WO2025190864A1

WO2025190864A1 - Improved cell lysis methods, reagents, and kits for viral vector release and production

Info

Publication number: WO2025190864A1
Application number: PCT/EP2025/056461
Authority: WO
Inventors: Nazguel WAGNER; Martin Saballus; Julia Niemann
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 relates to a method for releasing a viral vector from a cell culture, wherein a cell of the cell culture is not required to be separated from the surrounding liquid prior to contacting the cell culture with the lysis reagent. The present invention further relates to a method for producing a viral vector inter alia comprising releasing the viral vector from a cell culture according to disclosed method for releasing a viral vector from a cell culture. The present invention further relates to a cell lysis reagent for releasing a viral vector from a cell culture. The present invention further relates to a kit for releasing a viral vector from a cell culture, wherein the kit comprises a container that contains the disclosed cell lysis reagent.

Description

IMPROVED CELL LYSIS METHODS, REAGENTS, AND KITS FOR VIRAL VECTOR RELEASE AND PRODUCTION

TECHNICAL FIELD OF THE INVENTION

The present disclosure generally relates to the field of viral vector production. In particular, the present invention relates to a method for releasing a viral vector from a cell culture, wherein a cell of the cell culture is not required to be separated from the surrounding liquid priorto contacting the cell culture with the lysis reagent. The present invention further relates to a method for producing a viral vector inter alia comprising releasing the viral vector from a cell culture according to disclosed method for releasing a viral vector from a cell culture. The present invention further relates to a cell lysis reagent for releasing a viral vector from a cell culture. The present invention further relates to a kit for releasing a viral vector from a cell culture, wherein the kit comprises a container that contains the disclosed cell lysis reagent.

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 approvals 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 of 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 by improving the release of a viral vector from a cell culture. The method comprises providing a lysis reagent comprising a buffer having a concentration of 0.01 M to 3 M and a salt having a concentration of 0.01 M to 5 M. This is followed by contacting the cell culture with the lysis reagent to generate a lysis composition and incubating the lysis composition such that a viral vector is released from the cell culture, wherein a cell of the cell culture is not required to be separated from the surrounding liquid priorto contacting the cell culture with the lysis reagent in step (b). By not requiring the cell to be separated from the cell culture, the lysis reagent can be directly contacted without a cell separation step, simplifying the cell lysis workflow. The lysis reagent, comprising buffer and salt in the defined concentrations, allows for precise tuning the ionic strength of the lysis composition, ensuring that high amounts of viral vector are released and remain stable. Furthermore, the method can be flexibly applied to a wide range of samples, which include cell broth down to cell pellets, and sample sizes, allowing for broad applicability. In addition, the method according to the present invention allows for efficient release of the viral vector, maximizing extraction while being gentle to the viral vector to preserve its integrity and functionality, e.g. achieving high functional/transducing titers by using carefully designed lysis reagents as disclosed herein. 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, reagents, 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 comprising a buffer having a concentration of 0.01 M to 3 M and a salt having a concentration of 0.01 M to 5 M;

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

(c) incubating the lysis composition such that a viral vector is released from the cell culture, wherein a cell of 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).

The method according to the first aspect advantageously allows for the efficient release of a viral vector from a cell culture. Specifically, providing the lysis reagent comprising a buffer having a concentration of 0.01 M to 3 M and a salt having a concentration of 0.01 M to 5 M allowed for achieving improved yields of capsid and functional viral vector titers within short incubation times of less than 4 hours (see e.g. Figs. 3 to 10). Furthermore, ionic strength and the type of salt have been carefully selected and screened to ensure high lysis efficiencies and the stability of the viral vector. Moreover, the method advantageously does not require separating a cell from the cell culture prior to contacting the cell culture to the lysis reagent in step (b), i.e. can be applied to a wide variety of samples, including for example cell broth and other crude sample types (see e.g. Figs. 11 to 14). The elimination of the step of separating the cell from the surrounding liquid, e.g. via centrifugation advantageously simplifies the cell lysis and enables direct application to the cell culture, e.g. in a culture vessel such as bioreactor. At the same time, no biohazardous components or method steps are comprised which ensures safety for the operator and environment (see Figs. 15 to 17).

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 a cell culture according to the method according to the first aspect; and x.4 optionally, purifying the released viral vector.

According to a third aspect, a cell lysis reagent for releasing a viral vector from a cell culture is provided, preferably a cell broth, selected from one of the following: i) an acidic lysis reagent selected from:

I. lx acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate 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 sugar, preferably sucrose, having a concentration selected from 1% (w/v) to 10% (w/v);

II. 5x acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate 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 sugar, preferably sucrose, having a concentration selected from 10% (v/v) to 35% (w/v); or

III. lOx acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate 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 sugar, preferably sucrose, having a concentration selected from 20% (w/v) to 60% (w/v); ii) a detergent containing alkaline lysis reagent 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, 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 sugar, preferably sucrose, 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, 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 sugar, preferably sucrose, having a concentration selected from 10% (v/v) to 35% (w/v); or

III. 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, 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 sugar, preferably sucrose, having a concentration selected from 20% (w/v) to 60% (w/v); or iii) an alkaline lysis reagent containing no detergent 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, 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 sugar, preferably sucrose, 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, 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; optionally, a divalent cation, preferably Mg2+, having a concentration selected from 5 mM to 15 mM; and optionally, a sugar, preferably sucrose, having a concentration selected from 10% (v/v) to 35% (w/v); or

III. 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, 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; optionally, a divalent cation, preferably Mg2+, having a concentration selected from 15 mM to 50 mM; and optionally, a sugar, preferably sucrose, having a concentration selected from 20% (w/v) to 60% (w/v).

According to a fourth aspect, a kit for releasing a viral vector from a cell culture, preferably a cell broth, is provided, wherein the kit comprises a container that contains a cell lysis reagent according to the third aspect of the invention, and preferably one or more of the following:

(i) a mammalian cell, preferably selected from a HEK293 cell or a derivative of a HEK293 cell;

(ii) one or more plasmids for viral vector production;

(iii) a transfection reagent; and/or

(iv) a cell culture medium.

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 show a schematic process flow of a AAV vector production.

Fig. 2A shows a schematic process flow of a cell lysis and capsid and functional titer quantification.

Fig. 2B shows an exemplary overview of cell lysis study variables, including some but not all tested variables.

Fig. 3 shows AAV2 functional titers determined after cell lysis and clarification by transduction assay using Incucyte® Live-Cell Analysis system. N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N l, N2, N5, N6, N9 and N 10 relate to acidic lysis reagents according to the present disclosure. Standard cell lysis reagent, "prior art cell lysis reagent" (N20/21/22), is highlighted in black. Error bars represent standard deviation from the mean (n = 3).

Fig. 4 shows AAV2 capsid titers determined after cell lysis and clarification using Octet®

AAVX Biosensors. N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N l, N2, N5, N6, N9 and N 10 relate to acidic lysis reagents according to the present disclosure. Standard cell lysis reagent, "prior art cell lysis reagent" (N20/21/N22), is highlighted in black. <LOQ refers to values below the limit of quantitation. Error bars represent standard deviation from the mean (n = 3).

Fig. 5 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. 6 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 l and 13 stands for PageRuler™ unstained Protein ladder (Thermo Fisher Scientific).

Fig. 7 shows AAV2 functional titers determined after cell lysis and clarification by transduction assay using Incucyte® Live-Cell Analysis system. N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N7, N8, N5, N il, N 12, N 15, N 16, N25, N27, N29 and N31 relate to alkaline lysis reagents containing a detergent according to the present disclosure. Standard cell lysis reagent, "prior art cell lysis reagent" (N20/21/22), is highlighted in black. Error bars represent standard deviation from the mean (n = 3).

Fig. 8 shows AAV2 capsid titers determined after cell lysis and clarification using Octet®

AAVX Biosensors. N-number refers to the identification n umber of AAV2 production and cell lysis conditions, wherein N7, N8, N 11, N 12, N 15, N 16, N25, N27, N29 and N31 relate to alkaline lysis reagents containing a detergent according to the present disclosure. Standard cell lysis reagent, "prior art cell lysis reagent" (N20/21/N22), is highlighted in black. <LOQ refers to values below the limit of quantitation. Error bars represent standard deviation from the mean (n = 3).

