12-7750_STAR_AX_Tech_POS_Layout 1 2/15/12 12:39 PM Page 1
HyperCel™ STAR AX
Salt Tolerant Advanced Recovery Anion Exchange Chromatography Sorbent
A Novel, High Productivity Sorbent for Protein Capture and
Contaminant Removal from Undiluted Feedstocks
Sylvio Bengio, Magali Toueille, Jérôme Champagne, and René Gantier, Pall Life Sciences, Chromatography Group
ABSTRACT
RESULTS (continued)
HyperCel STAR AX sorbent is a novel salt-tolerant anion exchange chromatography sorbent designed for purification
of proteins from cell culture, plasma, or other feedstock, with minimal sample treatment. The sorbent is based on
the robust, industry-proven HyperCel matrix and can be operated in laboratory scale to large production scale
columns. HyperCel STAR AX sorbent has high dynamic binding capacity at short residence time (>100 mg/mL BSA
at 1 to 2 min residence time), and it maintains binding capacity at conductivities up to 15 mS/cm; therefore, it allows
direct feed processing without dilution or ultrafiltration/diafiltration (UF/DF). The sorbent is easy to operate and chemically stable (1 M NaOH). All combined, these features contribute to better productivity while decreasing downstream
processing costs.
Purification of HSA from Undiluted Plasma on HyperCel STAR AX Sorbent
Figure 3
Purification of HSA on 1 mL PRC Prepacked Column of HyperCel STAR AX Sorbent
Elution,
pH 4.0, 2 mS/cm
mAU
FT
2000
INTRODUCTION
Table 1
Main Properties of HyperCel STAR AX Sorbent
Average particle size
Ion exchange ligand
Dynamic binding capacity1
Typical range of feedstock conductivity
Recommended cleaning conditions2
60 – 90 µm
Primary amine
>100 mg BSA /mL within pH range 7.5 – 8.0
2 – 15 mS/cm
1 M NaOH
0
α-amylase DBC (mg/mL)
70
Residence Time = 2.5 min
18
Residence Time = 4 min
20 60
60
60
80
18
18
80
Conductivity
16
100
16
14
120
12
12
140
120
14
140
12
10
120
60
50
40
HyperCel STAR AX
30
20
160
8
180
6
180
180
6
200
4
200
4
8.25
8.5
7
7.25
pH
7.5
7.75
8
pH
8.5
7
8
9
10
Conductivity (mS/cm)
11
12
CCS CCS
CHOP:
413,000
CHOP:
413,000
ppm ppm
4
8.25
6
Synergistic Effect of HyperCel STAR AX and Protein A Sorbents for Removal of Host Cell Proteins (CHOP)
in Monoclonal Antibody Purification
Figure 5
Enhanced Removal of CHOP in a MAb Purification Process: Synergy with Protein A
160
10
8
5
140
160
8
Rigid Q agarose
Spiking in CHO cell culture supernatant (CCS) at 0.5 mg/mL; Capture of α-amylase in non-diluted CHO CCS and in
CCS diluted 2X and 4X; Data shows a 4-fold higher capacity for HyperCel STAR AX sorbent vs. rigid Q agarose,
when used with undiluted CCS.
100
100
7.75
7
7.25
7.5
7.75
8
8.25
8.5
pH
A Design of Experiments (DoE) study was done to explore the influence of various pHs (7.0 to 8.5), conductivities
(3 to 20 mS/cm) and residence times (1 to 4 minutes) on the dynamic binding capacity (DBC) for bovine serum
albumin (BSA) used as a model.
Data shows that the sorbent achieves a high DBC (> 100 mg/mL) over a wide range of pHs and conductivities at
short residence time.
CHOP content x1000 (ppm)
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 to 20 mS/cm.
Residence time: 1 to 4 min (0.25 to 1 mL/min). Numbers indicate binding capacity for BSA in mg/mL of sorbent.
