molecularly imprinted polymers - · pdf filesodium silicate had been polymerized in water...

Molecularly Imprinted Polymers

Professor Anthony R Rees MA, DPhil DScCSO, Biotage

2010年11月30日（東京）と12月2日（大阪）に開催した

ExploraSepセミナーのプレゼンテーション資料です。

Uppsala : Summer and Winter

バイオタージ本社があるスウェーデンの風景

Contents

Part I

� What are MIPs – a short history?

� How do they behave?

� Examples of selectivity

Agenda


� ExploraSep™ : a new screening concept

Part II

� ExploraSep and Genotoxins: Case Studies

The concept of ’Imprinting’

In 1931 the group of Polyakov (Kiev) reported some unusual adsorption properties in silica particles prepared using a novel synthesis procedure. Sodium silicate had been polymerized in water using (NH4)2CO3 as the gelating agent. After two weeks, additives (benzene, toluene or xylene) had been added. The silica was subsequently allowed to dry for 20–30 days, after which the additive was removed by extensive washing in hot water. Subsequent adsorption studies revealed a higher capacity for uptake of the additive by the silica than for capacity for uptake of the additive by the silica than for structurally related ligands, i.e. some kind of memory for the additive was apparent, at least in the cases of benzene and toluene.

In 1934-5 more detailed investigations of the phenomenon werereported and the observed selectivity was explained as resultingfrom structural changes in the silica reflecting the nature of theadditive.

1950,s Polyakov repeated and reiterated his founding experimentsthough they were not widely accepted or referenced

1970’s Gunther Wolff (Germany) took the concept to anotherlevel….

MIPは1930年頃から研究されていた技術です。共有結合で化合物を結合するポリマーの研

究を経て、より汎用性の高い現在のMIP（非共有結合により化合物を捕捉）が開発されました。

The Birth of Covalent ImprintingWolff (1972-78)

“Polymers with binding groups located in a definite spatial proximity andcooperativity in cavities of specific shape should show high selectivity inbinding. This arrangement is similar to those of natural receptor sites.”

Covalent imprinting – boronate esters with diol templates

Non- covalent Imprinting – the universal method

”According to this strategy .... a monomer mixture containing ...cross-linking units is

polymerized in the presence of a free substrate ..act as a template.... This is simply a mixing

process and no chemical attachment to the monomeric units is required. The monomers

are, however, chosen in such a way as to have non-covalent binding abilities

(ie ionic, hydrogen bond, hydrophobic, charge transfer etc) complementary to those of the(ie ionic, hydrogen bond, hydrophobic, charge transfer etc) complementary to those of the

guest template.”

Arshady & Mosbach, 1981

The Non-covalent Process simplified

MIP

MIPは、特定の化合物を“鋳型”にして作られます。

O

OH

OH

OF3C

N

NH

SO3H

O

NH

N

COOH

NN

N+

NH

NH2

O OH

OH

O

Methacrylic acid Trifluoro-methacrylic acid

Acrylamido-(2-methyl)-propane sulfonic acid

4-Vinylbenzoic acid

Cl-

Itaconic acid

The Functional Monomer Landscape

A C I D I C

B A S I C

O

NH2

N

COOH

COOH

O

O

OH O

O

O

OH

OH

OH

OO

O

CN

N+

N

N

O

4-Vinylpyridine 4-Vinylbenzyl-trimethyl-ammonium chloride

4-Vinylbenzyl-iminodiacetic acid

4(5)-Vinylimidazole 1-Vinylimidazole 4-Vinylbenzamidine

AcrylamideStyreneHydroxyethylmethacrylate Methylmethacrylate

Methyl-a-D-glucopyranoside-6-acrylate

Vinylpyrrolidone Acrylonitrile

Fluorescent reporter monomer

N E U T R A L

S P E C I A L

MIPは様々なモノマーで構成される特殊なポリマーです。

NH

NH

OO

N,N'-Methylene-bisacrylamide

NH

NH

O

O

Phenylene-diacrylamide

N N

OO

Bisacryloyl piperazine

The Polymer backbone

H Y D R O P H I L I C

Divinylbenzene

O

O

O

O

Ethyleneglycol dimethacrylate

OO

O

O

OO

Trimethylolpropane

trimethacrylate

H Y D R O P H O BI C

N

O

H

HN

H

H

OO

OO

H

H

-

Spatial site A

Definableinteractions

The MIP Binding Site(small molecule)

