Alpha-1 proteinase inhibitor (a1PI), also named alpha-1-antitrypsin (AAT) is a glycopeptide inhibitor of proteases, and is found in human serum and other fluids. Isolation of a1PI from human plasma is presently the most efficient practical method of obtaining a1PI in quantity and human plasma is the only FDA approved source Matthiessen (US 2007/0037270). 

AAT is an essential protease inhibitor found minly in the blood. AAT normally protects connective tissue, such as the elastic tissues of the lungs, from degradation by elastase, an enzyme released by neutrophils at sites of inflammation. (Kumpalum US 8,580,931).

By Alcohol/Ethanol Precipitation steps

Most published processes for AAT isolation begin with one or more fractions of human plasma known as Cohn fraction IV precipitates such as Cohn fraction IV, or more specifically fraction IV1 and fraction IV1-4 as well as precipitates of Kistler-Nitschmann supernatant A or A-I which are obtained from plasma as a paste after a series of ethanol precipitations and pH adjustments. Brinkman (WO2009/025754).  Cohn IV-1 precipitate is usually derived after ethanol fractionating of plasma by precipitation with 20% ETOH at pH 5.2 (Matthiessen (US 2007/0274976). 

In the Cohn-Oncley method 6 which consists of 3 precipitation steps, the first precipitation step, referred to as Fraction I precipitation, is performed at high pH (7.2) and low ethanol concentration (8-10%) to precipitate proteins such as fibrinogen and Factor XIII away from IgG and A1PI, which remain in the supernatant. IgG is then precipitated from the Fraction I supernatant in a second precipitation step, referred to as a Fraction II+III precipitation, performed at moderate pH (6.8) and high ethanol concentration (20-25). The bulk of the A1PI remains in the supernatant of the Fraction II+III precipitation, and is subseqeuntly separated from albumin in a third precipitation step, referred to as a Fraction I-IV-1 precipitation, performed at low pH (5.2) and moderate ethanol concentration (18%). 

Starting with Cryosupernatant

Bruckschwaiger (US13/776,448, published as US 2013/0224183) teaches that a single initial precipitation step that captures all of the proteins normally precipitated in the Fraction I, Fraction II+III and Fraction IV-1 precipitates combined using an initial low pH, high alcohol precipitation and that IgG and ApI could be efficiently extracted from this precipitate wihtout the use of subsequent protein precipitations. Bruckshwaiger teach that this is accomplished using a high inital ETOH precipitation step (20-30% ETOH at pH 5-6) without any prior alcohol precipitation steps resulting in co-precipitation of the immunoglobulins with A1PI. This precipitate can then be solubized, forming a suspension having an soluble porition where the immunoglobulins are located and an insoluble porition which A1PI is located for further processing.  

Starting with Effluent/Supernatant II+III

Bastek “Purification of alpha-1-proteinase inhibitor using ligands from combinatorial peptide libraries” dissertation, North Carolina State University, 2000) disclsoes that the effluent II+III in the Cohn fraction contains about 30 mg/ml totoal protein 3% of which is A1PI. Eff. II+III  main component is hSA, in addition to anti-thrombin III, IgG, IgA, Apo A-1, transferrin, haptoglobin and alpha-1 acid glycoprotein. Bastek teaches that Eff+III contains 87% of the total A1PI in plasma compared to only 49% in redissolved IV-1 paste (see starting with Fraction I-IV precipitate below) and thus has a distinct advantage over IV-1 paste for A1PI recovery.  

Kupalume (US2009/p292114) also teaches that Cohn fraction II+III supernatant (also known as supernatant A in the modified Cohn fractionation method described by Kistler and Nitschmann, 1962, Vox Sang, 7. p. 414 to 424) have been used as a source of AAT and that Cohn fraction II+III supernatant contains 2-3 times more active AAT than Cohn fraction Iv-1. 

Starting with Fraction I-IV1 Precipitate: 

Bastek “Pufication of alpha-1-proteinase inhibitor using ligands from combinatorial peptide libraries” dissertation, North Carolina State University, 2000) disclsoes that A1Pi has been purifed from redissolved Cohn fraction IV-1 paste as starting material. The Cohn process has 6 major precipitation steps starting with the centrifual harvest of cryoprecipitate from freshly thawed plasma. The effluent is brought to 8% ETOH at pH 7.2, -2.5C and the resulting fraction I precipitate is discarded. The supernatant I composition is modifed to 25% ETOH at pH 6.6, -6C. A second precipitation, Paste II+III, is collected by centrifugation and used as the intermediate for immunoglboulin renrichment. The II+III eflluent pH is decreased to 5 to yield fraction Iv-1. IV-1 paste frequently serves as the intermediate for isolation of A1pI and anti-thrombin III, while further fractionation of the eflluent yeidls albumuin (Fraction V). The IV-1 paste is redissolved at pH 7-8, 20C and contains 5-10% A1PI. The major contaminants are anti-thrombin III, albumin, lipoproteins, cerulpolasmin and trqansferrin. 

