Toyopearl Resins for HIC

650 series for most proteinsToyopearl Hexyl-650Toyopearl Butyl-650Toyopearl Phenyl-650Toyopearl Ether-650

600 series for monoclonal antibodiesToyopearl Butyl-600Toyopearl Phenyl-600 NEW!Toyopearl PPG-600

550 series for smaller proteinsToyopearl SuperButyl-550

TSK-GEL High-Performance Resins for HIC

TSKgel Phenyl-5PW TSKgel Ether-5PW

ToyoScreen Process Development Columns for HIC

Hydrophobic InteractionChromatography

05_hic.qxd 14.03.2008 12:46 Uhr Seite 27

Toyopearl resins for Hydrophobic InteractionChromatography

Hydrophobic interaction chromatography (HIC) is a powerful tool forthe process purification of biomolecules. The technique utilizes theaccessible hydrophobic regions located on protein surfaces and theirinteractions with a weakly hydrophobic stationary phase. HIC is anexcellent complement to ion exchange and size exclusionchromatography particularly when protein isoforms exist or whenfeedstock impurities are of similar isoelectric point or molecularweight. The selectivity differences exploited by HIC can also be usedafter affinity separations in which closely related proteins with similarrecognition sites are not distinguishable by the affinity ligand.

How does HIC work?Proteins and other molecules with hydrophobic surfaces are attracted

to the hydrophobic ligands of both reversed phase (RP) and HIC resins.RP resins have higher surface coverage and/or more hydrophobicligand compared to HIC resins. Because of this, in a RP separation thetarget binding readily occurs in an aqueous solution, and desorption ispromoted by the addition of an increasing amount of organic solvent.

In HIC, proteins are bound to the resin by employing an aqueous highsalt mobile phase. The salt conditions contribute to a lyotropic effectwhich allows the proteins to bind to the lower surface coverage of ahydrophobic ligand. Proteins are eluted by the simple technique ofdecreasing the salt concentration. Most therapeutic targets are elutedin a low salt or a no salt buffer.

During elution the energy of interaction for a HIC step is less thanthat of a RP step. One means of gauging the relative binding energybetween the two techniques is to measure the surface tension of thetwo sets of binding and elution conditions. Figure 1 provides acomparison of the surface tension generated by HIC and RP elutionsystems. Since HIC separates under milder eluting conditions,biological activity is typically retained.

Five different hydrophobic surfaces and selectivitiesTosoh Bioscience offers five HIC ligands featuring different

degrees of hydrophobicity and selectivity. The hydrophobicity ofToyopearl HIC resins increases through the ligand series: Ether, PPG(polypropyleneglycol), Phenyl, Butyl, and Hexyl (Figure 2).

28 www.toyopearl.com

Hydrophobic Interaction Chromatography

hydrophilic polymer resin matrix

• robust chemical stability between pH 1-13 • temperature range 4 °-60 °C autoclavable at 121 °C• compatible with organic solvents• constant bed volume over a wide range of

salt concentrations• low non specific protein binding• superior protein recovery

good mechanical stability• excellent flow characteristics in large industrial

size columns• direct scale-up from TSK-GEL HIC HPLC columns

FeaturesFeatures BenefitsBenefits

Mode Gradient (typical) Δ Surface tension (erg/cm2)

HIC 1.8 to 0 mol/L 4 (NH4)2SO4/ aqueous bufferRPC 10 to 50% ACN/ 0.1%TFA 23

C. Horvath et. al., Separation Processes in Biotechnology, (J. Asenjo, Ed.) 9, 447 (1990) Marcel Dekker

The surface tension of aqueous solutions used The surface tension of aqueous solutions used in HIC and RPCin HIC and RPC

Surfa

ce te

nsio

n (e

rg/c

m2 )

(NH4)2SO4 Concentration (mol/L)

% AcetonitrileHIC RPC

0 1 2 3 4

0 20 40 60 80 100

80

60

40

20

Desorb protein

Desorb protein

Bind protein

Bind protein

(NH4)2SO4

CH3CN

Figure 1

Available HIC ligandsAvailable HIC ligands

Ether

Phenyl

Butyl

O

OH

O n

PPG

Hydrophobic

Hydrophilic(OCH2CH2)nOH

OCH2CH2CH2CH3

Hexyl

OCH2CH2CH2CH2CH2CH3

Figure 2

05_hic.qxd 14.03.2008 12:46 Uhr Seite 28

Coordinating the hydrophobicity of the therapeutic target to theresin hydrophobicity is critical for the best overall purificationperformance. Too hydrophobic a resin for a given protein can result inits irreversible binding to the resin or a loss of enzymatic activity.Table I and II show typical mass recovery and biological activityrecovery data for Toyopearl HIC resins.

