Ion Exchange Chromatography

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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Ion Exchange Chromatography

TOYOPEARL Ion Exchange Chromatographic Resin

IEC is probably the most widely used liquid chromatographic method for the separation of proteins. It is based on the binding of proteins to positively or negatively charged groups which are immobilized on a stationary phase and which are in equilibrium with free counterions in the mobile phase. In the process of adsorption, the mobile ions are exchanged by the solute, the protein. The binding of proteins to the ion exchange matrix predominantly occurs via charged amino acid residues located at the surface of the protein molecule. with other, non-charge mediated, chromatographic techniques in the design of an efficient downstream purification process. IEC is often used in conjunction with hydrophobic interaction (HIC), size exclusion (SEC), or reversed phase (RPC) techniques in a logical and effective sequence. By careful selection of sequence, salt concentrations between steps can be matched, avoiding unnecessary addition or removal of salt which is time consuming and potentially costly.

Resin Selection and Process Economics Capture

Ion exchange chromatography is well known for its high capacity for charged biomolecules. It is one of the principal reasons why it is often chosen as the initial capture step in a process containing a series of chromatography steps.. The choice of an ion exchange resin will have a significant impact on the economics of a process. It is therefore important for the process designer to make an informed choice based on a knowledge both of the techniques available and the many ways of using the alternative resins. Tosoh Bioscience offers an extended range of product options in both TOYOPEARL and TSK-GEL-5PW polymeric IEC media. The essential choices fall into the following: Ion exchange mode (anion or cation exchange) Performance attributes (TOYOPEARL for routine use, TSK-GEL 5PW for methods requiring higher resolution) Particle Size (selectable on basis of productivity, or throughput) These resins may be easily used in scale up from analytical TSK-GEL-5PW and methods development (MD-G) columns, satisfying the analytical needs for resolution as well as manufacturing needs for productivity. Within each resin chemistry, method transfer between products is highly predictable. The entire range offers true seamless scalability, based on conserved selectivity. Specific product features of Tosoh Bioscience IEC resins are also listed in this section.

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.

Resolution and Selectivity

IEC also exhibits high resolving power, both binding and elution selectivity being controllable by a number of variables, such as salt concentration, salt type, pH, in addition to resin mode selection and specific chemistry. It is also extensively used for both intermediate and final stages of purification.

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

column bed stability mechanical stability

continuous selectivity

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TOYOPEARL ion exchange resins

TOYOPEARL ion exchange resins

The current TOYOPEARL group of ion exchangers offers four particle size ranges and five functional groups (Table I) to meet chromatographic needs across a broad spectrum of applications. The TOYOPEARL-650 ion exchange series is designed on the HW65, 1000 pore size, base resin. They provide outstanding performance in all stages of purification. High dynamic capacity for large and medium sized molecules, fast mass transfer kinetics, good selectivity and resolution are all important features of the product line. The TOYOPEARL-550 series features the HW-55, 500 pore, base resin offering higher internal surface area and consquently higher capacity for most proteins in early downstream capture situations. The more highly cross-linked structure imparts higher rigidity, which together with a coarse grade particle permits faster flow for high throughput capture. A high capacity SP Cation Exchange capture resin is available as an extra course grade (EC) product. Other resins can also be ordered as EC grade upon request.

Table I

Structure of TOYOPEARL ion exchange resins

TOYOPEARL resin pore size Functional group
DEAE-650S DEAE-650M DEAE-650C SuperQ-650S SuperQ-650M SuperQ-650C

1000

HW- -O-CH2-CH2-HN+-(C2H5)2 65 anion exchanger

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

CM-650S CM-650M CM-650C SP-650S SP-650M SP-650C

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
-

SP-550C MegaCap SP-550EC

500

Resins are available in 4 particle diameter ranges:

S M C EC 20 - 40 m (Superfine) 40 - 90 m (Medium) 90 - 120 m (Coarse) 100 - 300 m (Extra Coarse)

Note: R' = proprietary polymer

Please check the database on our website for numerous applications.

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17

Ion Exchange Chromatography

Chemical stability and routine cleaning

Cyclic cleaning is often accomplished by simply using a high salt wash, however the polymeric base resins of all TSK-GEL and TOYOPEARL ion exchangers are chemically and thermally stable. Caustic or acidic solutions may be used for cleaning, sanitization and depyrogenation, since the ion exchange capacity of our resins remain relatively unchanged for at least 20 days at extreme pH values (Figure 2). Although ten days of exposure to strong base (pH 12) decreases the small ion capacity of TOYOPEARL DEAE-650M, the bovine serum albumin adsorption capacity remains constant after 28 days of exposure. Overnight cleaning or sterilization procedures with strong acid or base are therefore possible with TOYOPEARL and TSK-GEL ion exchange resins.

