Purification method

Details Purification method Purification method

1999-01-25

2002-05-14

Low, Christopher S. F. (Department: 1654)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S002600, C530S384000, C530S395000, C530S412000, C530S414000, C530S416000, C530S417000, C530S427000, C530S829000, C530S831000, C424S078100, C424S078110

Reexamination Certificate

active

06387877

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to an improved method for the purification of alpha-1-acid glycoprotein, and to therapeutic uses of highly purified alpha-1-acid glycoprotein.
BACKGROUND OF THE INVENTION
Alpha-1-acid glycoprotein (AAG) is a plasma glycoprotein of approximate molecular weight 41 kD. It is an acute phase protein, present in plasma at a concentration of between 0.5-1 g/l in healthy people, rising in disease states, particularly inflammatory diseases, to levels up to about 2 g/l.
The physiological role of AAG is poorly understood. As an acute phase protein, its serum level increases in response to a number of stresses and insults including infection, trauma, burns, etc. AAG is known to act on a wide variety of cells and it has been suggested that AAG may play a role in the immune response. In addition, AAG has been shown to bind to a diversity of drugs, particularly basic and lipophilic drugs. Therapeutic uses of AAG based on this latter aspect have been suggested in the literature but none have been actually developed as far as the clinic.
We believe that one reason for this is the relatively high level of contaminants which remain even in so-called highly purified preparations. The endotoxin lipopolysaccharide (LPS) derived from bacterial cell walls, also known as pyrogen, is one such contaminant.
LPS is the causative agent of septic shock, which is a major cause of morbidity following gram-negative bacterial infection, particularly in hospitalised and immunocompromised patients. The presence of LPS in AAG preparations renders them unsuitable for human therapy.
Currently available methods of purifying AAG are laborious and time consuming, involving a large number of individual steps. Furthermore, they are unsuitable for large scale preparative processes. One such technique is described by Hao and Wickerhauser (Biochem. Biophys. Acta, 322, 99-108 (1973)). This involves adsorption and elution of a Cohn Fraction V supernatant from DEAE Sephadex, concentration, dialysis, adsorption and elution from carboxymethylcellulose, dialysis and finally freeze drying. With both dialysis steps taking 48 hours each, the whole process takes over a week. Furthermore, despite Hao and Wickerhauser's suggestion to the contrary, the technique is not amenable to scale up for the treatment of the volumes of starting material handled by commercial manufacturers (typically several batches per week of up to 10,000 1 per batch of Cohn Fraction V supernatant). Most importantly this process has not been able to reduce the levels of bound contaminating LPS to levels acceptable for clinical use.
Other prior processes for purifying AAG have not been successful in depleting LPS from AAG preparations to levels which are acceptable for clinical use. One such method involves adsorption and elution of AAG preparations from Detoxigel resins (Boutten et al Eur. J. Immunol. 22, 2687-2695 (1992)). The purpose of this method was to ensure LPS was depleted from an AAG preparation for use in in vitro studies examining the effects of added LPS on cytokine production. This chromatography medium is not however suitable for use in preparing products for human administration, and in any event, LPS levels were only reduced to 200 pg/mg (approx. 2 EU/mg) of protein (EU=endotoxin units). This level is still too high for products intended for human use, particularly at the AAG doses likely to be required clinically (for example from 10 g to 30 g per dose) e.g. in the treatment of drug toxicity.
SUMMARY OF THE INVENTION
We have now developed a new process for removing LPS from an AAG containing preparation.
Thus in its broadest aspect, the present invention provides a method of removing LPS from an AAG containing preparation comprising contacting said preparation with a finely divided non-toxic resin.
In this way, it is possible to deplete LPS from AAG containing preparations to levels which are compatible with therapeutic uses of the preparations.
Preferred resins are non-substituted resins.
Preferably, said resin is a particulate resin, especially an inorganic particulate resin and more preferably a hydrophilic resin. Resins with porous surfaces for example silane-based resins such as fumed silica are particularly suitable. One such fumed silica resin which may be used in the method of the invention is the commercially available fumed silica product AEROSIL™ fumed silica (Degussa AG, Frankfurt), which has siloxane and silanol groups on the surface of the particles.
AEROSIL™ fumed silica and similar resins have previously been used in the pharmaceutical industry both as a component, for example in the formulation of tablets and ointments, and also in purification processes such as the removal of lipid and lipid-like substances, and lipoprotein from plasma and plasma derived products. We are not aware of any previous suggestion to use AEROSIL™ fumed silica, or any other finely divided particulate resin as a depyrogenating agent for AAG. The non-toxic nature of AEROSIL™ fumed silica represents a distinct advantage over prior methods of purifying AAG which rely on separation techniques using materials which are not suitable for therapeutic applications.
For use in the process of the invention, the particles may have a high surface to weight ratio such as from 1 m
2
/g to 1000 m
2
/g, preferably from 50 m
2
/g to 700 m
2
/g, and more preferably from 330 m
2
/g to 430 m
2
/g, such as 380 m
2
/g.
We have also developed a new simple purification method for AAG which includes our new depyrogenation step and which produces a depyrogenated AAG preparation suitable for clinical use. The new purification method accordingly overcomes the aforementioned disadvantages associated with prior AAG purification processes.
Thus in another aspect, the present invention provides a method of purifying AAG comprising contacting an AAG-containing preparation with an anion exchange matrix, eluting an AAG-enriched fraction from said matrix and depyrogenating an AAG-enriched fraction by contact with a finely divided non-toxic particulate resin followed by elution of an LPS-depleted AAG fraction.
Using such a technique, AAG preparations containing as little as 0.016 EU/mg AAG protein can be obtained.
DETAILED DESCRIPTION OF THE INVENTION
A variety of AAG containing starting materials may be used, for example plasma, cryosupernatant, and plasma fractions for example Cohn Fraction V supernatant and Cohn Fraction IV supernatant. In the case of recombinant AAG production, the technique may also be used on cell cultures and cell culture supernatants and fractions thereof. For reasons of economy, Fraction V supernatant is a preferred starting material, since this enables maximum usage of donated plasma, the fraction essentially being a waste product in the purification of albumin, and being particularly rich in AAG. Fraction V supernatant typically contains 40% ethanol, 10 mM citrate, 50 mM acetate pH 4.8. It has a low protein content (<2 g/l); 80% of the UV absorbing material (OD280) has a molecular weight <10,000 daltons. AAG has a relatively low molecular weight and is extremely soluble; it does not precipitate during the Cold Ethanol Fractionation Process hence the majority (~60 to 80%) is found in solution in Fraction V supernatant. Typical AAG concentrations in Cohn Fraction V supernatant will be in the range 0.2 to 0.35 g/l.
Any conventional anion exchanger may be used, provided, of course, that it has the ability to bind AAG. Examples include inert substrates such as agarose, for example Sepharose carrying functional groups having the ability to bind AAG such as positively charged groups for example diethylaminoethyl (DEAE), diethyl-(2-hydroxypropyl)-aminoethyl (QAE) and quaternary ammonium (Q). High capacity resins are preferred, and preferably resins of larger particle size, in the range 100 to 300 &mgr;m. The increased bed stability of large beads is of advantage in treating viscous materials such as Fraction V supernatant allowing minimal back pressure when the process is carried out by column chromat

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