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Method for enhanced protein stabilization and production of cell liell lines useful for production of such stabilized proteins

A cell line, cell technology, applied in function, total, 1] This application requires the field submitted by the same inventor on May 28, 1999, which can solve the problem of improving the conventional production yield of various biological factors or products, insufficient Effective discovery of biological factors or products, time-consuming and expensive

Inactive Publication Date: 2002-07-17
PHOTOGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the methods and concepts taught in these paradigms, including methods for adding exogenously produced stress proteins and for inducing transient constitutive production of these proteins, are not sufficient to effectively and flexibly enhance multiple biological factors or The conventional production yield of the product is not enough to effectively discover new biological factors or products
For example, making and purifying stress proteins in useful quantities for adding or modifying products would often be too time-consuming and expensive

Method used

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  • Method for enhanced protein stabilization and production of cell liell lines useful for production of such stabilized proteins
  • Method for enhanced protein stabilization and production of cell liell lines useful for production of such stabilized proteins
  • Method for enhanced protein stabilization and production of cell liell lines useful for production of such stabilized proteins

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Embodiment 1

[0045] Generally, in this embodiment, transient stress is applied to eukaryotic cell lines to increase viral titers. Specifically, permissive eukaryotic cells of the desired virus are selected using conventional selection methods. These cells are grown to near confluence under standard conditions and then subjected to transient stress for a time sufficient to stimulate the production of one or more stress proteins. Applicable stress factors include heat stress (such as increasing or decreasing incubation temperature), chemical stress (such as increasing or decreasing pH), oxidative level stress (such as increasing or decreasing O 2 levels), nutrient alterations (such as reducing or increasing essential media components), and any other such factors that can induce transient stress and lead to expression of stress proteins (such as exposure to toxic substances). Following such stress, a stock solution of the desired virus is added to infect the stressed eukaryotic cells. Prefe...

Embodiment 2

[0055] More specifically, permissive eukaryotic cells of the desired virus are selected using conventional selection methods and grown to near confluence under standard conditions. By mixing stress protein expression vectors with lipid / phospholipid formulations to produce approximately 0.01-100 μg vector / 10 4 -10 6 A transfection agent for target cells, thereby preparing a DNA transfection agent for these cells. Preferably, such lipid / phospholipid formulations comprise cationic lipids or phospholipids, more preferably dioleoylphosphatidalethanolamine. This transfection agent is then delivered to a permissive cell culture, allowing transfection to occur. After transfection, cells were incubated in fresh medium. To increase the transfection efficiency, the transfection step can then be repeated. The resulting genetically heterogeneous cell cultures are then purified by selection of cell lines exhibiting high yields of expression of the transfected stress protein and increase...

Embodiment 3

[0062] In this embodiment, genetically modifying a non-permissive eukaryotic cell to become permissive, and the generation of a new permissive eukaryotic cell line is achieved by inserting a stress protein expression vector into the non-permissive eukaryotic cell line. The new permissive cell lines are then used to efficiently produce viral agents by inoculating the cell lines with infectious or potentially infectious material, followed by incubation and harvesting of the virus or virus products thus produced. This cell line is preferred for facilitating the replication of difficult-to-culture neural permissives and media that have never been cultured before. Thus, this cell line can be used both as a virus hunter and for the production or manufacture of useful quantities of the medium. Example 3. Generation of new permissive cell lines

[0063] Example 3 illustrates an example of the third embodiment. However, the invention and the third embodiment are not limited to the de...

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Abstract

A method for increasing production yield of viruses, viral proteins, and other related biological materials through enhanced control and stabilization of protein production via stress proteins and the resultant protein products. The present invention is also directed to methods for selection or engineering of cell lines yielding such enhanced stabilized products. More specifically, example embodiments of the present invention are directed to methods for enhancing production of a viral agent, production of cell lines exhibiting permanent genetic modification, production of permissive eucaryotic cell lines, enhancing functional recombinant product yield, and the products of such methods.

Description

Background of the invention [0001] This application claims the benefit of co-pending US Provisional Application Serial No. 60 / 136,676, filed May 28, 1999, by the same inventor. [0002] The present invention relates to the production and stabilization of functional biological substances, such as proteins, viral agents, or other biological agents or products, including, but not limited to, enzymes, hormones, growth factors, structural proteins, tumor suppressors, nucleic acids, and nucleic acid Probes, vaccines, antigens, antibiotics, lipids, simple and complex carbohydrates, alcohols and other solvents, and products of these methods. These functional biological substances are useful, for example, in the manufacture of vaccines and diagnostic assays or tests. [0003] However, the production of viruses, viral antigens, and other viral products useful in the manufacture of products such as vaccines and diagnostic assays is expensive, time consuming, and requires a high level of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/005C07K14/47C12N1/15C12N1/19C12N5/02C12N5/08C12N5/10C12N15/09C12N7/00C12N7/01C12P21/02C12P21/04
CPCC12N7/00C12N2760/18451C07K14/47
Inventor H·C·迪斯J·斯莫里克
Owner PHOTOGEN INC
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