, the lysis composition may further comprise the following: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v).

Step (c)

Step (c) according to the method of the first aspect defines incubating the lysis composition such that a viral vector is released from the cell culture. Different incubation conditions are suitable in frame of the present disclosure. Exemplary conditions include adjusting temperature of the lysis composition ranging from 5°C to 50°C, preferably between 25°C to 45°C, more preferably 30°C to 42°C, e.g. 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C. According to a particular embodiment, the temperature is 37°C. Exemplary conditions further include shaking or stirring the lysis composition. For example, the lysis composition may be shaken at 10 to 1000 rpm, preferably 20 to 500 rpm, more preferably 50 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 readily understood by the skilled person, different lysis conditions are suitable and can be flexibly adjusted according to the respective requirements.

According to a preferred embodiment, in step (c) the cell culture is partially lysed. While partial lysis is generally less preferred the method of the present disclosure allows only partial lysis, as the subsequent step (d) contacting the lysis composition with an acidifying reagent to lower the pH allows for completing the cell lysis. Hence, the partial lysis is acceptable, as the full lysis can be achieved in the second lysis step. As a result, less incubation time is required in the first step, rendering the release of the viral vector faster and thus overall more efficient.

According to a preferred embodiment, incubating step (c) comprises: incubating for less than 6 hours, preferably less than 5 hours or less than 4 hours, more preferably for less than 3 hours, such as for 2 hours, 1 hour, or 0.5 hour; and/or incubating for a time selected from the range of 15 min to 300 min, preferably 20 min to 250 min, 25 min to 200 min, or more preferably 30 min to 150 min or 30 to 100 min, most preferably 30 min to 90 min or 45 min to 75 min.

According to a preferred embodiment, the time required for conducting steps (c) and (d) is less than 360 min, preferably less than 300 min or less than 240 min, more preferably for less than 180 min or less than 120 min, most preferably less than 100 min, such as 90 min, 80 min or 70 min or less; and/or is a time selected from the range of 15 min to 360 min, preferably 20 min to 300 min, 25 min to 240 min, or more preferably 30 min to 180 min or 40 to 120 min, most preferably 45 to 90 min.

The method according to the present disclosure advantageously allows for a fast and efficient release of viral vector from the cell culture. As demonstrated in the Examples, the method according to the present disclosure efficiently releases the viral vector such as AAV in 70 min or less. Such quick release of viral vector improves efficiency of the overall process.

Step (d)

Step (d) according to the method of the first aspect defines contacting the lysis composition with an acidifying reagent to lower the pH. The step of acidifying advantageously allows for releasing any viral vector that has not been released within steps (b) and/or (c). As result, the incubation time for step (c) can be reduced, rendering the method more efficient. In addition, as is demonstrated in the Examples below, by including step (d), the released viral vector is of higher purity containing less host cell related impurities, such as host cell proteins (see Figs. 3, 11, and 15).

According to a preferred embodiment, in step (d) contacting the lysis composition with an acidifying reagent lyses the cell culture essentially completely. In some cases, after step (c) part of the cell culture is lysed such that some cells may remain viable. For instance, less than 90% of cells are not lysed, preferably less than 50%, more preferably less than 30%. These remaining non-lysed cells are then effectively lysed in step (d), such that the acidifying reagent essentially completely lyses the cell culture. Hence, more than 90%, preferably more than 95%, more preferably more than 98% or more than 99% of the cells are lysed after step (d). In one embodiment, the cells of the cell culture are completely lysed after step (d). As demonstrated in the Examples below, no viable cells can be detected anymore after step (d) (see Examples, e.g. Table 6). According to a preferred embodiment, contacting step (d) comprises titrating the acidifying reagent to the lysis composition. By titrating the acidifying reagent to the lysis composition, the amount of acidifying reagent added to the lysis composition can be closely adjusted. Furthermore, e.g. by in parallel measuring the pH, it is possible to flexibly and precisely adjust the pH and thus adjust the conditions in step (d).

According to a preferred embodiment, step (d) comprises one or more steps of measuring the pH. Measuring the pH advantageously allows for precisely adjusting the pH during step (d). According to a preferred embodiment, the measurement is performed using a pH probe. Such pH probe can be provided separately and included in the container or vessel in which the release of the viral vector can take place. The pH probe can also be part of a culture vessel, e.g. part of a bioreactor or other culture vessel (e.g. shake flask), such that the culture vessel is equipped with the pH probe for measuring the pH. As a pH probe is conventionally used throughout culture to monitor the pH, there is no need for additional equipment, such that step (d) and the measuring of the pH can be directly done in the culture vessel, such as a bioreactor.

According to a preferred embodiment, the released viral vector is purified. Preferably, purifying is performed after step (d), such that the viral vector is released. According to a preferred embodiment, purifying is performed by a single-step or multistep approach, preferably at least by depth filtration, centrifugation, and/or sterile filtration after step (d).

According to a preferred embodiment, wherein the purified released viral vector comprises 10% less host cell derived impurities, preferably 25% or 30% less host cell proteins, compared to a method wherein either step (b) or step (d) is not performed. As demonstrated in the Examples, the host cell derived impurities such that the host cell proteins can be significantly reduced (see Figs. 11 and 15).

According to a preferred embodiment, wherein the capsid viral vector titer and/or transducing viral vector titer is increased compared to a method wherein step (d) is not performed. According to a preferred embodiment, the capsid viral vector titer is increased by at least 10%, preferably at least 25%, compared to a method wherein step (d) is not performed. According to a preferred embodiment, the transducing viral vector titer is increased by at least 10%, preferably at least 25%, compared to a method wherein step (d) is not performed. According to a preferred embodiment, at least 10% more viral vector is released, preferably at least 25%, compared to a method wherein step (d) is not performed. Acidifying reagent

According to a preferred embodiment, in step (d) the acidifying reagent lowers the pH to an acidic pH, preferably a pH of less than 6.0, more preferably less than 5.0, most preferably less than 4.5. According to a preferred embodiment, in step (d) the acidifying reagent 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, such as e.g. 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5. Lowering the pH has the advantage that any residual cells that have not lysed yet are essentially completely lysed, allowing also for a reduction in incubation time but also obtaining high yields of viral vector. Furthermore, the lowering of the pH to the indicated ones has the advantage that less host cell related impurities are present, such that the released viral vector is of higher purity (see also Fig. 15).

According to a preferred embodiment, the acidifying reagent comprises an acid, preferably an organic acid. As demonstrated in the Example, an acid is particularly suitable as an acidifying reagent in order 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.

According to a preferred embodiment, the acidifying reagent is provided in liquid form or solid form, preferably in liquid form. According to a preferred embodiment, the acidifying reagent comprises an acid and a solvent, preferably but not limited to water. In such case, the acid is dissolved in the solvent such that it can be provided to the lysis composition in liquid form. It is typically easierto handle and perform the contacting step when the acidifying reagent is provided in liquid form. Also, this can be more exact than adding the acidifying reagent in solid form. Suitable solvents are readily known to the person skilled in the art. According to another embodiment, the acidifying reagent is provided in solid form, e.g. as a powder. The particular form of acidifying reagent shall not be limiting in scope of the present disclosure.

The acidifying reagent preferably comprises a concentrated acid such that only low volumes of acid need to be added in order to acidify the lysis composition. Preferably, the acidifying reagent comprises a concentrated organic acid, preferably a concentration carboxylic acid, most preferably a concentration acetic acid or citric acid. Preferably the concentrated acid, e.g. concentrated acetic acid or citric acid, has a concentration sufficient to reduce the pH of the lysis composition to a n acidic pH, preferably a pH of less than 6.0, more preferably less than 5.0, most preferably less than 4.5. Exemplary concentrations of acid in the acidic reagent are at least 100 mM, preferably at least 250 mM, at least 500 mM, or at least 750 mM, most preferably at least 1 M, such as 2 M, 3 M or 4 M. According to a particular embodiment, the acidifying reagent comprises a carboxylic acid, such as acetic acid or citric acid, having a concentration of at least 1 M.

Further embodiments

According to a preferred embodiment, at least one of steps (b), (c) and (d) is conducted in a bioreactor, preferably at least two of these steps are conducted in a bioreactor, most preferably, all the steps (b), (c), and (d) are conducted in a bioreactor. Conducting one or more steps of the method in a bioreactor has the advantage that the cell culture does not need to be transferred into another container or vessel, but the bioreactor can be used in order to perform the release of the viral vector from the cell culture, as also demonstrated below in Example 8.

According to a preferred embodiment, the bioreactor agitation facilitates mixing in these steps. By agitating 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 such that a more homogeneous composition is obtained and thus a more efficient release of the viral vector.

For example, the method may be performed in a tank bioreactor, such as a continuous stirred tank reactor, a rocking motion bioreactor, a wave bioreactor, an Erlenmeyerflask, a spinnerflask, a rotating wall bioreactor, and/or a multi parallel bioreactor.

According to a preferred embodiment, the bioreactor allows for temperature control and/or pH control. By including a temperature control, the temperature in any of steps (b), (c) and/or (d) can be controlled, such that a desirable condition is provided for the release of the viral vector. It may be advantageous to increase the temperature, e.g. above 30°C in order to increase the speed of the reaction and thus the release of the viral vector. On the other side, too high temperatures can be avoided which would impact the integrity of the viral vector. By controlling the pH, the pH of the lysis composition can be closely monitored and affected. For instance, if not enough acidifying reagent is added in step (d), the pH control can facilitate adjustment into the described pH regime.

In some embodiments, a controller may monitor and/or control one or more parameters associated with the process, like e.g. the critical process parameters (e.g. pH, DO and temperature). Mixing may e.g. be performed in stirred, rocking motion or orbital shaking mode.

According to a preferred embodiment, the bioreactor allows for pH adjustment, preferably by being configured to allowfor contacting the lysis composition with an acidifying reagent in step (d). In some embodiment, the bioreactor chamber may be a dimensionally stable vessel made e.g. from stainless steel, glass or plastic material. Alternatively, it is envisaged that the bioreactor chamber may be a flexible bag made from at least one suitable polymer, which may further be configured to be placed into a holder to safeguard dimensionally stability. In the art, the far most of the flexible bag containers as well as rigid plastic bioreactors are also known as single use bioreactors (SUB), which are encompassed herein.

According to one embodiment, in the method for releasing a viral vector from a cell culture the cell culture is a cell broth and all the steps (b), (c), and (d) are conducted in a bioreactor. In such an embodiment, advantageously the bioreactor (i) agitation facilitates mixing in these steps; (ii) allows for temperature control and (iii) a I lows for pH adjustment, preferably by being configured to a I low for contacting the lysis composition with an acidifying reagent in step (d). Preferably, the viral vector is AAV or Adenovirus and/or the cell culture comprises a mammalian cell which is selected from a HEK293 cell or a derivative of a HEK293 cell.

According to one embodiment, in the method for releasing a viral vector from a cell culture the cell culture is a cell broth and all the steps (b), (c), and (d) are conducted in a bioreactor. Preferably, the viral vector is AAV or Adenovirus and/or the cell culture comprises a mammalian cell which is selected from a HEK293 cell or a derivative of a HEK293 cell. In such an embodiment, the lysis reagent is a detergent containing alkaline lysis reagent, preferably selected from:

5x alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.3 M to 1.5 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.3 M to 1.5 M; a detergent, preferably a polysorbate, having a concentration selected from 1% (v/v) to 5% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 5 mM to 15 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 10% (v/v) to 35% (w/v); or preferably, lOx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 1 M to 3 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.8 M to 5 M or 1 M to 3 M; a detergent, preferably a polysorbate, having a concentration selected from 4% (v/v) to 10% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 15 mM to 50 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 20% (w/v) to 60% (w/v).

