CN118006688A

CN118006688A - Purification method of recombinant adeno-associated virus vector

Info

Publication number: CN118006688A
Application number: CN202311490231.0A
Authority: CN
Inventors: 施俊伟; 张子辉; 柯辰; 王宏伟
Original assignee: Beijing Solobio Genetechnology Co Ltd
Current assignee: Beijing Solobio Genetechnology Co Ltd
Priority date: 2022-11-10
Filing date: 2023-11-09
Publication date: 2024-05-10

Abstract

The invention provides a method for purifying recombinant adeno-associated virus (rAAV) vectors, by which highly purified rAAV can be obtained. The purification method provided by the invention not only can take scale into account, but also can meet clinical application of the purified rAAV sample.

Description

Purification method of recombinant adeno-associated virus vector

Technical Field

The invention relates to the field of biotechnology and gene therapy, in particular to a separation and purification method of a recombinant adeno-associated virus (rAAV) vector applied to gene therapy.

Background

Adeno-associated virus (AAV) belongs to the parvoviridae, a single-stranded DNA virus with a genome size of 4700bp, the genome mainly comprising two reading frames encoding Rep and Cap flanked by inverted terminal repeats (INVERTED TERMINAL REPEAT, ITR) with palindromic sequences. AAV has a plurality of serotypes, and tissue tropism of different serotypes is greatly different, for example AAV8 has stronger hepatic tropism, AAV9 can cross the blood brain barrier and has better central nervous system tropism. There is currently increasing research on adeno-associated virus (AAV) capsid protein engineering, including methods that utilize DNA shuffling (DNA shuffling) or error-prone PCR to obtain capsid proteins of novel AAV viral vectors that are functionally optimized. Among them AAVX is a novel AAV vector obtained by DNA shuffling technology (Xiao Xiao et al, US9186419B 2), and studies have shown that AAVX has a high transduction efficiency for liver targeting (see, e.g., WO2022/100748A 1). The AAV viral vector is used for delivering therapeutic gene fragments to specific tissues and organs, so that a plurality of monogenic genetic diseases or viral infectious diseases can be effectively treated, and the AAV viral vector is the most promising gene therapeutic vector at present.

Many methods for producing rAAV vectors are known in the art, including transfection, stable cell line production, and infectious virus production systems, among which three plasmid packaging systems (Xiao et al.,Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J.Virol.72(3):2224(1998)) and baculovirus expression systems are commonly used, and baculovirus expression systems have been widely used for AAV production due to their advantages of economy, efficiency, safety, scalability, etc. (Urabe M et al, 2002,Human Gene Therapy,13:1935-1943;US6723551;Smith RH et al, 2009, mol. Ther,17:1888-1896; chen H et al, 2008,Mol Ther,16:924-930, etc.). However, AAV products produced using baculovirus expression systems may contain impurities such as baculovirus proteins, baculovirus DNA, etc., which must be considered during AAV purification.

Currently, various purification means such as ultracentrifugation, affinity chromatography, ion exchange chromatography, mixed chromatography, molecular sieve chromatography, etc. are applied to AAV vector purification (Qu Weigong, 2015, china pharmacy, 26 (34): 4880-4883). In the early stages, the purification route based on PEG precipitation and ultracentrifugation was applied to AAV purification. While this approach is suitable for a variety of serotypes, it is advantageous in terms of empty shell removal, but it also has significant disadvantages: on the one hand, the ultracentrifugation treatment capacity is small, the single centrifugation takes longer time, and the method is not suitable for large-scale purification; on the other hand, cesium chloride is a heavy metal, and when cesium chloride is used as a super-ionic medium, there is a great risk in terms of sample safety (Benjamin Strobel et al 2015,Hum Gene Ther Methods,26 (4): 147-157; qu Weigong, 2015, china pharmacy, 26 (34): 4880-4883).

As the dosage of AAV-related gene therapy products continues to increase, mass production is becoming increasingly important for downstream purification processes of AAV, and various chromatographic column chromatography has shown its unique advantages, such as affinity chromatography and ion exchange chromatography, etc. (Benjamin Adams et al, 2020,Biotechnol Bioeng,117 (10): 3199-3211; qu Weigong, 2015, china pharmacy, 26 (34): 4880-4883; weihong Qu et al, 2015,Curr Pharm Biotechnol,16 (8): 684-695, etc.).

There are studies mentioning one or more ion exchange chromatography (IEX) combined with Ultracentrifugation (UC) purification routes (e.g. WO2019/094253a1; cn102947453a3, etc.), but such routes are too costly due to the use of ultracentrifugation and cannot be applied in large scale purification for industrial production. The purification routes of ion exchange and molecular sieve chromatography are mentioned in the prior art, however, the routes have poor AAV isolation specificity and are difficult to remove for residual proteins having isoelectric points and molecular weights close to AAV (Marc-Andr Robert et al, 2017,Biotechnol J,12 (3): 1600193,1-16).

Along with the continuous development of the gene therapy field, the requirements of clinical AAV products on quality indexes such as purity, activity, residual host cell protein, residual host cell DNA and the like are continuously improved, how to reasonably utilize different purification means to form a set of purification process, and the acquisition of clinical AAV is a great challenge, and most of the process routes in the existing literature and patents are laboratory-grade sample preparation, so that the requirements of clinical AAV quality can be rarely met; on the other hand, the dosage requirements of AAV-related gene therapy products are also increasing, and it is particularly important that the purification process is easy to amplify, while the purification process based on ultracentrifugation has a number of limitations in the amplification process.

In addition, most of the purification methods in the prior art are based on adeno-associated viral vectors such as AAV8 and AAV9, and there are few reports on purification methods of recombinant viral vectors based on AAVX. In addition, most of the purification methods in the prior art are aimed at traditional rAAV samples produced by taking adenovirus as a helper virus, and few reports are available for purifying rAAV samples produced by a baculovirus packaging system with relatively high purification difficulty.

Based on the above problems, there is still a great need for a large-scale purification process that meets the quality requirements of clinical grade AAV products, particularly AAVX products, while compromising scalability.

Disclosure of Invention

Based on the above, the invention provides a purification method of recombinant adeno-associated virus vector, the purified rAAV sample has high recovery rate, purity and activity, and various impurities (including residual host cell protein, residual host cell DNA and the like) related quality indexes are excellent. Meanwhile, the purification method has low cost, is easy for large-scale purification, and is an advantage which is not possessed by other purification methods in industrial production.

The invention provides a purification method of a recombinant adeno-associated virus (rAAV) vector, which is characterized by comprising the following steps in sequence:

(1) Obtaining a sample containing the rAAV vector to be purified;

(2) Subjecting the sample to affinity chromatography or cation exchange chromatography, and collecting eluate;

(3) Purifying the eluent by apatite chromatography;

(4) Carrying out molecular sieve chromatography or ultrafiltration on the sample purified in the step (3);

(5) Subsequently, carrying out anion exchange chromatography on the sample, and harvesting eluent;

(6) And carrying out molecular sieve chromatography or ultrafiltration on the eluent again.

Recombinant adeno-associated virus (rAAV) vectors

In certain embodiments, the rAAV vector has a dna sequence comprising any one of the capsid proteins from AAV clades a-F or hybrid/chimeric versions thereof, including but not limited to any one of the capsid proteins AAV1、AAV2、AAV3、AAV3A、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV2.5、AAV-DJ、AAV-DJ/8、AAVhu.32、AAVhu.68、AAVrh8、AAVrh10、AAVrh74、AAV-PHP.B、AAV-PHP.eB、AAV-PHP.S or AAVX or variants thereof. In a preferred embodiment, the rAAV has a capsid protein comprising a protein derived from AAVX. In some embodiments, the AAVX capsid protein has the amino acid sequence set forth in SEQ ID NO: 1.

In certain embodiments, the rAAV vector is prepared by conventional AAV vector packaging systems, including, but not limited to, two-plasmid packaging systems, three-plasmid packaging systems, baculovirus packaging systems, and AAV packaging systems helper with adenovirus (Ad) or herpes virus (HSV), preferably three-plasmid packaging systems and baculovirus packaging systems.

Affinity chromatography

The AAV is captured mainly by the affinity filler, so that the residual inactivating agent, the residual host cell protein, the residual host cell DNA, the residual baculovirus protein, the residual baculovirus DNA, the endotoxin and other relevant impurities in the sample can be effectively removed.

In certain embodiments, the affinity chromatography is heparin affinity chromatography.

In certain embodiments, the affinity chromatography comprises equilibration, loading, elution steps, and optionally, a wash step.

In certain embodiments, the affinity chromatography adjusts the conductance of the sample to be 5.5+ -1 ms/cm to 10.5+ -1 ms/cm, preferably 6.5+ -1 ms/cm to 9.5+ -1 ms/cm, more preferably 7.5+ -1 ms/cm to 8.5+ -1 ms/cm, prior to loading. In certain embodiments, the affinity chromatography adjusts the conductance of the sample to 5.5.+ -. 1ms/cm, 6.5.+ -. 1ms/cm, 7.5.+ -. 1ms/cm, 8.5.+ -. 1ms/cm, or 9.5.+ -. 1ms/cm, more preferably 8.5.+ -. 1ms/cm or 9.5.+ -. 1ms/cm, prior to loading. In certain embodiments, the affinity chromatography adjusts the pH of the sample to a pH of 6.0 to 7.6, preferably 6.5 to 7.0, prior to loading. In certain embodiments, the affinity chromatography adjusts the pH of the sample to 6.5, 6.6, 6.8, or 7.5, preferably 6.5, 6.6, or 6.8, prior to loading.

In certain embodiments, the equilibration solution A of the affinity chromatography is a PB Buffer (Phosphate Buffer) containing NaCl, wherein the concentration of PB Buffer is 10mM-60mM, preferably 20-50mM, more preferably 30-40mM; the concentration of NaCl is 10mM-50mM, preferably 20 mM-40 mM, more preferably 30 mM-40 mM, and the pH of the equilibration solution is 6.2-7.6, preferably 6.5-6.8, more preferably 6.6 or 6.8. In certain embodiments, the equilibration solution a of the affinity chromatography is one of 20mM PB+20mM NaCl、20mM PB+30mM NaCl、20mM PB+40mM NaCl、30mM PB+20mM NaCl、30mM PB+30mM NaCl、30mM PB+40mM NaCl、40mM PB+20mM NaCl、40mM PB+30mM NaCl、40mM PB+40mM NaCl、50mM PB+20mM NaCl、50mM PB+30mM NaCl、50mM PB+40mM NaCl, preferably 30mM PB+40mM NaCl or 40mM PB+30mM NaCl. In certain embodiments, the pH of the equilibration liquid a of the affinity chromatography is 6.2, 6.6, 6.8, or 7.6, preferably 6.6 or 6.8.

