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Serum-free VERO cell banking process

一种无血清、无血清培养基的技术，应用在动物细胞、细胞培养基、生物化学设备和方法等方向，能够解决组成改变、妨碍产物纯化、朊病毒污染等问题

Active Publication Date: 2011-02-09
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But serum-containing culture systems are becoming less popular for large-scale production of vaccines
Serum supplementation has many disadvantages, including compositional variation between batches, high protein content that prevents product purification, and potential for viral, mycoplasma, or prion contamination
Furthermore, recent threats to human health caused by unidentified factors of bovine spongiform encephalopathy (BSE) may limit the continued use of bovine serum in culture methods for the synthesis of health care products such as viral vaccines [Butler et al. Biotechnol.Prog., 16, 854-858 (2000)]

Method used

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  • Serum-free VERO cell banking process
  • Serum-free VERO cell banking process
  • Serum-free VERO cell banking process

Examples

Experimental program
Comparison scheme
Effect test

example

[0035] Several agents were tested as low temperature stabilizers in this study. Cell banks containing Hy-Soy, HyPep Rice, HyPep Wheat, and methylcellulose were compared to cell banks containing FBS and tested for cell bank stability. Hy-Soy and HyPep Rice proved to be as effective as FBS as low temperature stabilizers for the first year of the study. Thus, Hy-Soy can be used in serum-free cell banks and HyPep Rice can be used as a substitute.

[0036] Materials and methods

[0037] Personal protective equipment: sterile gloves, sterile cuffs, sterile coveralls, clean room hoods, clean room beards, and clean room foot covers.

[0038] Disposable Sets: Disposable Serological Pipettes; 1.8ml Sarstedt Cryotubes; Corning Coastar 75cm 2 and 150cm 2 Tissue Culture Flask; Ten-Disc Nunc Cell Factory [www.nuncbrand.com].

[0039] Glassware and Accessories: Glass cover with C-bend for aliquoting media from 10L and 20L total media; Gelman filter with C-bend; Cell Factory adapter; wit...

example 1

[0043] Thawing and Cell Growth

[0044] Put two containing 2×10 7 Frozen stock tubes of Vero cells were placed in a 37°C water bath. The tubes were kept in the water bath with agitation and checked frequently until the frozen cells were thawed. The outside of the tube was cleaned with 70% isopropanol and placed in a laminar flow hood. Quickly transfer thawed cells to a T-150cm 2 bottle and add 50 ml of growth medium dropwise while shaking the culture vessel. Growth medium consisted of VP-SFM with 4 mM L-glutamine. After about half of the medium was added, the remainder was added at a slightly faster rate. The cells were placed in a Forma incubator (37°C, 5% CO 2 ) and allowed to attach overnight. The medium was degassed and 50 ml of fresh growth medium was added to the bottle the next day.

example 2

[0046] cell delivery

[0047] Incubate cells in a Forma incubator at 37 °C with 5% CO 2 Under cultured T-150cm 2 Grow in bottles to confluency (4 or 5 days). The medium was vented and the bottle was washed twice with 20 ml PBS without magnesium and calcium. 5 ml of trypsin was added to the flask and the flask was incubated at room temperature for 2-3 minutes to remove cells from the flask. Trypsin was neutralized with 5 ml of STI and 10 ml of VP-SFM was added for nutritional support until the procedure was complete. The cells now in suspension were sampled for enumeration. Add 1 mL of trypan blue solution to 1 mL of the cell sample. Mix it gently with a vortex shaker. Place 10 µl of the mixture in the chamber of a hemocytometer. Cells that did not undergo staining were counted as viable and total cell numbers were determined.

[0048] The cell suspension was then divided into 4 × 10 4 The concentration of cells / cm2 was inoculated into five new T-150cm 2 in the bottle...

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Abstract

A serum-free Vero cell banking process provides a standardized and consistent way to generate reliable and stable cell banks for viral vaccine production. The substitution of animal-derived substances with plant-derived substances in the growth and freezing media increases the viability of the cells upon thawing and reduces their recovery time, thereby allowing a more precise schedule for manufacturing and more consistent processes.

Description

technical field [0001] The present invention relates to a serum-free cell freezing medium and the use of this medium in a method for generating a stable serum-free cell bank for virus vaccine production. Background technique [0002] Fetal bovine serum (FBS) is a low temperature stabilizer commonly used in cell banking. But serum-containing culture systems are becoming less popular for large-scale production of vaccines. Serum supplementation presents a number of disadvantages, including variation in composition from batch to batch, high protein content that prevents product purification, and the potential for viral, mycoplasma, or prion contamination. Furthermore, recent threats to human health caused by unidentified factors of bovine spongiform encephalopathy (BSE) may limit the continued use of bovine serum in culture methods for the synthesis of health care products such as viral vaccines [Butler et al. Biotechnol. Prog., 16, 854-858 (2000)]. Therefore, non-animal der...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N7/00C12N5/00
CPCC12N5/0031C12N2500/32C12N2500/76C12N5/0043C12N5/06C12N5/00
Inventor 埃内·阿利克梅茨艾米·海伦·尼科尔斯多萝西·琼·普鲁默
Owner WYETH LLC
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