In such an embodiment, the acidifying reagent may comprise a concentrated acid, preferably a concentrated organic acid, having a concentration sufficient to reduce the pH of the lysis composition to an acidic pH, preferably a pH of less than 6.0, more preferably less than 5.0, most preferably less than 4.5, optionally a concentration of at least 1 M. In such an embodiment, the method may not comprise a lysis step based on mechanical or physical lysis, such as repeated cycles of freezing and thawing for cell lysis or a step of microfluidization for cell lysis.

According to a particular embodiment, the cell culture of the method for releasing a viral vector from a cell culture is not required to be separated from the surrounding liquid prior to contacting the cell culture with the lysis reagent in step (b) and comprises a mammalian cell which is selected from a HEK293 cell ora derivative of a HEK293 cell. Preferably, the viral vector is AAV or Adenovirus. In such an embodiment, the lysis composition can have a pH of more than 7.2, preferably more than 7.5, most preferably more than 7.8 or 8.0, and may comprise: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v).

According to a particular embodiment, in the method for releasing a viral vector from a cell culture in step (c) the cell culture is partially lysed and in step (d) contacting the lysis composition with an acidifying reagent lyses the cell culture essentially completely. In such an embodiment, the lysis composition can have a pH of more than 7.2, preferably more than 7.5, most preferably more than 7.8 or 8.0, and may comprise: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v).

In such an embodiment, the acidifying reagent may comprise a concentrated acid, preferably a concentrated organic acid, having a concentration sufficient to reduce the pH of the lysis composition to an acidic pH, preferably a pH of less than 6.0, more preferably less than 5.0, most preferably less than 4.5, optionally a concentration of at least 1 M. Preferably, the viral vector is AAV or Adenovirus and/or the cell culture comprises a mammalian cell which is selected from a HEK293 cell or a derivative of a HEK293 cell. In such an embodiment, the method may not comprise a lysis step based on mechanical or physical lysis, such as repeated cycles of freezing and thawing for cell lysis or a step of microfluidization for cell lysis. Method for producing a viral vector

The method according to the second aspect advantageously allows for efficiently producing a viral vector by utilizing the improved release of the viral vector from a cell culture according to first aspect. Hence, the above disclosed advantages can also be found for the method according to the second aspect. Specifically, the method allows for producing viral vector with improved yields of capsid and functional viral vector within short incubation times. Furthermore, releasing the viral vector from the cell culture in a method according to the second aspect leads to less host cell related impurities, in particular host cell proteins, such that purer viral vector can be produced.

The individual steps and preferred embodiments of the method according to the second aspect correspond to the individual steps and embodiments of the method according to the first aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the method according to the second aspect. This particularly but not exclusively includes the viral vector, the cell culture, the lysis reagent, including the buffer, salt, divalent cation, detergent, cryo-protectant, nuclease, as well as the acidifying reagent, and steps (a), (b) and (c). Further features will now be described in detail.

According to a preferred embodiment, step x.3 releasing the viral vector from a cell culture is performed by the contacting the cell culture which is a cell broth in a cultivation container with the lysis reagent to generate a lysis composition and incubating the lysis composition in the cultivation container such that the cell culture is partially lysed and contacting the lysis composition with an acidifying reagent lyses the cell culture essentially completely. According to a preferred embodiment, at least one of the steps of x.2 and x.3 is conducted in a bioreactor, preferably both steps x.2 and x.3 are conducted in a bioreactor. Such embodiments have the advantage that the incubation conditions can be well defined, e.g. in the bioreactor, such that the temperature and/or mixing, stirring or shaking can be adjusted easily. Exemplary conditions include adjusting temperature of the cell culture and/or lysis composition ranging from 5°C to 50°C, preferably between 25°C to 45°C, more prefera bly 30°C to 42°C, e.g. 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C. According to a particular embodiment, the temperature is 37°C. Exemplary conditions further include shaking or stirring the cell culture and/or lysis composition. For example, the cell culture and/or lysis composition may be shaken at 10 to 1000 rpm, preferably 20 to 500 rpm, more preferably 50 to 250 rpm. In a particular embodiment, the cell culture and/or lysis composition is shaken at 120 rpm. According to a particular embodiment, the cell culture and/or lysis composition is incubated at 37°C and shaken at 120 rpm. However, as readily understood by the skilled person, different conditions are suitable and can be flexibly adjusted according to the respective requirements.

According to an alternative embodiment, step x.3 releasing the viral vector from a cell culture is performed by contacting the cell culture which is a cell broth outside a cultivation container with the lysis reagent to generate a lysis composition and incubating the lysis composition outside the cultivation container such that a viral vector is released from the cell culture. For instance, the cell culture could be transferred out of the culture container, e.g. bioreactor, via a sterile connection, e.g. tubing, and the lysis reagent can be added either inline, i.e. through a coupling to the sterile connection, or in another container, wherein the lysis composition is incubated.

According to a preferred embodiment, the method comprises step x.4 purifying the released viral vector, wherein purifying may comprise a single-step or multistep approach, preferably at least by depth filtration, centrifugation, and/or sterile filtration. As demonstrated in the Examples, downstream purification of the lysis compositions according to the present disclosure efficiently removes host cell related impurities, such as nucleic acids, e.g. DNA, and proteins. Accordingly, when using the acidifying reagent, a fraction of the impurities may precipitate, such that these can be removed in downstream purification processes, such as at least by depth filtration, centrifugation, and/or sterile filtration.

According to a preferred embodiment, the purified released viral vector comprises 10% less host cell derived impurities, such as host cell proteins, preferably 25% or 30% less host cell derived impurities, such as host cell proteins, compared to a method wherein either step (b) or step (d) is not performed. As is demonstrated in the examples, such lower content of host cell derived impurities, such as nucleic acids and proteins, is advantageous, as less downstream purification is required and a purer viral vector is produced. According to a preferred embodiment, the method further comprises step x.5 formulating the viral vector for gene therapy.

Kit for releasing a viral vector from a cell culture

(i) a lysis reagent; and

(ii) an acidifying reagent.

The kit according to the third aspect advantageously allows for efficiently releasing a viral vector from a cell culture and can be applied in scope of the methods disclosed herein. Hence, the above disclosed advantages can also be found for the cell lysis according to the third aspect. Specifically, the reagent allows for achieving improved yields of capsid and functional viral vector within short incubation times. Furthermore, releasing the viral vector from the cell culture using the kit according to the third aspect leads to less host cell related impurities, in particular host cell proteins, such that purer viral vector can be produced.

The individual and preferred embodiments of the kit according to the third aspect correspond to the embodiments of the methods according to the first and second aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the kit according to the third aspect. This particularly but not exclusively includes the viral vector, the cell culture, the lysis reagent, including the buffer, salt, divalent cation, detergent, cryo-protectant, nuclease, as well as the acidifying reagent. Further features will now be described in detail.

According to a preferred embodiment, the kit is for use in a method for releasing the viral vector according to the first aspect and/or for use in a method for producing a viral vector according to the second aspect.

According to a preferred embodiment, the lysis reagent is a detergent containing alkaline lysis reagent, preferably selected from: i. lx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or l,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v); ii. 5x alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or l,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.3 M to 1.5 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.3 M to 1.5 M; a detergent, preferably a polysorbate, having a concentration selected from 1% (v/v) to 5% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 5 mM to 15 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 10% (v/v) to 35% (w/v); or iii. preferably, lOx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or l,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 1 M to 3 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.8 M to 5 M or 1 M to 3 M; a detergent, preferably a polysorbate, having a concentration selected from 4% (v/v) to 10% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 15 mM to 50 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 20% (w/v) to 60% (w/v).

According to a preferred embodiment, the acidifying reagent comprises an acid, preferably an organic acid, more preferably a carboxylic acid, such as acetic acid or citric acid. As is demonstrated in the examples below, such acidifying reagent components are particularly suitable for releasing the viral vector.

According to a preferred embodiment, the acidifying reagent is provided in liquid form 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 by the skilled person in the art.

Kit for producing a viral vector

(i) the kit according to the third aspect of the invention;

(ii) either one of

II. a mammalian cell, which can be modified to produce the viral transient transfection with one or more plasmids for viral vector production allowing for transient viral vector production.

The kit according to the fourth aspect advantageously allows for efficiently producing a viral vector by utilizing the kit for improved release of the viral vector from a cell culture according to third aspect. Hence, the above disclosed advantages for the method according to the first and second aspect, as well as the kit according to the third aspect can also be found for the kit according to the fourth aspect. Specifically, the kit allows for producing viral vector with improved yields of capsid and functional viral vector within short incubation times. Furthermore, using the kit leads to less host cell related impurities, in particular host cell proteins, such that purer viral vector can be produced. The individual and preferred embodiments of the kit according to the fourth aspect correspond to the embodiments of the methods according to the first and second aspect and the kit according to the third aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the kit according to the third aspect. This particularly but not exclusively includes the viral vector, the cell culture, the lysis reagent, including the buffer, salt, divalent cation, detergent, cryo -protecta nt, nuclease, as well as the acidifying reagent. Furtherfeatures will now be described in detail.

According to a preferred embodiment, the kit is for use in a method for producing a viral vector according to the second aspect.

According to a preferred embodiment, the kit comprises one or more of the following components: a cell culture media suitable for culturing the mammalian cell; a transfection reagent; and/or one or more plasmids encoding at least part of a viral vector, preferably at least part of an adeno-associated virus (AAV) or Adenovirus (Ad).

Items according to the present disclosure

The following items provide further advantageous embodiments of the present disclosure:

1. A method for releasing a viral vector from a cell culture, the method comprising following steps:

(a) providing a lysis reagent;

2. The method according to item 1, wherein in step (c) the cell culture is partially lysed, and/or wherein in step (d) contacting the lysis composition with an acidifying reagent lyses the cell culture essentially completely.

3. The method according to item 1 or 2, wherein the method has one or more of the following characteristics: it does not comprise a lysis step based on mechanical or physical lysis, and/or it does not comprise a step of repeated cycles of freezing and thawing for cell lysis or a step of microfluidization for cell lysis.

4. The method according to one or more of items 1 to 3, wherein the cell culture is a cell broth or cell pellet, preferably a cell broth.

5. The method according to one or more of items 1 to 4, wherein the cell culture is not required to be separated from the surrounding liquid prior to contacting the cell culture with the lysis reagent in step (b).

6. The method according to one or more of items 1 to 5, wherein at least one of steps (b), (c) and (d) is conducted in a bioreactor, preferably at least two of these steps are conducted in a bioreactor, most preferably, all the steps (b), (c), and (d) are conducted in a bioreactor

7. The method according to item 6, wherein the bioreactor agitation facilitates mixing in these steps.

8. The method according to item 6 or 7, wherein the bioreactor allows for temperature control and/or pH control.

9. The method according to one or more of items 6 to 8, wherein the bioreactor allows for pH adjustment, preferably by being configured to allowfor contacting the lysis composition with an acidifying reagent in step (d).

10. The method according to one or more of items 1 to 9, wherein contacting step (d) comprises titrating the acidifying reagent to the lysis composition.

11. The method according to one or more of items 1 to 10, wherein step (d) comprises one or more steps of measuring the pH, preferably by measuring using a pH probe. 12. The method according to one or more of items 1 to 11, wherein in step (d): the acidifying reagent lowers the pH to an acidic pH, preferably a pH of less than 6.0, more preferably less than 5.0, most preferably less than 4.5; and/or the acidifying reagent 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.

13. The method according to one or more of items 1 to 12, wherein the acidifying reagent comprises an acid, preferably an organic acid.

14. The method according to item 13, wherein the acid is a carboxylic acid.

15. The method according to item 13 or 14, wherein the acid is acetic acid or citric acid, preferably acetic acid.

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

17. The method according to one or more of items 1 to 16, wherein the acidifying reagent is provided in liquid form or solid form, preferably in liquid form.