In certain embodiments, the affinity chromatography eluate is 10% to 100% (v/v) solution B+90% to 0% (v/v) equilibration solution A, preferably 50% to 100% (v/v) solution B+50% to 0% (v/v) equilibration solution A. Wherein, the solution B is PB buffer solution containing NaCl, the concentration of the PB buffer solution is 10mM-60mM, preferably 20-50mM, more preferably 30-40mM, the concentration of NaCl is 200mM-600mM, preferably 300-600mM, more preferably 400-500mM, and the pH of the eluent is 6.2-7.6, preferably 6.5-6.8. In certain embodiments, the eluent is 10% (v/v) solution b+90% equilibrium solution a, 20% (v/v) solution b+80% equilibrium solution a, 30% (v/v) solution b+70% equilibrium solution a, 40% (v/v) solution b+60% equilibrium solution a, 50% (v/v) solution b+50% equilibrium solution a, 60% (v/v) solution b+40% equilibrium solution a, 70% (v/v) solution b+30% equilibrium solution a, 80% (v/v) solution b+20% equilibrium solution a, 90% (v/v) solution b+10% equilibrium solution a, 100% (v/v) solution B, preferably 50% (v/v) solution b+50% equilibrium solution a, 60% (v/v) solution b+40% equilibrium solution a, 70% (v/v) solution b+30% equilibrium solution a, 80% (v/v) solution b+20% (v) solution b+50% (v/v) solution b+50% equilibrium solution B, preferably 100% (v/v) solution b+50% (v/v) solution b+50% (v/v) solution B/v) solution b+20% solution b+50%; the solution B of the affinity chromatography is 20mM PB+300mM NaCl、20mM PB+400mM NaCl、20mM PB+500mM NaCl、20mM PB+600mM NaCl、30mM PB+300mM NaCl、30mM PB+400mM NaCl、30mM PB+500mM NaCl、30mM PB+600mM NaCl、40mM PB+300mM NaCl、40mM PB+400mM NaCl、40mM PB+500mM NaCl、40mM PB+600mM NaCl、50mM PB+300mM NaCl、50mM PB+400mM NaCl、50mM PB+500mM NaCl、50mM PB+600mM NaCl or 50mM PB+600mM NaCl, preferably 30mM PB+400mM NaCl, 30mM PB+500mM NaCl, 40mM PB+400mM NaCl or 40mM PB+500mM NaCl, more preferably 30mM PB+400mM NaCl or 40mM PB+500mM NaCl. In certain embodiments, the pH of the eluate of the affinity chromatography is 6.2, 6.6, 6.8, or 7.6, preferably 6.6 or 6.8.

In certain embodiments, the buffer used in the wash step in the affinity chromatography is 10% to 100% (v/v) solution B+90% to 0% (v/v) equilibration solution A, preferably 20% to 40% (v/v) solution B+80% to 60% (v/v) equilibration solution A. In certain embodiments, the buffer used in the affinity chromatography wash step is 10% (v/v) solution B+90% (v/v) solution A, 20% (v/v) solution B+80% (v/v) solution A, 30% (v/v) solution B+70% (v/v) solution A, 40% (v/v) solution B+60% (v/v) solution A, 50% (v/v) solution B+50% (v/v) solution A, 60% (v/v) solution B+40% (v/v) solution A, 70% (v/v) solution B+30% (v/v) solution A, 80% (v/v) solution B+20% (v/v) solution A, 90% (v/v) solution B+10% (v/v) solution A, 100% (v/v) solution B, preferably 30% (v/v) solution B+70% (v) solution A.

In certain embodiments, the affinity chromatography flow rate is in the range of 50cm/h to 300cm/h, preferably 70cm/h to 200cm/h, more preferably 90 to 150cm/h. In certain embodiments, the affinity chromatography is performed at a flow rate of 50cm/h, 60cm/h, 70cm/h, 80cm/h, 90cm/h, 100cm/h, 110cm/h, 150cm/h, 200cm/h, 250cm/h, or 300cm/h, preferably 90cm/h, 100cm/h, or 150cm/h, more preferably 90cm/h or 150cm/h.

Cation exchange chromatography

The cation exchange chromatography mainly adopts the ion exchange principle, the cationic chromatography filler is matched with negative charge in a specific buffer solution, AAV is positively charged, and the AAV is captured in advance by the charge-acting filler, so that impurities related to the processes such as residual inactivating agent, residual host cell protein, residual host cell DNA, residual baculovirus protein, residual baculovirus DNA and endotoxin in a sample can be effectively removed.

In certain embodiments, the filler for cation exchange chromatography is one of POROS 50HE, POROS HS, bio-RAD Nuvia HR S, SP HP, capto S.

In certain embodiments, the cation exchange chromatography comprises equilibration, loading, elution steps.

In certain embodiments, the cation exchange chromatography adjusts the conductance of the sample to 5.5.+ -. 1ms/cm to 10.5.+ -. 1ms/cm, preferably 5.5.+ -. 1ms/cm to 7.5.+ -. 1ms/cm, prior to loading; the pH of the sample is adjusted to 6.0-7.6, preferably 6.5-7.0. In certain embodiments, the conductance of the sample used for the cation exchange chromatography is 5.5.+ -. 1ms/cm, 6.5.+ -. 1ms/cm, 7.5.+ -. 1ms/cm, 8.5.+ -. 1ms/cm, 9.5.+ -. 1ms/cm or 10.5.+ -. 1ms/cm, preferably 5.5.+ -. 1ms/cm, 6.5.+ -. 1ms/cm or 7.5.+ -. 1ms/cm, more preferably 7.5.+ -. 1ms/cm. In certain embodiments, the pH of the sample used for the cation exchange chromatography is 6.5, 6.6, 6.8 or 7.5, preferably 6.5, 6.6 or 6.8.

In certain embodiments, the equilibrium solution A of the cation exchange chromatography is PB buffer containing NaCl, wherein the PB buffer has a concentration of 10mM-60mM, preferably 20-50mM, the NaCl has a concentration of 20-40mM, and the pH of the equilibrium solution is 6.2-7.6, preferably 6.5-6.8. In certain embodiments, the equilibrium liquid a of the cation exchange chromatography is one of 20mM PB+20mM NaCl、20mM PB+30mM NaCl、20mM PB+40mM NaCl、30mM PB+20mM NaCl、30mM PB+30mM NaCl、30mM PB+40mM NaCl、40mM PB+20mM NaCl、40mM PB+30mM NaCl、40mM PB+40mM NaCl、50mM PB+20mM NaCl、50mM PB+30mM NaCl、50mM PB+40mM NaCl, preferably 30mM PB+40mM NaCl or 40mM PB+30mM NaCl. In certain embodiments, the pH of the equilibrium liquid a of the cation exchange chromatography is one of 6.2, 6.6, 6.8, 7.6, preferably 6.6 or 6.8.

In certain embodiments, the cation exchange chromatography eluate is 10% -100% (v/v) solution B+90% -0% (v/v) equilibrium solution A, preferably 50% -100% (v/v) solution B+50% -0% (v/v) equilibrium solution A, wherein solution B is a PB buffer containing NaCl, wherein the PB buffer has a concentration of 10mM-60mM, preferably 20-50mM, more preferably 30-40mM, and the NaCl has a concentration of 200mM-600mM, preferably 300-600mM, more preferably 400-500mM, and the pH of the eluate is 6.2-7.6, preferably 6.5-6.8. In certain embodiments, the eluate of the cation exchange chromatography is 10% (v/v) solution B+90% (v/v) solution A, 20% (v/v) solution B+80% (v/v) solution A, 30% (v/v) solution B+70% (v/v) solution A, 40% (v/v) solution B+60% (v/v) solution A, 50% (v/v) solution B+50% (v/v) solution A, 60% (v/v) solution B+40% (v/v) solution A, 70% (v/v) solution B+30% (v/v) solution A, 80% (v/v) solution B+20% (v/v) solution A, 90% (v/v) solution B+10% (v/v) solution A or 100% (v/v) solution B, preferably 50% (v/v) of solution B+50% (v/v) of equilibration solution A, 60% (v/v) of solution B+40% (v/v) of equilibration solution A, 70% (v/v) of solution B+30% (v/v) of equilibration solution A, 80% (v/v) of solution B+20% (v/v) of equilibration solution A, 90% (v/v) of solution B+10% (v/v) of equilibration solution A or 100% (v/v) of solution B, more preferably 60% (v/v) of solution B+40% of equilibration solution A or 80% (v/v) of solution B+20% of equilibration solution A; in certain embodiments, the solution B is 20mM PB+300mM NaCl、20mM PB+400mM NaCl、20mM PB+500mM NaCl、20mM PB+600mM NaCl、30mM PB+300mM NaCl、30mM PB+400mM NaCl、30mM PB+500mM NaCl、30mM PB+600mM NaCl、40mM PB+300mM NaCl、40mM PB+400mM NaCl、40mM PB+500mM NaCl、40mM PB+600mM NaCl、50mM PB+300mM NaCl、50mM PB+400mM NaCl、50mM PB+500mM NaCl、50mM PB+600mM NaCl or 50mM PB+600mM NaCl, preferably 30mM PB+400mM NaCl, 30mM PB+500mM NaCl, 40mM PB+400mM NaCl or 40mM PB+500mM NaCl, more preferably 30mM PB+400mM NaCl or 40mM PB+500mM NaCl. In certain embodiments, the pH of the eluate of the cation exchange chromatography is one of 6.2, 6.6, 6.8, 7.6, preferably 6.6 or 6.8.

In certain embodiments, the cation exchange chromatography is performed at a flow rate of 50cm/h to 300cm/h, preferably 70cm/h to 200cm/h. In certain embodiments, the cation exchange chromatography is performed at a flow rate of 50cm/h, 100cm/h, 150cm/h, 200cm/h, 250cm/h, or 300cm/h, preferably 50cm/h, 100cm/h, 150cm/h, or 200cm/h, more preferably 150cm/h.

Apatite chromatography

The filler used in apatite chromatography is mainly divided into Ceramic Hydroxyapatite (CHT) chromatography or Ceramic Fluorapatite (CFT), wherein the filler used in CHT chromatography is made of hydroxyapatite, the chemical structure is (Ca ₅(PO₄)₃OH)₂, wherein the structure of phosphate radical enables the filler to have cation exchange property, positive charge component can be combined through cation exchange, calcium ion structure enables the filler to have metal chelating property, and carboxyl component can be combined.

In certain embodiments, the apatite chromatography is Ceramic Hydroxyapatite (CHT) chromatography or Ceramic Fluoroapatite (CFT) chromatography. In certain embodiments, the apatite chromatography is CHT chromatography.

In certain embodiments, the apatite chromatography comprises equilibration, loading, elution steps.

