WO2018149809A1

WO2018149809A1 - Method for purification of whey proteins

Info

Publication number: WO2018149809A1
Application number: PCT/EP2018/053506
Authority: WO
Inventors: Ola Lind; James Mcrae; Max Mayr
Original assignee: Ge Healthcare Bioprocess R&D Ab
Priority date: 2017-02-16
Filing date: 2018-02-13
Publication date: 2018-08-23
Also published as: AU2018221613A1; CN117402229A; CN110267968A; EP3583114A1; AU2018221613B2; GB201702499D0; CN110267968B

Abstract

The invention describes a method of purifying alpha-lactalbumin from whey with a single chromatographic step. The method involves adjusting the pH of the whey and loading the whey onto a cation exchanger for a prolonged period of time/volume after which a highly pure alpha-lactalbumin can be eluted from the column. This method uses only a single chromatography step to achieve a high purity alpha-lactalbumin eluate and allows the alpha-lactalbumin depleted whey to be further processed for example pH adjusted back to a target set point, dried and recovered as a whey product or otherwise further processed.

Description

Title: Method for purification of whey proteins

Field of the invention

A method for purifying whey proteins and alpha-lactalbumin is described. This method involves separation and purification of the alpha-lactalbumin from whey (including different whey types, e.g. sweet, sour or ideal) to produce highly pure alpha-loactalbumin or alpha-lactalbumin-enriched whey protein isolate in a process utilizing a single chromatography step in which the nutritional and functional properties of the protein are retained allowing for use such as an ingredient in various food products, infant formulas, adult nutritional formulas, sport formulas, medical formulas enteral formulas and other specialist nutrition, nutraceutical and pharmaceutical products. In addition, this method also allows the alpha-lactalbumin depleted whey to be further processed to extract other whey proteins and produce other whey protein products.

Background of the invention

Whey is the liquid component of milk and is typically obtained during the manufacture of cheese or casein as a result of separation and clarification from curds or casein. Whey is composed of water, lactose, salts, residual fats and several proteins. Whey proteins possess interesting nutritional, functional, physiological, and pharmaceutical properties and are divided into two principal groups: the globulin protein fraction containing mainly beta-lactoglobulin and immunoglobulins; and the albumin fraction including alfa-lactalbumin and serum albumin.

Alpha-lactalbumin is a globular protein composed of 123 amino acids it is found in both human and bovine milk for which it has 74% conserved amino acid sequence homology. Alpha-lactalbumin occurs as an acidic single chain protein with a molecular mass corresponding to 14070 Da in human milk and 14178 Da in bovine milk. In humans alpha-lactalbumin is the dominant whey protein and makes up approximately -75% of total whey protein and -40% by weight of total protein in bovines however alpha lactalbumin is quantitatively the 2nd most important protein in whey making up -25% of total whey protein and -5% by weight of total protein superseded by the dominant protein present in bovine whey beta-lactoglobbulin which is considered to be absent in human milk REF.

Whey proteins have different utilities such as an ingredient in various food products, infant formulas, adult nutritional formulas, sport formulas, medical formulas enteral formulas and other specialist nutrition, nutraceutical and pharmaceutical products. Of the whey proteins alpha-lactalbumin has been shown to exhibit a number of important nutritional and functional characteristics due to its amino acid content and functional properties (Marshall, 2004, K. Lisak Jakopovic et al, 2015).

As there is a number of commercial applications and products that already use alpha-lactalbumin and new human health and nutrition applications are being generated, there are several processes for purification of whey proteins and alpha-lactalbumin including ultrafiltration, heat precipitation, and ion exchange methods.

Ultrafiltration methods use membranes which allow for separation of proteins and molecules based on their physical size allowing proteins and molecules up to a certain size to pass through into the permeate these methods can be effective at performing separations when no proteins or molecules of similar size exist in the feed material however to isolate molecules larger and smaller multiple steps are required this method is described in US. Pat. No. 5,008,376 (Bottomley) and further in US. Patent 6,613,377 (Chao Wu) which further applies adjustment of the pH to below 4 to further increase efficiency of the method. US 6,613,377 B2 describes a whey treatment process for specifically enriching alpha lactalbumin. A whey protein product is acidified to a pH below 4, concentrated and then the desired alpha lactalbumin is precipitated to form a low calcium product.

