Peptide Mapping. Hardware and Column Optimization

Transcription

1 Peptide Mapping Hardware and Column Optimization 1 Peptide Map: definition Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; ormass SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases. 2 1

2 ICH Guidelines for Peptide Map Peptide map Selective fragmentation of the product into discrete peptides is performed using suitable enzymes or chemicals and the resulting peptide fragments are analyzed by HPLC or other appropriate analytical procedure. The peptide fragments should be identified to the extent possible using techniques such as amino acid compositional analysis, N-terminal sequencing, or mass spectrometry. Peptide mapping of the drug substance or drug product using an appropriately validated procedure is a method that is frequently used to confirm desired d product structure t for lot release purposes. Source: 3 Innovation: 1290 Infinity Quaternary Pump Quaternary Pump with Binary Pump-like Performance 1290 Infinity Quaternary Pump Boost Performance Highest accuracy and precision for composition and flow, with exceptionally low delay volume Save Time Accelerates transfer of existing HPLC methods to UHPLC Reduce Costs Outstanding UHPLC performance at HPLC-like costs of ownership 4 2

3 Agilent 1290 Infinity Quaternary Pump Specifications & Benefits Power Range For any kind of analysis Composition Accuracy and Precision < 0.15 % RSD or 0.02 min SD ± 0.4 % (1-99 % Composition B) High RT precision in gradient runs Flow Accuracy and Precision < 0.07 % RSD or 0.01 min SD ±1.0 0%o or 10µ µl High RT precision in isocratic runs Composition Range 1-99 % Wide analytical range Delay Volume < 350 µl For fast quaternary gradients 1290 Quaternary Pump Flow Diagram 6 3

4 Composition Accuracy and Precision Step Gradient Analysis with Tracer Different mixtures (H2O, MeOH, ACN) at different pressures and different flow rates Full application range! 7 Multipurpose valve functions for highest comfort Extra Mixing Volume for lowest baseline ripple (TFA applications) 8 4

5 Why is delay/dwell volume important 1. Different dwell volumes result in a RT time shift (the time for the mobile phase to reach the column head) 2. Different dwell volume could effect resolution (peaks spends different time under isocratic/gradient conditions) 3. additionally, the dwell volume effects the gradient shape (dispersion effects, flush out behavior => the programmed gradient becomes deteriorated) 4. Therefore even with the same geometrical delay volume the chromatograms could look different on different systems 5. The dwell volume has an big impact for narrow bore applications, especially combined with fast gradient 9 Dwell volume determination Programmed W. Dolan LCGC Vol 24, No 5,

6 1290 Quaternary Pump with J380 Mixer and bypass J380 mixer Note different transition volumes Bypass Mixer 335 ul delay J380 Mixer 480 ul delay 11 TFA Mixing Noise: Effect of JetWeaver Mixer Without Mixer With Mixer 12 6

7 1290 Infinity Quaternary Pump - BlendAssist Simple tool for online-dilution of modifiers and gradient set-up You need different concentrations of modifiers in your analysis, would like to have just one stocksolution and do online dilution to profite from the quaternary mixing capability of your pump? Here is a simple tool BlendAssist! Water Water 1% TFA Desired method conditions - example: 1. 5 to 95% gradient of ACN with 0.1% TFA in Water and 0.08% TFA in ACN % gradient of ACN with 0.5% TFA in Water and 0.4% TFA in ACN ACN ACN 1% TFA Without BlendAssist you need to either pre-mix the required solvents or by using stock-solutions of TFA in Water and ACN to program complex gradients (%A, B, C, D). With BlendAssist: just program your binary organic/aqueous gradient and define the dilution factor! Infinity Quaternary Pump - BlendAssist Simple tool for online-dilution of modifiers and gradient set-up 14 7

8 Analysis of Glucocorticoids - Comparing dynamically mixed mobile phase vs. user influence Norm A:B=70: dynamically mixed mobile phases A:B=70:30 min Norm premixed mobile phases (same user) min Infinity Diode-Array Detector 16 8

