Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1995-10-13
1999-06-29
Feisee, Lila
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
530415, 210656, 210670, 210692, 522175, B01D 1508
Patent
active
059170220
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a process for the removal of endotoxins from biologically derived products particularly products such as proteins for therapeutic use. The present invention is particularly but not exclusively directed to the removal of endotoxins from blood plasma fractions such as albumin, as well as biological products derived from gram negative bacterial culture, such as Eschedchia coli culture.
BACKGROUND OF THE INVENTION
The endotoxins are lipopolysaccharides (LPS) of gram negative bacteria such as E.coli, and exist in the outer membrane of the cell envelope. They account for more than half the mass of the outer membrane of the cell envelope and they are constantly shed into the environment of the bacterium (Pearson 1985). The basic unit size of LPS is 10,000 to 20,000. However in aqueous solutions LPS generally exists in vesicles ranging in molecular weight from 300,000 to 1 million (Weary 1985).
The LPS molecule contains 3 distinct chemical regions, the Lipid A region, a central polysaccharide region and the O-antigen region. The Lipid A region resides in the cell membrane when endotoxins are contained within the cell wall. This is linked to a central polysaccharide core and this in turn is linked to the O-antigenic side chain, a repeating oligosaccharide structure which varies with different gram negative species.
The Lipid A region is composed of a glucosamine disaccharide containing phosphate groups and is highly substituted with long chain fatty acids. It is now known that Lipid A is responsible for most, if not all, activity associated with bacterial endotoxins and that endotoxins must be released from the bacterial surface to be effective (Rietschel and Brade 1992). The biological activities induced by endotoxins are extremely diverse. These are mediated through the activation of macrophages and other cellular components which lead to a wide range of biological effects. In mild doses, endotoxins produce moderate fever and stimulation of the immune system which in turn leads to microbial killing. In higher doses, they produce high fever, hypotension disseminated blood clotting and lethal shock.
The presence of endotoxins in biologically derived products (biologicals) prepared for therapeutic use is of major concern due to the diverse and potentially harmful biological activities of these molecules. Maintaining sterility in processes used in the manufacture of biologicals, together with stringent protocols for the preparation of equipment, helps to ensure products are free of endotoxins. However, raw materials used to manufacture biologicals are often not sterile. Indeed, when the source of a biological is from a gram negative bacterial culture (e.g. a method using an E.coli fermentation system to express recombinant protein), the endotoxin levels in the starting material will be very high. In practice, maintenance of sterility throughout an entire process is not always possible or cost effective. Therefore it is often desirable to have methods in place which either destroy or remove endotoxins while maintaining the integrity of the therapeutic biological component.
There have been numerous approaches to achieving destruction or removal of endotoxins (Pearson 1985, Weary 1985). These include hydrolysis with acid or base, oxidation, alkylation, heat treatment and treatment with polymicin B. However with each of these approaches the effect of the inactivation method on the desired biological product must be evaluated. Furthermore, while pyrogenic activity may be reduced, often endotoxin components remain and the presence of these endotoxin components may be of no benefit in the final product and could possibly be detrimental. It is therefore preferred to remove these endotoxin components from the final biological product.
Selective binding of endotoxins on charged, hydrophobic or affinity media, or separation on the basis of size can be performed. At pH levels greater than pH2, endotoxin aggregates are negatively charged and will bind to positively charged surf
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CSL Limited
Feisee Lila
Romeo David S.
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