Figure 2

Chemical stability of TOYOPEARL resins in alkaline and acidic solutions

A. TOYOPEARL CM-650M
.015

Ion Exchange Capacity (meq/ml)

.013 .011 .009 .007 .005 0 5 10 15 20

B. TOYOPEARL DEAE-650M
.015

Ion Exchange Capacity (meq/ml)

.013 .011 .009 .007 .005 0 .030 5 10 15 20

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)

Sanitization and alternative cleaning options

All TOYOPEARL ion exchange resins retain full capacity after 28 day storage in 1% sodium hypochlorite, another effective sanitizing agent. Concentrated urea, guanidine, and detergent solutions could be used to clean these resins. Alcohols may be used to remove lipid residues and the resins can be subjected to repeated autoclave cycles. Figure 4 shows the relatively unchanged capacity after eight hours at 121C. Figure 1

.028 .026 .024 .022 .020 0 5 10 20 30

Exposure Time (days)

Solution: 0.5 M NaOH T Temperature: 25 C

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

With Changing Salt Concentrations

1.1 1.0

Bed Height (cm)

DEAE-TOYOPEARL 650M

DEAE-TOYOPEARL 650M

Negligible volume change due to ionic strength T TOYOPEARL DEAE- 650M

110 100 90 80 0 0.1 0.2 0.3 NaCI (M) 0.4 0.5

dextran-based resin

dextran-based resin

Relative Bed Volume

1.4

1.2

1.1 1.0

1.0 1.1

Figure 4
DEAE acrylic resin

DEAE acrylic resin

1.1 1.0

T TOYOPEARL DEAE-650M can be autoclaved at 121C

Ion Exchange Capacity (meg/ml)

1.0 1.1 1.0

DEAE cellulose resin

1.1 1.0

DEAE cellulose resin

0.10 0.12 0.08 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

5 Exposure time (h)

10

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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

Pressure Drop (bar)

2,5 2,0 1,5 1,0 0,5

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

Linear Velocity (cm/h)

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

Recovery of mass and activity

TOYOPEARL and TSK-GEL ion exchange resins deliver exceptional protein mass recovery, as shown in Table II and IV. The mass recovery percentage of each protein was determined spectrophotometrically from the recovered fractions. Retention of activity indicates that protein-resin interactions do not disrupt the bioactive conformation of the product. Nonspecific protein/resin interactions, which can lead to protein inactivation or irreversible binding, are minimized with TOYOPEARL resins. Examples of recovered protein activity listed in Table II and IV show that hydrophilic TOYOPEARL ion exchange resins have the essential characteristics for high activity recovery of bioactive species, including large proteins. Table II

Recovery of enzymatic activity on TOYOPEARL DEAE-650M

Protein Phospholipase D Prolyl endopeptidase Alanine dehydrogenase Phenylalanine dehydrogenase Serine acetyltransferase Da ~56,000 79,000 240,000 310,000 650,000 % Activity recovery 92 96 79 95 95

Recovery of enzymatic activity on TOYOPEARL CM-650M

Protein Da % Activity recovery 87 91 99

Phospholipase D ~56,000 Lipid transfer protein 69,000 Purine nucleotide phosphorylase 68,000

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19

Ion Exchange Chromatography

Figure 6

Comparison of resin loading capacity

Resin:
% of Total Capacity:

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

Column: Sample: Sample:

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

Note: Total capacity = trypsin inhibitor frontal capacity o

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.

TSK-GEL 5PW bulk resins

It is frequently necessary to rapidly develop gram scale quantities of a product for preclinical tests or Phase I trials. Direct scale-up from an analytical method is the most convenient solution to this challenge. Tosoh Bioscience offers 20 and 30m particle size Figure 7

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.

Transfer of methods between TOYOPEARL and TSK-GEL resins

For larger scale manufacturing purposes, Tosoh Bioscience provides the TOYOPEARL line of process scale resins in 35, 65, and 100m particle sizes. Due to the similarity of both base resin chemistry and surface functionalization, TSK-GEL (pressures up to 20 bar) and TOYOPEARL (pressures up to 3 bar) offer similar selectivity. Methods can be transferred across series with a high degree of predictability. This is shown clearly in Figure 8. Figure 8

Process scale-up purification of -galactosidase with TOYOPEARL DEAE-650M

A. 23 ml column

Comparison of TSKgel DEAE-5PW and TOYOPEARL DEAE-650S resins

Absorbance at 254 nm B. 2.7 liter column

A. TSKgel DEAE-5PW

C. 113 liter column

B. TOYOPEARL DEAE-650S

10

15

Relative Elution Volume (Ve/Vv)

