Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Glycoprotein – e.g. – mucins – proteoglycans – etc.
Reexamination Certificate
1999-11-24
2002-10-08
Tate, Christopher R. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Glycoprotein, e.g., mucins, proteoglycans, etc.
C530S329000, C530S416000, C530S831000
Reexamination Certificate
active
06462180
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the purification of alpha-1 proteinase inhibitor (&agr;-1 PI) from aqueous solutions. More specifically, the invention relates to the purification of &agr;-1 PI from blood plasma or from plasma fractions produced from Cohn-Oncley fractionation with chromatography. Viral removal is accomplished by the addition of PEG and by nanofiltration. Inactivation of enveloped viruses is accomplished by addition of detergent prior to the chromatography process.
BACKGROUND
Alpha-1 proteinase inhibitor (&agr;-1 PI) is a glycoprotein with a molecular weight of about 55,000 Daltons. The protein is a single polypeptide chain to which several oligosaccharide units are covalently bound. Alpha-1 PI acts as an inhibitor of endogenous proteases, such as trypsin, chymotrypsin, pancreatic elastase, skin collagenase, renin, urokinase and proteases of polymorphonuclear lymphocytes.
Alpha-1 PI is currently used therapeutically to treat persons having a genetically caused deficiency of &agr;-1 PI. In such a condition, &agr;-1 PI is administered to inhibit lymphocyte elastase in the lungs. Lymphocyte elastase breaks down foreign proteins in the lungs. When &agr;-1 PI is not present in sufficient quantities to regulate elastase activity, the elastase breaks down lung tissue. In time, this imbalance results in chronic lung tissue damage and emphysema. Alpha-1 PI has been successfully used to treat this form of emphysema.
The demand for &agr;-1 PI typically exceeds the available supply. Alpha-1 PI for therapeutic use is currently purified from human plasma. This source of the protein is limited, which contributes to the low supply. In order to maximize the available supply of &agr;-1 PI, a process for purifying &agr;-1 PI from human plasma should have the highest possible yield. The purity of the &agr;-1 PI isolated from human plasma is also critical, because trace impurities can stimulate immune responses in patients who are receiving &agr;-1 PI. Finally, the process of purifying &agr;-1 PI from human plasma using current techniques requires an extensive amount of time, for the separation of the &agr;-1 PI from other proteins, viruses, etc. All of these factors (i.e., low yields, long production times, and low purity), contribute to the inadequate supply of &agr;-1 PI.
Various methods of purifying &agr;-1 PI from human plasma have been described. Bollen, et al., U.S. Pat. No. 4,629,567 (1986) used five different chromatography steps to purify the &agr;-1 PI from yeast,
E. coli
, and human plasma. The five steps involved DEAE ion exchange, thiol-disulfide exchange, heparin affinity, zinc-chelate chromatography, and amino hexyl ion exchange. No purity and yield data were shown.
Novika, et al.,
Gematol. Transfuziol
. 34:46-50 (1989) reported isolation methods from the by-products of the manufacture of blood products. They used affinity, DEAE cellulose, and gel filtration chromatographies. The purity and yield data were not available.
Podiarene, et al.,
Vopr. Med. Khim
. 35:96-99 (1989) reported a single step procedure for isolation of &agr;-1 PI from human plasma using affinity chromatography with monoclonal antibodies. Alpha-1 PI activity was increased 61.1 fold with a yield of 20%.
Burnouf, et al.,
Vox. Sang
. 52, 291-297 (1987) starting with plasma supernatant A (equivalent to Cohn Fraction II+III) used DEAE chromatography and size exclusion chromatography to produce an &agr;-1 PI which was 80-90% pure (by SDS-PAGE) with a 36-fold increase in purity. Recovery was 65-70% from the supernatant A.
Hein, et al.,
Eur. Respir. J
. 9:16s-20s (1990) and co-owned U.S. Pat. No. 4,697,003 present a process which employs Cohn Fraction IV-1 as the starting material and utilizes fractional precipitation of &agr;-1 PI with polyethylene glycol followed by anion exchange chromatography on DEAE Sepharose®. The final product has a purity of about 60% with 45% yield.