Fig. 9 shows AAV2 functional titers determined after cell lysis and clarification by transduction assay using Incucyte® Live-Cell Analysis system. N-number refers to the identification number of AAV2 production and cell lysis conditions, wherein N23, N24, N26, N28 and N30 relate to detergent-free alkaline lysis reagents according to the present disclosure. Standard cell lysis reagent, "prior art cell lysis reagent" (N20/21/22), is highlighted in black. Error bars represent standard deviation from the mean (n = 3).

Fig. 10 shows AAV2 capsid titers determined after cell lysis and clarification using Octet®

AAVX Biosensors. N-number refers to the identification n umber of AAV2 production and cell lysis conditions, wherein N23, N24, N26, N28 and N30 relate to detergent- free alkaline lysis reagents according to the present disclosure. Standard cell lysis reagent, "prior art cell lysis reagent" (N20/21/N22), is highlighted in black. <LOQ refers to values below the limit of quantitation. Error bars represent standard deviation from the mean (n = 3).

Fig. 11 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. 12 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. 13 shows AAV2 functional titers (see Set A,_cell pellet lysis) determined after cell 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. 14 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. 15 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. 16 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 X100- containing (TRT) lysis reagent. Error bars represent standard deviation from the mean (n = 3).

Fig. 17 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. 18 shows total cell concentrations and viability rates determined after lysis screening to compare different lysis approaches 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. 19 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 lysis approaches were compared. 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 AV capsid aggregates are contained in the samples.

Fig. 20 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 lysis approaches were compared. 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. 21 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 lysis approaches were compared. 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.

Method for releasing a viral vector from a cell culture

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 comprising a buffer having a concentration of 0.01 M to 3 M and a salt having a concentration of 0.01 M to 5 M;

(c) incubating the lysis composition such that a viral vector is released from the cell culture, wherein a cell of 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). The method according to the first aspect advantageously allows for efficiently releasing a viral vector from a cell culture. As demonstrated in the present disclosure, in particular the Examples, providing the lysis reagent comprising a buffer having a concentration of 0.01 M to 3 M and a salt having a concentration of 0.01 M to 5 M enabled achieving improved yields of capsid and functional viral vector within short incubation times of less than 4 hours (see e.g. Figs. 3 to 10). Furthermore, ionic strength and the type of salt have been carefully selected and screened to ensure high lysis efficiencies and the stability of the viral vector. Moreover, the method advantageously does not require separating a cell from the cell culture prior to contacting the cell culture to the lysis reagent in step (b), i.e. can be applied to a wide variety of samples, including for example cell broth and other crude sample types (see e.g. Figs. 11 to 14) but also different types of viral vectors including AAV2, AAV8 and Adenovirus (see Figs. 1 to 16 and 19). The elimination of the step of separating the cell from the surrounding liquid, e.g. via centrifugation advantageously simplifies the cell lysis and enables direct application to the cell culture, e.g. in a culture vessel such as bioreactor. At the same time, no biohazardous components or method steps are comprised which ensures safety for the operator and environment (see Figs. 15 to 17).

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.

"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 (see Figs. 1 to 21). 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, wherein a cell of 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. 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 HEK 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. 11, 12, 15, and 16 to 21), as well as for purified or processed samples, such as cell pellets (see Figs. 13 and 14). 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-T, HEK293-F, HEK293-FT, etc. 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, orany 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. 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 be (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. Lysis reagent

According to method step (a), the method comprises providing a lysis reagent comprising a buffer having a concentration of 0.01 M to 3 M and a salt having a concentration of 0.01 M to 5 M. The present disclosure provides various lysis reagents as illustrated in the Examples section. In general, however, all lysis reagents according to the present disclosure at least comprise a buffer having a concentration of 0.01 M to 3 M and a salt having a concentration of 0.01 M to 5 M. Hence, the method according to the present disclosure comprises providing a lysis reagent comprising a buffer having a concentration of 0.01 M to 3 M and a salt having a concentration of 0.01 M to 5 M. It was found that the combination of buffer and salt at such concentration allows to precisely adjust the ionic strength such that the viral vector is efficiently released such that high titers of viral vector are obtainable (see Figs. 3 to 16 and 19) without substantial aggregation. A salt according to the present disclosure is different from a buffer in that both cannot be the same or identical compound.

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".

According to a preferred embodiment, the lysis reagent comprises a buffer having 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, 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 (head) space to be filled by the lysis reagent.

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 salt is selected from potassium chloride and 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, a buffer concentration is 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 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 (head) 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.

According to a preferred embodiment, the lysis reagent does not comprise a substance of the group of 4-(l,l,3,3-tetramethylbutyl)phenol, ethoxylated. According to a preferred embodiment, the lysis reagent does not comprise Triton X-100. Avoiding such compounds has the advantage that no biohazardous compounds are added.

Divalent cation

According to a preferred embodiment, the lysis reagent further 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, especially when not applying a nuclease, e.g. for the below described acidic lysis reagent.

According to a preferred embodiment, the divalent cation in the lysis reagent has a concentration of 0.1 mM to 100 mM, preferably a concentration selected from the following ranges: i) 0.5 mM to 5 mM for a lx lysis reagent; ii) 5 mM to 15 mM for a 5x lysis reagent; or iii) preferably, 15 mM to 50 mM for a lOx lysis reagent.

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

Cryo-protectant

According to a preferred embodiment, the lysis reagent further 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. However, the cryo-protectant offers the advantage of improving the storability and freezing of the viral vector containing compositions after lysis and/or purification when optionally added to the lysis reagents according to the present disclosure. However, the cryo-protectant does not need to be present during cell lysis but may be added subsequently, e.g. after step (c) of the method according to the present disclosure. It can be advantageous to include the cryo-protectant in the lysis reagent to avoid subsequent addition steps, i.e. a lysis reagent comprising the cryo-protectant simplifies the lysis process, as no cryo-protectant needs to be added during or after lysis.

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. Acidic lysis reagent

According to a preferred embodiment, the lysis reagent is an acidic lysis reagent, preferably having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5. According to a particular preferred embodiment, the acidic lysis reagent has a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5. As demonstrated in the Examples (see Figs. 3, 4, 11-16), the acidic lysis reagent allows for efficiently releasing the viral vector from the cell culture resulting in high AAV yields when compared to state-of-the-art lysis reagents. Also, the acidic lysis reagent allows for efficient removal of host cell related impurities by aiding in the efficient precipitation of host cell relates impurities (see Fig. 6). Furthermore, the purity indicates high stability in the lysis composition, with the acidic lysis reagent minimizing AAV vector aggregates. Finally, the acidic lysis reagent is highly efficient, in that vector extraction can be achieved within 30 min to 120 min, which is considerably shorter than conventional lysis protocols.

According to a preferred embodiment, the acidic lysis reagent has a pH selected from the range of 1.8 to 6.0 or 2.0 to 5.5, preferably 2.75 to 5.0. Such pH range allows for efficient release of the viral vector. According to particular embodiments, the pH is selected from a range of 1.8 to 6.0, 1.9 to 5.9, 2.0 to 5.8, 2.0 to 5.7, 2.0 to 5.6, 2.0 to 5.5, 2.1 to 5.4, 2.2 to 5.3, 2.3 to 5.2, 2.4 to 5.1, 2.5 to 5.0. Such pH values include for instance a pH of about 2.0 (including values +/- 10%) or a pH value of about 4.0 (including values +/- 10%).

According to a preferred embodiment, the acidic lysis reagent comprises a buffer suitable for buffering the acidic lysis reagent at acidic pH, preferably between 1.0 to 7.0, more preferably 2.0 to 6.0. Buffers which efficiently buffer the acidic 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 acidic pH.

According to a preferred embodiment, the acidic lysis reagent comprises a buffer selected from an acetate buffer, hydrochloric acid-potassium chloride buffer (HCI-KCI), glycine buffer, citrate buffer, citrate-phosphate buffer, phosphate buffer, glycyl-glycine buffer, sodium formate buffer, succinate buffer, pyridine buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 2-[Bis(2- hydroxyethyl)amino]-2-(hydroxymethyl)propane-l,3-diol (BIS-TRIS) buffer, or combinations thereof, preferably an acetate buffer or glycine buffer. These buffer compounds have been found suitable for the acidic lysis reagent, as these efficiently buffer the lysis reagent and possibly the lysis composition at the acidic pH. In one embodiment, the pH of the acidic lysis reagent is selected from the range of 2.0 to 6.0 for which following buffers may be chosen from: i) Hydrochloric Acid-Potassium Chloride Buffer (HCI-KCI); pH Range 1.0 to 2.2, ii) Glycine-HCI Buffer; pH range 2.0 to 3.6, iii) Citrate Buffer; pH range 3.0 to 6.2, iv) Acetate Buffer; pH range 3.6 to 5.6, or v) Citrate-Phosphate Buffer; pH range 2.6 to 7.0.

