KR20240063694A - Method for Purifying AAV - Google Patents

Abstract

The present invention relates to a method for purifying Adenovirus-associated virus (AAV). The purification method of the present invention consists of a purification method with a different order and conditions than the conventional method, and can obtain AAV in high yield.

Description

Method for purifying AAV {Method for Purifying AAV}

The present invention relates to a method for purifying AAV. More specifically, it relates to a method for purifying AAV to improve the productivity of AAV.

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.

In order to develop AAV as a gene therapy, the productivity of AAV is a very important factor. In addition, since AAV is produced through culturing of cells and AAV, a large amount of impurities related to the AAV vector, including proteins and nucleic acids, are inevitably produced, so a purification process to remove impurities is essential. In particular, it is extremely difficult and important to develop a process to purify AAV to high purity in order to use it as a clinically usable human genetic therapeutic.

The present inventors have made extensive research efforts to develop a method for purifying AAV with improved AAV productivity. As a result, the present invention was completed by demonstrating that the AAV purification time can be shortened and production efficiency can be greatly improved by adjusting the cell disruption method and the type and conditions of chromatography that make up the AAV purification method.

According to one aspect of the present invention, the present invention provides a method for purifying Adenovirus-associated virus (AAV) comprising the following steps:

(a) disrupting the cells producing AAV;

(b) filtering the supernatant containing the disrupted cells;

(c) performing cation exchange chromatography on the filtrate filtered in the above step to produce a column effluent;

(d) producing a column effluent by subjecting the column effluent to affinity chromatography;

(e) producing a column effluent by subjecting the column effluent to anion exchange chromatography; and

(f) filtering the column eluate to produce purified AAV.

In this specification, “adeno-related virus” corresponds to a virus belonging to the genera 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 cells in step (a) are derived from a cell culture containing AAV-producing cells and their cell culture supernatant.

In one embodiment of the invention, the cells producing the AAV are human cells or insect cells. The human cells and insect cells may be cell lines such as HEK293 and sf9, but are not limited thereto, and any cell line available in the art may be used without limitation.

In one embodiment of the present invention, the cell disruption method is performed using a microfludizer.

In one embodiment of the present invention, the microfluidizer is performed under conditions of 1500-3500 psi, 1500-3000 psi, 1500-2500 psi, 1500-2000 psi, 1500 psi, 2000 psi, 2500 psi, or 3000 psi. It may be possible, but it is not limited to this.

In one embodiment of the present invention, the cell disruption is performed in one or more cycles. For example, the cell disruption may be performed in 1 cycle, 2 cycles, or 3 cycles.

In another embodiment of the present invention, the cell disruption method may use a freeze & thawing (F&T) method. However, according to a specific embodiment of the present invention, the yield of the method using the above-described microfluidizer appears to be superior to that of the conventional F&T method.

In one embodiment of the present invention, the filtration in step (b) is performed by a membrane filter.

In one embodiment of the present invention, the filtration in step (b) is performed one or more times.

In one embodiment of the present invention, the filtration of step (b) may be repeated one or more times, for example, once, twice, or three times, but is not limited thereto.

In one embodiment of the present invention, the filtration in step (b) is performed by a depth filtration device.

In one embodiment of the present invention, the deep filtration device is mainly used for the purpose of separating the culture fluid by removing cell fragments and animal cells from the culture fluid after the separation step after cell culture.

In one embodiment of the present invention, the filtration in step (b) is performed by an absolute grade filtration device.

In one embodiment of the present invention, absolute filtration grade means that more than 99.98% of particles with a size larger than that indicated on the filtration device (filter) can be removed.

In one embodiment of the present invention, the absolute filtration grade membrane filter includes Sartobran P, Bottle Top Vacuum Filter, Millipore Express® SHC, etc. may be used, but this is only illustrative and not limiting.

In one embodiment of the present invention, the filtration in step (b) is performed sequentially by a depth filtration device and an absolute filtration grade filtration device.

In one embodiment of the present invention, the membrane filter is a membrane filter made of cellulose acetate or polyethersulfone. In a specific embodiment of the present invention, the membrane filter is preferably made of polyether sulfone material.

In one embodiment of the present invention, the pore size of the membrane filter may be 0.2 to 0.7 μm. More specifically, 0.2 to 0.65 μm, 0.2 to 0.5 μm, 0.2 to 0.45 μm, 0.22 to 0.65 μm, 0.22 to 0.5 μm, or 0.22 to 0.45 μm, but is not limited thereto.

In a specific embodiment of the present invention, the pore size of the membrane filter may be the pore size of an absolute filtration grade membrane filter.

In one embodiment of the present invention, the filtrate obtained in step (b) is filtered by tangential flow filtration (TFF).

The 'tangential flow filtration' is a method of filtering a fluid by moving it parallel to the membrane filter surface. It is a filtration method that can separate dissolved or dispersed substances in a liquid by particle size or molecular weight, and uses the pressure difference as a driving force. do. The tangential flow filtration method is often used to remove impurities according to size, concentrate proteins, and exchange buffers for protein purification during the production of vaccines and biological drugs. During the process, conditions such as temperature, conductivity, flow rate, and pressure can be monitored.

In one embodiment of the present invention, the tangential flow filtration is performed using an elution buffer containing Tris, NaCl, or a combination thereof.

In one embodiment of the present invention, the Tris is 5-50mM, 5-40mM, 5-30mM, 5-25mM, 5-20mM, 7.5-50mM, 7.5-40mM, 7.5-30mM 7.5-25mM, 7.5-20mM, 8-50mM, 8-40mM, 8-30mM, 8-25mM, 8-20mM, 10-50mM, 10-40mM, 10-30mM 10-25mM, 10-20mM, 15-50mM, 15-40mM, 15-30mM, 15-25mM, 15-20mM, more specifically at a concentration of 10mM or 20mM. It is included in the elution buffer, but is not limited thereto.

In one embodiment of the present invention, the NaCl is included in the elution buffer at a concentration of 50-150mM, 50mM, 100mM, or 150mM, and more specifically, it is included in the elution buffer at a concentration of 100mM. However, it is not limited to this.

In one embodiment of the present invention, the elution buffer may be pH 7-10, pH 7-9.5, pH 7-9, pH 7-8.5, pH 7-8, pH 7-7.5, or pH 7.5. It is not limited to this.

In one embodiment of the present invention, the filtrate from step (b) is treated with nuclease.

In one embodiment of the present invention, the nuclease is incubated at 37°C for 10 minutes - 2 hours, 15 minutes - 2 hours, 20 minutes - 2 hours, 25 minutes - 2 hours, 0.5 - 2 hours, 0.5 - 1 hours. , 1-2 hours, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 0.5 hours, 1 hour, 1.5 hours, or 2 hours, but is not limited thereto.

In one embodiment of the present invention, the nuclease is benzoase.

Step (c) of the present invention is a step of performing cation exchange chromatography. Cation exchange chromatography functions to separate AAV particles from the filtered filtrate.

Resins constituting the cation exchange chromatography may include, without limitation, sulfonic acid-based resins, such as aryl and alkyl substituted sulfonate resins, and carboxylic acid-based resins. Examples include carboxymethyl (CM), methyl sulfonate (S) and sulfopropyl (SP) resins.

The cation exchange chromatography may undergo equilibration, washing, and elution steps with various buffer solutions.