Brinkman (WO2009/025754) discloses separating alpha-a-antitrypsin (AAT) from apolipoprotein A-I (ApoA-1) from Fraction IV1 (obtained by a first pricipitation using 8%ETOH, pH 7.2, a second precipitation using 20% ETOH, pH6.9, taking the supernatant (the precipitate FRaction II+III is set aside for other purposes) and adjusting the ph of the superntant to about 5 and ETOH to 21%. The precipitate that forms (Fraction IV1) in this 3rd precipitate contains AAT, ApoA-I and other contaminintng proteins/lipids. APOA-I and AAT can then be seaprated from each other  by suspending the fraction IV1 at pH 9.6, coooling and adding ETOH 12%, pH 5.4. The soluble AAT filtrate is then searpated form the insoluble ApoA-I material by filtration. 

Kee (WO2004/060528) discloses preparation of a Cohn Fraction IV1-4 fraction by 1st cooling human plasma to -2-2C, adjust pH 69-7.5, cold ETOH 6-10%, lower temp to -4-0C, the precipitate that forms (Fraction I) is removed by centrifugation or filtration, 2nd, the filtrate/sueprnatant adjusted to pH 6.77.1, ETOH added 18-22%, temp -7-03C, the precipitate that forms (Fraction II+III) is set aside for other purposes, 3rd, the filtrate/sueprnatant is adjsuted to pH 4.9-5.3, ETOH 16-20%, temp -7-03, after the supension settles, pH adjusted 40-44% and ETOH added 40-44%, the precipitate that forms (Fraction IV1-4) is removed. It contains AAT as well as contaminating proteins. The Fraction IV1-4 paste is resupended in buffer, pH 7.5-9.5 and treated with dithiothreitol (DTT) and fumed silica. The solutlbe AAT product is separated from the precipitated fumed silica using a filter press. The ATT final filtrate is then applied to an IEX, the eluate to HIC, it is stablized by pasteurization, then NF. 

By chromatography

Affinity Chromatography:

Buettner (US 15/770,111, published as US 2018/0305401) discloses a method for purifying a first protein of interest and second protein of interest which can be IgG and A1PI starting from a fibringoen depleted plasma solution (see plasma proteins) using one affinity chromatography such as CAPTURESECT taht binds to a CH3 domain of IgG  and purifying the AqPI in the flow-through fraction using a second affinity chromastography. The IgG is eluted from teh 1st affinity chromatography and polished by IEX and viral filtration. The A1PI is also eluted from the 2nd affinity chromatography. A flwoth-through fraction of the A1Pi purificaiton process can e further processed to isolate addtional proteins such as prothrombin complex followed by albumin. 

AEX: 

Coan (US4,697,003) teaches a method for separating alpha-1-proteinase inhibitor (AKA alpha-1 antitrypsin) starting with either Cohn Effluent II_III, Cohn Effluent I and cryosupernatant solution. According to the method, the aqueous solution is treated to lower the concentraiton of salts such as by DF, contacting the solution with an AEX in bind and elute mode. 

Glaser (Preparative Biochemistry, 5: 333-348 (1975) discloses isolating AAT from Cohn fraction IV1 paste. The paste is stirred in phosphate buffer at pH 8.5 in order to reactivate AAT which is largely inactivated by the pH of 5.2 employed in Cohn fractionation. After dialysis and centrifugation, the supernatant is subjected to two rounds of AEX. 

Schulz (US 2006/0194300) discloses a process for preparing A1AT from a solution containing A1AT such as a reconstituted plasma fraction IV1 (Cohn) which inlcues subjecting the solution to AEX (DEAE) , eluting the A1AT foloowed by solvent/Detergent treatment. 

AEX-CEX: 

Lebing and Chen (US5,610,285) describe an initial AEX followed by CEX at low pH and low ionic strenght to purify human AAT from plasma. 

Wood (CA2432641) discloses purification of alpha-1 proteinase inhibitor (alpha-1 PI) from aqueous solutions such as human plasma by precipitation of contaminating protens by adding PEG followed by AEX in bind and elute mode. Further purificaiton may be done by CEX which takes advantage of the fact that alpha-1-PI does not bind to the CEX under certain conditions. 

AEX-HA: Mattes (Vox Sanguinis, 81: 29-36 (2001) and US 6.974,792) discloses a method for isolating AAT from Cohn fraction IV that involves ethanol precipitation, AEX and adsorption on HA. 

Schulz (US2006/0194300) discloses a process for preparing A1AT by subjecting an A1At containing solution to IEX, adding detergents and optionally a solvent for inactivating lipid enveloped viruses followed by increasing the salt concentraiton to salt out the detergent.