Separation & Purification: Endotoxin Reduction Using Disposable Membrane Adsorption Technology in cGMP Manufacturing - - BioPharm International

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Separation & Purification: Endotoxin Reduction Using Disposable Membrane Adsorption Technology in cGMP Manufacturing


BioPharm International
Volume 20, Issue 5

ABSTRACT

The bioprocessing industry is increasing its use of membrane chromatography technology, especially for contaminant removal. The high flow rates, ease of use, disposability, and relatively low cost make it ideal for polishing applications. In a case study from Avecia Biotechnology, UK, the Sartobind Q (Sartorius) membrane chromatography product was used to remove endotoxin from a process stream. The result demonstrated advantages in time, product recovery, and cost over a traditional column step, leading to its use in a cGMP manufacturing process.



In biopharmaceutical manufacturing, it is essential that any products intended for injection into human subjects are produced virtually free of potentially harmful contaminants such as endotoxins and viruses. To ensure this, manufacturers incorporate certain contaminant removal steps into their processing procedures.

Endotoxins are typically associated with Gram negative bacteria, e.g., E. Coli. As the prefix "endo" suggests, this type of contaminant is not secreted by bacteria (as are exotoxins), but derives from the structure of the bacteria, specifically, from part of the outer monolayer of the outer membrane.1 When bacteria are lysed, whether intentionally or as a side effect of the process, they release these structural components.

Introducing even small amounts of certain endotoxins (e.g., lipopolysaccharides or LPS) into the bloodstream can cause a severe immune reaction. The presence of endotoxins triggers the immune system's signaling cascade, leading to the secretion of cytokines. The subject develops an abnormally high body temperature and respiration rate and low blood pressure. This can lead to endotoxic shock, which may be fatal. Therefore, it is essential to develop a robust process step to remove endotoxins from the process stream.

One mode of separating the target substance from endotoxin molecules is by size exclusion via ultrafiltration. But this requires a significant difference in size between the target molecule and the endotoxin, as when the target substance is a nucleotide. In this case, the smaller target molecule passes through the membrane while the endotoxin is retained. Often, however, the target molecule and the endotoxin molecule fall into a similar size range, so separation based on size exclusion is not applicable. The size of endotoxin molecules can range from <10 kDa in a monomeric form to >10,000 kDa in an aggregated form.2

An alternative mode of endotoxin removal is separation based on charge. This is possible because of the strong negative charge associated with endotoxin molecules under conditions around pH 8.0. This charged state is a result of the ethanolamine part of the endotoxin molecule having no charge at this pH, whereas the lipid-A and core polysaccharide regions are negatively charged at pH 8.0.

Column chromatography is commonly used to take advantage of charge as a tool for separating proteins. In this process, a micro-porous stationary phase, conventionally a gel or resin consisting of agarose or cellulose beads, is packed into a tube or column of glass or stainless steel. By coating the stationary phase with anionic groups (ion-exchange chromatography), it is possible to selectively adsorb certain charged components of a process stream, depending on the conditions of the mobile phase. For example, under the conditions of low conductivity (<20 mS/cm-1 ) and pH around 8.0, certain molecules (including endotoxin) have a net negative charge, and therefore, will bind to a positively charged stationary phase. Thus, they are retained in the column (removing them from the process stream), while the non-binding (target substance) molecules pass through the column. The outcome is a significant reduction of endotoxin levels in the process stream.

Such resin and column-based chromatography systems, however, suffer from drawbacks such as low flow rates, the need to pack the column with stationary phase, long regeneration times, channeling, and susceptibility to fouling.3 In the context of contaminant removal (i.e., binding impurities rather than binding target proteins), these drawbacks become more of an issue. Because contaminant removal is usually a "polishing" step, flow rates and convenience are priorities, and these are not strengths for resin or column chromatography. Even with relatively low levels of contaminant in the process stream, low flow rates mean that conventional resin-based columns might need to be quite large, thus further raising costs.4


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