4/4/2013. BioHPLC columns. Paul Dinsmoor Biocolumn Technical Specialist. April 23-25, Size Exclusion BioHPLC Columns

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1 BioHPLC columns Paul Dinsmoor Biocolumn Technical Specialist April 23-25, 2013 Size Exclusion BioHPLC Columns 1

2 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 Improved Efficiency With Smaller Particles Column Pressure Bio SEC-3 (7.8x300mm) Bio SEC-5 (7.8x300mm) 93 bar 45 bar Peak Protein SEC-3, 300Å (7.8x300mm) SEC-5, 300Å (7.8x300mm) Agilent Bio SEC-3, 300Å, 7.8x300mm Agilent Bio SEC-5, 300Å, 7.8x300mm Min 1 Thyroglobulin li BSA Dimer BSA Ribonuclease A Uracil Column: Bio SEC-3 300Å and Bio SEC-5 300Å Buffer: 150 mm Phosphate buffer, ph 7 Flow rate: 1.0 ml/min for 7.8x300 mm Temperature: Ambient (~23 C) Detection: UV 214nm Injection: 10 µl (3 L for 4.6x300 mm) Sample: 1) Thyroglobulin (1.0 mg/ml), 670 kd; 2) BSA dimer, 132 kd; 3) BSA (1.0 mg/ml), 66 kd; 4) Ribonuclease A (1.0 mg/ml), 13.7 kd, and 5) Uracil (2.5 g/ml), 120D. 2

3 Fast separation of dimer from monomer using the Agilent Bio SEC Å 7.8 x 150 mm column ml/m ml/min ml/min monomer Column: Agilent Bio SEC-3, 7.8 x150mm Sample: mab (2mg/ml) Injection: 5ul Flow rate: , and d2ml/min (56b bar, 75b bar, 105b bar) Eluent: 150mM sodium phosphate Detection: 220nm dimer Flow Rate Resolution Monomer/Dimer Monomer Efficiency Percentage Dimer 1.0ml/min , ml/min , ml/min , Fast SEC TB EN Column Choice: Resolving Ranges Peptides Insulin Myoglobin Proteins Ovalbumin BSA IgG Thyroglobulin 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 D 1kD 10kD 100kD 1,000kD 10,000kD 3

4 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 4

5 Agilent Bio IEX Columns Comparing Separations on Each Particle Size 1.7 m 3 m 5 m Peak N 10 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, NP 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-NP Min Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies Columns: Agilent Bio MAb, NP10, 4.6x250 mm Mobile phase: A, 10 mm phosphate, ph 7.5 B, A + 0.1M NaCl A Gradient: A) 15-75%B in 30 min B) 15-65%B in 30 min C) 15-55%B in 30 min B D) %B in 30 min E) 15-40%B in 30 min C Flow rate: 0.8 ml/min Sample: Monoclonal Antibody D Injection: 10 L (1.5 mg/ml) Temperature: 25 o C E Detection: UV 214 nm Min 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. 5

6 Reverse Phase BioHPLC Columns NEW! Zorbax 300 SB RRHD for Proteins and Peptides! Stablebond 300 silica C-18, C-8, C-3, 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 6

7 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* ,500 21,000 32, ,500 14,000 24, ,500 17, ,200 7,000 12, N.A. 4,200 6, N.A. 2,100 2,500-33% -50% -67% Analysis Time Peak Volume -80% Solvent -90% Usage * Reduction in analysis time compared to 150 mm column 300SB-C18 RRHD Protein Digest TB# EN 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 14 7

8 Fast Separation of Reduced and Alkylated MAb Conditions optimized for the ZORBAX RRHD 300SB-C3 Column mau CHO Cell Derived MAb Light chain Heavy chain 1 Heavy chain 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# EN Time %B (min) Comparison C3 and Diphenyl Phases Ribonuclease A, Cytochrome C and Lysozyme (3 mg/ml) mau ZORBAX RRHD 300SB-C3, 1.8 µm Time (min) %B mau min ZORBAX RRHD 300-Diphenyl, 1.8 µm Arrows indicate better separation resolution of ZORBAX RRHD 300-Diphenyl min TB# EN 16 8

9 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 17 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.. 9

10 Chromatographic Comparison BSA tryptic digest 2.7um AdvanceBio Peptide Mapping, 2.1 x 150mm, gradient adjusted for length mau Greater Rs, increased sensitivity peaks Bar min Vendor B, 1.7um, C18, 2.1 x 100mm, gradient adjusted for length mau Less Rs, decreased sensitivity peaks 476 Bar Conditions: water/acn (TFA), 40C,.3 ml/min min 19 Peptide Mapping for LC/MS TB# EN mau Digested EPO Protein Glycopeptide mapping Agilent LC/MS 100% sequence coverage 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 hl desired d 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 20 10

11 Poroshell 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-C 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 TB# EN Sample: 1. Angiotensin II 2. Neurotensin 3. Rnase 4. Insulin 5. Lysozyme 6. Myoglobin 7.Carbonic Anhydrase 8.Ovalbumin Time (min) 11

12 6 4/4/2013 Reversed Phase Heavy and light chain analytical characterization Use of 1200 LC, Poroshell 300SB columns and UV detection to characterize antibodies TB# EN 23 More Poroshell Bonded Phases Provide Selectivity Options to Enhance Resolution: mau mau 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 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 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! 12

13 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 PLRP-S Continued: Features ph 1-14 Extreme buffer concentrations High temperature stability Durable and Resilient Benefits Acid and base cleanup Typically y < 8M 200 C Long Lifetimes Inherently hydrophobic so does not require a bonded alkyl chain to confer hydrophobicity 1000A & 4000A pores Avoids typical silica problems of silanol group Analysis of very large biomolecules or high speed separations 13

14 25-bp Ladder Double Stranded DNA Ladder 1 HPLC % PAGE PLRP-S 100Å PLRP-S 300Å 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 Å 300Å bp Up to bp Å Up to bp Å >500 bp PLRP-S 4000Å 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. 14

15 NEED ASSISTANCE? LC Column help desk orders customer service (option 1,1) technical support applications assistance (option 3,3,2) 29 BioHPLC Columns on the Agilent Website To learn more and order online visit 15

16 Product Literature New BioHPLC Column Selection Guide # New AdvanceBio Peptide Column Brochure # EN 32 16

17 New BioHPLC SEC New Zorbax SB 300A and IEX Brochure 1.8um RRHD Brochure # # EN Biomolecule Purification Brochure # New Protein Identification And Impurity Profiling # EN 17

18 New LC Handbook # New Primer: Recombinant Protein Characterization # Part Numbers 18

19 Agilent Bio SEC-5 Ordering Information Agilent Bio SEC-3 Ordering Information 19

20 Agilent Bio IEX Ordering Information AdvanceBio Peptide Mapping Columns Ordering Info Dimension AdvancedBio Peptide Mapping Column Part # s #s 4.6X150mm X150mm X150mm X 250mm X100mm mm Guard column mm Guard column mm Guard Column

21 Poroshell 300 Ordering Information 41 Zorbax 1.8um 300SB RRHD Ordering Info 21

22 Zorbax 300SB Ordering Info 43 PLRP-S Ordering Info 44 22