chromatographytech note 6128

Workflow for Protein Purification from Whey Using Nuvia™ Ion
Exchange MediaPaul Ng, Mark Snyder, Process Chromatography Division, Bio-Rad
Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547

Materials and Methods

Introduction
Whey proteins play a vital role in the formulation of foodsupplements because of numerous desirable nutritionaland functional properties. Here we present a process forwhey purification using Nuvia S and Nuvia Q ion exchange
media. The major whey proteins are α-lactalbumin (ALA),
β-lactoglobulin (BLG), bovine serum albumin (BSA), and bovine
immunoglobulins (Hahn et al. 1998) (Table 1). Minor proteinssuch as lactoperoxidase, lactoferrin, and proteose-peptone
account for the other protein components in whey.

Whey was obtained as a concentrate from Hilmar
Ingredients. The solution was aliquoted, stored frozen at–20ºC, and thawed before the experiments. The whey
concentrate was diluted tenfold in equilibration buffer andfiltered through a 1.2 micron filter (Sartorius Group) toremove particulates prior to chromatography.

Whey

Table 1. Protein composition of bovine whey.
ProteinAverage
Concentration, g/L

Proportionin Whey, %Molecular
WeightIsoelectric
PointALA

1.5

22

14,200

4.7–5.1

BLG

3–4

51

18,400

5.2

BSA

0.3–0.6

6.6

65,0004.9

IgG, IgA, IgM

5.8–7.3

0.6–0.911

150,000–900,000

Lactoperoxidase

0.06<178,000

9.6

Lactoferrin

0.05<178,000

8.0

Proteose-peptone

0.5

74,000–20,000—

The objective of this study was to develop a separation
process to fractionate usable proteins from whey (Figure 1).
First, we purified a total whey protein isolate (WPI) becauseof its high-end functional advantages, as evidenced by ademand from nutrition and health segments. Secondly, ALAand BLG were purified because of their value in infant formulaand confections, respectively.
Whey

Purified Proteins and Reagents

Purified bovine proteins, including ALA, BLG, and BSA,
were purchased from Sigma-Aldrich. The proteins weresolubilized in the appropriate column equilibration buffer at
a concentration of 2 mg/ml on the day of the experiments.
All chemicals used for the preparation of buffers were ofanalytical grade. The buffers were sterile filtered prior to use.
Electrophoresis

For SDS-PAGE, the sample was first mixed with an equalvolume of loading buffer, then heated at 95ºC for 5 min.
Up to 30 μl of the mixture was loaded into the wells of a
4–20% Criterion™ or Criterion™ TGX™ precast gel(Bio-Rad Laboratories, Inc.) and separated at 200 V or
300 V, respectively. The gel was stained with Bio-Safe™
Coomassie stain and scanned with a GS-800™ USB calibrated
densitometer (both from Bio-Rad).
Column Chromatography

All chromatography columns and instruments were fromBio-Rad. Experiments were automated and executed using a
BioLogic DuoFlow™ system and software. Nuvia S or Nuvia Q
media was packed in a Bio-Scale™ MT2 column. For WPI
preparation, the system was programmed to repeatedly run thefollowing chromatography cycle (60 column volumes (CV)/hr)when using Nuvia S media:
Equilibration with 3 CV of 0.04 M Na lactate, pH 4.0
Loading with 20 CV of 1/10 diluted whey■■ Washing with 10 CV of 0.04 M Na lactate, pH 4.0■■ Elution with 20 CV of 0.04 M Na phosphate, pH 8.0■■ Washing with 3 CV of 1 M NaCl■■ Washing with 3 CV of 1 M NaOH■■ Pre-equilibration with 3 CV of 0.2 M Na lactate, pH 4.0■■ Equilibration with 5 CV of 0.04 M Na lactate, pH 4.0■■■■Nuvia S media

WPI

Nuvia Q media

ALA

BLG

Fig. 1. Workflow for protein purification from whey with Nuvia S andNuvia Q media.

To purify ALA and BLG from whey, a segmented gradientmethod using Nuvia Q media was developed:
Equilibration with 3 CV of 0.02 M Na phosphate, pH 6.0
Loading 5 ml of 10x diluted whey■■ Washing with 4 CV of 0.02 M Na phosphate, pH 6.0■■ Gradient elution with 10 CV 0–0.1 M NaCl■■ Step elution with 5 CV in 0.1 M NaCl■■ Gradient elution with 10 CV 0.1–0.3 M NaCl■■ Step elution with 5 CV in 0.3 M NaCl■■ Gradient elution with 10 CV 0.3–0.9 M NaCl■■ Step elution with 10 CV in 0.9 M NaCl

414— A 280 — pH

3.5

12382

61.5
140.5

20pH

102.5
A 280

To determine the elution profile of individual proteins ormixtures of proteins, chromatography was carried out witha gradient at the target pH. For example, the column wasequilibrated in 0.020 M Na phosphate, pH 6.0. After injecting
a protein sample of 100 μl, the column was washed with five
CV of equilibration buffer. The adsorbed protein was eluted
using 40 CV 0–0.3 M linear gradient of NaCl (buffer B). Thecolumn was cleaned with five CV of 1 M NaCl and 1 M NaOH,and stored in 0.1 M NaOH.

00

1,000

2,000

3,000
Time, sec

4,000

5,000

Fig. 2. Cation exchange chromatography of 80 mg of crude whey on a Nuvia Scolumn. Column: 7 x 27 mm; 2 ml fractions were collected; flow rate: 60 CV/hr; buffer A:0.04 M Na lactate, pH 4.0; buffer B: 0.04 M Na phosphate, pH 8.0.

