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Tosoh Biosciences 2003
Tosoh Biosciences 2003
TOYOPEARL Resins for IEC Anion Exchange TOYOPEARL DEAE-650 TOYOPEARL SuperQ-650 TOYOPEARL QAE-550 Cation Exchange TOYOPEARL CM-650 TOYOPEARL SP-650 TOYOPEARL SP-550 TOYOPEARL MegaCap SP-550EC
TSK-GEL-5PW Bulk Resins for IEC Anion Exchange TSK-GEL SuperQ-5PW TSK-GEL DEAE-5PW Cation Exchange TSK-GEL SP-5PW
Method Development Glass Columns (MD-G) TOYOPEARL MD-G SuperQ-650S TOYOPEARL MD-G DEAE-650S TOYOPEARL MD-G SP-650S TOYOPEARL MD-G CM-650S TSK-GEL MD-G Q-5PW (20) TSK-GEL MD-G DEAE-5PW (20) TSK-GEL MD-G SP-5PW (20)
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Sample Concentration
A major feature of ion exchange is its power to concentrate a bound component. It is often used to capture and concentrate very dilute feedstream components early in a process sequence.In addition, it is used as a concentration step following SEC. In both cases, the main aim of the step is to reduce sample volume and increase sample concentration.
Sequential Processing
Charge interactions between resin and target molecules are fundamental to the selective power of IEC. It may be complemented
Features
porous, hydrophilic polymer based resin
Benefits
suitable for laboratory scale and process chromatography autoclavable at 121C -60 C compatible with organic solvents constant packing volume over a wide range of salt concentrations excellent flow characterization also in large industrial columns (up to 3 bar) easy scale up from TSK-GEL-5PW IEC columns high yields of biologically active proteins
chemical stability
continuous selectivity
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Table I
1000
1000
HW65
-O-R'-N+-(CH3)3 strong anion exchanger -O-CH2-CH2-N+-(C2H5)3 strong anion exchanger -O-CH2-COO weak cation exchanger
-
QAE-550C
500
HW55
1000
HW65
1000
Applications:
Proteins, antibodies, plasma proteins, peptides, tryptic digests, nucleotides, oligonucleotides, viruses, antibiotics, glycoproteins
HW- -O-R-O-CH2-CH2-CH2-SO365 strong cation exchanger HW- -O-R-O-CH2-CH2-CH2-SO3 55 strong cation exchanger
-
500
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Figure 2
B. TOYOPEARL DEAE-650M
.015
Resin storage
The recommended storage solution is 20% ethanol. Long-term storage in strong base (0.5 M NaOH) conditions, however, is not advised.
C. TOYOPEARL SuperQ-650M
Ion Exchange Capacity (meq/ml)
O.5 M HCl
TOYOPEARL resins have more stable bed volumes than other ion exchange media
With Changing pH
1.1 1.0 1.6
Figure 3
DEAE-TOYOPEARL 650M
DEAE-TOYOPEARL 650M
dextran-based resin
dextran-based resin
1.4
1.2
1.1 1.0
1.0 1.1
Figure 4
DEAE acrylic resin
1.1 1.0
1.1 1.0
T TOYOPEARL DEAE-650M
0.2
0.4
As pH Varies:
As Salt Varies:
Columns: 16 mm I.D. x 35 to 45 cm L Solutions: pH experiment: 0.05 M phosphate (pH 5 to 10) salt experiment: 0.02 M phosphate (pH 7.5) 0M to 0.5 M NaCl
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Mechanical capacity
Mechanical stability
TOYOPEARL IEC resins maintain stable bed volumes during the pH and ionic strength changes that occur during IEC chromatography. Notice in Figure 1 that the bed volume of other anion exchangers may change several percent during the course of a salt or pH gradient. Multi-cycle gradient operation and re-equilibration is accomplished without volume changes in the TOYOPEARL resin. The mechanical stability of TOYOPEARL resins allows the use of longer column beds and/or higher flow rates for fast, efficient column washing and regeneration. Typical linear velocities for TOYOPEARL SP-550C packed in a 1.4 m I.D. process column are shown in Figure 5. The pressure/flow relationship remains linear up to 600 cm/h.
