KR20240002094A - Baculovirus Infected Cell Stock for AAV vector production and Method for manufacturing AAV vector - Google Patents

Abstract

The present invention relates to baculovirus-infected cell stocks for AAV vector production and methods for producing AAV vectors. When using a baculovirus-infected cell stock that produces the adeno-related viral vector of the present invention, the time required to produce the adeno-related viral vector can be reduced, storage stability can be greatly improved, and the productivity of the AAV vector can be greatly improved. .

Description

Baculovirus Infected Cell Stock for AAV vector production and Method for manufacturing AAV vector}

The present invention relates to baculovirus-infected cell stocks for AAV vector production and methods for producing AAV vectors.

Adenovirus-associated virus (AAV) is one of the most promising viral vectors used for human gene therapy. AAV has the ability to effectively infect human proliferating as well as non-proliferating cells, has a relatively harmless immunogenic profile, and is not associated with any disease. From this perspective, recombinant adeno-related viruses are being evaluated in clinical trials of gene therapy for various diseases such as hemophilia and cystic fibrosis. In 2012, Unicur's Glybera, an AAV-based drug, received marketing approval as a new drug from the European Medicines Agency (EMA), and treatments using AAV, such as Novartis' spinal muscular atrophy (SMA) treatment Zolgensma, are being developed.

HEK 293T cells, HeLa cells and other mammalian cell lines are used for the production of AAV. The production of these AAVs is provided by a DNA plasmid containing the genes for replication proteins Rep78, Rep68, Rep52, and Rep40 and the virion or structural proteins VP1, VP2, and VP3, as well as a therapeutic gene of interest (GOI). do. However, there are limitations to mass production and commercial scale production using these mammalian cells, and vectors cultured in mammalian cells are likely to be contaminated with unwanted pathogens present in mammalian cells. To overcome these problems of mammalian production systems, a production system using insect cells has recently been developed.

Baculovirus is a type of virus that infects insect cells, and is used for research on gene transfer and the production of various biological drugs, including the production of AAV of the present invention. Baculovirus has a membrane structure on its surface, so its stability may be somewhat reduced when the virus is preserved in liquid baculovirus stock (LBS). Therefore, rather than preserving the virus with a liquid baculovirus stock (LBS) containing baculovirus, the present inventors used a baculovirus infected cell stock (BICS) capable of producing baculovirus. ), and used it to improve the productivity of AAV.

The present inventors have made extensive research efforts to develop a mass production method suitable for mass production of AAV vectors. As a result, a baculovirus infected cell stock (BICS) was created to produce AAV vectors, and the stock and AAV production method were established to improve the productivity of AAV vectors.

Accordingly, the purpose of the present invention is to provide a method for producing a baculovirus infected cell (BIC) stock that produces an adeno-associated virus (AAV) vector.

Another object of the present invention is to provide a method for generating an AAV vector using the BIC stock prepared by the above-described method.

According to one aspect of the present invention, the present invention provides a method for producing a baculovirus infected cell (BIC) stock producing an adeno-associated virus (AAV) vector:

(a) culturing insect cells and infecting the cultured insect cells with a baculovirus containing an AAV vector expression construct; and

(b) cultivating the baculovirus-infected cells.

In this specification, “adeno-related virus” corresponds to a virus belonging to the genus Parvoviridae and Dependovirus, and is one of the smallest DNA viruses, with a virus particle diameter of 18 to 25 nm and single stranded DNA. It's a virus. Adeno-related viruses are non-pathogenic satellite viruses that require a helper adenovirus for replication and can carry relatively small genes of ~4.7 kb in size. The genome of AAV contains inverted terminal repeats (ITRs), which flank unique coding nucleotide sequences for non-structural replication (Rep) and structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the capsid. The terminal of the ITR has a length of about 100 to 150 nt and is self-complementary and therefore consists of an energetically stable intramolecular duplex structure that may be in the form of a T-type hairpin. This hairpin structure serves as the origin for viral DNA replication and as a primer for the cellular DNA polymerase complex.

The AAV has serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9, but is not limited thereto, and various other serotypes of natural or recombinant AAV are known. The ITR, Rep, or VP of these serotypes have at least 70%, 75%, 80%, 85%, 90%, or 95% nucleotide and/or amino acid sequence homology. AAV used in one embodiment of the present invention is a serotype of AAV1.

In one embodiment of the present invention, the method for producing the baculovirus infected cell (BIC) stock additionally includes the step of (c) freezing the baculovirus infected cells.

The freezing is preferably performed when the cell viability of the BIC is at least 95%.

The baculovirus-infected cells (BIC) are prepared by infecting insect cells with liquid baculovirus in the first passage, and the cells passaged by infecting new insect cells with the baculovirus-infected cells of the first passage are the second passage. of baculovirus infected cells (BIC). The freezing is preferably performed on first- or second-passage baculovirus-infected cells.

The liquid baculovirus is obtained by culturing insect cells such as Sf9, transfecting them with pDNA expressing the baculovirus, and recovering the supernatant in which the baculovirus is produced by expressing the pDNA. It refers to a liquid culture medium containing selected baculoviruses.

In one embodiment of the present invention, the AAV vector expression construct includes i) a Rep-Cap sequence expressing AAV replication protein and AAV capsid protein; and ii) a GOI sequence expressing a gene of interest (GOI).

In one embodiment of the present invention, the Rep-Cap sequence can be used by selecting and combining known nucleotide sequences depending on the serotype of AAV to be produced. For example, to produce AAV1, AAV2 Rep78 and AAV1 Cap sequences can be used, and the sequence information of each nucleotide sequence is SEQ ID NO: 2 or 3, SEQ ID NO: 4 or 5, but the sequence information is illustrative and limited thereto. That is not the case.

In one embodiment of the present invention, the GOI nucleotide sequence includes the HGF gene of SEQ ID NO: 1, but is not limited to this and nucleotide sequences of various genes of interest may be introduced.

In this specification, the term 'nucleotide' includes not only natural nucleotides, but also analogues in which sugar or base sites are modified (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

It is clear to those skilled in the art that the nucleotide sequence suffices to be a nucleotide sequence encoding a protein or polypeptide identical to the protein or polypeptide encoded by the gene of interest, and is not limited to any specific nucleotide sequence.

This is because even if a mutation in the nucleotide sequence occurs, when the mutated nucleotide sequence is expressed as a protein or polypeptide, there are cases where there is no change in the amino acid sequence of the protein or polypeptide. This is called codon degeneracy. Accordingly, the nucleotide sequence can be either a functionally equivalent codon, or a codon encoding the same amino acid (e.g., due to codon degeneracy, there are six codons for arginine or serine), or a codon encoding a biologically equivalent amino acid. It contains a nucleotide sequence containing.

Considering the mutations having the above-mentioned biological equivalent activity, the nucleotide sequence of the present invention encoding the protein or polypeptide is interpreted to also include sequences showing substantial identity thereto. The above substantial identity is achieved by aligning the sequence of the present invention and any other sequence to correspond as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art. 60% or more homology (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more specifically 70% or more homology (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), more specifically 80% or more homology (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), and even more specifically, at least 90% homology (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and most specifically refers to a sequence that exhibits at least 95% homology (e.g., 95%, 96%, 97%, 98%, or 99%). All integers between 60% and 100% and decimal numbers in between are included within the scope of the present invention with respect to % homology.

Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are discussed in Smith and Waterman, Adv. Appl. Math. 2:482(1981) ; Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is accessible from NCBI (National Center for Biological Information), etc., and can be accessed on the Internet using blastp, blastn, It can be used in conjunction with sequence analysis programs such as blastx, tblastn, and tblastx. BLAST can be accessed through the BLAST page on the ncbi website. The sequence homology comparison method using this program can be found on the BLAST help page of the ncbi website.

In one embodiment of the present invention, i) the Rep-Cap sequence and ii) the GOI sequence are included in different DNA fragments. For example, the baculovirus-infected cell according to one embodiment of the present invention is i) baculovirus containing a Rep-Cap sequence expressing AAV replication protein and AAV capsid protein. A first BIC is infected, and ii) a second BIC is infected with a baculovirus comprising a GOI sequence expressing a gene of interest (GOI). In one embodiment of the present invention, the DNA fragment containing the GOI sequence includes a promoter operably linked thereto. As used herein, a "promoter" or "transcription regulatory sequence" acts to regulate transcription of one or more coding sequences and is located upstream with respect to the direction of transcription of the transcription start point of the coding sequence, Structurally, DNA-dependent RNA polymerase binding sites, transcription initiation points and transcription factor binding points, repressor and activating protein binding points, and transcription from promoters directly or indirectly to those skilled in the art. It includes, but is not limited to, any other sequence of nucleotides known to act in dose control.

In one embodiment of the present invention, the promoter is a promoter derived from the genome of a mammalian cell (e.g., metallothioneine promoter, beta-actin promoter, EF-1alpha promoter, human heroglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, The promoters of Moloney virus (the promoter of Epstein Barr virus (EBV) and the promoter of Rouss sarcoma virus (RSV)) can be used and generally have a polyadenylation sequence as the transcription termination sequence.

In one embodiment of the present invention, the DNA fragment containing the GOI sequence includes at least one polyadenylation sequence. The polyadenylation sequence may range from about 500 nt in length.

In one embodiment of the present invention, the DNA fragment containing the GOI sequence includes ITR sequences on both sides. In one embodiment of the present invention, the insect cells are Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, Ha2302, Hz2E5, High Five (Invitrogen, CA, USA) and expresSF+® (US 6,103,526; Protein Sciences Corp., CT, USA), etc., but are not limited thereto.

In a specific embodiment of the invention, the insect cells are Sf9 cells.