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

Column: Sample: Elution: Flow rate: Detection:

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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Ion Exchange Chromatography

TOYOPEARL SuperQ, highest capacity from a unique chemistry

The Tosoh Bioscience anion exchange resin TOYOPEARL SuperQ features a quaternary amine functionalized polymer attached to TOYOPEARL HW-65. This polymeric technology provides significantly higher protein binding capacity, as shown in Table V. For example, the capacity of TOYOPEARL SuperQ for bovine serum albumin is more than double the capacity of a commonly used Q agarose-based material. TOYOPEARL SuperQ is available in three particle sizes to enable productivity optimization consistent with high capacity.

Figure 10

T TOYOPEARL ion exchange media selection chart

STRONG WEAK

pKa = 12.2

SuperQ- 650C QAE-550C CAPTURE

pKa = 11.5

DEAE- 650C

TOYOPEARL DEAE-650, a resin with a difference

TOYOPEARL DEAE-650 is the resin of choice when a high resolution anion exchanger is needed. The titration curve of TOYOPEARL DEAE-650, shown in Figure 9, indicates that it is a true monofunctional diethyl amino ethyl group with a single pKa value of 11.5, a value notably higher than those of other DEAE-type ion exchangers. This resin remains fully protonated within the pH range required for the purification of most biomolecules, and can be utilized at pH values up to 10.5 for basic proteins. The resulting rapid re-equilibration kinetics allow TOYOPEARL DEAE-650 to work as efficiently as a quaternary anion exchanger. The homogeneous functionality also contributes to enhanced resolution capabilities displayed by this particular DEAE resin. Figure 9
CATION A PURIFICATION A ANION PURIFICATION A

SuperQ- 650M

DEAE- 650M

SuperQ- 650S POLISHING DEAE- 650S

pKa = 1.2

SP- 650C SP- 550C CAPTURE MegaCap SP-550EC

pKa = 4.7

CM- 650C

SP- 650M

Titration curves of commercial anion exchangers

12 10 1 2 3 4 5

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

Different selectivities also available for cation exchange

Two types of TOYOPEARL cation exchangers, with alternative selectivities, are available. TOYOPEARL SP-650 resins are sulfonated with strongly acidic groups and can be used at ph values as low as 2-4. For users preferring a weakly acidic resin, the carboxymethyl functional groups of TOYOPEARL CM-650 resins provide a pKa of ~ 4.7.

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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TOYOPEARL SuperQ and DEAE

High capacity for capture with three special resins

Tosoh Bioscience also offers three high capacity capture resins: TOYOPEARL QAE-550C, TOYOPEARL SP-550C and TOYOPEARL MegaCap SP-550EC. TOYOPEARL QAE-550C is a quaternary amine strong anion exchange resin and TOYOPEARL SP-550C is a strong cation exchange material. The higher surface area of the HW-55 base resin result in approximately twice the protein binding capacity of the HW-65 derived resins, as shown in Tables V and VI. These coarse grade materials provide high linear velocities required for maximum throughput. They are the resins of choice for initial column or batch capture processes for peptides and small proteins. TOYOPEARL MegaCap SP-550EC extra coarse grade resin is available with a particularly high capacity for smaller protein molecules (e.g. insulin). Table V

TOYOPEARL SP-650M 20,000 magnification

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 ()

Strong anion exchange resins

TOYOPEARL SuperQ-650M TOYOPEARL QAE-550C Q agarose-based material# 143 mg/ml 70 mg/ml 56 mg/ml 7.0 mg/ml 25 mg/ml NA 9.1 mg/ml 9 mg/ml 3 mg/ml* 40 - 90 50 - 150 45 - 165 1,000 500 400

Weak anion exchange resins

TOYOPEARL DEAE-650M DEAE agarose-based material# 30 mg/ml 97 mg/ml 15 mg/ml 4.3 mg/ml 12 mg/ml 2 mg/ml* 40 - 90 45 - 165 1,000 500

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 ()

Strong cation exchange resins

TOYOPEARL SP-650M TOYOPEARL SP-550C SP agarose-based material# 50 mg/ml 110 mg/ml 101 mg/ml 42 mg/ml 111 mg/ml 50 mg/ml* ND 48 mg/ml 50 mg/ml* 40 - 90 50 - 150 45 - 165 1,000 500 400

Weak cation exchange resins

TOYOPEARL CM-650M CM agarose-based material# CM agarose-based material# 38 mg/ml 94 mg/ml 97 mg/ml 50 mg/ml 30 mg/ml* 4.3 mg/ml ND 15 mg/ml* 2 mg/ml* 40 - 90 45 - 165 45 - 165 1,000 400 500