Heat precipitation is another method and involves adjusting the pH and heating the whey to denature and precipitate the protein of interest US. Pat. No. 5,455,331 (Pearce) describes use of this method for producing an alphalactalbumin product. Ion exchange methods involve contacting the whey with an anion or cation exchanger so as to selectively retain a protein fraction. Such a process is described in US. Pat. No. 5,077,067 (Thibault which selectively removes lactoglobulins from whey and US 5,756,680 (Sepragen) which applies a sequential process to separate different proteins in the whey to other methods including El-Sayed & Chase 2009 and 2010 which describe a two- step chromatography process to produce alpha- lactalbumin and beta-lactoglobulin at room temperature to purify alpha-lactalbumin (ALA) and beta- lactoblobulin (BLG) from whey concentrate.

The above methods require multiple steps, long processing times, many buffers or chemical treatments, cause denaturation to the protein and are unable to deliver a product with as high purity and yield. As such there is a need for improved methods that allow for improved processing for purification of whey proteins, especially in producing alpha-lactalbumin.

Summary of the invention

The present invention provides a novel method for purification of alpha-lactalbumin (ALA). The invention describes a method of purifying alpha-lactalbumin to a high purity from whey in a single chromatographic step. The method involves adjusting the pH of the whey to a pH of 3.5-4 which can be achieved with a number of acids and adjusting the temperature to ~50C, such as 42-55C^°C. The whey is then loaded to a cation exchanger for a prolonged period of time/volume after which a highly pure alpha-lactalbumin can be eluted from the column. The novel aspect of the process is both in the simplicity of the process and the way in which the separation is achieved. The inventors have determined that at the described pH condition alpha- lactalbumin and beta-lactoglobulin both bind the cation exchanger. However, due to differences in charge and conformation at this condition alpha-lactalbumin is somewhat inhibited by mass transport limitations for which beta-lactoglobulin does not experience this effect means that the cation exchange medium must allow for equal access to available binding sites either by physical or kinetic limitation.

The inventors have found use of a large sized resin bead cation exchanger facilitates equal access at a defined contact or residence time with the cation exchanger. However, it could be estimated that other cation exchanger mediums and formats could facilitate the same capability including but not limited to chromatography membranes, open beds etc.

The other aspect of the invention that facilitates displacement of bound beta-lactoglobulin is by extension of the time/volume of whey loaded on to the cation exchanger. The combined effects of these allow separation by a single step and enable the replacement effect that occurs with alpha- lactalbumin displacing bound beta-lactoglobulin from the cation exchanger. The method of the invention uses only a single chromatography step using only simple low cost buffers to achieve a high purity alpha-lactalbumin eluate > 80% (range 70 -95%) pure at a yield of 75% (range 65- 85%). In addition the alpha-lactalbumin depleted whey can be further processed for example pH adjusted back to a target set point, dried and recovered as a whey product or otherwise further processed. There are several advantages with the method of the invention some of which are listed below:

Process simplicity - single chromatography step - also means less buffers/ less

programming requirement and shorter process time (though process time is sort of long) Works with simple buffers - providing pH and conductivity conditions are met almost any acid/base and salt can be used so low cost more economic options can be used

- High purity gives high value product with more utility can be used in more applications and high yield maximum recovery means less loss in process maximum return on investment and maximum utilization of material from feed material Whey can still be used - requires additional processing but essentially process takes low cost product stream and extracts a higher cost product stream leaving the lower cost stream still useful.

Thus, the invention relates to a method for purification of whey proteins, comprising the following steps: providing a cation exchanger allowing for equal access of available binding sites by both alpha-lactalbumin and beta-lactoglobulin and complete replacement effect;

acidifying a whey fraction;

equilibrating said cation exchanger;

- loading said whey fraction on said cation exchanger;

washing said cation exchanger; and

eluting whey proteins from said cation exchanger.

