Purification process for viral vectors

Abstract

Herein is reported a method for separating recombinant viral particles from other compounds of a mammalian cell culture broth comprising the steps of adding a silica-based filter aid like diatomaceous earth (DE) or synthetic silica-based filter aid to a mammalian cell culture broth comprising recombinant viral particles at a weight ratio of more than 1:2 DE or synthetic silica-based filter aid / biological wet mass (BWM) to obtain a pre-filtration mixture and subjecting the pre-filtration mixture to alluvial filtration, whereby recombinant viral particles are separated from other compounds of the mammalian cell culture broth.

Description

[0001] Purification Process for Viral Vectors

[0002] The current invention is in the field of gene therapy. In more detail, herein is presented a method for the purification, especially the clarification, of therapeutic recombinant adeno-associated viral particles. of the Invention

[0003] Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene, to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of said therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used gene therapy vectors.

[0004] Bioprocessing of rAAV is complex and requires systematic and coordinated steps both in upstream as well as downstream processing. However, the use of traditional cultivation processes for the production of therapeutic rAAV does not support effective commercial manufacturing strategies. This is even more pronounced by the large size of rAAV as compared to therapeutic biomolecules, such as, e.g., antibodies. Additionally, rAAV are much more complex.

[0005] The bio-manufacturing process of therapeutic rAAV requires the insertion of the therapeutic transgene into the recombinant AAV capsid shell resulting in full rAAV particles, i.e. recombinant AAV particles comprising an encapsidated nucleic acid. However, a percentage of rAAV that do not contain the desired transgene (empty recombinant AAV particles, i.e. rAAV particles not comprising an encapsidated nucleic acid) are concomitantly produced, as well as partly filled rAAVs (partly filled recombinant AAV particles).

[0006] Thus, rAAV purification processing has its unique challenges to generate clinical products of high titer, high potency and high purity. There is a need for the development of efficient, scalable and GMP compatible clarification and purification processes for therapeutic rAAV production.

[0007] As such, an efficient clarification process helps rAAV manufacturers to fulfill their current and future challenges in the development of high quality therapies.

[0008] Current practices for clarification and purification of recombinant viral particles include the for example filtration using depth filters (Adams, B. et al., 2020, Biotechnol Bioeng 117(10):3199- 3211).

[0009] In addition, the use of alluvial filtration has been reported for lentiviral vector clarification (Labisch et al., 2021, J Biotechnology, 326: 11-20). WOOl / 48155 describes the use of diatomaceous earth to purify viruses obtained from cell lysate. This disclosure requires a step of cell lysis which creates a culture containing significant amounts of contaminating cellular debris, such as host cell DNA and protein.

[0010] US 110271700 reports on a method of purifying a supernatant containing viral vectors from a cell culture, comprising removing cells by filtering said cell culture using diatomaceous earth. Here it is emphasized that the cells should not be lysed prior to filtering.

[0011] Meierrieks et al. (2023, J Biotechnology 367: 31-41) report alluvial filtration with DE concentration of half the wet-cell weight or less, i.e. a DE:BWM ratio of 1 :2 (AAV2: 6.25 g / L) or less, such as 1 :8. But it also reports on increased loss of viral particles especially with increased DE:BWM ratios. The authors conclude that 0.181 mg DE per 1E10 AAV particles shall not be exceeded as upper limit as otherwise the process gets worse again. That is, as long as a ratio of 0.181 mg DE / 1E10 AAV particles was not exceeded, the particle loss could be limited to < 2% during clarification. When the DE concentration and incubation time were increased, particle loss rose with a quadratic term for both factors.

[0012] Summary of the Invention

[0013] The present invention overcomes the problems associated with the separation or purification of recombinant viral particles from cell cultures.

[0014] Herein is reported a method for the separation or / and purification of recombinant viral particles, especially rAAV, from cell cultures. The separation or / and purification is achieved by alluvial filtration for example with diatomaceous earth (DE) as filter aid.

[0015] The current invention is, at least in part, based on the unexpected finding that the purification by alluvial filtration is especially efficient for cell cultures when the DE is added to the cell culture at a certain ratio of DE to cells / biological wet mass (BWM). The viral particles can be thereby effectively purified from e.g. host cell protein / debris and host cell DNA. Importantly, also the recovery of the viral particles that include the viral genome (full rAAV particles, i.e. the therapeutic product) is very high.

[0016] Thus, one exemplary aspect of the invention is directed to a method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of: a) adding a filter aid, for example diatomaceous earth (DE), to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 filter aid / biological wet mass (BWM) to obtain a pre-filtration mixture, b) subjecting the pre-filtration mixture to alluvial filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth.

[0017] One exemplary aspect of the invention is directed to a method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of a) adding diatomaceous earth (DE) to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1:2 DE / biological wet mass (BWM) to obtain a pre- filtration mixture, b) subjecting the pre-filtration mixture to alluvial filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth.

[0018] One exemplary aspect of the invention is directed to a method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of a) adding diatomaceous earth (DE) to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 DE / biological wet mass (BWM) to obtain a pre- filtration mixture, b) subjecting the pre-filtration mixture to filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth.

[0019] One exemplary aspect of the invention is directed to a method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of: a) adding diatomaceous earth (DE) to a mammalian cell culture broth, wherein the mammalian cell culture broth is a cellular lysate of the mammalian cell culture broth comprising the recombinant viral particles, intact cells and cell debris and the compounds of the mammalian cell culture broth are comprising intact cells and cell debris, at a ratio of more than 1 :2 DE / biological wet mass (BWM) to obtain a pre-filtration mixture, b) subjecting the pre-filtration mixture to alluvial filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth. One exemplary aspect of the invention is directed to a use of a filter aid, for example diatomaceous earth (DE), to separate recombinant viral particles from (other) compounds of a mammalian cell culture broth, wherein diatomaceous earth (DE) is added to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 DE / biological wet mass (BWM) to obtain a pre-filtration mixture and subjecting the pre-filtration mixture to alluvial filtration (and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth).

[0020] In one embodiment the DE is added at a ratio of about 2:3 to about 3:4 DE / BWM. In one embodiment the DE is added at a ratio of about 2:3 or about 3:4 DE / BWM. In one preferred embodiment the DE is added at a ratio of about 2:3 DE / BWM.

[0021] In one embodiment DE / BWM has the unit of DE / L / %BWM.

[0022] In one embodiment the mammalian cell culture broth is a cellular lysate. In one embodiment the mammalian cell culture broth is a cellular lysate of the mammalian cell culture broth comprising the recombinant viral particles, intact cells and cell debris and the compounds of the mammalian cell culture broth are comprising intact cells and cell debris.

[0023] In one embodiment the cellular lysate further comprises host cell DNA.

[0024] In one embodiment the separating is from compounds comprising intact cells, cell debris and nonsoluble particles. In one embodiment the separating is further from host cell DNA or wherein the clarifying further results in reducing of host cell DNA.

[0025] In one embodiment the separating is a purifying. In one embodiment the purifying or clarifying is a reduction of turbidity. In one embodiment the reduction of turbidity is by at least 75 %. In one embodiment the reduction of turbidity is by at least 80%. In one embodiment the reduction of turbidity is by at least 85 %. In a preferred embodiment the reduction of turbidity is by at least 90 %.

[0026] In one embodiment the purifying is a reduction of (the concentration or amount of) host cell DNA. In one embodiment the (concentration or amount of) host cell DNA is reduced by at least 80 %. In one embodiment the (concentration or amount of) host cell DNA is reduced by at least 85 %. In a preferred embodiment the (concentration or amount of) host cell DNA is reduced by at least 90 %. In one embodiment the (concentration or amount of) host cell DNA is reduced by at least 95 %.

[0027] In one embodiment the viral genome recovery is at least 75 %. In one embodiment the viral genome recovery is at least 80 %. In one embodiment the viral genome recovery is at least 85 %. In a preferred embodiment the viral genome recovery is at least 90 %. In one embodiment the viral genome recovery is at least 95 %.

[0028] In a preferred embodiment the viral capsid recovery is at least 80 %. In one embodiment the viral capsid recovery is at least 85 %. In one embodiment the viral capsid recovery is at least 90 %.

[0029] In one embodiment the turbidity reduction is by at least 90 % and the reduction of host cell DNA is by at least 90 %. In one embodiment the turbidity reduction is by at least 90 %, the reduction of host cell DNA is by at least 90 % and the viral genome recovery is at least 90 %. In one embodiment the turbidity reduction is by at least 90 %, the reduction of host cell DNA is by at least 90 %, the viral genome recovery is at least 90 % and the capsid recovery is at least 80 %.

[0030] In one embodiment the cellular lysate is obtained by lysis and wherein the lysis is a chemical lysis, an enzymatic lysis or a physical lysis or a mixture thereof.

[0031] In one embodiment the volume of the mammalian cell culture broth or the cellular lysate is 50 L or more. In one embodiment the volume of the (crude) mammalian cell culture broth is in the range of and including 50L to lOOOL. In one embodiment the volume of the mammalian (crude) cell culture broth or the cellular lysate is 50 L or more, 75 L or more, or 100 L or more. In one embodiment

[0032] In one embodiment the mammalian cell culture broth is obtained by cultivating transiently transfected mammalian cells producing recombinant viral particles. In one embodiment the (crude) mammalian cell culture broth is obtained by cultivating stably transfected mammalian cells producing recombinant viral particles.

[0033] In one embodiment the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle.

[0034] In one embodiment the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 2 or AAV 8 serotype. In one embodiment the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 2 serotype. In one embodiment the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 8 serotype.

[0035] In one embodiment the mammalian cell is a HEK(293) cell or a CHO cell. In a preferred embodiment the mammalian cell is a HEK(293) cell. In one embodiment the mammalian cell is a CHO cell. In one embodiment the mammalian cell is a CHO KI cell. In one embodiment after step b) the method further comprises the following step: c) subjecting the alluvial filtrate obtained in step b) to a sterile filtration.

[0036] In one embodiment step b) or / and step c) further comprises: recovering the filtrate.

[0037] In one embodiment steps a) or / and b) are performed in a fully closed system.

[0038] In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. As such, the particular features presented herein can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0039] Description of the Figures

[0040] Figure 1 Setup of process for addition of the filter aid (DE) to the bioreactor and rAAVp clarification using alluvial filtration.

[0041] Figure 2 Capacity in L m2and Flux in L m"2h-1for alluvial filtration with different DE:BWM ratios ranging from 1 :4, 1 :3 and 1 :2 up to a ratio of 2:3. For the ratio 1 :2, two filtrations were carried out. Therefore, the average and standard deviation were calculated and displayed in the figure. The “CH 33P” indicates the filter sheet and “C 300” indicates the DE grade which were used.

[0042] Figure 3 Turbidity and host cell (HC) DNA reduction in % for alluvial filtration with different DE:BWM ratios ranging from 1 :4, 1 :3 and 1 :2 up to a ratio of 2:3. For the ratio 1 :2, two filtrations were carried out. Therefore, the average and standard deviation were calculated and displayed in the figure. The “CH 33P” indicates the filter sheet and “C 300” indicates the DE grade which were used.

[0043] Figure 4 Turbidity reduction in percent of the different filters tested for AAVv clarification. For alluvial filtration (Purafix CH33P + Cel pure C300), the average values and standard deviation of experiments performed in various scales were calculated. A DE:BWM ratio of 2:3 was used in these experiments. Filters, where a white bar displays the turbidity reduction, were deemed not suitable, as the turbidity reduction is lower than 90 %. Figure 5 Host cell (HC) DNA reduction in percent of the different filters tested for AAVv clarification. For alluvial filtration, the average values and standard deviation of experiments performed in various scales were calculated. A DE:BWM ratio of 2:3 was used in these experiments. Filters, where a white bar displays the HC DNA reduction, were deemed not suitable, as the HC DNA reduction is lower than 90 %. The bar that is displayed grey hatched (PDP8), belongs to the filter which was already disqualified in the previous step (regarding turbidity reduction, see Figure 4). No HC DNA data is available for the TfDf module.

[0044] Figure 6 Viral genome (vg) recovery in percent of the different filters tested for AAVv clarification. For alluvial filtration, the average values and standard deviation of experiments performed in various scales were calculated. A DE:BWM ratio of 2:3 was used in these experiments. Filters, where a white bar displays the vg recovery, were deemed not suitable, as the vg recovery is lower than or equal to 80 %. The bar that is displayed grey hatched (TFDF-30), belongs to the filter which was already disqualified in the previous step (regarding turbidity and HC DNA reduction, see Figure 4 and Figure 5).

[0045] Figure 7 Capsid (cp) recovery in percent of the different filters tested for AAVv clarification. For alluvial filtration, the average values and standard deviation of experiments performed in various scales were calculated. A DE:BWM ratio of 2:3 was used in these experiments. Filters, where a white bar displays the cp recovery, were deemed not suitable, as the cp recovery is lower than or equal to 80 %. The bars that are displayed grey hatched (Clarisolve 20MS, Millistak+® HC Pro DOSP, PDP8 + V100P and TFDF-30), belong to filters which were already disqualified in the previous step (regarding turbidity and HC DNA reduction as well as vg recovery, see Figure 4, Figure 5 and Figure 6).

[0046] Detailed Description of the Invention

[0047] The current invention is in the field of gene therapy. More precisely herein is reported a method for the separation / purification / production / clarification of recombinant adeno-associated virus particles (rAAVp), wherein the producing cells (e.g., HEK 293 cells) have been cultivated for a transient or stable production of the rAAVp.

[0048] During the fermentation, i.e. production phase, the rAAVp producing cells express adeno- associated viral particles after transfection or induction. The rAAVp are partly released from the cells - but in most cases not completely. Therefore, the cells are lysed at the end of the production phase using, e.g., detergent agents. Thereby a lysed cell culture broth is obtained. The resulting lysed cell culture broth is a mixture of rAAVp, host cell proteins, DNA, cell debris and all other substances and particles derived from the producing cells (e.g. HEK cells), the cultivation medium, i.e. all compounds used during the production of the rAAVp. Most of the compounds, such as, e.g., host cell DNA, have a negative impact on the purification of the rAAVp, especially the chromatography steps.

[0049] After the cell lysis, the next step in downstream processing of rAAVp cultivation broths is the primary clarification, the so-called “harvest”. The purpose of this unit operation is the removal of preferably cell debris (measured as turbidity), but also host cell proteins and DNA.

[0050] Herein is reported a method for separating recombinant viral particles from other compounds of a mammalian cell culture broth comprising the steps of adding diatomaceous earth (DE) to a mammalian cell culture broth comprising recombinant viral particles at a weight ratio of more than 1 :2 DE / biological wet mass (BWM) to obtain a pre-filtration mixture and subjecting the prefiltration mixture to alluvial filtration, whereby recombinant viral particles are separated from other compounds of the mammalian cell culture broth.

