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Perfusion medium

A culture medium, cell culture technology, applied in the field of inhibiting cell growth in a perfused state, lipid-based additives, improved cell culture media, can solve problems such as no teaching or prompting, no teaching or prompting of cell growth

Active Publication Date: 2020-05-26
BOEHRINGER INGELHEIM INT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, this reference does not teach or suggest the use of linoleic acid or arachidonic acid as nutritional supplements for use in perfused cultures, nor does it teach or suggest the use of linoleic acid to inhibit the growth of cells in a perfused state

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0220] Example 1. Effect of temperature shift on viability, viable cell density and specific productivity.

[0221] In this example, a bioreactor model was used. Lower temperature shifts are commonly used in cell culture to limit or inhibit cell growth.

[0222] Therefore, this example investigates the effect of temperature shift on (A) percent viability, (B) viable cell density (e 5 cells / mL) and (C) specific productivity (pg / cell / day). See Figure 1. Perfusion was initiated on day 2 of cell culture and gradually increased to 2 vessel volume / day (2VVD) changes. Arrows indicate days (day 7) when the temperature transitioned (to low) from 37 °C to the value indicated in the legend (29 °C – square or 28 °C – triangle).

[0223] In this example, this approach was found to slow growth, but was not found to affect cell specific productivity. In one cell line (data not shown), temperature shifts negatively affected product quality, increasing light chain and basic species. As s...

Embodiment 2

[0224] Example 2. Cell growth inhibition and cell productivity increase by exogenous linoleic acid.

[0225] In this example, a deep well plate model was used. This example demonstrates that linoleic acid inhibits cell growth and increases cell specific productivity (q p ). See Figure 3.

[0226] Linoleic acid was added to cell culture medium at various concentrations of 500 μM, 900 μM, 1350 μM and 1800 μM, and the effect on (A) viable cell density (e 5 cells / ml), (B) percentage of cell viability (%) and (C) specific productivity (q p ) role. Higher concentrations of linoleic acid showed a significant effect on inhibiting cell growth and increasing specific productivity. For example, at day 7, the specific productivity of cells demonstrated with linoleic acid at 500, 900 and 1350 μM was about the same and increased significantly relative to normal perfusion medium. Linoleic acid at 1800 μM was similar to arachidonic acid at 500 μM in terms of viable cell density, viabili...

Embodiment 3

[0228] Example 3. Cell growth inhibition and cell productivity increase by exogenous arachidonic acid.

[0229] In this example, a deep well plate model was used. This example analyzes the effect of arachidonic acid when added to the perfusion medium. See Figure 4.

[0230] The effect of arachidonic acid on (A) viable cell density (e 5 cells / ml), (B) percentage of cell viability (%) and (C) specific productivity (q p ) role.

[0231] It can be seen that arachidonic acid added at a concentration of 500 μM in normal perfusion medium inhibited cell growth by up to 31% and increased specific cell productivity by 46% or more. Thus, arachidonic acid inhibited cell growth and increased cell specific productivity (q p ).

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Abstract

The present disclosure provides a method of culturing mammalian cells, e.g., by perfusion cell culture, expressing a heterologous protein in a cell culture, comprising culturing mammalian cells expressing a heterologous protein in a culture medium comprising an effective amount of one or more lipids or lipid metabolites selected from the group consisting of: linoleic acid, arachidonic acid, and prostaglandin E2, or derivatives and / or precursors thereof. The lipids or lipid metabolites or combinations thereof can lead to growth suppression and / or increased productivity with reduced cell bleed.The present disclosure also provides methods for increasing the productivity of a cell culture by culturing the cells in a culture medium comprising an effective amount of one or more lipids or lipidmetabolites selected from the group consisting of: linoleic acid, arachidonic acid, and prostaglandin E2, or derivatives and / or precursors thereof. The present disclosure also provides culture mediumfor use in producing therapeutic proteins with increased productivity, wherein the medium comprises one or more lipids or lipid metabolites selected from the group consisting of: linoleic acid, arachidonic acid, and prostaglandin E2, or derivatives and / or precursors thereof.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 571,915, filed October 13, 2017, which is incorporated herein by reference in its entirety. technical field [0003] The present invention relates to improved cell culture media and methods which achieve greater cell-specific productivity and better sustained and / or maintained viability relative to prior art methods. The present invention further relates to the use of new and improved lipid-based supplements for cell culture media that are more effective in part by effectively inhibiting cell growth during cell culture, for example during the production phase of perfusion cell culture. Maintain cell culture viability and increase cell specific productivity. More particularly, the invention relates to the use of effective amounts of one or more lipid or lipid metabolite additives, including linoleic acid, arachidonic acid, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12P21/00
CPCC12N5/0018C12N2500/36C12N2501/02C12N2511/00C12P21/02
Inventor H·林S·王郑黎黎
Owner BOEHRINGER INGELHEIM INT GMBH
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