Endotoxin Removal and Prevention for Pre-Clinical Biologics Production

The removal and prevention of endotoxin from protein solutions is key to the success of recombinant protein production, especially in the pre-clinical manufacturing setting. The facilities for protein production at this scale (mg to low gram quantities) occur mostly in a non-GMP-quality facility, lacking strict “clean-in-place” procedures and changeover procedures in-between the hundreds of requests placed per year. Often these productions are carried out in transient cell culture systems, introducing plasmid DNA from bacterial preparations, and media and buffers throughout the purification process are made at the bench, not in a clean room. With all of these opportunities for endotoxin introduction, specific prevention and removal strategies must be in place and evaluated to clear the endotoxin from the protein preparation. The failure to remove endotoxin to an acceptable level may cause a pyrogenic response in animals. Although the acceptable limit for humans is 5 endotoxin units (EU) per kg body weight per hour (Eurpoean Pharmacopoeia, 1997), very often material used for pre-clinical studies will be introduced into animal models that have a much higher sensitivity. Rabbits or swine, for example, have a much higher sensitivity to endotoxin as compared to rats or mice (C. Galanos, 1993) (Malyala P., 2008). Care must be taken to provide material that will fit the guidelines of the intended use. In the pre-clinical setting with tight timelines, stable cell lines are often not produced and many protein productions occur in transient cell culture systems where mammalian cells are transfected with plasmid DNA to produce the recombinant protein of interest. In most cases, these transient expressions have lower protein yields than stable pools. As endotoxin is reported in units EU/mg of protein, one would expect that when protein amounts are lower via a lower expressing cell line or transient production, the ratio of endotoxin to protein of interest would be driven higher. Given this concern, it is critical to establish protocols and evaluate tools to remove endotoxin in protein solutions. Here, we present our findings evaluating various options for endotoxin removal, and propose strategies for endotoxin prevention