Capture of an Acid-Sensitive Recombinant Protein from
E. coli Lysate Using Mixed-Mode Chromatography
Magali Toueille, Jérôme Champagne, Sylvio Bengio, René Gantier, Pall Life Sciences, Cergy, France
SUMMARY
This study highlights the use of mixed-mode chromatography sorbents for the capture of a model protein, recombinant
Figure 3
Glutathione-S-Transferase (rGST), expressed in the periplasm of E. coli.
rGST Breakthrough on Mixed-Mode vs. Anion Exchange Chromatography
3. REPRODUCIBILITY OF rGST CAPTURE ON HEA HYPERCEL SORBENT, 12 REPETITIVE RUNS (1 ML COLUMN)
rGST was chosen as a relatively difficult protein to purify, due to its lability at acidic pH, limiting elution conditions and
Figure 5
separation from acidic contaminants from E. coli.
Performance of HEA HyperCel Sorbent During 12 Successive Runs of rGST Capture Performed in Optimized Conditions
HEA HyperCel mixed-mode sorbent has a high selectivity for rGST and a capacity >10 mg rGST/mL.
In contrast, anion exchange was not efficient.
0.6
0.4
HEA HyperCel
Step robustness and a 300-fold scale-up of the separation performance on HEA HyperCel sorbent were also demonstrated.
0.2
INTRODUCTION
0
0
4
Figure 1
Periplasmic expression in E. coli strain BL-21
3D Structure of rGST.
E. coli lysate:
– pH 7, conductivity 4 mS/cm
– rGST concentration 2 mg/mL; total protein content 11 mg/mL
6
8
10
12
14
rGST Loaded (mg/mL sorbent)
pH
100
4000
11
10
80
rGST
DBC at 10%
breakthrough
0.8 mg/mL
9
3000
8
7
2000
1000
16
0
10
(Image from the RCSB PDB (www.pdb.org) of PDB ID 1GLT (Matsuda,
K., Mizuguchi, K., Nishioka, T., Kato, H., Go, N., Oda, J. (1996) Crystal
structure of glutathione synthetase at optimal pH: domain architecture
and structural similarity with other proteins. Protein Eng. 1083-1092.)
20
30
40
50
40
6
5
20
4
3
0
2
4
6
8
Run Number
60 mL
10
12
10
12
11.1 mg/mL
• ~ 10 times higher DBCs for mixed-mode vs anion exchange*
• No pH or conductivity adjustments needed
% of Load
Theoretical isoelectric point pI ~6.7
*Same DBC below 1 mg/mL were obtained for Q Ceramic HyperD F sorbent and rigid Q agarose (data not shown)
rGST Purity in Elution
100
100
80
80
60
40
2. rGST PURIFICATION ON HEA HYPERCEL SORBENT
2. MIXED-MODE CHROMATOGRAPHY: MEP, HEA
AND PPA HYPERCEL SORBENTS
60
rGST Elution Yield
MW 27.7 kDa
Protein unstable at acidic pH (< 4.0)
5000
% of Total Protein
1. rGLUTATHIONE-S-TRANSFERASE (rGST)
2
rGST Bound
Overlay of Chromatograms, Runs 1 to 12
UV280nm (mAU)
C/C0
Conditions for efficient capture step using HEA HyperCel sorbent were determined to protect the biological activity of rGST.
Q HyperCel
% of Load
0.8
Screening of mixed-mode and ion exchange sorbents was performed.
20
1 mL PRC Prepacked Column (Pall):
Analytics:
Exploits multiple, distinct protein-ligand interactions to
– Load pH: 7.0
– GST enzymatic activity assay (Sigma)
adsorb target proteins or impurities
– Load conductivity: 4 mS/cm
– Microfluidics electrophoresis (Bioanalyzer*, Agilent)
Offers new solutions to separations where traditional
– Total load: 7.5 mg/mL (60% of DBC at 10% breakthrough)
– GST quantification by ELISA (GE Healthcare)
chromatographic methods (ion exchange, HIC, affinity. . .)
– Elution pH 4.5 or 5.0, 50 mM Na acetate (+ strip at pH 3.0)
– Total protein quantification by Bradford Assay
are not effective
– CIP: 1 M NaOH, 60 min
60
40
20
0
0
2
4
6
8
Run Number
10
2
12
4
6
8
Run Number
• HEA HyperCel sorbent can be treated with 1 M NaOH for 60 minutes for
cleaning-in-place
(Thermo Scientific)
– Where feedstream conductivity is too high for efficient
• No modification of separation performance after 12 successive runs:
capture on traditional ion exchange sorbents
– Alternative/complement to conventional hydrophobic
interaction (HIC) or hydroxyapatite
Table 2
- Same chromatographic profile
Optimization of Elution Conditions for rGST Purification on HEA HyperCel Sorbent Used as Capture Step
- No significant variation of DBC, yield and purity
– Separations where affinity ligands are too expensive
3. RATIONALE OF USING MIXED-MODE SORBENTS
FOR CAPTURING rGST FROM E. coli LYSATE
rGST purity (%)
70
90
Yield (%)
65
50
Elution pH
4.5
5
rGST purification (fold)
3.2
4.9
Residual contaminating protein (mg/mg rGST)
0.4
0.1
4. PERFORMANCE OF HEA HYPERCEL SORBENT FOR rGST CAPTURE DURING 65 AND
300 TIMES SCALE-UP
Figure 6
Features are extensively documented from previously published
work on monoclonal antibodies and other proteins from various feedstocks.