In a more specific embodiment of the present invention, the Sf9 cell is a rhabdovirus-negative Sf9 cell.

In one embodiment of the present invention, the culture medium used for the culture may be Gibco's Sf-900 III SFM, or Lonza's Insect X-Press.

In one embodiment of the present invention, the culture is cultured under mixing conditions of 110 to 150 rpm. More specifically, it may be 110 to 140 rpm, 110 to 130 rpm, 110 to 120 rpm, 120 to 150 rpm, 130 to 150 rpm, or 140 to 150 rpm, but is not limited to this and is not limited to specific embodiments of the present invention. According to this, there is no significant difference in culture efficiency within the above range.

In one embodiment of the present invention, the culture is performed in a culture solution with a volume of 30 to 70 mL. More specifically, it may be 30 to 60 mL, 30 to 50 mL, 40 to 70 mL, 50 to 70 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, etc., but is not limited thereto. According to specific embodiments of the invention, there is no significant difference in culture efficiency within the above range.

In one embodiment of the present invention, the culturing is performed at 26 to 30°C. More specifically, the culturing may be carried out at 26 to 29°C, 26 to 28°C, 27 to 29°C, 28 to 29°C, 26°C, 27°C, 28°C, 29°C, or 30°C, but is limited thereto. That is not the case.

In one embodiment of the present invention, the culture is performed at pH 6.0 to 6.4, but is not limited thereto. In the insect cell culture and BIC production of the present invention, the pH conditions were maintained within the above range even without special adjustment.

In one embodiment of the present invention, the culture in step (a) has an initial cell density of 0.5 x 10 ⁶ to 1.2 x 10 ⁶ cells/mL.

In one embodiment of the present invention, the Osmolarity of the culture medium in the above culture is preferably 360-380 mOsm/kg.

In one embodiment of the present invention, the infection with the baculovirus is performed at a multiplicity of infection (MOI) of 0.1 to 3.

In a specific embodiment of the present invention, the infection of the baculovirus is 0.1 to 3, 0.1 to 2.5, 0.1 to 2, 0.1 to 1.5, 0.1 to 1.0, 0.1 to 0.5, 0.1 to 0.4, 0.1 to 0.3, 0.1 to 1.0. It consists of 0.2 or 0.1 MOI (multiplicity of infection), but is not limited thereto, and there is no significant difference in culture efficiency within the above range.

In one embodiment of the present invention, the culture time in step (b) is 18 to 24 hours after baculovirus infection, but is not limited thereto.

According to another aspect of the present invention, the present invention provides a method of producing an AAV vector using the BIC stock prepared by the above baculovirus infected cell (BIC) stock production method:

(a) BIC cells are taken out from the baculovirus infected cell (BIC) stock producing the adeno-associated virus (AAV) vector, and the host cells of the same type as the BIC are Adding to the culture containing the population and culturing in a bioreactor; and

(b) Isolating the AAV vector from the culture medium in which the culture was completed in step (a).

In one embodiment of the present invention, the baculovirus-infected cells include i) a baculovirus comprising a Rep-Cap sequence expressing an AAV replication protein and an AAV capsid protein; and ii) a baculovirus containing a GOI sequence expressing a gene of interest (GOI).

Cells infected with a baculovirus containing the Rep-Cap sequence and cells infected with a baculovirus containing a GOI sequence expressing a gene of interest (GOI) are thawed and cultured in a frozen and preserved state. It proliferates. These baculovirus-infected cells (BIC) co-infect non-baculovirus-infected host cells. The co-infected cells are cultured and propagated in a bioreactor to produce AAV particles.

In one embodiment of the present invention, the initial cell density of the uninfected host cells for AAV production is 0.5 x 10 ⁶ to 1.2 x 10 ⁶ cells/mL. In a specific embodiment of the present invention, when cells are cultured at a cell density higher than the above conditions, for example, an initial cell density of 1.5 x 10 ⁶ cells/mL, the cells initially die before sufficient production of AAV occurs and the medium My nutrients are depleted.

In one embodiment of the present invention, the volume of the bioreactor may be at least 1L, 3L, 5L, 10L, 20L, 50L, 100L, 150L, 200L, or 300L, but is not limited thereto.

In one embodiment of the present invention, the bioreactor is an automated bioreactor that uses a combination of pumps, filters, etc. to culture the cells in the bioreactor while continuously removing cell consumption products and performing cell metabolism. A medium depleted of nutrients can be replaced.

In one embodiment of the present invention, the culture in step (a) is performed by additionally adding cell nutrients during the culture.

In one embodiment of the present invention, the additionally added cell nutritional component includes glucose, glutamine, yeast extract, or a combination thereof.

In a specific embodiment of the present invention, the additionally added cell nutrient is added to supplement the glucose concentration in the medium up to 5 g/L, but is not limited thereto, and has the concentration or ratio contained in the medium before supplementation. May contain glucose, glutamine, and yeast extract.

In a specific embodiment of the present invention, the additionally added glucose, glutamine and yeast extract are at a concentration of 30 to 60 g/L of glucose, 10 to 60 g/L of glutamine, and 20 to 60 g/L of yeast extract or the above ratio. It can be supplemented by adding a supplementary feeding media containing, but is not limited to this.

In a specific embodiment of the present invention, the additionally added glucose, glutamine, and yeast extract include 30 to 60 g/L of glucose, 12 to 24 g/L of glutamine, and 20 to 50 g/L of yeast extract; Glucose 60 g/L, glutamine 12 to 18 g/L, and yeast extract 20 to 40 g/L; Alternatively, it may be supplemented by adding feeding media containing 60 g/L of glucose, 12 to 18 g/L of glutamine, and 20 to 30 g/L of yeast extract or in the above ratio, but is limited thereto. no.

In one embodiment of the present invention, the number of additions of the supplementary medium may be once, twice, or three times before AAV recovery, but is not limited thereto.

In one embodiment of the present invention, an anti-foaming agent may be added at a concentration of 1 to 5% during the culture in step (a). More specifically, 1%, 2%, 3%, 4%, or 5% of anti-foaming agent can be added, but there is no significant difference within the above range.

In one embodiment of the present invention, the anti-foaming agent may be a silicone-based or oil-based anti-foaming agent, but is not limited thereto. The silicone-based anti-foaming agent may include polydimethylsiloxane. The oil-based anti-foaming agent may include ethylene bis stearamide (EBS), paraffin wax, and ester wax.

In one embodiment of the present invention, during the culture in step (a), the BIC stock has a multiplicity of infection (MOI) of 0.01 to 0.05, 0.01 to 0.04, 0.01 to 0.03, 0.02 to 0.05, 0.02 to 0.04, and 0.02 to 0.03. ) is added to a host cell population homologous to BIC. The host cell population homologous to the BIC may be, for example, insect cells, more specifically Sf9.

In one embodiment of the present invention, the addition of the BIC during the culture in step (a) is done 8 to 18 hours after the start of the culture of the host cells of the same species as the BIC.

In a specific embodiment of the present invention, upon addition of the BIC stock, the productivity increased to 20 hours or less as a result of infection at 20 to 28 hours after culturing host cells of the same species as BIC, such as insect cells, and an additional 16 hours. As a result of infection from 22 hours to 16 hours or less, productivity has increased. Additionally, as a result of additional infection at 12 to 18 hours, productivity increased to 12 hours or less. Therefore, the above-mentioned infection time is suitable to be 10 to 18 hours after culturing insect cells, but the shorter the time, the higher the AAV production efficiency.

In one embodiment of the present invention, in the culture in step (a), the baculovirus-infected cells are 1 to 2 passages, or 1 passage baculovirus-infected cells. More specifically, the baculovirus-infected cells may be 1 to 2 passages old, but the lower the number of passages, the better AAV productivity.

The first-passage baculovirus-infected cells (BIC) were prepared by infecting insect cells with liquid baculovirus, and the second-passage baculovirus-infected cells were prepared by infecting the first-passage baculovirus-infected cells with new insects. It refers to cells that have been infected and passaged.

In one embodiment of the present invention, DO (dissolved oxygen, %) of the culture medium during the culture in step (a) is 8 to 16%.

In a specific embodiment of the present invention, as a result of culturing in step (a) under conditions where the DO of the culture medium was 20 to 40%, the productivity of AAV was lowest at 30%, and productivity improved at 20% or less. was confirmed, and additionally, as a result of culturing under DO conditions of 10 to 30%, it was confirmed that AAV productivity improved as DO decreased. Additionally, the results of culturing under DO conditions of 10 to 16% confirmed that AAV productivity improved as DO decreased.

In one embodiment of the present invention, the completion time of culture in step (b) is 96 to 132 hours after addition of BIC. In this specification, the time of completion of the culture is expressed as a recovery time or harvest time.

In a specific embodiment of the present invention, the culture completion time is 96 hours to 120 hours, 96 hours to 114 hours, 96 hours to 108 hours, 96 hours to 102 hours, 102 hours to 120 hours, and 102 hours after addition of BIC. It may be from 102 hours to 114 hours, 102 hours to 108 hours, 108 hours to 120 hours, or 108 hours to 114 hours, but is not limited thereto.

In one embodiment of the present invention, the culture medium in step (b) is a culture medium containing the produced AAV, and is a culture medium in which destruction and lysis of cells in the culture medium have been performed at least once.

In a specific embodiment of the present invention, the cell destruction and lysis may be accomplished by mechanical, physical or chemical lysis. The mechanical lysis may be, for example, lysis using a cell disruptor such as an ultra-high pressure emulsifier (Microfluidizer), the physical lysis may be, for example, freezing and thawing, and the chemical lysis may be treatment with cell digestion enzymes and nucleases. However, it is not limited to this.

In one embodiment of the present invention, the method for producing the AAV vector of the present invention may additionally include the step of purifying the produced AAV particles and the grown insect cell harvest.

