Purification of alpha-1 proteinase inhibitor

Details Purification of alpha-1 proteinase inhibitor Purification of alpha-1 proteinase inhibitor

1998-03-09

2001-02-27

Clark, Deborah J. R. (Department: 1633)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Separation or purification

C530S416000, C530S350000, C800S007000, C800S008000, C800S016000, C800S013000, C800S014000

Reexamination Certificate

active

06194553

ABSTRACT:

This invention concerns generally the purification of a therapeutically useful protein and specifically the purification to near homogeneity of alpha-1 proteinase inhibitor (&agr;
1
-PI), especially transgenic human &agr;
1
-PI from a non-human animal source.
Currently, &agr;
1
-PI (also known as &agr;
1
-antitrypsin inhibitor) derived from human plasma is commercially available (Prolastin® &agr;
1
-PI, Bayer Corporation) to treat congenital deficiencies of the protein. Such plasma-derived &agr;
1
-PI comprises about 85% &agr;
1
-PI of total protein on a wt/wt basis. Human plasma as a source, however, has disadvantages, including a limited supply, and the potential of viral contamination. These disadvantages have prompted investigations into a variety of recombinant sources so that the existing patient population could be more fully treated and the number of indications expanded without the above disadvantages.
Recombinant human &agr;
1
-PI has been produced in both
E. coli
and yeast (Courtney et al., 1984; Sleep et al., 1991), but the lack of post-translation glycosylation by the micro-organisms has led to unacceptably high pharmacokinetic clearance rates. While the conventional solution to this problem is to transfect mammalian cells to make a recombinant form of the protein, this approach is too costly given the large dose of &agr;
1
-PI needed for congenitally deficient patients (60 mg/kg/week).
Wright et al., 1991, describe transgenic sheep that express human &agr;
1
-PI with mammalian-like glycosylation in their milk at up to 30 g/l. Wright et al. (1994), without giving details, further report having isolated &agr;
1
-PI “of very high purity (>99%)”. However, our initial studies demonstrated that the major obstacles in preparing a pharmaceutical grade preparation of this protein for clinical use would be in effectively eliminating the remaining sheep whey protein. A particular problem involved selectively eliminating sheep &agr;
1
-PI from the transgenic human &agr;
1
-PI.
The prior art reveals a number of methods which have been used to purify &agr;
1
-PI. Bischoffet al. (1991) have isolated site-directed mutant &agr;
1
-PI produced by
E. coli.
Their method consisted of chromatography over a silica-based anion exchange substrate, zinc-chelate chromatography, ammonium sulfate fractionation, and hydrophobic interaction chromatography. The resultant product was reported to be “of high purity as determined by PAGE under reducing conditions in the presence of SDS” (pp. 3468-3469). HPLC analysis of the product showed no contaminant at greater than 1%, the detection limit of the instrumentation.
Hoylaerts et al. (1986) have used a small scale immunoaffinity chromatography process to obtain non-glycosylated recombinant &agr;
1
-PI which was about 80% pure. They also developed a large scale purification process for &agr;
1
-PI which consisted of precipitation with poly(ethylene glycol), DEAE-Sepharose® chromatography, zinc-chelate chromatography, a kappa-chain-agarose chromatography step, a heparin-agarose chromatography step, and an aminohexyl-agarose chromatography step. The six step process yielded material which was greater than 95% pure.
However, the &agr;
1
-PI isolated by Bischoff et al. and Hoylaerts et al. is non-glycosylated and therefore is of limited pharmacological use. Archibald et al. (1990) report characterization of transgenic human &agr;
1
-PI produced in the milk of transgenic mice. Mistry et al. (1991) have developed a purification process for native, glycosylated sheep &agr;
1
-PI which consists of ammonium sulfate precipitation, concanavalin A chromatography, anion exchange chromatography on a Mono Q® column, and preparative scale native-PAGE. A further step used was immunoadsorbant column chromatography to obtain a product which was greater than 98% pure as measured by SDS-PAGE.
However, even a purity of 98+% would be inadequate for parenteral drug use of &agr;
1
-PI isolated from a non-human source. The potential immune response to contaminants necessitates a product of extremely high purity. It is thus of primary importance to discover a method which results in a product of the requisite purity for parenteral use.
Such a high level of purity requires a multistep process, with a high yield at each step. The prior art reveals anion exchange chromatography steps, fractionation steps using either poly(ethylene glycol) or ammonium sulfate, and immunoaffinity chromatography steps. Bollen et al. (1986) report a multistep process for purifying &agr;
1
-PI which yields “highly purified” &agr;
1
-PI with “trace contaminants”. The Bollen process includes anion exchange, thiol exchange, heparin-affinity, and Zn-helate chromatography steps.
The use of an immobilized zinc affinity chromatography step for purification of &agr;
1
-PI is detailed in the prior art. See, for example, Kurecki, et al., 1979. The use of cation exchange to purify a complex of native human &agr;
1
-PI and proteinase-3 has been reported by Ballieux et al. (1993) The complex isolated by Ballieux et al. is too small to contain intact proteins, and is most likely a degradation product of the complexed proteins. The use of a cation exchange chromatography step in a scheme for purifying intact, native human &agr;
1
-PI derived from plasma also was described by Lebing and Chen (1994).
We have prepared an essentially homogenous transgenic human alpha-1 proteinase inhibitor (tg &agr;
1
-PI) which has a purity substantially greater than 99.99 g tg &agr;
1
-PI/100 g total protein. This product is preferably prepared by subjecting a solution containing impure tg &agr;
1
-PI to a series of chromatography steps comprising at least one cation exchange step. The preferred embodiment includes anion exchange, protein G affinity chromatography, immobilize nickel affinity chromatography, and hydrophobic interaction chromatography steps. Preferably, the contact with the cation exchange material (resin) is performed at about pH 5.5 with the salt concentration at or below about 10 mM. In the most preferred embodiment, the purified tg &agr;
1
-PI includes less than 40 pg of protein other than casein, native sheep &agr;
1
-PI, and tg &agr;
1
-PI per mg total protein.


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Ballieux, B.E.P.B., et al., “Isolation of a protein complex from purulent sputum consisting of proteinase-3 and &agr;10-antitrypsin reactive with anti neutrophil cytoplasmic antibodies,”J. Immun. Meth. 159:63-70 (Feb. 1993.
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