Preferably the cation exchanger comprises chromatography beads having an average bead size diameter of 130-300 μιτι, preferably about 200 μιτη. In one embodiment the cation exchanger is packed in a column run with radial flow. The chromatography beads may be of natural or synthetic origin, preferably natural origin made of polysaccharides such as cellulose or agarose.

In the method of the invention the whey is acidified to a pH of 3.5-4.0, preferably pH 3.7. The equilibration and wash may be with 0.05-0.2% HAc or just water, but larger volumes are required or weaker acids, but also works with buffer system e.g. citric acid + Na2HP04. Preferably the loading is performed at a linear velocity of 200 - 1000 cm/ and the method is preferably run at a temperature of 42-55^°C, preferably about 50^°C.

The elution is preferably a 1 step elution with 30-200 mM, preferably 75mM -125 mM NaOH or KOH. The eluate is preferably re-titrated from pH 12 to pH 4.5-8 This is to reconstitute the whey to its pH before acid titration, it is required to still use the rest of the whey protein that stayed in flow through.

The column may be cleaned in place (CIP) with 1M NaOH /KOH at 50-60°C for >3 hours at high flow rates > 800 cm/h. The range is 100 - 800. First the flow rate is slow to avoid overpressure, later the high flow rate is required to wash out dirt most likely minerals that were covered with protein first.

The eluate obtained with the method of the invention comprises alfa-lactalbumin in a purity 70-95%, preferably 85-95%, and a yield of over 65-85%, preferably 75-85%. Other whey proteins may be collected in the flow through, such as beta-lactoglobulin. Brief description of the drawings

Fig 1 is a schematic view of the one-step purification process of the invention.

Fig 2 is a chromatogram according to Experiment 1 in the Experimental section,

showing the UV absorbance at 280 nm over the course of the process extracting

alpha-lactalbumin from pH adjusted whey. The point at which beta-lactoblogulin

can be seen to start to be displaced by alpha-lactalbumin can be observed at

-400 L , at this point the level of beta in flow through exceeds that of the starting

feed material. Fig 3 is a chromatogram showing ion exchange analysis of the elution pool at 220 nm as described in Experiment 3 below.

FIG 4 is a chromatogram showing ion exchange analysis of the elution pool at 280 nm as described in Experiment 3 below.

Detailed description of the invention

The invention will now be described more closely in association with some non-limiting Examples and the accompanying drawings.

An overview of the process of the invention is described in Fig 1.

The invention described relates to a method of processing whey to obtain a purified alpha- lactalbumin or enriched alpha-lactalbumin whey protein isolate or fraction. The invention makes use of a cation exchanger that facilitates equal access to binding sites for alpha-lactalbumin and beta-lactoglobulin. In the examples given this is achieved by using a large bead cation exchange resin. However, other formats of chromatographic medium could be used that allow for equal access of available binding sites by both alpha-lactalbumin and beta-lactoglobulin. The cation exchange resin is appropriately packed into a column or suitable vessel in the example provided a radial flow column has been used to allow for high flow rates and decreased processing times. The cation exchanger is prepared for a run by use of a suitable equilibration buffer in the example 0.1% acetic acid is used.

The whey, ideal whey, acid whey, whey concentrate or whey isolates (all-inclusive whey protein product) are then adjusted to a pH between 3.5-4 with the optimum pH being 3.7. This pH adjustment can be performed using a number of acids however acetic, citric, phosphoric and HCI are suitable and compatible with the processing environment. This pH adjustment can be performed by several means either by in-line dilution, or by adjustment in a balance tank or other method. The required amount of acid needed to perform this adjustment can be easily determined by someone skilled in the art by performing a titration with the intended feed material. At this pH the proteins present in the whey undergo a number of changes due to the impacts of pH which interferes with tertiary and quaternary structural elements of protein structure causing change to function, denaturation and aggregation of some of the proteins. Under these conditions alpha-lactalbumin occurs in a molten globule form (Laureto et al, 2002, Rosner and Redfield, 2009).