According to a preferred embodiment, in step (b) of the method according to the present disclosure the lysis composition: i) has a pH of less than 5.0, preferably less than 4.5; and/or ii) has a pH selected from the range of 2.5 to 5.0, preferably 3.5 to 4.5 with the proviso that the lysis reagent is the acidic lysis reagent.

Lysis compositions having the indicated pH (see exemplary lysis compositions in Table 5) were found efficient for releasing the viral vector and additionally, reduce host cell related impurities (see Fig. 6).

According to a preferred embodiment, particularly concerning the acidic lysis reagent, no nuclease is added. Eliminating the need for nuclease is advantageous, as the nuclease is associated with high costs and additional processing steps, including addition of the nuclease and incubation, e.g. at particular temperatures.

According to a preferred embodiment, the released viral vector is purified, preferably at least by depth filtration, centrifugation, and/or sterile filtration after step (c), wherein the released viral vector comprises at least 20% less, preferably 25% less, more preferably 30% less or 35% less host cell derived DNA impurities compared to a method wherein the lysis reagent is not an acidic lysis reagent but a neutral or alkaline lysis reagent. Due to the low pH of the reagents the cell lysates exhibit reduced viscosity, potentially due to the precipitation of host cell proteins and nucleic acids. This allows for efficient filtration without requiring additional nuclease digestion (see Fig. 5 shown for setup N2, N5 and N6).

According to a preferred embodiment, the released viral vector is purified, preferably by at least depth filtration, centrifugation, and/or sterile filtration after step (c), wherein the released viral vector comprises at least 25% less, preferably 50% less, more preferably 60% less or 70% less host cell derived protein impurities compared to a method wherein the lysis reagent is not an acidic lysis reagent but a neutral or alkaline lysis reagent. Due to the low pH of the reagents the cell lysates exhibit reduced viscosity, potentially due to the precipitation of host cell proteins and nucleic acids. This allows for efficient filtration without requiring additional nuclease digestion (see Fig. 5 shown for setup N2, N5 and N6). In addition, the host cell related protein impurities are significantly reduced (see Fig. 6).

Exemplary acidic lysis reagents can be found in Table 1.

Alkaline 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 2 and 3, Figs. 7-10). 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. According to a particular embodiment, the alkaline lysis reagent has a pH selected from the range of 7.2 to 10, 7.3 to 9.9, 7.3 to 9.8, 7.4 to 9.7, 7.4 to 9.6, preferably 7.5 to 9.5. 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 alkaline 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 alkaline 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, in step (b) the lysis composition has a pH of i) more than 7.2, preferably more than 7.5, most preferably more than 7.8 or 8.0; and/or ii) selected from the range of 7.2 to 10.0, preferably 7.5 to 9.5, with the proviso that the lysis reagent is the alkaline lysis reagent.

Lysis compositions having the indicated pH (see exemplary lysis compositions Table 5) were found efficient for releasing the viral vector (see Figs. 7-10, 19).

According to a preferred embodiment, 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. In one embodiment, the nuclease is added to the lysis composition after step (b) (see also Examples 2 and 3), however, the nuclease may equally well be added to the lysis reagent before 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 M-SAN 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.

Exemplary alkaline lysis reagents can be found in Table 2 and 3.

Detergent

The lysis reagents according to the present disclosure can be detergent-containing or detergent free. It has in particular been found that acidic lysis reagents as disclosed herein containing a detergent effectively lyse the cells and thus release the viral vector from the cell culture (see e.g. Example 1, Table 1). Hence, according to a particular preferred embodiment, the acidic lysis reagent comprises further a detergent. On the other hand, the alkaline lysis reagent can be flexibly applied with or without the detergent still showing excellent release of the viral vector (see Examples 2 and 3, Tables 2 and 3). Hence, the alkaline lysis reagent can comprise a detergent or be detergent-free.

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 and/or a non-ionic detergent that is toxic and/or hazardous to the environment. 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, preferably Tween. 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 (N P-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 in a method 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. 17). 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 a preferred embodiment, the lysis reagent does not comprise a substance of the group of 4-(l,l,3,3-tetramethylbutyl)phenol, ethoxylated, , preferably the lysis reagent does not comprise any non-ionic detergent that is toxic and/or hazardous to the environment. According to a preferred embodiment, the lysis reagent does not comprise Triton X-100, preferably the lysis reagent does not comprise any non-ionic detergent that is toxic and/or hazardous to the environment. Avoiding such compounds has the advantage that no biohazardous compounds are added.

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.

Exemplary lysis reagents comprising the detergent can be found in Table 1 and 2.

According to a particular embodiment of the present method, the lysis reagent is an acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, 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. subcell 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 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. subcell 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. In such embodiment, the method may 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.

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 is detergent-free. 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. In such embodiment, the method may 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.

Further exemplary lysis reagents

According to a preferred embodiment, the lysis reagent is an acidic lysis reagent selected from:

I. lx acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate buffer or glycine 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 acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate buffer or glycine 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. lOx acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate buffer or glycine 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 lysis reagent is a detergent containing alkaline lysis reagent 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. 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 lysis reagent is an alkaline lysis reagent containing no detergent 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; 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; 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. 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; 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)

According to the method for releasing a viral vector from the cell culture as disclosed herein, step (b) 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 or joining 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 comprises the compounds in essentially the concentrations of a lx lysis reagent, which includes the lx acidic lysis reagent but also the lx detergent containing alkaline lysis reagent and the lx detergent-free 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 has the characteristics from one of the following:

I. the lysis composition has a pH of less than 5.0, preferably less than 4.5, and comprises: a buffer, preferably an acetate buffer or glycine 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. 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); or

III. 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; 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)

According to the method for releasing a viral vector from the cell culture as disclosed herein, step (c) 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, 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. According to a preferred embodiment, incubating step (c) comprises 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. 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 3.5 h, 2 h and 0.5 h. Such quick release of viral vector improves efficiency of the overall process.

Further embodiments

According to a preferred embodiment, 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 (c). 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. According to a particular embodiment, when using the acidic lysis reagent, a large fraction of the impurities precipitate, such that these can be readily removed in downstream purification processes, such as depth filtration, centrifugation, and/or sterile filtration.

According to a preferred embodiment, significantyields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell broth, wherein significant amounts are at least lxlO¹¹ capsids per mL and/or lxlO⁶ TU per mL. As demonstrated in the Examples below, the method according to the present disclosure advantageously allows for achieving such high yields.

According to a preferred embodiment, significantyields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell pellet, wherein significant amounts are at least 2xlOⁿ capsids per mL and/or lxlO⁶ TU per mL. As demonstrated in the Examples below, the method according to the present disclosure advantageously allows for achieving such high yields.

According to a preferred embodiment, the capsid viral vector titer and/or transducing viral vector titer is comparable or increased compared to a Triton-X based cell lysis buffer. As demonstrated in the Examples below, the method according to the present disclosure advantageously allows for achieving such high yields without any use of a Triton-based detergent. Hence, the methods according to the present disclosure advantageously avoid using a toxic and/or hazardous detergent, such as Triton X, which is below shown to drastically reduce cell viability (see Fig. 17).

According to one embodiment, the method comprises following features: i) the lysis reagent is a lx acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate buffer or glycine 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) in step (b) the lysis composition has a pH of less than 5.0, preferably less than 4.5; iii) no nuclease is added; iv) the cell culture is a cell pellet; v) the cell culture comprises a mammalian cell, preferably a HEK293 cell or a derivative of a HEK293 cell; and vi) the viral vector is a non-enveloped virus, preferably AAV or Adenovirus.

In such embodiment, preferably 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. In such embodiment, preferably the released viral vector com prises at least 20% less, preferably 25% less, more preferably 30% less or 35% less host cell derived DNA impurities compared to a method wherein the lysis reagent is not an acidic lysis reagent but a neutral or alkaline lysis reagent. In such embodiment, preferably significant yields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell pellet, wherein significant amounts are at least 2xl0ⁿ capsids per mL and/or lxlO⁶ TU per mL.