HH

Spatial site B

With the monomers employed interactions can involveH-bonding, charge-charge, van der Waals,

hydrophobic and charge-transfer

特定の化合物をかたどったキャビティ内で、水素結合やファンデルワールス力などが作用します。

Engineering binding and CapacityEngineering binding and Capacity

Template ratios: K and Capacity

Molecularly Imprinted Polymers – a Summary

• Highly cross-linked polymer based phases

• Pre-determined selectivity for a particular analyte or group of structurally related compounds

– Size exclusion

– Chemical interactions in highly defined positions (H-bonding/ionic, Van der Waals interactions, ππππ−−−−ππππinteractions)interactions)

• Selective target recognition

– Mimics of polyclonal antibodies or receptors

MIPは高度に架橋された特殊なポリマーで、特定の化合物をかたどったキャビティ内においてポジション限定的に化合物と相互作

用します。選択的に化合物を”認識”する仕組みは抗原抗体反応（ポリクローナル）に似ています。

Contents

Part I

� What are MIPs?



Agenda



Part II


Relative Selectivity of Sample Preparation Techniques

Non-Selective Dirty Extracts

Highly-Selective Clean Extracts (Lower LODs and LOQs)

MIPs

MIPを充填した固相抽出カラム（AFFINILUTE MIP）を例に挙げ、MIPの優れたターゲット選択性を説明します。

AFFINILUTE MIP SPE procedure

• MIP methodology differs from conventional SPE methodology

• Protocols for reversed phase, ionic-exchange resins etc CANNOT be used without careful comparison with the recommended MIP method

• Selectivity is typically introduced during the interference wash step with organic solvents

AFFINILUTE MIP: Selective extraction in complex matrices

• Technical features– MIPs generate stronger interaction between the sorbent

and the analyte

– Interfering substances can be washed away using harsher washing conditions

– The MIP material is stable at high temperatures, in organic solvents and at extreme pH (normally)

– The MIP material is stable at high temperatures, in organic solvents and at extreme pH (normally)

• Advantages– Cleaner extracts

– Simplified washing protocols with less extraction steps

– Minimized matrix effects and ion suppression

– Higher precision

MIPを充填した固相抽出カラム（AFFINILUTE MIP）では、超微量のターゲット化合物を極め

てクリーンな分析用サンプルとして抽出できます。

Drug-like

• Clenbuterol

• Beta agonists (class-selective)

• Beta blockers (class-selective)

• Beta receptors (class-selective, β-agonists & β-blockers)

• NSAIDs

Forensic

AFFINILUTE MIP Phases and Applications Commercially Available For…

Forensic

• Amphetamines

• NNAL

• TSNAs (Tobacco Specific Nitrosamines)

Food/Agricultural

• Chloramphenicol

• Fluoroquinolones

• Nitroimidazoles

• Triazine herbicides (class-selective)

• Polyaromatic hydrocarbons (PAH)

MIPを充填した固相抽出カラム（AFFINILUTE MIP）で抽出できる化合物の一覧です

Contents

Part I

� What are MIPs?



Agenda



Part II


Example 1: Amphetamine drug Family

• A class of compounds including amphetamine (alpha-methylphenethylamine) and substituted amphetamines

• CNS stimulants– Clinically used to treat ADHD, narcolepsy and

other sleeping disorders.

– Stimulants and hallucinogens illegally used as – Stimulants and hallucinogens illegally used as recreational club drugs and as performance enhancers.