[0051] DEFINITIONS

[0052] Unless otherwise defined herein, scientific and technical terms used in connection with the current invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0053] Useful methods and techniques for carrying out the current invention are described in e.g. Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N. Y., VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987). The content of which is incorporated herein by reference,

[0054] The use of recombinant DNA technology enables the generation of derivatives of a nucleic acid. Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can, for example, be carried out by means of site directed mutagenesis. Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0055] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0056] The term “about” denotes a range of + / - 20 % of the following numerical value. In certain embodiments, the term about denotes a range of + / - 10 % of the thereafter following numerical value. In certain embodiments, the term about denotes a range of + / - 5 % of the thereafter following numerical value.

[0057] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The term “comprising” also encompasses the term “consisting of’. The present disclosure also contemplates other embodiments “comprising”, “consisting of’ and “consisting essentially of’ the embodiments or elements presented herein, whether explicitly set forth or not.

[0058] The terms “empty recombinant AAV particle” and “empty rAAVp”, which can be used interchangeably, denote a protein shell composed of adeno-associated capsid polypeptides without a therein encapsidated / packaged functional nucleic acid (rAAVp = recombinant adeno-associated virus particle). That is, an empty rAAVp either may be free of encapsidated nucleic acid or comprises a nucleic acid or part thereof that is not transcribed at all or not transcribed into a functional transcript. Accordingly, an empty rAAVp does not function to transfer a nucleic acid that encodes a functional protein or is transcribed into a functional transcript of interest into a target cell. In certain embodiments of all aspects and embodiments, the functional protein or the functional transcript of interest has a therapeutic effect.

[0059] The terms “full recombinant AAV particle” or “full rAAVp”, which can be used interchangeably, denote a non-covalent complex formed of a protein shell composed of adeno-associated capsid polypeptides and a therein encapsidated / packaged functional nucleic acid sequence. That is, a full rAAVp comprises a nucleic acid that is transcribed into a functional transcript. Accordingly, the full rAAVp functions to transfer a nucleic acid that encodes a protein or is transcribed into a transcript of interest into a target cell. In certain embodiments, a functional nucleic acid comprises at least one coding nucleic acid sequence interspaced between two adeno-associated viral inverted terminal repeats (ITRs).

[0060] The term “full to empty ratio” denotes the mathematical ratio of the number of full recombinant AAV particles (full rAAVp) to the total number of recombinant AAV particles (sum of full rAAVp and empty rAAVp) in a sample or in a recombinant AAV particle preparation. As the number of full rAAVp can be at most the same as the total number of rAAVp, the ratio can be at most 1. Generally, the ratio is less than 1 and is expressed as a percentage. The number of full rAAVp can be determined by determining the number of nucleic acid sequences interspaced between two AAV ITRs in the sample or preparation. This can be done by PCR, especially digital droplet PCR (ddPCR) or quantitative PCR (qPCR). The total number of rAAVp can be determined by determining the number of protein shells formed of adeno-associated capsid polypeptides in the sample or preparation. This can be done by ELISA, especially by a capsid polypeptide specific ELISA.

[0061] The terms “recombinant AAV vector" or “rAAVv” or “transgene”, which can be used interchangeably herein, denote a nucleic acid derived from a wild-type genome of an adeno- associated virus, wherein except for the ITR (adeno-associated virus Inverted Terminal Repeat) sequences all endogenous AAV nucleic acids are replaced by one or more exogenous nucleic acid(s). For example, such an exogenous nucleic acid can be a nucleic acid transcribed into a transcript of interest or that encodes a therapeutic protein or a therapeutic nucleic acid. Typically, for a rAAVv one or both ITR sequences of the wild-type AAV genome are retained. Thus, a rAAVv can be distinguished from a wild-type AAV vector, since all or at least a part of the viral genome has been replaced with a non-native (i.e. exogenous) nucleic acid with respect to the virus. Incorporation of a non-native nucleic acid therefore defines the AAV vector as a "recombinant" vector. It has to be pointed out that the serotype of the ITRs in the rAAVv does not need to be the same as the serotype of the adeno-associated capsid polypeptides forming the shell of the rAAVp comprising said rAAVv.

[0062] In principle, any non- AAV nucleic acid can be packaged into a shell composed of adeno-associated capsid polypeptides resulting in a rAAVp, e.g. for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo.

[0063] As used herein, the term "serotype" is used to classify different wild-type and recombinant AAV particles based on the amino acid sequence of the polypeptides forming the protein shell (capsid) of the respective AAV particle. Originally, serologic distinctiveness was determined based on the lack of cross-reactivity between antibodies to one AAV particle as compared to another AAV particle. Such cross-reactivity differences are usually due to differences in capsid polypeptide sequences and the respective antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference or wild-type AAV or other AAV serotype, they differ by at least one amino acid residue compared to the reference or wild-type or other AAV serotype.

[0064] Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new AAV particle has no serological difference, this new AAV particle would be a subgroup or variant of the corresponding wild-type serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype.

[0065] The term “vector" denotes the portion of a larger nucleic acid, e.g. of a recombinant plasmid, that is ultimately packaged or encapsulated or encapsidated either directly or in form of a single strand or in form of RNA into a protein shell composed of adeno-associated virus capsid polypeptides to form a rAAVp. In cases where recombinant plasmids are used to construct or manufacture rAAVps, the viral particle does not include the portion of the "plasmid" that does not correspond to the vector part of the recombinant plasmid. For example, in case of a rAAVp the recombinant vector comprises that part of the recombinant plasmid that is interspaced between two AAV ITRs. The non-vector portion of the recombinant plasmid is referred to as the "plasmid backbone". The plasmid backbone is important for cloning and amplification of the plasmid, a process that is needed for propagation and recombinant virus production, but is not itself packaged or encapsulated or encapsidated into the recombinant AAV particle. Thus, a “vector" refers to the nucleic acid that is packaged or encapsulated or encapsidated by a protein shell composed of adeno-associated virus capsid polypeptides, i.e. in a rAAVp.

[0066] The term “filter aid”, also known as filtration aid, or filter material or filter media denotes substances or materials used to enhance the efficiency of filtration processes. A filter aid functions as an agent composed of solid particles that improve filtering efficiency by creating a porous, permeable, and rigid lattice structure known as the filter cake. Examples of filter aids are for example perlite, cellulose or silica-based filter aids, like diatomaceous earth (DE) or synthetic silica-based filter material / aid.

[0067] Silica-based filter aid comprise silicon dioxide (SiO2) as main component. Also mixtures of different filter aids are possible.

[0068] WO 1999 / 11764 reported methods for generating high titer helper-free preparations of recombinant AAV vectors. Not further defined AAV producer cells grown in suspension in bioreactors were infected with Adenovirus Type 5 (Ad5) at a multiplicity of infection (MOI) of 10 in low serum media at 1.5 L scale at different pH values. At a culture pH of 7.2, 4.7 E+12 total particles were obtained, at a culture pH of 7.4 1.95 E+13 total particles were obtained, at a culture pH of 7.6 1.84 E+13 total particles were obtained and at a culture pH of 8.0 1.63 E+13 total particles were obtained. The cultivation was performed in a 1.5 L bioreactor and, thus, the cultivation volume can be calculated (75 % of the nominal value) to have been about 1.125 L. Therefore the total particle number correspond to 4.2 E+09 vp / mL (pH 7.2), 1.7 E+10 vp / mL (pH 7.4), 1.6 E+10 vp / mL (pH 7.6) and 1.5 E+10 vp / mL (pH 8), respectively.

[0069] WO 2000 / 14205 reported the production of AAV particles in a non-defined cell type denoted as JL-14 cells by co-infection with adenoviral helper virus, whereby at a pH value of 7.4 the highest number of AAV particles (sum of intracellular and secreted AAV particles), at a pH value of 8 AAV particles with the highest infectivity and at a pH of 7.6 the highest ratio of number of AAV particles to infectivity was obtained. The cultivation was performed in a volume of 1.5 L medium and, thus, the total particle number correspond to 3.0 E+09 vp / mL (pH 7.2), 1.3 E+10 vp / mL (pH 7.4), 1.2 E+10 vp / mL (pH 7.6), 3.3 E+09 vp / mL (pH 7.8) and 1.1 E+10 vp / mL (pH 8), respectively. Based on the provided infectivity data it can be assumed that the full / empty ratio of the thereby produced rAAV particles is below 1 %.

[0070] Piras, B.A., et al. (Mol. Ther. Meth. Clin. Dev. 3 (2016) 16015) compared distribution of AAV8 in cell culture media and lysates on days 3, 5, 6 and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3.

[0071] Piras et al. employed adherent HEK293T / 17 cells cultured in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum supplemented with 2 mmol / 1 GlutaMAX (Life Technologies, Grand Island, NY). AAV was produced by two-plasmid transfection using PEIpro(TM) (Polyplus-transfection SA, Illkirch, France) 1 day after seeding cells at a density of 7.26x 1 E+04 cells / cm2.

[0072] Powers, A.D., et al. (Hum. Gene Ther. Meth. 27 (2016) 112-121) reported the development and optimization of AAV hFIX particle production by transient transfection in an iCELLis(R) fixed- bed bioreactor. A yield to as high as 9 E+14 viral particles per square meter of fixed bed were obtained. On day 3 after inoculation with HEK293T / 17 cells, the vessel was transfected with plasmid scAAV-LPl-hFIXco-helpv3 and plasmid CR21+LTAAV help 2-8 at a plasmid mass ratio of 3: 1, respectively, using polyethylenimine (PEIpro(TM) Transfection Reagent Cat #115-375; Polyplus) in IMDM (Lonza) or DMEM supplemented with 10% FBS and 6 mM GlutaMAX(TM). The PEI and DNA solutions were combined at a 2: 1 ratio.

[0073] Poulain, A., et al. (J. Biotechnol. 255 (2017) 16-27) reported rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch. Cells were transfected using linear polyethylenimine (PEIpro(TM)) from Polyplus-Transfection (Illkirch, France). On the day of transfection, cells were suspended at a density of 2 x 1 E+06 cells / mL in CD DG44 medium (Life Technologies Inc., Burlington, ON, Canada), supplemented with 4 mM glutamine and 0,1% Kolliphor® P 188. The cell suspension was distributed in 6-well plates (1.8 mL / well). The DNA:PEIpro(TM) complexes were prepared at a ratio of 1 :5 (w:w), with a total of 2 pg DNA per well to transfect in 100 pL of complete culture medium.

[0074] WO 2017 / 096039 reported scalable methods for producing recombinant AAV vectors in serum- free suspension cell culture systems suitable for clinical use. Production of rAAV vectors was performed in bioreactors with HEK293F cells using triple transfection at a cell density of 1 E+06 cells / mL (1.000.000 cells / mL) with a plasmid ratio of 1 : 1 : 1 and a PEI-based transfection reagent (PEI / DNA weight ratio of 2: 1 with 'A of PEI as free PEI) at a temperature of 37 °C and a pH value of 7.2.

[0075] Nyamay’antu, A., et al. (Cell Gen. Ther. Ins. 4 (2018) 71-79) reported that PEI is widely used due to its affordability and high DNA delivery efficiency, in both adherent and suspension cells grown in serum-free medium. PEIpro(TM) is suited for small- to large-scale production of various viruses, notably AAV particles. In stirred-tank bioreactors using HEK293 or HEK293T cells titers in the range of 0.8-1.5 E+09-E+10 vg / mL can be obtained.

[0076] Koo, T., et al. (Nat. Commun. 9 (2018) 1855) reported that CRISPR-LbCpfl prevents choroidal neovascularization in a mouse model of age-related macular degeneration. To produce AAV vectors, they were pseudotyped in AAV9 capsids. HEK293T cells (ATCC, CRL-3216) were transfected with pAAV-ITR-LbCpfl-crRNA, pAAV2 / 9 encoding for AAV2rep and AAV9cap, and helper plasmid. HEK293T cells were cultured in DMEM with 2% FBS. Recombinant pseudotyped AAV vector stocks were generated using PEI coprecipitation with PEIpro(TM) (Polyplus-transfection) and triple-transfection with plasmids at a molar ratio of 1 : 1 : 1 in HEK293T cells. After 72 h of incubation, cells were lysed and particles were purified by iodixanol stepgradient ultracentrifugation. The Rep proteins from AAV2 are commonly and nearly exclusively used in the production of rAAVps derived from the serotypes AAV1 to AAV13 (Daya, S., and Berns, K.I., Clin. Microbiol. Rev. 21 (2008) 583-593; Zincarelli, C., et al., Mol. Ther. 16 (2008) 1073-1080).

[0077] WO 2019 / 094253 reported means and methods for preparing viral vectors and uses thereof. Adherent HEK293 cells were cultivated in bioreactors at a pH value of 7.23 and triple transfected (plasmid ratio 1 :1 : 1) with PEI / DNA at a PEI-plasmid ratio of about 1 : 1 by weight.

[0078] Collaud, F. et al. (Mol. Ther. Meth. Clin. Dev. 12 (2019) 157-174) reported titers for recombinant AAV8 particles of 6.0 ± 1.89 E+04 vg / cell and 1.77 ± 1.37 E+04 vg / cell for (single stranded) and (self-complementary) AAV, respectively, for adherent HEK293 cells. A fully scalable method based on triple transfection of HEK293 cells cultured in suspension was also reported. Triple transfection of HEK293 cells was performed with polyethylenimine (PEIpro(TM), Polyplus) directly in 10 L bioreactors. AAV vectors were recovered from both supernatant and cells by mild detergent lysis followed by AVB Sepharose affinity column purification. Purified vectors were then concentrated and tested for quality and potency. No information about the pH value and obtained titers are provided.

[0079] Nyamay’antu, A., et al. (Cell Gen. Ther. Ins. 6 (2020) 655-661) reported that the efficiency of the delivery process is essential to obtain a high number of producing cells. Of the existing transfection methods, the use of PEI-based transfection reagent is predominant in gene therapy as it combines affordability and compatibility for transfection of adherent and suspension cells. In comparison to the gold standard PEIpro(TM) used for viral vector manufacturing, FectoVIR(TM)-AAV has been found to improve significantly recombinant AAV2 particle production yield of both viral genome production and packaging efficiency in suspension cells of an rAAV2-GFP of up to 10-fold compared to PEIMax(TM) and up to 2-fold compared to PEIpro(TM), respectively, when each transfection reagent is used under the recommended conditions. In more detail, suspension HEK293T cells were transfected using the respective transfection reagent under the recommended conditions. rAAV2-GFP were harvested 72 hours post transfection. The obtained titer with VectoVIR(TM) is in the range of 1 E+04 to 4.5 E+04 vg / cell depending on the used volume of complexation (l%-10%) corresponding to 1 E+12 vg / mL. The respective functional titers are about 2-8 E+08 TU / mL. The results are almost independent of the employed cultivation medium.