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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23

Ion Exchange Chromatography

TOYOPEARL MegaCap SP-550EC

TOYOPEARL MegaCap SP-550EC is designed as a capture resin for peptides and small proteins such as insulin. The excellent flow properties of this resin combine with enhanced separation efficiency Figure 11 Saturation Binding Capacity
Conditions: Column: 4.6 mm I.D. x 35 mm Eluent A: 20 mM phosphate buffer (pH 3.0) Eluent B: 20 mM phosphate buffer (pH 2.5)+0.5 M NaCl/ MeOH = 7/3 Flow rate: 0.54 mL/min Sample: Insulin (bovine) 28 mg/mL in Eluent A Injection Volume: 5 mL

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

Detector Responce (mV)

TOYOPEARL MegaCap SP-550EC (100-300m)

80 60 40 20 0 0 20

Agarose Media (100-300m)

T TOYOPEARL SP-550C (50-150-m)

T TOYOPEARL SP-650C (50-150m)

20

40

60

80

100

120

Binding Capacity (g/L) Figure 12

Pressure Flow Curve
0,4 TOYOPEARL SP-650M (40-90 m) TOYOPEARL SP-650C (50-150 m) 0,2 Agarose Media (100-300 m) 0,1 T SP-550EC (100-300 m) 0 1000 2000 Linear Velocity (cm/h) 3000

40

60

80

100

120

Elution Time (min)

Column:

Pressure drop (MPa)

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

Equilibrium adsorption capacity for analytes of different molecular weight

Carnosine1) Molecular weight (Da) Absorption capacity
1)

GGTA 2) 451 38

Bradykinin 3) 1,060 52

Insulin 5,800 102

Lysozyme 14,000 72

-ChymotrypsinogenA
25,000 54

226 4

Carnosine: -Alanyl-L-Histidine; 2) GGTA: Gly-Gly-Tyr-Arg; 3) Bradykinin: Arg-Pro-Pro-Gly Phe-Ser-Pro-Phe-Arg 2 AcOH 3H2O

Equilibrium adsorption capacity vs pore size and particle size

Resin TOYOPEARL MegaCap SP-550EC TOYOPEARL SP-550C TOYOPEARL SP-650C Agarose based media Particle size (m) 100 - 300 50 - 150 50 - 150 100 - 300 Pore Size () 300 500 1,000 300 Static Binding Capacity (g/L) Insulin Lysozyme 95 - 120 120 47 105 60 - 90 80 - 120 35 - 55 80

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TOYOPEARL MegaCap and MD-G

TOYOPEARL MD-G Series Columns

The Tosoh Bioscience Method Development Glass (MD-G) series is a new generation of preparative columns designed for methods development of downstream chromatographic processes. Packed with either 20 or 35m ion exchange resin, MD-G columns are used to develop purification protocols for scale up to any level. The MD-G column geometry and design is tailored for method optimization, and bed volume is sufficient to provide predictable information about production scale separations. Successful purification strategies can be developed with identical chemistry and selectivity throughout the TOYOPEARL and TSK-GEL product range. This scale-up strategy is shown in Figure 14 where a separation method for standard proteins was optimized on a TOYOPEARL SP-650S MD-P column, and then scaled more than 200-fold in a Merck Superformance pilot-scale column. TOYOPEARL and TSK-GEL MD-G columns (10 mm I.D. x 6.8 cm L, glass) feature the small particle versions of commercial 1,000 pore resins. These resins exhibit the same properties as those described previously within this section. MD-G columns are resilient to the mechanical and chemical conditions imposed by the rigors of biological feedstreams and the specific demands of the process environment. They are compatible with HPLC, FPLC, KTA, BioCAD, BioSYS and ProSYS systems.

Figure 14

Comparison of MD-P separation and scale-up separation on a pilot-scale column

A. TOYOPEARL SP-650S MD-P column
7.5 mm I.D.

Absorbance [280 nm]

136 cm/h Rs = 2.89

Minutes

B. TOYOPEARL SP-650S preparative column

10 cm I.D.

Absorbance [280 nm]

136 cm/h Rs = 3.21

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:

Elution: Flow rate: Gradient Detection:

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.

TOSOH BIOSCIENCE

For ordering information, see page 26, 27 and 28.

25

Ion Exchange Chromatography

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

26

TOSOH BIOSCIENCE

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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

25 250 1,000 5,000 25 250 1,000 5,000

15-25

20-40

0.06-0.12

20-40

TOSOH BIOSCIENCE

27

Ion Exchange Chromatography

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

28

TOSOH BIOSCIENCE

1-800-366-4875