• Heavily regulated worldwide

• Schedule I & II drugs as reported by the DEA, USA

AFFINILUTE MIPによるアンフェタミン抽出を例に、MIPのターゲット選択性について説明します。

Amphetamine Structures

NH2

Amphetamine Metamphetamine

NH2

Phentermine

NH2OHNO

HNO

HNNH2

Amphetamine Metamphetamine

NH2

Phentermine

NH2OHNO

HNO

HN

Common ‘core’ structure

O

MDA

O

MDMA

O

MDEA

O

MDA

O

MDMA

O

MDEA

Methamphetamine “crystal meth”, “ice” – potent stimulantMDA, MDEA & MDMA (Ecstasy) – psychedelic stimulant

MDA (3,4-Methylenedioxyamphetamine), MDMA (3,4-methylenedioxy-N-methamphetamine) MDEA (3,4-methylenedioxy-N-ethylamphetamine)

AFFINILUTE MIPアンフェタミンは”クラス選択性”があり、アンフェタミンに似た構造の他の化合物も捕捉することができます。

SupelMIP SPE Extract of Blank Urine (MRM 136/119)

700

1400 Polymer SPE Extract of Blank Urine (MRM 136/119)

700

1400SupelMIP SPE Extract of Blank Urine (MRM 136/119)

700


700

1400SupelMIP SPE Extract of Blank Urine (MRM 136/119)

700


700

1400Polymeric SPE (Oasis)

Blank urine sample

AFFINILUTE MIP SPE

Blank urine sampleLow

background

Urine extracts (MRM 136/118)

AFFINILUTE MIP : High Sensitivity

AFFINILUTE MIP vs Conventional Hydrophilic Polymer†

0

0 2 4 6 8 10

Min

0

0 2 4 6 8 10

Min

SupelMIP SPE Extract of Amphetamine Spiked Urine (15 pg/mL,

MRM 136/119)

0

700

1400

0 2 4 6 8 10

Min

Polymer SPE Extract of Amphetamine Spiked Urine (15 ph/mL,

MRM 136/119)

0

700

1400

0 2 4 6 8 10

Min

No peak

0

0 2 4 6 8 10

Min

0

0 2 4 6 8 10

Min


MRM 136/119)

0

700

1400

0 2 4 6 8 10

Min


MRM 136/119)

0

700

1400

0 2 4 6 8 10

Min

0

0 2 4 6 8 10

Min

0

0 2 4 6 8 10

Min


MRM 136/119)

0

700

1400

0 2 4 6 8 10

Min


MRM 136/119)

0

700

1400

0 2 4 6 8 10

Min

No peak

Polymeric SPE (Oasis)

Spiked urine sample (15 pg/mL)

AFFINILUTE MIP SPE

Spiked urine sample (15 pg/mL)

� ” AFFINILUTE MIP Amphetamines gives an Astronomically Low Background” –

�Texas Veterinary Diagnostic Lab

Amphetamine

15 ppt

MIPは、夾雑物が多量に存在する環境下でもpptレベルでターゲット化合物を捕捉することができます。

Higher recovery and reproducibility

% Recovery % RSD

AFFINILUTE MIP vs Conventional Hydrophilic Polymer†

Affinilute Affinilute

High

Reproducibility

using

AFFINILUTE

High

Recoveries for

all compounds

using

AFFINILUTE

† M.-R. Fuh, T.-Y. Wu and T.-Y. Lin,

Talanta, 2006, 68:987-991

Lowered Ion-Suppression

Standard solution

AFFINILUTE SPE extract

AFFINILUTE MIP vs Conventional Hydrophilic Polymer

Hydrophilic

Polymer SPE extract

Example 2 : NNAL

� Carcinogenic nicotine metabolite measured in urine of smokers and passive smokers

� A nitrosamine so within the genotoxic ’class’

� Current methods - elaborate and time-consumingconsuming

� Trace level determination required

AFFINILUTE MIPによるタバコ特異ニトロソアミン代謝物（NNAL）抽出を例に、MIPの選択性の高さを説明します。

N

NCH3

ON

O O

N

N

NCH3

OH

NNK NNAL

O

NNAL Structure

CH3N

N

O

OHOH

OHO

COOH

NNAL-O-Glucuronide NNAL-N-Glucuronide

NO

OHOH

OHO

COOH

OH

N+

NCH3

NO

Note: -N-N=O is a PGI (genotoxin) functional group

Conventional method Affinilute method

Affinilute™ NNAL

Over 20 laborious steps

Results in 3 days

Insufficient detection limits Results after 0.5 h!Results after 0.5 h!

