Pyrogens, removal

Nelson, L. Application of Reverse Osmosis in Pyrogen Removal, Depyrogenation, Technical Report No. 7 Parenteral Drug Association Philadelphia, 1985 28-36. 

Cross-flow filtration (CFF) also known as tangential flow filtration is not of recent origin. It began with the development of reverse osmosis (RO) more than three decades ago. Industrial RO processes include desalting of sea water and brackish water, and recovery and purification of some fermentation products. The cross-flow membrane filtration technique was next applied to the concentration and fractionation of macromolecules commonly recognized as ultrafiltration (UF) in the late 1960 s. Major UF applications include electrocoat paint recovery, enzyme and protein recovery and pyrogen removal. 

Typical UF performance for pyrogen removal with a polymeric and ceramic membrane is shown in Table 13. It can be seen that both types of UF membranes can adequately remove pyrogens. The choice of UF membrane (ceramic or polymeric) will depend on operating conditions or other special process requirements. Ceramic membrane ultrafiltration can achieve a 5 log reduction in pyrogen level. These UF membranes have been validated for the production of water meeting the requirements of pyrogen-free water for injection (WFI) standards.f ... 

Table 13. Pyrogen Removal with Ultrafiltration Membranes... 

The most significant claim is for the removal of "pyrogens" (endotoxins). Table 2.6 17 compares the pyrogen removal efficiency for a positively charged nylon 0.2 ju pore size membrane, Zetapor , and a conventional cellulosic 0.22 ju membrane. Normally, a 10,000 molecular weight cut-off UF membrane is required to remove pyrogens. (These membranes have an equivalent pore-size of 30 A or 0.003 ju). As expected, the conventional MF membrane shows no retention whatsoever, but the positively charged membrane shows better than 97% retention. 

Minobe S, Watanabe T, Sato T et al. Characteristics and application of adsorbents for pyrogen removal. Biotechnol Appl Biochem 1988 10 143-153. 

Other applications of ultrafiltration are scattered. Not surprisingly there is a growing market in microelectronics, where smaller and smaller integrated circuits require purer and purer water. There is also a market in pyrogen removal for pharmaceuticals. Ultrafiltration is used to sterilize beer without heating and hence to produce draft beer in cans. Enzyme recovery, gelatin concentration, and juice clarification are possible, but their success depends on specific situations. 

Pharmaceutical Industry. In the pharmaceutical industry, sterility of deionized water systems is maintained by using an ozone residual. The ozone residual concentration is maintained at >0.3 ppm ppm in the water recirculation loop. Prior to product compounding, the ozone residual is removed by contact with uvirradiaton for <1 s. Ozone also is used to oxidize pyrogens from distilled water destined for intravenous solutions. 

Ultra filtration. This process removes macromolecules, microorganisms, particulate matter, and pyrogens using a thin, selectively permeable membrane. Ultrafiltration caimot remove ions from water and is generally employed as a polishing process. 

The ability to remove particulates has made RO indispensable in the production of ultra-pure water for microchip washing. Its ability to remove large molecules enables it to produce pyrogen-free water for the pharmaceuticals industry. In the USA and elsewhere RO is permitted for producing the water used in making up injectable preparations. The European Pharmacopoeia still insists on distillation for this, but the larger amounts of water needed for ampoule washing, etc. are often purified by RO. 

The treatment of the crude penicillin extract varies according to the objective but involves formation of an appropriate salt, probably followed by treatment to remove pyrogens, and by sterilization. This last is usually achieved by filtration but pure metal salts of benzylpenicillin can be safely sterilized by dry heat if desired. 

Water produced by reverse osmosis (RO) is forced by an osmohc pressure through a semi-permeable membrane which acts as a molecular filter. The difiusion of solubles dissolved in the water is impeded, and those with a molecular weight in excess of 250 do not difftise at all. The process, which is the reverse of the natural process of osmosis, thus removes microorganisms and their pyrogens. Post-RO contaminahon m occur if the plant after the membrane, the storage vessel or the distribuhon system is not kept Ifee Ifom microorganisms. 

In the case of injectables and ophthalmic preparations which are manufactured aseptically but do not receive a sterilization treatment in their final container the packaging has to be sterilized. Dry heat at 170°C is often used for vials and ampoules. Containers and closures may also be sterilized by moist heat, chemicals and irradiation, but consideration for the destruction or removal of bacterial pyrogens may be necessary. 

Another concern with regard to the use of E. coli is the presence on its surface of LPS molecules. The pyrogenic nature of LPS (Chapter 7) renders essential its removal from the product stream. Fortunately, several commonly employed downstream processing procedures achieve such a separation without any great difficulty. 

Succinylated or phthalylinated LPS displays significant reduction in toxicity (up to 100000-fold) while retaining its adjuvanticity. Acid treatment (0.1 M HCl) of LPS obtained from various Salmonella species resulted in the production of an LPS-derivative termed monophos-phoryl lipid A (MPL). This also displays adjuvanticity, with little associated pyrogenicity or toxicity. This alteration of biological activity can also be achieved by removal of some of the fatty acids found in the LPS lipid A region. As LPS is effective in activating both cellular and humoral immune responses, research in this area continues to be pursued. 

Using aseptic technique, open the required number of vials, vor-texing each prior to removing an aliquot. Pool the samples aseptically in a sterile, pyrogen-free vial. Eor in-process sample, treat the bulk solution as a pooled sample. 

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