An optimum HIC process step will balance high dynamic bindingcapacity, adequate selectivity, good mass recovery and retention ofbiological activity.

The wide range of Toyopearl selectivities enables a developer tooptimize protein separations at the extremes of the hydrophobicspectrum. Highly retentive Toyopearl Hexyl-type and Toyopearl Butyl-typeresins are used to separate hydrophilic proteins. These two resins shouldalso be considered for separations requiring a low salt environment.

Toyopearl Ether-type resin is used for the purification of veryhydrophobic targets such as certain monoclonal antibodies andmembrane proteins. These proteins may bind irreversably to other morehydrophobic resins.

Toyopearl PPG-type and Toyopearl Phenyl-type phases complement theother HIC ligands available in the Toyopearl series and offer alternatives

for mid-range hydrophobic Toyopearl proteins.ToyoScreen prepacked columns for process development

ToyoScreen columns packed with the full range of our Toyopearl HICproducts are available in 1 mL and 5 mL resin volumes. They provide aconvenient way to screen different resins for both target retention andrecovery. Multiple columns can be connected in series for additionalseparation. Please see the ordering information at the end of thissection or contact us for more information on these products.

The retention and selectivity of protein standards on Toyopearl HICresins using the ToyoScreen process development columns are shownin Figure 3.

Influence of salt typeIn addition to the hydrophobicity of the ligand, the selectivity in HIC

is influenced by the eluent salt type. Figure 4 demonstrates the effectof salt type on the resolution factor of different protein pairs.

For ordering information, see pages 34 - 36. 29

Table I

High mass recovery (%) of proteins

Toyopearl HIC resin

Ether Phenyl Butyl-650M -650M -650M

Bovine serum albumin 84 62 76*α-Chymotrypsinogen 96 88* 90Cytochrome C — 81* 87*IgG 91 — —α-Lactalbumin 90 — —Lysozyme 94 92 85Ovalbumin 83 88 73Ribonuclease A — 72* 82*

Procedure: A 200 mL sample containing 200 mg of protein was loaded onto a 7.5 mm ID x 7.5 cm L column and eluted with a 60 minutegradient of 1.8 mol/L (*1.5 mol/L) to 0.0 mol/L ammonium sulfate in0.1 mol/L sodium phosphate (pH 7.0). The mass recovery wasdetermined spectrophotometrically at UV 280 nm and 25°C.

Table II

Recovery of enzymatic activity of proteins

Toyopearl Protein % ActivityHIC resin recovery

Phenyl-650 Phytochrome 79Butyl-650 Halophilic protease 85Butyl-650 Poly (3-hydroxybutyrate) depolymerase 88Butyl-650 Aculeacin-A acylase 82Butyl-650 Opine dehydrogenase 81

0

50

100

250

200

250

300

0 10 20 30 40 50

Elution time (min)

UV

@ 2

80nm

(mA

U)

Column: ToyoScreen (1 mL)Eluent A: 0.1 mol/L Phosphate buffer +1.8 mol/L Sodium sulfate (pH 7.0)Eluent B: 0.1 mol/L Phosphate buffer (pH 7.0)Gradient: 30 min linear gradient from A to BFlow rate: 1 mL/minInj. vol.: 50 µLSamples: Ribonuclease A, Lysozyme, α-Chymotrypsinogen, 1 mg/mL

Screening of Toyopearl HIC resins - Standard ProteinsScreening of Toyopearl HIC resins - Standard Proteins

Ether-650M

PPG-600M

Phenyl-650M

Butyl-650M

SuperButyl-550C

Hexyl-650C

Figure 3

Influence of salt-type on resolutionInfluence of salt-type on resolution

100

50

0

Reso

lutio

n fa

ctor

(Rs)

