Bacterial endotoxin test for the determination of endotoxins
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.
1. A method 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, wherein the method comprises the following steps in the following order:
(a) adding magnesium ions to the sample,
(b) diluting the sample, and
(c) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution,
wherein:
the antibody is a monoclonal therapeutic antibody,
said endotoxin masking and/or said LER effect is caused by endotoxin-binding proteins present in said sample and/or formulation ingredients or buffer components, and
the formulation ingredients or buffer components comprise an amphiphilic compound combined with citrate buffer or phosphate buffer.
2. The method of claim 1 , wherein said sample is a formulation sample.
3. The method of claim 1 , wherein said magnesium ions in step (a) are added in form of MgCl 2 .
4. The method of claim 1 , wherein in step (a) magnesium ions are added to a final concentration of about 10 to 100 mM.
5. The method of claim 1 , wherein the antibody is formulated with polysorbate 80 and with a citrate buffer.
6. The method of claim 5 , wherein the antibody is formulated with about 25 mM sodium citrate buffer and about 700 mg/l polysorbate 80 and has a pH value of about 6.5.
7. The method of claim 1 , wherein said method comprises additionally the production of a low endotoxin recovery (LER) positive control by spiking a known amount of endotoxin into an aliquot of the sample comprising the antibody.
8. The method of claim 7 , wherein said LER positive control exhibits a LER effect if steps (a) to (c) of the method of claim 1 has not been performed on the positive control.
9. The method of claim 8 , wherein said production of said low endotoxin recovery (LER) positive control comprises shaking of the endotoxin spiked aliquot.
10. The method of claim 9 , wherein said production of said low endotoxin recovery (LER) positive control is by spiking a known amount of endotoxin into an aliquot of the sample and shaking the endotoxin spiked aliquot of the sample for 60 min to 2 hours.
11. The method of claim 7 , wherein said production of said low endotoxin recovery (LER) positive control comprises spiking said aliquot of the sample comprising the antibody with Controlled Standard Endotoxin (CSE).
12. The method of claim 11 , wherein said CSE spiked to said aliquot is in a defined concentration.
13. The method of claim 1 , wherein the antibody is the anti-CD20 antibody rituximab.
14. The method of claim 1 , wherein in step (b) the pH-value of the sample is adjusted by diluting the sample with 10-50 mM Tris/HCl buffer pH 6.0-9.0.
15. The method of claim 1 , wherein in step (b) the sample is diluted at a ratio of 1:10.
16. The method of claim 1 , wherein during dialysis in step (c) the sample has a pH-value of 6.0-8.0.
17. The method of claim 1 , wherein in step (c) the dialysis takes about 24 hours at room temperature.
18. The method of claim 1 , wherein for the dialysis in step (c) a membrane with a molecular-weight cut-off of 10 kDa is used.
19. The method of claim 1 , wherein for the dialysis in step (c) a cellulose acetate membrane is used.
20. The method of claim 1 , further comprising changing water twice in dialysis step (c).
21. The method according to claim 1 , wherein said amphiphilic compound is a non-ionic detergent.
22. The method according to claim 21 , wherein said non-ionic detergent is polysorbate.
23. The method according to claim 22 , wherein said polysorbate is polysorbate 80.
24. The method according to claim 4 , wherein said magnesium ions are added to a final concentration of about 25 to 75 mM.
25. The method according to claim 9 , wherein said shaking is for about 45 min to about 2 hours.
26. The method according to claim 12 , wherein said defined concentration is about 0.5 or about 5 EU/ml.
27. The method according to claim 14 , wherein said buffer is at pH 6.0-8.0.