Genetically encoded polypeptide for affinity capture and purification of biologics
Inventors
Luginbuhl, Kelli M. • Chilkoti, Ashutosh
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12269847-B2
Publication Date
2025-04-08
Expiration Date
Abstract
The invention relates to genetically encoded fusion proteins comprised of a capture component that binds a target with high affinity and a peptide polymer, such as elastin-like polypeptides, that display phase behavior and can be used for purification. The invention further relates to methods for optimizing capture fusion proteins for individual biologic targets such that phase separation occurs under desirable conditions, such as at room temperature, lower concentrations of salt, and/or at suitable pH ranges and optimized capture domains and polypeptides with phase behavior that have been identified by the optimization methods.
Core Innovation
The invention relates to genetically encoded capture-fusion proteins that combine at least one capture domain with at least one elastin-like polypeptide (ELP). The capture domain binds an antibody or fragment thereof, while the ELP provides phase behavior enabling liquid-liquid phase separation under mild conditions. The system uses phase transition to form phase-separated coacervates or aggregates in the presence of antibodies or antibody fragments.
A central aspect is tuning the phase transition behavior and transition parameters, including transition temperature (Tt), ionic strength, and pH, by modifying features of the capture domain and the ELP or phase polypeptide composition. The document describes fusion protein variants that include capture-domain variants and ELP-like phase polypeptides, including different architectures and compositions intended to support controlled, reversible phase separation.
The invention also supports purification concepts in which biologic capture via phase separation is followed by separation of aggregates and recovery of purified material. The described approach includes contacting biologics with the fusion protein to form bound fusion protein, triggering aggregation or phase separation, separating aggregates from contaminants, and then separating the fusion protein from the antibody or fragment through changes in ionic strength or pH or competitive binding, with reversibility enabling recovery of the purification reagent.
Claims Coverage
The provided independent claim recites a fusion protein with two core components, a capture domain and an elastin-like polypeptide, and specifies antibody binding by the capture domain. Dependent claims refine this concept through specific capture-domain selections, ELP constraints, spacer constraints, and downstream purification workflow features. In total, the inventive coverage includes at least one independent claim and multiple dependent claims refining composition and operation.
Antibody-binding capture domain fused to elastin-like polypeptide (ELP)
A fusion protein comprising at least one capture domain and at least one elastin-like polypeptide (ELP), wherein the at least one capture domain binds to an antibody or fragment thereof.
ELP proline/glycine composition constraints
The fusion protein includes an ELP comprising at least 20% proline and at least 40% glycine.
Protein A/G/L capture domain selection
The fusion protein uses at least one capture domain selected from protein A, a domain thereof, protein G, a domain thereof, or protein L, a domain thereof.
Spacer length constraint
The spacer within the fusion protein has a length of between 1 and about 26 amino acids.
Non-repeating ELP subsequence constraint
Any 5 to 10 amino acid subsequence of the at least one ELP does not repeat.
Phase-transition purification workflow for antibody or fragment
Purifying an antibody or fragment comprises contacting it with the fusion protein to form a bound fusion protein; triggering a phase transition to aggregate bound fusion proteins; separating bound fusion protein from contaminants; and separating fusion protein from the antibody or fragment.
Overall, the claim set centers on a genetically encoded fusion protein that links antibody-binding capture domains with an ELP phase-forming component, with refinements that constrain ELP composition and sequence behavior, define spacer length, allow selection among protein A, G, or L capture domains, and support a purification workflow based on binding, induced phase transition and aggregation, contaminant separation, and subsequent separation of the fusion protein from the antibody or fragment.
Stated Advantages
Improved purity and impurity profiles versus Protein A in several assays.
Comparable or better yield versus Protein A in the reported examples.
Enables purification demonstrations in high-throughput formats.
Supports tangential flow filtration (TFF) flux and time performance as demonstrated in examples.
Provides reversible phase transition enabling recovery of the purification reagent.
Documented Applications
Purification or capture of biologics including monoclonal antibodies such as HERCEPTIN (trastuzumab), XGEVA (denosumab), and KEYTRUDA (pembrolizumab) using the fusion protein approach.
Purification or capture of viruses, including AAV (AAV8), using the fusion protein approach.
Purification involving cells or viruses as described conceptually, with phase separation used for aggregate formation and subsequent separation steps.
Demonstrations in high-throughput formats and in tangential flow filtration (TFF) formats for purification workflows.
Interested in licensing this patent?