Rapid Endotoxin Detection and Removal Technologies to Overcome Low Endotoxin Recovery

J. Yeng, J.J. Ston, D. Barua, S. Barua, S. Barua
Missouri University of Science and Technology,
United States

Keywords: antibody, endotoxin, pharmaceutical purification, bioseparation, low endotoxin recovery (LER), polymer, nanoparticles

Summary:

Endotoxins are considered as the major contributors to the pyrogenic response observed with contaminated pharmaceutical products. Recombinant biopharmaceutical products are manufactured using living organisms, including gram-negative bacteria. Upon the death of a gram-negative bacteria, endotoxin in the outer cell membrane is released into the lysate where it can interact with and form bonds with biomolecules. Once in the bloodstream, endotoxin induces fever, tissue damage, and sepsis, a systemic inflammatory response syndrome that can lead to disseminated intravascular coagulation, shock, and organ failure. Sepsis is possibly fatal, especially for those with a compromised immune system such as cancer patients. The incidence of sepsis is estimated to be 18 million cases per year worldwide including 0.75 million just within the U.S. which contributes to more than 0.2 million deaths per year. A pyrogenic reaction is caused by as little as 1 ng of endotoxin per kilogram of body weight per hour. Endotoxin contamination of biologic products may also occur through water, raw materials such as excipients, media, additives, sera, equipment, containers closure systems, and expression systems used in manufacturing. The incidence of Low Endotoxin Recovery has been reported in undiluted and spiked biopharmaceutical products. The manufacturing process is therefore in critical needs to reduce and remove endotoxin by monitoring raw materials and in-process intermediates at critical steps, in addition to final drug product release tests. We have developed a novel technology for the rapid detection of endotoxins using a fluorescence-based sensor (EndoDye) that is specific to endotoxin, highly sensitive and can detect endotoxin as low as 0.0001 ng/ml (300 fold lower than the LAL assay) in solutions. Sequential changes in the fluorescence spectrum of EndoDye are observed when a fixed amount of EndoDye is incubated with an increase molar ratio of endotoxins. We incubated endotoxin contaminated protein samples with EndoDye resulting in the quenching of EndoDye’s fluorescence intensity like a donor-quencher model between EndoDye and endotoxins. Its selectivity and capacity are unaffected by pH, conductivity, chelating agents, surfactants, and solvents. Another important feature of our method is the instantaneous detection time by a change in the fluorescence intensity after and before binding with endotoxins. This required time is faster than other existing techniques with an estimated required time of 24-48 h or even longer. With this level of accuracy and with a short testing time, there is no wonder why our fluorescence-based sensor technology creates an attractive alternative to standard testings. We have also manufactured polymer nanoparticles (~800 nm) without any surface modification, which are capable of adsorbing and removing endotoxin from protein solutions at an efficiency >99% while maintaining >95% protein recovery. We have termed the nanoparticle as PolyBall. PolyBall is effective in adsorbing and removing endotoxin from solutions regardless of the presence or absence of salts, pH, buffer and ionic strengths. In general, the endotoxin removal efficiency by PolyBall increases with increasing the nanoparticle concentration. PolyBall is proven to be effective on removing >99% endotoxins from a variety of protein solutions including monoclonal antibodies.