18. The method according to one or more of items 1 to 17, wherein the acidifying reagent comprises an acid and a solvent, preferably water.

19. The method according to one or more of items 1 to 18, wherein the lysis reagent is an alkaline lysis reagent, preferably having a pH of more than 7.0, preferably more than 7.5, most preferably more 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.

20. The method according to item 19, wherein the alkaline lysis reagent has a pH selected from the range of 7.2 to 10, preferably 7.5 to 9.5, more preferably 8 to 9 or 8.2 to 8.

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

22. The method according to one or more of items 1 to 21, wherein the lysis reagent comprises a buffer selected from Tris(hydroxymethyl)-aminomethan (TRIS) buffer, 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, phosphate buffer, glycine-sodium hydroxide buffer, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffer, TES buffer, DIPSO buffer, TAPSO buffer, triethanolamine buffer, HEPPSO buffer, POPSO buffer, TRICINE buffer, HEPPS buffer, EPPS buffer, BIGIN buffer, TAPS buffer, AMPSO buffer, taurine buffer, CHES buffer, AMP buffer, CAPSO buffer, or combinations thereof, preferably TRIS buffer or BTP buffer. 23. The method according to one or more of items 1 to 22, wherein the lysis reagent comprises a buffer having a concentration of 0.01 to 3 M, preferably a concentration selected from the following ranges: i) 0.05 to 1 M for a lx lysis reagent; ii) 0.3 to 1.5 M for a 5x lysis reagent; or iii) preferably, 1 to 3 M for a lOx lysis reagent.

24. The method according to one or more of items 1 to 23, wherein the lysis reagent comprises a salt, preferably selected from potassium chloride or sodium chloride.

25. The method according to item 24, wherein the salt in the lysis reagent has a concentration of 0.01 to 5 M, preferably a concentration selected from the following ranges: i) 0.05 M to 1 M for a lx lysis reagent; ii) 0.3 M to 1.5 M for a 5x lysis reagent; or iii) preferably, 0.8 M to 5 M or 1 M to 3 M for a lOx lysis reagent.

26. The method according to one or more of items 1 to 25, wherein the lysis reagent comprises a detergent, preferably a non-ionic detergent, more preferably a non-ionic detergent that is not classified as toxic and/or hazardous to the environment.

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

28. The method according to item 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).

29. The method according to one or more of items 26 to 28, wherein the detergent is a polyoxyethylen alkyl ethers such as Triton X-100, Nonidet P40, NP-40 and respective non-ionic detergents from the Brij class.

30. The method according to one or more of items 26 to 29, wherein the detergent has a concentration of 0.1% (v/v) to 20% (v/v), preferably selected from the following ranges: i) 0.1% (v/v) to 2% (v/v) for a lx lysis reagent; ii) 1% (v/v) to 5% (v/v) for a 5x lysis reagent; or iii) 4% (v/v) to 10% (v/v) for a lOx lysis reagent.

31. The method according to one or more of item 1 to 30, wherein the lysis reagent comprises a divalent cation, preferably Mg2+, Ca2+, Ba2+, Cu2+, Fe2+, Zn2+, Mn2+, Ni2+, or a combination thereof, more preferably Mg2+, Cu2+, Zn2+, Mn2+, Ni2+, or a combination thereof, most preferably Mg2+. 32. The method according to item 31, wherein the divalent cation in the lysis reagent has a concentration of 0.1 to 1000 mM, preferably a concentration selected from the following ranges: i) 0.5 to 5 mM for a lx lysis reagent; ii) 5 to 15 mM for a 5x lysis reagent; or iii) preferably, 15 to 50 mM for a lOx lysis reagent.

33. The method according to one or more of items 1 to 32, wherein the lysis reagent comprises a cryo-protectant, preferably a sugar, more preferably sucrose, trehalose or mannitol, most preferably sucrose.

34. The method according to item 33, wherein the cryo-protectant in the lysis reagent has a concentration of 1% (w/v) to 70% (w/v), preferably a concentration selected from the following ranges: i) 1% (w/v) to 10% (w/v) for a lx lysis reagent; ii) 10% (v/v) to 35% (w/v) for a 5x lysis reagent; or iii) 20% (w/v) to 60% (w/v) for a lOx lysis reagent.

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

36. The method according to item 35, wherein the nuclease is salt-tolerant.

37. The method according to one or more of items 1 to 36, wherein in step (b) the lysis composition has a pH of more than 7.2, preferably more than 7.5, most preferably more than 7.8 or 8.0.

38. The method according to one or more of items 1 to 37, wherein in step (b) the lysis composition has a pH selected from the range of 7.2 to 10.0, preferably 7.5 to 9.5.

39. The method according to one or more of items 1 to 38, wherein the lysis reagent is a detergent containing alkaline lysis reagent, preferably selected from: i. lx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v); ii. 5x alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.3 M to 1.5 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.3 M to 1.5 M; a detergent, preferably a polysorbate, having a concentration selected from 1% (v/v) to 5% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 5 mM to 15 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 10% (v/v) to 35% (w/v); or iii. preferably, lOx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 1 M to 3 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.8 M to 5 M or 1 M to 3 M; a detergent, preferably a polysorbate, having a concentration selected from 4% (v/v) to 10% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 15 mM to 50 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 20% (w/v) to 60% (w/v). The method according to one or more of items 1 to 39, wherein in step (b) the lysis composition comprises the compounds in essentially the concentrations of a lx lysis reagent, preferably essentially the concentrations according to item 39, and/or the lysis composition has the following characteristics: I. the lysis composition has a pH of more than 7.2, preferably more than 7.5, most preferably more than 7.8 or 8.0, and comprises: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or l,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v).

41. The method according to one or more of items 1 to 40, wherein incubating step (c) comprises: incubating for less than 6 hours, preferably less than 5 hours or less than 4 hours, more preferably for less than 3 hours, such as for 2 hours, 1 hour, or 0.5 hour; and/or incubating for a time selected from the range of 15 min to 300 min, preferably 20 min to 250 min, 25 min to 200 min, or more preferably 30 min to 150 min or 30 to 100 min, most preferably 30 min to 90 min or 45 min to 75 min.

42. The method according to one or more of items 1 to 41, wherein the time required for conducting steps (c) and (d) is less than 360 min, preferably less than 300 min or less than 240 min, more preferably for less than 180 min or less than 120 min, most preferably less than 100 min, such as 90 min, 80 min or 70 min or less; and/or is a time selected from the range of 15 min to 360 min, preferably 20 min to 300 min, 25 min to 240 min, or more preferably 30 min to 180 min or 40 to 120 min, most preferably 45 to 90 min.

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

44. The method according to one or more of items 1 to 43, wherein the cell culture is not subjected to centrifugation, cell pelleting or an exchange of the liquid surrounding the cells of the cell culture prior to steps (a), (b) and (c).

45. The method according to one or more of items 1 to 44, wherein the released viral vector is purified, preferably by a single-step or multistep approach, preferably at least by depth filtration, centrifugation, and/or sterile filtration after step (d). 46. The method according to item 45, wherein the purified released viral vector comprises 10% less host cell derived impurities, such as host cell proteins, preferably 25% or 30% less host cell derived impurities, such as host cell proteins, compared to a method wherein either step (b) or step (d) is not performed.

47. The method according to one or more of items 1 to 46, wherein the capsid viral vector titer and/or transducing viral vector titer is increased compared to a method wherein step (d) is not performed.

48. The method according to item 47, wherein the capsid viral vector titer is increased by at least 10%, preferably at least 25%, compared to a method wherein step (d) is not performed.

49. The method according to item 47 or 48, wherein the transducing viral vector titer is increased by at least 10%, preferably at least 25%, compared to a method wherein step (d) is not performed.

50. The method according to one or more of items 1 to 49, wherein at least 10% more viral vector is released, preferably at least 25%, compared to a method wherein step (d) is not performed.

51. A method for producing a viral vector, the method comprising following steps: x.l providing a cell culture capable of producing a viral vector; x.2 culturing the cell culture to produce a viral vector; x.3 releasing the viral vector from the cell culture according to one or more of items 1 to 50; and x.4 optionally, purifying the released viral vector.

52. The method according to item 51, wherein step x.3 releasing the viral vector from a cell culture is performed by the contacting the cell culture which is a cell broth in a cultivation container with the lysis reagent to generate a lysis composition and incubating the lysis composition in the cultivation container such that the cell culture is partially lysed and contacting the lysis composition with an acidifying reagent lyses the cell culture essentially completely.

53. The method according to item 51 or 52, wherein the method comprises purifying the viral vector, wherein purifying comprises a single-step or multistep approach, preferably at least by depth filtration, centrifugation, and/or sterile filtration.

54. The method according to item 53, wherein the purified released viral vector of step x.4 comprises 10% less host cell derived impurities, preferably 25% or 30% less host cell derived impurities, compared to a method wherein either step (b) or step (d) of step x.3 is not performed.

55. The method according to one or more of items 51 to 54, wherein the method further comprises step x.5 formulating the viral vector for gene therapy. 56. The method according to one or more of items 51 to 55, wherein at least one of the steps of x.2 and x.3 is conducted in a bioreactor, preferably both steps x.2 and x.3 are conducted in a bioreactor.

57. The method according to one or more of items 1 to 56, wherein the viral vector is predominantly present intracellularly and preferably is selected from an adeno-associated virus (AAV) or an adenovirus (Ad).

58. The method according to one or more of items 1 to 57, wherein the cell culture comprises a mammalian cell.

59. The method according to item 58, wherein the mammalian cell is modified to be configured to produce a viral vector, preferably AAV.

60. The method according to item 58 or 59, wherein the mammalian cell is selected from a HEK293 cell or a derivative of a HEK293 cell.

61. The method according to one or more of items 1 to 60, wherein the cell culture comprises a cell capable of being cultured in suspension.

62. A kit for releasing a viral vector from a cell culture, the kit comprising:

(i) a lysis reagent; and

(ii) an acidifying reagent.

63. The kit according to item 62, wherein lysis reagent is a detergent containing alkaline lysis reagent, preferably selected from: i. lx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v); ii. 5x alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.3 M to 1.5 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.3 M to 1.5 M; a detergent, preferably a polysorbate, having a concentration selected from 1% (v/v) to 5% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 5 mM to 15 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 10% (v/v) to 35% (w/v); or iii. preferably, lOx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 1 M to 3 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.8 M to 5 M or 1 M to 3 M; a detergent, preferably a polysorbate, having a concentration selected from 4% (v/v) to 10% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 15 mM to 50 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 20% (w/v) to 60% (w/v). The kit according to item 62 or 63, wherein the acidifying reagent comprises an acid, preferably an organic acid, more preferably a carboxylic acid, such as acetic acid or citric acid. The kit according to one or more of items 62 to 64, wherein the acidifying reagent is provided in liquid form or solid form, preferably in liquid form, wherein the liquid form preferably comprises an acid and a solvent, preferably water. A kit for producing a viral vector, the kit comprising:

(i) the kit according to one or more of items 62 to 65;

(ii) Either one of I. a mammalian cell capable of producing the viral vector by being modified by genome editing the mammalian cell with one or more nucleic acid molecules for viral vector production allowing for stable viral vector production; or

67. The kit according to item 66, wherein the kit comprises one or more of the following components: a cell culture media suitable for culturing the mammalian cell; a transfection reagent; and/or one or more plasmids encoding at least part of a viral vector, preferably at least part of an adeno-associated virus (AAV) or Adenovirus (Ad).

