Direct Capture of α-amylase from CHO Cell Culture
™
Supernatant Using Novel HyperCel STAR AX
Salt Tolerant Anion Exchange Chromatography Sorbent
René Gantier1, Magali Toueille2, Jérôme Champagne2 - 1. Pall Life Sciences, Northborough, MA, USA; 2. Pall Life Sciences, Cergy, France
OPTIMIZATION OF ELUTION
CONDITIONS BY NaCl GRADIENT
INTRODUCTION
CHO (Chinese Hamster Ovary) cell culture is well established for the production of recombinant proteins. Anion exchangers
(Q or DEAE sorbents) are commonly used as first purification steps. Using conventional anion exchangers requires cell
culture supernatant (CCS) dilution to lower ionic strength or diafiltration to achieve sufficient capacity. However, these
operations increase buffer consumption and processing time while limiting throughput. Using a “salt tolerant” anion
exchanger such as HyperCel STAR AX sorbent allows direct capture from undiluted feedstock and improves significantly
the process economics at production scale.
CAPTURE STEP PURIFICATION OF α-AMYLASE
α-amylase elution conductivities were 38 mS/cm on HyperCel STAR AX sorbent and 24 mS/cm on rigid Q agarose
(Figure 3).
HyperCel STAR AX sorbent allows the elimination of a large amount of contaminants in the flowthrough and washes
Efficient purification of α-amylase on HyperCel STAR AX sorbent with better resolution of target enzyme from
(SDS-PAGE A, lanes 2, 3 and 4)
nucleic acids contaminants.
Higher elution conductivity required for HyperCel STAR AX sorbent compared to conventional AEX, due to the stronger
interaction of its ligand with proteins in the presence of salt (“salt tolerance”). However, HyperCel STAR AX sorbent allows
This study describes the use of HyperCel STAR AX “salt tolerant” anion-exchange sorbent for the capture of an acidsensitive model protein (α-amylase) spiked in CHO CCS.
Chromatograms and SDS-PAGE on Figure 5 illustrate the capture of α-amylase in optimized conditions:
Higher A280nm / A260nm ratio in elution from rigid Q agarose confirms that nucleic acid elimination during capture is more efficient on
HyperCel STAR AX sorbent than on rigid Q agarose
complete elution of bound proteins during elution gradient to 1 M NaCl.
Figure 5
α-amylase Capture on HyperCel STAR AX Sorbent and Rigid Q Agarose in Optimized Conditions.
HyperCel STAR AX salt tolerant anion exchanger was developed on the HyperCel matrix that confers mechanical and
chemical stability. The ligand is based on primary amine chemistry, which provides salt tolerance to the sorbent with the
ability to capture proteins in a high conductivity environment (Table 1 and Figure 1).
Figure 3
OD280nm
OD260nm
Conductivity
pH
mAU
α-amylase 24 mS/cm
Strip
2 M NaCl
mAU
Properties of HyperCel STAR AX Sorbent
2000
Average particle size
80 µm
Ion exchange ligand
Primary amine
1600
>100 mg BSA/mL within pH range
7.5 – 8.0 at conductivity 15 mS/cm
1200
Recommended operating range of
feedstock conductivity
2 – 15 mS/cm
Recommended cleaning conditions (2)
1 M NaOH
2400
NaCl gradient
up to 1 M
Flowthrough
Eq
2000
3 4
5
6 7
E
62 kDa
49 kDa
2400
1600
38 kDa
1600
1200
Nucleic acids
50 mS/cm
800
400
800
800
400
0
0
0
0
20
40
60
80
100
120
140
0
160 mL
20
40
60
80
100
120
140
0
mL
Equilibration (Eq): 50 mM Tris-HCl pH 7.5, 8 mS/cm, load: 2-fold diluted CCS, wash: 50 mM Tris-HCl, pH 7.5, 8 mS/cm; elution: NaCl
gradient up to 1 M, strip: 50 mM Tris-HCl pH 7.5 + 2 M NaCl. (A) load: 26 mg α-amylase/mL sorbent; (B) load: 24 mg α-amylase/mL
sorbent.
(1) Determined using a 5 mg/mL BSA in 25 mM Tris-HCl , 0.14 M NaCl at 2 minute residence time
(2) Injection of 5 column volumes (CV) of 0.5 – 1 M NaOH, 1 hour contact time
W2
2
α-amylase
NaCl gradient
up to 1 M
Flowthrough
W1
1
Nucleic acids
>80 mS/cm
α-amylase 38 mS/cm
Eq
Flowthrough
Crude CCS
pH 7.5, 12 mS/cm
Eq
3200
2800
2400
Dynamic binding capacity
OD280nm
OD260nm
Conductivity
pH
4000
(B)
HyperCel STAR AX
Strip
2 M NaCl
2800
(1)
Strip
2 M NaCl
mAU
(A)
Rigid Q Agarose
Table 1
(A)
HyperCel STAR AX
NaCl Gradient Optimization of Elution Conductivity on HyperCel STAR AX Sorbent and Rigid Q Agarose.