Figure 5
Pressure drop vs. linear velocity for T TOYOPEARL SP-550C (1.4 m I.D. x 20 cm L)
3,0
100
200
300
400
500
600
Column packing
It is best to pack TOYOPEARL ion exchange resins by the application of gentle pressure from 0.5 - 3 bar. We do not recommend packing TOYOPEARL resin by gravitational settling. TOYOPEARL resins should not be packed under high pressure (> 7 bar). Typical packing mobile phases include phosphate, Tris, or acetate buffer containing inorganic salt at the highest ionic strength to which the column will be exposed during operation (including any clean-in-place). Typical packing and operational velocities for TOYOPEARL ion exchange resins are shown in Table III. For detailed packing instructions please refer to our website for downloads. Table III
Optimum packing and operational velocities for TOYOPEARL ion exchange resins
Column size (I.D. x L) Grade Packing flow rate (cm/h) Operating flow rate (cm/h)
TOYOPEARL DEAE-650, SuperQ-650, CM-650, SP-650 22 mm x 20 cm S M C TOYOPEARL QAE-550, SP-550 22 mm x 20 cm Table IV C 700 - 1,000 80 - 240 cm/h 400 - 600 800 - 1,000 800 - 1,200 50 - 65 cm/h 80 - 130 cm/h 80 - 650 cm/h
Phospholipase D ~56,000 Lipid transfer protein 69,000 Purine nucleotide phosphorylase 68,000
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Figure 6
Q agarose
72 %
TMAE-acrylic
111 %
TOYOPEARL QAE-550C
107 %
SuperQ 650M
40 %
30
60
90
30
60
90
30
60
90
30
60
90
Minutes
7.5 mm I.D. x 7.5 cm L ovalbumin, trypsin inhibitor 110 mg each protein in 2.0 ml
Elution: 100 min linear gradient of NaCl from 0 to 0.5 M in Tris/HCl buffer (pH 8.7) Flow rate: 136 cm/h Detection: UV @ 280 nm
Load
The ability to load large amounts of material on a column is important when developing a chromatography capture step. This is shown in Figure 6, as four competitive resins are each loaded with 33mg of protein per mL of resin bed. Although the mass loading and bed volume are identical for all four resins, TOYOPEARL SuperQ-650M provides the best separation because it is loaded to a lower percentage of its potential capacity. The same load is bound in a narrower, more concentrated zone within the column. TOYOPEARL size exclusion resins. In practice, however, large scale chromatography columns sometimes provide less efficient flow distribution than laboratory or pilot-scale columns. Column hardware design and wall effects with some resins do not always permit direct scale up. To restore column efficiency, bed height (z) is increased and column diameter decreased to obtain a target mass throughput with the same calculated final column volume, V (f). Resolution (Rs) in gradient elution ion exchange chromatography can be described by the equation: Rs = z1/2/[dp(u)]1/2[GS]1/2 particle size (dp); linear velocity (u); normalized gradient slope (GS); bed height (z) It is possible to preserve chromatographic resolution during scale up by keeping constant the terms in the equation. Linear velocity (u) and particle size (dp) are optimized at small scale, and are used in the large-scale process. The equation above may also be used to select an appropriate particle size grade of resin for an application. TOYOPEARL resins are available in three particle size grades to allow efficient process optimization. The gradient term in the equation, GS, refers to normalized gradient slope, and is dependent on resin volume. Further discussions on the gradient separation of proteins and the calculation of GS may be found in S. Yamamoto, K. Nakanishi and R. Matsuno, Ion Exchange Chromatography of Proteins, Marcel Dekker, New York, 1988.