Throughout the description, where methods, kits, compositions or uses are described as having, including, or comprising specific components or steps, it is contemplated that, additionally, there are methods, kits, compositions or uses of the present invention that consist essentially of, or consist of, the recited components or steps.

In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

Terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The use of the term "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should be understood generally as open- ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

Where the use of the term "about" or "approximately" is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

All citations are hereby incorporated by reference.

All individual embodiments and aspects as disclosed herein can be combined with each other within the framework and context of the present disclosure. It will be understood that the embodiments disclosed herein are only exemplary, and that any feature presented for a particular exemplary embodiment may be used with the present disclosure on its own or in combination with any feature presented for the same or another particular exemplary embodiment and/or in combination with any other feature not mentioned. It will further be understood that any feature presented for an example embodiment in a particular category may also be used in a corresponding manner in an example embodiment of any other category.

EXAMPLES

It should be understood that the following examples are for illustrative purpose only and are not to be construed as limiting this invention in any manner. The following examples demonstrate the advantages of the method of the present invention for releasing a viral vector, preferably adeno- associated virus (AAV) or adenovirus (Ad), from a cell culture, and the method for producing a viral vector, preferably adeno-associated virus (AAV) or adenovirus (Ad), as well as the kits according to the present disclosure. Specifically, the exam pies demonstrate that the herein disclosed two step lysis approach increases extraction of viral vectors, wherein the two-step lysis approach includes a step of contacting the cell culture with the lysis reagent to generate a lysis composition and incubating the lysis composition, followed by contacting the lysis composition with an acidifying reagent. Such increase in extraction of viral vectors is achieved by enhancing the efficiency of the cell lysis while achieving improved removal of host cell-related impurities.

In particular, the herein disclosed lysis method divides the process into two short lysis steps, applying different beneficial conditions in the cell broth that cannot be combined in a single step. In the first step, concentrated (e.g. lOx) high salt, alkaline lysis buffer with a detergent and salttolerant nuclease is added to the bioreactor to initiate cell lysis. The bioreactor agitation function is used to mix all components and keep the cells in suspension. In addition, the temperature control at 37°C is activated to active maximum nuclease activity. The adjusted lysis conditions are maintained for approximately lh. Afterwards, a large fraction of the cells is lysed, so that only a small fraction of cells (< 20%) is still viable. At this point, the pH value is abruptly reduced to pH 4 by titration with a concentrated acidifying reagent, such as an acid, e.g. 2 M acetic acid. In this second step, the low pH causes a loss in nuclease activity but also cause immediate lysis of the remained cells. Moreover, the low pH results in an almost immediate precipitation of impurities, which are predominantly host cell- related impurities, e.g. host cell proteins or host cell nucleic acids, and cell debris. Therefore, the subsequent clarification process can be started shortly, such as after a few minutes at pH 4. The addition of the acidifying reagent can also be realized using the pH controller of the bioreactor system, such that the acidifying reagent can be added in an amount sufficient to acidify the lysis composition while monitoring the pH, e.g. via a pH probe in the bioreactor. Alternatively, inline addition and mixing of acid can be used when the cell broth is transferred to the filter stages for clarification. Another benefit of downshifting to acidic pH, e.g. pH 4, is its applicability in the subsequent downstream steps. Conventional capture steps typically require the virus to be in a specific pH environment, which is usually achieved by adjusting pH though ultra- and diafiltration. Depending on the virus stability, neutralization of the pH can be done at a later step. However, this should only be done after clarification, which means after the separation of the precipitate from the supernatant. A potential setup of an integrated solution of the method according to the present disclosure into a bioprocessing solution is illustrated in Fig. 1.

Materials and Methods

Cell culture and viral vector production

The two-step cell lysis method was developed and compared with other lysis approaches using suspension-adapted HEK293 cells (Expi293F™, Thermo Fisher Scientific Inc.) that were able to transiently producing viral vectors, such as adeno-associated virus (AAV) or adenovirus (Ad). The HEK293 cells were cultivated in a stirred bioreactor in different batch sizes. At a defined time of cultivation, the HEK293 cells were modified by transient transfection to initiate viral vector production. Various viral vectors were produced, and the cell broth was used to demonstrate the feasibility of two-stage lysis for different products.

As models for AAV, two different serotypes, AAV2 and AAV8, were produced by transiently transfecting the cells with one or more plasmids. As a model for an adenovirus (Ad) vaccine, the ChAdOxl 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, University of Oxford (Joe et. al. 2021 (bioRxiv 2021.12.22.473478)). The transient production is initiated by infection of the HEK293 cell culture with the purified virus. After transfection (AAV) or infection (Ad) of the cells, the cultivation was continued for a defined time of viral vector production, usually 48 to 72 hours. Immediately afterwards, the cell cultures were used for the lysis experiments.

Lysis reagents

In order to adjust the lysis conditions in the whole cell culture while keeping the dilution of the cells through the lysis reagent addition at a minimum, concentrated lysis reagent solutions were prepared. Preferably, lOx concentrated lysis reagent solutions (in some cases lower concentrated) were applied, which is limited by the solubility of the components. To initiate the first lysis step, one volumetric part of lOx lysis reagent solution was added to nine volumetric parts of cell cultures resulting in about 11 % dilution of the viral vector.

The lysis reagent solution applied in the first step of the two-step lysis method can comprise buffer, salt, divalent cation, cryoprotectant and detergent, which is described in more detail below. It should be noted that a nuclease (e.g. San HQ 2.0, ArcticZymes, Norway) can also be added in the first lysis step but is preferably stored and added separately to avoid loss of enzymatic activity by the concentrated lysis reagents.

The lysis reagent solution contains a detergent, such as polysorbate 20, that enables efficient solubilization of cell membranes and thus facilitate lysis. To adjust and stabilize the pH value in the lysate, the lysis reagent contains a buffer component, such as TRIS-hydrochloride for pH 8.0 adjustment. However, a low amount of buffer reagent (e.g. 25 - 140 mM in final lysis composition) should be added to avoid a high acid consumption for the low pH shift in the second lysis step. Another important part of the lysis reagent solution is salt, such as potassium chloride (KCI) or sodium chloride (NaCI), aiming for osmotic cell lysis, suppression of virus aggregation, and precipitation of impurities. In case a nuclease is added to the cell broth, an appropriate divalent cation, such as magnesium chloride (MgCl2), can be part of the lysis reagent solution to enhance nuclease activity. Optionally, a cryoprotectant can be added by the lysis reagent solution to protect virus from degradation during storage, freeze, and thaw. However, in a preferred embodiment the cryoprotectant is added in a later process step, after ultrafiltration and diafiltration prior to freezing, to reduce fluid viscosity and product dilution during the filtration steps.

For reduction of the pH value (acidification) in the second lysis step, a separate solution is used. This solution contains a concentrated acid, such as 2 M acetic acid. The high concentration is applied to reduce the dilution of the virus product during titration. However, it is important to ensure that the type of acid, its concentration and the mixing of the system are selected in such a way that the virus is not damaged during acidification due to localized over acidification.

The two-step lysis approach was compared to in house single step lysis approaches as well as to publicly available single step lysis approaches as a reference standard, which are described in more detail below.

As a reference standard a publicly available buffer recipe and the corresponding lysis method was used in accordance with Fedosyuk et al, 2019. It has been published in e.g., Joe et. al. 2021 (bioRxiv 2021.12.22.473478) and has become a standard in laboratories. Another standard lysis reagent that has been used in this study is a 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol known as Triton X-100 containing lysis reagent ("TRT"). Triton X-100 and its derivatives were commonly used for AAV extraction in the past, however, because its being biohazardous, it has been listed on the REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals) list, which restricts or prohibits their use in the European Union. Single step lysis method

Different cell lysis reagents were tested using suspension-adapted HEK293 cells (Expi293F™, Thermo Fisher Scientific Inc.) that were transiently producing viral vectors, such as AAV2 and Adenovirus. The single-step cell lysis process is shown in Fig. 2 and is initiated by adding the cell lysis reagent to the HEK293 cells. As lysis reagent reference, a "prior art cell lysis reagent" (see e.g. Fedosyuk et al., 2019, Vaccine, Vol. 37, pp. 6951-6961) is used, requiring the addition of a nuclease for optimal performance. Following an incubation period of 0.5-3.5 hours, the next step involves purifying via sterile filtration using low-protein binding filtration units. The collected filtrates are then subjected to analysis using different quantification methods.

Two step lysis method

For investigation and development of the two-step lysis method, the following steps were conducted in all trials, independent of the different viral vectors, reagents, and additives:

The first lysis step was initiated by adding a concentrated cell lysis reagent and in some cases a defined amount of nuclease to the HEK293 suspension cell culture (Expi293F™, Thermo Fisher Scientific Inc.) producing a viral vector of interest. With the addition of the components, the incubation phase starts. During the addition and the incubation phase, the lysis composition was continuously mixed to avoid concentration gradients and keep the biomass (cells and cell debris) into suspension. Additionally, the lysis composition was temperature controlled in a range between 30°C and 37°C. After a defined incubation phase (e.g. 60 min), a second lysis step was initiated by acidification of the cell suspension. At this time point, a large proportion but not the complete cells are lysed and consequently the entire virus has not been released at this stage.

Acidification (the reduction of the pH value) was manually conducted by addition of a concentrated organic acid while the lysis composition continued to be mixed. After reaching a predefined low pH value, a second incubation time starts, which was shorter than the first incubation time (e.g. 5 min).

Finally, the lysis process was finished by sampling and/or clarification of the lysate. Samples for investigation of cell broth characteristics were immediately analysed. In addition, supernatant samples for virus titre and host cell related impurity analytic were generated by centrifuging the biomass down (5,000 xg, 5 min). Samples were stored at -80°C prior to analysis.

Clarification of In order to investigate the influence of the different lysis approach on the subsequent clarification operation, a small-scale filter setup was used. The filter arrangement consisted of a double layer depth filter with average retention rates of 8 pm for the first and 0.8 pm for the second filter layer (SartoClear® Cap DL60, 25 cm², Sartorius Stedim Biotech GmbH) and a 0.2 pm sterile filter (Sartopore® 2 XLG, 17.3 cm²; SartoScale 47, Sartorius Stedim Biotech GmbH). A peristaltic pump (SciLog®, Parker) with inline SU pressure sensors (17525SP-10, Sartorius Stedim Biotech GmbH) in front of each filter step was used for process control. Depth filters were pre-flushed with pure water (50 L/m²) using a volumetric flux of 150 L/m²/h. The filtration was performed in a constant flow mode at 6.3 mL/min (150 L/m²/h) until a pressure of 1.3 bar was reached. To increase mAb recovery, a buffer post-flush (20 L/m²) with the corresponding lx lysis buffer was performed in constant pressure mode with a pressure limit of 1.5 bar. Total filter throughputs were calculated dividing the filtered volume of harvested liquid by the depth filter area.

Functional titer quantification for AAV2

The determination of active AAV2 vector yields is carried out through an AAV2-transduction assay, also known as potency, activity, infectivity testing or assay. This quantitative method serves to measure the potency of AAV2 vectors. Regulatory bodies such as the FDA and Ph. Eur. require potency testing for cellular and gene therapy products, making it an essential aspect of the development process.

The potency test conducted in this study adheres to the FDA requirements for potency tests of cellularand gene therapy products. It is an in vitro assay that assesses the transfer of genetic material from the viral vector to a permissive cell line, followed by the quantification of transgene expression. This robust and reliable assay provides valuable insights into the effectiveness and functionality of the AAV vectors being evaluated.

For the AAV2-Transduction assay, adherent HEK293 cells were seeded in either 24- or 96- well plates and immediately transduced with serially diluted AAV2-filtrates. After a 72 hours incubation period, GFP expression was analyzed using Incucyte® Live-Cell Analysis system (Sartorius Stedim Biotech GmbH).