In one embodiment of the present invention, the cation exchange chromatography in step (c) is eluted with an elution buffer containing Tris, NaCl, or a combination thereof.

In one embodiment of the present invention, the Tris is 5-50mM, 5-40mM, 5-30mM, 5-25mM, 5-20mM, 7.5-50mM, 7.5-40mM, 7.5-30mM 7.5-25mM, 7.5-20mM, 8-50mM, 8-40mM, 8-30mM, 8-25mM, 8-20mM, 10-50mM, 10-40mM, 10-30mM 10-25mM, 10-20mM, 15-50mM, 15-40mM, 15-30mM, 15-25mM, 15-20mM, more specifically at a concentration of 10mM or 20mM. It is included in the elution buffer, but is not limited thereto.

In one embodiment of the present invention, the NaCl is included in the elution buffer at a concentration of 50-150mM, 50mM, 100mM, or 150mM, and more specifically, it is included in the elution buffer at a concentration of 100mM. However, it is not limited to this.

In one embodiment of the present invention, the elution buffer is pH 7-10, pH 7-9.5, pH 7-9, pH 7-8.5, pH 7-8, pH 7-7.5, pH 7-10, pH 7. It may be -9.5, pH 7-9, pH 7-8.5, pH 7-8, pH 7-7.5, or pH 7.5, but is not limited thereto.

Step (d) of the present invention is a step of performing affinity chromatography. Affinity chromatography functions to separate AAV particles contained in the eluate of the cation exchange chromatography.

Columns containing Sepharose and other materials can be used without limitation in the affinity chromatography.

The affinity chromatography may undergo equilibration, washing, and elution steps with various buffer solutions.

In one embodiment of the present invention, the affinity chromatography in step (d) is eluted with an elution buffer containing Sodium Acetate, NaCl, or a combination thereof.

In one embodiment of the present invention, the Sodium Acetate is 50-200mM, 50-180mM, 50-150mM, 50-140mM, 50-130mM, 50-125mM, 50-120mM, 50- 110mM, 50-100mM, 60-200mM, 60-180mM, 60-150mM, 60-140mM, 60-130mM, 60-125mM, 60-120mM, 60-110mM, 60- 100mM, 70-200mM, 70-180mM, 70-150mM, 70-140mM, 70-130mM, 70-125mM, 70-120mM, 70-110mM, 70-100mM, 80- 200mM, 80-180mM, 80-150mM, 80-140mM, 80-130mM, 80-125mM, 80-120mM, 80-110mM, 80-100mM, 90-200mM, 90- at concentrations of 180mM, 90-150mM, 90-140mM, 90-130mM, 90-125mM, 90-120mM, 90-110mM, 90-100mM, 80mM, 90mM, or 100mM. It is included in the elution buffer, but is not limited thereto.

In one embodiment of the present invention, the NaCl is 50-200mM, 50-180mM, 50-150mM, 50-140mM, 50-130mM, 50-125mM, 50-120mM, 50-110mM mM, 50-100mM, 60-200mM, 60-180mM, 60-150mM, 60-140mM, 60-130mM, 60-125mM, 60-120mM, 60-110mM, 60-100 70-180mM, 70-200mM, 70-180mM, 70-150mM, 70-140mM, 70-130mM, 70-125mM, 70-120mM, 70-110mM, 70-100mM, 80-200mM 80-180mM, 80-150mM, 80-140mM, 80-130mM, 80-125mM, 80-120mM, 80-110mM, 80-100mM, 90-200mM, 90-180mM at concentrations of 90-150mM, 90-140mM, 90-130mM, 90-125mM, 90-120mM, 90-110mM, 90-100mM, 80mM, 90mM, or 100mM. It is included in the elution buffer, but is not limited thereto.

In one embodiment of the present invention, the pH of the elution buffer used for elution of the column may be pH 2-5, pH 2-4, pH 2-3, pH 2-2.5, or pH 2.5, but is limited thereto. It doesn't work.

Step (e) of the present invention is a step of performing anion exchange chromatography. Anion exchange chromatography functions to separate AAV particles contained in the eluate of the affinity chromatography.

The anion exchange chromatography includes polyamine resins, trialkylbenzyl ammonium resins, aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE) and quaternary amino ethyl (QAE) resins and other materials. Columns can be used without restrictions.

The anion exchange chromatography may undergo equilibration, washing, and elution steps with various buffer solutions.

In one embodiment of the present invention, the anion exchange chromatography in step (e) is eluted with an elution buffer containing Bis-Tris-Propane (BTP), NaCl, or a combination thereof, but is limited thereto. no.

In one embodiment of the present invention, the Bis-Tris-Propane (BTP) is 5-100mM, 5-80mM, 5-60mM, 5-50mM, 5-40mM, 5-30mM, 5 -20mM, 10-100mM, 10-80mM, 10-60mM, 10-50mM, 10-40mM, 10-30mM, 10-20mM, 15-100mM, 15-80mM, 15 It is included in the elution buffer at a concentration of -60mM, 15-50mM, 15-40mM, 15-30mM, 15-20mM, or 20mM, but is not limited thereto.

In one embodiment of the present invention, the elution buffer is pH 8-10, pH 8.5-10, pH 9-10, pH 8-9.5, pH 8.5-9.5, pH 9-9.5, pH 8-9, pH 8.5. It may be -9, or pH 9, but is not limited thereto.

In one embodiment of the present invention, the anion exchange chromatography in step (e) is performed by eluting while gradually increasing the concentration of NaCl in the flowing elution buffer.

In one embodiment of the present invention, the concentration of NaCl in the eluate is 100mM to 300mM, 100mM to 280mM, 100mM to 260mM, 100mM to 250mM, 100mM to 240mM, 100mM to 230mM. 100mM to 220mM, 100mM to 210mM, 100mM to 200mM, 100mM to 190mM, 120mM to 300mM, 120mM to 280mM, 120mM to 260mM, 120mM to 250mM, 120mM to 240mM, 120mM to 230mM, 120mM to 220mM, 120mM to 210mM, 120mM to 200mM, 120mM to 190mM, 140mM to 300mM, 140mM to 280mM, 140mM to 260mM, 140mM to 250mM, 140mM to 240mM, 140mM to 230mM, 140mM to 220mM, 140mM to 210mM, 140mM to 200mM, 140mM to 190mM, 150mM to 300mM 150mM to 280mM, 150mM to 260mM, 150mM to 250mM, 150mM to 240mM, 150mM to 230mM, 150mM to 220mM, 150mM to 210mM, 150mM to 200mM, 150mM to 190mM, 160mM to 300mM, 160mM to 280mM, 160mM to 260mM, 160mM to 250mM, 160mM to 240mM, 160mM to 230mM, 160mM to 220mM, 160mM to 210mM, 160mM to 200mM, 160mM to 190mM, 170mM to 300mM, 170mM to 280mM, 170mM to 260mM, 170mM to 250mM, 170mM to 240mM, 170mM to 230mM 170mM to 220mM, 170mM to 210mM, 170mM to 200mM, or 170mM to 190mM, but is not limited thereto.

In one embodiment of the present invention, the eluate may be eluted by injecting 1-3 column volumes (CV) of elution buffer per step. More specifically, 1-3 CV, 1-2.5 CV, 1-2 CV, 1-1.5 CV, 1.5-3 CV, 1.5-2.5 CV, 1.5-2 CV, 1 CV, 1.5 CV, 2 CV, 2.5 CV. , or 3 CV, but is not limited thereto.