HyperCel
HyperCel
STARSTAR
AX AX
FT mode
FT mode
CHOP:
362,000
CHOP:
362,000
ppm ppm
Protein
A
Protein
A
(bind/elute
(bind/elute
mode)mode)
Protein
A
Protein
A
(bind/elute
mode)mode)
(bind/elute
Capture of Human Serum Albumin (HSA) from Undiluted Plasma
Figure 2
Dynamic Binding Capacity of HyperCel STAR AX Sorbent for HSA from Undiluted (11 mS/cm) and Diluted (7 mS/cm)
Plasma – Comparison with Conventional DEAE Rigid Agarose
40
7 mS/cm
11 mS/cm
DBC for HSA (mg/mL)
35
P = Plasma
mL
30
25
20
15
5
5
4.5
4.5
4
4
3.5
3.5
3
3
2.5
2.5
2
2
1.5
1.5
1
1
0.5
0.5
CHOP content x1000 (ppm)
20
7.5
20
Experimental conditions were optimized in AcroPrep™ 96-well filter plates filled with HyperCel STAR AX sorbent
(data not shown), and transferred to column chromatography. Chromatogram and SDS-PAGE analysis of fractions
confirmed that most contaminants were eliminated by high conductivity wash (W2), leading to a 99% pure HSA fraction in a single step (E), with 90% yield. HSA was eluted by a simple decrease of pH (4.0), without addition of salt,
allowing a direct load on an orthogonal cation exchange column (Pall S HyperCel sorbent, data not shown).
0
20
7.25
10
10
Residence Time = 1 min
7
.
0
80
Figure 1
Results Generated by DoE Study
6
Transferrin
Albumin
Figure 4
Capture of α-amylase Spiked in CHO at 5 to 12 mS/cm: DBC vs. Load Conductivity
Dynamic Binding Capacity vs. Residence Time, pH and Conductivity
8
IgG
Capture of an Acidic Protein (pI 3.5) Spiked in CHO Cell Culture Supernatant
RESULTS
10
W2
500
2. Injection of 5 column volumes (CV) of 1 M NaOH, 1 hour contact time.
14
Albumin
1000
1. Determined using a 5 mg/mL BSA in 25 mM Tris-HCl , 0.14 M NaCl at 2 minutes residence time.
80
W1
FT W1 W2 E P
1500
Anion exchange chromatography is a standard method for protein capture or impurity removal when used in
flowthrough mode. Typically, anion exchange sorbents with diethyl-amino-ethyl (DEAE) or quaternary amine (Q) functionalities are used. However, these conventional sorbents have low binding capacity for protein in relatively high
salt conditions associated with undiluted biological feedstock. In this case, the feedstock needs to be diluted, typically 2- to 5-fold, to adjust its conductivity around or below 5 mS/cm.
HyperCel STAR AX sorbent uses a primary amine ligand, able to maintain its dynamic binding capacity (DBC) at
conductivity up to 15 mS/cm, allowing direct feed processing.
16
Wash 2,
pH 7.5, 20 mS/cm
FT from neat
plasma load Wash 1
2500
E
413000413000
0
0
CCS CCS
CHOP:
400 ppm
CHOP:
400 ppm
CHOP:
CHOP:
3,3003,300
ppm ppm
90% MAb yield on HyperCel STAR AX sorbent as first step in
flowthrough (FT) mode.
362000362000
3300 3300
400
400
A
A HyperCel
HyperCel
HyperCel ProteinProtein
HyperCel
STAR STAR
AX AX
STAR STAR
AX AX
+ Protein
A
+ Protein
A
CCS, CHO cell culture supernatant; MAb: 1.7 g/L ; CHOP ~700 000 ng/mL (413,000 ppm),
load 20 mg/mL, pH :7.2, conductivity: 13.8 mS/cm. Equilibration: 50 mM Tris, pH 7.2, conductivity
13.8 mS/cm. Residence time: 1 minute.
Figure 5 illustrates the synergistic effect of HyperCel STAR AX sorbent when used before a conventional MAb capture
step, using a Protein A sorbent: when HyperCel STAR AX sorbent is used, the total combined clearance of CHOP
obtained is about 8-fold better, or a 3-Log reduction.
Using HyperCel STAR AX sorbent as a pre-purification step before protein A may eliminate HCPs that co-elute with
the MAb after the Protein A step, and improves the life span of this step.
CONCLUSIONS
10
5
0
HyperCel STAR AX
DEAE rigid agarose
Data shows that DBC is less affected by conductivity changes in the 7 – 11 mS/cm range, and significantly higher
than when using a conventional sorbent with undiluted plasma.
Contact Information: Phone: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • +65 6389 6500 (Asia/Pacific) • Email: biopharm@pall.com • Web: www.pall.com/biopharm
HyperCel STAR AX is a novel anion exchange sorbent from Pall, designed for large-scale purification of proteins
from various feedstocks.
Based on a robust, industry-proven, scalable matrix.
High dynamic binding capacity for proteins across a broad conductivity range (2 to 15 mS/cm) at short residence
time (1 to 2 minutes).
Allows direct feedstock processing and limits dilution and UF/DF.
Can be used for direct capture of proteins or intermediate steps for efficient contaminant removal.
Contributes to better process economics.
© 2012, Pall Corporation. Pall,
, AcroPrep, and HyperCel are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. 2/12, GN 12.7750