For capsid titer quantification, the binding rate of serially diluted clarified cell lysates were determined using the Octet® Bio Layer Interferometry platform (Sartorius Stedim Biotech GmbH). AAV samples were loaded for 1800 s on commercially available Octet® AAVX Biosensors (Sartorius Stedim Biotech GmbH). The binding rates were compared to the corresponding commercially available AAV2 and AAV8 standards (Progen).

Adenovirus (Ad) samples were loaded on Octet® ProA Biosensors (Sartorius Stedim Biotech GmbH) which were coated beforehand with Ad-specific antibodies (Abeam). The Ad probe preparation and titer quantification was conducted in accordance with a published protocol by Kruse et al., 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)

Analysis of host cell-related impurities (protein and DNA)

To quantify DNA, a Quant-iT™ PicoGreen™ dsDNA assay-kit (P7589, ThermoFisher Scientific, USA) was used according to the manufacturer's instructions. Samples were diluted with TRIS-EDTA buffer (10 mM TRIS, 1 mM EDTA, 0.1 % SDS) to reach the detection range of 3.13 to 200 ng/mL.

For the determination of HCP impurities, a HEK 293 HCP 3G ELISA kit (F650S, Cygnus Technologies, USA) was used according to the manufacturer's instructions.

A plate reader (infinite® 200, Tecan, Switzerland) was applied in both assays. All samples were analyzed at least in duplicates.

Other quantification methods

Viable cell concentrations (VCC), total cell concentrations (TGC), viability ratios, and average cell diameters were analyzed using an automated Trypan Blue assay-based cell counter system (Cedex HiRes Analyzer, Roche, Germany). For turbidity measurement a nephelometer (TL2350, Hach, Germany) with a small 10 mL cuvette was used.

Example 1: Single step lysis reagent for lysing HEK293 producing AAV2

In Example 1, single-step lysis reagents according to the present disclosure were used for lysing suspension adapted HEK293 cells after cultivation and production of AAV2. Cell lysis was conducted on cells, which were harvested at 300 g for 5 min. lx cell lysis buffer was added to the cell pellets followed by an incubation step for 0.5-3.5 h. Finally, a clarification step is performed using low protein binding filtration units. Examples of tested cell lysis reagents are listed in Table 1. Table 1: Examples of single-step cell lysis reagents

Filterability of the crude lysates

In order to test whether the crude lysates allow filtration, crude lysates of the HEK293 cells (Expi293, Thermo Fisher Scientific) producing AAV2 vectors were sterile filtered through 0.22 pm CA Spin-X filters (Sigma Aldrich). Variables of cell lysis conditions (pH, salt molarity, incubation time and the addition of a detergent) are shown in Fig.2, as well as pictures taken of the spin filters. Shown cell lysis setups are conducted without a nuclease digestion step.

The pictures in Fig.2 highlight that the single-step acidic lysis reagents when added to the cells form lysis compositions that are very easy to filter. Specifically, a clear permeate containing the AAV2 is formed, whereas precipitated impurities such as host cell-relating impurities remain on the filter. In contrast, a lysis reagent pH 8 results in a lysate having high viscosity, necessitating additional nuclease digestion to facilitate the filtration step (see Fig.2 shown for setup N25 without a nuclease digestion step).

Analysis of host cell impurities To determine the host cell-related impurities, an SDS-PAGE analysis of sterile filtered crude lysates of HEK293 (Expi293) cells producing AAV2 vectors under reducing and denaturing conditions was performed. The protein bands on the gel were visualized by Coomassie blue stain. Variables of cell lysis conditions (pH, salt molarity, incubation time and the addition of a detergent) are shown in Table 1 for the single-step cell lysis reagents. M in lanes 1 and 13 stands for PageRuler™ unstained Protein ladder (Thermo Fisher Scientific).

The gel bands shown in Fig. 3 demonstrate that the acidic pH significantly reduces host cell-related impurities compared to alkaline pH lysis reagents. The results thus show that a lower pH is advantageous to increase purity of the viral vector (here AAV2) and thus, a more efficient downstream purification but also higher safety of the viral vector product. Also, the gel bands correlate with the observed better filterability (see Fig. 2) in that the better filtered samples have less host cell-related impurities. In addition, this advantageous property of the acidic lysis reagents according to the preset disclosure were observed consistently for different salt concentrations ranging from 50 to 500 mM, wherein higher salt concentrations appear to contain slightly less host cell-related impurities.

Conclusions

Example 1 demonstrates that the single-step lysis reagents according address the following technical problems by implementing several key improvements: i) Facilitated removal of host cell impurities: the acidic cell lysis reagents aid precipitation and efficient removal of host cell-related impurities due to their low pH, resulting in improved purification and potentially higher quality final product. The removal of host cell proteins has been confirmed through densitometry analysis of SDS-PAGE gels stained with Coomassie blue (see Fig- 3 shown for setups N l, N5 and N9). The acidic reagents enable the precipitation of a majority of protein and nucleic acid impurities during the cell lysis step while maintaining high target molecule yield and functionality. As a result, the final product with higher purity can be potentially obtained with fewer steps required in downstream processing. This contributes to less product loss and more efficient purification. ii) Reduction of AAV vector aggregates: the improved filterability and purity indicate that the acidic cell lysis reagents minimize formation of AAV vector aggregates, enabling easier purification process and likely higher titers. iii) Efficient filtration without requiring additional nuclease digestion (see Fig. 2 shown for setup N2, N5 and N6). Conversely, when a pH 8 reagent is employed for cell lysis, the resulting lysate has high viscosity, necessitating additional nuclease digestion to facilitate the filtration step (see Fig. 2 shown for setup N25 without a nuclease digestion step). iv) Shorter incubation time: the single-step cell lysis reagents enable efficient cell lysis and AAV vector extraction within short time. Shortening the incubation time in the production process has the potential to lower overall production costs. v) Biohazard-free: The reagents are formulated using component that are safe for humans and the environment, eliminating potential biohazard risk associated with their usage, e.g. such as Triton-XIOO.

Overall, the improved viral vector extraction efficiency provided by the single-step lysis reagents of the present disclosure have the potential to significantly impact the manufacturing costs of viral vector-based drugs. By increasing yields and streamlining the production process, these technical and economic advancements have the capacity to make viral vector-based therapies more accessible and affordable to patients in need.

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

In Example 2, different single-step buffer systems and formulations of lysis reagents are tested. Typically, a concentrated lysis reagent was applied, e.g. a 5x or lOx lysis reagent in order to achieve a concentration comparable to the lx lysis reagents. As demonstrated below, the herein disclosed lysis reagents address the technical problems typically encountered in cell lysis, including a low cell lysis efficiency and/or long lysis duration, as well as high AAV vector aggregation, and potential need of physical or mechanical lysis steps (e.g. freeze-thaw cycle(s)).

In Example 2, the lysis reagents were used for lysing a whole cell culture and cells which were pelleted. Specifically, suspension adapted HEK293 cell, post cultivation and production of AAV2, were utilized for this purpose according to the present disclosure. For lysing the whole cell culture, typically a e.g. 5x or lOx concentrated lysis reagent was applied in order to achieve a concentration comparable to the lx lysis reagents in the prior Examples. For the lysis of cell pellets, an equivalent amount of whole cell broth was first subjected to centrifugation at 300 g for 5 min and the supernatant was removed. The resulting cell pellet was then subjected to a lx lysis reagent, such that the concentrations of the components in the cell lysis reagents are comparable between the whole cell broth and the cell pellet. The applied single-step cell lysis reagents are listed below in Table 2. As demonstrated below, the single-step lysis reagents address the technical problems typically encountered in cell lysis, including a low cell lysis efficiency and/or long lysis duration, as well as high AAV vector aggregation, and potential need of physical or mechanical lysis steps (e.g. freeze-thaw cycle(s)). In addition, it is below demonstrated that the lysis reagents are applicable for various sample types, such as a cell broth and cell pellets, allowing consistent results. This is particularly advantageous, as different types of viral vectors may be predominantly intracellularly or extracellularly or both. By allowing applicability for different sample types, the lysis reagents are suitable for intra- and/or extracellular viral vectors.

Table 2: Recipes of single-step cell lysis reagents used in Example 2

AAV2 functional titer in cell broth

The functional AAV2 titer was determined after cell lysis of a whole cell broth and clarification using the transduction assay described above on basis of the Incucyte. For comparison, the "prior art cell lysis reagent" was used as a reference lysis reagent, as well as the Cel Lytic™ M (Cl), Cel Lytic™ MT (C2, both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. The results are shown in Fig. 4.

As visible in Fig. 4, the CelLytic™ M (Cl), CelLytic™ MT (C2) and M-PER (C3) lysis reagents showed a very low functional titer of less than 0.5xl0⁶ TU/mL. Such results were expected, as these lysis reagents are only instructed to be suitable for cell pellets. On the other side, also the "prior art cell lysis reagent" (see N82) resulted in a relatively lowfunctional titer of about 4xl0⁶ TU/mL. The single- step cell lysis reagents of the present disclosure allowed for obtaining functional titers of almost 10⁷ TU/mL.

AAV2 capsid titer in cell broth

The capsid AAV2 titer was determined after cell lysis of a whole cell broth and clarification using Octet® AAVX Biosensors described above. For comparison, the "prior art cell lysis reagent" was used as a reference lysis reagent, as well as the CelLytic™ M (C l), CelLytic™ MT (C2, both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. The results are shown in Fig. 5.

As visible in Fig. 5, the CelLytic™ M (Cl), CelLytic™ MT (C2) and M-PER (C3) lysis reagents did not result in any measurable capsid titer. Such results were expected, as these lysis reagents are only instructed to be suitable for cell pellets. On the other side, also the "prior art cell lysis reagent" (see N82) resulted in a relatively low capsid titer of about 3xlOⁿ capsids/mL. The single-step reagents of the present disclosure allowed for obtaining up to 7xlOⁿ capsids/mL. Overall, the results indicate that the single-step lysis reagents are not only well applicable to cell broth but also lead to increased capsid titers, showing their beneficial effects and more efficient cell lysis.

AAV2 functional titer in cell pellets

The functional AAV2 titer was determined after cell lysis of a cell pellet and clarification using the transduction assay described above on basis of the Incucyte. For comparison, the "prior art cell lysis reagent" was used as a reference lysis reagent, as well as the CelLytic™ M (Cl), CelLytic™ MT (C2, both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. The results are shown in Fig.6.

As visible in Fig. 6, the CelLytic™ M (Cl) and M-PER (C3) lysis reagents, as well as the "prior art cell lysis reagent" (see N98) showed a very low functional titer of less than 0.5xl0⁶ TU/mL. Only the CelLytic™ MT (C2) yielded a measurably functional titer of about 4xl0⁶ TU/mL. The single-step reagents of the present disclosure allowed for obtaining functional titers of almost 6xl0⁶ TU/mL.

AAV2 capsid titer in cell pellet

The capsid AAV2 titer was determined after cell lysis of a cell pellet and clarification using Octet® AAVX Biosensors described above. For comparison, the "prior art cell lysis reagent" was used as a reference lysis reagent, as well as the CelLytic™ M (Cl), CelLytic™ MT (C2, both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. The results are shown in Fig. 7.

As visible in Fig. 7, the CelLytic™ M (Cl) and M-PER (C3) lysis reagents, as well as the "prior art cell lysis reagent" (see N98) did not result in any measurable capsid titer. Only the CelLytic™ MT (C2) yielded a measurably capsid titer of about 6xlOⁿ capsids/mL. The single-step cell lysis reagents of the present disclosure allowed for obtaining up to 7.5xlOⁿ capsids/mL.