[0080] In a blog article entitled “Optimization of AAV production for high-yielding and scalable GMP processes with Catalenf ’ (www.polyplus-transfection.com) different transfection reagent to DNA ratios were tested with the two serotypes AAV9 (1 : 1 and 2: 1) and AAV2 (3:1.5 and 5:2.5). The AAV2 vector yield was not affected as notably, with a 4-5-fold increase in the vector genome titer and a 3-6-fold increase in the viral particle titer with FectoVIR(TM)-AAV as compared to PEIpro(TM). These results show that improvement in yield may vary with the AAV serotype. In a further study comparing additional AAV2 and AAV5 vectors (different from the previous AAV2 and AAV5 vectors) and using a DoE approach to optimization, experiments were conducted varying transfection reagent to DNA ratios (3:2, 3: 1.5) and plasmid DNA molar ratios (1 : 1 : 1, 2: 1 :2, 1 :2: 1) were performed. A 3-5-fold increase for AAV2 and a 1.1-1.6-fold increase for AAV5 in the vector genome titer with FectoVIR(TM)-AAV compared to PEIpro(TM) was observed. The viral particle titer increased 3.5-4.5-fold for AAV2 and 2.5-3.75-fold for AAV5. Reagent-to-DNA ratios of 2: 1 and 1.5: 1 and plasmid ratios of 1 : 1 : 1 to 2: 1 :2 to 1 :2: 1 were used. The obtained titer with VectoVIR(TM) was in the range of 4 E+l 1 to 1 E+12 vg / mL.

[0081] Rossi, A. and Peigne, C-M. (Cell Culture Dish Article May 17, 2021) outlined that, typically, AAV production titers are around 1 E+l 1 to 1 E+12 in vg / mL and 1 E+08 to 1 E+09 TU / mL.

[0082] Wosnitzka, K., et al. (Cell Gen. Ther. Ins. 7 (2021) 1-7) reported that analysis of physical titers revealed a 3-fold increase in both viral particles (VP) and viral genome (VG) per ml of cell culture when using FectoVIR(TM)-AAV transfection reagent compared to PEIpro(TM).

[0083] Porte, M., et al. (poster entitled “Next-Generation Transfection Reagent for Large Scale AAV Manufacturing”, Polyplus, Illkirch, France) reported the transfection of suspension-HEK293T cells with the optimal conditions for the other PEI- based reagent (1.5 pg / million cells, ratio DNA : PEI of 1 pg : 4 pL) and FectoVIR(TM)-AAV (1 pg / million cells, ratio DNA : reagent of 1 pg : 1 pL) following the recommended protocol for each reagent. A titer of about 5 E+l 1 vg / mL versus 1.5 E+l l vg / mL using FectoVIR(TM) and PEI-based transfection reagent, respectively, with a packaging efficiency of 20 % vs. about 13.5 %, respectively, was obtained.

[0084] Nakamura et al. (Eur. J. Haematol. 73 (2004) 285-294) reported about the development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components. It is outlined that adeno-associated virus (AAV) vector systems have several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, Nakamura et al. explored the possibility of generating AAV packaging cell line, 293T R / C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep / Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. They showed that the 293T R / C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep / Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R / C. VA.E2A.E4. cells might be able to tolerate the Rep / Cap and E4 gene products, or have less copy numbers of the Rep / Cap and E4 genes than the traditional method. Moreover, they showed that the AAV vectors derived from 293T R / C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R / C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.

[0085] WO 2018 / 192983 reported an adeno-associated virus (AAV) producer cell comprising nucleic acid sequences encoding rep / cap gene; helper virus genes; and the DNA genome of the AAV vector particle, wherein said nucleic acid sequences are all integrated together at a single locus within the AAV producer cell genome. Reported are also nucleic acid vectors comprising a nonmammalian origin of replication and the ability to hold at least 25 kilo bases (kb) of DNA, characterized in that said nucleic acid vector comprises nucleic acid sequences encoding: rep / cap gene, and helper virus genes as well as uses and methods using said nucleic acid vector in order to produce stable AAV packaging and producer cell lines.

[0086] WO 2018 / 194438 reported a cell line for producing a non-replicating adenovirus, and a preparation method therefor and, more specifically, to: a cell line for producing a replicationdeficient adenovirus by expressing any one or more selected from an El protein, and an E1A protein or an E1B protein of an adenovirus; and a method for preparing the same. In addition, it is reported the use of the cell line, for expressing any one or more selected from an El protein, and an El A protein or an E1B protein of an adenovirus.

[0087] WO 2020 / 078953 reported adeno-associated virus (AAV) vector producer cell comprising nucleic acid sequences encoding AAV rep and cap genes, helper virus genes, and a DNA genome of the AAV vector; the AAV rep gene comprising an intron, the intron comprising a transcription termination sequence with a first recombination site located upstream and a second recombination site located downstream of the transcription termination sequence; and the nucleic acid sequences all integrated together at a single locus within the AAV vector producer cell genome.

[0088] WO 2020 / 132059 reported a mammalian cell line for producing adeno-associated virus (AAV), suitably including nucleic acids encoding helper genes and AAV genes, under the control of derepressible promoters. The disclosure also relates to isolated nucleic acid molecules that encode such genes, as well as methods of using the mammalian cells for producing AAVs. Especially is reported a mammalian cell for producing an adeno-associated virus (AAV), comprising (a) a nucleic acid molecule encoding a viral helper gene under control of a first derepressible promoter; (b) a nucleic acid molecule encoding an AAV gene under control of a second derepressible promoter; and (c) a nucleic acid molecule encoding a repressor element of the first and the second derepressible promoters. EP 3 822 346 reported the use of an engineered mammalian packaging cell line for producing recombinant virus particles, wherein the cell line is engineered to lack cell surface expression of heparan sulfate. Further disclosed are a method for producing recombinant virus particles and a recombinant virus particle obtainable by the method. Further disclosed is a mammalian packaging cell line deposited under number DSM ACC3355 or DSM ACC3356.

[0089] WO 2022 / 112218 reported methods for the production of Adeno-associated virus (AAV), comprising steps of providing a stable AAV producer cell line in which at least some or all genes encoding the components necessary for the production of AAV are stably integrated into the cell genome, and culturing said cells in perfusion culture during the AAV production step (i.e., during the N step), wherein said perfusion culture encompasses continuous replacement of spent media with fresh media, and wherein said continuous replacement of spent media with fresh media continues after the induction of AAV production. In the cell at least (a) a gene encoding the AAV Rep protein Rep78 or Rep68, (b) a gene encoding the AAV Rep protein Rep52 or Rep40; (c) the genes encoding the adenoviral helper functions E4orf6 and E2A stably integrated into the host cell genome. Further at least the following genes are stably integrated into the host cell genome (a) the genes encoding the AAV Cap proteins VP1, VP2, VP3; (b) a gene encoding the AAV Rep protein Rep78 or Rep68; (c) a gene encoding the AAV Rep protein Rep52 or Rep40; (d) the genes encoding the adenoviral helper functions E4orf6 E2A; (e) the gene of interest flanked by AAV ITRs.

[0090] WO 2022 / 173944 reported methods for producing an adeno-associated virus (AAV) in an El complementary producer cell. Especially is reported a method of producing an adeno-associated virus (AAV) in an El complementary producer cell, comprising (a) transfecting the El complementary producer cell with one or more vectors comprising (1) an El A adenovirus helper gene; (2) an adenovirus helper gene selected from E2A, E4, or both; (3) a viral-associated, noncoding RNA (VA RNA); and (4) an AA V gene selected from Rep, Cap, or both; (b) culturing the transfected El complementary producer cell under conditions suitable for producing the AAV; and (c) purifying the AAV from the cultured El complementary producer cell, thereby obtaining the AAV.

[0091] WO 2022 / 192261 reported compositions and methods for producing and characterizing stable viral vector producer cell lines that enable industrial scale production of viral vectors. Novel viral vector genome constructs, in which the constructs can be precisely mapped and viral vector genome constructs precisely quantified, are also disclosed for efficient production and characterization of viral vectors in mammalian cells.

[0092] WO 2023 / 077078 reported recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines which have been engineered to reduce expression and / or activity of one or more genes and / or proteins to increase rAAV titers. Especially is reported a recombinant adeno- associated virus (rAAV) packaging and / or producer cell line comprising cells in which the expression of a gene selected from the group consisting of RIG-1 (DDX58), IFIT3, MDA5 (IFIH1), CGAS (cGAS), CHUK (IKK-a), DDX41, DHX58 (LGP2), IFI6, IKBKB (IKK-a), IRF3, IRF7, MAVS, MYD88, NFKB1, NFKB2, TBK1, TRIP, and TRIM25, and any combination thereof is reduced compared to that in control parental cells.

[0093] WO 2023 / 102549 reported systems for increasing AAV particle production. These systems comprise producer cell lines adapted for the production of AAV particles, as well as methods of producing AAV particles using said producer cell lines. Also provided are AAV particles produced by said production systems, producer cell lines and methods. Especially it is reported a genetically engineered producer cell line in which the expression of at least one of TMED10, M0N2, TMED2, HS2ST1, C3orfS8, SPPL3, SURF4, LSMS, ARF1, and PI4KB is reduced as compared to a control cell line, and / or in which the expression of at least one of B4GALT7, B3GAT3, OAF, EXT2, C0MMD3, SLC3SD1, B3GALT6, SDC1, NDST1, RAC1, CSK, GLCE, PDCL, FAM20B, TM4SFS, DGAT2, POMT1, YY1, and DPF2 is increased as compared to a control cell line.

[0094] WO 2023 / 114897 reported methods for the production of recombinant adeno-associated virus (rAAV) particles. These methods are particularly useful for the large-scale production of AAV particles. Especially it is reported A method for producing recombinant AAV (rAAV) particles, comprising (a) introducing into a mammalian cell a first polynucleotide comprising an rAAV genome, to generate an AA V producer cell; (b) culturing the AA V producer cell in a first culture medium at a first temperature for a first period of time; (c) culturing the AAV producer cell in a second culture medium at a second temperature for a second period of time, wherein the second temperature is about 38°C to about 42°C, such that rAAV particles are produced by the AAV producer cell, wherein the rAAV particles comprise an rAAV genome comprising a transgene, and an AAV capsid comprising an AAV capsid protein.

[0095] WO 2023 / 166026 reported cell lines in which DNA fragmentation is inhibited, uses of cell lines in which DNA fragmentation is inhibited for the production of adeno-associated virus (AAV), related methods of producing AAV, and methods of producing AAV, comprising the step of exposing the cells in which AAV is produced to an inhibitor of DNA fragmentation during the AAV production phase.

[0096] WO 2023 / 171698 reported a producer cell for the production of an adeno-associated virus wherein cell damage is avoided or suppressed at the establishment of a cell line, a method for producing the producer cell, and a method for producing an AAV using the producer cell. In the producer cell a Cap gene under the control of a foreign promoter and a Rep gene under the control of a foreign promoter is integrated in the chromosome, that is free from a VA-RNA gene and / or an E4 gene, and that is a mammalian cell.

[0097] The content of all documents outlined in this section are expressly incorporated by reference herein.

[0098] RECOMBINANT CELL

[0099] Generally, for efficient as well as large-scale production of a rAAVp a cell expressing and, if possible, also secreting said rAAVp is used. Such a cell is termed “recombinant producer cell” or short “producer cell”.

[0100] For the generation of a recombinant producer cell a suitable mammalian cell is transfected with the nucleic acids required for producing said rAAVp, including the required AAV helper functions.

[0101] Generally, for expression of a coding sequence, i.e. of an open reading frame, additional regulatory elements, such as a promoter and a polyadenylation signal (sequence), are necessary. Thus, for functional transcription an open reading frame has to be and is operably linked to said additional regulatory elements. This can be achieved by combining these parts into a so-called expression cassette. The minimal regulatory elements required for an expression cassette to be functional in a mammalian cell are a promoter functional in said mammalian cell, which is located upstream, i.e. 5’, to the open reading frame, and a polyadenylation signal (sequence) functional in said mammalian cell, which is located downstream, i.e. 3’, to the open reading frame. Additionally a terminator sequence may be present 3’ to the polyadenylation signal (sequence). For expression, the promoter, the open reading frame / coding region and the polyadenylation signal sequence have to be arranged in an operably linked form.

[0102] Likewise, a nucleic acid that is transcribed into a non-protein coding RNA is called “RNA gene”. Also for expression of an RNA gene, additional regulatory elements, such as a promoter and a transcription termination signal or polyadenylation signal (sequence), are necessary. The nature and localization of such elements depends on the RNA polymerase that is intended to drive the expression of the RNA gene. Thus, an RNA gene is normally also integrated into an expression cassette.

[0103] In case of an rAAVp, which is composed of different (monomeric) capsid polypeptides and a therein encapsidated single stranded DNA molecule and which in addition requires other viral helper functions for production and encapsidation, a multitude of expression cassettes differing in the contained open reading frames / coding sequences are required. In this case, at least an expression cassette for each of the transgene, for the polypeptides forming the capsid of the rAAVp, for the required viral helper functions are required. Thus, individual expression cassettes at least for each of the helper functions El A, E1B, E2A, E4orf6, the rep and cap genes are required. HEK293 cells express the El A and E1B helper functions constitutively.

[0104] ADENO-ASSOCIATED VIRUS

[0105] For a general review of AAVs and of the adenovirus or herpes helper functions see, Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. In US 4,797,368 design considerations for constructing recombinant AAV vectors are described (see also WO 93 / 24641). Additional references describing AAV vectors are West et al., Virol. 160 (1987) 38-47; Kotin, Hum. Gene Ther. 5 (1994) 793-801; and Muzyczka J. Clin. Invest. 94 (1994) 1351. Construction of recombinant AAV vectors is described in US 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8 (1988) 3988-3996; Tratschin et al., Mol. Cell. Biol. 5 (1985) 3251-3260; Tratschin et al., Mol. Cell. Biol., 4 (1994) 2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al. J. Virol. 63 (1989) 3822-3828.

[0106] An AAV is a replication-deficient parvovirus. It can replicate only in cells, in which certain viral functions are provided by a co-infecting helper virus, such as adenoviruses, herpesviruses and, in some cases, poxviruses such as vaccinia. Nevertheless, an AAV can replicate in virtually any cell line of human, simian or rodent origin provided that the appropriate helper viral functions are present.

[0107] Without helper viral genes being present, an AAV establishes latency in its host cell. Its genome integrates into a specific site in chromosome 19 [(Chr) 19 (ql3.4)], which is termed the adeno- associated virus integration site 1 (AAVS1). For specific serotypes, such as AAV2 other integration sites have been found, such as, e.g., on chromosome 5 [(Chr) 5 (pl3.3)], termed AAVS2, and on chromosome 3 [(Chr) 3 (p24.3)], termed AAVS3.

[0108] AAVs are categorized into different serotypes. These have been allocated based on parameters, such as hemagglutination, tumorigenicity and DNA sequence homology. Up to now, more than 12 different serotypes and more than a hundred sequences corresponding to different clades of AAV have been identified.

[0109] The capsid protein type and symmetry determines the tissue tropism of the respective AAV. For example, AAV2, AAV4 and AAV5 are specific to retina, AAV2, AAV5, AAV8, AAV9 and AAV- rh.10 are specific for brain, AAV1, AAV2, AAV6, AAV8 and AAV9 are specific for cardiac tissue, AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 are specific for liver, AAV1, AAV2, AAV5 and AAV9 are specific for lung. Pseudotyping denotes a process comprising the cross packaging of the AAV genome between various serotypes, i.e. the genome is packaged with differently originating capsid proteins.