In a specific embodiment of the present invention, the eluate is eluted by injecting 1.5 column volumes (CV) of elution buffer per step, but is not limited thereto.

In a specific embodiment of the present invention, the eluate can be eluted by injecting 1-3 column volumes of elution buffer per step while increasing the NaCl concentration by 1-5mM from, for example, 170mM to 190mM. The concentration of NaCl raised per 1-3 column volumes is 1-5mM, 1.2-5mM, 1.3-5mM, 1.4-5mM, 1.5-5mM, 1.6-5mM, 1.7-5mM, 1.8-5M mM, 1.9-5mM, 2-5mM, 2.1-5mM, 2.2-5mM, 2.3-5mM, 2.4-5mM,2.5-5mM, 2.6-5mM, 2.7-5mM, 2.8-5mM mM, 2.9-5mM, 3-5mM, 3.2-5mM, 3.4-5mM, 3.6-5mM, 3.8-5mM, 4-5mM, 4.2-5mM, 4.4-5mM, 4.6-5 mM, 4.8-5mM, 1-4mM, 1.2-4mM, 1.3-4mM, 1.4-4mM, 1.5-4mM, 1.6-4mM, 1.7-4mM, 1.8-4mM, 1.9-4 mM, 2-4mM, 2.1-4mM, 2.2-4mM, 2.3-4mM, 2.4-4mM,2.5-4mM, 2.6-4mM, 2.7-4mM, 2.8-4mM, 2.9-4 mM, 3-4mM, 3.2-4mM, 3.4-4mM, 3.6-4mM, 3.8-4mM, 1-3.5mM, 1.2-3.5mM, 1.3-3.5mM, 1.4-3.5mM, 1.5-3.5 mM, 1.6-3.5mM, 1.7-3.5mM, 1.8-3.5mM, 1.9-3.5mM, 2-3.5mM, 2.1-3.5mM, 2.2-3.5mM, 2.3-3.5mM, 2.4-3.5mM,2.5-3.5 mM, 2.6-3.5mM, 2.7-3.5mM, 2.8-3.5mM, 2.9-3.5mM, 3-3.5mM, 3.2-3.5mM, 3.4-3.5mM, 1-3mM, 1.2-3mM, 1.3-3 1.4-3mM, 1.5-3mM, 1.6-3mM, 1.7-3mM, 1.8-3mM, 1.9-3mM, 2-3mM, 2.1-3mM, 2.2-3mM, 2.3-3mM mM, 2.4-3mM,2.5-3mM, 2.6-3mM, 2.7-3mM, 2.8-3mM, 2.9-3mM, 1-2.5mM, 1.2-2.5mM, 1.3-2.5mM, 1.4-2.5 1.5-2.5mM, 1.6-2.5mM, 1.7-2.5mM, 1.8-2.5mM, 1.9-2.5mM, 2-2.5mM, 2.1-2.5mM, 2.2-2.5mM, 2.3-2.5mM, 2.4-2.5mM mM, 1-2mM, 1.2-2mM, 1.3-2mM, 1.4-2mM, 1.5-2mM, 1.6-2mM, 1.7-2mM, 1.8-2mM, 1.9-2mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, or 5mM, but is not limited thereto.

The present invention provides a method for purifying AAV with improved productivity of AAV. When using the AAV purification method of the present invention, the productivity of AAV can be greatly improved.

Figure 1 is a diagram showing the yield of AAV vectors when the filtrate is subjected to cation exchange chromatography or anion exchange chromatography in relation to step (c) of the purification method of the present invention.
Figure 2 is a diagram showing the yield of AAV vectors according to a method of disrupting AAV producing cells (freeze and thawing; or microfluidization) in relation to step (a) of the purification method of the present invention.
Figure 3 is a diagram showing the vector yield according to the type of filter for filtering the cell lysate in relation to step (b) of the purification method of the present invention.
Figure 4 is a diagram showing the vector yield according to the type of tangential flow filtration filter and enzyme conditions for filtering the cell lysate in relation to step (b) of the purification method of the present invention.
Figure 5 shows the vector according to the pH (2.5/3.5) of the elution buffer used for elution of affinity chromatography and the concentration of sodium acetate salt (0.05 M, 0.1 M) in relation to step (d) of the purification method of the present invention. This is a diagram showing the yield.
Figure 6 is a diagram showing the yield of the vector according to the NaCl salt concentration (0.1 M, 0.25 M, 0.5 M) of the elution buffer used for elution of affinity chromatography in relation to step (d) of the purification method of the present invention. am.
Figure 7 is a diagram showing the vector yield according to the elution volume and salt concentration in the buffer in relation to step (e) anion exchange chromatography of the purification method of the present invention.
Figure 8 is a diagram showing the full/empty ratio of the vector according to the elution volume and salt concentration in the buffer in relation to step (e) anion exchange chromatography of the purification method of the present invention.
Figure 9 is a diagram showing the yield of AAV vector according to the overall process of the purification method of the present invention.
Figure 10 is a diagram showing the results of silver staining of AAV vectors purified according to each condition in an example of the present invention.
Figure 11 is a diagram showing the results of analyzing the quality of AAV purified by the final purification method in the present invention using an Analytical Ultracentrifuge.

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) %.

Example 1: AAV initial purification process

The present inventors established the initial purification process in the above order. Process conditions for each process are shown in Table 1 below.

Process Conditions based on 150 mL Process Step Input Process Conditions Equipment & Consumables Cell Pellet
[Input Titer (3 F/Ts)] 150 2500 g, 29 min and storage at -80℃, min 24hrs Centrifuge, Freezer Suspension 5 20mM Tris + 100mM NaCl, pH 7.5 Stirring Mixer Cell Disruption 150 -80℃, ~12 hours Freezer Supernatant 150 8000g, 30min Centrifuge Nucleic acid digestion 150 25 units/mL, 37℃, 30 min Stirring Mixer Filtration 1 150 ^180cm2 ~5 um Filter (Sartorius) Filtration 2 200 ^34cm2 0.22 um Bottle Top filter CEX 200 5mL Column Capto S (Cytiva) AEX 1 200 5mL Column Capto Q (Cytiva) Affinity 200 1mL Column Capto AVB (Cytiva) Buffer Adjustment 6 20mM BTP + 50mM NaCl, pH 9.0 Manual Shaking AEX 2 120 1mL CIMultus QA (Bia) 100 kDa Filtration 15 100 kDa Amicon (Merck) Frozen DS (Output Titer) 0.2 Freezer

Example 2: Selection of CEX and AEX columns

In order to improve the initial purification process of the AAV, the present inventors wanted to determine which of the cation exchange chromatography (CEX) and anion exchange chromatography (AEX) columns was more suitable. As shown in Table 2 below, the types of columns, The yield of the AAV vector was measured while changing the pH and composition of the buffer.