According to one embodiment, the method comprises following features: i) the lysis reagent is a 5x acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate buffer or glycine 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); ii) in step (b) the lysis composition has a pH of less than 5.0, preferably less than 4.5; iii) no nuclease is added; iv) the cell culture is a cell broth; v) the cell culture comprises a mammalian cell, preferably a HEK293 cell or a derivative of a HEK293 cell; and vi) the viral vector is a non-enveloped virus, preferably AAV or Adenovirus.

In such embodiment, preferably 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. In such embodiment, preferably the released viral vector com prises at least 20% less, preferably 25% less, more preferably 30% less or 35% less host cell derived DNA impurities compared to a method wherein the lysis reagent is not an acidic lysis reagent but a neutral or alkaline lysis reagent. In such embodiment, preferably significant yields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell broth, wherein significant amounts are at least lxlO¹¹ capsids per mL and/or lxlO⁶ TU per mL.

According to one embodiment, the method comprises following features: i) the lysis reagent is a lOx acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate buffer or glycine 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); ii) in step (b) the lysis composition has a pH of less than 5.0, preferably less than 4.5; iii) no nuclease is added; iv) the cell culture is a cell broth; v) the cell culture comprises a mammalian cell, preferably a HEK293 cell or a derivative of a HEK293 cell; and vi) the viral vector is a non-enveloped virus, preferably AAV or Adenovirus.

In such embodiment, preferably 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. In such embodiment, preferably the released viral vector com prises at least 20% less, preferably 25% less, more preferably 30% less or 35% less host cell derived DNA impurities compared to a method wherein the lysis reagent is not an acidic lysis reagent but a neutral or alkaline lysis reagent. In such embodiment, preferably significant yields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell broth, wherein significant amounts are at least lxlO¹¹ capsids per mL and/or lxlO⁶ TU per ml_.

According to one embodiment, the method comprises following features: i) the lysis reagent is a detergent containing alkaline lysis reagent which is a 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) 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; iii) the method further comprises providing a nuclease, preferably a salt-tolerant 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; iv) the cell culture is a cell pellet; v) the cell culture comprises a mammalian cell, preferably a HEK293 cell or a derivative of a HEK293 cell; and vi) the viral vector is a non-enveloped virus, preferably AAV or Adenovirus.

In such embodiment, preferably 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. In such embodiment, preferably significant yields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell pellet, wherein significant amounts are at least 2xl0ⁿ capsids per mL and/or lxlO⁶ TU per mL.

According to one embodiment, the method comprises following features: i) the lysis reagent is a detergent containing alkaline lysis reagent which is a 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); ii) 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; iii) the method further comprises providing a nuclease, preferably a salt-tolerant 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; iv) the cell culture is a cell broth; v) the cell culture comprises a mammalian cell, preferably a HEK293 cell or a derivative of a HEK293 cell; and vi) the viral vector is a non-enveloped virus, preferably AAV or Adenovirus.

In such embodiment, preferably 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. In such embodiment, preferably significant yields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell broth, wherein significant amounts are at least lxlO¹¹ capsids per mL and/or lxlO⁶ TU per mL.

According to one embodiment, the method comprises following features: i) the lysis reagent is a detergent containing alkaline lysis reagent which is a 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); ii) 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; iii) the method further comprises providing a nuclease, preferably a salt-tolerant 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; iv) the cell culture is a cell broth; v) the cell culture comprises a mammalian cell, preferably a HEK293 cell or a derivative of a HEK293 cell; and vi) the viral vector is a non-enveloped virus, preferably AAV or Adenovirus.

According to one embodiment, the method comprises following features: i) the lysis reagent is an alkaline lysis reagent containing no detergent which is a 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; 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) 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; iii) the method further comprises providing a nuclease, preferably a salt-tolerant 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; iv) the cell culture is a cell pellet; v) the cell culture comprises a mammalian cell, preferably a HEK293 cell or a derivative of a HEK293 cell; and vi) the viral vector is a non-enveloped virus, preferably AAV or Adenovirus.

In such embodiment, preferably 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. In such embodiment, preferably significant yields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell pellet, wherein significant amounts are at least 2xlOⁿ capsids per mL and/or lxlO⁶ TU per mL.

According to one embodiment, the method comprises following features: i) the lysis reagent is an alkaline lysis reagent containing no detergent which is a 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; 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); ii) 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; iii) the method further comprises providing a nuclease, preferably a salt-tolerant 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; iv) the cell culture is a cell broth; v) the cell culture comprises a mammalian cell, preferably a HEK293 cell or a derivative of a HEK293 cell; and vi) the viral vector is a non-enveloped virus, preferably AAV or Adenovirus.

According to one embodiment, the method comprises following features: i) the lysis reagent is an alkaline lysis reagent containing no detergent which is a 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; 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); ii) 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; iii) the method further comprises providing a nuclease, preferably a salt-tolerant 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; iv) the cell culture is a cell broth; v) the cell culture comprises a mammalian cell, preferably a HEK293 cell or a derivative of a HEK293 cell; and vi) the viral vector is a non-enveloped virus, preferably AAV or Adenovirus.

According to a preferred embodiment, the method has the following features: i) the cell culture comprises a mammalian cell, preferably selected from a HEK293 cell or a derivative of a HEK293 cell; ii) incubating step (c) comprises incubating for less than 4 hours, preferably for less than 2 hours; and iii) the lysis reagent does not comprise Triton X-100, preferably the lysis reagent does not comprise any non-ionic detergent that is toxic and/or hazardous to the environment. Method for producing a viral vector

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 a cell culture according to the method for releasing a viral vector from a cell culture according to the first aspect of the invention; and x.4 optionally, purifying the released 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 achieving improved yields of capsid and functional viral vector within short incubation times of less than 4 hours (see e.g. Figs. 3 to 10). Furthermore, ionic strength and the type of salt have been carefully selected and screened to ensure high lysis efficiencies and the stability of the viral vector. Moreover, the method advantageously does not require separating a cell from the cell culture prior to contacting the cell culture to the lysis reagent in step (b), i.e. can be applied to a wide variety of samples, including for example cell broth and other crude sample types (see e.g. Figs. 11 to 14 and 18 to 21). The elimination of the step of separating the cell from the surrounding liquid, e.g. via centrifugation advantageously simplifies the cell lysis and enables direct application to the cell culture, e.g. in a culture vessel such as bioreactor. At the same time, no biohazardous components or method steps are comprised ensuring safety for the operator and environment (see Figs. 15 to 17).

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 / no nuclease, as well as the acidic and alkaline lysis reagents, 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 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 a viral vector is released from the cell culture. Such embodiment has 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. 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. 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 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 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 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. According to a particular embodiment, when using the acidic lysis reagent, a large fraction of the impurities precipitate, such that these can be removed in downstream purification processes, such as at least depth filtration, centrifugation, and/or sterile filtration. According to a preferred embodiment, the method further comprises step x.5 formulating the viral vector for gene therapy.

Reagent for releasing a viral vector from a cell culture

The cell lysis reagent 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 of less than 4 hours (see e.g. Figs. 3 to 10). Furthermore, ionic strength and the type of salt have been carefully selected and screened to ensure high lysis efficiencies and the stability of the viral vector. Moreover, the reagent advantageously does not require separating a cell from the cell culture prior to contacting the cell culture to the lysis reagent, i.e. can be applied to a wide variety of samples, including for example cell broth and other crude sample types (see e.g. Figs. 11 to 14 and 18 to 21). At the same time, no biohazardous components are comprised which ensures safety for the operator and environment (see Figs. 15 to 17).

The individual and preferred embodiments of the cell lysis reagent according to the third aspect correspond to the individual embodiments of the method according to the first and second aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the cell lysis reagent 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, cryoprotectant, nuclease / no nuclease, as well as the acidic and alkaline lysis reagents. Further features will now be described in detail.

According to a preferred embodiment, the reagent is for use for releasing a viral vector, preferably adeno-associated virus (AAV) or adenovirus (Ad), from a cell culture, preferably a cell broth, optionally wherein the cell culture comprises a mammalian cell selected from a HEK293 cell or a derivative of a HEK293 cell. According to a particular embodiment, the reagent is for use for releasing a viral vector from a cell culture according to the method of the first aspect and/or for use for producing a viral vector according to the method of the second aspect.

Kit for releasing a viral vector from a cell culture

(ii) one or more plasmids for viral vector production;

(iii) a transfection reagent; and/or

(iv) a cell culture medium.

