BioHPLC Column Selection Guide Cover - Agilent Technologies

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Biomolecule Separations Tips & Tools 8 www.agilent.com/chem/biohplc Peptide Separations Peptide Mapping Peptide mapping is required for the characterization of proteins. It is used to confirm the identity of a protein and to identify and quantify post-translational modifications. The purified protein is first digested using an enzyme, such as trypsin, yielding a range of peptide fragments. The specificity of the enzyme cleavage produces a fingerprint of peptides which is characteristic of that protein. Identification of the peptide fragments confirms the identity of the protein, and changes in the profile of the peptide digest can be used to identify post-translational modifications to that protein that may have occurred during the manufacturing or purification processes. Reversed-phase UHPLC/HPLC is the preferred technique for the analysis of peptide digests with either MS or UV detection. LC/MS is used for the identification of the peptide fragments and determination of sequence coverage whereas LC/UV is more commonly used for peptide map comparisons in the monitoring/QC segments. To achieve sufficient resolution for quantification and identification, longer column lengths or higher efficiency particles such as the sub-2 µm ZORBAX RRHD, or superficially porous Poroshell are recommended. Peptide digests are complex mixtures, and for complete coverage, i.e. resolution of the individual peptides, a high efficiency/high resolution column is required. The peptide fragments can range in size and hydrophobicity, so Agilent offers several columns for peptide mapping. There are three options: pore sizes, particle sizes, and superficially porous and fully porous for UHPLC separations. Capillary electrophoresis is an alternative technique to liquid chromatography for the separation of complex peptide mixtures. Further information can be found in the following Case Study: An orthogonal view of peptide mapping – analysis of bovine serum albumin digest using capillary electrophoresis and quadrupole time-of-flight mass spectrometry (5990-7631EN) www.agilent.com/chem/library
Increased resolution for peptide mapping Column: ZORBAX 300SB-C18 858750-902 2.1 x 100 mm, 1.8 µm Mobile Phase: A: 0.1% TFA B: 0.01% TFA + 80% ACN Flow Rate: 0.5 mL/min Gradient: 2% B for 1 min, 2 to 45% B for 8.8 min, 45 to 95% B for 0.2 min, 95% B for 2 min, 95 to 2% B for 0.2 min Temperature: 50 ºC Detector: 1290 Infinity LC with diode array detector at 280 nm Sample: Enzymatic protein digest (MAb) Peptide Mapping Column Selection Application Technique Agilent Columns Notes Large peptide fragments/hydrophobic peptide core Small hydrophobic peptides Biomolecule Separations 400 bar HPLC Poroshell 300 SB-C18 ZORBAX 300SB-C18, 3.5 µm Agilent 1200 LC 600 bar UHPLC Poroshell 300 SB-C18 Agilent 1260 Infinity LC and 1260 Infinity Bio-inert Quaternary LC 1200 bar UHPLC ZORBAX RRHD Poroshell 300 SB-C18, 1.8 µm Agilent 1290 Infinity LC 400 bar HPLC Poroshell 120 EC-C18 Poroshell 120 SB-C18 Agilent 1200 LC 600 bar UHPLC Poroshell 120 EC-C18 Poroshell 120 SB-C18 Agilent 1260 Infinity LC and 1260 Infinity Bio-inert Quaternary LC 1200 bar UHPLC Poroshell 120 EC-C18 Poroshell 120 SB-C18 Agilent 1290 Infinity LC If you have an Agilent 1290 Infinity LC in your lab we recommend starting with a ZORBAX RRHD 300SB-C18 column to screen your peptide map. Norm 350 300 250 200 150 100 50 0 -50 1 2 3 4 5 6 7 8 9 min The longer 100 mm Agilent ZORBAX RRHD 300SB-C18 column provides maximum resolution for protein digests – in this sample the total run time, including washing and equilibration, is under fifteen minutes. Recommended column choices determined by system/column pressure maximum and peptide size/hydrophobicity. BioHPLC COLUMNS 9
Page 1 and 2: Agilent BioHPLC Column Selection Gu
Page 3 and 4: BIOCOLUMN SELECTION GUIDELINES From
Page 5 and 6: Column Selection Flow Chart Biocolu
Page 7 and 8: Protein Column Selection Guide Appl
Page 9: Separation of charge variants of hu
Page 13 and 14: Preparative scale purification of L
Page 15 and 16: DNA and RNA Oligonucleotide Column
Page 17 and 18: Broad Distribution Biomolecules Car
Page 19 and 20: ReveRseD-PHase HPLC Confidently per
Page 21 and 22: Sterically Protected 300StableBond
Page 23 and 24: Improved reproducibility of monoclo
Page 25 and 26: Short-chain ZORBAX 300SB-C3 is stab
Page 27 and 28: ZORBAX 300Å StableBond Hardware De
Page 29 and 30: LC/MS analysis of angiotensin on Ex
Page 31 and 32: Tips & Tools ZORBAX 300Å Extend-C1
Page 33 and 34: Poroshell 300 columns separate prot
Page 35 and 36: Monoclonal IgG1 chains: Separation
Page 37 and 38: Poroshell 120 • 120Å pore size f
Page 39 and 40: 0 µm 0 µm 0 µm A B C Scanning el