2

3

4

Ion

Ra

tio

Affinilute™ NNAL - sensitivity

0

1

2

0 0.4 0.8 1.2 1.6 2.0

NNAL Concentration, ng/ ml urine

Ion

Ra

tio

R2 = 0.9981

Sensitivity down to 0.1-0.2 ppb

”Using MIP in SPE was a

success for NNAL

measurement”

John Bernert, CDC Atlanta

Affinilute™ NNAL – the sensitivity LIMIT

John Bernert, CDC Atlanta

Faster and more efficient SPE

by using MIP allows higher

sample throughput

Allows high sensitivity Allows high sensitivity –– detection of detection of 2 ppt 2 ppt possible!possible!

Contents

Part I

� What are MIPs?



Agenda



Part II


目的のターゲット化合物を捕捉できるMIPを探す方法として、ExploraSepプレートによるスクリーニングを説明します。

ExploraSepTM : Exploiting Similarity

Target compounds are screened on multiwell plates containing a large number of different MIPs generated against many different templates and containing different monomer chemistries

How and why does ExploraSep work?

ExploraSepは96ウェルプレートフォーマットの製品で、

様々な化合物をテンプレート（鋳型）にして作成した様々なMIPが搭載されています。

Similarity – the Concept

メディシナルケミストリーにおける”ファーマコフォア”に良く似た概念で、MIPの交差反応性（クラス選択性）を

“セレクトフォア” と定義して説明します。

Similarity leads to cross-reactivity

Other molecules that share common features may also fit into the MIP

Chemical group functionalitiesfixed in space

Other molecules that share common features may also fit into the MIP binding site

”Selectophore”Hydrophobic siteH-bond donorH-bond acceptor

Selectophore concept Summary

• Comparable to pharmacophore concept in medicinal chemistry*

• Pharmacophore =‘‘a set of structural/chemical features in a molecule that is recognized at a receptor site and is responsible for the biological activity’’activity’’

• Similar molecules can bind to the same site –otherwise Pharma would have no business!

• It is to be expected that the selectivity ‘profile’ of a MIP binding site will be at least as flexible as a biological receptor site

*Gund, P., Prog. Mol. Subcell. Biol. 1977, 5: pp 117–143

MIPには交差反応性（クラス選択性）があるので、鋳型にした化合物そのもの以外でも、構造と化学的特性が似て

いれば保持（結合）することができます。

ExploraSep Chemistries

128 resins

Plate A (acidic functionalities)• Hydrophobic and carboxylic acidic moieties• Successful target analytes - amines, amides, nitrosamines, esters, carboxylic acids

ExploraSepは4種類あり、それぞれに32種類のポリマーが載っています（n=3）。

• Successful target analytes - amines, amides, nitrosamines, esters, carboxylic acids

Plate U (proprietary ’urea’ functionalities)• Hydrophobic moieties and urea groups• Successful target analytes - phosphates, phosphonates, sulphates, sulphonates, anions of carboxylic acids

Plate H (aromatic functionality)• Hydrophobic aromatic moieties• Successful target analytes - non-polar and aromatic compounds

Plate C (CHO or multi-OH binding functionality)• Hydrophobic moieties and hydroxy functionalities• Successful target analytes - 1,2- and 1,3-diols, and α-hydroxy carboxylic acids

Select appropriate ExploraSep 96-well

Plate(s)

Screen ExploraSep Plate to Find “Hit”

Conduct Binding Curve using

cartridges packed with Selected

MIP/NIP Phases

Hit Found?

Adjust Screening

Conditions

Adjust Binding

NO

YES

Overview of ExploraSep Process

Is MIP/NIP

Binding

Different?

Develop preliminary method using

buffer/solvents (no sample matrix)

Apply sample matrix and confirm

method. Optimize wash/elution steps

for maximum selectivity and recovery.

