resin solutions for peptide solid phase synthesis and ... · resin solutions for peptide solid...

Resin solutions for peptide solid phase synthesis and purification

Alessandra Basso, PhDLifetech ManagerCPhI, Frankurt 24 October 2017

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Over three decades, Purolite has grown into the world’s premier resin technology manufacturer and innovation leader, with production plants and advanced research labs across the globe. 1,400 Employees

45+ Global Sales Offices

5 R&D Centers

4 Production Plants

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Purolite Life Sciences include the widest portfolio of resins for biotech applications.

We develop standard and customized solutions for our customer needs.

Purolite Life Sciences product portfolio

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Resins for Solid Phase Peptide Synthesis

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Core resinPolystyrene is the most common core resin in solid phase chemistry:

- High swelling in apolar solvents

- Chemically stable

Swelling factor of 1% crosslinked polystyrene in various solvents (mL/g of resin)

Solvent Swelling (ml/g) Solvent Swelling (ml/g)

THF 5.5 Et2O 3.2

Toluene 5.3 CH2CN 4.7

CH2Cl2 5.2 EtOH 5.0

Dioxane 4.9 MeOH 1.8

DMF 3.5 Water 1.0 (no swelling)

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Chloromethyl Resin (Merrifield Resin)

Particle size: 100-200 mesh (75 -150 micron)Loading: 1.0 - 1.6 mmol/g (after coupling with AA 0.4 -.0.6 mmol/g)

Advantages and use:- Substrates are attached to Merrifield resin by nucleophilic displacement

of chlorine- Resin-substrate bond is acid stable and requires strong acid conditions for cleavage- Useful for SPPS and different applications as scavenger resins and polymer-supported reagents

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Aminomethyl Resin (AM Resin)


Advantages and use:- Aminomethyl (AM) resin has long been used in solid phase peptide synthesis as a core

resin - various linkers can be attached through a stable amide bond

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2-Chlorotrityl Chloride Resin (2-CTC)


Advantages and use:- widely used in both solid phase organic and peptide chemistry- cleaved with acetic acid - particularly useful when less acid labile protecting groups are required on the substrate

following cleavage - Bulky triphenylmethyl group prevents cyclization or racemization through steric

hindrance- used to immobilize acids, alcohols and also amines or thiols- 2-chlorotrityl resin has better stability than the trityl resin

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4-Benzyloxybenzyl Alcohol Resin (Wang resin)


Advantages and use:- most widely used solid phase support for acid substrates

- linker attached to the polystyrene core is 4-hydroxybenzyl alcohol - linkage has good stability to a variety of reaction conditions, but can be readily cleaved by moderate treatment with an acid, generally trifluoroacetic acid

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4-Methylbenzhydrylamine Hydrochloride Salt Resin (MBHA Resin)


Advantages and use:- originally developed for the formation of peptide amides using the Boc-N

protection/TFA deprotection strategy- form very stable amide or amine linkages to either carboxylic or electrophilic alkyl substrates structurally similar to Rink- strong acid conditions are required to cleave substrates from these resins (hydrofluoric acid or trifluoromethanesulfonic acid (TFMSA)); therefore they are also used as base resins for anchoring linkers

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Resins for peptide purification

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Chromalite® synthetic resinsProducts available

• Chromalite CGC and CGA – cation and anion gel type activated styrene/polydivinylbenzene copolymer

• Chromalite MN - hyper-crosslinked polystyrene adsorbent

• Chromalite PCG - macroporous polydivinylbenzene adsorbent

• Chromalite AD - macroporous polydivinylbenzene adsorbent for analytical chromatography (5, 10 and 15 micron only)

• Chromalite MDEA – anion activated methacrylate resin

• HPLC pre-packed columns

• Purophase SPE Reverse Phase NEW

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Chromalite: particle size selectionChromalite size range Application

5 micron Analytical (HPLC)

10, 15 micron Preparative (HPLC)

15, 35, 50, 75 micron Process/polishing of proteins, small molecules and synthetic biomolecules, recombinant proteins, front-end capture, desalting, purification, SPE

75 and 150 micron Large scale purification, desalting, polishing of proteins and synthetic biomolecules

• Chromalite are offered in a particle size range from 5 micron up to 200 micron (larger particle size available in the standard range of products from Purolite)

