Purification and labeling of extracellular vesicles using a mixed mode resin composition
Inventors
Jones, Jennifer C. • Welsh, Joshua A. • McKinnon, Katherine M. • Berzofsky, Jay A.
Assignees
US Department of Health and Human Services
Publication Number
US-12083448-B2
Publication Date
2024-09-10
Expiration Date
2038-12-28
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Abstract
Disclosed is a method of purifying extracellular vesicles in a sample comprising extracellular vesicles and molecules that are not bound to the extracellular vesicles. The method includes (a) providing a mixed mode resin composition containing a first resin having pores with a pore size that traps unbound molecules by at least by a size exclusion mechanism, and a second resin containing at least one affinity ligand; (b) contacting the sample with the mixed mode resin composition to trap at least a portion of the unbound molecules; and (c) separating the sample from the mixed mode resin composition and obtaining a sample containing extracellular vesicles at a higher concentration than prior to step (b). Further disclosed is a method of labeling an extracellular vesicle with a fluorophore that labels proteins which includes the use of a mixed mode resin composition.
Core Innovation
The invention provides a method of purifying extracellular vesicles (EVs) in a sample comprising EVs and molecules that are not bound to the EVs. The method includes providing a mixed mode resin composition comprising a first resin with pores sized to trap unbound molecules having a size less than or equal to 1,000,000 Da by at least a size exclusion mechanism, and a second resin comprising at least one affinity ligand. The sample is contacted with the mixed mode resin composition to trap at least a portion of the unbound molecules in the first resin, followed by separating the sample from the resin to obtain a sample containing EVs at a higher concentration than prior to contacting.
The invention further provides a method for labeling EVs with a fluorophore that labels proteins. The method involves contacting an EV and a fluorophore to produce labeled EV and unbound fluorophore, removing unbound fluorophore by contacting the mixture with a mixed mode resin composition wherein the first resin removes the unbound fluorophore by size exclusion and the second resin transiently and reversibly binds the labeled EV in a calcium-dependent manner, first eluting unbound molecules, and second eluting selectively the EV with an additive.
The problem being solved arises from the limitations of existing EV purification methods. Standard purification methods are not suitable due to the small size of EVs (30-100 nm). Existing size exclusion chromatography methods remove some proteins but leave residual proteins and other undesirable molecules, are limited to processing samples individually, and are not broadly applicable for biofluid processing or high-throughput (HTS) or good manufacturing practice (GMP) scale production necessary for therapeutic applications. Thus, there is a need for novel methods that effectively purify EVs and are compatible with robotic, high-throughput systems.
Claims Coverage
There are multiple inventive features related to methods of purifying extracellular vesicles using a mixed mode resin composition, including features about specific resin compositions, binding mechanisms, elution steps, and labeling methods.
Mixed mode resin composition for selective purification of extracellular vesicles
The composition comprises a first resin having pores that trap unbound molecules ≤1,000,000 Da by size exclusion, incorporating at least one affinity ligand which is a quaternary ammonium compound or trialkylamine, and a second resin comprising a non-porous bead made of a synthetic polymer with an outer surface coated with at least one affinity ligand.
Affinity binding of the second resin to unbound molecules or extracellular vesicles
The second resin can bind at least a portion of unbound molecules or can transiently bind to a phospholipid membrane on the extracellular vesicle in a calcium-dependent manner.
Two-step elution after binding in calcium-dependent manner
Following binding, first eluting molecules not bound to either resin, then second eluting extracellular vesicles selectively in presence of at least one additive.
Structure of the first resin bead
The first resin bead comprises a porous core with a matrix material and an affinity ligand, a porous shell, and optionally at least one second affinity ligand coated on the exterior surface.
Surface coating of the second resin bead
The second resin bead comprises a matrix material with at least one ligand coated on the exterior surface that has affinity either for unbound molecules or for extracellular vesicles.
Use of synthetic polymers in resin composition
The second resin bead synthetic polymer can be selected from polystyrene, polyalkylene, polyester, polydivinylbenzene, acrylamide polymer, polyacrylate, vinyl ester polymer, and vinylamide polymer among others.
Implementation in high-throughput systems
The method can be performed with high-throughput, cartridge-based, or multi-well filtering systems.
Use of nanoparticle, nucleic acid, or proteomic assays for analysis
The method can include utilizing nanoparticle analysis, nucleic acid assays, or proteomic assays to analyze characteristics or composition of the purified extracellular vesicle sample.
Affinity ligands for the first resin and second resin
The first resin's affinity ligand is a quaternary ammonium compound or trialkylamine; the second resin can have a calcium-dependent phospholipid ligand with a phosphatidylserine receptor such as a transmembrane immunoglobulin and mucin domain (TIM) protein, or be heparin.
The claims define inventive methods of purifying extracellular vesicles from samples using a tailored mixed mode resin composition that combines size exclusion and affinity capture mechanisms, with specific resin structures, binding ligands, elution steps, and optional labeling and analytical methods, optionally implemented in high-throughput systems.
Stated Advantages
The method is simple to use and fast.
Does not dilute extracellular vesicle concentration during purification.
Provides extracellular vesicles with high purity and increased yield relative to other known methods.
Enables high-throughput processing compatible with robotic systems and preparative scale-up.
Allows use of small sample volumes (microliters).
Labeling method with fluorophores improves detection sensitivity by increasing signal-to-noise ratio for downstream analyses.
Compatible with downstream proteomic assays such as gel electrophoresis, western blotting, and mass spectrometry.
Documented Applications
Purification of extracellular vesicles from biofluids such as plasma, serum, urine, cerebrospinal fluid, saliva, tears, ascites, and pleural effusion, as well as cell culture supernatants and purified EVs conjugated with labels.
Labeling of extracellular vesicles with fluorophores for enhanced detection in proteomic, cytometric, and nanoparticle analyses.
Downstream analyses of purified EVs including nanoparticle tracking analysis, flow cytometry, imaging cytometry, fluorescence microscopy, mass spectrometry, gel electrophoresis, and nucleic acid sequencing (e.g., miRNA profiling).
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