Validate MIP Extraction Procedure

Adjust Binding

Curve Conditions

NO

YES

ExploraSepによるMIPスクリーニングから、

最終的に実用のためのプロトコルを決定する

までのプロセスの概要です。

ExploraSep Screening: The Process

50%

60%

70%

80%

90%

100%

% R

em

ov

ed

Na

ph

az

oli

ne

fro

m M

IP/N

IP r

el.

ExploraSep screen of Naphazoline on Plate A

Remaining on column

3% HAc wash

0.5% HAc wash

H2O wash

0%

10%

20%

30%

40%

% R

em

ov

ed

Na

ph

az

oli

ne

fro

m M

IP/N

IP r

el.

s

td. H2O wash

Load

MIP NIP

Alpha adrenergic activity

ExploraSepには、各種MIPと、それぞれのMIPに対応するNIP(non-imprinted polymer)が載っています。NIPは、MIPと

全く同じモノマー組成で、テンプレート（鋳型）化合物を使わずに作成したポリマーです。

ExploraSep Screening Examples

Atrazine

0

5

10

15

20

25

30

35

40

45

50

EA1

EA2

EA3

EA4

EA5

EA6

EA7

EA8

EA9

EA10

EA11

EA12

EA13

EA14

EA15

EA16

EA17

EA18

EA19

% Bound

Melatonine

0

20

40

60

80

100

EA1

EA2

EA3

EA4

EA5

EA6

EA7

EA8EA

9EA

10EA

11EA

12EA

13EA

14EA

15EA

16EA

17EA

18EA

19EA

20EA

21EA

22

% b

ou

nd

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

EU001

EU002

EU003

EU004

EU005

EU006

EU007

EU008

EU009

EU010

EU011

EU012

EU013

EU014

EU015

EU016

EU017

EU018

EU019

EU020

EU021

EU022

EU023

EU024

EU025

EU026

EU027

EU028

EU029

EU030

EU031

EU032

Phase

% B

ou

nd

Selective Sorbent

Human Insulin

EA1

EA2

EA3

EA4

EA5

EA6

EA7

EA8

EA9

EA10

EA11

EA12

EA13

EA14

EA15

EA16

EA17

EA18

EA19

EA1

EA2

EA3

EA4

EA5

EA6

EA7

EA8EA

9EA

10EA

11EA

12EA

13EA

14EA

15EA

16EA

17EA

18EA

19EA

20EA

21EA

22

Metoprolol

0

10

20

30

40

50

60

70

EA1

EA2

EA3

EA4

EA5

EA6

EA7

EA8

EA9

EA10

EA11

EA12

EA13

EA14

EA15

EA16

EA17

EA18

EA19

% Bound

棒グラフの縦軸はポリマーに捕捉（保持）された量（%）です。

N

N

HN

N

O

O

Theophylline

Theophylline

0

10

20

30

40

50

60

70

80

90

100

EA1 EA2 EA3 EA4 EA5 EA6 EA7 EA8 EA9 EA10 EA11 EA12 EA13 EA14 EA15 EA16 EA17 EA18 EA19

% Bound

Caffeine 100,0

Selective Sorbent

ExploraSep: Sensitive to small differences

N

N N

N

O

O

CH3

Caffeine

Caffeine

0,0

20,0

40,0

60,0

80,0

100,0

No Selective Sorbent was found

A MIP that binds theophylline but not caffeineDifferential binding with only one methyl group difference!

Summary

• MIPs are artificial binding sites in polymers and can be made to operate in SPE or chromatography mode

• MIPs are stable to all organic solvents and normally extremes of pH (depends on monomer chemistry)

• MIPs can be made selective for a restricted group of • MIPs can be made selective for a restricted group of highly related molecules OR a ‘CLASS’ of chemically similar compounds

• MIPs can be manufactured for use at process scale

• New separation materials for most compounds of interest to pharma can be discovered using ExploraSep

End of Part IEnd of Part I

Contents

Part I

� What are MIPs?