In one embodiment of the present invention, the purification may be performed by one or more chromatography and purification processes including affinity chromatography and ion exchange chromatography (AEX or CEX).

In another embodiment of the present invention, the purification may be performed using one or more nanofiltration, ultrafiltration, or microfiltration processes. The nanofiltration, ultrafiltration, or microfiltration processes include, for example, EMD Millipore Express SHC XL10 0.5/0.2 μm filter, EMD Millipore Express SHCXL6000 0.5/0.2 μm filter, EMD Millipore Express SHCXL150 filter, EMD Millipore Millipak Gamma Gold 0.22 μm filter, Pall It may be Supor EKV, 0.2 μm sterile grade filter, Asahi Planova 35 N, Asahi Planova 20 N, Asahi Planova 75 N, Asahi Planova BioEx, Millipore Viresolve NFR or Sartorius Sartopore 2 XLG, 0.8/0.2 μm.

In another embodiment of the present invention, the purification may be performed by a size exclusion chromatography process.

In one embodiment of the invention, the titer of the AAV particles is measured according to genome copy number (vg/mL). The concentration of genomic particles may be based on the results of qPCR of vector DNA, but the method of measuring titer is not limited thereto. The vg/mL represents vector genome per millimeter.

Since the method for producing an AAV vector according to another aspect of the present invention described above involves culturing insect cells, which are host cells, to produce the AAV vector, in relation to the method for producing a BIC stock according to an aspect of the present invention, Contents that can be commonly applied between the two inventions, such as culture conditions for insect cells, culture methods, and baculovirus infection methods, are equally applied.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing a baculovirus infected cell (BIC) stock producing an adeno-associated virus (AAV) vector.

(b) The present invention provides a method of producing an AAV vector using the BIC stock prepared by the above baculovirus infected cell (BIC) stock production method.

(i) Time required to produce an adeno-associated virus vector when using a baculovirus infected cell (BIC) stock producing the adeno-associated virus (AAV) vector of the present invention It can reduce storage stability and greatly improve the productivity of AAV vectors.

Figure 1 is a diagram showing the effect of the type of culture medium of the present invention on the growth of insect cells in terms of specific growth rate, viable cell density, glucose, and glutamine concentration.
Figure 2 is a diagram showing the effect of the type of culture medium of the present invention on insect cell density according to culture time.
Figure 3 is a diagram showing the effect of the type of culture medium of the present invention on cell growth rate and maximum cell density.
Figures 4 and 5 are diagrams showing the effect of the feeding time of the culture medium of the present invention on insect cell growth, such as specific growth rate, viable cell density, osmolarity, glucose and glutamine concentration.
Figure 6 is a diagram showing the effect of nutrient feeding time of the culture medium of the present invention on insect cell growth in terms of lactate concentration.
Figure 7 is a diagram showing the change in titer of AAV according to the initial cell concentration and MOI of the present invention.
Figure 8 is a diagram showing the change in titer of AAV according to the initial cell concentration of the present invention.
Figure 9 is a diagram showing the AAV production titer according to the recovery time of baculovirus-infected cells of the present invention.
Figure 10 is a graph showing the effect on the productivity of the AAV1 vector of the present invention when baculovirus-infected cells of the present invention were cultured according to DO, MOI, infection time, and concentration of anti-foam agent.
Figure 11 is a graph showing the effect on the productivity of the AAV1 vector of the present invention when baculovirus-infected cells of the present invention were cultured according to DO, infection time, and rpm speed.
Figure 12 is a graph showing the effect on the productivity of the AAV1 vector of the present invention when baculovirus-infected cells of the present invention are cultured according to DO and infection time.
Figure 13 is a graph showing the effect on the productivity of the AAV1 vector of the present invention when baculovirus-infected cells of the present invention are cultured according to DO and MOI.
Figure 14 is a graph showing the effect on the productivity of the AAV1 vector of the present invention when baculovirus-infected cells of the present invention are cultured according to MOI and infection time.
Figure 15 is a graph showing the regression analysis equation for AAV1 titer derived from the examples of the present invention and the influence (normalization effect) on the productivity of the AAV1 vector of the present invention for each condition (DO, infection time, and MOI). .
Figure 16 is a graph showing the regression analysis equation for the maximum cell number derived from the examples of the present invention and the influence (normalization effect) on the productivity of the AAV1 vector of the present invention for each condition (DO, infection time, and MOI) am.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

<Development of AAV culture process and AAV production using BICS>

Equipment and experimental materials

(1) Equipment

Equipment Productor Part No. Company BSC #AC2-6G8-TAPGB Esco Centrifuge #5942 000 315 Effendorf Incubator 160i Thermofisher Shaker NB101-SRC N-Biotect Automated Cell Counter #Countess II(AMQAXIOOO) Thermofisher

(2) Materials

Materials Product No. Company SF-RVN Cell #SFRVNLIC-1EA Merck 1000mL Erlenmeyer Flask with Vented Cap 431147 Corning 125mL Erlenmeyer Flask with Vented Cap 431403 Corning 50mL Conical Tube 50050 S.P.L. 5mL Pipet Aid Tip 91005 S.P.L. 10mL Pipet Aid Tip 91010 S.P.L. 50mL Pipet Aid Tip 91050 S.P.L. Pipe Aid 075232 Drummond 1000P Pipet QN71810 Glison 1000P Pipet Tip BT1000.96 Neptune 20P pipet RD73364 Gilson 20P Pipet Tip BT20 Neptune Insect-XPRESS media BEBP12-730Q Lonza Merck CD 74405CP Merck Sf-900 II SFM 10902088 Thermofisher Sf-900 III SFM 12658027 Thermofisher Trypan blue solution, 0.4% (Trypan blue) 15250061 ThermoFisher 1.7mL microtube MCT-175-C Axygen Cell Counting Chamber Slides (C-Chip) Invitrogen C10283

Example 1. Selection of basic culture conditions for Sf-RVN cells

AAV is produced by co-infecting insect cells with Rep-Cap baculovirus and GOI baculovirus.

The present inventors performed the following experiment to establish basic culture conditions for Sf-RVN cells, which are insect cells used for production of the AAV vector of the present invention.

Sf-RVN® Insect Cell Line is a rhabdovirus-negative Sf9 cell line that can be grown in both adherent and suspension cultures. The Sf-RVN® insect cell line is used for the production of adeno-associated viruses (AAV) of the invention, as well as recombinant proteins, and virus-like particles (VLPs).

Basic culture conditions according to the Sf-RVN culture guidelines distributed by Merck are shown in Table 3 below.

Cell cut volume (mL) RPM Temperature Initial Cell Density
(e ⁶ cells/mL) 20-50mL in 125mL Flask 130-140 27-28 One 30-100mL in 250mL Flask 130-140 27-28 One 60-150mL in 500mL Flask 130-140 27-28 One 100-300mL in 1L Flask 90-100 27-28 One

The volume of the cell culture vessel and culture medium was fixed by culturing at 50 mL in a 125 mL flask. Based on the recommended RPM of 130, it was changed to 110 and 150 RPM, and the recommended temperature of 28℃ was changed to 26, 27, and 28℃, and the initial cell density was changed to 0.5 based on the recommended density of 1 e ⁶ cell/mL. , 0.75, 1.0, 1.5 e ⁶ cell/mL were changed to check the effect on cell growth. To account for experimental errors, culture was performed using two flasks per experimental group.

Experimental
Group (n=2) RPM Temperature Cell cut volume (mL) Initial pH initial Cell Density (e6 cells/mL) Control 130 27 50 6.25 1.0 One 110 27 50 6.25 1.0 2 150 27 50 6.25 1.0 3 130 26 50 6.25 1.0 4 130 28 50 6.25 1.0 5 130 27 30 6.25 1.0 6 130 27 70 6.25 1.0 7 130 27 50 6.10 1.0 8 130 27 50 6.40 1.0 9 130 27 50 6.25 0.50 10 130 27 50 6.25 0.75 11 130 27 50 6.25 1.50

For each experimental group, 1 ml was sampled every 24 hours to measure cell count, viability, and concentrations of glucose and glutamine.

(1) Viable cell density and cell viability according to conditions

As a result of measuring viable cell density and cell viability according to the above conditions, when culturing with an initial cell density of 1.5 e ⁶ cells/mL, the initial cell density was lower at 0.5, 0.75, and 1 e ⁶ cells/mL. Cells died faster than in culture, and other conditions had no significant effect.

(2) Viable cell density and specific growth rate according to conditions

As in (1) above, there were no conditions that had a significant effect on the specific growth rate except for the initial cell density.

(3) Average specific growth rate according to conditions

In the case of RPM conditions, there was no significant difference in average specific growth rate among the tested 110 to 150 RPM, so the general 130 RPM was selected.

Since the culture volume (working volume) did not show a significant difference in average specific growth rate among the tested culture volumes of 30 to 70 mL, it was decided to culture at a volume of 50 mL, which is the middle value among 30-70 mL.

The initial pH conditions did not show a significant difference in average specific growth rate between pH 6.1 and pH 6.4, so it was decided not to adjust them.

Since there was no significant difference in average specific growth rate depending on the culture temperature of 26-28°C, the general culture temperature of 28°C was selected.

When the initial cell density was 1.5 e ⁶ cells/mL, the Average Specific Growth Rate increased dramatically, but nutrients in the medium were quickly consumed, causing rapid cell death. In the remaining conditions of 0.5 to 1 e ⁶ cells/mL, the Average Specific Growth Rate was similar in all conditions, so, except for the 1.5 e ⁶ cells/mL condition, the middle value of 0.75 e ⁶ cells/mL was selected among the remaining conditions.