The kit for releasing a viral vector from a cell culture according to the fourth 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 kit for releasing a viral vector from a cell culture according to the fourth aspect. Specifically, the kit allows for achieving improved yields of capsid and functional viral vector within short incubation times of less than 4 hours (see e.g. Figs. 3 to 10). Furthermore, ionic strength and the type of salt have been carefully selected and screened to ensure high lysis efficiencies and the stability of the viral vector. Moreover, the kit advantageously does not require separating a cell from the cell culture prior to contacting the cell culture to the lysis reagent, i.e. can be applied to a wide variety of samples, including for example cell broth and other crude sample types (see e.g. Figs. 11 to 14 and 18 to 21). At the same time, no biohazardous components are comprised which ensures safety for the operator and environment (see Figs. 15 to 17).

The individual steps and preferred embodiments of the kit according to the fourth aspect correspond to the individual steps and embodiments of the methods according to the first and second aspect, as well as the cell lysis reagent according to the third aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the kit according to the fourth 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 / no nuclease, as well as the acidic and alkaline lysis reagents. Furtherfeatures 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 kit is for use for releasing a viral vector, preferably adeno- associated virus (AAV) or adenovirus (Ad), from a cell culture, preferably a cell broth, optionally wherein the cell culture comprises a mammalian cell selected from a HEK293 cell or a derivative of a HEK293 cell. According to a particular embodiment, the kit is for use for releasing a viral vector from a cell culture according to the method of the first aspect and/or for use for producing a viral vector according to the method of the second aspect.

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;

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

2. The method according to item 1, wherein the cell culture is a cell broth or cell pellet, preferably a cell broth.

3. The method according to items 1 or 2, wherein in a cell of the cells of 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 items 1 to 3, wherein the lysis reagent comprises a buffer having a concentration of 0.01 M to 3 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, 1 M to 3 M for a lOx lysis reagent.

5. The method according to one or more of items 1 to 4, wherein the lysis reagent comprises a salt, preferably selected from potassium chloride and sodium chloride.

6. The method according to item 5, wherein the salt in the lysis reagent has a concentration of 0.01 M 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.

7. The method according to one or more of items 1 to 6, 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+.

8. The method according to item 7, wherein the divalent cation in the lysis reagent has a concentration of 0.1 mM to 100 mM, preferably a concentration selected from the following ranges: i) 0.5 mM to 5 mM for a lx lysis reagent; ii) 5 mM to 15 mM for a 5x lysis reagent; or iii) preferably, 15 mM to 50 mM for a lOx lysis reagent. 9. The method according to one or more of items 1 to 8, wherein the lysis reagent comprises a cryo-protectant, preferably a sugar, more preferably sucrose, trehalose or mannitol, most preferably sucrose.

10. The method according to item 9, 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.

11. The method according to one or more of items 1 to 10, wherein the lysis reagent is an acidic lysis reagent, preferably having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5.

12. The method according to item 11, wherein the acidic lysis reagent has a pH selected from the range of 1.8 to 6.0 or 2.0 to 5.5, preferably 2.75 to 5.0.

13. The method according to item 11 or 12, wherein the acidic lysis reagent comprises a buffer suitable for buffering the acidic lysis reagent at acidic pH, preferably between 1.0 to 7.0, more preferably 2.0 to 6.0.

14. The method according to one or more of items 11 to 13, wherein the acidic lysis reagent comprises a buffer selected from an acetate buffer, hydrochloric acid-potassium chloride buffer (HCI-KCI), glycine buffer, citrate buffer, citrate-phosphate buffer, phosphate buffer, glycylglycine buffer, sodium formate buffer, succinate buffer, pyridine buffer, 2-(N- morpholino)ethanesulfonic acid (MES) buffer, 2-[Bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)propane-l,3-diol (BIS-TRIS) buffer, or combinations thereof, preferably an acetate buffer or glycine buffer.

15. The method according to one or more of items 11 to 14, wherein in step (b) the lysis composition: i) has a pH of less than 5.0, preferably less than 4.5; and/or ii) has a pH selected from the range of 2.5 to 5.0, preferably 3.5 to 4.5.

16. The method according to one or more of items 11 to 15, wherein no nuclease is added.

17. The method according to one or more of items 11 to 16, wherein the released viral vector is purified, preferably at least by depth filtration, centrifugation, and/or sterile filtration after step (c), wherein the released viral vector comprises at least 20% less, preferably 25% less, more preferably 30% less or 35% less host cell derived DNA impurities compared to a method wherein the lysis reagent is not an acidic lysis reagent but a neutral or alkaline lysis reagent. 18. The method according to one or more of items 11 to 17, wherein the released viral vector is purified, preferably by at least depth filtration, centrifugation, and/or sterile filtration after step (c), wherein the released viral vector comprises at least 25% less, preferably 50% less, more preferably 60% less or 70% less host cell derived protein impurities compared to a method wherein the lysis reagent is not an acidic lysis reagent but a neutral or alkaline lysis reagent.

19. The method according to one or more of items 1 to 10, 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.

21. The method according to item 19 or 20, wherein the alkaline lysis reagent comprises a buffer suitable for buffering the alkaline 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 19 to 21, wherein the alkaline 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 19 to 22, 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.

24. The method according to one or more of items 19 to 23, 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.

25. The method according to one or more of items 19 to 24, 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.

26. The method according to item 25, wherein the nuclease is salt-tolerant.

27. The method according to one or more of items 1 to 26, 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 and/or a non-ionic detergent that is not toxic and/or hazardous to the environment. The method according to item 27, wherein the detergent is selected from one or more of Tween, Triton, Nonidet, Igepal or Tergitol, preferably Tween. The method according to item 27 or 28, 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). The method according to one or more of items 27 to 29, 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. The method according to one or more of items 27 to 30, 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. The method according to one or more of items 1 or 31, wherein the lysis reagent is one of the following: i) an acidic lysis reagent selected from:

II. 5x acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate buffer or glycine 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. lOx acidic lysis reagent having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5, comprising: a buffer, preferably an acetate buffer or glycine 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); ii) a detergent containing alkaline lysis reagent selected from:

III. 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); or iii) an alkaline lysis reagent containing no detergent 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; 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; 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. 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; 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 32, 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 32, and/or the lysis composition has the characteristics from one of the following:

III. 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; 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). The method according to one or more of items 1 to 33, 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.

35. The method according to one or more of items 1 to 34, wherein the cell culture is not subjected to sonication, high-pressure homogenization, or freeze-thaw.

36. The method according to one or more of items 1 to 35, 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).

37. The method according to one or more of items 1 to 36, 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 (c).

38. The method according to one or more of items 1 to 37, wherein significant yields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell broth, wherein significant amounts are at least lx 10¹¹ capsids per mL and/or lxlO⁶ TU per mL.

39. The method according to one or more of items 1 to 38, wherein significant yields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell pellet, wherein significant amounts are at least 2x10¹¹ capsids per mL and/or lxlO⁶ TU per mL.

40. The method according to one or more of items 1 to 39, wherein the capsid viral vector titer and/or transducing viral vector titer is comparable or increased compared to a Triton-X based cell lysis buffer.

41. 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 a cell culture according to one or more of items 1 to 39; and x.4 optionally, purifying the released viral vector.

42. The method according to item 41, wherein step x.3 releasing the viral vector from a cell culture is performed by 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 a viral vector is released from the cell culture.

43. The method according to one or more of items 41 to 42, wherein the method comprises purifying the viral vector, wherein purifying comprises a single-step or multistep approach, preferably by at least depth filtration, centrifugation, and/or sterile filtration.

44. The method according to one or more of items 41 to 43, wherein the method further comprises step x.5 formulating the viral vector for gene therapy. 45. The method according to one or more of items 41 to 44, 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.

46. The method according to one or more of items 1 to 45, wherein the viral vector is predominantly present intracellularly and preferably is selected from an adeno-associated virus (AAV) or an adenovirus (Ad).

47. The method according to one or more of items 1 to 46, wherein the cell culture comprises a mammalian cell.

48. The method according to item 47, wherein the mammalian cell is modified to be configured to produce a viral vector, preferably AAV.

49. The method according to items 47 or 48, wherein the mammalian cell is selected from a HEK293 cell or a derivative of a HEK293 cell.

50. The method according to one or more of items 1 to 49, wherein the cell culture comprises a cell capable of being cultured in suspension.