[0110] The wild-type AAV genome has a size of about 4.7 kb. The AAV genome further comprises two overlapping genes named rep and cap, which comprise multiple open reading frames (see, e.g., Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral. 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The Rep protein encoding open reading frame provides for four proteins of different size, which are termed Rep78, Rep68, Rep52 and Rep40. These are involved in replication, rescue and integration of the AAV. The Cap protein encoding open reading frame provides four proteins, which are termed VP1, VP2, VP3, and AAP. VP1, VP2 and VP3 are part of the proteinaceous capsid of the AAV particles. The combined rep and cap open reading frames are flanked at their 5'- and 3'-ends by so-called inverted terminal repeats (ITRs). For replication, an AAV requires in addition to the Rep and Cap proteins the products of the genes E1A, E1B, E4orf6, E2A and VA of an adenovirus or corresponding factors of another helper virus.

[0111] In the case of an AAV of the serotype 2 (AAV2), for example, the ITRs each have a length of 145 nucleotides and flank a coding sequence region of about 4470 nucleotides. Of the ITR’s 145 nucleotides 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure has the function of a primer during viral replication. The remaining 20, non-paired, nucleotides are denoted as D-sequence.

[0112] The wild-type AAV genome harbors three transcription promoters P5, Pl 9, and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571) for the expression of the rep and cap genes.

[0113] The ITR sequences have to be present in cis to the coding region. The ITRs provide a functional origin of replication (ori), signals required for integration into the target cell’s genome, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. The ITRs further comprise origin of replication like-elements, such as a Rep-protein binding site (RBS) and a terminal resolution site (TRS). It has been found that the ITRs themselves can have the function of a transcription promoter (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0114] For replication and encapsidation, respectively, of the viral single-stranded DNA genome an in trans organization of the rep and cap gene products is required.

[0115] The rep gene locus comprises two internal promoters, termed P5 and Pl 9. It comprises open reading frames for four proteins. Promoter P5 is operably linked to a nucleic acid sequence providing for non-spliced 4.2 kb mRNA encoding the Rep protein Rep78 (chromatin nickase to arrest cell cycle), and a spliced 3.9 kb mRNA encoding the Rep protein Rep68 (site-specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence providing for a nonspliced mRNA encoding the Rep protein Rep52 and a spliced 3.3 kb mRNA encoding the Rep protein Rep40 (DNA helicases for accumulation and packaging).

[0116] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV duplex DNA replication, whereas the smaller Rep proteins, Rep52 and Rep40, seem to be essential for progeny and singlestrand DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0117] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit defined enzyme activities, which are required for resolving replication at the AAV termini. Expression of Rep78 or Rep68 could be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0118] It is deemed that all Rep proteins, primarily Rep78 and Rep68, exhibit regulatory activities, such as induction and suppression of AAV genes as well as inhibitory effects on cell growth (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894; Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virology, 181 (1991) 738-741).

[0119] Recombinant overexpression of Rep78 results in phenotype with reduced cell growth due to the induction of DNA damage. Thereby the host cell is arrested in the S phase, whereby latent infection by the virus is facilitated (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0120] Tratschin et al. reported that the P5 promoter is negatively auto-regulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of expression of the Rep protein, only very low expression has been reported for certain cell lines after stable integration of AAV (see, e.g., Mendelson et al., Virol. 166 (1988) 154-165).

[0121] The cap gene locus comprises one promoter, termed P40. Promoter P40 is operably linked to a nucleic acid sequence providing for 2.6 kb mRNA, which, by alternative splicing and use of alternative start codons, encodes the Cap proteins VP1 (87 kDa, non-spliced mRNA transcript), VP2 (72 kDa, from the spliced mRNA transcript), and VP3 (61 kDa, from alternative start codon). VP1 to VP3 constitute the building blocks of the viral capsid. The capsid has the function to bind to a cell surface receptor and allow for intracellular trafficking of the virus. VP3 accounts for about 90 % of total viral particle protein. Nevertheless, all three proteins are essential for effective capsid production.

[0122] It has been reported that inactivation of all three capsid proteins VP1 to VP3 prevents accumulation of single-strand progeny AAV DNA. Mutations in the VP1 amino-terminus ("Lip-negative" or "Inf-negative") still allows for assembly of single-stranded DNA into viral particles whereby the infectious titer is greatly reduced. The AAP open reading frame is encoding the assembly activating protein (AAP). It has a size of about 22 kDa and transports the native VP proteins into the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein encoding sequence.

[0123] In individual AAV particles, only one single-stranded DNA molecule is contained. This may be either the "plus" or "minus" strand. AAV particles containing a DNA molecule are infectious. Inside the infected cell, the parental infecting single stranded DNA is converted into a double stranded DNA, which is subsequently amplified. The amplification results in a large pool of double stranded DNA molecules from which single strands are displaced and packaged into capsids.

[0124] Adeno-associated viral (AAV) vectors can transduce dividing cells as well as resting cells. It can be assumed that a transgene introduced using an AAV vector into a target cell will be expressed for a long period. One drawback of using an AAV vector is the limitation of the size of the transgene that can be introduced into cells.

[0125] Parvovirus particles, including AAV serotypes and variants thereof, provide a means for ex vivo, in vitro and in vivo delivery of nucleic acid, which encode proteins, into cells such that the infected cells express the encoded protein. AAVs are viruses useful as gene therapy vectors as they can penetrate cells and introduce nucleic acid / genetic material so that the nucleic acid / genetic material may be stably maintained in the infected cells. Because AAV are not associated with pathogenic disease in humans, AAVs are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV-related pathogenesis or disease.

[0126] AAV particles used as vehicles for effective gene delivery possess a number of desirable features for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also demonstrated no sustained toxicity and immune responses were minimal or undetectable. AAV are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints and hematopoietic stem cells.

[0127] Recombinant AAV particles do not typically include viral genes associated with pathogenesis. Such particles typically comprise a genome, wherein one or more of the wild-type AAV genes have been deleted in whole or in part, for example, rep and / or cap genes, but retain at least one functional flanking ITR sequence, as necessary for the rescue, replication, and packaging of the recombinant vector into an rAAV. Thus, an AAV vector includes sequences required in cis for replication and packaging (i.e. functional ITR sequences). Recombinant AAV particles, as well as methods and uses thereof, can be based on any wild-type AAV genome or serotype or combination thereof. As a non-limiting example, a rAAV can be based upon any wild-type AAV genome, i.e. comprise the respective ITR sequences, such as AAV1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, rh.74, rh.10 or 7m8 for example. Such particles can be based on the same strain or serotype (or subgroup or variant), or be different from each other. As a non-limiting example, a rAAV based upon one wild-type genome can be identical or different to one or more of the capsid proteins that package the vector. In addition, a recombinant AAV vector can be based upon an AAV (e.g., AAV2) wild-type serotype genome distinct from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector can be based upon AAV2, whereas at least one of the three capsid proteins could be an AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10 or AAV-7m8 or a variant thereof, for example. AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10 and AAV-7m8 capsids.

[0128] In certain embodiments of all aspects and embodiments of the invention, the rAAVp is derived from a wild-type AAV particle selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10 and AAV-7m8, as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof, for example, as set forth in WO 2013 / 158879, WO 2015 / 013313 and US 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0129] In certain embodiments of all aspects and embodiments of the invention, the rAAVp comprises a capsid polypeptides with an amino acid sequence having 70 % or more sequence identity to an wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.10, AAV-rh.74, or AAV-7m8 capsid sequence.

[0130] In certain embodiments of all aspects and embodiments of the invention, the rAAVp comprises one or two ITR sequence having 70 % or more sequence identity to a wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 ITR sequence.

[0131] Recombinant AAV particles can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for, among other things, administration and delivery to a subject in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity. Protocols for the generation of adenoviral vectors have been described in US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94 / 17810 and WO 94 / 23744, which are incorporated herein by reference in their entirety.

[0132] In one embodiment the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 2 or AAV 8 serotype. In one embodiment the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 2 serotype. In one embodiment the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 8 serotype.

[0133] RECOMBINANT ADENO-ASSOCIATED VIRAL PARTICLES

[0134] Different methods are known in the art for generating recombinant AAV particles. For example, transfection with an AAV vector comprising plasmid and a plasmid comprising AAV helper sequences (rep and cap) in conjunction with co-infection with one AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) or transfection with a recombinant AAV vector comprising plasmid, an AAV helper plasmid (comprising rep and cap), and an helper function plasmid. Non-limiting methods for generating rAAV are described, for example, in US 6,001,650, US 6,004,797, WO 2017 / 096039, and WO 2018 / 226887. Following rAAV production (i.e. particle generation in cell culture systems), rAAV can be obtained from the host cells and / or cell culture supernatant and purified.

[0135] For the generation of recombinant AAV particles, expression of the Rep and Cap proteins, the helper proteins E1A, E1B, E2A and E4orf6 as well as optionally the adenoviral VA RNA in a single mammalian cell is required. The helper proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), especially the CMV IE promoter. Thus, any promoter can be operably linked to said genes for functional expression.

[0136] Generally, to produce rAAV, different, complementing plasmids are co-transfected into a host cell. One of the plasmids comprises the transgene sandwiched between the two cis acting AAV ITRs. The missing AAV elements required for replication and subsequent packaging of progeny recombinant genomes, i.e. the open reading frames for the Rep and Cap proteins, are contained in trans on a second plasmid. The overexpression of the Rep proteins results in inhibitory effects on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236-5243). Additionally, a third plasmid comprising the genes of a helper virus, i.e. El, E4orf6, E2A and VA from adenovirus, is required for rAAV production.

[0137] To reduce the number of required plasmids, rep, cap and the adenovirus helper genes may be combined on a single plasmid. Alternatively, the host cell may already stably express the El gene products. Such a cell is a HEK293 cell. The human embryonic kidney clone denoted as 293 was generated back in 1977 by integrating adenoviral DNA into human embryonic kidney cells (HEK cells) (Graham, F.L., et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line comprises base pair 1 to 4344 of the adenovirus serotype 5 genome. This encompasses the El A and E1B genes as well as the adenoviral packaging signals (Louis, N., et al., Virology 233 (1997) 423-429).

[0138] When using HEK293 cells the missing E2A, E4orf6 and VA genes can be introduced either by coinfection with an adenovirus or by co-transfection with an E2A-, E4orf6- and VA-expressing plasmid (see, e.g., Samulski, R.J., et al., J. Virol. 63 (1989) 3822-3828; Allen, J.M., et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K., et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, T.R., et al., Gene Ther. 2 (1995) 29-37; Conway, J.E., et al., J. Virol. 71 (1997) 8780-8789; Chiorini, J. A., et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, F.K., et al., J. Virol. 70 (1996) 3227-3234; Salvetti, A., et al., Hum. Gene Ther. 9 (1998) 695-706; Xiao, X., et al., J. Virol. 72 (1998) 2224- 2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors can be used (see, e.g., Conway, J.E., et al., J. Virol. 71 (1997) 8780-8789; Johnston, K.M., et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, A.J., et al., Gene Ther. 2 (1995) 481-485; Fisher, J.K., et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, K.M., et al., Hum. Gene Ther. 8 (1997) 359-370).

[0139] In order to limit the transgene activity to specific tissues, i.e. to limit the site of action, the transgene can be operably linked to an inducible or tissue specific promoter (see, e.g., Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0140] The coding sequences of E1A and E1B (open reading frames) can be derived from a human adenovirus, such as, e.g., in particular of human adenovirus serotype 2 or serotype 5. An exemplary sequence of human Ad5 (adenovirus serotype 5) is found in GenBank entries X02996, AC 000008 and that of an exemplary human Ad2 in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequences encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146 as reported in EP 1 230 354, as well as plasmids pGS119 and pGS122 as reported in WO 2007 / 056994, can also be used as a source for the El A and E1B open reading frames.

[0141] El A is the first viral helper gene that is expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes the 12S and 13S proteins, which are based on the same E1A mRNA by alternative splicing. Expression of the 12S and 13S proteins results in the activation of the other viral functions E1B, E2, E3 and E4. Additionally, expression of the 12S and 13S proteins force the cell into the S phase of the cell cycle. If only the El A-derived proteins are expressed, the cell will die (apoptosis).

[0142] E1B is the second viral helper gene that is expressed. It is activated by the El A-derived proteins 12S and 13S. The E1B gene derived mRNA can be spliced in two different ways resulting in a first 55 kDa transcript and a second 19 kDa transcript. The E1B 55 kDa protein is involved in the modulation of the cell cycle, the prevention of the transport of cellular mRNA in the late phase of the infection, and the prevention of ElA-induced apoptosis. The E1B 19 kDa protein is involved in the prevention of ElA-induced apoptosis of cells.

[0143] The E2 gene encodes different proteins. The E2A transcript codes for the single strand-binding protein (SSBP), which is essential for AAV replication

[0144] In addition, the E4 gene encodes several proteins. The E4 gene derived 34 kDa protein (E4orf6) prevents the accumulation of cellular mRNAs in the cytoplasm together with the E1B 55 kDa protein, but also promotes the transport of viral RNAs from the cell nucleus into the cytoplasm.

[0145] The viral associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad), regulating translation. The adenoviral genome comprises two independent copies: VAI (VARNAI) and VAII (VA RNAII). Both are transcribed by RNA polymerase III (see, e.g., Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289) from a type 2 polymerases III promoter. For recombinant AAV particle production, the adenoviral VA RNA gene can be driven by any promoter.

[0146] The structure, function, and evolution of adenovirus-associated RNA using a phylogenetic approach was investigated by Ma, Y. and Mathews, M.B. (J. Virol. 70 (1996) 5083-5099). They provided alignments as well as consensus VA RNA sequences based on 47 known human adenovirus serotypes. Said disclosure is herewith incorporated by reference in its entirety into the current application.

[0147] VA RNAs, VAI and VAII, are consisting of 157-160 nucleotides (nt).

[0148] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is believed to play the dominant pro-viral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, V.K. and Conn, G.L., Virus Res. 212 (2016) 39-52).

[0149] The VA RNAs are not essential, but play an important role in efficient viral growth by overcoming cellular antiviral machinery. That is, although VA RNAs are not essential for viral growth, VA RNA-deleted adenovirus cannot grow during the initial step of vector generation, where only a few copies of the viral genome are present per cell, possibly because viral genes other than VA RNAs that block the cellular antiviral machinery may not be sufficiently expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0150] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported efficient production of adenovirus vector lacking genes of virus-associated RNAs that disturb cellular RNAi machinery, wherein HEK293 cells that constitutively and highly express flippase recombinase were infected to obtain VA RNA-deleted adenovirus by FLP recombinase-mediated excision of the VA RNA locus.

[0151] The human adenovirus 2 VA RNAI corresponds to nucleotides 10586-10810 of GenBank entry AC_000007 sequence. The human adenovirus 5 VA RNAI corresponds to nucleotides 10579- 10820 of GenBank entry AC_000008 sequence.

[0152] GENERAL DESCRIPTION OF RECOMBINANT AAV PARTICLE PRODUCTION

[0153] After entry into the host cell nucleus, AAV can follow either one of two distinct and interchangeable pathways of its life cycle: the lytic or the lysogenic. The former develops in cells infected with a helper virus such as Ad or herpes simplex virus (HSV) whereas the latter is established in host cells in the absence of a helper virus.