Selection of column candidates between CEX and AEX Process Step Process Candidates (CEX or AEX prior to Affinity) CEX, pH7.5_10mM CEX, pH7.5_20mM CEX, pH9.0_10mM CEX, pH9.0_20mM AEX, pH7.5_10mM AEX, pH7.5_20mM AEX, pH9.0_10mM AEX, pH9.0_20mM Cell pellet 100 100 100 100 100 100 100 100 Input titer (3 F/Ts) 1.05E+12 1.18E+12 1.05E+12 1.18E+12 1.05E+12 1.18E+12 1.05E+12 1.18E+12 Suspension ㅡ ㅡ ㅡ ㅡ ㅡ ㅡ ㅡ ㅡ Cell disruption (1 F/T) Supernatant 90 91 90 91 90 91 90 91 Nucleic acid digestion ㅡ ㅡ ㅡ ㅡ ㅡ ㅡ ㅡ ㅡ Filtration I 74 74 74 74 74 74 74 74 Filtration II 59 59 59 59 59 59 59 59 CEX 48 48 50 50 N/A N/A N/A N/A AEX I N/A N/A N/A N/A 15 13 47 50 Affinity 15 17 21 18 5 4 16 17 Buffer adjustment ㅡ ㅡ ㅡ ㅡ ㅡ ㅡ ㅡ ㅡ AEX II 9 12 16 13 One One 9 11 100 kDa filtration 6 7 11 8 0 0 6 8 Frozen DS 5.7 7.2 11.2 8.1 0.4 0.3 5.9 7.5 Output titer 6.0E+10 8.5E+10 1.2E+11 9.5E+10 3.3E+09 3.7E+09 6.2E+10 7.9E+10

After filtration under the same conditions as above, CEX or AEX was performed to measure the yield of the AAV vector.

Specifically, a sample that had completed Filtration step 2 was prepared, and a buffer was manufactured under the following four conditions.

pH 7.5 pH 9.0 10mM Tris + 100mM NaCl 20mM Tris + 100mM NaCl 10mM Tris + 100mM NaCl 20mM Tris + 100mM NaCl

Capto S 5 mL Column (CEX, cation exchange chromatography) was connected to AKTA Avant. Next, Column Equilibrium was performed using the buffer prepared under the conditions shown in Table 3 above. The sample that had undergone filtration was bound to the CEX Column using the Sample Pump. Elution was performed by flowing the buffer prepared using the flow through method. When elution was completed, column CIP (cleaning in place) was performed. By repeating the above process, all four types of buffers were confirmed. When the experiment with all four types of buffers was completed, the Capto S 5mL Column was filled with Storage Buffer and separated from the AKTA Avant.

Experiments were conducted on four types of buffers in the same manner as above using Capto Q 5mL Column (AEX, anion exchange chromatography).

All subsequent processes for a total of eight samples recovered through the above process were performed, and the output titer of the final sample was measured and compared. The results are shown in Figure 1.

The IEX Column is divided into a Cation Exchange Column (CEX) and an Anion Exchange Column (AEX). As shown in FIG. 1, the experimental results show that the Vg Yield when using CEX is on average about 10% lower than when using AEX. It was confirmed that it was 2.3 times higher.

In addition, the buffer composition when using CEX showed that the Vg Yield was about 1.5 times higher in a weak base environment of pH 9.0 than in a neutral environment of pH 7.5. However, pH 7.5 was selected because the recommended pH environment for using the subsequent process, Affinity Chromatography, is neutral. Based on the results, the CEX Column was used as a pretreatment step before using the Affinity Column (AC), and the buffer composition at this time was selected as 20mM Tris + 100mM NaCl, pH 7.5.

Based on the above results, Capto S 5 mL Column (CEX), 20mM Tris + 100mM NaCl, pH 7.5 Buffer conditions were selected, and the experiment was repeated three times. As a result, the Vg Yield was 15.1, 11.5, respectively. It was measured at 10.9%, the average was 12.5, the standard deviation was 2.3, and the CV was confirmed to be about 18%. Based on the results, it was determined that the process was reproducible on a 150 mL scale.

In addition, it was confirmed that there was reproducibility at the 150 ml scale, and the Filter Area in the Filtration 2 step was doubled from 33 cm ² to 55 cm ² and the CEX Volume was doubled from 5 mL to 10 mL to ensure reproducibility at the 300 ml scale. Confirmed. The experiment was repeated a total of three times, and as a result of the experiment, each Vg Yield was measured as 13.5, 10.5, and 13.1%, with an average of 12.4, standard deviation of 1.6, and CV of about 13%.

Example 3: Cell disruption method

The present inventors also measured the yield of the AAV vector while changing the cell disruption method in order to improve the initial purification process of the AAV.

Cell disruption method Process Stage Partial Disruption (1 time F/T) Complete Disruption (MF) Cell pellet 100 100 100 100 Input titer (3 F/Ts) 6.65E+11 1.36E+12 6.65E+11 1.36E+12 Suspension ㅡ ㅡ ㅡ ㅡ Cell disruption ㅡ ㅡ Supernatant 99 91 249 176 Nucleic acid digestion ㅡ ㅡ ㅡ ㅡ Filtration I 80 76 212 146 Filtration II 71 60 127 96 CEX 56 51 105 72 Affinity 25 15 25 18 Buffer adjustment ㅡ ㅡ ㅡ ㅡ AEX II 16 13 16 15 100 kDa filtration 12 11 12 12 Frozen DS 11.5 10.9 12.4 12.3 Output titer 7.70E+10 1.48E+11 7.90E+10 1.68E+11

Specifically, the samples prepared through the Sample Preparation process are divided 1:1, half of the samples undergo Cell Disruption through one cycle of Freezing & Thawing (F/T) process, and the remaining half of the samples are subjected to High Pressure Homogenizer ( Cell disruption was performed through one cycle under 2500 psi conditions using microfluidization. By performing the above process twice, all subsequent processes were performed on a total of 4 samples recovered, and the output titer of the final sample was measured and compared.

The results are shown in Figure 2.

As shown in Figure 2, in the first experiment, the Vg Yield values of the F/T method and the MF method were 11.5 and 12.4, respectively, showing that the MF method was about 8% higher. In the second experiment, the Vg Yield values of the F/T method and MF method were 10.9 and 12.3, respectively, showing that the MF method was about 13% higher. Based on the experimental results, the MF method was selected as the cell disruption method.

Example 4: Filtration Method Selection

The present inventors also measured the yield of the AAV vector while changing the filtration method in order to improve the initial purification process of the AAV.

Process Stage Runs Satobran P Bottle Top Vacuum Filter Millipore Express® SHC Cell pellet 100 100 100 Input titer (3 F/Ts) 1.64E+12 1.64E+12 1.64E+12 Suspension ㅡ ㅡ ㅡ Cell disruption Supernatant 99 99 99 Nucleic acid digestion ㅡ ㅡ ㅡ Filtration I 79 79 79 Filtration II 71 67 67 CEX 63 57 56 Affinity 13 16 18 Buffer adjustment ㅡ ㅡ ㅡ AEX II 10 13 16 100 kDa filtration 9 11 12 Frozen DS 8.5 10.8 11.8 Output titer 1.40E+11 1.59E+11 1.98E+11

Specifically, the sample prepared through Filter 1 (Depth) process was first divided into three equal parts.

Three types of Filter 2 (Absolute) were prepared as follows and filtration was performed for each sample.

Filter Sartobran P Bottle Top Vacuum Filter Millipore Express®SHC Pore size (μm)* 0.65/0.45 0.22 0.5/0.2 Surface Area ( ^cm2 ) 17.3 33 17.3 Membrane Cellulose Acetate Poly Ether Sulfate Poly Ether Sulfate

In the description of the pore size, a designation such as a/b means that the filter is a double filter configuration including both a filter with a pore size of a μm and a filter with a pore size of b μm. All subsequent processes for a total of three samples recovered through the above process were performed, and the output titer of the final sample was measured and compared. The results are shown in Figure 3.