9 Dispersion volume - Why it makes no sense to specify a geometrical volume Simple example: disperision in a straight tube Dispersion: 2 4 r F L 24 D m Volumetric peak variance F: Flow rate D m : Molecular diffusion coefficient L: Capillary length Derived from Aris, Taylor and Golay equation Same geometrical volume (V 1 = V 2 ), but totally different dispersion volumes V 1 V 2 R. Tijssen, Mechanism and Importance of Zone-Spreading, in Handbook of HPLC, Vol 78, 1998, Marcel Dekker, NY 17 Why do we need a new cell technology? Short 2.1 mm ID column 10 mm pathlength 13 µl volume + = short path-length (3 mm, 2 µl) How to achieve smaller cell volume? long path-length (10 mm, 0.5 µl) High light transmission => low noise Optofluidic Waveguides - Long path length - Small cell volume - High light transmission Low S/N Low light transmission => high noise Highest S/N Low S/N 18 9

10 Max-Light Cartridge Cell - Optofluidic waveguides Non-coated fiber (fused silica) High Light Transmission due to Total-Internal Reflection (TIR) principle (~ 100 % Light efficiency) Benefits: Highest Sensitivity (S/N) with small cell volumes (dispersion effects) More reliable and robust peak integration (automated) due to nearly no Refractive Index and thermal effects (solvent temperature) Coating free fused silica (no special care instructions or smiling baseline effects) Easy cell selection (one cell for all major applications) Cartridge design for ease of use 19 Reversed-Phase Separation brief overview Reversed phase HPLC is used for the analysis of complex biologics (protein therapeutics, cell lysates, serum, etc) intact proteins peptide mapping amino acid analysis Detailed information about the primary structure Including disulphide shifting AdvanceBio Peptide Mapping 120 Mass spec compatible (no salt buffer) The pore size of the media must be matched to the size of the molecule Intact Protein Analysis s 300Å Peptide Mapping 120Å Confirm protein identity (primary structure analysis) Look for impurities Develop impurity profiles Peptide mapping Amino acid analysis ZORBAX RRHD 300SB-C

11 Reversed-Phase Product Families Particle Porosity Functionalities Particle Pore Size Application Sizes ZORBAX Stable Bond Silica Fully Porous C18, C8, C3, CN 3.5um, 5um 300A Low ph ZORBAX 300Extend Silica Fully Porous Extend-C18 3.5um, 5um 300A High ph ZORBAX RRHD Silica Fully Porous SB-C18, SB-C8, 1.8um 300A High speed SB-C3, Diphenyl High resolution ZORBAX Eclipse AAA Silica Fully Porous C18 3.5um, 5um 80A Amino Acids AdvanceBio Peptide Mapping Silica Superficially EC-C18 2.7um 120A Peptide Mapping Poroshell 300 Silica Superficially SB-C18, SB-C8, SB-C3, Extend-C18 PLRP-S PS/DVB Fully Porous 3um, 5um, 8um, 10um, 12um, 18um PLRP-S PS/DVB Fully Porous 5um, 8um, 10um, 30um 5um 300A High speed High resolution 100A, 300A Peptides, oligos, proteins 1000A, 4000A Proteins and DNA VariTide RPC PS/DVB Fully Porous 6um 200A Synthetic peptides Emphasized column technology Page Protein/Peptide Separations by Reversed-phase Larger Molecules = Slower Diffusion So, a need to decrease the diffusion time for macromolecules! To improve, we can increase the Diffusion Rate by: elevating operating temperature decreasing solvent viscosity and, or

12 Decrease the Diffusion Distance! 1. Rapid Resolution High Definition (RRHD) 300 Family (C3, C8, C18, diphenyl) Developed very small (totally porous) particles (<2 um) UHPLC RRHD 1.8um,, Superior efficiency Very narrow bands Ultra high resolution Fast flows Short column lengths (pressure constraint) 2. Poroshell 300 and AdvanceBio120 Peptide Mapping Limit diffusion distance into a particle (shell particle) 5um Poroshell um AdvanceBio120 Peptide HPLC Superior efficiency Very narrow bands Ultra high resolution Fast flows Long column lengths (no pressure constraint) ZORBAX RRHD 300Å 1.8 µm Columns Application Objectives: The ZORBAX RRHD 300Å 1.8 µm columns are designed for primary structure analysis - confirming protein identity, quantifying post translational modifications and fingerprinting impurity profiles by reversed-phase UHPLC. With UHPLC, 1200 bar stability, faster separations are achieved to improve productivity but with no compromise in data accuracy. Primary benefit of these Agilent RRHD reversed-phase columns for separations of proteins is increased resolution with shorter analysis times. Resolution is critical in these separations due to the small differences in structure between the candidate protein and the impurities. RRHD provides HPLC and UHPLC level productivity for large molecules EN 24 12