Performance of HEA HyperCel Sorbent for rGST Capture During 65- and 300-fold Scale-up
• Active rGST is recovered after mildly acidic elution (pH > 4.0) from HEA HyperCel sorbent
Selectivity: unique separation mechanism, different from ion exchange, HIC or affinity
• Separation of rGST from contaminants is improved at pH 5.0 vs. pH 4.5
– Usually better recoveries than HIC
• Further steps of the study performed using pH 5.0 elution
Yield
100
– Near physiological conditions: binds primarily by hydrophobic interactions; no need for pH or conductivity adjustments of
the feedstock
– Elution at mild acidic pH favors biological activity of the protein (rGST is unstable at pH < 4.0)
90
4.5
Figure 4
80
4
Chromatogram and Gel Electrophoresis Profiles of rGST Capture Using HEA HyperCel Sorbent in Optimized Conditions on
1 mL HEA HyperCel PRC Prepacked Column
70
3.5
60
3
50
2.5
40
2
30
1.5
20
1
10
0.5
Flowthrough
Ligand structure of HyperCel mixed-mode sorbents.
Elution
pH 5
Elution
pH 3
Yield (%)
Figure 2
CIP
Uncharged at
near-neutral pH
CH2 CH2 CH2 S
CH2 CH2
(Spacer)
H+
N CH2 (CH2)4 CH3
pKa ~ 8
(Spacer)
PPA HyperCel
H+
N CH2
Positive charge can contribute
to binding of acidic proteins or
inhibits binding of basic proteins.
It can moderate attractive or
repulsive interactions based
on pH and conductivity.
T
S
G
r
11.0
4000
pKa = 4.8
HEA HyperCel
te
pH
UV280nm (mAU)
O
MEP HyperCel
5
a
lys
Elu
t
ion
pH
5
MW
10.0
GST
3000
rGST Purification Factor (fold)
Scalable to manufacturing scale
rGST Purification Factor
9.0
8.0
0
7.0
2000
1
6.0
5.0
1000
0
rGST
65
Column Volume (mL)
300
• Purification performance and yield are consistent during 65- and 300-scale-up
4.0
Micro-fluidic electrophoresis gel analysis
0
0
20
40
60
80 mL
CH2 CH2
pKa ~ 8
Table 3
CONCLUSION
Performance of HEA HyperCel Sorbent for rGST Purification During Capture Step
rGST PURIFICATION
1 .rGST CAPTURE FROM E. coli LYSATE: SCREENING OF CHROMATOGRAPHY SORBENTS
Direct capture of rGST from E. coli lysate
Yield (%)
50
rGST purity (%)
90
rGST purification (fold)
4.9
rGST activity yield (%)
51.1
GST activity enrichment (fold)
4.7
Load: 7.5 mg rGST (15 mL E. coli lysate); Elution: 50 mM Na acetate, pH 5.0.
Screening of anion exchange and mixed-mode sorbents:
- MEP, HEA and PPA HyperCel sorbents
®
- Q Ceramic HyperD F, Q HyperCel sorbents, rigid Q agarose
Table 1
Run Conditions for Sorbent Screening Based on Dynamic Binding Capacity (DBC)
Sorbent
Load pH
Load conductivity (mS/cm)
HEA HyperCel
7.0
4
Q HyperCel
8.5
2
• Direct load of cell lysate (no pH or conductivity adjustment)
• Efficient resolution of rGST from E. coli acidic proteins
Mixed-Mode chroMatography on hea hypercel sorbent is an efficient
solution for capturing rgst froM E. COLI
lysate can be loaded directly onto the column, simplifying process.
capture and elution of rgst on hea hypercel sorbent is selective and yields to dbc
>10 mg/ml with 4 to 5-fold purification factor.
• Selective elution at mildly acidic pH (pH 5.0) protects biological activity
a mild selective elution (ph 5.0) preserves acid-sensitive protein biological activity.
• Elution recovery in line with that generally obtained using GST
affinity sorbents1,2
robustness and scalability of the separation performance were also demonstrated.
• DBC >10 mg/mL
efficient regeneration using 1 M naoh cip.
the model could be extended to other E. coli proteins.
1. Arca, P., et al. Purification and study of a bacterial glutathione S-transferase. FEBS Lett. 263 (1) (04/1990) 77.
2. Hee-Joong Park, et al. Purification and Biochemical Properties of Glutathione S-Transferase from Lactuca sativa. J.
Biochem. and Mol. Biol. Vol. 38, No. 2 (03/2005) 232.
Column: 1 mL PRC prepacked with sorbent
Note: Detailed description of the present study can be found in USD 2824, Pall Application Note
Phone: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • Email: biopharm@pall.com • Web: www.pall.com/biopharm
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