Methods of producing membrane vesicles

Details Methods of producing membrane vesicles Methods of producing membrane vesicles

2000-04-27

2004-11-02

Saunders, David (Department: 1644)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S355000, C435S371000, C435S372000, C435S372100, C435S820000

Reexamination Certificate

active

06812023

ABSTRACT:

FIELD OF INVENTION
The present invention relates to methods of preparing biological material, for use in various experimental, diagnostic or therapeutic uses, including immunotherapy treatment or prophylaxy of tumors. More particularly, the present invention relates to methods of preparing membrane vesicles (in particular exosomes) released by various types of mammalian cells, comprising diafiltration and/or density cushion centrifugation. The invention also provides novel methods for characterizing and analysing exosome preparations, which can be used in quality control assay for the purpose of pharmaceutical product production. The invention is suitable to produce pharmaceutical grade preparations of such membrane vesicles and to fully characterize said preparations, for use in human beings.
BACKGROUND OF THE INVENTION
Membrane vesicles are essentially spherical vesicles, generally less than 130 nm in diameter, composed of a lipid bilayer containing a cytosolic fraction. Particular membrane vesicles are more specifically produced by cells, from intracellular compartments through fusion with the plasmic membrane of a cell, resulting in their release in biological fluids or in the supernatant of cells in culture. Such vesicles are generally referred to as exosomes. Exosomes arc more particularly between about 30 and about 120 nm, preferably 50 and 90 nm, more specifically between about 60 and 80 nm in diameter and, advantageously, carry membrane proteins (particularly major histocompatibility complex proteins or other protein which directly or indirectly participate in antigen presentation). In addition, depending on their origin, exosomes comprise membrane proteins such as MHC I, MHC II, CD63, CD81 and/or HSP70 and have no endoplasmic reticulum or Golgi apparatus. Furthermore, exosomes are void of nucleic acids (e.g. DNA or RNA).
Exosome release has been demonstrated from different cell types in varied physiological contexts. In particular, it has been demonstrated that B lymphocytes release exosomes carrying class II major histocompatibility complex molecules, which play a role in antigenic presentation (Raposo et al., J. Exp. Med. 183 (1996) 1161). Similarly, it has been demonstrated that dendritic cells produce exosomes (i.e., dexosomes, Dex), with specific structural and functional characteristics and playing a role in immune response mediation, particularly in cytotoxic T lymphocyte stimulation (Zitvogel et al., Nature Medicine 4 (1998) 594). It has also been demonstrated that tumor cells secrete specific exosomes (i.e., texosomes, Tex) in a regulated manner, carrying tumor antigens and capable of presenting these antigens or transmitting them to antigen presenting cells (patent application No. WO99/03499). It is also known that mastocyte cells accumulate molecules in intracellular vesicular compartments, which may be secreted under the effect of signals (Smith and Weis, Immunology Today 17 (1996) 60). Therefore, as a general rule, cells appear to emit signals and communicate with each other via membrane vesicles that they release, which may carry antigenic proteins (or polypeptides or peptides), MHC molecules or any other signal (cytokine, growth factor, etc.) with specific structural and functional characteristics, produced in different physiological situations. These vesicles, particularly exosomes, thus represent a product of particular interest for diagnostic, vaccination or therapeutic applications or to deliver molecules of interest. Therefore, it would be of particular interest to have an effective method that could be used at an industrial scale to prepare membrane vesicles compatible with biological use, particularly pharmacological use.
Conventional methods to prepare membrane vesicles (e.g. exosomes) involve a series of differential centrifugation steps to separate the vesicles from cells or cell debris present in the culture medium. In this regard, the documents mentioned above essentially describe the preparation of vesicles with a series of centrifugations at 300 g, 10,000 g and 70,000 g or 100,000 g, upon which the resulting pellet at the bottom of the tube is resuspended to {fraction (1/1000)}
th
its original volume with a saline solution to constitute a concentrated exosome solution. However, these methods are essentially unsuitable for clinical applications for a number of reasons: 1) length of time, 2) scale-up and validation in GMP environment, 3) significant risk of contamination by cell debris, 4) poor reproducibility due to operator variability, 5) aggregation of exosomes resulting from pelleting (high localized exosome concentration in pellet) and 6) low recovery at end of processing. There is therefore a need for improved methods of preparing membrane vesicles, suitable with industrial constraints and allowing production of vesicle preparations of therapeutic quality.
International application n° PCT/FR00/00105 discloses methods of preparing membrane vesicles through chromatographic techniques, such as anion exchange chromatography and/or gel permeation chromatography.
SUMMARY
The present invention now provides novels methods of preparing membrane vesicles in high yields, high purity, and in relatively short periods of time. The present invention also discloses methods of characterizing (or analyzing or dosing) a membrane vesicle preparation, which can be used in pharmaceutical production to determine the activity, phenotype and/or quantity of vesicles. The invention now allows the production and characterization of clinically acceptable lots of membrane vesicles, with reproducibility, limited operator variation, and increased product quality. This invention further relates to methods of removing particulate bodies, such as haptoglobin, from various medium or compositions, the resulting compositions and media and their uses.
More specifically, an aspect of the present invention resides in methods of preparing membrane vesicles using density cushion centrifugation.
Another aspect of the present invention resides in methods of preparing membrane vesicles using a series of ultrafiltration steps and/or clarification step, more specifically a combination of a concentration and diafiltration by ultrafiltration, preferably preceded by a clarification.
Another aspect of this invention resides in methods of preparing membrane vesicles using a combination of density cushion centrifugation and ultrafiltration and/or clarification step, more specifically a combination of a concentration and diafiltration by ultrafiltration, preferably preceded by a clarification, followed by density cushion centrifugation.
In a particular aspect, the method of this invention comprises a density cushion centrifugation preceded or followed by a diafiltration.
The method of this invention can be applied to various biological samples containing membrane vesicles, including a biological fluid, a culture supernatant, a cell lysate or a pre-purified solution. In a particular embodiment, the method is used to prepare (e.g., purify or separate or isolate) membrane vesicles from a biological sample enriched with membrane vesicles.
A particular aspect of the present invention resides in a method of preparing membrane vesicles from a biological sample, comprising:
a. the culture of a population of membrane vesicle-producing cells under conditions allowing the release of the vesicles,
b. a membrane vesicle enrichment step, and
c. the treatment of said enriched biological sample by density cushion centrifugation.
In a further preferred embodiment, the membrane vesicle-producing cells are cultured in a culture medium with reduced particulate bodies' content, preferably a medium deprived of haptoglobin aggregates. As will be demonstrated in this application, the use of such a medium allows increased production yields and/or higher purity and quality levels to be achieved.
The enrichment step may comprise one or several centrifugation, clarification, ultrafiltration, nanofiltration, affinity chromatography and/or diafiltration steps. More preferably, the enrichment step comprises a c

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