The pH-adjusted whey protein product is then loaded onto the cation exchanger. In the example provided this is done using a large bead size cation exchange chromatography resin packed in a radial flow column. This can be done by use of a pump, positive displacement or other means so long as a consistent residence contact time is maintained. The flow rate required in the example is that required to achieve a contact time of 3-6 minutes, preferably about 4 minutes. This a critical element and is dependent on the properties of the cation exchanger used in other embodiments using other cation exchanger formats different requirements may exist. The period of time or volume of the loading phase is also a critical aspect of the invention as this aspect of the process allows alpha-lactalbumin to displace the bound beta-lactoglobulin and any other bound proteins. Fig 2 shows that the process of replacement takes place gradually over the loading phase. It is acknowledged by the inventors that a longer loading phase will result in higher purity alpha- lactalbumin and shorter loading phase will give an alpha-lactalbumin enriched whey protein isolate.

Table 1 below shows the amount of β-Lactaglobulin in flowthrough increases over the course of the run and exceeds the amount in the starting material as it is displaced from the column by a- Lactalbumin. a-Lactalbumin present in the flow through increases over the course of the run also however does not reflect a traditional breakthrough curve. Analysis of composite samples of flow through analysed using the method described in Experiment 2. The table shows the amount of alpha-lactalbumin relative to beta-lactoglobullin via sample analysis area vs starting feed whey area, area is calculated by mL x mAU as some UV Absorbance values were greater than 2 AU. It can be seen that both alpha-lactalbumin and beta-lactoglobulin initially bind the cation exchanger and then after a period of loading alpha-lactalbumin displaces bound beta-lactoglobulin in a

replacement or displacement effect. This is seen as the level of beta-lactoglobulin in flow-through exceeds that of the starting feed material. Table 1

The level of alpha-lactalbumin in the whey flow through can be measured by performing analysis using an analytical anion exchange or other suitable method however following process optimization time or volume set points can be used for simplified process operation.

Following the loading phase, a wash of any unbound materials in the column is performed this is a standard operation for any chromatographic method in the example presented as per the equilibration phase 0.1% acetic acid is used however there is many other suitable buffers including water. Thereafter the elution of the bound alpha-lactalbumin is performed. Elution of the bound alpha- lactalbumin can be achieved by either displacement using an elution buffer a containing a suitable level of free positive ions or level of conductivity or by changing the charge of the bound protein by shifting the pH (with salt .e.g. NaCL or citrate or P0₄ ). In the provided example this is achieved by use of KOH buffer at 22 mSc and pH 11. However there are many other suitable buffers including NaOH and others, the critical aspect being that the conductivity be > 10 mSc and pH > 5. However use of an elution buffer at these lower limits will elute the product much more slowly and a greater level of elution buffer will be required impacting upon the process economics and any downstream processing steps.

Following elution the eluate containing the highly pure alpha-lactalbumin can be pH adjusted back to 4-7 for further processing. EXPERIMENTAL SECTION

Experiment 1: Extraction of Alpha-Lactalbumin using SP Sepharose Big Beads in Radial Flow Column

This experiment was performed using SP Sepharose Big Beads (GE Healthcare) packed in a 21 L radial flow column. The column was first equilibrated with 0.1% Acetic acid, pH Adjustment of Whey was performed by the addition of 8.5% Phosphoric acid at 5% Whey volume. The pH adjusted Whey was then loaded onto the column at 5.25 L/min for an approximate 4 min residence time with a total of 105 column volumes. Following loading of the column a wash step prior to elution was performed with 0.1% Acetic acid with Elution of bound materials then performed with 125 mM NaOH. The point at which beta can be seen to start to be displaced by alpha can be observed at -400 L on the above Chromatogram, at this point the level of beta in flow through exceeds that of the starting feed material.

Experiment 2 Analysis of the flow through stream- alpha lactalbumin depleted whey Samples of the flow through from Experiment 1 were collected at intervals and analysed as per below. Parameter

FPLC AKTA Pure 25M, with 10 mm path length multi- wavelength UV detector (GE Healthcare).

Tubing and connectors as required.

