A New Method for Antibody Purification
Gurpreet Kaur, Venugopal Narne, Hongshan Li, Lisa Bradbury; Pall Corporation, Bangalore, India and Woburn, MA, USA
ABSTRACT
MATERIALS AND METHODS
RESULTS (continued)
Figure 2b
IgG Purification From Albumin Depleted Plasma Using AcroSep Column With MEP HyperCel Resin
Purified antibodies form the basis of many diagnostic tests targeting specific proteins. Antibody purification protocols frequently employ Protein A or
Protein G ligands due to the high specificity and degree of purification in a single step. However, there are limitations to using Protein A or G, including
weak or poor capture of antibodies from some species or isotypes1. Additional concerns include Protein A or G leaching, antibody aggregation due to
low pH elution conditions, and high resin cost. Ideally, alternative methods will have similar performance without the disadvantages listed above.
Analysis of IgG Purity
Pall Corporation has developed a novel method for efficient antibody purification. The method offers selective binding of antibodies irrespective of class
and species, and is cost effective. The method uses two simple chromatography steps, achieves > 90% purity, and uses a relatively higher elution pH
that minimizes antibody aggregation.
Reference graph is generated using 25 µg to 1000 µg standard IgG diluted in 100 mM sodium phosphate buffer, pH 6.8. Area under the peak is plotted
against the IgG concentration. 10 µL of each test sample is run with the same conditions. The following formula is used for calculating IgG recovery in
All fractions are analyzed by SDS-PAGE and HPLC for purity estimations.
mAU
Analysis of IgG Recovery
8.0
Pure lgG
100
test samples taking into consideration the volumes of each fraction obtained during chromatography.
The method provides a cost effective approach to antibody purification for diagnostic and other purposes without the disadvantages of Protein A and
Protein G purification methods.
total IgG
in test sample
Figure 4
IgG Recovery Analysis From IgG Purification on Columns With MEP HyperCel Resin
Chromatogram from AcroSep column with MEP HyperCel resin run, with OD280 (total
protein) in green and pH in orange. Unbound fraction from the AcroSep column with Blue
Trisacryl M resin is loaded on the AcroSep column with MEP HyperCel resin. Each step in
the purification is indicated with arrows.
The total IgG and percent recovery values from HPLC analysis are an average of three individual
IgG purifications. The percent std deviation values of each fraction are MEP load = 26; MEP eluate
pH 5.0 = 9; and MEP eluate pH 4.0 = 3.5.
250
7.5
200
7.0
150
OD280 nm
80
Blue Trisacryl® M resin is used first to significantly reduce albumin content. Then, antibody is purified with MEP HyperCel™ resin. Bound antibody is
eluted at pH 5.0 after two simple wash steps. Pre-packed AcroSep™ chromatography columns simplify the entire process.
RESULTS (continued)
area) x std. IgG concentration
=[ (test sample peak
] X total fraction volume
(std. IgG peak area )
pH
6.5
100
60
6.5
50
RESULTS
MATERIALS AND METHODS
0
5.0
Pall AcroSep chromatography column, 1 mL, with pre-packed Blue Trisacryl M resin (PN 25896-C001) equilibrated with PBS.
Pall AcroSep chromatography column, 1 mL, with pre-packed MEP HyperCel resin (PN 12035-C001) equilibrated with 50 mM Tris, pH 8.0.
Pall Acrodisc syringe filter with GxF/0.45 µm Supor membrane (PN AP-4424)
®
5.5
40
®
Pooled EDTA plasma from healthy human donor
Pure human IgG as a standard for HPLC (Sigma Chemicals)
20 mM phosphate buffer saline pH 7.2 (PBS)
Automated chromatography system (AKTA◆ Explorer, GE Healthcare) for IgG purification
Depletion of Albumin Using AcroSep Chromatography Column with Blue Trisacryl M Resin
Neat plasma is diluted ~6.5 times (300 µL diluted to 2.0 mL) with PBS and filtered using syringe filter to remove particulates. The 2 mL diluted and filtered
sample is injected on the AcroSep column with Blue Trisacryl M resin at a flow rate of 0.3 mL/min. Unbound fraction depleted of albumin containing IgG
is collected.
mAU
mS/cm
OD280 nm, Total Protein
Conductivity
Chromatogram from the AcroSep column with Blue Trisacryl M resin run, with OD280
(total protein) in green and conductivity (salt) in orange. Unbound fraction from the
AcroSep column with Blue Trisacryl M resin (first peak) is collected for the AcroSep
column with MEP HyperCel resin run.
