KR-20260068075-A - Milk extracellular vesicle concentration process

Abstract

A process for obtaining an extracellular vesicle (EV)-rich product comprising: i. a step of obtaining an EV-containing liquid; ii. a step of applying the EV-containing liquid to ultrafiltration (UF) to provide an EV-rich UF residue (UFR) and a UF permeate (UFP), wherein the abundance is an increase in the number of EVs relative to the total protein level in the UFR compared to that in the EV-containing liquid; wherein the step of chelating divalent cations with EDTA in the preparation of the EV-containing liquid is not used, and preferably, EDTA is not used to prepare the EV-containing liquid.

Inventors

• 파이츠마, 아눅 레오니
• 반 더 로, 알폰스 아드리아누스 야코바
• 아른츠, 아르놀두스 야코부스

Assignees

• 프리슬랜드캄피나 네덜란드 비.브이.

Dates

Publication Date

20260513

Application Date

20240911

Priority Date

20230912

Claims (14)

1. As a process for obtaining extracellular vesicle (EV)-rich products, i. Step to obtain an EV-containing liquid; ii. A step comprising applying the above EV-containing liquid to ultrafiltration (UF) to provide an EV-rich UF residue (UFR) and a UF permeate (UFP), preferably wherein the UF is performed in a volumetric filtration (DF) mode, and Abundance is an increase in the number of EVs relative to the total protein level in the UFR compared to EV-containing liquids; In the preparation of an EV-containing liquid, the step of chelating divalent cations with EDTA is not used, and preferably, EDTA is not used to prepare the EV-containing liquid; The amount of protein in the EV-rich product is less than 4.0 femtograms/EV, preferably less than 3.0 fg/EV, more preferably less than 2.0 fg/EV, process.
2. A process according to claim 1, comprising an additional step iii to be performed after step ii, wherein step iii is a filtration step, preferably a size exclusion chromatography (SEC) step, and more preferably a SEC step in a mock moving bed (SMB) chromatography setting.
3. A process according to claim 1 or 2, wherein the EV-containing liquid is a milk fraction, preferably a skim milk fraction.
4. A process according to any one of claims 1 to 3, wherein the EV-containing liquid is whey, preferably cow's milk whey.
5. A process according to any one of claims 1 to 4, wherein the UF is performed at a differential pressure of 50 to 200 kilopascals (kPa), more preferably 70 to 150 kPa.
6. A process according to any one of claims 1 to 5, wherein the EV-containing liquid has a dry material content of 0.1 to 15%, preferably 1.0 to 15%, more preferably 2.0 to 10%, particularly preferably 3.0 to 9.0%, and most preferably 5.0 to 8.0%.
7. A process according to any one of claims 1 to 6, wherein the EV-containing liquid has a pH greater than 5.2, preferably 5.2 to 8.0, more preferably 6.0 to 7.5, and most preferably 6.0 to 6.6.
8. A process according to any one of claims 1 to 7, wherein the UF step is performed using a polymer membrane, preferably a polymer spiral-wound membrane.
9. In claim 8, the polymer membrane has a cut-off size of 600 kD to 1000 kD, in the process.
10. A process according to any one of claims 1 to 7, wherein the UF step is performed using a ceramic film; preferably, the ceramic film has a cut-off size of 0.15 to 0.25 microns, preferably 0.19 to 0.21 microns.
11. A process according to any one of claims 1 to 10, wherein the EV-containing liquid is an acidic whey.
12. A composition comprising extracellular vesicles (EVs) and proteins, wherein the amount of protein is less than 4.0 femtograms/EV, preferably less than 3.0 fg/EV, more preferably less than 2.0 fg/EV.
13. Use of an EV-rich product obtained by the process of any one of claims 1 to 11 or a composition of claim 12 in food or pharmaceuticals, preferably synthetic foods.
14. An extracellular vesicle-rich product obtained by the process of any one of claims 1 to 11.