In certain embodiments, the apatite chromatography adjusts the conductance of the sample to between 5+ -1 ms/cm and 12+ -1 ms/cm, preferably between 5+ -1 ms/cm and 9+ -1 ms/cm, more preferably between 6+ -1 ms/cm and 8+ -1 ms/cm, prior to loading. In certain embodiments, the apatite chromatography adjusts the conductance of the sample to 5+ -1 ms/cm, 6+ -1 ms/cm, 7+ -1 ms/cm, 8+ -1 ms/cm, 9+ -1 ms/cm, 10+ -1 ms/cm, 11+ -1 ms/cm or 12+ -1 ms/cm, preferably 5+ -1 ms/cm, 6+ -1 ms/cm, 7+ -1 ms/cm or 8+ -1 ms/cm prior to loading.

In certain embodiments, the equilibration solution A of the apatite chromatography is a PB buffer comprising NaCl, wherein the PB buffer has a concentration of 10mM-60mM, preferably 20-50mM, more preferably 30-40mM, the NaCl has a concentration of 10mM-50mM, preferably 20-40mM, more preferably 30-40mM, the pH of equilibration solution A is 6.2-7.6, preferably 6.5-6.8. In certain embodiments, the apatite chromatography equilibrium solution A is one of 20mM PB+20mM NaCl、20mM PB+30mM NaCl、20mM PB+40mM NaCl、30mM PB+20mM NaCl、30mM PB+30mM NaCl、30mM PB+40mM NaCl、40mM PB+20mM NaCl、40mM PB+30mM NaCl、40mM PB+40mM NaCl、50mM PB+20mM NaCl、50mM PB+30mM NaCl、50mM PB+40mM NaCl, preferably 30mM PB+40mM NaCl or 40mM PB+30mM NaCl. In certain embodiments, the apatite chromatography balance a has a pH of 6.2, 6.6, 6.8 or 7.6.

In certain embodiments, the apatite chromatography eluate is 10% -100% (v/v) solution B+90% -0% (v/v) equilibration solution A, preferably 20% -50% (v/v) solution B+80% -50% (v/v) equilibration solution A, more preferably 30% -40% (v/v) solution B+70% -60% (v/v) equilibration solution A, wherein solution B is a PB buffer comprising NaCl, wherein the PB buffer has a concentration of 10mM-60mM, preferably 20-50mM, more preferably 30-40mM, and the NaCl has a concentration of 200mM-600mM, preferably 300-600mM, more preferably 400-500mM, and the pH of the eluate is 6.2-7.6, preferably 6.5-6.8; in certain embodiments, the apatite chromatography eluate is 10% (v/v) solution B+90% (v/v) solution A, 20% (v/v) solution B+80% (v/v) solution A, 30% (v/v) solution B+70% (v/v) solution A, 40% (v/v) solution B+60% (v/v) solution A, 50% (v/v) solution B+50% (v) solution A, 60% (v/v) solution B+40% (v/v) solution A, 70% (v/v) solution B+30% (v/v) solution A, 80% (v/v) solution B+20% (v/v) solution A, 90% (v/v) solution B+10% (v/v) solution A or 100% (v/v) solution B, preferably 20% (v/v) solution B+80% (v/v) solution B+40% (v) solution A, 70% (v/v) solution B+30% (v/v) solution A, 50% (v/v) solution B+30% (v/v) solution A, more preferably 40% (v/v) of solution B+60% (v/v) of equilibration liquid A or 30% (v/v) of solution B+70% (v/v) of equilibration liquid A. In certain embodiments, the solution B is 20mM PB+300mM NaCl、20mM PB+400mM NaCl、20mM PB+500mM NaCl、20mM PB+600mM NaCl、30mM PB+300mM NaCl、30mM PB+400mM NaCl、30mM PB+500mM NaCl、30mM PB+600mM NaCl、40mM PB+300mM NaCl、40mM PB+400mM NaCl、40mM PB+500mM NaCl、40mM PB+600mM NaCl、50mM PB+300mM NaCl、50mM PB+400mM NaCl、50mM PB+500mM NaCl、50mM PB+600mM NaCl or 50mM PB+600mM NaCl, preferably 30mM PB+400mM NaCl, 30mM PB+500mM NaCl, 40mM PB+400mM NaCl or 40mM PB+500mM NaCl, more preferably 30mM PB+400mM NaCl or 40mM PB+500mM NaCl. In certain embodiments, the pH of the apatite chromatography eluate is 6.2, 6.6, 6.8, or 7.6.

In certain embodiments, the apatite chromatography is performed at a flow rate of 50cm/h to 300cm/h, preferably 100cm/h to 250cm/h, more preferably 150cm/h to 200cm/h. In certain embodiments, the apatite chromatography is performed at a flow rate of 50cm/h, 100cm/h, 150cm/h, 200cm/h, 250cm/h or 300cm/h, preferably 50cm/h, 100cm/h, 150cm/h or 200cm/h, more preferably 150cm/h or 200cm/h.

Molecular sieve chromatography

Molecular sieve chromatography, also known as gel filtration chromatography or size exclusion chromatography, is a chromatographic technique that uses porous gel with a certain pore size range as a stationary phase to separate components in a mixture according to molecular size.

In certain embodiments, the molecular sieve in step (4) is G25 chromatography and the molecular sieve in step (6) is S200 chromatography.

G25 chromatography is mainly based on the molecular sieve principle, wherein large molecules flow out of an outer water channel, small molecules such as salt molecules flow out of an inner water channel after a short path, and the like, and flow out after a long path, so that the purpose of replacing buffer solution is realized, and preparation is made for the next purification.

The S200 chromatography is mainly used for fine purification of AAV by a molecular sieve principle, and the AAV has a molecular weight larger than that of common residual host cell proteins and residual baculovirus proteins, so that the two residual proteins can be further removed by the S200 chromatography. At the same time, this step can accomplish buffer displacement.

In certain embodiments, the molecular sieve comprises equilibration, loading, and elution steps.

In certain embodiments, the equilibration solution for G25 chromatography comprises Tris buffer and NaCl, wherein the concentration of Tris buffer is in the range of 10mM-60mM, preferably 30mM-60mM, more preferably 40mM-50mM, and the concentration of NaCl is 10mM-60mM, preferably 30mM-60mM, more preferably 40-50mM, pH 7.0-9.0, preferably 8.0-9.0. In certain embodiments, the G25 chromatography equilibration buffer species is 30mM Tris+30mM NaCl、30mM Tris+40mM NaCl、30mM Tris+50mM NaCl、30mM Tris+60mM NaCl、40mM Tris+30mM NaCl、40mM Tris+40mM NaCl、40mM Tris+50mM NaCl、40mM Tris+60mM NaCl、50mM Tris+30mM NaCl、50mM Tris+40mM NaCl、50mM Tris+50mM NaCl、50mM Tris+60mM NaCl、60mM Tris+30mM NaCl、60mM Tris+40mM NaCl、60mM Tris+50mM NaCl or 60mM Tris+60mM NaCl, preferably 40mM Tris+40mM NaCl, 40mM Tris+50mM NaCl, 50mM Tris+40mM NaCl or 50mM Tris+50mM NaCl. In certain embodiments, the pH of the equilibration buffer for G25 chromatography is 7.0, 7.5, 8.0, 8.5, or 9.0.

In certain embodiments, the G25 chromatography is performed at a flow rate of 30cm/h to 180cm/h, preferably 50cm/h to 150cm/h, more preferably 90 to 120cm/h. In certain embodiments, the G25 chromatography is performed at a flow rate of 30cm/h, 60cm/h, 90cm/h, 120cm/h, 150cm/h or 180cm/h, preferably 90cm/h or 120cm/h.

In certain embodiments, the equilibration solution for S200 chromatography comprises KCl, KH ₂PO₄、Na₂HPO₄, and NaCl, wherein the concentration of KCl is 1-3mM, preferably 2.67mM, the concentration of KH ₂PO₄ is 1-2mM, preferably 1.47mM, the concentration of na ₂HPO₄ is 7-9mM, preferably 8.06mM, the concentration of NaCl is 100mM-200mM, preferably 120-160mM. In certain embodiments, the equilibration solution for S200 chromatography is one of 2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+100mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+117.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+127.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+137.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+147.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+157.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+167.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+200mM NaCl, preferably 2.67mM KCl+1.47mM KH ₂PO₄+8.06mM Na₂HPO₄ +137.93mM NaCl. In certain embodiments, the equilibration liquid of S200 chromatography further comprises a concentration of F68. In certain embodiments, the final concentration of F68 is from 0.0001% to 1%, preferably from 0.001% to 0.1%, and in certain embodiments, the concentration of F68 is one of 0.0001%, 0.001%, 0.01%, 0.1%, 1%, preferably 0.001% or 0.01%.

In certain embodiments, the flow rate of the S200 chromatography is 15cm/h to 120cm/h, preferably 30cm/h to 60cm/h. In certain embodiments, the flow rate of the S200 chromatography is 15cm/h, 30cm/h, 45cm/h, 60cm/h, 75cm/h, 90cm/h, 105cm/h or 120cm/h, preferably 30cm/h, 45cm/h or 60cm/h.

Ultrafiltration (TFF ultrafiltration)

TFF (tangential flow ultrafiltration) is mainly based on the principle of tangential flow ultrafiltration, wherein macromolecules are trapped and small molecules such as salt molecules penetrate, so that the purpose of concentrating and/or replacing buffer solution is achieved, preparation is carried out for next chromatography or final purified stock solution is obtained, and residual host cell proteins and residual baculovirus proteins can be further removed in the step.

In certain embodiments, the ultrafiltration in step (4) and step (6) is TFF ultrafiltration.

In certain embodiments, the TFF ultrafiltration comprises concentration and washing steps.

In certain embodiments, the medium used for TFF ultrafiltration is a conventional membrane pack or hollow fiber, preferably hollow fiber. In certain embodiments, the hollow fiber pore size is 100KD.

In certain embodiments, the wash filtrate of the TFF ultrafiltration in step (4) comprises Tris buffer, naCl and F68, wherein the concentration of Tris buffer is 30mM-60mM, preferably 40-60mM, the concentration of NaCl is 100mM-300mM, preferably 150-250mM, the concentration of F68 is 0.001% -0.1%, preferably 0.01% -0.1%, the pH is 6.5-9.5, preferably 6.5-9.0, and in certain embodiments, the wash filtrate of the TFF ultrafiltration in step (4) is one of 30mM Tris+200mM NaCl+0.01％ F68、40mM Tris+200mM NaCl+0.01％ F68、50mM Tris+200mM NaCl+0.01％ F68、60mM Tris+200mM NaCl+0.01％ F68、50mM Tris+100mM NaCl+0.01％ F68、50mM Tris+200mM NaCl+0.01％ F68、50mM Tris+300mM NaCl+0.01％F68、50mM Tris+200mM NaCl+0.001％ F68、50mM Tris+200mM NaCl+0.005％ F68、50mM Tris+200mM NaCl+0.01％ F68、50mM Tris+200mM NaCl+0.02％ F68、50mM Tris+200mM NaCl+0.05％ F68、50mM Tris+200mM NaCl+0.1％ F68, preferably one of 50mM Tris+200mM NaCl+0.001％ F68、50mM Tris+200mM NaCl+0.005％ F68、50mM Tris+200mM NaCl+0.01％ F68、50mM Tris+200mM NaCl+0.02％ F68、50mM Tris+200mM NaCl+0.05％ F68、50mM Tris+200mM NaCl+0.1％ F68, more preferably 50mM Tris+200mM NaCl+0.01% F68. In certain embodiments, the pH of the washing solution of TFF ultrafiltration in step (4) is one of 6.5, 7.5, 8.5, 9.5, preferably one of 6.5, 7.5, 8.5.

