1. Kasey Mackay 1, Giulia Weissenberger 2, Michael Lihon 1, Anne London 1 1 Novartis Institutes for Biomedical Research, NBC/PPA, 250 Massachusetts Ave, Cambridge, MA, 02139 USA 2 SUNY Brockport, Dept of Biology, 350 New Campus Drive Brockport, NY, 14420 USA Endotoxin, stable, heat-resistant lipopolysaccharides (LPS), are released by gram negative bacteria upon cell death and cell wall lysis. LPS have a hydrophobic lipid group which is covalently bonded to a long polysaccharide tail. This structure makes endotoxin difficult to remove from some biological substances. Endotoxin can enter a biological manufacturing process at virtually any step, especially in a non-GMP environment. In the NBC/PPA Midscale Protein Production group, protein production requests are increasingly made requiring lower and lower endotoxin amounts (measured in EU/mg protein) for use in in-vivo pre-clinical studies. To meet these requests, practices need to be implemented not only to prevent the introduction of endotoxin, but remove endotoxin if the final product has unacceptable levels. Introduction Results Protein Characteristics A single monoclonal antibody, IgG1 with a pI = 7.98, (Protein X) was used to test these technologies on their endotoxin removal capabilities and protein recovery. Table 1 shows the starting load characteristics for each of the 3 loads containing increasing endotoxin amounts. Protein concentration was measured by the Absorbance at 280nm by a Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific). Endotoxin Removal Technologies Table 2 shows the commercially available endotoxin removal technologies tested in these experiments. It also lists the mode of actions (membrane filter or resin), ligand, and technology conditioning procedure used as recommended by the vendors instructions for use. Endotoxin Detection Samples were analyzed using an Endosafe R - PTS TM spectrophotometer (Charles River Laboratories). Single-use Limulus amoebocyte lysate (LAL) test cartridges containing precise amounts of licensed LAL reagent, chromogenic substrate, and control standard endotoxin (CSE) (Charles River) were used for detection. The cartridges operate in ranges from 10-0.1 to 5-0.005 EU/mL. CSE, purified E. coli extract (Associates of Cape Cod, Inc) was used to spike endotoxin into the pooled starting materials on a log scale.  There are many technologies available that are capable of removing >95% of endotoxin from a biological sample.  9 out of the 11 technologies tested gave a yield >90%. Mustang E gave a yield >85% and DNA Etox a yield <50%.  Major concerns should be protein recovery for samples with low amounts of protein load and high endotoxin levels since it’s increasingly difficult to separate endotoxin from the sample.  Every protein interacts differently with endotoxin, thus an appropriate removal technology needs to be determined by experimentation. Endotoxins, small molecules present in gram negative bacterial membranes, pose a risk of contamination for biological products. If not cleared during the manufacturing process, tissue injury, fever, or fatal septic shock can occur if injected into mammals. For both clinical and commercial manufacturing, technologies have been developed to clear this impurity. Here, 11 commercially available technologies are evaluated at small scale and compared for pre-clinical protein production. Evaluation of Endotoxin Removal Technologies Division of Biochemical Technology BIOT483  243rd ACS National Meeting  March 28, 2012 Abstract Conclusion TechnologyVendorMode of ActionLigandConditioning Procedure Mustang Q Pall Life Sciences Membrane: AIEX (0.2μm pore size, 3 layer bed height), manual Positively charged modified PES 1M NaOH, 1X dPBS, 1M NaCl, 1X dPBS, load, 1X dPBS Mustang E Pall Life Sciences Membrane: AIEX (0.8μm pore size, 16 layer bed height), manual Positively charged modified PES 1X dPBS, load, 1X dPBS Sartobind Q Sartorius Stedim Membrane: AIEX (>3μm, 0.8mm bed height, 3 layers), manual Quaternary ammonium 1X dPBS, load, 1X dPBS Detoxi-GelPierce Resin: Affinity (45-165μm bead diameter), gravity drip Immobilized polymixin B 1% sodium deoxycholate, 1X dPBS, load, 1X dPBS ActiClean Etox Sterogene Bioseparations Resin: Affinity (60-160μm bead diameter), manual Nontoxic and non- mutagenic (Proprietary) 1M NaOH (2 hrs), 1X dPBS, load, 1X dPBS ReductEtox Sterogene Bioseparations Resin: Affinity (100- 200μm bead diameter), manual Nontoxic and non- mutagenic (Proprietary) 1M NaOH (2 hrs), 1X dPBS, load, 1X dPBS DNA Etox Sterogene Bioseparations Resin: Affinity (60-160μm bead diameter), manual Nontoxic and non- mutagenic (Proprietary) 1M NaOH (2 hrs), 1X dPBS, load (1M NaCl), 1X dPBS EndoTrap RedHyglos GmbH Resin: Affinity (90μm bead diameter), gravity drip Protein ligand (bacteriophage derived) Regeneration buffer (provided), 1X dPBS, load, 1X dPBS EndoBind-RBioDtechResin: Affinity, gravity drip DADPA-agarose- conjugated Sushi peptide Milli-Q water, 1X dPBS, load, 1X dPBS Cellufine ET- Clean S Chisso Corp Resin: Affinity (40-130μm bead diameter), manual Cellulose beads. Poly (ε-lysine) Milli-Q water, 0.2M NaOH in 95% ethyl alcohol (3 hrs), Milli- Q water, 1X dPBS, load, 1X dPBS Cellufine ET- Clean L Chisso Corp Resin: Affinity (40-130μm bead diameter), manual Cellulose beads. Poly (ε-lysine) Milli-Q water, 0.2M NaOH in 95% ethyl alcohol (3 hrs), Milli- Q water, 1X dPBS, load, 1X dPBS Table 2- Endotoxin Removal Technologies & Conditioning Procedures Figure 1- Ability of Technologies to Remove Endotoxin Figure 2- Product Recovery post Endotoxin Removal Technology Target Endotoxin Amount100EU1,000EU10,000EU Concentration (mg/mL)0.981.011.00 Sample Volume (mL)10.0 Actual Starting Endotoxin (EU)971,11013,553 Product Quality (% Monomer)96.998.998.7 Conductivity (mS/cm)15.7815.8315.64 pH7.427.627.57 Table 1- Starting Load Characteristics  Mustang Q, Mustand E, Sartobind Q, Detoxi-Gel, EndoBind-R, Cellufine ET Clean-S, and Cellufine ET Clean-L consistently remove >95% endotoxin.  ActiClean Etox, ReductEtox, DNA Etox, and EndoTrap do not remove endotoxin consistently.  All of the technologies allow for a yield of >90% except for DNA Etox.  ReductEtox, EndoTrap Red, EndoBind-R, and Cellufine ET-Clean S consistently had >95% product recovery. Methods Acknowledgements I would like to thank the efforts of the PPA/midscale laboratory in this research. A special thanks goes out to our summer intern Giulia for opening this can of worms. Other noteworthy contributors are Michael Lihon and Midscale Lab Manager, Anne London. Figure 3- Test Mustang Q using Protein X and Y for Endotoxin Removal (left) and Product Recovery (right)  Endotoxin removal characteristics are protein dependent; no two proteins are the same.