IgA – Ypatent

IgA on mucosal surfaces is produced locally and not dervied from circulating IgA. IgA is one of the gamma globulins on the basis of its electrophoretic mobility. It is composed of two H chains and 2 L chians. it may be monomeric, dimeric or trimeric. IgA mononomers are joined together as dimers at the constant regions of thier H chians by a J chain. IgA is secreted as one of two subclasses, IgA1 and IgA2. IgA1 predominates in the circulating blood wehrein most of it occurs as a monomer. Most IgA on mucosal surfaces, sucha s the surfaces of the trachea, bronchi, and bronchioles in the lungs, occurs as dimers or trimers joined by J chains. IgA dimers and timers have an increased ability to bind to and agglutinate target molcuels. (Simon, US 6,932,967).

Starting Preparations/Samples

IgA can be frationated from plasma or other biological fluid using various combinations of precipitations and chromatographic techniques. (Menyawi US 14/377,535).

IgA is a byproduct in the cold ethanol fractionation process to prepare immunologlobulin G. In the fractionation method, pooled human plasma is first treated to produce a cryoprecipitate and cryo-supernatant. The cryo-supernatant is subjected to a first ethanol fractionation to yeild a supernatant I which is subjected to a second ethanol fractional to yield fraction II+III. Fraction II+III is subjected to a third ethanol fractionation procedure to yield a supernatant III and fraction III precipitation. The fraction III precipitate enriched in IgA is generally discarded as an unwanted byproduct. Simon (US 15/205, 359, published as US 2016/0319039).

Fraction III precipitate:

Fraction III precipitate, contains the majority of serum IgA, but also a variety of other proteins as well as lipidic material which is difficult to process. Further purification procedures have thus been developed to obtain IgA (Leibl, J. Chromatography B, 678 (1996) 173-180).

Fraction III is obtained as by-product of large scale fractionation of human plasma. This fraction corresonds to the insoluble proteins after extraction of fraction II+III. (Pejaudier, Vox Sang, 23: 165-175 (1972).

The fraction III precipitate enriched in IgA is generally discarded as an unwanted by-product. (Simon, US 6,932,967).

Particular Purification Schemes

Affinity Chromatography:

Corthesy (US 2015/0056180) disclosses preparation of human plasma IgA by affinity chromatography using CaptureSelect Human IgA resin using 3 different sources of plasma IgA; cryo-depleted plasma, resolubilised cold ethanol fractionation paste or a strip fraction from ACX. Secretory IgA was latter obtained by combining in vitro IgA with recombinant human secretory component.

Jacalin: Roque-Barreira (J Immunology, 134(3), 1985) disclose that jacalin can precipitate IgA and describe an affinity chromatography procedure using jacalin.

Anion Exchance (AEX):

Chtourou (US2004/0132979) discloses a prepurification step (e.g., a fraction I+II+III precipitate obtained from plasma is resuspended in acetate buffer and octanoic acid is added, precipitate is separated , subjected tto virus inactivation) and a single AEX step carried out at alkaline pH (e.g., TMAE-Fractogen quilibrated with a glycine-NaCl mixture pH 9), washed with the equilibrating buffer and then a sequenctial elution is performed using a first phosphate buffer at pH 6.2 to elute the IgGs, then a second elution with the same buffer to which has been added 150 mM NaCl which elutes an IgA and IgG4 enriched fraction.

Leung, (Kindey International, 59, 2001, pp. 277-285) discloses separation of polymerica nd monomeric IgA1 pooled fractions by AEX which include eluting with 12.5 mL of elution buffer (a linear salt gradient from 0-1 mol/L NaCL in 20 mmol/L Tris-HCL, pH8.0. Subfractions of 250 ul were collected throughout the elution.

Menyawi (US 14/377,535, published as US 2015/0005476) discloses a process for enriching IgA from a composition, such as one that has been obtained from plasma, by loading the composition on an AEX, either strong or weak, under conditions that allow the IgA to bind, optionally applying a pre-elution step by applying a low conductivity soltuion, preferably at a weakly acidic to neutral pH and then applying an alkaline first elution solution with a substance having at east 2 acid groups such as a multivalent carboxylic acid wherein the protein eluted is enriched for monomeric IgA, but essentially devoid of IgM. An additional optional step of applying an acidic elution solution that comprises a strong competitor for the AEX is then performed so as to enrich dimeric IgA.

Moller (US5,410,025) discloses precipitation of a Cohn Fraction II/III or III in a buffer, elimination of impurities by precipitation with octanoic acid at pH 4-6, then treating at a low conductivity with an AEX, attaching most of the IgA and IgM Since the anticomplementary activity of the IgG fractio nnot attached to the AEX is low, the fraction can be employed in conjunction with the fractions taht contain IgA and/or IgM to prepare mixture that can be converted into IVIG preparations.

Pejaudier, (Vox Sang, 23: 165-175 (1972) disclsoes various schemes of preparation of IgA from a Fraction III recipitate by resuspending the fraction III precipitate with acetate buffer, pH 5.7, stirring for 30 min and keeping overnight at 4C, the precipitate was discarded and the supernatant passed on DEAE cellulose equilibrated at pH 5.7 with acetate buffer. Elution was done with acetate, pH 5.7. Then ethanol 32%, pH 5.7, superntant revmoed and precipitate dissolved in 0.9% NaCl, pH 5 with caprylic acid, recipitate remove (alpha2M) and supernatnt contains IgA.

