Protein-based purification matrices and methods of using the same
Inventors
Luginbuhl, Kelli M. • DZURICKY, Michael
Assignees
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Publication Number
US-11591576-B2
Publication Date
2023-02-28
Expiration Date
Abstract
Provided herein are protein-based purification matrices and methods of use thereof to purify biologics and/or to remove contaminants from a composition. Methods of bringing two or more biologics in close proximity are also provided. The disclosed compositions and methods allow for faster, more efficient purification of a biologic compared to traditional affinity chromatography.
Core Innovation
The disclosure describes a protein-based purification matrix comprising a fusion protein. The fusion protein includes at least one lentivirus-binding domain and at least one polypeptide with phase behavior, and the lentivirus-binding domain comprises defined amino acid regions or specified SEQ ID NOs.
The polypeptide with phase behavior includes elastin-like or resilin-like polypeptides, and the phase behavior is associated with a transition temperature and liquid-liquid phase separation behavior using LCST and UCST concepts. Environmental factors reversibly trigger phase transitions and aggregation that change complex size and morphology.
A lentiviral particle binds to the protein-based purification matrix to form a complex. The complex is contacted with a first environmental factor to increase complex size, separated from at least one contaminant based on size, and the viral particle is released from the protein-based purification matrix by contacting the complex with a second environmental factor. The document further describes using the matrix to stabilize biologics during production, purification, and storage.
Claims Coverage
The provided set includes two independent claims, directed to a protein-based purification matrix and to a method of purifying a viral particle. The core inventive coverage is a fusion protein having a lentivirus-binding domain defined by SEQ ID regions or identities and a phase-behaving polypeptide, together with a workflow using first and second environmental factors to increase complex size, enable size-based contaminant separation, and release the lentiviral particle from the matrix.
Protein-based purification matrix with lentivirus-binding domain and phase-behavior polypeptide
A protein-based purification matrix comprising a fusion protein, wherein the fusion protein comprises at least one lentivirus-binding domain and at least one polypeptide with phase behavior; wherein the lentivirus-binding domain comprises amino acids 2-860 of SEQ ID NO: 73, amino acids 2-768 of SEQ ID NO: 74, amino acids 2-40 of SEQ ID NO: 75, amino acids 2-38 of SEQ ID NO: 76, SEQ ID NO: 168, or SEQ ID NO: 169.
Method of purifying a lentiviral particle using environmental-factor-driven complex size increase and release
A method of purifying a viral particle comprising contacting the viral particle with a protein-based purification matrix, wherein the protein-based purification matrix comprises a fusion protein that comprises at least one lentivirus-binding domain and at least one polypeptide with phase behavior; wherein the lentivirus-binding domain comprises the sequence of any one of SEQ ID NOs: 73-76 or 168-169, or a sequence with at least 90% identity thereto; wherein the viral particle is a lentiviral particle, and wherein the viral particle binds to the protein-based purification matrix to form a complex; contacting the complex with a first environmental factor to increase the size of the complex; separating the complex from at least one contaminant on the basis of size; and separating the viral particle from the protein-based purification matrix by contacting the complex with a second environmental factor.
Overall, the claims tie purification to a fusion-protein matrix that combines a lentivirus-binding domain defined by specific SEQ ID regions or identities with a polypeptide with phase behavior, and to a purification workflow that uses first and second environmental factors to increase complex size, perform size-based contaminant separation, and release the lentiviral particle from the matrix.
Stated Advantages
Stabilize biologics during production, purification, and storage.
Improve yield/titer.
Extend shelf-life.
Enable separation of the complex from at least one contaminant on the basis of size.
Enable separating the viral particle from the protein-based purification matrix by contacting the complex with a second environmental factor.
Documented Applications
Purifying a viral particle, specifically a lentiviral particle, using a protein-based purification matrix with a lentivirus-binding domain and a polypeptide with phase behavior.
Purifying a lentiviral particle using the disclosed protein-based purification matrix and an environmental-factor-based complex enlargement and release sequence.
Stabilizing biologics during production, purification, and storage.
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