As shown in Figure 3, the Vg Yield for each filter type was 8.5, 10.8, and 11.8. From the above results, it was found that the filter made of poly ether sulfate had a higher Vg Yield than the filter made of cellulose acetate. Therefore, the Millipore Express SHC ^® Filter with the highest Vg Yield was selected as the filter used in filtration 2. .

Based on the above results, while maintaining the determined process conditions, the sample volume was increased four times to confirm process reproducibility on a 600 mL sample scale.

For Filtration 2, Millipore Express® SHC Filter was used and the filter area was scaled up from 17 cm ² to 34 cm ^2. For CEX, the column volume was scaled up from 5 mL to 20 mL. In the case of AC (affinity chromatography), the column volume was scaled up from 1 mL to 2 mL or 5 mL. The above process was repeated 4 times to recover a total of 4 samples, all subsequent processes were performed, and the output titer of the final sample was measured and compared.

As a result of the experiment, each Vg Yield was measured as 12.3, 18.8, 11.9, and 14.8, with an average of 12.5, standard deviation of 3.2, and CV of about 22%. Based on the results, it was determined that the process was reproducible on a 600 mL scale.

Example 5: Selection of Tangential Flow Filtration Filter

The present inventors also measured the yield of the AAV vector while changing the filter used in the tangential flow filtration method in order to improve the initial purification process of the AAV.

Based on the results of Examples 2 to 4, the process up to Filtration 2 was performed. A hollow fiber filter (1, 2 times: mPES 30 kDa hollow fiber 235 cm ² / 3, 4 times: mPES 100 kDa hollow fiber 235 cm ² ) was connected to the TFF (Tangential Flow Filtration) System. The TFF System and hollow fiber were cleared in place (CIP) using 0.2M NaOH and distilled water (DW). The sample was injected into the sample tank using a transfer pump. The buffer was replaced with the buffer prepared by performing TFF (20mM Tris + 100mM NaCl, pH 7.5). Samples for which buffer replacement was completed were recovered and Nucleic acid Digestion (25 U, 50 U, or 100 U/30 min or 60 min) was performed. All subsequent processes for a total of four samples recovered through the above process were performed, and the output titer of the final sample was measured and compared.

Process Stage Runs 30 kDa
25U, 30min 30 kDa
50U, 30min 100 kDa
100 U, 30 min 100 kDa
100 U, 60 min Culture broth 100 100 100 100 Input titer (3 F/Ts) 1.11E+13 9.07E+12 6.28E+12 6.23E+12 Cell disruption (MF) ㅡ ㅡ ㅡ ㅡ Supernatant 109 109 98 107 Filtration I 75 102 81 92 Filtration II 120 142 89 88 T.F.F. 136 174 99 82 Nucleic acid digestion 120 153 83 85 CEX 83 135 87 66 Affinity & Neutralization 53 66 30 30 Buffer adjustment ㅡ ㅡ ㅡ ㅡ AEX II 22 18 10 10 100 kDa filtration 21 22 10 8 Frozen DS 20.6 21.7 10.1 8.1 Output titer 2.14E+12 1.92E+12 6.34E+11 5.07E+11

The results are shown in Figure 4.

As shown in Figure 4, the TFF Step Yield for Runs 1 and 2 was confirmed to be more than twice as large as that for Runs 3 and 4. However, in Runs 1, 2, 3, and 4 before and after TFF, the Vg Yeild was about 95-110%, and in fact, a hollow fiber filter with a membrane cutoff of 30 kDa and a hollow fiber filter with a 100 kDa membrane cutoff were used. ), the difference in process efficiency was not significant. Nevertheless, a 100 kDa hollow fiber filter was selected because the 100 kDa filter shortened the process time by about 2 hours.

Example 6: Affinity Chromatography Improvement

The present inventors sought to improve the affinity chromatography method in order to improve the initial purification process of AAV.

Example 6-1. Determination of pH and sodium acetate concentration of buffer

In the first experiment, Vg Yield was confirmed according to buffer pH and sodium acetate concentration. Specifically, affinity chromatography (Hitrap Capto AVB 5 mL Column) was performed using a buffer with pH 2.5 or 3.5 and a sodium acetate concentration of 0.05 M or 0.1 M.

Comparison by buffer pH Process Stage Runs One 2 3 4 Culture broth 100 100 100 100 Input titer (3 F/Ts) 1.24E+12 1.24E+12 1.24E+12 1.24E+12 Cell disruption (MF) ㅡ ㅡ ㅡ ㅡ Supernatant 135 135 135 135 Filtration I 143 143 143 143 Filtration II 130 130 130 130 T.F.F. 111 111 111 111 Nucleic acid digestion ㅡ ㅡ ㅡ ㅡ CEX 53 53 53 53 Affinity & Neutralization 34 One 38 One Buffer adjustment ㅡ ㅡ ㅡ ㅡ AEX II ㅡ ㅡ ㅡ ㅡ 100 kDa filtration ㅡ ㅡ ㅡ ㅡ Frozen DS ㅡ ㅡ ㅡ ㅡ Output titer ㅡ ㅡ ㅡ ㅡ

The results are shown in Figure 5.

As shown in Figure 5, it was confirmed that a very high Vg Yield was observed at pH 2.5 compared to pH 3.5, regardless of the concentration of sodium acetate. In addition, under conditions with the same pH 2.5 environment, it was confirmed that the Vg Yield was approximately 10% higher in the 0.1 M Sodium Acetate buffer environment than in the 0.05 M Sodium Acetate buffer environment.

From the above results, 0.1 M Sodium Acetate, pH 2.5, was selected as the buffer used for affinity chromatography.

Example 6-2. Determination of salt concentration of buffer

In the second experiment, based on the results of the first experiment, Vg Yield was confirmed when different salt concentrations were used in 0.1 M Sodium Acetate, pH 2.5 buffer.

Specifically, Vg Yield was measured at salt concentrations of 0.1 M NaCl, 0.25 M NaCl, or 0.5 M NaCl.

Comparison by salt concentration Process Stage Runs One 2 3 Culture broth 100 100 100 Input titer (3 F/Ts) 1.71E+12 1.71E+12 1.71E+12 Cell disruption (MF) Supernatant 80 80 80 Filtration I 86 86 86 Filtration II 86 86 86 T.F.F. 74 74 74 Nucleic acid digestion ㅡ ㅡ ㅡ CEX 51 51 51 Affinity & Neutralization 38 33 37 Buffer adjustment ㅡ ㅡ ㅡ AEX II ㅡ ㅡ ㅡ 100 kDa filtration ㅡ ㅡ ㅡ Frozen DS ㅡ ㅡ ㅡ Output titer ㅡ ㅡ ㅡ

The results are shown in Figure 6.

As shown in Figure 6, it was confirmed that Vg Yield was 38, 33, and 37 depending on the concentration of each salt.

From the above results, the optimal buffer composition for performing AC was selected as 0.1 M Sodium Acetate, 0.1 M NaCl, pH 2.5.

Example 7: Improvement of anion exchange chromatography

In order to improve the initial purification process of AAV, the present inventors sought to improve the process yield by finding appropriate Elution Buffer conditions in anion exchange chromatography. Based on the affinity chromatography improvement results of Example 6, a common process including affinity chromatography was performed.