Cytochrome c/Conalbumin Conalbumin/β-Glucosidase

Na 3 C

itrat

e

Na 2SO

4

(NH 4) 2SO

4

NaC

l

NH 4SC

N

Na 3 C

itrat

e

Na 2SO

4

(NH 4) 2SO

4

NaC

l

NH 4SC

N

Chromatography on a Butyl-substituted supportColumn dimensions: 4.1 mm ID x 4 cm LElution: Linear gradient, 20 min., 1.0 mol/L to 0 mol/L of indicated salt in 20 mmol/L phosphate buffer (pH 7.0). Flow rate, 1 mL/min.J. Fausnaugh, L. Kennedy and F. Regnier, J. Chromatography 317, 141 (1984)

Figure 4

05_hic.qxd 14.03.2008 12:46 Uhr Seite 29

30 www.toyopearl.com

Hydrophobic Interaction Chromatography

The Hofmeister lyotropic salt series shown in Figure 5 ranks anionsand cations by their ability to promote protein precipitation. Ions on theleft are referred to as “lyotropic” while the ions on the right are called“chaotropic”. Lyotropic salts will precipitate or “salt out” proteins athigh salt concentrations due to increased hydrophobic interaction,while chaotropic salts will promote protein denaturation at high saltconcentrations. Figure 5 indicates that different salt systems maygenerate a variety of adsorption and desorption selectivities for eachresin. This feature of HIC provides an additional parameter for theoptimization of a process step.

Particle size optimizationToyopearl and TSK-GEL PW-type methacrylic base beads incorporate

the same polymer chemistry (see Figure 6 or refer to the Size Exclusionsection for a more detailed discussion) and are available in a variety ofparticle sizes:

100 µm Toyopearl C-grade Capture65 µm Toyopearl M-grade Intermediate Purification35 µm Toyopearl S-grade Intermediate Purification/Polishing

30 and 20 µm TSK-GEL PW-type High Resolution In many cases analytical columns are available packed with 10 µm

particles having the same selectivity as the process media. Figure 7 shows the variety of ligands and particle sizes available for

HIC process-scale applications. This figure also depicts where eachparticle size is used in a chromatographic manufacturing train (i.e. forcapture, intermediate purification, or polishing).

Methacrylic base beads available for HICMethacrylic base beads available for HIC

Pore size (Å) 50 125 400-500 750 1000 >1000 >1700

Toyopearl HW-type: 40 50 55 60 65 75 80

TSK-GEL PW-type: G1000 G2000 G4000 G5000 G6000

Increasing pore surface area

Product name

Figure 6

Feedstock

Pure Product

Capture

IntermediatePurification

Polishing

QCSame selectivity HPLC columns are available for most process media

Process stepProcess step Bead sizeBead size Process media Process media

100 µm

Toyopearl SuperButyl-550CToyopearl Hexyl-650CToyopearl Butyl-650CToyopearl Phenyl-650C

65 µmToyopearl Butyl-600MToyopearl Phenyl-600MToyopearl PPG-600M

Toyopearl Butyl-650MToyopearl Phenyl-650MToyopearl Ether-650M

Toyopearl Butyl-650SToyopearl Phenyl-650SToyopearl Ether-650S

35 µm

30 µm TSKgel Phenyl-5PW (30)TSKgel Ether-5PW (30)

TSKgel Phenyl-5PW (20)TSKgel Ether-5PW (20)

20 µm

10 µm TSKgel Phenyl-5PW 2 mm ID x 7.5 cm LTSKgel Ether-5PW 2 mm ID x 7.5 cm L

Figure 7

Hofmeister lyotropic salt seriesHofmeister lyotropic salt series

For cations

Ammonium and sodium sulfate are the most commonly usedsulfate salts in HIC. NaCl is often used as well.

SO42- > HPO4

2- > CH3COO- > halide > NO3- > CIO4

- > SCN-

(CH3)4N+ > NH4+ > K+ > Na+ > Cs+ > Li+ > Mg2+ > Ca2+ > Ba2+

For anions

Figure 5

05_hic.qxd 14.03.2008 12:46 Uhr Seite 30

Figure 11 compares the selectivities of the new Toyopearl Phenyl-600M,Toyopearl Phenyl-650M and an agarose Phenyl resin.

For smaller molecules such as lysozyme (12,000 Da) the even narrowerpore diameter Toyopearl SuperButyl-550C (derived from the 500 Å porediameter Toyopearl HW-55) is recommended. A comparison of the DBCof Toyopearl SuperButyl-550C with other Toyopearl HIC resins is shown inFigure 12.