In certain embodiments, the equilibration solution of TFF ultrafiltration in step (6) comprises KCl, KH ₂PO₄、Na₂HPO₄ and NaCl, wherein the concentration of KCl is 1-3mM, preferably 2.67mM, the concentration of KH ₂PO₄ is 1-2mM, preferably 1.47mM, the concentration of Na ₂HPO₄ is 7-9mM, preferably 8.06mM, the concentration of NaCl is 100mM-200mM, preferably 120-160mM. In certain embodiments, the equilibrium solution for TFF ultrafiltration in step (6) is one of 2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+100mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+117.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+127.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+137.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+147.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+157.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+167.93mM NaCl、2.67mM KCl+1.47mM KH₂PO₄+8.06mM Na₂HPO₄+200mM NaCl, preferably 2.67mM KCl+1.47mM KH ₂PO₄+8.06mM Na₂HPO₄ +137.93mM NaCl.

In certain embodiments, the equilibrium solution for TFF ultrafiltration in step (6) further comprises a concentration of F68, in certain embodiments the final concentration of F68 is from 0.0001% to 1%, preferably from 0.001% to 0.1%, in certain embodiments the final concentration of F68 is one of 0.0001%, 0.001%, 0.005%, 0.008%, 0.01%, 0.1%, 1%, preferably 0.005% or 0.008%. In certain embodiments, the balance of TFF ultrafiltration in step (6) further comprises a concentration of sucrose in the final concentration of 0.1% -3%, preferably 1-2%, in certain embodiments, the final concentration of sucrose is one of 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, preferably 1.5% or 2%.

In certain embodiments, the inlet pressure of the TFF ultrafiltration in step (4) is from 3 to 15psi, preferably from 6 to 12psi, more preferably from 10 to 12psi; the flow rate of TFF ultrafiltration in step (4) is 53ml/min-159ml/min, preferably 80-120ml/min, more preferably 90-110ml/min; the concentration factor of TFF in step (4) is 3-7, preferably 3-5; the washing filtration multiple of TFF in the step (4) is 5-9 times, preferably 6-8 times. In certain embodiments, the TFF ultrafiltration in step (4) is performed at a flow rate of one of 53ml/min, 79ml/min, 106ml/min, 132ml/min, 159ml/min, preferably 106ml/min. In certain embodiments, the inlet pressure of the TFF ultrafiltration in step (4) is one of 3psi, 5psi, 7psi, 10psi, 12psi, 15psi, preferably 10psi. In certain embodiments, the concentration of TFF ultrafiltration in step (4) is one of 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, preferably one of 3-fold, 4-fold, 5-fold, more preferably 4-fold. In certain embodiments, the TFF ultrafiltration in step (4) is performed at one of a 5-fold, 6-fold, 7-fold, 8-fold, and 9-fold, preferably at one of a 6-fold, 7-fold, and 8-fold, and more preferably at 8-fold.

In certain embodiments, the inlet pressure of the TFF ultrafiltration in step (6) is from 3 to 15psi, preferably from 6 to 12psi, more preferably from 10 to 12psi, and in certain embodiments, the inlet pressure of the TFF ultrafiltration in step (6) is one of 3psi, 5psi, 7psi, 10psi, 12psi, 15psi, preferably 10psi. In certain embodiments, the TFF ultrafiltration in step (6) is performed at a flow rate of 53ml/min to 159ml/min, preferably 80 to 120ml/min, more preferably 90 to 110ml/min. In certain embodiments, the TFF ultrafiltration in step (6) is performed at a flow rate of one of 53ml/min, 79ml/min, 106ml/min, 132ml/min, 159ml/min, preferably 106ml/min.

Anion exchange chromatography

Anion exchange chromatography is mainly used for further fine purification of AAV by anion exchange principle, and the step is mainly used for removing residual host cell proteins, residual baculovirus proteins and endotoxin; in addition, due to the difference between the isoelectric points of the empty shell and the solid isoelectric point, the empty shell can be removed through the step, and the solid heart rate of the final product is improved. After the anion exchange chromatography fine purification step, the residual host cell protein and the residual baculovirus protein exceed the quality standard, and other detection items basically accord with the stock solution quality standard.

In certain embodiments, the anion exchange chromatography species include, but are not limited to Poros HQ、Poros PI、HiTrap Q、UnoQ、Mono Q HR、DEAE Macroprep、Q-sepharose、CIM-QA monolithic disk、Source 15Q、Q HP, and the like.

In certain preferred embodiments, the anion exchange chromatography is Q HP chromatography.

In certain embodiments, the anion exchange chromatography comprises steps of equilibration, loading, elution, and the like, and optionally, comprises a wash step.

In certain embodiments, the equilibration solution A of the anion exchange chromatography is a Tris buffer containing NaCl, wherein the concentration of the Tris buffer is between 10mM and 60mM, preferably between 30 and 60mM, more preferably between 40 and 50mM, the concentration of NaCl is between 10mM and 60mM, preferably between 30 and 60mM, more preferably between 40mM and 50mM, the pH of equilibration solution A is between 7 and 9, preferably between 8 and 9; in certain embodiments, the equilibration buffer a species of anion exchange chromatography is 30mM Tris+30mM NaCl、30mM Tris+40mM NaCl、30mM Tris+50mM NaCl、30mM Tris+60mM NaCl、40mM Tris+30mM NaCl、40mM Tris+40mM NaCl、40mM Tris+50mM NaCl、40mM Tris+60mM NaCl、50mM Tris+30mM NaCl、50mM Tris+40mM NaCl、50mM Tris+50mM NaCl、50mM Tris+60mM NaCl、60mM Tris+30mM NaCl、60mM Tris+40mM NaCl、60mM Tris+50mM NaCl or 60mM Tris+60mM NaCl, preferably 40mM Tris+40mM NaCl, 40mM Tris+50mM NaCl, 50mM Tris+40mM NaCl or 50mM Tris+50mM NaCl. In certain embodiments, the pH of the equilibrium liquid a of the anion exchange chromatography is one of 7.0, 7.5, 8.0, 8.5, 9.0, preferably one of 8.0, 8.5, 9.0.

In certain embodiments, the eluate of the anion exchange chromatography is 10% -100% (v/v) solution B+90% -0% (v/v) equilibrium solution A, preferably 80% -100% (v/v) solution B+20% -0% (v/v) equilibrium solution A, wherein solution B is Tris buffer containing NaCl, wherein the concentration of Tris buffer is 10mM-60mM, preferably 30-60mM, more preferably 40-50mM, the concentration of NaCl is 100mM-500mM, preferably 100-300mM, more preferably 200-300mM, and the pH of the eluate is 7-9, preferably 8-9; in certain embodiments, the eluate of anion exchange chromatography is 10% (v/v) solution B+90% (v/v) solution A, 20% (v/v) solution B+80% (v/v) solution A, 30% (v/v) solution B+70% (v/v) solution A, 40% (v/v) solution B+60% (v/v) solution A, 50% (v/v) solution B+50% (v) solution A, 60% (v/v) solution B+40% (v/v) solution A, 70% (v/v) solution B+30% (v/v) solution A, 80% (v/v) solution B+20% (v/v) solution A, 90% (v/v) solution B+10% (v/v) solution A or 100% (v/v) solution B, preferably 80% (v/v) solution B+20% (v/v) solution A, more preferably 80% (v/v) solution B+20% (v/v) solution A+20% (v/v) solution B. In certain embodiments, the solution B species is one of 30mM Tris+100mM NaCl、30mM Tris+200mM NaCl、30mM Tris+300mM NaCl、40mM Tris+100mM NaCl、40mM Tris+200mM NaCl、40mM Tris+300mM NaCl、50mM Tris+100mM NaCl、50mM Tris+150mM NaCl、50mM Tris+200mM NaCl、50mM Tris+250mM NaCl、50mM Tris+300mM NaCl、60mM Tris+100mM NaCl、60mM Tris+200mM NaCl、60mM Tris+300mM NaCl, preferably 50mM Tris+200mM NaCl. In certain embodiments, the pH of the eluate of the anion exchange chromatography is one of 7.0, 7.5, 8.0, 8.5, 9.0, preferably one of 8.0, 8.5, 9.0, more preferably 8.5.

In certain embodiments, the wash solution in the optional wash step of the anion exchange chromatography comprises Tris buffer of NaCl, wherein the concentration of Tris buffer is 10mM-60mM, preferably 30-60mM, more preferably 40-50mM, the concentration of NaCl is 100mM-500mM, preferably 100-200mM, and the pH is 7-9, preferably 8-9. In certain embodiments, the wash liquor in the optional wash step in the anion exchange chromatography is one of 30mM Tris+100mM NaCl、30mM Tris+200mM NaCl、40mM Tris+100mM NaCl、40mM Tris+200mM NaCl、50mM Tris+100mM NaCl、50mM Tris+150mM NaCl、50mM Tris+200mM NaCl、50mM Tris+250mM NaCl、50mM Tris+300mM NaCl、60mM Tris+100mM NaCl、60mM Tris+200mM NaCl, preferably 40mM Tris+100mM NaCl, 40mM Tris+200mM NaCl, 50mM Tris+100mM NaCl, 50mM Tris+150mM NaCl, or 50mM Tris+200mM NaCl, more preferably 50mM Tris+100mM NaCl. In certain embodiments, the pH of the wash liquor is one of 7.0, 7.5, 8.0, 8.5, 9.0, preferably one of 8.0, 8.5, 9.0, more preferably 8.5.

In certain embodiments, the anion exchange chromatography is performed at a flow rate of 50cm/h to 400cm/h, preferably 50cm/h to 300cm/h, more preferably 100 to 200cm/h. In certain embodiments, the anion exchange chromatography is performed at a flow rate of one of 50cm/h, 100cm/h, 150cm/h, 200cm/h, 250cm/h, 300cm/h, 350cm/h, 400cm/h, preferably one of 50cm/h, 100cm/h, 150cm/h, 200cm/h, more preferably 100cm/h, 150cm/h, or 200cm/h.

Pretreatment of harvest before purification

In certain embodiments of the application, the virus harvest may also be subjected to a pre-purification treatment prior to purification. The pretreatment comprises the steps of high-pressure homogenization, deep filtration, virus inactivation and the like.

High pressure homogenization

High pressure homogenization breaks cells by mechanical shear force, and releases AAV in cells sufficiently, which is a common means for breaking cells in industry and is well known to those skilled in the art.