13 Four Rapid Resolution High Definition Phases Family of Four Reversed-Phase Ligands C18 C8 C3 Diphenyl Lysates Increasing protein size/hydrophobicity Small Proteins ZORBAX 300Å, 1.8 µm Ligand C18 C8 C3 Diphenyl Application Small intact proteins/peptide maps/ cell lysates Intact proteins Larger /hydophobic proteins, including MAbs Unique selectivity Agilent 1290 Infinity um Poroshell 300 and 2.7um AdvanceBio 120 Poroshell 300 AdvanceBio Peptide Mapping um 0.50 um um um 1.70 um 2.70 um Use AdvanceBio Peptide Mapping 120 for peptide maps containing small proteins and peptides Peptide digests of proteins or monoclonal antibodies Highly compatible with TFA and formic acid mobile phases for efficient LC/MS analysis Use Poroshell 300 for intact protein analysis and large peptide fragments Does the sample contain all polypeptides or larger? Poroshell 300 can be used with very large proteins/monoclonal antibodies, complex biologicals 26 13

14 4 4/4/2013 Reversed-Phase Intact Protein Separations RRHD 300 High Efficiency ultra fast protein separations 1.8 um RRHD 300 columns for the separation of large peptides, proteins and antibodies. RRHD 300 particles provide HPLC and UHPLC level productivity for large molecules Sharper peaks and fast, efficient separations with large proteins and peptides Stable Bond phase technology 27 RRHD 300 SB-C18 Intact protein Analysis - Fast Separation of Insulin Separation of Isoforms Heat- degraded Characterization mau A mau 80 B Oxidized insulin Chain B insulin Heat-treated insulin (55C for 24 h) Oxidized insulin Chain A Subspecies of oxidized Insulin chain B Forced degradation products Injection 2ul, flow: 1.0mL/min Press: 680 bar Gradient 35-50%B, 0-4 min. 33%B, 4-5min. 10 Injection 4ul flow:1.0ml/min Press: 680 bar Gradient 35-50%B, 0-5 min min min. min min Insulin, a small protein biotherapeutic, was used in this study together with its isoforms and degradation products to demonstrate utility for rapid separation profiling and characterization of a biopharmaceutical API. Using a ZORBAX RRHD300 SB-C18 column (2.1 x 50mm) and optimized chromatographic condition for ultra fast analysis, chromatograms in A and B show excellent resolution and selectivity for resolving insulin and its products in run times under 3 minutes. Additionally, low TFA addition (0.01% ) in ACN (see Experimental) make the separations highly transferable to LC/MS analysis

15 Reversed-Phase Peptide Separations AdvanceBio Peptide Mapping 120 Porous Shell 0.5um SOLID CORE 1.7um 2.7 um AdvanceBio Peptide Mapping 120 columns for the separation of small proteins, polypeptides and peptides. AdvanceBio Peptide Mapping 120 particles have a solid silica core with a superficially porous surface with a 120Å pore size This results in more efficient mass transfer, sharper peaks and fast, efficient separations with small proteins and peptides 29 What Parts of the Knox/VanDeemter Equation Are Affected by the Column Particle A term eddy diffusion and flow distribution Impacted by the column particle Column particle size and column packing quality impact this A tight particle size distribution improves the A term B term longitudinal diffusion Impacted by column particle This is impacted by column pore size C term mass transfer component Impacted by the column particle and its properties Mass transfer is improved by using smaller particles with shorter diffusion paths Improved with a superficially porous particle Allows high efficiency at high flow rates 30 15

16 31 Comparison of Three Columns Tryptic Digest Zorbax 1.8um, 2.1mm x 100mm PoroShell 300 AdvanceBio Peptide Mapping

17 Premixed versus Blend Assist; 0.05%TFA-Water/0.04%TFA-ACN Premixed Note Fine Structure Blend Assist 33 Duplicate Runs Premixed 0.05%TFA-Water/0.04%TFA-ACN 34 17

18 Triplicate Runs Blend Assist: Stock 1%TFA; 0.05%TFA Water/0.04%TFA ACN 35 Overlay of Blend Assist TFA percent Differences: Blue: 0.05%TFA-Water/0.04%TFA-ACN Red: 0.1%TFA-Water/0.08%TFA-ACN 36 18

19 Maximum Peaks Plotted for Three Columns 37 Lab Session Goals Setup 1290-quaternary system to run tryptic digest gradient on AdvanceBio Peptide Mapping 120 column Use Blend dassist to create Gradient Use Openlab Chemstation to create virtual sequence set of data to compare columns 38 19

20 Questions 39 20