■■■■

pH and A 280 Measurements

The pH and A280 of the chromatography fractions weremonitored using the BioLogic DuoFlow system instrumentation.
Total protein was determined by the Quick Start™ Bradford
protein assay (Bio-Rad).
Results and Discussion
Preparation of Whey Protein Isolate

As defined by the Whey Protein Institute (www.wheyoflife.org),
WPI is the most pure and concentrated form of whey proteinwith little or no lipids and lactose. Since the majority of theproteins have isoelectric points above 4.7, loading of whey at
pH 4.0 onto Nuvia S media resulted in the capture of theseproteins (Etzel 2004, Hahn et al. 1998). Lipids, lactose, andunbound proteins remain in the flowthrough and the wash. As
shown in Figure 2, a single peak was collected upon elution
with pH 8.0 elution buffer. Capacity of the Nuvia S column,
based on the measurement of the protein content in theeluate pool, was 60 ± 2 g/L (60 CV/hr, n = 4). As shown inFigure 3, proteins in WPI and in whey are similar. Interestingly,
densitometry of the major species, including ALA, BLG, andBSA in whey and WPI, showed nearly identical distributions.

© 2011 Bio-Rad Laboratories, Inc.

MW
200,000
116,250
97,40066,200

BSA

45,00031,00021,500BLG
ALA14,4006,500Standard

WPI

WheyFig. 3. SDS-PAGE analysis of WPI and whey. Ten mg of whey protein
and WPI along with Bio-Rad SDS-PAGE standards were run on a
4–20% Criterion TGX precast gel. Proteins were stained using BioSafeCoomassie stain. MW, molecular weight.
Anion Exchange Media Selection and Optimization

ALA and BLG account for about 75% of the proteins inwhey. These two model proteins were therefore selectedfor further experimentation. In an initial screen, Nuvia Q
media was compared to two media with high binding
capacity (Media A and Media B). The media were tested
using a small mixture of ALA and BLG (10% CV) thatwas eluted using a shallow NaCl gradient (0–0.3 M).
This permits rapid and uniform evaluation of processparameters but further optimization will be needed for theselected media. The resulting chromatograms from thisscreening experiment were compared (Figure 4). All threemedia produced two equally well-resolved peaks. Peaks ofMedia A eluted sooner, suggesting weaker anion exchange
properties than Nuvia Q media and Media B. The bead sizeof Media A is the smallest of the three media, which maylead to higher column pressure upon scale-up. Additionally,rigidity, cleanability, capacity, and cost will have to befactored in when choosing the appropriate media for largescale purification.

Bulletin 6128

0.100AU0.0250.050

AU0.050

0.075

0.000

0.025

–0.0250.000
01020Min, tenth3040Whey
Pool 1Pool 2				

Fig. 4. ALA and BLG purification chromatograms on three anion
exchangers. Comparison of Nuvia Q media (—), Media A (—), and Media B (—)
performance. AU, absorbance units.

Fig. 6. SDS-PAGE analysis of whey, purified ALA (Pool 1), and purified
BLG (Pool 2) against protein standards.

Purification of ALA and BLG from Whey

To minimize processing costs, a purification scheme for
ALA and BLG from whey ideally should require a singlechromatographic step to purify both proteins. Initial studies(Kim et al. 2003, Gerberding and Byers 1998) demonstrated
the removal of the major whey proteins (ALA, BSA, and BLG)
in a segmented gradient (data not shown). Further work wasperformed to optimize these conditions. The purification
profile of each protein was first identified by injecting thepurified protein alone and then a mixture containing thethree purified proteins. As expected, separation of themixture paralleled that of the individual proteins. When whey
was used as the feed stock, two major pools were resolved
upon adsorption and elution with the segmented gradient(Figure 5).
0.70.6
Pool 1A 280

0.5

Conclusions
Nuvia S and Nuvia Q media, two novel high-capacity media
for ion exchange chromatography, allow efficient enrichmentof proteins from crude mixtures. The feasibility to prepare
WPI, purified ALA, and purified BLG has been demonstrated.
Because of the high-throughput properties of Nuvia media, arapid single-step purification of proteins from whey is possible.
References
Etzel MR (2004). Manufacture and use of dairy protein fractions. J Nutr 134,996S-1002S.
Gerberding SJ and Byers CH (1998). Preparative ion-exchange
chromatography of proteins from dairy whey. J Chromatogr A 808, 141-151.
Hahn R et al. (1998). Bovine whey fractionation based on cation-exchange
chromatography. J Chromatogr A 795, 277-287.
Kim JI et al. (2003). Separation of whey proteins by anion-exchange
membranes. Korean J Chem Eng 20, 538-541.
Whey Protein Institute. www.wheyoflife.org, accessed April 18, 2011.

140— A 280 — Conductivity, mS/cm

120100Pool 2800.4600.3

400.2

Conductivity, mS/cm

0.8ALA
BLG
standard standard

Coomassie is a trademark of BASF.

200.1
000

1,000

2,000Time, sec

3,000Fig. 5. Anion exchange chromatography of 8 mg of crude whey on a7 x 27 mm Nuvia Q media column. Fractions of 2 ml were collected; flow
rate: 60 CV/hr; buffer: 0.02 M Na phosphate, pH 6.0; segmented gradient:
0–0.1 M NaCl, 0.1–0.3 M NaCl, and 0.3–0.9 M NaCl.

SDS-PAGE analysis (Figure 6) of the fractions showed
two peaks, one containing predominantly ALA (Pool 1)
and the other containing predominantly BLG (Pool 2). Thecorresponding ALA and BLG standards are also shown in thegel scan. Purities of the two whey-derived proteins in eachpeak were estimated by densitometry to be at least 85%.

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Bulletin 6128

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