Column Guard Column - Source 0 (GE Healthcare)

Mono 0 5/50 GL (GE Healthcare)

Buffers Buffer A - 50 mM Tris pH 7.5

Buffer B - 50 mM Tris 450 mM NaCI pH 7.5

Equilibration Buffer A - 50 mM Tris pH 7.5 unless stated

3CV @ 2.5 mL/min.

Loading Sample loaded by sample pump to 100 μί

sample loop.

Sample loop emptied with 1 mL sample.

Sample injected from loop with 1 mL Buffer A onto column @ 2.5 mL/min, with pressure flow control.

Temp 25C

Wash Buffer A - 50 mM Tris pH 7.5 unless stated

3CV @ 2.5 mL/min.

Elution Gradient Step 1 Buffer B -50 mM Tris 450 mM NaCI pH 7.5

0-63% gradient, Buffer B over 25 CV @ 2.5 mL/min.

Elution Gradient Step 2 Buffer B - 50 mM Tris 450 mM NaCI pH 7.5

63-100% gradient Buffer B over 3 CV @ 2.5 mL/min.

Elution - Regen Buffer B - 50 mM Tris 450 mM NaCI pH 7.5

3 CV @ 2.5 mL/min.

Equilibration Buffer A - 50 mM Tris pH 7.5

5CV @ 4 mL/min.

The results of this experiment (Table 3) show how the amount of β-Lactaglobulin in flowthrough increases over the course of the run and exceeds the amount in the starting material as it is displaced from the column by a-Lactalbumin. a-Lactalbumin present in the flow through increases over the course of the run also however does not reflect a traditional breakthrough curve. In this AEX assay there is enough resolution to differentiate between the two forms of a-Lactalbumin, molten (resolution at 11.5-12.5 mL) and native (resolution at 14.7 mL). Additionally, β-Lactaglobulin is also resolved into the two different forms. Table 2 below shows the purity of eluate pool of alpha-Lactalbumin as assessed using the technique described in Experiment 2 as measured at 220 nm and 280 nm UV absorbance.

Table 3 below shows the yield of material as assessed using the technique described in Experiment 2 as measured at 220 nm UV absorbance.

Experiment 3 Analysis of eluate pool purity by analytical ion exchange.

Samples of the composite eluate pool generated from Experiment 1 were analysed for purity

Parameter

Tubing and connectors as required.

Column Guard Column - Source 0 (GE Healthcare)

Mono 0 5/50 GL (GE Healthcare)

Buffers Buffer A - 50 mM Tris pH 7.5

Buffer B - 50 mM Tris 450 mM NaCI pH 7.5

Equilibration Buffer A - 50 mM Tris pH 7.5 unless stated

3CV @ 2.5 mL/min.

Loading Sample loaded by sample pump to 100 μί

sample loop. Sample loop emptied with 1 mL sample.

Temp 25C

Wash Buffer A - 50 mM Tris pH 7.5 unless stated

3CV @ 2.5 mL/min.

Elution Gradient Step 1 Buffer B -50 mM Tris 450 mM NaCI pH 7.5

0-63% gradient, Buffer B over 25 CV @ 2.5 mL/min.

Elution Gradient Step 2 Buffer B - 50 mM Tris 450 mM NaCI pH 7.5

63-100% gradient Buffer B over 3 CV @ 2.5 mL/min.

Elution - Regen Buffer B - 50 mM Tris 450 mM NaCI pH 7.5

3 CV @ 2.5 mL/min.

Equilibration Buffer A - 50 mM Tris pH 7.5

5CV @ 4 mL/min.

The data shown in FIG 3 and FIG 4 was collected using the above method.

References

Moyyodo El-Soyed, Howard Chose, Single ond two-component cotion-exchonge adsorption of the two pure majorwhey proteins, Journal of Chromatography A, 1216 (2009) 8705-8711

M.M.H. El-Soyed, HA.Chose. Purification of the two major proteins from whey concentrate using a cotion-exchonge selective adsorption process. J Biotechnology Progress, Vol.26, No.l, 2010, pp. 192- 199.

