Methods for purification of recombinant AAV vectors
Inventors
Sheldon, Paulene Mclean Quigley • Gagnon, Peter S. • Nichols, Gina • Thorne, Barbara A.
Assignees
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Publication Number
US-10696952-B2
Publication Date
2020-06-30
Expiration Date
Abstract
Provided herein are methods for the purification of recombinant adeno-associated virus (rAAV) vectors that can be used for gene transfer and specifically for gene therapy or vaccination. Recombinant AAV vectors of the invention are substantially free of in-process impurities, including production components such as cellular nucleic acids, cellular proteins, helper virus, and media components.
Core Innovation
The patent describes methods for isolating a population of recombinant adeno-associated virus (rAAV) particles from in-process impurities in a feedstream. The stated goal is to reduce or remove cellular nucleic acids, cellular proteins, helper virus proteins, and media components while maintaining isolation of rAAV particles, to provide preparations substantially free of in-process impurities.
A first purification step is orthogonal capture on an apatite chromatography medium in the presence of polyethylene glycol (PEG), where the rAAV particles bind to the apatite chromatography medium and are then eluted using an elution buffer containing less than 3% (w/v) PEG. The disclosure specifies ceramic hydroxyapatite (CHT) and ceramic fluoroapatite (CFT), and indicates that the binding approach can include weak anionic binder serotypes such as AAV-1/4/5/8.
A second purification step is hydrophobic interaction chromatography (HIC) in a high salt buffer comprising citrate, followed by elution with a medium salt buffer. The disclosure states that rAAV particles and in-process impurities bind to the HIC medium under the high-salt citrate condition, and that subsequent elution with a medium-salt citrate condition releases the rAAV particles.
Additional unit operations are described to support upstream and downstream impurity management, including flow-through anionic exchange filtration, tangential flow filtration (TFF), heat inactivation of helper virus in the feedstream eluted from apatite, and downstream SEC and final anion exchange chromatography. The disclosure also describes upstream clarification and Benzonase digestion, and downstream polishing and viral clearance filtration options.
Claims Coverage
The partial content provided includes two independent claims. Across these independent claims, the core coverage is a multi-step workflow that uses apatite chromatography with PEG for rAAV binding and low-PEG elution, combined with HIC using citrate high-salt and medium-salt buffers, with one claim further adding specific upstream filtration/TFF, helper-virus heat inactivation, and downstream SEC and anionic exchange steps.
Apatite PEG binding with low-PEG elution
Contacting a feedstream containing rAAV particles with an apatite chromatography medium in the presence of polyethylene glycol (PEG) wherein the rAAV particles bind to the apatite chromatography medium, and eluting the rAAV particles bound to the apatite chromatography medium with an elution buffer containing less than 3% (w/v) PEG.
High-salt citrate HIC capture and medium-salt elution
Contacting the feedstream eluted from the apatite chromatography medium containing the rAAV particles with a hydrophobic interaction chromatography (HIC) medium in a high salt buffer comprising citrate wherein the rAAV particles and the in-process impurities bind to the HIC medium, and eluting the rAAV particles bound to the HIC medium with a medium salt buffer.
Upstream flow-through anionic exchange filtration and TFF
Subjecting the feedstream containing the rAAV particles to flow-through anionic exchange filtration, and subjecting the feedstream containing the rAAV particles to TFF.
Helper virus heat inactivation after apatite
Heat inactivating a helper virus in the feedstream eluted from the apatite chromatography medium.
SEC and final anionic exchange chromatography after HIC
Subjecting the feedstream containing the rAAV particles eluted from the HIC medium to SEC, and subjecting the feedstream containing the rAAV particles eluted from the SEC medium to anionic exchange chromatography.
Overall, the claim coverage centers on an orthogonal purification sequence: apatite chromatography with PEG for rAAV binding and elution using less than 3% (w/v) PEG, followed by HIC in citrate high-salt conditions with elution under medium-salt citrate conditions. The second independent claim further adds specified upstream filtration/TFF, helper-virus heat inactivation, and downstream SEC and anionic exchange chromatography steps.
Stated Advantages
Achieves preparations substantially free of in-process impurities, including cellular nucleic acids, cellular proteins, helper virus proteins, and media components.
Provides improved capture/reproducibility behavior associated with PEG increasing apatite binding capacity and breakthrough reproducibility.
Provides impurity and helper virus clearance effects described via glucan clearance and reduced infectious Ad5 helper virus.
Documented Applications
Purification and isolation of recombinant adeno-associated virus (rAAV) particles from in-process impurities in a feedstream for gene therapy and gene vaccination.
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