Overall, the results indicate that the single-step lysis reagents according to the present disclosure are not only well applicable to cell pellets but also lead to increased capsid and functional titers, showing their beneficial effects and more efficient cell lysis.

Beneficial but non-essential components

Additionally, the data depicted in Fig.4, Fig.5, Fig.6 and Fig.7 indicatethat thefunctional and capsid titers obtained with single-step lysis reagents lacking sucrose, i.e. no cryo-protectant (see N80, N81, N96 and N97) are essentially comparable to those achieved with its inclusion (see N78, N79, N94 and N95). These results highlight that the cryo-protectant such as a such, e.g. sucrose, are only optionally added to the single-step lysis reagents according to the present disclosure. Specifically, these are added in order to improve the storability and freezing of the viral vector containing compositions after lysis and/or purification. However, the cryo-protectant does not need to be added for cell lysis. As an optional compound of the lysis reagent, it can be advantageous in order to avoid a subsequent step (e.g. after cell lysis and/or purification) of adding a cryo-protectant, such as a sugar, e.g. sucrose.

Furthermore, concerning the detergent, polysorbate 20, it was observed that depending on the lysis conditions for both whole culture and cell pellet lysis, the inclusion of polysorbate 20 resulted in up to 30% higher capsid and functional titers. An additional advantage of polysorbate 20 is its ability to prevent cavitation effects during subsequent downstream processes like filtration, thereby reducing the risk of product loss.

Filterability and pH of lysis compositions according to the present disclosure lOx concentrated reagents were initially applied to the whole cell culture to achieve a final concentration of the lx lysis reagent. Subsequently, a specific nuclease, as indicated in the Table 2, was added, and the mixture was incubated for one hour. After incubation, the filterability and pH of the crude lysis compositions were evaluated. The results are shown below in Table 3:

Table 3: Evaluation of filterability and pH of crude lysis compositions from example 2. Filterability was assessed qualitatively and categorized into four grades: A for excellent filterability, B for good filterability, C for fair filterability and D for moderate filterability. "Nuc." stands for nuclease, "n.a." stands for not applicable, as the composition of commercially available lysis reagents are unknown.

Table 3 illustrates how filterability of crude lysis compositions can vary depending on the cell lysis conditions. These variations are linked to different cell lysis efficiencies and alterations in host cell impurities. The acidic lysis reagents N70 to N77 led to excellent filterability and were thus categorized into the group A. In contrast, alkaline lysis reagents yielded good to moderate filterability. Notably, N78, identified as the most efficient lysis reagent in this disclosure (Fig. 4 and Fig. 5), has shown moderate filterability.

Conclusions Example 1 demonstrates that the lysis reagents according to the present disclosure led consistently to high capsid and functional titers for different types of cell culture samples. Specifically, it was shown that the single-step lysis reagents of the present disclosure can be used for a whole cell broth and cell pellets. Comparison of the cell lysis efficiencies for the best performing alkaline reagent yields following results: - Basic reagent: 100% (cell broth) and 100% (cell pellet)

Acidic reagent: -80% (cell broth) and -45% (cell pellet) prior art reagent: -45% (cell broth) and -0% (cell pellet)

- CelLytic™ MT: -0% (cell broth) and -80% (cell pellet) Hence, the cells can either undergo a step of separation from the surrounding liquid (e.g. by centrifugation/sedimentation and removal of the supernatant) or the cell broth without any processing step can be used. This is particularly advantageous, as the cell lysis reagents of the present disclosure can be flexibly used with broad applicability. Also, such application allows for using the cell lysis reagents according to the present disclosure in situ, i.e. the cell lysis reagent can be added directly to the culture container, e.g. bioreactor or shake flask, after production of the viral vector, e.g. AAV.

Example 3: Lysis reagents are effective and non-toxic

In Example 3, the efficacy of a Triton X-100 containing lysis reagent and the single-step lysis reagents were evaluated for whole cell culture lysis. While Triton X-100 containing reagents are known for effectiveness and were widely used historically for cell lysis and intracellular product extraction. However, their usage has become restricted, as Triton X-100 is listed by the European Union in the REACH list of particularly substances that are of very high concern, i.e. substances that should be avoided for use to protect human health and the environment from the risks that can be posed by chemicals. For a whole culture lysis, suspension adapted HEK293 cell, post cultivation and production of AAV2, were utilized for this purpose according to the present disclosure. Typically, a e.g. 5x or lOx concentrated lysis reagent was applied in order to achieve a concentration comparable to the lx lysis reagents in the prior Examples. The applied cell lysis reagents are listed below in Table 4.

Table 4: Recipes of cell lysis reagents used in Example 3

AAV2 functional titer in cell broth for different lysis reagents

The functional AAV2 titer was determined after cell lysis of a whole cell broth and clarification using the transduction assay described above on basis of the Incucyte. For comparison, the "prior art cell lysis reagent" was used as a reference lysis reagent, as well as the Triton X-100 containing lysis reagent. The results are shown in Fig. 8.

As shown in Fig. 8, the single-step cell lysis reagents allowed for obtaining up to 9xl0⁶ TU/ml, while the "prior art cell lysis reagent" (see N82) resulted in 4xl0⁶ TU/ml. Triton X-100 containing cell lysis reagent (see TNT), whose use is restricted due to the cytotoxicity mentioned earlier and further exemplified later, being up to 7xl0⁶ TU/ml, demonstrated equivalent efficiency to the reagents disclosed in this example. Hence, the results indicate that the single-step lysis reagents allow for highly efficient cell lysis and increased yields of functional titer without containing any cytotoxic components.

AAV2 capsid titer in cell broth for different lysis reagents

The capsid AAV2 titer was determined after cell lysis of a whole cell broth and clarification using the Octet® AAVX Biosensors described above. For comparison, the "prior art cell lysis reagent" was used as a reference lysis reagent, as well as the Triton X-100 containing lysis reagent. The results are shown in Fig. 9.

As shown in Fig. 9, the single-step cell lysis reagents allowed forobtaining of up to 9xlOⁿ capsids/ml, while the "prior art cell lysis reagent" (see N82) resulted in almost 4xlOⁿ capsids/ml. The commonly used Triton X-100 containing cell lysis reagent (see TNT), whose use is restricted due to the cytotoxicity mentioned earlier and further exemplified later, being about 8xlOⁿ capsids/ml demonstrated equivalent efficiency to the reagents disclosed in this example. Hence, the results indicate that the single-step lysis reagents allow for highly efficient cell lysis and increased yields of capsid titer without containing any cytotoxic components. Toxicity tests

To investigate potential toxic effects of Triton X-100 compared to a detergent used in the lysis reagents according to the present disclosure. AAV crude cell lysis compositions, derived from using Triton X-100 or polysorbate 20 containing lysis reagents as outlined in Table 4, were initially at lx concentration in the final crude lysate after undergoing a sterile filtration step. These were then further diluted in ranges from 1:10 to 1:320. The resulting diluted samples were applied to adherent HEK293 cells following the transfection assay protocol previously described. Subsequently, the phase contrast confluence of these cells was analyzed using Incucyte® Live-Cell Analysis system The results are shown in Fig. 10.

The acquired phase contrast images in Fig. 10 show that the cells are affected dramatically by the presence of Triton X-100 (see TRT), leading to a reduction of phase contrast confluence from more than 90% to 12%. In contrast, even at the highest concentration of Tween 20, regardless of the buffer component being acidic or alkaline (see N70A, N74A or N78A), there was no reduction in cell confluence remaining high at 94%. The results demonstrate the strong cytotoxic effect of Triton X- 100 on cells.

Conclusions

Example 3 demonstrates that the single-step lysis reagents led consistently to high capsid and functional titers for different types of cell broth and allows for obtaining similar results (or in some cases improvements) over a Triton X-100 based lysis reagent. On the other side, Triton X-100-based lysis reagents are toxic and thus pose subsequent problems when purifying the viral vector but also for waste management. On the other side, the herein used polysorbate-based lysis reagents do not raise such concerns, rendering these advantageous for safety and waste management reasons.

Example 4: Comparison of two-step and single-step lysis for AAV2

In Example 4, the method for releasing a viral vector from a cell culture according to the present disclosure is compared to a single-step lysis, wherein in particular no second step of acidifying the lysis composition is performed. As found above in Examples 1 to 3, the alkaline lysis reagents achieved high capsid and functional AAV titers but at the same time led to increased contents of host cell-related impurities. On the other hand, acidic lysis reagents tested above were able to effectively remove host cell-related impurities and improve filterability in downstream purification but were not as efficient in releasing highest viral vector capsid and functional titers. However, the acidic lysis reagents were in most cases still more effective than the tested reference lysis reagents. - T2. -

Nevertheless, the inventors decided to follow a different approach in combining the beneficial aspects of the two different single-step lysis approaches (Examples 1 and 2) into two separate steps with the object to maximize viral vector yield but keeping host cell-related impurities at a low level. As demonstrated below this process advantageously led to consistently high viral vector capsid and functional titers and low contents of host cell-related impurities.

As material for lysis HEK293 cell from a fed-batch process producing AAV2 with a viable cell density of 1.7xl0⁶ cells/mL and an end viability of 59% (after AAV2 production) was used. The lysis reagents were added to the fed-batch culture of about 320 mL and the lysis composition was incubated for 60 min at 30 to 37°C. For the herein disclosed method for releasing a viral vector from a cell culture in two steps, a step of acidifying the lysis composition was performed subsequently by adding a 2 M acetate solution to adjust the pH to 4. In order to test the effect of the lysis approaches on the subsequent clarification operation, the lysates were clarified using a depth filter followed by a sterile filter as described above. Afterwards, the sample was quantified for the capsid titer, functional titer and host cell protein and DNA content as described above. The tested reagents are disclosed in Table 5. As nuclease, 50 U/mL San HQ 2.0 salt tolerant nuclease (ArcticZymes, Norway) was used for the single step pH 8 and the two-step lysis approach. Since the nuclease is not stable at pH 4 and quickly denatured, it was not added to single step pH 4 approach.

Table 5: Overview of single and two step lysis approaches including the used composition and whether a nuclease was added or not. In the single step pH 4 and two step approach the pH was adjusted to pH 4 using 2 M acetate solution after starting of the lysis.

Results after lysis

After lysis as described above (see in particular Table 5), the total cell concentrations (TOO), cell viability, turbidity, host cell protein (HOP), DNA, AAV capsid titer and functional AAVtiter of the lysed cell broth were measured as described above. The results of these measurements can be found in Table d below. Table6: Results of total cell concentrations (TCC), cell viability ratio (Viab.), turbidity, host cell protein (HCP), DNA, AAV capsid titer and functional AAV titer after lysis.

The results in Tabled show that the single step lysis using the alkaline lysis reagent (pH 8) still resulted in viable cells (63%) indicating that the cells were insufficiently lysed, which is likely due to the short incubation time of 60 min, whereas longer incubation times e.g. of 120 min resulted in complete lysis. On the other hand, the single step pH 4 lysis but also the two-step approach resulted in 0% viability indicating an essentially complete lysis of the cells. The lysis of the cells not only cause the release AAV, but also significantly increases the HCP content and DNA content. However, the change of pH and conductivity in the lysate cause potentially precipitation of at least some impurities which facilitating their subsequent removal, e.g. by filtration of centrifugation. The precipitation is indicated by the high turbidity of 115 NTU compared to 50 and 39 NTU for the single step pH 8 and two-step approach, respectively. Finally, the measured capsid and functional AAV titer for the single-step pH 8 lysis were very low compared to the single-step pH 4 and two-step lysis, which is likely due to the insufficient lysis achieved within the 60 min. On the other hand, the two-step lysis approach led to further enhancements over the single-step pH 4 lysis in that higher capsid and functional titers were achieved - despite a further dilution of 25 % due to the pH 4 adjustment. Without this dilution, the titers were even higher. Hence, the two-step approach allowed for complete cell lysis and highest AAV yields (capsid and functional titer), showing the advantages over the single-step approaches. Moreover, the very short time of about 70 min for lysis (60 min alkaline cell lysis and 10 min acidification) is very efficient compared to commonly applied lysis protocols in the art, which typically require a few hours (e.g. 4 to 6 hours).

