CN-122029284-A - Purification of AAV particles using anion exchange chromatography with improved elution system

Abstract

The present invention relates to a method for the purification and enrichment of intact adeno-associated virus particles by anion exchange chromatography using basic amino acids in an elution buffer.

Inventors

• H. Mary
• Edelman B.
• O. Rammo

Assignees

• 默克专利股份公司

Dates

Publication Date

20260512

Application Date

20240731

Priority Date

20230801

Claims (15)

1. 1. A method of enriching for intact adeno-associated virus (AAV) particles by contacting a sample comprising intact and empty AAV particles with a chromatography matrix comprising anion exchange groups such that the intact AAV particles bind to the matrix and eluting the particles with an elution buffer comprising one or more basic amino acids.
2. 2. The method according to claim 1, wherein the method comprises the steps of: a) Contacting a sample comprising AAV particles with a chromatography matrix comprising anion exchange groups; b) Optionally washing the chromatographic matrix; c) AAV particles bound to a chromatographic matrix are eluted with an elution buffer comprising one or more basic amino acids.
3. 3. The method according to claim 1 or 2, wherein the sample is a clarified lysate pre-purified by filtration and/or centrifugation.
4. 4. A method according to one or more of claims 1 to 3, wherein the sample is a prepurified lysate prepurified by affinity chromatography.
5. 5. The method according to one or more of claims 1 to 4, wherein in step c) the conductivity of the elution buffer is higher than the conductivity of the sample in step a).
6. 6. The method according to one or more of claims 1 to 5, wherein the chromatography matrix is a resin, a membrane or a monolith.
7. 7. The method according to one or more of claims 1 to 6, wherein the chromatography matrix comprises Trimethylammonioethyl (TMAE) groups.
8. 8. The method according to one or more of claims 1 to 7, wherein the anion exchange groups are attached to the chromatography matrix by polymer chains made by grafting monomers containing the anion exchange groups to the substrate of the chromatography matrix.
9. 9. The method of one or more of claims 1 to 8, wherein a majority of empty AAV capsids elute prior to a majority of intact AAV particles.
10. 10. The method according to one or more of claims 1 to 9, wherein the pH of the elution buffer is equal to the pH of the loading buffer.
11. 11. The method according to one or more of claims 1 to 10, wherein the basic amino acid is arginine and/or lysine.
12. 12. The method according to one or more of claims 1 to 11, wherein the concentration of basic amino acids in the elution buffer is between 2 and 800 mM.
13. 13. The method according to one or more of claims 1 to 12, wherein during the elution buffer is applied in a linear gradient.
14. 14. The method according to one or more of claims 1 to 13, wherein elution is performed with a linear conductivity gradient in the range between 2 and 40mS/cm while maintaining the pH at a constant level.
15. 15. The method according to one or more of claims 2 to 14, wherein steps a) to c) are repeated two to four times.

Description

Purification of AAV particles using anion exchange chromatography with improved elution system The present invention relates to a method for the purification and enrichment of intact adeno-associated virus particles by anion exchange chromatography using basic amino acids as additives in elution buffers. Adeno-associated viruses (AAV) have been identified and developed as potent viral vectors to deliver genes in cultured cells in vitro and in vivo. At the same time, AAV is the leading platform for in vivo gene therapy delivery. AAV is a small non-enveloped virus containing a single-stranded DNA genome of about 4.7kb, consisting of two Inverted Terminal Repeats (ITRs) capable of forming a T-shaped secondary structure and acting as origins of genome replication, a Rep region encoding four overlapping replication proteins Rep78, rep68, rep52 and Rep40, and a cap region encoding three structural proteins VP1, VP2 and VP3 and Assembly Activating Protein (AAP). AAV viruses naturally isolate serotypes 1-9 with the same genomic structure, but these serotypes may exhibit different tissue tropism. AAV is considered one of the most promising gene delivery vehicles, since it appears to be nonpathogenic, shows high transduction efficiency, and is stably expressed. AAV vectors can be produced in a variety of cell lines in an adherent or suspension cell culture mode using transient transfection or co-transfection methods. Depending on the particular serotype and time of production, viral particles comprising complete, partial and empty types may be secreted from the cells into the culture medium in varying proportions or contained within the cells. Initially, human cells (e.g., heLa or HEK293 cells) were transfected and selected with the ITR cassette-containing rAAV transfer vector and the Rep and Cap-containing packaging construct to generate stable AAV producer cells. Production of recombinant AAV vectors (rAAV) is then achieved by infection with helper viruses, such as adenovirus (AdV), which provide helper functions. After identifying the AdV genes required for AAV vector packaging, a helper-free approach was established using a double or triple transfection protocol consisting of two or three plasmids, including the constructed helper plasmid, rather than the helper virus. The system is widely applied to scientific research and drug development. In addition, the development of baculovirus expression vectors provides another method to produce rAAV viruses in insect Sf9 cells. These different techniques have been shown to be capable of producing sufficient rAAV virus for laboratory and clinical trials. Cell lysis steps are typically required at harvest to release the viral particles into the supernatant. For this application, common cell lysis reagents such as Triton X-100, tween 20 and NaCl are widely used. After cell lysis, AAV needs to be purified. Typical AAV purification processes include clarification, concentration and diafiltration (using tangential flow filtration), chromatographic purification (using affinity chromatography and ion exchange chromatography). In some processes, ultracentrifugation and gradient ultracentrifugation are used in place of or in addition to chromatography. The final step in AAV purification typically involves concentration and diafiltration into a suitable excipient buffer composition and sterile filtration. Despite the increasing demand for viral vectors as a vehicle in gene therapy, current production processes have shortcomings in purification methods that are simultaneously scaled up to meet production requirements while reducing impurity burden. Adeno-associated viruses (rAAV) are attractive vectors in this field due to their ability to induce dividing and non-dividing tissues, patient safety, and control for cell-specific applications. However, production of these viruses while increasing efficiency and reducing manufacturing costs is a difficult task for downstream processing. The major challenge of the current AAV process is the production of genome-free particles. Although the mechanism of action is not fully understood, empty rAAV capsids are considered major process impurities, potentially compromising the safety and effectiveness of the final formulation drug. Whether produced in mammalian cell lines or insect cell lines, the resulting rAAV feed stream typically contains empty capsids at levels of 10-90% and needs to be further removed downstream. A typical downstream protocol consists of an initial affinity capture step to capture all rAAV particles from the feed stream and remove other process related impurities, and a subsequent polishing (polish) step to separate the intact and empty rAAV particles (to separate full of EMPTY RAAV PARTICLES). It was found that the complete capsids have a higher charge density due to the negatively charged genome they carry. Although this negatively charged genome only slightly changes the isoelectric point (pI) (empty shell: pi=5.9, an