The basic culture conditions selected according to the culture results for each condition above are shown in Table 5.

RPM Temperature (°C) Cell Culture volume (mL) Initial pH initial Cell Density
(e ⁶ cells/mL) 130 28 50 Not control 0.75

(4) Fed-batch culture medium and culture time settings

First, as a result of measuring changes in glucose and glutamine concentration according to the above conditions, the lower the initial cell density, the slower the rate of glucose depletion, and the depletion rate of glucose was faster than that of glutamine.

The ingredients of the feed media used were glucose, glutamine, and yeast extract.

As mentioned above, since glucose is consumed faster than glutamine, the timing of feeding injection was adjusted according to the concentration of glucose.

Additionally, as a result of measuring the consumption rate of glucose and glutamine, when 1 g/L of glucose was consumed, 0.34 g/L of glutamine was consumed.

The ratio of Yeast Extract and Glucose needs to be tested in the future, but for now it was set at 1:1. At this time, since the solubility of Yeast Extract is approximately 100g/L, it was temporarily made with Yeast Extract 60g/L, Glucose 60g/L, and Glutamine: 18g/L to prevent saturation.

Research on Feeding Time is necessary, but the current analysis results show that all Glutamine is consumed in 72 hours, so the feed was replenished at 48 hours to bring the initial medium glucose concentration to 4-6g/L.

(5) Selection of type of culture medium

The present inventors attempted to select a medium in which insect cells grow well using insect cells adapted to Lonza's Insect-Xpress, Merck's CD, Gibco's Sf-900 II, and Gibco's Sf-900 III SFM.

The culture conditions were selected the same as those in (3) above, and considering the error of the experiment, each experimental group was cultured in 3 flasks, and 1 ml was sampled every 24 hours to measure Viable Cell Density, Viability, Glucose, and Glutamine Concentration. did.

Experimental
Group (n=3) Media RPM Temperature
(°C) Initial Cell Density Volume (mL) One Gibco Sf-900 II 130 28 0.75 e6 cells/mL 50 2 Lonza Company: Insect-Xpress 3 Merck iCD 4 Gibco Sf-900 III SFM

The results are shown in Figures 1 to 3.

As shown in Figures 1 to 3, there was no significant difference between Average Specific Growth Rate and Media, but Gibco's Sf-900 III had the best Max Viable Cell Density, so Gibco's Sf-900 III was selected as the Growth Media. .

(6) Selection of feed conditions

Based on the randomly selected values of Glucose: 60g/L, Yeast Extract: 60g/L, and Glutamine: 24g/L in the preliminary experiment, in order to know the concentration ratio of Glucose, Glutamine, and Yeast Extract, Glucose was fixed at 6Og/L and Glutamine and Yeast extract concentration was adjusted. Since the solubility of Yeast Extract was 100g/L, it was changed to 90g/L, 60g/L, and 30g/L, and the concentration of Glutamine was changed to 12g/L and 18g/L based on a rate of 24g/L. As shown in Figures 1 and 2, to prevent glutamine depletion at 72 hours after culture, feed was supplemented at 48 hours. To account for experimental errors, culture was performed in two flasks per experimental group.

The experimental conditions are shown in Table 7, and 1 mL of culture medium was sampled every 24 hours to measure viable cell density, viability, glucose concentration, glutamine concentration, osmotic pressure, and lactic acid concentration.

Feed composition and concentration conditions Experimental group (n=2) Glucose (g/L) Glutamine (g/L) Yeast extract (g/L) Control group (No feeding) - - - One 60 12 30 2 60 18 30 3 60 24 30 4 60 12 60 5 60 18 60 6 60 24 60 7 60 12 90 8 60 18 90 9 60 24 90

The results are shown in Figures 4 and 5.

As shown in Figures 4 and 5, when feeding was performed at 48 hours, the specific growth rate of the experimental group increased compared to the control group, but the difference between the experimental groups was not significant.

Since the optimal osmolarity of insect cells is 360-380 mOsm/kg, it is recommended that the osmolarity be maintained at 360-380 mOsm/kg during culture. Osmolarity increased slightly during feeding, but it is acceptable as it is between 360-380 mOsm/kg in all experimental groups.

If a large amount of lactic acid, a by-product of cell culture, is produced, it has a negative effect on the growth and production of insect cells, so the lactic acid concentration was measured, and as a result, compared to when feed containing 30 g/L of yeast extract was added. Since the lactic acid concentration was high when 60 g/L and 90 g/L of yeast extract were added, the concentration of yeast extract in the feed was set to 30 g/L.

Example 2. Selection of basic culture conditions for AAV production

Comparison of AAV production according to MOI (multiplicity of infection) and CDI (cell density at infection) conditions

The present inventor conducted the following experiment to determine multiplicity of infection (MOI), cell density at infection (CDI), and feeding time, which are process parameters that affect AAV production.

First, to prepare baculovirus infected cell stock (BICS), Sf-RVN cells were subcultured at a concentration of 0.6 e ⁶ cells/mL in 50 ml of a 125 mL flask using X-PRESS or SFM III media. Cultivation was performed for 1 day at 28°C at 130 rcf (relative centrifugal force). When the concentration of the cells reached 1.2 e ⁶ cells/ml, liquid baculovirus containing the Rep/Cap gene and liquid baculovirus containing the GOI gene were added to the Sf-RVN cells at a concentration of 3 MOI, respectively, and incubated for 18 hours. was cultured. Afterwards, cells were collected, the pellet was diluted with X-press media or SFM III media, 10% DMSO was added, and finally stored in LN2 tank. Among the BICS manufactured in this way, the BICS using X-press media were named BICCX0001 and BICCX0002, and the BICS using SFM III media were named BICCX0007 and BICCX0008. The odd-numbered BICS, named BICCX, contains Rep-Cap genes (SEQ ID NO: 3 and 5) and the even-numbered BICS contains the GOI gene (SEQ ID NO: 1).

Among the BICCXs, BICCX0001 and BICCX0002 were inoculated at MOIs of 0.01, 0.025, 0.05, and 0.075, and AAV production was compared. At this time, AAV production was compared with CDI (cell density at infection) set at 1.5, 3, and 6 e ⁶ cells/mL.

Harvest time showed that the highest titer was observed at 120 hours after infection in preliminary experiments, so cells were harvested at 120 hours after infection. To recover AAV, the culture medium containing the cells was centrifuged at 2500xg for 40 minutes, AAV was recovered from the supernatant, and the titer of AAV was detected through qPCR.

Each culture experimental condition is shown in Table 8 below.

experimental group MOI CDI (e ⁶ cells/mL) Control - - One 0.01 1.5 2 0.025 1.5 3 0.05 1.5 4 0.075 1.5 5 0.05 1.5 6 0.01 3 7 0.025 3 8 0.05 3 9 0.05 6

The results are shown in Figure 7.

As shown in Figure 7, it was confirmed that the productivity of AAV decreased when the cell density of CDI was increased to 3 e ⁶ cells/mL or more. Additionally, it was confirmed that productivity was excellent in the MOI range of 0.01 to 0.05.

From the above results, CDI of 1.5 e ⁶ cell/mL or less and MOI of 0.01 to 0.05 were excellent, so 0.02 MOI was selected.

The present inventors additionally observed changes in AAV productivity according to CDI conditions shown in Table 9 below.

experimental group MOI CDI (e ⁶ cells/mL) Volume (mL) One 0.02 1.2 250 2 0.02 1.2 100 3 0.02 0.8 50 4 0.02 1.0 50 5 0.02 1.2 50

The results are shown in Figure 9.

As shown in Figure 9, there was no significant effect on AAV productivity depending on the culture volume of 50 to 250 mL or CDI change between 0.8 and 1.2 e ⁶ cells/mL.

In conclusion, CDI was selected as 0.8 to 1.2 e ⁶ cells/mL and MOI as 0.02.

<Development of Viral Stock Manufacturing Method for AAV Production>

Baculovirus is a type of virus that infects insect cells, and is used for research on gene transfer and the production of various biological drugs, including the production of AAV of the present invention. Baculovirus has a membrane structure on its surface, so its stability may be somewhat reduced when the virus is preserved using Liquid baculovirus stock (LBS). Therefore, rather than preserving the virus with Liquid baculovirus stock (LBS) containing baculovirus, the present inventors attempted to produce a baculovirus infected cell stock (BICS) capable of producing baculovirus, and produced BIC as in Examples 3 to 5 below. Stability was confirmed according to conditions and passage.

Example 3. Confirmation of AAV productivity of baculovirus infected cell stock (BICS)

The present inventors confirmed the productivity of AAV according to the infection concentration of BICS as follows.

To produce AAV, Sf-RVN cells were subcultured at a concentration of 0.6 e ⁶ cells/mL in 50 ml of a 125 mL flask using X-PRESS media. Cultivation was performed for 1 day at 28°C at 130 rcf (relative centrifugal force). Cultured Sf-RVN cells were counted and when the cell density reached approximately 1.2 e ⁶ cell/ml, two types of BIC stocks, BICCX0001 and BICCX0002, were added to the flask at 0.05, 0.1, and 0.5 MOI (total 3 sets). Sampling was performed from each flask for 6 days at 24-hour intervals, and each sample was stored in a Deepfreezer at -80°C until AAV titer analysis.

For titer analysis, the samples were repeated three times by freezing/thawing using liquid nitrogen, centrifuged at 3000 rcf for 25 minutes, and the supernatant was recovered. The titer of the supernatant was analyzed using the AAV pro titration kit (TAKARA, Cat. 6233).

The results were as shown in Table 10 and Figure 9 below.