51. A cell lysis reagent for releasing a viral vector from a cell culture, preferably a cell broth, selected from one of the following: i) an acidic lysis reagent selected from:

III. 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, 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 sugar, preferably sucrose, having a concentration selected from 20% (w/v) to 60% (w/v); or iii) an alkaline lysis reagent containing no detergent 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, 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 sugar, preferably sucrose, 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, 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; optionally, a divalent cation, preferably Mg2+, having a concentration selected from 5 mM to 15 mM; and optionally, a sugar, preferably sucrose, having a concentration selected from 10% (v/v) to 35% (w/v); or

7.5, most preferably more than 7.7, comprising: a buffer, preferably a Tris(hydroxymethyl)-aminomethan (TRIS) 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; optionally, a divalent cation, preferably Mg2+, having a concentration selected from 15 mM to 50 mM; and optionally, a sugar, preferably sucrose, having a concentration selected from 20% (w/v) to 60% (w/v). Reagent according to item 51 for use for releasing a viral vector, preferably adeno-associated virus (AAV) or adenovirus (Ad), from a cell culture, preferably a cell broth, optionally wherein the cell culture comprises a mammalian cell selected from a HEK293 cell or a derivative of a HEK293 cell. A kit for releasing a viral vector from a cell culture, preferably a cell broth, wherein the kit comprises a container that contains a cell lysis reagent according to item 50. The kit according to item 53 further comprising one or more of the following:

(ii) one or more plasmids for viral vector production;

(iii) a transfection reagent; and/or

(iv) a cell culture medium. Throughout the description, where methods, reagents, kits or uses are described as having, including, or comprising specific components or steps, it is contemplated that, additionally, there are methods, reagents, kits 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 lysis reagents of the present invention, as well as their application in frame of the methods disclosed herein, their use for releasing a viral vector, preferably adeno-associated virus (AAV) or adenovirus (Ad), from a cell culture, and the kit according to the present disclosure. Specifically, the examples demonstrate that an increased extraction of viral vectors can be achieved compared to reference lysis reagents such as the "prior art lysis reagent". Such increase is achieved by enhancing the efficiency of the cell lysis while the formation of viral vector aggregates, such as AAV vector aggregates, is reduced and the removal of host cell-related impurities is facilitated by their precipitation.

Materials and Methods

Cell lysis workflow

The 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. An exemplary and in the Examples followed workflow of viral vector production, cell lysis, and downstream processing, including clarification and purification can be found in Fig. 1. The cell lysis process is shown in Fig. 2 and is initiated by adding the cell lysis reagent to the HEK293 cells. Notably, the majority of cell lysis reagents, including reference lysis reagents such as the "prior art cell lysis reagent" (see e.g. Fedosyuk et al., 2019, Vaccine, Vol. 37, pp. 6951-6961), require the addition of a nuclease for optimal performance. For the lysis reagents according to the present disclosure, nuclease is in some cases not required, e.g. for an acidic lysis reagent (see below Tables). Following an incubation period of 0.5-3.5 hours at 37°C and 120 rpm, 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.

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.

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 AAV2 capsid titer quantification, the binding rate of serially diluted AAV2 clarified cell lysates were determined using Octet® AAVX Biosensors (Sartorius Stedim Biotech GmbH) at 1800 sec on the Octet® Bio Layer Interferometry platform (Sartorius Stedim Biotech GmbH). The binding rates were compared to those of 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). is rea

A common example for a standard lysis reagent is the "prior art cell lysis reagent", which is a widely used detergent-mediated cell lysis buffer that has become a standard in laboratories. It has been published in e.g., Joe et. al. 2021 (bioRxiv 2021.12.22.473478) and has become a standard in laboratories. The recipe for the "prior art cell lysis reagent" can be found in Table 1. This buffer has been used as reference lysis reagent for the comparison to the novel lysis reagents disclosed herein.

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. The recipe for the "TRT" cell lysis reagent can be found in Table 1.

Further reference cell lysis reagents used in Examples below are selected from CelLytic™ M (Cl, Sigma Aldrich) and CelLytic™ M (C2, both from Sigma Aldrich) and M-PER® (Thermo Fisher Scientific), all of which are used according to the manufacturer's handbooks.

The novel lysis reagents according to the current disclosure are typically formulated by combining stock solutions of buffering salt adjusted to the appropriate pH (e.g. acetate buffer at pH 4) and neutral salt (e.g. potassium chloride and magnesium chloride) solutions, taking into account the individual solubilities of the salts. For sucrose and polysorbate 20 supplementations, stock solutions of sucrose and polysorbate 20 are used.

Purification

The clarified AAV2 lysate was loaded onto a 1 ml prepacked POROS™ CaptureSelect™ AAVX column. The affinity purification process has been conducted according to the manufacturer's instructions on AKTA™ pure 150 system. Following a buffer exchange step of the eluate to 20 mM BisTRISPropane, pH 9, an anion exchange chromatography has been performed using a 1 ml prepacked POROS™ GoPure™ HQ column. The anion exchange purification has been conducted according to the manufacturer's instructions on AKTA™ pure 150 system. SDS-PAGE

The clarified AAV2 lysates were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Mini-PROTEAN® TGX Precast 4-20% gels according to the manufacturer's instructions.

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: Acidic lysis reagent for lysing HEK293 producing AAV2

In Example 1, acidic 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 acidic cell lysis reagents are listed in Table 1 - all of which have an acidic pH well below pH 7.0. Furthermore, the acidic lysis reagents contain a detergent, specifically polysorbate 20. However, other detergents, in particular other polysorbates are applicable as detergent for the acidic lysis reagent disclosed herein. As demonstrated below, the herein disclosed acidic lysis reagents address the technical problems typically encountered in cell lysis, including a low cell lysis efficiency and/or long lysis duration, high AAV vector aggregation, high host cell impurity contents, as well as potential need of a nuclease treatment and/or other physical or mechanical lysis steps (e.g. freeze-thaw cycle(s)).

Table 1: Examples of acidic lysis reagents

AAV2 functional titer

The functional AAV2 titer was determined 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. The results are shown in Fig. 3.

As visible in Fig. 3, the functional titers are very high for the acidic lysis reagents according to the present disclosure, being between 4xl0⁷ to 6xl0⁷ TU/mL compared to functional titers of less than lxlO⁷TU/mL for the reference lysis reagent. These results were obtained consistently for different salt concentrations ranging from 50 to 500 mM, incubation duration ranging from 0.5 to 2 h. In the tested acidic lysis reagents, Tween20 was used as a detergent and the pH of the reagent was set to 4.0. Hence, the results indicate that the acidic lysis reagents according to the present disclosure allow for highly efficient cell lysis and increased yields of functional titer.

AAV2 capsid titer

The AAV2 capsid titer was determined after cell lysis and clarification using Octet® AAVX Biosensors as described above. For comparison, the "prior art cell lysis reagent" was used as a reference lysis reagent. The results are shown in Fig. 4.

As visible in Fig. 4, the capsid titers are very high for the acidic lysis reagents according to the present disclosure, being between l.OxlO¹³ to 1.5xl0¹³ capsids/mL compared to capsid titers of less than 2.0xl0¹² capsids/mL for the reference lysis reagent. These results were obtained consistently for different salt concentrations ranging from 50 to 500 mM, incubation duration ranging from 0.5 to 2 h. In the tested acidic lysis reagent, Tween20 was used as a detergent and the pH of the reagent was set to 4.0. Overall, the results indicate that the acidic lysis reagents according to the present disclosure allow for highly efficient cell lysis and increased yields of capsid titer. Filterability of the crude

In order to test whether the crude lysates a I low 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. 5, as well as pictures taken of the spin filters. Shown cell lysis setups are conducted without a nuclease digestion step.

The pictures in Fig. 5 highlight that the acidic lysis reagents according to the present disclosure 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 contract, a lysis reagent pH 8 results in a lysate having high viscosity, necessitating additional nuclease digestion to facilitate the filtration step (see Fig. 5 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 acidic lysis reagents. M in lanes 1 and 13 stands for PageRuler™ unstained Protein ladder (Thermo Fisher Scientific).