[0154] When a latently infected cell is super-infected with a helper virus, the AAV gene expression program is activated leading to the AAV Rep-mediated rescue (i.e., excision) of the provirus DNA from the host cell chromosome followed by replication and packaging of the viral genome. Finally, upon helper virus-induced cell lysis, the newly assembled virions (particles) are released. Thus, the lytic phase of the AAV life cycle is induced.

[0155] Therefore, in the presence of Ad helper functions, the rAAV vector is subjected to the wild-type AAV lytic processes by being rescued from the plasmid backbone, replicated and packaged into preformed AAV capsids as single-stranded molecules (Goncalves, M.A.F.V., Virol. J., 2 (2005) 43).

[0156] Generation of a recombinant AAV particle involves replacing a majority of the AAV's wild-type genome with a desired transgene and providing the viral genes that are essential for virus packaging in-trans on a separate plasmid. Once all components are transfected together into a packaging cell line, recombinant AAV particles are assembled using the cell’s cellular machineries. The process of viral assembly and encapsulation takes roughly two days, after which the cells are lysed to release the rAAV for further purification and concentration (https: / / old.abmgood.com / marketing / knowledge_base / Adeno_Associated_Virus_Production_an d Modifi cati on_of_A A V . php) . AAV is not released very efficiently from the cells, although major differences have been observed between serotypes (see, e.g., Strobel, B., et al., Lamia T. Comparative Analysis of Cesium Chloride- and lodixanol -Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications. Hum. Gene Ther. Methods 26 (2015) 147-157). When harvesting the culture, a cell disruption method is usually applied to recover the vectors entrapped in the cells.

[0157] Historically, manufacturing of rAAVps was performed by double transfection of a plasmid containing the rep and the cap ORFs and a plasmid with the gene of interest flanked by ITRs. Then, a helper virus, typically Adenovirus, was co-infected (see, e.g., Aponte-Ubillus, J. J., et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Muzyczka, N., Curr. Top. Microbiol. Immunol. 158 (1992) 97-129). In this setting, the separation of the helper virus from the final product was difficult, but a critical element to avoid induction of inflammatory responses after injection into patients (see, e.g., Schnell, M.A., et al., Mol. Ther. 3 (2001) 708-722.). Therefore, production of rAAVps nowadays moved towards an adenovirus-free approach by utilizing triple transfection (see, e.g., Large, E.E., et al., Viruses 13 (2021) 1336). To this end, three components are needed: one plasmid encoding the genes for Rep and Cap without the ITRs, a second plasmid with the transgene of interest flanked by ITRs, and a helper plasmid to provide the helper genes of the helper virus (see, e.g., Aponte-Ubillus, J. J., et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Farris, K.D. and Pintel, D.J., Hum. Gene Ther. 19 (2008) 1421-1427; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Ferrari, F.K., et al., Nat. Med. 3 (1997) 1295-1297). For example, the Adenovirus helper bears the minimal required adenoviral genes E2A, E4 and VA. It is important to note, that the Human Embryonic Kidney cells 293 (HEK293) constitutively express the adenoviral genes El A / B, which are also required for production of rAAVps. Therefore, HEK293 cells are classic producer cells for rAAVps and for manufacturing. Other cell types require a supplementation of El A / B.

[0158] Carter et al. have shown that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with a transgene (Carter, B. J., in "Handbook of Parvoviruses", ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). Further, it has been reported that the ITRs have to be maintained to retain the function of replication, rescue, packaging, and integration of the transgene into the genome of the target cell.

[0159] When cells comprising the respective viral helper genes are transduced by an AAV vector, or, vice versa, when cells comprising an integrated AAV provirus are transduced by a suitable helper virus, then the AAV provirus is activated and enters a lytic infection cycle again (Clark, K.R., et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, R.J., Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0160] Producer cells contain the rep and cap gene sequences, as well as the transgene cassette flanked by ITR sequences on one or more plasmids that are retained, e.g., via drug selection. Production of rAAV in these cell lines generally occurs after their infection with the required helper functions. Therefore, cells are infected either with replication-competent adenovirus (usually wild type Ad5) or a plasmid comprising the respective helper genes to supply helper virus proteins and initiate rAAV production. A packaging cell line differs from a producer cell line as it only contains the rep and cap genes.

[0161] More generally, cells transfected or transduced with DNA for the recombinant production of AAV particles can be referred to as a "recombinant cell". Such a cell can be any mammalian cell that has been used as recipient of a nucleic acid (plasmid) encoding packaging proteins, such as AAV packaging proteins, a nucleic acid (plasmid) encoding helper proteins, and a nucleic acid (plasmid) that encodes a protein or is transcribed into a transcript of interest, i.e. a transgene placed between two AAV ITRs. The term includes the progeny of the original cell, which has been transduced or transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total nucleic acid complement as the original parent, due to natural, accidental, or deliberate mutation.

[0162] Numerous cell growth media appropriate for sustaining cell viability or providing cell growth and / or proliferation are commercially available. Examples of such medium include serum free eukaryotic growth mediums, such as medium for sustaining viability or providing for the growth of mammalian (e.g., human) cells. Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI- 1640 (Thermo-Fisher Scientific) and mixtures thereof. Such media can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0163] For producing rAAV, three plasmids are co-transfected into a mammalian cell. The transgene plasmid encodes the expression cassette, which is cloned between the AAV ITRs, whereas rep and cap genes are provided in trans by co-transfecting a second, packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. The third plasmid, also referred to as helper plasmid, contains the minimal helper virus factors, commonly adenoviral E2A, E4orf6 and VA genes, but lacking AAV ITRs.

[0164] Diverse methods for the DNA transfer into mammalian cells have been reported in the art. These are all useful in the methods according to the current invention. In certain embodiments of all aspects and embodiments, electroporation, nucleofection, or microinjection for nucleic acid transfer / transfection is used. In certain embodiments of all aspects and embodiments, an inorganic substance (such as, e.g., calcium phosphate / DNA co-precipitation), a cationic polymer (such as, e.g., polyethylenimine, DEAE-dextran), or a cationic lipid (lipofection) is used for nucleic acid transfer / transfection is used. Calcium phosphate and polyethylenimine are the most commonly used reagents for transfection for nucleic acid transfer in larger scales (see, e.g., Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), whereof polyethylenimine is preferred.

[0165] The growth in serum-free suspension culture and improvement of efficiency and reproducibility of transfection conditions using PEI as a transfection reagent permits ready scale-up the AAV production using shake-flasks, wave, or stirred-tank bioreactors.

[0166] The composition may comprise further plasmids or / and cells. Such plasmids and cells may be in contact with free PEI.

[0167] In addition to PEI, valproic acid (VP A) can be used to improve transfection efficiency. VP A, a branched short-chain fatty acid and inhibits histone deacetylase activity. Due to this reason, it is commonly added to mammalian cell culture as an enhancer of recombinant protein production.

[0168] Encoded AAV packaging proteins include, in certain embodiments of all aspects and embodiments, AAV rep and / or AAV cap. Such AAV packaging proteins include, in certain embodiments of all aspects and embodiments, AAV rep and / or AAV cap proteins of any AAV serotype.

[0169] Encoded helper proteins include, in certain embodiments of all aspects and embodiments, adenovirus El A and E1B, adenovirus E2 and / or E4, VA RNA, and / or non-AAV helper proteins.

[0170] The cultivation can be performed using the generally used conditions for the cultivation of eukaryotic cells of about 37 °C, 95 % humidity and 8 vol.-% CO2. The cultivation can be performed in serum containing or serum free medium, in adherent culture or in suspension culture. The suspension cultivation can be performed in any fermentation vessel, such as, e.g., in stirred tank reactors, wave reactors, rocking bioreactors, shaker vessels or spinner vessels or so called roller bottles. Transfection can be performed in high throughput format and screening, respectively, e.g. in a 96 or 384 well format.

[0171] Methods according to the current invention can include AAV particles of any serotype, or a variant thereof. In certain embodiments of all aspects and embodiments, a recombinant AAV particle comprises any of AAV serotypes 1-12, an AAV VP1, VP2 and / or VP3 capsid protein, or a modified or variant AAV VP1, VP2 and / or VP3 capsid protein, or wild-type AAV VP1, VP2 and / or VP3 capsid protein. In certain embodiments of all aspects and embodiments, an AAV particle comprises an AAV serotype or an AAV pseudotype, where the AAV pseudotype comprises an AAV capsid serotype different from an ITR serotype.

[0172] Expression control elements include constitutive or regulatable control elements, such as a tissuespecific expression control element or promoter. ITRs can be any of AAV2 or AAV6 or AAV8 or AAV9 serotypes, or a combination thereof. AAV particles can include any VP1, VP2 and / or VP3 capsid protein having 75 % or more sequence identity to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV12, AAV 2i8, AAV rh.10, AAV rh.74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or comprises a modified or variant VP1, VP2 and / or VP3 capsid protein selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV-2i8, AAV-rh.10, AAV-rh.74 and AAV-7m8 AAV serotypes.

[0173] Following production of recombinant viral (e.g., AAV) particles, if desired, the viral (e.g., rAAV) particles can be purified and / or isolated from host cells using a variety of conventional methods. Such methods include column chromatography, CsCl gradients, iodixanol gradient and the like.

[0174] For example, a plurality of column purification steps such as purification over an anion exchange column, an affinity column and / or a cation exchange column can be used. (See, e.g., WO 02 / 12455 and US 2003 / 0207439). Alternatively, or in addition, an iodixanol or CsCl gradient steps can be used (see, e.g., US 2012 / 0135515; and US 2013 / 0072548). Further, if the use of infectious virus is employed to express the packaging and / or helper proteins, residual virus can be inactivated, using various methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C for, e.g., 20 minutes or more. This treatment effectively inactivates the helper virus since AAV is heat stable while the helper adenovirus is heat labile.

[0175] An objective in the rAAV production and purification systems is to implement strategies to minimize / control the generation of production related impurities such as proteins, nucleic acids, and vector-related impurities, including wild-type / pseudo wild-type AAV species (wtAAV) and AAV-encapsulated residual DNA impurities.

[0176] Considering that the rAAV represents only a minor fraction of the biomass, rAAV need to be purified to a level of purity, which can be used as a clinical human gene therapy product (see, e.g., Smith P.H., et al., Mo. Therapy 7 (2003) 8348; Chadeuf G., et al, Mo. Therapy 12 (2005) 744; report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0177] In certain embodiments of all aspects and embodiments of the method according to the current invention, as an initial step, typically the cultivated cells that produce the rAAVps are harvested, optionally in combination with harvesting cell culture supernatant (medium) in which the cells (suspension or adherent) producing recombinant AAV particles have been cultured. The harvested cells and optionally cell culture supernatant may be used as is, as appropriate, lysed or concentrated. Further, if infection is employed to express helper functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C for, e.g., 20 minutes or more, which inactivates only the helper virus since AAV is heat stable while the helper adenovirus is heat labile.

[0178] The cells in the harvested cultivation broth can be lysed using methods now in the art, such as, e.g., detergent lysis or freeze-thaw cycles, to release the rAAV particles. Concurrently during cell lysis or subsequently after cell lysis, a nuclease, such as, e.g., benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris, e.g. by filtering or centrifuging, to render a clarified cell lysate. In a particular example, the lysate is filtered with a micron diameter pore size filter (such as a 0.1-10.0 pm pore size filter, for example, a 0.45 pm and / or pore size 0.2 pm filter), to produce a clarified lysate.

[0179] The lysate (optionally clarified) contains recombinant AAV particles (comprising full as well as empty rAAVps) and production / process related impurities, such as soluble cellular components from the host cells that can include, inter alia, cellular proteins, lipids, and / or nucleic acids, and cell culture medium components. The optionally clarified lysate is then subjected to purification steps to purify the rAAV (comprising rAAV vectors) from impurities using chromatography. The clarified lysate may be diluted or concentrated with an appropriate buffer prior to the first chromatography step.

[0180] After cell lysis, optional clarifying, and optional dilution or concentration, a plurality of subsequent and sequential chromatography steps can be used to purify the rAAV.

[0181] The first chromatography step is preferably an affinity chromatography step using an AAV affinity chromatography ligand.

[0182] If the first chromatography step is affinity chromatography the second chromatography step can be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV purification is via affinity chromatography, followed by purification via anion exchange chromatography or / and cation exchange chromatography or / and size exclusion chromatography, in any order or sequence or combination.

[0183] The removal of empty capsids from full ones, for example, during downstream processing is based on their different isoelectric points (pl) in anion exchange chromatography. The average calculated pl across all serotypes is 5.9 for full capsids and 6.3 for empty capsids (Venkatakrishnan, B., et al., J. Virol. 87 (2013) 4974-4984).

[0184] Cation exchange chromatography functions to separate the AAV from cellular and other components present in the clarified lysate and / or column eluate from an affinity or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV over a wide pH range include, without limitation, any sulfonic acid based resin as indicated by the presence of the sulfonate functional group, including aryl and alkyl substituted sulfonates, such as sulfopropyl or sulfoethyl resins. Representative matrices include but are not limited to POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Additional examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and commercial DOWEX®, AMBERLITE®, and AMBERLYST® families of resins available from Aldrich Chemical Company (Milliwaukee, WI, USA). Weak cation exchange resins include, without limitation, any carboxylic acid based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on the phosphate functional group), methyl sulfonate (S) and sulfopropyl (SP) resins.

[0185] Anion exchange chromatography functions to separate rAAV from proteins, cellular and other components present in the clarified lysate and / or column eluate from an affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thereby control the amount of empty rAAV in the eluate. For example, the anion exchange column having full and empty rAAV bound thereto can be washed with a solution comprising NaCl at a modest concentration (e.g., about 100-125 mM, such as 110-115 mM) and a portion of the empty rAAV can be eluted in the flow through without substantial elution of the full rAAV. Subsequently, full rAAV bound to the anion exchange column can be eluted using a solution comprising NaCl at a higher concentration (e.g., about 130-300 mM NaCl), thereby producing a column eluate with reduced or depleted amounts of empty rAAVps and proportionally increased amounts of full rAAV comprising an rAAV vector.

[0186] Exemplary anion exchange resins include, without limitation, those based on polyamine resins and other resins. Examples of strong anion exchange resins include those based generally on the quatemized nitrogen atom including, without limitation, quaternary ammonium salt resins such as trialkylbenzyl ammonium resins. Suitable exchange chromatography materials include, without limitation, MACRO PREP Q (strong anion-exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion-exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion-exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion-exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion-exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak anion-exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong anion-exchanger available from GE healthcare, Marlborough, MA, USA); DEAE SEPHAROSE (weak anion- exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q SEPHAROSE (strong anion-exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE) and quaternary amino ethyl (QAE).

[0187] A commercial manufacturing process to purify recombinant AAV particles intended as a product to treat human disease should achieve the following objectives: 1) consistent particle purity, potency and safety; 2) manufacturing process scalability; and 3) acceptable cost of manufacturing.

[0188] Exemplary processes for recombinant AAV particle purification are reported in WO 2019 / 006390.

