Novel Mixed-Mode Chromatography Media
for Protein Separations Using New 1 mL AcroSep™ Columns
Gurpreet Kaur, Masilamani Selladurai, Matt Hymes, Hongshan Li, Lisa Bradbury
Pall Corporation; Bangalore, India and Woburn, MA, USA
Abstract
Materials and Methods
The separation of proteins with similar pI’s has been a protein purification challenge for some time. To address this problem,
Pall Life Sciences has developed two new unique resins whose interaction with proteins is based on both hydrophobicity and ion
exchange. HEA and PPA HyperCel™ resins offer researchers an opportunity to achieve multi-dimensional protein separation in
a single chromatographic step.
Pall AcroSep 1 mL Pre-packed HEA and PPA Columns
• HEA HyperCel resin, PN 20250-C001
• PPA HyperCel resin, PN 20260-C001
HEA and PPA HyperCel resins have strong hydrophobic moieties, which
bind proteins under lower salt conditions than typical hydrophobic
interaction chromatography (HIC) media. This has several advantages
including reduced probability of protein loss due to instability in high
salt. Additionally, HEA and PPA HyperCel resins have an amine group
(pKa ~8) which can act as a weak ion exchanger to improve recovery
during the elution step. In moderate to low pH conditions most
proteins will be charged such that an electrostatic repulsion occurs
prompting elution. This combination of hydrophobic and charge
interaction allows for the separation of proteins having similar pl or
hydrophobicity. The hydrophobicity of HEA and PPA is sufficiently strong
that the entire process can be performed under the protein friendly
conditions of moderate to low salt.
The performance of HEA and PPA HyperCel media is demonstrated using a simple protein mixture. The HEA and PPA HyperCel
AcroSep pre-packed 1 mL columns are used in combination with an automated chromatography system and a manual syringe
set up to show flexibility of use and similarity in results. The selectivity differences between HEA and PPA HyperCel resins are
discussed. The ability to readily control the strength of hydrophobic and charge interactions during protein separation allows
for multi-dimensional separations in a single chromatography step. Thus, these new chromatography media have the potential
to significantly improve the overall purification process of many proteins.
Protein Load for AcroSep Columns
HEA HyperCel Column – 1 mg bovine serum albumin (BSA) + 1 mg α-Chymotrypsinogen A + 0.5 mg lysozyme (2.5 mg total).
PPA HyperCel column load was the same except α-Chymotrypsinogen A was decreased to 0.75 mg (2.25 mg total protein).
Pure proteins purchased from Sigma.
Protein Separation Method Using Automated Chromatography System or Syringe Pump
Table 1
Volume
Flow Rate
Chrom System
Syringe*
H2O wash
10 CV
0.5 mL/min
1 mL/min
Equilibrate
10 CV
0.5 mL/min
1 mL/min
Protein load
1 mL
0.2 mL/min
0.1 mL/min
Wash
5 CV
0.5 mL/min
0.5 mL/min
Elute
3 or 5 CV
0.2 mL/min
0.5 mL/min
*A syringe pump was used for these experiments to maintain a controlled flow rate, however, this is not required.
The syringe can be operated manually. 1 mL fractions were collected for analysis.
Buffers for Protein Separation Method
Table 2
Step Elution Buffers
Results
Figure 1
Protein Separation with PPA HyperCel AcroSep
Columns on an Automated Chromatography System
Figure 2
Protein Separation with HEA HyperCel AcroSep
Columns on an Automated Chromatography System
Peak 3
Peak 1
Peak 4
AcroSep Column Equilibration Buffer
1
2
3
4
HEA HyperCel
100 mM carbonate +
150 mM NaCl
pH 10.0, 21 mS
50 mM
NaPhosphate
pH 7.0
25 mM
25 mM
25 mM
NaCitrate pH 5.0 NaCitrate pH 4.0 NaCitrate pH 3.0
PPA HyperCel
Phosphate buffered
25 mM
saline, pH 7.2, 15.5 mS NaCitrate pH 5.0
25 mM
25 mM
N/A
NaCitrate pH 4.0 NaCitrate pH 3.0
Note that protein binding for HEA HyperCel resin was done at pH 10 to eliminate the charge on the resin (pKa ~8) which
otherwise prevents binding of lysozyme (pI 10-11). This was not an effective strategy for PPA HyperCel, so PBS was used
instead. As a result the pH 7.0 elution buffer was not appropriate for PPA HyperCel resin. Step elution was used for both
systems. Gradients were not used for these methods.
Peak 4
pH 7.0
Peak 1
Peak 2
Peak 2
pH 5.0
pH 5.0
Peak 3
pH 4.0
pH 4.0
pH 3.0
Discussion
pH 3.0
HEA and PPA HyperCel resins operate on the basis of a mixed-mode mechanism, where both hydrophobic and ionic effects
are present.
AKTA chromatography system programmed as indicated in Table 1. Blue trace is OD280, green trace is pH, orange trace is conductivity. MALDI analysis used
for protein identification.
