Sampling for endotoxin analysis
Because of the high viscosity, it was crucial to mix the formulation thoroughly after elution through the Sartobind Q15 anion
exchanger. As the absorption efficiency of the Q15 will decline rather sharply when the capacity is exceeded, a marked difference
in the endotoxin level can be observed between the start and end of the removal process.
Analysis of endotoxin content
All reagents and standards were supplied by Lonza (Basel, Switzerland). The chromogenic method was used for determination
of the endotoxin content with a sensitivity of 0.005 EU/mL, as described in the pharmacopeia. A dilution of 1:200 was prepared
by diluting 50 µl formulation in 9.95 mL WFI, and the sample was analyzed on the kinetic QCL instrument together with a positive
control. If the positive control was positive, the test was considered valid and the results were automatically calculated
by the software. No endotoxin present would give a result of NMT 1 EU/mL.
Table I: Values of endotoxin, pH and Gd/Iodine concentrations of the formulations before and after treatment with Sartobind
Q15. Note that the values of endotoxin are the measured ones and differ from the theoretical values cited in the experimental
Application to formulations containing Na
Occasionally, unintentional contamination of sterile formulations takes place. The following describes such an example where
the method was used to depyrogenize formulations of X-ray agents having endotoxin levels far above values accepted for injection.
The formulations were made with different ratios of Na+ and Ca2+ that were important to preserve. In addition to the equilibration and sterilization procecures described above, the anion
exchange membranes were equilibrated with TRIS-buffers having the same Ca2+/Na+ ratios as the formulations. The volumes of formulations were 100 mL, and Sartobind Q100 (100 cm2 ) membrane filters having a theoretical capacity for endotoxin of 4750 EU were used.
Figure 1: The capacity of Sartobind Q15 for lipopolysaccharide (LPS) of Escherichia Coli. Comparison of the endotoxin content
pre- and post-filtration with the anion exchange membrane disclose that the approximate capacity for each cartridge is around
450–460 European Units (EU). Within this capacity range the filtrate has a measured endotoxin concentration < 0.5 EU/mL that
is safe for intravenous injection into experimental animals.