Scale-up
Ion exchange chromatography is used at all scales, from research and development through manufacturing. The hallmark of a successful scale up is the ability to maintain resolution as the chromatographic bed volume is increased in direct proportion to the volume of feedstock. The focus of production scale is to increase the throughput of the separation system to handle the increase in sample mass. Factors such as mobile phase composition and sample load (mg sample/ml bed volume) are optimized at small scale during methods development. Thus column volume (CV (initial): CV (final) ) is increased in direct proportion to the increase in sample mass (M (initial): M (final) ), while the sample load or concentration (mg/ml) remains constant as in the equation below: M(i) / M(f) = CV(i) / CV(f) Scale-up of column volume is typically achieved by increasing the cross-sectional area of the column, while maintaining the bed height (z). This assumes that the efficiency of the large scale chromatographic bed equals that of the small scale bed. Such performance has been demonstrated in column efficiency studies with
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Scale-up
A 5000-fold scale-up of the -galactosidase enzyme purification was accomplished on TOYOPEARL DEAE-650M with the strategy described in the previous paragraph. The chromatograms in Figure 7 demonstrate the excellent scale-up characteristics of TOYOPEARL ion exchange media. The operational parameters for the scale up are listed in Figure 8. Gradient slope (GH) and particle diameter (dp) remained unchanged. Linear velocity (u) was reduced by 15% in the largest scale separation, and resolution actually improved relative to the smallest scale separation. This may be partly attributed to increased bed height. Although column volume was increased in part by increasing the bed height (z), the principal change in column volume was a result of the greater column diameter (1.4cm to 60cm). This example illustrates how TOYOPEARL media can be conveniently scaled up from laboratory to production scale columns.
versions of TSKgel DEAE-5PW, TSKgel SuperQ-5PW, and TSKgel SP5PW resins for those researchers who need direct scale-up from analytical resins of the same type and who need to pack their own columns. TSK-GEL 5PW preparative resins are made from the same polymer, (G5000PW), which is used in the prepacked TSK-GEL 5PW analytical ion exchange columns, and provide identical selectivity. Designed for high performance medium scale separations, or for final stage product polishing, TSK-GEL 5PW resins have been used in columns up to 30 cm in diameter. The enhanced mechanical stability and excellent permeability of TSK-GEL 5PW resins permit high linear flow rates. For example, bed compression was not observed at the highest flow rate studied, 540 cm/h (20 bar or 300 psi), with a 55 mm I.D. column. TSK-GEL 5PW preparative LC resins allow process development groups to directly scale up methods developed on TSK-GEL analytical ion exchange columns with a pressure maximum around 20 bar for the resin.
A. TSKgel DEAE-5PW
B. TOYOPEARL DEAE-650S
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15
TOYOPEARL DEAE-650M A. 14 mm I.D. x 15 cm L B. 108 mm I.D. x 30 cm L C. 600 mm I.D. x 40 cm L 1 % -galactosidase: A. 8 ml; B. 1 liter; C. 40 liters Sample: linear gradient from 0 to 0.03 M NaCl Elution: in 0.014 M Tris-HCl (pH 7.7) A. 1.0 ml/min; B. 60 ml/min; C. 1,600 ml/min Flow rate: Linear velocity: A. 39 cm/h; B. 40 cm/h; C. 34 cm/h UV @ 254 nm Detection: Column:
60
Minutes
120
55 mm I.D. x 20 cm L calf liver acetone powder, 94 mg in 4.7 ml in 0.02 M Tris-HCl (pH 8) 100 min linear gradient from 0 M to 0.25 M NaCl followed by a 20 min linear gradient from 0.25 M to 0.5 M NaCl in 0.02 M Tris-HCl (pH 8) 50 cm/h UV @ 280 nm
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Figure 10
pKa = 12.2
DEAE- 650C
SuperQ- 650M
DEAE- 650M
CM- 650C
SP- 650M
CM- 650M
SP- 650S
8
POLISHING
6 4
CM- 650S
pH
2 0 0.1 6 0.2 0.3 7 0.4 0 0.1 0 0.1 8 0.2 0 0.1 9 0.2 0 0.1 10 0.2
12 10 8 6 4 2 0 0.1
0.2
0.1
0.2 0
0.1
0.2
0.3
0.4 0
0.1 0
0.1
0.2
Titration curves of commercial anion exchangers in 0.5 M 1 = DEAE-Sephadex A-25; = DEAE-Sephadex A-50; 3 4 ; 5 7 8 risaeryl M; 9 = DEAEBio-Gel A; 10
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Equilibrium protein adsorption capacities and properties of commercial ANION exchange resins
BSA (67 kDa) Ferritin (444 kDa) Thyroglobulin (660 kDa) Particle size (m) Pore diameter ()
Notes: Adsorption capacities listed are batch adsorption capacities except where noted by *, indicating results reported by the manufacturer; # This material is not a Tosoh Bioscience product.
