BioHPLC columns Tim Rice Biocolumn Technical Specialist
AU Typical Application Areas Size Exclusion: Aggregation Analysis Ion Exchange: Charge Isoform Analysis 0.035 Monomer 0.030 0.025 0.020 0.015 Dimer 0.010 0.005 0.000-0.005 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 Minutes Reversed Phase: Separation of light and heavy chains Peptide Mapping: Post-translational modifications 2
Size Exclusion BioHPLC Columns
NEW Size Exclusion Columns 5 m Particle 100Å, 150Å, 300Å, 500Å, 1000Å, 2000Å pore sizes High stability and long lifetime Great reproducibility Unique, 3 m particle 100Å, 150Å, 300Å pore sizes Highest resolution Highest efficiency Faster SEC separations
Smaller Particles for Higher Resolution Agilent Bio SEC-3, 300Å, 7.8x300mm Column Bio SEC-3 (7.8x300mm) Bio SEC-5 (7.8x300mm) Pressure 1350 psi 650 psi Peak Protein SEC-3, 300Å (7.8x300mm) SEC-5, 300Å (7.8x300mm) 1 Thyroglobulin 2460 1120 2 BSA Dimer 5100 2720 3 BSA 13090 6590 4 Ribonuclease A 22000 11160 5 Uracil 38500 27860 Agilent Bio SEC-5, 300Å, 7.8x300mm 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Min Technology: Small Particle SEC Columns Results: Higher Resolution SEC Separations 5
Use Higher Resolution to Achieve Faster Separations Increase flow rate and use a shorter column for faster separations. 2.0ml/min 1.5ml/min 1.0 ml/min Column: Agilent Bio SEC-3, 7.8 x150mm Sample: mab (2mg/ml) Injection: 5ul Flow rate: 1.0, 1.5 and 2ml/min (56 bar, 75 bar, 105 bar) Eluent: 150mM sodium phosphate + 100mM Na-sulfate Detection: 220nm monomer dimer 4 Minutes Technology: Small Particle SEC Columns Results: Faster SEC separations 6
Mechanism of SEC Separation Pore Size Determines Linear Separation Range FLOW Page 7
Insulin Myoglobin Ovalbumin BSA IgG Thyroglobulin Column Choice: Resolving Ranges Peptides Proteins Globular proteins Agilent Bio SEC 100Å Agilent Bio SEC 150Å Agilent Bio SEC 300Å Agilent Bio SEC 500Å Agilent Bio SEC 1000Å ProSEC 300S Agilent Bio SEC 2000Å Zorbax GF-250 Zorbax GF-450 100D 1kD 10kD 100kD 1,000kD 10,000kD
Ion Exchange BioHPLC Columns
NEW Ion Exchange Columns Non-porous PS/DVB particles (polystyrene divinylbenzene) Uniform polymeric coating with SCX, WCX, SAX, WAX layers, designed for protein and peptide separations Available in 10 µm, 5 µm, 3 µm, 1.7 µm particle sizes High surface area High capacity
Agilent Bio IEX Columns Comparing Separations on Each Particle Size 1.7 m 3 m 5 m Peak N Column: Bio WCX-NP, 4.6x50mm Buffer A: 20 mm PBS Buffer B: A+1.0 M NaCl Gradient: 0-100%B (20 min) Flow rate: 1.0 ml/min for NP10, NP5, NP3 0.75 ml/min for NP1.7 Sample 1) Ribonuclease A 2) Cytochrome C 3) Lysozyme Concentration: 1.0 mg/ml Detector: 280 nm Average N ~80,000 for WCX-NP1.7 Peak N 10 m 0 2 4 6 8 10 12 14 16 18 20 22 Min
AU Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 (Acidic Isoforms) weakly bound (Basic Isoforms) Strongly bound 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 Minutes Column: Agilent Bio MAb, NP5, 4.6mm x 250mm Buffer A: 10 mm Sodium Phosphate, ph 7.50 Buffer B: A + 100 mm NaCl, ph 7.50 Gradient: 15-95% B in 60 min Flow rate: 0.8 ml/min. Sample: 5 L, 5 mg/ml, mab 12
Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies A B C D E 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Min Columns: Agilent Bio MAb, NP10, 4.6x250 mm Mobile phase: A, 10 mm phosphate, ph 7.5 B, A + 0.1M NaCl Gradient: A) 15-75%B in 30 min B) 15-65%B in 30 min C) 15-55%B in 30 min D) 15-47.5%B in 30 min E) 15-40%B in 30 min Flow rate: 0.8 ml/min Sample: Monoclonal Antibody Injection: 10 L (1.5 mg/ml) Temperature: 25 o C Detection: UV 214 nm Optimization of method conditions for the isoform characterization of a monoclonal antibody. Changes in the buffer conditions, ph and gradient conditions sharpen peaks and increase resolution of acidic and basic isoforms.
