See also purification of IgA from plasma

Immunoglobulin A is the dominant immunoglobulin in human secretions including breast milk. Due to the low levels of IgA normally present in cows’ milk, the established method to increase the yield of IgA on a commercial scale is by immunization regimes to boost the levels in the milk. An inherent problem with this procedure is that it generally takes up to 3 months for the imminization regime to generate a hyperimmune response in the cows and then a further month to harvest the hpperimmune milk in sufficinet quantities. (WO2006/119560).

IgA is the most abundant of the five classes of immunoglobulin and is primarily found in the nucosa of the gastrointestinal tract and the respiratory tract. There are two types of IgA, secretory IgA (sIgA) and serum IgA. Serum IgA is generally a monomer found in the serum wehre it functions as a seocnd line of defense whereas sIgA is dimeric or polymeric and contains a scretroy component in addition to IgA. SIgA is a common constituent of breast milk. Sand (US 15/249811, published as (US 2016/0362477)

Starting Sources/Samples for IgA

Isolation of IgA from Seminal and Vaginal Fluids

(donadoni (“setting of methods for analysis of mucosal antibodies in seminal and vaginal fluids of HIV seropositive subjects from Cambodian and Italian Cohorts” 2010, 5(3)) discloses techniques for isolation of samples from vaginal fluid and semen. The IgA was subsequently isolated from the samples using a scheme of 1st IgG  purificaiton by Protein G affinity chromatography (Protein G binds to IgG fc region but not IgA and IgM), 2nd: IgM-IgA residual fractions from the IgG purificaiton underwent an AEX step where the fractions were applied to a HiTrap Q HP column, washed and eluted with buffer B (340 mM NaCl, 20 mM TrisHCL, pH7.2), 3rd: IgM and IgG were separated using gel filtration chromatography. 

Starting Sources/Samples for sIgA

See also isolation of sIgA from plasma

Intestinal luminal fluid/mucosal of pigs and cows:

Sand (US 15/249811, published as (US 2016/0362477) discloses methods for the purificaiton of sIgA from the intestinal luminal fluid or mucosa of pigs and cows. The lumen is the interior caviy of the intestine wehre digested food passes through and nutrients are absorbed. In one emboidment, the prcoess includes treating the intestinal luminal fluid with polyethylene glycol (PEG) having a molecular weight of 3-30k and isoalting the precipitated sIgA. The sIgA may also be pastuerized. 

Purification Schemes

Affinity Chromatography:

Jacalin: Ttotal IgA1 fraction can be purified from the precipitate using the well established jacalin affinity chromatography method for the separation of IgA1 from IgA2, IgG and IgM from human sera. Agarose bound jaclin with a binding capability of 1-3 mg IgA/mL gel slurry is obtained commercially (perice). Briefly, the Cohn Fraction III precipitate is resuspended in phosphate buffered saline (PBS) and applied to a bed of jaclin-agarose. The column is washed with PBS until protein in the flow through portion is no longer detected. The jacalin bound portion cotaining the IgA1 is eluted with 0.1 M Galactose. (Simon, US2008/0260822). 

Affinity purification methods have a long established history isoalating various specific ligand binding macromolecules form complex biofluid and tissue extracts.Preparations of IgA and IgM dervied form human plasma or serum are enriched in C. difficle toxin A and toxin B specific antibodies by affinity purificaiton methods.  In this case, C. diffcile toxins A and B are immobilized to an appropriate solid-phase support with which re-suspended Cohn fraction II precipitate or jacalin-purified IgA solutions will be incubated, then washed. Proteins binding only C. diffcile toxins A and B will bthen be eluted. (Simon, US 2008/0260822)

–Affinity Tagged Secretory Component as the ligand: 

Brown (US 14.476,559, published as US 2014/0371431; see also US 16/401,322, published as US 20190256577) discloses a method of islating IgA by application of an IgA source to secretory component that is modified to contain an affinity tag so as to form secretory IgA containg the affinity tag which can then be captured by a solid support resin. Purification of secretory IgA is advantageous because the secretory componant tag protects IgA proteins from digestion in the gastrointestinal tract. Affinity tags which can be used include peptide tags such as AviTag which allows biotinylation by the enzyme BirA so the protein can be silated by streptavidine, a calmodulin tag, and a FLAG tag. In some embodiments plasma dimeric IgA in the naturally occurring monomer-dimer mixture is convalently bound to the recombinant peptide tagged secretory component in vitro. In other embodiments, native secretory component is covalently bound to one or more amino acid residues through conventional synthetic techniques. As an example using a histidine tag, a single histidine residue or a poly histidine is added to secretory components, regardless of whether produced by recombinant, synthetic addition or other techniques. The secretory IgA that is now tagged can then be purified by affinity binding of the tag to a binding moiety immobilized on a resin. In the case of a FLAG tag, an antibody to the FLAG peptide is immobilized on the support as described in US 4,703,004. 