Table VI
Equilibrium protein adsorption capacities and properties of commercial CATION exchange resins
Lysozyme (14.4 kDa) Hemoglobin (64 kDa) IgG (155 kDa) Particle size (m) Pore diameter ()
Notes: Adsorption capacities listed are Equilibrium adsorption capacities except where noted by* (reported by the manufacturer). # This material is not a Tosoh Bioscience product. ND=not determined.
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and capacity to provide a highly cost effective initial step. Low flow-induced pressures within the packed bed and the large particle size permit the passage of crude biological feed materials. Figure 13
120 100
1
80 60 40 20 0 0 20
20
40
60
80
100
120
40
60
80
100
120
Column:
0,3
TOYOPEARL MegaCap SP-550EC (7.5 mm I.D. x 75 mm) Eluent: A. 20 mM Phosphate buffer + 1 M Na CI (pH 6.0) B. 20 mM Phosphate buffer + 1 M NaCI (pH 6.0) gradient: a) to b) 180 min linear Flow rate: 1 mL/min Detection: UV @ 280 nm Samples: 1. Insulin, 2. Cytochrome C, 3. Lysozyme (1 mg/mL) Injection: 500 L
Table VII
GGTA 2) 451 38
Bradykinin 3) 1,060 52
Lysozyme 14,000 72
-ChymotrypsinogenA
25,000 54
226 4
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Figure 14
Minutes
Minutes Column: A. MD-P SP-650S, 7.5 mm I.D. x 8.5 cm L B. Merck Superformance column packed with T Toyopearl SP-650S (10 cm I.D. x 10 cm L) A. 0.5mL of 5 mg/mL each of cytochrome C and ribonuclease A B. 20mL of 25 mg/mL each of cytochrome C and ribonuclease A A. 0.02 M phosphate (pH 6.8) B. 0.02 M phosphate (pH 6.8 with 0.5M NaCl) 136cm/h linear velocity to 100% B in ten column volumes UV @ 280 nm
Sample:
Note:
The MD-P column is an earlier version of the MD-G columns. The same 35 m resin now packed in glass columns was formerly packed in 7.5 mm I.D x 8.5 cm PEEK columns.
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Ordering Information
TSK-GEL LABPAK: Part # 43380 43280 Product description IEXPAK PW (20) SP-5PW (20), DEAE-5PW (20), SuperQ-5PW (20) IEXPAK PW (SP-5PW, DEAE-5PW, SuperQ-5PW) Contents Particle size (m) 15-25 20-40
3 x 25mL 3 x 25mL
TOYOPEARL LABPAK: 19817 43210 43220 IEXPAK HP CM-650S, SP-650S, DEAE-650S, SuperQ-650S AIEXPAK (DEAE-650M, SuperQ-650M, QAE-550C) CIEXPAK (CM-650M, SP-650M, SP-550C) 4 x 25mL 3 x 100mL 3 x 100mL Dimensions (ID x L) 10mm x 6.8cm 10mm x 6.8cm 10mm x 6.8cm 10mm x 6.8cm 20-50 40-90 and 50-150 40-90 and 50-150 Particle size (m) 35 35 35 35
TOYOPEARL MD-G series columns: Part # Product description 22231 22232 22233 22234 TOYOPEARL MD-G SuperQ-650S TOYOPEARL MD-G DEAE-650S TOYOPEARL MD-G SP-650S TOYOPEARL MD-G CM-650S
TSK-GEL MD-G series columns: 22241 22242 22243 TOYOPEARL MD-G Q-5PW (20) TOYOPEARL MD-G DEAE-5PW (20) TOYOPEARL MD-G SP-5PW (20) 10mm x 6.8cm 10mm x 6.8cm 10mm x 6.8cm 20 20 20