Deep filtration

The feed liquid which is homogenized under high pressure contains a large amount of cell fragments, the cell fragments are removed mainly through a commercial depth filter by depth filtration, so that the feed liquid becomes clear, preparation is made for subsequent column chromatography, and meanwhile, a large amount of impurities such as residual host cell DNA, residual baculovirus DNA and the like can be removed by depth filtration.

Virus inactivation

Virus inactivation: the virus inactivation is mainly S/D inactivation, and the S/D inactivation is mainly used for inactivating baculovirus in a sample and other possible lipid-enveloped viruses, so that the safety of the product is ensured.

In certain embodiments, the method further comprises subjecting the sample to viral inactivation prior to performing the step (2) affinity chromatography.

In certain embodiments, the viral inactivation is S/D inactivation. In certain embodiments, the inactivating agent in the S/D inactivation is a combination of polysorbate 80 and tributyl phosphate or a combination of Triton-100 and tributyl phosphate. In certain embodiments, the S/D inactivation is at a temperature of from 2 to 39deg.C, preferably from 2 to 8deg.C, from 22 to 25deg.C, and one of from 35 to 39deg.C, more preferably from 22 to 25deg.C. In certain embodiments, the S/D inactivation is for a period of time ranging from 1h to 6h, optionally from 3h to 4h, and in certain embodiments, the S/D inactivation is for one of 1h, 2h, 3h, 4h, 5h, 6h, preferably 3h or 4h.

Nuclease treatment

The nuclease treatment mainly degrades DNA and RNA in the sample into DNA fragments through nuclease, so that residual host cell DNA and residual baculovirus DNA are effectively removed, the residual DNA level in the sample can be reduced to a qualified level after the treatment of the step, and nuclease residues are newly introduced in the step. Common nucleases include ribonucleases, deoxyribonucleases or omnipotentiases, including, for example, but not limited to, benzonase.

In certain embodiments, the eluate obtained in step (2) is subjected to nuclease treatment, followed by apatite chromatography in step (3).

In certain embodiments, the components used for nuclease digestion are nuclease and magnesium ions. In certain embodiments, the nuclease is at a concentration of 15U/ml to 150U/ml, preferably 20 to 90U/ml, and in certain embodiments, the nuclease is at a concentration of 15U/ml, 20U/ml, 30U/ml, 60U/ml, 90U/ml, 120U/ml, or 150U/ml, preferably 30U/ml. In certain embodiments, the magnesium ion is at a concentration of 1mM-12mM, preferably 1mM-10mM, and in certain embodiments, the magnesium ion is at a concentration of 1mM, 2mM, 4mM, 6mM, 8mM, 10mM, or 12mM, preferably 2mM. In certain embodiments, the temperature of digestion of the nuclease is 2-39 ℃, preferably 2-8 ℃, 22-25 ℃, or 35-39 ℃, more preferably 2-8 ℃. In certain embodiments, the nuclease has a digestion time of 0.5h to 18h, preferably 12h to 18h, and in certain embodiments, the nuclease has a digestion time of one of 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, or 18h, preferably 12h, 13h, 14h, 15h, or 16h, more preferably 12h.

Compared with the existing purification method, the purification method has the following advantages:

The invention provides a large-scale AAV product purification method, which is suitable for large-scale production and purification of AAV products in industry. In addition, the purification method of the invention makes the purity, activity, residual host cell protein, residual host cell DNA and other quality indexes of AAV products purified by the method more excellent by carrying out specific selection on each purification step and the sequence thereof, and meets clinical grade standards, thereby meeting clinical requirements and ensuring excellent recovery rate. In addition, the purification method provided by the invention is particularly suitable for rAAVX products, and fills the technical gap that the rAAVX products can not be effectively purified in the prior art. The purification method provided by the invention can be widely applied to rAAV products obtained by packaging various packaging systems, including rAAV products prepared by packaging with a baculovirus packaging system, wherein the purification difficulty is high.

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. To facilitate an understanding of the disclosure, a number of terms and phrases are defined below.

As used herein, "adeno-associated virus (AAV)" refers to a small replication-defective, non-enveloped virus that is capable of infecting humans and some other species. AAV is known to not cause disease and to elicit only a very mild immune response. AAV can infect both dividing and resting cells, and can remain episomal without integration into the host cell's genome. These characteristics make AAV a powerful viral vector for gene therapy.

As used herein, "recombinant" as used in reference to a recombinant nucleic acid molecule refers to a sequence that has a non-naturally occurring sequence, or that has been prepared by artificial combination of two or more sequence fragments. Such artificial combination may be achieved by chemical synthesis or by artificial manipulation of isolated nucleic acid molecule fragments (e.g., by genetic engineering techniques). The term "recombinant" also includes nucleic acids, proteins and viruses that are altered by the addition, substitution or deletion of only a portion of the native nucleic acid molecule, protein or virus.

The term "recombinant virus" as used herein encompasses viruses that are not naturally occurring sequences or sequences made by artificial combination of at least two sequences of different origin. As used herein, "recombinant adeno-associated virus (rAAV)" or "recombinant adeno-associated virus (rAAV) vector" refers to an AAV particle having a recombinant nucleic acid molecule packaged therein. It includes at least one AAV capsid protein and a packaged recombinant nucleic acid molecule comprising a heterologous polynucleotide sequence (i.e., a polynucleotide sequence other than a wild-type AAV genomic sequence, such as a transgene sequence to be delivered to a mammal). The rAAV may have any serotype of AAV capsid protein or variant thereof, such as any of AAV clades a-F capsid proteins or hybrid/chimeric versions thereof, including but not limited to any of the capsid proteins of AAV1、AAV2、AAV3、AAV3A、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV2.5、AAV-DJ、AAV-DJ/8、AAVhu.32、AAVhu.68、AAVrh8、AAVrh10、AAVrh74、AAV-PHP.B、AAV-PHP.eB、AAV-PHP.S or AAVX or variants thereof. Such AAV capsid proteins or variants thereof, and related methods of preparation, are known in the art. As referred to herein, rAAVX or rAAVX vector refers to a rAAV or rAAV vector (e.g., rAAV 2/X) whose capsid protein comprises or is derived from a AAVX capsid protein (e.g., whose amino acid sequence is shown in SEQ ID NO: 1).

As used herein, "vector" refers to any molecule or entity capable of transporting, transducing or otherwise acting as a carrier for a heterologous molecule.

As used herein, "viral vector" refers to a virus or viral particle capable of transferring a nucleic acid molecule into a cell. Viral plasmid vectors refer to the nucleic acid itself that is capable of transfer (e.g., transfer plasmid, packaging plasmid, etc.). Viral vectors and viral plasmid vectors contain structural and/or functional genetic elements derived primarily from viruses.

The terms "a" or "an" as used herein mean one or more. Thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

The terms "comprising," "having," "including," and "containing" as used herein are to be construed as inclusive and not exclusive. The terms "consisting of … …" or "consisting essentially of … …" should be construed to be exclusive rather than inclusive.

The term "about" as used herein refers to an approximate range of 10% (±10%) from a given reference value, unless otherwise specified.

The term "and/or" as used herein is meant to encompass any and all possible combinations of one or more of the associated listed items, as well as be interpreted as having no combination in the alternative ("or").

The term "or" as used herein refers to any one member of a listed item and also includes any combination of members of said item.

The term "isolating" or "purifying" as used herein refers to preparing rAAV particles free of at least some impurities or contaminants.

The term "impurity" or "contaminant" as used herein refers to (without limitation) undesired impurities or contaminants present in the rAAV sample, including: media formulations known in the art for supporting the production of rAAV vectors such as salts, bovine serum, amino acid supplements, vitamin supplements, growth factors, serum albumin, and the like; a host cell, host cell protein or host cell DNA for expressing and packaging the viral particle; helper virus, helper virus protein or helper virus DNA (e.g., baculovirus protein or DNA); empty capsids or empty particles of non-authentic rAAV particles; and other non-rAAV vector materials (e.g., dextran) introduced during purification or chromatographic buffers used in purifying rAAV vectors.

A true rAAV vector refers to a rAAV particle comprising a heterologous nucleic acid (e.g., a transgene) capable of infecting a target cell.

Empty capsid or empty particle refers to an AAV particle or viral particle comprising an AAV capsid but lacking all or part of a genome comprising a heterologous nucleic acid sequence flanking one or both sides of an AAV ITR. Such empty capsids do not function to transfer the heterologous nucleic acid sequence into a host cell or cell within an organism.

Methods of producing rAAV vectors

Many methods for producing rAAV vectors are known in the art, including transfection, stable cell line production, and infectious hybrid virus production systems. Production culture systems for the production of rAAV viral particles all require: 1) Suitable host cells include, for example, a cell line of human origin such as HeLa, A549 or 293 cells, or, in the case of baculovirus production systems, an insect-derived cell line such as SF-9; 2) Suitable helper functions are provided by wild-type or mutant adenoviruses (e.g., temperature sensitive adenoviruses), herpes viruses, baculoviruses, or plasmid constructs providing helper functions; 3) AAV rep and cap genes and gene products; 4) Transgenes flanked by AAV ITR sequences (e.g., therapeutic transgenes); and 5) suitable media and media components to support rAAV production. Suitable media are known in the art and may be used to produce rAAV vectors. These media include, but are not limited to, those produced by Hyclone laboratories and JRH, including modified Eagle media (Modified Eagle Medium, MEM), dulbecco's modified Eagle media (Dulbecco's Modified Eagle Medium, DMEM), custom formulations such as those described in U.S. Pat. No. 6,566,118, and Sf-900II SFM media such as described in U.S. Pat. No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for the production of recombinant AAV vectors.

Various methods of producing rAAV are known in the art, including, but not limited to, expression plasmid expression/packaging systems (e.g., three plasmid packaging systems, two plasmid packaging systems, etc.), viral expression/packaging systems (e.g., baculovirus expression/packaging systems), and AAV expression/packaging systems with Ad or HSV helper virus, and the like. An expression plasmid system generally refers to a plasmid comprising an expression cassette comprising a nucleic acid sequence encoding a molecule of interest, structural and non-structural proteins of AAV, and other components that aid in rAAV production. In some embodiments, the packaging system includes a helper virus. rAAV is a replication-defective virus, i.e. it lacks replication capacity itself. Helper viruses allow replication of the rAAV by providing components that assist in replication of the rAAV. Examples of helper viruses include, but are not limited to, adenovirus (Ad) and Herpes Simplex Virus (HSV). For example, a plasmid comprising cap and rep genes carrying a nucleic acid sequence encoding a molecule of interest, AAV, can be transfected into cells that already contain Ad. The cells are then maintained for rAAV production prior to the acquisition of the rAAV. In some embodiments, the expression plasmid system can include a two plasmid AAV packaging system. For example, the expression cassette may be cloned into one plasmid and the cap, rep and helper genes cloned into a second plasmid. Helper virus genes refer to DNA sequences of helper viruses that are necessary for AAV production. This plasmid was transfected into cells suitable for rAAV production. In further embodiments, the expression plasmid system can comprise a three-plasmid AAV packaging system. For example, the expression cassette may be cloned into a first plasmid, the rep and cap genes of AAV may be cloned into a second plasmid, and the helper virus genes cloned into a third plasmid. Three plasmids were transfected into a cell expression system for the production of rAAV. In other embodiments, a baculovirus production system is also included.