[0189] Methods to determine infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for assaying for empty rAAV and full rAAV with packaged transgenes are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0190] To determine the presence or amount of degraded / denatured capsid, purified rAAV can be subjected to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel, then running the gel until sample is separated, and blotting the gel onto nylon or nitrocellulose membranes. Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). A secondary antibody that binds to the primary antibody contains a means for detecting the primary antibody. Binding between the primary and secondary antibodies is detected semi-quantitatively to determine the amount of capsids. Another method would be analytical HPLC with a SEC column or analytical ultracentrifuge.

[0191] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0192] The invention is based, at least in part, on the unexpected finding that the purification by alluvial filtration is especially efficient for cell cultures when the filter aid, e.g. DE, is added to the cell culture at a certain ratio of filter aid to cells / biological wet mass (BWM).

[0193] In more detail and surprisingly, it has been found that the methods according to the current invention provide for, amongst other things, improved processes for the separation or / and purification of recombinant adeno-associated viral particles from cultivation broths, especially lysates of mammalian cell cultivation broths. Although it was reported earlier that high DE concentrations of half the wet-cell weight (i.e. DE / BWM of 1 :2) resulted in a significant loss of particles and an optimum ratio of 1 :8 was found (Meierrieks et al., 2023), it was found by the inventors that concentrations above 1 :2 DE / BWM lead to good separation and low particle loss. Especially, ratios of about 2:3 to about 3 :4 DE / BWM provide for good separation and good particle recovery. Thus, the invention encompasses at least the following independent and dependent embodiments:

[0194] 0. A method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of: a) adding a filter aid to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 filter aid / biological wet mass (BWM) to obtain a prefiltration mixture, b) subjecting the pre-filtration mixture to alluvial filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth.

[0195] 0a. A method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of: a) adding a filter aid to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 filter aid / biological wet mass (BWM) to obtain a prefiltration mixture, b) subjecting the pre-filtration mixture to filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth.

[0196] Ob. A method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of: a) adding a silica-based filter aid, like diatomaceous earth (DE) or synthetic silica-based filter aid to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 silica-based filter aid / biological wet mass (BWM) to obtain a pre- filtration mixture, b) subjecting the pre-filtration mixture to alluvial filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth.

[0197] 0c. A method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of: a) adding a silica-based filter aid, like diatomaceous earth (DE) or synthetic silica-based filter aid to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 silica-based filter aid / biological wet mass (BWM) to obtain a pre- filtration mixture, b) subjecting the pre-filtration mixture to filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth.

[0198] Od. The method according to any one of embodiments 0 or 0a, wherein the filter aid is diatomaceous earth (DE) or perlite or synthetic silica-based filter aid, or cellulose or a mixture of any of these.

[0199] Oe. The method according to any one of embodiments 0 to Od, wherein the filter aid is diatomaceous earth (DE) or synthetic silica-based filter aid, or a mixture thereof.

[0200] Of. The method according to any one of embodiments 0 to Oe, wherein the filter aid is diatomaceous earth (DE).

[0201] 1. A method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of a) adding diatomaceous earth (DE) to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 DE / biological wet mass (BWM) to obtain a pre-filtration mixture, b) subjecting the pre-filtration mixture to alluvial filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth.

[0202] 2. A method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth comprising the steps of a) adding diatomaceous earth (DE) to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 DE / biological wet mass (BWM) to obtain a pre-filtration mixture, b) subjecting the pre-filtration mixture to filtration, and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth.

[0203] 2a. A method for clarifying a mammalian cell culture broth comprising recombinant viral particles, wherein the method comprises the steps of: a) adding diatomaceous earth (DE) to a crude mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 DE / biological wet mass (BWM) to obtain a pre-filtration mixture, b) subjecting the pre-filtration mixture to filtration, and thereby clarifying the mammalian cell culture broth.

[0204] 3. The method according to any one of embodiments 1 to 2a, wherein the DE is added at a ratio of about 2:3 to about 3:4 DE / BWM.

[0205] 4. The method according to any one of embodiments 1 to 3, wherein the DE is added at a ratio of about 2:3 or about 3:4 DE / BWM.

[0206] 5. The method according to any one of embodiments 1 to 4, wherein the DE is added at a ratio of about 2:3 DE / BWM.

[0207] 5a. The method according to any one of embodiments 1 to 5, wherein DE / BWM has the unit of DE / L / %BWM.

[0208] 6. The method according to any one of embodiments 1 to 5a, wherein the mammalian cell culture broth is a cellular lysate of the mammalian cell culture broth comprising the recombinant viral particles, intact cells and cell debris and the compounds of the mammalian cell culture broth are comprising intact cells and cell debris.

[0209] 7. The method according to embodiment 6, wherein the cellular lysate further comprises host cell DNA.

[0210] 8. The method according to any one of embodiments 1 to 7, wherein the separating is from compounds comprising intact cells, cell debris and non-soluble particles or wherein the clarifying results in removing compounds comprising intact cells, cell debris and nonsoluble particles.

[0211] 9. The method according to embodiment 8, wherein the separating is further from host cell DNA or wherein the clarifying further results in reducing of host cell DNA. 10. The Method according to any one of embodiments 1 to 9, wherein the separating is a purifying.

[0212] 11. The method according to embodiment 10, wherein the purifying is a reduction of turbidity.

[0213] 12. The method according to any one of embodiments 1 to 9, wherein the clarifying results in a reduction of turbidity.

[0214] The method according to any one of embodiments 11 to 12, wherein the reduction of turbidity is by at least 75 %.

[0215] 14. The method according to any one of embodiments 11 to 13, wherein the reduction of turbidity is by at least 80 %.

[0216] 15. The method according to any one of embodiments 11 to 14, wherein the reduction of turbidity is by at least 85 %.

[0217] 16. The method according to any one of embodiments 11 to 15, wherein the reduction of turbidity is by at least 90 %.

[0218] 17. The method according to any one of embodiments 10 to 16, wherein the purifying is a reduction of (the concentration or amount of) host cell DNA.

[0219] 18. The method according to any one of embodiments 1 to 17, wherein the (concentration or amount of) host cell DNA is reduced by at least 80 %.

[0220] 19. The method according to any one of embodiments 1 to 18, wherein the (concentration or amount) of host cell DNA is reduced by at least 85 %.

[0221] 20. The method according to any one of embodiments 1 to 19, wherein the (concentration or amount) of host cell DNA is reduced by at least 90 %.

[0222] 21. The method according to any one of embodiments 1 to 20, wherein the (concentration or amount) of host cell DNA is reduced by at least 95 %.

[0223] 22. The method according to any one of embodiments 1 to 21, wherein the viral genome recovery is at least 75 %.

[0224] 23. The method according to any one of embodiments 1 to 22, wherein the viral genome recovery is at least 80 %. 24. The method according to any one of embodiments 1 to 23, wherein the viral genome recovery is at least 85 %.

[0225] 25. The method according to any one of embodiments 1 to 24, wherein the viral genome recovery is at least 90 %.

[0226] 26. The method according to any one of embodiments 1 to 25, wherein the viral genome recovery is at least 95 %.

[0227] 27. The method according to any one of embodiments 1 to 26, wherein the viral capsid recovery is at least 80 %.

[0228] 28. The method according to any one of embodiments 1 to 27, wherein the viral capsid recovery is at least 85 %.

[0229] 29. The method according to any one of embodiments 1 to 28, wherein the viral capsid recovery is at least 90 %.

[0230] 30. The method according to any one of embodiment 11 to 29, wherein the turbidity reduction is by at least 90 % and the reduction of host cell DNA is by at least 90 %.

[0231] 31. The method according to any one of embodiments 11 to 30, wherein the turbidity reduction is by at least 90 %, the reduction of host cell DNA is by at least 90 % and the viral genome recovery is at least 90 %.

[0232] 32. The method according to any one of embodiments 11 to 31, wherein the turbidity reduction is by at least 90 %, the reduction of host cell DNA is by at least 90 %, the viral genome recovery is at least 90 % and the capsid recovery is at least 80 %.

[0233] 33. The method according to any one of embodiments 6 to 32, wherein the cellular lysate is obtained by lysis and wherein the lysis is a chemical lysis, an enzymatic lysis or a physical lysis.

[0234] 34. The method according to any one of embodiments 1 to 33, wherein the volume of the (crude) mammalian cell culture broth or the cellular lysate is 50 L or more.

[0235] 35. The method according to any one of embodiments 1 to 34, wherein the volume of the (crude) mammalian cell culture broth is in the range of and including 50L to lOOOL.

[0236] 36. The method according to any one of embodiments 1 to 35, wherein the volume of the mammalian (crude) cell culture broth or the cellular lysate is 50 L or more, 75 L or more, or 100 L or more. 37. The method according to any one of embodiments 1 to 36, wherein the (crude) mammalian cell culture broth is obtained by cultivating transiently transfected mammalian cells producing recombinant viral particles.

[0237] 37a. The method according to any one of embodiments 1 to 36, wherein the (crude) mammalian cell culture broth is obtained by cultivating stably transfected mammalian cells producing recombinant viral particles.

[0238] 38. The method according to any one of embodiments 1 to 37a, wherein the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle.

[0239] 39. The method according to any one of embodiments 1 to 38, wherein the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 2 serotype or of AAV 8 serotype.

[0240] 39a. The method according to any one of embodiments 1 to 39, wherein the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 2 serotype.

[0241] 39b. The method according to any one of embodiments 1 to 39, wherein the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 8 serotype.

[0242] 40. The method according to any one of embodiments 1 to 39b, wherein the mammalian cell is a HEK cell or a CHO cell.

[0243] 41. The method according to any one of embodiments 1 to 40, wherein the mammalian cell is a HEK293 cell.

[0244] 42. The method according to any one of embodiments 1 to 40, wherein the mammalian cell is a CHO cell.

[0245] 43. The method according to any one of embodiments 1 to 40 and 42, wherein the mammalian cell is a CHO KI cell.

[0246] 44. The method according to any one of embodiments 1 to 43, wherein after step b) the method further comprises the following step: c) subjecting the (alluvial) filtrate obtained in step b) to a sterile filtration .

[0247] 45. The method according to any one of embodiments 1 to 44, wherein step b) or / and step c) further comprises: recovering the filtrate. 46. The method according to any one of embodiments 1 to 45, wherein step a) or / and b) are performed in a fully closed system.

[0248] 47. The method according to any one of embodiments 1 to 46, wherein the method comprises the further step of

[0249] - evenly distributing the DE in the mammalian cell culture broth or in the lysed mammalian cell culture broth.

[0250] 48. The method according to any one of embodiments 1 to 47, wherein

[0251] - the DE and the biomass are (good / thoroughly) mixed by (intense) stirring (in the bioreactor).

[0252] 49. The method according to any one of embodiments 1 to 48, wherein the filter sheet is (Purafix) CH 33P.

[0253] 50. The method according to any one of embodiments 1 to 49, wherein the DE grade is (Cellpure) C 300.

[0254] 51. The method according to any one of embodiments 1 to 50, wherein the filter sheet is (Purafix) CH 33P and the DE grade is (Cellpure) C 300.

[0255] 52. Use of a filter aid, for example diatomaceous earth (DE), to separate recombinant viral particles from (other) compounds of a mammalian cell culture broth, wherein the filter aid, for example diatomaceous earth (DE) is added to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 filter aid / biological wet mass (BWM) to obtain a pre-filtration mixture and subjecting the pre-filtration mixture to alluvial filtration (and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth).

[0256] 53. Use of diatomaceous earth (DE), to separate recombinant viral particles from (other) compounds of a mammalian cell culture broth, wherein DE is added to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 DE / biological wet mass (BWM) to obtain a pre-filtration mixture and subjecting the pre-filtration mixture to alluvial filtration (and thereby separating recombinant viral particles from (other) compounds of the mammalian cell culture broth). (Primary) Clarification / Harvest

[0257] Most viral vectors like of the type AAV are considered according to the german genetic engineering (Gesetz zur Regelung der Gentechnik “Gentechnikgesetz” - GenTG) act as “biosafety level 1 - without biological safety measure”. According to the ZKBS (Zentrale Kommission fur Siologische Sicherheit), the grouping of AAVv by October 2021 is as follows:

[0258] AAV-1 to AAV-3, AAV-3b, AAV-5, AAV-6, AAV-8, AAV-9 und AAV-rhlO:

[0259] Biosafety level 1

[0260] • AAV-4, A AV-7, AAV- 10 to AAV- 13 :

[0261] Biosafety level 2

[0262] Based thereon there are certain safety requirements for the handling and use of equipment during the downstream processing of rAAVp containing solutions:

[0263] • Genetically modified organisms, GMOs, have to be handled in fully contained, i.e. closed, systems. Transfer operations also completely closed. No open handling is allowed.

[0264] • The working area has to be designed in such a way that it is not possible that GMOs leave the area in the event of an accident (e.g., barriers, airlocks).

[0265] • The release of aerosols into the work area or the environment has to be avoided / prevented. Aerosol-forming devices must be fitted with a high-performance particle filter.

[0266] This is also applicable to the harvest equipment resulting in certain requirements:

[0267] 1. Air relief valves have to be connected to an exhaust air system to prevent entering the lab or ways to flush the equipment prior filling with non-harmful liquid (which leads to the dilution of the product solution).

[0268] 2. Disassembly of equipment need to made without any spill of non-deactivated liquid.

[0269] Any procedure that is used to harvest rAAVp has to fulfil at least these requirements.

[0270] The work done underlying the current invention was performed with rAAV particles of the serotype AAV 2 and AAV 8 serotype. The depth filtrations were performed with rAAV particles of the AAV 8 serotype. The tangential flow depth filtration was performed with rAAV particles of the AAV 2 serotype. The alluvial filtrations were performed with rAAV particles of the AAV 8 and AAV 2 serotype.

[0271] One widely used method for rAAVp clarification is depth filtration as there are easily scalable single-use options available. Depth filters have a high capacity to hold back solids. The filtration can be performed directly after fermentation (and lysis). After primary depth filtration, a (two- step) microfiltration can follow.

[0272] There are reports of several depth filters being used for rAAVp clarification. Parker et al. (2020; Pharma's Almanac 6(2): 124-127) from Merck KGaA (more specifically MilliporeSigma in the US) reported high rAAVp yields for several Clarisolve® and Millistak+® depth filters. The rAAVp yield was highest for the Clarisolve® 20MS with about 95 %. The Millistak+® D0HC showed the second highest yield with almost 90 %. Third, came the Millistak+® HC Pro D0SP with roughly 85 % yield. For the Millistak+® C0HC and Millistak+® HC Pro C0SP, the AAVv yield was lower with circa 78 %. Raghavan et al. (2019; Cell & Gene Therapy Insights 5(9): 1311- 1322) from Pall Corporation showed an rAAVp recovery of around 88 % for the filter combination of PDP8 and Bio 10. This combination also had the highest throughput with almost 230 L / m2While the V100P also had a rather high rAAVp recovery of 84 %, it had a much lower throughput of only 40-45 L / m2. The PDK11 was also tested and showed an AAVv recovery of 70 % and roughly 190 L / m2throughput.