The gel bands shown in Fig. 6 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. 5) 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 acidic lysis reagents according to the present disclosure address the following technical problems by implementing several key improvements: i) Enhanced efficiency of cell lysis: the acidic cell lysis reagents ensure effective disruption of cells, enhancing the extractability of intracellular AAV vectors. Their usage resulted in up to 4.5-fold higher yields of fully active AAV vectors compared to those obtained using reference lysis reagents (see Fig. 3). 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. ii) 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. 6 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. iii) 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. iv) Elimination of the need for nucleases: the acidic cell lysis reagents eliminate the nuclease addition during or after cel I lysis. Due to the low pH of the reagents the cell lysates exhibit reduced viscosity, potentially due to the precipitation of host cell proteins and nucleic acids. This allows for efficient filtration without requiring additional nuclease digestion (see Fig. 5 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. 5 shown for setup N25 without a nuclease digestion step). Furthermore, nuclease digestion can contribute to the high costs associated with the manufacturing of biopharmaceuticals. Elimination of the need for nucleases thereby reduces associated expenses. v) Shorter incubation time: the acidic cell lysis reagents enable efficient cell lysis and AAV vector extraction within 30 minutes, which is considerably shorter than the standard cell lysis protocols. Shortening the incubation time in the production process has the potential to lower overall production costs. The optimized cell lysis reagents 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. vi) 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 acidic lysis reagents of the present disclosure have the potential to significantly impact the manufacturing costs of viral vectorbased 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: Alkaline lysis reagent containing a detergent for lysing HEK293 producing AAV2

In Example 2, alkaline lysis reagents containing a detergent 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 supplemented with a salt active nuclease 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 alkaline cell lysis reagents with recommended nucleases are listed in Table 2 - all of which have an alkaline pH well above pH 7.0. Furthermore, the alkaline lysis reagents contain a detergent, specifically polysorbate 20, and a salt tolerant nuclease was added to the lysis composition. However, other detergents, in particular other polysorbates are applicable as detergent for the alkaline lysis reagent disclosed herein. As demonstrated below, the herein disclosed alkaline 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)).

Table 2: Recipes of alkaline cell lysis reagents with recommended nucleases

AAV2 functional titer

The functional AAV2 titer was determined 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. The results are shown in Fig. 7.

As visible in Fig. 7, the functional titers are very high for the alkaline lysis reagents according to the present disclosure, being between 5xl0⁷ to 8.5xl0⁷TU/mLcompared to functional titers of less than lxlO⁷TU/mL for the reference lysis reagent. These results were obtained consistently for different salt concentrations ranging from 275 to 1000 mM, incubation duration ranging from 0.5 to 3.5 h, and a pH ranging from 8 to 9. In the tested alkaline lysis reagents, Tween20 was used as a detergent and a salt tolerant nuclease were added to the lysis composition. Hence, the results indicate that the alkaline lysis reagents containing a detergent according to the present disclosure allow for highly efficient cell lysis and increased yields of functional titer. AAV2 capsid titer

The AAV2 capsid titer was determined after cell lysis and clarification using Octet® AAVX Biosensors as described above. For comparison, the " prior art cell lysis reagent" was used as a reference lysis reagent. The results are shown in Fig. 8.

As visible in Fig. 8, the capsid titers are very high for the alkaline lysis reagents containing a detergent according to the present disclosure, being between 4xl0¹² to 16xl0¹² capsids/mL compared to capsid titers of less than 2. OxlO¹² capsids/mL for the reference lysis reagent. These results were obtained consistently for different salt concentrations ranging from 275 to 1000 mM, incubation duration ranging from 0.5 to 3.5 h, and a pH ranging from 8 to 9. In the tested alkaline lysis reagents, Tween20 was used as a detergent and a salt tolerant nuclease was added to the lysis composition. Overall, the results indicate that the alkaline lysis reagents containing a detergent according to the present disclosure allowfor highly efficient cell lysis and increased yields of capsid titer.

Conclusions

Example 2 demonstrates that the alkaline lysis reagents containing a detergent according to the present disclosure address the following technical problems by implementing several key improvements: i) Increased yield: the alkaline cell lysis reagents containing a detergent ensure effective disruption of cells, enhancing the extractability of intracellular AAV vectors (see Fig. 7). 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. ii) Reduction of AAV vector aggregates: the higher titers indicate that the alkaline cell lysis reagents minimize formation of AAV vector aggregates, enabling easier purification process. iii) 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. iv) Shorter cell lysis process: The optimized cell lysis reagents 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. Overall, the improved viral vector extraction efficiency provided by the alkaline lysis reagents containing a detergent 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 3: Detergent-free alkaline lysis reagent for lysing HEK293 producing AAV2

In Example 3, alkaline lysis reagents without a detergent 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 supplemented with a salt active nuclease 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 alkaline cell lysis reagents are listed in Table 3 - all of which have an alkaline pH well above pH 7.0. Furthermore, the alkaline lysis reagents contain no detergent, and a salt tolerant nuclease was added to the lysis composition. As demonstrated below, the herein disclosed detergent-free alkaline 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)).

Table 3: Recipes of detergent-free alkaline cell lysis reagents with recommended nucleases AAV2 functional titer

The functional AAV2 titer was determined 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. The results are shown in Fig. 9.

As visible in Fig. 9, the functional titers are very high for the detergent-free alkaline lysis reagents according to the present disclosure, being between 6xl0⁷ to 9xl0⁷ TU/mL compared to functional titers of less than lxlO⁷ TU/mL for the reference lysis reagent. These results were obtained consistently for different salt concentrations ranging from 275 to 500 mM, incubation duration ranging from 0.5 to 3.5 h, and a pH of 8. In the tested alkaline lysis reagents, a salt tolerant nuclease were added to the lysis composition. Hence, the results indicate that the detergent-free alkaline lysis reagents according to the present disclosure allow for highly efficient cell lysis and increased yields of functional titer.

AAV2 capsid titer

The AAV2 capsid titer was determined after cell lysis and clarification using Octet® AAVX Biosensors as described above. For comparison, the "prior art cell lysis reagent" was used as a reference lysis reagent. The results are shown in Fig. 10.

As visible in Fig. 10, the capsid titers are very high for the detergent-free alkaline lysis reagents according to the present disclosure, being between 8xl0¹² to 15xl0¹² capsids/mL compared to capsid titers of less than 2xl0¹² capsids/mL for the reference lysis reagent. These results were obtained consistently for different salt concentrations ranging from 275 to 500 mM, incubation duration ranging from 0.5 to 3.5 h, and a pH of 8. In the tested alkaline lysis reagents, a salt tolerant nuclease was added to the lysis composition. Overall, the results indicate that the detergent-free alkaline lysis reagents according to the present disclosure allow for highly efficient cell lysis and increased yields of capsid titer.

Conclusions

Example 3 demonstrates that the detergent-free alkaline lysis reagents according to the present disclosure address the following technical problems by implementing several key improvements: i) Increased yield: the detergent-free alkaline cell lysis reagents ensure effective disruption of cells, enhancing the extractability of intracellular AAV vectors (see Fig. 9). 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 higheryields 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. ii) Reduction of AAV vector aggregates: the higher titers indicate that the alkaline cell lysis reagents minimize formation of AAV vector aggregates, enabling easier purification process. iii) Detergent-free: The reagents are formulated without any added detergents enabling their use in detergent-sensitive analytical and preparative applications. Furthermore, the detergent-free nature of the reagents eliminates the risk of detergent-related extraction and leaching of contaminants from packaging materials, tubing, vessels, and other equipment used in the manufacturing process. iv) 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. v) Shorter cell lysis process: The optimized cell lysis reagents 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.

Overall, the improved viral vector extraction efficiency provided by the detergent-free alkaline 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 4: Lysis reagents for whole cell culture lysis and cell pellet lysis

In Example 4, the lysis reagents according to the present disclosure 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 forthis 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 cell lysis reagents according to the present disclosure are listed below in Table 4. 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 addition, it is below demonstrated that the lysis reagents according to the present disclosure 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 4: Recipes of cell lysis reagents used in Example 4

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 CelLytic™ M (Cl), CelLytic™ MT (C2, both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. The results are shown in Fig. 11.

As visible in Fig. 11, 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 low functional titer of about 4xl0⁶ TU/mL. The 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 (Cl), CelLytic™ MT (C2, both Sigma Aldrich) and M-PER (C3, Thermo Fisher Scientific) lysis reagents. The results are shown in Fig. 12.

As visible in Fig. 12, 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 3x10¹¹ capsids/mL. The reagents of the present disclosure allowed for obtaining up to 7xl0ⁿ capsids/mL.

Overall, the results indicate that the lysis reagents according to the present disclosure 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. 13.

As visible in Fig. 13, 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 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. 14.

As visible in Fig. 14, 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 6x10¹¹ capsids/mL. The reagents of the present disclosure allowed for obtaining up to 7.5xlOⁿ capsids/mL.