The following two buffers were manufactured.

Normal 20mM BTP, pH 9.0 Elution 20 mM BTP + 1 M NaCl, pH 9.0

First, 1 mL of CIMmultus QA monolith column was connected to AKTA Avant. Column Equilibrium was performed using the Normal Buffer in Table 10 above. The sample that had undergone affinity chromatography was bound to the AEX Column using the Sample Pump. Elution was performed using the Gradient Elution (Conc B: 10~20%, 60 CV) method. Specifically, the above two buffers (20mM BTP, pH9.0 / 20mM BTP, 1M NaCl, pH9.0) were flowed using Pump A and Pump B, respectively, to adjust the concentration of NaCl in the buffer. The eluted sample was collected, and column CIP was performed when elution was completed. After filling the storage buffer inside the column, store it separately from the device. Lastly, the Empty/Full Ratio in the collected sample was checked. The empty/full ratio was determined using the ELISA (Progen AAV 1 Titration kit) analysis method. Specifically, after collecting fractions according to peak on FPLC, the AAV1 Capsid ratio was expressed as Empty/Full Ratio based on the ELISA results for the corresponding fraction.

The experimental conditions and results are shown in Tables 11 and 12 and Figures 7 and 8.

Process Stage Runs One 2 3 Culture broth 100 100 100 Input titer (3 F/Ts) 5.98E+12 1.90E+13 9.49E+12 Cell disruption (MF) ㅡ ㅡ ㅡ Supernatant 81 111 191 Filtration I 66 129 183 Filtration II 63 135 114 T.F.F. 36 126 98 Nucleic acid digestion 37 ㅡ ㅡ CEX 43 85 107 Affinity & Neutralization 13 27 35 Buffer adjustment ㅡ ㅡ ㅡ AEX II 7 20 15 100 kDa filtration 6 20 11 Frozen DS 5.5 20.3 11.0 Output titer 3.43E+11 3.88E+12 1.03E+12

As a result of the experiment, when the CV for each step was 5 CV, peak separation was not performed properly, but when the CV for each step was lowered to 1.5 CV, it was confirmed that peak separation was achieved. It was confirmed that the Empty/Full Ratio in the area behind the peak dividing area was recovered at a very high ratio of 82%. In particular, the empty/full ratio was confirmed to be high in the eluate starting from a NaCl concentration of 170mM and increasing the NaCl concentration to 1.5 CV per 2mM NaCl until the concentration reached 190mM.

Example 8: AAV purification process setup

Process procedure

Process Condition Process Step Input (mL) Output (mL) Scale Cell Pellet 600 600 Cell Disruption 600 600 Supernatant 600 600 Filtration I (Full recovery: 20mM + Tris 100mM NaCl, pH 7.5) 600 650 Sartorius PP3 (PP)
5 μm 180 cm ² Filtration II 650 650 Merck SHC (PES)
0.5-0.2 μm 17 cm ² T.F.F.
with 20mM Tris + 100mM NaCl, pH 7.5 650 200 Repligen
100 kDa 225 cm ² Nucleic acid digestion 200 200 50 U 1 hr 37℃ Volume restoration 200 600 With 20mM Tris + 100mM NaCl, pH 7.5 CEX 600 600 Cytiva Capto S 20mL Affinity (100mM Sodium Aceate + 100mM NaCl,pH 2.5)
& Neutralization (60mM BTP, pH 9.0) 650 20 Cytiva Capto AVB 5 mL Buffer adjustment 20 250 With 20mM BTP + 50mM NaCl, pH 9.0 AEX 250 27 BIA CIM QA 1 mL 100 kDa filtration 27 0.8 Sartorius Vivaspin 100 kDa Frozen DS 0.8 0.8

Example 8-1. Cell Pellet

The culture medium was taken out from the SF9-derived insect cells stored at -80°C, placed in a 37°C water bath to dissolve the culture medium, and then stirred using a magnet at 100 to 150 rpm gently enough to prevent the formation of air bubbles for about 10 to 20 minutes. Afterwards, 0.5 mL was sampled for sample analysis.

Example 8-2. Cell Disruption Using Microfluidizer

Cells were disrupted at 2500 psi using a microfluidizer. The cell lysate was dispensed into 12 50 mL conical tubes and centrifuged at 10,000 rpm (approximately 13,000 g) for 1 hour and 10 minutes at 4°C. The supernatant of the sample after centrifugation was collected, and 0.6 mL of sample for analysis was collected separately and stored at -80°C.

Example 8-3. Filtration

After wetting DW on a Sartorius PP3 Depth filter at 20 mL/min for about 5 minutes, all water was removed. The sample was added and filtrated at 20 mL/min. When the sample was almost completely in the filter, 50 mL of Tris buffer (20mM tris + 100mM NaCl, pH 7.5) was added to completely recover the sample. 0.6 mL of sample for analysis was collected separately and stored at -80°C.

DW Wetting was also performed on the Merck Optiscale SHC filter at a speed of 20 mL/min for 5 minutes. The sample was added and filtrated at 20 mL/min. 0.6 mL of sample for analysis was collected separately and stored at -80℃, and the remaining sample was stored overnight at 2 to 8℃ after filtration.

Example 8-4. TFF (mPES 100 kDa Hollow Fiber 235 cm ² )

After washing the tube for TFF use with water, the tube and pressure sensor were connected to the hollow fiber. Retentate tubes and permeate tubes are collected into the waste liquid container. Approximately 4.5 L of the prepared buffer (20mM Tris + 100mM NaCl, pH 7.5) and the tube were connected to the TFF configuration. TFF was performed by flowing the sample, the recovered sample was refrigerated, and 0.6 mL of sample for analysis was sampled and stored at -80 degrees.

Example 8-5. Benzonase treatment

The solution after TFF was treated with 1/5000 volume of Benzonase (e.g. for 200 mL of sample, 40 μl of Benzonase was treated) and reacted in a 37°C water bath for 1 hour. Afterwards, it was diluted to 600 mL with 20mM Tris + 100mM NaCl, pH 7.5 Buffer and stored in the refrigerator.

Example 8-6. Tablet preparation

<CEX Buffer Manufacturing>

20mM Tris + 100mM NaCl, pH 7.5 (Trisma HCl 50.8 g/L, Trisma base 9.44 g/L, NaCl 116.88 g/L prepared (x20 solution) then diluted and used, Base buffer)

2 M NaCl (Column Wash)

1 M NaOH (Column Wash)

0.2 M Sodium Acetate, 20% EtOH (Column storage)

<Manufacture of Affinity Chromatography Buffer>

20mM Tris + 100mM NaCl, pH 7.5 (Base buffer)

0.1 M sodium acetate + 0.1 M NaCl, pH 2.5 (Elution buffer)

20% EtOH (Column storage)

0.1 M Citric acid (Column wash)

10mM NaOH (Column wash)

PBS pH 7.5 (Tablet, Column wash)

20% EtOH (Column storage)

60mM BTP, pH 9.0 (for neutralization of Elution Sample)

20mM BTP + 50mM NaCl, pH 9.0 (for sample dilution)

<Manufacturing of AEX Buffer>

20mM BTP pH 9.0 (Base buffer)

20mM BTP + 1M NaCl, pH 9.0 (Elution buffer)

1 M NaOH, 2 M NaCl (Column wash)

0.5 M Ammonium acetate (Column wash)

20% EtOH (Column storage)

<FPLC device CIP>

Lines (A1, A3, B1, B2, B3, S1, out1) used in AEX were immersed in DW.