Description

Milk extracellular vesicle concentration process The present invention relates to an extracellular vesicle (EV) concentration process, in particular to a process for obtaining a milk stream rich in milk-derived extracellular vesicles (mEVs), more preferably rich in bovine mEVs. The present invention further relates to a product comprising such an mEV-rich milk stream and to uses of such an mEV-rich milk stream. US 10,729,159 (US 2019/0150474) relates to a method for purifying exosomes, comprising using a whey composition as an exosome source. Exosomes are isolated by treating the whey composition with a first ultrafiltration to obtain a first permeate and a first residue. Subsequently, the first residue may be treated with a second ultrafiltration to obtain a second permeate and a second residue. During the second ultrafiltration process, the first residue may be treated with carbon dioxide. Subsequently, the second residue may be treated with a third ultrafiltration to obtain a third permeate and a third residue. Subsequently, the third permeate may be optionally dried to obtain exosome powder. Moleirinho et al. disclose challenges in the manufacturing of biotherapeutic particles, including upstream and downstream steps and a manufacturing platform (Moleirinho, M. G., (2019) Current challenges in biotherapeutic particles manufacturing. Expert Opinion on Biological Therapy, 20(5), 451-465. https://doi.org/10.1080/14712598.2020.1693541). BJ Benedikter et al. disclose a method for isolating extracellular vesicles from cell culture media for compositional and functional studies (BJ Benedikter et al Sci Rep 7, 15297 (2017). https://doi.org/ 10.1038/ s41598-017-15717-7). The term "extracellular vesicle" or "EV" is defined herein as a general term for particles surrounded by a lipid bilayer released from cells; unlike cells, EVs cannot replicate. The diameter of EVs ranges from near the size of the smallest physically possible single-layer liposome (approximately 20–30 nanometers) to over 10 microns, but most EVs are smaller than 200 nm. They transport cargo from parent cells, including proteins, nucleic acids, lipids, metabolites, and even organelles. Most cells studied to date are believed to release EVs, including some bacteria, fungi, and plant cells that are surrounded by cell walls. A wide variety of EV subtypes have been proposed, defined differently based on size, biosynthetic pathways, cargo, cellular source, and function. As used herein, the term “integrated extracellular vesicle” refers to an extracellular vesicle (EV) in which the vesicle membrane is not ruptured and/or otherwise degraded, and thus the vesicle size can be determined using the methods described in other parts of this invention. Endogenous cargoes inherently present in milk-derived extracellular vesicles (mEVs), namely bioactivators, therapeutic agents (e.g., miRNA), and/or other biomolecules, are maintained in an active form in the intact EV. Extracellular vesicles (EVs) include exosomes (<100 nm) and microvesicles (100 nm to 10 microns). EVs exist in biological fluids and are involved in various physiological and pathological processes. EVs are considered an additional mechanism for intercellular communication that enables cells to exchange proteins, lipids, and genetic material. Numerous studies have provided strong evidence that EVs are involved in the regulation of immune responses and act as both enhancers and buffers of the immune system, depending on the source and type of the vesicles. Studies have shown that milk-derived EVs, using human breast milk as well as bovine colostrum and commercially pasteurized milk, exhibit anti-inflammatory effects both in in vitro systems and therapeutically in animal models. Strategies targeting the gut, particularly the gut microbiome, are currently under investigation and hold potential as therapeutic interventions for these conditions. In recent years, the use of milk-derived EVs as standalone drugs or drug carriers has been frequently proposed. Due to their composition, milk-derived EVs possess high biocompatibility and limited immunogenicity across species. When absorbed into the gastrointestinal tract, milk-derived EVs have been demonstrated to exhibit excellent stability, remaining intact after absorption. Due to these characteristics, milk-derived EVs are not only highly suitable as drug carriers but also possess significant immunomodulatory functions in themselves; these vesicles can act as therapeutic agents even without loading. Therefore, it is desirable to have an efficient and scalable method for isolating or at least concentrating milk streams with EVs. Such a process contributes to a sustainable method of resolving and/or treating problems or defects in the subject's immune response, and contributes to sustainable and environmentally friendly drug carriers. Research in the field of milk-derived EVs (mEVs) has grown rapidly over the past few years, but a standard protocol for repr