10
20
30
40
50
60
70 mL
Strip
2 M NaCl
(B)
Rigid Q Agarose
mAU
1
E
2 3 4
5
6
7
4000
Figure 1
OPTIMIZATION OF WASH CONDITIONS: IMPACT ON
ENZYME ACTIVITY, CHOP ELIMINATION AND YIELD
Dynamic Binding Capacity (DBC) of HyperCel STAR AX Sorbent for BSA vs. pH / Conductivity. Contour Plot
3200
62 kDa
49 kDa
α-amylase
2400
from Response Surface Modeling Analysis.
Flowthrough 2X-diluted CCS, pH 7.5, 8 mS/cm
Eq
W1
38 kDa
W2
1600
Residence Time = 1 min
Residence Time = 2.5 min
20
20
18
20 60
60
60
80
18
Four different wash conditions tested to address their impact on separation of α-amylase from CHOP
using crude and 2-fold diluted CCS loads (Table 2).
Residence Time = 4 min
80
18
800
Initial purity of α-amylase in CCS = 62%.
100
100
80
16
Conductivity
16
100
14
12
12
140
0
16
14
120
10
0
120
14
140
12
10
160
Table 2
140
Screening of Wash Conditions
160
10
6
8
8
180
6
180
7
7.25
7.5
7.75
180
6
200
4
8
8.25
8.5
7
7.25
7.5
7.75
pH
8
No wash
Equilibration conditions
pH wash
pH 4.5, 2 mS/cm
High salt wash
pH 7.5, 15 mS/cm
pH + moderate salt wash
pH 4.5, 5 mS/cm
α-amylase recovery (% of load)=
α-amylase in elution (mg) x 100
α-amylase in load (mg)
8.5
80
100
120
140
mL
α-amylase (mg) x 100
CHOP (mg) + α-amylase (mg)
α-amylase purity (%)
Results shown in Table 3 indicate that due to its “salt tolerance” HyperCel STAR AX sorbent can purify α-amylase from either
undiluted or diluted feed with equivalent productivity and purification performance. In contrast, using undiluted feedstock on
conventional rigid Q agarose would result in productivity about 4 times lower (data not shown). Therefore, the use of HyperCel
STAR AX salt-tolerant sorbent provides more process flexibility compared to a conventional sorbent.
4
8.25
60
(A) Equilibration (Eq): 50 mM Tris-HCl pH 7.5, 12 mS/cm, load undiluted CCS (12.5 mg α-amylase/mL sorbent), wash 1 (W1): 50 mM Tris-HCl, pH 7.5, 12
mS/cm; wash 2 (W2): 50 mM Na acetate, pH 4.5, 2 mS/cm, elution (E): 50 mM Tris-HCl, pH 7.5, 48 mS/cm. (B) Equilibration (Eq): 50 mM Tris-HCl pH 7.5, 8
mS/cm, load 2-fold diluted CCS (26 mg α-amylase/mL sorbent), wash 1 (W1): 50 mM Tris-HCl, pH 7.5, 8 mS/cm; wash 2 (W2): 50 mM Na acetate, pH 4.5,
2 mS/cm, elution (E): 50 mM Tris-HCl, pH 7.5, 34 mS/cm. SDS-PAGE: (1) CCS + α-amylase, (2) flowthrough, (3) wash 1, (4) wash 2, (5) elution, (6) strip, (7)
molecular weight marker.
200
4
40
120
160
8
20
7
7.25
7.5
pH
7.75
8
8.25
8.5
pH
Improvement of final purity by 20 to 30%, with various impacts on yield and activity.
With 2-fold diluted CCS (Figure 4, A and B):
Column: 0.5 cm I.D. x 5 cm bed height (~1 mL); Sample: 5 mg/mL BSA in equilibration buffer; Equilibration buffer: 25 mM Tris-HCl,
pH 7.0 – 8.5; Conductivity 3 – 20 mS/cm; Residence time: 1 – 4 min (0.25 – 1 mL/min).
– pH wash: allows highest CHOP elimination for HyperCel STAR AX sorbent and rigid Q agarose (purity ~93%);
Better elution yield on HyperCel STAR AX sorbent.
Table 3
– High salt wash: significant decrease of yield on rigid Q agarose (α-amylase eluted during wash).
Performance of HyperCel STAR AX Sorbent for Capture of α-amylase under Optimized Conditions.
– pH + moderate salt wash: strong decrease in elution yield on both sorbents due to loss of α-amylase activity
(sensitive to combination of low pH + high salt).