All lines were purged with DW using System CIP. To perform purging, enter Flow Rate: 50 mL/min, Volume per Position: 35 mL/min and perform it in the 'System CIP' tab.

Example 8-7. FPLC (CEX, Hitrap capto S 5 mL 4ea)

Buffer and Line are connected as shown in Table 13 below.

Line Buffer A1 20mM Tris + 100mM NaCl, pH 7.5 A2 20% EtOH A3 D.W. B1 2M NaCl B2 1M NaOH B3 0.2 M Sodium Acetate + 20% EtOH S1 Sample after TFF Out1 Storage Bottle

While flowing 20% EtOH through the line at 0.5 mL/min, four Hitrap capto S 5 mL columns were connected to AKTA to prevent air bubbles from entering. (Column Position 4)

To perform CEX, the method was set as follows.

Column Volume 20mL Pressure Limit Delta Column 0.4 MPa Flow Rate 20mL/min Wavelength UV280 & UV260

Equilibration was performed by flowing 5 CV of A1 Buffer (20mM Tris + 100mM NaCl, pH 7.5) at a flow rate of 20 mL/min.

Sample application was performed by flowing all of S1 (sample after TFF) into the column at a flow rate of 20 mL/min. At this time, all samples that passed without binding to the column were collected as Out1. (The sample is a target sample containing AAV.)

Reequilibration was performed again by flowing 2.5 CV of A1 Buffer (20mM Tris + 100mM NaCl, pH 7.5) at a flow rate of 20 mL/min.

Materials bound to the column were eluted by flowing 3 CV of B1 Buffer (2 M NaCl) at a flow rate of 20 mL/min.

Column wash was performed by flowing 5 CV of B2 Buffer (1 M NaOH) at a flow rate of 20 mL/min.

NaOH present in the column was removed by flowing 5 CV of A3 Buffer (DW) at a flow rate of 20 mL/min.

Equilibration was performed by flowing 6 CV of A1 Buffer (20mM Tris + 100mM NaCl, pH 7.5) at a flow rate of 20 mL/min.

After filling the column with the storage solution by flowing 5 CV of B3 Buffer (0.2 M Sodium Acetate + 20% EtOH) at a flow rate of 15 mL/min, the column was separated from the device and stored in an appropriate environment.

All lines used in CEX were immersed in DW.

All lines were washed with DW using the CIP method described above.

All lines used in CEX were immersed in 20% EtOH Buffer.

Fill all lines with 20% EtOH and store them using the CIP method described in 3.11.12.

Example 8-8. FPLC (AC, Cytiva Capto AVB 5 mL 1ea)

All lines used in AC were dipped in DW. All lines were washed with DW using the CIP method. Additionally, the buffer and line are connected as shown in Table 16 below.

Line Buffer A1 20mM Tris + 100mM NaCl, pH 7.5 A2 20% EtOH A3 PBS B1 0.1 M Sodium Acetate + 0.1 M NaCl, pH 2.5 B2 0.1 M Citric acid B3 10mM NaOH S1 Samples after CEX (refrigerated storage) Out1 Storage Bottle

A refrigerated Hitrap Capto AVB 5 mL column was connected to AKTA to prevent air bubbles from entering while flowing 20% EtOH at 0.5 mL/min.

Next, a tube for sample fraction collection was prepared. 5 mL of 60 mM BTP, pH 9.0 buffer was added to a 15 mL tube, and 6 tubes were mounted in AKTA in numerical order. To perform AC, the Method Setting was set as follows.

Column Volume 5mL Pressure Limit Delta Column 0.4 MPa Flow Rate 5mL/min Wavelength UV280 & UV260

Equilibration was performed by flowing 10 CV of A1 Buffer (20mM Tris + 100mM NaCl, pH 7.5) at a flow rate of 5 mL/min.

Sample application was performed by flowing S1 (sample after CEX (refrigerated storage)) into the column at a flow rate of 5 mL/min. At this time, all samples that passed without binding to the column were collected as Out1.

Reequilibration was performed again by flowing 20 CV of A1 Buffer (20mM Tris + 100mM NaCl, pH 7.5) at a flow rate of 20 mL/min.

Materials bound to the column were eluted by flowing 5 CV of B1 Buffer (0.1 M Sodium Acetate + 0.1 M NaCl, pH 2.5) at a flow rate of 5 mL/min. At this time, 2 to 3 fractions from the peak were selectively collected and combined, and then 0.6 mL was sampled from the combined sample and stored at -80°C.

Column wash was performed by flowing 8 CV of B2 Buffer (0.1 M Citric acid) at a flow rate of 5 mL/min.

Column wash was performed by flowing 6 CV of A3 Buffer (PBS) at a flow rate of 5 mL/min.

Column wash was performed by flowing 10 CV of B3 Buffer (10mM NaOH) at a flow rate of 5 mL/min.

Column wash was performed by flowing 10 CV of A3 Buffer (PBS) at a flow rate of 5 mL/min.

After filling the column with the storage solution by flowing 15 CV of A2 Buffer (20% EtOH) at a flow rate of 4 mL/min, the column was separated from the device and stored in an appropriate environment.

All lines used in AC were soaked in DW.

All lines were washed with DW using the CIP method described above.

Soak all lines used in AC in 20% EtOH Buffer. All lines were filled with 20% EtOH and stored using the CIP method.

Example 8-9. FPLC (AEX, CIMmultus QA 1 mL monolithic column (2 um), 1 ea)

The sample recovered after AC was sampled to the extent of 0.6 mL and stored at -80°C.

The recovered sample was diluted 12 to 13 times with a 20mM BTP + 50mM NaCl pH 9.0 solution. (e.g. 20 mL Affinity solution -> diluted to 250 mL)

All lines used in AEX were dipped into DW.

All lines are cleaned with DW using the CIP method.

Connect Buffer and Line as shown in the table below.

Line Buffer A1 20mM BTP, pH9.0 A2 20% EtOH A3 D.W. B1 20 mM BTP + 1 M NaCl, pH 9.0 B2 1 M NaOH + 2 M NaCl B3 0.5 M Ammonium Acetate S1 Diluted samples after AC (refrigerated) Out1 Storage Bottle

CIMmultus QA 1 mL Monolith Column was connected to AKTA with 20% EtOH flowing at 0.5 mL/min.

To perform AEX, the Method Setting was set as follows.

Column Volume 1mL Pressure Limit Delta Column 2 MPa Flow Rate 3mL/min Wavelength UV280 & UV260

Equilibration was performed by setting A1 Buffer (20 mM BTP, pH 9.0) and B1 Buffer (20 mM BTP + 1 M NaCl, pH 9.0) to Conc B 6% and flowing 15 CV at a flow rate of 3 mL/min. Sample application was performed by flowing S1 (sample after AC (refrigerated storage)) into the column at a flow rate of 3 mL/min.

Reequilibration was performed by setting A1 Buffer (20 mM BTP, pH 9.0) and B1 Buffer (20 mM BTP + 1 M NaCl, pH 9.0) to Conc B 6% and flowing 10 CV at a flow rate of 3 mL/min.