For ordering information, see pages 34 - 36. 31

Mechanical stabilityThe semi-rigid polymeric backbone of Toyopearl and TSK-GEL HIC

resins permits high flow rates for maximum throughput andproductivity. Toyopearl resins may be operated up to 3 bar and TSK-GEL PW-type resins may be operated up to 20 bar. The pressure-flowcharacteristics for each particle size grade of Toyopearl Phenyl-650resins are shown in Figure 8. The beads are compatible with both fixedbed and dynamic axial compression columns.

Chemical stabilityThe polymeric structure of these products also makes them resistant

to a wide range of pH conditions and ionic strengths. In addition, thehydroxylated surface of the base bead reduces non-specific binding ofproteins.

Pore size optimizationMost Toyopearl HIC products are derived from the versatile size

exclusion resin, Toyopearl HW-65 (1,000 Å mean pore size), as thebase bead for the majority of protein separations. But the pore sizeand accessible surface area of Toyopearl can be optimized for a givenprotein. More accessible surface area increases the dynamic bindingcapacity (DBC) of the bead for a particular therapeutic target. This hasled to the development of two specialty lines of HIC materials withhigher dynamic binding capacities.

High dynamic binding capacity Toyopearl process resinsFor monoclonal antibodies a pore size of 750 Å is optimum. A specially

made base resin, Toyopearl HW-60, has this pore size. Three ligands areavailable on Toyopearl HW-60: Toyopearl PPG-600M, Toyopearl Phenyl-600M and Toyopearl Butyl-600M. A comparison of their DBCs withToyopearl Phenyl-650M is shown in Figure 9. The selectivities of ToyopearlButyl-600M, Toyopearl PPG-600M and the new Toyopearl Phenyl-600M areshown in Figure 10.

Pressure-flow curve for Pressure-flow curve for Toyopearl Phenyl-650 resinsToyopearl Phenyl-650 resinsof various particle sizeof various particle size

Flow (cm/h)

0

Column:Eluent:

0

Phenyl-650M

Pres

sure

(bar

)

Phenyl-650C

Phenyl-650S

Toyopearl Phenyl-650C, M and S, 25 mm ID x 25 cm L 2 mol/L (NH4)2SO4

1.5

2

2.5

3

400 600 800200

Figure 8

0

0.05

0.1

0.15

0 10 20 30 40 50

C/C 0

IgG loaded (g/L-resin)

Breakthrough curves of polyclonal IgG on various HIC resinsBreakthrough curves of polyclonal IgG on various HIC resins

Phenyl-600M

Agarose type Phenyl

Butyl-600M

Phenyl-650M

Column: 7.8 mm ID X 20 cm L Sample: polyclonal human IgGBinding buffer: 1 g/L IgG in 0.8 mol/L (NH4)2SO4 + 0.1 mol/L sodium phosphate (pH 7.0)Linear velocity: 300 cm/hrTemperature: 25°C

DBC was calculated at 10% of breakthrough.

Figure 9

Comparison of Toyopearl 600M series resinsComparison of Toyopearl 600M series resins

Column: 7.5 cm ID X 7.5 cm LSample: 1 g/L RNase A (1), lysozyme (2) and a-chymotrypsinogen A (3) Sample load: 100 µLGradient: 60 min linear gradient from buffer A to BBuffer A: 1.8 mol/L (NH4)2SO4 + 0.1 mol/L sodium phosphate (pH 7.0)Buffer B: 0.1 mol/L sodium phosphate (pH 7.0)Linear velocity: 136 cm/hrTemperature: 25°C

0 10 20 30 40 50 60 70

UV

@ 2

80 n

m (m

AU)

Elution volume (mL)

Toyopearl Phenyl-600M

Toyopearl PPG-600M

Toyopearl Butyl-600M

1

23

0

50

100

150

Figure 10

05_hic.qxd 14.03.2008 12:46 Uhr Seite 31

The new Toyopearl Phenyl-600M also has a high DBC for lysozyme.(Figure 13).

The engineered higher dynamic binding capacity of the 600 and 550series HIC products for their specific targets and the selectivitydifferences induced by the smaller mean pore size of the respectivebeads, can have a dramatic impact on process economics. Improvingtarget feedstock concentration and purity for downstream steps cangreatly enhance throughput and reduce process costs.

