The following applications were developed to demonstrate endotoxin removal from plasma derivatives. The final endotoxin levels reflect application needs. Lower final endotoxin levels can be achieved by increasing residence times on the column or increasing resin-to-sample ratios.
All solutions were prepared in endotoxin-free water (LAL Reagent Water, Bio-Whittaker Inc., Walkersville, MD). Endotoxin was measured using the Pyrogent Plus and chromogenic QCL-1000 LAL test kits from Bio-Whittaker. Sterile, disposable plasticware and glassware were used at all times to reduce the chance of endotoxin contamination. ActiClean Etox resin was packed into 2 ml bed volume columns. The columns were cleaned by perfusion with 30 ml of 1M NaOH, allowed to stand at 40C overnight, and washed with LAL water until neutrality. A sample of the final wash must test negative for endotoxin.
1. Sterile, 25% human serum albumin solution (HSA, Alpha Therapeutic Corp., Los Angeles, CA) was spiked with standard E. coli endotoxin to 30 EU/ml and adjusted to pH 7.0. Ten ml of the HSA solution were perfused through two columns set in series at approximately 0.3 ml/min. Five fractions were collected at 2.0 ml increments, then diluted 1 to 10 and heat inactivated (70 0C for 10 min) to prevent any enhancement or inhibition of the endotoxin assay. The fraction with the highest protein concentration was selected, and further dilutions were made to the maximum of expected lysate sensitivity.
A significant reduction in endotoxin was achieved while more than 99% of HSA was recovered.
2. A pyrogenic lot of human Intravenous Immunoglobulin solution (5%) was treated with ActiClean Etox. Ten ml of the IgG sample were perfused through the column at approximately 0.3 ml/min. Five fractions were collected at 2.0 ml increments, then diluted 1 to 3 and heat inactivated at 700C for 10 min. The fraction with the highest protein concentration was selected, and further dilutions made to the maximum of expected lysate sensitivity.
A 10-fold reduction in endotoxin was achieved while more than 98% of IgG was recovered.
3. A sterile, affinity-purified Factor VIII solution (Alpha Therapeutic Corp., Los Angeles, CA) containing 100 units Factor VIII/ml was spiked with E. coli endotoxin to 20 EU/ml final concentration and adjusted to pH 7.0. Ten ml of the Factor VIII solution were perfused through the column at 0.3 ml/min. Five fractions were collected at 2.0 ml increments, then diluted 1 to 3 and heat inactivated as above. The fraction with the highest protein concentration was selected, and further dilutions were made to the maximum of expected lysate sensitivity.
Endotoxin was reduced below 0.1 EU/ml while Factor VIII recovery, determined by a chromogenic assay, was found to be greater than 95%.
4. A sterile, affinity-purified Factor IX solution (Alpha Therapeutic Corp., Los Angeles, CA) containing 100 units Factor IX/ml was spiked with E. coli endotoxin to 40 EU/ml and adjusted to pH 7.0. Ten ml of the Factor IX solution were perfused through the column at 0.3 ml/min. Five fractions were collected at 2.0 ml increments, then diluted 1 to 3 and heat inactivated. The fraction with the highest protein concentration was selected, and further dilutions were made to the maximum of expected lysate sensitivity.
Endotoxin was reduced to 0.5 EU/ml while Factor IX recovery, determined by a chromogenic assay, was found to be greater than 98%.
5. Sterile fetal calf serum (Biocell Laboratories, Rancho Dominguez, CA) was spiked with E. coli endotoxin to 10 EU/ml final concentration and adjusted to pH 7.0. Ten ml of the serum were perfused through the ActiClean Etox column at 0.3ml/min. Five fractions were collected at 2.0 ml increments, and then diluted 1 to 3 and heat inactivated. The fraction with the highest protein concentration was selected, and further dilutions made to the maximum of expected lysate sensitivity.
The treatment reduced the endotoxin level in the serum down to 0.12 EU/ml.