Baculovirus production systems have many advantages over other production systems, such as being scalable, safe, significantly increasing AAV yields (Urabe M et al, 2002,Human Gene Therapy,13:1935-1943;US6723551;Smith RH et al, 2009, mol. Ther,17:1888-1896; chen H et al, 2008,Mol Ther,16:924-930; NC 101522903A et al). The main process of AAV production by the exemplary baculovirus packaging system is: co-infecting insect cells with three recombinant baculoviruses: a recombinant baculovirus for producing AAV Rep protein, a recombinant baculovirus for producing AAV Cap structural protein, a third recombinant baculovirus carrying exogenous gene and AAV ITR, wherein after the three recombinant baculovirus are infected with sf9 insect cells together, the recombinant AAV vector can be obtained after 3 days, and the yield of 5 multiplied by 10 ⁴ recombinant AAV vectors can be produced by each sf9 insect cell. As research progresses, it also appears that AAV viruses are produced by co-infecting insect cells with only two recombinant baculoviruses: a recombinant baculovirus for producing AAV REPCAP protein, a baculovirus carrying exogenous gene and AAV-ITR, the two recombinant baculovirus being used for co-infection of sf9 cells to package recombinant AAV vector; and expressing Rep and Cap by artificially synthesized promoters which can act in insect cells and are contained in introns, wherein the Rep and Cap can be positioned in the same Baculovirus Expression Vector (BEV) or respectively positioned in different BEVs to produce rAAV, thus not only maintaining the stability of baculovirus, but also achieving good effect in rAAV production.

Suitable rAAV production media of the invention can be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% -20% (v/v or w/v). Alternatively, as known in the art, rAAV vectors may be produced under serum-free conditions (possibly also referred to as culture medium free of animal-derived products). It will be appreciated by those skilled in the art that commercial or custom media designed to support production of rAAV vectors may also be supplemented with one or more cell culture components known in the art (including but not limited to glucose, vitamins, amino acids, and or growth factors) to increase rAAV titres in production culture.

RAAV production cultures can be grown under a variety of conditions (over a wide temperature range, different lengths of time, etc.) appropriate for the particular host cell used. As known in the art, rAAV production cultures include adhesion-dependent cultures (which can be cultured in suitable adhesion-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed bed or fluidized bed bioreactors). rAAV vector production cultures may also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways including, for example, rotating bed flasks, stirred tank bioreactors, and disposable systems such as wave-balloon systems.

RAAV serotypes

Adeno-associated viruses (AAV) can be divided into serotypes, e.g., AAV1-AAV12 (high gloss equivalent, 2004,J Virolm 78:6381-6388; mori S et al, 2004,Virology 330:375-383; schmidt M et al, 2008,J Virol 82:1399-1406), primarily human and primate hosts. The capsid proteins of AAV viruses of different serotypes are diverse in sequence and spatial conformation, such that there is a significant difference in cell surface binding receptors and infectivity of cells (Timpe J et al 2005,Curr Gene Ther 5:273-284). For example, AAV2 has a broad spectrum of infections, particularly with respect to neuronal cells, in terms of infectivity; AAV1 and AAV7 are more efficient in transduction in skeletal muscle; AAV3 readily transduces megakaryocytes; the advantages of AAV5 and AAV6 infection of airway epithelial cells are remarkable; whereas AAV8 transduces hepatocytes more efficiently than other serotypes.

In addition, there is increasing research on modification of adeno-associated virus (AAV) capsid proteins, and the purpose of modification is to enhance targeting of viral vectors on the one hand and reduce immunogenicity of viral vectors on the other hand. The engineering methods include methods utilizing DNA shuffling (DNA shuffling) or error-prone PCR to obtain capsid proteins of the novel AAV viral vectors that are functionally optimized. AAVX is a novel AAV vector obtained using DNA shuffling technology (Xiao et al, US9186419B 2), and studies have shown that AAVX has a good transduction efficiency for liver targeting (see, e.g., WO2022/100748 A1).

The specific embodiment is as follows:

the examples below are given for the purpose of illustration only and are not intended to limit the scope of the application.

The recombinant adeno-associated viral vector used in the following examples is rAAVX, and the shRNA coding sequence is an exemplary foreign gene. The construction information of the recombinant adeno-associated virus vector is described in WO2022/100748A 1. The packaging and preparation methods are described with reference to CN101522903a and Xiao et al.,Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J.Virol.72(3):2224(1998). Except for the special source, the other reagent raw materials are common raw materials sold in the market, and the preparation of the reagent adopts a conventional method. Methods not described in detail in the examples are all routine in the art.

EXAMPLE 1 purification of rAAVX vector samples

The three plasmid transfection method rAAVX virus vector was used (see Xiao et al.,Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J.Virol.72(3):2224(1998)), for a sample containing rAAVX virus vector. The above samples were purified by the purification methods of the present application and the prior art, respectively, to compare the purification effects.

Experimental example 1: the purification process of the application is adopted to purify a sample containing rAAVX carriers

(1) Pretreatment of harvest liquid (high pressure homogenate and inactivation)

After harvesting the stock solution of the sample containing the viral vector, homogenizing under high pressure of 300bar to 400bar, and detecting the turbidity of the sample after homogenizing to about 715NTU. Turbidity was reduced to <5NTU. Then adding the inactivating agent polysorbate 80 and tributyl phosphate into the sample for inactivation, wherein the inactivation temperature is 22-25 ℃ and the inactivation time is 3h. After inactivation, ultrapure water was added to the sample for dilution, and the electrical conductivity was adjusted to 7.97ms/cm, and the pH was 6.88. Then, the mixture was filtered through a 0.22 μm PES filter.

(2) Affinity chromatography

The filtered sample obtained in the above step was subjected to heparin affinity chromatography (product No. 17012-065100, soviet micro technologies Co., ltd.). The flow rate during the affinity chromatography was 90cm/h. Heparin affinity chromatography column was equilibrated with 2-3 column volumes of affinity chromatography equilibration solution A (30mM PB+40mM NaCl,pH6.8) prior to loading. After loading, the column is washed with 2-3 column volumes of equilibration solution, and eluted with an eluent, wherein the eluent is 100% (v/v) solution B, and wherein the solution B is 30mM PB+400mM NaCl (pH 6.6).

(3) Enzymatic digestion

After heparin affinity chromatography, nuclease (GMP-SSNP, inc. of GmbH, yinqiao, technology Co., ltd.) and magnesium chloride were added to the sample, and digested at 2-8deg.C for 16h, wherein the concentration of nuclease was 30U/ml and the concentration of magnesium ion was 2mM.

(4) CHT chromatography

The enzyme digested sample was diluted with 40mM PB (pH 6.6), its conductivity was adjusted to 7.64ms/cm, the pH was 6.54, and the sample was filtered through a 0.22 μm filter and subjected to CHT chromatography (Bio-Rad, cat. No. 157-0040). The flow rate during CHT chromatography was 150cm/h. The column was equilibrated beforehand with 2-3 column volumes of CHT equilibration solution a, equilibration solution a 40mM PB+30mM NaCl (ph 6.6), and then loaded. Washing with 2-3 column volumes of balance liquid after loading, and eluting after washing. The eluent was 40% (v/v) solution B+60% (v/v) equilibrium solution A, wherein solution B was 40mM PB+500mM NaCl (pH 6.6). The eluate containing the peak of interest was collected.

(5) G25 chromatography

The sample obtained in the above step was subjected to G25 chromatography (product number SEC0035, hemsl and Biotech Co., ltd.) at a flow rate of 120cm/h and a equilibration solution of 40mM Tris+50mM NaCl (pH 8.5). The sample was equilibrated with 2-3 column volumes of equilibration solution prior to loading. After loading, the column was washed with equilibration solution and eluted, and the peak eluate of interest was collected.

(6) Anion exchange chromatography (Q HP chromatography)

The sample obtained in the above step was subjected to Q HP chromatography (Boglaung (Shanghai) Biotechnology Co., ltd., product No. AI 0072) at a flow rate of 100cm/h. The equilibrium solution for Q HP chromatography was 50mM Tris+50mM NaCl (pH 8.5). The chromatographic column is washed with 2-3 column volumes of equilibration solution in advance, then the sample is loaded, after loading is finished, the chromatographic column is washed with 2-3 column volumes of equilibration solution, then elution is carried out, the eluent is 80% (v/v) of solution B+20% (v/v) of equilibration solution A, wherein the solution B is 50mM Tris+200mM NaCl (pH 8.5), and the peak eluent of the order is collected.

(7) S200 chromatography

The sample obtained in the above step was subjected to S200 chromatography (product number SEC0092, hemsl and Biotech Co., ltd.) at a flow rate of 30cm/h. The equilibration solution for S200 chromatography was a solution containing 2.67mM KCl,1.47mM KH ₂PO₄,8.06mM Na₂HPO₄, 137.93mM NaCl and 0.001% F68. And S200, balancing the chromatographic column by using 2-3 column volumes of balancing liquid in advance, loading samples, continuously flushing the chromatographic column by using the balancing liquid after loading samples, collecting liquid containing a target peak when the target peak appears, and sterilizing and filtering the finally collected liquid by using a 0.22 mu m filter membrane to obtain a final sample.

Experimental example 2: purifying sample containing rAAVX carrier by the second purifying process

(1) Pretreatment of harvest liquid (high pressure homogenate and inactivation)

After harvesting the sample, high pressure homogenization was performed at a pressure of 300bar and the turbidity of the homogenized sample was measured to be approximately 622NTU. Turbidity was reduced to <5NTU. Then adding the inactivating agent polysorbate 80 and tributyl phosphate into the sample for inactivation, wherein the inactivation temperature is 22-25 ℃ and the inactivation time is 3h. After inactivation, ultrapure water was added to the sample for dilution, and the electrical conductivity was adjusted to 8.03ms/cm, and the pH was 6.84. Then, the mixture was filtered through a 0.22 μm PES filter.

(2) Affinity chromatography

The filtered sample obtained in the above step was subjected to heparin affinity chromatography (product No. 17012-065100, soviet micro technologies Co., ltd.). The flow rate during the affinity chromatography was 150cm/h. The heparin affinity chromatography column was equilibrated with 2 column volumes of affinity chromatography equilibration solution A (40mM PB+30mM NaCl,pH6.8) prior to loading. After the sample loading was completed, the column was washed with 2 column volumes of equilibration solution, washed with 30% (v/v) of solution B+70% (v/v) of equilibration solution A, washed with 50% (v/v) of solution B+50% (v/v) of equilibration solution A, and eluted, wherein solution B was 40mM PB+500mM NaCl (pH 6.6). The eluate containing the peak of interest was collected.

