BioHPLC columns. Tim Rice Biocolumn Technical Specialist

Transcription

1 BioHPLC columns Tim Rice Biocolumn Technical Specialist

2 AU Typical Application Areas Size Exclusion: Aggregation Analysis Ion Exchange: Charge Isoform Analysis Monomer Dimer Minutes Reversed Phase: Separation of light and heavy chains Peptide Mapping: Post-translational modifications 2

3 Size Exclusion BioHPLC Columns

4 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

5 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 BSA Dimer BSA Ribonuclease A Uracil Agilent Bio SEC-5, 300Å, 7.8x300mm Min Technology: Small Particle SEC Columns Results: Higher Resolution SEC Separations 5

6 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

7 Mechanism of SEC Separation Pore Size Determines Linear Separation Range FLOW Page 7

8 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 D 1kD 10kD 100kD 1,000kD 10,000kD

9 Ion Exchange BioHPLC Columns

10 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

11 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, 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-NP1.7 Peak N 10 m Min

12 AU Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies (Acidic Isoforms) weakly bound (Basic Isoforms) Strongly bound Minutes Column: Agilent Bio MAb, NP5, 4.6mm x 250mm Buffer A: 10 mm Sodium Phosphate, ph 7.50 Buffer B: A 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

13 Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies A B C D E 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) %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.

14 Agilent Bio WCX: ph An increase in ph reduced the retention time. An increase in ph increased resolution of earlier peaks (circled). Increased resolution of early peaks 14

15 Reverse Phase BioHPLC Columns

16 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

17 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

18 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% -80% -90% Analysis Time Peak Volume Solvent Usage * Reduction in analysis time compared to 150 mm column

19 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 19

20 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 (min) 0 1 %B

21 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 min mau 12 ZORBAX RRHD 300-Diphenyl, 1.8 µm Arrows indicate better separation resolution of ZORBAX RRHD 300-Diphenyl min TB# EN 21

22 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

23 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..

24 Chromatographic Comparison BSA tryptic digest 2.7um AdvanceBio Peptide Mapping, 2.1 x 150mm, gradient adjusted for length mau peaks 319 Bar Greater Rs, increased sensitivity min Vendor B, 1.7um, C18, 2.1 x 100mm, gradient adjusted for length mau peaks 476 Bar Less Rs, decreased sensitivity Conditions: water/acn (TFA), 40C,.3 ml/min min 24

25 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 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

26 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

27 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)

28 Reversed Phase Heavy and light chain analytical characterization Use of 1200 LC, Poroshell 300SB columns and UV detection to characterize antibodies TB# EN 28

29 More Poroshell Bonded Phases Provide Selectivity Options to Enhance Resolution: mau DAD1 A, Sig=215,16 Ref=360,100 ( D) 1 5, DAD1 A, Sig=215,16 Ref=360, ( D) 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!

30 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

31 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

32 In Summary Agilent has many options for the analysis of bio-molecules and will continue to develop new products to address this growing market.

33 NEED ASSISTANCE? LC Column help desk orders customer service (option 1,1) technical support applications assistance (option 3,3,2) 33

34 BioHPLC Columns on the Agilent Website To learn more and order online visit