Monoclonal antibodiesHydrophobic interaction is a very useful technique for the purification of

monoclonal antibodies. The diverse hydrophobic nature of mAbs is seenin Figure 14 . This figure measures the hydrophobicity (using elution timeas a surrogate measurement) of 51 different mouse IgGs on a TSK-GELPhenyl-5PW analytical column. Some of the IgGs have elution times 2-3times longer than others indicating greater hydrophobicity.The Toyopearl series of HIC ligands (Figure 2) with their differenthydrophobicities gives chromatographic developers a range of options forfinding the right ligand for their target molecule.

For a very hydrophobic mAb, such as mouse anti-chicken 14 kDa lectin,the less hydrophobic Toyopearl Ether ligand works quite well. Thepurification from ascites fluid (Figure 15) was performed with a 10 µmTSKgel Ether-5PW semi-preparative column. Identical selectivity forscale-up was found with corresponding 65 µm Toyopearl Ether-650M.

32 www.toyopearl.com

Hydrophobic Interaction Chromatography

Toyopearl Phenyl-600M Breakthrough curve (lysozyme)Toyopearl Phenyl-600M Breakthrough curve (lysozyme)

Breakthrough curve (lysozyme)

Phenyl-600M 58Phenyl-650M 27

Column: 7.8 mm ID x 20 cm LSample: 1 mg/mL lysozyme in 0.1 mol/L phosphate buffer (pH 7.0) + 1.8 mol/L (NH4)2SO4Linear velocity: 300 cm/hr

0

0.05

0.1

0.15

0 20 40 60 80

C/C

0

Lysozyme loaded (mg/mL-resin)

Phenyl-650M Phenyl-600M

Binding capacity (mg/mL - gel) (10% Breakthrough)

Figure 13

Typical dynamic binding capacities for LysozymeTypical dynamic binding capacities for Lysozyme

Binding capacity (mg/mL - gel) (10% Breakthrough)

Ether-650M 12.5Phenyl-650M 27.5Butyl-650M 32.2Hexyl-650C 33.2PPG-600M 44.2SuperButyl-550C 54.3 ConditionsColumn size: 7.8 mm ID x 20 cm LSample: 1 mg/mL Lysozyme in 0.1 mol/L Phosphate buffer + 1.8 mol/L Sodium Sulfate (pH 7.0) Linear velocity: 100 cm/hDetection: UV @ 280 nm

0

0.01

0.02

0.03

Abso

rban

ce U

V @

280

nm

0 20 40 60 80Lysozyme loaded (mg/mL-resin)

Phenyl-650M

Butyl-650M

Ether-650M

PPG-600MHexyl-650C

SuperButyl-550C

Figure 12

Selectivity comparison of phenyl-type resinsSelectivity comparison of phenyl-type resins

Column: 7.5 cm ID X 7.5 cm LSample: 1 g/L RNase A (1), lysozyme (2) and α-chymotrypsinogen A (3) Sample load: 100 µLGradient: 60 min. linear gradient from buffer A to BBuffer A: 1.8 mol/L (NH4)2SO4 + 0.1 mol/L sodium phosphate (pH 7.0)Buffer B: 0.1 mol/L sodium phosphate (pH 7.0)Linear velocity: 136 cm/hrTemperature: 25°C

Toyopearl Phenyl-650M

00

50

100

150

10 20 30 40 50 60 70

UV

@ 2

80 n

m (m

AU)

Elution volume (mL)

1

2

3Toyopearl Phenyl-600M

Agarose type Phenyl

Figure 11

05_hic.qxd 14.03.2008 12:46 Uhr Seite 32

Protein aggregate removalThe larger pore Toyopearl products such as Butyl-650 and Phenyl-650

are very useful for protein aggregate separation and removal. Early workby Karger et al1 in 1989 involving proteins and aggregates larger than200 kDa demonstrated the effectiveness of HIC for this application.

GlycoproteinsToyopearl HIC resins can purify glycoproteins, which often bind

irreversibly to saccharide-based chromatographic media. Figure 16 showsthe purification of a large glycoprotein on Toyopearl Butyl-650S resin.

