Bacterial endotoxin test for the determination of endotoxins

Inventors

Alexander, Christian • DEUTSCHMANN, Sven • Lang, Pierre • VON WINTZINGERODE, Friedrich • Zaehringer, Ulrich

Assignees

Foschungszentrum Borstel Leibniz Lungenzentrum • Roche Diagnostics GmbH • Hoffmann La Roche Inc

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-flee aqueous solution, and iv) determining bacterial endotoxin in the sample using a bacterial endotoxin test, particularly the Limulus amoebocyte lysate assay.

Core Innovation

A method is provided for the reduction of endotoxin-masking and/or for overcoming Low Endotoxin Recovery (LER) effect in a limulus amoebocyte lysate (LAL) assay of a sample comprising an antibody. The method is performed with steps in a specified order: adding magnesium ions to the sample, diluting the sample, and dialyzing the sample at a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution.

The endotoxin-masking and/or LER effect is attributed to endotoxin-binding proteins present in the sample and/or formulation ingredients or buffer components. The formulation ingredients or buffer components comprise an amphiphilic compound combined with citrate buffer or phosphate buffer.

The approach is specifically directed to monoclonal therapeutic antibodies and is applicable where the LAL assay system is based on limulus amoebocyte lysate reactivity. By performing magnesium-ion addition followed by dilution and then dialysis within the defined pH range against an endotoxin-free aqueous solution, the method reduces masking and/or overcomes LER effect during the LAL assay for antibody-containing samples.

Claims Coverage

The claim coverage presents one independent claim reciting a complete ordered workflow with three core inventive steps: magnesium-ion addition, dilution, and dialysis at a specified pH window, applied to an antibody-containing sample in an LAL assay. The claim also links the LER/masking problem to endotoxin-binding proteins and specifies formulation/buffer composition features involving amphiphilic compounds combined with citrate or phosphate buffer.

Across the independent claim content, the coverage centers on an ordered three-step sample-preparation workflow for LAL testing, with the assay issue linked to endotoxin-binding proteins and with formulation/buffer composition defined as amphiphilic compounds combined with citrate buffer or phosphate buffer.

Stated Advantages

Documented Applications