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Method of producing membrane vesicles

A technology of membrane capsules and purification membranes, applied in biochemical equipment and methods, chemical instruments and methods, microorganisms, etc., can solve the problems of cell debris pollution, low recovery, poor reproducibility of operator changes, etc., and achieve high occupancy, Effect of increasing immunogenic potential, increasing efficiency of direct loading

Inactive Publication Date: 2003-06-25
阿诺塞斯公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are largely unsuitable for clinical use for a number of reasons: 1) length of time, 2) scale-up and availability in a GMP environment, 3) significant risk of contamination by cellular debris, 4) operator Poor reproducibility of changes in , 5) due to exosome aggregation at the time of pellet formation (locally high exosome concentration in the pellet), and 6) low recovery at the end of final processing

Method used

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  • Method of producing membrane vesicles
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preparation example Construction

[0201] In the preparation method of the present invention, other techniques, such as ion exchange and affinity chromatography and flow field-flow fractionation, can also be used to concentrate Tissa body. percolation

[0202] Diafiltration can be used on concentrated Tissa system preparations to reduce the concentration of contaminating media and cellular proteins. Diafiltration is performed according to several techniques that allow exchange of sample buffer with formulated buffer, including ultrafiltration, chromatography, ultracentrifugation, and through dialysis bags.

[0203] In a preferred embodiment, diafiltration is performed using an ultrafiltration system. This particular embodiment is effective as demonstrated in the experimental section. Furthermore, if the capsule preparation has been concentrated by ultrafiltration, the diafiltration steps can easily be combined using the same methodology.

[0204] In this regard, in certain embodiments, exosomes are diafilte...

Embodiment 1

[0265] Other aspects and advantages of the present invention are shown in the following examples, which should be considered as illustrations and not as limitations of the invention. Embodiment 1. Preparation of culture medium

[0266] Prior to use, the medium has been treated by ultrafiltration (in this example, clinical grade AIM V is a serum-free cell culture medium from Life Technologies, Inc.) in order to remove clumped proteins, removal of clumped proteins It does not affect cell growth, but considerably contributes to the subsequent isolation of pure Disa bodies.

[0267] Ultrafiltration of the medium was performed using a 500 kDa hollow fiber membrane (from A / G Technology, NeeDham, Mass or from a vendor with related products). The size of the membrane retains protein aggregates in the retentate while allowing non-aggregated proteins to pass through with the filtrate. The filtrate was collected, sterile filtered through 0.22 micron filter paper into vials, and stored ...

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Abstract

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 analyzing 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.

Description

field of invention [0001] The present invention relates to methods for the preparation of biological substances for various experimental, diagnostic or therapeutic uses, including immunotherapeutic treatment or tumor prevention. More specifically, the present invention relates to methods for preparing membrane vesicles, in particular exosomes, released by various types of mammalian cells, including diafiltration and / or density pad centrifugation. The present invention also provides novel methods of characterizing and analyzing exosome preparations, which can be used in quality control assays for pharmaceutical manufacturing purposes, as well as methods of loading immunogenic compounds into membrane vesicles. The present invention is suitable for producing pharmaceutical grade preparations of such capsules and fully characterizing the preparation for use in humans. Background of the invention [0002] Membrane vesicles are essentially spherical sacs, usually less than 130 nm...

Claims

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Application Information

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IPC IPC(8): C12N5/02A61K39/00A61P11/06A61P29/00A61P35/00A61P37/02A61P37/04A61P37/08C12N5/0784C23C16/44C23C16/509G01N33/543H01J37/32
CPCC12N2500/99A61K2039/55555A61K39/0011H01J37/32431C12N2501/23C23C16/4401C12N2501/22A61K2039/5154C12N5/0639G01N33/5432C23C16/509C12N2501/24C12N2500/90A61P11/06A61P29/00A61P35/00A61P37/02A61P37/04A61P37/08A61K39/4644A61K39/4615A61K39/4622
Inventor 亨利·兰帕斯基可帝斯·路格珍-柏纳德·莱佩克迪-威·徐珍奎-尤恩·耀
Owner 阿诺塞斯公司
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