800
150
0.0
10.0
Sample
20.0
PBS
30.0
40.0
mL
H2O Caprylate pH 5.0
pH 4.0
Excellent IgG Purity Demonstrated by SDS-PAGE and HPLC Analysis
SDS-PAGE analysis of starting plasma, depleted plasma (MEP load), and MEP fractions is shown in Figure 3. The most prominent band in the plasma
sample is albumin, at ~67 kDa. This correlates with the very high concentration of albumin in plasma relative to all other proteins. Although the MEP
HyperCel resin is selective for antibody, the high amount of albumin results in some binding. Thus, to eliminate the albumin, plasma is first passed
through the column with Blue Trisacyl M resin, which removes most of the albumin (lane 3, MEP load). The albumin depleted sample then passes through
the column with MEP HyperCel resin and all the IgG is bound as seen by the absence of IgG in the flow through fraction (MEP FT). There is no loss of
IgG in the water or caprylate washes (lane 5, data not shown). The eluate at pH 5.0 contains pure IgG (pH 5.0 lane). The eluate fraction at pH 4.0 contains
MW
100
Plasma MEP load MEP FT
H2O
pH 5.0
pH 4.0
Reduced 12% SDS-PAGE (0.5-4.0 µg total protein). MW = molecular weight markers, FT =
unbound fraction, H2O = water wash, pH 5.0 and 4.0 = sample from elution at indicated pH.
Red boxes indicate location of IgG heavy and light chains. Coomassie◆ Brilliant Blue R-250
stain (GE Healthcare) used for detection.
97
Figure 1
Schematic of IgG Purification Using AcroSep Chromatography Columns with Blue Trisacryl M and MEP HyperCel Resins
45
5.0
10.0
15.0
20.0
25.0
mL
Water wash to remove residual albumin
• In the second column, the IgG is actually purified.
lgG Light Chain
Pass sample through Blue Trisacryl AcroSep column
lgG and other proteins bind
A new two step method for the purification of IgG is fast and relatively simple.
lgG Heavy Chain
• In the first column run (Blue Trisacry M resin) IgG merely passes through the column.
0.0
Add EDTA. Pass sample through MEP AcroSep column
resulting from low pH induced aggregation, can be largely eliminated with the less harsh elution pH of 4.0-5.5. The structure of the MEP ligand also
allows for binding of all IgG isotypes from all species tested thus far. Additionally, some of the other Ig proteins have been shown to bind3, 4. Taken
together, these performance characteristics make MEP HyperCel resin as a good choice for antibody purification upstream of use in diagnostic
applications. However, it is not as specific as Protein A or G. Albumin will bind, albeit not as strongly as IgG and, hence, an additional step of AcroSep
columns with Blue Trisacryl M resin to remove albumin is needed. This is especially important since so many samples containing antibody also contain
albumin. The total cost of this procedure is approximately half as compared to Protein A.
CONCLUSION
0
Albumin and other proteins bind
9.2
23
MEP HyperCel resin facilitates antibody binding through hydrophobic interactions and antibody elution via an ionizable group present on the pyridine
ring. Elution is accomplished by charge repulsion which is largely dependent on the pKa of the MEP ligand with some contribution of the pl of the target
molecule. Elution based on charge repulsion from MEP resin is best accomplished at a pH < 5.5. The MEP ligand has many advantages as compared
to Protein A or G. In particular Protein A leaching into the purified antibody can lead to numerous problems for specific applications. Additionally some
monoclonal antibodies are particularly sensitive to low pH elution conditions used with Protein A and G, typically ~3.0. Loss of antibody activity, often
50
Dilute and filter plasma sample
85
213
The affinity properties of Cibacron Blue dye for albumin and other plasma proteins are well established2. The AcroSep column with Blue Trisacryl M resin
is used to effectively remove albumin and some other proteins from plasma, thereby improving the performance of the MEP HyperCel resin. Additionally,
the pre-packed column is very easy to use and works with pumped chromatography systems or a syringe.
66
200
0
1
MEP
pH 4.0
DISCUSSION
Figure 3
SDS-PAGE Analysis of Chromatography Fractions
400
100
250
MEP
pH 5.0
0
less IgG along with other proteins.
Unbound
fraction
containing lgG
600
% recovery
Total lgG µg
4.5
The chromatogram from the Blue Trisacryl M purification step (Figure 2a) shows removal of a significant amount of protein (green trace, second peak).
Data in Figure 3 (lane 3, MEP load) shows that the major protein in plasma (albumin) is depleted by virtue of its affinity for Cibacron◆ Blue dye. The
unbound fraction collected from the AcroSep column with Blue Trisacryl M resin (Figure 2a, first peak), which contains ~88% of the IgG along with other
plasma proteins, is used for IgG purification on AcroSep columns with MEP HyperCel resin.
