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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)
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:- 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)
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:- 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)
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:- 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)
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:- 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
CGC100X2CGC100X4CGC100X8
CGC200X2CGC200X4CGC200X8
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
10.0 mm x 250 mm HPLC Column Preparative
21.2 mm x 50 mm HPLC Column Preparative
21.2 mm x 100 mm HPLC Column Preparative
21.2 mm x 150 mm HPLC Column Preparative
21.2 mm x 250 mm HPLC Column Preparative
30.0 mm x 50 mm HPLC Column Preparative
30.0 mm x 100 mm HPLC Column Preparative
30.0 mm x 150 mm HPLC Column Preparative
30.0 mm x 250 mm HPLC Column Preparative
50.0 mm x 50 mm HPLC Column Preparative
50.0 mm x 250 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
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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
CGC50x4 Sulphonic acid 60 - 75 35 - 75 1.1 min 5 min
CGC50x8 Sulphonic acid 45 - 60 35 - 75 1.7 min 5 min
CGC100x2 Sulphonic acid 75 - 90 50 - 175 0.6 min 5 min
CGC100x4 Sulphonic acid 60 - 75 50 - 175 1.1 min 5 min
CGC100x8 Sulphonic acid 45 - 60 50 - 175 1.7 min 5 min
CGC200x2 Sulphonic acid 75 - 90 150 - 300 0.6 min 5 min
CGC200x4 Sulphonic acid 60 - 75 150 - 300 1.1 min 5 min
CGC200x8 Sulphonic acid 45 - 60 150 - 300 1.7 min 5 min
CGA50x2 Quaternary Ammonium 70 - 85 35 - 75 0.6 min 4 min
CGA50x4 Quaternary Ammonium 55 - 70 35 - 75 1.0 min 4 min
CGA50x8 Quaternary Ammonium 40 - 55 35 - 75 1.2 min 3.5 min
CGA100x2 Quaternary Ammonium 70 - 85 50 - 175 0.6 min 4 min
CGA100x4 Quaternary Ammonium 55 - 70 50 - 175 1.0 min 4 min
CGA100x8 Quaternary Ammonium 40 - 55 50 - 175 1.2 min 3.5 min
CGA200x2 Quaternary Ammonium 65 - 80 150 - 300 0.7 min 4 min
CGA200x4 Quaternary Ammonium 55 -70 150 - 300 1.0 min 4 min
CGA200x8 Quaternary Ammonium 40 - 55 150 - 300 1.2 min 3.5 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)
CGC50x2 Sulphonic acid 75 - 90 35 - 75 0.6 min 5 min
CGC50x4 Sulphonic acid 60 - 75 35 - 75 1.1 min 5 min
CGC50x8 Sulphonic acid 45 - 60 35 - 75 1.7 min 5 min
CGC100x2 Sulphonic acid 75 - 90 50 - 175 0.6 min 5 min
CGC100x4 Sulphonic acid 60 - 75 50 - 175 1.1 min 5 min
CGC100x8 Sulphonic acid 45 - 60 50 - 175 1.7 min 5 min
CGC200x2 Sulphonic acid 75 - 90 150 - 300 0.6 min 5 min
CGC200x4 Sulphonic acid 60 - 75 150 - 300 1.1 min 5 min
CGC200x8 Sulphonic acid 45 - 60 150 - 300 1.7 min 5 min
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