Set A1 Buffer (20mM BTP, pH9.0) and B1Buffer (20mM BTP + 1M NaCl, pH9.0) to the initial Conc B of 17%, and Conc B rises by 0.2% in steps every 1.5 CV, finally Conc B was set to increase to 24%.

At this time, samples coming out through the column were collected in 15mL tubes of 1.5 CV each. Afterwards, we looked at the peak, collected only the fractions collected from the part where the peak rises, and did 0.5 mL sampling. Before the point where the peak rises, collect it as AAV Empty Fraction and store it at -80℃, and store one rising point separately. Afterwards, everything was collected starting from the rising part of the peak (AAV Full). AAV full fractions were combined using a pipette, then 0.6 mL was sampled and stored in a deep freezer. The remaining solution was refrigerated until concentrated.

Column wash was performed by flowing 10 CV of A3 Buffer (DW) at a flow rate of 3 mL/min.

Column wash was performed by flowing 10 CV of B2 Buffer (1 M NaOH + 2 M NaCl) at a flow rate of 2 mL/min.

Column wash was performed by flowing 20 CV of A3 Buffer (DW) at a flow rate of 3 mL/min.

Column wash was performed by flowing 10 CV of B3 Buffer (0.5 M Ammonium Acetate) at a flow rate of 3 mL/min.

Column wash was performed by flowing 15 CV of A3 Buffer (DW) at a flow rate of 3 mL/min.

After filling the column with the storage solution by flowing 15 CV of A2 Buffer (20% EtOH) at a flow rate of 2 mL/min, the column was separated from the device and stored in an appropriate environment.

All lines used in AEX were dipped in DW.

All lines were washed with DW using the CIP method.

All lines used in AEX were soaked in 20% EtOH Buffer.

All lines were filled with 20% EtOH and stored using the CIP method.

Examples 8-10. concentration

A rough sample up to AEX was put into the Sartorius Viva Spin and spun at 2000 g for about 2 to 3 minutes. The remaining sample was filled with pH 8.0 PBS up to 15 mL and then rotated at 2500 g for about 2 to 3 minutes. The above process was repeated one more time. After concentrating so that the final volume was 0.8 to 1 mL, samples for analysis and samples for storage were divided and stored. For storage samples, 0.2~0.3 mL was dispensed, and for analysis samples, 0.1 mL was dispensed.

Process Stage Input (mL) Output
(mL) Scale Runs One 2 3 4 Culture broth 600 600 100 100 100 100 Input titer (3 F/Ts) 2.19E+13 3.04E+13 4.61E+12 6.93E+12 Cell disruption (MF) 600 600 ㅡ ㅡ ㅡ ㅡ Supernatant 600 600 117 88 125 110 Filtration I (full recovery: 20mM Tris 100mM NaCl pH7.5) 600 650 Sartorius PP3(PP) 5㎛
180cm2 87 77 134 105 Filtration II 650 650 Merck SHC (PES) 0.5-0.2 ㎛ 17 cm2 101 88 127 97 TFF with 20mM Tris 100mM NaCl pH7.5 650 200 Repligen
100 kDa
225 cm2 128 76 98 74 Nucleic acid digestion 200 200 50U 1hr 37℃ 128 76 98 74 Volume restoration 200 600 With 20mM Tris 100mM NaCl pH7.5 ㅡ ㅡ ㅡ ㅡ CEX 600 600 Cytiva Capto S 20ml 83 54 77 49 Affinity (100mM Sodium Acetate, 100mM NaCl, pH2.5) & Neutralization (60mM BTP, pH9.0) 650 20 Cytiva Capto AVB 5 ml 39 27 10 17 Buffer adjustment 20 250 With 20mM BTP 50mM NaCl pH9.0 ㅡ ㅡ ㅡ ㅡ AEX 250 27 BIA CIM QA 1ml 16 13 7 10 100 kDa filtration 27 0.8 Sartorius Vivaspin 100 kDa 23 20 8 8 Frozen DS 0.8 0.8 23.0 20.0 8.1 7.5 Output titer 5.03E+12 6.00E+12 3.83E+11 5.18E+11

A filtration process with a cut-off of 100 kDa was performed and concentration was performed. The results are shown in Table 20 and Figure 9.

As shown in Table 20 and Figure 9, there was little change in yield when filtration was performed with a cut-off of 100 kDa, and it was found that the yield of AAV was maintained during the concentration process.

The present inventors silver stained the AAV vector obtained as a result of purification under various conditions performed in the above-described examples for VP1, VP2, and VP3 proteins, and the results are shown in Figure 10. Additionally, Analytical Ultra-centrifuge was performed and the results are shown in Figure 11.

As shown in Figure 10, the VP1, VP2, and VP3 proteins of the AAV vector were clearly identified, confirming that the AAV vector was successfully purified in the purification experiment according to each condition of the present invention.

In addition, as shown in Figure 11, as a result of quality analysis of the AAV vector purified by the purification method of the present invention using an Analytical Ultracentrifuge, when measured at a concentration of 2.5 E11 vg/ml, Full AAV 66% / Empty 8.7% It was confirmed that AAV was purified to good quality through distribution.

Claims (17)

Method for purification of Adenovirus-associated virus (AAV) comprising the following steps:
(a) disrupting the cells producing AAV;
(b) filtering the supernatant containing the disrupted cells;
(c) performing cation exchange chromatography on the filtrate filtered in the above step to produce a column effluent;
(d) performing affinity chromatography on the column eluate to produce a column eluate;
(e) producing a column effluent by subjecting the column effluent to anion exchange chromatography; and
(f) filtering the column eluate to produce purified AAV.
The method of claim 1, wherein the cells in step (a) are derived from a cell culture containing AAV-producing cells and their cell culture supernatant.
The method of claim 1, wherein the AAV-producing cells are human cells or insect cells.
The method of claim 1, wherein the cell disruption method is performed using a microfludizer.
The method of claim 1, wherein the filtration in step (b) is performed using a membrane filter.
The method of claim 1, wherein the filtration in step (b) is performed one or more times.
The method of claim 1, wherein the filtration in step (b) is performed by a depth filtration device.
The method of claim 1, wherein the filtration in step (b) is performed by a filtration device of absolute filtration grade.
The method of claim 5, wherein the membrane filter is a membrane filter made of cellulose acetate or polyethersulfone.
The method of claim 1, wherein the filtrate from step (b) is filtered by tangential flow filtration (TFF).
The method of claim 10, wherein the tangential flow filtration is performed using an elution buffer containing Tris, NaCl, or a combination thereof.
The method of claim 1, wherein the filtrate from step (b) is treated with nuclease.
The method of claim 12, wherein the nuclease is benzoase.
The method of claim 1, wherein the cation exchange chromatography in step (c) is eluted with an elution buffer containing Tris, NaCl, or a combination thereof.
The method of claim 1, wherein the affinity chromatography in step (d) is eluted with an elution buffer containing Sodium Acetate, NaCl, or a combination thereof.
The method of claim 1, wherein the anion exchange chromatography in step (e) is eluted with an elution buffer containing Bis-Tris-Propane (BTP), NaCl, or a combination thereof.
The method of claim 1, wherein the anion exchange chromatography in step (e) is carried out at a concentration of 1-3 column volumes (CV) per step while increasing the concentration of NaCl from 170 to 190mM in increments of 1-5mM. A method for purifying AAV, which involves eluting by injecting an elution buffer.