DNA plasmid purification and endotoxin removalToyopearl Hexyl-650C resin was successfully used for plasmid DNA

purification by Cambrex, Baltimore, MD (US patent 6,953,686). Hexyl-650C was shown to be the most effective among HIC resins for endotoxinremoval with capacities exceeding 2 million EU/mL of resin. Additionally,RNA and protein impurities were effectively eliminated. Hexyl-650C wasalso effective in separating the supercoiled and open circular forms ofplasmid DNA.

Removal of misfolded proteinsBecause misfolded proteins will generally be more hydrophobic than

the native protein, Toyopearl Butyl-650M resin is used frequently for theremoval of misfolded proteins. In many cases flow throughchromatography can be accomplished under eluent conditions binding themisfolds while allowing the target to flow through the column.

Please check the database on our website, www.tosohbioscience.de,for additional applications.

1 Grinberg, N. Blanco, R. Yarmush, D. Karger, B. L. Protein Aggregation in High PerformanceLiquid Chromatography: Hydrophobic Interaction Chromatography of β-Lactoglobin, Anal.Chem. 1989, 61, 514-520

33

Purification of mAbs from ascites fluidPurification of mAbs from ascites fluidA. 10 µm TSKgel Ether-5PW

A. TSKgel Ether-5PW, 7.5 mm ID x 7.5 cm L B. Toyopearl Ether-650M, 7.5 mm ID x 7.5 cm L anti-chicken 14 kDa lectin, diluted ascites fluid,A. 1.5 mg in 100 µL; B. 0.76 mg in 50 µL60 min linear gradient from 1.5 mol/L to 0 mol/L (NH4)2SO4in 0.1 mol/L phosphate buffer (pH 7.0)136 cm/hUV @ 280 nm

Column:

Sample:

Elution:

Linear velocity:Detection:

Minutes0 15 30

B. 65 µ m Toyopearl Ether-650M

0 15 30Minutes

mAb

mAb

Figure 15

For ordering information, see pages 34 - 36.

Large glycoprotein purified on Toyopearl Butyl-650SLarge glycoprotein purified on Toyopearl Butyl-650S

Fraction number (8 mL/fraction)

Abso

rban

ce, 2

80nm

Antib

acte

rial a

ctiv

ity (%

-Inhi

bitio

n)2.0

1.0

02010 30 70 100

Column:Sample:Elution:

Toyopearl Butyl-650S, 22 mm ID x 26 cm L crude protein from sea hare Aplysia kurodaimulti-step (NH4)2SO4 in 50 mmol/L phosphate buffer (pH 7.0)A. load & wash: 40% saturated (NH4)2SO4B. 20% saturated (NH4)2SO4C. 0% saturated (NH4)2SO4

40 50 60 80 90

A. C.B.

100

50

0

Figure 16

0

10

20

30

40

50

60

70

80

90

No.

1N

o.3

No.

5N

o.7

No.

9N

o.11

No.

13N

o.15

No.

17N

o.19

No.

21N

o.23

No.

25N

o.27

No.

29N

o.31

No.

33N

o.35

No.

37N

o.39

No.

41N

o.43

No.

45N

o.47

No.

49N

o.51

Antibody

Elut

ion

time

of e

ach

antib

ody

on

TSKg

el P

heny

l-5PW

(min

)

Column: TSK-GEL Phenyl-5PWEluent : (A) 0.1 mol/L phosphate buffer containing 1.8 mol/L ammonium sulfate (pH 7.0) (B) 0.1 mol/L phosphate buffer (pH 7.0)Flow rate : 1 mL/minGradient : (B) 0% (0 min)--0% (5 min)--100% (65 min) linearSamples : 51 kinds of mouse monoclonal antibodies

Hydrophobic diversity of mouse monoclonalsHydrophobic diversity of mouse monoclonals Plot of chromatographic elution times for 51 different mouse mAbs