(3) Enzymatic digestion

Nuclease (GMP-SSNP (R) and magnesium chloride (America Co., ltd.) are added to the sample after heparin affinity chromatography, and digested at 2-8deg.C for 12-16h, wherein the enzyme concentration is 30U/ml, and the magnesium ion concentration is 2mM.

(4) CHT chromatography

The enzyme digested sample was diluted with 40mM PB pH6.6, adjusted to a conductivity of 7.7ms/cm, pH 6.55, and filtered through a 0.22 μm filter before performing CHT chromatography (Bio-Rad, cat. No. 157-0040). The flow rate during CHT chromatography was 200cm/h, the column was equilibrated with 2 column volumes of CHT equilibration solution A, 30mM PB+40mM NaCl (pH 6.6) and then loaded. After the completion of the loading, the column was washed with 2 column volumes of equilibration solution, and eluted after washing, with 30% (v/v) of solution B+70% (v/v) of equilibration solution A, wherein solution B was 30mM PB+400mM NaCl (pH 6.6). The eluate containing the peak of interest was collected.

(5) TFF ultrafiltration (hollow fiber ultrafiltration)

The sample obtained in the previous step was added with F68 at a final concentration of 0.01% followed by hollow fiber ultrafiltration (Repligen, cat. D02-E100-05-N) at a flow rate of 106ml/min and an inlet pressure of 10psi. The sample will be concentrated during ultrafiltration and washed with 50mM Tris+200mM NaCl+0.01% F68 (pH 8.5) at a concentration factor of 4 and 8 times. The hollow fibers are then rinsed with a wash filtrate and the filtrates are combined.

(6) Anion exchange chromatography (Q HP chromatography)

First, the filtrate obtained in the above step was diluted 4-fold with 50mM Tris pH8.5 to perform Q HP chromatography (Boglong (Shanghai) Biotechnology Co., ltd., product No. AI 0072). The flow rate of the QHP chromatography was 200cm/h. The Q HP chromatographic balance liquid is 40mM Tris+40mM NaCl (pH 8.5), the chromatographic column is washed by the Q HP chromatographic balance liquid with 3 column volumes in advance, then the sample is loaded, and after the loading is finished, the chromatographic column is washed by the balance liquid with 3 column volumes continuously. Then, the mixture was washed with 50mM Tris+100mM NaCl,pH of a washing buffer solution of 8.5. Subsequent to elution, the eluent was 50mM Tris+200mM NaCl,pH 8.5 and the eluent containing the elution peak was collected.

(7) TFF ultrafiltration (hollow fiber ultrafiltration)

The sample obtained in the above step was added with F68 at a final concentration of 0.005% and subjected to hollow fiber ultrafiltration (Repligen, cat# D02-E100-05-N), at a flow rate of 106ml/min, and an inlet pressure of 10psi. Both the equilibrium liquid and the washing liquid are 2.67mM KCl+1.47mM KH ₂PO4+8.06mM Na₂HPO₄ +137.93mM NaCl+0.005% of F68+1.5% of sucrose, and the washing and filtering times are 7 times. After the washing and filtering are completed, the hollow fiber is washed by the washing and filtering solution, and the filtrates are combined. The filtrate is sterilized and filtered by a 0.22 mu m filter membrane to obtain the stock solution.

Comparative example: purifying sample containing rAAVX carrier by using prior art purification process

Taking the purification method in CN102803478A as an example, the purification process in the prior art is adopted to purify the sample, and the method specifically comprises the following steps:

(1) The sample containing rAAVX carriers is filtered and purified by a series of filters connected in series; the effluent is digested by Benzonase;

(2) Removing production contaminants from the digested sample by an anion exchange MQ filter;

(3) Concentrating the digested sample by TFF ultrafiltration;

(4) Purifying the concentrated sample of step (3) by apatite chromatography: the equilibration solution was 20mM borate (pH 9.0) +5% (w/v) PEG6000; flow rate: 96cm/hr. And 4 times of continuous washing are carried out, wherein the washing solutions are respectively as follows: 50:50 (volume: volume) 20mM borate (pH 9.0) +5% (w/v) PEG6000:40mM borate (pH 9.0) +10% (w/v) PEG6000, 150mM potassium phosphate+20 mM borate (pH 9) +5% (w/v) PEG6000, 20mM borate (pH 9) +5% (w/v) PEG6000, and 20mM HERES (pH 7.0) +150mM NaCl buffer; the eluent is as follows: 50mM potassium phosphate+20 mM HERES (pH 7.0) +150mM NaCl buffer;

(5) Heat inactivating at 52 ℃ for 10min;

(6) Hydrophobic Interaction Chromatography (HIC): adopting HIC butyl column; the balancing liquid is as follows: 75:25 (volume: volume) 1M citrate+20 mM sodium phosphate: equilibrating the HIC butyl column with a 20mM sodium phosphate mixture; the flow rate is: 106cm/hr; washing liquid: 75:25 (volume: volume) 1M citrate+20 mM sodium phosphate: 20mM sodium phosphate buffer mixture; eluent: 0.35M citrate+20 mM sodium phosphate;

(7) Size Exclusion Chromatography (SEC), eluent 20mM NaCl+20mM Tris (pH 8.0);

(8) The virus removing filter is used for removing the virus of the sample in the last step;

(9) Anion exchange chromatography: adopting Unosphere Q anion exchange columns, and balancing liquid is as follows: 20mM NaCl+20mM Tris (pH 8.0) buffer; flow rate: 309cm/hr; washing liquid: 60mM NaCl; eluent: 130mM NaCl.

Purified rAAV sample detection

The genome titer of the original harvest before purification in example 1, and the genome titer, residual host DNA, etc. of each sample (experimental example 1, experimental example 2, and comparative example) after purification by different methods were separately examined by Q-PCR. Wherein the purified residual host cell protein is detected by ELISA, the purified purity is detected by SEC-HPLC, 2005,J Virol Methods,127 by reference such as Ulrich-Peter Rohr et al: the method described in the 40-45 document detects the titer of infection after purification, and the real heart rate after purification is detected by UPLC-fluorescence. The test results are shown in Table 1.

TABLE 1

Note 1: the amount of residual host cell DNA per 3.5e+12 (vg) genome titer in the purified sample was calculated as: 3.5E+12 (vg). Times.residual host cell DNA (ng/ml)/sample genomic titre after purification (vg/ml).

And (2) injection: the amount of residual host cell protein per 3.5E+12 (vg) genome titer in the purified sample was calculated as: 3.5E+12 (vg). Times.residual host cell protein (ng/ml)/sample genomic titre after purification (vg/ml).

And (3) injection: viral infection titer corresponding to genome titer per 3.5e+12 (vg) in the purified sample was calculated as: 3.5E+12 (vg). Times.infectious titer (IU/ml)/genome titer (vg/ml) of purified sample.

The results show that the rAAVX products purified by the method of the application have various indexes meeting the related standards and clinical requirements. In particular, in the purified sample, the total recovery rate of the rAAV virus vector is 45-50%, the purity is above 99%, the solid rate is as high as 64.8% -72.6%, and each parameter is obviously superior to that of the sample obtained by the purification method in the prior art (comparative example).

Example 2 purification of rAAVX viral vector samples prepared from baculovirus expression System

To verify the applicability of the purification method of the application to rAAVX viral vector samples obtained from other expression systems, a baculovirus expression system was used in this example to express and package rAAVX.

Experimental example 3: purification of rAAVX viral vector samples

Packaging and preparation of rAAVX viral vectors using baculovirus expression systems (see example 5 of CN101522903a for specific methods of packaging preparation), samples containing rAAVX viral vectors were obtained. The sample was purified by the method of experimental example 2.

Purified rAAV sample detection

The genome titer of the original harvest of the sample rAAVX before purification and the genome titer, residual host DNA, residual baculovirus DNA and other indicators of the purified rAAVX sample (experimental example 3) in example 2 were detected by Q-PCR, respectively, and the remaining residual host cell proteins, the purity of the purified sample, the infectious titer of the purified sample and the real heart rate were detected by the indicator detection method described in example 1. The correlation detection results are shown in Table 2.

TABLE 2

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Note 1: the residual baculovirus DNA includes residual baculovirus DNA erroneously packaged into the interior of AAV virus and free residual baculovirus DNA. "digestion" refers to digestion with nucleases (to remove free residual baculovirus DNA) prior to detection of the purified sample.

And (2) injection: non-digestion refers to the absence of nuclease digestion prior to detection of the purified sample.

And (3) injection: the amount of residual host cell DNA per 3.5e+12 (vg) genome titer in the purified sample was calculated as: 3.5E+12 (vg). Times.residual host cell DNA (ng/ml)/sample genomic titre after purification (vg/ml).

And (4) injection: the amount of residual baculovirus DNA per genomic titer of 1e+4 (vg) in the purified sample was calculated as: 1E+4 (vg). Times.residual baculovirus DNA (copies/ml)/post-purification sample genome titer (vg/ml).

And (5) injection: the amount of residual host cell protein per 3.5E+12 (vg) genome titer in the purified sample was calculated as: 3.5E+12 (vg). Times.residual host cell protein (ng/ml)/sample genomic titre after purification (vg/ml).

And (6) injection: viral infection titer corresponding to genome titer per 3.5e+12 (vg) in the purified sample was calculated as: 3.5E+12 (vg). Times.infectious titer (IU/ml)/genome titer (vg/ml) of purified sample.

The above results indicate that the use of the purification method of the present application can effectively purify rAAVX viral vectors prepared by using a baculovirus expression system, and that excellent results are obtained from the viewpoints of recovery rate, purity, residue, real heart rate, and the like.

The embodiments of the present application described above are not intended to limit the present application, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present application should be included in the scope of the present application.

Claims

1. A method of purifying a recombinant adeno-associated virus (rAAV) vector, comprising the steps of, in order:

(1) Obtaining a sample containing the rAAV vector to be purified;

(3) Purifying the eluent by apatite chromatography;

2. The method of claim 1, wherein the rAAV vector has a dna sequence comprising any one of the capsid proteins from AAV clades a-F or hybrid/chimeric versions thereof, including but not limited to any one of the capsid proteins AAV1、AAV2、AAV3、AAV3A、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV2.5、AAV-DJ、AAV-DJ/8、AAVhu.32、AAVhu.68、AAVrh8、AAVrh10、AAVrh74、AAV-PHP.B、AAV-PHP.eB、AAV-PHP.S or AAVX or variants thereof, preferably AAVX.

3. The method of claim 1 or 2, wherein the rAAV vector is prepared from a conventional AAV vector packaging system, including, but not limited to, a two plasmid packaging system, a three plasmid packaging system, a baculovirus packaging system, and an Ad or HSV helper AAV packaging system, preferably a three plasmid packaging system and a baculovirus packaging system.

