Recombinant adeno-associated virus compositions and methods for producing same
Inventors
Dai, Yong • Zhou, Jingmin • DANIELS, Garrett • Chan, Jonathan • Haller, Jorge • Nelson, Stuart
Assignees
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Publication Number
US-12729385-B2
Publication Date
2026-09-08
Expiration Date
Abstract
Disclosed herein are compositions comprising recombinant adeno-associated virus (rAAV), as well as recombinant baculovirus systems and methods of using the same for producing and purifying such compositions. Also disclosed herein are assays for testing the titer and potency of such compositions.
Core Innovation
A therapeutic composition comprises recombinant adeno-associated virus (rAAV) particles having an AAV9 capsid protein and an expression cassette encoding human glucocerebrosidase (GCase), wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO: 2. The composition comprises more than 1E+13 viral genomes per milliliter (vg/mL), less than 15% empty rAAV particles, and at least 80% full rAAV particles.
The composition also comprises less than or equal to 1.3 ng residual host cell DNA from the insect cells per 1E+14 vg/mL of the rAAV particles. The document further characterizes physical and impurity-related properties, including residual impurities and physicochemical parameters such as osmolality and pH, and includes testing concepts such as sterility and microbial tests.
Production is described by infecting Sf9 insect cells with a population of baculovirus vectors and lysing the insect cells to produce a cellular lysate comprising the rAAV particles. The lysate is subjected to affinity chromatography with a binding agent specific for a capsid protein, followed by anion-exchange chromatography to produce a second eluate with fewer empty rAAV particles than the first eluate, and the second eluate is concentrated by tangential flow filtration using a flow buffer comprising Tris, MgCl2, NaCl, and Poloxamer 188 to produce the therapeutic composition.
Claims Coverage
The provided partial content centers on one independent claim defining an AAV9 rAAV therapeutic composition encoding human GCase with quantitative particle-content and impurity constraints, together with a purification workflow. Four inventive features are identified.
High-titer AAV9 GCase therapeutic composition with low empty capsids
A therapeutic composition comprising rAAV particles comprising an AAV9 capsid protein and an expression cassette encoding human glucocerebrosidase (GCase) (SEQ ID NO: 2), wherein the composition comprises more than 1E+13 vg/mL, less than 15% empty rAAV particles, and at least 80% full rAAV particles.
Low residual host cell DNA per viral genome amount
The therapeutic composition comprises less than or equal to 1.3 ng residual host cell DNA from the insect cells per 1E+14 vg/mL of the rAAV particles.
Sf9 insect cell baculovirus production with capsid-specific affinity and empty-reducing anion-exchange
The therapeutic composition is produced by infecting Sf9 insect cells with a population of Baculovirus vectors, lysing to produce a cellular lysate comprising the rAAV particles, contacting an affinity chromatography column with the cellular lysate using a binding agent specific for a capsid protein to produce a first eluate, and performing anion-exchange chromatography on the first eluate to produce a second eluate comprising fewer empty rAAV particles than the first eluate.
Tangential flow filtration concentration using a specified Tris/MgCl2/NaCl/Poloxamer buffer
The second eluate is concentrated by performing tangential flow filtration using a flow buffer comprising Tris, MgCl2, NaCl, and Poloxamer 188 to produce the therapeutic composition.
The claim coverage centers on an AAV9 rAAV therapeutic composition encoding SEQ ID NO: 2 GCase with high vg/mL, low empty particle fraction, and low residual insect host cell DNA, together with a purification workflow that includes capsid-specific affinity chromatography, anion-exchange chromatography to reduce empty particles, and tangential flow filtration using a Tris/MgCl2/NaCl/Poloxamer 188 buffer.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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