[0273] Tangential Flow Depth Filtration (TfDf, more specifically TFDF™ by Repligen Corporation) combines the less fouling for tangential flow filtration with a depth filter’s particle retention capacity. Repligen has shown rAAVp clarification with 70 % recovery yield at 450 LMH using their KrosFLo® TFDF® system (Dryden, W. et al., 2021; Biochemical Engineering Journal 167:107892).

[0274] THE METHOD ACCORDING TO THE CURRENT INVENTION

[0275] The clarification of crude cultivation broths via alluvial filtration is known from the art (see above). Depending on the BWM the respective amount of filter aid is added. The filter aid used in the current method was diatomaceous earth (DE). Other filter aids like perlite, synthetic silica-based filter material / aid or cellulose are available.

[0276] Recommendation from the DE vendor and from the art was to add the DE powder directly to the bioreactor, e.g. via a powder bag and tri-clamp connection. However, this is not possible for rAAVp processes as a tri-clamp on the bioreactor bag cannot be opened anymore as soon as the bioreactor contains rAAVp. Connecting the powder bag before the bioreactor contains rAAVp is also problematic due to sterility concerns. A variation of this procedure based on established antibody production methods has also significant drawbacks as an additional vessel for the addition of diatomaceous earth as filter aid is required and the filter cassettes needs to be disassembled. This exposes the environment to the rAAVp. Likewise, adding the DE to a separate mixing vessel and afterwards pumping the lysed cell culture broth into said vessel to mix both requires additional equipment and time.

[0277] It has now been found by the current inventors that suspending the DE in water with a fixed concentration and only after a homogeneous slurry of DE and water is obtained, adding the slurry of this DE suspension to the bioreactor, where it is mixed with the lysed cell culture (see Figure 1) allows for good mixing between DE and biomass by intense stirring within the bioreactor. This is an easier and faster solution compared to other methods known from the art.

[0278] In more detail, in the first step a suspension of DE is generated in a first vessel. Therefore, the necessary amount of water is filled into the vessel and the DE is added slowly while stirring. For smaller scales, a magnetic stirrer can be used to stir the suspension of DE as can be seen in Figure 1. For larger scales, other mixing methods known in the art can be used. After a homogeneous slurry of DE and water is obtained, the suspension is transferred into the second vessel (the bioreactor), where it is mixed with the lysed cell culture. It has to be pointed out that good mixing between DE and biomass needs to be ensured which can be achieved by the stirrer in the bioreactor.

[0279] In one embodiment the method comprises the further step of evenly distributing the DE in the mammalian cell culture broth or in the lysed mammalian cell culture broth.

[0280] In one embodiment the DE and the biomass are (good / thoroughly) mixed by (intense) stirring (in the bioreactor).

[0281] In an optional second step, the filter module(s) is (were) flushed with water or buffer, e.g. about 50 L / m2During the flushing process, the filters should be also vented. After flushing, a pressure test can be performed with a higher pressure than the intended upper working pressure for filtration to ensure that there are no leakages. Thereafter the filter is connected to the harvest tube (of the bioreactor).

[0282] In the next step, the filtration is started by pumping the mixture of the reactor content, i.e. the cultivation broth, and DE from the bioreactor through an alluvial filter. The DE will remain on the membrane in the alluvial filter module and the soluble parts of the mixture will be obtained in the flow-through. It has been found that the flow-through was already clarified by > 90 % (regarding turbidity). Herein is reported a method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth. Herein is reported a method for clarifying a mammalian cell culture broth comprising recombinant viral particles.

[0283] In one embodiment, the purifying or clarifying is a reduction of turbidity.

[0284] In one embodiment the reduction of turbidity is by at least 75 %. In one embodiment the reduction of turbidity is by at least 80 %. In one embodiment the reduction of turbidity is by at least 85 %. In a preferred embodiment the reduction of turbidity is by at least 90 %.

[0285] During the filtration, a constant flux, e.g., of 300-500 LMH can be kept until a defined pressure, e.g. 1.5 bar, is reached. Reaching this pressure threshold, the flow can be either stopped completely or slowly reduced to zero while keeping the pressure constant.

[0286] In an optional further step, to further reduce process risks, a sterile filtration using a sterile filter, e.g. a combination of a 0.5 pm pre-filter and a 0.2 pm main-filter, can directly follow the alluvial filtration, i.e. is placed downstream of the alluvial filtration step.

[0287] In one embodiment the method further comprises a step of subj ecting the (alluvial) filtrate obtained before to a sterile filtration. In one embodiment the method further comprises recovering the filtrate.

[0288] In a final step, the alluvial filter module after completion of the filtration is emptied by pumping air into the filter module and, thus, reducing the hold-up volume and loss of product. Thereafter, the inlet and outlet tubing of the filter module are disconnected in a closed manner, e.g. by welding. This will allow to ensure a fully closed transport of the filter module to the autoclave for inactivation in line with legal provisions.

[0289] In one embodiment the steps of the method are performed in a fully closed setting / system.

[0290] A fully closed system in the context of this application means: during or after the process the system is completely closed. Cells and viral vectors are fully isolated from the environment. Any sampling / disassembly or other manipulation does not lead to exposure of environment or personnel to viral vectors, (based on Safety Levels of German Genetic Engineering Act: “a system that completely separates the process from the environment” - see “Bundesgesetzblatt: Neufassung des Gentechnikgesetzes”). This definition excludes the requirement for fully closed handling of non-toxic raw materials. Alluvial filtration

[0291] In the alluvial filtration step the amount of DE added as filter aid depends on the biological wet mass of the cultivation broth to be processed (BWM). A small scale experiment was carried out with different DE:BWM ratios to assess the effect of the DE:BWM ratio. The DE:BWM ratios tested were 1 :4, 1 :3, 1 :2 and 2:3. A ratio of 2:3 means that each 2 g DE were added per each 3 g BWM of the cell culture lysate. Converted to a concentration, a DE:BWM ratio of 1 :4 equals 2.50 g DE / L / % BWM, 1 :3 equals 3.33 g DE / L / % BWM, 1 :2 equals 5.00 g DE / L / % BWM and 2:3 equals 6.67 g DE / L / % BWM and 3 :4 equals 7.5 g DE / L / % BWM. These experiments were carried out with a higher flux than the usual target range of 300-500 LMH. The results are shown in Figure 2 and Figure 3.

[0292] It has been found that the highest filter capacity was achieved with the highest DE:BWM ratio of 2:3 (Figure 2), while the impact on impurity reduction was neglectable (Figure 3). When increasing the DE:BWM ratio, there always has to be a balance between using more DE to increase filter capacity and the maximum cake volume the filter module can accommodate. Thus, a DE:BWM ratio ranging from more than 1 :2 to 3 :4 has been found to be most suitable.

[0293] In the method reported herein the filter aid (DE) is added to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 DE / biological wet mass (BWM). In one embodiment the DE is added at a ratio of about 2:3 to about 3:4 DE / BWM. In one embodiment the DE is added at a ratio of about 2:3 or about 3:4 DE / BWM. In one embodiment the DE is added at a ratio of about 2:3 DE / BWM.

[0294] It has been found that alluvial filtration can effectively be used to reduce turbidity (i.e. intact cells, cell debris or / and non-soluble particles) and host cell DNA. The most suitable ratio DE / BWM is 2:3 or more. Both classes of impurities (HC DNA, turbidity) can be removed to a high degree.

[0295] Herein is reported a method for separating recombinant viral particles from (other) compounds of a mammalian cell culture broth. Herein is reported a method for clarifying a mammalian cell culture broth comprising recombinant viral particles.

[0296] In one embodiment the purifying or clarifying is a reduction of turbidity.

[0297] In one embodiment the purifying is a reduction of (the concentration or amount of) host cell DNA.

[0298] In one embodiment the reduction of turbidity is by at least 75 %. In one embodiment the reduction of turbidity is by at least 80 %. In one embodiment the reduction of turbidity is by at least 85 %. In a preferred embodiment the reduction of turbidity is by at least 90 %. In one embodiment the (concentration or amount of) host cell DNA is reduced by at least 80 %. In one embodiment the (concentration or amount of) host cell DNA is reduced by at least 85 %. In a preferred embodiment the (concentration or amount of) host cell DNA is reduced by at least 90 %. In one embodiment the (concentration or amount of) host cell DNA is reduced by at least 95 %.

[0299] In the following, results from the conducted separation and clarification experiments are collected with DE / BWM ratios of 2:3. The experiments were performed with lysed cell culture once per filter in a small scale for all depth filters and tangential flow depth filters. For the alluvial filters, experiments in various scales were performed. Shown in the figures is the average value and standard deviation of alluvial filtration (with Purafix CH33P + Cel pure C300) experiments ranging from 10 L to 100 L scale. As can be seen in Figure 4, most of the filters that were tested reached a sufficient turbidity reduction of over 90 %. Only the KI OOP and PDP8 filter medium as well as the TfDf module did not perform well regarding turbidity reduction.

[0300] In the next step, the ability to reduce host cell (HC) DNA was tested. It has been found (Figure 5) that only six filters were able to reduce host cell (HC) DNA by over 90 %. However, the PDP8 filter medium was already deemed not suitable concerning turbidity reduction. For the TfDf, no HC DNA data is available. Again, alluvial filtration performed very well with over 95% of removal ofHC DNA.

[0301] In the next step, the ability to recover the viral genome (vg) for the different filters was tested. The vg recovery is regarded to be 80 % or higher for a filter to be categorized as suitable. It has been found (Figure 6) that this is only the case for the alluvial filtration, the VI OOP filter medium and the TFDF-30. The TfDf however was already deemed not suitable regarding turbidity reduction.

[0302] In one embodiment the viral genome recovery is at least 75 %. In one embodiment the viral genome recovery is at least 80 %. In one embodiment the viral genome recovery is at least 85 %. In a preferred embodiment the viral genome recovery is at least 90 %. In one embodiment the viral genome recovery is at least 95 %.

[0303] In the next step, the ability to recover the capsid (cp) for the different filters was tested. The cp recovery is regarded to be 80 % or higher for a filter to be categorized as suitable. It has been found (Figure 7) that five different filters or filter combinations were suitable when only cp recovery is concerned. All other filters except the alluvial filtration however, were already deemed not suitable regarding turbidity reduction, HC DNA reduction and / or vg recovery. For the TfDf, a cp recovery of more than 100 % was observed, which is due to the variances of the ELISA that was used for determining the cp amount.

[0304] In a preferred embodiment the viral capsid recovery is at least 80 %. In one embodiment the viral capsid recovery is at least 85 %. In one embodiment the viral capsid recovery is at least 90 %. In the following Table 1, the results from the previous four figures (Figures 4 to 7) are summarized. Every value for impurity reduction and product recovery that is above the aforementioned respective limit for suitability has a grey background. Every value below this limit is displayed with a white background. It has been found that the only filter that was suitable regarding all criteria was the alluvial filter.

[0305] For the TfDf, a cp recovery of more than 100 % was observed, which is due to the variances of the ELISA that was used for determining the cp amount.

[0306] Table 1: Collection of the tested filtermedia including filter type and vendor with the corresponding results forturbidity reduction, HC DNA reduction, viral genome (vg) recovery and capsid (cp) recovery in percent.

[0307] Alluvial filtration scale up

[0308] As the alluvial filtration was the most promising technology in smaller scales, it was scaled up to clarify cell culture lysate from a 500 L bioreactor in order to test scalability. A Filtrodisc™ BIO SD 16” D module with 8 filter lenses and a DE:BWM ratio of 2:3 was used for clarification. It has been found that the scale-up can be seen as successful as a high turbidity reduction of 99% was observed and product recovery was equally high with 94% vg recovery and 107 % cp recovery. Cp recovery measurement of more than 100 % is due to the variances of the ELISA assay that was used for determining the cp amount.

[0309] In one embodiment the volume of the (crude) mammalian cell culture broth or the cellular lysate is 50 L or more. In one embodiment the volume of the (crude) mammalian cell culture broth is in the range of and including 50L to lOOOL. In one embodiment the volume of the mammalian (crude) cell culture broth or the cellular lysate is 50 L or more, 75 L or more, or 100 L or more.

[0310] Lysis vs. non-lysis

[0311] In a last step, the impact of the lysis of the cells before alluvial filtration was assessed. As expected, the turbidity and host cell protein values for lysed cell culture were significantly higher. It could also be shown that a significantly higher amount of viral vectors is released when the cell culture was lysed. Importantly, it has been found that the alluvial filtration works with lysed cells or for cells without lysis. There is no substantial loss regarding the product titer by alluvial filtration for lysed cells.

[0312] Further it has been found that, although alluvial filtration is possible for non-lysed cells, viral genome recovery is much worse for non-lysed cells (53%) than for lysed cells (87%) likely as viral vectors still trapped inside the cells and are removed together with the cells.

[0313] *** The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0314] Examples Materials and Methods

[0315] Filter aid, filter holders and filter modules that were used are shown in the following table. The actual volume that can be filtered with each module depends on the cell culture lysate and does not always have to be in the range given in the “Scale” column.

[0316] Table 2: List of used filter type and name including manufacturer, article number and additional information for use like filter area, maximum cake volume and scale. In some cases, article and manufacturer names have changed. The new names are indicated in grey with brackets behind the old name. Within the rest of the document, the old names may be used.

[0317] Example 1

[0318] Method according to the current invention for separating rAAVp from crude cultivation broths using alluvial filtration

[0319] Generally, the clarification of crude mammalian cultivation / cell culture broths containing rAAVp of the AAV2 or AAV8 serotype using alluvial filtration was performed directly from the bioreactor, which contains lysed cell culture broth. The cell culture was lysed with a detergent, in this case Triton was used, but other detergents are also possible for cell lysis. In addition to that, a nuclease treatment with 50 U / mL Benzonase® endonuclease was performed. After lysis and nuclease treatment, a samples were drawn, e.g. for BWM determination. Depending on the BWM the respective amount of filter aid, e.g. 2 g filter aid per 3 g BWM, was added to the lysed cell culture broth. The filter aid in this case was diatomaceous earth (DE) and was evenly distributed within the lysed cell culture.

[0320] In more detail, first, the suspension of DE with a concentration of 200 g / L was generated in a first vessel, e.g. a glass or plastic bottle / tank with a magnetic stirrer or a container with a single-use bag and mixer. Therefore, the necessary amount of water was filled into the vessel and the DE was added slowly via a tube using a pump while continuously stirring. After a homogeneous slurry of DE and water was obtained, the slurry of this DE suspension was transferred into the second vessel (a bioreactor for large scale; or a bottle for small scales respectively), where it was mixed with the lysed cell culture (see Figure 1). In small scale the cell culture lysate is in a bottle / flask, and the DE was directly added to said bottle / flask, e.g. in a safety cabinet which enables safe handling according to the regulations. Good mixing between DE and biomass was ensured by intense stirring in the second vessel.