Agilent Bio WCX: ph 7.0 5.8 An increase in ph reduced the retention time. An increase in ph increased resolution of earlier peaks (circled). Increased resolution of early peaks 14
Reverse Phase BioHPLC Columns
O Zorbax Stablebond for Reverse Phase Separations of Proteins 300 A pore size available in C-3, C-8, C-18, and CN non-endcapped particle sizes: 3.5, 5, and 7 R1 R1 R1 R Si R R Si R R Si R OH O OH O 16
NEW! Zorbax 300 SB RRHD for Proteins and Peptides! Stablebond 300 silica C-18, C-8, C-3, HILIC and diphenyl bonded phase 1.8 um particle size 1200 Bar pressure limit for uhplc 2.1 x 50 mm and 2.1 x 100 mm
UHPLC Columns Increase Resolution and Increase Speed Column Length (mm) Resolving Power N(5 µm) Resolving Power N(3.5 µm) Resolving Power N(1.8µm) Analysis Time* 150 12,500 21,000 32,500 100 8,500 14,000 24,000 75 6000 10,500 17,000 50 4,200 7,000 12,000 30 N.A. 4,200 6,500 15 N.A. 2,100 2,500-33% -50% -67% -80% -90% Analysis Time Peak Volume Solvent Usage * Reduction in analysis time compared to 150 mm column
300SB-C18 RRHD Protein Digest TB# 5990-7989EN Column: Zorbax 1.8um 300SB-C18 RRHD, 2.1x100mm A :.1% TFA in water, B:.085% TFA in ACN Gradient: 20%B 1min, 2-45%B 8.8min, 45-95%B.2min, 95%B 2min, 98-2%B.2min, 20%B 1.8min Flow rate:.5 ml/min, Temp: 50C, Pressure: ~640bar Sample: Protein digest, 5ul inj vol, 1 mg/ml 19
0.454 0.501 0.766 3.897 0.569 4.010 Fast Separation of Reduced and Alkylated MAb Conditions optimized for the ZORBAX RRHD 300SB-C3 Column mau 120 100 80 CHO Cell Derived MAb Light chain Heavy chain 1 Heavy chain 2 60 40 20 0 1 2 3 4 5 min Columns: ZORBAX RRHD 300SB-C3, 2.1 x 100 mm, 1.8 µm Sample: Reduced MAb (IgG1) (1.0 mg/ml)- BioCreative IgG1 Sample injection: 2 µl Mobile phase A: 0.1% TFA in water Mobile phase B: 80% n-propyl alcohol, 10% ACN, 9.9% water and 0.1% TFA Temperature: 74 C Flow rate: 0.5 ml/min Detection: UV, 280 TB# 5990-9667EN Time (min) 0 1 %B 2 20 5 50 6 90 6.1 1 20
1.646 1.345 1.564 1.787 1.846 1.666 1.337 1.587 1.754 1.811 Comparison C3 and Diphenyl Phases Ribonuclease A, Cytochrome C and Lysozyme (3 mg/ml) mau 12 10 8 6 4 2 ZORBAX RRHD 300SB-C3, 1.8 µm Time (min) %B 0 10 2.5 70 1 1.2 1.4 1.6 1.8 2 2.2 2.4 min mau 12 ZORBAX RRHD 300-Diphenyl, 1.8 µm 10 8 6 4 Arrows indicate better separation resolution of ZORBAX RRHD 300-Diphenyl 2 1 1.2 1.4 1.6 1.8 2 2.2 2.4 min TB# 5990-9668EN 21
NEW! AdvanceBio Peptide Mapping Column for HPLC and UHPLC: 2.7um Superficially Porous 120A pore size 600 bar pressure limit 2um frit to reduce clogging Greater analytical confidence: Each batch is tested with a rigorous peptide mix to ensure suitability and reproducibility Save Time: 2 to 3 times faster than fully porous particles Increased Flexibility: Highly compatible with TFA and formic acid mobile phases for efficient LC/MS analysis 22
Superficially Porous Particle Technology Decrease the diffusion time for macromolecules and limit the diffusion path! The particle has a solid core (1.7um) and porous outer layer with a 0.5um diffusion path Reduces secondary interactions & enables selective separation for a wide range of peptides..