Post infection
Time 24 hours 48hrs
(feeding) 72 hours 96hrs 120hrs 144 hours BICS 0.05 MOI 1.78 E+10 9.81 E+11 6.63 E+12 6.89 E+12 1.01 E+13 1.00 E+13 BICS 0.1 MOI 3.92 E+09 2.62 E+12 8.65 E+12 8.94 E+12 1.04 E+13 8.43 E+12 BICS 0.5 MOI 3.19 E+10 4.10 E+12 1.03 E+13 8.83 E+12 1.17 E+13 1.22 E+13

As shown in Table 10 and Figure 9, there was no significant effect on AAV titer within the MOI range of 0.05 to 0.5, and the virus titer was confirmed to be excellent at 120 hpi. Hereafter, culture conditions were set to 0.02 MOI infection and 120 hr recovery as selected in Example 2 above.

Example 4. Confirmation of AAV productivity according to BICS production and passage of BICS using Lonza insect X-PRESS media

In order to set optimal BICS production conditions for increasing passage of BICS, the present inventors confirmed the productivity of AAV according to the number of passages of BICS in Lonza insect X-PRESS media as follows.

Two types of Rep/CAP BIC and GOI BIC were used for AAV production.

The process variables were as shown in Table 11 below. Infection was performed assuming an infectivity of 100 pfu per BICS cell, and AAV production was performed on a 50 mL scale.

- process variable Set No. Media Passage No. MOI hpi (hr) One Lonza 2 0.1 22 2 Lonza 2 0.1 24 3 Lonza 2 0.5 22 4 Lonza 2 0.5 24 5 Lonza 2 One 22 6 Lonza 2 One 24 7 Lonza 3 0.1 24 8 Lonza 2 0.1 24 9 Lonza 2 One 24 10 Lonza 3 One 24

AAV was produced using BICS prepared under the same conditions as above. At this time, the MOI was set to 0.02 MOI, which is the existing AAV production condition, but the MOI was adjusted if additional testing was necessary. AAV production was carried out in a 50 mL flask, glucose concentration was measured 48 hours after infection, and feed was added to reach a concentration of 5 g/L. Each sample was recovered at 108 (±12) hours and stored in a -80°C deepfreezer until analysis.

Samples were processed in the same manner as in Example 3 to measure the titer of AAV, and the results are shown in Table 12.

Passage BICS MOI BICS Harvest time (hr) Media AAV MOI Titer(vg/L) One 3 18 X-PRESS 0.02 1.0 E+13 2 0.1 22 X-PRESS 0.1 6.6 E+12 2 0.5 22 X-PRESS 0.1 2.9 E+12 2 One 22 X-PRESS 0.1 2.6 E+12 2 0.1 24 X-PRESS 0.1 6.2 E+12 2 0.5 24 X-PRESS 0.1 2.2 E+12 2 One 24 X-PRESS 0.1 1.8 E+12

Specifically, the BICS MOI refers to the MOI of the baculovirus inoculated for the production of BIC. Additionally, the AAV MOI refers to the MOI of baculovirus-infected cells (BICS) added to produce AAV.

As mentioned above, during BICS production, the highest AAV production was achieved at a baculovirus MOI concentration of 0.1, and there was no significant difference in harvest time under conditions of 22 to 24 hours.

Based on the above results, AAV production was performed using BICS produced with baculovirus infected at passage 2 and MOI of 0.1 and 1, and the BICS concentration was treated over a wide range of 0.05 to 0.2 MOI concentration, and the results were as follows.

Passage BICS MOI BICS Harvest time (hr) Media AAV MOI Titer(vg/L) 2 0.1 22 X-PRESS 0.01 9.7 E+11 2 0.1 22 X-PRESS 0.01 1.0 E+12 2 0.1 22 X-PRESS 0.02 1.4 E+12 2 0.1 22 X-PRESS 0.02 1.2 E+12 2 0.1 22 X-PRESS 0.05 6.4 E+12 2 0.1 22 X-PRESS 0.05 7.1 E+12 2 0.1 22 X-PRESS 0.1 6.4 E+12 2 0.1 22 X-PRESS 0.1 8.3 E+12 2 0.1 22 X-PRESS 0.2 5.6 E+12 2 0.1 22 X-PRESS 0.2 4.8 E+12 2 One 24 X-PRESS 0.05 3.7 E+12 2 One 24 X-PRESS 0.05 3.1 E+12 2 One 24 X-PRESS 0.01 5.0 E+12 2 One 24 X-PRESS 0.01 3.2 E+12 2 One 24 X-PRESS 0.02 3.6 E+12 2 One 24 X-PRESS 0.02 4.1 E+12 2 One 24 X-PRESS 0.05 2.7 E+12 2 One 24 X-PRESS 0.05 1.9 E+12 2 One 24 X-PRESS 0.1 2.1 E+12 2 One 24 X-PRESS 0.1 2.0 E+12 3 0.1 24 X-PRESS 0.02 2.2 E+10 3 0.1 24 X-PRESS 0.02 2.7 E+10 3 0.1 24 X-PRESS 0.1 1.5 E+11 3 0.1 24 X-PRESS 0.1 2.4 E+11

As shown above, passage 2 (P2) BICS produced by infection with baculovirus at 0.1 MOI showed an AAV production of at least 5 E+12 vg/L when BICS was infected with 0.05 MOI or more, and baculovirus In the case of P2 BICS produced at an MOI of 1, the titer was slightly higher when BICS was infected at a lower concentration of 0.02 MOI or less.

In the case of AAV production using P3 BICS, the titer increased to about 2 E+11 vg/L when infected with BICS at an MOI of 0.1 MOI or more, but AAV productivity was at least ten times lower than that of P1 or P2. It shows that even if the passage increases under equal conditions, the productivity of AAV decreases as the passage increases.

When SFM III media, which is a more nutritious media than the existing

Passage BICS MOI BICS Harvest time (hr) Media AAV MOI Titer(vg/L) 2 0.1 24 SFM III 0.02 2.7 E+11 2 0.1 24 SFM III 0.02 5.6 E+11 2 0.1 24 SFM III 0.1 9.0 E+12 2 0.1 24 SFM III 0.1 4.5 E+12 2 One 24 SFM III 0.02 1.1 E+13 2 One 24 SFM III 0.02 7.1 E+12 2 One 24 SFM III 0.1 6.3 E+12 2 One 24 SFM III 0.1 5.5 E+12 3 One 24 SFM III 0.02 3.2 E+12 3 One 24 SFM III 0.02 2.3 E+12 2 One 24 SFM III 0.05 5.3 E+12 3 One 24 SFM III 0.05 6.1 E+12 3 One 24 SFM III 0.1 5.2 E+12 3 One 24 SFM III 0.1 3.6 E+12

As described above, when the number of passages of BICS was increased using SFM III media, it was confirmed that when BICS was produced at 1 MOI, which is a higher concentration than X-PRESS, the productivity of AAV was similar or slightly higher. The AAV production concentration was also slightly lower than the passage number 1 condition, but showed productivity of about 7.5E+12 vg/L.

Even at passage number 3, when SFM III media was used, the produced AAV showed higher titers than when X-PRESS media was used. However, in this case as well, the productivity of AAV was lower than at passage number 2, and it appears that sufficient AAV productivity can be maintained only by adding a larger amount of BICS.

In terms of overall trend, the passage stability of BICS using SFM III was confirmed to be higher than that of X-PRESS. However, in the case of this experiment, since

Example 5. Confirmation of AAV productivity according to BICS production and BICS passage using SFM III media

As mentioned in Example 4 above, initial BICS was produced using SFM III to compare passage stability according to X-PRESS and SFM III media. The lot numbers of BICS produced using SFM III were BICCX0007 and BICCX0008. This experiment was conducted based on BICCX0007 (Rep-Cap BICS) and BICCX0008 (GOI (HGF gene) BIC), and the present inventors produced BIC with the process variables shown in Table 15 below and passed the BICS on SFM III media. The productivity of AAV was confirmed.

Set No. Batch record Media Passage MOI hpi (hr) One BICCX0001, BICCX0002 SFM III 2 0.1 18 2 22 3 24 4 0.5 18 5 22 6 24 7 One 18 8 22 9 24 10 3 One 24 11 BICCX0007, BICCX0008 SFM III 2 0.1 18 12 24 13 0.5 24 14 One 18 15 24 16 1.5 24 17 3 24 18 3 One 24 19 1.5 24 20 3 24

AAV was produced using BICS prepared under the same conditions as above. At this time, the MOI was set at 0.02 MOI based on BICS, the existing AAV production conditions, but the MOI was adjusted if additional testing was necessary. AAV production was carried out in a 50 mL flask, glucose concentration was measured 48 hours after infection, and feed was added to reach a concentration of 5 g/L. Each sample was recovered at 108 (±12) hours and stored in a -80°C deepfreezer until analysis. Samples were processed in the same manner as in Example 3 to measure the titer of AAV.

As a first test, AAV production was conducted for BICCX0007 and BICCX0008 at an AAV production infection concentration of 0.02 to 0.1 MOI. The results for AAV production are shown in Table 16 below.

Batch record Passage BICS MOI BICS harvest time (hr) Media AAV MOI vg/L BICCX0007, 0008 One 3 18 SFM III 0.02 1.6 E+13 0.02 2.3 E+13 0.05 1.2 E+13 0.05 1.7 E+13 0.1 1.4 E+13 0.1 1.4 E+13

As described above, AAV production was stable over a wide range regardless of the AAV production infection concentration, and production was also confirmed to be higher (approximately 1 E+13 vg/L) compared to BICCX0001 and BICCX0002.

In addition, AAV production at passage 2 of BICCX0007-0008 was as follows. Only the BICS infection concentration condition was used as a variable, and the infection concentration during AAV production was unified at 0.02.

