Methods for the purification of viral vectors
Inventors
Mayani, Mukesh • Liu, Tongyao • Ismail, Ayman
Assignees
Fondazione Telethon • Ospedale San Raffaele SRL • Bioverativ Therapeutics Inc
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Publication Number
US-12173307-B2
Publication Date
2024-12-24
Expiration Date
Abstract
Viral vector production processes and methods of purifying a viral vector from a host cell are provided herein.
Core Innovation
The invention relates to purifying enveloped viral vectors, including lentiviral vectors (LVV), to remove transgene protein contaminants that co-purify during conventional downstream processing. In particular, the method addresses an LVV comprising a factor VIII (FVIII) transgene and an FVIII transgene protein contaminant, including FVIII/FVIIIXTEN transgene protein contaminant, present in a composition obtained from a host cell.
A first chromatography step is introduced by contacting a composition comprising the LVV and the FVIII transgene protein contaminant with a first chromatography matrix that is an affinity chromatography column having an affinity ligand that specifically binds the FVIII transgene protein contaminant. The LVV is recovered in the flow-through of the affinity chromatography matrix, thereby separating the LVV from the FVIII protein contaminant.
In embodiments, the workflow includes an additional chromatography step in which an LVV-selective chromatography matrix is used to selectively bind and elute the LVV, including anionic exchange (AEX) chromatography before or after the affinity transgene-protein capture step. The document further describes process elements supporting the affinity capture and overall purification, including repeating affinity capture cycles, performing the capture in the presence of calcium chloride (CaCl2), adjusting salt concentration to a defined range, and optionally combining substantially purified LVV with pharmaceutical excipients and performing UF/DF with tangential flow filtration (TFF).
Claims Coverage
The document provides one independent claim directed to producing a lentiviral vector drug product using a first affinity chromatography matrix that selectively captures an FVIII transgene protein contaminant while recovering the LVV in the flow-through. Dependent claims refine this workflow with additional LVV-selective chromatography, specific buffer and pH conditions, an optional CaCl2 stabilizing agent, salt concentration targeting, and downstream UF/DF with TFF formulation steps. Overall coverage focuses on inventive separation of LVV from FVIII transgene protein contaminants via affinity flow-through capture, potentially integrated with upstream AEX and downstream formulation and purification operations.
Affinity capture of FVIII transgene protein contaminant with flow-through LVV recovery
Contacting a composition comprising the LVV and a FVIII transgene protein contaminant with a first chromatography matrix comprising an affinity chromatography column having an affinity ligand that specifically binds the FVIII transgene protein contaminant, and recovering the LVV in the flow-through of the chromatography matrix to separate the LVV from the FVIII protein contaminant.
Upstream LVV-selective anionic exchange capture prior to affinity separation
Contacting the composition with a second anionic exchange (AEX) chromatography matrix or membrane that selectively binds the LVV and eluting the LVV before the contacting step with the first affinity chromatography matrix.
Loading buffer with Tris-HCl or phosphate at defined pH range
Carrying out the contacting/loading of the first chromatography matrix in a loading buffer comprising Tris-HCl buffer or phosphate buffer at a pH about 7.0 to about 7.5.
Stabilization of FVIII transgene protein contaminant using CaCl2 during affinity contacting
Carrying out step (i) with an agent that stabilizes the FVIII transgene protein contaminant, wherein the agent is CaCl2.
Salt concentration adjustment to a target range
Adjusting the salt concentration of a composition containing the LVV and the FVIII transgene protein contaminant to a target level from about 0.2 M to about 0.6 M.
Downstream UF/DF with tangential flow filtration for formulated LVV
Using ultrafiltration/diafiltration (UF/DF) with tangential flow filtration (TFF) in a formulation buffer to further purify the LVV separated from the FVIII transgene protein contaminant.
The claim set centers on separating LVV from FVIII transgene protein contaminant by affinity chromatography using an affinity ligand that specifically binds the FVIII transgene protein contaminant, with LVV recovered in the affinity flow-through. Dependent claims further define optional integration with an upstream LVV-selective AEX step, specify loading buffer identity and pH range, optionally add CaCl2 to stabilize the contaminant, constrain salt concentration adjustment to a defined range, and include UF/DF with TFF for downstream formulation and purification.
Stated Advantages
Separates the LVV from the FVIII protein contaminant by recovering the LVV in the flow-through of an affinity chromatography matrix.
Reduces contaminating FVIII/FVIIIXTEN activity and impurity levels while retaining functional vector recovery.
Provides substantially contaminant-free LVV acceptance criteria after purification and formulation steps.
Documented Applications
Producing a lentiviral vector (LVV) drug product by purifying an LVV from a host cell composition containing an FVIII transgene protein contaminant.
Formulating purified LVV into pharmaceutical compositions, optionally including UF/DF with tangential flow filtration (TFF) and pharmaceutical excipients, consistent with substantially free of transgene protein contaminant acceptance criteria.
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