Uemura (US 5,258,177) discloses a method of prepararing an IgA rich preparation froma plasma fraction which incorporates DEAE Sepahdex condition with 0.005 mM naCl, pH 7.0, washing several times and then eluting the IgA using 100 mM NaCl which was substqeuntly concentrated by UF.

AEX-HA:

Leibl (US6,646,108 and WO97/25352) discloses a method for the separation of IgG and IgA form an immunoglublin containing starting material by mxing the sample with Fractogen EMD TMAE 650 suspended in 50 mM sodium acetateaceitic acid buffer, pH 5.0 (non-bound material separated over a suction filter), washing twice with 50 mM sodium acetate/acetic acid buffer, pH 5.0, then stirring the gel for 2 h at 4C with 50 mM sodium acetate/acetic acid buffer +0.5 M NaCL, pH 5.5 (elution carrier out twice). The eluted material was dialysed against buffer pH 7.5. 3% of buffer B (0.5 M NaH2PO4/Na2HPO4 +150 mM NaCl, pH 6.8) was added to the retained material and this material was mixed with hydroxylapatite (ceramic) which had been equilibrated with 975% buffer A (PBS, pH 7.4 and 3% buffer B (0.5 M NaH2PO4/Na2HPO4 + 150 mM NaCl, pH 6.8) and suspended in the same buffer mixture. The sueprnatant was suction filtered and the HA washed which was combined with the first sueprnatant. The eluates of HA were mixed by stirring with ammmonium sulfate, the precipitate resuspended. analysis shoed that IEX brought about a partial enrichment of IgA but still contained HMWPs and/or aggregates. The HA treatmetn increased the ration of IgA to IgG.

AEX-thiophilic resin-Metal chelate chromatography:

disclose that IgA can be separated by other proteins such as alpha anti-trypsin, IgG and IgM contained in a fluid such as plasma or serum or mucosal secretions (milk, colostrum, tears and saliva) by metal chelate chromatography. The IgA is absorbed to the metal ion matrix (ions include zinc, copper, nickel, iron, manganese, chromium, cadmium, calcium, magnesium) and then selectively eluted. Alternatively, the fluid containing IgA is applied to the metal ion matrix under conditions such that at least one of the others proteins is absorbed while the IgA is not substantially absorbed. According to the disclosed scheme, ethanol (8%) was added to plasma, the suerpnatant removed by UF, then delipidated with aerosil, the delipidated solution loaded onto a DEAE Sepharose Fast Flow (euqilibrated with 10 mM sodium acetate at pH 5.2), IgG and transferrin were removed in the void volumen and albumin eluted with 25 mM sodium acetate pH 4.5. IgA, haptoglobin, ceruloplasmin, alpha antitrypsin, IgM complement (C3) and small amounts of IgG and albumin co-eluted with 0.5 M sodium chloride pH 5.2, a solution of 1.7M ammonium sulfate, 0.1M sodium acetate was added to the eluate from the DEAE column with 0.5 M sodium chloride at pH 5.2, the solution loaded onto a thiophilic resin, IgA rich material eluted with sodium acetate, ammonium sulfated and dialyzed solution loaed onto a chemalte agarose column after Zn2+ had been immobilized.

Gel permeation

(Leibl, J. Chromatography B, 678 (1996) 173-180) discloses a quick separation method of IgG and IgA by starting with a saline extract of fraction III precipitate treated with 2 M ammonium sulfate, dissolving the precipitate and running directly on a Superdex gel permeation column. Six peaks were obtained and the amterial fractionated in the maximum of peak 4 consisted of rather pure IgA.

Hydroxyapatite:

–HA-AEX:

(Leibl, J. Chromatography B, 678 (1996) 173-180) disscloses a method of purifying IgA by extraction of a fraction III precipitate, removal of coagulation proteins and lipoprotines by heparin-Sepharose and precipitation with dextran sulfate (Heparin Sepharose also adsorbed two thirs of the alpha macroglobulin), removal of remaining alpha macroglobulin by HA, Fractogel TMAE to further increase the IgA content and then gel permeation.

Purification of dimeric/polymeric IgA containing Secretory Component (SC)

Simon (US 15/205, 359, published as US 2016/0319039) discloses a method of purifying IgA with recombinant secretory component. According to the procedure a fraction III precipitate that is produced as a byproduct from production of IgG by ethanol fractionation of pooled human plasma is further purified by ion exchange adsorption purificaiton follwed by incubation with immobilized hydrolases to inactivate viruses and vasoactive substances. From 4-22% of plasma IgA is dimeric and polymeric IgA. The resulting dimeric IgA is further coupled to recombinant secretory comonent that is produced by recombinant techniques. the coupling is accomplished by forming disulfide bonds under mildly oxidizing conditions. Dimeric IgA cotnaining both J chain and SC is again purified by ion-exchange and size exclusion chromatography and/or UF. The purified dimeric and polymeric IgA containing SC is iptionally stablized for example by the addition of human serum albumin to a final concentraiton of 5%.