Batch record Passage BICS MOI BICS harvest time (hr) Media AAV MOI vg/L BICCX0007, 0008 2 0.1 18 SFM III 0.02 4.4 E+10 0.1 18 5.2 E+10 One 18 1.4 E+11 One 18 1.0 E+11 0.1 24 1.8 E+2 0.1 24 2.1 E+12 0.5 24 4.1 E+12 0.5 24 5.1 E+2 One 24 7.4 E+12 One 24 8.9 E+12 1.5 24 1.2 E+13 1.5 24 6.3 E+12 3 24 6.1 E+12 3 24 8.5 E+12

As shown in the table above, when producing BICS, the recovery time was more than 24 hours and the infection MOI was 1, which showed the best productivity. AAV productivity was approximately 8.2 E+12 vg/L, which showed a tendency to slightly decrease compared to BICS passage 1, but showed a slightly higher AAV titer than BICS passage 2 using X-PRESS. AAV production in BICS passage 3 is shown in the table below. Shown in 18.

Batch record Passage BICS MOI BICS harvest time (hr) Media AAV MOI vg/L BICCX0007, 0008 3 0.5 24 SFM III 0.02 1.4 E+12 0.5 1.1 E+12 One 1.6 E+12 One 1.4 E+12 3 3.3 E+12 3 3.8 E+12

As shown in the table above, in passage 3, it was confirmed that AAV production was approximately 1.5 E+12 vg/L, which was significantly reduced compared to the previous passage number 2. When the MOI of BICS was increased, it increased to about 4 E+12 vg/L. Compared to the case of using the X-PRESS medium confirmed in Example 4, no rapid decrease in AAV production was confirmed at passage 3. Therefore, it was found that BICS stability was better in SFM III medium than in X-PRESS medium.

<Setting of bioreactor production conditions for AAV production>

The present inventors conducted research in a 15 ml Ambr vessel to set the conditions for AAV1 production in a bioreactor using the BICS established in the above example.

laboratory equipment equipment Model No. Company clean bench AC2-6G8-TAPGB ESCO Ambr15 Ambr® 15 cell culture generation 2 Satorius Centrifuge 5910R Effendorf Incubator 160i Thermofisher Shaker NB101-SRC N-biotech Automated Cell Counter Countess II (AMQAX1000) Thermofisher bioreactor Biostat-B-DCI-2FL Satorius

Example 6. Setting of AAV1 production conditions in Ambr 15

The present inventors confirmed productivity for four conditions: DO (dissolved oxygen), infect time, MOI, and antifoam. The BICS used at this time were BICCX0011B and BICCX0012B, manufactured in the same process as BICCX0007 and BICCX0008.

(1) Variable setting

- DO: 20%, 30%, and 40%;

- infect time: 20 hr, 24 hr, and 28 hr after incubation,

- MOI: 0.01, 0.02, and 0.03;

- Antifoam agent: Add 20 μl of 1%, 3%, and 5% antifoam agent per 15 ml in the culture stage.

(2) Fixed value setting

The remaining culture conditions were set the same as those used in the previous test.

- Initial cell density: 6 E+5 cells/mL

- Badge: Lonza Insect X-PRESS

- Feed time: 48, 96 hpi

- Feed media: Glucose/Glutamine/yeast extract=60/12/30 (g/L)

- Feed up to 5 g/L at timepoint

-harvest time: 120 hr hpi

- RC:HGF = 1:1

- RPM 947 (200 rpm in Bioreactor)

- pH 6.2 (However, due to Ambr settings, pH cannot be set below 6.5, so it is not adjusted)

(3) Test condition settings and results

The test conditions and test results of Ambr 15 operated according to the above variables are shown in Table 20 below.

std order Run order PT type Block DO% Infection time MOI Antifoam% vg/L 8 One 2 One 20% 28 0.01 3% 3.21 E+13 10 2 One One 20% 20 0.02 One% 6.20 E+13 6 3 2 One 20% 28 0.0 One% 4.07 E+13 5 4 2 One 40% 28 0.02 5% 3.89 E+13 4 5 2 One 20% 24 0.01 5% 4.87 E+13 7 6 2 One 30% 24 0.02 3% 4.22 E+13 3 7 2 One 40% 28 0.01 One% 2.45 E+13 2 8 2 One 30% 20 0.01 One% 3.85 E+13 11 9 One One 40% 24 0.03 One% 4.65 E+13 12 10 One One 20% 20 0.03 5% 4.59 E+13 13 11 0 One 30% 24 0.02 3% 4.22 E+13 One 12 2 One 30% 28 0.03 5% 3.17 E+13 9 13 One One 40% 20 0.01 5% 4.24 E+13

A graph regarding the productivity of AAV1 for the above conditions and results is shown in Figure 10.

As shown in Figure 10, in the case of DO, productivity increased in a range lower than 20%, and productivity increased in infection time below 20 hours.

Productivity was highest at MOI of 0.02 to 0.03, and in the case of antifoam agent, there was no change in productivity depending on concentration.

Additionally, it was confirmed that the pH gradually decreased from 6.2 to about 6.0 during cultivation.

Example 7. Setting of AAV1 production conditions at Ambr 15 2

The present inventors attempted to set additional production conditions based on the results of Example 6. In this example, productivity was confirmed for DO (dissolved oxygen), infect time, and rpm conditions.

(1) Variable setting

- DO: 10%, 20%, and 30%;

-infect time: 16 hr, 19 hr, and 22 hr after incubation,

- RPM: 100, 200 rpm (based on bioreactor)

(2) Fixed value setting

The remaining culture conditions were set the same as those used in the previous test.

- Initial cell density: 6 E+5 cells/mL

- Badge: Lonza Insect X-PRESS

- Feed time: 48, 96 hpi

- Feed media: Glucose/Glutamine/yeast extract=60/12/30 (g/L)

- Feed up to 5 g/L at timepoint

-harvest time: 120 hr hpi

- RC:HGF = 1:1

- RPM 947 (200 rpm in Bioreactor)

- Antifoam agent 5% fixed

- MOI: fixed at 0.02

(3) Test condition settings and results

The test conditions and test results of Ambr 15 operated according to the above variables are shown in Table 21, Table 22, and Figure 11 below.

std order Run order PT type Block Infection time DO% RPM vg/L 11 One One One 22 30 100 2.49 E+13 14 2 0 One 19 20 200 5.12 E+13 One 3 2 One 19 30 200 5.45 E+13 5 4 One One 22 30 100 2.24 E+13 12 5 One One 16 10 200 6.09 E+13 13 6 0 One 19 20 100 4.04 E+13 3 7 2 One 22 20 200 4.51 E+13 4 8 2 One 16 20 100 5.21 E+13 10 9 One One 16 30 200 4.98 E+13 7 10 One One 22 10 200 5.16 E+13 9 11 One One 22 10 100 4.25 E+13 6 12 One One 16 10 200 7.45 E+13 2 13 2 One 19 10 100 5.69 E+13 8 14 One One 16 30 100 4.64 E+13

std order Run order Viable cell no. at infection time
(cells/ml) Total cell No.
(cells/ml, at harvest) Viable cell no.
(cells/ml, at harvest) Viability
(at harvest) vg/l 11 One 1.30 E+06 5.42 E+06 1.41 E+06 26% 2.49 E+13 14 2 1.16 E+06 5.05 E+06 3.74 E+06 74% 5.12 E+13 One 3 1.20 E+06 5.46 E+06 4.59 E+06 84% 5.45 E+13 5 4 1.05 E+06 6.06 E+06 1.88 E+06 31% 2.24 E+13 12 5 1.26 E+06 4.72 E+06 3.92 E+06 83% 6.09 E+13 13 6 1.20 E+06 4.14 E+06 2.73 E+06 66% 4.04 E+13 3 7 1.07 E+06 4.29 E+06 2.53 E+06 59% 4.51 E+13 4 8 1.01 E+06 3.65 E+06 2.30 E+06 63% 5.21 E+13 10 9 1.16 E+06 3.71 E+06 2.89 E+06 78% 4.98 E+13 7 10 1.34 E+06 5.20 E+06 4.47 E+06 86% 5.16 E+13 9 11 1.30 E+06 4.70 E+06 3.43 E+06 73% 4.25 E+13 6 12 1.13 E+06 5.05 E+06 4.04 E+06 80% 7.45 E+13 2 13 1.20 E+06 4.86 E+06 3.89 E+06 80% 5.69 E+13 8 14 1.23 E+06 4.08 E+06 2.45 E+06 60% 4.64 E+13

As shown in Figure 11, in the case of DO, productivity increased in a range lower than 10%, and in the case of infection time, productivity increased in a range of 16 hours or less. In the case of RPM, productivity increased at rpm higher than 200.

Example 8. Setting of AAV1 production conditions in Ambr 15 3

The present inventors attempted to set additional production conditions based on the results of Examples 6 and 7 above. In this example, productivity was confirmed for DO (dissolved oxygen), infect time, MOI, and harvest time conditions.

(1) Variable setting

- DO: 8%, 12%, and 16%;

- infect time: 12 hr, 15 hr, and 18 hr after incubation,

- MOI: 0.015, 0.0225, 0.03

-harvest time: 117, 120, 123 hr

(2) Fixed value setting

The remaining culture conditions were set the same as those used in the previous test.