Purification of IgG Using AcroSep Chromatography Column with MEP HyperCel Resin
1.5 mL of the unbound fraction from the AcroSep column with Blue Trisacryl M resin is loaded on an AcroSep column with MEP HyperCel resin for IgG
purification. Prior to this chromatography step, 10 mM EDTA is added to the sample to prevent binding of transferrin and, thus, improve purity of eluted
IgG. The MEP load sample (~2.5 mg total protein) is injected at 0.2 mL/min on an MEP HyperCel column equilibrated with 50 mM Tris, pH 8.0. The
column is washed first with high purity water then with 25 mM sodium caprylate in 50 mM Tris, pH 8.0. Bound proteins are eluted in two steps with
50 mM sodium acetate buffer, first pH 5.0 then pH 4.0. The eluted fractions are immediately neutralized to pH 7.0 using 1.5 M Tris buffer. The column is
stripped of remaining proteins with 5.2 mL 50 mM sodium acetate, pH 3.0. A schematic representation of the entire procedure is shown in Figure 1.
20
Human Antibody Purification Using AcroSep Chromatography Columns with Blue Trisacryl M and MEP
HyperCel Resins
Figure 2a
Albumin Depletion From Human Plasma Using AcroSep Column With Blue Trisacryl M Resin
MEP Load MEP FT
Purity of IgG achieved is > 95%.
30
1.5 mL of the AcroSep column with Blue Trisacryl M resin flow through fraction is loaded onto the MEP HyperCel column. The green trace in Figure 2b’s
chromatogram shows plasma protein separation on an AcroSep column with MEP HyperCel resin. The unbound fraction contains proteins other than
IgG, mostly albumin and transferrin (Figure 3, MEP FT). If albumin binds to MEP HyperCel resin via hydrophobic interaction, it can be eluted with water
wash. The sodium caprylate wash removes other proteins bound to the column via weak interactions. Elution at pH 5.0 removes most of the IgG (tall
blue peak) from the column due to electrostatic repulsion (MEP becomes partially charged). Tightly bound proteins and residual IgG are eluted at pH 4.0
(last green peak, Figure 2a).
Yield of the purified IgG from the column with MEP HyperCel resin is ~85%.
The total cost of AcroSep columns with Blue Triscryl M and MEP HyperCel resins is less than half the price of Protein A-based resin required to purify
an equivalent amount of IgG.
The 10 µL samples from fractions run on SDS-PAGE are subjected to HPLC analysis for confirmation of product purity. A size exclusion column (BioSep◆ SEC-S3000, 300 mm X 4.6 mm column) with 100 mM sodium phosphate buffer, pH 6.8 is used and IgG is detected at 280 nm. The sample is run at
0.35 mL/min. The HPLC data is consistent with the SDS-PAGE results. Based on SDS-PAGE and HPLC results, the IgG obtained from the column with
MEP HyperCel resin at pH 5.0 is estimated to be > 95% pure.
This method can be used with chromatography systems, a peristaltic pump set up, or manually with a syringe.
REFERENCES
Good IgG Recovery After This Two-Step Purification
Caprylate wash to remove other proteins
pH 5.0 elution – lgG specifically elutes
HPLC analysis of samples used for the purification steps show that ~88% and 85% of the IgG is recovered in the Blue Trisacryl M flow through fraction
and MEP HyperCel resin pH 5.0 elution fractions respectively (Figure 4, data not shown). An additional 9% is found in the pH 4.0 column with MEP
HyperCel resin fraction, but it is more dilute and contains several contaminants. Overall 75% IgG recovery from a two-step purification method compares
well to other methods, especially with plasma as a starting material.
1. Thurston, C.F. and Henley, L.F., (1988). Walker, J.M., ed. Methods in Molecular Biology, Vol. 3 - New protein techniques. Humana Press: Clifton, N.J.,149-158.
2. Gianazza, E. and Arnaud, P., (1982). Biochemistry Journal Vol. 203 - Chromatography of plasma proteins on immobilized Cibacron Blue F3-GA.
Mechanism of the molecular interaction.
3. Subramanian, A., (2005). Papers in Biotechnology – Chromatographic purification of mAbs with non-affinity supports
pH 4.0 elution – lgG and other protein elute
Contact: Phone: 800.521.1520 (USA and Canada) • (+)800.PALL.LIFE (Outside USA and Canada) • www.pall.com/oem • E-mail: LabCustomerSupport@pall.com
4. Guerrier, L., Flayeux, I., and Boschetti, E., (2001). Journal of Chromatography B Vol. 755 – A dual mode approach to the selective separation of
antibodies and their fragments.
© 2009, Pall Corporation. Pall,
, Acrodisc, AcroSep, HyperCel, Supor, and Trisacryl are trademarks of Pall Corporation. ® indicates a trademark registered in the USA.
AKTA is a trademark of GE Healthcare. Cibacron is a trademark of CIBA-GEIGY Ltd. Coomassie is a trademark of Imperial Chemical Industries. BioSep is a trademark of
Phenomenex, Inc.
7/09, GN09.3008
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