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Improved bacterial endotoxin test for the determination of endotoxins

A bacterial endotoxin and endotoxin technology, applied in the preparation of test samples, measuring devices, and resistance to vector-borne diseases, etc., can solve the problems of underestimation and undetection of endotoxin pollution, so as to improve health status and improve Detection, life-saving effects

Active Publication Date: 2018-05-11
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Potential endotoxin contamination that occurs during manufacturing remains underestimated or undetected when using conventional LAL assays due to the LER effect

Method used

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  • Improved bacterial endotoxin test for the determination of endotoxins
  • Improved bacterial endotoxin test for the determination of endotoxins
  • Improved bacterial endotoxin test for the determination of endotoxins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0269] Example 1: Technical Equipment and Reagents

[0270] 1. Technical equipment

[0271] 1.1 Microplate reading system (also referred to as "reader" herein)

[0272] · PRO, Multimode Microplate Reader; Tecan / Tecan Deutschland GmbH, Switzerland, P / N: 30050303, Germany.

[0273] ·Magellan V.7.1 software

[0274] ·Costar TM Cell culture plate, 96-well, Fisher Scientific, P / N: 07-200-89.

[0275] 1.2 Shaker system and glass vials

[0276] • Multi Reax; Heidolph, Germany, P / N: 545-10000-00.

[0277] • 1.5 ml screw top glass bottle (N8); Macherey-Nagel GmbH & Co. KG, Germany, P / N: 702004 (Qty. 100).

[0278] · N 8PP nut, black, top closed; Macherey-Nagel GmbH & Co. KG, Germany, P / N: 70250 (Qty. 100).

[0279] • 4 ml screw top glass bottle (N13); Macherey-Nagel GmbH & Co. KG, Germany, P / N: 702962 (Qty. 100).

[0280] · N 13PP nut, black, top closed; Macherey-Nagel GmbH & Co. KG, Germany, P / N: 702051 (Qty. 100).

[0281] 1.3 Dialysis equipment

[0282] ·Rotary Dialyz...

Embodiment 2

[0318] Embodiment 2: The present invention overcomes the method for LER effect

Embodiment 21

[0319] Example 2.1: A solution to overcome the LER effect

[0320] In this example, rituximab and rituximab placebo were used as samples. However, as described below, the protocols described herein can be used to overcome LER in all drug antibody typical formulations.

[0321] Materials used in this example

[0322] -membrane:

[0323] 10kDa cellulose acetate (CA) membrane from Harvard Apparatus, USA, P / N: SP1 7425-CA10K

[0324] - Dialyzer:

[0325] FastSpin DIALYZER, chamber volume 1000 μl, Harvard Apparatus, USA, P / N 740510 (Qty.1) or 740504 (Qty.5)

[0326] - Sample vials:

[0327] ·1.5ml screw top glass bottle (N8); Macherey-Nagel GmbH&Co.KG, Germany, P / N: 702004

[0328] · N 8PP nut, black, top closed; Macherey-Nagel GmbH&Co.KG, Germany, P / N: 70250

[0329] - Crystallization dish:

[0330] ·900ml, Duran, VWR Germany, P / N:216-1817

[0331] -MgCl 2 - stock solution:

[0332] · 1M MgCl2 (magnesium chloride hexahydrate) in water for analysis ACS, ISO, Reag. P...

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Abstract

Herein is reported a method for determining bacterial endotoxin at low concentrations in a sample of an antibody (that has been produced using bacterial cells) comprising the following steps in the following order: i) adding magnesium ions to the sample, ii) diluting the sample, iii) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution, and iv) determining bacterial endotoxin in the sample using a bacterial endotoxin test, particularly the limulus amoebocyte lysate assay.

Description

technical field [0001] This paper reports a bacterial endotoxin test (BET) sample preparation method that overcomes the low endotoxin recovery (LER) effect due to endotoxin shielding. Background technique [0002] Protein therapeutics such as monoclonal antibodies are often produced using genetically transformed eukaryotic and prokaryotic cells such as bacteria. For bacterial production fast growing bacteria such as Escherichia coli (Escherichia coli) are used. However, highly toxic lipopolysaccharide (LPS) is secreted into the medium during growth and culture of recombinant proteins. These components are known as bacterial endotoxins (abbreviated endotoxins). Gram-negative bacteria have LPS as an important component of their cell wall. Gram-negative bacterial cells contain approximately 3.5x 10 5 LPS molecules occupy a total area of ​​about 4.9 μm 2 (Rietschel, 1994, FASEB J. 8:217-225). In the case of E. coli, this means that LPS makes up about three-quarters of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/579
CPCG01N33/579G01N33/56911G01N1/38Y02A50/30G01N2333/195G01N2400/50G01N33/6854
Inventor C·亚历山大S·多伊奇曼P·朗F·范温钦杰罗德U·策林格
Owner F HOFFMANN LA ROCHE & CO AG
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