- Initial cell density: 6 E+5 cells/mL

- Badge: Lonza Insect X-PRESS

- Feed time: 48, 96 hpi

- Feed media: Glucose/Glutamine/yeast extract=60/12/30 (g/L)

- Feed up to 5 g/L at timepoint

-harvest time: 120 hr hpi

- RC:HGF = 1:1

- RPM 947 (200 rpm in Bioreactor)

- Antifoam agent 5% fixed

- rpm: fixed at 200 (based on bioreactor)

(3) Test condition settings and results

The test conditions and test results of Ambr 15 operated according to the above variables are shown in Table 23, Table 24, and Figures 12 to 16 below.

std order Run order PT type Block DO% Infection time(h) MOI Harvest time(h) CS1-3 10 One 2 One 13 18 0.015 120 CS1-4 4 2 2 One 16 18 0.0225 120 CS1-5 13 3 0 One 13 15 0.0225 120 CS1-6 One 4 2 One 10 12 0.0225 120 CS1-6 123 CS1-7 8 5 2 One 16 15 0.03 120 CS1-8 7 6 2 One 10 15 0.03 120 CS1-9 15 7 0 One 13 15 0.0225 120 CS1-10 11 8 2 One 13 12 0.03 120 CS1-11 9 117 CS1-11 3 2 One 10 18 0.0225 120 CS1-11 123 CS1-12 12 10 2 One 13 18 0.03 120 CS2-3 14 11 0 One 13 15 0.0225 120 CS2-4 6 12 2 One 16 15 0.015 120 CS2-5 2 13 2 One 16 12 0.0225 120 CS2-6 9 14 2 One 13 12 0.015 120 CS2-7 5 15 2 One 10 15 0.015 120 CS2-12 117 CS2-12 10 15 0.0225 120 CS2-12 123

Viable cell no. at infection time
(cells/ml) Total cell No.
(cells/ml, at harvest) Viabler cell No.
(cells/ml, at harvest) Viability
(at harvest) vg/L average CS1-3 1.11 E+06 4.51 E+06 3.69 E+06 82% 4.08 E+13 CS1-4 1.09 E+06 4.46 E+06 3.30 E+06 74% 4.54 E+13 CS1-5 1.02 E+06 3.93 E+06 3.14 E+06 80% 4.90 E+13 CS1-6 7.80 E+05 3.76 E+06 2.92 E+06 77% 5.16 E+13 CS1-6 4.02 E+06 3.42 E+06 85% 4.81 E+13 CS1-7 9.60 E+05 2.61 E+06 1.83 E+06 70% 3.58 E+13 CS1-8 9.40 E+05 3.39 E+06 2.85 E+06 84% 4.19 E+13 CS1-9 1.04 E+06 3.88 E+06 3.18 E+06 82% 4.84 E+13 CS1-10 8.68 E+05 3.25 E+06 2.32 E+06 71% 3.69 E+13 CS1-11 3.77 E+06 3.13 E+06 83% 4.70 E+13 CS1-11 1.02 E+06 3.71 E+06 3.04 E+06 82% 4.27 E+13 CS1-11 3.43 E+06 2.64 E+06 77% 4.13 E+13 CS1-12 1.02 E+06 4.28 E+06 3.34 E+06 78% 4.21 E+13 CS2-3 9.70 E+05 4.01 E+06 3.25 E+06 81% 5.25 E+13 CS2-4 1.10 E+06 3.91 E+06 3.00 E+06 77% 4.00 E+13 CS2-5 8.97 E+05 3.48 E+06 2.82 E+06 81% 4.04 E+13 CS2-6 8.80 E+05 8.94 E+06 1.43 E+06 16% 4.41 E+13 CS2-7 1.16 E+06 3.89 E+06 2.84 E+06 73% 3.78 E+13 CS2-12 3.91 E+06 2.74 E+06 70% 3.63 E+13 CS2-12 9.70 E+05 3.38 E+06 2.47 E+06 73% 3.81 E+13 CS2-12 4.09 E+06 3.07 E+06 75% 4.49 E+13

As shown in Table 23-24 and Figure 12-16, when the MOI was fixed at 0.021, the maximum AAV titer could be expected at 10-11% DO and an infection time of 10-11 hours (Figure 12), infection time When fixed at 12 hours, the maximum AAV titer could be expected at 10-11% DO and 0.02-0.0225 MOI (Figure 13), and when DO was fixed at 11%, infection time was 10-11 hours and 0.02-0.0225 MOI. The maximum AAV titer could be expected (Figure 14). Through this, it was found that among the process variables, DO, infection time, and MOI have a great influence on the production process. Figure 15 shows that MOI has the greatest influence, and Figure 16 shows that among them, MOI and infection time This was shown to have the greatest impact. Ultimately, it was confirmed that optimal AAV production was achieved at DO 10.8%, BIC infection time after culture (12 hours), and MOI 0.0215.

Example 9. Bioreactor test

The present inventors conducted a productivity test for AAV1 in a bioreactor under the conditions obtained from the results of Examples 6 to 8 above.

(1) Condition setting

- scale: 1 L

- MOI: 0.0215

-infection time: 12hr

- DO: 10.8%

- Antifoam agent: 5%

- feed time: 48h, 96,

- Other conditions were the same as Example 8.

(2) Bioreactor operation results

The bioreactor operation results according to the above conditions are shown in Table 25 below.

- - 48hrs 96hrs 120hrs AAV titer Name Scale Live cell No.
(cells/ml) Viability Live cell No.
(cells/ml) Viability Live cell No.
(cells/ml) Viability vg/l Reactor 1 1 L 1.75 E+06 71% 1.23 E+06 29% 6.10 E+05 15% 2.99 E+13 Reactor 2 1 L 1.76 E+06 68% 9.50 E+05 23% 5.70 E+05 14% 3.02 E+13 Flask 1 50mL 3.45 E+06 98% 3.40 E+06 84% 5.20 E+05 11% 2.48 E+13 Flask 2 50mL 3.33 E+06 98% 3.55 E+06 78% 8.80 E+05 18% 3.39 E+13 Flask 3 50mL 3.28 E+06 95% 2.91 E+06 71% 5.20 E+05 12% 2.80 E+13

(3) Final culture conditions

Final culture conditions were as follows:

