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Novel process for separating and determining the viral load in a pancreatin sample

A viral load and trypsin technology, applied in biochemical equipment and methods, trypsin, microbial determination/testing, etc., can solve the problem that no reliable method for detecting or isolating viral pollutants has been developed, and it is impossible to measure trypsin, etc. question

Active Publication Date: 2010-09-01
ABBOTT LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Until recently, reliable methods for the quantitative detection or isolation of viral contaminants in trypsin samples have not been developed
This may be due to the incompatibility of the enzymatically active components of trypsin with the cell lines typically used to propagate viruses using techniques known to those skilled in the art, thus making it more difficult or even impossible to assay for virus in samples of trypsin Titer

Method used

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  • Novel process for separating and determining the viral load in a pancreatin sample

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Experimental program
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Effect test

Embodiment 1

[0077] Example 1: Study of deleterious effects of trypsin on different cell lines

[0078] In order to detect viruses in test samples of material using cell culture, the deleterious effect on the cells of the trypsin sample under study should be determined so that false negative results can be ruled out when evaluating CPE. Therefore, studies were performed on different cell lines to determine the deleterious effects of trypsin test sample suspensions, as described below.

[0079] A 0.5 ml portion of the PAN suspension prepared above was taken for testing for deleterious effects and was named "pancreatin suspension test sample".

[0080] Low-speed centrifugation: Spin the remaining PAN suspension in a conventional refrigerated centrifuge ( 22R Heraeus , with fixed angle rotor no. 3745) at 10,000 rpm (10,800 xg) and 4°C for 15 minutes. The supernatant after slow centrifugation was then centrifuged at 10,000 rpm and 4°C for an additional 15 minutes and named "supernatant a...

Embodiment 2

[0086] Embodiment 2: the test of different trypsin addition amounts to viral load

[0087] The two-step ultracentrifugation method of the present invention as described above was applied to isolate virus from different suspensions loaded with trypsin powder. Isolation of virus from trypsin is a prerequisite for detection of low viral loads by titration on susceptible cell lines:

[0088] - Mix 8 g of drug substance with 8 ml of antibiotic solution (1.0 g streptomycin sulfate and 1.2 g penicillin G per 20 ml of sterile PBS) and 64 ml of PBS and stir for 60 minutes in an ice-water bath

[0089] - Centrifuge the resulting suspension at 4°C and 10,800xg for 15 minutes

[0090] - Centrifuge the supernatant after the first centrifugation again at 4°C and 10,800xg for 15 minutes

[0091] The obtained supernatant after low speed centrifugation was used for the first ultracentrifugation. The first ultracentrifugation was performed using a discontinuous sucrose gradient (four centr...

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Abstract

Processes for separating a viral load from a pancreatin sample and for quantitatively determining the viral load in a pancreatin sample with high sensitivity are described herein.

Description

technical field [0001] The present invention relates to a method for the substantial quantitative isolation of viral load from a trypsin sample, and a method for the quantitative determination of the viral load of such a trypsin sample with high sensitivity. Background technique [0002] Pancreatin is a long-known mixture of various physiologically active components derived from the mammalian pancreas. The major components of pancreatin are digestive enzymes, especially pancreatic lipase, but also contain amylase and protease. Due to its valuable therapeutic properties and high safety level in use, pancreatin has long been used very successfully as a pharmaceutical preparation in enzyme replacement therapy. Of these pancreatic lipases are of greatest importance, but amylases and proteases also contribute significantly to the therapeutic benefits of pancreatic enzymes. Pancreatin used for therapeutic purposes is usually obtained from bovine ("bovine pancreatin") or porcine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/94C12Q1/24C12Q1/04
CPCC12Q1/24
Inventor D·贝歇尔L·德纳F·吕费尔M·弗林克
Owner ABBOTT LAB INC
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