Kunz C, Lonnerdol B. Re-evaluation of the whey protein/casein ratio of human milk. Acta Paediatr 1992;81:107-12.

Heike I. Rosner ond Christina Redfield, The Human a-Lactalbumin Molten Globule:

Comparison of Structural Preferences at pH 2 and pH 7 J Mol Biol. 2009 Nov 27; 394(2-3): 351-362.

Maria Joao Santos, Jose A. Teixeira, Ligia R. Rodrigues, Fractionation of the major whey proteins and isolation of b-Lactoglobulin variants by anion exchange chromatography, Separation and Purification Technology 90 (2012) 133-139.

Samuel Mburu Kamau, Seronei Chelulei Cheison, Wei Chen, Xiao-Ming Liu, and Rong-Rong Lu, Alpha-Lactalbumin: Its Production Technologies and Bioactive Peptides, Vol. 9, 2010—

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY.

NICOLETA STANCIUC, GABRIELA RAPEANU, AN OVERVIEW OF BOVINE a-LACTALBUMIN STRUCTURE AND FUNCTIONALITY, The Annals of the University Dunarea de Jos of Galati Fascicle VI - Food Technology, 34(2).

Katarina Lisak Jakopovic*, Irena Barukcic, Rajka Bozanic, Physiological significance, structure and isolation of a-lactalbumin, Mljekarstvo 66 (1), 3-11 (2016)

Marshall K. 2004. Therapeutic applications of whey protein. Altern Med Rev 9:136-56.

Laureto PPD, Frare E, Battaglia F, Mossuto MF, Uversky VN, Fontana A. 2005. Protein dissection enhances the amyloidogenic properties of a-lactalbumin. FEBS J 272:2176- 88.

PATRIZIA POLVERINO DE LAURETO.l ERICA FRARE.l ROSSELLA GOTTARDO.l HERMAN VAN

DAEL.2 AND ANGELO F0NTANA1, Partly folded states of members of the Lysozyme/lactalbumin superfamily: A comparative study by circular dichroism spectroscopy and limited proteolysis, Protein Science (2002), 11:2932-2946.

Claims

1. A method for purification of whey proteins, comprising the following steps:

- providing a cation exchanger allowing for equal access of available binding sites by both alpha-lactalbumin and beta-lactoglobulin and complete replacement effect;

- acidifying a whey fraction;

- equilibrating said cation exchanger;

- loading said whey fraction on said cation exchanger;

- washing said cation exchanger; and

- eluting whey proteins from said cation exchanger.

2. Method according to claim 1, wherein the cation exchanger comprises

chromatography beads having an average bead size diameter of 130-300 μιη, preferably about 200 μιη.

3. Method according to claim 1 or 2, wherein the cation exchanger is packed in a

column run with radial flow.

4. Method according to claim 1, 2 or 3, wherein the whey is acidified to a pH of 3.5-4.0, preferably pH 3.7.

5. Method according to claim 4, wherein the acidifying is with H3PO4 , HCI, HAc, or Citric acid.

6. Method according to one or more of the above claims, wherein equilibration and wash is with 0.05-0.2% HAc.

7. Method according to one of more of the above claims, wherein the loading is

performed at a linear velocity of 200 - 1000 cm/h.

8. Method according to one of more of the above claims, wherein the process is run at a temperature of 42-55^°C, preferably about 50^°C.

9. Method according to one or more of the above claims, wherein the elution is 1 step elution with 30-200 mM, preferably 75mM -125 mM NaOH or KOH.

10. Method according to one or more of the above claims, wherein the eluate is re- titrated from pH 12 to pH 4.5-8.

11. Method according to one or more of the above claims, wherein the column is

cleaned in place (CIP) with 1M NaOH /KOH at 50-60°C for >3 hours at high flow rates > 800 cm/h.

12. Method according to one or more of the above claims, wherein the eluate comprises alfa lactalbumin in a purity 70-95%, preferably 85-95%, and a yield of over 65-85%, preferably 75-85%.

13. Method according to one or more of the above claims, wherein other whey proteins are collected in the flow through, such as beta-lactoglobulin.