- DO: 10.8%

-infection time: 12 hours

- MOI: 0.0215

- R-C:HFG=1:1

- Initial cell count: 6 E+5 cells/ml

- Culture medium: Lonza insect X-PRESS

- feed time: 48, 96 hr hpi, feed up to 5g/L at timepoint

-harvest time: 120 hr hpi

- rpm: 200 rpm in bioreactor

- pH: not controlled

<110> Helixmith Co., Ltd <120> Baculovirus Infected Cell Stock for AAV vector production and Method for manufacturing AAV vector <130> PN220161 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 2879 <212> DNA <213> Artificial Sequence <220> <223> HGF-X7d4(opti) <400> 1 atgtgggtca ccaagctgct tcctgctctg ctgctccagc acgtgctgct gcacctgctg 60 ctgctgccta tcgccatccc ctacgccgag ggccagagaa agcggagaaa cacaatccac 120 gagttcaaga aaagcgccaa gacgaccctg atcaagatcg accccgccct gaagatcaag 180 accaagaagg tgaacaccgc cgaccagtgc gccaaccggt gcaccagaaa caagggcctg 240 cctttcacct gtaaagcctt cgtgttcgac aaggccagaa agcagtgtct gtggttcccc 300 ttcaacagca tgagcagcgg cgtgaagaag gaattcggcc acgagtttga tctgtacgag 360 aacaaggact acatcagaaa ttgcatcatc ggcaagggca gaagctacaa gggtacagtg 420 tccatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480 aggtaagaac agtatgaaga aaagagatga agcctctgtc ttttttacat gttaacagtc 540 tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600 tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660 tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720 tatttgtgga tccttataag aaaagcaata aacaaacaag taatgatctc aaataagtaa 780 tgcaagaaat agtgagattt caaaatcagt ggcagcgatt tctcagttct gtcctaagtg 840 gccttgctca atcacctgct atcttttagt ggagctttga aattatgttt cagacaactt 900 cgattcagtt ctagaatgtt tgactcagca aattcacagg ctcatctttc taacttgatg 960 gtgaatatgg aaattcagct aaatggatgt taataaaatt caaacgtttt aaggacagat 1020 ggaaatgaca gaattttaag gtaaaatata tgaaggaata taagataaag gatttttcta 1080 ccttcagcaa aaacataccc actaattagt aaaattaata ggcgaaaaaa agttgcatgc 1140 tcttatactg taatgattat cattttaaaa ctagcttttt gccttcgagc tatcggggta 1200 aagacttgca ggagaactat tgccggaacc ccagaggcga ggaaggcggc ccctggtgct 1260 tcaccagcaa ccccgaggtg agatacgagg tgtgcgacat cccccagtgc agcgaggtcg 1320 agtgcatgac gtgcaacggc gagagctacc ggggcctgat ggaccatacc gagtctggga 1380 agatctgcca gagatgggac caccagacac ctcaccggca caagttcctg ccagaaagat 1440 atcctgacaa gggcttcgac gataactact gcagaaaccc tgacggccaa cctagacctt 1500 ggtgttacac gctgggacccc cacaccagat gggaatactg tgctattaag acatgtgccg 1560 acaataccat gaacgacaca gacgtgcctc tcgagacaac agaatgcatc cagggccagg 1620 gagaaggata tagaggtaca gtgaacacca tctggaacgg aattccatgt cagcggtggg 1680 acagccaata cccccacgag cacgatatga cccctgagaa cttcaagtgt aaagacctgc 1740 gggaaaacta ttgcagaaac ccagatggct ctgaatctcc ttggtgcttc acaaccgacc 1800 ctaatatcag agtgggctac tgcagccaga tccccaattg cgacatgagc cacggacagg 1860 actgctacag gggcaatggc aagaactaca tgggaaacct gagccagacc agaagcggcc 1920 tgacctgcag catgtgggat aagaacatgg aagatctgca cagacacatc ttctgggagc 1980 ctgatgcttc taagctgaat gagaactact gccggaaccc tgatgatgat gcccacggcc 2040 cctggtgcta cactggcaac cccctgatcc catgggacta ctgtcctatc agcagatgtg 2100 aaggcgacac cacacctacc atagttaacc tggaccaccc cgtgatcagt tgcgccaaga 2160 ccaagcagct gagagtggtg aacggcatcc ctacaagaac caacatcgga tggatggtgt 2220 ccctgagata cagaaacaag cacatctgcg gcggcagcct gatcaaggaa agctgggtcc 2280 tcaccgccag acagtgtttt cctagccggg acctgaagga ctacgaggcc tggctgggca 2340 tccacgacgt gcacggcaga ggcgacgaga agtgcaagca ggtgcttaac gtgtcccaac 2400 tggtgtacgg ccccgagggc agtgatctgg tgctgatgaa actggccaga cccgccgtgc 2460 tcgacgactt cgtgtctaca atcgacctgc ctaactacgg ctgtaccatc cccgagaaga 2520 ccagctgcag cgtgtacggc tggggctata caggcctcat taactacgac ggactgctga 2580 gagtggctca cctgtacatc atgggcaacg agaaatgctc ccagcaccac agaggcaagg 2640 tgaccctgaa cgagagcgag atctgtgcag gcgccgagaa gatcggcagc ggcccttgcg 2700 agggcgatta cggaggacct ctggtctgcg agcagcacaa gatgcggatg gtcctgggag 2760 tgatcgttcc tggcagagga tgtgccatcc ctaacagacc aggaatcttt gtgcgggtgg 2820 cctactacgc caagtggatc cacaaaatca tcctgaccta caaggtgcca cagtcttga 2879 <210> 2 <211> 1866 <212> DNA <213> Artificial Sequence <220> <223> AAV2 Rep78 original <400> 2 atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 60 ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat 120 tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 180 cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg 240 caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg 300 aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 360 taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc 420 gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaaa 480 acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg 540 aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 600 gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact 660 tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 720 cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 780 tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 840 cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 900 attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc 960 acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1020 accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1080 aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1140 aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1200 gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1260 aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg 1320 ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1380 gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg 1440 gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1500 gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1560 gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1620 aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1680 ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1740 tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1800 ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1860 caataa 1866 <210> 3 <211> 1866 <212> DNA <213> Artificial Sequence <220> <223> AAV2 Rep78 example <400> 3 ctggcggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 60 ggcatttctg acagctttgt gaactgggtg gccgagaagg agtgggagtt gccgccagat 120 tctgacttgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 180 cgcgactttc tgacggagtg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg 240 caatttgaga agggagagag ctacttccac ttacacgtgc tcgtggaaac caccggggtg 300 aaatccttag ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 360 taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaacggc 420 gccggaggcg ggaacaaggt ggtggacgag tgctacatcc ccaattactt gctccccaaaa 480 acccagcctg agctccagtg ggcgtggact aatttagaac agtatttaag cgcctgtttg 540 aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 600 gagcagaaca aagagaatca gaatcccaat tctgacgcgc cggtgatcag atcaaaaact 660 tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 720 cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 780 tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 840 cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 900 attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc 960 acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1020 accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1080 aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1140 aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1200 gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1260 aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg 1320 ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1380 gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg 1440 gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1500 gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1560 gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1620 aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1680 ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1740 tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1800 ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1860 caataa 1866 <210> 4 <211> 2211 <212> DNA <213> Artificial Sequence <220> <223> AAV1 CAP original <400> 4 atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60 gagtggtggg acttgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120 gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180 aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240 cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300 caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420 ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcatcggc 480 aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540 tcagtccccg atccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600 actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660 gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720 accaccagca cccgcacctg ggccttgccc acctacaata accacctcta caagcaaatc 780 tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840 gggtattttg atttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900 atcaacaaca attggggatt ccggcccaag agactcaact tcaaactctt caacatccaa 960 gtcaaggagg tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg 1020 gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080 ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140 ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga atatttccct 1200 tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260 ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320 caatacctgt attacctgaa cagaactcaa aatcagtccg gaagtgccca aaaacaaggac 1380 ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440 ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaat 1500 tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560 ggcactgcta tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc 1620 atgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680 acagacgaag aggaaattaa agccactaac cctgtggcca ccgaaagatt tgggaccgtg 1740 gcagtcaatt tccagagcag cagcacagac cctgcgaccg gagatgtgca tgctatggga 1800 gcattacctg gcatggtgtg gcaagataga gacgtgtacc tgcagggtcc catttgggcc 1860 aaaattcctc acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc 1920 aagaacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggcg 1980 gagttttcag ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagt 2040 gtggaaattg aatggggagct gcagaaagaa aacagcaagc gctggaatcc cgaagtgcag 2100 tacacatcca attatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160 tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211 <210> 5 <211> 2211 <212> DNA <213> Artificial Sequence <220> <223> AAV1 CAP example <400> 5 acggctgccg acggttatct acccgattgg ctcgaggaca acctctctga gggcattcgc 60 gagtggtggg acttgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120 gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180 aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240 cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300 caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420 ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcatcggc 480 aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540 tcagtccccg atccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600 actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660 gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720 accaccagca cccgcacctg ggccttgccc acctacaata accacctcta caagcaaatc 780 tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840 gggtattttg atttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900 atcaacaaca attggggatt ccggcccaag agactcaact tcaaactctt caacatccaa 960 gtcaaggagg tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg 1020 gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080 ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140 ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga atatttccct 1200 tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260 ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320 caatacctgt attacctgaa cagaactcaa aatcagtccg gaagtgccca aaaacaaggac 1380 ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440 ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaat 1500 tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560 ggcactgcta tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc 1620 atgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680 acagacgaag aggaaattaa agccactaac cctgtggcca ccgaaagatt tgggaccgtg 1740 gcagtcaatt tccagagcag cagcacagac cctgcgaccg gagatgtgca tgctatggga 1800 gcattacctg gcatggtgtg gcaagataga gacgtgtacc tgcagggtcc catttgggcc 1860 aaaattcctc acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc 1920 aagaacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggcg 1980 gagttttcag ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagt 2040 gtggaaattg aatggggagct gcagaaagaa aacagcaagc gctggaatcc cgaagtgcag 2100 tacacatcca attatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160 tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

Claims (22)

Method for preparing baculovirus infected cell (BIC) stocks producing adeno-associated virus (AAV) vectors:
(a) culturing insect cells and infecting the cultured insect cells with a baculovirus containing an AAV vector expression construct; and
(b) cultivating the baculovirus-infected cells.
The method of claim 1, further comprising the step of (c) freezing the baculovirus-infected cells.
The method of claim 1, wherein the AAV vector expression construct is
i) Rep-Cap sequence expressing AAV replication protein and AAV capsid protein; or ii) a method for producing a BIC stock for producing an AAV vector, comprising a GOI sequence expressing a gene of interest (GOI).
The method of claim 3, wherein the i) Rep-Cap sequence and ii) GOI sequence are contained in different DNA fragments.
The method of claim 1, wherein the insect cells are Sf9 cells.
The method of claim 1, wherein the culture is performed under mixing conditions of 110 to 150 rpm.
The method of claim 1, wherein the culturing is performed in a culture solution with a volume of 30 to 70 mL.
The method of claim 1, wherein the culturing is performed at 26 to 30°C.
The method of claim 1, wherein the culturing is performed at pH 6.0 to 6.4.
The method of claim 1, wherein the baculovirus infection is performed at a multiplicity of infection (MOI) of 0.1 to 3.
A method of producing an AAV vector using the BIC stock prepared by the method according to any one of claims 1 to 10:
(a) BIC cells are taken out from the baculovirus infected cell (BIC) stock producing the adeno-associated virus (AAV) vector, and the insect cells of the same species as the BIC are Adding to the culture containing the population and culturing in a bioreactor; and
(b) Isolating the AAV vector from the culture medium in which the culture was completed in step (a).
The method of claim 11, wherein the culture in step (a) has an initial cell density of 0.5 x 10 ⁶ to 1.2 x 10 ⁶ cells/mL.
The method of claim 11, wherein the culturing in step (a) is performed by additionally adding cell nutrients during the culturing.
The method of claim 13, wherein the cell nutritional component includes glucose, glutamine, yeast extract, or a combination thereof.
The method of claim 11, wherein the baculovirus-infected cell is: i) a baculovirus comprising a Rep-Cap sequence expressing an AAV replication protein and an AAV capsid protein; and ii) a method of producing an AAV vector using a BIC stock, which is two types of cells each infected with a baculovirus containing a GOI sequence expressing a gene of interest (GOI).
The method of claim 11, wherein an anti-foaming agent is added at a concentration of 1 to 5% during culturing in step (a).
The method of claim 11, wherein during the culture in step (a), the BIC is infected with the same insect cell population as the BIC at an MOI (multiplicity of infection) of 0.01 to 0.05. An AAV vector is generated using the BIC stock. How to.
The method of producing an AAV vector using a BIC stock according to claim 11, wherein the addition of the BIC during the culture in step (a) is performed 8 to 18 hours after the start of culture of the insect cells of the same species as the BIC. .
The method of claim 11, wherein the baculovirus-infected cells (BIC) in the culture in step (a) are 1 to 2 passage baculovirus-infected cells.
The method of claim 11, wherein DO (dissolved oxygen, %) of the culture medium during the culture in step (a) is 8 to 16%.
The method of claim 11, wherein the completion time of culture in step (b) is 96 to 132 hours after adding BIC to the culture medium containing the same host cell population as BIC in step (a), using a BIC stock. How to generate AAV vectors.
The method of claim 11, wherein in step (b), the culture medium is a culture medium containing the produced AAV, and an AAV vector is produced using a BIC stock, which is a culture medium in which cell destruction and lysis of cells in the culture medium have been performed at least once. method.