Agenda



� Part II


Potential Genotoxic Impurities (PGI): Structural Alerts

(From Müller et al, Regulatory Toxicology and Pharmacology 44 (2006) 198–211)

Genotoxic impurities and ExploraSepTM

The Analytical Challenge

• Validated methods for multiple API’s and PGI are required• The genotoxic compounds may be closely related to

the API

The Process Chemistry challengeThe Process Chemistry challenge

• Investigate process conditions that may create PGI• Map the effect of all reaction conditions that effect PGI

formation• Investigate process changes required to control PGI

concentrations• Develop back-up strategy to scavenge PGI with high

yield of API

PGI Screening Process

• ExploraSep– Screen each of 4 plates in clean solvent mixture

containing API and PGI

– Take ‘hits’ and develop separation method (SPE or chromatography

• Comprehensive data– Raw data supplied with analysis showing polymer of – Raw data supplied with analysis showing polymer of

choice for further development

• Rapid Results– Projects completed on agreed schedule at fixed

price

• Proven Scale-up– MIP production already at 500 Kg/year. Option of

bulk resin, SPE cartridges, pre-packed Flash cartridges or HPLC preparative columns

Case Study 1

A large US PharmaA large US Pharma

ExploraSep Plate A: loaded in toluene

50.0

60.0

70.0

80.0

90.0

100.0

% R

em

ov

ed

fro

m M

IP/N

IP r

el.

std

R600127

CS 1: Screening for Genotoxin over API

17%v59%

12%v47%

API

0.0

10.0

20.0

30.0

40.0

50.0

EA077

EA078

EA003

EA004

EA005

EA006

EA007

EA006

EA051

EA052

EA047

EA048

EA037

EA038

EA041

EA042

EA045

EA046

EA021

EA022

EA039

EA040

EA025

EA026

EA027

EA028

EA029

EA030

EA043

EA044

EA IPN

EA RP

EA TSNA

EA BaP

MIPs/NIPs

% R

em

ov

ed

fro

m M

IP/N

IP r

el.

std

T003113

Weak‘hits’

12%v47%

PGI

ExploraSep Plate C: loaded in toluene

50.0

60.0

70.0

80.0

90.0

100.0

% R

em

ov

ed

fro

m M

IP/N

IP r

el.

std

R600127

%re

tain

ed

CS 1: Selectivity for Genotoxin over API

5%v65%

API

0.0

10.0

20.0

30.0

40.0

50.0

EC001

EC002

EC003

EC004

EC005

EC006

EC007

EC008

EC009

EC010

EC011

EC012

EC013

EC014

EC015

EC016

EC017

EC018

EC019

EC020

EC021

EC022

EC023

EC024

EC025

EC026

EC027

EC028

EC029

EC030

EC031

EC032

MIPs/NIPs

% R

em

ov

ed

fro

m M

IP/N

IP r

el.

std

T003113

%re

tain

ed

Several hits that differentiates PGI (T003113) and API (R600127)

PGI

Elution profile of R600244 (5ug/mL) and R250025 (10ug/mL)

99,3%99,6%99,6%

60

70

80

90

100

% N

on

-re

tain

ed

on

MIP

re

lati

ve

to

sta

nd

ard

CS 1: API binds and PGI elutes

API5µg/mL

High Selectivity forR600244 (API) over R250025 (PGI)

(0% = non-detectable by lc/ms/ms)

1,1%0,0%0,0%0

10

20

30

40

50

EA005 EA007 EA091

% N

on

-re

tain

ed

on

MIP

re

lati

ve

to

sta

nd

ard

R600244

R250025

PGI10µg/ｚmL

CS 1: Conclusion

• First API-PGI set screened on Plate C identifies several candidates with selectivity for PGI for further evaluation. EC 029 selected for further evaluationevaluation

• Second API-PGI set screened on plate A shows selectivity for API over PGI. EA 005 and EA 007 selected for further evaluation.

Case Study 2

A large European PharmaA large European Pharma

Case Study 2

• Structures confidential but:

– PGI contains amide and piperazine moieties.

– API contains piperazine, indole and dione moieties.

• Log P

– PGI ~ 1.3

– API ~ 3.3.

