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-free 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 preparation of a sample, wherein the sample comprises an antibody, for a limulus amoebocyte lysate (LAL) assay, wherein the method comprises the following steps in the following order:
(a) adding magnesium ions,
(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.
2. A method for determining bacterial endotoxin in a sample comprising an antibody exhibiting the LER effect, wherein the method comprises the following steps in the following order:
(a) adding magnesium ions to the sample,
(b) diluting the sample,
(c) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution, and
(d) determining bacterial endotoxin in the sample by using a LAL assay.
3. The method of claim 1 , wherein the antibody is formulated with polysorbate 80.
4. The method of claim 1 , wherein the antibody is formulated with a citrate buffer.
5. The method of claim 1 , wherein the antibody is formulated with 25 mM sodium citrate buffer and 700 mg/l polysorbate 80 and has a pH value of 6.5.
6. The method of claim 1 , wherein the antibody is the anti-CD20 antibody rituximab.
7. The method of claim 1 , wherein in step (a) magnesium ions are added to a final concentration of 2.5 to 75 mM.
8. 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.
9. The method of claim 1 , wherein in step (b) the sample is diluted at a ratio of 1:10.
10. The method of claim 1 , wherein during dialysis in step (c) the sample has a pH-value of 6.0-8.0.
11. The method of claim 1 , wherein in step (c) the dialysis takes 24 hours at room temperature.
12. 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.
13. The method of claim 1 , wherein for the dialysis in step (c) a cellulose acetate membrane is used.
14. The method of claim 1 , wherein for the dialysis in step (c) the aqueous solution is changed twice.
15. The method of claim 1 , further comprising the step of producing a low endotoxin recovery (LER) positive control 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.
16. A method for rendering a sample comprising an antibody reactive to factor C in a LAL enzymatic cascade, comprising the steps of:
(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.
17. The method of claim 2 , wherein the antibody is a therapeutic antibody.
18. The method of claim 2 , wherein the antibody is formulated with polysorbate 80.
19. The method of claim 2 , wherein the antibody is formulated with a citrate buffer.
20. The method of claim 2 , wherein the antibody is formulated with 25 mM sodium citrate buffer and 700 mg/l polysorbate 80 and has a pH value of 6.5.
21. The method of claim 2 , wherein the antibody is the anti-CD20 antibody rituximab.
22. The method of claim 2 , wherein in step (a) magnesium ions are added to a final concentration of 25 to 75 mM.
23. The method of claim 2 , 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.
24. The method of claim 2 , wherein in step (b) the sample is diluted at a ratio of 1:10.
25. The method of claim 2 , wherein during dialysis in step (c) the sample has a pH-value of 6.0-8.0.
26. The method of claim 2 , wherein in step (c) the dialysis takes 24 hours at room temperature.
27. The method of claim 2 , wherein for the dialysis in step (c) a membrane with a molecular-weight cut-off of 10 kDa is used.
28. The method of claim 2 , wherein for the dialysis in step (c) a cellulose acetate membrane is used.
29. The method of claim 2 , wherein for the dialysis in step (c) the aqueous solution is changed twice.
30. A method for rendering a sample comprising an antibody reactive to factor C in a LAL enzymatic cascade, comprising the steps of:
(a) adding magnesium ions to the sample,
(b) diluting the sample,
(c) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution, and
(d) determining bacterial endotoxin in the sample by using a LAL assay,
wherein the antibody is a monoclonal therapeutic antibody.
31. The method according to claim 1 , wherein said magnesium ions are MgCl 2 .
32. The method according to claim 2 , wherein said magnesium ions are MgCl 2 .
33. The method according to claim 8 , the pH-value of the sample is adjusted by diluting the sample with 10-50 mM Tris/HCl buffer 6.0-8.0.
34. The method according to claim 16 , wherein said magnesium ions are MgCl 2 .
35. The method according to claim 23 , wherein the pH-value of the sample is adjusted by diluting the sample with 10-50 mM Tris/HCl buffer pH 6.0-8.0.
36. The method according to claim 30 , wherein said magnesium ions are MgCl 2 .
37. A method for preparation of a sample, Wherein the sample comprises an antibody, for a limulus amoebocyte lysate (LAL) assay, wherein the method comprises the following steps in the following order:
(a) adding magnesium ions,
(b) diluting the sample,
(c) dialyzing the sample having a pH-value of 5.7-8.0 against an endotoxin-free aqueous solution, and
(d) determining bacterial endotoxin in the sample by using a LAL assay,
wherein the antibody is a therapeutic antibody.
38. The method of claim 37 , wherein the antibody is formulated with polysorbate 80.
39. The method of claim 37 , wherein the antibody is formulated with a citrate buffer.
40. The method of claim 37 , wherein the antibody is formulated with 25 mM sodium citrate buffer and 700 mg/l polysorbate 80 and has a pH value of 6.5.
41. The method of claim 37 , wherein the antibody is the anti-CD20 antibody rituximab.
42. The method of claim 37 , wherein in step (a) magnesium ions are added to a final concentration of 25 to 75 mM.
43. The method of claim 37 , 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.
44. The method of claim 37 , wherein in step (b) the sample is diluted at a ratio of 1:10.
45. The method of claim 37 , wherein during dialysis in step (c) the sample has a pH-value of 6.0-8.0.
46. The method of claim 37 , wherein in step (c) the dialysis takes 24 hours at room temperature.
47. The method of claim 37 , wherein for the dialysis in step (c) a membrane with a molecular-weight cut-off of 10 kDa is used.
48. The method of claim 37 , wherein for the dialysis in step (c) a cellulose acetate membrane is used.
49. The method of claim 37 , wherein for the dialysis in step (c) the aqueous solution is changed twice.
50. The method of claim 37 , further comprising the step of producing a low endotoxin recovery (LER) positive control 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.
51. The method according to claim 37 , wherein said magnesium ions are MgCl 2 .
52. The method according to claim 43 , the pH-value of the sample is adjusted by diluting the sample with 10-50 mM Tris/HCl buffer 6.0-8.0.