Dubin, et al.,
Prep. Biochem
. 20:63-70 (1990) have shown a two step chromatographic purification. First &agr;-1 PI, CI inhibitor, &agr;-1 antichymotrypsin, and inter &agr;-1 trypsin inhibitor were eluted from Blue Sepharose® and then &agr;-1 PI was purified by gel filtration. Purity and yield data were not available.
Ballieux, et al., purified an &agr;-1 PI and proteinase-3 complex from purulent sputum using 4-phenylbutylamine affinity chromatography, cation exchange, and a final immunoaffinity step (Ballieux, B. E., et al.,
J. Immunol. Methods
159:63-70 (1993)). The pH of the buffer used in the cation exchange step was 7.0. Under the conditions used, most of the sputum proteins bound to the resin, but &agr;-1 PI and proteinase-3 passed through without binding.
Jordan, et al., U.S. Pat. No. 4,749,783 (1988) described a method where biologically inactive proteins in a preparation were removed by affinity chromatography after a viral inactivation step. The basis of the separation between the native and denatured forms of the protein was the biological activity of the native protein towards the affinity resin and not physical differences between the native and denatured proteins.
Lebing and Chen, co-owned U.S. Pat. No. 5,610,285, described a method where &agr;-1 PI was captured from IV-1 paste suspension using a DEAE chromatography step. The collected solution was ultrafiltered then applied to an S-cation column for initial purification. Alpha-1 PI was collected as the flow-through fraction. The product, in a sucrose solution, was then treated with TNBP/cholate in order to inactivate viruses. Following filtration and ultrafiltration, the product was applied to a second S-cation column for final purification. Once the product was formulated and freeze dried, it was virally inactivated a second time by heating to 80° C. for 72 hours. Product purity and virus safety were greatly improved versus the Hein process, described above, but, in practice, the Lebing and Chen process was too resource intensive for large-scale manufacturing.
A process for the purification of &agr;-1 PI that improves the yield and purity of the &agr;-1 PI, that requires a shorter production time and uses less resources (such as reagents, water, resins, and column size) is needed. The present invention provides a process of purifying &agr;-1 PI from a blood plasma fraction with a higher yield, higher purity, shorter production time, and use of less resources than known methods.
SUMMARY OF THE INVENTION
The present invention provides methods for purifying &agr;-1 PI from an aqueous solution containing &agr;-1 PI. A portion of contaminating proteins is removed from the aqueous solution, so that a partially purified solution containing &agr;-1 PI is obtained. The contaminating proteins may, for example, be precipitated by adding polyethylene glycol (PEG) to the aqueous solution and adjusting the pH of the solution to from about 5.0 to about 6.0. The conductivity of the partially purified solution is then adjusted, such as by diluting it to reduce its conductivity, for example. The purified solution is diluted with water, for example, and the water may contain sodium phosphate. The conductivity of the solution is adjusted so that &agr;-1 PI will bind to an anion exchange resin. This conductivity is typically between about 2.0 milliSiemens (mS) and about 6.0 mS, for example. The method further includes passing the purified solution over an anion exchange resin so that the &agr;-1 PI in the solution binds to the anion exchange resin. The &agr;-1 PI can then be eluted from the anion exchange resin to obtain an eluted solution containing &agr;-1 PI.
In another embodiment of the invention, a portion of contaminating proteins is removed by washing the anion exchange resin, to which &agr;-1 PI is bound, with a buffer solution. The buffer solution removes a portion of contaminating proteins that are bound to the anion exchange resin without removing &agr;-1 PI bound to the anion exchange resin.
In another embodiment of the invention, viruses are deactivated prior to dilution of the purified solution and its addition to an anion exchange resin. The vir
Chavez Mark D.
Lebing Wytold
Ownby David W.
Trukawinski Susan
Wood Woody D.
Bayer Corporation
Massey, Jr. Carl B.
Tate Christopher R.
Winston Randall
Womble Carlyle Sandridge & Rice PLLC
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