Endotoxins, also called lipopolysaccharides (LPS), can be a major impurity of proteins or biologically active substances. The presence of small amounts of endotoxin in a recombinant protein can cause serious side effects in patients including endotoxin shock, tissue injury, and death. Due to these side effects, it is essential that endotoxin is removed from drugs, injectables, and other biological and pharmaceutical products. It is advantageous to remove endotoxin from research samples to insure accurate screening data.
Efficient and cost-effective removal of endotoxins from pharmaceutical and biological preparations can be challenging. Ion exchange chromatography during downstream processing is frequently used, however affinity chromatography such as ActiClean Etox may also be used. ActiClean uses a high affinity ligand for endotoxin removal and helps to dissociate protein-endotoxin interactions, leading to a more efficient recovery and higher yields of protein recovery.
Mammalian cell culture-derived therapeutics has the risk of cellular DNA contamination. Regulatory authorities have set strict standards for DNA contamination and require reduction by orders of magnitude from the fermenter to the final product. The FDA recommends the DNA in the final product be less than 100 pg/dose.
Most lysis methods cause the release of large quantities of nucleic acids. These contaminants must be removed because they can increase the viscosity of the feed stream as well as interfere with subsequent chromatographic steps.
Cell culture clarification processes, centrifugation or tangential flow filtration are primarily used as a clarification steps prior to chromatography. These technologies operate under high shear conditions and can increase cell disruption, resulting in increased contamination by host cell DNA that can be carried through the purification steps. Direct CaptureTM of proteins using CellthruTM resins can reduce the DNA load of downstream purification. The cells pass through the resin bed of large beads with lower shear forces and less disruption of cellular integrity.
Different methods exist to remove DNA from protein preparations. Affinity chromatography using Cellthru™ Protein A resin typically removes 95-98% of contaminating DNA, CHOP, virus and other cellular debris. Ion exchange chromatography can also be used although affinity chromatography typically gives a higher log reduction of DNA contamination. Heparin affinity chromatography has also been used for DNA removal as heparin binds DNA very efficiently.
Contaminating Host Cell Proteins (CHOP)
Using some of the expression systems proteins are secreted into the culture media leading to a reduced HOP contamination. Cell culture clarification systems, centrifugation or tangential flow filtration are used as a primary clarification steps prior to chromatography. These technologies produce high shear forces and can increase cell disruption, resulting in increased host cell protein contamination, which can be carried through the downstream purification. Direct CaptureTM of proteins from cell culture using CellthruTM resins can reduce the DNA load on downstream purification as the cells gently pass through between the large beads with less disruption while protein is captured on the beads.
The downstream processing of any biologically-derived therapeutics requires effective viral clearance. Biological products have the potential risk of carrying and amplifying adventitious viruses that may be present or introduced into the materials. Typically a number of processing steps are necessary to provide adequate viral clearance. Some methods such as virus removing filters and chromatography provide physical means to separate and remove the. Other methods include irradiation, high heat, or chemical treatment to inactivate the virus. Nanofiltration can remove almost all viral species, while exposure to a low pH or detergents are also robust steps for inactivating enveloped viruses. Chromatographic or ion exchange resins show greater variances and decreased capacity in the removal of virus. Several of our stable affinity chromatography resins allow inactivation of viruses and may be sanitized with sodium hydroxide.