Anion exchange resins: Part # Product description Container size (mL) Particle size (m) Ion exchange capacity (meq/mL resin) 0.12-0.18 Adsorption capacity (mg/mL resin) 52-88
TSK-GEL bulk media 43383 SuperQ-5PW (20) 18535 18546 18547 43283 SuperQ-5PW (30) 18536 18548 18549 43381 DEAE-5PW (20) 14710 14711 18436 43281 DEAE-5PW (30) 14712 14713 18370
25 250 1,000 5,000 25 250 1,000 5,000 25 250 1,000 5,000 25 250 1,000 5,000
15-25
20-40
0.12-0.18
52-88
15-25
0.05-0.11
25-45
20-40
0.05-0.11
20-40
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Ordering Information
Anion exchange resins: Part # Product description Container size (mL) Particle size (m) Ion exchange capacity (meq/mL resin) 0.28-0.38 Adsorption capacity (mg/mL resin) 60-80
TOYOPEARL 43271 14026 14704 14027 18365 19823 17223 17224 17225 19679 43205 17227 17228 17229 43275 17231 17232 17233 19804 07472 14692 07973 43201 07473 14693 07974 18367 07988 14694 07989
QAE-550C
SuperQ-650S
SuperQ-650M
SuperQ-650C
DEAE-650S
DEAE-650M
DEAE-650C
100 250 1,000 5,000 50,000 25 250 1,000 5,000 50,000 100 250 1,000 5,000 100 250 1,000 5,000 25 250 1,000 5,000 100 250 1,000 5,000 50,000 250 1,000 5,000
50-150
20-50
0.20-0.30
105-155
40-90
0.20-0.30
105-155
50-150
0.20-0.30
105-155
20-50
0.08-0.12
25-35
40-90
0.08-0.12
25-35
50-150
0.05-0.11
25-35
Conditions: samples for adsorption capacity are: SuperQ-650, QAE-550C, DEAE-650, SuperQ-5PW, and DEAE-PW, bovine serum albumin.
Cation exchange resins: Part # Product description Container size (mL) Particle size (m) Ion exchange capacity (meq/mL resin) 0.06-0.12 Adsorption capacity (mg/mL resin) 20-40
TSK-GEL bulk media 43382 SP-5PW (20) 14714 14715 18435 43282 SP-5PW (30) 14716 14717 18384
15-25
20-40
0.06-0.12
20-40
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Cation exchange resins: Part # Product description Container size (mL) Particle size (m) Ion exchange capacity (meq/mL resin) 0.14-0.18 Adsorption capacity (mg/mL resin) 80-120
TOYOPEARL 43272 14028 14705 14029 18366 19822 08437 14698 08438 43202 07997 14699 07998 18369 07994 14700 07995 19803 07474 14695 07971 43203 07475 14696 07972 07991 14697 07992 19828 19829 19830 19831 19832
SP-550C
100 250 1,000 5,000 50,000 SP-650S 25 250 1,000 5,000 SP-650M 100 250 1,000 5,000 50,000 SP-650C 250 1,000 5,000 CM-650S 25 250 1,000 5,000 CM-650M 100 250 1,000 5,000 CM-650C 250 1,000 5,000 MegaCap SP-550EC* 100 250 1,000 5,000 50,000
50-150
20-50
0.13-0.17
40-60
40-90
0.13-0.17
40-60
50-150
0.12-0.18
35-55
20-50
0.08-0.12
30-50
40-90
0.08-0.12
30-50
50-150
0.05-0.11
25-45
100-300
0.14-0.18
60-90*
* Adsorption capacity for insulin: 90-120 mg/mL resin Conditions: samples for adsorption capacity are: SP-650, SP-550C, MegaCap SP-550EC, CM-650, and SP-5PW, Lysozyme
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