Column-based fully scalable rAAV manufacturing process
Inventors
Qu, Guang • Oh, YoungHoon • Lu, Lin • Wright, John Fraser
Assignees
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Publication Number
US-11702639-B2
Publication Date
2023-07-18
Expiration Date
Abstract
In accordance with the invention, provided herein are methods for purifying recombinant adeno-associated (rAAV) vector particles.
Core Innovation
The invention relates to a method for purifying recombinant adeno-associated (rAAV) vector particles. The method comprises harvesting cells and cell culture supernatant comprising rAAV vector particles to produce a harvest, optionally concentrating the harvest, and lysing the harvest or concentrated harvest to produce a lysate.
The lysate is treated to reduce contaminating nucleic acid, filtered to produce a clarified lysate, and optionally diluted to produce a diluted clarified lysate. The clarified lysate or diluted clarified lysate is then subjected to anion exchange column chromatography to separate rAAV vector particles from protein impurities.
The resulting column eluate or concentrated column eluate is subjected to size exclusion column chromatography, and the second column eluate or diluted second column eluate is subjected to cation exchange column chromatography to separate rAAV vector particles from protein or other production impurities. After the final chromatography step, the third column eluate or concentrated third column eluate is filtered to produce purified rAAV vector particles.
The document describes a column-based, scalable purification platform for recombinant AAV that addresses separation from protein impurities and other production impurities while reducing contaminating nucleic acid. It further reports recovery of rAAV vector particles of about 40%–70% and a final product that is formulated and concentrated to high particle concentrations, while excluding cesium chloride gradient ultracentrifugation.
Claims Coverage
The partial content includes two independent claims, each describing a sequential multi-column purification workflow with optional concentration and dilution, nucleic-acid reduction, intermediate filtration, and final filtration to obtain purified rAAV vector particles. Across the independent claims, the inventive features focus on reducing contaminating nucleic acid in a lysate, clarifying by filtration, and separating impurities using anion exchange, size exclusion, and cation exchange column chromatography in sequence.
Sequential multi-column purification workflow
Subjecting the clarified lysate or diluted clarified lysate to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles; subjecting the column eluate or concentrated column eluate to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles; and subjecting the second column eluate or diluted second column eluate to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities and other production impurities.
Nucleic acid reduction in a lysate
Treating the lysate to reduce contaminating nucleic acid in the lysate thereby producing a nucleic acid reduced lysate.
Clarification by filtration after nucleic acid reduction
Filtering the nucleic acid reduced lysate to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate.
Final filtration to yield purified rAAV vector particles
Filtering the third column eluate or the concentrated third column eluate thereby producing purified rAAV vector particles.
Harvest-to-product processing with optional concentration and intermediate dilution
Harvesting cells and cell culture supernatant comprising rAAV vector particles to produce a harvest; optionally concentrating the harvest to produce a concentrated harvest; optionally concentrating the column eluate; optionally diluting the clarified lysate; optionally diluting the second column eluate; and optionally concentrating the third column eluate before final filtration.
Overall claim coverage is centered on a harvest-to-purified-product method that combines nucleic-acid reduction, intermediate filtration, and sequential anion exchange, size exclusion, and cation exchange column chromatography to separate rAAV vector particles from protein impurities and other production impurities, ending with filtration to produce purified rAAV vector particles.
Stated Advantages
Separating rAAV vector particles from protein impurities and other production impurities.
Reducing contaminating nucleic acid in the lysate.
Yielding purified rAAV vector particles and reporting a recovery of about 40%–70%.
Excluding cesium chloride gradient ultracentrifugation as a purification step.
Documented Applications
No documented applications found
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