COMPARISON OF DYNAMIC BINDING
CAPACITY AT 10% BREAKTHROUGH
HyperCel STAR AX sorbent capacity performances for α-amylase capture were compared to two conventional AEX sorbents:
rigid Q agarose and Q polymeric.
With crude CCS (Figure 4, C and D):
– Benefit from using the salt tolerant HyperCel STAR AX sorbent at 12 mS/cm due to 5-fold higher DBC compared to
conventional AEX.
Feedstock
DBC 10%BT
(mg/mL)
Yield*
Purity*
Productivity*
(g/L/hr)
Crude CCS
(12 mS/cm)
21
96%
94%
8
2-fold diluted
CCS (8 mS/cm)
40
94%
93%
9
– Better wash condition on HyperCel STAR AX sorbent was pH wash, providing best yield and CHOP elimination.
DBC with crude feedstock
– Clear difference between HyperCel STAR AX sorbent and conventional AEX sorbents: HyperCel STAR AX sorbent
– On rigid Q agarose, capacity is too low to consider a productive scalable process in these conditions.
* Values obtained with loads of samples at 60% of the DBC.
provided a 2-fold higher capacity compared to the other sorbents (Figure 2A).
Figure 4
– For the remainder of the study, rigid Q agarose was kept for comparison.
α-amylase Yield and Purity at Elution vs. Wash Conditions after Capture on HyperCel STAR AX Sorbent and Rigid Q Agarose
During Runs Loaded at 60% of DBC 10% breakthrough.
Influence of feedstock dilution on DBC
– HyperCel STAR AX sorbent provided the highest capacity (20 mg/mL) with crude feedstock (Figure 2B), approximately
2X diluted CCS: 0.25 g/L α-amylase; pH 7.5, 8 mS/cm
5-fold higher than that of rigid Q agarose. This confirms the “salt tolerance” of HyperCel STAR AX sorbent.
– Capture using crude and 2-fold diluted feedstock was further investigated.
Yield
(%)
100
Figure 2
80
DBC at 10% Breakthrough (BT) of α-amylase on HyperCel STAR AX Sorbent, AEX Sorbents, and Rigid Q Agarose
60
B
A
25
α-amylase DBC 10% BT (mg/mL)
% breakthrough α-amylase (%)
80
HyperCel STAR AX
Rigid Q agarose
Q polymeric
20
80 mg/mL
15
38 mg/mL
44 mg/mL
10
5
0
0
10
20
30
40
50
60
70
80
α-amylase loaded (mg/mL sorbent)
90
100
Purity
(%)
100
84 82
94 93
84
89
89
0
Rigid Q Agarose
60
40
HyperCel STAR AX
30
50
20
No wash
72
84
High salt wash pH+ moderate
salt wash
Rigid Q Agarose
88
93
93
6
7
8
9
10
Conductivity (mS/cm)
11
12
DBC at 10% BT of α-amylase on HyperCel STAR AX and: (A) AEX sorbents using crude CCS (pH 7.5, 12 mS/cm) containing α-amylase at 2.0
mg/mL in CCS to enhance binding (B) Rigid Q agarose using undiluted (12 mS/cm), 2-fold (8 mS/cm) and 4-fold (5 mS/cm) diluted CCS,
with a more challenging α-amylase concentration (0.5 mg/mL) mimicking that of a real feedstock. Each data point is the mean of 3 separate
measurements.
0
(A)
92
91 89
HyperCelSTARAXsalt-tolerantanionexchangesorbentcanefficientlycaptureandpurify
biologically-activeα-amylase,frombothcrudeanddilutedCHOfeedstock.
53
60
65
pH wash
High salt wash pH+ moderate
salt wash
No wash
Crudeorundilutedfeedstockcanbeappliedwithequivalentproductivity,bringingprocess
flexibilityandrobustness.
pH wash
High salt wash pH+ moderate
salt wash
Rigid Q Agarose
98
96
80
49
No wash
0
(%)
100
92
20
5
83
96 94
40
pH wash
40
10
97
20
80
60
CONCLUSION
60
(%)
100
50
Purity
HyperCel STAR AX
80
20
70
0
Yield
HyperCel STAR AX
40
30
Crude CCS: 0.5 g/L α-amylase; pH 7.5, 12 mS/cm
74
61
40
(B)
DBCofHyperCelSTARAXsorbentatshortresidencetimeof1minutewithcrudeCCSis
5-foldhigherthanthatofaconventionalrigidQagarosesorbent.
Capturefromcrudefeedeliminatestime/bufferconsumingoperationssuchasdilution.
93
Casestudycouldberepresentativeoftypicalsituationsencounteredwithvarious
recombinantproteinexpressionsystems.
70
32
20
0
97
(C)
No wash
pH wash
High salt wash pH+ moderate
salt wash
(D)
(Run conditions: See Figure 5 legend).
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