Clarification Results

The three different lysates were clarified using an industrial relevant and scalable filtration setup as described above. The filtration performance and final filtrate composition was measured and compared. Specifically, the DNA remove, HCP removal and functional AAV recovery was measured for the filtration performance. For the final filtrate composition, the HCP content, relative HCP content (related to functional AAV), DNA content, relative DNA content (related to functional AAV), as well as the total number of functional AAVs was measured. The results are shown in Table 7 and

Tabled below.

Table 7: Filtration performance of the lysed cell broth.

*ongoing release of virus throughout filtration lead to artificial higher functional recoveries.

During filtration, no filter blockage was observed, and a low final pressure was measured. The low pressure indicates a depth filter capacity of more than 150 L/m². The HCP and DNA removal for the single step pH 4 and the two-step approach were high, whereas the single step pH 8 approach resulted in only low HCP removal. Furthermore, the functional AAV recovery for single step pH 4 and the two-step approach was at 100%. Note that the functional AAV recovery for single step pH 8 is artificially increased and to be considered as an artifact, as the lysis continued throughout the filtration such that more functional AAV was released. This is to be expected that the single step lysis at pH8 requires at the tested conditions longer, particularly 2 hours or more.

Tabled: Final filtrate composition including relative values for HCP and DNA related to the functional AAV concentrations.

After clarification, low concentrations of residual DNA impurities were detected with all three tested lysis approaches. The single step pH 4 lysis approach achieved the highest DNA depletion, however, the values achieved with the two-step approach are also at a low level that they can be potentially easy removed by a subsequent polishing step. - 1 -

The other measurements for the final filtrate composition show the advantages of the two-step lysis approach compared to the single-step approaches. Specifically, highest functional AAV titers were obtained while keeping the host cell related impurities in particular the HCP content low. The two- step approach led to a doubling in functional titer compared to single step pH 8 lysis and a purer composition in relative HCP content compared to single step pH 8. Hence, the two-step lysis approach led to fast (< 70 min) and complete (100% recovery) lysis, highest functional titer, and low impurity levels due to precipitation of DNA and HCP's.

Conclusions

Example 4 demonstrates that the two-step lysis approach disclosed herein achieved high functional viral vector yields compared to single step methods (+50% compared to pH 4 single step lysis) with low contents of host cell related impurities, including digestion and subsequent precipitation of DNA results in more than 60% reduction and precipitation of HCP results in an approx. 50% lower HCP impurity level compared to pH 8 single step lysis. In addition, the two-step approach ensures robust and complete lysis of cells in short time (< 70 min compared to conventional > 120 min). Moreover, the precipitation of impurities facilitates their removal which is indicated by significantly lower final pressures over the filter setup for the pH 4 lysis approaches. Therefore, acidification has a high potential to enable higher filter capacities compared to high-salt lysis with pH8 (data not shown). Therefore, the two-step lysis approach overall leads to a highly efficient lysis yielding more and purer viral vector.

Example 5: Two-step lysis without cryo-protectant

In Example 5, the two-step lysis approach of Example 4 was further tested. Specifically, it was tested whether the cryo-protectant in the lysis reagent is required. The inventors hypothesized that sucrose is not required, as this compound is primarily added in order to stabilize the viral vectors more for freezing. Hence, sucrose can also be added prior to freezing but it has potentially minor effects for cell lysis. Therefore, a condition, wherein 0.5% (v/v) Tween20, 25 mM Tris/HCI, 400 mM KCI, 5 mM MgCI2 and a salt tolerant nuclease (San HQ (50 U/mL)) at pH 8 was tested. The lysis reagent was added as a 5x concentrate to the cell broth (TCC 2.7xl0⁶ c/mL, 78% viability) and 60 min after lysis the pH was adjusted to pH 4 using 2 M acetate buffer for 5 min. Afterwards, filtration was performed.

The two-step lysis approach without a cryo-protectant in the lysis reagent resulted in essentially complete lysis by measured 0% viability. Large flocs were obtained during mixing indicating precipitation of the host cell-related impurities (HCP, DNA), such that these could be readily filtered out. Specifically, a filter capacity of 106 L/m² was measured and 82% DNA removal and 30% HCP removal. The composition of the final concentrate was 5.2E+03 pg/L HCP, 2.3E+02 pg/L DNA and 1.8E+06 Vp/mLfunctional AAV. The high functional AAV titer and low measured impurities indicate that the sucrose is not required for lysis and can be added at a later step as cryo-protectant before freezing in order to render the viral vector more stable for freezing.

Example 6: Comparison of two-step and single-step lysis for AAV8

In Example 6, HEK293 cells producing AAV8 were lysed with the two-step lysis approach. As reference further single step protocols were used. The example demonstrates that the two-step lysis approach is applicable to another AAV serotype (compared to Examples 1 to 5 wherein AAV2 was produced). The two-step lysis approach yielded high AAV8 titers while keeping the host cell related impurities at a low level. Hence, the two-step cell lysis approach is widely applicable and efficient for obtaining large amounts of pure viral vectors.

For this example, HEK293 cells were transiently transfected with plasmids to produce AAV8 with a GFP coding sequence as transgene. After production of the AAV8, 9 mL of the cell broth were mixed with the respective reagents as described in Table 9. As another reference, the cell pellet was lysed. Since AAV8 is mostly secreted into the culture media and only few viral vectors remain intracellularly, this example demonstrates that conventional lysis approaches applicable for cell pellets would not allow obtaining sufficient amounts of AAV8.

Table 9: Lysis conditions for obtaining AAV8. After lysis, samples were analysed for TCC, cell viability ratio and cell diameter, as well as the pH was measured. The crude lysed samples were then centrifuged (5,000 xg, 5 min) and DNA, HCP, and AAV8 capsid titer were measured. The results are disclosed in Table 10.

Table 10: Results of total cell concentrations (TCC), cell viability ratio (Viab.), cell diameter, and pH of the crude lysed samples, as well as host cell protein (HCP), DNA, and AAV8 capsid titer after lysis and clarification. *pH was adjusted after incubation to pH 4.

The results in Table 10 show that most AAV8 is present in the supernatant, as the capsid titers for the cell broth (a, b) are considerably higher than after lysis of the cell pellet (i). Without lysis the viability for the cell broth references almost did not change during the experiment (initially 69.6% for a and after the 60 min 68% for b). For the single-step lysis approach at pH 8 (see d) viable cells were still measured after the 60 min incubation (1.2%) indicating incomplete cell lysis. Similarly, using a lysis reagent without a detergent only resulted in insufficient cell lysis (see g) with 13.6% viable cells after lysis. All other conditions resulted in essentially in full lysis. The pH measured in the compositions is around pH 8 (d, e, f, d, h) or around pH 4 (c, i). For the two step lysis approaches (e, f, g, h) acetic acid was added to adjust the pH at around 4.

Due to high AAV8 capsid titers in the non-lysed cell culture supernatants (see a and b), only a small increase in the AAV8 titer was achieved by lysing the cells. Nevertheless, the model process can be used to obtain some information about the lysis efficiency of the cells and the release or reduction of impurities. Overall, highest AAV8 capsid titers were measured for single step, pH 8 lysis, however, along with highest impurity levels (1.14xl0⁴ pg/L HOP and 9.36xl0² pg/L DNA). On the other hand, the single step, pH 4 lysis yielded low AAV8 levels close to the reference materials (see c compared to a, b) but low impurity levels for DNA and HOP. The two-step lysis approaches overall yielded higher AAV8 capsid titers (ranging from 2.98 to 3.13xl0¹⁰ capsids/mL) compared to single-step, pH 4 but lower impurity levels compared to single-step, pH 4 (see e, f, g, h).

For better comparability of the two-stage lysis approaches using different lysis reagent reagents in the first lysis step, an excerpt of the data from Table 10 (e, f, g, h) is shown in Fig. 11. Again, Fig. 11, [A] demonstrates that a detergent is required for fast and complete lysis. Furthermore, it was shown that nuclease is not required for a fast cell lysis but has a positive effect on the reduction of DNA impurities despite subsequent pH 4 precipitation (Fig. 11, [A, B]). As a negative control, the DNA concentration was approximately twice as high without nuclease (f) compared to the approaches with addition of nuclease (e, h). Another aspect shown in Fig. 11, [A,B,C] is that not adding the cryoprotectant sucrose with the lysis reagent (h) has no disadvantages for the lysis and can therefore be performed at a later step.

In this Example, the conditions for the two-step cell lysis approach were further varied. Specifically, the cell broth was mixed with a lOx lysis reagent comprising buffer, salt, divalent cation, cryoprotectant and detergent (e) as well as a nuclease compared to a condition without nuclease (f), without detergent (g) and without a cryoprotectant (h). Results show that it is in principle all conditions yielded high capsid titers and low impurities. Hence, different lysis compositions are suitable for the initial lysis step. Noteworthy, the presence of the detergent appears to be required in order to obtain a full cell lysis and as such higher yields of viral vector, especially for viral vectors that are present intracellularly. On the other hand, the addition of a nuclease facilitates reduction of DNA and thus allows for obtaining viral vectors with lower DNA impurities. The presence or absence of the cryo-protectant (sucrose) did not have an effect on the yield or purity supporting that this is an optional compound for cell lysis.

In conclusion, the example demonstrates applicability of the two-step cell lysis for different AAV serotypes, indicating applicability for different viral vectors. The two-step cell lysis resulted in high yields of viral vectors while keeping the host cell related impurities at a low level.

Example 7: Comparison of two-step and single-step lysis for Adenovirus In Example 7, different lysis protocols were applied to cell broth producing adenovirus (Ad) and various parameters were measured. The example demonstrates that the two-step lysis approach is applicable to Ad-producing host cells and achieves consistently high Ad yields and keeps the host cell related impurities at a low level.

For this Example, a cell broth of HEK293 cells producing Adenovirus was used having a TCC of 25.6xl0⁶ cells/mLwith a viability of 80.4%. For each lysis condition, 27 mL of the cell broth were lysed with 3 mL of a lOx lysis reagent in 50 mL tubes and incubated at 37 °C inside an incubator. Mixing was achieved by placing the tubes on a disc rotator. After 90 min some lysis conditions require addition of an acidifying reagent or salt. After 120 min sampling was done and different properties were measured. The lysis conditions as disclosed in Table 11 were applied.

Table 11: Lysis conditions applied to cell broth for yielding Adenovirus.

Chanqes of cell broth properties by lysis

The TCC and viability was measured after performing the lysis as described above. The results are shown in Fig. 12. The viability is generally low except for the reference samples SO, SI, as well as S7 wherein a single step lysis at pH 4 was performed without the addition of a nuclease. A slight viability was still measured for S5 wherein the samples were lysed by a single step, using an alkaline lysis reagent. In contrast, the two-step cell lysis approach disclosed herein resulted in efficient cell lysis without any detectable viability remaining (see S9).

Impact on capsid titer

Furthermore, the capsid titer of the Ad was measured for the crude lysed sample, as well as after 0.2 pm filtration of the samples. As shown in Fig. 13, overall high capsid titers were measured, except for S7, wherein samples were insufficiently lysed. Slightly lower capsid titers were obtained for the single step cell lysis, pH 4 condition 2 (see S8). For all other lysis methods, capsid titers between 1.5 to 2 x 10¹² capsids/mL were measured. Hence, the two-step cell lysis approach yielded Ad capsid titers comparable to conventional methods used in the art (see S2 and Sil). It should be noted, that the lysis time of 2 h was equal for all samples, however, as indicated from examples 3 and 4 it can be potentially shortened for the two-step lysis approach (S9).