• Chromalite are all spherical, synthetic resins made in uniform particle size range (U.C. < 1.5)

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Chromalite particle size selectionParticle size (m) 5 10 15 35 50 75 100 >200

Ion Exchange - Cation CGC10x6CGC10x8

CGC50X2CGC50X4CGC50X8



Ion Exchange - Anion CGA10x6CGA10x8

15SBG

15AD2Q

CGA50X2CGA50X4CGA50X850SBM50SBG

CGA100X2CGA100X4CGA100X8

CGA200X2CGA200X4CGA200X8

Reversed Phase (RP) 5AD15AD25MN

10AD110AD210MN

15AD115AD215MN

PCG1200F15 PCG1200FPCG900FPCG600F

PCG1200MPCG900MPCG600M

PCG1200CPlus

PCG1200CPCG900CPCG600C

Solid Phase Extraction (SPE)

70MNPCG1200MHEMA

Macroporous (MP) type: AD, PCG, SBM - Obtained with porogens, DVB x-linking usually high. High porosity.Hypercrosslinked polystyrene adsorbents : MN - Hypercrosslinked adsorbents (MN) with extremely high surface area values (>1200 m2/g) and presence of nanopores. High surface areas optimal for adsorption of many organic substances (SPE). Non-porous (or gel-type): CGA/CGC, SBG - Obtained without porogen, with different DVB cross-linking. MDEA – Highly porous methacrylate functionalized with diethylenamine.

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Jetting: process for producing uniform particle size beads

• UC <1.2

• Simple operation

• Higher yields

• Particle sizes from 150μm to 600μm

• Patented membrane technology

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From analytical to preparative scale5AD1, 5AD2, 10AD1, 10AD2, 15AD1, 15AD2, 15AD2Q

4.6 mm x 150 mm HPLC Column Analytical

4.6 mm x 250 mm HPLC Column Analytical

10.0 mm x 150 mm HPLC Column Preparative












All size for all demands

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CONTROL OF POROSITY FOR EFFICIENT SEPARATIONS

Source 15 RP SA: 423 m2/g

PV: 0.62 ml/gPorosity: 200-1000 A

PCG1200 15 umSA: 730 m2/gPV = 1.42 ml/gPorosity: 1200 A

Absence of nanopores in the region of 30A

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CONTROL OF POROSITY FOR EFFICIENT SEPARATIONS

PLRP-S 100 A (50 micron)

SA: 507 m2/g

PV: 1.05 ml/g

Porosity: 300 A

PCG1200F15 (15 micron)

SA: 730 m2/g

PV = 1.42 ml/g

Porosity: 1200 A

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Particle size control for efficient performance

Source 15 RPCD50=14.7um, UC=1.00

PCG1200F15D50= 15.2 um, UC=1.04

PLRP-S 300 AD50=10.3, UC=1.12 10 AD2

D50=10.7, UC=1.0

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Optimal pressure stability

Excellent Stability

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Optimal resolution from analytical to preparative

CHP 55A 18 um (Mitsubishi)

AMBERCHROM™ HPR10 10 micron (Dow)

YMCbasic 15 micron (YMC Co.)

Excellent Scale-up purification

Insulin: 6.7 KDa

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AD1 and AD2 materials optimal for RP separation of hydrophobic molecules

Column length: 150 X4.6 mmMobile Phase: 65/35 (ACN/KPi)Phosphate buffer 50 mM PO43- pH 3.2Flow rate: 1 mL/minT: 21°CDetection: 254 ηmInjection volume: 10 μL

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PLRP-S has different properties than AD1 material

PLRP versus AD1

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PLRP-S 10 um 300 A Column 4.6x150 mmIsocratic elution at 27% BА- 0,05% ТFA, В - 0,05% ТFA in 80% acetonitrileFlow rate- 0,5ml/min, pressure – 1350 psi Resolution R=0.76

15AD1Column 4.6x150 mmIsocratic elution at 27% BА- 0,05% ТFA, В - 0,05% ТFA in 80% acetonitrile.Flow rate- 1ml/min, pressure – 1515 psi Resolution R=1.09

Separation of peptides

octapeptide and its impurity (with 1 aminoacid missed)Good efficiency also with larger PS