Case Study 2

EXSEPA15

40.0

60.0

80.0

100.0

120.0

Cu

mu

lati

ve

Re

co

ve

ry %

A15D7

A15D8

NIP

MIP

0.0

20.0

Inj. of

Analyte

Wash with

ACNL

Wash with

1 %

Methanol

Wash with

2 %

Methanol

Wash with

5 %

Methanol

Wash with

10 %

Methanol

Wash with

20 %

Methanol

Wash with

50%

Methanol

Wash with

Methanol

Cu

mu

lati

ve

Re

co

ve

ry %

Binding curve for genotoxin (d7) and API (D8) on MIP EA15 derived

from ExploraSep A Plate.

Arrow shows difference between imprinted (MIP) and non-imprinted (NIP) polymer

Case Study 2 (cont)

Conclusion

– Quite good selectivity on several of the polymers on the A plate : EA07, EA15 and EA23

– Polymers were packed in HPLC columns and – Polymers were packed in HPLC columns and supplied to customer

Case Study 3

A European CMOA European CMO

Case Study 3

• All genotoxin structures known but only a few of the APIs.

• Log P of Methyl p-Toluenesulfonate (PGI) ~ 2.0, log P of 21-chlorodiflorasone (API) ~ 2.7.

• Log P of 1,3-diisopropylurea (PGI) ~ 0.6, log P of API • Log P of 1,3-diisopropylurea (PGI) ~ 0.6, log P of API ~ 2.7.

• All screened with ExploraSep

O

CH3

F

CH3

HO

Cl

O

OH

CH3

API or intermediate:

Case Study 3 (cont)

GTI:

O

NH

NH

O

F

Methyl p-Toluenesulfonate 1,3-diisopropylurea

Case Study 3 (cont)

20.00

25.00

30.00

35.00

40.00

45.00

50.00

% B

ind

ing

of

GT

I/A

PI

GTI

API

Case Study 3 (cont)

0.00

5.00

10.00

15.00

20.00

IPA 1% HAc in IPA 2% HAc in IPA

% B

ind

ing

of

GT

I/A

PI

Selectivity graph of methyl p-toluenesulfonate (PGI)/API in loading on EU064

Selectivity Plot of Diisopropylurea vs. API on ExploraSep plate A in Loading

60.00

70.00

80.00

90.00

100.00

% B

ou

nd

AP

I o

n p

oly

me

r

EA091: 100/57

Case Study 3 (cont)

0.00

10.00

20.00

30.00

40.00

50.00

60.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

% Bound Diisopropylurea on polymer

% B

ou

nd

AP

I o

n p

oly

me

r

Load: 95:5 Toluene/MeCN

EA090: 99/47

EA091: 100/57

EA088: 77/5

EA085: 90/28

Further evaluation of additional A plate candidates

Binding curve for Diisopropylurea and API: Toluene

80.00

90.00

100.00

110.00

120.00

130.00

140.00

% R

ec

ov

ery

of

an

aly

tes

fro

m p

oly

me

r re

l. s

td.

DIPU: EA088

DIPU: EA090

DIPU: EA091

Case Study 3 (cont)

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

Load:

toluene/MeCN

95:5

W1:

toluene/MeCN

95:5

W2 W3 W4 W5 W6

% R

ec

ov

ery

of

an

aly

tes

fro

m p

oly

me

r re

l. s

td.

DIPU: EA091

API: EA088

API: EA090

API: EA091

Arrow shows API versus PGI extraction on EAO88= Starting point for optimized separation

API

Case Study 3 (cont)

Separation in LC mode on EA088Strong retention of toxic contaminant obtained

PGI

CS 3: Conclusions

• Initial screening showed weak hits

• Additional synthesis and screen showed stronger hits

• Packing of strongest hit in HPLC mode showed satisfactory separationsatisfactory separation

Case Study 4

European consortium studyEuropean consortium study

Case Study 4

Cumulative Recovery

60%

80%

100%

120%

Musk Ketone

0%

20%

40%

60%

Load

Hep

tane

0.05

% F

A0.

075%

FA

0.10

% F

A0.

15%

FA0.

25%

FA

McC

N

2,4-DNT

Recognition of molecules carrying the same functional group (-NO2)

Summary

• The cross-reactivity of MIPs allows selective binding of other molecules

• This ’Similarity’ concept has been used to separate API’s from PGI’s

• All polymers identified in ExploraSep screening • All polymers identified in ExploraSep screening can be produced at pilot and/or Process scale

• ExploraSep is a powerful tool in separation method discovery

Additional InformationAdditional Information

Contents

� How can MIPs be scaled up?