Impact on DNA and HCP impurities

Furthermore, the impurities (HCP and DNA) of the Ad were measured for the crude lysed sample, as well as after filtration. In Figs. 14 a nd 15 the impurities were set in relation to achieved titers. As shown in Figs. 14 and 15, the two-step lysis approach yielded lowest HCP contents (see S9) - only achieved by S8, which, however, resulted in lower capsid titers (see Fig. 13). Also, the DNA content was at a relatively low level for the two-step approach (see S9) similar to S2, S3, S8 and Sil. Only the condition, wherein the inhouse lysis reagent was doped with pDADMAC (polydiallyldimethylammonium chloride; substitution of nuclease by cationic flocculant) lower DNA levels were obtained (see S4). However, in the two-step approach hazardous substances such as Triton X-100 and pDADMAC are avoided, which is advantageous, as such compounds are a hazard to the environment and should be avoided to be used for pharmaceutical composition, such as viral vector formulations envisioned for gene therapy. Conclusions

Example 7 demonstrates that the two-step lysis approach is applicable for lysing cells producing adenovirus, highlighting the broad applicability for different viral vectors. Hence, the two-step lysis approach is very efficient in cell lysis the lysis time can be potentially further decreased. Furthermore, the results highlight again that the two-step lysis approach provides the best compromise between obtaining high viral vector yields and minimizing host cell related impurities. Specifically, a high Adenovirus capsid titer was obtained while simultaneously keeping the HCP but also DNA levels at a low level.

Example 8: Implementation of the two-step cell lysis in a bioprocess

In Example 8, a preferred setup and workflow of the two-stage lysis is described in a more detail. The process setup is shown in Fig. 1 and aims to simplify the workflow, increase safety, and enhance product yields.

In the first step, concentrated (e.g. lOx) high salt, alkaline lysis buffer with a detergent and salt tolerant nuclease is added to the bioreactor to initiate cell lysis. Even if the bioreactor has reached its maximum working volume at the end of cultivation, the lOx concentrated lysis reagents (approximately 11% additional volume) can be added to the cell culture by stopping the culture aeration and using the bioreactor headspace (usually additional 25% of the working volume) for this purpose. In addition, the bioreactor agitation function is used to mix all components and keep the cells in suspension. Moreover, the temperature control at 37°C remains active to achieve maximum nuclease activity. The adjusted lysis conditions are maintained for approximately 1 hour. Afterwards, a large fraction of the cells are lysed, so that only a small fraction of cells (< 20%) is still viable. At this point, the pH value is abruptly reduced to pH 4 by titration with a concentrated acidifying reagent, such as an acid, e.g. 2 M acetic acid. For this purpose, the pH probe and pH controller which is already used during the cultivation can also be used for the titration. In addition, the acidifying reagent can be added by using the pH dosing pump of the bioreactor.

In this second step, the low pH causes a loss in nuclease activity but also cause immediate lysis of the remained cells. Moreover, the low pH results in an almost immediate precipitation of impurities, which are predominantly host cell-related impurities, e.g. host cell proteins or host cell nucleic acids, and cell debris. Therefore, the subsequent clarification process can be started shortly, such as after a few minutes at pH 4. Alternatively, inline addition and mixing of acid can be used when the cell broth is transferred to the filter stages for clarification. The precipitation of impurities also has the advantage that higher filter capacities can be achieved and consequently less filter material is required in the clarification step.

After lysis and clarification, it is recommended to perform the next capture step directly afterwards to avoid degradation of the viral vector. Prior to this, it might be necessary to perform a tangential flow filtration (TFF) step to adapt the puffer conditions for the capture chromatography, remove some of the dissolved impurities, and concentrate the product. In a streamlined setup, TFF can be performed in a single-pass (SP) mode. By removing most of the remaining contaminants, such as proteases, in the subsequent capture step the viral vector is stabilized. If the product needs to be stored after the capture, e.g. by freezing it at -80°C, a further adaption of the buffer conditions is recommended. This includes the adjustment/ neutralization of the pH value and the addition of a cryoprotectant such as sucrose. As an alternative to storage or after storage, the viral vector product can be further processed by polishing chromatography and transferred to the fill and finish step.

Conclusions

The Examples above demonstrate that the methods of the present disclosure are very efficient in lysing cells to obtain increased capsid and functional viral vector titers and reducing host cell-related impurities. Notably, the achieved functional virus yield of the two-step approach is higher compared to the known single step methods (see e.g. Example 3: +50% compared to pH4 high salt lyse w/o nuclease). Also, the two-step lysis ensures robust and complete lysis of cells. The digestion and subsequent precipitation of DNA results in more than 60% reduction. In addition, the lysis time can be shortened by almost 50% (< 70 min compared to conventional 120 min+), improving efficiency. Precipitation of HCP results in an approx. 50% lower HCP impurity level compared to pH 8 high salt lysis. In addition, the precipitation of impurities by acidification facilitates their removal by filtration indicated by significantly lower filtration pressure compared to high-salt lysis with pH 8. Also, by using a lysis reagent that avoids toxic compounds, such as Triton X-100, the detergent containing lysis reagents according to the present disclosure comprising a polysorbate detergent, such as Tween 20, toxic side effects and improved waste management are achieved. Finally, examples are provided that show implementation of the two-step lysis approach into a bioprocess, allowing for simplified release of a viral vector from a cell culture and exact process control.

Claims

(a) providing a lysis reagent;

2. The method according to claim 1, wherein in step (c) the cell culture is partially lysed and/or in step (d) contacting the lysis composition with an acidifying reagent lyses the cell culture essentially completely.

3. The method according to claim 1 or 2, wherein the cell culture is a cell broth or cell pellet, preferably a cell broth, and/or wherein the cell culture is not required to be separated from the surrounding liquid prior to contacting the cell culture with the lysis reagent in step (b).

4. The method according to one or more of claims 1 to 3, wherein the method has one or more of the following characteristics: it does not comprise a lysis step based on mechanical or physical lysis, and/or it does not comprise a step of repeated cycles of freezing and thawing for cell lysis or a step of microfluidization 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 conducted in a bioreactor, preferably at least two of these steps are conducted in a bioreactor, most preferably, all the steps (b), (c), and (d) are conducted in a bioreactor, optionally, wherein the bioreactor has one or more of the following characteristics: the bioreactor agitation facilitates mixing in these steps; the bioreactor allows for temperature control and/or pH control; and/or the bioreactor allows for pH adjustment, preferably by being configured to allowfor contacting the lysis composition with an acidifying reagent in step (d).

6. The method according to one or more of claims 1 to 5, wherein in step (d): the acidifying reagent lowers the pH to an acidic pH, preferably a pH of less than 6.0, more preferably less than 5.0, most preferably less than 4.5; and/or the acidifying reagent 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.

7. The method according to one or more of claims 1 to 6, wherein the acidifying reagent comprises an acid, preferably an organic acid, more preferably a carboxylic acid, 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: a pH of more than 7.0, preferably more than 7.5, most preferably more 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 a pH selected from the range of 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 lysis reagent comprises a detergent, preferably a non-ionic detergent, more preferably a non-ionic detergent that is not classified as toxic and/or hazardous to the environment.

10. The method according to 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 detergent has a concentration of 0.1% (v/v) to 20% (v/v), preferably selected from the following ranges: i) 0.1% (v/v) to 2% (v/v) for a lx lysis reagent; ii) 1% (v/v) to 5% (v/v) for a 5x lysis reagent; or iii) 4% (v/v) to 10% (v/v) for a lOx lysis reagent.

11. The method according to one or more of claims 1 to 10, wherein the method further comprises providing a nuclease which 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 lysis reagent is a detergent containing alkaline lysis reagent, preferably selected from: i. lx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v); ii. 5x alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.3 M to 1.5 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.3 M to 1.5 M; a detergent, preferably a polysorbate, having a concentration selected from 1% (v/v) to 5% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 5 mM to 15 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 10% (v/v) to 35% (w/v); or iii. preferably, lOx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 1 M to 3 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.8 M to 5 M or 1 M to 3 M; a detergent, preferably a polysorbate, having a concentration selected from 4% (v/v) to 10% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 15 mM to 50 mM; and optionally, a cryo-protectant, preferably a sugar, having 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 lysis composition has one or more of the following characteristics: it has a pH of more than 7.2, preferably more than 7.5, most preferably more than 7.8 or 8.0; it has a pH selected from the range of 7.2 to 10.0, preferably 7.5 to 9.5; and/or it comprises the compounds in essentially the concentrations of a lx lysis reagent, preferably essentially the concentrations according to claim 11, optionally the lysis composition has the following characteristics:

I. the lysis composition has a pH of more than 7.2, preferably more than 7.5, most preferably more than 7.8 or 8.0, and comprises: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or l,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having 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: incubating step (c) comprises incubating for less than 6 hours, preferably less than 5 hours or less than 4 hours, more preferably for less than 3 hours, such as for 2 hours, 1 hour, or 0.5 hour; and/or incubating for a time selected from the range of 15 min to 300 min, preferably 20 min to 250 min, 25 min to 200 min, or more preferably 30 min to 150 min or 30 to 100 min, most preferably 30 min to 90 min or 45 min to 75 min; and/or wherein the time required for conducting steps (c) and (d) is less than 360 min, preferably less than 300 min or less than 240 min, more preferably for less than 180 min or less than 120 min, most preferably less than 100 min, such as 90 min, 80 min or 70 min or less; and/or is a time selected from the range of 15 min to 360 min, preferably 20 min to 300 min, 25 min to 240 min, or more preferably 30 min to 180 min or 40 to 120 min, most preferably 45 to 90 min.

15. A method for producing a viral vector, the method comprising following steps: x.l providing a cell culture capable of producing a viral vector; x.2 culturing the cell culture to produce a viral vector; x.3 releasing the viral vector from the cell culture according to one or more of claims 1 to 14; and x.4 optionally, purifying the released viral vector.

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

17. A kit for releasing a viral vector from a cell culture, the kit comprising:

(i) a lysis reagent; and

(ii) an acidifying reagent, preferably the acidifying reagent comprises an acid, more preferably an organic acid, most preferably a carboxylic acid, such as acetic acid or citric acid; optionally wherein lysis reagent is a detergent containing alkaline lysis reagent, preferably selected from: i. lx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.05 M to 1 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.05 M to 1 M; a detergent, preferably a polysorbate, having a concentration selected from 0.1% (v/v) to 2% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 0.5 mM to 5 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 1% (w/v) to 10% (w/v); ii. 5x alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 0.3 M to 1.5 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.3 M to 1.5 M; a detergent, preferably a polysorbate, having a concentration selected from 1% (v/v) to 5% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 5 mM to 15 mM; and optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 10% (v/v) to 35% (w/v); or iii. preferably, lOx alkaline lysis reagent having a pH of more than 7.0, preferably more than 7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) buffer or 1,3- bis(tris(hydroxymethyl)methylamino)propane (BTP) buffer, having a concentration selected from 1 M to 3 M; a salt, preferably potassium chloride or sodium chloride, having a concentration selected from 0.8 M to 5 M or 1 M to 3 M; a detergent, preferably a polysorbate, having a concentration selected from 4% (v/v) to 10% (v/v); optionally, a divalent cation, preferably Mg2+, having a concentration selected from 15 mM to 50 mM; and - optionally, a cryo-protectant, preferably a sugar, having a concentration selected from 20% (w/v) to 60% (w/v).