Overall, the results indicate that the 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. 11, Fig. 12, Fig.13 and Fig. 14 indicate that the functional and capsid titers obtained with 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 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 acidic and alkaline lysis 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 4, 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 5:

Table 5: Evaluation of filterability and pH of crude lysis compositions from example 4. 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 5 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. 11 and Fig. 12), has shown moderate filterability.

Conclusions Example 4 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 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 5: Lysis reagents are effective and non-toxic

In Example 5, the efficacy of a Triton X-100 containing lysis reagent and the lysis reagents of the present disclosure 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 according to the present disclosure are listed below in Table 6. 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)).

Table 6: Recipes of cell lysis reagents used in Example 5

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. 15.

As shown in Fig. 15, the reagents of the present disclosure allowed for obtaining of 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 lysis reagents according to the present disclosure a I low 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. 16.

As shown in Fig. 16, the reagents of the present disclosure allowed for obtaining 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 lysis reagents according to the present disclosure 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 6, 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. 17.

The acquired phase contrast images in Fig. 17 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 5 demonstrates that the lysis reagents according to the present disclosure 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 7: Cell lysis for AAV8

In Example 7, HEK293 cells producing AAV8 were lysed in a single step protocol as described herein. The example demonstrates that the lysis approach is applicable to another AAV serotype (compared to Examples 1 to 6 wherein AAV2 was produced). The lysis approach yielded high AAV8 titers while keeping the host cell related impurities at a low level. Hence, the 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 7. 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 7: 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 8. Table 8: 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 8 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 lysis approach at pH 8 (see d) viable cells were still measured after the 60 min incubation (1.2%) indicating incomplete cell lysis. All other conditions resulted in essentially in full lysis. The pH measured in the compositions is around pH 8 (d) or around pH 4 (c, e).

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 HCP 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 HCP.

In conclusion, the example demonstrates applicability of the cell lysis approaches of the present disclosure for different AAV serotypes, indicating applicability for different viral vectors. Example 8: Applicability of cell lysis approach for Adenovirus

In Example 8, different lysis protocols were applied to cell broth producing adenovirus (Ad) and various parameters were measured. The example demonstrates that the lysis methods according to the present disclosure are 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 m Lof 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 9 were applied.

Table 9: Lysis conditions applied to cell broth for yielding Adenovirus.

Changes 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. 18. The viability is generally low except for the reference samples SO, SI. 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. 19, overall high capsid titers were measured. Slightly lower capsid titers were obtained for the single step cell lysis, pH 4 (see S8). For all other lysis methods, capsid titers between 1.5 to 2 x 10¹² capsids/mL were measured. Hence, the cell lysis approach yielded Ad capsid titers comparable to conventional methods used in the art (see S2 and Sil).

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 afterfiltration. In Figs.20 and 21 the impurities were set in relation to achieved titers. As shown in Figs. 20 and 21, the acidic lysis approach yielded lowest HCP contents (see S8). Also, the DNA content was at a relatively low level.. However, in the approaches disclosed herein hazardous substances such as Triton X-100 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 8 demonstrates that the lysis approaches are applicable for lysing cells producing adenovirus, highlighting the broad a ppi icability for different viral vectors. Hence, the lysis approaches are very efficient in cell lysis. Furthermore, the results highlight again that the alkaline lysis reagents maximize yields, whereas the acidic lysis reagents allow for minimizing host cell related impurities.

Conclusions

The Examples above demonstrate that the lysis reagents are very efficient in lysing cells to obtain increased capsid and functional viral vector titers. The lysis reagents are widely applicable to different cell cultures or samples, including cell broth and cell pellet but also various viral vector types. This allows for applying the lysis reagents directly to the cell culture, e.g. in the culture container, such as a bioreactor or shake flask. In addition, it was shown that relatively short incubation times between 0.5 to 3.5 h are sufficient for cell lysis, improving efficiency. At the same time, the results of the lysis reagents according to the present disclosure revealed different properties. Specifically, the alkaline lysis reagents (with and without the detergent, here Tween 20) allowed for obtaining maximal capsid and functional titers, wherein the titers are highest at higher salt concentrations, e.g. higher than 50 mM such as 275 mM, 500 mM and 1000 mM. Also, the acidic lysis reagents yielded high capsid and functional titers compared to reference lysis reagents but not as high as the alkaline lysis reagents. On the other side, the acidic lysis reagents have the advantage that these are filterable without using a nuclease and result in particularly low host cell-related impurities. Hence, while the acidic lysis reagents may not achieve highest capsid and functional titer, these lead to a purified viral vectorthat had less impurities compared to the alkaline based lysis reagents. In addition, the acidic lysis reagents do not require a nuclease digestion step and thus lead to simplified and less costly lysis processes. Finally, 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.

Claims

2. The method according to claim 1, wherein the cell culture is a cell broth or cell pellet, preferably a cell broth.

3. The method according to one or more of claims 1 or 2, wherein:

I. the buffer has a concentration selected from the following ranges:

0.05 M to 1 M for a lx lysis reagent;

0.3 M to 1.5 M for a 5x lysis reagent; or preferably, 1 M to 3 M for a lOx lysis reagent; and/or

II. the salt has a concentration selected from the following ranges:

0.05 M to 1 M for a lx lysis reagent;

0.3 M to 1.5 M for a 5x lysis reagent; or preferably, 0.8 M to 5 M or 1 M to 3 M for a lOx lysis reagent.

4. The method according to one or more of claims 1 to 3, 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+, having a concentration of 0.1 mM to 100 mM, preferably a concentration selected from the following ranges: i) 0.5 mM to 5 mM for a lx lysis reagent; ii) 5 mM to 15 mM for a 5x lysis reagent; or iii) preferably, 15 mM to 50 mM for a lOx lysis reagent.

5. The method according to one or more of claims 1 to 4, wherein the lysis reagent is an acidic lysis reagent, preferably having a pH of less than 6.0, preferably less than 5.0, most preferably less than 4.5.

6. The method according to claim 5, wherein in step (b) the lysis composition: i) has a pH of less than 5.0, preferably less than 4.5; and/or ii) has a pH selected from the range of 2.5 to 5.0, preferably 3.5 to 4.5.

7. The method according to one or more of claims 1 to 4, 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 higherthan 8.5.

8. The method according to claim 7, 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.

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 toxic and/or hazardous to the environment, having 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.

10. The method according to one or more of claims 1 or 9, wherein the lysis reagent is one of the following: i) an acidic lysis reagent selected from:

III. 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; 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).

11. The method according to one or more of claims 1 to 10, 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 claim 10, and/or the lysis composition has the characteristics of one of the following:

II. 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); or

III. 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; 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).

12. The method according to one or more of claims 1 to 11, wherein the method has one or more of the following features: i) 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; ii) wherein significant yields of capsid viral vector titer and/or transducing viral vector titer are obtained for a cell culture being a cell broth, wherein significant amounts are at least lxlO¹¹ capsids per mL and/or lxlO⁶ TU per mL; iii) wherein the viral vector is predominantly present intracellularly and preferably is selected from an adeno-associated virus (AAV) or an adenovirus (Ad); and/or iv) wherein the cell culture comprises a mammalian cell, preferably selected from a HEK293 cell or a derivative of a HEK293 cell.

13. The method according to one or more of claims 1 to 12, wherein the method has the following features: i) the cell culture comprises a mammalian cell, preferably selected from a HEK293 cell or a derivative of a HEK293 cell; ii) incubating step (c) comprises incubating for less than 4 hours, preferably for less than 2 hours; and iii) the lysis reagent does not comprise Triton X-100, preferably the lysis reagent does not comprise any non-ionic detergent that is toxic and/or hazardous to the environment.

14. 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 a cell culture according to one or more of claims 1 to 13; and x.4 optionally, purifying the released viral vector.

15. The method according to claim 14, 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.

16. A cell lysis reagent for releasing a viral vector from a cell culture, preferably a cell broth, selected from one of the following: i) an acidic lysis reagent selected from:

17. A kit for releasing a viral vector from a cell culture, preferably a cell broth, wherein the kit comprises a container that contains a cell lysis reagent according to claim 16, and preferably one or more of the following:

(i) a mammalian cell, preferably selected from a HEK293 cell or a derivative of a HEK293 cell; (ii) one or more plasmids for viral vector production;

(iii) a transfection reagent; and/or

(iv) a cell culture medium.