HEA HyperCel − the amine includes an n-hexyl substituent
CH2-CH2-CH2-
CH2-(CH2)4-CH3
:
The chromatogram in Figure 1 demonstrates protein separation using PPA HyperCel resin and shows that at pH 7.2 more than
90% of lysozyme does not bind, partly due to charge repulsion (peak 1). This peak is quite broad, possibly suggesting that the
lysozyme might be slightly retarded due to a weak interaction with the PPA HyperCel media. The small amount that is bound
elutes in the first elution step (peak 2, pH 5). The α-Chymotrypsinogen A is eluted with pH 4.0 buffer (peak 3) while BSA is eluted
with pH 3.0 buffer (peak 4). Note that protein binding occurred in buffer containing physiologic salt (~150 mM), good for most
proteins but quite low for typical hydrophobic interaction.
PPA HyperCel − the amine includes a phenylpropyl substituent
:
BSA
97
66
97
pH 5.0
pH 3.0
pH 7.0
pH 4.0
Wash
FT
pH 3.0
Load
pH 4.0
MW
pH 5.0
Wash
FT
Load
MW
Figure 3
Protein Separation on PPA and HEA HyperCel AcroSep Columns Using the Syringe Method
BSA
66
45
α-Chymotrypsinogen A
30
20
45
H+
N
:
Binding of very basic proteins such as lysozyme (pI 10-11) to HEA and PPA HyperCel resin may require increasing the pH close
to or above the resin pKa of 8, as demonstrated with HEA HyperCel (Figure 2). At pH 10, lysozme binds due to elimination of charge
repulsion and a likely increase in hydrophobic interaction due to a pH induced structural change. The proteins elute from HEA
HyperCel resin in the same sequence. Lysozyme elutes first with pH 7.0 buffer (peak 1) followed by α-Chymotrypsinogen A (peak
2) and BSA (peak 4) with pH 5.0 and pH 3.0 buffers, respectively. A small fraction of α-Chymotrypsinogen B contaminant is eluted
with pH 4.0 buffer (peak 3). The order of elution reflects the charge state of the resin and proteins and changes in hydrophobic
properties due to the combined influence of salt and pH.
Both include an amine group with pKa ~8
The PPA aromatic ligand is more hydrophobic than the aliphatic HEA ligand, thus providing two selectivity options for protein
purification. Protein binding is predominantly hydrophobic while elution is driven by electrostatic repulsion. This dual interaction
mechanism has several advantages over traditional hydrophobic chromatography methods (HIC or reverse phase). Both ligands
are more hydrophobic than traditional HIC chemistries, thus less salt is required for protein capture. The use of charge repulsion
in the presence of low salt significantly improves the elution efficiency. The mixed protein separation data (Figures 1, 2, and 3)
illustrate some of the performance differences between PPA and HEA, with slightly later elution of α-Chymotrypsinogen A from
PPA (pH 4) as compared to HEA (pH 5). Although the order in which these proteins elute is consistent with their pIs, in the case
of α-Chymotrypsinogen A (pI 8.8-9.6) the elution requires a further drop in pH to alter the hydrophobic interaction as well. Thus,
purification methods using HEA or PPA HyperCel resin are likely to result in less protein structure alteration (less extreme salt
and pH conditions) and higher recovery as compared to other HIC or reverse phase methods.
α-Chymotrypsinogen A
30
Lysozyme
14
20
Lysozyme
Conclusions
14
Fractions from PPA (left) and HEA (right) run on 12% SDS-PAGE stained with Coomassie Brilliant Blue G-250 (GE). Load lane has ~8 µg protein; eluate, flow
through (FT) and wash lanes have ~3 µg protein/lane except fractions containing little or no protein (maximum volume loaded).
PPA and HEA HyperCel fractionation of the same four proteins with the same buffers using a syringe pump (to mimic manual
use) demonstrates consistency between pumped and syringe-based methods. Fractionation on PPA HyperCel resin shows that
lysozyme does not bind at pH 7.2, while BSA and α-chymotrypsinogen are retained. The α-Chymotrypsinogen A and BSA are
eluted with pH 4.0 and pH 3.0 buffers, respectively. All four proteins are retained on HEA HyperCel resin at pH 10 (Figure 3). The
bound proteins are eluted with pH 7.0 (lysozyme), 5.0 (α-chymotrypsinogen A), and 4.0 and 3.0 (BSA) buffers. This data shows
good consistency with fractionation on the automated chromatography system.
© 2008, Pall Corporation. Pall,
, AcroSep, and HyperCel are trademarks of Pall Corporation. ® indicates a trademark registered in the USA.
• HEA and PPA HyperCel chemistries offer protein scientists two new mixed-mode separation media with the capacity for
multi-dimensional separation in a single chromatography run.
• The degree to which hydrophobic and ionic interactions contribute to each step in the purification is controlled by salt and pH.
• Although similar, there are clear performance differences between HEA and PPA allowing the user to determine the best
choice for their target protein.
• The HEA and PPA ligands are highly hydrophobic resulting in high efficiency protein binding as compared to conventional HIC
resins. As a result, effective protein binding usually only requires physiologic NaCl concentrations rather than high concentrations
of lyotropic salts typical for traditional HIC methods. This reduces the probability of protein precipitation or unfolding.
• HEA and PPA HyperCel resins are available in an easy to use disposal 1 mL pre-packed column compatible for use on an
automated chromatography system or for manual use with a syringe.
is a service mark of Pall Corporation. Coomassie is a trademark of Imperial Chemicals Industries, Ltd.
12/08, GN 08.2628