# US 12,758,896 B2 · High-throughput chromatography screening for extracellular vesicles

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- Number: US 12,758,896 B2
- Document ID: US12758896B2
- Status: Granted
- Kind: B2
- Application number: 17/774,774
- Filed: 2020-11-05
- Published: 2022-12-08
- Granted: 2026-10-13
- Earliest claimed: 2019-11-05
- Assignees: Lonza Sales AG
- Applicants: Lonza Sales AG
- Inventors: Michael Doherty; Aaron Noyes; Andrew Wood
- Web page: https://patentgrep.com/patent/US12758896B2

## CPC

- G01N 33/5076: {involving cell organelles, e.g. Golgi complex, endoplasmic reticulum}
- B01D 15/3828: {Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography}
- G01N 21/49: within a body or fluid
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- B01D 2221/10: Separation devices for use in medical, pharmaceutical or laboratory applications, e.g. separating amalgam from dental treatment residues
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)

## Abstract

The present disclosure relates to high-throughput screening methods for identifying one or more chromatography operational parameters (e.g., binding parameters) and/or reagents for purifying EVs (e.g., exosomes) from a sample using chromatography. Also disclosed herein are methods for improving one or more aspects of EV (e.g., exosome) purification, e.g., improving EV yield, increasing EV ligand density, and/or reducing impurity recovery.

## Claims

### Claim 1

A method of purifying an extracellular vesicle (EV) comprising a prostaglandin F2 receptor negative regulator (PTGFRN) protein or a fragment thereof expressed in or on the EV from a sample, the method comprising
(i) contacting the sample to an ion-exchange chromatography resin, a size exclusion chromatography resin, an ion-exchange chromatography medium, or a size exclusion chromatography medium, wherein the resin or medium is not a mixed mode chromatography resin or medium, under a plurality of chromatography operational parameters,
(ii) collecting a fraction comprising the EV from step (i), and
(iii) determining an EV yield, impurity recovery, and/or EV ligand density from step (ii),
wherein the contacting of the sample to the resin or medium occurs in a microplate or in miniature columns, and
wherein the PTGFRN protein or fragment thereof anchors a payload or an exogenous biologically active molecule to the EV.

### Claim 2

A method of identifying one or more chromatography operational parameters suitable for purifying an extracellular vesicle (EV) comprising a prostaglandin F2 receptor negative regulator (PTGFRN) protein or a fragment thereof expressed in or on the EV from a sample comprising the EV and an impurity with a chromatography, the method comprising:
(i) contacting the sample to an ion-exchange chromatography resin, a size exclusion chromatography resin, an ion-exchange chromatography medium, or a size exclusion medium, wherein the resin or medium is not a mixed mode chromatography resin or medium, under a plurality of chromatography operational parameters,
(ii) collecting a fraction comprising the EV from step (i),
(iii) determining an EV yield, impurity recovery, and/or EV ligand density from step (ii), and
(iv) identifying the one or more chromatography operational parameters, wherein the one or more identified chromatography operational parameters are suitable for purifying the EV from the sample if (a) the EV yield is increased, (b) the impurity recovery is reduced, and/or (c) the EV ligand density is increased, as compared to a corresponding value in the sample prior to step (i),
wherein the contacting of the sample to the resin or medium occurs in a microplate or in miniature columns, and
wherein the PTGFRN protein or fragment thereof anchors a payload or an exogenous biologically active molecule to the EV.

### Claim 3 (depends on 1)

The method of claim 1, wherein the collecting of the fraction in (ii) is performed under a flow through mode, bind and elute mode, or weak-partition mode.

### Claim 4 (depends on 1)

The method of claim 1, further comprising (iv) adjusting at least one of the chromatography operational parameters and repeating steps (i) to (iii), if (a) the EV yield is decreased, (b) the impurity recovery is increased, and/or (c) the EV ligand is decreased, as compared to a desired corresponding value.