Chromatographic Comparison BSA tryptic digest 2.7um AdvanceBio Peptide Mapping, 2.1 x 150mm, gradient adjusted for length mau 70 60 50 40 30 20 10 104 peaks 319 Bar Greater Rs, increased sensitivity 0 0 2.5 5 7.5 10 12.5 15 17.5 20 min Vendor B, 1.7um, C18, 2.1 x 100mm, gradient adjusted for length mau 40 30 97 peaks 476 Bar Less Rs, decreased sensitivity 20 10 0 0 2 4 6 8 10 12 Conditions: water/acn (TFA), 40C,.3 ml/min min 24
Peptide Mapping for LC/MS TB# 5991-1813EN mau 300 250 Digested EPO Protein Glycopeptide mapping Agilent LC/MS 100% sequence coverage 200 150 100 50 0 0 5 10 15 20 25 30 Digested EPO (recombinant humanized) separated on a 2.1 x 250mm AdvanceBio Peptide Mapping Column Mobile phase: A-water (0.1%FA), B- ACN (0.08%FA), temp: 55C, Flow: 0.5mL/min min Excellent separation capability for generating peptide maps of highly desired proteins, i.e., glycopeptide mapping during fast analysis times Provides compatibility with TFA and Formic acid mobile phases for LC/MS analyses Pressure maintained below 600bar for 250mm column length 25
Poroshell 300 300 A pore size Stablebond chemistry available in C-3, C-8,C-18, and C-18 Extend 5 um particle size Porous Shell SOLID CORE 5 m
High Flow Rates with 2.1 mm ID Poroshell for High Resolution and Fast Separations Columns: Poroshell 300SB-C18 2.1 x 75 mm, 5 m MP: A: 0.1% TFA B: 0.07% TFA in ACN Gradient: 5 100% B in 1.0 min. Flow Rate: 3.0 ml/min. Temperature: 70 C Pressure: 250 bar Detection: UV 215 nm 1 2 3 4 5 6 7 8 TB# 5989-9899EN Sample: 1. Angiotensin II 2. Neurotensin 3. Rnase 4. Insulin 5. Lysozyme 6. Myoglobin 7.Carbonic Anhydrase 8.Ovalbumin 0 0.5 1.0 Time (min)
Reversed Phase Heavy and light chain analytical characterization Use of 1200 LC, Poroshell 300SB columns and UV detection to characterize antibodies TB# 5989-0604EN 28
1.449 1.739 1.036 1.354 1.837 0.901 1.264 1.430 1.497 3.012 1.741 1.395 1.860 1.077 1.289 0.976 1.463 1.523 More Poroshell Bonded Phases Provide Selectivity Options to Enhance Resolution: mau 400 300 200 100 0 DAD1 A, Sig=215,16 Ref=360,100 (05080223.D) 1 5, 6 2 3 4 0DAD1 A, Sig=215,16 Ref=360,100 0.5 (05080225.D) 1 1.5 2 2.5 mau 400 300 200 100 Poroshell 300SB-C18 Poroshell SB-C18, 2.1 x 75 mm 2.1 x 75 mm Poroshell 300SB-C3 Poroshell SB-C3, 2.1 x 75 mm 2.1 x 75 mm 7 9 8 5 1 3 2 4 9 7 6 8 5, 6 4 5 4 6 1.5 7 7 Samples: 1. Angiotensin II 2. Neurotensin 3. RNase A 4. Insulin B Chain min 5. Insulin 6. Cytochrome C 7. Lysozyme 8. Myoglobin 9. Carbonic Anhydrase 0 0 0.5 1 1.5 2 2.5 1.5 min Column: Agilent Poroshell (2.1 x 75 mm); Temp.: 70 0 C; Flow: 0.5 ml/min; Det: UV 215 nm Mobile Phase: A= 0.1% TFA/H 2 O, B= 0.07% TFA/ACN; Gradient: 5-100% B in 3.0 min Changing from SB-C18 to SB-C3, within the Poroshell family results in resolution of peaks 5 and 6, still in 3 min!
PLRP-S Polystyrene divinylbenzene bead Available in 100, 300, 1000, and 4000A pore sizes Particle sizes 3, 5, 8, 10, and higher Various geometries from Capillary to preparative ph 2-14 stable
25-bp Ladder Double Stranded DNA Ladder 1 HPLC 100 50 12% PAGE PLRP-S 100Å PLRP-S 300Å 25 100 50 25 1 1 Column: PLRP-S 150x2.1mm ID Eluent A: 100mM TEAA Eluent B: 100mM TEAA, 50% ACN Gradient: 12.5%-50%B in 150 mins Flow Rate: 200µl/min 1 1 Pore Size Resolving Range PLRP-S 1000Å 100 50 100Å 300Å 50-70 bp Up to 250-300 bp 25 1000Å Up to 400-450 bp 1 1 4000Å >500 bp PLRP-S 4000Å 100 50 25 time
In Summary Agilent has many options for the analysis of bio-molecules and will continue to develop new products to address this growing market.
NEED ASSISTANCE? LC Column help desk 1-800-227-9770 orders customer service (option 1,1) technical support applications assistance (option 3,3,2) lc-column-support@agilent.com 33
BioHPLC Columns on the Agilent Website To learn more and order online visit www.agilent.com/chem/biohplc