The process path

A Stage Gate approach to Production

ScreeningActivities Activities

Transfer of

polymeri

ActivitiesOptimization• Process• Performance

ActivitiesPilot scale up

Production ��

ActivitiesProcess scale-

up

Phase 1

MIP Base

Phase 1

MIP Base

Phase 2A

Process

Phase 2A

Process

Phase 2B

Optimizati

on

Phase 2B

Optimizati

on

Phase 2C

Scale up

Pilot/

Production

Phase 2C

Scale up

Pilot/

Production

Phase 3

Process

Plant

Scale up

Phase 3

Process

Plant

Scale up

Commer

cialization

Commer

cialization

1 2 3 4 5 6

ActivitiesFull scale

productionScreeningActivitiesScreeningActivities Activities

Transfer of

polymeri

ActivitiesTransfer of

polymeri




Production ��


Production ��


up


up

Phase 1

MIP Base

Phase 1

MIP Base

Phase 2A

Process

Phase 2A

Process

Phase 2B

Optimizati

on

Phase 2B

Optimizati

on

Phase 2C

Scale up

Pilot/

Production

Phase 2C

Scale up

Pilot/

Production

Phase 3

Process

Plant

Scale up

Phase 3

Process

Plant

Scale up

Commer

cialization

Commer

cialization

11 22 33 44 55 66


production


production

� Applying stage-gate process for efficiency and risk reduction

� Utilising DoE (Design of Experiments) tool to attain robust product and control

Deliverable

Cost effective MIP

Deliverable

Scalable process

Deliverable

Optimized MIP

from scaled up

process

Deliverable

kg MIP deliveryDeliverable

Full scale process approval

polymeri

zation

process

Robustness

• Performance Production ��kg Full scale

sampling

Deliverable

Cost effective MIP

Deliverable

Scalable process

Deliverable

Optimized MIP

from scaled up

process

Deliverable

kg MIP deliveryDeliverable

Full scale process approval

polymeri

zation

process

Robustness

polymeri

zation

process

Robustness

• Performance• Performance Production ��kg

Production ��kg Full scale

samplingFull scale

sampling

Tools in materials development

• A select group of experts

• Innovative MIP chemistry � large IP

portfolio

• Polymer chemistry at the research frontierfrontier

– Novel concepts

– New methods

• Modeling

Chemometric predictions and Monte Carlo models

Scale up – DOE Starting point

Focus– Polymer properties

– PSD (particle size distribution)

0,5 L reactorsParallel 50ml

Transfer to pilot scale …

2 L 10 L 30 L 50 L

Example – Selective removal of nitrosamines in presence of nicotine

• To selectively remove a group of 4 nitrosamines at a total concentration of 70 ng/ml from a tobacco extract

• The structurally related

• Nitrosamines to be removed

N

NNO

N

O

N

NO

N

N

NON

N

NO

NNK NNN NAB NAT

• The structurally related nicotine should be retained in the mixture -at a concentration of 700 µg/ml

• Nitrosamines are structurally related and are at 10000x lower concentration

• Nicotine to be retained

N

N

(S)-(-)-Nicotine

Optimization of the MIP recipe

• Final DOE on “best” existing recipe

• 5 Parameters checked

DOE computer aided optimization of the recipe

• 5 Parameters checked

• Coefficients analyzed with software MODDE

– contour plots for the responses

DoE driven process scale-up: Optimisation/model

The DOE Responses

75

60

80

100R

em

ov

al

(%)

Compound 1

Compound 2

The Final Design: Pilot Scale (25kg)

Increasing Performance

Target Performance Reached

0

20

40

60

50 mL 500 mL 2 L 10 L 30 L

Reactor Volume

Re

mo

va

l (%

)

Compound 2

Compound 3

Compound 4

Increasing scale

molecularly imprinted polymers - · pdf filesodium silicate had been polymerized in water...

Documents