PLRP versus AD1

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PLRP-S 10 um 300 A Column 4.6x150 mmIsocratic elution at 27% BА- 0,05% ТFA, В - 0,05% ТFA in 80% acetonitrileFlow rate- 0,5ml/min, pressure – 1350 psi Resolution R=0.76

10AD2Column 4.6x150 mmIsocratic elution at 27% BА- 0,05% ТFA, В - 0,05% ТFA in 80% acetonitrileFlow rate- 1ml/min, pressure – 1900 psi Resolution R=1.07

Separation of peptides

octapeptide and its impurity (with 1 aminoacid missed)Same performance of competitor

PLRP versus AD2

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Waters Symmetry 5 um silica C184.6x150 mm columnEluent 1 ml/minLinear gradient 10 – 40% acetonitrile in 0.05% TFA in waterRetention factor R=2.9

10AD24.6x150 mm columnEluent 1 ml/minLinear gradient10 – 40% acetonitrile in 0.05% TFA in water Retention factor R=2.8

Separation of peptidesSeparation of hexa- (1st peak) and octapeptide (2nd)

Performances of 15 micron are same as 5 micron silica

SILICA versus AD2

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1- phenol, 2- acetophenol, 3 –nitrobenzene, 4 – benzene, 5- toluene

PLRP-S 300 A 10 um4.6X150 mm column Isocratic Elution – 60% acetonitrileFlow rate – 1ml/min

Separation of benzene derivatives

15AD24.6x150 mm column Isocratic elution – 60% acetonitrileFlow rate – 1ml/min

PLRP versus AD2

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Separation of sulfanilamide compounds

Conditions:

Column length: 150 x 4.6mm

Mobile phase: 85/15 (Sodium

Tetraborate·10H2O pH 9.30 / ACN)

Flow rate: 1mL/min

Detection: 254 nm

Injection volume: 1 μL

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Chromalite CGC and CGA

CGC: Gel Strong Acid Cation Chromatographic ResinsCGA: Gel Strong Base Anion Exchange Chromatographic Resins

Matrix: Gel polystyrene crosslinked with divinylbenzene (2, 4 and 7%)Application: For Fine Chemical and Pharmaceutical Column Separations pH Stability: 1-14. Appearance CGC: Orange to dark brown spherical beads. Appearance CGA: Pale yellow to dark yellow spherical beadsExpiry date from DOM: 5 years. Supplied we in H+ from (CGC) and Cl- from (CGA

Product Functional group Total moisture % 90% in Range µm Volume capacity (eq/liter) Weight capacity (eq/Kg)

CGC50x2 Sulphonic acid 75 - 90 35 - 75 0.6 min 5 min









CGA50x2 Quaternary Ammonium 70 - 85 35 - 75 0.6 min 4 min


CGA50x8 Quaternary Ammonium 40 - 55 35 - 75 1.2 min 3.5 min





CGA200x4 Quaternary Ammonium 55 -70 150 - 300 1.0 min 4 min


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Chromalite CGC in acetate form

CGC: Gel Strong Acid Cation Chromatographic ResinsCGA: Gel Strong Base Anion Exchange Chromatographic Resins

Matrix: Gel polystyrene crosslinked with divinylbenzene (2, 4 and 7%)Application: For Fine Chemical and Pharmaceutical Column Separations pH Stability: 1-14. Appearance CGC: Orange to dark brown spherical beads. Appearance CGA: Pale yellow to dark yellow spherical beadsExpiry date from DOM: 5 years. Supplied we in H+ from (CGC) and Cl- from (CGA

Product Functional group Total moisture % 90% in Range µm Volume capacity (eq/liter) Weight capacity (eq/Kg)










Peptide TFA Peptide AcetateCGC/acetate

Purolite can provide the ready to use CGC resins in acetate formRSF available

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Resins for Solid Phase Peptide Synthesis

Why Purolite for SPPS

- world’s premier resin technology manufacturer and innovation leader

- Largest resin manufactures in Life Sciences

- EU made resins: quality, regulatory, application specialists

Official launch of resins for SPPS at Tides 2018 (Boston May 7-10, 2018)

Request now your free sample for evaluation

Thank you

Come and visit us at Purolite stand80K10

25th October – 16.30 -Fred Ghanem

resin solutions for peptide solid phase synthesis and ... · resin solutions for peptide solid...

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