Whey can be characterized as milk from which 90-95% of the casein and the fat have been removed. Whey comprises a variety of proteins such as alpha-lactablumin, beta-lactalbumin, immunoglobulins, albumin, enzymes, growth factors and hormones.

Specific Proteins in Whey

Alpha-lactalbumin: is a protein found in the milk of mammals. However, beta-lactoglobulin does not occur in human milk but is found in bovine milk. Some infants show different degrees of intolerance to bovine milk. Alpha-lactalbumin is largely used both in preparation of humanized milk and compositions of non-allergenic milk products for infants who are allergic to beta-lactoglobulin. Accordingly, there is interest to fractionate bovine whey proteins resulting in a whey protein isolate suitable for infant formulas.

Caseinomacropeptide (CMP also known as GMP (glycomacropeptide) and CDP (casein derived peptide): is a complex mixutre of macropeptides found in sweet wheys (rennet and chesse wheys) derived from kappa-casein by the action of the enzymes chymosin and/or pepsin. CMP is rich in sialic acid and has a number of potential therpaeutic uses. Ayers (WO02/28194)

Human alpha-lactalbuin (HAMLET) has been shown to induce cell death in tumor cells by interacting with specific histone proteins and with nucleosomes (Svanborg, US 2006/0233807A1). 

Purification Schemes Used

–Ion Exchange:

Ion-Exchange: chromatographic methods, particularly IEX, for whey protein separation has been developed and used successfully on a commerical scale. 

(i) Cation Exchange

Ahmed (US5756680) discloses a method for the sequential separation of why proteins. In one embodiment, when is passed through a chromatographic column prepacked with a strong S cation exchange resin, equilibrated with acetate buffer at pH 3.8 where all the whey proteins bind, washing and then sequentially eluting immunoglobulin and beta-lactoglobulin at pH4, followed by alpha lactalbumin at pH 5, followed by BSA at pH 7 and then lactoferrin at pH 7.5.

Etzel (US 5,986,063) discloses a process to yield a stream enriched in beta-lactoglobulin and a second stream enriched in alpha-lactalbumin from a solution containing whey proteins by contacting the solution with a cation exchanger and selectively eluting the bound beta-lactogobulin fraction and the bound alpha-lactalbumin protein fraction by changes in the eluting pH. More specifically, the pH of the solution containing why proteins is adjusted to a pH of less than about 4.5, then the pH of the bound fraction is adjusted to about 4.9, elution of the beta-lactoglobulin fraction and adjusting the pH to about 6.5 and eluting the alpha-lacalbumin fraction.  

Mozaffar (US 6096870) discloses separation of whey proteins through the use of chromatography such as cation and anion exchange. Included in the procedure is pH adjustment to 3.8 prior to loading onto cationic exchanger washing and two sequential elutions the first elution for beta-Lactoglobulin and the second for alpha-Lactalbumin.

Hansen (US 13/063579) discloses a method for isolation of a b-lactoglobulin product and an alpha-enriched whey prtoein isolate from whey which provides the steps of (i) providing whey obtain from an animal, (ii) adjusting the pH value of the whey to pH 4.5 or below, (iii) loading the whey to a chromatographic support, (iv) optionally washing the chromatographic support, (v) eluting the beta-lactoglobulin from the support using a first elution buffer having a pH of 4 or less and a salt concentration of at least 0.1M and (vi) eluting the alpha renriched whey protein isolate with a second eltuion buffer. 

Hansen (US 2007/0092960) also discloses that the combination of high operating temperature and high flow rate applied upon loading biomolecule containing fluids onto a chromatographic column significantly improves the adsorptive capactiy and the productivity of the adsorbent of the column. In one embodiment sweet whey is pumped through a heat exchanger to reach 50C before being loaded onto a chromatography column and the pH adjusted to 4.7. Non bound material was washed out and the bound proteins eluted in two separate steps. In elution 1, all the bound beta-lactoglobulin is recovered and in elution 2 all the bound immunoglobulin G, bovin serum albumin and alpha lactalbumin is recovered.

–Overload Mode:

Ayers (US6,592,905 and WO/1997/027757) discloses a process for producing an immunoglobulin enriched whey protein fraction from a whey protein solution using CEX.  Accordingly to the invention, the CEX is contacted with an excess of adsorabel protein at a pH level below about 4.5 which causes the CEX to adsorb preferentially whey proteins with the immunoglobulin being displcaed. 

(ii) Anion Exchange (AEX):

Ayers (WO02/28194) discloses a process for producing a CMP isolate and an acid and heat stable beta-lactoglobuilin enriched WPI from a feedstock containing whey proteins including CMP and beta-lactoglublin by coacting the feestock with an AEX under conditions in which CMP and beta-lactoglobuilin are both adsorbed, eluting the beta-lactoglobuilin and the CMP from the AEX and contacting the eluate with a second AEX under conditions whereby CMP is selectively adsorbed, collecting the beta-lactoglobulin continaing flow through from the second anion exchanger and eluting CMP from the second AEx.

Mozaffar (US 6096870) discloses subjecting whey to an anionic exchange resin at about pH 7.5 wherein the beta-lactoglobulin adsorbs, collecting the flow through which is alpha-lactalbumin, immunoglobulins, bovine serum albumin and lactoferrin and eluting the adsorbed beta-lactoglobulin with a buffer at a pH of about 7.5.

Outinen (WO 95/19714) discloses fractionating alpha-lactalbumin and beta-lactoglobulin by passing a whey solution through an anion exchange resin, washing the column with deinonized water to obtain alpha-lactalbumin and eluting with a weak acqueous NaCl solution to recover beta-lactoglobulin.

–AEX-CEX: 

Gerberding & Byers, “Preparative ion-exchange chromatography of proteins from dairy whey” J. Chromatography A, 1998(808): 141-151). discloses separating proteins such as alpha-lactalbumin, beta-lactoglobulin, BSA and IgG and lactose from a sweet dairy whey mixture using AEX and then CEX. The AEX was most effective in separating beta-lactoglobuilin. CEX was used to further recover the IgG. 

(iii) Filtration

Ultrafilitration: UF is the most widely used commercial method of whey fractionation. It is employed after an initial chromatographic prcoessing step to further separate the whey proteins. However, it suffers form the drawback of high capital and operating costs, membrane fouling, incomplete removal of low Molecular mass solutes thereby requiring in place cleaning and sanitation of the membrane to minimize microbial problems. (Gerberding, J. Chromatography A 808 (1998) 141-151)

(Cheang J. Membrane Science 231 (2004) 159-167) discloses use of a two stage tangential flow filtration system for the purificaiton of alpha LA and beta LG from whey protein isolate. Separation was aheived using 100 and 30 kDa membranes in series in both the order of 100kDa followed by 30 kDa and 30 kDa followed by 100 kDa. In the second strategy, the 30kDa membrane was used to obtain purified alpha LA in the permeate solution while retaining the beta LG and BSA. The collected retentate was then searpated in stage 2 using a 100 kDa membrane.

(iv) Membrane Separation

EP-A 0 311 283 teaches the use of UF membrane for achieving an alpha-La/beta-Lg ratio of at the most 3/1 in the permeate.

(iv) Precipitation

–Ammonium Sulfate-IEX: (Lozano, , International Dairy J. 18(1), 2008, p. 55-63) discloses differential precipitation with ammonium sulfate to isolate beta-lactoglobulin from other whey proteins using 50% ammonium sulfate. The precipitate was dissolved and separated again using 70% ammonium sulfate, leaving a supernatant liquid enriched in beta-lactoglobulin. After dialysis and lyophilization, isolation of the protein was performed by IEX.