Page 41 and 42: Exploiting chemical stability - TFA
Page 43 and 44: Temperature as a tool to enhance ma
Page 45 and 46: ZORBaX amino acid analysis (aaa) Co
Page 47 and 48: High resolution of 24 amino acids u
Page 49 and 50: Ion-Exchange Column Selection Appli
Page 51 and 52: Consistent ion-exchange MAb separat
Page 53 and 54: Column Specifications Tips & Tools
Page 55 and 56: Weak cation-exchange chromatography
Page 57 and 58: PL-SAX Strong Anion-Exchange Column
Page 59 and 60: Analysis of representative whey pro
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Standard protein separation Column:
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Agilent Bio-Monolith HPLC Column Se
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Baseline expansion of a separation
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size exclusion Chromatography (seC)
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Column Specifications Agilent Bio S
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Comparison of Agilent Bio SEC-3 and
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Column Length size exclusion Chroma
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Column Specifications Agilent Bio S
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Calibration curves - Bio SEC-5 Colu
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ProSEC 300S • Mechanically robust
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Overlay of UV and light scattering
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Retention volume versus log (MW) fo
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Agilent PL aquagel-OH SEC Columns f
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Heparin Column: 2 x PL aquagel-OH 3
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Polyethylene Glycol/Oxide standards
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aFFiniTY CHROMaTOGRaPHY Agilent Bio
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Rapid human polyclonal IgG quantifi
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Multiple Affinity Removal System af
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Illustration of high abundance prot
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mRP-C18 High-Recovery Protein Colum
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MeTHOD DeveLOPMenT ZORBAX Column Me
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Reversed-Phase LC/MS Methods Method
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Method Development Ionic Strength:
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Method Development After the initia
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Tips & Tools Agilent offers a varie
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Human serum: Low abundance protein
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2-D LC/MS Analyses Using ZORBAX Cap
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ZORBAX Bio-SCX Series II Capillary
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ZORBAX HPLC Capillary Columns (glas
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Microbore HPLC for sensitive peptid
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BioPharmaceutical Lifecycle Reverse
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ZORBAX 300Å StableBond ZORBAX Prep
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PLRP-S Prep to Process Application
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Prep to Process PLRP-S Size (mm) Pa
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PL-SAX AND PL-SCX FOR PREP TO PROCE
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Prep to Process PL-SAX and PL-SCX D
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Crude peptide screen Column: VariTi
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CARTRIDGE COLUMN SYSTEMS Cartridge
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USP DESIGNATIONS - BIOHPLC COLUMNS
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BioHPLC Columns Literature Title Ag
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BioHPLC Columns Literature Title An
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BioHPLC Columns Literature Title Co
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BIOPHARM DEFINITIONS A active start
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dimer A polymer made up of two iden
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K-L ligase An enzyme that causes mo
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S secondary structure In proteins,
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Poroshell 300 Citations Matilainen,
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Agilent 1260 Infinity Bio-inert Qua
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