4. A method according to any one of claims 1 to 3, wherein the affinity chromatography is heparin affinity chromatography.

5. The method of claim 4, wherein the affinity chromatography comprises the steps of equilibration, loading, elution, and the like, and optionally, a washing step, wherein,

The conductance of the sample is adjusted to be 5.5+/-1 ms/cm-10.5+/-1 ms/cm, preferably 6.5+/-1 ms/cm-9.5+/-1 ms/cm before loading; the pH of the sample is adjusted to 6.0-7.6, preferably 6.5-7.0;

The balance solution A used in the affinity chromatography is PB buffer containing NaCl, wherein the concentration of the PB buffer is 10mM-60mM, preferably 20-50mM, the concentration of the NaCl is 10mM-50mM, preferably 20-40mM, and the pH of the balance solution A is 6.2-7.6, preferably 6.5-6.8;

The eluent used in the affinity chromatography is 10% -100% (v/v) of solution B+90% -0% (v/v) of balance solution A, preferably 50% -100% (v/v) of solution B+50% -0% (v/v) of balance solution A, wherein the solution B is PB buffer containing NaCl, the concentration of the PB buffer is 10mM-60mM, preferably 20-50mM, the concentration of the NaCl is 200mM-600mM, preferably 300-600mM, and the pH of the eluent is 6.2-7.6, preferably 6.5-6.8;

The flow rate of the affinity chromatography is 50cm/h to 300cm/h, preferably 70cm/h to 200cm/h;

The buffer solution used in the impurity washing step in the affinity chromatography is 10% -100% (v/v) of solution B+90% -0% (v/v) of balance solution A, preferably 20% -40% (v/v) of solution B+80% -60% (v/v) of balance solution A.

6. The method of any one of claims 1-5, wherein the cation exchange chromatography packing is one of POROS 50HE, POROSHS, bio-RAD Nuvia HR S, SP HP, capto S.

7. The method of claim 6, wherein the cation exchange chromatography comprises equilibration, loading, elution, and the like, wherein the conductance of the sample is adjusted to 5.5±1ms/cm to 10.5±1ms/cm, preferably 5.5±1ms/cm to 7.5±1ms/cm, prior to loading; the pH of the sample is adjusted to a range of 4.5 to 9.5, preferably 6.0 to 7.5, more preferably 6.5;

The balance solution A of the cation exchange chromatography is PB buffer solution containing NaCl, wherein the concentration of the PB buffer solution is 10mM-60mM, preferably 20-50mM, the concentration of the NaCl is 10mM-50mM, preferably 20-40mM, and the pH of the balance solution A is 6.2-7.6, preferably 6.5-6.8; the eluent used in the cation exchange chromatography is 10% -100% (v/v) of solution B+90% -0% (v/v) of balance solution A,

Preferably 50% to 100% (v/v) of solution B+50% to 0% (v/v) of equilibration solution A, wherein solution B is a PB buffer comprising NaCl, wherein the PB buffer has a concentration of 10mM-60mM, preferably 20-50mM, the NaCl has a concentration of 200mM-600mM, preferably 300-600mM, and the pH of the eluate is 6.2-7.6, preferably 6.5-6.8;

The flow rate of the cation exchange is 50cm/h to 300cm/h, preferably 70cm/h to 200cm/h.

8. The method of any one of claims 1-7, wherein the apatite chromatography is CHT chromatography or CFT chromatography.

9. The method of claim 8, wherein the apatite chromatography comprises equilibration, loading, elution, etc., wherein the conductance of the sample is adjusted to 5±1ms/cm-12±1ms/cm, preferably 5±1ms/cm-9±1ms/cm, prior to loading;

The balance solution A of the apatite chromatography is PB buffer solution containing NaCl, wherein the concentration of the PB buffer solution is 10mM-60mM, preferably 20-50mM, the concentration of the NaCl is 10mM-50mM, preferably 20-40mM, and the pH of the balance solution A is 6.2-7.6, preferably 6.5-6.8; the eluent used in the apatite chromatography is 10% -100% (v/v) of solution B+90% -0% (v/v) of balance solution A, preferably 20% -50% (v/v) of solution B+80% -50% (v/v) of balance solution A, wherein the solution B is PB buffer containing NaCl, the concentration of the PB buffer is 10mM-60mM, preferably 20-50mM, the concentration of the NaCl is 200mM-600mM, preferably 300-600mM, and the pH of the eluent is 6.2-7.6, preferably 6.5-6.8;

the flow rate of the apatite chromatography is 50cm/h to 300cm/h, preferably 100cm/h to 250cm/h.

10. The method of any one of claims 1-9, wherein the molecular sieve employed in step (4) is G25 chromatography, comprising equilibration, loading and elution steps; the ultrafiltration is TFF ultrafiltration, which includes concentration and washing steps.

11. The method of claim 10 wherein said medium used for TFF ultrafiltration is hollow fiber having a pore size of 100kD.

12. The method according to claim 10 or 11, wherein in step (4),

The equilibrium liquid of the G25 chromatography is Tris buffer containing NaCl, wherein the concentration of the Tris buffer is 10mM-60mM, preferably 30mM-60mM, the concentration of the NaCl is 10mM-60mM, preferably 30mM-60mM, the pH range is 7.0-9.0, preferably 8.0-9.0, and the flow rate is 30cm/h-180cm/h, preferably 50cm/h-150cm/h;

The washing filtrate of the TFF ultrafiltration is Tris buffer containing NaCl and F68, wherein the concentration of the Tris buffer is 30mM-60mM, preferably 40-60mM, the concentration of the NaCl is 100mM-300mM, preferably 150mM-250mM, the concentration of the F68 is 0.001% -0.1%, preferably 0.01% -0.1%, the pH range is 6.5-9.5, preferably 6.5-9.0, and the inlet pressure of the TFF ultrafiltration is 3-15psi, preferably 8-12psi; the flow rate of the TFF ultrafiltration is 53ml/min-159ml/min, preferably 80-120ml/min; the concentration factor of TFF is 3-7 times, preferably 3-5 times; the TFF has a wash filtration factor of 5 to 9, preferably 6 to 8.

13. The method of any one of claims 1-12, wherein the anion exchange chromatography species includes, but is not limited to Poros HQ、Poros PI、HiTrap Q、UnoQ、Mono Q HR、DEAE Macroprep、Q-sepharose、CIM-QA monolithic disk、Source 15Q、Q HP,, preferably the anion exchange chromatography is Q HP chromatography.

14. The method of claim 13, wherein the anion chromatography comprises the steps of equilibration, loading, elution, and the like, and optionally, a washing step, wherein,

The balance A of the anion exchange chromatography is Tris buffer containing NaCl, wherein the concentration of the Tris buffer is 10mM-60mM, preferably 30-60mM, the concentration of the NaCl is 10mM-60mM, preferably 30-60mM, and the pH of the balance A is 7-9, preferably 8-9;

the eluent of the anion exchange chromatography is 10% -100% (v/v) of solution B+90% -0% (v/v) of balance solution A, preferably 80% -100% (v/v) of solution B+20% -0% (v/v) of balance solution A, wherein the solution B is Tris buffer solution containing NaCl, the concentration of the Tris buffer solution is 10mM-60mM, preferably 30-60mM, the concentration of the NaCl is 100mM-500mM, preferably 100-300mM, and the pH of the eluent is 7-9, preferably 8-9;

The flow rate of the anion exchange chromatography is 50cm/h to 400cm/h, preferably 50cm/h to 300cm/h;

The washing solution in the washing step of the anion exchange chromatography is Tris buffer containing NaCl, wherein the concentration of the Tris buffer is 10mM-60mM, preferably 30-60mM, the concentration of NaCl is 100mM-500mM, preferably 100-200mM, and the pH value is 7-9, preferably 8-9.

15. The method of any one of claims 1 to 14, wherein the molecular sieve employed in step (6) is S200 chromatography, comprising equilibration, loading and elution steps; the ultrafiltration adopts TFF ultrafiltration, and comprises a washing and filtering step.

16. The method of claim 15 wherein said medium used for TFF ultrafiltration is a conventional membrane pack or hollow fiber, wherein the hollow fiber has a pore size of 100kD.

17. The method according to claim 15 or 16, wherein in step (6),

The balancing solution for the S200 chromatography comprises KCl, KH ₂PO_4、Na₂HPO₄ and NaCl, wherein the concentration of KCl is 1-3mM, preferably 2.67mM, the concentration of KH ₂PO₄ is 1-2mM, preferably 1.47mM, the concentration of Na ₂HPO₄ is 7-9mM, preferably 8.06mM, and the concentration of NaCl is 100mM-200mM, preferably 120-160mM; the S200 chromatographic balance liquid also comprises F68 with a certain concentration, wherein the concentration of the F68 is 0.0001-1%, preferably 0.001-0.1%; the flow rate of the S200 chromatography is 15cm/h to 120cm/h, preferably 30cm/h to 60cm/h;

the balancing solution of the TFF ultrafiltration comprises KCl, KH ₂PO₄、Na₂HPO₄ and NaCl, wherein the concentration of KCl is 1-3mM, preferably 2.67mM, the concentration of KH ₂PO₄ is 1-2mM, preferably 1.47mM, the concentration of Na ₂HPO₄ is 7-9mM, preferably 8.06mM, and the concentration of NaCl is 100mM-200mM, preferably 120-160mM; the balance liquid also contains F68 with a certain concentration, and the concentration of the F68 is 0.0001-1%, preferably 0.001-0.1%; the balance liquid also contains a certain concentration of sucrose, wherein the concentration of the sucrose is 0.1% -3%, preferably 1-2%; the TFF ultrafiltration inlet pressure is 3-15psi, preferably 6-12psi; the flow rate of the TFF ultrafiltration is 53ml/min-159ml/min, preferably 80-120ml/min.

18. The method of any one of claims 1 to 17, further comprising the step of inactivating the virus in the sample prior to performing the affinity chromatography of step (2).

19. The method of claim 18, wherein the virus is inactivated by an S/D inactivation method, and the inactivating agent is polysorbate 80 and tributyl phosphate, or Triton-100 and tributyl phosphate; the inactivation temperature is 2-39deg.C, preferably 22-25deg.C; the S/D inactivation time is 1h-6h, preferably 3h-4h.

20. The method according to any one of claims 1 to 19, wherein the eluate obtained in step (2) is subjected to nuclease treatment followed by apatite chromatography in step (3).

21. The method of claim 20, wherein the nuclease treatment is performed by adding nuclease and magnesium ions to the eluate, the nuclease having a concentration of 15U/ml to 150U/ml, preferably 20 to 90U/ml; the concentration of the magnesium ions is 1mM-12mM, preferably 1 mM-10 mM; the temperature of the nuclease digestion is 2-39 ℃, preferably 2-8 ℃; the nuclease treatment time is 0.5h-18h, preferably 12h-18h.