### Claim 5 (depends on 1)

The method of claim 1, wherein the chromatography operational parameters comprise a plurality of pHs, a plurality of weak acids and/or conjugate bases, a plurality of alcohols, a plurality of carbohydrates, a plurality of detergents, a plurality of chaotropic agents, a plurality of kosmotropic agents, a plurality of mass challenge, a plurality of residence time, a plurality of temperatures, a plurality of salt concentrations, a plurality of buffers, or any combination thereof.

### Claim 6 (depends on 5)

The method of claim 5, wherein: (i) the plurality of pHs is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or about 13; (ii) the plurality of salt concentrations is between about 0 M to about 4 M; or (iii) both (i) and (ii).

### Claim 7 (depends on 1)

The method of claim 1, wherein the EV yield is determined by one or more of the following:
(1) measuring an EV particle count of the fraction in (ii);
(2) measuring an absorbance of the fraction in (ii);
(3) measuring an intrinsic fluorescence of the fraction in (ii);
(4) measuring a light scattering emission signal of the fraction in (ii);
(5) measuring a turbidity of the fraction in (ii);
(6) measuring a static light obscuration of the fraction in (ii); and
(7) measuring a static refractive index of the fraction in (ii).

### Claim 8 (depends on 7)

The method of claim 7, wherein:
(a) the light scattering emission signal is generated using an excitation wavelength ranging from about 280 nm to 700 nm and is detected by measuring an emission wavelength that is 0-20 nm longer or shorter than the excitation wavelength and ranging from 260 nm to 720 nm;
(b) the absorbance is measured at a wavelength from about 200 nm to about 1,100 nm;
(c) the intrinsic fluorescence is measured at an excitation wavelength of about 460 nm and an emission wavelength of about 470 nm; or
(d) any combination of (a) to (c).

### Claim 9 (depends on 1)

The method of claim 1, wherein the impurity recovery is determined using an assay comprising an ELISA, Alphalisa, flow cytometry, Western blot, immunoprecipitation, fluorescence spectroscopy, chemiluminescence, mass spectrometry, absorbance spectroscopy, polymerase chain reaction (PCR), or combinations thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the chromatography resin is Poros XS.

### Claim 11 (depends on 1)

The method of claim 1, wherein the sample is derived from a cell culture or from a body fluid of a subject.

### Claim 12 (depends on 1)

The method of claim 1, wherein the EV comprises an exosome.

### Claim 13 (depends on 1)

The method of claim 1, wherein the EV further comprises a targeting moiety.

### Claim 14 (depends on 2)

A method of purifying an extracellular vesicle (EV) from a sample, comprising identifying one or more chromatography operational parameters according to the method of claim 2, and further purifying the EV from the sample using the one or more identified chromatography operational parameters.

### Claim 15 (depends on 2)

A method of increasing a (PTGFRN) protein or fragment density of an extracellular vesicle (EV) present in a sample, the method comprising identifying one or more chromatography operational parameters according to the method of claim 2, and further contacting the sample to a chromatography resin or medium under the one or more identified chromatography operational, wherein the one or more identified chromatography operational parameters are capable of selecting for EVs with high amounts of PTGFRN protein or fragment.

### Claim 16 (depends on 2)

A method of decreasing an amount of impurity in a sample comprising an extracellular vesicle (EV), the method comprising identifying one or more chromatography operational parameters according to the method of claim 2, and further contacting the sample to a chromatography resin or medium under the one or more identified chromatography operational parameters, wherein the one or more identified chromatography operational parameters are capable of decreasing the amount of impurity in the sample.

### Claim 17 (depends on 2)

A method of increasing an extracellular vesicle (EV) yield in a sample, the method comprising identifying one or more chromatography operational parameters according to the method of claim 2, and further contacting the sample to a chromatography resin or medium under the one or more identified chromatography operational parameters, wherein the one or more identified chromatography operational parameters are capable of increasing the EV yield of the sample.