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SEPABEADS® FUNCTIONALISED POLYMERS

FOR CHROMATOGRAPHY

Via Roma, 55 – 20082 Binasco (MI) Italy Phone: 0039.02.900130223 – 221 Fax: 0039.02.90090774 e-mail: marketing@resindion.com - technical@resindion.com

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Chap. 1. Introduction 3

Chap. 2. Morphological polymer structure 3

Chap. 3. General characteristics summary 5

Chap. 4. SEPABEADS FP/EB area of application 6

Chap. 5. SEPABEADS FP and EB product line 7

Chap. 6. Chromatographic techniques 13

6.1. Ion exchange chromatography 14

6.2. Affinity chromatography 15

6.3. Hydrophobic interaction chromatography 16

6.4. Size exclusion chromatography 17

Chap. 7. Chromatography: from laboratory to industrial scale 18

Chap. 8. Regulatory support file 20

Chap. 9. Custom-made products 20

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1. INTRODUCTION Resindion is now proposing a wide range of specially activated functionalized polymer beads based on hydrophilic highly porous polymer matrix, which offers a very attractive solution for almost every conceivable work-up problem at large scale chromatography application: SEPABEADS FP and EB SERIES: Available in two particle size ranges:

FP grade for fixed-packed bed column application

EB grade for expanded bed column application

2. MORPHOLOGICAL POLYMER STRUCTURE

CO

O R O CO

CO

O

C O

OR ' X

C OO

R O CO

C OO

R ' X

R ' X

O

OR '

X

O

OR

O

O

X = A c t iv e F u n c t io n a l G r o u p

+

S u s p e n s io n P o ly m e r iz a t io n

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POROSITY

PORE DIAMETER (Ǻ)

STANDARD POROSITY GRADE 800 – 1500

HIGH POROSITY GRADE > 5000

Conventional micro porous support

SEPABEADS FP/EB Non conventional

highly porous support

High mechanical resistance

Very rigid: stable internal morphology in water, in organic media and in “dry state”

Conventional macro porous support

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3. GENERAL CHARACTERISTICS SUMMARY

Chemically stable polymer structure

Incompressible spherical beads

Low swelling tendency in high molar solution and in common solvents

High stability in alkalis, acids and solvents

High mechanosmotic stability

High resistance to micro-organism

Available with different porosity

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4. SEPABEADS FP/EB AREA OF APPLICATION The following simplified scheme is given as a guide for the possible application point in an ideal production process. SEPABEADS FP / EB SEPABEADS EC

PRODUCT FERMENTATION

PURIFICATION

PURIFICATION

PURIFICATION

IMMOBILISATION

ENZYME FERMENTATION

BIOCATALYSIS

FINAL PRODUCT

FINAL PRODUCT

FINAL PRODUCT

FINAL PRODUCT

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5. SEPABEADS FP AND EB PRODUCT LINE

The recent progress of biotechnology greatly depends on the development of biomolecules separation technology, and methods for large-scale capture, purification and polishing are of increasing importance.

Among a number of separation methods, Column Liquid Chromatography (LC) is regarded as the most practical process for large-scale industrial applications, because various separation modes can be employed and automatic operations are easily performed.

To achieve a high resolution at industrial-scale, column-packing materials should be carefully designed.

At present time, Resindion R&D developed polymeric sorbent beads usable for industrial applications of Ion Exchange Chromatography (IEC), Affinity Chromatography (AC), Metal Chelate Affinity Chromatography (IMAC), Hydrophobic Interaction Chromatography (HIC) and Size Exclusion Chromatography (SEC).

It has to be noted that the type of functional group identifies SEPABEADS FP/EB Product line, while the particle size derives from the application technique, either fixed bed (FP product line) or expanded bed (EB product line).

Pressure Drop In Water At 25°C

0

2

4

6

8

10

12

14

16

18

20

0 1 2 3 4 5 6 7 8 9 10

LV (m/h)

Pres

sure

dro

p (m

H2O

/m b

ed) SEPABEADS FP SERIES

SEPABEADS EB SERIES

Due to the perfect spherical form of SEPABEADS FP/EB and to the reduced particle size distribution range (uniformity coefficient: max 1.5), the pressure drop produced by a resin bed is very low, as shown in the figure.