Corthesy (WO 2013/132052) discloses that it is possible to combine plasma-derived J chain-containing immunolgoublin, in particular IgA and/or IgM, with secretory component without the need to first purify the J chain containing immunoglobulin. The method includes the steps of obtaining a bood dervied protein composition that includes J chain containing IgA and/or IgM and admixing the composition with secretory component. The SC binds to the J-chain containing immunoglobulin. The SC may also contain a tag such as a hexa-histidne tag, which can aid in the purificaiton of the resulting protein. If such a tag is attached via a cleavalbe linker, the tag may be cleaved off prior to use in the invention. 

Jones (J of Immunoglogical Methods, 104 (1987) 237-243) discloses humans secretory component bound covalently to Sepharose 4B as an affinity adsorbent to isolate and purify polymeric immunoglobulin from cell culture sueprnatants. The method was used to isolate murine IgM isotype. Gel filtration of the eluted antibodies followed by enzyme immunoassay showed that all recovered IgM was of pentameric molecular size. Murine IgA were also isolated form cell culture supernatants. Most of the IgA recovered following affinity adsorption with SC was greater molecular size than dimer. 

Cation Exchange: 

IgA has an acidic isoelectic point in the range of about 4.5-6.5 due to variable glycosylation and is generally considered not to be able to adsorb to cation exchange resins in an amount that would be of commercial value. However, Brown (WO2006/119560A1) discloses fractionation of IgA from milk products such as skim milk by cation exchange by modifying the loading and elution conditions.

Hydroxyapatite (HA): 

(Luellau, J. Chromatography A, 796 (1998) 165-175 disclsoes using hydroxyapatite (HA) elution chromatography for the separation of molecular variants of monoclonal IgA from cell culture supernatants.

Metal chelate chromotography: 

Bertonlini (WO00/41721) discloses a method for separating IgA from other proteins such as IgG and IgM by metal chelate chromotography where a metal ion is attached via a chelate forming ligand to a matrix. The metal ion include ions such as zinc, copper, nickel, iron, manganese etc. The starting fluid can be any material containing IgA such as serum plasma, plasma fractions or mucosal secretions (e.g., milk, tears, saliva).

Ion Exchange:

–AEX Exchange:

(Lullau, Biotechnology techniques, 12(6), 1998, pp.; 425-430) dislsoes mIgA produced under serum free conditions by a murine hybridoma cell lineina stirred tank reactor, binding the IgA to DEAE Sepharose FF under low salt conditions and performing salt elution steps of 100-500 mM NaCl. . To separate dimeric IgA form monomeric and polymeric forms, the igA containing fractions from the DEAE IEX were applied to type I cereamic hydrozapatite (CHT). 

Microfiltration: 

Couto (US 2004/01673200 disclsoes a prcoess for seaprating moelcules of interest such as antibodies which emplys three filtraiton unit operations, clarification using microfiltration, concentration using UF. 

Hensgens (US 2011/0130545) disclsoes a a process for separating secretory IgA which includes separating the fat from the mild such as by skimming followed by subjecting the low-fat milk to microfiltration yeilding an S-IgA containing prempeate and a casein rich retentat. 

–acidifying skim milk first: Gregory (US5670196) teaches a method of preparing a whey product with immunoglobulins such as IgG by first seprating cream or fat, then acidifying (lower pH from about 6.0 to about 4.5-4.7) to precipitate casein and then smicrofiltering the supernantant milk or milk serum using a charged depth filter. 

Salt solutions: Uemura (US5258177) discloses using low inoic strenght salt solutions such as sodium caprylate or zinc caprylate to provide an IgA rich preparation. The starting solution can be a Cohn’s Fraction III or Fraction II + III.