[0321] Shortly before the filtration, the alluvial filter modules were flushed with 50 L / m2water or buffer. If there was a secondary filter, e.g. a sterile filter, which required a certain minimum flow rate for flushing, this was applied. Otherwise, the flow which is also used during filtration was used. If the secondary filter required a different minimum flushing volume than the alluvial filter, the higher volume was used for flushing both filters. During the flushing process, the filters were also vented. Afterwards, a pressure test was performed with a pressure higher than the critical pressure for filtration, i.e. for example 1.8-2.0 bar, to ensure that the system is tight. Afterwards, the water or buffer vessel was disconnected and the filter was connected to the harvest tube of the second vessel. If clamp-on flow sensors were used, which have to be zeroed with the fluid of which the flow rate is to be measured, the tubing was filled with the mixture of cell culture lysate and DE up until after the flow sensor. Then, the tubing was closed after and in front of the sensor to stop any flow and the flow sensor was zeroed. The pressure sensors were zeroed without pressure before the filtration was started. In case there was a scale to weigh the filtrate, this was also tared. When everything was ready, the filtration was started. The lysed cell culture mixed with DE was pumped from the second vessel through an alluvial filter (see Figure 1). The DE remained on the membrane in the alluvial filter module and the supernatant was obtained in the flow-through. The flow- through was already clarified by >90 % (regarding turbidity).

[0322] During the alluvial filtration, a constant flux of 300-500 LMH was kept until a defined pressure, e.g. 1.5 bar, was reached. At this pressure threshold, the flow was either stopped completely or slowly reduced to zero while keeping the pressure constant.

[0323] To further reduce process risks, a sterile filtration over a sterile filter combination of (for example) a 0.5 pm pre-filter and a 0.2 pm main-filter membrane can directly follow the alluvial filtration.

[0324] After successful filtration, the alluvial filter module was emptied by pumping air into it and, thus, reducing the hold-up volume and loss of product. For smaller scales using the 2”, 5” or 10” alluvial filter module, this step was optional. Then, the inlet and outlet tubing of the filter module were disconnected in a closed manner by welding or using Luer locks depending on the scale. This allowed a fully closed transport to the autoclave for inactivation.

[0325] The following Table 3 lists several example runs of the process (some in different scales) with information about the clarified lysate weight and the respective filter module size that was used.

[0326] Table 3: Example runs of the process with information about the clarified lysate, like serotype and lysate weight, as well as information about the alluvial filter, such as module size, filter sheet and DE grade. Example 2

[0327] Determination of biological wet mass

[0328] The determination of biological wet mass (BWM) was required to calculate the amount of diatomaceous earth needed as filter aid. To measure the BWM, two samples, e.g. of 9 mL each, were drawn from the vessel containing the cell culture lysate. Therefore, two 15 mL falcon tubes were first weighed on an analytical balance and afterwards each tube was filled with one of the two samples drawn. The filled falcon tubes were weighed again followed by centrifugation at 4500 g for 10 min. During centrifugation, a cell pellet was formed in each tube. The supernatant was discarded. The tubes with the pellets but without the supernatant were weighed again. From the weights noted down during the process, the concentration of BWM and the total BWM in the bioreactor were calculated using equations (1) and (2), which are described below.

[0329] The concentration of biological wet mass CBWM in g kg1was calculated by dividing the weight of the cell pellet mpeiiet in gram after centrifugation and decantation of the supernatant by the weight of the sample msampie in kilogram prior to centrifugation.

[0330] The total biological wet mass BWM in gram was obtained by multiplying the concentration of biological wet mass CBW in [g-kg-1] with the weight of the cell culture lysate niLysate in kilogram. This value was later used to determine how much filter aid was needed to be added for alluvial filtration.

[0331] BWM — CBWM■ Tl^ysate

[0332] The following Table 4 shows exemplary process data for concentration of BWM CBWM from which the total BWM and necessary amount of DE could be calculated.

[0333] Table 4: Example runs of the process with values for the concentration of BWM CBWM, total BWM, the used ratio ofDE:BWMand the concentration of DE in relation to the lysate weight CDE. Examole 3

[0334] Determination of filtration parameters

[0335] Pressure

[0336] Pressure is one of the filtration parameters which was measured or / and used as a setpoint for process control. This was done using a PendoTECH Single-Use Pressure Sensor™ (article number: PREPS-N-000 or PREPS-N-038) in between pump and filter and in between filters.

[0337] Flow

[0338] Flow is a filtration parameter which was measured and used as a setpoint for process control. This was done using a LeviFlow® Clamp-On Sensor LFSC-12D-007 (article number: 100-30390) from Levitronix ® in between the pump and the filter. For small scales, the flow for a specific tube at a specific pump speed was determined to correlate rpm to flow.

[0339] Flux

[0340] Flux was a setpoint from which the flow setpoint for the filtration process was calculated via equation (3). The flux J in L m“2h-1, also abbreviated as LMH, through the filter is defined as the filtrate volume Vputrate in L per filter area AF in m2and per filtration time t in h (Liderfelt and Royce, 2018, Chapter 14 Biopharmaceutical Processing. Elsevier: 279-293) and can also be calculated from the filter capacity CF in L m“2and filtration time t in h using equation (4).

[0341] Determination of filter performance indicators (filter capacity, impurity reduction and product recovery)

[0342] Filter

[0343] The filter capacity CF in L-m“2describes the filtrate volume Vpiitrate in L that flowed through the filter area A in m2until the pressure drop increases to a set maximum (constant flux operation) or the flux declines to a set minimum (constant pressure drop operation) (Bolton et aC 2006, Journal of Membrane Science 279(l):625-634). The following equation (4) was used to calculate the filter capacity:

[0344] , Filtrate (4)

[0345] F Turbidity reduction

[0346] Before and after filtration, the turbidity was measured with a turbidimeter. The turbidity reduction and indicate impurity removal and filter performance was calculated using equation (5). The turbidity reduction Redr in percent is calculated from feed turbidity Tpeed in Nephelometric Turbidity Units (NTU) and filtrate turbidity Tpntrate in NTU. 100 (5)

[0347] HC DNA reduction

[0348] Before and after filtration, the residual HC DNA concentration was measured, e.g. using qPCR. The HC DNA reduction and indicate filter performance was calculated using equation (6). The HC DNA reduction Redpc DNA in percent was calculated using either the concentration of HC DNA in the feed CHC DNA, Feed in ng / mL for HCP or pg / mL for HC DNA and the concentration of HC

[0349] DNA in the filtrate CHC DNA, Filtrate in pg / mL, or the mass of HC DNA in the feed mpc DNA, Feed in pg and the mass of HC DNA in the filtrate mnc DNA, Filtrate in pg.

[0350] Viral genome and capsid recovery

[0351] Before and after filtration, the viral genome titer was measured, e.g. using ddPCR, and the capsid titer was measured, e.g. via ELISA or ECLIA. From this, the product recovery was calculated using equation (7). The recovery Rectin percent, with either viral genome (vg) or capsid as i, was calculated either from the concentration of i in the feed Ci,Feed in vg / mL or capsid / mL and the concentration of i in the filtrate Ci, Filtrate in vg / mL or capsid / mL, or from the number of i in the feed ii i. Feed in and the number of i in the filtrate i, Filtrate-

[0352] Exemplary data The following Table 5 shows the filtration performance indicators across different example runs and scales. It can be seen that these indicators were very consistent across the example runs with only some variation in product recovery, which still was 80% or more for all examples.

[0353] Table 5: Example runs of the process with measured values for filter performance indicators like filter capacity, turbidity reduction in relation to initial turbidity in the lysate, host cell DNA (HC DNA) reduction, viral genome (vg) and capsid (cp) recovery in percent.

[0354] Example 5

[0355] Scale-Up of the process

[0356] For scaling up the process, the filter area was determined using the filter capacity determined in a smaller scale (see description and equation (8) below). In addition to the filter area, the cake volume was also considered when scaling the alluvial filter (see description and equation (9) below). The flux and pressure setpoints were kept the same as in smaller scales. Thereby comparability was ensured. Apart from this, bioreactor size, mixing and filtrate vessel size, tubing diameters, sensors and potentially the pump were adapted, where required for the higher flow, which was necessary to keep the flux constant while using a higher filter area. As the flush buffer volume retained in the system after filter flushing was larger for larger systems, dilution can play an impact in the samples drawn after filtration. Therefore, this effect was compensated by a dilution factor (see description and equation (11) below).

[0357] Scaling by Capacity

[0358] To scale the filter area for alluvial and depth filtration, the filter capacity was used. The necessary filter area A2 for the new scale was calculated using the capacity determined in previous small scale experiments CF,I in L-m“2and the intended filtrate volume VFHtrate,2 in L that is to be scaled- up / -down to. In addition to that, a safety factor .s was applied.

[0359] Taking example run 1 as an example for scale-up from a 100 L bioreactor harvest to a 500 L bioreactor harvest, the calculation would be the following: In example run 1, filter capacity was 268 L / m2In example run 6, the volume to be filtered is 398 L. Without considering a safety factor, this would mean that a filter area of at least 1.49 m2is necessary. The closest suitable filter module size would be the 16” module as, with its 1.81 m2, it has a filter area slightly larger than the minimum estimate from the calculated 1.49 m2Choosing the 16” filter module for example 6 means that a safety factor of 1.22 is included in the filter scaling.

[0360] Scaling by Cake Volume

[0361] For scaling the alluvial filtration, the filter cake volume Vcake in L was considered. The filter cake volume was calculated using equation (9). The diameter of the filter capsule dpnter in meter and the filter cake height hcake in meter, which were measured after filtration, were needed. 1000 (9)

[0362] The expected cake volume Vcake, 2 for the new scale was calculated using equation (10) with the cake volume determined in previous experiments Vcake, 1 in liter, the filtrate volume from previous small scale experiments VFHtrate,i in liter and the desired permeate volume VFHtrate,2 in liter that is intended to be scaled-up / -down to (Daumke et al., 2019 Alluvial Filtration, Single-Use Technology in Biopharmaceutical Manufacture: 271-277). In addition to that, a safety factor .s was applied.

[0363] To scale the cake volume, results from filtrations with 2”, 5” or 10” alluvial filter modules have to be used as filter cake height can best be determined with the structure of these modules in contrast to the 12” and 16” module which have a different structure that makes an accurate estimate challenging. Therefore, example run 4 is taken as an example for scaling cake volume from a 10 L bioreactor harvest with a 10” filter capsule to a 500 L bioreactor harvest. In example run 4, a filter cake height of 0,014 m was measured which corresponds to a filter cake volume of 0.71 L considering the filter capsule diameter of 0.254 m. Filtrate volume of example run 4 were 11.3 L and desired filtrate volume of example run 6 were 398 L. This leads to an expected cake volume of at least 25 L without safety factor. The next size of filter module able to accommodate this cake volume is the 16” filter module and therefore was chosen for example run 6. In this case, a safety factor of 1.84 is included.

[0364] Dilution factor

[0365] In some cases, when there is concern that the filtrate might be strongly diluted from rinsing the filter, e.g. when the filtrate volume is small and / or the hold-up volume of the filter is large, the dilution factor DF was calculated via equation (11) using the concentration of glucose in the feed CGlucose.Feed (feed can be the cell culture lysate) and the concentration of glucose in the filtrate CGiucose, Filtrate as glucose is expected to pass the filter without any loss. Using this dilution factor, the concentration of i without dilution (ct, undiluted), with i being e.g. HC DNA concentration or viral genome titer or else, was calculated from the concentration of i in the diluted sample (ct, diluted).

[0366] Example 6

[0367] Comparison lysis vs. no lysis prior filtration

[0368] To compare the filtration performance and product recovery for lysed versus non-lysed culture, the same cell culture from one bioreactor was used. One part of the material (circa 2 L) was lysed with the standard protocol used for all example runs while the other part of the material (circa 1 L) stayed non-lysed. A sample of both the lysed and non-lysed cell culture was drawn. Both the lysed and non-lysed material were then filtered with the same system and parameters in a small scale using 2” alluvial filter capsules. Afterwards, samples were drawn from both filtrates and analysed. The impurity reduction and product recovery were calculated using the sample from the lysate prior filtration to have the same reference prior filtration for both scenarios and calculate the product recovery in relation to the total amount of product contained inside and outside of the cells which can only be measured using a lysate sample. Thereby it was ensured that a potential loss of product contained in the non-lysed cells which can be filtered out, could be visible. As Table 6 shows, it could indeed be seen that there was a high loss of product after filtration of the non-lysed cell culture, especially regarding the viral genome titer where almost half of it was lost and only 53% could be recovered. For the non-lysed material, the pressure limit of 1.5 bar was not reached before all of the non-lysed cell culture was filtered and therefore it can only be said that the filter capacity is at least 538 L / m2but more could be possible. For the lysed cell culture, the pressure limit of 1.5 bar was reached and thus the maximum filter capacity of 905 L / m2could be determined. Table 6: Results from the filtrations to comparing lysed and non-lysed cell culture. Important read-outs such as measured values for filter capacity, turbidity reduction and product recovery are listed.

Claims

- 62 -Patent Claims1. A method for separating recombinant viral particles from compounds of a mammalian cell culture broth comprising the steps of: a) adding diatomaceous earth (DE) or synthetic silica-based filter aid to a mammalian cell culture broth comprising recombinant viral particles at a ratio of more than 1 :2 DE or synthetic silica-based filter aid / biological wet mass (BWM) to obtain a pre-filtration mixture, b) subjecting the pre-filtration mixture to alluvial filtration, and thereby separating recombinant viral particles from compounds of the mammalian cell culture broth.

2. The method according to claim 1, wherein the DE or synthetic silica-based filter aid is added at a ratio of about 2:3 to about 3:4 DE or synthetic silica-based filter aid / BWM.

3. The method according to any one of claims 1 to 2, wherein the mammalian cell culture broth is a cellular lysate of the mammalian cell culture broth comprising the recombinant viral particles, intact cells and cell debris and the compounds of the mammalian cell culture broth are comprising intact cells and cell debris.

4. The method according to claim 3, wherein the cellular lysate further comprises host cell DNA.

5. The method according to any one of claims 1 to 4, wherein the separating is from compounds comprising intact cells, cell debris and non-soluble particles.

6. The Method according to any one of claims 1 to 5, wherein the separating is a purifying.

7. The method according to any one of claims 3 to 6, wherein the cellular lysate is obtained by lysis and wherein the lysis is a chemical lysis, an enzymatic lysis or a physical lysis.

8. The method according to any one of claims 1 to 7, wherein the volume of the mammalian cell culture broth or the cellular lysate is 50 L or more.

9. The method according to any one of claims 1 to 8, wherein the mammalian cell culture broth is obtained by cultivating transiently transfected mammalian cells producing recombinant viral particles.

10. The method according to any one of claims 1 to 9, wherein the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle.

11. The method according to any one of claims 1 to 10, wherein the recombinant viral particle is a recombinant adeno-associated viral (rAAV) particle of AAV 2 or AAV 8 serotype.

12. The method according to any one of claims 1 to 11, wherein the mammalian cell is a HEK293 cell or a CHO cell.

13. The method according to any one of claims 1 to 12, wherein after step b) the method further comprises the following step: c) subjecting the alluvial filtrate obtained in step b) to a sterile filtration .

14. The method according to any one of claims 1 to 13, wherein step b) or / and step c) further comprises recovering the filtrate.

15. The method according to any one of claims 1 to 14, wherein steps a) or / and b) are performed in a fully closed system.

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