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SEPABEADS FP AND EB FOR CHROMATOGRAPHY

BASIC

DA QA

O

O

OHNEt2 O

O

OHNMe3+

Cl-

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ACIDIC

CM IDA

SP

O

O

OHO

O

O

OH O

O

OHN COOH

COOH

O

O

OHSO3H

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BASIC

DA QA

O

O

OHNEt2 O

O

OHNMe3+

Cl-

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ACTIVE GROUPS

HG BU

EP

O

O

OHOH O

O

OHO

O

O

O

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SEPABEADS FP AND EB PRODUCTS LINE

Particle Size (µm) Basic mechanism of separation Product Name

Functional Group

Total Exchange Capacity FP Line

Fixed Bed EB Line

Expanded Bed IEC MCAC (IMAC) AC HIC SEC

CM Carboxymethyl min. 0.1 meq/ml 90 – 250 150 - 300 X X

SP Sulphopropyl min. 0.1 meq/ml 90 – 250 150 – 300 X

DA Diethylamino min. 0.25 meq/ml 90 – 250 150 – 300 X

QA Quaternary ammonium min. 0.2 meq/ml 90 – 250 150 – 300 X

IDA Iminodiacetic min. 50 µmol Zn++/ml 90 – 250 150 – 300 X

HG Diol - 90 – 250 150 – 300 X

BU Butyl - 90 – 250 150 - 300 X

EP Epoxy min. 70 µmol/ml 90 – 250 150 - 300 X

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6. CHROMATOGRAPHIC TECHNIQUES

1. ION EXCHANGE CHROMATOGRAPHY (IEC)

2. AFFINITY CHROMATOGRAPHY (IMAC)

3. HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC)

4. SIZE EXCLUSION CHROMATOGRAPHY (SEC)

On the basis of the hydrophilic nature of the acrylic polymers of SEPABEADS,

different kinds of interactions could be used to perform a chromatographic

separation of a compounds mixture applying different chromatographic

techniques.

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6.1. ION EXCHANGE CHROMATOGRAPHY

Different grade of interaction:

Neat charge

Charge density

Charge distribution

Bound sample selectivity removed by changing salt concentration or pH

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6.2. AFFINITY CHROMATOGRAPHY

Separation is achieved by a reversible interaction between a target molecule and a

specific “ligand” blocked to the matrix through a spacer arm.

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6.3.HYDROPHOBIC INTERACTION CHROMATOGRAPHY

Target molecules are charged on the matrix through a high concentration buffer,

condition that allows hydrophobic interaction between the molecule hydrophobic

functional groups present on the matrix surface. Elution takes places applying a

buffer decreasing concentration.

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6.4.SIZE EXCLUSION CHROMATOGRAPHY

Larger MW: shorter retention

time (exclusion limit)

Smaller MW: longer

retention time

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7. CHROMATOGRAPHY:

FROM LABORATORY TO INDUSTRIAL SCALE SEPABEADS FP and EB products are also available in pre-packed columns or bulks1

PRE-PACKED COLUMNS BULKS SEPABEADS

CQK31S (10 µm) CQK31P (10 and 30 µm) FP/EB-CM

CQK30S (10 µm)2 CQK30P (10 and 30 µm) FP/EB-SP

CQA31S (10 µm) CQA31P (10 and 30 µm) FP/EB-DA

CQK35S (10 µm) CQA35P (10 and 30 µm) FP/EB-QA

CQP30 (10 µm) CQP30P (10 and 30 µm) FP/EB-HG

CQH3BS (10 µm) CQH3BS (10 and 30 µm) FP/EB-BU

1 See separately the MCI-GEL catalogue 2 Available also as Protex DEAE and SP at 5 µm

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PARTICLE SIZE RANGE COMPARATIVE TABLE

SEPABEADS FP

SEPHAROSE FF and XL

TOYOPEARL “5…C”

TOYOPEARL “6…M”

EMD FRACTOGEL

SEPABEADS FP

TOYOPEARL “6…C”

EMD FRACTOPREP

STREAMLINE / SEPHAROSE BIG BEADS

SEPABEADS EB

MCI-GEL

PORE SIZE 300 – 600 Å

PORE SIZE 800 - 1200 Å

SEVERAL PORE SIZE

0 10 20 30 40 50 90 100 150 165 250 300 PARTICLE SIZE (µm)

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Neither this publication, nor any products in this brochure shall be construed as recommending the infringement of any patent, nor extending any license, express or implied, nor assuming any liability under any issued or pending patent. The data presented have been carefully compiled from our records which we believe to be accurate and reliable. We make, however, no warranties or representations with respect hereto, nor is freedom from any patent to be inferred

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8. REGULATORY SUPPORT FILE

RDN commitment to the quality of SEPABEADS products is ruled by ISO9001:2000 as

part of RDN corporate strategy for the highest customer satisfaction degree.

The monomers applied to manufacture SEPABEADS comply with the Resolution

AP(97)1, which rules the use of certain products application in the Food processes.

As Regulatory support file, for each standard product, is released the following

documentation:

• product description;

• material safety data sheet;

• storage conditions and limitations (if any);

• standard specification;

• detailed quality control procedures;

• pre-conditioning, regeneration and standard cleaning in place procedure;

• lot-to-lot certificate of analysis.

9. CUSTOM-MADE PRODUCTS

→ special quality control testing;

→ special Regulatory support studies;

→ beads modification (size and porosity);

→ special ligand development

are offered under request.

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