Figure 14

05_hic.qxd 14.03.2008 12:46 Uhr Seite 33

TOSOH BIOSCIENCE34 www.toyopearl.com

Hydrophobic Interaction Chromatography

Ordering Information

ToyoScreen process development columns for HICPart # Product description Package21372 ToyoScreen Ether-650M 1 mL x 6 each21373 ToyoScreen Ether-650M 5 mL x 6 each21374 ToyoScreen Phenyl-650M 1 mL x 6 each21375 ToyoScreen Phenyl-650M 5 mL x 6 each21376 ToyoScreen Butyl-650M 1 mL x 6 each21377 ToyoScreen Butyl-650M 5 mL x 6 each21378 ToyoScreen Hexyl-650C 1 mL x 6 each21379 ToyoScreen Hexyl-650C 5 mL x 6 each21380 ToyoScreen PPG-600M 1 mL x 6 each21381 ToyoScreen PPG-600M 5 mL x 6 each21380 ToyoScreen Butyl-600M 1 mL x 6 each21381 ToyoScreen Butyl-600M 5 mL x 6 each21494 ToyoScreen Phenyl-600M 1 mL x 6 each21495 ToyoScreen Phenyl-600M 5 mL x 6 each21382 ToyoScreen SuperButyl-550C 1 mL x 6 each21383 ToyoScreen SuperButyl-550C 5 mL x 6 each21398 ToyoScreen HIC Mix Pack 1 mL x 6 Grades x 1 each21399 ToyoScreen HIC Mix Pack 5 mL x 6 Grades x 1 each

ToyoScreen column accessoriesPart # Product description Comment21400 ToyoScreen Column Holder

TSK-GEL LabPakPart # Product description Container size (mL) Particle size (µm)43278 HICPAK PW (20) (Ether-5PW, Phenyl-5PW) 2 x 25 mL 10-3043175 HICPAK PW (30) (Ether-5PW, Phenyl-5PW) 2 x 25 mL 20-40

05_hic.qxd 14.03.2008 12:46 Uhr Seite 34

For ordering information, see pages 34 - 36. 35

Toyopearl HIC resins

Part # Product description Container size (mL) Particle size (µm) Typical capacity (mg lysozyme/mL resin)

43151 Toyopearl Ether-650S 25 20-50 10-3016172 10016174 1,00016176 5,00019805 Toyopearl Ether-650M 25 40-90 10-3016173 10016175 1,00016177 5,00021301 Toyopearl PPG-600M 25 40-90 45-5521302 10021303 1,00021304 5,00021305 50,00021887 Toyopearl Phenyl-600M 25 40-90 45-6521888 10021889 1,00021890 5,00020891 50,00043152 Toyopearl Phenyl-650S 25 20-50 30-5014477 10014784 1,00014935 5,00019818 Toyopearl Phenyl-650M 25 40-90 30-5014478 10014783 1,00014943 5,00018364 50,00043126 Toyopearl Phenyl-650C 25 50-150 30-5014479 10014785 1,00014944 5,00043153 Toyopearl Butyl-650S 25 20-50 30-5007476 10014701 1,00007975 5,00018826 50,00019802 Toyopearl Butyl-650M 25 40-90 30-5007477 10014702 1,00007976 5,00018355 50,00043127 Toyopearl Butyl-650C 25 50-150 30-5007478 10014703 1,00007977 5,00021448 Toyopearl Butyl-600M 25 40-90 -21449 10021450 1,00021451 5,00021452 50,00019955 Toyopearl SuperButyl-550C 25 50-150 52-7019956 10019957 1,00019958 5,00019959 50,00044465 Toyopearl Hexyl-650C 25 50-150 30-5019026 10019027 1,00019028 5,000

05_hic.qxd 14.03.2008 12:46 Uhr Seite 35

www.toyopearl.com36

Hydrophobic Interaction Chromatography

Part # Product description Container size (mL) Particle size (µm) Adsorption capacity (mg lysozyme/mL resin

TSK-GEL 5PW HIC resins for high resolution 43276 TSKgel Ether-5PW (20) 25 10-30 10-3016052 25016053 1,00018437 5,00043176 TSKgel Ether-5PW (30) 25 20-40 10-3016050 25016051 1,00018439 5,00043277 TSKgel Phenyl-5PW (20) 25 10-30 10-3014718 25014719 1,00018438 5,00043177 TSKgel Phenyl-5PW (30) 25 20-40 10-3014720 25014721 100017210 5,000

05_hic.qxd 14.03.2008 12:46 Uhr Seite 36

For further information please contact:

Hichrom Limited1 The Markham Centre, Station Road, Theale

Reading, Berkshire, RG7 4PE

Tel: 0118 930 3660 Fax: 0118 932 3484Email: technical@hichrom.co.uk www.hichrom.co.uk